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Viral Pathogenesis
OP128
A single amino acid change in the matrix protein VP40 of Marburg virus improves viral fitness
A. Köhler1, U. Welzel1, G. Schudt1, A. Herwig1, S. Becker1, L. Kolesnikova1
1Philipps University Marburg, Institute of Virology, Marburg, Germany
Marburg virus (MARV, family Filoviridae) induces a severe haemorrhagic fever in humans and non-human primates but only transient non-lethal disease in rodents. However, sequential passages of MARV in rodents boost infection to a lethal disease. Selection of adapted highly pathogenic MARV variants in guinea pigs needed only 8 passages. Adapted MARV contained amongst others one mutation in the matrix protein VP40 at position 184 (D184N). VP40 generally plays an important role in viral assembly, budding and suppression of interferon signaling. We compared functions of wtVP40 and VP40D184N in human and guinea pig cell lines using different approaches. Both wtVP40 and VP40D184N suppressed the interferon response to the same extent in human and guinea pig cells. However, when tested in an infectious virus-like particles (iVLP) assay, VP40D184N led to enhanced transcription and replication in guinea pig compared to human cells. Although the amount of VP40D184N -induced iVLPs was reduced in guinea pig cells, the relative amount of NP in these iVLPs was increased. We cloned, rescued and analysed recombinant MARVs containing either wtVP40 (recMARV_wt) or guinea pig-adapted VP40 (recMARV_D184N). Compared to recMARV_wt, recMARV_D184N grew to higher titers in guinea pig cell lines but not in human cells. Furthermore, recMARV_D184N showed higher infectivity and displayed efficient assembly in guinea pig cells at ultrastructural level. Our data suggests that in guinea pig cells VP40D184N enhances infectivity, viral replication and transcription as well as assembly of viral particles which might improve viral fitness and contribute to the high pathogenicity of guinea pig adapted MARV. Corresponding author: Alexander Köhler
Viral Pathogenesis
OP129
TMPRSS2 and TMPRSS4 facilitate proteolytic activation of H3N2 Influenza A virus hemagglutinin in mice
N. Kühn1, B. Hatesuer1, R. L. O. Stricker1, S. Bergmann2, N. Kasnitz3, A. Keppner4, E. Hummler4, S. Weiß3, K. Schughart1
1Helmholtz Centre for Infection Research, Infection Genetics, Braunschweig, Germany 2University of Tennessee Health Science Center, Department of Microbiology, Immunology and Biochemistry, Memphis, TN, United States 3Helmholtz Centre for Infection Research, Department of Molecular Immunology, Braunschweig, Germany 4University of Lausanne, Department of Pharmacology and Toxicology, Lausanne, Switzerland
Introduction: Influenza epidemics and pandemics are responsible for serious global morbidity and mortality. Due to the high genetic variability of the virus, selection pressure results increasingly in development of resistance. Objectives: Host cell factors essential for viral spread but not for cellular survival are attractive targets for novel antiviral therapy in humans. Cleavage of the influenza virus hemagglutinin (HA) requires host proteases for viral infectivity. A multitude of candidates like TMPRSS2, TMPRSS4 and TMPRSS11d (HAT) has been suggested in vitro, but the proteases that activate influenza viruses are unknown. We reported recently that Tmprss2-deficient mice are protected against H1N1 virus spread, but just to a minor extend to H3N2 virus spread and pathogenesis. Results: In this study we present first in vivo data for the HA-cleaving Trypsin-like protease TMPRSS4. TMPRSS2 and TMPRSS4 were both expressed in the same cell types of the murine lung. This co-localization of host proteases supports reciprocal compensation of these proteins. However, deletion of Tmprss4 did not protect mice from body weight loss and death upon infection with H3N2 influenza virus. In contrast, Tmprss2-/-Tmprss4-/- double knock-out mice showed a strongly reduced viral spread and lung pathology in addition to a reduced body weight loss and mortality. Furthermore, only a minor fraction of the HA precursor protein was processed into HA1 and HA2. Conclusion: Summarizing, our results identify TMPRSS4 as the second host cell factor next to TMPRSS2, responsible for the cleavage activation of H3N2 Influenza viruses in vivo and demonstrate a low specificity of proteases for the H3 cleavage loop in contrast to H1N1 viruses. Corresponding author: Nora Kühn
Viral Pathogenesis
OP130
Cryptic virulence potential in H5 and H7 field strains of avian influenza virus
J. Veits1, E.- S. M. Abdelwhab1, T. C. Mettenleiter1
1Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Institute of Molecular Virology and Cell Biology, Insel-Riems, Greifswald, Germany
Based on their virulence avian influenza viruses (AIV) are classified as low pathogenic (LP) or highly pathogenic (HP). Whereas LPAIV specify all 16 known avian HA subtypes, HPAIV are naturally restricted to subtypes H5 and H7. A major virulence determinant is the polybasic cleavage site (PCS) within HA, which allows proteolytic HA activation by ubiquitous proteases and systemic disease in poultry. To investigate the requirements to transform an LPAIV to an HPAIV in nature, we created reverse genetics systems of recent German LPAIV field isolates, H5N3 and H7N7. Whereas the introduction of a PCS into H5N3 LPAIV did not result in the induction of clinical disease, the introduction of a PCS in H7N7 yielded a highly virulent phenotype causing lethal disease. Thus, whereas in field virus H5N3 introduction of a PCS was not sufficient for virulence, in H7N7 it transformed the LPAIV into HPAIV. These data demonstrate a difference in cryptic virulence potential in H5 and H7 AIV field strains and further emphasize the need to collect molecular data for a robust risk analysis on emergence of HPAIV from circulating LPAIV. Corresponding author: Jutta Veits
Viral Pathogenesis
OP131
Pathogenic Old World hantaviruses cause podocyte damage by utilizing actin stress fibers for viral release
S. Hägele1, J. Reiser2, M. Zeier1, E. Krautkrämer1
1University of Heidelberg, Department of Nephrology, Heidelberg, Germany 2Rush University Medical Center, Department of Medicine, Chicago, United States
Hemorrhagic fever viruses are associated with bleeding disorders due to enhanced permeability. Old World hantaviruses mainly affect the kidney causing hemorrhagic fever with renal syndrome (HFRS). Previously, we showed that human renal cells are permissive for Old World hantaviruses. We revealed that tight junctions were disrupted by hantaviruses in vitro as well as in vivo, correlating with the clinical picture. How hantaviruses interfere with cell-to-cell contacts is not well characterized. Therefore, we analyzed the role of the cytoskeleton, which integrity is crucial for tight junctions, during hantaviral infection. A human kidney podocyte cell line was used as a relevant infection model for the Old World hantavirus Hantaan (HTNV). Cytoskeletal proteins, actin and tubulin, were analyzed by confocal microscopy and disrupted by cytochalasin D or nocodazole, respectively. We observed in HTNV-infected podocytes a punctate and a filamentous pattern of the hantaviral nucleocapsid (N) protein. The filamentous N protein co-localized with actin stress fibers but not with microtubules. This localization of N protein depends on the integrity of the actin cytoskeleton, because the filamentous pattern disappeared after actin depolymerization. The amount of cells which showed filamentous N protein increased during infection and correlated with viral release. Infection kinetic studies revealed that the filamentous pattern and viral egress occurred simultaneously with the disruption of tight junctions in podocytes during infection. In conclusion, these results indicate that hantaviruses use actin stress fibers for assembly and/or egress. As a consequence, this association may cause podocyte damage leading to renal failure. Corresponding author: Stefan Hägele
Viral Pathogenesis
OP132
Analysis of cell death during human cytomegalovirus infection of macrophages
G. Frascaroli1, C. Tauber1, I. Cobres1, T. Mertens1
1Ulm University Medical Center, Institute of Virology, Ulm, Germany
Macrophages (Mφ) are multifunctional immune cells well equipped to kill pathogens and to promote a specific immune reaction. However, Mφ are susceptible to infection by human cytomegalovirus (HCMV), a virus that has evolved various strategies to interfere with cell viability and to extend the survival of infected cells by expressing different anti-apoptotic proteins. In order to investigate the impact of HCMV infection on the apoptotic process, uninfected and HCMV-infected Mφ were treated with inducers of extrinsic as well as intrinsic apoptosis. Apoptosis was revealed by TUNEL assays and FACS analysis of phosphatidylserine exposure on the cell membrane (Annexin V-FITC/7AAD assays). Surprisingly, Mφ showed a great resistance against all tested apoptosis inducers. Only the treatment with the protein kinase inhibitor staurosporine efficiently induced apoptosis in Mφ. Irrespectively on the multiplicity of infection (MOIs) as well as the times after infection, HCMV-infected Mφ exhibited very low levels of apoptosis thus suggesting that Mφ are constitutively resilient and poorly undergo apoptosis. More recently, another type of cell death named pyroptosis has been described in association with antimicrobial and inflammatory responses. In order to investigate whether HCMV infection triggers pyroptosis, Mφ were infected with TB40E prior to analysis of interleukin-1ß (IL-1ß) secretion, caspase-1 activation and formation of pyroptotic pores. Preliminary results indicate that HCMV infection leads to an increase of the active form of caspase-1 in M1-Mφ but not in M2-Mφ, IL-1ß secretion and pore formation. Whether IL-1ß secretion is a consequence of pyroptotic cell death triggered by HCMV or instead inflammasome formation, remains to be confirmed in additional studies. Corresponding author: Giada Frascaroli
Viral Pathogenesis
OP133
Recruitment of an Innate Immune DNA Sensor by the Cytoplasmic Isoforms of KSHV LANA
G. Zhang1, B. Chan2, A. Buch1, J. Rückert1, A. Pich3, B. Sodeik1, M. M. Brinkmann1,2, T. F. Schulz1
1Hannover Medical School, Institute of Virology, Hannover, Germany 2Helmholtz Centre for Infection Research, Braunschweig, Germany 3Hannover Medical School, Institute of Toxicology, Hannover, Germany
The latency associated nuclear antigen (LANA) of Kaposi’s Sarcoma-associated Herpesvirus (KSHV) is mainly localized and functions in the nucleus of the latently infected cells and plays a pivotal role in the replication and maintenance of latent viral episomal DNA. Here we used mass spectrometry to dissect the composition of KSHV LANA containing protein complexes and identify new cellular proteins interacting with LANA. We found that the cyclic GMP-AMP synthase (cGAS), an innate immune DNA sensor, to be one of the cellular partners of LANA. cGAS is known to detect foreign DNA in the cytoplasm. We found that a previously reported N-terminally truncated cytoplasmic isoform of LANA that results from internal translation initiation within the LANA N-terminal domain (Toptan T et al. J Virol. 2013) interacts with cGAS. We could show that cGAS restricts the reactivation of KSHV from latency. KSHV LANA, and in particular its cytoplasmic variant, antagonize cGAS function. We hypothesize that, by antagonizing cytoplasmic cGAS, the N-terminally truncated cytoplasmic form of LANA promotes the reactivation of the lytic replication cycle. Corresponding author: Guigen Zhang
Viral Pathogenesis
P386
Activation of latent chromosomally integrated Human Herpes Virus 6 (HHV-6) and its possible role in human disease development
B. Prusty1
1University of Wuerzburg, Department of Microbiology, Wuerzburg, Germany
Introduction and Objectives: Human herpesvirus 6 (HHV-6) is a neurotropic virus that achieves latency by integrating into telomeric ends of human genome (ciHHV-6). Approximately, 0.2-5% of human population carry genetically inherited ciHHV-6. HHV-6 has been associated with several human disorders including multiple sclerosis, chronic fatigue syndrome, mesial temporal lobe epilepsy, schizophrenia, bipolar disorder, Alzheimer’s. However, because of its nearly ubiquitous nature, HHV-6 is assumed to be a silent commensal. To negate this myth, we asked a few questions- Is HHV-6 latency a dead end for the virus? Can activated, partial or full-length viral genome re-integrate into human chromosome? Can partial transcription and/or translation from the latent or activated HHV-6 genome be associated with human diseases? Materials and methods: To find an answer to our questions, we utilized primary blood cells from individuals having genetically inherited HHV-6 to study molecular mechanism behind viral activation. Results: Preliminary data shows that chromosomally integrated HHV-6 is not always silent. Common drugs and environmental stimuli can activate it through modulation of human telomere. Due to its telomeric localization, whole or parts of viral genome can be excised during t-circle formation. Excised viral genome can possibly shuffle in and out of host chromosomes resulting in genomic instability. Further, the “semi-latent” virus is functionally active enough to influence host cell without forming infectious viral particles. Conclusions: Over 95% of human population carry latent HHV-6 that can get activated while exposed to specific, timely, relevant triggers. Hence, it might constitute a prime factor in effectuating several human diseases, with thus far unidentified and unexplored origins. Corresponding author: Bhupesh Prusty
Viral Pathogenesis
P388
Nipah virus envelope proteins stimulate primary porcine brain microvascular endothelial cells in the absence of virus replication
T. C. Freitag1, S. Erbar1, A. Maisner1
1Philipps University Marburg, Institute of Virology, Marburg, Germany
Nipah virus (NiV) is a BSL-4 classified paramyxovirus that establishes a systemic endothelial infection. Clinical NiV encephalitis is mostly the result of a prominent infection of microvascular endothelial cells in the CNS that causes vasculitis, thrombosis, and inflammatory cell infiltration. In agreement with the view that these processes are mediated by cytokines, experimentally NiV-infected hamsters showed upregulation of several cytokines in CNS tissues. It is however not clear, if the cytokines detected in vivo were secreted from immune cells or rather from endothelial cells. To address this question, we analyzed cytokine production in primary porcine brain microvascular endothelial cells (PBMEC) upon infection with NiV. Aside of a minor upregulation of type I IFNs, qPCR analyses revealed an upregulation of IL 1α, IL 1β and IL 6. To determine if NiV replication is needed for the observed cytokine upregulation in PBMEC, cells were incubated with UV-inactivated viruses and virus-like particles (VLPs). With both we found an upregulation of several cytokines indicating that virus replication and viral RNA are not essentially needed for this effect. Additionally, we observed an increased expression of adhesion molecules in NiV-infected PBMEC. As supernatants of NiV-infected cells that were cleared from infectious viruses as well as VLPs could induce an upregulation of adhesion molecules in naïve indicator PBMEC, we conclude that this upregulation is not directly induced by viral replication steps but rather by secreted cytokines and/or binding of the NiV surface glycoproteins to the PBMEC. Corresponding author: Tanja Christiane Freitag
Viral Pathogenesis
P389
Role of a Legionella pneumophila Protease in Influenza HA Activation
A. Arendt1, W. Garten1, H.- D. Klenk1, E. Böttcher-Friebertshäuser1
1Philipps-University Marburg, Institute of Virology, Marburg, Germany
Bacterial coinfections are one of the major causes for complications of influenza and contribute significantly to morbidity and mortality. Viral infection has been shown to prime the respiratory epithelium for attachment and colonisation of bacteria such as S. pneumonia and S. aureus. Interestingly, certain bacteria have been demonstrated to support proteolytic activation of influenza virus directly by secreting a protease that is able to cleave the hemagglutinin (HA) precursor HA0 in its fusion competent form (HA1 and HA2). Thereby bacteria may contribute to virus spread and enhanced viral pathogenicity. Here, we were able to show that Legionella pneumophila secretes a protease in liquid culture that is able to cleave the HA of influenza virus in vitro. Interestingly, different cleavage patterns were observed for distinct virus subtypes. Some subtypes of HA were cleaved similar to trypsin, that was used as a positive control. Furthermore the infectivity of these viruses was increased after treatment with L. pneumophila supernatant. For other subtypes, atypical cleavage fragments were observed, and infectivity was not enhanced. Taken together we could show that Legionella pneumophila secretes a protease which is able to cleave and functionally activate the influenza virus HA of specific subtypes. Identification and further characterization of the protease is currently ongoing. Corresponding author: Annika Arendt
Viral Pathogenesis
P390
Kaposi Sarcoma Herpesvirus (KSHV) K15 protein contributes to the increased invasiveness and angiogenic properties of infected
endothelial cells
S. gramolelli1, M. Weidner-Glunde1, B. Abere1, T. F. Schulz1
1medizinische hochschule hannover, virology, hannover, Germany
Kaposi sarcoma herpesvirus (KSHV), a γ2-herpesvirus, is the causal agent of Kaposi sarcoma (KS), a highly vascularized tumour. We have previously shown that the KSHV ORF K15 contributes to virus-driven angiogenesis in endothelial cells by recruiting and activating PLCγ1and thereby triggering the NFAT pathway (Bala et al. Plos Pathogens 2012). In this study, we report that recruitment and activation of PLCγ1 by K15 is also responsible for the increased invasiveness of KSHV-infected endothelial cells. We found that the cytoplasmic domain of K15 interacts with the n- and c-terminal SH2 domains in the γ-specific array (γSA) of PLCγ1. We identified a small protein fragment capable of inhibiting the K15- PLCγ1 interaction in a dominant negative manner. We could also show that this protein fragment antagonizes K15-dependent NFAT activation specifically without affecting the activation of the NF-kB pathway also triggered by K15. We also found that this protein fragment inhibits KSHV-mediated angiogenesis and invasiveness of infected endothelial cells. Our ability to block the increased angiogenesis and invasiveness of KSHV-infected endothelial cells with a protein fragment acting in a dominant negative manner suggests that it may be possible to target K15-dependent signalling pathways therapeutically and encourage the search for a small molecule interfering with the recruitment and/or activation of PLCγ1 by K15. Corresponding author: silvia gramolelli
Viral Pathogenesis
P391
Mass spectrometry-based protein expression analysis for the elucidation of pathogenic mechanisms of cowpox viruses
M. Bürger1, J. Döllinger1, P. W. Dabrowski1, A. Nitsche1
1Robert Koch Institute, ZBS 1, Berlin, Germany
Cowpox viruses (CPXV) are complex dsDNA viruses coding for about 200 proteins. The pathogenic as well as zoonotic potential may vary from strain to strain but the molecular mechanisms are hardly known so far. To elucidate the mechanisms underlying the varying virulence of CPXV, comparative protein expression analysis of a reference CPXV strain Brighton Red (BR) and a higher virulent zoonotic wild-type CPXV strain isolated from a rat (WT) were carried out. Human keratinocytes (HaCaT) were used as a model close to the regular entry route of CPXV infections and were infected with MOI = 5. Cytoplasmic and secreted proteins were quantified with nLC-ESI MS/MS (LTQ Orbitrap) 2, 6 and 10 h p.i. using Stable Isotope Dimethyl Labeling. Functional annotation of host cell proteins was carried out with Cytoscape GeneMANIA. Furthermore, the amount of released virus particles in the supernatant as well as cell status during the time course of infection of different cell lines were observed using plaque assay and xCELLigence system. It could be shown that CPXV WT infected cells release higher amounts of infectious particles in contrast to CPXV BR leading to faster cell death. Furthermore, non-modified as well as differently regulated host cell proteins reflecting various cellular processes during CPXV BR and CPXV WT infection could be identified. From this study it can be hypothesized that differences in the virulence of both CPXV strains may result from a differently regulated host immune response during infection with both CPXV strains. Corresponding author: Marica Bürger
Viral Pathogenesis
P392
Comparison of the infection of porcine precision-cut lung slices by porcine influenza A virus H3N2 and porcine coronaviruses
T. Krimmling1, C. Schwegmann-Weßels1
1University of Veterinary Medicine Hannover, Institute for Virology, Hannover, Germany
Ex vivo systems are a useful tool to analyze virus infection. Here, we established coronavirus infection studies in porcine precision cut lung slices (PCLS). Porcine coronaviruses as well as porcine influenza A viruses (SIV) can be involved in the porcine respiratory disease complex (PRDC). PRDC is caused by a combined infection of several viruses and/or bacteria. We compared single infection by SIV H3N2, transmissible gastroenteritis coronavirus (TGEV), and porcine respiratory coronavirus (PRCoV) - a respiratory variant of TGEV emerging in the 1980s in Belgium. Single PRCoV infection mainly causes mild disease of the respiratory tract, whereas TGEV can infect the respiratory and the gastrointestinal tract. The influenza virus H3N2 has been proven to infect ciliated and mucus-producing cells of the respiratory epithelium in the bronchioles. Finally as part of the PRDC, co-infection of the PCLS with both influenza and coronavirus was analyzed. The vitality of PCLS was checked by monitoring ciliary activity, live-dead staining, and measurement of LDH release. Vital PCLS were infected for one or two days and the site of infection was analyzed by confocal microscopy after staining of viral antigen. So far, our results have shown infection of PCLS by TGEV and PRCoV, but a different distribution of TGEV protein compared to SIV H3N2. Furthermore, co-infection of PCLS with H3N2 and PRCoV suggests an increase in PRCoV-infected cells. Infectious titers of released viral particles and the effect of co-infection on ciliary activity will be determined. Coronavirus infection was shown in porcine PCLS. Future co-infection studies will show how the different viruses influence each other during viral replication. Corresponding author: Tanja Krimmling
Viral Pathogenesis
P393
Revealing the competition of influenza virus NS1 and host cell PKR for dsRNA binding sites in living cells - a FLIM-FRET and
Anisotropy approach
F. Jolmes1, K. Schierhorn2, M. Mielke1, C. Seidel3, T. Wolff2, A. Herrmann1
1Humboldt Universität zu Berlin, Institut for Biology/ Molecular Biophysics, Berlin, Germany 2Robert Koch Institute, Division 17 - Influenza Viruses and Other Respiratory Viruses, Berlin, Germany 3Heinrich-Heine Universität, Molecular Physical Chemistry , Düsseldorf, Germany
Introduction: The influenza A Virus (IAV) protein NS1 is the major antagonist of the host cellular innate immune response. In this study we focused on the interaction between NS1 and the double stranded (ds)RNA-dependent protein kinase (PKR) which is an important immune effector protein. Objectives: To study the interaction between NS1 and PKR in presence or absence of dsRNA in living cells. Materials & Methods: To obtain the objective we usedfluorescencelifetimeimaging withFörsterresonanceenergytransfer (FLIM-FRET) as a high resolution confocal laser microscopy method. Therefore we transfected tagged proteins and polyIC_Rhodamine (Rhd) into HEK293T cells. As FRET pairs we used Turquoise - GFP (NS1-PKR) and GFP-Rhd (PKR-dsRNA) to measure NS1-PKR and PKR-dsRNA simultaneously. Furthermore we used the FRET pair YFP-Rhd (NS1-dsRNA) to measure the interaction between NS1 and dsRNA. Results: We showed that NS1wt-Turquoise led to a reduction of the FRET efficiency between PKR-GFP and the dsRNA-Rhd. This indicated that NS1 modulates the interaction between PKR and dsRNA. Furthermore, we could show an enhanced interaction between PKR and NS1 by adding dsRNA. That led to the hypothesis that dsRNA is necessary for the interaction between NS1 and PKR. Mutation of NS1 at the position R35 and R46 led to a complete loss of interaction between NS1 and PKR or dsRNA. In addition, we had hints that NS1 and PKR are competing for RNA binding sites. Conclusion: Our study identified for the first time in a cell-based FLIM-FRET system organisational principles of the PKR-NS1-dsRNA complex by analysing different combinations of PKR, various NS1 mutants and dsRNA. In upcoming experiments we will analyse changes in binding affinity using Biacore experiments. Corresponding author: Fabian Jolmes
Viral Pathogenesis
P394
Prevention of intra-host evolution of pandemic H1N1 influenza A virus (A(H1N1)pdm09) mpJena/5258 with hemagglutinin 222D/G (HA-
222D/G) polymorphism in ribavirin-treated mice
N. Seidel1, H. Braun1, M. Richter1, E. Walther1, A. Hoffmann1, P. Wutzler1, A. Sauerbrei1, M. Schmidtke1
1Jena University Hospital, Department of Virology and Antiviral Therapy, Jena, Germany
Introduction: The HA-222D/G polymorphism was frequently detected in clinical A(H1N1)pdm09 isolates. Recently, we demonstrated that it facilitates viral intra-host evolution of the A(H1N1)pdm09 mpJena/5258 (mpJena/5258) in BALB/c mice. Selection of the HA-G222 variant after day 4 p. i. correlated with strong symptom worsening (body weight loss and clinical score) and severe pneumonia. Objectives: Here, we address the question whether severe influenza caused by HA-G222 selection might be prevented by the neuraminidase inhibitor oseltamivir or the nucleoside analog ribavirin. Methods: mpJena/5258-infected BALB/c mice were treated with 100 mg/kg/d oseltamivir or ribavirin for 5 and/or 7 days (starting 4 h before infection). The effects of both drugs on survival, body weight loss, clinical score, mean time to death, lung viral titers on day 4 p. i., and intra-host evolution were analyzed. Results: Oseltamivir had neither an effect on viral replication in the lung on day 4 p. i. nor on the other study parameters. In contrast, ribavirin significantly decreased the lung viral titers, prevented lethality and significantly reduced body weight loss as well as clinical score. Furthermore, ribavirin hampered the selection of the HA-G222 variant, whereas oseltamivir did not affect the intra-host evolution of mpJena/5258. Conclusion: Our results demonstrate that anti-influenza drugs inhibiting viral replication in the lung, like ribavirin, might prevent severe influenza caused by clinical A(H1N1)pdm09 isolates with HA-222D/G polymorphism because they affect viral intra-host evolution. Corresponding author: Nora Seidel
Viral Pathogenesis
P395
Depletion of CpG dinucleotides in small dsDNA viruses genome: role of divergent evolutionary pressure
M. Upadhyay1, N. Sharma1
1Indian Institute of Technology, New Delhi, Kusuma School of Biological Sciences, New Delhi, India
Introduction: Dinucleotide frequencies may provide important clues on virus evolution. The CpG dinucleotide in particular has recently received much attention. Dinucleotide frequencies for small double-stranded DNA (ds-DNA) viruses infecting vertebrates have not been systematically studied. The evolutionary forces contributing to the differences in the relative abundance of dinucleotides in this group of viruses remain unclear. Objective: To investigate the differences, if any, in the relative abundance of dinucleotides among small ds-DNA viruses and to identify the evolutionary forces factors contributing to the evolution of this group of viruses. Materials and Methods: We analyzed a total of 185 full-length small ds-DNA viruses sequences available in GenBank; this includes 128 sequences of viruses belonging to the family Papillomaviridae and 57 sequences of viruses belonging to the family Polyomaviridae. The relative abundance of dinucleotides, effective number of codon (ENC), relative synonymous codon usage (RSCU) and GC1,2 and GC3 values for coding regions were analysed. Results: The relative abundance of CpG dinucleotides was significantly lower in polyomaviruses as compared to that of papillomaviruses (0.21±0.14 vs 0.51±0.11; P<0.0001). Analysis of the codon usage bias, neutrality plots and the relative abundance of CpG dinucleotides within the codon suggest that mutational pressure plays a major role in the depletion of CpG dinucleotide among papillomaviruses; while translational selection against CpG containing codons accounts for the loss of CpG dinucleotides among polyomaviruses. Conclusion: Depletion of CpG dinucleotides may contribute to the evolution of small ds-DNA viruses. Both polyomaviruses and papillomaviruses are significantly CpG depleted. We demonstrate fundamental differences in evolutionary forces underlying CpG depletion among small ds-DNA viruses. Corresponding author: Mohita Upadhyay
Viral Pathogenesis
P396
Identification of lung stem/progenitor cells that survived acute influenza A virus infection using a Cre recombinase-encoding virus
A. Dudek1, P. Reuther1, M. Heiner2, S. Herold2, M. Schwemmle3
1Albert-Ludwigs-Universität, Institute of Virology, Freiburg, Germany 2Justus-Liebig-Universität, Infektiologie, Gießen, Germany 3Institute of Virology, Freiburg, Germany
Recovery from severe Influenza A virus (IAV) infection requires the regeneration of damaged airway epithelium. This regeneration depends on stem and progenitor cell populations residing within the lung epithelium. It is still under debate whether these cells are targeted by IAV and, if so, whether there is a subset of stem/progenitor cells surviving acute viral infection. To address this question, we generated a mouse adapted A/SC35M (H7N7) virus encoding a Cre-recombinase within the NS genome segment. This modified IAV replicated efficiently in cell culture and in the mouse model and, importantly, did not lose the Cre-recombinase gene upon passaging in cell culture or mice. To show that the Cre-encoding IAV can be utilized to control gene expression in virus-infected cells, we established a human lung-derived Calu-3 cell line comprising a loxP_dsRed_loxP_eGFP cassette. Indeed, viral infection of these cells induced the irreversible elimination of the dsRed gene resulting in a switch from red to green fluorescence. Infecting mice harboring a similar reporter cassette within the ubiquitously active rosa26 locus, we could visualize long term surviving cells at a time point where viral infection was cleared by the animal. To elucidate the identity of these cells, we performed flow cytometric analysis and could thereby reveal the presence of EpCamhighCD24low bronchiolar/alveolar progenitor cells. This approach offers the future opportunity to analyze to which extend surviving stem/progenitor cells contribute to the reconstitution of IAV-induced lung damage and whether viral infection results in epigenetic changes inherited to their progeny, which might influence susceptibility to viral and bacterial lung pathogens, respectively. Corresponding author: Alexandra Dudek
Viral Pathogenesis
P397
Expression levels of the attachment protein G differ between strains of a murine pneumovirus and determine the virulence
A. Adenugba1, C. Krempl1
1Institute for Virology and Immunobiology, University of Wuerzburg, Wuerzburg, Germany
Background: Strains of pneumoviruses, e.g. of respiratory syncytial virus (RSV) or of pneumonia virus of mice (PVM), are defined by the sequence of the putative attachment protein G, respectively G gene. In the case of PVM, the G genes of the major strains, i.e. 15 and J3666, differ in their organization in that a small upstream open reading frame (uORF) appears to precede the main J3666-G ORF in frame (GJ366665A). In addition a 2nd form of the J3666-G gene has been described in which the stop codon of the uORF is deleted by a point mutation (GJ366665U), thus, extending the major G ORF. Objective: In the present study, we analyzed the influence of the three forms of the G genes on protein expression, replication and virulence of PVM. Method: We replaced the G gene of recombinant PVM 15 (rPVM) with either of the two forms described for PVM J3666, thus, generating rPVM-GJ366665A and rPVM-GJ366665U, respectively. The recombinant viruses were analyzed with respect to the expression of G as well as to the effect on replication and virulence. Results: The uORF in the G gene of rPVM-GJ366665A somewhat reduced the expression of the main ORF but caused no major disadvantages on replication in tissue culture or the lungs of mice. However, rPVM-GJ366665A was found to be less virulent than the parental rPVM or rPVM-GJ366665U. In comparison, extension of the major G ORF in rPVM-GJ366665U increased the expression of the protein without affecting replication in tissue culture or in vivo. However, rPVM- GJ366665U was significantly more virulent in mice than rPVM or rPVM-GJ366665A. Conclusion: Thus, the G gene organization modulates G protein levels that in turn determine the virulence of PVM strains. Comparable mechanisms may determine the virulence of RSV strains. Corresponding author: Akinbami Adenugba
Viral Pathogenesis
P398
Low-dose Prednisolone Attenuates Disease Progression in Antiretroviral-Untreated HIV-Infection: Sex-based differences
C. Kasang1, S. Kalluvya2, C. Majinge2, G. Kongola2, M. Mlewa2, I. Massawe2, R. Kabyemera2, K. Magambo2, A. Ulmer3, H. Klinker4, E. Gschmack5, A. Horn5, E. Koutsilieri5, W. Preiser6, D. Hofmann5, J. Hain7, A. Müller1, B. Weissbrich5, A. Rethwilm5, A. Stich5, C. Scheller5
1Medical Mission Institute Würzburg, Würzburg, Germany 2Bugando Medical Centre, Mwanza, Tanzania, United Republic of 3HIV Schwerpunktpraxis, Stuttgart, Germany
4University Clinic of Würzburg, Würzburg, Germany 5University of Würzburg, Institute of Virology and Immunobiology, Würzburg, Germany 6University of Stellenbosch, Institute of Virology, Stellenbosch, Germany 7University of Würzburg, Institute for Mathematics, Würzburg, Germany
Background: HIV infection causes a chronic and general activation of the immune system, accompanied by a progressive loss of CD4 T cells and the development of AIDS. There is accumulating evidence that this hyperimmune activation is a causative factor in HIV disease pathogenesis. Methods: We conducted a double-blind, randomized, placebo-controlled clinical trial including 326 HIV patients. Patients eligible for the study were HAART-naïve, free of AIDS-defining symptoms and with CD4 counts > 300 cells/µl. Patients received 5 mg prednisolone per day for 24 months or placebo. The primary study endpoint was time to progression to an AIDS-defining condition or a drop of CD4 counts below 200 cells/µl. Results: Prednisolone treatment caused a reduction in chronic immune activation and had no significant effects on HIV viral load. CD4-counts increased under prednisolone treatment and decreased under placebo. In a per protocol analysis that included only patients with at least 80% study drug adherence, females treated with prednisolone progressed significantly slower to AIDS compared to placebo. No differences between prednisolone and placebo were observed in males. Conclusion: Our results demonstrate that the chronic immune activation is a key factor in HIV disease pathogenesis. Sex-based differences in the effects of prednisolone were observed. Corresponding author: Christa Kasang
Viral Pathogenesis
P399
Isolation and characterization of H9N2 avian influenza virus from a poultry flock co-infected with an avian infectious bronchitis virus
in Egypt
M. AboElkhair1,2
1Faculty of Veterinary Medicine, Virology, Sadat City, Egypt 2University of Sadat city, Virology, Sadat, Egypt
In the last decade, frequent incidence of H9N2 outbreaks have caused high mortality in poultry farms resulting in great economic losses in several countries of Asia and the Middle East. In Egypt, co-infection of H9N2 with infectious bronchitis virus (IBV) has been observed extensively during these outbreaks. The current study reports isolation and characterization of H9N2 virus co-infecting with IBV a broiler chicken flock in Egypt. The genomic RNA segments of the current isolate were subjected to RT-PCR amplification followed by sequencing analysis. The deduced amino acid sequences of the eight segments of our isolate were compared with those of Egyptian H9N2 viruses isolated from healthy and diseased chicken flocks during the period of 2011-2013. Phylogenetic analysis revealed that the isolate of the current study was closely related to other Egyptian H9N2 viruses isolated from healthy and diseased chicken flocks. The comparison of deduced amino acid sequences of all isolates indicated that there was no particular molecular characteristic difference among all Egyptian isolates whether they were isolated from apparently healthy, diseased or co-infected with IBV chicken flocks. The finding of the current study could suggest that the most important factor for enhancement of H9N2 pathogenicity could be co-infection with other pathogens. Corresponding author: Mohammed AboElkhair
Viral Pathogenesis
P400
Invasion of HPV8-E7 expressing keratinocytes is associated with fibronectin and integrin α3 overexpression.
S. Heuser1, M. Hufbauer1, P. Zigrino1, C. Mauch1, B. Akgül1
1University of Cologne, Institute of Virology, Cologne, Germany
Introduction: βHPV types (e.g. HPV8) are associated with the development of cutaneous squamous cell carcinomas (SCC). We could show, that 8E7 induces invasion of keratinocytes in organotypic skin cultures (OSC) based on de-epidermalised dermis but not on collagen type I composites. Objectives: We hypothesized that invasion of E7+ keratinocytes is triggered through interaction with extracellular matrix proteins. Materials and Methods: Invasion was measured in Boyden chamber assays and expression of cellular proteins was determined by Western blot and FACS analysis. Results: The junctional bridging proteins ZO-1 and β-catenin are strongly upregulated in E7+ OSC, which coincided with downregulation of E-cadherin expression. E7 mediated cadherin-switch (‾E-cadherin, N-cadherin) could be linked to the presence of a fibronectin matrix in monolayer cells. The significant role of fibronectin in stimulating E7 mediated invasion was confirmed in Boyden chamber assays. Enhanced fibronectin mRNA expression and protein secretion was detected in E7+ keratinocytes grown on laminin and fibronectin. Increased levels of fibronectin were also detected in peritumoral areas of HPV8-positive cutaneous squamous cell carcinomas. 8E7+ cells cultured on fibronectin showed upregulation of integrin α3 chain. Knockdown of integrin α3 abolished the invasive character of 8E7+ cells. In addition, we recently identified a single aa residue in E7, which is necessary for induction of cell invasion. Conclusion: These data indicate that fibronectin triggers HPV8-E7 mediated cell invasion which is mediated through upregulation of integrin α3. Corresponding author: Baki Akgül
Viral Pathogenesis
P401
Characterization of two Austrian field isolates of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV)
L. Sinn1, B. Lamp1, G. Mößlacher2, I. Hennig-Pauka3, T. Ruemenapf1
1University for Veterinary Medicine Vienna, Institute of Virology, Wien, Austria 2Oberösterreichischer Tiergesundheitsdienst, Linz, Austria 3University for Veterinary Medicine Vienna, Clinic for Swine, Wien, Austria
Porcine reproductive and respiratory syndrome virus (PRRSV) is a member of the Arteriviridae and of major economical importance for the swine industry worldwide. PRRSV outbreaks occur frequently in formerly free livestocks without epidemiological links and available vaccines are not satisfyingly successful. To obtain information about current PRRSV strains in Austria, we investigated field samples from recent outbreaks with different severity of pathology. For virus isolation, we obtained serum samples from Upper Austrian pig farms experiencing mild to severe PRRSV problems. Porcine alveolar macrophages (PAMs) and MARC-145 cells were inoculated with these field samples. Upon passaging and cloning, viral growth was monitored by immunostaining. After sequencing, viral genomes were assembled and classified. To characterize the pathogenicity of these strains in detail, an animal trial with eight-week-old piglets was performed. Whole genomes of two current PRRSV isolates could be determined. These strains from two different farms belonged to PRRSV type 1 subtype 1 and were completely independent, but closely related with each other and with a South Corean isolate. Their virulence could be confirmed in an animal trial: In necropsy lung lesions were found for both strains. Respiratory symptoms were only observed for one strain, matching the reported differences in virulence in the field. Taken together we could isolate two recent Austrian PRRSV type 1 subtype 1 strains from field outbreaks and characterize them by their complete genome showing that both isolates clustered with a South Corean isolate in phylogenetic analyses. Furthermore we could prove their pathogenic potential and their difference in virulence in an animal trial. Corresponding author: Leonie Sinn
Viral Pathogenesis
P402
Multibasic proteolytic cleavage site and mutations in the HA2 protein are essential for full virulence and transmissibility of H7N1 virus
in chickens
E.- S. M. Abdelwhab1, J. Veits1, K. Tauscher2, J. Teifke2, J. Stech1, T. C. Mettenleiter1
1Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Institute of Molecular Virology and Cell Biology, Insel-Riems, Greifswald, Germany 2Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Department of Experimental Animal Facilities and Biorisk Management, Insel-Riems, Greifswald, Germany
In 1999, low pathogenic (LP) avian influenza (AI) H7N1 virus in Italy caused widespread outbreaks in poultry. After few months the virus transformed into highly pathogenic (HP) form causing severe economic losses. Comparison of full genome sequences of LP and HP viruses indicated a considerable number of mutations in all gene segments. The hemagglutinin (HA) protein of the HPAI virus has a peculiar multibasic proteolytic cleavage site motif (mCS). To date, the genetic markers responsible for increased virulence and transmissibility of this virus has not been fully understood. In this study, recombinant LPAI and HPAI H7N1 viruses of chicken-origin were generated using reverse genetics. A panel of mutants carrying specific mutations in the HA of LP and HP viruses were used for experimental infection of chickens as well as replication kinetics in cell culture. HPAI H7N1 virus with monobasic CS or carrying the whole HA of LPAI H7N1 virus induced very mild disease, if any, in chickens resembling the low pathogenic virus. On the contrary, LPAI virus with the whole HA of HPAI virus was as virulent and transmissible as the HPAI strain where all infected and contact chickens died. Meanwhile, recombinant LPAI virus with mCS alone was significantly less virulent than the HPAI virus. Mutations in the HA2, in the vicinity of the cleavage site, were required for the exhibition of full virulence in chickens although it did not alter tissue tropism and virus excretion of infected birds or the replication efficiency of the HPAI virus on cell culture. Keywords: Avian influenza virus, H7N1, virulence, transmissibility, chickens Corresponding author: El-Sayed M. Abdelwhab