Trial®

Help to Hold the Hope1R13AI115934-01 to Mariano Garcia-Blanco and Carlos A. Sariol

Dear Participants,

The main objective of this meeting is to promote the exchange of scientific information between academia, government and industry in the area of dengue research. This meeting has acquired an essential niche as one of the conferences available to the international Dengue community. We expect it will serves a distinct function by providing a mid-format meeting at which the latest unpublished research can be discussed in depth.

Particularly relevant are the unpublished results of all five dengue vaccine candidates presently in clinical trials, including result from Phase III safety study recently completed in our Latin American Region. We encourage all of you to promote the networking and to establish new collaborations and to open new opportunities for the students and young investigators.

Dear Participants,

The Pan American Dengue Research Network (Pan Dengue Net) welcomes all of you and appreciates your time and contributions, which have made the Fourth Meeting possible.

We feel it is essential to continue to have meetings that give the Pan American dengue research community the opportunity to integrate key aspects from different areas of dengue research (virology, immunology, epidemiology, vector biology, modeling among others), and attract participants from other disciplines as well, allowing valuable cross-fertilization.

These interactions are likely to result in recruitment opportunities for participants who are seeking post-doctoral fellowships or new faculty positions, as well as in fostering North-South productive collaborations. We hope this meeting will contribute to meet these goals.

This year, our event has participants from at least 25 countries from the Americas, Europe, Asia, and Australia. By the end of September, we had 295 registered participants but more significant, 67% are females, Congratulations!

This meeting would not be possible without the contribution of our Sponsors, particularly the Brazil Ministry of Health, the Instituto Evandro Chagas, and the Chief of Arbovirology and Hemorrhagic Fevers Section and Chairman of this Meeting Dr. Pedro Vasconcelos and his team. Also the contribution of Dr. Maurício Lacerda Nogueira as Coordinator and member of the Brazilian crew has been key to the meeting organization.

The scientific success and quality of this meeting is largely attributable to Dr. Eva Harris (just Eva), Chairperson of the Scientific Committee, who as in previous meetings devoted a lot of energy to select the Speakers, to select the members of the scientific committee and to put together the exciting Agenda of this meeting.

We are deeply in debt to all of you, members of our community, who took time away form your daily activities (academic, administrative, scientific, clinical, etc.) to be part of the Organizing and Scientific committees (listed on this agenda) to guarantee the success of this Fourth meeting.

We are already working on the 2016 Pan Dengue Net Fifth meeting. Volunteers are more than welcome! Don't be shy, we will need a lot of help.

If you are a potential Sponsor, please add us to your contributions list for 2016!!

If you think you can help, please contact us with your comments, suggestions and ideas at contactus@denguemeeting.com or through our web site wwwpandenguenet.org

Pedro F.C. Vasconcelos (Brazil) Chairman

Eva Harris (USA) Chairperson

Carlos Sariol (USA/PR) Acting Secretary Jorge Osorio (USA/Colombia) Aravinda de Silva (USA) Jorge Muñoz Jordan (CDC, USA) Cristina Cassetti (USA, NIH) Margarita Ossorio (USA, NIH)

Pedro F.C. Vasconcelos (Brazil) Chairman

Mauricio Lacerda Nogueira (Brazil) Coordinator

Carlos Sariol (USA/PR) Acting Secretary Adriano Mondini (Brazil) Daniele B. A. Medeiros (Brazil) Eunice Lima (Brazil) João Carlos Lopes (Brazil) Sueli G. Rodrigues (Brazil) Giovanini Evelin Coelho (Brazil) Maria Guadalupe Guzman (Cuba) Rosa M. del Angel (Mexico) Cristina Cassetti (USA, NIH) Margarita Ossorio (USA, NIH) Jorge Muñoz Jordan (CDC, USA) Mariano Garcia-Blanco (USA) Janiffer Oliveira Chiang (Brazil)

Eva Harris (USA) Chairperson Pedro F.C. Vasconcelos (Brazil) Mauricio L Nogueira (Brazil) Laura White (USA) Andrea Gamarnik (Argentina) Irene Bosch (USA) Juan Ernesto Ludert (Mexico) Aravinda de Silva (USA) Jorge Osorio (USA/Colombia) Alan D. Barrett (USA) Anna Fernadez Sesma (USA) Cristovam P. Diniz (Brazil) Duane Gubler (Singapore) Elizabeth Hunsperger (USA) Ernesto Marques Jr. (USA) Mauro M. Teixeira (Brazil) Nikkos Vasilakis (USA) Ana B Perez (Cuba) Luiz Tadeu Figueiredo (Brazil) Márcio Teixeira Nunes (Brazil) Jorge Muñoz Jordan (CDC, USA) Maria Guadalupe Guzman (Cuba)

Scott Halstead

PROGRAMMonday 10/20/14 - Morning8:30 am - 12:45 pm

Virology / Antivirals - 85 minRichard Kuhn (USA)Andrea Gamarnik (Argentina)Guilherme Ferreira Silveira- Claudia Santos (Brazil)Sebastian Aguirre (USA)Andrea da Poain (Brazil)

Highlighted Posters (Poster Walk)

ImmunologyEmily Gallichotte (USA)Raphael Zellweger (USA)Cintia Marinho (Brazil)Mariana Gandini (Brazil)Daniel Limonta (Cuba)

Clinical ResearchCynthia Rodriguez (Honduras)Crystyan Siles (Peru)Carlos Narvaez (Colombia)Mayara Marques Carneiro da Silva (Brazil)

Virology/Antivirals

Pathogenesis

COFFEE BREAK 15 minutes

Virology / Antivirals - 95 minM. Garcia-Blanco (USA / Singapore)Aruna Sampath (Unither Virology, USA)Mariana Leguia (Peru)Elsa Damonte (Argentina)Maria Flechas (Colombia) Devika Sirohi (USA)

Monday 10/20/14 - Afternoon2:00 - 5:10 pmPathogenesis - 80 minPedro Vasconcelos (Brazil)Beatriz Sierra (Cuba)Irene Bosch (USA)Yerly Useche (Colombia)Henry Puerta Guardo (USA)

Immunology - 85 minErnesto Marques (Brazil)Eva Harris (USA)Daniela Weiskopf (USA) AmeIia Pinto (USA)Davis Beltran (Panamá)

LUNCH 12:45-2:00 PM

COFFEE BREAK 25 minutes

Tuesday 10/21/14 - Morning8:30 am - 12:40 pm

Immunology/Diagnostics/ Prognostics - 85 minGuadalupe Guzman (Cuba)Elizabeth Hunsperguer (USA)Allan Brasier (USA)Monique da Rocha Queiroz Lima (Brazil)Parichat Duangkhae (USA)

Epidemiology/Phylogenetics - 85 minM. Nogueira Lacerda (Brazil)Jorge Muñoz (USA/Puerto Rico/CDC)Leah Katzelnick (USA)Magelda Montoya (USA)Rosmari Rodriguez (Cuba)

Tuesday 10/21/14 - Afternoon2:00 - 5:10 pmVaccines - 75 minNicholas Jackson (Sanofi)Jorge Osorio (Takeda)Stephen Whitehead/Ricardo Palacios (USA/Brazil)Alexander Schmidt (GSK)Beth-Ann Coller (Merck)

Vaccine/Modeling - 100 minAnna Durbin (USA)Aravinda de Silva (USA)Alex Perkin (USA)Diana Rojas (University of Florida/USA-Colombia)Leah Katzelnick (UK/USA)Laura White (USA)

Diagnostics/PrognosticsIrene Bosch (MIT/USA)Claire Huang (CDC/USA)Angela Argolo (Brazil)Diego Allonso (Brazil)Stalin Vilcarromero (Peru)

VectorNatapong Jupatanakul (USA)José Joaquín Carvajal Cortés (Brazil)Luciana dos Santos Dias (Brazil)Kacey C. Ernst (USA)

Highlighted Posters (Poster Walk)

Epidemiology/PhylogeneticsRaquel Lana (Brazil)Adrienne Guinard (GSK/Belgium)Cynthia Jimenez (Colombia)Mabel Carabali (Colombia)

Vaccine/ModelingHarry Partidos (Takeda)Clemente Diaz (GSK/Puerto Rico)Guido Camargo (Colombia)Milene Silveira Ferreira (Brazil)Melissa Mattocks (USA)Alienys Izquierdo (Cuba)

LUNCH 12:45-2:00 PM

20 minutesCOFFEE BREAK

COFFEE BREAK 15 minutes

Wednesday 10/22/14 - Morning8:30 am - 12:40 pm

Dengue in Brazil Round Table - 80 minKleber Luz (Brazil)Marcio Nunes (Brazil)Giovanini Coelho (Brazil)

Tuesday 10/22/14 - Afternoon2:00 - 5:10 pm

COFFEE BREAK 20 minutes

Clinical Research/Cost Studies - 90 minLuis Villar (Colombia)Federico Narvaez (Nicaragua)Eduardo Undurraga (USA/Chile)Ivony Yireth Agudelo (Colombia)Joao Bosco Siquiera (Brazil)

LUNCH 12:45-2:00 PM

Vector Biology/Ecology/Surveillance - 65 minSarah Anne Jablonski Guagliardo (USA)Catalina Alfonso Parra (USA)George Dimopholous (USA)Amy Morrison (USA/Perú)

COFFEE BREAK 20 minutes

Vector Control II - 105 min

Andrew R. McKemey (USA)Margaret de Lara Capurro (Brazil)Peter Ryan (Australia)Luciano Moreira (Brazil)

Round Table I

Roberto Barrera (USA/CDC/Puerto Rico)Alvaro Eiras (Brasil)Dawn Wesson (USA)

Round Table II

19:00-21:00Welcome Cocktail

Meeting Sessionsand Invited Speakers

SundayOctober 19th

18:00-18:15Opening Ceremony

18:15-19:00Keynote

POSTER SESSIONS5:15-6:45 pm

POSTER SESSIONS5:15-6:45 pm

BELÉM, BRAZIL | OCTOBER 19-22 | 2014

Silvio Urcuqui (Colombia)Viviana Castilla (Argentina)Antonio Dias (UK)Cinthia L. Dionisio (Mexico)Juan Ludert (Mexico)Maria Paulina Cabarcas (Colombia)Ruben Soto Acosta (Mexico)

Marciano Paes (Brazil)Emiliana Silva (Brazil)Victor Fiestas (Brazil)Monique Trugilho (Brazil)Jonas Conde (Brazil)

VIROLOGY/ANTIVIRALSINVITED SPEAKERS

STRUCTURE-FUNCTION ANALYSES OF THE DENGUE VIRUS NS1 PROTEIN1,2 1 1 1 3 3 4 3Richard J. Kuhn , Joyce Jose , Jinsam Chang , Andrew Miller , David Akey , Clay Brown , Theodore C. Pierson , & Janet Smith

1 2 3Markey Center for Structural Biology, Dept. of Biological Sciences, Bindley Bioscience Center, Purdue University, West Lafayette, IN; Life Sciences Institute, 4University of Michigan, Ann Arbor, MI, Laboratory of Viral Diseases, NIAID, NIH, Bethesda, MD

We have recently solved the high-resolution x-ray crystal structures of the dengue virus and the West Nile virus NS1 proteins. The NS1 protein is found as a functional dimer with a hydrophobic and hydrophilic face separated by an extended set of beta strands. The hydrophobic face is suggested to play an intimate role in RNA synthesis by directly contacting membrane replication proteins. As NS1 is also secreted in the form of a lipoprotein complex, this face also serves to organize lipids in the hexamer form of NS1. Several additional structural features of NS1 suggest a role in evading immune surveillance. Mutational and functional studies are being done to interrogate the relationship between the structure and function of the NS1 protein. Surprisingly, many single point mutations render the virus restricted in replication. Many of these detrimental substitutions were made in the beta roll and greasy nger domains. This region is suggested to play a role in RNA synthesis. Insights into the functional differences between the West Nile and dengue NS1 proteins are being pursued by exchanging regions or specic residues that differ between the two proteins. Certain chimeras, while not expected to have structural differences, do present phenotype differences. Structural features will be presented in the context of biological functions.

RNA STRUCTURE DUPLICATIONS IN THE DENGUE VIRUS GENOME FACILITATES HOST ADAPTATION

Sergio Villordo*, Claudia Filomatori* and Andrea Gamarnik

Fundación Instituto Leloir-CONICET, Buenos Aires, Argentina

Dengue virus cycles in nature between humans and mosquitoes, however, it is still unclear how the virus adapts to use different cellular machineries for replication and overcome different types of antiviral responses. RNA viruses in general have high capacity to adapt to different environments due to the genetic diversity of viral populations. Using dengue virus, we recently found specic RNA sequences in the viral 3'UTR that are essential for viral replication in mosquito cells but dispensable for replication in mammalian cells. These studies provided direct evidence for host-specic functions of viral RNA elements and raised the question whether viral RNA structures are under specic selective pressures during host adaptation. To further these studies, we evaluated how RNA cis-acting elements evolve under single-host selective pressures in mosquito and mammalian cells and during host switch. Deep sequencing of dengue virus populations subjected to host adaptation showed a strong selection of different mutations in the viral 3'UTR in each host, while cis-acting elements present at the 5' end of the genome remained unchanged. Secondary RNA structure analysis showed that viruses selected in mosquito cells contained mutations mapping to a single stem-loop structure, which was found to be duplicated in mosquito-borne aviviruses. Using recombinant viruses and evaluation of tness parameters, we found that RNA duplication is required to tolerate mosquito-adaptive mutations for replication in mammalian cells. Our ndings provide a novel model supporting the role of RNA duplications for efcient dengue virus replication in multiple hosts.

THE LOVE-HATE RELATIONSHIP BETWEEN HOST PROTEINS AND DENGUE VIRUS RNAS1 2,3,4,5 1,2 1 3,4 3,4Mariano A. Garcia-Blanco , Katell Bidet , Alex Ward , Brandt Levitt , and Shelton Bradrick

1Program in Emerging Infectious Diseases, Duke-NUS Graduate Medical School, Singapore, 2NUS Graduate School for Integrative Sciences and Engineering, National 4University of Singapore, Singapore, 3Center for RNA Biology, Duke University Medical Center, Durham, North Carolina, USA, Department of Molecular Genetics and

Microbiology, Duke University Medical Center, Durham, North Carolina, USA, and 5Department of Medicine, Duke University Medical Center, Durham, North Carolina, USA.

Viral RNA-host protein interactions are critical for replication of aviviruses, a genus of positive-strand RNA viruses comprising major vector-borne human pathogens including dengue viruses (DENV). We examined three conserved host RNA-binding proteins (RBPs) G3BP1, G3BP2 and CAPRIN1 in dengue virus (DENV-2) infection and found them to be novel regulators of the interferon (IFN) response against DENV-2. The three RBPs were required for the accumulation of the protein products of several interferon stimulated genes (ISGs), and for efcient translation of PKR and IFITM2 mRNAs. This identies G3BP1, G3BP2 and CAPRIN1 as novel regulators of the antiviral state. Their antiviral activity was antagonized by the abundant DENV-2 non-coding subgenomic aviviral RNA (sfRNA), which bound to G3BP1, G3BP2 and CAPRIN1, inhibited their activity and lead to profound inhibition of ISG mRNA translation. This work describes a new and unexpected level of regulation for interferon stimulated gene expression and presents the rst mechanism of action for an sfRNA as a molecular sponge of anti-viral effectors in human cells. We also examined the function of ERI3, a host 3'-5' RNA exonuclease, which is required for dengue virus replication. We determined that this enzyme is required, paradoxically, to stabilize viral RNAs. In addition to presenting mechanistic data on the role of ERI3 in dengue virus infection we will present the results of our high throughput screen for ERI3 inhibitors and their action as anti-dengue compounds.

DEVELOPMENT OF AN IMINOSUGAR-BASED THERAPEUTIC FOR DENGUE VIRAL INFECTION1 3 1,2 2 3 1A Sampath , E. Plummer , K. Wareld , S. Enterlein , S. Shresta ; U. Ramstedt

1 2 3Unither Virology, Silver Spring, MD, IBT, Gaithersburg, MD, LIAI, La Jolla, CA.

Unither Virology has identied a lead iminosugar candidate, UV-4B for the treatment of Dengue viral (DENV) infection. UV-4B shows in vitro antiviral activity against all four DENV serotypes, including clinical isolates, when tested in a yield reduction assay. In vivo, UV-4B has shown efcacy in an antibody dependent enhancement model of DENV infection. In this model, the minimal effective dose for UV-4B was determined to be 20 mg/kg/dose, TID, via oral gavage with signicant protection observed when administered as late as 24-48 hours post infection.

The proposed mechanism of action of UV-4B is that it inhibits the enzymatic activities of ER α-glucosidases. Since the proposed mechanism of action is via a host glycosylation pathway that affects infectivity and virus particle assembly, it is anticipated that development of viral resistance to UV-4B will be less likely to occur than with directly acting antiviral agents. In an in vitro study that evaluated the potential for DENV-2 to develop drug resistance to UV-4B, 38 cycles of treatment with UV-4B did not demonstrate any clear pattern for causing drug induced resistance when compared to the vehicle control. In vivo, the antiviral effect of UV-4B was sustained through 5 passages of DENV-2 virus in a STAT1x2 knockout mouse model. In vitro and in vivo safety pharmacology studies have been conducted. In vitro assessments of protein binding, blood-to-plasma distribution, metabolic stability and inhibition potential of human cytochrome P450 enzymes have been performed. The pharmacokinetic characteristics of UV-4B have been studied in mice, rats, and dogs. The toxicological prole of UV-4B has been evaluated in mice, rats, and dogs have been found acceptable to proceed to rst in human (FIH) studies. An investigational new drug application (IND) for a FIH study has been approved with clinical testing initiated mid-2014. UV is also planning Phase 2 clinical trials to demonstrate efcacy of UV-4B in DENV infection. Acknowledgements: The project was supported by Award from the National Institute of Allergy and Infectious Diseases, HHSN272201100030C

ABSTRACTS ORAL PRESENTATIONSVIROLOGY/ANTIVIRALS

SINGLE POINT MUTATIONS IN THE HELICASE DOMAIN OF NS3 ENHANCE DENGUE VIRUS TYPE 1 REPLICATION AND BYPASS TYPE I IFN RESPONSE IN HUMAN MONOCYTE-DERIVED DENDRITIC CELLS

Guilherme Ferreira Silveira, Daisy Maria Strottmann, Luana de Borba, Daniel Santos Mansur, Juliano Bordignon, Claudia Nunes Duarte dos Santos

Dengue is one of the most prevalent arboviral disease worldwide, with 2.5 billion people living in areas under risk of infection. Although dengue pathogenesis is not yet completely understood, several factors seem to be associated with dengue disease and the outcome of the infection is determined by the interplay of viral and host factors. In this study we evaluated the infection prole of two recombinant strains of dengue virus serotype 1 (DV-1) carrying single point mutations at NS3hel protein L435S or L480S, in human monocyte-derived Dendritic Cells (mdDCs), targets for DV infections. Both mutant viruses could infect and replicate more efciently producing higher viral progeny in infected mdDCs, compared to parental non-mutated virus (vBACDV1). Mutated DVs also induces a differential gene expression response on mdDCs compared to vBACDV1 virus, with up-regulation of IFN signaling and pattern recognition receptors (PRR) canonical pathways. mdDCs infected with DV-1 NS3 mutated viruses expressed higher levels of TLR3 and 7 mRNAs and induce a pronounced production of type I IFN compared to parental virus. The type I IFN produced by the mdDCs can reduce infection rate, especially in DV-1 NS3 mutated strains. These results suggest that the type I IFN function was effective to reduce but not sufcient to prevent viral replication of mutated strains and demonstrate that single point mutations in NS3hel protein of DV-1 are sufcient to modify the viral tness and bypass the type I IFN function in human primary cells.

DENGUE VIRUS COUNTERACTS THE CGAS/STING/IRF3 PATHWAY IN INFECTED HUMAN CELLS.1 1,2 1 1 1,2 1,2Sebastian Aguirre , Jenish Patel , Ana Maestre , Dabeiba Bernal-Rubio , Adolfo Garcia-Sastre , and Ana Fernandez-Sesma .

1Department of Microbiology and the Global Health and Emerging Pathogens Institute, Icahn Mount Sinai School of Medicine and Icahn Mount Sinai Graduate School of 2Biomedical Sciences , New York, NY, USA

Dengue virus (DENV) has become a global threat infecting near to 390 million humans per year. To establish a productive infection in humans, DENV needs to counteract the host innate immune system, particularly the type I interferon (IFN) system. It was demonstrated by our group and others that DENV interferes with both, the production and signaling of type I IFN pathways through the expression of viral proteins that specically target adaptor molecules involved in these essential host responses to pathogens. We showed that the DENV protease complex NS2B3 is able to interact with and cleave the adaptor molecule STING, inhibiting the activation of IRF3 and subsequent type I IFN induction in infected cells. A recent report showed an anti avivirus activity of the newly described PRR cGAS, which after activation generate a second messenger (cGAMP) that in turn activates the adaptor STING and the subsequent induction of type I IFN. In order to characterize the role of cGAS during DENV infection we evaluated the ability of this protein to be activated and induce type I IFN during DENV infection in primary cells. We also investigated the role of the NS2B3 protease complex in the inhibition of type I IFN induction induced by cGAS. We observed that DENV infection inhibits the sensing of cytosolic DNA in dendritic cells. This new mechanism of inhibition by interfering with the cGAS/STING/IRF3 pathway is dependent of the DENV protease complex NS2B3. Furthermore, silencing of cGAS in human dendritic cells (DCs) resulted in a higher accumulation of DENV RNA after infection. These results suggest an active role of cGAS as a sensor during DENV infection and conrm the role of DENV protease as a master regulator of the type I IFN response in DENV infected cells.

DENGUE VIRUS CAPSID PROTEIN ABILITY TO TRANSLOCATE NUCLEIC ACIDS ACROSS MEMBRANES: IMPLICATIONS IN VIRUS ENTRY INTO HOST CELLS1 2 1 3 3 2 1Christine Cruz-Oliveira , João Miguel Freire , Thaís M. Conceição , Daisy M. Strottmann , Claudia N. Duarte dos Santos , Miguel A. R. B. Castanho , Andrea T. Da Poian

Instituto de Bioquímica Médica Leopoldo de Meis, UFRJ, Rio de Janeiro Brasil; Instituto de Medicina Molecular, Universidade de Lisboa, Lisboa, Portugal.

Instituto Carlos Chagas Filho, Fiocruz-PR, Curitiba, Brasil

For all enveloped viruses, genome access to the cytoplasm depends on the fusion of viral envelope with a cellular membrane, a process triggered by a viral fusion protein anchored at the virion envelope. In the case of dengue virus (DENV), it is currently established in the literature that, despite the different entry routes of viral particle internalization, genome release into the cytoplasm occurs through envelope (E) protein-mediated membrane fusion. However, based on recent results, we hypothesize that DENV capsid (C) protein may cooperate with E protein during the fusion stages. We characterized the capsid proteins of all aviviruses as supercharged proteins, which are proteins with a high net charge/molecular weight (MW) that are able to efciently penetrate cells and deliver functional cargoes. We found that recombinant DENV C protein, as well as two synthetic peptides comprising C protein residues 45-72 and 67-100, were able to translocate short oligonucleotide sequences (ssDNA and siRNA) and large nucleic acids (GFP-encoding plasmid) across cellular membranes. The GFP-encoding plasmid as well as the siRNAs were not only transported into the cells, but also expressed efciently. In addition, DENV 2 C protein was able to internalize DENV 1 RNA (derived from DENV1 infectious clone vBACDV1) in hepatic cells. After C protein-mediated transfection, DENV1 RNA expression was monitored using antibodies against DENV E protein through microscopy and ow citometry studies. Besides efcient viral RNA expression and translation, transfection of DENV1 RNA with C protein promotes virus assembly, since infectious DENV particles were detected in the medium at 96 hours post transfection. Taken together, these results reinforce the role of C protein in viral genome release into the cytoplasm.

ASSESSING DENGUE VIRUS-INDUCED CHANGES IN GENE EXPRESSION PROFILES VIA RIBOSOME PROFILING1 1 1 1 1 2Mariana Leguia , Diana Juarez , Michael Torres , Eric S. Halsey , Daniel G. Bausch , Anton Vila-Sanjurjo .

1 2U.S. Naval Medical Research Unit No. 6, Universidade de A Coruña, Peru

A majority of the research on dengue (DENV) virus-induced changes in gene expression has focused on the role of the adaptive immune response, which is undeniably important. However, epidemiological data suggest that the host's genetic background may also contribute important susceptibility factors that could exert their effect in a manner independent from the adaptive immune response. Cellular responses with the potential to make a difference between life and death outcomes are ultimately mediated by the actions of proteins encoded in the genome. Thus, an understanding of differential global gene expression at the proteome level is essential to understand how DENV infection can result in dramatically different disease outcomes. Ribosome proling is a new technique that enables direct measurements of protein expression at the whole cell level. In so doing, it generates all the information needed for a comprehensive understanding of how global gene expression may inuence particular disease phenotypes. We have recently completed the rst ribosome proling-based study of DENV-2 infected human cells. Our results indicate that ribosome proling is a powerful tool to study changes in cellular dynamics upon DENV infection. Specically, we are able to pinpoint differentially regulated genes and corroborate previously identied putative predictors of disease progression. As a whole, these data sets elucidate differentially regulated genes in the context of the host's genetic background. Furthermore, this approach has the potential to enable discovery of genes not previously associated with particular disease states, and in so doing, lead to the development of improved vaccines, diagnostics and therapeutics.

DENGUE-3 VIRUS (DENV-3) ENTRY INTO THE HOST CELL: MECHANISM AND ANTIVIRAL TARGET FOR HEPARAN SULFATE-MIMICKING AGENTS

Elsa B. Damonte, Luana E. Piccini, Viviana Castilla.

Laboratorio de Virología, Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, IQUIBICEN- CONICET, 1428 Buenos Aires, Argentina

Dengue virus (DENV) entry into the host cell represents an attractive antiviral strategy for chemotherapy to suppress the beginning of infection. However, it appears to be a very complex process regulated by several cell- and virus-dependent factors that may affect antiviral susceptibility. Here, we studied rst the mode of entry of DENV-3 strain H87 into the host cell by analyzing the effect of pharmacological and molecular inhibitors of different endocytic pathways on viral infectivity and antigen expression. Together, the results obtained demonstrated that DENV-3 entered mosquito C6/36 cells by clathrin-mediated endocytosis whereas the entry into monkey Vero and human A549 cells occurred via a clathrin-independent, dynamin and acid pH-mediated route with a partial involvement of caveolae. The kinetics of viral entry, analyzed by using a resistance to ammonium chloride assay, indicated that virion penetration started at 5 min post-binding and the time for viral escape from endosomes was about 15 min. The antiviral susceptibility of DENV-3 to entry inhibitors was next analyzed in both Vero and A549 cells. Diverse classes of sulfated polysaccharides (SP), compounds mimicking the cellular heparan sulfate residues, exhibited a potent and selective antiviral activity against DENV-3. Carrageenans were the most active inhibitors with effective concentration 50% (EC50) values around 1 µg/ml and selectivity indices (ratio cytotoxicity/antiviral activity) higher than 1000. The target of these compounds was DENV-3 entry by dual blockade of virus adsorption and internalization of the nucleocapsid from endosomes.

The comparison with our previous studies on Vero cell entry with DENV-1 (clathrin-mediated endocytosis and resistance to SP) and DENV-2 (non classical clathrin- and caveola-independent pathway and susceptibility to SP) conrms that diverse viral and cellular factors affect DENV entry and should be considered for evaluation of safe antiviral agents reactive against all DENV serotypes.

IDENTIFICATION OF TERPENES INHIBITORS OF DENGUE VIRUS 1 2 2 1Camila Flechas-Alarcón , Nerlis Pájaro-Castro , Jesus Olivero-Verbel , Sindi Velandia-Cruz1, Raquel Ocazionez-Jimenez .

Centro Nacional de Investigaciones para la Agroinsdustrialización de Especies Vegetales Aromáticas y Medicinales Tropicales (CENIVAM), 1Laboratorio de Arbovirus, Centro de Investigaciones en Enfermedades Tropicales (CINTROP), Universidad Industrial de Santander, Bucaramanga, Colombia. 2Grupo de Química Ambiental y Computacional, Facultad de Ciencias Farmacéuticas, Universidad de Cartagena, Cartagena, Colombia.

Introduction - An antiviral drug is not currently available for treatment of dengue. Essential oils from aromatic plants can inhibit dengue virus infection and it has been attributed to its chemical constituents. Hence, the identication of terpenes inhibitors of the virus could contribute to discovery of anti-dengue drugs. Methods - The concentration that inhibited 50% (IC50) DENV-2 replication was determined by using a virus-HepG2-based assay measuring the NS1-protein level. Potential cytotoxic effects (CC50 values) were evaluated in ve human cell types by using MTT assays. Docking of the compounds in viral proteins was performed using AutoDock Vina. Identication of protein residues interacting with the compound was carried out with LigandScout 3.0 software. Results - β-Caryophillene, Citral and Estragole were identied as inhibitors of DENV-2 replication at IC50 of 22.5 ± 5.6, 31.3 ± 4.5 and 30.0 ± 1.0 µM, and with a SI value of 71.7, 8.0 and 6.7, respectively. Time-of-drug-addition studies revealed that the compounds act at the very early stages of the viral replication cycle. Preincubation of high-titer stocks of virus with compounds at the highest non-toxic concentration did not resulted in inactivation of the virus. (R)-(-)- and (S)-(-)-Carvone, Geranyl-acetate, Nerol, Carvacrol, p-Cymene, Thymol and β-Mircene did not exhibit promising antiviral activity. All compounds were not cytostatic for human cells derived from kidney, liver and skin (CC50 between 200 and 902 µM). The afnity scores for the compounds on C, prM-E, E, NS3-NS2B and NS5 viral proteins varied between -4.0 and -7.3 kcal/mol. β-Caryophyllene interacting with NS3-NS2B had the highest afnity (-7.2 kcal/mol). Conclusion - Promising terpenes inhibitors of dengue virus were identied which could be used as a starting point for consideration in designing effective drugs for dengue. This research was carried out thanks to nancial support from the Colombian Institute of Science and Technology (Colciencias), Grant RC-245-2011 (Patrimonio Autónomo del Fondo Nacional de Financiamiento para la Ciencia, Tecnología e Innovación, Francisco José de Caldas).

DENGUE VIRUS MATURITY DETERMINED BY MASS SPECTROMETRY AND INFLUENCE OF PRM CONTENT ON VIRUS BIOLOGY1, 2 2 2 1 3 1,2Devika Sirohi , Victoria Hedrick , Ernesto S. Nakayasu , Valorie D. Bowman , Catherine P. Riley & Richard J. Kuhn

1 2 3Markey Center for Structural Biology, Dept. of Biological Sciences, Bindley Bioscience Center, Purdue University, West Lafayette, IN, USA; Pathology Associates Medical Laboratory, Coeur d'Alene, ID, USA

Maturation of Dengue Virus (DENV), driven by structural rearrangements of Envelope (E) protein and furin protease-mediated cleavage of the precursor membrane (prM) protein, is required for particle infectivity. However, studies indicate that furin cleavage is inefcient in DENV compared to other aviviruses. As a consequence, DENV preparations are structurally diverse and comprise a continuum of particles with variable maturity from immature 'spiky' (180 prM/0 M) to fully mature 'smooth' particles (0 prM/180 M). The presence of prM on particles alters the representation and accessibility of epitopes and inuences virus-antibody interactions, perhaps unfavorably for the host. Antibodies against prM exhibit weak neutralization activity and can potentially enhance infection. In fact, levels of prM antibody in patients have been shown to have a positive correlation with disease severity. While the signicance of prM in disease pathogenesis is clear, the prM content of DENV virions and how it modulates infectivity and disease outcome remains unknown. One of the bottlenecks is the lack of a robust method for quantitative determination of prM content on virions. We have developed mass spectrometry based sensitive assays for measuring the maturity of DENV virions. These are being used to probe the prM content of DENV produced in cell culture and we are developing protocols to study virus produced from natural infection of mosquitoes (vector) and humans (host). In parallel, cryo-electron microscopy has been employed to provide a physical assessment of virus particles under analysis. Using these methods, we have compared the efciency of cleavage among different serotypes of DENV and are identifying factors that modulate the cleavage efciency. This presentation will focus on the methods developed for estimating DENV maturity, its applications in vaccine development, serotypic differences in maturity and our future efforts geared towards establishing a correlation between prM cleavage efciency and protective versus enhancing antibody response.

HIGHLIGHTED POSTERSVIROLOGY/ANTIVIRALS

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Dengue virus inhibits miR-133a expression through its 3'UTR but miR-133a overexpression blocks dengue virus replication1 1 1,2 1 1,3 2 1Jorge Andrés Castillo , Juan Camilo Castrillón , Mayra Diosa-Toro , Juan Guillermo Betancur , St Laurent G 3rd , Jolanda M Smit , Silvio Urcuqui-Inchima

1 2Grupo Inmunovirología, Facultad de Medicina, Universidad de Antioquia; Department of Medical Microbiology, University Medical Center Groningen, University of 3Groningen, The Netherlands. St Laurent Institute, 9 Lewis St, Providence, RI 02906, USA

Dengue virus (DENV) is the most common vector-borne viral infection worldwide with approximately 390 million cases and 25,000 reported deaths each year. Despite the high economic and social impact of DENV, neither vaccine nor antiviral drug therapy exists for any of the four dengue serotypes. MicroRNAs (miRNAs) are non-coding small RNA molecules responsible for the regulation of gene expression by repressing mRNA translation or inducing its degradation. miRNAs have been described to poses antiviral activity towards several mammalian-infecting viruses. However, the role of miRNAs during DENV replication is not yet understood and well-studied. Here, we analyzed miR-133a expression in Vero cells during DENV infection and we observed a rapid down-regulation of miR-133a early in infection. The 3'UTR of DENV participates in this process by altering the expression of proteins involved in the miRNA biogenesis pathway. In addition, we observed that reduced

levels of miR-133a augment expression of the polypyrimidine tract binding protein during the rst 12 hours after infection. Furthermore, transfection of Vero cells with mature miR-133a was found to inhibit DENV replication, while inhibition of miR-133 expression with miR-133a antisense mimics, increased the viral replication. Taken together, our results indicate that miR-133a has anti-DENV activity. To the best of our knowledge, this is the rst study demonstrating that a cellular miRNA has anti-DENV activity.

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The heterogeneous nuclear ribonucleoprotein K is a cell factor required for dengue virus type 2 multiplication

Viviana Castilla, Jesús E. Brunetti, Luis A. Scolaro

Laboratorio de Virología, Departamento de Química Biológica, Facultad de Ciencias, Universidad de Buenos Aires, Argentina

Heterogeneous nuclear ribonucleoproteins (hnRNPs) A2 and K are cellular RNA-binding proteins that participate in mRNA splicing, trafcking and translation and have been implicated in the multiplication of several cytoplasmic RNA viruses. To identify cellular factors involved in dengue virus (DENV) replication, here we characterized hnRNP A2 and K expression in Vero and A549 cells infected with DENV type 2 and we evaluated the effect of hnRNP K silencing on DENV-2 multiplication. We analyzed hnRNP intracellular localization by indirect immunouorescence assays, using anti-hnRNP and anti-viral glycoprotein E antibodies, at 2 or 5 days post- infection (dpi). Whereas hnRNP A2 showed a clear nuclear localization, similar to that observed in uninfected cells, the cytoplasmic translocation of hnRNP K was evident at 5 dpi in both cell types. We also performed A549 cell transfection with a plasmid that allows the expression of T7-tagged hnRNP K and the re-localization of transiently overexpressed hnRNP K to the cytoplasm was observed at 2dpi. The analysis by western blot showed no signicant differences in the total levels of hnRNP K expression between uninfected and DENV-2 infected Vero or A549 cells. Finally, A549 cells were transfected with small interfering RNAs (siRNAs) that target hnRNP K or small non-interfering RNA used as control, and at 24 h post-transfection cells were infected. The effectiveness of siRNA silencing was corroborated by western blot and at 1 and 5 dpi, virus yield and the amount of glycoprotein E expressing cells were assessed by plaque assay and immunouorescence technique, respectively. A signicant reduction of 66.6% and 96.0% of virus yield was achieved at 1 and 5 dpi, respectively, and the amount of uorescent cells was also inhibited. The results obtained indicate that DENV-2 infection alters hnRNP K intracellular distribution and this hnRNP would play an important role in DENV-2 multiplication in cell cultures.

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MicroRNA profiling associates miR-21 and miR-146a with dengue virus-induced apoptosis and replication in human hepatoma cells 1 1 1,2 1Antonio Gregorio Dias Junior ; Daniele Barbosa de Almeida Medeiros ; Ana Cecília Ribeiro Cruz ; Márcio Roberto Teixeira Nunes ; Evonnildo Costa Gonçalves3;

3 1 1,2Antônio Rosário Carlos Vallinoto ; Edna Cristina Santos Franco ; Pedro Fernando da Costa Vasconcelos 2

1Instituto Evandro Chagas/SVS/MS - Seção de Arbovirologia e Febres Hemorrágicas - Ananindeua, Pará, Brazil. Universidade do Estado do Pará - Belém, Pará, Brazil 3

Universidade Federal do Pará - Belém, Pará, Brazil

Dengue virus (DENV 1-4) is a mosquito-borne agent recognized by its impact on public health in tropical and sub-tropical countries. Infected individuals may present severe forms of the disease, namely dengue hemorrhagic fever and dengue shock syndrome. These are often associated with a subversion of the innate immune responses (e.g. due viral proteins inhibition of interferon - IFN - type I pathway; antibody dependent enhancement; host polymorphisms, etc.) along with an inammatory cytokine storm and organ impairment, where apoptotic cells might be found. To exploit possible mechanisms involved in these events, we infected IFN-decient Huh7.5 and IFN competent HepG2 human hepatoma cell lines with DENV2. In comparison, Huh7.5 produced the highest virus titers with caspase 3 production and decreased cellular viability over time. In addition, an microRNA proling revealed miR-21 and miR-146a were upregulated at 48 hours post-infection (hpi) in Huh7.5 cells. Hence, we show DENV replication affects cellular microRNA proling in an IFN-decient environment, concomitant with peaks of detectable infectious particles and caspase 3 detection.

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Effect of dengue virus infection on calcium homeostasis in hepatic cultured cells1 2 3 1 4 2 1 1Cinthia L. Dionicio , Franshelle Peña ., Eda Benitez , Fernando Medina , José L. Zambrano , Marie C. Ruiz , Rosa M. Del Angel , Juan E. Ludert

1 3Department of Infectomics and Molecular Pathogenesis and Department for Biochemistry, Center for Research and Advanced Studies (CINVESTAV), Mexico City, Mexico; 2Center for Biochemistry and Biophysis and 4Center for Microbiology and Cell Biology, Venezuelan Institute for Scientic Research (IVIC), Caracas, Venezuela. email: jludert@cinvestav.mx

Dengue is the mosquito-borne viral disease most important to humans. It produces a wide range of clinical conditions ranging from mild forms (dengue fever) to the hemorrhagic forms, known as severe dengue (DS). The dengue virus (DENV) belongs to the family Flaviviridae and 4 serotypes have been described. Ca2+ is a second messenger controlling many intracellular processes in mammalian cells. Substantial evidence exists indicating that Ca2+ plays an important role in the replication cycle of several viruses, including some Flavivirus. However, the importance of Ca2+ during the replication cycle of DENV is unknown. This study aims to explore the possible involvement of Ca2+ and various mediators of this ion levels during DENV infection. Experiments carried out in Hep-G2 cells infected with DENV-4 (MOI=3) and loaded with Fura-2, showed that infection signicantly (p≤0.005) increased the permeability of the plasma membrane to Ca2+, and causes a

signicant (p≤0.005) reduction in the levels of Ca2+ releasable from the endoplasmic reticulum, at 12 and 18 hpi. In addition, we observed an increase in the mRNA levels of the proteins involved in capacitive entry, STIM1 and Orai1, at 18 and 24 hpi. However, WB analysis of Orai1 and STIM1 protein levels showed no changes in the levels of these proteins along infection. The results suggest that the observed increase in plasma membrane permeability is due to an increase in the activation of channels, rather than an increase in protein production. Finally, it was observed that manipulation of the intra and extracellular levels of Ca2+ in infected Huh-7 cells through treatments with Ca2+ chelators, such as BAPTA-AM and EGTA, reduced viral particle production in more than 1 log (p<0.005), without affecting cell viability. This result suggests that the changes induced in Ca2+ homeostasis during infection are required for an efcient viral replication.

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The dengue virus non-structural protein 1 (NS1) is secreted from the mosquito cell line C6/36 following a non-classical secretory pathway.

Ana C. Alcalá, Fernando Medina, Rebeca Basave, Rosa María del Ángel, Juan E. Ludert

Department of Infectomics and Molecular Pathogenesis, Center for Research and Advanced Studies (CINVESTAV), Mexico City, D.F., Mexico. C.P. 07360. Email: jludert@cinvestav.mx

The dengue virus non-structural protein 1 (NS1) is a glycoprotein of 46-50Kd found mainly associated to intracellular membranes and organelles. NS1 is essential for viral replication, and has been found to co-localize with the viral replication complexes, although its roles in virus replication are not well understood. A small fraction of NS1 can also be found associated with lipid rafts on the plasma membrane or soluble, secreted to the supernatant. Early during infection, NS1 circulates in high levels in patients´ sera and evidence exists indicating that circulating NS1 plays a role in dengue pathogenesis. The notion in the eld is that during infection, NS1 is secreted only by cells of vertebrates and not by insect or mosquito cells. In this work, however, evidence is presented indicating that the Aedes albopictus cell line C6/36 and its variant C6/36HT, infected with DENV-4 or DEN-2 (MOI=3) secrete NS1 to the media starting at 10-12 h.p.i. The presence of NS1 in the cells supernatants was determined using a commercial assay (PLATELIA™, BIO-RAD). Secretion of NS1 takes place in the absence of any signicant (less than 5%) cell lysis as indicated by several cytotoxicity and cell viability assays. Cell treatment with brefeldin A decreases the secretion of NS1 from infected Vero cells by about 40%, but it does not affect NS1 secretion from C6/36 cells. Similarly, C6/36 cells treatment with drugs that affect the cytoskeleton (cytochalasin D and nocodazol) or inhibit exosome release (spiroepoxide) had no effect on NS1 secretion. In contrast, the inhibition of the caveolin-1 chaperone complex transport by the use of siRNAs to silence caveolin-1 expression signicantly (over 60%) reduces NS1 secretion from infected C6/36 cells. These results suggest that NS1 is indeed secreted by infected insect cells but following a secretion route different from vertebrate cells.

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Small molecule inhibitors for dengue virus NS2B/NS3 protease serotype 2 1 1 2Maria Cabarcas-Montalvo , Jesus Olivero-Verbela *, Raquel Ocazionez-Jimenez .

1Environmental and Computational Chemistry Group. University of Cartagena, Cartagena, Colombia. *Corresponding Author. joliverov@unicartagena.edu.co. 2Laboratorio de Arbovirus. Centro de Investigaciones en Enfermedades Tropicales. (Cintrop). Universidad Industrial de Santander. Bucaramanga. Colombia.

Dengue virus is a mosquito-borne avivirus responsible for millions of human infections worldwide each year. Until today little progress has been made in nding therapeutic compounds to control dengue disease and the treatment is supportive. NS2B-NS3 protease (NS2B-NS3pro) of dengue fever virus is essential for viral polyprotein processing and hence for viral replication, therefore, the development of small molecule inhibitors of the viral protease is of considerable interest for the treatment. Virtual screening of approximately two hundred thousand compounds from PubChem database was conducted by in silico blind docking to identify novel inhibitors against the dengue protease using NS2B/NS3 crystal structure (2FOM). Near two hundred compounds presented great theoretical afnity binding to 2FOM. From this group, ve compounds were purchased and submitted to assays to conrm the inhibitory protease activity using two methods: The rst was a uorometric competitive assay performed against a recombinant NS2B-NS3pro expressed in E. coli and puried by afnity chromatography, employing Boc-Gly-Arg-Arg- AMC as a substrate. The second one, an antiviral activity test consisted of the yield reduction assay in HepG-2 cells infected with dengue virus serotype 2. Three strong NS2BNS3pro inhibitors were conrmed for both approximations. IC50 values estimated for uorometric assays were19.9 �M, 17.5 �M and 9.1 �M for CID54681617, CID54692801 and CID54715399, respectively. For the same chemicals, IC50 values and percentages of NS1 viral title reductions for the in vitro antiviral assays corresponded to 61.5 �M, 79.9%; 14.9 �M, 69.8%; and 1.8 �M, 73.9 %, respectively. Similarity searches based on calculated molecular descriptors and multivariate methods revealed that two of these compounds, CID54681617 and CID54715399, are structurally different from those already reported in the literature. These data show the protocol used in this work allows the discovery of promising molecules against dengue virus infection. UniCartagena-Colciencias (Grant 110751929058: Contract 256-2010). UniCartagena-CENIVAM (RC-0572-7-2012).

HP07

Nordihydroguaiaretic acid (NDGA) inhibits replication and viral morphogenesis of dengue virus1 1 2 2 1Rubén Soto-Acosta , Patricia Bautista-Carbajal , Gulam H. Syed , Aleem Siddiqui , Rosa M. Del Angel

1 2Departmento de Infectómica y Patogénesis molecular, CINVESTAV-IPN, México, D.F., Mexico. Department of Medicine, Division of Infectious Disease, University of California, San Diego, La Jolla, CA, United States.

Dengue is the most common mosquito borne viral disease in humans. The infection with any of the 4 dengue virus serotypes (DENV) can either be asymptomatic or manifest in two clinical forms, the mild dengue fever or the more severe dengue hemorrhagic fever that may progress into dengue shock syndrome. A DENV replicative cycle relies on host lipid metabolism; specically, DENV infection modulates cholesterol and fatty acid synthesis, generating a lipid-enriched cellular environment

necessary for viral replication. Thus, the aim of this work was to evaluate the anti-DENV effect of the Nordihydroguaiaretic acid (NDGA), a hypolipidemic agent with antioxidant and anti-inammatory properties. A dose-dependent inhibition in viral yield and NS1 secretion was observed in supernatants of infected cells treated for 24 and 48h with different concentrations of NDGA. To evaluate the effect of NDGA in DENV replication, a DENV4 replicon transfected Vero cells were treated with different concentrations of NDGA. NDGA treatment signicantly reduced DENV replication, reiterating the importance of lipids in viral replication. NDGA treatment also led to reduction in number of lipid droplets (LDs), the neutral lipid storage organelles involved in DENV morphogenesis that are known to increase in number during DENV infection. Furthermore, NDGA treatment resulted in dissociation of the C protein from LDs. Overall our results suggest that NDGA inhibits DENV infection by targeting genome replication and viral assembly.

POSTERS SESSION VIROLOGYV-P01

THE DENGUE VIRUS NON-STRUCTURAL PROTEIN 1 (NS1) INTERACTS WITH THE HUMAN RIBOSOMAL PROTEIN L18 IN HUH-7 INFECTED CELLS

Margot Cervantes-Salazar, Ariana Hurtado-Monzón, Antonio Ángel-Ambrocio, Rubén Soto-Acosta, Patricia Bautista-Carbajal, Juan E. Ludert, 1Rosa María del Ángel1Departamento de Infectómica y Patogénesis Molecular. Centro de Investigación y de Estudios avanzados del instituto politécnico Nacional (CINVESTAV-IPN), México City, México

The persisting epidemics of dengue disease in the world as well as the increasing number of cases, especially of the severe forms of the infection, have augmented the interest in the study of dengue virus (DENV) replicative cicle. The austerity of the viral genome makes it dependent on the participation of differents molecules and organelles from the host for entry and replication. The NS1 protein plays important roles in viral replication and pathogenesis, however, the function of NS1 are not fully understand. Thus the amein of this work was to identify proteins that interact with NS1 in infected cells. Using afnity chromatography and immunoprecipitation assay, approaches a total of 64 cell proteins were isolated and identied by mass spectrometry (MALDI-ToF MS / MS) from Huh-7 cells. Using the string 9.1 data bases it was found that many of them are functionally related. One of the proteins identied was the ribosomal protein L18. The L18 protein is part of the large ribosomal subunit and it has been described that it is involved in translation initiation. Western-blot and confocal microscopy analysis indicated that the expression of L18 protein is not altered during DENV infection, however L18 protein silencing by siRNA inhibited viral infection determined by viral yiel, NS1 secretion and qRT-PCR without any effect in total cellular protein synthesis. Additionally, silencing of L18 in Vero cells, stable trasfected of DENV 4 replicon, induce an inhibition in luciferase activity. All these results suggest that the ribosomal protein L18 is involved the replicative cycle of DENV.

V-P02

CALMODULIN INHIBITION REDUCES DENGUE VIRUS REPLICATION IN HEPATIC CELLS

Patricia Bautista-Carbajal, Rubén Soto-Acosta, Margot Cervantes-Salazar, Antonio H. Angel-Ambrocio, Juan E. Ludert, Rosa Ma. del Ángel1

1Departamento de Infectómica y Patogénesis Molecular. Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN), México City, México

Dengue is the most important mosquito borne-viral disease in global public health.The virion contain an RNA genome of positive polarity that encodes 3 structural and 7 nonstructural proteins. DENV replication occurs in close association with membranes, in particular derived from the endoplasmic reticulum (ER). The association of the replication complex to the ER has focused our attention on calcium sensor proteins such as calmodulin (CaM). This protein can turn on approximately 30 systems with different targets, including calcium extrusion pumps and translate modest increases of intracellular Ca2+ in physiological responses. The aim of this work was to analyze if CaM may have a direct role during dengue virus replicative cycle. Western blot analysis indicated that CaM levels increased up in Huh7 cells at 48 hours post infection. Afterwards, infected cells were treated with the CaM antagonist W7 (25 µM) for 48 hours after infection. Cell treatment resulted in a decrease in viral yield by 1 log. The reduction in viral yield was accompanied by a reduction in mRNA levels and NS1 secretion. In addition, confocal microscopy and ow cytometry analysis of treated cells showed a reduction by about one third in the percentage of infected cells. Interestingly, confocal microscopy analysis of treated cells indicated that the expression levels of NS3 are reduced and the intracellular distribution of the E protein altered. Finally, W7 treatment of a VeroR cell line, stably transfected with a DENV4 replicon results in a reduction in luciferase activity used as an indicator of translation and replication. These results suggest the CaM is necessary for dengue virus replication and presumably play a role in viral translation and replication.

V-P03

BIOLOGICAL ASPECTS OF THE INFECTION BY WMEL AND WMELPOP STRAINS OF WOLBACHIA IN BRAZILIAN AEDES AEGYPTI GENOTYPE1 2 1Heverton Dutra , Rafael de Freitas , Luciano Moreira

1 2Mosquitos Vetores: Endossimbiontes e Interação Patógeno Vetor, Centro de Pesquisas René Rachou - Fiocruz, Belo Horizonte, MG; Laboratório de Transmissores de Hematozoários, Instituto Oswaldo Cruz, Fiocruz, Rio de Janeiro, RJ; Brazil

Wolbachia are obligate, intracellular, maternally-transmitted bacterial endosymbionts infecting around 70% of all known arthropods, causing a variety of biological effects including "reproductive parasitism", which favors it spread in wild populations. Aedes aegypti do not harbor Wolbachia, but when wMel and wMelPop strains were independently inserted into this species, they signicantly reduced the mosquito vectorial competence for DENV. Wolbachia has been trialed as a novel strategy for reducing dengue incidence (ED = www.eliminatedengue.org). However, Wolbachia-based control will only be effective if the infection generated in the laboratory is

able to xate into wild mosquitoes. Here we evaluated the effects of wMel and wMelPop strains on key biological parameters of recently backcrossed Brazilian A. aegypti, in preparation for eld releases, reared under different stress conditions. For both strains, there was signicant difference in larval development time only in highest stress condition, where infected individuals developed more rapidly than uninfected, regardless of sex. Regarding wMel infection, we observed several phenotypic changes, ranging from no effect at all (fecundity, fertility), to partially detrimental (longevity and body size in highest stress condition), and partially benecial (increased levels of glycogen under the highest stress condition). About infection levels, there was generally, a higher bacterial concentration in the Brazilian mosquitoes over time for wMel strain compared to Australian mosquitoes, exhibiting maternal transmission and cytoplasmic incompatibility rate of 96% and 99,51% respectively. For wMelPop, there were benecial phenotypic effects: larval development time was lower in infected individuals, which showed an increased body size and glycogen content in highest stress condition compared to uninfected females. In conclusion, we highlight key phenotypic effects of Wolbachia on the biology of Brazilian A. aegypti. We also think it contributes with relevant information regarding the benecial effects of wMel strain, being suitable for application in the eld, possibly controlling dengue outbreaks in Brazil.

V-P04

STRUCTURAL AND MOLECULAR CHARACTERIZATION OF DENV2 CAPSID PROTEIN AND ITS MUTANTS1 1 1 1Janaina Figueira Mansur , Renata Morgado Pereira , Rafael Mesquita Stoque , Ronaldo da Silva Mohana Borges

1Laboratório de Genômica Estrutural - Instituto de Biofísica Carlos Chagas Filho - Universidade Federal do Rio de Janeiro- Rio de Janeiro- Brazil.

The process of DENV nucleocapsid assembly is not well understood. There are some studies that showed the presence of capsid protein at the cytoplasmic side of the endoplasmic reticulum (ER) membranes and that some substitutions and deletions at hydrophobic regions of capsid protein can disrupt its association with membranes, suggesting that capsid-membrane interaction is mediated by conserved regions among residues 45 to 65. Furthermore, it was also shown that in the absence of capsid protein there is production of subviral particles without RNA. These ndings suggest that the production of infectious viral particle is dependent on the interaction between viral genome and capsid protein associated with ER membranes. The comprehension of the fundamental processes of the viral infection cycle can provide potential targets for design of antiviral drugs. In this context, the present work aims to study the molecular and structural aspects of capsid protein and characterize its interaction with membranes. DENV2 capsid protein is a highly basic protein, composed exclusively of alpha-helix, that has about 12 KDa and is dimeric in solution. In this work, we have cloned, expressed and puried ve mutants of capsid protein (L81N, I88N, L50S, L54S, L50S/L54S). The oligomeric state of these proteins was analyzed by gel ltration chromatography and they showed to be dimeric as the wild type protein. L81N and I88N mutants were also analyzed by EGS cross-linking assay and the results corroborate with those obtained in the chromatography. The secondary structure of L81N and I88N were studied by circular dichroism and presented alpha-helix structure; however, the I88N mutant showed loss of secondary structure. Stability of these two mutants was analyzed by urea denaturation assays monitoring the tryptophan uorescence. L81N and I88N mutants were much less stable than wild type, suggesting that hydrophobic residues at the alpha-helix 4 are important to protein stability.

V-P05

DENGUE VIRUS INTERACTOME: INTERACTION OF NONSTRUCTURAL PROTEIN 5 (NS5) AND HUMAN PROTEINS1 1Estefania A. Aguilera1, Emiliana M. Silva , and Ronaldo Mohana-Borges

1 Laboratório de Genômica Estrutural- IBCCF - Universidade Federal do Rio de Janeiro, RJ, Brazil

Among DENV nonstructural proteins, NS5 is the most conserved protein in DENV serotypes. It is a bifunctional enzyme that contains the S-adenosyl methionine transferase and RNA-dependent RNA polymerase (RdRp) catalytic domains. Although all suggested functions of NS5 are generally to occur in the cytosol NS5 has been located in the cell nucleus. There is no role for nuclear localization of dengue virus NS5, however the interaction study of NS5 and cellular proteins it's really important for pathogenesis understanding of DENV infection. Thus, the objective of this work is to verify the role of NS5 and its RdRp and MTase domains on the recruitment and interaction with host proteins during the replicative cycle, since we believe that the understanding of this process is essential to development of antiviral treatments and new therapies. In this study, we employed the Matchmaker™ Gold Yeast Two-Hybrid System to identify human proteins that interact with NS5 protein. Y2HGold yeast cells were transformed with the pGBKT7-NS5 construct using lithium acetate method and mixed with cDNA human library provided in yeast Y187, together, they mate to create diploids cells that contain four reporter genes: HIS3, ADE2, MEL1, and AUR1-C, that are activated in response to two-hybrid interactions. As result we screened 132 putative interactions between DENV NS5 protein and human proteins. We demonstrated that NS5 interacts with several proteins that have diverse functions; including metabolic, regulatory, transcriptional factors and cytoskeletal proteins. Among the screened proteins our results showed that NS5 interacts with tumor necrosis factor receptor superfamily member 6, cyclin-D-binding Myb-like transcription factor 1 and centromere protein F. The next step of this work will conrm some of these putative interactions by co-immunoprecipitation, pull-down, co-immunolocalization and mass spectrometry and then investigate the role of these interactions in viral replication and pathogenesis.

V-P06

VERTICAL TRANSMISSION OF DENGUE VIRUS IN AEDES AEGYPTI CAPTURED IN PUERTO IGUAZÚ, MISIONES, ARGENTINA1 2 2Manuel Espinosa , Sergio Giamperetti , Marcelo Abril1, Alfredo Seijo

1 2 Fundación Mundo Sano, Servicio de Zoonosis, Hospital F.J. Muñiz; Buenos Aires, Argentina

In recent years, there have been reports of transovarial or vertical transmission of the dengue virus in Aedes aegypti and A. albopictus. While it is still uncertain how this can currently impact the incidence of outbreaks - where the typical form of transmission (human with viremia-vector-susceptible human) prevails --, this

phenomenon could play a crucial role in maintaining the virus in the ecosystem during interepidemic periods, mainly in regions where circulation of the virus is particularly seasonal in nature. As part of the Entomological Vigilance of Dengue Program implemented by the Mundo Sano Foundation in Puerto Iguazú, Misiones Province, Argentina, genetic material for serotype DEN 3 was detected in male specimens of A. aegypti captured in the urban area, in the months following the dengue epidemic that affected the Tropic of Capricorn region during the summer-fall of 2009. This event represents the rst evidence of the vertical transmission phenomenon documented in Argentina. The existence of this alternative way of transmission in a region that is close to the international border, with the presence of A. aegypti and A. albopictus, high human transit due to the great number of tourists visiting the Iguazú Falls National Park, and the history of circulation of the four serotypes of the dengue virus, as well as yellow fever, should be given epidemiological importance in order to stay vigilant regarding the incidence of dengue virus and other related avivirus.

V-P07

MODULATION OF DENV-2 REPLICATION VIA BIOCHEMICAL AND GENETIC INHIBITION OF CHOLESTEROL SYNTHESIS1 3 1 1 2 1Bryan L. Owen , Román Vidaltamayo , Augusto Rojas , Hugo Barrera , Javier Ramos , Ana Rivas

1 2Biochemistry Department, Faculty of Medicine, Nuevo Leon Autonomous University; Infectology Department, Universitary Hospital José Eleuterio Martínez, 3Monterrey, Nuevo León; Research Department of Health Sciences at Universidad de Monterrey; Mexico

Pathogenesis of dengue virus (DENV) infection is still, not completely understood. Although Antibody dependent enhancement and immunology related factors have been appointed as causative for severe Dengue, several cases are not consistent with this hypothesis, suggesting that other mechanisms could be involved. Cholesterol levels have been reported to regulate DENV replication in vitro and proved to be necessary to successfully complete the viral life cycle. Also, Lovastatin treatment reduced viral RNA on a Dengue replicon system. The aim of this work is to evaluate the effect of cholesterol regulation on DENV replication cycle. Cholesterol synthesis was biochemically (statins lovastatin, pravastatin and atorvastatin) and genetically (iRNA against HMG-CoA R) inhibited in non-infected and DENV infected HuH-7 Hepatoma cells. Statin concentration was maintained by applying treatment every 24 h. Supernatant was collected every 24h up to 96h for cholesterol and DENV evaluation and replaced with fresh media containing treatment. DENV replication was evaluated using Anti Flavivirus Group antigen 4G2 and diaminobenzidine (DAB) ourophore. Viral titter was measured with the Plaque formation unit (PFU) assay in BHK-21 cells and cholesterol levels were evaluated using Caymans cholesterol uorometric assay. A reduction in 4G2 antigen was observed in all statin treated cells compared with the untreated control. DENV inhibition percentages for LOV, PRA and ATO were 30%, 40% and 20% respectively at 72h p.i. 12h pre-treatment with statins slightly reduced virus titter while post-infection continuous treatment result on a marked reduction on DENV infective particles up to 40% for PRA, 30% for LOV and 20% for ATO at 96h p.i. Statin treatment also reduced Intracellular cholesterol levels in a time dependent manner simultaneously to DENV reduction. Genetic inhibition of cholesterol synthesis also reduces DENV titer. Our results indicate that cholesterol inhibition causes a reduction of DENV infectious particles and 4G2 aviviral antigen signal. Our results suggest that cholesterol is particularly involved on DENV later stages of replication.

V-P08

TRANSPLACENTAL TRANSFER PROFILE OF DENGUE VIRUS-SPECIFIC IGG 1, 2 1 2 1 1 2 1, 2 2Priscila Castanha , Eduardo J. M. Nascimento , Marli Cordeiro , Robyn Konicki , Guangchao Gu , Claudeir Silva Jr. , Ernesto Marques Jr. , Cynthia Braga

1Center for Vaccine Research, University of Pittsburgh, Pittsburgh, PA, USA; 2Oswaldo Cruz Foundation, Aggeu Magalhaes Research Center, Recife, Brazil

Placental transfer of maternal dengue virus (DENV)-specic IgG to the fetus likely plays an essential role in immunity and pathogenesis of DENV infection in infants. We investigated the placental transfer prole of DENV-specic IgG antibodies in maternal and umbilical cord blood sera pairs from parturients who took part of an ongoing birth cohort study in Recife, Brazil. Serotype-specic antibody prole was determined by Plaque reduction neutralization test. In-house virus-capture ELISA was used to estimate the levels of DENV-specic IgG (total as well as IgG1 and IgG4). Antibody titers were log-transformed and the transplacental antibody transfer calculated as ratio (value infant/value mother). From 200 parturient examined, 10.5% did not have detectable neutralizing DENV-specic antibodies to any of the DENV serotypes, 62.2% showed monotypic infection by DENV3, 22.8% the dual infection by DENV3/DENV4 and 4.5% other DENV serotype combinations. Dengue-specic IgG titers in the cord sera were higher than maternal levels (p=0.026), which is consistent with an active transport mechanism across the placenta. A negative correlation between DENV-specic IgG maternal titers and placental transfer ratio of anti-DENV3 (r= -0.1846, p=0.022) and anti-DENV4 (r= -0.3249, p=0.015) antibodies was found, demonstrating that high levels of maternal anti-dengue IgG antibodies reduce the maternofetal transfer of DENV serotype-specic IgG antibodies. Additionally, the titers of both isotypes IgG1 (r=0.6944, p<0.0001) and IgG4 (r=0.6875, p<0.0001) were highly correlated among the maternal-cord pairs, showing that both isotypes are efciently transferred via placenta. Further analysis is underway to assess the efciency of the transplacental transfer of other DENV-specic IgG isotypes.

V-P09

PRESENCE OF DENGUE IGA ANTIBODIES IN CASES OF DENGUE HEMORRHAGIC FEVER AND DENGUE SHOCK SYNDROME IN HONDURAN PATIENTS DURING THE DENGUE OUTBREAK-2013

1 1 2 1Ivette Lorenzana , Leda Parham , Carlos Sánchez , María José López1 2Virology Unit, Microbiology School, National Autonomous University of Honduras; Hospital Escuela Universitario-Materno Infantil, Tegucigalpa, Honduras

Dengue fever is one of the most serious arthropod-borne viral diseases. The clinical manifestations of dengue infection include severe and life -threatening forms dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). The laboratory diagnosis of dengue infection could be achieved (among other test) by the

detection of specic antibodies. Dengue IgA antibodies (Ab) could be a promising approach as an indicator of recent infection, and moreover, as possible marker for disease severity. The purpose of this study was to determine the presence of dengue IgA-Ab and to correlate it with primary and secondary infection and disease severity. The study population was 59 patients, clinically classied as DHF (n=55) and DSS (n=4) according to the WHO criteria and laboratory conrmed. Twenty samples were classied as primary infection and 39 as secondary infection. Dengue specic IgA-Ab was performed in plasma samples by IgA antibody-capture EIA (VIRCELL, S.L. Spain). The overall percentage of IgA-Ab and IgM-Ab positivity was 60% (n=35) and 86% (n=51) respectively. The presence of IgA-Ab was statistically signicant for secondary infections (31/39; 80%) when was compared with primary infection (4/20; 20%) OR=15.5, p=0.0001. Regarding disease severity, 56% of IgA-Ab was found in DHF vs 100% in SSD and 51% of IgM-Ab in DHF vs 47% in DSS. Our ndings are in agreement with other studies, which have reported higher positivity of IgA-Ab detection in samples from secondary infections when compare with primary cases. There is not statistical signicant difference of IgA-Ab according to disease severity, there is a tendency to nd higher presence of IgA-Ab positivity in DSS than in DHF (OR=7). Further analysis should be done to determine the utility of IgA-Ab detection as a marker of diagnosis and/or prognosis in dengue disease.

V-P10

EXPRESSION LEVEL EVALUATION OF DGCR8 IN HUMAN CELLS DURING VIRAL INFECTION BY DENGUE VIRUS 41 1 1 1 1 1 1 1Samir Casseb , Karla de Melo , Gustavo Holanda , Bruno Tardelli , Maria Helena Mendonça , Ana Celila Cruz , Pedro Fernando da Costa Vasconcelos

1 Instituto Evandro Chagas /SVS/MS, Ananindeua, Pará, Brazil

Dengue virus and its four serotypes (DENV-1 to DENV-4) infect 390 million people and are implicated in at least 25,000 deaths annually, Dengue virus is the most common disease in tropical and subtropical regions. In Brazil, DENV-4 reemerged in 2010 in Boa Vista and Cantá municipalities Roraima State. Virus spread to other geographic regions of Brazil with cases of infection start to be registered in the Northern (Roraima, Amazonas and Pará State), Northeast (Bahia, Pernambuco and Piauí State) and Southeast (Rio de Janeiro and São Paulo State). The Drosha-DGCR8 complex (Microprocessor) is required for microRNA (miRNA) biogenesis. DGCR8 recognizes the RNA substrate, whereas Drosha works as an endonuclease. High-throughput sequencing and crosslinking immunoprecipitation (HITS-CLIP) were used to identify DGCR8 RNA targets in human cells. Unexpectedly, miRNAs were not the most abundant targets. DGCR8-bound RNAs also comprised several hundred mRNAs as well as small non-codius RNAs and long non-coding RNAs. In the present study, we investigated the correlate expression of DGCR8 during viral infection by Dengue virus 4 in A549 cells. We used the method described by Johnson et al. 2005 for quantication of viral load, together with method for absolute quantication curve by RT-qPCR, quantication mRNA used for DGCR8 target and endogenous control RPL38 by RT-qPCR using SYBR Green method. Quantication of mRNA related to DGCR8, showed no changes from uninfected cells. Since its reduced expression during viral infection after three days post-infection, and its lower expression on the third day, might be related with the low expression of DGCR8 with high viral replication rate. The low expression of DGCR8 may help the viral replication process; the possible low expression of DGCR8 might be related not only with the biogenesis of miRNAs, but possibly to others small RNAs.

V-P11

STRUCTURAL STABILITY OF DENGUE VIRUS RECOMBINANT NS1 PROTEIN

Diego R. Coelho¹, Diego Allonso¹, Ronaldo Mohana-Borges¹

¹Laboratório de Genômica Estrutural, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, RJ, Brazil.

The current literature about Dengue suggests that the recombinant NS1 protein expressed in bacteria (NS1bac) may have almost the same conformation of the wild type form, except for the post-translational modications. Thus, the aim of this study was to characterize the structural stability of NS1bac in order to compare, hereafter, to the glycosylated protein. First, the NS1bac protein was expressed in E. coli, puried and refolded. Its monomeric population was then puried by size exclusion chromatography. The stability of tertiary and secondary structures was analyzed by uorescence spectroscopy and circular dichroism (CD), respectively. The results suggested that the structure of NS1bac is very stable against thermal denaturation, acidic pH, and increasing concentration of urea and guanidine hydrochloride. The secondary structure analysis by circular dichroism also revealed a typical spectrum of α-helix and β-sheet contents. In order to perform a future comparison, it was also developed a protocol for expression and purication of recombinant hexameric glycosylated NS1 protein (NS1mam). The NS1mam was puried from BHK-21 cell culture transfected with recombinant vector containing the NS1 gene. The preliminary structural analysis also revealed a typical CD spectrum of α-helix and β-sheet, in agreement with recent crystallographic results published.

V-P12

LIVER INJURY IN EXPERIMENTAL DENGUE VIRUS INFECTION IN CALLITHRIX PENICILLATA1 2 3 3 1Daniele Henriques , Vera Barros , Paulo Castro , Gilmara Silva , Milene Ferreira1 and Pedro Vasconcelos

1 2 3Seção de Arbovirologia e Febres Hemorrágicas, Instituto Evandro Chagas, Ananindeua, Pará; Seção de Patologia, Instituto Evandro Chagas, Ananindeua, PA; Centro Nacional de Primatas, Instituto Evandro Chagas, Ananindeua, PA; Brazil

Dengue viruses are globally the most important arboviruses causing human disease in more than 100 countries, and none vaccine is licensed for them. The lacks of an experimental model that mimic human disease is of concern, missing information on the disease pathogenesis. This study aimed to evaluate pathological changes in the liver of marmoset Callithrix penicillata sequentially infected with DENV3 and DENV2 Brazilian isolates obtained from fatal cases. C6/36 cells supernatant were used to infect 26 marmosets subcutaneously with DENV3 (primary infection-PI) (3.23 x 103 PFU/ml); uninfected control animals (CA) were reserved up the end of experiment. Daily for seven days and at intervals of 10, 15, 20, 30, 45 and 60 days post-infection experimented animals were anesthetized and sacriced; secondary

infection-SI with DENV2 (4.47 x 104 PFU/ml) was performed two months after PI of the remaining 13 non-sacriced animals. Liver of infected and CA animals were processed for histopathological and immunohistochemical assays. Both PI and SI marmoset livers showed sinusoidal congestion; portal spaces presented discrete inammatory response in most PI liver samples, while for SI the inammatory inltrate was more evident in the parenchyma. In both experiments, the inammatory inltrate was predominantly mononuclear; foci of hepatocellular necrosis were also observed. Other histological changes sporadically observed at different time intervals include apoptotic Councilman corpuscles in midzone, hyperplasia and hypertrophy of Kupffer cells, hepatocellular swelling in the lobe areas I and III, and megakaryocytes in both experiments; focal hemorrhage was only observed in a SI animal showing acute hepatic hemorrhagic failure. Samples of both experiments showed mild acute hepatitis. No pathologic changes were observed on CA livers. These ndings were consistent with those described for livers of human fatal dengue, indicating marmosets as suitable model to study dengue pathogenesis and thus may be a useful experimental model for dengue vaccine evaluation.

V-P13

CO-CIRCULATION OF DENGUE VIRUS 1, 2, 3 AND 4 IN SYMPTOMATIC PATIENTS DURING AN OUTBREAK, CONTAGEM, MINAS GERAIS, BRAZIL, 2013

Elisa Helena Paz Andrade, Ana Paula Pessoa Vilela, Julio Cesar Camara Rosa, Leandra Barcelos Figueiredo, Jaquelline Germano de Oliveira, Paulo Cesar Peregrino Ferreira, Claudio Antonio Bonjardim, Erna Geessien Kroon

Universidade Federal de Minas Gerais, Belo Horizonte, Brazil

In dengue endemic countries the co-circulation of multiple Dengue virus (DENV) serotypes in the same area has been described. Contagem is a city located in the State of Minas Gerais, in the Southest region of Brazil. In 2013, Contagem underwent a dengue epidemic with 23.572 notied cases, of which three patients died. In this study, we report the co-circulation of DENV1-4 in symptomatic patients during this outbreak. A total of 49 acute phase plasma samples from patients clinically suspected of dengue admitted to a hospital, located in Contagem, were collected during three weeks of May, 2013. All patients were residents of Contagem or neighboring cities. RNA of DENV serotypes 1, 2, 3 and 4 were detected by RT-semi-nested PCR targeting the C-prM region of its genome. Positive semi-nested PCR samples were puried and used directly as template in sequencing reactions. Of 49 samples, 33 (67.3%) tested positive for DENV RNA by RT-semi-nested PCR. Of these, 20 DNA fragments were sequenced and 8 (40%) were identied as DENV-3, 5 (25%) as DENV-4, 4 (20%) as DENV-2 and 3 (15%) as DENV-1. The data conrmed the co-circulation of the four DENV serotypes in one outbreak in a short time period. Financial support: CAPES, CNPq, MS/ SCTIE/ Decit/ FAPEMIG/ CNPq/ PRONEX Dengue / INCT-Dengue

VP-14

MODULATION OF DENGUE VIRUS INFECTION BY STRESS GRANULES.

Araujo D.F.F., Assunção-Miranda I., Carneiro L.A.

Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.

Stress granules (SGs) are cytoplasmic RNA granules that form in response to various types of cellular stress such as heat shock, hypoxia and radiation. Their formation results in inhibition of translation initiation of new proteins with the phosphorylation of eukaryotic initiation factor elF2, as a major event. The role of SGs suppression in translation and in RNA silencing suggests that such structures may have an impact on viral replication. Several studies have demonstrated interactions of these structures with several viruses during the course of infection. Recently, it was shown that dengue virus (DENV) infection negatively regulates the formation of SGs. Furthermore, co-localization between viral replication sites and proteins of SGs was also evidenced by immunouorescence, suggesting a possible interaction. Thus, we propose to investigate the role of SG formation in the viability of the host cell, the viral replication and assembly of new viral particles in the course of infection by DENV2. Initially, we evaluated the replication cycle of DENV2 comparing infected wild type cells with infected cells in which no phosphorylation of elF2 and consequently no formation of SGs, takes place. The study was performed by comparing cell viability, production of infectious particles, viral replication and the production of viral proteins in both cell types. It was found that in cells in which no phosphorylation of eIF2 takes place, there is a decrease in cell viability and in the production of infectious particles, despite a higher production of viral mRNA and proteins. Next stages of the project involve the study of the production of inammatory cytokines produced by DENV2 infection such as IL-6, KC and RANTES in both cell types and also to check the type of death induced by the virus and to identify which structures present in SGs are important for the completion of the viral life cycle.

V-P15

THE HETEROGENEOUS NUCLEAR RIBONUCLEOPROTEIN K IS A CELL FACTOR REQUIRED FOR DENGUE VIRUS TYPE 2 MULTIPLICATION

Viviana Castilla, Jesús E. Brunetti, Luis A. Scolaro.

Laboratorio de Virología, Departamento de Química Biológica, Facultad de Ciencias, Universidad de Buenos Aires, Argentina.

Heterogeneous nuclear ribonucleoproteins (hnRNPs) A2 and K are cellular RNA-binding proteins that participate in mRNA splicing, trafcking and translation and have been implicated in the multiplication of several cytoplasmic RNA viruses. To identify cellular factors involved in dengue virus (DENV) replication, here we characterized hnRNP A2 and K expression in Vero and A549 cells infected with DENV type 2 and we evaluated the effect of hnRNP K silencing on DENV-2 multiplication. We analyzed hnRNP intracellular localization by indirect immunouorescence assays, using anti-hnRNP and anti-viral glycoprotein E antibodies, at 2 or 5 days post- infection (dpi). Whereas hnRNP A2 showed a clear nuclear localization, similar to that observed in uninfected cells, the cytoplasmic translocation of hnRNP K was evident at 5 dpi in both cell types. We also performed A549 cell transfection with a plasmid that allows the expression of T7-tagged hnRNP K and the re-localization of transiently overexpressed hnRNP K to the cytoplasm was observed at 2dpi. The analysis by western blot showed no signicant differences in the total levels of hnRNP

K expression between uninfected and DENV-2 infected Vero or A549 cells. Finally, A549 cells were transfected with small interfering RNAs (siRNAs) that target hnRNP K or small non-interfering RNA used as control, and at 24 h post-transfection cells were infected. The effectiveness of siRNA silencing was corroborated by western blot and at 1 and 5 dpi, virus yield and the amount of glycoprotein E expressing cells were assessed by plaque assay and immunouorescence technique, respectively. A signicant reduction of 66.6% and 96.0% of virus yield was achieved at 1 and 5 dpi, respectively, and the amount of uorescent cells was also inhibited. The results obtained indicate that DENV-2 infection alters hnRNP K intracellular distribution and this hnRNP would play an important role in DENV-2 multiplication in cell cultures.

V-P16

VIRAL GROWTH CURVE FOR DETERMINING DAYS OF HARVEST OF DENGUE VIRAL STOCK IN VERO CELLS

Vivian Regina Silveira, Iray Maria Rocco, Rezolina Pereira dos Santos, Adriana Yurika Maeda, Fernanda Gisele da Silva, Terezinha Lisieux Moraes Coimbra, Ana Lucia Rodrigues Oliveira, Maria do Carmo Sampaio Tavares Timenetsky.

Insituto Adolfo Lutz, Coordenadoria de Controle de Doenças, Sao Paulo, Brazil.

Faced with the need for a dengue vaccine, Adolfo Lutz Institute directs its efforts to implement techniques to measure neutralizing antibodies. According to WHO, the correlation between the presence of neutralizing antibodies and virus protection against infection is not absolute and suggests that the Plaque Reduction Neutralization Test (PRNT) is the most widely accepted approach for measuring virus neutralization and protective antibodies. The deployment of PRNT implies the production of viral antigen. Knowing the growth curve of these viruses is an important parameter to PRNT results as a large amount of inactive particles can result in a falsely low antibodies titers. Therefore, the best harvest day must occur during the late exponential growth phase. The aim of this study was to observe the growth curve of these viruses and determine the best harvests days with titers around 105PFU/ml, verify the possibility of two different harvests in one bottle. Standards strains DENV1(Hawaii), DENV2(NGC), DENV3(H87) and DENV4(H241) adapted in Vero cells were used. Viral stocks were grown in Vero cells from the ATCC. The conditions for amplication were standardized using a Multiplicity of Infection of about 10-2 to DENV3 and 10-3 to DENV1, 2 and 4. The contents of this viral preparation was quantied by titration in 24 wells plates and revealed by immunostaining using monoclonal antibodies. The results demonstrate that it is possible to make two harvests and suggest that the best days to harvest of DENV3 is the day 7 (104.90 PFU/ml) and a second harvest on day 12 (104.70PFU/ml). For DENV1, 2 and 4 the best for the harvests days were the day 5 (105.69PFU/ml, 104.93PFU/ml and 105.87PFU/ml) and second harvest day 10 (105.38PFU/ml, 104.58PFU/ml e 105.13PFU/ml) respectively.

V-P17

EXPRESSION OF THE DENGUE VIRUS NS1 PROTEIN IN HEPG2 CELLS AND ULTRASTRUCTURAL ASPECTS1 1 1 1 2 1Kíssila Rabelo , Marciano Viana Paes , Adriana de Souza Azevedo , Edson Roberto A. de Oliveira , Cecília Jacques G. de Almeida , Ada Maria de Barcelos Alves and

1Simone Morais da Costa1 2Laboratório de Biotecnologia e Fisiologia de Infecções Virais, Instituto Oswaldo Cruz, Fiocruz,- RJ, Brasil. Laboratório de Imunofarmacologia, Instituto Oswaldo Cruz, Fiocruz,- RJ, Brasil

Dengue is an important viral disease, with epidemics in tropical and subtropical regions of the world. The disease is complex, with different manifestations, in which the liver is normally affected. The dengue non-structural protein 1 (NS1) is essential for virus viability, although its function is not yet elucidated. It is found in infected cells associated with plasma membrane and secreted into the circulation. Some reports indicate that NS1 can be used as a protective antigen, while others suggest its involvement in viral pathogenesis. Therefore, studies concerning the effect of the NS1 in hepatic cells can elucidate some aspects of its role during the virus infection. In this work, we standardized an efcient protocol of HepG2 transfection, a human hepatocarcinome cell, with a plasmid encoding the dengue-2 virus NS1 protein and evaluated the effect of this protein in the cell ultrastructures. Such assays were necessary, since HepG2 cells are particularly difcult to be transfected. First, we evaluated three different transfection methods: Lipofectamine, FuGENE 6 and nucleofection, and the expression of NS1 were detected by immunouorescence and ow cytometry. Subsequently, we analyzed ultrastructural changes in HepG2 cells expressing the recombinant protein by electron microscopy. Low levels of NS1 expression were observed in cells transfected with Lipofectamine and FuGene, 24h post transfection. On the other hand, the efciency of nucleofection was remarkable higher when compared to the others methods, in which approximately 59% of cells expressed the NS1. The analysis by electron microscopy suggested that NS1 appears to induce some morphological alterations in HepG2 involving organelles as Endoplasmic Reticulum and Mitochondria, as well as in Lipid Bodies production. Funding support: IOC-Fiocruz, PAPES-Fiocruz, FAPERJ, CNPq.

V-P18

THE DENGUE VIRUS NS3 PROTEIN IS LOCATED IN DETERGENT RESISTANT MICRODOMIANS OF HMEC-I INFECTED CELLS1 1 2 1García-Cordero J , Bueno-Cruz K , Gutiérrez-Castañeda B , González-Y-Merchand J3, Cedillo-Barrón L .

1 2 3Depto. Biomedicina Molecular, CINVESTAV, D.F.; Facultad de Estudios Superiores IZTACALA UNAM; Estado de México; Dpto. de Microbiología Molecular ENCB-IPN D.F.; México

Dengue is a viral disease transmitted by mosquitoes, the causative agent is an enveloped virus of positive-strand RNA and there are four serotypes (DEN-1 to 4). The interaction of virus with the host cell is still a matter of study. It is proposed that there are different kinds of cellular receptors including: heparan sulfate, lectins, DC-SIGN, heat shock proteins, among others. Several reports have shown that different life cycle events, including the process of virus replication, are held in

membranous organelles. It has been shown that the complex formed by replicative NS3 and NS5 proteins is associated with membranes of the endoplasmic reticulum. Cell membranes are characterized by the presence of dynamic structures of proteins and lipids known as lipid rafts or Resistant Microdomains Detergents (RMD) where events such cell signaling are take place. The goal of this work was to evaluate whether the dengue virus uses the lipid rafts during the events following the entry in a model of endothelial cells (HMEC-I). HMEC-I cells were infected with Dengue-2 virus and analyzed at 48h post-infection to evaluate the level of colocalization of a nonstructural protein NS3 (as part of the replication complex) and lipid rafts markers residents such as caveolin and otillin. Confocal microscopy results show a high level of colocalization between lipid rafts resident proteins and viral NS3 protease, but not for the transferrin receptor as no resident marker of lipid rafts. However when the extraction of detergent resistant microdomains (MRD) was performed through sucrose gradients and by evaluating NS3 distribution in each of the MRD fractions, it was observed that NS3 localises to the lipid rafts region. Furthermore, when NS3 was immunoprecipitated with caveolin, it was observed that it is indeed associated only with caveolar rafts. Moreover, it was found that lipid rafts could be playing an important role during viral assembly due to the fact that when cells were treated with an inhibitor of cholesterol synthesis (Lovastatin) it was observed that there was a reduction in viral infectious progeny. Finally, it was observed that NS5 protein did not colocalize with caveolar lipid rafts, which suggests that lipid rafts may only participate during the processing of the polyprotein. In conclusion, it was found that NS3 viral protein is localized in a protein complex associated to caveolin and that this complex affects the assembly of dengue virus in HMEC-I cells.

POSTERS SESSION ANTIVIRALSA-P01

VITAMIN D INHIBITS DENGUE VIRUS INFECTION IN THE HUMAN MONOCYTIC CELL LINE U937-DCSIGN BY INDUCING AUTOPHAGY.1 1 1 1 1Antonio H. Angel-Ambrocio , Margot Cervantes-Salazar , Rubén Soto-Acosta , Patricia Bautista-Carbajal , Rosa M Del Angel

1Centro de Investigación y de Estudios Avanzados Del Instituto Politécnico Nacional. México D. F.

The 1α,25-dihidroxy-calcitriol, vitamin D3 (VD3) has widely spread effects on cellular differentiation and proliferation, and immune responsiveness in monocytes/macrophages, dendritic cells and lymphocytes. The use of VD3 has been proposed for the treatment of autoimmune, bacterial and viral infectious diseases. Previous work from our group showed that treatment with VD3 resulted in a signicant reduction in the number of DENV U937 and HUH7 infected cells. The mechanism involved in the antiviral action of VD3 is not fully understood but it is known that VD3 induces autophagy in many cells lines and this has been the mechanism proposed to explain VD3 reduction in virus replication. Autophagy has been shown to facilitate DENV replication in some cells lines but to impede it in others. In this study, we sought to characterize VD3-induced antiviral mechanisms in DENV infected monocityc cells, using the highly permissive cell line U937-DCSIGN. Cells in culture were infected with DENV and treated either with VD3, rapamycin (an inductor of autophagy), 3-methyladenine (3MA, an inhibitor of autophagy), VD3 plus 3-methyl adenine or left untreated as controls. Treatment with either VD3 or rapamycin at non-toxic concentrations induced autophagy and resulted in an inhibition of DENV replication, while 3MA treatment has not effect. Interestingly, the combination of VD3 with 3MA treatment only partially revert the antiviral effect observed with VD3. We also found that VD3 induces the secretion of human cathelicidin, a protein that has direct antimicrobial activity. Our results support the idea that autophagy is one of the mechanisms induced by VD3 to inhibit DENV infection; however, the secretion of cathelicidin may also be an anti-DENV mechanism induced by VD3 in monocytic cells. These ndings provide insights into novel approaches to treat the severe forms of dengue disease.

A-P02

SYNTHETIC COMPOUNDS OBTAINED IN HOUSE INHIBIT DENGUE VIRUS INFECTION IN VITRO1 2 1 2 1 1Andrea C. Koishi , Taís A. Corrêa , Paula R. Zanello , Mauro V. Almeida , Juliano Bordignon , Claudia N.D. dos Santos

1 2Instituto Carlos Chagas, Brazil; Universidade Federal de Juiz de Fora, Brazil

Dengue is a signicant public health problem worldwide caused by a mosquito-borne Flavivirus. In Brazil, until the 18º epidemiological week of 2014 there were 394.614 cases registered, being 176 cases of severe dengue, 2.302 cases of dengue with warning signs and 104 conrmed deaths. Currently the four virus serotypes are circulating in the country in the proportion of DENV1 (85.5%), DENV4 (11.7%), DENV2 (2.4%) and DENV3 (0.4%). Despite the important social and clinical impact of the disease, there are no vaccines or specic antiviral therapies for its prevention and treatment. Considering the above, drug discovery research for dengue is of utmost importance. Here we propose the evaluation of the antiviral activity of synthetic compounds obtained in house. The antiviral activity screening was performed using an in situ ELISA assay that uses the human-derived hepatoma Huh7.5 lineage infected with DENV-1/FGA/89 as substrate. This assay was developed and validated to this purpose. Compound cytotoxicity was assessed by the MTT and Neutral Red assays. Selected hits were tested in human peripheral blood mononuclear cells (PBMCs) infected with DENV-4/TVP360, and infection and cell viability (7-AAD and Anexin V staining) were evaluated by ow cytometry. Out of 49 compounds screened with the maximum non-toxic dose (MNTD) in Huh7.5, fteen were able to reduced DENV-1 infection in at least 70%. The ones that presented SI values greater than 5 were chosen for further analysis. In order to evaluate the antiviral activity in a more relevant physiological system, PBMCs were infected with DENV-4 and treated for 120h. Treatment with three compounds were able to lower infection to levels compared to IFN-α 2A (reference compound) with little cytotoxic effect. This study shows that compounds obtained in house reduced DENV infection in human cell lineage and primary cell culture, being promising for the development of a dengue antiviral drug.

A-P03

MARCGRAVIACEAE-ORIGINATED COMPOUNDS DEMONSTRATED ANTIVIRAL ACTIVITY AGAINST DENV-2 IN PRIMARY HUMAN MONOCYTES INFECTED IN VITRO 1 1 1 1 1Luciana Gomes Fialho ; Cíntia da Silva Mello ; Vagner Pereira da Silva ; Maria Raquel Figueiredo ; Claire Fernandes Kubelka

1Fiocruz, Rio de Janeiro - RJ - Brasil

Introduction: The discovery of medicinal plants with antiviral activity against Dengue virus (DENV)-2 is important since the viral load and replication generate immune responses leading to severe disease outcomes. The use of the virus main target cells in this study, like primary monocytes, reproduces events occurring with dengue patients. Marcgraviaceae, has been evaluated for its biodynamic activities, due to its almost complete chemical and pharmacological uniqueness. The aim of this work was to determine the antiviral potential of a Marcgraviaceae sp. Methods and results: Primary human monocytes were infected with DENV-2 (strain 16681). We analyzed for three days the iNOS expression by ow cytometry, while cell culture supernatants were evaluated for NS1, by ELISA and for NO2-, by Griess reaction. The virus induced expression/production of all factors evaluated at 48 hours post-infection and this time-point was adopted for antiviral assays with plant fractions. Monocytes treated after infection either with buthanolic fraction or with 89-98 subfraction, both derived from a leave crude ethanolic extract were able to reduce signicantly the NS1 production (50% and 79%, respectively). The 89-98 subfraction increased NO production in 55%. Afterwards, we evaluated if these compounds could be inhibiting the infection to new cells. Monocytes were infected and incubated, simultaneously, with fractions; after two hours fresh medium was added and the cells analyzed for the infection rate by ow cytometry at 48 hours. The results showed that only 89-98 subfraction presented a strong reduction in monocytes infection rate (60%) compared with cells infected without compounds, suggesting that this fraction would be blocking the infection of new monocytes. Conclusion: Compounds studied are good candidates for the development of herbal medicines for dengue treatment, since they generate an antiviral response and possible prevent the infection of new cells. Financial support: PROEP-IOC & RPT 11D/FIOCRUZ; CAPES; CNPq and FAPERJ.

A-P04

IN VITRO STUDY OF A MEDICINAL PLANT WITH IMMUNOMODULATORY AND ANTIVIRAL POTENTIAL IN CONTINUOUS LINES OF HUMAN HEPATOCYTES INFECTED WITH DENGUE VIRUS-2.

1 1 1 2 3 1Cíntia da Silva Mello ; Fialho, Luciana Gomes ; Marinho, Cintia Ferreira ; Siani, Antonio C ; Valente, Ligia M ; Kubelka, Claire F .1 2 3Instituto Oswaldo Cruz, Farmanguinhos, FIOCRUZ; Instituto de Química, UFRJ; Rio de Janeiro, Brasil

Introduction: Infection with Dengue virus (DENV) in Brazil is characterized as an endemic disease with periodic epidemics. Phytotherapy is considered an alternative for the immunomodulation of the immune system through the dynamic regulation of signaling molecules such as cytokines, which interfere in their cellular effector functions and in humoral components. The aim of this study is to investigate compounds originated from medicinal plants that could modulate the innate immune response associated with immunopathological features in DENV infection using an in vitro infection model with continuous lines of human hepatocytes (Huh-7). Methods and Results: After target cell infection by DENV-2, cultures were treated with the aqueous and hydroalcoholic extracts of Uncaria spp. (UGC/UGFEEA) and tested at different concentrations. The infected cell rate was determined after incubation periods by virus specic immunouorescence and ow cytometry analysis. Nonstructural viral proteins (NS1) were detected in the cell culture supernatants as was the chemokine MIF, all determined by ELISA assays. Antiviral effects of UGC/UGFEEA were observed as compared to untreated cells in concentrations that reduced the frequency of viral antigen positive and the amount of NS1 secreted. Immunomodulatory effects were shown by reduced MIF secretion. In UGC, the optimal concentration was 10 μg/mL as antiviral activity by ow cytometry(DENV= 57.45 vs DENV+UGC 10 μg/mL= 46.74) and immunomodulatory effect (72h: DENV=3084.71 vs DENV+UGC 10 μg/mL=1874.54). In UGFEEA, 1μg/mL showed better antiviral activity by ow cytometry (72h): DENV=44.67 vs DENV+UGFEEA 1μg/mL=25.02 and NS1(24h): DENV=2.56 vs DENV+UGFEEA 1μg/mL=0.75), and 0.1μg/mL greater imunonodulador effect (72h): DENV=3084.71 vs DENV+UGFEEA 0.1 μg/mL=1232.86).Values shown are means of DO. Conclusion: These preliminary results showing a possible antiviral and immunomodulatory activities from UGC/UGFEEA in human hepatocytes, open perspectives to advanced studies about the mechanisms of action related to these extracts and their assets components such as endothelial cell permeability and the modulation of cell activation. Supported by FIOCRUZ/RPT11D;IOC/PROEP;CNPq;FAPERJ;CAPES.

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SCREENING FOR ANTI-DENGUE VIRUS ACTIVITY OF QUINIC ACID DERIVATIVES.

Paula Rodrigues Zanello, Andrea Cristine Koishi, Fabricio Klerynton Marchini, Mauro Vieira de Almeida, Claudia Nunes Duarte dos Santos, Juliano Bordignon

Instituto Carlos Chagas, FIOCRUZ, Rio de Janeiro, RJ, Brazil

Introduction: Among all human arthropod-vector diseases, Dengue (genus Flavivirus) is the most important as it is a health threat worldwide. Despite the large number of cases and their severity, to date there is neither specic dengue treatment nor approved vaccine to prevent infection. Substances obtained from quinic acid have demonstrated antiviral acitivity against HIV, Hepatitis B Virus, Herpes Simplex-1, Enterovirus 71 and Inuenza H1N1. Here we test the anti-dengue virus activity of quinic acid derivatives against the four dengue serotypes. Methods: Prior to the antiviral screening, compound cytotoxicity was assessed by MTT and Neutral Red assays. Quinic acid derivatives had their antiviral activity (against four dengue serotypes) screened in Huh7.5 cells through in situ ELISA. Foci-forming assays validated ELISA ndings. Two promising compounds were also validated in human peripheral blood mononuclear cells from healthy volunteers (PBMCs) infected in vitro with DENV-4. Results: Out of 10 tested compounds, two (AAP17 and ALB34) were able to reduced DENV-1 to -4 infection. PBMCs infected with DENV-4 had similar low infection rates Further results showed that AAP17 and ALB34 did not act as virucidal, nor affect the binding and internalization of viral particles. Discussion: To our knowledge, this is the r st report on the use of quinic acid derivatives against DENV. Data demonstrated the anti-DENV action of two promising drugs in both Huh7.5 and human primary cells. Additional experiments using DENV subgenomic replicon system may determine the mode of action of hit substances. These results contribute for the development of a unique dengue treatment against the virus.

A-P06

ANTIVIRAL ACTIVITY OF FUNGI AND PLANT EXTRACTS AGAINST DENGUE VIRUS1 2 1 2 2 3 1 3Emerson C. Barbosa ; Fernanda L. M. Francisco ; Gabriela C. Mota ; Tânia M. A. Alves ; Carlos L. Zani ; Luiz H. Rosa ; Carlos Eduardo Calzavara-Silva ; Erna G. Kroon

1and Jaquelline G. Oliveira1 2 Laboratório de Imunologia Celular e Molecular - Centro de Pesquisas René Rachou (CPqRR) – FIOCRUZ; Laboratório de Química de Produtos Naturais, Centro de

3Pesquisas René Rachou (CPqRR) - FIOCRUZ; Departamento de Microbiologia - Universidade Federal de Minas Gerais (UFMG), Brasil

Natural products could be, in the future, a source of antiviral drugs for dengue treatment and also for other diseases caused by viruses of the Flaviviridae family, or even by a wide spectrum of viruses. In this study, were screened 2940 plant and fungi extracts obtained from the Fiocruz collection of plant and fungi extracts (COLAB) for antiviral activity against Dengue virus (DENV). Ethanolic extracts of different anatomical parts of a wide range of plant and from cultures of fungi isolates were evaluated in vitro against Dengue virus 2 (DENV-2) by the cytopathic effect (CPE) denoted by degree of inhibition upon treating DENV2-infected BHK-21 cells. The potency of these extracts at 25 µg/mL on DENV-2 inhibition were further examined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay, in which the percentage of viability of cells alone, cells infected with DENV-2 and infected cells treated with extracts were measured and compared. A total of 114 out of the 2940 extracts tested presented antiviral activity against DENV-2. Thirty two out of the those 114 extracts were obtained from plants belonging to 17 distinct families: Malpighiaceae (6), Erythroxylaceae (1), Melastomataceae (2), Myrtaceae (1), Rubiaceae (2), Fabaceae (1), Clusiaceae (1), Combretaceae (1), Leguminosae (2), Lythraceae (2), Asteraceae (4), Amaryllidaceae (3), Sapindaceae (2), Ochnaceae (1), Begoniaceae (1), Annonaceae (1), Primulaceae (1). All the 82 fungal extracts with antiviral activity were obtained from cultures of endophytic fungi, which are in the process of taxonomic identication. Eight fungal extracts presented interesting effective concentration 50 (EC50) values, ranging between 3,1 µg/mL and 12,5 µg/mL, with no cytotoxicity at 100 µg/mL. Our results indicate that fungi extracts can be a promising source of antiviral agents. Financial support by FIOCRUZ, CPqRR, CNPq and FAPEMIG.

PATHOGENESISABSTRACTS INVITED SPEAKERS

PATHOGENESIS OF DENGUE VIRUS INFECTIONS: UPDATE AND CHALLENGES

Pedro F. C. Vasconcelos

PAHO/WHO Collaborating Center for Arbovirus Research and Diagnostic Reference; National Reference Laboratory for Dengue and other arboviruses; National Institute for Science and Technology on Viral Hemorrhagic Fevers (INCT-FHV); Department of Arbovirology and Hemorrhagic Fevers, Instituto Evandro Chagas, SVS/MoH, Rodovia BR-316, km-07, Ananindeua, Pará, Brazil

The pathogenesis of dengue virus infections remain poorly understood and therefore a challenge to scientists around the world. Despite several discoveries on participation of antibodies and different viral lineages in the disease severity, it is not clear the role of both and also of cells and their cytokines in the pathophysiology of dengue, mainly on severe disease (Yacoub et al., 2013). Many of difculties for a clear understanding of pathogenesis of dengue are the lack of an ideal model to study the dengue disease. The ideal model is the one reproducing the human disease, and no one animal has demonstrated this quality. In the absence of a perfect model, the Old World monkey especially rhesus monkey were used not only to test potential vaccine candidates but rather to observe viremia and humoral response particularly antibodies response, as previously used for yellow fever and yellow fever 17D vaccine (Theiler & Smith, 1937). Only recently New World monkeys, especially marmosets were investigated their susceptibility to dengue virus infections (Omatsu et al., 2011; Omatsu et al., 2012; Ferreira et al., 2014). The results showed these animals as promissory model to study dengue viruses. Several lineages of mice have been infected with dengue viruses and different degrees of susceptibility were achieved. More robust results were obtained on humanized mice and interferon decient animals, and also immunocompetent mice challenged several times with DENV3 (Kuruvilla et al., 2007; Zompi & Harris, 2013; Diniz et al., 2014). This is easily understood since mice are naturally resistant to dengue viruses and despite the interesting results obtained these animals cannot be considered as ideal model to study dengue pathogenesis. Considering the difculties of a good model to study dengue viruses and the preliminary results indicating that marmosets are susceptible to those viruses, and also the availability of these animals in the National Primate Center (CENP) at the Evandro Chagas Institute (IEC), we performed three distinct experiments on marmosets (Callithrix penicillata) with dengue viruses: Experiment 1. 26 animals were subcutaneously infected with Brazilian isolates of DENV3 and 60 days later 13 remaining animals (not euthanized) were infected with DENV2; experiment 2. Three animals were infected with DENV2 and one year and 7 months later challenged with DENV3; experiment 3. Three animals were infected weekly with DENV3 and 24 hours later received anti-DENV2 antibodies, four animals were mock no infected controls and two received only anti-DENV2 antibodies. The results were interesting: Experiment 1 - animals developed high viremia titers and robust antibody responses; many cytokines were increased; Interesting, secondary infections after 2 months of primary infections revealed some discrete alteration characterized by pathologic changes, antigen detection and expression of selected cytokines. Experiment 2 – viremia was high and antibody response robust; secondary infection was accompanied by important pathologic changes and they developed thrombocytopenia, leukopenya, discrete increased levels of aminotransferases and a strong in situ response in liver, kidney, lung and dermal tissues mimicking nding obtained on human fatal dengue disease. Experiment 3 – animals presented low viremia titles and they remained all timing experiment apparently without alterations; nonetheless, changes were observed on liver, brain, lung and lymph nodes. In conclusion, marmosets that are closed to human on evolutionary aspect showed to be a handle, cheaper, and feasible model to investigate the pathogenesis of dengue virus infections and perhaps also to potential vaccine candidates, but interval between primary and secondary infections should be longer; nally other New World primate species should be investigated the potential susceptibility of them to dengue viruses using the new available virologic and immunologic tools. Financial support: this study had the nancial support of INCT-FHV (grant CNPq/CAPES/FAPESPA nº 573739/2008-0), CNPq (grant 301641/2010-2), IEC, CENP and SVS/MoH

ABSTRACTS ORAL PRESENTATIONSEUROPEAN ANCESTRY: A RISK FACTOR FOR SEVERE DENGUE DISEASE. GWAS STUDY IN CUBAN POPULATION

1 2 2 1 1 4 2,3 1Beatriz Sierra ; Peter Triska ; Pedro Soares , Ana B. Perez , Gissel Garcia , Anavaj Sakunthabai , Luisa Pereira , María G. Guzman 1 2Virology Department, PAHO/WHO Colaborating Center for the study of dengue and its vector, Institute of Tropical Medicine Pedro Kourí (IPK), Havana, Cuba. Institute

3 4of Molecular Pathology and Immunology of the University of Porto (IPATIMUP), Porto, Portugal. Faculty of Medicine, University of Porto, Porto, Portugal. Functional Genetics of Infectious Diseases Unit, Pasteur Institute, Paris, France

The proclivity showed by certain populations and ethnic groups to develop severe disease, while others remain asymptomatic, support the impact of human genetic factors on DV disease outcome. Particularly Cuban dengue outbreaks have provided evidence of a reduced risk of people with main African ancestry for severe dengue disease compared to people predominantly of European descent. We conducted a genome-wide association study of 2.5 million single nucleotide polymorphisms in 274 Cuban individuals collected during the 2006 dengue epidemic, from two cities, Havana and Guantanamo, from the western and eastern sides of the island, respectively. This screening included DF, DHF and asymptomatic cases, and controls geographically matched. The genomic proportions of European, African, Native American and Asian ancestry were evaluated for all the Cuban groups by using the program ADMIXTURE. A local ancestry analysis was performed by using the algorithm RFMix, using two parental populations from 1000 Genomes database to represent the European and African components. Our results showed that Cuban current population have a mixed composition, mostly from African and European descent, markedly different to other populations of Latin America. The mean proportions of African and Native American-ancestries are higher in Guantanamo, while the European-ancestry component predominates in Havana. Different patterns in the mean proportions of ancestry among the studied groups are observed, especially in Havana, with a signicantly higher European ancestry proportion in the DHF group compared to the asymptomatic group, while this last one showed a signicantly higher proportion of African ancestry compared to the DHF group. Notably, local ancestry analysis showed some chromosomal regions having a considerable higher proportion of African ancestry in asymptomatic versus DHF, both in Havana and in Guantanamo. Our results provide insights on how the genetic ancestry can condition the susceptibility to dengue severe disease, thus facilitating the identication of potential dengue susceptibility/resistance genes.

GENOME-WIDE TRANSCRIPTOME ANALYSIS AND GENE SILENCING SCREENING IDENTIFIES LYSOSOMAL FUNCTION AS A MAJOR REGULATOR OF DENGUE VIRUS REPLICATION IN HUMAN MYOBLASTS

1 1 2 3 4 5 6 1Irene Bosch , Sowmya Shivanna , Andrea Gamarnik , Cybele García , Katherine Martin , Jonathan Dinsmore , Alan Derr , Lee Gehrke1 2 3 Massachusetts Institute of Technology, MA, USA; Leloir Institute, Buenos Aires Argentina, Universidad de Buenos Aires, Argentina; 4 BioarrayUSA, Boston, MA, USA; 5 Beth Israel Deaconess Hospital, Boston, MA, USA; 6 Massachusetts University Medical School, Worcester, MA, USA

RNA inhibition based screenings against avivirus represent a powerful approach to identify host factors involved in the virus lifecycle. In the present study we utilized human myoblasts and a gene expression primary screen to assess the genes with differential expression during infection. By using a reporter virus we measured luciferase to assess virus replication in the shRNA screen of 75 selected genes. In addition, the dengue virus genome copy number and uorescent microscopy image analysis of virus NS3 protein were utilized to cross-validated the effective hits. We determined that Cathepsin S (CTSS) and Rab7A (p<0.05) increased virus replication pointing towards lysosomal function as a major site for dengue virus control. Increased virus replication was conrmed using specic lysosomal CTSS inhibitors in treated and infected myoblasts, therefore conrming the novel ndings of the shRNA screen. Inhibitors of the Jak-STAT pathways were used as positive controls to show expected increase in viral replication of treated and infected myoblasts. The implications of increasing lysosomal antiviral activity against a avivirus may contribute to new therapeutic options. This work was in part supported by NIH AI10019 and by The Broad Institute siRNA platform.

THE HUMAN GENETIC COMPONENT OF SUSCEPTIBILITY TO DENGUE VIRUS INFECTION AND DISEASE PROGRESSION IN A SAMPLE OF COLOMBIAN POPULATION

1 1 1 1 2 3 3 1Yerly Useche , Juan-C Chacón , Omer Campo , Alonso García , Berta-N Restrepo , Carlos Narváez , Doris-M Salgado , Gabriel Bedoya1 2 3 GENMOL, Facultad de Ciencias Exactas Naturales, Universidad de Antioquia; Instituto Colombiano de Medicina Tropical ICMT, Universidad CES; Facultad de Medicina, Universidad Surcolombiana

Differences in Dengue susceptibility and severity have been attributed to many factors including ethnicity and host genetics. In fact, several polymorphisms in candidate genes, focused on immune related pathways, have been associated to dengue infection and/or dengue severity. Moreover, epidemiological studies have shown high morbidity and mortality in individuals of European ancestry compared to individuals of African ancestry and studies on Brazilian and Colombian populations showed African ancestry, as protective for DHF. We assessed the effect of genetic ancestry, estimated with 30 Ancestry Informative Markers, and 49 variants in 27 candidate genes on DENV infection and progression of dengue in children (172 cases and 144 controls) from Huila, Colombia. Cases included 128 with dengue haemorrhagic fever/dengue shock syndrome and 76 with dengue fever. Ancestry and genetic associations adjusted by sex and ancestry were calculated using Admixmap and Plink, respectively. We found that mean ancestry fractions of Huila's sample are: European 62.0%±6.4%, Amerindian 28.3%±6.0% and African 9.4%±4.6% and were similar within cases and between cases and controls, except the low African ancestry in controls compared to cases (P<0.0001). The case-population study revealed association with Amerindian and African ancestry effects. Eight variants in IL6, TNFSF10, IL17F, RIPK2, IL18, NFKBIA, IL4R y CD209 genes were associated with susceptibility to DENV infection (ORs = 1.92-17.68; P = 0.0001-0.01). Conversely, seven variants in IKBKE, IL1B, IL12B, HLAB, IL6 y CSF3 genes were associated with resistance to DENV infection (ORs = 0.26-0.57; P = 0.0026-0.01). Otherwise, two variants rs1805016 (IL4R) and rs17127114 (JAK1) were associated with occurrence of haemorrhages (OR 2.539; P<0.020 and OR 1.882; P<0.032, respectively), without ancestry effects. In conclusion, this

population has variants of susceptibility and resistance to DENV infection and only risk variants to occurrence of haemorrhages. These associations are consistent with previous studies. Financial support: COLCIENCIAS (Grant Code 1115-493-26145; Contract 441.2009).

DENGUE VIRUS NON-STRUCTURAL PROTEIN-1 (NS1) INCREASES HUMAN PULMONARY ENDOTHELIAL CELL PERMEABILITY IN VITRO

Henry Puerta-Guardo, Dustin Glasner, and Eva Harris

Division of Infectious Diseases and Vaccinology, School of Public Health, University of California, Berkeley, CA

Dengue is the most prevalent arboviral disease in humans and a major public health problem worldwide. Systemic plasma leakage and increased vascular permeability leading to profound shock and potentially fatal complications are critical determinants of dengue severity. Dengue pathogenesis involves a complex interaction of the virus and host immune response, including cross-reactive antibodies and T cells, complement activation, and elevated levels of cytokines and other soluble mediators that correlate with severe disease. However, the mechanism of vascular dysfunction in dengue disease is still unclear. Secreted and cell-surface-associated dengue virus (DENV) nonstructural protein 1 (NS1) and anti-NS1 antibodies are implicated in contradictory roles of protection and pathogenesis, and how NS1 contributes to dengue pathogenesis remains uncertain. Here we evaluated the role of soluble NS1 (sNS1) in inducing endothelial dysfunction. Cultures of human pulmonary microvascular endothelial cells grown on a transwell permeable membrane system as a model of barrier function in vitro were exposed to sNS1 (0.2-20 µg/mL), and endothelial permeability was examined by continuously measuring the trans-endothelial electrical resistance (TEER). DENV2 sNS1 induced a signicant dose-dependent increase in endothelial permeability starting 2 hours post-treatment (hpt), with a ~20 and ~50% decrease in TEER at 5 and 20 µg/mL, respectively. This effect persisted for more than 24 h as compared to the TEER baseline values exhibited by untreated controls and treatment with unrelated protein (20 µg/mL OVA). Lower concentrations (0.2 and 1 µg/mL) showed less dramatic but still signicant decreases in TEER that returned to baseline ~12 hpt. When the sNS1 hexameric structure was disrupted by boiling, this effect was lost, but TEER reduction was restored as the hexamer reformed over time. sNS1 from DENV1, DENV3, and DENV4 yielded similar results. Confocal microscopy revealed concomitant alterations in intercellular junctional proteins. Our ndings suggest a new mechanism of sNS1 directly triggering endothelial vascular dysfunction that occurs in severe dengue disease.

HIGHLIGHTED POSTERSPathogenesis

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HISTOPATHOLOGICAL CHANGES AND VIRAL REPLICATION IN MULTIPLE ORGANS FROM DENGUE FATAL CASES IN BRAZIL 1 1 2 3 1Paes, M.V ; Póvoa, T.F ; Basílio-de-Oliveira C.A ; Chagas V.L.A ; Alves, A. M. B

1 2 3Laboratório de Biotecnologia e Fisiologia de Infecç ões Virais, IOC, Fiocruz, RJ, Brazil; Anatomia Patológica, Hospital Gaffrée Guinle, UNIRI O, RJ, Brazil; Anatomia Patológica, Hospital Universitário Clementino Fraga Filho, RJ, Brazil.

The pathogenesis of dengue hemorrhagic fever (DHF) is complex and not well dened. In the present work, we initiated a study with tissues obtained from different organs (liver, lung, heart and kidney) of four dengue fata l cases. In this work, we analyzed lesions in different organs of four dengue fatal cases, occurred in Brazil. Tissues were prepared for visualization in optical and electron microscopy, with damages quantication. Cases presented necrotic areas in the liver and diffuse macro and microsteatosis, which were more accentuated in case 1, who also had obesity. The lung was the most affected organ, with hyaline membrane formation associated with mononuclear inltrates in patients with pre-existing diseases such as diabetes and obesity (cases 1 and 2, respectively). These cases had also extensive acute tubular necrosis in the kidney. Infection induced destruction of cardiac bers in most cases, with absence of nucleus and loss of striations, suggesting myocarditis. Spleens revealed signicant destruction of the germinal centers and atrophy of lymphoid follicles, which may be associated to decrease of T cell number. Circulatory disturbs were reinforced by the presence of megakaryocytes in alveolar spaces, thrombus formation in glomerular capillaries and loss of endothelium in several tissues. Besides histopathological and ultrastructural observations, virus replication were investigated by detection of dengue antigens, especially the non-structural 3 protein (NS3), and conrmed by the presence of virus RNA negative strand (in situ hybridization ), with second staining for identication of some cells. Results showed that dengue had broader tropism comparing to what was described before in literature, replicating in hepatocytes, type II pneumocytes and cardiac bers, as well as in resident and circulating monocytes/macrophages and endothelial cells. These studies may contribute to a better understanding of the key cells involved in pathogenesis and replication of dengue. Financial support: Fiocruz/IOC, CNPq and Faperj.

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DENGUE VIRUS NONSTRUCTURAL 3 PROTEIN INTERACTS WITH HUMAN GLYCERALDEHYDE-3- PHOSPHATE DEHYDROGENASE (GAPDH) AND ALTERS ITS GLYCOLYTIC ACTIVITY.

1 1 2 1 1Emiliana M. Silva ; Jonas N. Conde1; Diego Allonso ; Manuela Leal ; Gustavo T. Ventura ; Ronaldo Mohana-Borges .1 2Laboratório de Genômica Estrutural, Instituto de Biofísica Carlos Chagas Filho, UFRJ, Brazil Laboratório de Biologia Computacional, Instituto Nacional de Metrologia, Qualidade e Tecnologia - INMETRO, Brazil

The multifunctional NS3 protein forms a non-covalent complex with the NS2B cofactor, functioning as serine-protease that is responsible for proteolytic processing of the viral polyprotein at the NS2A/NS2B, NS2B/NS3, NS3/NS4A and NS4B/NS5 junctions. It has also an ATPase/helicase and RNA triphosphatase domain at its C-terminal, which is essential for RNA replication. In this study, we employed yeast two-hybrid (Y2H) system to identify liver human proteins that interact with NS3 protein, which was performed using the Matchmaker GAL-4 Two-Hybrid System 3 (Clontech). The Y2H screening identied 49 colonies that grew in the absence of Trp,

Leu, His, Ade and were tested for the LacZ phenotype. Among the NS3- interacting partners, we found the glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The human GAPDH directly interacted with NS3 helicase and protease domains as demonstrated by ELISA and molecular docking. Surface plasmon resonance measurements revealed the high avidity and afnity between these interactions. In addition, NS3 protein co-localizes with GAPDH in hepatocytes cell lineage infected with dengue virus using mouse polyclonal anti-NS3 helicase and rabbit polyclonal anti-GAPDH. Glycolytic activity for GAPDH decreased in the presence of NS3 helicase and protease domains. As GAPDH is a well characterized glycolytic enzyme, displaying multiple functions in transcriptional coactivation, DNA repair, oxidative stress, apoptosis induction and regulation, we suggest that the GAPDH-NS3 interaction is associated with the viral replication and apoptosis processes. Financial Support: FAPERJ, CNPq and CAPES.

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EVALUATION OF CLINICAL AND INFLAMMATORY PARAMETERS IN PATIENTS INFECTED WITH DENGUE VIRUS (DENV)-41 1 1 2 3 4Victor Fiestas Solórzano , Elzinandes Leal de Azeredo , Luzia Maria de Oliveira- Pinto , Rivaldo Venâncio da Cunha , Paulo Vieira Damasco , Luis José de Souza , Claire

1Fernandes Kubelka1 2Laboratório de Imunologia Viral, Instituto Oswaldo Cruz/FIOCRUZ, RJ, Brazil Departamento de Clínica Médica, FM, Universidade Federal do Mato Grosso do Sul,

3Campo Grande, MS, Brazil Hospital Hospital Rio-Laranjeiras, RJ, Brazil 4Centro de Referência em Dengue e Faculdade de Medicina, Campos de Goytacazes, RJ, Brazil

Background: Dengue is considered the main human arbovirus and is a cause of morbidity and mortality throughout the world. DENV- 4 was reintroduced into Brazil in 2010 and then spread to the different regions of Brazil. The aim of this study was to evaluate the clinical and inammatory prole of D ENV-4 infection. Methods: During a DENV-4 outbreak in 2013, dengue suspected patients were enrolled in the states of RJ and MS, Brazil. Plasma levels of TNF-α, IL-1β, IL-6 , IL-10, IFN-γ, IL-8 and MCP-1 were evaluated by Luminex® and ELISA. Plasma levels of brinogen and sTF were also determined. Results: 207 patients with conrmed dengue infection were included. 146 (70.5%) had dengue without warning signs (DwoWS), 59 (28.5%) had dengue with warning signs (DwWS) and two (1.0%) had severe dengue. Three commonest warning signs were abdominal pain/tenderness, mucosal bleeding and increase in hematocrit concurrent with rapid decrease of platelet count. DwWS patients had signicantly lower platelet count (p<0.0001), lower brinogen levels (p=0.017) and signicantly higher AST and ALT levels (p<0.0001 and p=0.014 respective ly) than those with DwoWS. Concentrations of circulating cytokines and chemokines were signicantly elevated in dengue patients compared to healthy controls. IL-10 was signicantly higher (p=0.0008) in DwWS patients compared with DwoWS. Interestingly, plasma levels of IL-10 were associated signicantly and inversely with platelet (r=-0.28, p=0.002) and lymphocytes counts (r=-0.34, p=0.0006), but positively with AST levels (r=0.29, p=0.005). However, IL-10 did not show a good predictive value in discriminating DwoWS and DwWS patients (ROC curve: 0.66, 95% IC: 0 .58-0.75). No signicant difference was observed in levels of sTF between patients with DwoWS and DwWS. Conclusions: DENV-4 infections had a milder clinical presentation. DwWS patients had signicantly lower brinogen levels. In contrast, these patients showed elevated IL- 10 levels supporting the importance of this inammatory marker in the pathogenesis of dengue.

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PROTEOMIC ANALYSIS OF PLATELETS FROM DENGUE PATIENTS: CONTRIBUTIONS TO THE PATHOGENESIS ELUCIDATION 1 2 3 1 1 1 2 4Monique Trugilho ; Eugenio Hottz ; Paulo Carvalho ; Giselle Brunoro ; André Teixeira-Ferreira ; Ana Gisele Neves-Ferreira ; Patrícia Bozza ; Fernando Bozza ; Jonas

1Perales . 1 2 3Laboratório de Toxinologia, Laboratório de Imunofarmacologia, Instituto Oswaldo Cruz (IOC); Laboratório de Proteômica e Engenharia de Proteínas, Instituto Carlos Chagas (ICC); 4Instituto de Pesquisa Clínica Evandro Chagas (IPEC); FIOCRUZ, Brazil.

Dengue is the most frequent hemorrhagic viral disease and re-emerging infection in the world, affecting millions of people and causing thousands of deaths every year. Infection can be asymptomatic or may lead to sickness whose intensity vary from undifferentiated fever up to severe cases with bleeding and shock. Although thrombocytopenia is characteristically observed in mild and severe forms of dengue, the role of platelet activation in dengue pathogenesis has not been fully elucidated. Here we studied platelets isolated from healthy donors and dengue virus (DV)-infected patients, using label-free mass spectrometry-based quantication in a proteomic approach. Isolated platelets were lysed, trypsinized and fractioned on the OFFGEL system, followed by MS/MS analysis (ESI-LTQ-Orbitrap XL). Protein identication/quantication was performed using the PatternLab for Proteomics software. As preliminary results, we identied more than 3000 proteins in two conditions (control and dengue) and several proteins were found as differentially expressed with q-value of 0.05. As examples, we can mention upregulated proteins in dengue samples, such as disulde isomerase (PDI), required for platelet adhesion/aggregation; anti-oxidant superoxide dismutase (SOD1) and glucose-6- phosphate isomerase (GPI), involved in the glycolytic pathway activation. Thrombospondin-1 (THBS1), a glycoprotein which mediates platelet adhesion to endothelium upon injury/inammation and presents anti-angiogenic function when secreted by activated platelets, was downregulated in dengue. Another downregulated protein was platelet glycoprotein V (GP5), a component of the von Willebrand factor (vWF) receptor that is cleaved from the platelet surface during activation. Secretion of THBS1 and GP5 by activated platelets explains their lower levels in platelets from DV patients. Also, we identied, exclusively in dengue samples, proteins PCAM1 that mediates platelet-endothelial adhesion and BID, a pro-apoptotic protein. These ndings corroborate our previous data suggesting platelet activation, mitochondrial dysfunction and apoptosis induction in platelets from patients with dengue and may further improve our knowledge on the pathogenesis of dengue-associated vasculopathy.

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DENGUE VIRUS NON STRUCTURAL 1 (NS1) PROTEIN INTERACTS WITH HUMAN VITRONECTIN

Conde, J.N., Silva, E.M.1, Menezes, J.L., Allonso, D., Coelho, D.R., Mohana-Borges, R.

Instituto de Biofísica Carlos Chagas Filho, UFRJ, Rio de Janeiro, Brazil.

Dengue virus (DENV) nonstructural protein 1 (NS1) is a 50 kDa intracellular homodimeric glycoprotein that plays a pivotal role in DENV replication. NS1 associates with membranes and is also secreted into the plasma as a lipid-associated barrel-shaped hexamer that is detectable in patient serum in the rst few days after the onset of clinical symptoms. Several studies demonstrated that NS1 protein has a possible involvement in dengue pathogenesis and liver dysfunction. It is of fundamental importance to understand the interactions between DENV proteins and host cell proteins in order to develop new methods to control its infection. Using the full-length NS1 as bait, we performed a yeast two-hybrid screening against a human liver cDNA library and the nucleotide sequence of one of the true positive clones was identied as the vitronectin protein (VN). We performed a coimmunoprecipitation and colocalization experiments in HepG2 cells that were infected with DENV2 strain 16681 to conrm two-hybrid results. DENV- or mock-infected HepG2 cell extracts were incubated with anti-NS1 polyclonal antibody immobilized to a resin. The co-IP eluted fractions showed two bands of approximately 65 and 50 kDa corresponding to VN and NS1, respectively. To evaluate whether these proteins colocalize during DENV infection in vivo, we performed an immunouorescence assay of DENV- or mock-infected HepG2 cells using both anti-NS1 and anti-VN antibodies. We observed that infected cells were labeled for both VN and NS1 proteins. When the images were merged, distinct yellow regions were observed, indicating the colocalization of NS1 with VN in these areas. We also performed binding experiments with ELISA and SPR approaches conrming the directly interaction between these two proteins. NS1 and host proteins interactions will allow a better understanding of the viral replication process and also provide means for elucidating the molecular mechanisms of pathogenesis caused by DENV.

POSTERS SESSION PATHOGENESISP-P01

EFFECTS OF DENV2 IN BALB/C MICE INFECTED BY THE INTRACEREBRAL ROUTE: VIRUS SPREAD AND TISSUE DAMAGES1 1 1 1 1Juliana Amorim , Marciano Paes , Adriana Azevedo , Antonio Gonçalves , Simone Costa1, Ada Alves

1Laboratório de Biotecnologia e Fisiologia de Infecções Virais, IOC-FIOCRUZ, RJ, Brazil.

One of the most widely used mouse model for testing vaccines against dengue is based on inoculation of mice by the intracerebral route with a mouse brain adapted virus. Under this approach, the observation of morbidity and mortality are often considered as the main parameters to assess the protective effect of vaccine candidates. However, few studies have been conducted to evaluate the effect of DENV infection in different organs considering this mouse model. Therefore, in the present work we evaluated the kinetic of infection in order to analyze viremia and histopathologic features in liver and central nervous system of BALB/c animals intracerebrally inoculated with DENV2. Plaque assays in Vero cell culture revealed virus spread in serum and homogenates of peripheral organs. Histopathological studies in the liver and brain of infected animals, staining with hematoxylin and eosin, revealed hemorrhage and inammatory inltrates in both organs. Moreover, liver also exhibited edema, parenchyma degeneration and hepatocyte necrosis. In the liver, DENV antigens were detected in hepatocytes, endothelium and macrophages by immunohistochemistry. Damages were also evidenced by increase of serum hepatic enzymes (ALT and AST). Consistent with these ndings, ultrastructural analysis performed by electron microscopy showed swollen hepatocytes presenting enlarged mitochondria. These data revealed that, even with inoculation of a neuroadapted DENV by the intracerebral route, animals presented some damages which are also found in human infection. Furthermore, results showed alternative parameters which may be evaluated in tests of vaccines or antivirals against the dengue disease. Financial Support: IOC-FIOCRUZ; PDTIS-FIOCRUZ; INCTV; CNPq and FAPERJ

P-P02

MIR-21 AND MIR-146 EXPRESSION IN MONOCYTES INFECTED BY DENGUE VIRUS1,2 1 2 1Caroline Carvalho ; Paula Cezar-De-Mello ; Claire Kubelka ; Milton Moraes .

1 2Laboratório de Hanseníase. Instituto Oswaldo Cruz-FIOCRUZ, Rio de Janeiro; Laboratório de Imunologia Viral. Instituto Oswaldo Cruz- FIOCRUZ, Rio de Janeiro.

Background: Dengue infection is a great threat in South America countries,especially in Brazil. Host factors play an important role in immunopathogenesis of dengue. MicroRNAs (miRNAs) are non-coding small RNAs that can regulate protein production by binding to the 3'untranslated regions of mRNAs. Several miRNAs are involved in the regulation of immune response in various infectious diseases, including dengue. Aims and methods: The current investigation aims to study the regulation of miRNA expression during dengue virus infection. For that purpose, we selected miRNAs that may regulate TNF and IL-10 levels, miR-146a and miR-21, respectivelly. Monocytes were isolated from peripheral blood mononuclear cells (PBMCs) and infected by dengue virus type-2 (Thailand strain 16681/1984) for 24 hours. After RNA extraction, both mature miR-146a and miR-21 expression were determined by steem-loop RT-PCR method. Results and conclusions: Monocytes were able to produce high levels of miR-146a and miR-21 in response to dengue virus infection (p-value < 0.05). We can conclude that dengue virus induces these miRNAs expression and, as a consequence, it should be dampening genes of host immune response facilitating the viral replication.

P-P03

AN EMBRYONIC HEART CELL LINE IS SUSCEPTIBLE TO DENGUE VIRUS INFECTION

Arianna M Hurtado-Monzón; Antonio Angel-Ambrocio; Rubén Soto-Acosta; Eshwar R Tammineni; Elba D Carrillo; Patricia Bautista-Carbajal; Jorge A Sánchez; Rosa 1M. Del Angel .

1 Centro de Investigación y de Estudios Avanzados del Insituto Politécnico Nacional. México, D.F.

Dengue Virus (DENV), a positive-single strain RNA virus of the Flaviviridae family, is the causative agent of dengue fever. In most cases, the symptoms of dengue fever are self-limited. However in a small portion of people the disease progresses to the severe form of the infection called severe dengue, previously called dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). In recent years, patients with more severe form of the disease, with acute heart failure or progression to cardiogenic shock and death have been reported, however the pathogenesis of myocardial lesions has not been elucidated. Moreover the susceptibility of cardiomyocytes to DENV infection has not been described. Under this perspective, the susceptibility of the myoblast cell line H9c2, obtained from embryonic rat heart, to DENV infection was analyzed. In order to demonstrate the ability of DENV to infect this cell line and the infection efciency the presence of viral antigens in infected cells was analyzed by confocal microscopy, ow cytometry and focus assay. Additionally, the ability of DENV to replicate within H9c2 cells was determined by detection of viral RNA by RT-PCR and viral proteins by Western-blot. Finally, the formation of viral infective particles was evaluated by the infection of U937 DC-SIGN cells, a highly susceptible monocytic derived cell line, which expresses the dendritic cell receptor DC-SIGN with supernatants of infected H9c2 cells. Our ndings conrm that H9c2 cells are susceptible to DENV infection. Our results support the view that DENV may target heart cells as evidenced by infection of H9c2 cells. This cell line may thus represent an excellent model for the study and characterization of cardiac physiopathology in DENV infection.

P-P04

EXPERIMENTAL MURINE MODEL FOR THE PATHOGENESIS STUDY OF DENGUE VIRUSES1 1 1 1 1Debora Ferreira Barreto-Vieira , Fernanda Cunha Jácome , Arthur da Costa Rasinhas , Marcos Alexandre Nunes da Silva , Ortrud Monika Barth

1Laboratory of Morphology and Viral Morphogenesis, Instituto Oswaldo Cruz, Fiocruz, Rio de Janeiro, Brazil

A great difculty to study dengue virus (DENV) infection in humans and for a virus vaccine developing is the absence of a suitable animal model which presents a disease with similar aspects of the Dengue haemorrhagic fever and Dengue shock syndrome. In the majority of models the animals are immunocompromised and/or inoculated by routes like the intracerebral, with neuroadapted DENV. Tissues of adult BALB/c mice infected with non-neuroadapted DENV-1 and DENV-2 serotypes from patient sera were analyzed. The tissue fragments were processed following the standard techniques of fotonic and transmission electron microscopy. In primary infection with DENV-1 and DENV-2 morphogical alterations were observed inside hepatic, lung, kidney and cerebellum tissues. DENV-1 particles and specic DENV antigen was observed in C6/36 cells inoculated with the supernatant of spleen and lung macerates and with the animal sera. Ultrastructural studies of alveolar macrophages of animals infected with DENV-2 showed DENV-like particles inside the rough endoplasmic reticulum and Golgi complex, suggesting viral replication. DENV particles were ultrastructurally identied, and immunolocalized inside C6/36 cells, inoculated with the supernatant (liver, lung kidney and cerebellum) of tissue macerates. The corporal temperature in the majority of mice increased after the second day post-infection. Elevated enzyme levels of alanine aminotransferase and aspartate aminotrasferase were observed. In secondary infections morphological alterations were observed in liver, lung and heart. The tissue injuries were more severe than those seen in animals with signs of primary infection. DENV-1 particles, specic DENV-1 antigen and DENV-1 RNA were present in C6/36 cells inoculated with the animal sera. These studies conrm the susceptibility of BALB/c mice to infection and reinfection by DENV-1 and DENV-2 and those they can be used as a model for testing of drugs and vaccine candidates against DENV.

P-P05

HISTOLOGICAL CHANGES AND ACTIVATION PATTERN OF DENDRITIC CELLS IN THE SKIN OF THE CALLITHRIX PENICILLATA UNDERWENT SEQUENTIAL DENV-3 AND DENV -2 INFECTION

Daniele Freitas Henriques, Edna Cristina Santos Franco, Vera Lucia Reis Souza de Barros, Carla Pagliari, Milene Silveira Ferreira, Paulo Henrique Gomes de Castro, Gilmara Abreu da Silva Cavalcante, Pedro Fernando da Costa Vasconcelos

Instituto Evandro Chagas, Ananindeua, Pará, Brazil

INTRODUCTION: Dengue is an infectious disease caused by dengue virus (DENV sorotypes 1, 2, 3 and 4) and transmitted by bite of infected mosquitoes of the genus Aedes that release DENV in the skin of the host during the pasture. Dendritic cells have the function to present antigens to effectors' cells, shaping the adaptative immune response. In epidermis these cells express S100 glycoprotein and at dermis they express procoagulant factor XIIIa. Few immunopathological studies have evaluated the role of immune cells present in the skin after the rst moments following DENV infection. OBJECTIVE: To describe the histological changes and the activation pattern of dendritic cells in the skin of the Callithrix penicillata underwent sequential infection of DENV-3 and DENV-2. MATERIAL E METHODS: Primary infection was performed in 26 non-human primates with DENV-3 and secondary infection was performed with DENV-2 two months later. The animals' skin was collected 7 days and at intervals of 10, 15, 20, 30, 45 and 60 days post infection (dpi). Skin were subjected to pathological and immunohistochemistry for anti-S100 (Dako Z0311) and anti-FXIIIa (biogenex AN516-5M) examination. The dendritic cells were counted in three different elds of epidermis and reticular dermis. RESULTS: Pathological changes found were focal perivascular inammatory inltrate, dermatitis, interstitial edema, and miositis. FXIIIa cells number was 11.67 (±1.20) in control animals. These cells peak were 26.67 (±7.68) 7dpi and 30 (±4.58) 10dpi after primary and secondary infection, respectively. S100 cells number were 8.33 (±0.33) in control animals. These cells peak were 18.33 (±1.20) 2dpi and 19.67 (±2.40) 10dpi in primary and secondary infection, respectively (ANOVA+ t Test

p<0.01). CONCLUSION: The dendritic cells number increase at skin of animals subjected to sequential infection with DENV-3 AND DENV-2, suggesting that viral infection of these cells play important role in disease pathogenesis.

P-P06

EFFECT OF THE NS2B COFACTOR ON THE STABILITY AND HYDROPHOBICITY OF THE DENGUE VIRUS NS3 PROTEASE

Gustavo Ventura1, Emmerson Costa1 and Ronaldo Mohana Borges1

1Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil

NS3 is a multifunctional enzyme with two domains: a serine protease (NS3Pro) and a helicase, NTPase and RTPase (NS3Hel). NS3Pro requires a hydrophilic segment of 40 residues from the NS2B protein (NS2BCF40) as a cofactor, which is essential for this activity. Mutations in the catalytic site of the NS3Pro abolish proteolytic activity and viral infectivity, suggesting that this domain is a promising drug target. In this work, we investigated how NS2BCF40 could interfere in the structure of NS3Pro by evaluating two different constructs: the isolated NS3Pro and the NS3Pro fused with NS2BCF40 by a exible linker. We carried out intrinsic uorescence experiments monitoring the Tryptophan (Trp), which is used as a probe to evaluate protein conformational changes, and we used acrylamide and bis-ANS to determine the solvent exposure of Trp and hydrophobic clefts, respectively. We have also carried out circular dichroism (CD) assays to monitor the secondary structure of the constructs. Our results have shown that the NS2BCF40 causes drastic structural changes on the NS3Pro, which turns from a α + β to a random coil secondary structure, with signicant increase in the Trp residues exposure and decrease in the bis-ANS binding. We suggest that the NS3Pro adopts an extremely opened conformation in the presence of NS2BCF40, with evident loss of secondary structure that could favor the substrate binding and consequently increase the efciency of cleavage.

P-P07

IDENTIFICATION OF THE INTERACTION BETWEEN DENGUE NS1 PROTEIN AND CD14 IN HUMAN MONOCYTIC CELLS1 1 1 1 2 2Iamara da Silva Andrade ; Daniele Cristina Passos da Rocha ; Jonas Nascimento Conde ; Diego Allonso ; Lissa Catherine Reignault ; Wanderley de Souza ; Emiliana

1 1Mandarano da Silva ; Ronaldo Mohana-Borges1 2Laboratório de Genômica Estrutural - IBCCF- UFRJ, Brazil; Laboratório de Ultraestrutura Celular Hertha Meyer- IBCCF- UFRJ, Brazil

NS1 exists in different oligomeric forms, which are found in different cellular locations and as a soluble secreted hexameric lipoparticle. It is known that this protein is related to a paradoxical immune response, involving monocytes/macrophages and endothelial cells during the early stages of DENV infection. Our group has mapped interactions between DENV-2 NS1 and human liver proteins using a two-hybrid system and identied CD14 as an interacting partner. CD14 is expressed in several cells, especially in monocytes/macrophages, and is known as co-receptor for several Toll-like Receptors (TLRs).This molecule takes part in recognition of low doses of LPS, facilitating cellular responses. It is able to activate NF-κB-dependent cytokine production, TLR4 endocytosis resulting in type-I interferon expression and activation of NFATc transcription factors family members by autonomous signaling. CD14 also cooperates with DENV entry in monocytes. Our aim is to conrm the interaction between NS1 and CD14 in monocytic cells, and to understand the modulation of signaling pathways inuenced by this interaction. We conrmed the interaction between NS1 and CD14 by ELISA assay. Furthermore, we found that NS1 produced by DENV2 infected cells and recombinant NS1 incubated with THP-1 colocalizes with CD14 by confocal microscopy analyses. In addition, we observed by FACS assays that recombinant NS1 is able to bind to the surface of THP-1 cells using CD14 as a target for interaction. We concluded that NS1 that interacts with CD14 and this interaction might be important in modulating the cellular response. From now on we intend to better understand the role of this interaction in cell signaling pathways related to cytokine production. Financial support: CNPq and FAPERJ.

P-P08

NATURAL INFECTION WITH DENGUE VIRUS AFFECTS THE VIABILITY AND FUNCTION OF CRYOPRESERVED PERIPHERAL BLOOD MONONUCLEAR CELLS1 1 1 1,2 1,2 1Federico Perdomo , Diana M. Castañeda , Jairo A. Rodríguez , Rocío Vega , Doris M. Salgado and Carlos F. Narváez

1 2 Programa de Medicina, Facultad de Salud, Universidad Surcolombiana, Neiva, Colombia; Departamento de Pediatría, Hospital Universitario de Neiva, Colombia

The cryopreservation of peripheral blood mononuclear cells (PBMC) is a widely used technique to preserve phenotypic and functional cell characteristics long times. However, several factors such as used reagents, quality of stored and illness of patients source of PBMC, affect its efciency. Dengue is a viral vector-borne disease endemic in tropical areas and studies using cryopreserved PBMC in this eld are routine. It has been shown that in acute dengue infection an elevated frequency of PBMC, particularly T cells, undergo cell death. Thus, this dengue-related effect could modify the viability of cryopreserved PBMC when compared with healthy children and others pediatrics infections. Here, we evaluated the viability (trypan blue and amine dye staining) and function (frequency of IFN-γ producing T cells after a polyclonal stimulus) of cryopreserved PBMC from 14, 10, and 15 healthy, dengue infected and febrile non-dengue children, respectively. To dengue cases, pared acute and convalescent samples were also included. Frequency of non-viable PBMC detected by both methods, trypan blue and amine dye staining, was comparable (r=0.8, P<0.001). Percentage of non-viable PBMC in acute dengue but not in febrile non-dengue children was signicantly higher than healthy children (P<0.01, Dunn's post-hoc test) and the most of dead cells corresponded to CD3+ median (range) 65% (25-78), CD3-CD19- 34% (21-72) and nally CD19+ 1% (0.6-1.1). PBMC viability was restored in convalescent dengue at same levels than healthy children. When functional aspect was evaluated, acute dengue infection dramatically decreased the frequency of IFN-γ producing T cells and signicant higher levels were obtained in healthy and convalescent dengue (P<0.01, Dunn's post-hoc test). In short, dengue infection affects the viability and function of cryopreserved PBMC and could to alter the reliability of subsequent analysis carried out with these cells. Funded By Colciencias.

P-P09

ROLE OF CC CHEMOKINE RECEPTOR 1 AND TWO OF ITS LIGANDS IN HUMAN DENGUE INFECTION. THREE APPROACHES UNDER THE CUBAN SITUATION

Beatriz Sierra, Ana B. Perez, Gissel Garcia, Eglys Aguirre, Mayling Alvarez, Daniel Gonzalez, Maria G. Guzmán

Virology Department, PAHO/WHO Collaborating Center for the Study of Dengue and its Vector, Institute of Tropical Medicine “Pedro Kouri”, Autopista Novia del Mediodia, Km 61/2, La Lisa, Habana, Cuba

Any of the four dengue serotypes can cause a severe disease, partly due to systemic inammation orchestrated by mediators like cytokine and chemokines. We addressed the role of CCR1 and its ligands CCL3/MIP-1a and CCL5/RANTES in dengue infection using three different approaches: an ex vivo model exploring memory immune response in subjects with a well characterized dengue immune background, an in vivo study in patients with primary or secondary dengue infection, and an approach in fatal dengue. CCR1 and CCL3/MIP-1a gene expression showed differences after homotypic and heterotypic challenge according to dengue immune background of subjects, in correspondence with previous observations in Cuban dengue outbreaks. CCL5/RANTES gene expression was higher after homotypic challenge. CCR1 and CCL3/MIP-1a gene expression was higher in patients with secondary infection during critical days of the dengue disease, while the increase in RANTES expression started earlier than the observed for CCR1 and CCL3/MIP-1a. CCR1 and CCL3/MIP-1a gene expression was as high in brain as in spleen tissue from necropsy. Our results conrm the strong inuence of previous immunity in subsequent dengue infections, and confer a possible pathogenic role to CCR1 and CCL3/MIP-1a in dengue disease and a possible protective role for CCL5/RANTES, probably through CCR5 interaction.

P-P10

DENGUE VIRUS NONSTRUCTURAL 3 PROTEIN INTERACTS WITH HUMAN GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE (GAPDH) AND ALTERS ITS GLYCOLYTIC ACTIVITY.

1 1 1 2 1 1Emiliana M. Silva ; Jonas N. Conde ; Diego Allonso ; Manuela Leal ; Gustavo T. Ventura ; Ronaldo Mohana-Borges .1 2Laboratório de Genômica Estrutural, Instituto de Biofísica Carlos Chagas Filho, UFRJ, Brazil; Laboratório de Biologia Computacional, Instituto Nacional de Metrologia, Qualidade e Tecnologia - INMETRO, Brazil

The multifunctional NS3 protein forms a non-covalent complex with the NS2B cofactor, functioning as serine-protease that is responsible for proteolytic processing of the viral polyprotein at the NS2A/NS2B, NS2B/NS3, NS3/NS4A and NS4B/NS5 junctions. It has also an ATPase/helicase and RNA triphosphatase domain at its C-terminal, which is essential for RNA replication. In this study, we employed yeast two-hybrid (Y2H) system to identify liver human proteins that interact with NS3 protein, which was performed using the Matchmaker GAL-4 Two-Hybrid System 3 (Clontech). The Y2H screening identied 49 colonies that grew in the absence of Trp, Leu, His, Ade and were tested for the LacZ phenotype. Among the NS3-interacting partners, we found the glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The human GAPDH directly interacted with NS3 helicase and protease domains as demonstrated by ELISA and molecular docking. Surface plasmon resonance measurements revealed the high avidity and afnity between these interactions. In addition, NS3 protein co-localizes with GAPDH in hepatocytes cell lineage infected with dengue virus using mouse polyclonal anti-NS3 helicase and rabbit polyclonal anti-GAPDH. Glycolytic activity for GAPDH decreased in the presence of NS3 helicase and protease domains. As GAPDH is a well characterized glycolytic enzyme, displaying multiple functions in transcriptional coactivation, DNA repair, oxidative stress, apoptosis induction and regulation, we suggest that the GAPDH-NS3 interaction is associated with the viral replication and apoptosis processes. Financial Support: FAPERJ, CNPq and CAPES.

IMMUNOLOGYABSTRACTS INVITED SPEAKERS

DENGUE VIRUS NON-STRUCTURAL PROTEIN 1 TRIGGERS ENDOTHELIAL PERMEABILITY AND VASCULAR LEAK THAT IS INHIBITED BY NS1-SPECIFIC ANTIBODIES

1 1 1 1 2 2 1Eva Harris , Sarah Killingbeck , Henry Puerta Guardo , Dustin Glasner , Zachary MacMillen , Tomer Hertz , and P. Robert Beatty1 2Division of Infectious Diseases and Vaccinology, School of Public Health, University of California, Berkeley; Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, WA

The four dengue virus serotypes (DENV1-4) are mosquito-borne aviviruses that cause annually ~100 million cases of dengue and 500,000 hospitalizations from severe disease characterized by vascular leakage leading to shock. DENV nonstructural protein 1 (NS1) is secreted by infected cells and is found at high levels in patient serum during acute infection. We examined the protective efcacy of NS1 immunization against lethal DENV2 infection in a mouse model of vascular leak. Interferon α/β receptor-decient C57BL/6 (Ifnar-/-) mice were immunized with DENV2 recombinant NS1 combined with different adjuvants, including alum, Sigma adjuvant system (SAS), CpG DNA, Addavax and/or monophosphoryl lipid A (MPLA). Mice vaccinated with DENV2 NS1 with either SAS+CpG or Addavax+MPLA were fully protected against lethal peripheral challenge with DENV2, whereas mice vaccinated with DENV2 NS1 with alum were not protected. Screening of mouse sera on an array of overlapping 15-mer peptides spanning NS1 identied two peptides that correlated with protection. In addition, heterologous cross-protection was observed, as 60-75% of mice vaccinated with DENV1, DENV3, or DENV4 NS1 survived lethal DENV2 challenge. These results led us to hypothesize that NS1 itself may have direct pathogenic effects. We then found that inoculation of mice with NS1 alone increased both vascular leakage and key inammatory cytokines, while simultaneous administration of NS1 with a sublethal dose of DENV2 resulted in a lethal vascular leak syndrome. In separate experiments, we showed that DENV NS1 increases permeability of human pulmonary microvascular endothelial cells in vitro in a dose-dependent manner. We next demonstrated that lethal NS1-mediated

toxicity in vivo and disruption of endothelial integrity by NS1 in vitro were inhibited by NS1-immune serum. In addition, a MAb targeting one of the NS1 peptides that correlated with protection in vaccination experiments completely blocked NS1-triggered endothelial permeability and protected mice from NS1-mediated toxicity in vivo. Thus, physiologically relevant levels of DENV NS1 can directly contribute to increased vascular permeability, which can be blocked by anti-NS1 antibody. These ndings add an important and previously overlooked component to the causes of dengue vascular leak and support inclusion of NS1 in dengue vaccines.

COMPLEMENT MODULATION OF CELLULAR RESPONSES AND DENGUE-ASSOCIATED VASCULOPATHY1 2 2 1 1,2,3Eduardo J. M. Nascimento , Eugenio D. Hottz , Fernando Bozza , Simon Barratt-Boyes , and Ernesto T. A. Marques

1 2 3University of Pittsburgh, Center for Vaccine Research , Fiocruz, Instituto Osvaldo Cruz , Instituto Aggeu Magalhaes .

The dengue-associated vasculopathy starts around day 4 after onset of symptoms while the virus is disappearing from the blood and lasts for 3 or 4 days. During this period we observe high levels of complement activation associated with B-cell activation and platelet depletion. We propose that complement activation have an important role modulating the immune responses during this phase of the dengue disease. We, and others have observed signicant alterations in soluble complement factors (C3, C4 and CFD) and regulatory molecules (CFH and clusterin) in the plasma and also on the expression of complement receptors (CD46, CD55 and CD59) on cells from infected patients and also in in vitro infected cells. These complement alterations may play a role inducing high levels of plasmablast responses and platelet activation that together induces exacerbated inammatory responses in the microvasculature that leads to the dengue-associated vasculopathy.

ABSTRACTS ORAL PRESENTATIONSDENGUE VIRUS-SPECIFIC T CELL RESPONSES IN THE GENERAL POPULATION OF NICARAGUA VARY AS A FUNCTION OF THE INFECTING SEROTYPE AND ARE DOMINATED BY HLAB35-RESTRICTED EPITOPES

1 1 2 1 3 2 4 1Daniela Weiskopf , Ruklanthi de Alwis , Cristhiam Cerpas , Bjoern Peters , Lionel Gresh , Angel Balmaseda , Eva Harris , and Alessandro Sette1 2Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology; Laboratorio Nacional de Virología, Centro Nacional de Diagnóstico y Referencia,

3 4Ministerio de Salud, Managua, Nicaragua; Sustainable Sciences Institute, Managua, Nicaragua; Division of Infectious Diseases and Vaccinology, School of Public Health, University of California, Berkeley

Infections with any of the four dengue virus serotypes (DENV1-4) in more than 100 countries around the world account for as many as 390 million infections each year. All four DENV serotypes have circulated extensively in Nicaragua in recent years and as a result, the adult population at large has been exposed to more than one DENV serotype. To assess the T cell response against all four DENV serotypes, we obtained buffy coat (PBMC) samples from 130 Red Cross blood donors from an adult population in Managua, Nicaragua. These PBMC were HLA typed and the immune response to HLA-matched, predicted peptides was measured by ex vivo, IFNγ ELISPOT assay. This proteome-wide screen identied a total of 314 CD8+ T cell epitopes across all 10 DENV proteins (C, M, E, NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5). Interestingly, we observed different patterns of immunodominance to DENV antigens depending on the serotype. DENV3-specic responses predominantly targeted structural proteins whereas DENV1-, DENV2- and DENV4-specic were disproportionately found to (>90%) target non-structural (NS) proteins, (particularly NS3, NS4B and NS5). Additionally, we observed a striking dominance of HLA B-restricted responses: particularly, HLA B*35. The immunodominance of HLA B*35 was evident both in terms of breadth as well as magnitude of the DENV-specic CD8+ T cell response. Interestingly, this allele has been associated with protection from disease in a different population study in Malaysia. We found that the majority of responses were produced by CD8+ T cells displaying an effector memory phenotype (TEMRA and TEM). In terms of cytokine production, the majority of cells produced both IFNγ and TNFα, indicating a multifunctional phenotype. Our results provide novel insights into HLA-restricted T cell responses against all four DENV serotypes and are of particular relevance towards vaccine design and the identication of robust correlates of protection in natural immunity to DENV challenge.

A NEW MOUSE MODEL OF DENGUE SHOCK SYNDROME IDENTIFIES COMPLEMENT ACTIVATION AS A NOVEL TARGET FOR PHARMACOLOGICAL INTERVENTION1* 1* 4 1,2,3 4 2 3Amelia K. Pinto , James D. Brien , Xiaobo Wu1, William Tang , William E. Eddy4, John P. Atkinson , Sujan Shresta , and Michael S. Diamond1, ,

1 2 3Departments of Medicine , Pathology & Immunology , and Molecular Microbiology , Washington University School of Medicine, St Louis, MO 63110; La Jolla Institute 4for Allergy & Immunology , La Jolla, California 92037. * Co-rst author

Over 3.9 billion people are at risk for contracting Dengue virus (DENV) with approximately 390 million people becoming infected each year. While the majority of infected individuals have inapparent or mild infections, a signicant proportion develop Dengue fever (DF) or Dengue hemorrhagic fever (DHF)/Dengue shock syndrome (DSS). The number of these cases are increasing every year. The sepsis and cytokine storm associated with DHF/DSS are substantial clinical problems because of their propensity for high morbidity and mortality and considerable utilization of health care resources. A better understanding of the pathways involved in the induction of DHF/DSS may allow for the development of new biomarkers that predict disease severity at early stages and lead to pharmacological inhibitors to improve disease outcomes. Previously, we established a mouse model for viral sepsis employing animals that lacked type I IFN receptor (Ifnar) only on dendritic cells. Here, we apply this approach to DENV infection and demonstrate that the lack of Ifnar expression, specically on myeloid cells (LysMCre+Ifnarf/f), resulted in enhanced DENV replication, with levels of virus in serum similar to that observed for Ifnar-/- mice. Administration of a poorly-neutralizing cross-reactive avivirus antibody (4G2) to LysMCre+Ifnarf/f mice prior to DENV infection resulted in antibody-dependent enhancement of disease with many characteristics of human DHF/DSS. Antibody-enhanced infection of LysMCre+Ifnarf/f mice was associated with plasma leakage, hypercytokinemia, complement activation, and elevated serum liver enzymes. Analysis of complete blood counts revealed hemoconcentration and thrombocytopenia as is observed with human DHF/DSS. Remarkably,

pathogenesis of DENV-infected LysMCre+Ifnarf/f mice was blocked by administration of anti-complement antibodies, suggesting a key role for complement activation in DENV disease pathogenesis in this model. Our ndings establish a more immunocompetent mouse model of severe DENV pathogenesis that reects many facets of human disease and identies novel targets for pharmacological control of sepsis.

DENGUE INFECTION INDUCES EXPRESSION OF NKG2D LIGANDS IN HUMAN IMMATURE DENDRITIC CELLS IN VITRO

Davis Beltrán1,2,3, Simona Zompi4, Yamilka Diaz1, Janice Arakawa-Hyot5, Juan Miguel Pascale1, Eva Harris4, Lewis L. Lanier5, Sandra López-Vergès1

1Gorgas Memorial Institute for Health Studies, Panama city, Panama; 2INDICASAT-AIP, Panama city, Panama; 3Acharya-Nargayuna Univesity, Guntur, India; 4University of California Berkeley, Berkeley, California; 5University of California San Francisco, San Francisco, California

Immune response towards dengue virus (DENV) infection is mediated by the innate immune response in conjunction with the T-cell response and antibodies produced by B cells. Natural Killer (NK) cells may play a crucial role in the limitation of viral replication at early times of DENV infection, and this could result in mild dengue symptoms. NK cells can mediate antibody-dependent cell cytotoxicity (ADCC) of infected cells coated with DENV-specic antibodies; however in vitro experiments suggest that they are also capable of direct recognition of DENV-infected cells in the absence of antibodies. We hypothesized that ligands of NK activating receptors, such as NKG2D, can be induced by DENV infection allowing for recognition of infected cells by NK cells. This hypothesis is supported by genetic studies that showed an association between some MICB ad MICA alleles and dengue disease severity. To determine if DENV infection alters NKG2D ligand expression, we performed in vitro experiments in which immature dendritic cells (imDCs) derived from peripheral blood monocytes of volunteer donors were infected with DENV isolates from Panama. Twenty-four hours and 48 hours post-infection, we evaluated the expression of NKG2D ligand mRNA by RT-PCR/Q-PCR and the expression of the proteins at the cell surface by ow cytometry. DENV-infection of imDCs induces or increases the mRNA of many NKG2D ligands; however, we did not detect induction of ULBP-4 transcripts. Moreover we observed an increase of surface expression of NKG2D ligands MICA, ULBP-1, 2, -3, -5, and -6, whereas MICB and ULBP-4 did not show any signicant increase. These preliminary results suggest that NKG2D ligands are induced by DENV-infection and that this could allow for recognition of infected cells by NK cells. Further studies are needed to characterize the recognition of DENV-infected cells by NK cells and to study NK cell-mediated responses during dengue disease.

HIGHLIGHTED POSTERSHP13

USE OF CHIMERIC RECOMBINANT DENGUE VIRUSES TO MAP THE SEROTYPE 2 NEUTRALIZING HUMAN ANTIBODY RESPONSE

Emily Gallichotte1, Douglas Widman2, Scott Royal2, Aravinda de Silva1, Ralph Baric1,2

1Department of Microbiology and Immunology, University of North Carolina School of Medicine, Chapel Hill, NC; 2Department of Epidemiology, School of Public Health, University of North Carolina, Chapel Hill, NC

Primary infection with one of the four dengue virus (DENV) serotypes (DENV1-4) results in antibodies that neutralize the infecting serotype, but not other serotypes. Our group has previously reported on the isolation of serotype specic, strongly neutralizing monoclonal antibodies (hMAbs) from people exposed to natural DENV infections. We have demonstrated that these hMAbs bind to complex quaternary structure epitopes that are only expressed on intact virus particles. Recently we reported that it is possible to create viable recombinant DENVs in which these complex epitopes have been transplanted between serotypes. By using DENV3/4 chimeras, we observed that the hinge region between domains I/II of the envelope (E) protein contains epitopes that are the main target of type-specic antibodies that neutralize serotypes 3 and 4. In the current study we have used a similar approach to map sites on DENV2 recognized by neutralizing hMAbs and primary DENV2 human immune sera. Our studies have led to the identication of a novel quaternary structure-dependent DENV2 epitope that is distinct from EDI/II hinge region epitopes previously dened for serotypes 3 and 4. Importantly, we use gain and loss of function studies to demonstrate that different locations in the DENV1-4 E glycoprotein encode unique long-lived neutralizing epitopes, which are portable between serotypes. We will present data on the location of DENV2 epitope and its relative importance as a target of neutralizing antibodies in people exposed to natural infections and vaccines.

HP14

T CELLS PREVENT ANTIGEN-INDUCED ANTIBODY-DEPENDENT ENHANCEMENT OF DENGUE DISEASE IN MICE

Raphael M. Zellweger, William E. Eddy, William W. Tang, Robyn Miller and Sujan Shresta

La Jolla Institute for Allergy and Immunology, CA

Dengue virus (DENV) causes pathologies ranging from the febrile illness dengue fever to the potentially lethal severe dengue disease. A major risk factor for developing severe dengue disease is the presence of sub-protective DENV-reactive antibodies from a previous infection (or from an immune mother), which can induce antibody-dependent enhancement of infection (ADE). However, infection in the presence of sub-protective anti-DENV antibodies does not always result in severe disease, suggesting that other factors inuence disease severity. This study investigates how cellular responses inuence the outcome of antibody-mediated severe dengue disease. Mice were immunized with alum-adjuvanted UV-inactivated DENV prior to challenge with DENV. Immunization failed to induce robust T cell responses, and induced non-neutralizing antibody responses that increased disease severity upon infection. Transfer of DENV-primed T cells into immunized mice prior to infection prevented antibody-induced enhancement and dramatically reduced viral load upon infection. Our results suggest that in the presence of sub-protective anti-DENV

antibodies, efcient T cell responses reduce the risk of antibody-mediated severe dengue disease, while inefcient (or absent) T cell responses increase disease severity.

HP15

COMPLEMENT RECEPTORS ARE DOWNREGULATED IN DENGUE PATIENTS AND MODULATES THE VIRAL INFECTION IN HUMAN MONOCYTES IN VITRO.1 1 1 2 1 3Cintia Ferreira Marinho , Elzinandes Leal Azaredo , Amanda Torrentes de Carvalho , Alessandro Marins dos Santos , Claire Fernandes Kubelka , Luiz José de Souza ,

4 1Rivaldo Venâncio Cunha , Luzia Maria de Oliveira Pinto .1 2Laboratory of Viral Immunology-Oswaldo Cruz Institute, RJ-Brazil. Flow Cytometry Sector, Center for Cell Sorting and Analysis -Oswaldo Cruz Institute, RJ-Brazil. 3 4Referral Center for Dengue, RJ-Brazil. Department of Clinical Medicine, UFMS, MS-Brazil

Complement System (CS) activation inhibits infection of many viruses augmenting the neutralizing activity of antiviral antibodies. In Flavivirus infection, it appears to have both protective and pathogenic roles depending on immune status of the host, the specic virus and infectious phase. Dengue virus (DENV) is a member of the Flavivirus genus that comprises four closely related and antigenically distinct serotypes (DENV1-4). In severe dengue fever (DF), the levels of DENV non-structural-1 protein and of the products of complement activation, including C3a, C5a and SC5b-9, are higher before vascular leakage occurs, supporting the hypothesis that complement activation contributes to unfavourable outcomes. The clinical manifestations of DF range from asymptomatic to severe and even fatal. The risk of developing the most severe forms is related to the cross-reactive nature of the immune response to infection with heterologous serotypes of DENV. Here, we aimed to characterise CS by their receptors or activation product, in vivo in DF patients and in vitro by DENV-2 stimulation on monocytes. In comparison with healthy controls, DF patients showed lower expression of CR3(CD11b), CR4(CD11c) and, CD59 on monocytes. The DF patients who were high producers of SC5b-9 were also those that showed more pronounced bleeding or vascular leakage. Those ndings encouraged us to investigate the role of CS in vitro, using monocytes isolated from healthy subjects. Prior blocking with CR3 alone (CD11b) or CR3(CD11b/CD18) reduced viral infection, as quantied by the levels of intracellular viral antigen expression and soluble DENV non-structural viral protein. However, we found that CR3(CD11b) or CR3(CD11b/CD18) blocking did not inuence major histocompatibility complex presentation neither active caspase-1 on monocytes, thus probably ruling out inammasome-related mechanisms. Although it impaired TNF-α and IFN-α secretion. Our data provide strategies of blocking CR3(CD11b) pathways could have implications for the treatment of viral infection by antiviral-related mechanisms.

HP16

DENGUE-INDUCED TRAIL EXPRESSION ON NK CELLS DURING INFECTION1 1 1 1 1 1 1Mariana Gandini ; Fabienne Paiva ; Cintia Marinho ; Gladys Correa ; Luzia Pinto ; Claire Kubelka *; Elzinandes Azeredo *

1 Laboratório de Imunologia Viral, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, Brasil

Dengue is an arboviral disease and 2.5 billion people are at risk in tropical and subtropical regions of the world. Dengue virus can cause a febrile syndrome, in which severe cases are characterized by vascular permeability and effusions. A high viral burden in the acute phase may enhance the production of inammatory mediators, leading to plasma leakage. Innate immunity is an importante barrier, which limits viral spread. NK cells are one of main actors in early infection because of their antiviral and cytotoxic features against infected cells. NK cells can be activated directly, by target recognition, or indirectly, by cytokines (like interferons) and become cytotoxic, expressing TRAIL, an apoptotic inducer. Despite its antiviral role, the function of this cell population during dengue fever (DF) is not largely known. We studied NK cells involvement in DF immunopathology. The frequency of NK cells population (CD56+CD16+/-) and also TRAIL+NK cells were signicantly higher in mild DF cases (n=11) as compared to severe cases (n=4) or healthy controls (n=18). NK activation markers such as CD107a degranulation marker and pattern recognition receptor TLR3 were upregulated in patients' cells (n=10) as compared to healthy donors (n=10). Cytokines – IFNα, IL12 – related to cell activation were signicantly upregulated in mild DF cases. PBMCs cultured in vitro show that DENV-stimulated and IFNα-stimulated NK cells (n=8) were able to express TRAIL, suggesting an indirect activation of cells, regarding TRAIL expression. Type I IFN receptor blockage (n=6) on DENV-stimulated PBMCs showed TRAIL expression on NK cells was partially IFNα dependent (50% blockage). Also, during PBMC stimulation, TRAIL expression on NK cells was inversely correlated to DENV positive monocytes. Therefore, we observed DENV-induced activation of NK cells populations. A higher activation of NK cell would promote limited viral spread, resulting in dimished inammatory response and contributing to protection against severity during dengue fever. C.F.K. and E.L.A. contributed equally to this work. Supported by FIOCRUZ/RPT11D; IOC/PROEP;CNPQ; FAPERJ; CAPES.

HP17

REGULATION OF LEUKOCYTES APOPTOSIS DURING DENGUE INFECTION1 2 2 2 3Daniel Limonta , Amanda Torrentes-Carvalho , Cíntia Ferreira Marinho , Elzinandes Leal de Azeredo , Rivaldo Venâncio da Cunha , Claire Fernandes Kubelka2, Rita

2Maria Nogueira4, Luzia Maria de-Oliveira-Pinto1 2Arbovirus Laboratory, Pedro Kourí Institute of Tropical Medicine (IPK), La Habana, Cuba.; Laboratório de Imunologia Viral, Instituto Oswaldo Cruz (IOC), FIOCRUZ, Rio

3 4de Janeiro, Brasil; Setor Hemonúcleo, Universidade Federal do Mato Grosso do Sul, Mato Grosso do Sul, Brasil; Laboratório de Flavivírus, Instituto Oswaldo Cruz (IOC), FIOCRUZ, Rio de Janeiro, Brasil.

Background: Despite 390 million infections of dengue are estimated worldwide annually, dengue has no vaccine, antiviral treatment or reliable severity predictors. It has been shown that apoptotic cells from blood and tissues may be involved in the complex pathogenesis of dengue. However, very little is known about the interplay

between blood proapoptotic and antiapoptotic regulators in this viral hemorrhagic fever. Methods: Plasma levels of the three proapoptotic mediators Fas ligand (FasL), tumor necrosis factor-alfa (TNF-alfa), and TNF-related apoptosis-inducing ligand (TRAIL) were measured by ELISA in Brazilian dengue patients. Patients were classied according to the World Health Organization classication of dengue revised in 2009. Additionally, inhibitors of apoptosis protein (IAPs) were determined in plasma (Survivin) by ELISA and peripheral blood mononuclear cells (PBMCs) lysates (cIAP-1, cIAP-2, XIAP) by an apoptosis array kit. Levels of apoptotic proteins in plasma were correlated with counts of PBMCs obtained by ow cytometry. Results: FasL and TRAIL levels were elevated in dengue patients without warning signs when compared to patients with severe dengue and controls. The highest level of FasL was measured in a fatal case. Dengue patients with warning signs showed decreased levels of Survivin compared to patients with severe dengue and controls. Survivin was positively correlated with leukocyte counts. In contrast, TRAIL was inversely correlated with counts of lymphocyte subsets. There was a trend of elevated IAPs levels in PBMCs of patients with severe dengue. No signicant differences were found in TNF-alfa levels among the dengue patients studied. Conclusion: Our ndings support the likely antiviral effect of TRAIL in dengue patients. It appears that TRAIL might be involved with apoptosis induction of lymphocytes, whereas IAPs might participate in protecting leukocytes from apoptosis. Further research is needed to explore the interactions between pro and antiapoptotic molecules and their implications in dengue pathogenesis.

POSTERS SESSION INMUNOLOGYI-P01

EFFECT OF SERA OBTAINED FROM DENGUE PATIENTS ON TRIGGERING ACTIVATION OF PLATELETS AND ENDOTHELIA1 1 1 2 2 1Ma. Isabel Salazar-Sánchez , Daniel Núñez Avellaneda , Yesica Garcia-Lozano , María Alba Loroño-Pino , Carlos Machain-Williams , María Eugenia Castro-Mussot ,

3Norma Angélica Corona-de la Peña1 2Laboratory for Cellular Immunology and Immunopathogenesis, Departamento de Inmunología, Escuela Nacional de Ciencias Biológicas-IPN. Laboratorio de

3Arbovirología, Centro de Investigaciones Regionales Dr. Hideyo Noguchi, Universidad Autónoma de Yucatán. Unidad de Investigación en Trombosis, Hospital Carlos MacGregor, Instituto Mexicano del Seguro Social. MÉXICO.

During the pathological processes mediated by DENV, various factors seem to be involved in disease severity. Thrombocytopenia is commonly observed in the dengue fever and severe dengue syndromes and it correlates with the clinical outcome. The humoral immune response might be of major importance in the dengue immunopathogenesis. Antibodies raised against viral proteins are known to be reactive against some host proteins, including PDI (Protein disulde-isomerase) on platelets. In this current study, we analyzed the effect of sera from patients with dengue fever (DF) and dengue hemorrhagic fever (DHF) collected between 3 and 8 days after symptoms. Here, we established the effect of these sera on platelets and endothelia activation, measuring glycoprotein IIb/IIIa and E-selectin. We observed a signicant increment in the platelet activation mediated by glycoprotein IIbIIIa when platelets are pre-treated with sera from DHF patients.

I-P02

DETERMINATION OF IGG SUBCLASSES IN SERUM SAMPLES FROM PATIENTS WITH DIFFERENT CLINICAL FORMS OF DENGUE

Junqueira, Isabela C.; Silva, Priscila T.F.; Guilarde, Adriana O.; Rocha, Benigno M.; Siqueira-Júnior, João B.; Féres,Valéria C.R.; Martelli, Celina, T.; Silveira, Lucimeire A.

Universidade Federal de Goiás, Goiânia, Brasil

Variations in concentrations of antigen-specic IgG subclasses are associated with numerous infectious diseases. Observations on dengue hemorrhagic fever pathogenesis suggest a high degree of immune stimulation with extensive activation of complement system preceding the onset of shock, signaling a possible role of complement activation products in endothelium damage and plasma leakage. Antibodies IgG1 and IgG3 are known to activate the complement system efciently while IgG2 and IgG4 are weak activators. In the present study, IgG subclasses antibodies non DENV specic were determined in dengue seropositive patients through quantitative enzyme immunoassay “in house” procedure, whose values were extrapolated from standard curves constructed with known concentration of human commercial IgG1, IgG2, IgG3. Serum samples of individuals participating in this study were collected in the epidemic period of October 2005 to March 2006 on dengue reference health care units in the city of Goiânia-GO in a previously characterized cohort (Guilarde et al. 2008; Féres et al. 2006). Study groups were divided as follows: 30 - dengue fever (DF); 30-dengue with complications (DCC) and 30- dengue hemorrhagic fever (DHF). Medium concentration stratied analysis of non-dengue specic IgG subclasses per collect period and/or age group, considering the different clinical forms of dengue, indicates a signicant statistical difference in IgG1, IgG2 e IgG3 average concentrations between patients with classical and serious disease, being the major signicance presented in samples collected later during the convalescence period. It is even possible to dene different ranges of IgG1 concentration (< 1000 g/l, 1001- 2000 g/l e > 2000 g/l) that characterizes individuals among these different clinical forms. This study's results indicate that it may be a correlation between IgG subclasses concentrations and dengue prognosis.

I-P03

IMMUNOREGULATORY AND PROINFLAMMATORY CYTOKINES PROFILE IN PATIENTS WITH DENGUE

Silva, P.T.F.; Junqueira, I.C.J; Rocha, B.A.M., Tassara, M.; Guilarde, A. O.; Féres, V.C.R; Silveira, L.A.; Martelli, C.M.T.

Universidade Federal de Goiás, Goiânia, Brasil

Immunoregulatory and proinammatory cytokines in excess or produced under unregulated conditions has been implicated as one of the mechanisms involved in dengue immunopathogenesis. This study aimed to determine the prole of cytokines IL-10, IL-21, IFN-γ and TGF-β in patients' serum with different clinical forms of dengue and investigate its possible role as gravity biomarker. Participants were selected from a cohort of 647 patients recruited from basic health units, hospitals and/or ambulatory in 2012-2013 in the city of Goiânia - Goiás. Cases were laboratory conrmed by serological techniques (IgM, IgG, NS1) and/or RT-PCR and

classied as DF, DCC and DHF by specialist clinicians. Then, 160 samples were selected from patients in acute phase of infection, effervescence period, to perform citokynes´quantitative assays using commercial kits (ELISA-Biolegend, USA). The average levels of IL-21 had slightly elevated in patients with DHF (69.2 pg/mL) compared to DF (55.3 pg/mL) and DCC (40.5 pg/mL), but there was no difference statistically signicant. A similar distribution of TGF-β in DF (40294.2 pg/mL), DCC (42108.6 pg/ml) and DHF patients was found (32289.4 pg/ml) and alike, an increase of IFN-γ serum levels in all analysis groups: DF (136.9 pg/mL) DCC (131.6 pg/ml) and DHF (85.1 pg/mL). However, IL-10 median levels in severe clinical forms, DHF (131.5 pg/mL) and DCC (106.0 pg/ml), were signicantly higher (p = 0.027) than those with DF (89.8 pg/ml). These results demonstrate IL-10 as a possible biomarker to predict dengue's potential severity cases, and contribute for follow-up strategies or clinical intervention in order to improve the prognosis of dengue.

I-P04

LEVELS OF DENGUE VIRUS-SPECIFIC IGG1 AND IGG4 DO NOT CORRELATE WITH BOTH COMPLEMENT ACTIVATION AND DISEASE SEVERITY

Eduardo J. M. Nascimento; Priscila Castanha; Robyn Konichi; Marli Cordeiro; Ernesto T. A. Marques, Jr.

University of Pittsburgh, Pittsburg, USA

Complement activation plays an important role on disease severity caused by dengue virus (DENV). Despite the mechanism driving complement activation is still unclear, it is believed that antibodies contribute to the overall complement activation through classical pathway. IgG isotypes vary on their capability to activate complement, where IgG1 is the most efcient while IgG4 lacks this property. In this study, we measured the levels of DENV-specic IgG1 and IgG4 in plasma samples collected from dengue fever (DF) and dengue hemorrhagic fever (DHF) patients. In-house virus-capture ELISA was used to estimate the levels of DENV-specic IgG1 and IgG4 present on plasma samples from DF (n = 43) and DHF (n = 20) cases 3-fold serially diluted. The area under the curve (AUC) was calculated for each patient and compared among clinical outcomes. Man-Whitney test was used to compare the medians in between groups. According to the results, 74.5% and 65% of DF and DHF cases, respectively, have detectable levels of DENV-specic IgG1, while 23.2% of DF cases and 30% of DHF cases have DENV-specic IgG4 titers. Levels of DENV-specic IgG1 differed between primary and secondary infections (p<0.0001), while IgG4 levels were similar (p = 0.5). Levels of neither IgG1 (p=0.3) nor IgG4 (p=0.5) differed between DF and DHF. Additionally, correlation analyses of complement proteins (C3, factor H and factor D) that are correlated with disease severity indicate that only IgG1 is weakly correlated with C3 (r = 0.33, p = 0.008). Altogether, the results suggest that levels of antigen-specic IgG1 are not directly involved on increased complement activation in severe disease, as IgG4 is not involved on protection against severe disease. However, they may indirectly inuence on complement activation by triggering and/or controlling the complement cascade.

I-P05

PROMINENT EXPANSION OF A CD4+T CELL SUBSET IS ASSOCIATED WITH A PROTECTIVE ROLE IN DENGUE VIRUS INFECTION

Daniela Weiskopf, Derek J. Bangs, Ravi V. Kolla, John Sidney, Aruna D. de Silva, Aravinda de Silva, Bjoern Peters and Alessandro Sette

Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, USA

Dengue virus infections are a major cause of morbidity and mortality in tropical and sub-tropical regions of the world. The occurrence of four distinct antigenic serotypes (DENV1-4) adds complexity to the understanding of pathogenesis and immunity. While DENV-specic CD8+ T cell responses have been extensively studied, the breadth and specicity of HLA-restricted CD4+ T cell responses remains to be dened. The denition of these responses are important as CD4+ T cells are well characterized to contribute to host protection from viral and bacterial pathogens in a number of ways including: cytokine production, mediation of CD8+ T- and B- cell activation, and execution of cytotoxic functions to lyse virally infected cells. In an effort to dene HLA-restricted CD4+ T cell responses resulting from natural infection with DENV, we endeavored to map T cell responses in individuals from Sri Lanka where DENV is hyper-epidemic. To identify HLA class II candidates, we utilized a panel of algorithms for eight common HLA DR molecules (DRB1*0101, DRB1*0301, DRB1*0401, DRB1*0701, DRB*0802, DRB1*1101, DRB1*1302 and DRB1*1501) representative of the main DR supertypes to generate a library of DENV-derived, HLA DR binding peptides. While we could identify epitopes derived from all 10 DENV proteins, the highest number of responses was associated with the structural capsid protein, followed by nonstructural NS5, NS2A, and NS4B. Interestingly, while NS3 was the immunodominant protein for CD8+ responses, only 6 CD4+ T cell epitopes were targeted against this protein. Characterization of the phenotype of the responding CD4+ T cells revealed a DENV specic T cell subset that is specically expanded in donors carrying an allele associated with protection from severe DENV disease. We believe that our results will help shed light on the specic role of CD4+ T cells in DENV infection and may help in nding a correlate of protection.

I-P06

IMMUNODOMINANCE CHANGES AS A FUNCTION OF THE INFECTING DENGUE VIRUS SEROTYPE AND PRIMARY VERSUS SECONDARY INFECTION

Daniela Weiskopf, Michael A. Angelo, John Sidney, Bjoern Peters, Sujan Shresta, and Alessandro Sette

Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, USA

Dengue virus (DENV) is the causative agent of dengue fever (DF), the most prevalent arthropod-borne viral illness in humans. Disease can be caused by any of the four DENV serotypes (DENV1-4), which share 67-75% sequence homology with one another. The effect of subsequent infections with different serotypes on the T cell repertoire is not fully understood. We utilized mice transgenic for human leukocyte antigens (HLA) lacking the IFN -/ receptor to study T cell responses to heterologous DENV infection. First, we dened the primary immune response to DENV3 in the context of a wide range of HLA class I and class II molecules. The primary DENV3

immune response recognized epitopes derived from all 10 DENV proteins, with a signicant fraction of the response directed against the structural proteins. This is in contrast to primary DENV2 infection where structural proteins are a very minor component of the response. Together, these data suggest a different hierarchy of immune dominance as a function of the infecting serotype. We also investigated the effect of secondary heterologous DENV infection on the T cell repertoire. We found that while primary DENV3 infection elicited responses prevalently directed towards serotype specic epitopes, the responses following secondary heterologous infection with DENV3 followed by DENV2 were more frequently directed towards epitopes conserved between the two serotypes and resembling DENV2 primary infection. Our results provide new insights into HLA-restricted T cell responses in primary and secondary infection with DENV, which are of relevance for both vaccine design and DENV immuno-pathogenesis.

I-P07

ACTIVATED HUMAN PBMCS WITH INCREASED EXPRESSION OF CELL DEATH MARKERS ARE ASSOCIATED WITH DENGUE FEVER DISEASE

Amanda Torrentes-Carvalho; Cintia Ferreira Marinho; DØbora Batista de Oliveira; Paulo Vieira Damasco; Luiz Jose de Souza; Elzinandes Leal de Azeredo; Claire Fernandes Kubelka

Fundacao Oswaldo Cruz - Instituto Oswaldo Cruz, io de Janeiro / Brazil

Dengue Fever, a public health problem in Brazil, may present severe clinical manifestations as a

result of increased vascular permeability and coagulation disorders. Phagocytes are important targets for DENV replication, which induce inammatory mediators. DENV may be inducing apoptosis and disseminating it´s virus progenies to neighbor cells through phagocytized apoptotic bodies. Monocytes may have an essential role in developing disease severity. T cell activation is a crucial event for an effective immune response against infection, including the production of a range of cytokines. We aim to reveal processes that modulate the virus-cell interaction, with an emphasis on cell death mechanism. PBMCs from healthy individuals and DENV infected patients clinically classied were obtained from heparinized venous blood. Extra and intracellular staining were made by FACS. The analysis of apoptotic proteins prole expression was done using PBMCs lysates obtained from patients and controls. Levels of soluble factors were determined by ELISA. As results, we observed up regulation of CD29 and CD107a expression indicated that in patients the majority of T cells express activation and cytotoxic phenotype. Higher frequency of CD95 was presented in both monocytes and T lymphocytes specially in those T cells with cytotoxic activation prole. We also demonstrated high expressed low levels of anti-apoptotic molecule Bcl-2, corroborating this data. DNA fragmentation was observed in PBMCs from patients during Dengue infection, conrming apoptosis of T lymphocytes. Analysis of the apoptotic-related proteins expression prole showed that some molecules are over expressed in PBMCs from DENV infected patients at acute phase. High levels of pro inammatory cytokines TNF and IFNgama in patients` plasma might predispose cells to death. Our data support virus modulation of apoptotic factors in PBMCs from patients at acute phase of Dengue. The immune scenario generated as a result of interactions and/or virus itself may be interfering in activation and cell death. These mechanisms are relevant to understand and clarify the immunopathogenesis and disease severity. Financial Support: FIOCRUZ-IOC, CNPq and FAPERJ

I-P08

IMPLICATION OF CHEMOKINES ON THE ENDOTHELIAL BARRIER HOMEOSTASIS DURING DENGUE FEVER

Cipitelli, Márcio da Costa¹; Marinho, Cintia Ferreira¹; Faria, Nieli Rodrigues da Costa²; Kubelka, Claire¹, Luis José de Souza, Rivaldo Venâncio da Cunha³; De-Oliveira-Pinto, Luzia Maria¹

¹Laboratory of Viral Immunology - Oswaldo Cruz Institute, RJ-Brazil. ²Laboratory of Flavivirus -Oswaldo Cruz Institute, RJ-Brazil. ³Department of Clinical Medicine, UFMS, MS-Brazil.

Introduction: Endothelial dysfunction contributes to the pathogenesis of the dengue fever. Because endothelial activation often precedes overt endothelial dysfunction, biomarkers may be detectable before, and therefore, useful as biomarkers of disease severity or prognosis. Herein, we investigated the cytokines and chemokines as biomarkers of the activated endothelium in serum from Dengue Fever without (DF) or with Warning Signs (WS) plus Severe (WS/Severe) patients and their role in the modulation of endothelial barrier function using primary human endotelial cells (HMVEC). Methods: Patients from DENV-4- outbreaks were studied. Serum cytokines and chemokines levels were measured by ELISA. For measuring TEER (Trans-endothelial electrical resistance), HMVEC were plated at 2x105 cells/well on bronectin-coated transwell inserts at 37°C in 5% CO2 for two days. Untreated serum or pretreated serum with chemokines ligands blocking were added to the top chamber. Electrical resistance across HMVEC monolayers was measured by using an EndOhm-6 chamber and EVOM volt-ohmmeter. Results: The WS/Severe patients had show even higher frequency of vascular leakage and bleeding than those of DF patients. Moreover, the platelet counts were lower among these WS/Severe, while serum aminotransferase levels were signicantly higher in the WS/Severe as compared to DF. Both DF and WS/Severe patients showed higher TNF-a, IL-10, CCL2, CXCL10, CX3CL1, CXCL8 levels than normal controls. However, IL-1b and CCL5 did not differ signicantly from those found in controls. We had that after early time points (5min), serum from 3 to 4 DENV-patients decreased the HMVEC TEER and TEER recovery in only one patient. Our results showed that the HMVEC monolayers became more permeable upon in vitro blocking of CCL4 or CXCL10 serum DENV-patients, but not with blocking of CCL5 or CX3CL1, than untreated serum. Conclusion: Our preliminary ndings suggest that chemokines has been involved in the endothelial barrier homeostasis during dengue fever. Support: FIOCRUZ/IOC; FAPERJ; CAPES.

I-P09

LYMPHOCYTE ACTIVATION IN PRIMARY IMMUNE RESPONSE OF MICE INFECTED BY THE INTRACEREBRAL ROUTE WITH DENV21 1 1 1 1 2 1 1Oliveira ERA , Veltri ERP , Gonçalves AJS , Mantuano-Barradas M , Azevedo AS , de Meis J , Nogueira ACMA and Alves AMB

1 2Laboratory of Biotechnology and Physiology of Viral Infections, Fiocruz, Rio de Janeiro, Brazil; Laboratory of Thymus Research, Fiocruz, Rio de Janeiro Brazil

Dengue disease is considered a great burden in an extensive area of the globe. Mouse models for dengue infection represent a major tool to research anti-dengue strategies regarding vaccine candidates or antivirals. A widely used mouse model for this purpose is the intracerebral inoculation of a brain adapted virus into immunocompetent mice that results in lethality. Under this approach, mortality and morbidity are often seen as endpoints to determine efcacy of anti-dengue vaccines. Nevertheless, little is known about the immune mechanisms involving lymphocyte responses in intracerebrally infected mice. Here, a kinetic study of lymphocyte responses was made analyzing samples from BALB/c animals infected with DENV2 NGC strain by the intracerebral route. Activated T cell phenotypes CD3+CD4+CD45RBlow and CD3+CD8+CD45RBlow, peaked between days 3 and 7 post-infection in blood and spleen, and effector T cells (CD44hiCD62L-) were also seen in liver samples after 3 days of infection, as measured by ow cytometry technique. Lymphocyte inltrates were characterized in SNC of infected mice by the time of morbidity manifestations. Blood and spleen samples collected 8 to 11 days post-infection also showed increased B220+IgD- B cell phenotype, that were consistent with the presence of IgM and IgG anti-NS1 virus protein in serum of mice. Cytokines related with inammatory response, IL-12p70, TNF-α, MCP-1 and IL-10 were detected in serum samples of animals in the rst day post-infection, while on day 7 we found increased levels of IL-12p70, TNF-α, IL-10 and IFN-γ.These data provide new insights and a better knowledge about the primary immune response in mice infected with DENV by the intracerebral route and may contribute with the establishment of new parameters to evaluate the efcacy of anti-dengue candidate vaccines. Financial support: PDTIS-Fiocruz, INCTV, CNPq and FAPERJ.

IMMUNOLOGY/DIAGNOSTICS/PROGNOSTICSABSTRACTS INVITED SPEAKERS

DENGUE DIAGNOSTICS

Maria G. Guzman

"Pedro Kouri" Tropical Medicine Institute, PAHO/WHO Collaborating Center, Havana, Cuba lupe@ipk.sld.cu

Dengue diagnosis is important for clinical care, surveillance support, pathogenesis studies, vaccine and drug development and clinical trials. Direct (virus isolation, RNA and antigen detection) and indirect methods (serological investigations) constitute the dengue diagnostic tools. Direct methods show the highest condence, indirect methods show the highest opportunity for diagnosis being the widest applied to routine practice. Tools available include virus isolation and identication, IgM ELISA for the serological diagnosis, RT/PCR and real-time RT/PCR for genome detection and quantication and NS1 detection. Dengue diagnostic studies allow conrming acute, early and late convalescent dengue infection. Early diagnosis is of importance for case management. As part of the epidemiological surveillance, dengue diagnosis allows the conrmation of transmission, identication of circulating serotypes and genotypes as well as severe and fatal cases. There are still some needs requiring attention for dengue diagnosis: greater reagent availability, prociency should be improved and extended. Also important are the development of tests for early diagnosis, serological tests to differentiate dengue from other aviviruses, easy and inexpensive protocols for genomic characterization and viral quantication, simplify specimen handling and transportation and recombinant antigens for serological tests as well as tools supporting dengue vaccine evaluation. Finally, enhanced information and experience exchange between endemic areas as well as training and capacity building are needed.

EVALUATION OF THE PERFORMANCE OF AVAILABLE DENGUE DIAGNOSTIC TESTS FOR THE DEVELOPMENT OF AN OPTIMAL DIAGNOSTIC ALGORITHM FROM A SINGLE SPECIMEN

1 1 1 1 1 1 1 1Elizabeth Hunsperger , Jorge Munoz-Jordán , Manuela Beltran , Candimar Colon , Jessica Carrion , Jesus Vazquez , Raziel Rojas , Nereida Acosta , Juan Francisco 1 2 1Medina-Izquierdo , Kalanthe Horiuchi , Harold Margolis

1 2Centers for Diseases Control and Prevention (CDC), Division of Vector-Borne Diseases (DVBD), Dengue Branch, San Juan, Puerto Rico; CDC, DVBD, Ofce of the Director, Fort Collins, Colorado

Dengue diagnostic testing is a challenge because no single test detects all the analytes needed to conrm the diagnosis of dengue in a single specimen obtained during the febrile phase of the illness. However, the use of two diagnostic tests; one to detect dengue virus (DENV) RNA or antigen (NS1), and one to detect IgM anti-DENV conrms a high proportion of suspect dengue cases. This study was conducted to provide an estimate of the performance of dengue diagnostic tests during the rst 10 days after onset of fever and to develop a diagnostic testing algorithm for single specimen testing. The course of dengue from 1 to 10 days post onset of illness (DPO) was reconstructed using a panel (n=1234) of archived acute and convalescent serum samples obtained from previously conrmed dengue cases in Puerto Rico between 2005 through 2010. The panel contained all four DENV serotypes. Specimens were tested by the CDC developed real time RT-PCR and IgM anti-DENV tests, and commercial ELISAs for NS1 antigen and IgM anti-DENV. The evaluation showed that two IgM anti-DENV tests (Focus and InBios) had a sensitivity of 79% (CI95:70-85) and 58% (CI95:49-67) respectively on DPO=4 indicating that IgM anti-DENV adds diagnostic value in the acute phase of dengue. An analysis of the sensitivity of NS1 ELISAs compared to RT-PCR showed that they ranged from 78-93% (CI95: 68-98) during the rst 3 days of illness. Results indicated that either RT-PCR or NS1 provide the best denitive result in the rst 3 days of illness, whereas a 2-test format with IgM anti-DENV and RT-PCR/NS1 was best used after the rst 3 days. These results determined an algorithm of diagnostic testing that provided the highest likelihood of a denitive result in a single serum/plasma specimen obtained during the acute phase of dengue as dened by the 2009 WHO Dengue Case Classication.

ABSTRACTS ORAL PRESENTATIONSDISCOVERY AND QUALIFICATION OF A PREDICTIVE BIOMARKER PANEL FOR SEVERITY OF DENGUE SEROTYPE 3 INFECTION

1,2,3 1,2,3 2,3,5 2, 3, 5 8 6 2, 7 5Allan R. Brasier , Yingxin Zhao , John E. Wiktorowicz , Heidi M. Spratt , Eduardo J. M. Nascimento , Marli T. Cordeiro , Kizhake V. Soman , Hyunsu Ju , Adrian 1 7 7 6,8 9,10,11Recinos III , Susan Stafford , Zheng Wu , Ernesto T.A. Marques, Jr , and Nikos Vasilakis

1 2 3 Department of Internal Medicine, University of Texas Medical Branch, Galveston, TX; Sealy Center for Molecular Medicine, UTMB; Institute for Translational 4 5 6Sciences, UTMB; Department of Biochemistry and Molecular Biology, UTMB; Department Preventive Medicine and Community Health, UTMB; Laboratorio de

7 8Virologia e Terapie Experimental do Centro de Pesquisas Aggeu Magalhaes-CPqAM, Fiocruz, Recife, Pernambuco, Brazil; Biomolecular Resource Facility, UTMB; 9Department of Infectious Diseases and Microbiology and Immunology, University of Pittsburgh, United States of America; Department of Pathology and Center for

10 11Biodefense and Emerging Infectious Diseases, UTMB; Center for Tropical Diseases, UTMB; Institute for Human Infection and Immunity, UTMB.

Dengue viral (DENV) infection is a signicant health and economic risk to over 0.4 B inhabitants in 125 subtropical countries, including endemic transmission in the US Gulf Coast. Although the majority of DENV infections are self-limited, severe cases are associated with delayed vascular leakage and hemorrhage, a syndrome known as dengue hemorrhagic fever (DHF). Because intervention early in the course of disease can prevent signicant mortality, identication of biomarker panels and classication models will impact the clinical management of epidemic DENV outbreaks. We apply an approach using a proteomic discovery pipeline combined with a targeted quantitative proteomics study in an adult cohort of epidemic DENV-3 infection from Recife, Brazil. 120 differentially expressed proteins were identied in DHF vs dengue fever (DF) using an integrated discovery platform with size exclusion chromatography of denatured plasma proteins followed by depletion and saturation uorescence quantitation in 2DE. Quantitative selective reaction monitoring was used to measure a 19 member candidate biomarker panel containing the most informative features from the discovery study and from prior knowledge, representing proteins in the complement cascade (complement factor D), acute phase response (a2 macroglobulin, brinogen), and cytoskeletal proteins from cellular leak. The abundance of 15 proteins showing differential abundance were combined with clinical features in a random forest classier predict 3 distinct outcomes of DENV-3 severity: DF, DHF, and DF complicated (representing those with plasma leak but did not meet strict WHO criteria for DHF). The resulting classier produced overall 86% accuracy of prediction and correctly discriminates amongst the three outcomes of DENV-3 severity with an area under the receiver operating curve of >0.9 in both cases. This qualied dataset will be of value for informing point-of-care diagnostics to elucidate objective disease subtypes, and evaluate clinical interventions to reduce the impact of DENV-3 infections. Supported by NIAID Clinical Proteomics Center, HHSN272200800048C (ARB) and UL1TR000071 UTMB CTSA.

DENGUE VIRUS NS1 ANTIGEN: PERFORMANCE OF COMMERCIALLY AVAILABLE TESTS AND ALTERNATIVE APPLICATIONS FOR EARLY DIAGNOSIS OF DENGUE INFECTIONS.

1 1 1 1 1 1 1 1Monique Lima , Rita Nogueira , Ana Maria de Filippis , Priscila Nunes , Manoela Heringer , Nieli Faria , Fernanda Nogueira , Flávia dos Santos

Flavivirus Laboratory, Oswaldo Cruz Institute/FIOCRUZ, Av Brasil, 4365, Rio de Janeiro

In 2008 the Brazilian Ministry of Health established sentinel units in strategic cities using NS1 capture tests as a screening method of dengue suspected cases, however without a previous evaluation. The present study aimed to meet the demands of evaluating and conrming the role of these tests. The performance of 3 kits commercially available was evaluated in 450 samples. The NS1 Ag Strip test was the most sensitive (89%), followed by the Platelia NS1 ELISA (84%). A lower sensitivity was observed in DENV-3 cases by all three kits. The comparison of two generations of the NS1 Ag ELISA from PanBio after a test improvement by the manufacturer, showed a signicant increase in the sensitivity (72.3% to 80%). The sensitivity of the tests as an alternative tool for dengue diagnosis in tissues from fatal cases (n=23) were 34.7%, 60.8% and 91.3%, respectively. In the analysis of 74 tissues, the NS1 Ag Strip test showed a signicantly higher sensitivity (78.3%). This assay was more sensitive in the liver, lung, kidney, brain, spleen, and thymus. The usefulness of those tests was also evaluated in entomological surveillance after DENV-4 introduction in Rio de Janeiro. The NS1 Ag Strip rapid test and the Platelia NS1 ELISA conrmed the infection in Ae. aegypti collected in the eld. Reports of a low sensitivity of the NS1 tests in diagnosing DENV-4 cases occurred after the introduction of this serotype in the country. Two methods for immune complexes dissociation were tested. The sensitivity observed in non-dissociated samples (46.6%) was signicantly improved when the samples were submitted to acid (70.4%) and heat dissociations (77.5%). The results of this study the usefulness of the NS1 protein in the early diagnosis of dengue infections and demonstrate the alternative applications in conrming fatal cases and in the surveillance in the vectors.

DEFINING THE TARGETS OF DENGUE VIRUS INFECTION IN HUMAN SKIN1,5 1,5 1,5 2,5 4 1,3,5Parichat Duangkhae , Amanda P. Smith , Ernesto T.A. Marques, Jr. , Kate D. Ryman , Simon C. Watkins and Simon M. Barratt-Boyes

1 2 3 4Departments of Infectious Diseases and Microbiology ; Microbiology and Molecular Genetics ; and Immunology ; Center for Biologic Imaging and Center for Vaccine 5Research ; University of Pittsburgh, Pittsburgh, PA

Dengue is the most important arboviral infection of humans. Dengue virus (DENV) is introduced into human skin by the bite of infected mosquitoes. While Langerhans cells (LCs) have been implicated as the main targets of DENV infection the dynamics of infection of human skin remain ill-dened. Here, we exposed skin explants from healthy human donors to DENV to determine the targets of infection. Skin explants were inoculated with DENV and analyzed by immunouorescence after intervals of incubation using antibodies to cell-specic markers and viral protein NS3. Time course experiments showed that the rst targets of DENV infection were basal keratinocytes, with infection detected within 8 h. From 12-48 h of infection abundant virus replication was detected in epidermal LCs and dermal macrophages (MØs) as well as tubular structures consistent with lymphatic endothelium. Quantitative analysis indicated that exposure to DENV resulted in signicant inltration of MØs into dermis. These preliminary data suggest that DENV initially infects basal keratinocytes which may release factors promoting the mobilization of infected LCs

out of the epidermis, and the inux of MØs into the dermis, which subsequently replicate DENV to high levels. Ongoing experiments are designed to determine the mechanism for MØ recruitment. These studies are revealing that DENV infection of human skin is a dynamic process involving sequential interactions and recruitment of distinct cellular targets.

HIGHLIGHTED POSTERSHP32

A RAPID, MULTIPLEXED, MOBILE PHONE-ENABLED POINT-OF-CARE DIAGNOSTICS1,7 2,6 4,7 7 7 4,7 3,4 5Tam, Justina ; Yen, Chunwan ; de Puig Guixé, Helena ; Miyazaki, Hikaru ; Fiegen, Ann ; Phillips, Elizabeth ; Gómez-Márquez, Jose ; Hamad-Schifferli, Kimberly ;

6 7Regan, Patrick , Clavet, Charles6, Bosch, Irene2,6; and Gehrke, Lee 1 2 3 4 5FDA Commissioner's Fellow, FDA ORISE Fellow, Little Devices Lab @ MIT; MIT/SUTD International Design Center, Massachusetts ; Institute of Technology; Lincoln

6 7Labs, Massachusetts Institute of Technology; FDA Winchester Engineering and Analytical Center; Institute for Medical Engineering and Science, Massachusetts Institute of Technology, and Department of Microbiology and Immunobiology, Harvard Medical School

Background: Medical countermeasures surveillance and reporting during and after a public health emergency event require sensitive and specic detection/ diagnostic methods and devices. We are designing, building, and testing a rapid, multiplexed, mobile phone-enabled diagnostic device to detect dengue virus and Ebola virus, Category A bioterror agents, in the eld. The goal is to deliver a device that will permit screening for multiple pathogen markers without the need for refrigeration, specialized training, specialized equipment or chemicals. Mobile phone technology is used to analyze the lateral ow data, quantify the results, and upload the results for real time epidemiology. Methods: The device is based on lateral ow chromatography, an established technology. Current multiplexing permits assay for up to eight pathogen markers concurrently using one hundred microliters of sample. Monoclonal antibodies have been screened using ow cytometry and lateral ow chromatography to dene functional pairs when conjugated to gold nanoparticles and bound to nitrocellulose paper. Nanoparticle surface chemistries are being evaluated to identify low cost approaches toprepare conjugated nanoparticles. A mobile phone app has been coded to record the image of the multiplexed diagnostic, correct the image for user photography errors, quantify the signal intensities, and upload data to a server, with GIS. Results: A prototype device that detects and distinguishes the four serotypes of dengue virus, dengue IgG/IgM, Ebola glycoprotein, and ST2 protein has been built and tested. Initial specicity and sensitivity tests using laboratory proteins and human patient serum samples are favorable. The phone app records the data, measures signal intensities, and uploads data for real time epidemiology. Conclusion: A multiplexed rapid lateral ow diagnostic for eld use detects Category A pathogens and uploads data for real time epidemiology. The goal of this project is build a rapid, multiplexed, point of care diagnostic device that is paired with a mobile phone reader/analytic for real time epidemiology.

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USE OF FAST-GROWTH DENGUE-LIKE VIRUSES FOR DEVELOPMENT OF FAST NEUTRALIZATION ASSAY AND LETHAL MOUSE CHALLENGE STUDY1 1 1 1 2 1Claire Huang , Karen Boroughs , Janae Stovall , Betty Luy , Elizabeth Hunsperger , and Richard Kinney

1 2Division of Vector-Borne Diseases, Centers for Disease Control and Prevention, Fort Collins, CO 80521, USA & San Juan, Puerto Rico

Dengue virus (DENV) presents a substantial threat to public health globally, yet there is no approved DENV vaccine or effective anti-viral treatment. Wild-type DENVs usually grow slowly in cell cultures and do not cause signicant illness in weanling mice following peripheral challenge with virus. Therefore, many assays or diagnostics requiring live DENVs are time consuming, and it is difcult to establish lethal viral challenge mouse models for all 4 serotypes of DENV. We have successfully engineered chimeric West Nile/Dengue viruses for all 4 DENVs, which have been further genetically modied to enhance their viability and genetic stability in cell cultures. These chimeric viruses express the pre-membrane (prM) and envelope (E) proteins of DENV in the replicative genetic vector of WNV. Due to the robust replication ability of the WNV vector, these chimeric viruses exhibit faster and more vigorous growth in cell cultures than the wt DENVs. Because the viral envelope consists of DENV prM and E proteins, these chimeras can be used as surrogate DENVs in multiple applications, including diagnostics, vaccine development, vaccine testing, and virology research. This report focuses on results of using these chimeras in Ab neutralization assays with human clinical samples and in a mouse challenge study for a tetravalent DENV vaccine. Our results demonstrated that these DEN-like viruses are reliable substitutes for wt DENVs to establish a fast Ab neutralization assay and to be used as lethal challenge viruses in mouse studies of DENV vaccines by the most convenient intraperitoneal route of injection.

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DENGUE SURVEILLANCE IN CENTRAL BRAZIL: IMPLEMENTATION OF NS1AG TEST AS SCREENING TOOL FOR EARLY DIAGNOSIS OF DENGUE

Angela Argoloa, Vinícius Lemesa, Lucileis Fernandesa, Carmen Ramosa, Maysa Silvaa, Valéria Féresb.

a Laboratório de Saúde Pública Dr. Giovanni Cysneiros (LACEN-GO); b Faculdade de Farmácia da Universidade Federal de Goiás (FF/UFG)

Dengue is considered a major public health issue in South American countries. During the last three decades, Brazil has experienced an increase in the incidence of dengue, dengue hemorrhagic fever and, also, a sharp increase in the number of deaths. In Brazil, viral isolation has been used as part of viral surveillance of dengue. Serologic NS1Ag test was implemented in several public health laboratories to improve the early dengue diagnosis as well to increase the positivity of viral isolation. This study aims to describe the results of the NS1Ag test as screening tool for DENV monitoring in the reference public health laboratory of Goias, Central Brazil, 2013. Routinely, samples from patients with dengue suspicious were collected by epidemiological surveillance and performed by LACEN-GO: (1) Blood collected <5 days after onset of symptoms (DOS) tested by viral isolation (C6/36 cell culture followed by immunouorescence); (2) Serum with >6 DOS tested by ELISA IgM test. To

evaluate NS1Ag test as screening procedure, 570 paired samples (serum and blood) collected <5 DOS were analyzed. Data were extracted from GAL lab software and analyzed using Excel 2010. 17,877 samples were tested. 64.3% and 21% were positive for IgM or NS1Ag tests, respectively. Viral isolation was performed in 1,514 samples yielding 38% (n=575) of positivity. Among paired samples, 299/570 were positive by NS1Ag. Moreover, 243 were also positive by viral isolation (81.3% of co-positivity). Among NS1Ag negative samples, 27 had DENV-4 isolated. There was high concordance between NS1Ag test and viral isolation. NS1Ag test used as a screening test before viral isolation improved its positivity. However lower sensitivity of NS1Ag for DENV-4 was observed in agreement with other studies. NS1Ag seems to be feasible as screening tool to be performed before viral isolation in routine of DENV surveillance. Further cost-effectiveness studies of NS1Ag screening tool are necessary before its implementation in routine.

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ASSESSING POSITIVITY AND CIRCULATING LEVELS OF NS1 IN SAMPLES FROM 2012 DENGUE OUTBREAK IN RIO DE JANEIRO, BRAZIL1 2 2 1Diego Allonso , Marcelo D. F. Meneses , Davis F. Ferreira , Ronaldo Mohana-Borges

1 2 Laboratório de Genômica Estrutural, Instituto de Biofísica Carlos Chagas Filho, UFRJ, Rio de Janeiro, Brazil. Instituto de Microbiologia Paulo de Góes, UFRJ, Rio de Janeiro, Brazil.

Ideal early dengue diagnosis should not only detect the infection but also identify patients with higher likelihood to develop severe cases. Previous studies have suggested the potentiality of NS1 as viral marker for dengue severity. However, further studies using different sera panels are necessary to conrm such hypothesis. We aimed at analyzing qualitatively and quantitatively NS1 antigenemia in serum samples from DENV-infected patients collected during the 2012 outbreak occurred in Rio de Janeiro, Brazil. We developed a home-made ELISA able to detect similarly NS1 protein from the four DENV serotypes and from primary and secondary cases. This approach was used to calculate circulating NS1 concentration in previously identied positive samples. In parallel, we compared the NS1 positivity using the Platelia Dengue NS1 Ag assay from Bio-Rad with our system. A total of 128 samples were positive to DENV infection and were classied according to the new WHO guideline. The overall NS1 positivity was 68% according to Platelia assay, whereas all samples were NS1-positive when analyzed by our home-made ELISA (100% positivity). Fifty-four samples were positive to PCR revealing a co-circulation of DENV1 and DENV4. Not surprisingly, the NS1 positivity for DENV4 samples was lower than that obtained for DENV1. The circulating NS1 concentration varied from 7 to 284 ng/mL. However, no signicant difference was observed between primary and secondary cases, and between mild and severe cases. Our results support previous data that demonstrated the low efciency of Platelia assay to detect DENV4 infection. We did not observe any difference in NS1 levels in all conditions analyzed, indicating that NS1 antigen is not a suitable marker for disease severity.

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WHITE BLOOD CELL COUNTS IN THE DIAGNOSES OF DENGUE FEVER AND LEPTOSPIROSIS1 2 3 1 1 4 1 5Stalin Vilcarromero , Connie Fernandez , Manuel Cespedes , V. Alberto Laguna-Torres , Robert D. Hontz , Eduardo Gotuzzo , Tadeusz Kochel , Daniel G. Bausch1,

1 2 3 4U.S. Naval Medical Research Unit No. 6, Lima, Peru; Hospital Santa Gema de Yurimaguas,Yurimaguas, Peru; Instituto Nacional de Salud, Iquitos, Peru; Universidad 5Peruana Cayetano Heredia, Iquitos, Peru; Tulane School of Public Health and Tropical Medicine, New Orleans, USA

Dengue fever and leptospirosis are signicant causes of acute febrile illness in tropical regions of low and middle income countries. Differentiation between these two diseases on clinical grounds is difcult, and pathogen-specic laboratory diagnostics are not uniformly or rapidly available. However, white blood cell (WBC) and platelet counts are usually feasible. We explored the utility of these clinical parameters in distinguishing dengue fever from leptospirosis. We analyzed data from one year (2006) of a febrile surveillance program at the Public Hospital of Yurimaguas, a city in the Peruvian Amazon. WBC and platelet counts were performed on all study patients on the rst or second day of enrollment by the hospital's laboratory staff. Acute and convalescent serum samples were tested for dengue virus (virus isolation, PCR, ELISA IgM) and leptospirainfection (microagglutination, IgM) at the laboratory of the U.S. Naval Medical Research Unit No. 6 in Lima, Peru. A total of 337 patients were enrolled, of whom 51 (15%) were conrmed to have dengue fever and 51 (15%) leptospirosis. Although normal cell counts were frequently observed in both diseases, lymphocytosis (16/51, 31%), leukocytopenia (17/51, 33%) and thrombocytopenia (41/51, 80%), were common in dengue fever while normal values forleucocytes (31/51, 61%) and lymphocytes (32/51, 63%) predominated in leptospirosis. We conclude that leukocytopenia, lymphocytosis, and thrombocytopeniaareimportant early features in dengue fever, while normal leukocyte and lymphocytes counts aretypical in leptospirosis. These laboratory parameters are readily available in most hospitals, including in low and middle income countries, and may serve as valuable tools and early indicatorsto distinguish between dengue fever and leptospirosis.

POSTERS SESSION IMMUNOLOGY/DIAGNOSTICS/PROGNOSTICSDP-P01

EVALUATION OF A SIMULTANEOUS IGM-IGG RAPID DIAGNOSTIC ASSAY

NOGUEIRA, J.S.; SUZUKI, A.; SILVEIRA, V.R.; MONTORO, M.G.; BARBOSA, V.M.; CARVALHO, B.R; OLIVEIRA, C.H.; BISORDI, I.

Instituto Adolfo Lutz, Sao Paulo, Brazil.

Dengue virus infection is caused by four distinct serotypes of dengue virus (DENV 1-4) and can result in a range of clinical manifestations from asymptomatic infection to severe dengue fever. The detection of dengue-specic immunoglobulins in serum is a useful tool for routine diagnostic. Although the M antibody capture ELISA (MAC-ELISA) is adopted as the method of choice to diagnosis, rapid tests have been developed as quick and easy methods for clinical use. With the aim of evaluating

the performance of a rapid immunochromatographic test for simultaneous IgM and IgG detection (Panbio® Dengue Duo Cassette/ Inverness Medical), we used characterized sera tested by MAC-ELISA, hemagglutination inhibition (HI), qPCR and virus isolation. To establish the sensitivity and specicity of the assay relative to dengue IgM status, we used 44 seropositive samples and 64 seronegative samples by MAC-ELISA. We obtained 100% of sensitivity, 89% of specicity and 93.5% of serological agreement. The kappa value obtained was 0.915 indicating optimal agreement. To establish the sensitivity and specicity relative to dengue IgG status, we used 8 seropositive samples and 7 seronegative samples by HI. We obtained 100% of sensitivity, specicity and serological agreement. The kappa value obtained was 1.0. In order to determine the analytical specicity, we used 31 samples from patients with conrmed diseases other than dengue. Samples from patients with antibodies against Herpesvirus 6 and Parvovirus showed false-positive results on the Duo Cassette: 22.5% for IgM and 6.4% for IgG. We obtained indications that Panbio® Duo Cassette shows a good performance, being suitable for clinical use; however, other studies are required to evaluate consistently the potential of cross-reactivity.

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UTILITY OF A COMMERCIAL NON-STRUCTURAL PROTEIN 1 (NS1) ELISA FOR THE DIAGNOSIS OF DENGUE IN PROSPECTIVELY COLLECTED ACUTE FEBRILE ILLNESS SERUM SPECIMENS

B. Katherine Poole-Smith, Elba Caraballo, Brenda Torres-Velasquez, Kay M. Tomashek, and Elizabeth A. Hunsperger

Dengue Branch, Centers for Disease Control and Prevention, San Juan, Puerto Rico 00920

Dengue is an acute febrile illness (AFI) caused by one of four dengue viruses (DENV1-4) that presents similarly to inuenza, leptospirosis, or measles during the acute phase [0-5 days post onset (DPO)] of illness. DENV RT-PCR and anti-DENV IgM enzyme-linked immunoassays (ELISA) are used 0-5 DPO to conrm a dengue clinical diagnosis. DENV non-structural protein 1 (NS1) ELISA can be used 0-9 DPO to conrm a case. Most studies measuring sensitivity of DENV NS1 assay used archived serum specimens. We hypothesized that freeze-thaw effect on archived specimens increases the bioavailability of NS1 yielding articially high sensitivity. We evaluated the performance of the PanBio Dengue Early (NS1) ELISA using prospectively collected, never frozen specimens. Patients with AFI ≤7 days were enrolled at our Sentinel Enhanced Dengue Surveillance System (SEDSS) site in Puerto Rico from May 2012-March 2013 (n=1560). Acute specimens were tested by DENV RT-PCR. Acute and convalescent specimens (6-14 DPO) were tested by DENV IgM and Panbio Dengue Early ELISA. The sensitivity of Panbio Dengue Early ELISA was 74% (95CI 70-78) and specicity 90% (95CI 88-92) compared to RT-PCR (391 positive patients). The majority of the patients were infected with DENV1 (n = 386, 74% NS1 positive); 5 patients were infected with DENV4 (40% NS1 positive). DENV RT-PCR or IgM-positive patients <15 years old were 1.8 times more likely to have a positive NS1 test (OR = 1.9 95CI 1.3-2.7) than patients ≥ 15. NS1 positivity did not vary by patient gender. PanBio Dengue Early ELISA sensitivity was low relative to ndings from a recent meta-analysis and supports our hypothesis. Patients <15 years old may be more likely to have a primary DENV infection and less likely to have anti-NS1 antibodies to reduce NS1 bioavailability. Differential assay sensitivity by serotype needs further evaluation with a larger sample size.

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EVALUATION OF DENGUE DIAGNOSTIC ASSAYS IN GUARULHOS COUNTY, SÃO PAULO STATE, BRAZIL, 20141 1 1 1 1 2Fernanda Gisele da Silva , Sarai Joaquim dos Santos Silva , Iray Maria Rocco , Mariana Sequetim Cunha , Margarida G. Montoro , Regina A. Nunes Romano , Ana de

2 1Nazaré Melo Mesquita e Ivani Bisordi .1 2Núcleo de Doenças de Transmissão Vetorial, Centro de Virologia, Instituto Adolfo Lutz; Laboratório Municipal de Guarulhos, Brazil. E-mail: fernanda_sbio@yahoo.com.br

Dengue virus (DENV) is a Flavivirus with four serotypes (DENV-1, DENV-2, DENV-3, and DENV-4). Infections in humans may range from asymptomatic to mild or severe manifestations. Dengue is transmitted by Aedes mosquitoes and is considered the most important arboviral disease. Rapid diagnostic methods in samples obtained in the early days of symptoms are fundamental to the epidemiological surveillance and vector control. We evaluated the results of 56 patients (Guarulhos County, São Paulo State, Brazil) with negative response in NS1 detection essay and subsequent positive response in an IgM capture assay. Two blood samples were collected from each patient after suspicion of dengue. The rst sample was collected in the acute phase (3 days after onset of symptoms) and a second sample taken after the sixth day of illness. Samples of initial acute phase were processed by detection of NS1 protein using a commercial enzyme immunoassay. Samples collected after the sixth day of illness were processed by a commercial ELISA for IgM capture. The rst 56 samples were negative for NS1 and the second samples were all positive for IgM. The samples were subsequently submitted to RT-qPCR, virus isolation, IFA and in house MAC-ELISA assays. The qPCR technique conrmed 21 (37.5%) samples positive for DENV-1 and 7 (12.5%) samples positive for DENV-4. Virus isolation conrmed 7 (12.5%) positive for DENV-4 and 14 (25%) positive for DENV-1. The in-house MAC-ELISA tested positive for 43 of 56 samples (77%). Identication of serotypes was possible in 28 samples. The results suggest that DENV-1 and DENV-4 are currently circulating in the studied area and that there is a high percentage of false negative tests for the detection of NS1 protein for these serotypes. It suggests that the NS1 detection kits, as diagnostic tool, must be used with precaution.

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DEVELOPMENT OF ANTI-DENGUE VIRUS NS1 MONOCLONAL ANTIBODIES FOR DIAGNOSTIC OF ACUTE DENGUE INFECTIONS1 1 1 1 2 1, 2Marli Tenório Cordeiro , Klécia Marília Soares Melo , Rafael Dhalia , Cheysa A. Biondo , Eduardo J. M. Nascimento , Ernesto T. A. Marques

1 2Centro de Pesquisas Aggeu Magalhães, FIOCRUZ-PE, Brazil ; Center for Vaccine Research, University of Pittsburg, Pittsburg, Pennsylvania.

Detection of circulating dengue non-structural protein 1 (NS1) has been widely used as a reliable marker of acute infection. However, limited availability of antibodies specic to NS1 halts the advance of development of immune-assays for detection and quantication of this protein. Our aim was to develop a panel of mouse monoclonal antibodies (mAb) specic to dengue virus (DENV) NS1 for use as a tool for research and development of diagnostic of acute dengue infection. Thus, gene

sequences encoding DENV-NS1 isolated in Latin American countries were selected in public databases and aligned to generate a consensus sequence for each virus serotype (DENV1-4). These sequences were then optimized for bacterial expression, synthesized and cloned into prokaryotic expression vectors for ultimately transform Escherichia coli. After bacterial induction and recombinant protein purication, Balb/c mice were immunized and splenocytes were used for development and selection of hybridomas producing mAb specic to DENV NS1. A total of 168 hybridomas generated were selected and stored. These monoclonal antibodies reacted against both recombinant and native (from culture supernatants of DENV-infected C6/36 and serum samples collected from subjects during acute DENV infection) NS1 proteins. Among these, we selected 30 clones to determine the IgG subclass and we are using these monoclonal antibodies for development of immune-assays for dengue diagnosis. In order to select the best mAb pairs for NS1 antigen capture, screening tests are being performed with serum samples that were positive by RT-PCR (for each DENV serotype) and in commercial capture ELISA. Initial results indicate several pairs of monoclonals with potential for diagnostic development. Further tests still are necessary to improve assays sensitivity and specicity. Besides the immune-assays diagnosis these mAb will be used in several studies undertaken in our laboratories.

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EVALUATION OF REAL TIME RT-PCR ASSAY FOR DETECTION AND SEROTYPING OF DENGUE VIRUS

Natália Barbosa do Vale Alves, Bruno Tardelli Diniz Nunes, R.gis Bruni Andriolo, Daniele Barbosa de Almeida Medeiros, Val.ria Lima Carvalho, Pedro Fernandes da Costa Vasconcelos, Ana Cec.lia Ribeiro Cruz.

Instituto Evandro Chagas - Seção de Arbovirologia e Febres Hemorrágicas, Belem- Para, Brazil

Global public health highlights dengue as a major viral disease transmitted by arthropods. Dengue virus species harbors four serotypes (DENV 1, 2, 3, and 4) with increasingly geographic expansion along with its main vector, Aedes aegypti mosquitoes. It was estimated a 30 times increase over the past 50 years, thus threatening almost half of worldwide population living in risk areas for infection. Given its relevance, it is necessary improvements at the health surveillance through specic and sensitive diagnostic methods. To this end, this study evaluated a real time PCR assay (RT-qPCR) for DENV detection developed by the Centers for Disease Control and Prevention (CDC) and approved by the Food and Drug Administration (FDA): the CDC DENV-1-4 Real-Time PCR. Was tested 606 human patient-derived serum, total blood, and organ samples. Out of this total, 207 samples formed a panel with recognized DENV positive, DENV negative, and other viruses non-DENV positive samples, as determined by virus isolation in clone C6/36 mosquito cell line. Using DENV positive controls, the assay presented an efciency of amplication of 103% for DENV-1 (R. 0.99), 101% for DENV-2 (R. 0.98), 98% for DENV-3 (R. 0.98), and 99% for DENV-4 (R. 0.99). Hence, DENV RNA was detected in 100% of DENV positive samples only. The assay sensibility per serotype was 96.67% for DENV-1, 100% for VDEN-2, 95.45% for DENV-3, and 100% for DENV-4. The specicity was 87.85% with lacking of cross-reactivity reactions. In order to evaluate the RT-qPCR efcacy in a diagnostic routine basis, the remaining samples (399) were randomly selected without previous knowledge of virus isolation data. For this, the sensibility was 98.7% and the specicity was 94.1% with positive and negative predictive values of 80 and 99.67%, respectively. The accuracy was 95%. The observed concordance between RT-qPCR and cell culture virus isolation was 94.9% with kappa value of 0.85 (p < 0,0001). Thus, the data here presented for RT-qPCR assay demonstrated excellent values for coecient of correlation (R.) and efciency of amplication for DENV RNA. In addition, the assay was highly sensitive when detecting each virus serotype without cross-reacting reactions. Finally, statistic values observed at the random study demonstrated RT-qPCR as a precise and efcient assay for DENV serotypes detection and identication. In sum, RT-qPCR assay can be suggested as a routine method for a rapid and reliable detection and identication of DENV suspected cases, generating improvements for dengue surveillance.

DP-P06

EVALUATING THE LABORATORY DIAGNOSTIC RESPONSE AGAINST SEVER CASES OF DENGUE INFECTION, 2012-2013, SÃO PAULO STATE, BRAZIL

Mariana Sequetin Cunha, Vivian Regina Silveira, Adriana Maeda, Sarai Joaquim Santos Silva, Renato Pereira de Souza, Iray Maria Rocco, Ivani Bisordi, Akemi Suzuki

Instituto Adolfo Lutz, Sao Paulo, Brazil.

Dengue fever is the most prevalent arthropod-borne viral infection in the world, with an stimated 2.5 billion people living in areas under risk of infection. The virus belongs to the family Flaviviridae, genus Flavivirus, and has four serotypes: DENV-1, DENV-2, DENV-3, and DENV-4. Due to unplanned and uncontrolled urbanization, sewer and waste management systems, substandard housing and water accumulation, which have created ideal conditions for its primary vector Aedes aegypti, dengue fever incidence has increased in the past years. Albeit it has a low mortality rate, dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS), which are more serious manifestations often associated with secondary dengue virus infections may occur, mainly in areas where more than one serotype circulates simultaneously. DHF is characterized by plasma leakage and hemorrhagic diathesis

that is apparent by the time of defervescence typically after ve days of fever. During the period between June 2012 and July 2013, 16.033 dengue diagnosis requests were sent to the Instituto Adolfo Lutz, São Paulo, Brazil, from different municipalities. A total of 378 were death cases with dengue suspicious, and therefore IgM MAC-ELISA assay in-house, real-time PCR (qRT-PCR) and indirect immunouorescense assay (IFA) were performed. Samples used for diagnosis were serum, blood, liver, spleen or lungs. 78 cases were conrmed by at least one of these tests, and one tested positive for Hantavirus IgM. MAC-ELISA tested positive in samples varying from day 1 to 46 after onset of symptoms. qRT-PCR was able to amplify dengue virus from day 1 to 12, while IFA from days 1 to 4. qPCR and IFA demonstrated 5 DENV-1 and 15 DENV-4. Adolfo Lutz Institute is a reference laboratory, and therefore plays an important role on dengue diagnosis and surveillance in Brazil and its differential diagnosis.

DP-P07

GRO-ALPHA, A POTENTIAL DENGUE SEVERITY MARKER

Renato A S Oliveira; Marli Tenório Cordeiro; Patrícia Muniz Mendes Freire de Moura; Paulo Neves Baptista Filho; Ernesto Torres de Azevedo Marques Júnior; Laura Helena Vega Gonzales Gil

Aggeu Magalhães Research Center/Fiocruz. LaViTE, Recife, Brazil

It is believed that the development of dengue hemorrhagic fever (DHF) is the result of a complex interaction between the virus and host immune factors. The mechanisms of dengue pathogenesis is not fully understood, and the identication of biomarkers that identify patients at high risk of developing severe dengue is a major challenge. A wide preliminar screening for relative quantication of 36 circulating cytokines and chemokines levels, namely G-CSF, GM-CSF, IFN- gamma , IL-1beta, IL-1 beta , IL1ra, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12 p70, IL-13, IL-16, IL-17, IL17E, IL-23, IL-27, IL-32 , TNF- alpha , IL-8, RANTES, SDF-1, IP-10, I-TAC, MCP-1, GRO- alpha, I-309, MIP-1, MIP-1 alpha , Serpin E1, C5a, CD40 ligand, MMIF, sICAM-1 and sTREM-1, was evaluated, using serum/plasma of dengue patients at acute phase of infection and health donors. Sequential quantication by ELISA demostrated that GRO-alpha was signicantly increased in DF patients when compared to DHF patients. Our data indicate that we determine the prole of cytokines expression for DF and DHF patients, and GRO-alpha was identied for the rst time as a good prognostic marker.

EPIDEMIOLOGY/PHYLOGENETICSABSTRACTS INVITED SPEAKERS

AN OVERVIEW OF THE CIRCULATION OF DENGUE VIRUS IN SÃO JOSÉ DO RIO PRETO AND IN BRAZIL

Mauricio Lacerda Nogueira

Faculdade de Medicina de São José do Rio Preto (FAMERP), Brazil

Dengue virus (DENV) is a virus of the Flaviviridae family. There are four antigenically distinct DENV serotypes; DENV-1, DENV-2, DENV-3 and DENV-4 and each serotype show phylogenetically distinct genotypes. Over the last 8 years we were sampling and analyzing DENV transmission in São José do Rio Preto (SJRP), São Paulo and comparing with sequences obtained from the rest of the country. In this presentation we will overview the DENV introduction and circulation in Brazil and in Sao Jose do Rio Preto. Using NGS and conventional sequencing we were able to genotype several different linages that circulated in SJRP over the years. Since 2006 SJRP had 3 major outbreaks (linked to DENV-3, DENV-1 and DENV-4) and an endemic DENV-2 circulation. At least 2 different linages of DENV-3 and 2 linages of DENV-1 circulated in the city with differences in their immunogenic potential. Only one linage of DENV2 and DENV-4 were detected. When compared with Brazilian data we could infer the introductions of these viruses in the country, their spatial and temporal distribution and the occurrence of major outbreaks in the country. We can conclude that the phylodinamics of dengue circulation can be much more complex than expected even in a small city, with circulation not only of different serotypes but also different strains. These data provide us more information about the dynamics of DENV circulation and its role in emergence of outbreaks and endemic circulation.

PHYLOGENETIC EVIDENCE FOR THE EMERGENCE OF A NORTH AMERICAN LINEAGE OF DENGUE VIRUS SUBTYPE 1

Jorge L. Munoz-Jordan, Gilberto A. Santiago

Centers for Disease Control and Prevention, Dengue Branch, 1324 Canada Street, San Juan, PR 00920

Dengue, one of the most important re-emerging tropical diseases, is caused by four dengue virus subtypes (DENV-1-4). Transmission in previously non-epidemic areas is documented every year. Frequent international travel probably contributes to the increased distribution of DENV, as evidenced by emergence of new phylogenetic lineages. In 2009-2010, Nuevo Leon, Mexico, a region that had reported only limited dengue cases since 2005, reported 2271 laboratory-positive cases, with a high proportion of DENV-1 identications; and Key West, Florida experienced endemic DENV-1 transmission. Consequently, in 2013 autochthonous transmission of DENV-1 was reported in Hidalgo and Cameron Counties in Texas and in the Martin and Saint Lucie counties in Florida in 2013. These occurrences indicate local DENV transmission in new areas of North America. To assess the genetic relatedness of DENV-1 in North America and understand the increasing endemicity of DENV-1 in this region. We performed sequencing, maximum likelihood, and Bayesian phylogenetic analyses on the envelope gene from 103 DENV-1 isolates from the Americas, including 9 sequences obtained from clinical cases during the 2010 Nuevo Leon outbreak, 8 from Key West, 4 from Martin/St. Lucie counties, and 4 from other counties from cases during the 2009-2013 outbreaks in Florida, and 5 sequences from Southern Texas. We plotted this new dataset together with sequence data of DENV-1 isolates from the last 10 years obtained in Central America and the Caribbean. We identied the emergence of a new DENV-1 lineage currently propagating in North America. This North American lineage emerged from the Central American and Caribbean-South American lineages of the American-African genotype evolving in a short period of time following a northbound transmission trajectory. The recent DENV-1 emergence into non-endemic areas in North America seems to have driven the divergence of new monophyletic lineages associated with outbreaks in Northern Mexico/Southern Texas and Southern Florida. In situ microevolution of these emerging lineages has been identied using genomic sequence data from DENV-1 identied in non-travelers from these areas, conrming the establishment of these lineages in new regions of North America. Current efforts are in place to further understand DENV transmission among residents in continental US.

ABSTRACTS ORAL PRESENTATIONSA NATURALLY OCCURRING SINGLE POINT MUTATION IN E PROTEIN CHANGES THE NEUTRALIZATION PROFILE OF DENV-2 ASIAN 1 VIRUSES

1 1 2 1Magelda Montoya , Chunling Wang , Leah Katzelnick , and Eva Harris1 2Division of Infectious Diseases and Vaccinology, School of Public Health, University of California, Berkeley; Centre for Pathogen Evolution, University of Cambridge, United Kingdom

The four serotypes of dengue virus (DENV1-4) cause the most medically important arthropod-borne viral disease worldwide. Each serotype is comprised of several genotypes. A DENV2 genotype replacement event in Vietnam, where the previously dominant Asian-American (AA) genotype was entirely replaced by the Asian 1 (A1) genotype, was observed during 2003-6. We investigated the possible role of pre-existing immunity in driving clade replacement by generating reporter virus particles (RVPs) representing the two genotypes and analyzing their neutralization proles by patient sera collected in Vietnam prior to (1997-8) and after (2006-7) the replacement event. The Vietnamese 2007 samples neutralized the A1 better than the AA genotype, as did sera from post-secondary DENV2 infections in a Nicaraguan cohort. However, among isolates that shared the ve amino acid changes in charge or side chain between the E genes of A1 and AA, one A1 isolate had a different neutralization prole when analyzed using antigenic cartography with non-human primate antisera. Six A1 isolates were poorly neutralized by both homologous and heterologous antisera, but the outlier A1 was strongly cross-neutralized by DENV1 and DENV3 antisera and had a distinct substitution, K160Q. This mutation was observed in strains isolated at the end of the Vietnam DENV2 A1 epidemic, in a clade that persisted for multiple years. To test if this amino acid substitution determined differences in neutralizing antibody titers with human antisera, the A1 RVP with 160Q was mutated to K. Interestingly, the dominant neutralizing phenotype was lost in the Q160K A1 RVP when tested with Nicaraguan cohort antisera from post-secondary DENV2 infections (1.7-fold reduction, p<0.001), post-primary DENV3 infections (2.4-fold reduction, p<0.001), and post-primary DENV1 infections (1.5-fold reduction, p<0.05). We are analyzing changes in neutralization titer to 160K and 160Q variants by a panel of monoclonal antibodies sensitive to virion structure to determine if mutations at position 160, located in a valley on the surface of E Domain I, alter the global structure of the virus. Thus, our ndings from independent analyses indicate that a clade of viruses circulating for multiple years in Vietnam had a single point mutation in the E protein (K160Q) that signicantly altered polyclonal neutralization, in particular by heterologous antisera.

INCREASING CLINICAL SEVERITY DURING A DENGUE VIRUS TYPE 3 CUBAN OUTBREAK: DEEP SEQUENCING OF EVOLVING VIRAL POPULATIONS

Rosmari Rodriguez-Roche, Marco Vignuzzi, Hervé Blanc, Antonio V. Bordería, Gissel Díaz, Emidalys Santana and Maria G Guzman

Pedro Kouri Tropical Medicine Institute (IPK), Havana, Cuba

Dengue epidemics in Cuba have repeatedly demonstrated a month-to- month increase in clinical severity during secondary infections, expressed by a higher ratio of DHF/DF toward the end of the epidemic. During the DENV-3 epidemic occurred in Havana City in 2001-2002, the severe disease was associated with the sequence DENV-1/DENV-3, whilst the sequence DENV-2/DENV-3 was associated with mild and asymptomatic infections. To determine the role of the virus in the increasing severity, isolates collected at different point times during the epidemic were studied using a deep sequencing approach. A total of 22 full-length DENV-3 sequences were obtained using the Illumina technology and data processed through a computational pipeline, termed ViVan (Viral Variance Analysis), which facilitated the analysis of viral populations. Bayesian phylogenetic analysis revealed that two lineage were circulating in Havana at that time, although both lineage grouped within the genotype III introduced in Latin-America in 1994. The predominant lineage (20 isolates) is close related to strains that circulated in Nicaragua and Brazil, whilst the minor lineage (two isolates) is related to virus that circulated in Venezuela. According to the analysis of consensus sequences relatively few non-synonymous mutations were observed, only one of them was xed during the epidemic period at position 4380 in the NS2B gene. The quasispecies analysis indicated that a signicant minor population present in the isolate collected at the beginning of the epidemic was selected and become predominant at the end of the epidemic. On the other hand, the analysis of RMSD indicated that greater variability occurred at the non-structural genes compared with structural genes, in terms of signicant minor viral population; particularly, toward the end of the epidemic, involving mainly NS2B, NS3 and NS5 genes. Our results support that specic changes in non-structural genes could enhance viral tness in the context of heterotypic antibodies.

CROSS-REACTIVITY TO HETEROLOGOUS DENGUE VIRUS TYPES INCREASES IN THE YEARS FOLLOWING PRIMARY INFECTION AND IS MAINTAINED FOLLOWING SECOND INFECTION IN A PEDIATRIC DENGUE COHORT STUDY IN NICARAGUA

1 2 3 4 2Leah Katzelnick , Magelda Montoya , Lionel Gresh , Angel Balmaseda , Eva Harris1 2Department of Zoology, University of Cambridge, Cambridge, United Kingdom; Division of Infectious Diseases and Vaccinology, School of Public Health, University of

3 4California, Berkeley, CA, USA; Sustainable Sciences Institute, Managua, Nicaragua; Laboratorio Nacional de Virología, Centro Nacional de Diagóstico y Referencia, Ministry of Health, Managua, Nicaragua

The four dengue virus (DENV) serotypes infect an estimated 390 million individuals each year. Following a rst DENV infection, the neutralizing antibody response is thought to become increasingly specic to the infecting serotype, and over time, is expected to remain only to the infecting serotype. After secondary infection, neutralization of all serotypes is thought be relatively balanced and persist over time. However, these hypotheses have not been tested rigorously in a longitudinal cohort. We used reporter viral particles (RVPs) to measure changes in the type-specicity of neutralizing antibody responses to all four DENV serotypes in healthy annual samples from a cohort of Nicaraguan children for 1-6 years after their natural rst, second, and, in some cases, third infections. Post-infection neutralizing titers were ranked from highest to lowest in the year after infection, and fold-difference in neutralization between the best-neutralized serotype and each heterologous serotype was measured every year until subsequent infection. As expected, the trajectories of neutralizing responses after infection varied by individual in magnitude

and degree of cross-reactivity between serotypes. However, when rst-infection neutralizing antibody responses were analyzed as a group, increasing cross-reactivity was observed over time. Specically, the difference between the best and second-best neutralized serotypes decreased over time, with a mean decline of 1.3-fold/year (p<0.01), from an average difference of 8.5-fold in the rst year after infection (p<0.001). This effect remained signicant when serotype, year, and infection outcome were taken into account. Indeed, while the titer to the infecting type did not change signicantly over time, the second-best neutralized serotype and third-best neutralized serotype increased by 1.3 fold/year (p<0.01) and 1.2 fold/year (p<0.05), respectively. When neutralizing antibody responses were analyzed in the same subjects following subsequent second infections, cross-reactivity between serotypes did not change signicantly over time, although the magnitude of titers to all four serotypes decreased over time. We nd that children living in Nicaragua become more cross-reactive to heterologous DENV serotypes over time following rst infection and maintain cross-reactive responses following second infection.

HIGHLIGHTED POSTERSHP22

DENGUE INVASION IN ACRE: IMPORTANCE OF HUMAN MOBILITY ON THE SPREAD OF DISEASES

Raquel Martins Lana, Nildimar Alves Honório, Cláudia Torres Codeço

Fundação Oswaldo Cruz-RJ, Rio de Janeiro, Brazil

Since its reemergence in 1986, dengue fever has spread from the large centers to the peripheries of Brazil. By the end of the 1990's , most states were already affected, except the extremes - Rio Grande do Sul and Santa Catarina in the South region and Acre in the Northern region. This work describes the invasion of dengue in Acre, following its transportation networks since 2001, when the rst autochthonous cases were reported. Since then, dengue has concentrated in Southern Acre, where the capital Rio Branco is located. Cases have been reported annually at this location since then and, in 2009, dengue incidence exceeded 2800 cases per 100,000 inhabitants representing the largest epidemic in the state. Until recently, the northwest region of Acre, Alto Juruá, was dengue free due to the absence of its vector Aedes aegypti, although occasional imported cases were reported. However, investments in infrastructure since the year 2000 are changing the landscape. Commutation within the state increased drastically with the pavement of the BR-364 road, linking Rio Branco to Alto Juruá municipalities and the daily ights between these two regions have intensied; furthermore others conditions such as urbanization, availability of breeding sites and climate, created a scenario with favorable conditions for the spread of Ae. aegypti. In 2014, the rst dengue epidemic in Alto Juruá was reported. To understand the spatial diffusion of dengue and its association with increased human and commodities commutation, we calculated the effective reproductive number of dengue over the years for each municipality of Acre and related time of arrival to accessibility by transport.

HP23

DENGUE SEROPREVALENCE, FORCE OF INFECTION AND AVERAGE AGE OF FIRST INFECTION IN A SCHOOL-BASED COHORT OF CHILDREN, 5 -13 YEARS OF AGE, IN FORTALEZA, BRAZIL

Adrianne Patricia Guignard, Hogea C, Araújo FMC, Coelho ZCB, Colares JK, Feng Y, Innis BL, Coelho ICB

GlaxoSmithKline Vaccines, Belgium

Dengue is endemic in Fortaleza, Northeast Brazil, where periodical outbreaks have occurred since 1986. Dengue virus serotype 4 (DENV-4) was rst isolated in 2011 and associated with a large outbreak in 2012. An urban paediatric school-based cohort study with longitudinal surveillance of febrile illness was initiated in 2011 to estimate the dengue burden and characterize clinical and epidemiological patterns. We describe here the seroprevalence of DENV immunoglobulin G (IgG) antibodies in study participants at enrolment, the estimation of the force of infection (FOI) and the average age of rst infection. This study (NCT01391819) enrolled 2117 children, 5-13 year-olds, from 9 schools. Enrolment took place in three phases: September 2011-January 2012 (N=1253), May-June 2012 (N=394), April-June 2013 (N=470). Sera collected at enrolment were tested for DENV serum antibodies using an indirect commercial IgG enzyme-linked immunosorbent assay (ELISA). Overall seroprevalence was 50.3% (95%CI 48.2-52.5) and increased from 32.6% (95%CI 27.4-38.1) in the 5-year-olds to 71.4% (95%CI 62.7-79.1) in the 13-year-olds. Seroprevalence was lower in the 5-9 year-olds enrolled in 2011 compared to same-age children enrolled in 2013: 33.3% (95%CI 29.7-37.0) versus 46.5% (95%CI 41.2-51.8) (p<0.0001). The FOI based on seroprevalence was estimated with an exponentially damped linear catalytic model previously used for dengue in Brazil (Amaku, 2012). The model-estimated average age of rst infection was 9 years based on pooled seroprevalence data between 2011 and 2013, corresponding to an average FOI (rate of primary infection) of 11% per year. These preliminary results indicate that dengue transmission in children of Fortaleza is high and suggest high attack rates in 2012 when DENV-4 was introduced in a fully susceptible population. Fitting the model separately on seroprevalence data for children enrolled in 2011 and in 2012-2013 resulted in a subsequent shift downwards in the age of rst infection following the 2012 DENV-4 outbreak. Funding: GlaxoSmithKline Biologicals SA

HP24

THE PHYLOGENETIC RELATIONSHIPS OF DENGUE VIRUS TYPE 2 CIRCULATING IN COLOMBIA BASED ON COMPLETE GENOME 1,2 1,3*Cinthy Jimenez-Silva , Raquel E Ocazionez

1 2 3Centro de Investigaciones en Enfermedades Tropicales (CINTROP), Laboratorio de Sistemática y Biogeografía de la Escuela de Biología, Grupo de Investigación en Enfermedades

Infecciosas y Metabólicas (GINEM), Universidad Industrial de Santander, Bucaramanga, Colombia

Introduction. Colombia is one of the most affected American countries for Dengue viruses. In Santander, the dengue virus is endemic and this departament is one of the principal hot zone in outbreaks during the last 20 years. The serotype 2 has been caused the mayority of hemorrhagic dengue cases in Santander which has been related with secondary infections. However, the phylogenetic relationships of DENV-2 strains that has been circulated in Santander and Norte de Santander are unknown. Objective.To describe phylogenetic relationships of Dengue virus type 2 circulating in Colombia based on Complete Genome. Methods. In this study, we present a robust reconstruction of the phylogenetic relationships of DENV- 2 based on the complete genome of all available complete genome sequences from the Genbank, which included sixteen sequences of Colombia isolated in Antioquia, Guaviare, Norte de Santander and Santander from 1986 to 2007. We reconstructed the phylogenetic trees using the maximum-likelihood and the Bayesian analysis. Results. Our results showed that two genotypes (American and American/Asian) circulated in Colombia in different periods of time. These genotypes were subdivided in VI lineages. The lineage III and VI have circulated in Norte de Santander and Santander departments since 1998 to 2007. The Colombian sequence in the lineage III was related with Venezuela, Puerto Rico and Brazil strains from 90's. The Colombian lineage IV were closely related with Venezuelan isolates from 1996 to 2008. At the same time, this clade was subdivided into ve groups that evidence a temporal structure. Sequences of Colombia isolated in Guaviare in 2005 and Antioquia in 2004 were included in this lineage, for this reason, we supposed that this lineage was introduced since Venezuela through Norte de Santander / Santander and it has been circulated in different areas of Colombia since 1998 to 2008. Conclusion. Two genotypes of DENV-2 were presented in Colombia during 1986 to 2007. The phylogenetic relationships of DENV-2 was associated to changes in the virus predominance. Acknowledgments- This research was carried out thanks to nancial support received from the Universidad Industrial de Santander, Bucaramanga, Colombia (Grant # 5658)

HP25

THE DENGUE VACCINE INITIATIVE PROJECT: SERO-PREVALENCE STUDY IN CHILDREN AND ADULTS OF SANTA CRUZ COMUNA OF MEDELLIN 1 1 2 2 2 3Mabel Carabali , Jacqueline Lim , Carolina Velez , Andrea Trujillo-Correa , Kang Sung-Lee, Iván D. Velez , Jorge Osorio

1 2Dengue Vaccine Initiative, International Vaccine Institute, Seoul, Korea; Programa de Estudio y Control de Enfermedades Tropicales (PECET), Universidad de 3Antioquia, Medellín, Colombia; and Department of Pathobiological Sciences, University of Wisconsin

Dengue is a major public health problem in Colombia. During the last 10 years, there has been a signicant increase in the number cases of dengue, with circulation of all four serotypes and epidemic waves occurring every 3-4 years. Dengue Vaccine Initiative (DVI) conducted a serological survey to determine the true burden of dengue due to inapparent infections in the Santa Cruz comuna of Medellin, Colombia. The serosurvey included two years of follow up with ve bleedings, with six-month intervals from Nov. 2011 to Feb. 2014. More than 2000 randomly selected residents were enrolled in the study. To estimate the incidence and the age-specic sero-conversion rates on samples that showed a rise in IgG antibody titers by IgG Indirect ELISA. Through 5 bleedings, replacements were made to keep an open cohort of 2000 randomly selected residents. The sero-prevalence of IgG antibodies in each sero-survey were: 59.02% in S1; 63.9% in S2; 64.9% in S3; 64.7% in S4, and 64.7% in S5. The overall sero-conversion rate during the rst year was of 4.8%. The highest sero-conversion rate between S1 and S3 was found among children 5-9 years of age (7.3%), followed by young adults between 35-44 years-of-age (6.9%) and children 1-4 years old (5.6%). From the preliminary PRNT performed on a subset of sero-converted samples (n=48), 83.7% showed an immune response against all 4 serotypes, with DENV-3 (24.5%) and DENV-1 (16.3%) as the predominant serotypes. These sero-prevalence results may indicate an elevated dengue transmission in Santa Cruz and a higher risk for severe forms with increase in age. Results of further analyses of the immunological status of individuals in this geographic area will be presented at the conference.

POSTERS SESSION EPIDEMIOLOGY/PHYLOGENETICSEP-P01

INCREASE OF DENGUE ASSOCIATED WITH SOCIAL FACTORS: A SPATIAL-TEMPORAL EVALUATION

Marco Aurelio Horta; Cristina Maria Souza Catita; Aldo Pacheco Ferreira

Fiocruz - Fundação Oswaldo Cruz, Brazil

Background. Many social indicators have been proposed as potential determinants or risk factors of dengue fever (DF) transmission in several recent studies. Along with these social factors, geospatial modeling has provided an added dimension to better understanding and interpreting the complex eco-social-health factors involved in dengue transmission. However, the dengue transmission ecosystem is a complex combination not only of these social factors, which represent economic, sanitation and presence of high-risk environmental locations but also the implementation of preventative dengue control measures and thus human behavior. Methodology. We used spatial analysis to differentiate areas for the years 2008 and 2009 of increasing incidence of dengue fever (Group A) and areas of decreasing incidence (Group B) in the city of Coronel Fabriciano, Brazil. For each district we obtained information regarding patient residence (latitude and longitude), date of onset of symptoms, and related social indicators at the district level related to income, dependents, sewage and water systems, and education. The two regions were

found to be statistically different in LISA and one-way ANOVA test and varied signicantly in various social factors: percentage of households with garbage collection (F=5.36; p=0.02), dependency ratio (F=3.80; p=0.05), poverty index (F=28.46; p<0.001) and life condition index (F=8.95; p=0.003). The districts in group B showed higher values of population density, poverty index and dependency ratio, with less children and a lower dependency ratio. Conclusions. While social factors were found to be signicantly associated with either increasing or decreasing trends of dengue incidence, our results indicate several implications for the human behavior associated with preventative dengue control measures as those indices which indicated or factors associated with a “caretaker's presence” offered a factor of protection. Further renement of geospatial modeling may be useful to better capture the complex nature of dengue transmission.

EP-P02

IDENTIFICATION AND ANALYSIS OF DENGUE VIRUS GENETIC VARIATION AND ITS RELATIONSHIP WITH CLINICAL MANIFESTATIONS IN PATIENTS FROM AN ENDEMIC AND NON-ENDEMIC ZONES IN MEXICO

Galán-Huerta Kame Alberto; Loroño-Pino María Alba; Sauceda-Garza Jessica; Muñoz-Jordan Jorge; Santiago Gilberto Antonio; Pérez-Villarreal Jesús Zacarías; Ramos-Jiménez Javier; Rivas-Estilla Ana María

Faculty of Medicine - Universidad Autónoma de Nuevo León, Mexico

Objective: Our aim was to investigate if there is a relationship between clinical manifestations and DENV genetic variation in strains isolated from infected subjects from an endemic and non-endemic zone in Mexico. Methods: The study was conducted at the southern state of Yucatan and the northern state of Nuevo Leon in Mexico, from October to November 2012. Dengue infection diagnosis was made according to the new WHO guidelines and serum samples were obtained. Viral RNA was detected by RT-PCR to conrm DENV infection. Further, DENV viruses were isolated, amplied in C6/36 cell culture and E gene was sequenced. The relationship between clinical manifestations and E gene mutations were investigated for each strain isolated. Phylogeny reconstruction was inferred in MEGA5 using maximum likelihood approach. Results: Twenty-eight patients were analyzed:12 from Yucatan and 16 from Nuevo Leon. Clinical manifestations were different among the patients of both regions. The circulating viruses in both states were DENV-1 and DENV-2, with different frequencies. Three E gene sequences were obtained from Yucatan and 7 from Nuevo Leon. Twenty mutations were found among the DENV-1 E gene sequences and 11 at DENV-2. When the sequences were compared with themselves, two non-synonymous substitutions were found. One was at E475 (L�F) in DENV-1 and the other at E344 (N�Y) in DENV-2. DENV-1 viruses from both states grouped in the same clade, separated from 2011 viruses. Nuevo Leon's DENV-2 grouped at the same clade with the DENV-2 isolated in 2011. Conclusions: We found no relationship between clinical manifestations and E gene mutations in the analyzed patients. Due to the homology between DENV-1 from Nuevo Leon and Yucatan, we can infer they had the same origin. There was a DENV-1 reintroduction in Nuevo Leon and Yucatan in 2012 and DENV-2 is circulating in Nuevo Leon since 2011.

EP-P03

DESCRIPTIVE EPIDEMIOLOGY OF DENGUE TRANSMISSION IN A MEDIUM-SIZED CITY FROM BRAZIL, 2008 - 2012

Aline Chimello Ferreira, Francisco Chiaravalloti-Neto, Adriano Mondini

Faculdade de Ciências Farmacêuticas - Universidade Estadual Paulista, Brazil

Dengue has been a public health problem in tropical regions for decades but autochthonous transmission has now been reported in countries like USA, France and Croatia. In 2013, 2.35 million dengue cases were reported in the Americas and Brazil alone was responsible for more than 50%. The country has been presenting outbreaks for more than three decades and important lessons can be learned. Thus, a detailed analysis of dengue transmission in Araraquara, a medium-sized city at the central portion of São Paulo, which is a critical area for dengue transmission, may clarify transmission patterns and entail effective control measures. Dengue has been circulating in the city since 1990s at low incidences. However, the number of cases has increased in recent years and we will be describing the scenario of ve years of transmission herein. Ofcial data on dengue reports from 2008 to 2012 were recovered from the Information System on Diseases of Compulsory Declaration. Data from 5,253 reported cases were analyzed. The majority occurred at the rst ve months of the year - January to May - which is nal portion of the rainy season. Severe dengue or dengue with complications was a rare event in the city. The incidence in Caucasians (78%) was higher than in other ethnic groups, a pattern that has been described worldwide. Another important observation is that dengue transmission has become endemic in the city, with cases being ofcially reported in all months of the year, with the exception of 2009, which was atypical in the whole state of São Paulo. This is part of an ongoing project that is also focused on classical and spatial epidemiology involving a survey of risk factors such as socioeconomic and climatic variables. Financial Support: Fapesp grant 2013/02338-9.

EP-P04

CIRCULATION OF DENV-4 IN ARGENTINA" THIS WORK CHARACTERIZES THE DENV-4 STRAINS WITH AUTOCHTHONOUS CIRCULATION IN ARGENTINA, FROM 2010 TO THE PRESENT

Luppo VC, Morales MA, Fabbri CM, Levis S and Enría D.

Instituto Nacional de Enfermedades Virales Humanas, "Dr. Julio I. Maiztegui", INEVH, Argentina

During the beginning of 2010, circulation of DENV-4 was conrmed in Rosario, Santa Fe, the virus was isolated from the serum of a woman without history of recent travel, representing the southernmost detection of this virus in America. Epidemiological research also identied a group of 4 people with specic IgM and IgG antibodies to dengue, one of which had a history of travel to Colombia and his home was near the work site of the index case. In 2013 were identied by molecular

techniques in provincial laboratories of the Laboratory Network for the diagnosis of Dengue and other Arboviruses, 11 strains of DENV-4, which were later conrmed by the National Reference Center for the diagnosis of dengue and other Arbovirus, INEVH . 8 strains were amplied, obteining amplicons in 3 of them (Salta 2, Córdoba 1). In 2014 indigenous circulation of this serotype was detected in the province of Salta. This work characterizes the DENV-4 strains with autochthonous circulation in Argentina, from 2010 to the present. In the serum samples the viral RNA extraction was performed using a commercial kit (QIAGEN) and serotype was conrmed by RT-PCR , qRT-PCR and virus isolation. The complet gene E (envelope protein) was amplied and the obtained sequence was compared with other strains of different genotypes and geographic areas of DENV-4 virus in the Genebank. Phylogenetic analysis shows that the circulating strains of DENV-4 in Argentina grouped with genotype II strains, with a nucleotide difference of 1% in relation to the nearest American strains in the phylogenetic tree obtained. Only genotypes I and II have been detected in America. This shows an expansion, in recent years, the circulation of this serotype in our territory, alerting the possibility of future epidemics in the region due to the high availability of susceptible population.

EP-P05

VIRUS SURVEILLANCE IN THE SERUM OF PATIENTS WITH CLINICAL DIAGNOSIS OF DENGUE FROM A MEDIUM-SIZED CITY OF RONDONIA STATE, BRAZIL

Walquer Vinicius Esteves Gonçalves Pereira, Arianne Fagotti, Felipe Guioti, Aline Chimello Ferreira, Adriano Mondini

Universidade Estadual Paulista "Julio Mesquita Filho", Brazil

The infection by any of the four serotypes of the dengue virus (DENV 1-4) can be asymptomatic orproduce a disease that ranges from a mild febrile illness to life threatening hemorrhagicmanifestations. In Brazil, the majority of dengue disease diagnostics are obtained by serology andclinical and epidemiological criteria. Virus isolation, identication of DENV by polymerase chainreaction or the detection of the nonstructural protein 1 are usually performed in reference laboratories for a limited number of cases. No further tests to detect other arthropod borneviruses (arboviruses) are performed in febrile patients. In our study, we are testing samples withclinical diagnostic of dengue from a medium sized city from the west part of Brazil for DENV 1-4and other aviviruses, along with orthobunyaviruses and alphaviruses. We collected bloodsamples from febrile patients that had clinical diagnostic of dengue from August 2013 to January2014 in Ji-Paraná (Rondonia), which is allegedly an area with the circulation of severalarboviruses. Viral RNA was extracted and a Multiplex-Nested-RT-PCR with genus-specic primersto detect members of the Flavivirus, Alphavirus and Orthobunyavirus genera and species-specic primers to identify DENV 1-4, yellow fever virus, mayaro virus, oropouche virus and seven otherviruses was performed. We were able to collect 103 blood samples for the study. So far, we havetested 12 samples and we could not detect DENV or any other arboviruses. However, 89% of thesamples still remain untested. It is undeniably important to provide differential diagnostic forfebrile illnesses, not only for a better management of the patients but also to understand the epidemiology of the viruses that may be circulating in a certain location and the control measuresthat should be taken to control their spread.

EP-P06

FIRST DENGUE HAEMORRHAGIC FEVER EPIDEMIC IN THE AMERICAS, 1981: INSIGHTS INTO THE CAUSATIVE AGENT

Rosmari Rodriguez-Roche, Yoandri Hinojosa and Maria G. Guzman

Pedro Kouri Tropical Medicine Institute (IPK), Havana, Cuba

Historical records describe a disease in North America that clinically resembled dengue haemorrhagic fever during the latter part of the Slave Trading period. However, the dengue epidemic that occurred in Cuba in 1981 was the rst laboratory conrmed and clinically diagnosed outbreak of dengue haemorrhagic fever in the Americas. This totally unexpected and explosive epidemic was characterised by rapid dispersal of the virus and the accompanying disease, throughout Cuba, with extraordinarily high transmission rates. At that time, the predicted source of the dengue type 2 strain isolated during this epidemic was considered controversial, partly because of the limited sequence data and partly because the origin of the virus appeared to be Southern Asia. Bayesian phylogenetic analysis using genomic-length sequences revealed the relatively close genetic relatedness of the Cuban strains isolated in 1981 with the prototype NGC Asian strain isolated in 1944, all of which grouped within the Asian 2 genotype. Indeed, the 1981 strains are very distant from strains grouped in the Asian/American genotype, which have been circulating in the Americas since 1983 to date, and have displaced the American genotype. Evidence of viral evolution during the 1981 epidemic is presented, including non-conserved amino acid substitutions in both structural and non-structural proteins. In addition, the Cuban isolates exhibited particular amino acid substitutions that differentiate them from the prototype strain as well as from dengue type 2 strains isolated globally. Possible mechanisms of viral evolution and its implications for disease severity are discussed.

EP-P07

ECONOMIC IMPACT OF DENGUE EPISODE: MULTICENTER STUDY ACROSS FOUR BRAZILIAN REGIONS

Martelli CMT, Oliveira CS, Mendes MCO, Bahia LR, Parente MPPD, Cortes FJM, Siqueira Filha NT, Zara AL, Siqueira Junior JB

Centro de Pesquisa Aggeu Magalhães CPqAM - Fiocruz PE, Brazil

Objective: to evaluate the economic burden of dengue from public payer and societal perspectives in Brazil. Methods: investigation was designed as a multicenter cost study. Suspected dengue cases were recruited during 2012/2013 in four endemic regions (city): Midwest (Goiania), Southeast (Belo Horizonte and Rio de Janeiro), Northeast (Teresina and Recife), North (Belem). Participants were suspected or laboratory conrmed dengue cases, treated in ambulatory or hospital settings (private and public sectors). A household interview was scheduled 20-30 days after the onset of clinical symptoms. The time horizon was one-year

considering dengue seasonality. We calculated the direct cost, public payer perspective and direct/indirect costs for societal perspective. Estimation of annual national dengue costs took into account cases reported by notication system (SINAN) with possible under-reporting of passive surveillance. Results: we screened 2,223 patients and 2,097 (94.3%) symptomatic dengue cases were included. The majority of patients were adults. 1,661 cases were treated in ambulatory and 436 cases in hospitals. In the ambulatory cohort, the average number of medical visits ranged from 1.2 to 4.2. A higher number of medical visits were recorded among inpatients (3.2 to 5.0). For the public payer perspective, estimated cost per case was USD 43 (95% CI: 39-47) ambulatory and USD 237 (95% CI: 202-248) hospital. Dengue illness in Brazil was estimated to cost USD 126 million (95% CI: 112-135), ambulatory and hospitalized cases considering the reported cases (SINAN). Outpatients cost account for 62% of the total costs. For the societal perspective, the estimated cost per ambulatory case was USD 163 (95% CI: 142-169) and USD 465 (95% CI: 407-591) hospital. Dengue illness was estimated to cost USD 389 million (95% CI: 339-425), ambulatory and hospitalized cases. Our results show evidence of substantial economic impact in Brazil. We have provided a timely economic evaluation of dengue. Financial support: Sano.

EP-P08

CHARACTERIZATION OF A DENGUE VIRUS TYPE 4 OUTBREAK IN SOUTH-CENTRAL MATO GROSSO, BRAZIL1 2 3,4,5 3,4,5 3,4,5 2 6Nayara Zuchi , Steven G. Widen , Rubing Chen , Hilda Guzman , Letícia B. S. Heinen1, Sandra V. Mayer , Thomas G. Wood , Marcelo A. M. dos Santos , Jonathan

7 3,4,5 3,4,5E. Allen , Robert B. Tesh , Renata Dezengrini-Slhessarenko1, Nikos Vasilakis1 2Post-Graduation Program in Health Sciences, Medicine Faculty, Federal University of Mato Grosso, Cuiabá, Mato Grosso, Brazil; Department of Biochemistry and

3 4 5Molecular Biology, Center for Biodefense and Emerging Infectious Diseases and Department of Pathology, Center for Tropical Diseases, and Institute for Human 6Infections and Immunity, The University of Texas Medical Branch, Galveston TX, USA; MT-Laboratory, State Secretary of Health, Cuiabá, Mato Grosso, Brazil;

7Computing Application & Research Department, Lawrence Livermore National Laboratory, Livermore CA, USA

Dengue viruses (DENV) are by far the most important arboviral pathogens in the tropics globally, putting at risk of infection nearly a third of the global human population.. In the current study we characterize the phylogeny and intrahost variation of 26 isolates of dengue virus type 4 (DENV-4) from acute serum samples obtained during an outbreak in South-Central Mato Grosso State (MT), Brazil, in 2012. All 26 isolates located within genotype II in two distinct lineages forming a monophyletic clade. Further conrmation of the co-circulation of two distinct lineages is obtained by analysis of the intrahost virus variation in the acute serum samples. Based on our phylogenetic analyses, there are 6 independent introductions of DENV-4 in Brazil, presumably from Venezuela, Puerto Rico, China, and Southeast Asia. The DENV-4 isolates of the 2012 outbreak in South-Central Mato Grosso State were closely related with two 2010 isolates from the geographically close regions of Amazonas and Roraima and were closely related with strains sampled from Venezuela 2007, indicating the potential origin introduction. The extent and severity of the 2012 DENV-4 outbreak is likely attributed to the lack of immunity in the population.

EP-P09

TWENTY-FIVE YEARS OF DENV-1 IN BRAZIL: MOLECULAR EPIDEMIOLOGY OF STRAINS ISOLATED FROM 1986 TO 2011

Fernanda de Bruycker Nogueira¹, Flavia Barreto dos Santos¹, Nieli Rodrigues da Costa Faria¹, Jaqueline Bastos Santos Simões¹, Márcia Gonçalves de Castro², Priscila Conrado Guerra Nunes¹, Simone Alves Sampaio¹, Ana Maria Bispo de Filippis¹, Rita Maria Ribeiro Nogueira¹

¹Laboratory of Flavivirus, ²Laboratory Transmitters Hematozoa, Oswaldo Cruz Institute-FIOCRUZ, Rio de Janeiro, RJ, Brazil

E-mail: nandanog@ioc.ocruz.br

This study presents the phylogeny and molecular characterization based on envelope gene (E) analysis of DENV-1 (n=48) isolated during epidemics occurred since this serotype introduction in 1986 to 2011. From those strains, six were fully sequenced (coding region) and possible genomic recombination events were analyzed. The results of the phylogenetic analysis based on the E gene showed that DENV-1 isolates from the states of RJ, ES, MG, MS, AL, CE, PI and RN belong to genotype V (American/Africa), but grouping in distinct clades. Three groups were identied, one dating from 1986 to 2002 (lineage 1a), a second group isolated between 2009 and 2011 and a representative strain isolated in 2002 (lineage 2) and a group of strains isolated in 2010 and 2011 (lineage 1b). Lineage 2 has a high bootstrap support ensuring their separation from the other lineages. The lineages 1a and 1b, despite in distinct branches, were characterized in a same group supported by a bootstrap of 75%. Furthermore, the lineages 1a and 1b were more closely related to the American strains, while lineage 2 to the Asian strains. Amino acids (aa) substitutions were observed in the ectodomains I and III of the E protein and were associated to the lineages separation. A substitution on E297 differentiated the lineage 1a from the lineages 1b and 2. Changes observed in E338, E394 (ectodomain III), E428 and E430 (stem region) differentiated lineages 1a, 1b and 2. The complete coding region analysis showed a large number of specic aa changes (n=82) distributed among the six strains studied, however lineage 2 presented approximately 50% of the total detected. With the exception of the C gene, all the others genes analyzed allowed the DENV-1 classication into the distinct genotypes. However, only the E gene and the entire coding region were able to group those into the distinct lineages. No recombinant event was detected but a sample belonging to lineage 1a was closely related to the known recombinant strain AF513110/BR/2001.

EP-P10

EVIDENCES OF RECENT INTRODUCTION OF AFRICAN LINEAGES OF DENGUE 1 VIRUS IN BRAZIL

Adriana Yurika Maeda, Iray Maria Rocco, Ivani Bisordi, Akemi Suzuki, Vivian Regina Silveira,

Margarida Maria G. Montoro, Mariana Sequetin Cunha, Renato Pereira de Souza, Instituto Adolfo Lutz, Brazil

DENV is a RNA virus with a genome of approximately 11 Kb of the family Flaviviridae, genus Flavivirus with four related serotypes of Dengue (DENV 1-4). Each serotype presents its own range of genotypes. The present report describes the phylogenetic relationship of DENV-1 strains isolated in Sªo Paulo State, in 2010. DENV-1 virus was isolated from ve patients samples with suspected of Dengue infection, by inoculation into cell culture lineage Aedes albopictus C6/36 and identication by indirect immunouorescence. A fragment of 953 bp from the Envelope gene was amplied by one step RT-PCR for all strains. The fragments were sequenced and aligned with representative sequences of the genotypes of DENV-1, retrieved from GenBank. A Bayesian inference method applying a relaxed molecular clock model, available in the software BEAST v. 1.6.2, was used to estimate phylogenetic relationship between the sequences. All available data indicates that the DENV-1 strains analyzed in this study belong to two different clades or lineages in the genotype V. The rst lineage (Clade A) represents an older introduction of this genotype, with the studied strains representing the continuous circulation of a virus previously encountered in past epidemics of 2007 to 2009. The second lineage ( Clade B) is formed by samples that became associated with a lineage detected only after 2010. This lineage is the same associated with an outbreak of the disease detected rst in Reunion Islands in the Indic Ocean moving to East Africa. This relationship suggested that this lineage was probably introduced in Brazil after the African epidemics. Studies with a relaxed molecular clock model were performed pointing to a recent differentiation, within the last 300 years for DENV-1 and a relatively greater diversity of genotypes in the last 30 years. It is also indicated that the Clade B circulated previously in Africa and Reunion Islands before its diversication in Brazil.

EP-P11

PHYLOGEOGRAPHIC ANALYSIS OF PATTERNS OF INTERCHANGE OF DENGUE 3 VIRUS IN THE SOUTH AMERICAN COUNTRIES

Renato Pereira de Souza, Adriana Yurika Maeda, Iray Maria Rocco, Fernanda Gisele Silva, Sarai Joaquim dos Santos Silva, Ana Loecia R. Oliveira, Juliana Silva Nogueira, Ivani Bisordi

Instituto Adolfo Lutz, Brazil

DENV is a RNA Flavivirus with four related serotypes (DENV 1-4). Each serotype presents its own range of genotypes. The present report describes the patterns of interchange of Dengue 3 Virus in South America by means of a discrete phylogeograpic analysis. DENV-3 virus was isolated from 07 patients from Sªo Paulo State, in 2010, by inoculation into cell culture lineage Aedes albopictus C6/36 and identication by indirect immunouorescence. A fragment of 953 bp from the Envelope gene was amplied by one step RT-PCR for all strains. The fragments were sequenced and aligned with 60 representative sequences of the genotypes of DENV-3, retrieved from GenBank representing 15 countries. Bayesian phylogenetic reconstructions were performed using Markov chain Monte Carlo analysis implemented by BEAST version 1.6.2. The internal nodes were inferred under a GTR model with Gamma-distributed rate variation ( ) and a proportion of invariable sites (I), using a relaxed ( uncorrelated lognormal) molecular clock. Four independent MCMC runs of four chains each were run for 10 million generations, performing also a discrete phylogeographic analysis. All available data indicates that the DENV-3 strains analyzed in this study belong to tree different clades forming a monophyletic group originally introduced in the Caribbean region circa 1964-1968. From the Caribbean DENV-3 reached Central and South America at the same period of time. The analysis suggests that the virus reached South America via the Andean countries and Venezuela. After that the virus were introduced in Brazil, where undergone an important evolutionary differentiation, that accelerated its spreading throughout South America. The data suggests the existence of two exchange routes, one between Brazil, Peru, Bolivia and Paraguay and other linking Venezuela and the northern portion of South America with the Caribbean.

EP-P12

ISOLATION OF DENV STRAINS FROM INFECTED PATIENTS IN NORTHEAST MEXICO AND FURTHER EVALUATION OF ITS CAPACITY TO MODULATE STAT-1 EXPRESSION ON HUH-7 INFECTED CELL LINE

Arellanos-Soto Daniel, Ramos-Jiménez Javier, Fernández-Salas Ildefonso, Del Ángel-Núñez Rosa María, Rivas-Estilla Ana Maria

Universidad Autonoma de Nuevo Leon, Mexico

INTRODUCTION: Replication of DENV in patients seems to be inuenced by the ability of the infecting strain to modulate the host's signaling pathway required to activate antiviral response of IFN-type I, then its important to know modulation of genes like STAT-1, in response to infection with circulating DENV-strains. OBJECTIVE: To evaluate the levels of STAT-1 in Huh-7 cell-line, infected with clinical isolates of DENV circulating in Mexico from 2010-2014. METHODS: DENV-NS1 positive sera from subjects from Nuevo Leon were evaluated by qPCR to identify serotypes and then virus were isolated and amplied in C6/36 cells. The infected cultures were monitored by microscopy (7-14 days) and RT-PCR and then titrated in BHK-21 cells. Further, Huh-7 cells were infected (moi 0.1) for 36h. Afterwards cells were treated with IFN(1000UI/mL) before total protein extraction. STAT-1 expression was evaluated by Western-Blot. RESULTS: From 1079 DENV-NS1 positive sera, 13 DENV were isolated: 6 (46%) DENV-1, 6 (46%) DENV-2 and 1(8%) DENV-4. Viral strains isolated from patients had no co-infections or contamination with different serotypes. No advantages related to infecting serotype, Ct or NS1 levels were observed in production of viral particles. Induction of CPE on Huh-7 cells infected with the clinical isolates was similar regardless infecting serotype (granulation, vacuolation, multikaryons, lysis). After IFN-1 treatment, most of the strains negatively modulated the production of STAT- 1 on Huh-7 cells. There was a differential STAT-1 expression: sample 6870 (30%), 692 (7.2%), 694 (34.7 %) and sample 696 (35.4%). Meanwhile strain 4904 did not reduce the production of STAT-1 (112 %). CONCLUSION: Infection of Huh-7 cells with viral isolates from mexican patients decreased levels of STAT-1, coinciding with previous reports, however there is no information on the effect of Mexican-circulating DENV-strains from patients. This is the rst study of IFN modulation response using Mexican dengue viral strains.

EP-P13

DIFFERENT LINEAGES OF DENV-1 CIRCULATING SIMULTANEOUSLY IN THE AMERICAS DURING 2010-2011.

Díaz Y, Brenner H, Mamani E, Vázquez C, Regato M, Nina A, Méndez J, Franco L.

Instituto Conmemorativo Gorgas de Estudios de la Salud. Panama

DENV is a RNA Flavivirus with four related serotypes (DENV 1-4). Each serotype presents its own range of genotypes. The present report describes the patterns of interchange of Dengue 3 Virus inSouth America by means of a discrete phylogeograpic analysis. DENV-3 virus was isolated from 07patients from Sªo Paulo State, in 2010, by inoculation into cell culture lineage Aedes albopictus C6/36and identication by indirect immunouorescence. A fragment of 953 bp from the Envelope gene wasamplied by one step RT-PCR for all strains. The fragments were sequenced and aligned with 60representative sequences of the genotypes of DENV-3, retrieved from GenBank representing 15countries. Bayesian phylogenetic reconstructions were performed using Markov chain Monte Carloanalysis implemented by BEAST version 1.6.2. The internal nodes were inferred under a GTR modelwith Gamma-distributed rate variation ( ) and a proportion of invariable sites (I), using a relaxed (uncorrelated lognormal) molecular clock. Four independent MCMC runs of four chains each were runfor 10 millions generations, performing also a discrete phylogeographic analysis. All available dataindicates that the DENV-3 strains analyzed in this study belong to tree different clades forming amonophyletic group originally introduced in the Caribbean region circa 1964-1968. From the Caribbean DENV-3 reached Central and South America at the same period of time. The analysis suggests that thevirus reached South America via the Andean countries and Venezuela. After that the virus were introduced in Brazil, where undergone an important evolutionary differentiation, that accelerated itsspreading throughout South America. The data suggests the existence of two exchange routes, one between Brazil, Peru, Bolivia and Paraguay and other linking Venezuela and the northern portion of South America with the Caribbean.

EP-P14

CHARACTERIZATION OF A DENGUE VIRUS TYPE 4 OUTBREAK IN SOUTH-CENTRAL MATO GROSSO, BRAZIL1 2 3,4,5 3,4,5 1 3,4,5 2 6Nayara Zuchi , Steven G. Widen , Rubing Chen , Hilda Guzman , Letícia B. S. Heinen , Sandra V. Mayer , Thomas G. Wood , Marcelo A. M. dos Santos , Jonathan E.

7 3,4,5 1 3,4,5Allen , Robert B. Tesh , Renata Dezengrini-Slhessarenko , Nikos Vasilakis1 2Post-Graduation Program in Health Sciences, Medicine Faculty, Federal University of Mato Grosso, Cuiabá, Mato Grosso, Brazil; Department of Biochemistry and

3 5Molecular Biology, Center for Biodefense and Emerging Infectious Diseases and Department of Pathology, 4Center for Tropical Diseases, and Institute for Human 6Infections and Immunity, The University of Texas Medical Branch, Galveston TX, USA; MT-Laboratory, State Secretary of Health, Cuiabá, Mato Grosso, Brazil;

7Computing Application & Research Department, Lawrence Livermore National Laboratory, Livermore CA, USA

Dengue viruses (DENV) are by far the most important arboviral pathogens in the tropics globally, putting at risk of infection nearly a third of the global human population.. In the current study we characterize the phylogeny and intrahost variation of 26 isolates of dengue virus type 4 (DENV-4) from acute serum samples obtained during an outbreak in South-Central Mato Grosso State (MT), Brazil, in 2012. All 26 isolates located within genotype II in two distinct lineages forming a monophyletic clade. Further conrmation of the co-circulation of two distinct lineages is obtained by analysis of the intrahost virus variation in the acute serum samples. Based on our phylogenetic analyses, there are 6 independent introductions of DENV-4 in Brazil, presumably from Venezuela, Puerto Rico, China, and Southeast Asia. The DENV-4 isolates of the 2012 outbreak in South-Central Mato Grosso State were closely related with two 2010 isolates from the geographically close regions of Amazonas and Roraima and were closely related with strains sampled from Venezuela 2007, indicating the potential origin introduction. The extent and severity of the 2012 DENV-4 outbreak is likely attributed to the lack of immunity in the population.

EP-P15

DIFFERENT LINEAGES OF DENV-1 CIRCULATING SIMULTANEOUSLY IN THE AMERICAS DURING 2010-20111 2 3,4 5 6 7 8 9Yamilka Díaz , Hebleen Brenner , Enrique Mamani , Cinthia Vázquez , Mary Regato , Aleida Nina , Jairo Méndez , Leticia Franco

1 2 3 4 5Instituto Conmemorativo Gorgas de Estudios de la Salud-Panamá. INCIENSA-Costa Rica, Universidad San Marcos-Perú. Instituto Nacional de Salud-Perú, Laboratorio 6 7 8 9Central de Salud Pública-Paraguay, Instituto Leopoldo Izquieta-Guayaquil-Ecuador, INCIENSA-Bolivia. Instituto Nacional de Salud-Colombia. Centro Nacional de

Microbiología-Instituto Carlos III-España.

Dengue is a disease caused by one of the four serotypes of dengue virus (DENV1 to 4). Since 1980 the disease has broadly expanded throughout the Americas. During the 80's, DENV-1 was the predominant serotype in the region, replaced by DENV 2 in the 90's and subsequently for DENV 3 in 2000. Despite it, DENV 1 has caused several outbreaks in the America's, leading as the serotype more detected and reported in American countries and in imported dengue cases in other continents. During 2010, a highest incidence of dengue occurred in the Americas, several countries registered historically high case fatality rate with the co-circulation of at least two serotype of the virus, including DENV 1. In order to explain this situation we analyzed 50 DENV 1 isolates from 7 American countries collected during 2010 and 2011 through a collaborative study by the VIRORED labs-network. Phylogenetic analysis was performed using MEGA 5 based on 421 nucleotides fragment of the E/NS1 region. In our analyses the strains were mainly grouped in the American/Africa genotype (V) that is widely distributed across the continent. We found at least 5 different lineages circulating from Costa Rica to Paraguay. Strains from Panama and Costa Rica belongs to the Centro American lineages close to strains from Nicaragua and Mexico, except for 2 Panamanian strains that fall into a different clade, close to Asian strains. Viruses circulating in Colombia, Bolivia, Ecuador, Peru and Paraguay are grouped into two different South American lineages. Our nding suggest that during 2010-2011, DENV 1 has great diversity, with the circulation of at least one or two different lineages at the same time and place, and depending of its geographical location could have contributed with the high morbidity observed in 2010 when dengue cases in the region reach 1,6 mil with 1194 deaths.

VACCINE/MODELINGABSTRACTS INVITED SPEAKERS

RECENT SCIENTIFIC AND CLINICAL ADVANCES IN SANOFI PASTEUR'S DENGUE VACCINE PROGRAM

Nicholas Jackson

Sano Pasteur, Global Headquarters, 2 Pont Pasteur, Lyon, 69007, France

In 2012, results from the rst clinical efcacy study of a dengue vaccine, a phase 2b efcacy trial in Ratchaburi, Thailand, demonstrated clinical protection against dengue virus (DENV) serotypes 1, 3, and 4, but not DENV2, despite similar antibody neutralization levels observed for all four serotypes. This triggered the initiation of a broad investigational programme with emerging conclusions suggesting that several factors may have contributed to the observations in Thailand. In parallel to these investigations, the active surveillance phase of two Phase III efcacy trials in more than 30,000 children and adolescents in Asia and in Central and South America has concluded, and the clinical safety database now counts over 28,000 subjects who have received at least one dose of vaccine. The rst data analyses from the Asian Phase III study are available demonstrating that the primary endpoint was successfully achieved, with an overall efcacy of 56.5% against virologically-conrmed symptomatic disease, regardless of severity, against any serotype in children aged 2 to 14 years old (Capeding et al, 2014). Additional sub-analyses have demonstrated that efcacy varies by serotype, age and dengue baseline status. Importantly, preplanned observational analyses show an 88.5% reduction of dengue haemorrhagic fever, and a 67% reduction in hospitalization after 3 doses of vaccine. A favourable safety prole consistent with prior trials was observed. The results of this rst efcacy study will be supplemented by results from the second phase III study in Latin America including more than 20,000 children and adolescents aged 9 to 16 years old.

CLINICAL DEVELOPMENT OF A RECOMBINANT LIVE ATTENUATED TETRAVALENT DENGUE VACCINE

Pedro Garbes

Takeda Vaccines, Inc, Madison, WI, USA

Takeda is developing a tetravalent, live attenuated dengue vaccine (LADV) consisting of a molecularly characterized, attenuated DENV-2 strain and three chimeras in which the prM and E genes of the attenuated DENV-2 were substituted with those of DENV- 1, -3 or -4 viruses. The results of our Phase 1 and 2 clinical trials in healthy subjects support the safety and immunogenicity of the tetravalent vaccine. Administration of LADV was generally well-tolerated with mostly mild and transient local or systemic reactions. There were no related serious or severe adverse events (AEs), and no discontinuations due to vaccine-related AEs. The LADV induced neutralizing antibody responses to all four dengue viruses after one or two administrations. These studies highlight the safety and immunogenicity of the tetravalent LADV vaccine in children and adults in dengue endemic countries. Based on our results from Phase 1 and 2 studies, the LADV warrants further evaluation in Phase 3 efcacy studies in children and adults in dengue-endemic countries.

CLINICAL EVALUATION OF THE NIAID / BUTANTAN INSTITUTE LIVE ATTENUATED TETRAVALENT DENGUE VACCINE1 2Stephen Whitehead , Ricardo Palacios , João Luiz Miraglia, Beatriz Thomé, Neuza Maria Frazatti Gallina, Beth Kirkpatrick, Anna Durbin, Neuza Maria Frazatti

Gallina, Alexander Precioso.1 2Laboratory of Infectious Diseases, NIAID, NIH. Bethesda, MD, USA. Instituto Butantan, Sao Paulo, Brazil.

The Laboratory of Infectious Diseases at the NIAID has developed live attenuated dengue vaccine candidates shown to be both safe and immunogenic in monovalent and tetravalent studies in humans. These studies have enabled the down-selection of vaccine candidates to an optimal mixture of rDEN130, rDEN2/430, rDEN330/31, and rDEN430. In admixture TV003, each component is delivered at a potency of 1000 PFU and neutralizing antibody data collected 90 days post-vaccination in avivirus-naïve US adults showed seroconversion to DENV1, DENV2, DENV3, and DENV4 in 92%, 76%, 97%, and 100% of vaccinees, respectively, after a single subcutaneous dose. In this cohort, 74% of vaccinees achieved a tetravalent antibody response. When the potency of the DENV2 component of the vaccine was increased 10-fold in admixture TV005 and administered in the same manner as TV003, frequencies of seroconversion in vaccinees to the individual serotypes 1 - 4 reached 92%, 97%, 97%, and 97%, respectively, after a single dose, with 90% of vaccinees achieving a tetravalent antibody response. The safety prole remained similar for both TV003 and TV005. Following a second dose of vaccine given 180 days after the rst dose, vaccine viremia, adverse clinical signs, or boosts in neutralizing antibody titers were not observed in any vaccinee, indicating that sterilizing immunity was elicited following the rst dose. Importantly, the US data suggest that admixtures can be administered as a single dose. This is unprecedented among dengue vaccines and has positive implications for vaccine safety, compliance, cost, and dose sparing. Phase II studies of the vaccine produced at the Butantan Institute with potency of 1000 PFU per virus are currently underway in Sao Paulo, Brazil and include both dengue-naïve and dengue-experienced adults. Results from these studies will be presented and represent the rst time the vaccine has been produced and evaluated in a dengue-endemic area.

TETRAVALENT DENGUE PURIFIED INACTIVATED VACCINE (DPIV): STATUS OF THE GSK/FIOCRUZ/U.S. ARMY DENGUE VACCINE CANDIDATE

Alexander Schmidt

GlaxoSmithKline Vaccines, Rixensart, Belgium

GSK, Fiocruz, and the U.S. Army are collaborating to develop a Tetravalent Dengue Puried Inactivated Vaccine (DPIV). All three parties contribute to epidemiology studies, preclinical, clinical, and technical development. Two prospective cohort studies for dengue surveillance have been initiated in Brazil: a pediatric school-based

study in Fortaleza (NCT01391819) and a household-based study in Rio de Janeiro, Salvador and Manaus (NCT01751139). Two Phase I vaccine studies are ongoing in non-endemic (Maryland, United States, NCT01666652) and endemic (Puerto Rico, NCT01702857) regions. In these studies, DPIV, adjuvanted with either alum, AS01 or AS03, induced a robust immune response in both dengue naïve and in dengue primed subjects following 2 doses of vaccine administered 4 weeks apart. AS03 adjuvanted vaccine induced the highest titers of neutralizing antibodies, with Day 56 GMTs against the four serotypes ranging from 406 to 526 in unprimed subjects, and from 2150 to 3840 in primed subjects. Conclusion: The observed difference in GMTs between primed and unprimed subjects suggests that the analysis of immunogenicity (and at a later stage efcacy) data should be stratied by priming status since an efcacious vaccine will need to protect both primed and unprimed populations. The GSK/FIOCRUZ/U.S. Army collaboration is committed to develop a safe and efcacious dengue vaccine for use in infants, children, and adults.

PRECLINICAL AND CLINICAL TESTING OF A RECOMBINANT SUBUNIT VACCINE FOR DENGUE1 1 2 1 1 1 2 2Beth-Ann Coller , Susan Manoff , Andrew Bett , Michele Sausser , Tyler Finn , Amy Russell , Govindarajan Dhanasekaran , Danilo Casimiro

1 2 Merck Research Laboratories, North Wales, PA, 19454. Merck Research Laboratories, West Point, PA, 19486, USA.

Dengue viruses are a major cause of morbidity and mortality throughout the tropics and subtropics. It is estimated that more than 120 countries currently have endemic dengue virus transmission. Each year there are at least 100 million infections, of which more than a million are clinically severe, resulting in approximately 21,000 deaths. While a licensed dengue vaccine is not yet available, several vaccine candidates designed to protect individuals against dengue virus-induced disease are currently being evaluated in clinical trials, including a tetravalent recombinant subunit vaccine. Preclinical studies of this recombinant subunit vaccine have been conducted in non-human primates to evaluate the immunogenicity and efcacy of tetravalent formulations. Vaccine formulations have been evaluated in both dengue naïve and experienced animals. These preclinical studies have shown the capacity of the recombinant proteins to induce balanced tetravalent responses without evidence of interference. Data from these preclinical non-human primate studies will be presented. The vaccine candidate is also being tested in a Phase 1 clinical trial in healthy, avivirus-naïve, adults. An update on clinical trial status will also be provided.

A DENGUE HUMAN INFECTION MODEL (DHIM) TO EVALUATE THE PROTECTIVE EFFICACY OF THE LIVE ATTENUATED TETRAVALENT DENGUE CANDIDATE VACCINE TV003

1 2 1 1 2 1 1 2 1 1Anna Durbin , Beth Kirkpatrick , Dan Elwood , Beulah Sabundayo , Cathy Larssen , Cecilia Tibery , Paltama Grier , Kristen Pierce , Janece Lovchik , Matthew Jo , Sean 2 2 3Diehl , Marya Carmoli , and Stephen Whitehead

1 2 3Johns Hopkins Bloomberg School of Public Health, University of Vermont College of Medicine, National Institute of Allergy and Infectious Diseases, National Institutes of Health

Dengue virus (DENV) has become the most important arbovirus worldwide with estimates of as many as 400 dengue infections occurring annually resulting in 100 million symptomatic and 21,000 fatal infections. Neutralizing antibody, the accepted immunologic endpoint, was not predictive of protection against DENV illness in two recently completed efcacy trials of the lead DENV candidate vaccine CYD-TV. These results make it difcult for vaccine manufacturers to determine which candidates should be further evaluated in large efcacy trials in endemic areas where vaccine failure could predispose subjects to more severe disease upon subsequent DENV infection. A dengue human infection model (DHIM) would be useful in down-selecting candidate vaccines prior to testing in endemic areas as well as identifying putative correlates of protection. We have developed a DHIM to evaluate the protective efcacy of the live attenuated tetravalent dengue vaccine TV003 developed by the U.S. National Institutes of Health. In Phase I clinical trials, a single dose of TV003 induced neutralizing antibody to DENV-1, DENV-2, DENV-3, and DENV-4 in 92%, 76%, 97%, and 100% of vaccinees, respectively. It induced seroconversion to all four DENV serotypes in 75% of subjects and to ≥ 3 serotypes in 98%. On study day 0, 24 avivirus-naïve subjects received TV003 and 24 subjects received a placebo. Six months later, all 48 subjects received 1,000 PFU of rDEN230, an under-attenuated DENV-2 vaccine candidate. rDEN230 was previously demonstrated to induce viremia in 100% of inoculated subjects with a mean peak virus titer of 2.5 log10 PFU/mL. In addition, 80% of subjects developed a maculopapular rash and 40% developed neutropenia. The study is powered to detect 60% efcacy of TV003 to prevent viremia caused by rDEN230 (the primary outcome). Secondary outcomes include protection against rash and neutropenia. Preliminary clinical and virological outcomes of the trial will be presented.

Structure-function relationships of human antibodies that neutralize dengue viruses1 1 1 1 1 1 2 3 3 3Aravinda de Silva , Rukie de Alwis , Emily Gallichotte , Ralph Baric , Scott Royal , Doug Widman , William Messer , James Crowe , Scott Smith , Gopal Sapparapu ,

4 4 4Shee-mei Lok , Joannne Tan and Guntur Fibriansah1 2Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, USA; Department of Medicine, Oregon Health & Sciences University,

3 4Portland, Oregon, USA; Department of Pediatrics, Vanderbilt University, Nashville, USA; Program for Emerging Infectious Diseases, Duke-NUS, Singapore

Recent studies from our groups provide new insights into the properties of human antibodies (Abs) responsible for serotype-specic, durable neutralizing immunity against dengue virus (DENV). In people exposed to DENV, only a minor fraction of the total DENV-specic Ab response is responsible for neutralization. These rare but potently neutralizing Abs are produced by both memory B-cells and long-lived plasma cells. We have mapped the epitopes recognized by strongly neutralizing human monoclonal Abs (nHMAbs) and obtained high-resolution structures of nHMAbs bound to different serotypes of DENV. nHMAbs bind to multiple overlapping epitopes that are often quaternary epitopes because the Ab footprint extends into adjacent E protein molecules. Indeed a common theme emerging from our studies is that proper display of nHMAb epitopes requires the assembly of E protein molecules to form intact virus particles. As an alternate strategy, we have created recombinant DENVs with targeted mutations in the epitopes. Despite the structural complexity of these epitopes, it is possible to genetically transplant these epitopes between

serotypes. Studies with recombinant viruses demonstrate that epitopes dened by nHMAbs are also the target of neutralizing Abs in polyclonal dengue immune sera. nHMAbs neutralize DENVs by blocking DENV attachment to cells and steps after attachment, most likely low pH mediated DENV fusion with endosomes. We will discuss the implications of our work for evaluating vaccines in the current pipeline and developing next generation DENV vaccines. Supported by NIAID U54 AI057157 from SERCEB (AMds, WBM, SAS, JEC Jr, RSB), R01 AI107731-01 (AMds), 1 R56 AI097560-01 (AMds).

A NEW SIMULATION MODEL OF DENGUE VIRUS TRANSMISSION AND CONTROL IN IQUITOS, PERU1,2 2,3 4 5 6 5 7 8 8Alex Perkins , Bobby Reiner , Amit Verma , Steve Stoddard , Valerie Paz-Soldan , Amy Morrison , Gonzalo Vazquez-Prokopec , Helvio Astete , Eric Halsey , Tad

U 7 2,9 2,5Kochel8, riel Kitron , David Smith , Thomas Scott1 2 3 4 University of Notre Dame, IN USA. Fogarty International Center, National Institutes of Health, MD USA. Indiana University, Bloomington, IN USA. Center for Disease

5 6Dynamics, Economics, and Policy, Washington, DC. University of California, Davis, CA USA. Tulane University School of Public Health and Tropical Medicine, LA USA. 7 8 6 9 Emory University, GA USA. United States Naval Medical Research Unit No. , Lima, Peru. University of Oxford, UK

It is becoming increasingly recognized that mathematical models are useful, if not essential, tools for understanding the dynamics of transmission and control of dengue. Models can play important roles in study design, interpretation of eld-collected data, evaluation of control strategies, and forecasting future outbreaks. Given the general utility of models for dengue research, we developed a suite of models that are capable of incorporating various levels of epidemiological and entomological detail. Parameterization of these models leveraged 15 years of eld studies on the entomology, epidemiology, and human dimensions of dengue virus transmission in the city of Iquitos, Peru. Particularly novel features of these models include a sub-model of human movement that is the most realistic description of ne-scale movement in resource-poor cities to date, and a spatiotemporal surface of adult female Aedes aegypti densities that leverages a data set comprised of over 150,000 individual mosquito captures. Here, we show that predictions based on a mathematical model that combines this information into estimates of block-level seroprevalence are consistent with realized patterns of transmission during the invasion of DENV-3 to Iquitos. We then show predictions from a more rened model that can be used to disentangle the contributions of different types of heterogeneity to transmission. Finally, we show some preliminary results about the effectiveness of interventions featuring a combination of vaccination and vector control.

MODELING TRANSMISSION AND CONTROL OF DENGUE WITH VACCINES1 2,3 2 1 1Ira Longini , M. Elizabeth Halloran , Dennis Chao , Tom Hladish , Diana Rojas-Alvarez

1 2 3University of Florida, Fred Hutchinson Cancer Research Center, University of Washington

With tetravalent dengue vaccines on the near-time horizon, we will nally have an effective intervention strategy for dengue. We construct a detailed stochastic dengue transmission model for a population in Latin America. The model is used to project the transmission of dengue in this population over a twenty year time horizon. We then evaluate the overall vaccine effectiveness (i.e., reduction in dengue incidence, comparing vaccinated to unvaccinated populations) of different vaccination strategies for a plausible dengue vaccine. Our work captures the potential heterogeneity in protection of these vaccines with respect to different levels of circulating serotypes, prior and evolving population immunity, force of infection, vaccine action, and age structure. We show that the overall effectiveness of the best vaccine strategies would vary from 70-85% over time, space and the strategy employed.

ABSTRACTS ORAL PRESENTATIONSUSE OF ANTIGENIC CARTOGRAPHY TO CHARACTERIZE NEUTRALIZATION OF DIVERSE DENGUE VIRUSES IN THE MONTHS FOLLOWING PRIMARY DENGUE VIRUS INFECTION

1,2 1 2 2 2 1Leah C. Katzelnick , Judith M. Fonville , Gregory D. Gromowski , Jose Bustos Arriaga , Stephen S. Whitehead , Derek J. Smith1 2Department of Zoology, University of Cambridge, Cambridge, United Kingdom; National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA

The four dengue virus types, DENV1-4, elicit early neutralizing antibody responses with high titers to homologous as well as heterologous DENV types, which have been observed to narrow to the homologous type in the ensuing months. However, research on changes in the neutralizing response over time often use single representatives of each DENV type, and thus the degree to which changes are due to the absolute quantity or specicity of neutralizing antibodies is not well understood. Here, we use antigenic cartography to describe how 17 primary-infection African green monkeys (AGM) with antisera drawn one, three and ve months after inoculation neutralize a panel of 38 diverse DENV isolates assembled by the Dengue Antigenic Cartography Consortium. The neutralizing antibody response to the DENV panel over the four-month period signicantly declined for eight AGMs, was stable for eight AGMs, and dramatically increased for one AGM. We made antigenic maps of the three data sets, and nd that at each time point, some viruses were as close to viruses of a heterologous type as to some viruses of the homologous type. Based on the positions of antisera on the antigenic maps, moderate changes in neutralizing reactivity were observed between one and three months, but less change between three and ve months. A few AGM antisera shifted to the periphery of homologous antigenic cluster, suggesting increasing type-specicity, while the other antisera moved toward the center of the antigenic map over time, thus transitioning to more cross-reactive responses. We nd that only a subset of primary infection AGM antisera become more type-specic over time, and that few changes in neutralization breadth are observed between three and ve months after infection.

UTILITY, LIMITATIONS AND FUTURE OF NON-HUMAN PRIMATES FOR DENGUE RESEARCH AND VACCINE DEVELOPMENT1 2Laura J. White and Carlos A. Sariol

1 2Global Vaccine Inc., Research Triangle Park, North Carolina, USA . Caribbean Primate Research Center, Department of Microbiology and Medical Zoology, Department of Internal Medicine, University of Puerto Rico-Medical Sciences Campus, San Juan, Puerto Rico.

One foremost constraint to test the efcacy of a dengue vaccine candidate is the lack of an adequate animal model to reproduce dengue symptoms or its hemorrhagic or shock manifestations upon challenge. In spite of this, Non Human Primates (NHP) are considered the best model due to their genetic relatedness to humans and their ability to develop neutralizing antibodies and viremia similar to humans. Therefore, data obtained in NHP have been the gatekeeper for vaccines advancing to clinical trials, based on neutralizing activity in serum and reduction of post-challenge viremia, which indicates to vaccine developers and regulatory agencies of the potential for efcacy in humans. However, how predictive NHP are has been questioned recently. The results of a comprehensive review of published studies on dengue vaccines tested in NHP will be presented in the light of recent clinical data. Vaccine performance in NHP and humans will be compared, and the NHP model will be re-evaluated for its usefulness and potential for improvement. The replication and immunogenicity of vaccines tested in NHP have parallels to the human responses to the same vaccines, specically regarding under-attenuation, relative serotype dominance and immunogenicity in tetravalent formulations. We also found parallels in the induction of anamnestic responses upon second vaccine doses, sero-conversion to all 4 serotypes after a single dose, and serotype interference. Our analysis suggests that the predictive capacity of the NHP model may improve by examining additional immunological parameters in depth after dengue infection and vaccination. Also, the model may be improved by standardization of the stringency of the challenge, and the methods used for evaluating the magnitude and quality of the immune response.

HIGHLIGHTED POSTERSHP26

CHARACTERIZATION OF CELLULAR RESPONSES TO A TETRAVALENT LIVE-ATTENUATED DENGUE VACCINE IN FLAVIVIRUS-NAÏVE HUMAN VOLUNTEERS1 1 2,3 1 1Charalambos D. Partidos, Haiyan Chu, Sarah George, Dan T. Stinchcomb, Jorge E. Osorio1 2Takeda Vaccines, Inc, Madison, WI, USA, Department of Internal Medicine, Division of Infectious Diseases, Allergy and Immunology, Saint Louis University School of Medicine

We have developed a live attenuated tetravalent dengue candidate vaccine (TDV) based on an attenuated dengue 2 virus (TDV-2) and three chimeric viruses containing the pre-membrane and envelope genes of dengue viruses (DENV) -1, -3 and -4 expressed in the context of the attenuated TDV-2 genome (TDV-1, TDV-3, and TDV-4, respectively). The cellular responses elicited by this vaccine were analyzed in avivirus-naïve human volunteers vaccinated twice (day 0, 90) via the subcutaneous or intradermal routes. Using peptide arrays and intracellular cytokine staining, we demonstrated that the vaccine elicits CD8+ T cells targeting the non-structural NS1, NS3 and NS5 proteins of TDV-2. The cells were characterized by the production of IFN-γ, and TNF-α, and to a lesser extend IL-2. Responses were the highest on day 90 after the rst immunization and were still detectable on day 160 post-secondary immunization. In addition, CD8+ T cells were multifunctional, producing ≥ 2 cytokines simultaneously, and cross-reactive to the NS proteins of the other serotypes. Overall, these ndings highlight the immunogenic prole of our candidate dengue vaccine and support the further evaluation of clinical samples from ongoing phase II clinical trials.

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PHASE I STUDY OF DIFFERENT FORMULATIONS OF A TETRAVALENT DENGUE PURIFIED INACTIVATED VACCINE (DPIV): DAY 56 SAFETY AND IMMUNOGENICITY DATA IN HEALTHY ADULTS FROM PUERTO RICO

1 2 2 3 2 3 3 2Clemente Diaz, Luis J. Martinez, Maribel Campos,1 Leyi Lin, Elodie Tournay, Kenneth H. Eckels, Jean-François Toussaint, Edith Lepine, Rafael De La Barrera, 2 4 2 3Richard G. Jarman, Bruce L. Innis, Stephen J. Thomas, Alexander Schmidt

1 2 3University of Puerto Rico School of Medicine, San Juan, Puerto Rico; Walter Reed Army Institute of Research, Silver Spring, Maryland USA; GlaxoSmithKline Vaccines, 4Rixensart, Belgium; GlaxoSmithKline Vaccines, King of Prussia, Pennsylvania, USA

Background: We assessed the safety and immunogenicity of an investigational tetravalent dengue puried inactivated vaccine (DPIV) formulated with different adjuvant systems administered as a 2-dose schedule in Puerto Rico. Methods: In this Phase I, observer-blind study (NCT01702857), 100 healthy adults aged 18–39 years were randomized 1:1:1:1:1 to receive one of four DPIV formulations (1µg per dengue virus [DENV] type adjuvanted with either aluminum hydroxide [Alum], AS01E or AS03B, and 4µg per DENV type adjuvanted with Alum) or saline placebo, at Day (D) 0 and D28. Primary safety endpoints included solicited and unsolicited adverse events (AEs) recorded for 7 and 28 days post-vaccination, respectively, and serious AEs (SAEs) and potential immune-mediated diseases (pIMDs) through D56. Primary immunogenicity endpoints were neutralizing antibody titers per DENV type at D56 by 50% microneutralization assay. Results: No SAEs were reported through D56. A pre-existing pIMD (rheumatoid arthritis) was reported for one subject in the 1µg+AS03B group. Injection site pain was more common in the vaccine groups than in the placebo group. Headache and myalgia were the most frequent solicited general AEs across all groups. The rates of unsolicited AEs were similar across all groups. The according-to-protocol cohort for immunogenicity included 94 participants, of whom 89 were seropositive for one or more DENV type at D0, with geometric mean titers (GMTs) between 394 and 1020. By D56, GMTs had increased approximately 2-fold in the 1µg+Alum group, 3-fold in the 4µg+Alum group, and ≥4-fold in the 1µg+AS01E and 1µg+AS03B groups. D56 GMTs against DENV-1, -2, -3, and -4, respectively, were 1896, 1916, 2471 and 2569 in the 1µg+AS01E group, and 2634, 2338, 2473 and 4733 in the 1µg+AS03B group. Conclusions: All DPIV formulations were generally well-tolerated. AS01E- and AS03B-adjuvanted vaccines were more immunogenic than Alum-adjuvanted vaccines at D56. Funding: US Army and GlaxoSmithKline Biologicals SA.

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A NATIONAL AGENT-BASED MODEL OF DENGUE TRANSMISSION IN COLOMBIA 1,2 1 2 4 1 3 3 1Guido Camargo , Willem Van Panhuis , Hernando Diaz , Carlos Castaneda-Orjuela , Ernesto Marques , Nathan Stone , Shawn Brown , John Grefenstette

1 2 3 4University of Pittsburgh , Universidad Nacional de Colombia , Pittsburgh Supercomputer Center , Observatorio Nacional de Salud de Colombia

Dengue is the most signicant vector-borne disease throughout the globe. Almost three billion people live in risk areas and more than 300 million are infected each year. In Colombia, the four serotypes of the dengue virus circulate heterogeneously across the country. In the 2010, the country reported more than 140,000 cases. Vector control programs have been unable to stop the spread of this epidemic but a new dengue vaccine is expected in the near future. The public health impact and optimal vaccination strategies remain unclear, including the potential role for integrated dengue control that include vaccine and vector control components. We created a large-scale agent-based model of dengue transmission in Colombia that represents each of the almost 50 million people living in this country. For this we modied the existing Framework for Reconstructing Epidemic Dynamics (FRED) developed at the University of Pittsburgh. We used Colombia census microdata, population density grids, and transport data to develop a synthetic population and developed a vector density grid based on climate and population data. We used a four serotype deterministic dengue SEIR transmission model for dengue transmission. This model was parameterized based on historical dengue surveillance data provided by the Colombia Ministry of Health. This model for dengue transmission in Colombia is currently the largest scale agent-based model for dengue transmission worldwide, representing an entire country of 50 million inhabitants. This model can be used to simulate spatially explicit dengue dynamics and the impact of interventions strategies such as vaccination, vector control, or both. This model can be scaled up to represent larger populations in different countries or across the entire PAHO region. Using dengue simulation models will greatly support decision-making on the introduction of new dengue vaccines.

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EXPERIMENTAL SEQUENCIAL DENGUE VIRUS INFECTION IN MARMOSETS CALLITHRIX PENICILLATA: HISTOPATHOLOGICAL CHANGES AND VIRAL ANTIGEN DETECTION

1 1 2 2 1Ferreira, Milene Silveira ; Barros, Vera Lucia Reis Souza de ; Silva, Gilmara Abreu ; Castro, Paulo Henrique Gomes de ; Casseb, Samir Mansuor Moraes ; Domingues, 3 1Ricardo Augusto de Barros ; Vasconcelos, Pedro Fernando da Costa

1 2 3Evandro Chagas Institute, Ananindeua-Pará-Brazil. National Primate Center, Ananindeua - Pará- Brazil. Metropolitan College of Amazon.

Dengue is the most widespread arbovirus in the world. Presently, approximately 3.0 billion people live in areas at risk of infection. Several studies on the pathogenesis of DENV has been performed, however, to date there is not an animal model that reproduces the signs of severe disease observed in humans. To investigate the susceptibility of the marmoset Callithrix penicillata to dengue vírus infection DENV-2 and DENV-3 isolates recovered from fatal human cases were sequentially intraperitoneally inoculated. Three marmosets were infected with DENV-2 (4.47 × 104 PFU/mL) (primary infection) and after one year and seven months they were reinfected with DENV-3 (3.23 × 103 PFU/mL) (secondary infection). Tissue (lymph node, liver, spleen and kidney) were collected at 3, 5 and 10 dpi. to histopathological and immunohistochemical assays. The result showed no signicant change in the lymph nodes; liver presented reactive hyperplasia (3 and 5 dpi), giant cells in the sinusoid, similar to megakaryocytes (3 dpi), scattered necrotic Kupffer cells and slight mononuclear inammatory reaction (10 dpi); It was observed in the spleen red pulp a large number of lymphocytic elements, giant cells, similar to megakaryocytes (3 dpi) and hyperplasia in the white pulp (5 dpi) and hypoplasia (10 dpi); the kidney showed acute interstitial nephritis (5 dpi) and acute necrosis (bilateral) in small medullary areas (10 dpi). The result of immunohistochemical assay showed large amounts of viral antigens in all examined samples. The anatomical and physiopathological changes found in the marmosets are similar to those observed in human severe disease such as reactive hyperplasia of megakaryocytes giant cells, mononuclear inammatory inltrate and necrosis. Nonetheless the three infected marmosets showed no clinical signs of illness. The results indicate that the species Callithrix penicillata is a good model for studying the pathogenesis of dengue.

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A TETRAVALENT DENGUE VACCINE FOR EARLY LIFE IMMUNIZATION 1 2 1 1Melissa D. Mattocks , Syed Muaz Khalil1, , Daniel R. Tonkin1 , Robert E. Johnston , and Laura J. White

1 2Global Vaccines, Inc., Research Triangle Park, NC, USA. Department of Microbiology & Immunology, The University of North Carolina-Chapel Hill, Chapel Hill, NC, USA.

Preexisting immunity resulting from a previous DENV infection is the major risk factor for severe dengue during secondary heterologous infections. During primary infections in infants, maternal antibodies pose an analogous risk, therefore the need to vaccinate early in life. At the same time, maternal antibodies are likely to prevent induction of endogenous anti-DENV antibodies in response to current live, attenuated vaccine (LAV) candidates. Any effective early life dengue vaccine has to overcome maternal antibody interference (leading to ineffective vaccination) and poor induction of antibody responses (increasing the risk of severe dengue disease upon primary infection). Here we demonstrate the feasibility of a non-propagating VEE virus replicon vector (VRP) expressing DENV E protein as an early life vaccine platform for dengue. First, DENV-VRP vaccine is immunogenic even in the presence of maternal antibodies that otherwise interfere with a live virus vaccination in weanling BALB/c mice. Second, we observed that a single immunization in 7-day-old BALB/c mice with a VRP vaccine expressing E ectodomain of DENV induced neutralizing antibody (NAb) titers by 6 weeks, which remained stable until at least 15 weeks post-immunization. Third, DENV-specic cell-mediated immunity was also induced in these immunized mice. Fourth, the NAb levels induced to each serotype by a tetravalent VRP formulation were equivalent to those of each monovalent vaccine components, suggesting that this vaccine modality can overcome serotype interference. Fifth, VRP immunization in neonatal mice was durable and could be boosted later in life to further increase NAb and T-cell responses. Finally, although the neonatal immune response was lower in magnitude than responses in adult

BALB/c mice, we demonstrate that, both monovalent and tetravalent VRP vaccines generated protective immunity from a lethal intracranial challenge after a single neonatal immunization.

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A TETRAVALENT DENGUE VACCINE CONTAINING A MIX OF DOMAIN III-P64K AND DOMAIN III-CAPSID PROTEINS INDUCES A PROTECTIVE RESPONSE IN MICE1 1 2 2 1 2 2 1Alienys Izquierdo , Angélica García , Laura Lazo , Lázaro Gil , Mayling Alvarez , Lisset Hermida , Gerardo Guillén , María G. Guzmán

1PAHO/WHO Collaborating Center for the Study of Dengue and its Vector, Department of Virology, Tropical Medicine Institute “Pedro Kourí” (IPK), Autopista Novia del 2Mediodía Km 6 ½ P.O. Box 601 Marianao 13, Havana, Cuba; Center for Genetic Engineering and Biotechnology (CIGB), Avenue 31, P.O. Box 6162, Havana 6, 10 600,

Cuba.

Recombinant fusion proteins containing domain III of the dengue envelope protein fused to the P64k protein from Neisseria meningitidis and domain III of dengue 2 fused to capsid protein of this serotype were immunogenic and conferred protection in mice against lethal challenge, as previously reported. Combining the domain III-P64k recombinant proteins of dengue 1, 3 and 4 with the domain III-capsid protein from dengue 2, we obtained a novel tetravalent formulation containing different antigens. Here, the IgG and neutralizing antibody response, the cellular immune response and the protective capacity against the lethal challenge in mice immunized

with this tetravalent formulation were evaluated. The neutralizing antibody response obtained against D1, D2 and D3 together with the high levels of IFNγ secretion

induced after stimulation with the four dengue serotypes support the strategy of a new tetravalent formulation containing the domain III of envelope protein fused to the capsid protein of each dengue serotype.

VACCINE/MODELING POSTERS SESSIONVM-P01

THE DENGUE VACCINE INITIATIVE (DVI) PROJECT: PASSIVE FACILITY-BASED FEVER SURVEILLANCE IN CHILDREN AND ADULTS OF MEDELLIN, COLOMBIA 1 1 2 2 2 1 2 3Jacqueline Lim , Mabel Carabali , Catalina Le Cacheux , Diana Carolina Vélez , Andrea Trujillo-Correa , Kang Sung-Lee , Iván D. Velez , Jorge Osorio

1 2Dengue Vaccine Initiative, International Vaccine Institute, Seoul, Korea; Programa de Estudio y Control de Enfermedades Tropicales (PECET), Universidad de 3Antioquia, Medellín, Colombia; and Biomedical Science Laboratories, School of Veterinary Medicine, University of Wisconsin.

Dengue virus (DENV) infection is a major public health problem in Colombia, causing more human illness than any other arbovirus. Dengue Vaccine Initiative (DVI) conducted a passive facility-based fever surveillance to determine the true burden of dengue fever in the Santa Cruz comuna of Medellin, Colombia. The surveillance was based in Santa Cruz Hospital and three other primary health care centers afliated to METROSALUD E.S.E. Individuals between 1-65 years-of-age were evaluated for febrile episodes (≤7 days of evolution). Two paired samples were obtained from all participants; an acute sample (0-7 days of fever) and a convalescent sample 10-14 days after the rst visit. All samples were tested with rapid diagnostic test SD® Dengue Duo during the acute phase, and both acute and convalescent samples were analyzed with IgM/IgG capture ELISA. The positive cases on ELISA were tested with RT-PCR for serotyping. From Oct. 2011 until Feb. 2014, there were 579 enrollees in the surveillance with 150 (26%) laboratory-conrmed with dengue fever. Higher proportions of dengue-conrmed cases were found in children between 5-9 (20%) and in young adults between 15-24 (18.7%) years of age. The mean fever duration was longer for those dengue cases at 5.2 days compared to non-dengue cases 4.5 days (p-value=0.007). The majority of our dengue cases were outpatients (n=137) with only 9% of hospitalized patients. About 25% of the dengue cases had secondary infection where as 75% had primary infection. The RT-PCR results show that DENV-3 with47.6% is the most commonly circulating serotype, followed by DENV-1 with 9.5%. All signs and symptoms were similar between dengue patients and non-dengue febrile patients, except for rash and retro-orbital pain found more commonly among dengue cases. Our study results show patterns of the elevated dengue transmission in Santa Cruz comuna. Also, we observed some correlation between the national surveillance data from Medellin and our study ndings. More information will be presented at the conference.

VM-P02

THE DENGUE VACCINE INITIATIVE PROJECT: SERO-PREVALENCE STUDY IN CHILDREN AND ADULTS OF SANTA CRUZ COMUNA OF MEDELLIN 1 1 2 2 2 3Mabel Carabali , Jacqueline Lim , Carolina Velez , Andrea Trujillo-Correa , Kang Sung-Lee, Iván D. Velez , Jorge Osorio

1 2Dengue Vaccine Initiative, International Vaccine Institute, Seoul, Korea; Programa de Estudio y Control de Enfermedades Tropicales (PECET), Universidad de 3Antioquia, Medellín, Colombia; and Department of Pathobiological Sciences, University of Wisconsin

Dengue is a major public health problem in Colombia. During the last 10 years, there has been a signicant increase in the number cases of dengue, with circulation of all four serotypes and epidemic waves occurring every 3-4 years. Dengue Vaccine Initiative (DVI) conducted a serological survey to determine the true burden of dengue due to inapparent infections in the Santa Cruz comuna of Medellin, Colombia. The serosurvey included two years of follow up with ve bleedings, with six-month intervals from Nov. 2011 to Feb. 2014. More than 2000 randomly selected residents were enrolled in the study. To estimate the incidence and the age-specic sero-conversion rates on samples that showed a rise in IgG antibody titers by IgG Indirect ELISA. Through 5 bleedings, replacements were made to keep an open cohort of 2000 randomly selected residents. The sero-prevalence of IgG antibodies in each sero-survey were: 59.02% in S1; 63.9% in S2; 64.9% in S3; 64.7% in S4, and 64.7% in S5. The overall sero-conversion rate during the rst year was of 4.8%. The highest sero-conversion rate between S1 and S3 was found among children 5-9 years of age (7.3%), followed by young adults between 35-44 years-of-age (6.9%) and children 1-4 years old (5.6%). From the preliminary PRNT performed on a

subset of sero-converted samples (n=48), 83.7% showed an immune response against all 4 serotypes, with DENV-3 (24.5%) and DENV-1 (16.3%) as the predominant serotypes. These sero-prevalence results may indicate an elevated dengue transmission in Santa Cruz and a higher risk for severe forms with increase in age. Results of further analyses of the immunological status of individuals in this geographic area will be presented at the conference.

VM-P03

RECOMBINANT DENGUE 2 VIRUS NS3 PROTEIN CONSERVES STRUCTURAL ANTIGENIC AND IMMUNOLOGICAL PROPERTIES RELEVANT FOR DENGUE VACCINE DESIGN1 1 1 1 1 2 2 1Rosa Ramírez , Rosabel Falcón , Alienys Izquierdo , Oney Ortega , Mayling Alvarez , Emiliana Mandarano da Silva , Ronaldo Mohana-Borges , María G. Guzmán

1PAHO/WHO Collaborating Center for the Study of Dengue and its Vector, Department of Virology, Tropical Medicine Institute “Pedro Kourí” (IPK), Autopista Novia del 2Mediodía Km 6 ½ P.O. Box 601 Marianao 13, Havana, Cuba; Laboratorio de Genômica Estrutural, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do

Rio de Janeiro, Ilha do Fundão, Rio de Janeiro, RJ 21949-900, Brazil

The NS3 protein is a multifunctional non-structural protein of aviviruses implicated in the polyprotein processing. The predominance of cytotoxic T cell lymphocytes epitopes on the NS3 protein suggests a protective role of this protein in limiting virus replication. In this work, we studied the antigenicity and immunogenicity of a recombinant NS3 protein of the Dengue virus 2. The full-length NS3 gene was cloned and expressed as a His-tagged fusion protein in Escherichia coli. The pNS3 protein was puried by using two chromatography steps. The recombinant NS3 protein was recognized by anti-protease NS3 polyclonal antibody and anti-DENV2 HMAF by Western Blot. This puried protein was able to stimulate the secretion of high levels of gamma interferon and low levels of interleukin-10 and tumor necrosis factor-� in mice splenocytes, suggesting a predominantly Th-1 type T cell response. Immunized BALB/c mice with the puried NS3 protein showed a strong induction of anti-NS3 IgG antibodies, essentially IgG2b, as determined by ELISA. Immunized mice sera with recombinant NS3 protein showed specic recognition of native dengue protein by western blotting and immunouorescence techniques. The successfully puried recombinant protein was able of preserving the structural and antigenic determinants of the native dengue protein. The antigenicity shown by the recombinant NS3 protein suggests its possible inclusion into future DENV vaccine preparations.

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A FRAMEWORK FOR MODELING AND SIMULATING AEDES AEGYPTI AND DENGUE FEVER DYNAMICS1 2 2 3 4 5 2 1 6Tiago Lima , Raquel Lana , Cláudia Codeço , Tiago Carneiro1, Raian Maretto , Liliam Medeiros , Leonardo Santos , Izabel Reis , Gabriel Machado , Flávio Coelho ,

3Antônio Monteiro .1 2 3 Federal University of Ouro Preto (UFOP). Ouro Preto, MG, BRAZIL; Oswaldo Cruz Foundation (Fiocruz). Rio de Janeiro, RJ, BRAZIL; National Institute for Space

4 5Research (INPE). São José dos Campos, SP, BRAZIL; São Paulo State University (UNESP). São José dos Campos, SP, BRAZIL; National Centre for Monitoring Natural 6Disasters and Alerts (Cemaden). Cachoeira Paulista, SP, BRAZIL; Getúlio Vargas Foundation (FGV). Rio de Janeiro, RJ, BRAZIL.

Controlling dengue fever and its vector, the Aedes aegypti mosquito, is a public health challenge. It presents a complex dynamic, governed by interaction of multiple agents in a complex space. While environmental factors as temperature, rainfall and humidity interfere in developmental stages of dengue vector, social aspects could also contributed for increasing dengue incidence. Modeling is a powerful tool to understand epidemic dynamics and to evaluate costs, benets and effectiveness of control strategies. Several models have been developed aiming to understand dynamics of vector population and transmission disease. In order to support decision-makers and researchers to evaluate different methodologies, we present Dengue Modeling Environment (DengueME), an open source tool aiming at supporting the development and simulation of spatio-temporal models of dengue and its vector. DengueME was developed as a tool for helping the designing of site-specic and population-specic control strategies for dengue. It consists of a series of compartmental and individual-based models, implemented over a GIS database, that represents the Aedes aegypti's life cycle, human demography, human mobility, urban landscape and dengue transmission. The platform is designed to allow easy simulation of intervention scenarios. A Graphical User Interface (GUI) was developed to control parameters and data, and run simulations.

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A DNA VACCINE ENCODING THE ENVELOPE AND NS1 PROTEINS OF DENV2 – PROTECTION AGAINST DENGUE VIRUS INFECTION IN MICE

Paolla Beatriz de Almeida Pinto, Tamiris Azamor da Costa Barros, Kíssila Rabelo, Antonio José da Silva Gonçalves, Simone Morais da Costa e Ada Maria de Barcelos Alves

Laboratório de Biotecnologia e Fisiologia de Infecções Virais, Instituto Oswaldo Cruz, Fiocruz

Dengue represents a world health problem with recurrent epidemics in tropical and subtropical regions. The development of a dengue vaccine to protect against the four viral serotypes is considered a priority for the World Health Organization. The envelope (E) and the non-structural (NS1) proteins are identied as promising antigens to integrate a vaccine against DENV. The E protein interacts with receptors present on cell surfaces, allowing virus endocytosis. Therefore, neutralizing antibodies against this protein can prevent virus entry inside host cells. Other studies indicate that the NS1 protein is also able to induce a protective immune response. Previous reports with DNA vaccines developed by our group based on the expression of these two proteins individually demonstrated the induction of protective immune responses against dengue virus. In order to decrease the number of plasmids that may compose a tetravalent DNA vaccine based on E and NS1 proteins, we constructed one single plasmid containing both genes, named pNS1/E/D2. Recombinant clones were evaluated by restriction enzyme digestions and sequencing of the NS1 and E regions. After conrmation of correct open reading frames, BHK-21 cells were transfected with different clones of this plasmid. The immunouorescence analysis revealed that all plasmids mediated expression of the recombinant proteins in vitro in eukaryotic cells, detected with specic

antibodies. We evaluated the protector potential of pNS1/E/D2 and compared with the vaccines that encode the proteins E (pE1D2) and NS1 (pcTPANS1) individually. BALB/C mice were immunized with the vaccine pNS1/E/D2, as well as with pE1D2 and pcTPANS1, administered individually or simultaneously, and challenged with DENV2. Two pNS1/E/D2 clones and the mix pcTPANS1+pE1D2 induced complete protection, with 100% survival rates and no signs of infection.

Funding Support: IOC-FIOCRUZ, PDTIS-FIOCRUZ, INCTV, CNPq e FAPERJ

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MORPHOLOGICAL AND VIREMIA ANALYSIS OF MURINE MODEL INFECTED WITH LINEAGES OF DENGUE VIRUS TYPE 2

Fernanda Cunha Jácome, Débora Ferreira Barreto-Vieira, Marcos Alexandre Nunes, Arthur da Costa Rasinhas, Priscila Conrado Guerra Nunes, Thais Chouin Carneiro, Flávia Barreto dos Santos

Fundacao Oswaldo Cruz, Rio de Janeiro, Brasil.

The serotype 2 of dengue virus, circulating in Brazil belongs to the Southern Asia genotype. It was introduced in the country in 1990 when the rst cases of dengue hemorrhagic fever were reported. After seven years without activity, dengue virus type 2 (DENV-2) re-emerged in 2007 causing in 2008 the most notorious epidemics concerning mortality and morbidity. After reemergence, logenetic studies showed two distinct lineages within this genotype. Although molecular characterization of complete viral genome showed no genetic differences to support higher virulence of the 2007 emerging Lineage, studies showed a higher viremia in sera of patients infected with this lineage. In this study we infected BALB/c mice with representative strains of both DENV-2 lineages for susceptibility, viremia and morphological analysis. Groups of ve 2 months old male BALB/c mice were infected with strains of both DENV-2 lineages by the intravenous or intraperitoneal route. 72 hour post-infection, euthanasia was performed. Brain, heart lung, spleen and liver were harvested and divided into two parts. Part of the tissue was xated and processed by transmission electronic microscope techniques for morphological analysis and part was macerated with L-15 medium for RNA extraction and viral detection and quantitation using real time RT-PCR assay. Our preliminary results demonstrated the susceptibility of BALB/c mice to the DENV-2 lineages. Moreover, we observed, in the tissue where the viral genome was detected, morphological alterations similar to the ones reported in human cases of dengue infection.

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DESIGN AND PRODUCTION OF A TETRAVALENT CHIMERICAL PROTEIN USEFUL TO DENGUE VACCINE AND DIAGNOSIS1 1 1 2 1 1 1 1Izabella Batista, Camila de Oliveira, Eneida de Oliveira, Erna Kroon, Rodrigo Corrêa-Oliveira, Jaquelline de Oliveira, Bárbara Quinan, Carlos Eduardo Calzavara-Silva. 1 2 Lab. Imunologia Celular e Molecular, Centro de Pesquisas Rene Rachou - Fiocruz/MG; Lab. Vírus, Departamento de Microbiologia - ICB – UFMG.

Dengue virus (DENV) consists of four antigenically distinct serotypes, known as DENV 1 to 4. Nowadays, no vaccines or antiviral drugs for dengue prevention and treatment are available. A safe vaccine demands a balanced tetravalent immune response, since antibodies produced after a rst infection may not neutralize subsequent heteroserotype infections, increasing viral replication. In this study, an alternative vaccine or diagnosis strategy was proposed by the construction of an articial chimerical protein containing known immunogenic epitopes for all serotypes of DENV. Using bioinformatics tools, the most potential immunogenic regions derived from the known antigenic DENV proteins (envelope, capsid, membrane and non-structural protein 1), were selected. Regions sharing high homology among the four DENV serotypes were preferentially included. Antigenic regions from single or pairs of DENV serotypes were also included. All regions were spaced by amino acid residues to maintain the epitopes structure when the protein is expressed. The nal amino acid sequence of the chimerical protein was back translated and the sequence was optimized to be expressed in bacteria and used to design a 1400 bp minigene, named qDV, coding for a 50 KDa chimerical protein. The qDV minigene was submitted to several others in silico analyses and further commercially synthesized. The qDV was cloned into the expression vector pET TEV 28, able to express in E. coli BL21 a 53 kDa recombinant protein in fusion with a 6 his tagged, qDV-his. The qDV-his was puried by afnity chromatography and used in Western blot to test its reactivity with sera of DENV-infected patients and DENV-negative sera as negative control. Our results show a specic recognition of the protein qDV-his by antibodies in sera DENV-1, DENV-2, DENV-3 or DENV-4 infected patients, indicating that articial proteins can be designed to be used in the vaccine and/or diagnosis development of infectious diseases.

ROUND TABLE BRAZILEPIDEMIOLOGY OF DENGUE IN BRAZIL

Giovanini Coelho1 (SVS)Ministério da Saúde, Brazil

NEW APPROACHES FOR DENGUE DIAGNOSTIC 1 1 1 2 1Marcio Nunes , Clayton Silva , Sandro Patroca , and Pedro Vasconcelos Joao Lidio Vianez jr

1Center For Technological Innovations (CTI) , Molecular Diagnostic and Nanothecnology Core, Evandro Chagas Institute, Brazil.2Department of Arbovirology and Hemorrhagic Fevers (SAARB) , Evandro Chagas Institute, Brazil

Dengue is so far the most import arboviral disease that infect humans worldwide. Despite the absence of a vaccine and lack of an efcient vector control program, the development of novel, high sensitive approaches for early dengue diagnostic is essential for a better patient clinical management. In this context, many different methods have been developed aiming the Dengue virus (DENV) nucleic acid and antigen detection, such as RT-PCR and derived methods (e.g. RT-Nested-PCR, RT-real Time PCR, NS1 antigen detection) using genetic information for several DENV serotypes and genotypes available at the most importante genetic databanks (e.g. GenBank). At the CIT and SAARB, we have developed a new approach for dengue viruses (serotypes) detection employing nanoparticulated assay. This approach was able to capture and distinguish all four DENV serotypes. The method used four Iron Oxyde nanoparticles (10nm of diameter) surrounded with DENV (1-4) monoclonal antibodies against the envelope protein. The nanoimmunecomplexes were able to capture specically each of the DENV serotypes and were used in three distinct assays: 1) Viral genome recovering; 2) Immunosorbent nanoparticulated ELISA assay, and 3) Nanoparticulated-nanosensor assay. In the rst case, viral genomes were completely recovered from clinical samples from a initial volume of 250 uL and a RNA concentration of at least 20 ng/uL. In the second case, the nanoimmune complex was perfectly adapted to a ELISA format and able to detect the presence of envelope protein of DENV (1-4) by optical density reading at 405 nm lter; Finaly, the nanosensors were adapted in a small computer device and were able to read the uorescence emmited by positive samples. In this case, the positive samples were detected by nanosensors adapted to a minicomputer device composed by microchip which display the results in a integrated platform using the Google Earth (https://earth.google.com) allowing the dengue cases surveillance at real time. These aproaches could be helpfull on further dengue outbreaks and perfectly adapted to other arboviriral disease such as Yellow fever, Oropouche fever, Mayaro fever and the Chickungunya fever, recently detected in Brazil

Key words: Dengue virus, Dengue diagnostic, Nanoimmuneassay.

CLINICAL ASPECTS OF DENGUE1 2 2 2 2 2Luz, Kleber , Araujo, Ana ; Souza , Luisa ; Eberlin, Maria ; Guerra, Samia ; Almeida, Yngra .

1Institute of Tropical Medicine of Rio Grande do Norte.2Graduate students of UFRN.

Dengue is the most important arboviral disease in the world. About two to seven days after the bite of the Aedes the patient may develop the disease clinically. Early a backache in association or not with fever arises. The onset of fever marks the onset of the disease. The clinical course of Dengue may be divide in periods. The rst period corresponds to the rst three days. It has been characterized by high fever, headache, arthralgias, myalgias, abdominal pain, vomiting and sometimes rash. In the second period, there will be an apparent improvement with reduced levels of temperature. After this period the patient will be in the convalescence. It is exactly in the second period that capillary leakage may occur. In this case along with capillary leakage, if there is the perception of trends bleeding you may be evolving into a more severe form of the disease. This clinical form is mainly responsible for the causes of death. However, clinical other manifestations may be present and pose a greater risk of death. Severe organic impairment is therefore a risk of death. Myocarditis with decreased ejection fraction of the left ventricle, a signicant increase in levels of transaminases, especially above 1,000 international units, impaired consciousness, but especially a picture of respiratory distress should be interpreted as danger signals and an attitude should be taken in these patients. Is very important search for signs of bleeding. The tourniquet test should be performed, and the search for bleeding in mucosa. An important clinical aspect is the evaluation of hemodynamic and research of clamping pressure, which may be present in severe forms, it must be researched. Classication of cases can facilitate detection of severe cases, but it can be confusing to the clinician the basic health network. We reviewed 1320 cases of dengue were reported from 2011 to 2013, only 295 of the total classication was concordant in 347 and there was no agreement between the classications. In 678 the information did not allow a proper classication.

CONTROL OF DENGUE VECTORS IN BRAZIL1 1Rodrigo Lins Frutuoso , Ima Braga

1Ministério da Saúde, Brazil

Dengue is presently one of the most important reemerging diseases in the world. In Brazil, since the 1980's, there is an intense virus circulation with epidemic bursts affecting all the regions of the country. Nowadays, about 70% of the Brazilian municipalities are infested with the dengue vector, Aedes aegypti, and the four serotypes of dengue are circulating in the country.

In Brazil the activities of prevention and control of transmissible diseases are executed by the municipalities and nanced by the Federal Government through the National Health Fund. Dengue National Control Program is coordinate by the National Health Surveillance Secretariat who establishes guidelines that normalizes the activities executed by health municipalities programs including dengue vector control. Currently, about 60,000 vector control agents working in the municipal dengue control programs.

The main vector control actions are targeted for elimination or treatment of mosquito breeding sites. Since 1999 it was established in Brazil the management of Aedes aegypti resistance to insecticides using rotation of products because the resistance to temephos. Currently, growth regulators are used as larvicidas. When occur dengue transmission additional measures using space spray application of insecticides are used.

Some initiatives such as the LIRAa, a sampling methodology of the vector in a short time to direct efforts to control and disposal of tires with the support of Tire Manufacturers Association (ANIP) has contributed to improve vector control actions.

Despite the great effort expended to the vector control actions there are several challenges to the implementation of these control activities in large and medium-sized cities where the complexity of contemporary urban life generates factors that facilitate Aedes aegypti's proliferation and constraints on the reduction of its infestation rates.

CLINICAL RESEARCH/COST STUDIESABSTRACTS INVITED SPEAKERS

CLINICAL PREDICTORS OF SEVERITY IN DENGUE; AN APPROACH TO THE FIRST MOMENT OF THE MEDICAL EVALUATION1 2 2 2 1 2 3 4 5Villar L , Parra B , Bonelo A , Osorio L , Ocazionez R Martinez E , Romero C , Bosch I , Salgado D and AEDES Study Group, Colombia

1 2 3 4 5Universidad Industrial de Santander , Universidad del Valle Universidad del Norte Massachusetts Institute of Technology, USA, Universidad Surcolombiana.

Dengue is potentially fatal disease. To identify early clinical ndings in patients with higher risk of complications we conducted a multicentric prospective cohort study in different cities in Colombia including during 2003 -2008 ( endemic period) and 2009 – 2011 ( epidemic period) acute febrile patients in the rst 48 to 96 hours of illness and serologic or virologic evidence of dengue. Data collected followed a common methodology. Socio-demographics, warning signs (WHO 2009 ) were ascertained at baseline. Patients were followed for complications ( composite outcome) dened as new-onset hypotension (age-specic tachycardia and pulse pressure <20 mmHg or systolic or mean blood pressure below PAHO's age-specic cut-points for hypotension) or severe hemorrhage hematemesis, melena, hematochezia or hematuria). From a total of 1,198 at risk patients, we observed 178 (14.9%) composite outcomes among patients at risk: 159 (13.3%) due exclusively to hypotension, 15 (1.3%) due exclusively to major hemorrhage, and 4 (0.3%) due to the both . Somnolence or irritability was the most common clinical sign of alert at baseline, followed by abdominal pain or tenderness, minor hemorrhage, persisting vomiting, clinical uid accumulation, and liver enlargement. All six clinical signs of alert were consistently and statistically more prevalent among patients who progressed toward the composite outcome during follow-up . Nausea, and dehydration also were more prevalent among incident than non-incident cases. A high negative predictive value was observed when all signs of alert were absent (95.1%, 95%CI: 90.1 – 98.0%). After adjusting for patient's age and febrile syndrome's duration at enrollment, the presence of somnolence or irritability ad others than dehydration body mass index and standing mean blood pressure at baseline increased the likelihood of progression to hypotension and/or major hemorrhage.

PROGNOSTIC VALUE OF WARNING SIGNS IN RELATION TO DENGUE SHOCK1 1,2 1 1 2 3 4Gamaliel Gutiérrez , Federico Narváez , Lionel Gresh , Douglas Elizondo , Maria Angeles Pérez , Angel Balmaseda , Eva Harris

1 2 3Sustainable Sciences Institute, Managua, Nicaragua; Hospital Infantil Manuel de Jesús Rivera, Ministerio de Salud, Managua, Nicaragua; Laboratorio Nacional de 4Virología, Centro Nacional de Diagóstico y Referencia, Ministerio de Salud, Managua, Nicaragua; Division of Infectious Diseases and Vaccinology, School of Public

Health, University of California, Berkeley, CA, USA

In 2009, the World Health Organization revised its dengue guidelines, introducing a new severity classication as well as the notion of warning signs (namely, abdominal pain, persistent vomiting, clinical uid accumulation, mucosal bleeding, restlessness/lethargy, enlarged liver, and haematocrit increase concurrent with rapid decrease in platelets). According to the guidelines, patients with warning signs require strict observation and medical intervention. Here, we analyzed the prognostic value of warning signs, using data from a prospective study of dengue based at the National Pediatric Reference Hospital in Managua, Nicaragua. Shock, one of the most common severe manifestations in dengue patients, was used as a marker of severe outcome. A total of 812 laboratory-conrmed dengue cases were identied from August 2005 to January 2013, of which 221 (27.2%) progressed to shock (compensated or hypotensive). All warning signs, except persistent vomiting, were associated with shock (p<0.05). In contrast, when only considering warning signs that appeared before the onset of shock, warning signs were not associated with shock. In fact, patients who exhibited at least one warning sign were less likely to progress to shock than those who didn't (relative risk (RR) 0.42, 95%CI: 0.34-0.52). Moreover, the higher the number of warning signs identied in the patient, the lower the risk of shock (p<0.0001). These results suggest that the recognition of the warning signs leads to effective medical intervention to prevent shock. Consistent with this hypothesis, the reduction in the risk of developing shock was greater in the period after the revision of the WHO guidelines (2009-13: RR 0.32, 95%CI: 0.24-0.43) when warning signs received greater emphasis, than in the period before (2005-08: RR 0.62, 95%CI: 0.41-0.92). Further analysis of the association of warning signs and medical intervention are ongoing. In conclusion, these results suggest that the identication of warning signs is useful for early medical intervention, helping clinicians to prevent shock in patients with dengue.

ECONOMIC AND DISEASE BURDEN OF DENGUE IN MEXICO

Eduardo A. Undurraga,1 Miguel Betancourt-Cravioto,2 José Ramos-Castañeda,3,4 Ruth Martínez-Vega,3,5 Jorge Méndez-Galván,6 Duane J. Gubler,7 María G. Guzmán,8 Scott B. Halstead,9 Eva Harris,10 Pablo Kuri-Morales,11 Roberto Tapia-Conyer,2 and Donald S. Shepard1

1Brandeis University, Waltham, Massachusetts; 2Carlos Slim Health Institute, Mexico City, México; 3Instituto Nacional de Salud Pública, Cuernavaca, Mexico; 4Center for Tropical Diseases, University of Texas-Medical Branch, Galveston, Texas; 5Organización Latinoamericana para el Fomento de la Investigación en Salud, Bucaramanga, Colombia; 6Hospital Infantil de México Federico Gómez, Mexico City, Mexico; 7Duke-NUS Graduate Medical School, Singapore; 8Pedro Kouri Tropical Medicine Institute, Havana, Cuba; 9Pediatric Dengue Vaccine Initiative, Rockville, Maryland; 10University of California, Berkeley, California; 11Ministry of Health, Mexico City, Mexico

Dengue incidence and severity have dramatically increased in Mexico during the past decades. Having objective and comparable estimates of the economic burden of dengue is essential to inform health policy, increase disease awareness, and assess the impact of dengue prevention and control technologies. More so, considering recently available preliminary results from a phase III clinical efcacy trial of a dengue vaccine candidate in the Americas. We estimated the annual economic and disease burden of dengue in Mexico for years 2010-2011. We merged multiple data sources, including a prospective cohort study in Morelos, patient interviews and macro-costing from major hospitals, surveillance, budget, and health data from the Ministry of Health, WHO cost estimates, and available literature. Results suggest that Mexico had about 139,000 (95%CL: 128,000-253,000) symptomatic and 119 (95%CL: 75-171) fatal dengue episodes annually on average (2010-2011), compared to an average of 30,941 symptomatic and 59 fatal dengue episodes reported. The annual cost, including surveillance and vector control, was US$170 million (M) (95%CL: 151M-292M), or $1.56 (95%CL: 1.38-2.68) per capita, comparable to other countries in the region. Of these, 87 M (95%CL: 87M-209M) or $0.80 per capita (95%CL: 0.62-1.12) correspond to illness. Annual disease burden averaged 65 (95%CL: 36-99) disability-adjusted life years (DALYs) per million population. Inclusion of long-term sequelae, co-morbidities, impact on tourism, and health system disruption during outbreaks would further increase estimated economic and disease burden. With this study, Mexico joins Panama, Puerto Rico, and Thailand as the only countries or areas worldwide with comprehensive (illness and preventive) empirical estimates of dengue burden. In sum, Mexico's potential economic benets from dengue control would be substantial.

ABSTRACTS ORAL PRESENTATIONSASSOCIATED FACTORS TO PLASMA LEAKAGE IN DENGUE PATIENTS FROM ANTIOQUIA AND CHOCÓ BETWEEN 2000 AND 2013

1 1 2 3 3 1Ivony Yireth Agudelo , Leidy Diana Piedrahita , Luis Gonzalo Álvarez , Efrén Avendaño , Gabriel Bedoya , Berta Nelly Restrepo1 2 3Instituto Colombiano de Medicina Tropical- Universidad CES; Universidad CES, Medellín, Colombia; Universidad de Antioquia, Grupo de Genética Molecular, Medellín, Colombia

Introduction: Plasma leakage is the most severe complication caused by dengue virus infection and is also the mechanism that frequently leads to dengue shock syndrome. Plasma leakage is characterized by ascites, pleural and pericardial effusion and increased haematocrit level. Objetive: To identify clinical and laboratory factors that explains the presence of plasma leakage in dengue infected patients. Materials and methods: A cross sectional analytic study was performed on clinical records from dengue infected patients using the case-control methodology. Pearson's chi-square test or Fisher´s exact test, Mann-Whitney's non-parametric U test and a multivariate logistic regression model with explicative purpose were used to evaluate the data. Odd Ratios with 95 % condence intervals were calculated and signicance level of 5 % was considered. Results: A total of 350 clinical records were analyzed, 128 cases (36.6 %) presented signs of plasma leakage. After adjustments by confounding factors, we observed that abdominal pain (OR=2.46; IC95 % 1.13-5.4), leukopenia (OR=2.24; IC95 % 1.05-4.74) and melaena (OR=3.09; IC95 % 1.16-8.21) were the variables that explain the presence of plasma leakage in dengue infected patients. Conclusion: During the follow-up of dengue infected patients priority should be given to the survey of these clinical and laboratory ndings in order to identify the cases with higher probabilities of developing plasma leakage, allowing achieving more timely and appropriate management.

DENGUE IN PREGNANT WOMEN: CHARACTERISTICS OF THE REPORTED CASES IN BRAZIL, 2007 - 2013

Nascimento LB, Siqueira CM, Siqueira KPG, Siqueira Jr JB

Universidade Federal de Goiás, Brazil

Objective: To describe the characteristics of the reported dengue cases among pregnant women. Methods: We conducted a retrospective descriptive study of the reported dengue cases among pregnant women using the data from the national surveillance system from 2007 to 2013. Duplicated cases and inconsistent data were excluded. Cases were described according to age, year of onset of symptoms, trimester of pregnancy, nal classication (dengue, dengue with complications (DCC), dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS)) and hospitalization. Case fatality rate (CFR) was calculated as the number of deaths due to dengue among severe cases (DCC and DHF/DSS). Results: A total of 30,085 probable dengue cases in pregnant women were reported in the study period, ranging from 2,278 in 2012 to 7,143 in 2013. In general, 42,4% of the cases were classied as probable by laboratory tests (IgM+) and 57,6% by clinical/epidemiological criteria. The age interval ranged from 12 to 45 years of age with 11,630 cases in the 20 to 29 age group. The distribution of the cases according to the trimester of pregnancy was rst 26.7%, second 32.0%, third 23.0% and 18.4% missing information. 5.6% of the cases were hospitalized, although only 1.8% and 0.6% of the total cases were classied as DCC and DHF/DSS respectively. The main clinical outcomes of DCC cases related were suspected DHF cases that do not fulll all denition criteria (41.2%) and platelets count <20.000 mm3 (39.7%). There were 48 deaths observed among the severe cases yielding a CFR of 6.5%. Conclusion: Our results highlight that the number of pregnant women with symptomatic dengue infection follows the national trends of incidence and increase in severity. It is necessary to prepare the health system to recognize and manage this group of patients.

Financial support by Sano.

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DYNAMIC OF PLASMA CYTOKINES AND CYTOKINES RECEPTORS IN PEDIATRIC DENGUE AND SEPSIS: A COMPARISON

Doris M. Salgado, Martha Rocío Vega, Jairo A. Rodríguez and Carlos F. Narváez

Universidad Surcolombiana, Colombia

Pediatric dengue and sepsis are life-threatening infections. Shock and hemorrhage are serious and common symptoms for both conditions. Cytokines and their receptors play a critical role in their pathophysiology and some of them such as TNF-α, IL-6 and IL-10 have been associated with severe clinical outcome in dengue and sepsis studies. Due the high use potential of these biomarkers for diagnosis, clinical follow-up and treatment, it is necessary to know the plasma cytokines and cytokines receptor pattern for each condition. We determine the patterns of cytokines and their receptors in 180 children with several clinical ranges of dengue infection classied according the 2009 WHO guide, and 60 children (all from 1 to 14 years old) with conrmed sepsis. In most cases, two samples in acute as well as one in convalescent phase were collected. Plasma levels of VEGF, IL-6, IL-8, IL-10, VEGF RII, TNF RII, sIL-2Ra and ST2, were evaluated by beads based array or ELISA. Compared with convalescence, acute dengue and sepsis signicantly increased all tested markers (P<0.01, Wilcoxon test), except the VEGF and VEGF RII, whose levels were higher in convalescence. Evaluated biomarkers were early induced (3-4 days after fever onset) and lower levels of them were detected in acute late time (5-7 days). In acute period, plasma VEGF, IL-6, IL-8, VEGF RII and sIL-2 Rα were signicantly higher in sepsis than dengue. Interestingly, only IL-10 (a regulatory cytokine) and TNF RII were higher in acute dengue (P<0.02, Mann-Whitney test). Plasma ST2 was not different in both. When levels of biomarkers were analyzed in dengue-infected children, VEGF, IL-6, IL-8 IL-2 Rα, TNF RII and ST2 were related with clinically severe forms. Thus, pattern of plasma cytokines circulating in pediatric dengue and sepsis is highly different and it could have a potential as therapeutic target. Funded by Colciencias.

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VITAMIN D RECEPTOR-352 AND TNFA-308 SHOWED PROTECTION/SUSCEPTIBILITY ROLES IN DENGUE INFECTION IN HONDURAS

Cynthia M. Rodríguez, Ivette Lorenzana de Rivera

National Autonomous University of Honduras

Introduction. Dengue is a problem of health public in tropical and sub-tropical countries, where dengue cases increase every year. Variations in immune response could be related to polymorphisms in regulatory regions of cytokine genes and other molecules such as Vitamin D receptor and the immune regulatory effects, a thus is in turn may have inuence on the disease severity. Objective. To evaluate the association of polymorphic variants in Vitamin D receptor 352 and TNF alpha 308, among individuals with different dengue disease presentation. Material and methods. The study was carried out in Tegucigalpa, Honduras. The study population consisted of 500 participants: 285 dengue cases (154 with alarm signs) and 215 asymptomatic controls (AC), all of them laboratory conrmed. The dengue classication was done following the new WHO criteria. The selected single-nucleotide polymorphisms (SNPs) were carried out by a Real Time PCR methodology, using sequence specic primers, obtaining the allelic genotype for each investigated genetic marker. Results. In VDR-352 polymorphism, the TT genotype was observed in 59% of in the AC and this was decreasing as disease severity increases, and TC genotype showed a signicant risk in severe forms of the disease compared with the AC (OR: 1.6, p=0.02). For TNFa-308 polymorphisms it was observed that 75% of severe cases had GG genotype. Conclusion. These ndings are in agreement with previous studies performed by our group with VDR-352 and TNFa-308. A study by Loke et al, in VDR-352 shows how the T allele is associated with severity in Vietnamese. For TNFa-308 in opposition to Fernandez-Mestre that nds that the A allele is associated with severity in Venezuelan population. We believe that these are the rst steps to obtain a prognosis of the severity of disease in our population.

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DENGUE 2 VIRUS INFECTION WITH ACUTE MYOCARDITIS IN THE PERUVIAN AMAZON: A CASE REPORT1 2 2 1 1 1, 3Crystyan Siles , Moises Sihuincha , Carlos Vidal-oré , Julia S. Ampuero , Stalin Vilcarromero , Amy C Morrison Robert D Hontz1, Daniel G Bausch1, 4

1 2 3 4 U.S. Naval Medical Research Unit No. 6, Lima, Peru; Hospital Apoyo de Iquitos, Iquitos, Peru; University of California, Davis. USA, Tulane School of Public Health and Tropical Medicine, New Orleans, USA

Cardiac involvement is uncommon in dengue fever. We report a case of a 28-year-old woman with no history of cardiovascular or other chronic disease who developed acute myocarditis with dengue 2 virus infection. The patient presented with a history of four days of worsening fever, chest and joint pain, dyspnea, persistent vomiting and vaginal bleeding. On physical examination, she presented with a axillary temperature (T) of 39 ° C, heart rate (HR) of 48 beats per minute (bpm), respiratory rate (RR) of 24 per minute, and blood pressure (BP) of 80 / 60 mmHg. She further presented marked pallor, a generalized rash, and a positive Murphy sign. Initial routine blood testing showed mild thrombocytopenia (122000xmm³). Cardiomegaly was notice on a chest X-ray (cardiothoracic ratio 0.5) and an electrocardiogram revealed sinus bradycardia (45bpm). Echocardiography demonstrated mild mitral insufciency with an ejection fraction of 50% and normal diastolic function. Subsequent cardiac-specic analyses revealed elevated troponin I (0.19ng/mL) and B-type natriuretic peptide (4556pg/mL) on 7th day of illness. Ultrasound of the abdomen showed thickening of the gallbladder wall. Dengue virus serotype 2 was conrmed by real-time PCR. The patient was diagnosed with dengue fever with acute myocarditis. During hospitalization she showed persistent bradycardia (45-50xmin) with arterial hypotension (80/50mmHg) requiring the use of dobutamine for two days. Her symptoms resolved and she was discharged on the 13th day of illness in good health with normal vital signs, electrocardiogram, chest x-ray, and cardiac biomarkers were normalized as documented during follow-up at 3 months. Cardiac complications during dengue disease such as myocarditis have been documented

and physicians should be aware of heart manifestations even without specic symptoms upon initial presentation. Specic clinical exams and laboratory analysis in these patients could help to establish an early diagnosis and prevent excessive morbidity and mortality among dengue patients.

HP21

DENGUE HEMORRHAGIC FEVER IN PATIENTS FROM NORTHEAST OF BRAZIL IS ASSOCIATED WITH HIGH LEVELS OF INTERFERON-α AT ACUTE INFECTION.

Mayara Marques Carneiro da Silva; Renato A S Oliveira; Carlos Eduardo Calzavara-Silva; Marli Tenório Cordeiro; Patrícia Muniz Mendes Freire de Moura; Paulo Neves Baptista Filho; Ernesto Torres de Azevedo Marques Júnior; Laura Helena Vega Gonzales Gil

Aggeu Magalhães Research Center/Fiocruz, Brazil

Dengue is an acute febrile disease caused by the mosquito-borne dengue viruses. Fifty million new cases are reported each year, mainly in tropical regions. Interferon- α and β are expressed to different levels in dengue fever (DF) compared with dengue hemorrhegic fever (DHF) patients. Interferon-α�/�β�can be secreted�by all nucleated cells and are the main cytokines involved in the antiviral responses. Our results demonstrated that high levels of circulating interferon- � and low-level viremia were associated with primary DHF, suggesting that, in patients from Northeast Brazil, DENV3 infection elicits an innate response characterized by an increased secretion of IFN- � in the primary dengue hemorrhagic variant. These ndings shed lights of the immune response role in the dengue hemorrhagic fever development, and contribute to a better understanding of dengue pathogenesis.

POSTERS SESSION CLINICAL RESEARCH/COST STUDIESCR-P01

DENGUE VIRUS INFECTION WITH NEUROLOGICAL MANIFESTATIONS, BRAZIL

Campos, M.A., Oliveira, D.B. Machado, G.; Almeida G.M.F. , Abrahão, J.S., Kroon, E.G.

Instituto Rene Rachou, Fiocruz, Brazil

Dengue virus (DENV) is most an important mosquito-borne disease of humans. In Brazil more than 200.000 cases were reported in the rst 45 days of 2013. The different DENV (1,2,3,4 and 5) might be usually associated to a systemic and dynamic disease. Among the plethora of clinical symptoms caused by DENV, neurological manifestations has been undernotied or neglected. In this work we describe a case report of a patient presenting neurological manifestations likely associated to DENV-3. This case occurred in Belo Horizonte, Minas Gerais State, Brazil. In October 2012, our lab was contacted by a local Hospital to perform the diagnosis of a 21 old patient that presented dengue symptoms and also neurological symptoms. The patient stayed in hospital for 27 days presented wide range of neurological manifestation as confusion and behavior alterations. With the aim of characterizing the etiological agent, the samples were submitted to RNA extraction (Viral RNA QIAamp Extraction Kit, Qiagen, USA) followed by reverse transcription using random primers (MMLV, Promega, USA). The cDNA was used as the template in a real time PCR designed to amplify the NS5 region of virus of genus Flavivirus. qPCR was performed with commercial mix (SYBR® Green PCR Master Mix. Life Technologies,USA). Specic amplication in the real time PCR with a melting temperature of 79,5ᴼ C was observed for the sample (CSF). To identify the member of genus Flavivirus that was the etiological agent of such infection, a specic PCR for Dengue virus was performed conrming DENV as the etiological agent.The optimal alignment of the C-prM region showed high identity among the nucleotide sequences of the sample and other DENV 3 sequences deposited in GenBank. The levels of cytokines IL-6, IL-10, IL-8 and TNF alpha measured in CFS by CBA kit, did not show alterations when compared with CSFs from uninfected individuals.

CR-P02

DENGUE INFECTION A COMMON CAUSE OF FEBRILE ILLNESS IN PATIENTS AFRODESCENDANTS OF QUIBDO, CHOCO, COLOMBIA1 1 1 2 1Berta Nelly Restrepo , Leidy Diana Piedrahita , Ivony Agudelo , Katherine Marín , Ruth Ramírez

1Instituto Colombiano de Medicina Tropical, Universidad CES, Sabaneta, Colombia.2Institución Universitaria Colegio Mayor de Antioquia, Medellín, Colombia.

Introduction. Quibdo town in Chocó, has all the conditions that allow dengue transmission, poor public services, climatic and social requirements, however there are few studies about this problem in that place. In this city more than 90% of the population of African descent. Objective. To determine frequency of dengue infection in patients with acute febrile illness and compare the clinical features among dengue infected patients and other febrile cases. Materials and methods. A cross-sectional study was conducted from January 2008 to March 2010. The study population consisted of patients with febrile syndrome of less than 7 days of evolution, recruited from hospitals of the town. Dengue diagnosis was made in serum samples by detection of IgM antibodies, NS1 antigen, RT- PCR and virus isolation. Clinical and laboratory information were obtained. Results. During the study period were recruited 469 patients with acute febrile illness; of these 98.3 % were African descent. Dengue fever was found in 28.4 % of the cases. The four dengue serotypes were identied with predominance OF DENV -1. The age range was 0 to 76 years. The 70.7 % of cases were classied as dengue without warning signs. Nausea, vomiting, abdominal pain, cough, nasal congestion and sore throat were signicantly more frequent in patients with other febrile illness. Conclusions. Active surveillance of acute febrile syndrome allowed detection of undiagnosed cases of dengue and the observed frequency of this infection suggests that the study area has a high risk of dengue infection.

CR-P03

DYNAMIC OF PLASMA CYTOKINES AND CYTOKINES RECEPTORS IN PEDIATRIC DENGUE AND SEPSIS: A COMPARISON

Doris M. Salgado, Martha Rocío Vega, Jairo A. Rodríguez and Carlos F. Narváez

Universidad Surcolombiana, Colombia

Pediatric dengue and sepsis are life-threatening infections. Shock and hemorrhage are serious and common symptoms for both conditions. Cytokines and their receptors play a critical role in their pathophysiology and some of them such as TNF-α, IL-6 and IL-10 have been associated with severe clinical outcome in dengue and sepsis studies. Due the high use potential of these biomarkers for diagnosis, clinical follow-up and treatment, it is necessary to know the plasma cytokines and cytokines receptor pattern for each condition. We determine the patterns of cytokines and their receptors in 180 children with several clinical ranges of dengue infection classied according the 2009 WHO guide, and 60 children (all from 1 to 14 years old) with conrmed sepsis. In most cases, two samples in acute as well as one in convalescent phase were collected. Plasma levels of VEGF, IL-6, IL-8, IL-10, VEGF RII, TNF RII, sIL-2Ra and ST2, were evaluated by beads based array or ELISA. Compared with convalescence, acute dengue and sepsis signicantly increased all tested markers (P<0.01, Wilcoxon test), except the VEGF and VEGF RII, whose levels were higher in convalescence. Evaluated biomarkers were early induced (3-4 days after fever onset) and lower levels of them were detected in acute late time (5-7 days). In acute period, plasma VEGF, IL-6, IL-8, VEGF RII and sIL-2 Rα were signicantly higher in sepsis than dengue. Interestingly, only IL-10 (a regulatory cytokine) and TNF RII were higher in acute dengue (P<0.02, Mann-Whitney test). Plasma ST2 was not different in both. When levels of biomarkers were analyzed in dengue-infected children, VEGF, IL-6, IL-8 IL-2 Rα, TNF RII and ST2 were related with clinically severe forms. Thus, pattern of plasma cytokines circulating in pediatric dengue and sepsis is highly different and it could have a potential as therapeutic target. Funded by Colciencias.

CR-P04

THE DENGUE VACCINE INITIATIVE (DVI) PROJECT: PASSIVE FACILITY-BASED FEVER SURVEILLANCE IN CHILDREN AND ADULTS OF MEDELLIN, COLOMBIA1 1 2 2 2 1 2 3Jacqueline Lim , Mabel Carabali , Catalina Le Cacheux , Diana Carolina Velez , Andrea Trujillo-Correa , Kang Sung-Lee , Ivan D. Velez , Jorge Osorio

1 2Dengue Vaccine Initiative, International Vaccine Institute, Seoul, Korea; Programa de Estudio y Control de Enfermedades Tropicales (PECET), Universidad de Antioquia, 3Medellin, Colombia; and Biomedical Science Laboratories, School of Veterinary Medicine, University of Wisconsin

Dengue virus (DENV) infection is a major public health problem in Colombia, causing more human illness than any other arbovirus. Dengue Vaccine Initiative (DVI) conducted a passive facility-based fever surveillance to determine the true burden of dengue fever in the Santa Cruz comuna of Medellin, Colombia. The surveillance was based in Santa Cruz Hospital and three other primary health care centers afliated to METROSALUD E.S.E. Individuals between 1-65 years-of-age were evaluated for febrile episodes (≤7 days of evolution). Two paired samples were obtained from all participants; an acute sample (0-7 days of fever) and a convalescent sample 10-14 days after the rst visit. All samples were tested with rapid diagnostic test SDR Dengue Duo during the acute phase, and both acute and convalescent samples were analyzed with IgM/IgG capture ELISA. The positive cases on ELISA were tested with RT-PCR for serotyping. From Oct. 2011 until Feb. 2014, there were 579 enrollees in the surveillance with 150 (26%) laboratoryconrmed with dengue fever. Higher proportions of dengue-conrmed cases were found in children between 5-9 (20%) and in young adults between 15-24 (18.7%) years of age. The mean fever duration was longer for those dengue cases at 5.2 days compared to non-dengue cases 4.5 days (p-value=0.007). The majority of our dengue cases were outpatients (n=137) with only 9% of hospitalized patients. About 25% of the dengue cases had secondary infection where as 75% had primary infection. The RT-PCR results show that DENV-3 with47.6% is the most commonly circulating serotype, followed by DENV-1 with 9.5%. All signs and symptoms were similar between dengue patients and non-dengue febrile patients, except for rash and retro-orbital pain found more commonly among dengue cases. Our study results show patterns of the elevated dengue transmission in Santa Cruz comuna. Also, we observed some correlation between the national surveillance data from Medellin and our study ndings. More information will be presented at the conference.

CR-P05

THE DENGUE VACCINE INITIATIVE PROJECT: SERO-PREVALENCE STUDY IN CHILDREN AND ADULTS OF SANTA CRUZ COMUNA OF MEDELLIN1 1 2 2 2 3Mabel Carabali , Jacqueline Lim , Carolina Velez , Andrea Trujillo-Correa , Kang Sung-Lee, Iván D. Velez , Jorge Osorio

1 2Dengue Vaccine Initiative, International Vaccine Institute, Seoul, Korea; Programa de Estudio y Control de Enfermedades Tropicales (PECET), Universidad de Antioquia, 3Medellín, Colombia; and Department of Pathobiological Sciences, University of Wisconsin

Dengue is a major public health problem in Colombia. During the last 10 years, there has been a signicant increase in the number cases of dengue, with circulation of all four serotypes and epidemic waves occurring every 3-4 years. Dengue Vaccine Initiative (DVI) conducted a serological survey to determine the true burden of dengue due to inapparent infections in the Santa Cruz comuna of Medellin, Colombia. The serosurvey included two years of follow up with ve bleedings, with six-month intervals from Nov. 2011 to Feb. 2014. More than 2000 randomly selected residents were enrolled in the study. To estimate the incidence and the age-specic sero-conversion rates on samples that showed a rise in IgG antibody titers by IgG Indirect ELISA. Through 5 bleedings, replacements were made to keep an open cohort of 2000 randomly selected residents. The sero-prevalence of IgG antibodies in each sero-survey were: 59.02% in S1; 63.9% in S2; 64.9% in S3; 64.7% in S4, and 64.7% in S5. The overall sero-conversion rate during the rst year was of 4.8%. The highest sero-conversion rate between S1 and S3 was found among children 5-9 years of age (7.3%), followed by young adults between 35-44 years-of-age (6.9%) and children 1-4 years old (5.6%). From the preliminary PRNT performed on a subset of sero-converted samples (n=48), 83.7% showed an immune response against all 4 serotypes, with DENV-3 (24.5%) and DENV-1 (16.3%) as the predominant serotypes. These sero-prevalence results may indicate an elevated dengue transmission in Santa Cruz and a higher risk for severe forms with increase in age. Results of further analyses of the immunological status of individuals in this geographic area will be presented at the conference.

VECTOR BIOLOGYABSTRACTS INVITED SPEAKERS

TARGETING DENGUE IN THE MOSQUITO

George Dimopholous

W. Harry Feinstone Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland, USA

The understanding of Aedes aegypti - dengue virus interactions is crucial for the development of novel disease control strategies. The virus relies on numerous conserved host factors for its propagation from the mosquito gut to the salivary gland. Upon ingestion of an infected blood meal, the virus encounters the mosquito's innate immune system and the gut microbiota that can inhibit infection. We have explored the potential of conserved virus host factors, the mosquito immune system and the mosquito microbiota, for the development of novel dengue control strategies. The dengue virus uses the human and Ae. aegypti cellular machinery to establish infection. Many of the dengue virus host factors are conserved between the two host species and could be chemically inactivated to block virus infection. Several host factor inhibitors have already been developed to block virus infection in the human. We have explored the antiviral properties of human Dengue-targeting compounds in adult mosquitoes, and the feasibility to develop dengue virus transmission blocking strategies with such chemicals. We have shown that the Ae. aegypti JAK-STAT and Toll immune signaling pathways are implicated in anti-dengue defenses through the control of various anti-viral effectors. We have genetically manipulated the JAK-STAT pathway to confer resistance to dengue virus infection, thereby providing proof-of-principle that the mosquito's natural antiviral immunity can be engineered for resistance to the virus. The intestinal microbiota of Ae. aegypti can greatly inuence the mosquito's capacity to transmit the virus. We have identied a specic bacterial isolate that has mosquitocidal activity and mediated inhibition of the virus through bacteria-produced metabolites. These types of bacteria therefore represent powerful tools for the development of dengue biocontrol strategies. Here we discuss different complementary approaches for blocking dengue virus transmission by their mosquito vectors.

METHODS FOR MEASURING NATURAL DENGUE TRANSMISSION FROM HUMANS TO MOSQUITOES1,2 1,3 2 4 2 2 5 2 1 6Amy C. Morrison , Kanya C. Long , Juan Sulca , Valerie Paz-Soldan , Helvio Astete , Isabel Bazan , Hugo Jaba , Robert Hontz , Thomas W. Scott , Louis Lambrechts .

1 2 3Department of Entomology, University of California, Davis; Department of Virology and Emerging Infections, Naval Medical Research Unit No. 6 (NAMRU-6), Current 4afliation, Department of Biology, Andrews University; Department of Global Health Systems and Development, Tulane University School of Public Health and Tropical

5 6Medicine; Department of Entomology, NAMRU-6, Department of Genomes and Genetics, Insect-Virus Interactions Group, Institut Pasteur, Centre National de la Recherche Scientique URA 3012, Paris, France.

Despite substantial evidence showing that dengue virus (DENV) infection of Aedes aegypti is dependent on virus dose and that disease severity in humans is positively correlated with higher viremia levels, few studies have examined the ability of viremic humans with milder disease outcomes to infect mosquitoes. We present human-to-mosquito transmission studies for 51 naturally infected people (10-73 years-old) with symptoms from clinically inapparent to requiring hospitalization captured through active and passive febrile surveillance, and contact cluster investigations. DENV viremic participants fed 25-50 laboratory reared (F2) Ae. aegypti mosquitoes directly on their arms or legs, and provided blood samples with no additive and with EDTA for exposure of mosquitoes in an articial membrane feeder. Mosquitoes were then incubated for 14d and tested for DENV infection (midgut) and dissemination (head) by RT-PCR. Serum virus titers were quantied by focus-forming assay (FFA). Overall, the direct feeding method was well tolerated and accepted by study participants, an observation consistent with results from 12 focus groups, 7 of which had respondents participating in ongoing longitudinal cohort studies and the remainder not. When proper information is provided direct mosquito feeding was acceptable to community members and in many cases preferable to blood draws. Overall, feeding rates were signicantly higher for direct (79%) compared to indirect (42-48%) methods. Serum titers ranged from 8x102 to 2x106 FFU/ml, however among 17 people who had no detectable titer by FFA, 11 (65%) infected mosquitoes of which 8 (73%) resulted in disseminated infections. Although the number of comparisons with samples containing EDTA were low, there is a trend indicating that infection rates are highest by this method, an observation that would contraindicate its use in larger scale studies. Infection rates for direct feeds were signicantly higher than for indirect feeding with no additive (p=0.0032) and varied by severity of illness. The implications of these ndings for future studies will be discussed.

CHANGING PARADIGMS IN THE SURVEILLANCE AND CONTROL OF DENGUE VECTORS

Roberto Barrera

Dengue Branch, Centers for Disease Control and Prevention, San Juan, Puerto Rico.

Accurate estimation of vector populations is necessary to determine the effectiveness of vector control programs. However, establishing how many individuals exist in a local population of dengue vectors has been challenging. There are three sampling techniques that allow an approximate estimation of the total numbers of Aedes aegypti in a given area (absolute density) but they are rarely used in operative vector control programs. They are mark-release-recapture, indoor/outdoor aspiration of adult mosquitoes, and pupal surveys. More commonly used are estimations of presence / absence data, such as larval surveys and its derived Stegomyia indices (House, Container, and Breteau indices), and measures of relative density such as oviposition (eggs per ovicup, percentage of positive ovicups) and number of females attempting to bite human subjects (landing rates). A number of new traps are now available that allow more practical estimates of the relative density of adult mosquitoes, such as the electro-mechanical BG-Sentinel trap for host seeking females and a variety of passive traps for gravid females. The availability of these devices allows shifting the main emphasis of vector surveillance to the adult stage, which is the most closely related to virus transmission. Likewise, novel vector control techniques are targeting the adult stages of dengue vectors, including genetically modied mosquitoes, biological control of adults (bacteria, fungi),

insecticide impregnated surfaces (curtains, bednets, covers for water-storage containers, lethal ovitraps, auto dissemination devices), and sticky gravid traps. Thus, greater emphasis is being placed on both surveillance and control of the adult stages of dengue vectors.

PLATFORM MI-DENGUE: A COST-BENEFIT MOSQUITO SURVEILLANCE1 2 3 4Alvaro E. Eiras , Cecília M. Toledo , Tina Stutzman & Brett Ellis

1 2 3 4Universidade Federal de Minas Gerais, Brazil; Ecovec Tld., Belo Horizonte, Brazil; Massachusetts Institute of Technology (MIT), USA; Virgin Islands Department of Health, USA

Monitoring adult Aedes aegypti abundance with xed position traps has been considered as an alternative surveillance method that shows promise for directing vector control and predicting when and where dengue outbreaks will take place. With support from Massachusetts Institute of Technology (MIT) and a partnership between university and a ”spin-off” company, we developed the MI-Dengue Platform. The Platform consist of an integration of continuous vector monitoring, at ne spatial and temporal scales, and information technology platform for near real-time data collection, analysis, and decision-making. The surveillance data generated from the system is used to calculate weekly vector indices and easily discernible geographical hot spots to target vector control resources. Once identied, directing control efforts to high priority areas with virus detection may reduce future virus circulation. Citywide analysis also facilitates the detection of novel serotype introduction to the city, which can be an indicator for dengue epidemics. Furthermore, the viral analysis of mosquitoes is low-cost and can be implemented with a simple laboratory protocol. The results of two years of MI-Dengue platform 21 cities in Minas Gerais State, Brazil, indicated that over 27,000 dengue cases were prevented (2.7 times lower than conventional methods) and about US$ 7 million were saved in direct and indirect costs. MI-Dengue Platform is an alternative for monitoring other urban vector diseases such as Chikungunya. The Platform was used over 50 Brazilian cities and has been implemented recently in Lee County, Florida (USA).

CAN LETHAL OVITRAPS CONTRIBUTE TO DENGUE PREVENTION AND CONTROL? CHALLENGES TO IMPLEMENTATION AND ACCEPTANCE OF LETHAL OVITRAPS IN PERU AND ELSEWHERE

1 1 2,3Dawn Wesson , Valerie Paz Soldan , and Amy C. Morrison1 2 3Tulane University School of Public Health and Tropical Medicine Department of Entomology, University of California, Davis; Department of Virology and Emerging Infections, Naval Medical Research Unit No. 6 (NAMRU-6)

The objective of this study was to develop a lethal ovitrap (Attractive Lethal OviTrap = ALOT) for dengue prevention. From June 2011 through June2013, we tested the ALOT in dengue-endemic Iquitos, Peru. The study design was a prospective non-randomized controlled trial, where dengue incidence was compared in two adjacent neighborhoods, one with ALOT traps (intervention) and one with standard Ministry of Health (MOH) control procedures (control). Traps were placed at a density of ~3 per residence, with ~85% participation in the intervention area. MOH fumigation to control adult mosquitoes was ongoing in both areas during the study. Entomological indices were monitored in participating households at 3 month intervals, and individuals were monitored serologically through active febrile surveillance (home visits 3x/wk). One year into the trial, dengue incidence as measured by febrile surveillance was 75% lower (0.28 vs. 1.24 per 100 person years) in the intervention area compared to the control area (p<0.0001). After two years, dengue incidence was still signicantly lower in the treatment area (0.38 vs. 0.85 per 100 person years; p=0.0013). The proportion of nulliparous females (compared to parous and/or gravid females) was signicantly higher in the ALOT area, supporting the removal of older egg-laying mosquitoes from the population. Likewise, the Ae. aegypti house index and the ratio of male:female mosquitoes collected were signicantly different between treatment and control areas, suggesting that the ALOTs were selectively removing female mosquitoes from the treatment area. These results suggest that area-wide application with the ALOT could signicantly lower dengue transmission by selectively removing gravid female mosquitoes. Although the study results were promising, challenges remain to expanding ALOT usage and potential for commercialization. These challenges will be discussed in the context of large scale ALOT implementation.

PAT - AEDES TRANSGENIC PROJECT

Margareth L. Capurro and Danilo Oliveira Carvalho

Department of Parasitology, Instituto de Ciências Biomédicas, Universidade de São Paulo

New strategies for mosquito control are under development; and the genetic manipulation is one choice to the ght against the mosquito. The Sterile Insect Technique (SIT), which involves sterilization, mass rearing and release of a great number of sterile male insects on a target area, was the base for RIDL (Release of Insect carrying a Dominant Lethal gene) technology development, once it also comprises mass rearing and release of male mosquitoes, but this time it transfers a lethal gene to their offspring. The OX513A line of Aedes aegypti was evaluated and compared to the wild-type population under open eld conditions. Many parameters were evaluated under lab and eld conditions such as compatibility, competitiveness, longevity and other biological aspects. For eld experiments it was used ovitraps for monitoring (ovitrap index and number of egg/trap) and adult collection (aspiration or BG Sentinel). We demonstrated compatibility among wild-type population and OX513A line with presence of uorescent larvae, a male dispersal up to 200m, recapture along 3.2 days (longevity) and a competitiveness of 0.031. Similar parameters were found in other studies or for similar SIT programs with different species. The 21 million males released in two treated areas were able to suppress Ae. aegypti population in around 70-80% during the study period (number of eggs/trap and ovitrap index respectively). The suppression phase started when 50% of the larvae were transgenic, and population dropdown when it reached 70-80% uorescent larvae. However when releases stopped, the number of eggs/trap and also the ovitrap index increased 1.2 to 4.6 times respectively. No transgenic larvae were found after 6 months after release interruption.

WOLBACHIA IN A DENGUE CONTROL STRATEGY - CHALLENGE BRAZIL

ED Brasil Team, Luciano A. Moreira*

Centro de Pesquisas René Rachou, FIOCRUZ-MG* / IOC-RJ, Brazil

Vector-borne diseases such as dengue, malaria and other arboviruses impose serious burden towards public health in tropical regions of the world. Current control methods mainly rely on insecticides for mosquito control and because of that, resistance against commonly used chemicals is increasingly widespread. Our project involves the use of a naturally occurring bacterium called Wolbachia as a novel biological control agent (www.eliminatedengue.org/riodejaneiro). Wolbachia manipulates the reproduction of their host in order to be vertically transmitted from the mother to offspring. This bacterium is believed to be present in up to 70% of all insect species worldwide but it has never been found in Aedes aegypti, the main vector for dengue. When stably introduced into A. aegypti, Wolbachia was able to block dengue virus transmission and signicantly reduced Chikungunya virus and Plasmodium gallinaceum load in these mosquitoes. Currently, eld tests are been carried out in Australia, Indonesia and Vietnam (as part of the Eliminate Dengue Program), where Wolbachia-containing mosquitoes were able to invade local populations of A. aegypti. Since 2012 we have been working to implement this project in Brazil, involving the introgression of Wolbachia into Brazilian A. aegypti background, eld entomology activities to study mosquito abundance and population structure as well as community engagement activities. Here we will discuss several aspects towards the implementation of the Eliminate Dengue Project in Brazil, showing recent results of mosquito vector competence, eld entomology, community engagement and communication. This strategy has the potential to signicantly reduce the burden of dengue in Brazil and in other endemic countries and is perfectly complementary to current control methods, including vaccines. Eliminate Dengue Brasil is funded in part by the Ministry of Health in Brazil (SCTIEs/ SVS/ CNPq) and by a grant from the Foundation of the National Institutes of Health through the Grand Challenges in Global Health initiative of the Bill & Melinda Gates Foundation (www.eliminatedengue.org).

ABSTRACTS ORAL PRESENTATIONSAEDES AEGYPTI POPULATION STRUCTURE IS DRIVEN BY BOAT TRAFFIC IN THE PERUVIAN AMAZON

1 2 3 4 5 1 1 6Sarah Anne J. Guagliardo , Yoosook Lee , Jacklyn Wong , Yui Yin Chu , Helvio Astete , Gonzalo Vazquez-Prokopec , Thomas W. Scott6, Uriel Kitron , Steven T. Stoddard1 2Department of Environmental Sciences, Emory University, Atlanta, GA; Department of Pathology, Microbiology and Immunology School of Veterinary Medicine,

3 4University of California, Davis; Centers for Disease Control and Prevention, Atlanta, GA; Department of Comparative Medicine, Stanford School of Medicine, Palo Alto, 5 6CA; Naval Medical Research Unit No.6 (NAMRU-6) Iquitos Laboratory, Iquitos, Peru; Department of Entomology and Nematology, University of California, Davis,

In the Americas, as in much of the rest of the world, the dengue vector Aedes aegypti is predominantly found in urban areas. Its presence in rural areas is more limited, and the factors favoring its potential geographic expansion to rural and smaller urbanized settings are poorly understood. In the Peruvian Amazon, this vector has been expanding its range into rural communities over the last 5-10 years. Understanding Ae. aegypti dispersal patterns is important for anticipating the future range expansion of dengue and other viruses transmitted by this mosquito. To examine if human transportation networks play a signicant role in the population expansion of this mosquito, we analyzed Ae. aegypti population structure using a panel of 10 microsatellite markers. Adult and immature Ae. aegypti (>20 individuals per site) were collected from the city of Iquitos (pop: 380,000) and several neighboring communities that are connected by river transport, including Nauta (pop: 14,000), Indiana-Mazan (pop: 7,000), Barrio Florida (pop: 750), Tamshiaco (pop: 4,500), and Aucayo (pop: 800). We detected signicant departures from Hardy-Weinberg Equilibrium in all study sites due to lower than expected number of heterozygotes. FST statistics show signicant differentiation for the majority of study site pairs. Population structure among Ae. aegypti in different towns is not correlated with the geographic distance between towns, suggesting that human transportation networks may be responsible. Furthermore, Ae. aegypti gene ow among sub-populations is greatest between locations with heavy boat trafc such as Iquitos-Nauta (which also has heavy road trafc) and Iquitos-Indiana-Mazan, and lowest between locations with little or no boat (or road) trafc such as Barrio Florida-Iquitos interior. Bayesian clustering analysis using Structure program suggested denite admixture, with 5-6 genetic clusters. Our results provide strong evidence for the hypothesis, especially via boats, is responsible for Ae. aegypti spread in the Peruvian Amazon.

NEW INSIGHTS INTO THE MATING BIOLOGY OF AEDES AEGYPTI1 1 2 3 2,4 2 1Catalina Alfonso-Parra , Ethan Degner , Frank Avila , Yasir Ahmed , Laura K. Sirot , Mariana F. Wolfner and Laura C. Harrington

1 2 3Department of Entomology, Cornell University, Ithaca, NY. Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY. Department of Biology, 4University of Rochester, Rochester, NY. Department of Biology, College of Wooster, Wooster, OH

Understanding mosquito reproductive biology is an important step towards identifying novel targets to control mosquito populations. From the male side, we have been investigating the role of seminal uid proteins (SFPs) in Ae. aegypti reproductive success. SFPs are a heterogeneous group of proteins produced primarily in the accessory glands (AGs) of the male reproductive tract. These proteins are transferred, along with sperm, to females during mating and induce multiple physiological and behavioral changes in the mated female. Because of the importance of these proteins in Aedes fertility, they are intriguing targets for new control strategies. We will discuss the possible functions of the most promising SFP targets. Additionally, to gain insight into the male AG --its role in SFP production and secretion --we used the novel SFP AAEL010824 as a marker for AG activity. Finally, to further comprehend mosquito reproduction, we are analyzing the lower female reproductive tract transcriptome, characterizing how gene expression changes with time after mating. Our results suggest a wealth of new targets for the control these important disease vectors.

GENETICALLY ENGINEERED MOSQUITO FOR CONTROL OF DENGUE VECTOR - AEDES AEGYPTI

Andrew McKemey, Simon Warner, Glen Slade

Oxitec Ltd., United Kingdom

Dengue fever presents a global health concern with disease incidence having grown 30 fold in te last 50 years and recent estimate of up to 390 million infections annually. Control of Aedes aegypti, the principle mosquito vector of the dengue virus, remains the primary approach in the prevention of dengue. Oxitec have genetically engineered a strain of Aedes aegypti, known as OX513A that dies without a dietary antidote. Sustained release of OX513A male leads to suppression of wild populations as progeny from matings with wild females inherit the transgene and die prematurely due to lack of antidote. In 2010 a trial in the Cayman Islands over 3 million OX513A males were released over a 6 month period, successfully reducing the mosquito population by 85% compared to a control site. Subsequent studies in Brazil have suppressed Aedes population by over 80% in inner-city suburbs of 2000 people, and over 95% in a village of almost 3000 people. The use of OX513A males release has now moved form proof of principle to optimization and deployment on an operational scale with start of project in June 2013 to control Aedes aegypti in a Brazilian town of 50,000 people. In addition OX513A recently received regulatory approval for commercial release in Brazil.

USING INHERITED BACTERIA AS A WAY TO STOP MOSQUITOES FROM TRANSMITTING DENGUE: THE ELIMINATE DENGUE PROGRAM

Peter Ryan and Scott O'Neill

Monash University, Australia

Our group is examining the potential use of inherited bacterial symbionts of insects known as Wolbachia as a novel method to interfere with dengue transmission. This work has now progressed from basic bench studies into open eld trials in Australia, Vietnam and Indonesia. I will give an overview of Wolbachia-mosquitio-pathogen interactions as well as the current status of the global eliminate dengue program that aims to deploy Wolbachia infections as a cost effective and sustainable approach to dengue control.

HIGHLIGHTED POSTERSHP37

JAK/STAT SUPER-IMMUNE AEDES AEGYPTI AS A TOOL FOR DENGUE DISEASE CONTROL

Natapong Jupatanakul, Shuzhen Sim, Jayme Souza-Neto, Jared Balaich, and George Dimopoulos

W. Harry Feinstone Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland, USA

The JAK/STAT pathway is a cytokine signaling pathway involve in insect development and anti-viral immunity. Our previous result showed that dengue virus (DENV) infection in A. aegypti resulted in JAK/STAT pathway activation and expression of anti-viral effectors. A. aegypti was more susceptible to DENV infection when the JAK/STAT pathway was compromised through RNAi-mediation gene silencing of the JAK/STAT pathway components. These data suggest the JAK/STAT pathway as a key player for mosquito anti-DENV responses. In this study, we generated transgenic A. aegypti that over-express the JAK/STAT pathway components, the Domeless receptor (Dome) and Hop receptor adaptor protein, upon bloodfeeding. Over-expression of the JAK/STAT pathway components resulted in the transcriptional induction of the JAK/STAT pathway-regulated putative anti-DENV effectors, DVRF1 and DVRF2. We then checked vector competence to DENV and found that these transgenic mosquitoes had lower midgut DENV titers, and lower DENV dissemination to other organs compared to the wildtype. The tness cost of these mosquitoes, for example, longevity and fecundity will also be discussed.

HP38

COEXISTENCE AND DISPLACEMENT OF AEDES (STEGOMYIA) ALBOPICTUS (SKUSE, 1894) BY THE INVASION OF AEDES (STEGOMYIA) AEGYPTI (LINNAEUS, 1762) IN THE LETICIA COUNTRY (AMAZONAS-COLOMBIA)

1 2 3 4 5 3 3 1Carvajal JJ , Codeço CT , Alvarado LA , Frois LF , Rodriguez MH , Murcia LM , Falla C , Honório NA1 2Laboratório de Transmissores de Hematozoários - LATHEMA/FIOCRUZ, Rio de Janeiro, Brasil; Programa de Computação Cientíca - PROCC/FIOCRUZ, Rio de Janeiro,

3 4 Brasil; Laboratorio Departamental de Salud Pública, Leticia, Colombia; Programa de Estatística, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, Brazil;5Dirección de Ciencia, Tecnologia e Innovación, Gobernacion de Amazonas, Leticia, Colombia

The Dengue fever viruses are transmitted naturally by the mosquito Ae. aegypti, its main vector in the world, and in some regions, also by the mosquito Ae. albopictus. Dengue is endemic of tropical regions like the South-East Asia, Pacic South, Oriental Africa, Caribbean and Latin America. The rst register of Ae. albopictus in Colombia was in the Leticia county (Amazonas) in 1998, when Leticia was Ae. aegypti free. Ae. aegypti arrived a decade later, found in 2009 in the neighborhood of La Union in Leticia, near the frontier with the Tabatinga county (Amazonas, Brasil). Data from periodic entomological surveys of positive breeding sites of Ae. albopictus (2000-2012) and Ae. aegypti (2009-2012) in Leticia were analyzed using descriptive methods and models for spatial and time series analyses, as well as thematic maps with Radial Basis Functions. Ae. albopictus presented preference for disposable containers and low tanks, while Ae. aegypti was more frequently found in low tanks and disposable containers. Ae. albopictus showed a less seasonal behaviour than Ae. aegypti. It was observed an important reduction of positive breeding sites and infestation rates of Ae. albopictus, after the introduction of Ae. aegypti in 2009, moreover, it was found signicant evidence of displacement of Ae. albopictus to the periphery of the urban area of Leticia. Ae. albopictus populations were reduced and geographically displaced to the periphery of Leticia, after the introduction of Ae.

aegypti in 2009. The difcult control and surveillance along the frontier belt will keep being a challenge to reduce the infestation rates, by reinfestations, by precarious sanitation services, among other factors, which contribute for keeping the virus of Dengue.

HP39

EFFECTIVENESS OF SPINOSAD - AN ALLOSTERIC MODULATOR OF ACETYLCHOLINE RECEPTORS – ON BRAZILIAN POPULATIONS OF AEDES AEGYPTI RESISTANT TO THE ORGANOPHOSPHATE TEMEPHOS

1,2 3 3 3 1,2 1,2 1,2Dias, LS , Macoris, Maria LG , Andrighetti, Maria TM , Otrera, VG , Dias, AS , Martins, AJ , Lima, JBP1 2Laboratório de Fisiologia e Controle de Artrópodes Vetores, Rio de Janeiro, Brasil, Laboratório de Entomologia, Instituto de Biologia do Exército, Rio de Janeiro, RJ,

3Brasil, Superintendência de Controle de Endemias (Sucen) Marília, SP - Brasil.

In Brazil, control of dengue vector larvae was performed for decades with the organophosphate temephos, what led to resistance selection to this insecticide. Studies of new tools to control Ae. aegypti, which are less harmful to the environment and safer for human, have become of main importance. Spinosad, a product of the aerobic fermentation of Sacharopolyspora spinosa (an actinobacteria isolated from soil), presents insecticidal property, an action mechanism different from the classical insecticides, low toxicity to humans and animals. In the present work, we (i) standardized the dose response assay with L3 larvae of Ae. aegypti (Rockefeller strain) with mortality scored after 24, 48 and 72 hours of exposure to spinosad; (ii) evaluated, in laboratory, the efcacy of spinosad (NatularTM 20EC - 20,6%) on the Rockefeller strain and seven populations from different regions of Brazil with mortality scored after 24 hours of exposure to the chemical, and (iii) evaluated, with eld simulation, the efcacy of spinosad (NatularTM DT - 7,48%) on the Rockefeller strain and four eld populations using water tanks (capacity of 300 L) and mortality scoring every 24 hours. All eld populations used showed different levels of resistance to the organophosphate temephos. During the standardization of the assay, no signicant differences in mortality between different exposure times were observed. Spinosad, both in laboratory and eld simulation, was effective against all populations, regardless their status of temephos resistance. In eld simulation, the mortality of Ae. aegypti larvae was above 80% for up to 8 weeks. This persistence was similar for the susceptible strain Rockefeller and eld populations. The data presented here suggest that the evaluated populations do not exhibited cross-resistance between spinosad and temephos, demonstrating that this product may be a promising alternative to combat Ae. aegypti, especially in populations that are resistant to other neurotoxic compounds used in the control.

HP40

COMMUNITY AWARENESS AND SUPPORT OF THE CONTROVERSIAL RELEASE OF OX513A AEDES AEGYPTI IN KEY WEST, FL1 2 3 4 5 3Kacey C. Ernst , Steven Haenchen , Katherine Dickinson , Michael Doyle , Kathleen Walker and Mary H. Hayden

1 2University of Arizona, College of Public Health, Division of Epidemiology and Biostatistics (Tucson, AZ, USA); University of Arizona, College of Public Health, Division of 3 4Epidemiology and Biostatistics (Tucson, AZ, USA); National Center for Atmospheric Research (Boulder, CO, USA); Florida Keys Mosquito Control District (Key West,

5FL); University of Arizona, College of Agriculture, Department of Entomology (Tucson, AZ, USA)

Innovative control strategies for mosquito-borne disease include the release of genetically modied mosquitoes. Release of OX513A Aedes aegypti mosquito has demonstrated success in inter-mixing among natural Ae. aegypti populations to reduce vector indices within several eld sites. Following the dengue outbreak in 2009/2010, the Florida Keys Mosquito Control District began exploring the release of OX513A Ae. aegypti to prevent further outbreaks of dengue. This proposal was met with controversy. As part of a comprehensive knowledge, attitudes and practices cross-sectional survey conducted in Key West and neighboring Stock Island, FL in 2012 we investigated the proportion of the community that was aware of the proposed release and their current level of support. Of the four-hundred participants approximately half of the community had heard of the proposed release (50.1%). In multivariable analyses those who had heard of the release were more likely to be older, male, white non-Hispanic, educated and with an awareness of local dengue prevention campaigns. Of those who had prior awareness of the release (n=195) about half were supportive (n=68, 34.9% strongly supportive; somewhat supportive, n = 43, 22.1%). However, there was a fairly large proportion that was neutral (n = 49, 25.1%) and a smaller proportion that was opposed (n=16, 8.2%) or strongly opposed (n=19, 9.7%). Primary reasons for a neutral attitude were uncertainty about risks/ benets of the proposed release and lack of enough information to make a decision. Approximately one-third of individuals who expressed support of the release also expressed remaining concerns. As novel public health strategies are proposed, it is important to monitor and obtain feedback from the community. Efforts to maintain public communication and target outreach towards select groups are recommended and standards must be set to determine the level of awareness and support that are required prior to implementation.

POSTERS SESSION VECTOR BIOLOGYVB-P01

EVALUATION OF DENGUE ENTOMOLOGICAL INDEXES IN REGIONS OF THE STATE OF SÃO PAULO, BRAZIL - COMPARISON AMONG DIFFERENT ENTOMOLOGICAL INDEXES.

1 2 3 1 4 3 3 3Virgília Lima , Expedito Luna , Marylene Arduino , Valmir Andrade , Gerson Barbosa , Luis Filipe Mucci ,Ligia Serpa ,Gisela Marques . 1 2Superintendência de Controle de Endemias – SUCEN– Regional Campinas, SP,Brazil, Instituto de Medicina Tropical da Universidade de São Paulo IMT/USP, Brazil, 3Superintendência de Controle de Endemias – SUCEN – Laboratório de Culicídeos, Taubaté, SP, Brazil.

Introduction- The lack of understanding of the meaning of different entomological indexes to estimate Aedes aegypti density, casts doubt on their usefulness. This study was designed to improve the knowledge about dengue entomological indexes, in order to better use them in planning vector control activities. Objective: To evaluate and relate indexes of different stages of the mosquito vector (traditional, eggs, pupal productivity and adult using aspiration and also using traps). Methods- The entomologic indexes were measured monthly in selected areas of two municipalities located in the State of São Paulo, Brazil, during 18 months, using a random sample for each month and activity. Curves were used to describe indexes trends over time. Spearman correlations coefcient was used to analyze their relation. Results- 16143 houses were submitted to inspections to assess traditional indexes and 5671 to assess the adult vector ones. 2545 adult traps and 2452 traps were installed. Eggs indexes curves presented higher values than the others in summer. In winter they dropped sharply in both municipalities. The indexes of the same stage of the vector presented strong correlation coefcients higher than 0.60. The Breteau and house indexes pointed out infestations differences between the two cities, as also did pupal productivity and mean eggs number among positive traps. Discussion- The strong correlation and curves similarity of different states Ae. aegypti indexes suggest that they probably indicate patterns and level of infestation. The robust time series data collected in the same locations has made possible to assess these correlations, which differed from those of previous studies. Conclusion- The different stages indexes seem to measure the same phenomenon: the areas infestation. Eggs and adult indexes may be used for vector detection during poor infestation periods.

VB-P02

PRODUCTIVITY OF BREEDING SITES OF AEDES AEGYPTI AND AEDES ALBOPICTUS (DIPTERA, CULICIDAE) DURING THE DRY AND RAINY SEASON IN THE CITY OF CAXIAS, MARANHÃO, BRAZIL

1 2 1Carine Fortes Aragão Wanderli Pedro Tadei , Valéria Cristina Soares Pinheiro1Centro de Estudos Superiores de Caxias/Universida de Estadual do Maranhão-CESC/UEMA, Pça. Duque de Caxias, S/N – Morro do Alecrim. CEP: 65.604-380.

2Caxias/MA; Laboratório de Malária e Dengue. Instituto Nacional de Pesquisa da Amazônia-INPA-Manaus-AM

Dengue is a serious public health problem worldwide transmitted by Aedes aegypti and Aedes albopictus. Currently population growth and inappropriate housing conditions increase the availability of containers that are used as breeding sites for these species. Considering the continuous occurrence of dengue cases and dispersion of A. albopictus in the municipalities of the state of Maranhão, it is necessary to conduct studies to characterize breeding sites of these vectors. Three neighborhoods were selected and 600 properties were randomly selected and were surveyed for inspection of water containers. In containers with immature were collected all specimens to calculate the productivity and they were characterized regarding to: presence of organic matter; volume of water; exposure of breeding site and water turbidity. Of the total houses visited, 41 (6.8%) were positive for these species in the dry season and 61 (10.2%) in the rainy season. The storage type containers had higher productivity in both seasons. It were collected 877 immature of A. aegypti and 1 A. albopictus during the dry season. In the rainy season were collected 2212 immature of A. aegypti and 489 A. albopictus. The positive containers had the following characteristics: 43% consisted of plastic and 42% of cement, in the most of them 83% there was organic matter and had clear water (84%). The predominant volume was above 100 liters of water (40%) and partially sealed containers (60%). It is found that the storage of water by residents favors the great positivity of breeding sites found. From the data, it was found that the prevalence of A. aegypti compared to A. albopictus may be related to the habit of A. aegypti in residing urban areas, unlike the other species. It was found that the rainy season was most productive for both species and that storage containers are the most productive.

VB-P03

INFLUENCE OF CONTAINERS WATER VOLUME ON THE CHOICE OF SITES FOR OVIPOSITION BY MOSQUITOES UNDER NATURAL CONDITIONS IN THE CITY OF RIO DE JANEIRO, BRAZIL

1 1 2Thais Irene Souza Riback , Cláudia Torres Codeço , Nildimar Alves Honório1Programa de Computação Cientíca, Fundação Oswaldo Cruz, Av. Brasil 4365, Rio de Janeiro, 21040-360, Rio de Janeiro/RJ, Brazil. thaisriback@hotmail.com; claudia.codeco@gmail.com 2Laboratório de Transmissores de Hematozoários, Instituto Oswaldo Cruz, Fundação Oswaldo

Cruz, Rio de Janeiro/RJ, Brazil. nahonorio1@gmail.com

The theory of vector control population implies that the control is maximized when applied to reduction of adult survival rates and population density below a critical value. This theory is based on the assumption that new mosquitoes recruitment occurs in proportion to the actual adult population. In the case of Aedes aegypti, which presents eggs with characteristics of latency and endurance, the theoretical control effect is challenged by the population ability of quickly replenish their population

density, independent of the local population. Thus, the present study evaluated, under natural conditions: A) the inuence of abiotic factors upon oviposition dynamics and egg hatching; B) evaluate locally how aquatic microclimate conditions are inuenced/impacted by abiotic factors, and which impact on the choice of ideal sites for oviposition, eggs hatching and immature development; C) and contribute to the discussion of strategies for Ae. aegypti population control from a new perspective of the eggs in the population resilience. Therefore, were conducted experiments to evaluate the oviposition sites choice according to the availability of breeding sites containing different water volume, in order to test the following hypotheses: water volume affects the choice of oviposition breeding sites choice; oviposition rate would vary as a function of previously oviposition; there is an interaction between the container water volume and the number of laid eggs; and eggs hatching depends on the water line relationship.

VB-P04

DENGUE SURVEILLANCE IN MOSQUITOES FROM A MEDIUM-SIZED CITY IN SÃO PAULO STATE, BRAZIL 1 2 1 1 1 3 4Felipe Guioti Eduardo Sterlino Bergo , Aline Chimello Ferreira , Arianne Fagotti , Talita Motta Quiarim , Betania Paiva Drumond , Roberta Vieira de Moraes Bronzoni ,

5 1Mauricio Lacerda Nogueira , Adriano Mondini1 2 3Universidade Estadual Paulista “Júlio de Mesquita Filho” – NESP/FCFAR. Superintendência de Controle de Endemias – SUCEN/SR-06. Universidade Federal de Juiz

4 5de Fora – UFJF. Universidade Federal do Mato Grosso – UFMT – Campus Sinop. Faculdade de Medicina de São José do Rio Preto – FAMERP.

Infections by the four dengue serotypes (DENV 1-4) have been reported in more than 100 countries and DENV is considered one of the most important arthropod borne viruses in terms of public health. Aedes aegypti is the main vector of DENV in Brazil due to its ability to adapt to the urban environment. Araraquara, located in the center of the state of São Paulo, Brazil, has a high quality of life (Human Development Index: 0,830), which is comparable to many developing countries. Until now, no studies on DENV circulation have been available for this city. The aim of this study is to evaluate the presence of DENV 1-4 in samples of mosquitoes collected in Araraquara from February 2014 to January 2015. We have installed 150 ovitraps in a pre-established grid of census tracts that covered the entire municipality. The ovitraps were collected ve days after installation and the eggs in the cardboard paddles were hatched. Mosquitoes were identied and pooled according to collection site, species and gender. A Multiplex-Nested-RT-PCR was used to detect DENV in the mosquitoes. So far, approximately 800 mosquitoes – divided in 241 pools – have been collected. We have assessed 20 pools (10 pools of each gender). We could not detect the presence of DENV in any of the pools. It is important to note that DENV is actively circulating in the city and only 10% of the pools have been analyzed. We are still collecting eggs and we will be collecting samples from febrile patients with clinical diagnosis of dengue. These are preliminary data from a study that is being conducted in Araraquara to detect DENV in mosquitoes and in humans.

VB-P05

MICROBIOTA ISOLATED FROM MOSQUITOES AE. AEGYPTI AND AE. ALBOPICTUS FROM ENDEMICS AREAS OF PANAMA1,2 3 1 1 1 2 1,2 3Ortiz A , Ramirez JL , Torres R , Rovira J , Burgos R , Gonzalez C1, , Pascale JM , Dimopoulos G

1 2 3Instituto Conmemorativo Gorgas, Universidad de Panamá, W.H. Feisntone Dep. of Molecular Microbiology and Inmunology, Bloomberg School of Public Health, Johns Hopkins University, USA.

Background: Mosquitoes are the most important vectors of human and animal diseases affecting millions of people around the world, such as the case of dengue in which there are 50 millions of dengue cases per year. Panama ranks fth in the number of dengue cases in Central America with DENV-1, DENV-2 and DENV-3 co-circulating throughout the country. In the absence of good sanitary control and a reliable dengue vaccine, we need to use all strategies to control the spread of the disease. One of the ways to control the spread of dengue is by understanding the mosquito vector and its microbiota; nding potential microbial agents that have the ability to confer resistance to infection by dengue virus in mosquitoes. Methods: We collected females mosquitoes from dengue-endemics areas. About 237 mosquitoes were collected. Their midguts were dissected and used for the isolation of bacteria. Characterization of bacterial isolates was done via sequencing of its 16rs RNA gene. Currently, we are analyzing the prevalence and localization of at least 28 bacterial species that have been characterized. Results: Bacteria isolated from eld mosquitoes were heterogenous withbacteria isolated in Aedes aegypti presenting a prevalence of: Staphylococcus and Chryseobacterium 12 % ,Pantoea 8%, Asaia 7%, Microbacterium 7%, Asaia and Bacillus 7%, Enterobacter 1% ; Diversity in Aedes albopictus showed Chryseobacterium 25% , Staphylococcus and Comamonas 15% , Acinetobacter and Pantoea 10% ; Asaia, Pseudomona, Microbacterium, Halophic bacterium 5%. Conclusions: The characterized bacteria are currently being tested for its anti-dengue effect both in the mosquito as well as in vitro. Tests are also being conducted for its moquitocidal activity against Ae. aegypti and Ae. albopictus, two competent vectors of dengue virus in Panama.

VB-P06

EVALUATION OF INNATE IMMUNE RESPONSE MECHANISMS ASSOCIATED WITH SUSCEPTIBILITY TO DENGUE VIRUS IN DIFFERENT FIELD POPULATIONS OF AEDES AEGYPTI

1,2 1,3 1Idalba Mildred Serrato Pomar , Paola Andrea Caicedo Burbano , Clara Beatriz Ocampo Durán1 2Centro Internacional de Entrenamiento e Investigacion es Médicas, CIDEIM, Biology and vector control laboratory. Master student in biomedical sciences,

3Universidad del Valle. Doctorade student biomedical sciences, Universidad del Valle

The main vector of dengue virus (vD) is Aedes aegypti, mosquito widely adapted to the urban environment. Their ability to become infected and transmit the virus (vector competence-VC) varies between populations and depends on genetic and environmental characteristics. Understanding the genetic characteristics of VC will

identify potential targets to inhibit virus replication strategies. Preliminary studies on differential expression in D-2 selected strains from eld collected individuals in Cali, susceptible (Cali-S) and refractory (Cali-MIB), suggest that immune response genes associated with apoptosis play a role in the virus replication. This study evaluated if VC of the select strains is similar compared with other serotypes of vD. And was assessed if the molecular mechanisms identied in select strains are a characteristic of eld populations. The VC of the selected strains compared to 4 virus serotypes was measured by articial infection and immunouorescence. The VC of A. aegypti collected from 6 locations in Cali was measured. Three different populations with different VC were selected to evaluate the expression by qPCR of Dronc, Caspase-16, Cathepsin-B and Niemann genes at 0, 24, 36, 48h post-infection. No statistically signicant differences in the VC of the selected strains against the four serotypes were observed. The mosquitoes collected in Cali showed differences on VC at the different sites, however did not show a high susceptibility (ranges between 68 and 34%). When the midgut infection barrier-MIB (percentage of uninfected midguts) was evaluated, variation was also observed (11 to 38%). Increased expression of the genes evaluated when exposed to the virus compared to the blood was observed in all populations, mainly between 24 and 36 hours post-infection. Although there is variation between biological replicates (possibly by genetic variability), this pattern is maintained, demonstrating that these inhibition mechanisms are specic from the eld populations and explain the observed intermediate susceptibility.

VB-P07

ASSOCIATED FACTORS WITH REFRACTORINESS OR SUSCEPTIBILITY TO INFECTION WITH DENGUE-2 VIRUS OF AEDES AEGYPTI FIELD-DERIVED STRAINS FROM COLOMBIA, USING COMPARATIVE MICROARRAY-BASED ANALYSIS

1 2 3 1 1 1Paola A. Caicedo , Carl Lowenberger , George Dimopoulos , Idalba Serrato , Neal Alexander , Clara Ocampo1 2Centro Internacional de Entrenamiento e Investigaciones Médicas (CIDEIM), Santiago de Cali-Colombia; Department of Biological Sciences, Simon Fraser University,

3Burnaby BC, Canada; Department of Molecular Microbiology and Immunology, Malaria Research Institute, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD

BACKGROUND: Selected strains of Aedes aegypti, from Cali, Colombia with different susceptibility to dengue infection: Susceptible (Cali-S) and refractory with midgut infection barrier (Cali-MIB), have suggested apoptosis mechanisms as one of the innate immune responses to eliminate virus infection. OBJECTIVES: Compare the global gene expression of the midguts of Cali-S and Cali-MIB after ingestion of sugar, a bloodmeal, or a bloodmeal containing Dengue-2 virus using microarrays. METHODS: We fed Cali-R or Cali-S with blood or blood+DENv-2. We dissected midguts at 30 hours post feeding (time at what most of the changes have been observed according to previous studies), extracted RNA and generated cDNAs. We used microarrays, and Quantitative Real time PCR (qPCR) to identify differential gene expression in Cali-S and Cali-R Ae. aegypti. RESULTS: Preliminary results from the microarrays indicated the expression of a total of 3761 genes. Of these, a total of 165 immune-related genes have been identied. A differential expression between the two strains exposed to DENv-2 virus included genes in different functional groups; immunity, metabolism, proteolysis, redox, replication, transport, and unknown function. We have identied 15 genes with differential gene expression at 0, 8, 24 and 36 hours post-infection. Future studies will evaluate gene function using RNAi technology to evaluate the effects of gene knockdown on the phenotype. CONCLUSION: This study will provide a global overview of gene expression in susceptible and refractory mosquitoes and will be compared with other studies that have looked at specic molecules and pathways. This study will also validate the use of our eld derived strains as an important biological model to study Dengue-vector relationships.

VB-P08

EFFECT OF NS1 DENGUE VIRUS PROTEIN ON THE IMMUNE RESPONSE SIGNALING PATHWAYS IN AEDES AEGYPTI MOSQUITOES1 1 2 3 4Ma. Isabel Salazar-Sánchez , Luis Antonio Alonso-Palomares , Jorge I. Castañeda-Sánchez , Tereza Magalhães , Humberto Lanz-Mendoza .

1 2Laboratorio de Inmunología Celular e Inmunopatogénesis. Departamento de Inmunología- Escuela Nacional de Ciencias Biológicas-IPN. México; Departamento de 3Sistemas Biológicos. Universidad Autónoma Metropolitana - Unidad Xochimilco. México; Department of Virology and Experimental Therapeutics. Fiocruz-Recife.

4Brazil; Centro de Investigación sobre Enfermedades Infecciosas-Instituto Nacional de Salud Pública. México.

Dengue virus NS1 protein constitutes not only the virus replication complex inside the host cell, but it is also secreted to the extracelular milleu. Recently, de novo production of NS1 protein cells have been demonstrated in infected Aedes aegypti mosquitoes. Considering that NS1 protein is uptaken along with virus particles and plasma components during blood feeding, we considered important to determine its role in modulating immune response in mosquitoes. In this investigation we used Aag2 cell as well as Aedes aegypti mosquitoes to establish the effect of NS1 recombinant protein on the mRNAs expression levels of different effectors in immune response signaling pathways. Experimental design included untreated group for mRNA basal levels and positive controls. Our results from the time course reveal that effectors related to Toll signaling pathway in the immune response are upregulated by dengue virus NS1 protein.

VB-P09

MALE ACCESSORY GLAND SUBSTANCES FROM AEDES ALBOPICTUS AFFECT THE LOCOMOTOR ACTIVITY OF AEDES AEGYPTI FEMALES1,2,3 1 4 2,5 2,5† 6Tamara Lima-Camara , Claudia Codeço ,Nildimar Honório ,Rafaela Bruno , Alexandre Peixoto , Leon Lounibos

1 2Programa de Computação Cientíca – PROCC. Fundação Oswaldo Cruz/FIOCRUZ; Laboratório de Biologia Molecular de Insetos, Instituto Oswaldo Cruz, IOC-3 4FIOCRUZ, Departamento de Epidemiologia da Faculdade de Saúde Pública da USP, Laboratório de Transmissores de Hematozoários, Instituto Oswaldo Cruz, IOC-5 6FIOCRUZ; Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular – CNPq/Brazil, Florida Medical Entomology Laboratory, University of Florida, Vero

†Beach, Florida, USA. Deceased

Dengue is one of the world's most important mosquito-borne diseases and is usually transmitted by one of two vector species: Aedes aegypti or Aedes albopictus. These two diurnal mosquitoes are frequently found coexisting in similar habitats, enabling interactions between adults, such as cross-mating. The objective of this study was to assess cross-mating between Ae. aegypti females and Ae. albopictus males under articial conditions and evaluate the locomotor activity of Ae. aegypti virgin females injected with male accessory gland (MAG) homogenates to infer the physiological and behavioural responses to interspecic mating. After seven days of exposure, 3.3-16% of Ae. aegypti females mated with Ae. albopictus males. Virgin Ae. aegypti females injected with conspecic and heterospecic MAGs showed a general decrease in locomotor activity compared to controls and were refractory to mating with conspecic males. The reduction in diurnal locomotor activity induced by injections of conspecic or heterospecic MAGs is consistent with regulation of female reproductive activities by male substances, which are capable of sterilising female Ae. aegypti through satyrisation by Ae. albopictus

VB-P10

BIOLOGICAL AND CHEMICAL CHARACTERIZATION OF NEW PLASTIC OVITRAPS CONTAINING PYRIPROXYEN FOR AEDES AEGYPTI (DIPTERA: CULICIDAE) CONTROL

1 1 1,2 1Harburguer Laura , Seccacini Emilia , Zerba Eduardo , and Licastro Susana1Centro de Investigaciones de Plagas e Insecticidas (CIPEIN-CITEDEF –UNIDEF) (MINDEF/ CONICET) Juan Bautista de La Salle 4397, Villa Martelli, Buenos Aires,

2Argentina. 3iA, UNSAM, Buenos Aires, Argentina.

Aedes aegypti is an urban mosquito adapted to using articial containers for breeding. In the absence of a vaccine, controlling the dengue vector is essential to avoid epidemics. In Argentina larvae control is performed by applying the organophosphate larvicide temephos in containers that cannot be eliminated while adult control is performed by ground application of space sprays with pyrethroids. Ovitraps are widely used for monitoring mosquito populations. Its improvement for population control purposes could introduce a new tool with low environmental impact that will allow an integrated vector control. In this work ovitraps made of black synthetic plastic materials containing 0.1% of the larvicide pyriproxyfen were developed. It was found that these ovitraps had larvicidal activity against Ae. aegypti obtaining an emergence inhibition (EI) of 100% which implies that pyriproxyfen incorporated into the plastic was successfully released to the water. An accelerated aging test of these plastic ovitraps was performed, this test is internationally standardized to simulate at least two years of aging. The results indicate that after 14 days plastic ovitraps with pyriproxyfen that suffered accelerated aging produce 100% of EI of the treated larvae, and its behaviour was the same as not aging ovitraps. Finally ovitraps resistance to successive washes was evaluated and was found an EI of 100% for more than 25 washes. Polyethylene strips containing 0.5% pyriproxyfen that can oat (white) or that can be sunken (black) were also developed and an accelerated aging test performed, replacing water every day. Results of this work indicate the feasibility of using a long lasting material containing pyriproxyfen as larvicidal ovitrap. This tool together with the use of curtains or jar covers treated with insecticides could improve Ae. aegypti population control and potentially reduce dengue transmission

VB-P11

DOES WOLBACHIA INTERFERE WITH DENGUE VIRUS VERTICAL TRANSMISSION?

Etiene Casagrande, Eric P. Caragata, Luciano A. Moreira

Centro de Pesquisas René Rachou, FIOCRUZ-MG / IOC-RJ, Brazil

Dengue is a growing medical problem in subtropical and tropical areas, and disease transmission is particularly severe in Central and South American countries. Aedes aegypti is a highly competent vector for dengue, and is extremely procient at colonizing urban environments. The heavy burden of disease that persists indicates that current control strategies are insufcient. Therefore, there is a need to seek new strategies to supplement existing forms of control. Our work is based on the use of a strain of the bacterium Wolbachia pipientis (wMel) in Brazilian A. aegypti mosquitoes (Eliminate Dengue Project: Brazil). Despite several studies showing the interference of Wolbachia strains towards pathogens (wMel, wMelPop, wAlbA and wAlbB) on different mosquito hosts (A. aegypti, A. albopictus and A. stephensi), there is no evidence Wolbachia is able to block dengue in the progeny of infected mosquitoes, the so called vertical transmission. The aim of this study is to evaluate whether Wolbachia has an effect on the vertical transmission of dengue virus in Brazilian A. aegypti. Here, we compare Wolbachia-infected mosquitoes with those treated with antibiotics to remove Wolbachia. We utilised two modes of dengue infection. (1) By oral feeding with an infected blood meal, to simulate a natural dengue infection in mosquitoes. (2) Articial infection by nanoinjection of dengue into the thorax to increase the chance that the ovaries would become infected. Dengue virus was detected in the progeny from infected females through quantitative PCR (RT-qPCR), using primers and probes for the mosquito, dengue and Wolbachia in a multiplex Taqman assay. The present study could demonstrate another facet of the contribution of Wolbachia in restricting dengue transmission in A. aegypti.