ACTAMICROBIOLOGICABULGARICA

Volume 32, Issue 2, June 2016

Bulgarian Society for MicrobiologyUnion of Scientists in Bulgaria

ISSN 0204-8809

ACTAMICROBIOLOGICABULGARICA

Acta MicrobiologicaBulgarica

The journal publishes editorials, original research works, research reports, reviews, short communications, letters to the editor, historical notes, etc from all areas of microbiology

An Official Publicationof the Bulgarian Society for Microbiology

(Union of Scientists in Bulgaria)

Volume 32 / 2 (2016)

Editor-in-ChiefAngel S. Galabov

Press Product LineSofia

Editor-in-ChiefAngel S. Galabov

EditorsMaria AngelovaHristo Najdenski

Editorial BoardI. Abrashev, SofiaS. Aydemir, Izmir, TurkeyL. Boyanova, SofiaE. Carniel, Paris, France M. Da Costa, Coimbra, Portugal E. DeClercq, Leuven, Belgium S. Denev, Stara ZagoraD. Fuchs, Innsbruck, Austria S. Groudev, Sofia I. Iliev, PlovdivA. Ionescu, Bucharest, RomaniaL. Ivanova, VarnaV. Ivanova, PlovdivI. Mitov, Sofia I. Mokrousov, Saint-Petersburg, Russia P. Moncheva, SofiaM. Murdjeva, PlovdivR. Peshev, Sofia M. Petrovska, Skopje, FYROM J. C. Piffaretti, Massagno, SwitzerlandS. Radulovic, Belgrade, SerbiaP. Raspor, Ljubljana, SloveniaB. Riteau, Marseille, FranceJ. Rommelaere, Heidelberg, GermanyG. Satchanska, Sofia E. Savov, Sofia A. Stoev, Kostinbrod S. Stoitsova, Sofia T. Tcherveniakova, SofiaE. Tramontano, Cagliari, ItalyA. Tsakris, Athens, GreeceF. Wild, Lyon, France

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Small Interfering RNAs against Human Enteroviral Infections

Nikolay M. Petrov1,2*, Angel S. Galabov2

1Institute of Soil Science, Agrotechnologies and Plant Protection “Nikola Pushkarov”, Sofia/ Kostinbrod2The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, Sofia

ACTA MICROBIOLOGICA BULGARICA

Abstract Much is known about the enteroviral life cycle, however, because of the emergence of resistant mu-

tants, no effective specific drugs are available to treat enteroviral infections. RNA interference (RNAi) is a post-transcriptional, sequence-specific, gene-silencing defense mechanism of eukaryotic cells against viruses and transposons. It is an evolutionarily conserved mechanism for regulating gene expression which has the potential to be a therapeutic alternative to antiviral small molecule drugs. Small interfering RNAs (siRNAs) induce RNAi and are critical for the inhibition of RNA virus replication in the host cell. siRNAs are also important regulators of virus-host cell interactions. In this investigation we describe a relatively novel approach for using RNAi against CBVs by creating a siRNA pool covering different conserved CBV gene sequences. Many viruses have evolved mechanisms to inhibit RNAi cell defenses. A single nucleotide substitution in the RNAi target site within the viral genome can result in complete loss of interference, de-pending on the location and the nature of the resulting mismatch. Using polls of siRNAs inhibit the viruses which may escape single-site siRNA silencing by point mutations, overlapping the mutated target region. The polymerase complex of bacteriophage phi 6 is used to synthesize specific dsRNAs complementary to target CBV genes. The dsRNAs are cleaved using Dicer to siRNA pool and introduced to cells by transfec-tion. We produced specific dsRNAs and siRNA pools targeted to 5’UTR, 2A and 3D genes of CVB3 that inhibit completely virus replication in vitro in HEp-2 cells. Cytotoxic effects were not observed in the used concentrations.Key words: RNAi, siRNAs, enteroviruses

РезюмеМного се знае за ентеровирусния жизнен цикъл, обаче, поради появата на резистентни

мутанти, няма ефективни специфични лекарства на разположение за лечение на ентеровирусните инфекции. РНК интерференция (RNAi) е пост-транскрипционен, комплементарно-специфичен защитен механизъм на генно мълчание на еукариотните клетки, срещу вируси и транспозони. Това е еволюционно консервативен механизъм за регулиране на генната експресия, който има потенциала да бъде терапевтична алтернатива на нискомолекулните антивирусни средства. Малките интерфериращи РНК (миРНКи, siRNAs) индуцират RNAi и са критични за инхибиране на РНК вирусна репликация в клетката-гостоприемник. миРНКи са също важни регулатори на взаимодействията между вируса и клетката гостоприемник. В това изследване представяме сравнително нов подход за използване на RNAi срещу CBV чрез създаване на миРНК пул, обхващащ различни консервативни CBV генни последователности. Много вируси имат еволюирали механизми за инхибиране на RNAi клетъчната защита. Единично нуклеотидно заместване в прицелното RNAi място във вирусния геном може да доведе до пълна загуба на генно мълчание, в зависимост от разположението и естеството на полученото несъответствие. Използването на пулове от миРНКи инхибира вирусите, които могат да избегнат генното мълчание чрез единична място специфична точкова мутация, като припокриват мутиралия прицелен регион. Полимеразният комплекс на бактериофага Phi6 се използва за синтезиране на специфични двРНКи, комплементарни на прицелни CBV гени. двРНКи се нарязват

*Corresponding author: m_niki@abv.bg

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с помощта на Dicer до пул от миРНКи и се въвеждат в клетките чрез трансфекция. Получихме специфични прицелни двРНКи и миРНКи пулове спрямо 5’UTR, 2A и 3D гени на CVB3, които инхибират напълно вирусната репликация in vitro в HEp-2 клетки. При използваните концентрации не бяха наблюдавани цитопатични ефекти.

IntroductionCoxsackieviruses belong to the Enterovirus

genus within the Picornaviridae family. (Van Re-genmortel et al., 2000). Clinical manifestations of the Coxsackievirus group B (CVB) infections in-clude a wide range of diseases and tend to infect the heart, pleura, pancreas, and liver, causing pleuro-dynia, myocarditis, pericarditis, nonspecific febrile illnesses, rashes, upper respiratory tract disease, insulin-dependent diabetes, aseptic meningitis and hepatitis (Lim et al., 2013). Approximately 10 mil-lion symptomatic enteroviral infections are esti-mated to occur annually in the United States. From 2002-2004, an estimated 16.4-24.3% of these ill-nesses were attributed to coxsackievirus serotypes. For 2 of the 3 years, coxsackievirus B1 was the pre-dominant serotype. Enteroviruses are responsible for approximately 30,000-50,000 hospitalizations per year (Kadambari et al., 2014). The disease is often self-limiting and subclinical, but some acute infections are severe and lethal. Enteroviruses, and CBVs in particular, may have a role in the patho-genesis of type 1 diabetes (Hyoty and Taylor, 2002).

The coxsackievirus genome is a linear mole-cule of ssRNA, owing to its positive polarity. The ssRNA comprises an open reading frame (ORF), flanked on both 3′ (∼100 bases) and 5′ (∼800 bases) termini untranslated regions (UTRs). The ORF contains genes encoding 11 proteins (Huber et al., 1998). The protein capsid is composed of 4 structural proteins - VP1, VP2, VP3 and VP4. There are seven other non-structural proteins like two viral proteases (2A, and 3C), an RNA-depen-dent-RNA-polymerase (3D), two proteins involved in RNA synthesis (2B, and 2C), a primer of initi-ation of RNA synthesis (3AB) and a small poly-peptide (VPg) of 20–24 amino acids derived from gene 3B. The 5′ UTR of viral RNA is not linked to eukaryotic 7-methylguanosine triphosphate cap structure associated with eukaryotic mRNA, but the 5′ UTR covalently binds to VPg (Flanegan et al., 1977).

There are no virus-specific therapies for en-terovirus infections. Galabov and colleagues ex-amined a novel approach consisting of a consecu-tive and alternating, not simultaneous, application of the compounds in a combination on the model of an enterovirus infection in newborn mice (Vas-

sileva-Pencheva and Galabov, 2010). They used 3 compounds differing in their mode of antiviral action selected for consecutive and alternating ap-plication: disoxaril, guanidine hydrochloride and oxoglaucine. The triple combination for consecu-tive application of disoxaril, guanidine. HCl and oxoglaucine (DGO), applied in the order of men-tioning, shows a marked in vivo efficacy, expressed in a reduction of the mortality rate and lengthening of the mean survival time (Vassileva-Pencheva and Galabov, 2010). Thus, it has been demonstrated for the first time that preservation of the antiviral ac-tivity is possible if a triple combination of anti-en-teroviral inhibitors is applied in a consecutive and alternating manner (not simultaneously) (Vassile-va-Pencheva and Galabov, 2010).

Enterovirus infections, such as poliovirus and CBV3 infections have been successfully inhibited using RNAi in cell culture and in animal models (Gitlin et al., 2002, Merl et al., 2005; Tan et al., 2007; Fechner et al., 2008; Kim et al., 2008). These studies have employed sequence specific siRNAs, synthesized chemically.

Since identifying target sequence candidates for RNAi can be difficult and expensive, it would be beneficial to find an alternative way of produc-ing siRNAs. One mechanism would be to digest long dsRNA into siRNA pools representing multi-ple sequences of the viral gene or genome.

Materials and MethodsVirus

Coxsackievirus В3, neurotropic strain Nancy stock for in vitro experiments, was obtained from the collection of the Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, Sofia, Bulgaria. It was grown in monolayer HEp-2 cells with an infectious titer of 104.5 CCID50. Cells

Monolayer cell cultures of human epithelial carcinoma cells (HEp-2) were grown in DMEM medium containing 5 % fetal bovine serum in 96-well plates in CO2 incubator at 37 °C/ 5% CO2.Reference compounds

dsRNAs and siRNAs of the S segment of bacteriophage phi6 (2948bp) (obtained from Den-nis Bumford, University of Helsinki, Finland) were

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applied. Extraction of total RNA

We used RNEasy Mini Kit (Qiagen, Germa-ny) according to the manufacturer’s instructions.Touch-down

RT-PCR (Don et al., 1991) for amplification of a specific conserved fragment from 5’UTR, 2A and 3D of CVB3 genome. Used primers: 5’UTR For - ACC TTT GTG CGC CCT GTT; 5’UTR Rev - CAC GGA CAC CCA AAG TA; 2A For - GGA CAA CAA TCA GGG GCA; 2A Rev - TCC ATT GCA TCA TCT; 3D For- TCT CAT AGC ATT TGA TTA C; 3D Rev – ACG TGA CAC GTT CGG AGA AT; 3Da Pol For - TGG GGA TCC ATG TTG GCG GGA; 3Da Pol Rev - ACC CCC ACT GCA CCG TTA TCT.Synthesis of copy

DNA: included denaturation of RNA (0.05-0.5 µg) at 95°С for 5 min with 7 µl of appropri-ate primer in a final volume of 10 µl; incubation of a master mix (5 μl 5×MMLV-buffer, 2 μl dNTPs (2mM), 0.5 μl M-MuLV reverse transcriptase (200 U/μl), 7.5 μl DNase and RNase free H2O at 42°С for 60 min.

PCRPreparation of a master mix at a final volume

of 25 µl (6 μl cDNA, 2.75 μl10 × PCR buffer, 2.2 μl MgCl2 (25 mM), 2.2 μl dNTPs (2 mM), 1 μl of each primers (10 μM), 1 μl proofreading Pfu DNA-poly-merase (5 U/μl), 8.85 μl H2O) in Auto-Q Server (LKB, UK) Gel electrophoresis

According to Sambrook and Rassel (2001):

Fig. 1. Promoter sequences of the primers

DNA fragments were separated in 2% agarose gel in ТАЕ buffer with ethidium bromide (0.2 µg/ml) at 80-150V for 1 h. PCR products were visualized in trans-illuminator GenoPlex (VWR) with λ = 315 nm.In vitro dsRNA production system

The produced PCR fragments were used as templates. Polymerase promoter sequences needed for the dsRNA synthesis were added to both sides of the target sequence using PCR. The PCR primers were designed so that they contained RNA poly-merase promoter sequences at their 5’-ends. Thus, in the PCR product, RNA polymerase promoter sequences flanked the target sequence. In addition to promoter sequences, each primer should contain 17-22 nucleotides of target gene-specific sequence at the 3’-end (Fig.1).

dsRNA was synthesized by the combination of in vitro transcription and replication of DNA template (according to the instructions of Replica-tor RNAi Kit, Finnzymes, Finland). Generation of siRNA pools by Dicer- digestion

We used the enzyme Power Cut Dicer, which is a recombinant endo-ribonuclease from Giardia

intestinalis. It effectively cut dsRNA, yielding frag-ments with a length of 25-27 nucleotides, which re-sulted in accumulation of a pool of siRNAs. Transfection of cells

We transfected a cell monolayer with Oligo-fectamine™ Reagent (Invitrogen, USA), according to the manufacturer’s instructions.

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Determination of cytotoxicity of dsRNAs and siRNA pools on monolayer culture HEp-2 by staining with neutral red (NR)

Cells were cultured in 96 well plates (Cell-star) having an output density 2.5x105 cells/ml. Cells were incubated at 37°C/5% CO2 until con-fluent monolayer formation. Each of the samples was repeated four times. Cells were incubated for 72 hours, and then washed with 0.15 ml/well NR. Cells were incubated for 3 hours at 37°C/5% CO2 for the dye to penetrate viable cells. The dye was removed and cells were washed again with 0.15 ml/well of PBS, then were added in 0.1 ml/well of ex-traction solution (1% glacial acetic acid, 50% eth-anol and 49% distilled water). With this solution, the plates were placed on a shaker for 10 minutes, and afterwards the absorbance of the samples from each well was read on ELISA Reader (Organon, Germany) at a wavelength of 540 nm and a refer-ence value of 620 nm. The survival of the cells of the analyzed samples was calculated as % relative to control cells (which was assumed to be 100%) by the formula:

cell viability% = number of living cells per sample number of live cells in control x 100

The obtained values were used to build “dose-survival” curves, which identify 50% cyto-toxic concentration (CC50) of the individual sub-stances.Determination of the antiviral effect of the dsRNAs and siRNA pools obtained by inhibition of the cyto-pathic effect and staining with NR

Cells were cultured in 96-well plates with an output frequency of 2.5x105 cells/ml in the forma-tion of a confluent monolayer. The growth medium was aspirated and the cells were inoculated with a range of virus dilution corresponding to a multi-plicity of infection (MOI) 0.004. After expiration of adsorption, the unabsorbed virus was removed and 0.1 ml/well of the appropriate dilution of the substance was added. As controls, we used non-in-fected cells cultured under the same conditions as the viral control, untreated control substance and toxicity for each dilution of the substance-free vi-rus. Each of the samples was repeated four times. The plates were incubated for 72 hours at 37°C/ 5% CO2. The inhibition of the cytopathic effect was determined by NR uptake by viable cells the dye uptake of NR viable cells. Decant the middle of the plate and the cells were washed once with 0.15 ml /well of PBS (without Ca2+ and Mg2+). The phos-phate buffer solution was decanted and treated drop wise with a 0.1 ml/well of dye (1% glacial acetic

acid, 50% ethanol and 49% distilled water). The plate was incubated for 10 min with this solution, and afterwards the light absorbance by the sam-ple was read at 540 nm (Borenfreund and Purner, 1985). The obtained values were used to calculate the percentage of protection of the cells, using the formula:[OD (sample) – OD (virus control) / OD (toxicity control) – OD (virus control)] х 100,

where: OD – optical density (absorption)

The resulting value was used to determine the concentration of the substance that inhibited the preparation of an infectious viral progeny by 50% (IC50).Statistics

The results were processed by determining the standard deviation using the program Microsoft Office Excel 2007 and Clustal Omega.

Results and DiscussionWe optimized a new method that can produce

dsRNAs, specific to the conserved parts of different gene regions of CVB3, for induction of posttran-scriptional gene silencing and blocking the transla-tion of the target genes. Thereby, the replication and spread of the virus into the host cells were blocked. From the analyzed sequences from our virus collec-tion of CVB3, compared with the sequences from NCBI data base, we decided to target comparative-ly conserved regions from 5’UTR, 2A, and 3D. The designed primers flanked 396 bp and 1544 bp (3Da) fragments from 3D, 440 bp fragment from 2A and 520 bp fragment from 5’UTR genes of CVB3 (Fig. 2).

Fig. 2 dsRNAs of the conserved fragments from CVB3 genome

Legend: 1 - 100 bp ladder, 2 - 3D (396bp), 3 - 3Da (1544bp), 4 - 2A (440bp), 5 - 3Da

(1544bp), 6 - 3Da (1544bp), 7 - 2A (440bp), 8 - 5’UTR (520bp)

M 3D 3Da 2A 3Da 3Da 2A 5’UTR

1 2 3 4 5 6 7 8

Fig. 2. dsRNAs of the conserved fragments from CVB3 genomeLegend: 1 - 100 bp ladder, 2 - 3D (396 bp), 3 - 3Da (1544 bp), 4 - 2A (440 bp), 5 - 3Da (1544 bp), 6 - 3Da (1544 bp), 7 - 2A (440 bp), 8 - 5’UTR (520 bp)

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The choice of gene fragment plays a crucial role in target specific gene silencing. The gene frag-ments ranging from 50bp to 1000bp were used to successfully silence genes (Helliwell et al., 2002). Two factors can influence the choice of length of the fragment the shorter the fragment, the less ef-fective silencing will be achieved, but very long fragments increase the chance of recombination. The effectiveness of silencing also appears to be gene dependent and could reflect accessibility of target mRNA or the relative abundance of the tar-get mRNA. Fragment length of between 300 and 600bp is a suitable size to maximize the efficiency of silencing. The other consideration is the part of the gene to be targeted (Helliwell et al., 2002).

We chose 3D, 2A and 5’UTR to silence for the following reasons: the enteroviral RNA-dependent RNA polymerase 3D is one of the major compo-nents of the viral RNA replication complex and ex-hibits elongation activity (Van Dyke and Flanegan, 1980). It can uridylylate VPg and use VPg-pUpU as a primer during viral RNA replication (Paul et al., 2003). The viral genome contains also a con-served 5’UTR, which is important to translation and RNA replication (Rohll et al., 1994). Gama-rnik and Andino (1998) have suggested that the binding of 3CD to the cloverleaf at the 5’ end of the viral genome promotes the replication of RNA, rather than its translation. Furthermore, they found that PCBP2 binds to stem-loop IV in enterovirus translation (Gamarnik and Andino, 2000). 2A pro-tease cleaves dystrophin protein, which is the factor leading to CVB3 cardiomyopathy (Badorff et al., 1999). It cleaves also TBP in-vitro (Yalamanchili et al., 1997) and cleaves eIF4GI and eIF4GII, which lead to the shut off of host translation (Sommergru-ber et al., 1994). The cleavages of poly(A) binding protein (PABP) by 2A protease contributes to the inhibition of cellular translation (Kuyumcu-Marti-nez et al., 2002).

The produced dsRNA fragments we digested with Power Cut Dicer and received siRNA pools (Fig. 3).

To determine the cytotoxicity of the produced fragments we use 12 different concentrations of ds-RNAs (Fig. 4) and siRNAs (Fig.5): 72, 24, 8, 2.7, 0.9, 0.3, 0.2, 0.15, 0.1, 0.05, 0.03, 0.01 µM to si-lence 4 target genes of CVB3.

All the concentrations of the four used frag-ments (5’UTR, 2A, 3D and larger 3D called 3Da), even the highest (72 µM) showed no significant toxicity for HEp-2 cell monolayer (Fig. 4, 5). Even CC50 cannot be calculated. From 0.01 to 0.35 µM

Fig. 3. siRNA pools received from dsRNAsLegend: 1 - 100 bp ladder,2 - empty, 3- 3D, 4 - 3Da, 5 - 2A, 6 – PCR mix, 7 - 5’UTR

Fig.4 Cytotoxicity assay of sdRNAs for HEp-2 cells

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Fig.5 Cytotoxicity assay of siRNA pools for HEp-2 cells

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Fig.4. Cytotoxicity assay of sdRNAs for HEp-2 cells

Fig. 5. Cytotoxicity assay of siRNA pools for HEp-2 cells

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absolutely no cytotoxicity was observed for the used dsRNAs (Fig. 4) and siRNAs (Fig. 5). Weak cytotoxicity was observed in the range of 10 to 25 % when concentrations of dsRNAs of the tar-get gene fragments above 25 µM were used (Fig. 4). Even the highest used concentration (72 µM) induced very litle cytotoxicity of 12 % (Fig. 5).

In our experiments aiming to determine the antiviral effect of both dsRNAs and siRNA pools we used only seven concentrations that gave abso-lutely no cytotoxic effect on cell monolayer: 0.3, 0.2, 0.15, 0.1, 0.05, 0.03, 0.01 µM (Fig. 6, 7).

Using 0.3 µM of all tested dsRNAs (Fig. 6) and siRNA pools (Fig. 7) completely inhibit CVB3 replication in vitro in HEp-2 cells. It was established a 100% inhibition of the virus content at MOI 0.04. 5’UTR and 2A siRNA pools at concentration of 0.2 µM also inhibited by 100 % the virus titer of CVB3

Fig.6 Antiviral effect of dsRNAs against CVB3

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Fig. 7 Antiviral effect of siRNA pools against CVB3

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Fig. 7. Antiviral effect of siRNA pools against CVB3

Fig. 6. Antiviral effect of dsRNAs against CVB3

(Fig. 7). These are the most efficient fragments.Various RNAi applications have been used to

investigate gene functions, to target disease-related genes and as antiviral agents in cell culture and in vivo. The RNAi approach also has potential for an-tiviral therapy of virus infections. Treatment with siRdRP2 reduced the number of CBV3-infected cells. No off-target effects were detected that could be attributed to the w6–siRNA pools, probably due to the low concentration of each individual siRNA within the pool (Nygardas et al., 2009).

ConclusionWe optimized a new technique for produc-

tion of specific dsRNAs and siRNA pools target-ed at 5’UTR, 2A and 3D genes of CVB3 genome. With these small RNA pools we efficiently inhibit CVB3 replication in vitro in HEp-2 cells. 0.3 µM of all tested sdRNAs and siRNA pools completely supress CVB3 replication in vitro in HEp-2 cells: a 100% inhibition of the virus titer at MOI 0.04. 5’UTR and 2A siRNA pools at concentration of 0.2 µM also inhibited by 100 % the virus titer of CVB3. Cytotoxic effects were not observed in the used concentrations. It seems that the use of pooled siRNAs is a favorable means to target viral infec-tions and may offer a viable alternative for sin-gle-site siRNAs.

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Van Regenmortel, M., C. Fauquet, D. Bishop (2000). Virus Taxonomy. Classification and Nomenclature of Viruses. Seventh Report of the International Committee on Taxon-omy of Viruses. Academic Press, UT, USA.

Vassileva-Pencheva, R., A. Galabov (2010). Avoiding drug-resistance development by novel approach of com-bining anti-enteroviral substances against coxsackievirus B1 infection in mice. Antiviral Res. 85: 366-372.

Yalamanchili, P., R. Banerjee, A. Dasgupta (1997). Poliovi-rus-encoded protease 2APro cleaves the TATA-binding protein but does not inhibit host cell RNA polymerase II transcription in vitro. J. Virol. 71: 6881-6886.

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Diversity and Phylogeny of Begomovirus Populations and their Management

Chitra Nehra*, Avinash Marwal*, Rajarshi K. Gaur#Department of Bioscience, College of Arts, Science and Humanities, Mody University of Science and Technology, Lakshmangarh, Sikar-332311, Rajasthan, India

ACTA MICROBIOLOGICA BULGARICA

#Corresponding author: gaurrajarshi@hotmail.com;rkgaur.fasc@modyuniversity.ac.in*Equal contribution

AbstractBegomoviruses are plant-infecting viruses, which are transmitted by the whitefly vector Bemisia

tabaci and have been known to cause extreme yield reduction in a number of economically important vegetables around the world. They are abundant in tropical and subtropical environments, where insects that transmit these viruses are abundant. Identification of plant viruses, monitoring for new viral diseases, understanding the vectors that transmit viruses, and determining viral and vector impacts on the growth and development of crop cultivars and lines is vital to managing and controlling these diseases. In addition to damaging crops and causing yield losses, plant viruses interact with vectors and other diseases to increase the damage from the diseases/pests. The crops and weeds growing close to the crop fields are potential reservoirs of begomoviruses, but it is not known whether the same viruses infect several host species or co-infect any of the hosts. This increases the difficulty of controlling both the plant virus and the interacting pathogen or vector.

In a survey carried out in 2011-14 we observed several cultivated crops, ornamental plants and weeds with leaf curling, vein yellowing and growth stunting - typical symptoms of begomovirus. The DNA was isolated from the infected samples and the presence of begomovirus was confirmed by PCR using com-ponent specific primers. Further, rolling circular amplification (RCA) was performed to get the full-length sequence of the isolates. The full length sequence showed genome arrangement typical of monopartite/ bipartite begomovirus. Some of the infected samples showed association of a betasatellite responsible for the epidemic diseases in the case of monopartite. Phylogenetic analysis confirmed the diversity of bego-movirus in India. Our result also indicates that begomovirus has broaden its host range by recombination process. Expression of various full length or truncated or defective proteins of the virus has been effective in accomplishing pathogen-derived resistance. We analyzed the recombination parmeters of begomovirus strains and developed the RNAi strategy for the disease management. Key words: begomoviruses, RCA, RNAi, recombination

РезюмеБегомовирусите са растителни вируси, които се предават от вектор - белокрилката Bemisia

tabaci и са известни, че причиняват намаляване на добива в редица икономически важни зеленчуци по света. Те са в изобилие в тропическите и субтропическите местообитания, където насекомите, техни вектори са в изобилие. Идентификацията на растителните вируси, мониторинга на нови вирусни заболявания, установяването на вирусните вектори и определянето на вирус-векторните взаимодействия върху растежа и развитието на растителните линии и сортове е от жизненоважно значение за управлението и контрола на тези заболявания. В допълнение към нанасянето на щети на посеви и причиняването на загуби на продукция, растителните вируси взаимодействат с вектори и други патогени за увеличаване на щетите от тези заболявания / вредители. Културните видове и плевели, растящи в близост до посевите са потенциални резервоари на бегомовируси, но не е известно дали същите вируси заразяват няколко гостоприемни вида или ко-инфектират някои от

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гостоприемниците. Това увеличава трудността за контролиране както на растителния вирус, така и взаимодей-

ствието на патогена или вектора.По време на проучване от 2011 до 2014 г. наблюдавахме типични бегомовирусни симптоми

по културни видове, декоративни растения и плевели като листно завиване, жилкова хлороза и закърняване на растежа на растенията. ДНК беше изолирана от заразените проби, което потвържда-ва присъствието на бегомовирус чрез PCR, използвайки специфични праймери. Освен това, проведохме амплификация на въртящия се кръг (RCA), за да получим пълната дължина на геномите на вирусните изолати. Секвенциите на пълните геноми показаха типичното геномно подреждане на бегомовирусите като монопартидни / бипартидни. Някои от заразените проби показаха асоциацията на бета-сателитите, отговорни за епидемията от болести в случай на монопартиден вирусен геном. Филогенетичният анализ потвърждава разнообразието на бегомовирусите в Индия. Нашите ре- зултати показват също, че бегомовирусите разширяват своя кръг от гостоприемници чрез рекомбинационни процеси. Експресията на пълната или скъсена дължина на различни вирусни протеини или дефектни такива е ефективен процес за осъществяването на патоген-индуцирана устойчивост. Анализирахме рекомбинационните параметри на бегомовирусни щамове и разработихме стратегия на генно мълчание за контрол на болестта.

IntroductionThe genus Begomovirus (Family: Geminivri-

dae) is a major group infecting weeds, ornamental plants and economically significant crops in trop-ical and subtropical regions (Faquet et al., 2008; Adams et al., 2013). Begomoviruses are exclusive-ly transmitted by the white fly (Bemisia tabaci) and are divided into New World and Old World virus-es according to their geographic origins. The New World Begomovirus consists two genomic compo-nents, DNA-A and DNA-B, whereas the Old World begomoviruses are monopartite associated with ssDNA satellite molecules betasatellites and/or al-phasatellites (Seat el al., 2006; Brown el al., 2012).

Betasatellite molecules are half the size of DNA-A (~1.4Kbp) and enhance the symptoms severity along with other functions, i.e., transmis-sion, replication, encapsidation and movements in plants (Briddon et al., 2001; Nawaz-ul-Raham et al. 2009; Nawaz-ul-Rahman et al. 2010; Patil and Faquet, 2010). In contrast, alphasatellites may at-tenuate symptoms caused by helper begomoviruses beta satellites (Idris et al., 2011).

Begomoviruses (family Geminiviridae) and their associated satellite DNAs form complexes that cause severe diseases in agricultural systems (Mansoor et al., 2003; Mansoor et al., 2006; Zhou, 2013; Leke et al., 2015). These begomovirus-sat-ellite complexes infect a wide range of dicotyle-donous plants within ~37 different genera in 17 families of vegetable and fiber crops, ornamentals and uncultivated vegetation (Zhou, 2013). Various begomovirus-satellite complexes have been iden-tified in all major dicotyledonous plant crops and evidence advocates that these complexes are rapid-ly increasing their host and biogeographical rang-

es, thus threatening agriculture in tropical and sub-tropical regions worldwide (Mansoor et al., 2003, 2006; Leke et al., 2015).

RNA interference (RNAi) or post-transcrip-tional gene silencing (PTGS) occurs in a wide variety of organisms, including animals, fungi and plants (Bass, 2000; Saunders et al., 2004). Virus-derived small-interfering RNAs (siRNAs) are the hallmark of an innate immune response in plants that targets invading viruses through PTGS. RNA silencing is a sequence specific RNA degra-dation process that is triggered either by the forma-tion of dsRNA or alternatively by aberrant RNAs associated with transgenic viruses and transposons (Vaucheret, 2004). RNAs with hairpin with a loop structures are particularly actual inducers of PTGS in plants (Kon and Sharma, 2011).

During a survey carried out in 2011-2014, typ-ical begomovirus symptoms, such as leaf curling, vein yellowing, growth stunting, were observed in several crops, ornamental plants and weeds. In this study, we analyzed phylogeny and recombination break points of begomovirus isolates from different host plants, using different bioinformatics tools.

Materials and MethodsDNA isolation and viral genome amplification

Plant leaves showing geminivirus-like symp-toms such as leaf curling, leaf distortion, and stunt-ed growth were collected during 2011-2014 from Sikar district, Rajasthan, India. Collected samples included rose, radish, Petunia hybrida and Ca-tharant husroseus plant samples. Total DNA was extracted from the infected samples followed by rolling circular amplification (RCA) by using Tem-

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pliPhiTM kit (GE Healthcare) as per the manu-facturer’s instruction. RCA products were digest-ed with five different restriction enzymes (EcoR I, Hind III,PstI, SalI and BamHI) and cloned in pUC19 vector followed by sequencing by Xcelris Genomics, Ahmedabad, India. Sequence analysis, phylogenetic and recombination analysis

Sequences of each DNA-A were analyzed and initially submitted to a BLASTn search and on the basis of similarity score Begomovirus isolates were selected (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The studied sequences were further analyz-ed using the ORF (open reading frame) finder tool (http://www.ncbi.nlm.nih.gov/projects/gorf/) and the ExPASy tool (http://www.expasy.org/tools/). Phylogenetic analysis was done using other be-gomovirus isolates, obtained from NCBI database and trees were constructed by using the neighbor joining method in MEGA6.0 (Tamura et al., 2011). Bootstrap values from 1000 replicates were ana-lyzed. The identification of potential recombinant breakpoints and parents was carried out using RDP, recombination detection methods as implemented

in RDP 4.23 (Martin et al., 2010) with default set-ting.Engineering the plant virus for silencing

Engineering the plant virus for silencing can be studied with the help of RNA-mediated resist-ance against Croton yellow vein mosaic begomo-virus (CYVMV) infection as an example. CYVMV and its associated betasatellite from croton infested weeds have been characterized. Betasatellites are small circular ssDNA satellites containing a single open reading frame, ORF (termed as βC1), have been found to be associated with various plant dis-eases exclusively caused by monopartite begmo-viruses in the Old World. Effective gene silencing of croton yellow vein mosaic beta satellite encod-ed βC1 in Nicotiana benthamiana infiltrated with Agrobacterium tumefaciens that harbours the intron hairpin RNA (ihpRNA) construct aimed at the βC1

Fig. 1. Schematic diagram of the binary construct used for plant transformation. LB: Left border; RB: Right border.

(Sahu et al., 2014).A transgene consisting of the promoter region

of CYVMV DNA-β was designed to produce dou-ble stranded RNA (Fig. 1). The Nicotiana benthami-ana plants were transformed by Agrobacteium-me-diated gene transfer and were tested for tested for siRNA expression. CYVMV-infected transgenic lines showed small RNAs of approximate sizes, 23 nt higher expression intensities.

Results and DiscussionA total of four of DNA-A clones isolat-

ed from different host plants were sequenced and submitted to the NCBI databank. The se-quence of DNA-A clone KF218188 (host chilli), KJ700653 (host Pitunia hybrida), KF584008 (host rose) and KP698313 (host Catharanthusroseus) showed 2732nts, 2732nts, 2736nts and 2741nts in length, respectively. The full length sequences of KF218188, KJ700653, KF584008 and KP698313 shared similarity with RaLCuV: HQ698591(99), ChLCuV: HM007104 (99%), RLCuV:KR052159 (93%) and PLCrV: GQ478342 (93%). Hence, they can be considered as isolates of the Radish leaf curl

virus, Chilli leaf curl virus, Rose leaf curl virus and Papaya leaf crumple virus respectively.

The phylogenetic analysis of DNA-A clones (Fig. 2) constructed using the neighbor joining method showed three different clusters.

ChLCuV isolates KJ700653 and RaLCuV: KF218188 shared different clusters while PL-CrV:KP6698313 and RLCuV: KF584008 placed in similar cluster. ChLCuV: KJ700653 show maximum similarity with ChLCuV: KJ700653 and HM007104, while RaLCuVs: KF218188 demonstrated maximum proximity with RaLCuV: HQ698591. Isolate RLCuV: KF584008 shared maximum similarity with RLCuV: GQ478342 and PLCrV: KP698313 showed similarity with PLCrV: HM140368. For detection of recombination events, all DNA-A sequences were analyzed using RDP4 package. The recombination breakpoint analysis

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Fig. 2. Phylogenetic tree showing the relationships among the DNA-A described in this work and other begomoviruses obtained from GeneBank. The tree was construct-ed by the neighbor-joining method using MEGA 6 with a bootstrap values 1000 rep-licates. Horizontal distances are relative to calculated mutation distances, vertical branches are arbitrary. Bootstrap values are given away at nodes.

also provided strong evidence for presence of past recombination events in the analyzed sequences. A new begomovirus strain can arise by different inter and intraspecific recombination events (Patil

and Faquet, 2009). For all DNA-A clones, possible breakpoints, sequence fragments, parental geno-types and supported methods with P-Value are list-ed in Table 1.

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Table 1. Detection of recombination breakpoints

R-RDP, G-GENECONV, B-BOOTSCAN, M-MAXIMUM CHISEQURE, C-CHIMERA, S-SISSCANP-values are shown in bold.

№ Breakpoints Majorparent

Minor parent

Recombinant ORF P-value Supporting methods

1 1871-585 EU194194 JQ411026 KF218188 CR, V1, V2,C1 3.909× 10-36 R, G,B, M,C,S2 2553-246 GQ478342 FN794198 KJ700653 CR, V2,C4 1.395× 10-04 R, B,M,C3 1522-2099 GQ478342 LM645009 KJ700653 C1,C2 4.493× 10-13 R, G,B,M,C,S4 486-987 HM140368 LK028571 KP698313 V1 1.132× 10-38 R,G,B,M.C,S5 1053-1419 GQ478342 AM948961 KP698313 C3 7.420× 10-31 R,G,B,M,C,S6 1430-1869 LK028571 FN794198 KP698313 C2,C1 7.907× 1013 R,GB,M,C,S7 1911-2130 HM140368 GQ478342 KP698313 C1 7.962× 10-20 R,G,B,M,C,S8 1385-1893 LK028571 FN794198 KF584008 C1,C2,C3 7.907× 10-13 R,G,B,M,C,S

There is evidence for recombination events in all six ORFs V1, V2, C1, C2, C3, C4 and CR regions. Our analysis also indicates that the high-est frequency of recombination apparently occurs in the portion of the Rep encoding C1/AC1 ORF, with minimum breakpoint located in the N-termi-nal portion and/or the 5’-end of the common region. In begomoviruses, recombination breakpoints are nonrandomly distributed amongst mono- and bi-partite genomes, with hot spots in the Rep N-termi-nal portion and in the 5’-end of the common region (Lefeuvre et al., 2007; Lefeuvre et al., 2007a]. The recombination analysis in our study also shows that the most unique recombination event occurs in the C1/Rep and conserved region of the genome. This explains that studying DNA-A is contributed by other begomovirus isolates and increasing its host range by recombination process.

The available data and our results reveal the parallel importance of the bC1 gene in the bipar-tite begomovirus life cycle. Producing siRNA in N. benthamiana plants may be significantly important for producing antiviral resistance. We evaluated the silencing of bC1 in transgenic plants by analysis of siRNA. RNA isolated from a wild type N. benthami-ana plant and from transgenic plants (challenged with over 30 viruliferous whiteflies per plant and after 3 wpi) and subjected to siRNA detection by northern blot hybridization by using specific probe to bC1 transgene. This led to easy detection of re-sistance in these transgenic lines, i.e. no symptoms. The siRNA was absent in the non-transgenic plant showing disease symptoms.

ConclusionConsiderable resistance in transgenic plants

against viruses can be created by exploiting the

phenomenon of RNAi. Silencing specific genes by RNAi is a desirable natural solution to this problem as disease resistant transgenic plants can be pro-duced within a regulatory framework. Transgenic plants expressing RNAi vectors, as well as dsRNA containing crop sprays have been successful for efficient control of plant pathogens affecting eco-nomically important crop species. Begomoviruses are increasing their host range by a recombination process which is a major threat to economically im-portant plant species.

AcknowledgementsThe author/s are expressing thanks to the

Council of Scientific & Industrial Research (file no 09/1064 (0002)/2013-EMR-1) for financial sup-port.

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cation vote on taxonomic proposals to the International Committee on Taxonomy of Viruses. Arch. Virol. 158: 2023-2030.

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Briddon, R.W., S. Mansoor, I.nD. Bedford, M. S. Pinner, K. Saunders, J. Stanley, Y. Zafar, K. A. Malik, P. G. Markham (2001). Identification of DNA components required for in-duction of cotton leaf curl disease. Virology 285: 234-243.

Brown, J. K., C. M. Fauquet, R. W. Briddon, M. Zerbini, E. Moriones, J. Navas-Castillo (2012). Geminiviridae. In: King, A. M. Q., M. J. Adams, E. B. Carstens, E. J. Lefkowitz S (eds) Virus Taxonomy. Ninth Report of the ICTV. Elsevier/Academic Press, London, pp 351-373.

Cui, X. F., G. X. Li, D. W. Wang, D. W. Hu, X. P. Zhou (2005). A Begomovirus DNAβ-encoded protein binds DNA, func-tions as a suppressor of RNA silencing, and targets the cell nucleus. J. Virol. 79: 10764-10775.

Fauquet, C. M., R. W. Briddon, J. K. Brown, E. Moriones, J. Stanley, M. Zerbini, X. Zhou (2008). Strain demarcation

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Zhu, J. K. Brown (2011). An unusual alphasatellite asso-ciated with monopartite begomoviruses attenuates symp-toms and reduces betasatellite accumulation. J. Gen. Virol. 92: 706-717.

Kon, T., P. Sharma (2011). RNA silencing and viral encod-ed silencing suppressors. In: Gaur, R. K., Y. Gafni, V. K. Gupta, P. Sharma (eds) RNAi technology. CRC Press, USA, pp 209-240.

Lefeuvre, P., J. M. Lett, B. Reynaud, D. P. Martin (2007a). Avoidance of protein folds disruption in natural virus re-combinants. PLoS Pathog. 3: e181.

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Tomato Spotted Wilt Virus on Pepper (Capsicum annuum L.) Plants in Hungary. Molecular Characterization of Wild Type and Resistance Breaking Isolates. Searching for Resistance in Capsicum Genus

Asztéria Almási1, Gábor Csilléry2, Katalin Nemes1, Katalin Salánki1,László Palkovics3, István Tóbiás1*

1 Plant Protection Institute, Centre for Agricultural Research, HAS, Budapest, Hungary2Budakert Ltd, Budapest, Hungary3Corvinus University of Budapest, Department of Plant Pathology, Budapest, HungaryTechnology, Lakshmangarh, Sikar-332311, Rajasthan, India

ACTA MICROBIOLOGICA BULGARICA

*Corresponding author: e-mail: tobias.istvan@agrar.mta.hu

AbstractIn Hungary resurgence of Tomato spotted wilt virus (TSWV) has been frequently causing heavy crop

losses in pepper production since the mid-nineties. Management of TSWV was at first directed against thrips (using different insecticides or plastic traps), and against weeds as host plants of the virus and the thrips. Later on, Tsw resistance gene was introduced into different types of pepper. In 2010 and 2011, spo-radically, but in 2012 more frequently a resistance breaking (RB) strain of TSWV on resistant pepper culti-vars was observed in the Szentes region (South-East Hungary). The presence of a new resistance breaking strain was demonstrated by virological (test-plant, serological and RT-PCR) methods.

Previously, the non-structural protein (NSs) encoded by small RNA (S RNA) of TSWV was verified as the avirulence factor for Tsw resistance, therefore we analyzed the S RNA of the Hungarian RB and wild type (WT) isolates, and compared to previously analyzed TSWV strains with RB properties from different geographical origins. Phylogenetic analysis demonstrated that the different RB strains had the closest re-lationship with the local WT isolates and there was no conserved mutation present in any of the NSs genes of RB isolates from different geographical origins. According to these results, we concluded that the RB isolates evolved separately from a geographic point of view, and also according to the RB mechanism. The gene-silencing suppressor function of NSs protein is also discussed.

In order to find new genetic sources of resistance in Capsicum species 89, lines of Capsicum annuum, C. chinense, C. frutescens, C. chacoense, C. baccatum var. baccatum, C. baccatum var. pendulum and C. praetermissum were tested with TSWV-RB strain isolated in Hungary.Key words: Tomato spotted wilt virus (WT and RB strains), NSs protein, resistance

РезюмеПериодичната поява на Tomato spotted wilt virus (TSWV) често причинява тежки загуби

в производството на пипер от средата на деветдесетте години на миналия век. Контролът срещу TSWV първоначално включва употреба на инсектициди и инсектицидни уловки против трипсовете, пренасящи заразата. Атакувани са и плевелите, които са гостоприемници за вируса и неговите преносители. По-късно в някои типове пипер е внесен ген за устойчивост против TSWV. През 2010 и 2011 спорадично, а в 2012 по-често е наблюдавано преодоляване на устойчивостта (RB) на пиперови сортове в района на Szentes (Югоизточна Унгария). Чрез проучвания, включващи растителен тест, серология и RT-PCR, е доказано присъствието на щам, който преодолява устойчивостта при пипера.

Първоначално неструктурен протеин (NSs), кодиран от малка РНК (S RNA) на TSWV, е проверен като авирулентен фактор за Tsw устойчивост. Анализирани са S RNA на унгарски preodoljá-

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vasti na ustojcsivosta (RB) и диви типове (ili normalnie stamove) (WT) изолати. Направено е сравняване с предишни анализирани изолати на щамове на TSWV, способни да преодоляват устойчивостта (RB), които имат различен географски произход. Филогенетичният анализ показва, че различните RB щамове имат тясно родство с местните WT без консервативна мутация във всички NSs гени на RB изолатите от различните региони. Според резултатите е направено заключение, че изолатите RB частично се различават както по географски произход, така и по механизма на преодоляване на устойчивостта.

Обсъдена е също супресорната функция на NSs протеин. В опита да бъдат намерени нови генетични източници на устойчивост в пипера 89 линии от C.

annuum, C. chinense, C. frutescens, C. chacoense, C. baccatum var. baccatum, C. baccatum var. pendulum and C. praetermissum са тестирани с изолати TSWV-RB от Унгария.

IntroductionIn Hungary, Tomato spotted wilt virus

(TSWV) was described in tobacco in 1972 (Ligeti and Nagy, 1972), but the virus was not considered as an important pathogen in vegetable crops. Se-vere damage to the tomato and pepper production in the Szentes vegetable growing region (South-East Hungary) caused by TSWV infection was fir-st observed in 1995 (Gáborjányi et al., 1995). The symptoms were characterized by chlorotic spots, rings, patterns, occasional necrosis on the leaves and fruits, and malformations of the plants (Fig. 1). The introduction and spread of western flower thrips (Frankliniella occidentalis), a very efficient TSWV vector, played an important role in TSWV emergence (Jenser and Tusnádi, 1989; Jenser, 1995). Management of TSWV control was at first directed against thrips using different insecticides or plastic traps, and against weeds as host plants of the virus and the thrips. Later on, the Tsw re-sistance gene (Black et al., 1996) was introduced into different types of pepper (conical white, long pale green hot and sweet, tomato shape, spice pep-per and blocky types) (Csilléry, unpublished). Pep-per cultivars carrying the Tsw resistance gene upon TSWV inoculation showed necrotic local lesions on the leaves or other parts of the plant without sys-temic infection (Fig. 2). Thanks to the multicompo-nent management approaches - the efficient control against thrips and reservoir weed plants, and use of the Tsw resistance gene (originated from Capsi-cum chinense) there has been no economical loss caused by TSWV in pepper productions for a long time in Hungary. TSWV (wild type strain, WT) was present in some years and regions without econom-ical effect. For the first time in 2009, again in the Szentes vegetable growing region, virus infection was observed in TSWV resistant pepper varieties, from which resistance breaking isolates of TSWV (TSWV-RB strain) were detected (Salamon et al., 2010; Bese et al., 2012; Csilléry et al., 2012). The

reasons for the appearance of new resistance break-ing isolates were that some elements of multicom-ponent control management were neglected and also some effective chemicals (like Unifos 50 EC) were withdrawn from practice by EU regulation. Since that time TSWV infection could have caused heavy economic losses again. Since it was demons-tated that TSWV is able to adapt very rapidly to plant resistance, the Tsw resistance gene was bro-ken down only a few years after its deployment in pepper crops (Roggero et al., 2002; Thomas-Caroll and Jones, 2003; Margaria et al., 2004; Sharman and Persley, 2006).

Tomato spotted wilt virus (TSWV) is the type member of the genus Tospovirus (family Bunyavi-ridae) causing an important disease in horticultural and agronomic crops across temperate, subtropi-cal and tropical regions of the world (German et al., 1992; Goldbach and Peters, 1994). The virus was first described in Australia at the beginning of the 20th century (Brittlebank, 1919; Samual et al., 1930), and is now distributed worldwide, having an extremely broad host range that includes more than 900 plant species (Hanssen et al., 2010). High infection rates led to considerable economic loss-es worldwide, so that TSWV is considered one of the ten most economically destructive plant virus-es (Tomlinson, 1987; Goldbach and Peters, 1994). TSWV is transmitted by thrips species in a per-sistent manner (Whitfield et al., 2005). The larval thrips can aquire the virus, which is multiplying in the vector and adult thrips specimens transmit the virus (van der Wetering et al., 1996). TSWV has a spherical virion that varies in size from 80 to 120 nm with an enveloped structure. The TSWV genome is composed of three single-stranded lin-ear segments, large (L), medium (M), and small (S), named according to their lengths (Prins and Goldbach, 1998). The L RNA (~9 kb) contains an RNA-dependent RNA polymerase (RdRp) in a negative-sense orientation (Hann et al., 1991). In

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contrast, the M (~4.8 kb) and S (~3 kb) RNAs each contain two genes, one in positive- and the other in negative-sense orientation (Heinze et al., 2001). The M RNA encodes the NSm protein and Gn-Gc glycoproteins, while the S RNA encodes the NSs nonstructural and N nucleocapsid proteins (de Haan et al., 1990, Kormelink et al., 1992). According to de Ronde et al. (2013), NSs is the suppressor prote-in of the host plant gene silencing mechanism and it is responsible for breakdown of the plant’s resistan-ce (avirulence factor, avr). The contribution of the different domains of the protein to the gene silenc-ing function and as avr factor was recently analyzed in detail (de Ronde et al., 2014).

Our aim was to characterize the molecular differences between the WT and the recently emer-ged RB isolates in the S RNA to determine the po-tential origin of the RB strains and to identify the mutations in the avr factor responsible for break-down of the Tsw resistance. Moreover, our aim was to find genetic sources of resistance in Capsicum species against resistance breaking strain of TSWV (TSWV-RB).

Materials and MethodsVirus isolates

Fruit samples with typical TSWV symptoms from infected pepper plants (Capsicum annuum cv. Brendon and cv. Cibere) were collected in the main pepper growing region of Hungary (Szentes, Szeg-vár) in 2012 and were tested for presence of TSWV. Two TSWV isolates from cv. Brendon containing the Tsw resistance gene (HUP1-2012-RB, HUP2-2012-RB) and one WT (HUP4-2012-WT) isolate derived from cv. Cibere were mechanically inocu-lated and maintained on different test plants (Nico-tiana tabacum cv. Xanthi-nc, C. annuum cultivars ‘Celtic’, ‘Censor’, ‘Carma’, ‘Century’, ‘Dimentio’, ‘Skytia’, ‘Karakter’, ‘Brendon’, ‘Bronson’, and ‘Bravia’) to observe macroscopic symptoms and maintain the isolates. For long-time storage, sam-ples were kept in a deep freezer at -70 oC.Viral RNA extraction

RT-PCR, nucleotide sequence determination. Total RNA was isolated with the Spectrum Plant Total RNA Kit (Sigma) according to the manufac-turer’s instructions from pepper fruit samples or sy-stemically infected leaves of the test plants. The S RNA was cloned in two segments with overlapping regions. The first strand cDNAs were synthesized with Revert Aid H Minus First Strand cDNA Sy-nthesis Kit (Thermo Science) using the NSs-Re-verse (50-GGA CAT AGC AAG ATT ATT TTG

ATC CTG-30) and N-Reverse (50-GGG GAT CCA GAGCAA TTG TGT CAA TTT T-30) primers, res-pectively. The PCR amplification of the 1,404 bp fragment of NSs region was carried out with the primers NSs-Forward (50-GG CTGTAG CAG AGA GCA ATT GTG TCA TAA TTT T-30) and NSs-Reverse (50-GGA CAT AGC AAG ATT ATT TTG ATC CTG-30), while for the amplification of the 1,720 bp 30 fragment containing the N gene and the noncoding regions, N-Forward (50-AAT TTC TCC GCA ATC TAT TTC AGT TG-30) and N-Re-verse (50-GGG GATCCA GAG CAA TTG TGT CAA TTT T-30) primers were used. PCR was car-ried out in 50 µl final reaction volume as follows: amplification consisted of 5 min at 94°C followed by 35 cycles of 1 min of denaturation at 94°C, 30 s of annealing at 51°C, and 3 min of extension at 72°C, and a final extension cycle for 5 min at 72°C. PCR products were separated by electrophoresis in 1 % agarose gel stained with ethidium bromide and purified using Silica Bead DNA Gel Extraction Kit (Thermo Science) and cloned into CloneJet (Ther-mo Science) or pGEM-T Easy Vector (Promega, Madison USA). The sequence determination of the clones was carried out by BAYGEN (Szeged).Phylogenetic and sequence analysis

The nucleotide homology of the Hungarian and other TSWV strains retrieved from the Gen-Bank was examined by the BLAST program of NCBI. The nucleotide and deduced amino acid sequences were aligned with the ClustalW algo-rithm of the MEGA 6.06 program (Kumar et al., 2008). Phylogenetic trees were composed by the Neighbor-Joining method with 1,000 bootstrap replications (MEGA 6.06 program) with the entire viral proteins. The amino acid sequences of the N and NSs proteins of the Groundnut ringspot virus (GRSV) gained from the NCBI GenBank (acces-sion numbers JN571117.1) were incorporated into the phylogenetic trees as outgroup.Agrobacterium infiltration

NSs genes of TSWV RB and WT strains were cloned into pBin19 vector and Agrobacteri-um tumefaciens cells were transformed with them. Agrobacteria were cultivated overnight at 28°C in the presence of appropriate antibiotics. The cul-tures were harvested by centrifugation, and the pellet was resuspended in MES buffer containing 0.01M MgCl2 and acetosyringon. P14 was added to the infiltration solution (OD600 0.4). Final opti-cal density of the Agrobacterium cultures contain-ing NSs genes was adjusted at 600 nm (OD600) to 0.5. Agrobacterium-mediated transient expression

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P195_ESP

P114_ESP

P229_ESP

P228_ESP

P71-1_ESP

P125_ESP

P67-2_ESP

P65-2_ESP

P155_ESP

P90_ESP

P203_ESP

P86-1_ESP

VE427_RB_ESP

VE430_WT_ESP

P259_ESP

P267_RB_ITA Northern

P105-43.14_RB_ITA Northern

P105-44.7_RB_ITA Northern

p105/2006RB ITA Northern

P272_RB_ITA Northern

p105-RB-MaxII_ITA Northern

P166_RB_ITA Northern

P105 WT ITA Northern

p105-RB-MaxI_ITA Northern

p105-RB-Mar_ITA Northern

Br20_WT_BRA

Br20RB_BRA

TSWV-Gneung_KOR

TSWV-Njc_Kor

TSWV-Ghae_KOR

France81_FRA

TSWV-Pap_KOR

p202/3RB_ITA Sicily

p202_RB_ITA Sicily

p202/3WT_ITA Sicily

CAA19_FRA

GD98_BUL

HUP1-2012-RB

HUP2-2012-RB

BS97_WT_BUL

DH37_RB_BUL

HUP4-2012-WT

GRSV

92

99

88

67

40

87

95

83

78

52

98

96

88

100

70

63

64

97

94

68

52

65

63

70

43

66

0.02

Fig. 1. Phylogenetic tree based on the deduced amino acid sequences of the NSs protein of TSWV. Abbre-viations and accession numbers: HUP1-2012-RB : KJ649608; HUP2-2012-RB : KJ649609; HUP4-2012-WT: KJ649611; BS97: AJ418777; DH37: AJ418779; p202/3WT: HQ830187; p202/3RB: HQ830186; p202: DQ398945; GD98: AJ418780; CAA19: FR692822; VE430: DQ376184; VE427: DQ376185; p105: DQ376178; p105-RB-MaxI: HQ839730; p105/2006RB: DQ915946; p267: DQ376180; p105-RB-Mar: HQ839729; p105-44.7: DQ376183; p105-43.14: DQ376182; p105-RB-MaxII: HQ839731; Br20: DQ915948; Br20RB: DQ915947; p166: DQ376179; p272: DQ376181; France81: FR692829; TSWV-Pap: AB643674; TSWV-Ghae: AB643672; TSWV-Gneung: AB643671; TSWV-Njc: AB643673; p86-1: FR693020; p259: FR692932; p65-2: FR693005; p67-2: FR693007; p203: FR692900; p155: FR692871; p125: FR692857; p90: FR693023; p71-1: FR693811; p228: FR692917; p229: FR692918; p195: FR692895; p114: FR692852

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on Capsicum annuum cv Brendon leaves was con-ducted by pressure infiltration into the abaxial air space of 4- to 6-week-old plants using a needleless 2-ml syringe. P14 suspension was used for negative control.Resistance test

89 Capsicum items [Capsicum annuum (8), C. chinense (50), C. frutescens (8), C. chacoense (2), C. baccatum var. baccatum (4), C. baccatum var. pendulum (11) and C. praetermissum (6)] were inoculated at cotyledon stage with TSWV-RB strain. Symptoms were observed in the next 4 weeks.

ResultsTSWV isolates were tested on TSWV-sus-

ceptible pepper cultivars (‘Carma’, ‘Century’, ‘Di-mentio’, ‘Skytia’), and pepper cultivars carrying Tsw resistance gene (‘Celtic’, ‘Censor’, ‘Karakter’, ‘Brendon’, ‘Bronson’, ‘Bravia’). TSWV isolates causing necrotic local lesions (HR) on resistant pepper cultivars belonged to a wild type (TSWV-WT) strain, and isolates causing systemic symp-toms (chlorotic mosaic and ringspot pattern on the leaves, stunting) on all pepper cultivars belonged to a resistance breaking (TSWV-RB) strain. Three TSWV isolates were selected (HUP1-2012-RB, HUP2-2012-RB and HUP4-2012-WT) for further study. All the three virus isolates induced systemic symptoms (chlorotic or necrotic ringspot) on the in-oculated leaves of N. tabacum cv. Xanthi-nc plants.

Sequence similarities of the NSs genes were compared among the sequences of WT and RB isolates, originated from pepper from distinct geo-

graphical locations. Nucleotide sequence identi-ty among the Hungarian isolates was 99%, while compared to other isolates this value varied be-tween 95 and 99%. Amino acid (aa) sequences of the NSs protein (467 aa) were compared among the WT and RB isolates

Several mutations/changes were present only in the three Hungarian isolates at positions 122 (A to D), 137 (T to K), 174 (M to T), 450 (G to R), and 459 (P to S). The Hungarian RB isolates (HUP1-2012-RB, HUP2-2012-RB) had two aa substitutions compared to the WT Hungarian iso-late (HUP4-2012-WT) at positions 104 and 461 (A instead of T). Substitution at position 104 occurred only in the case of the Hungarian RB isolates. A phylogenetic tree was constructed based on the de-duced amino acid sequences of the NSs genes of the Hungarian and the selected isolates from the GenBank (Fig. 1).

One of the two main clusters consists of Spanish, the Northern Italian, and the two Brazilian l strains (further divided into different subgroups) regardless of the strain type, i.e., RB or WT. The other main branch contains the Korean, Hungari-an, Bulgarian and Italian strains from Sicily. The phylogenetic analysis supported the hypothesis that TSWV RB strains have been developed locally, and the worldwide trade and transport of plant propa-gating material do not seem to contribute to the ex-pansion of RB strains.

The NSs proteins were tested for their aviru-lence (Avr) activity by triggering of HR (necrosis) on Capsicum annuum cv Brendon (Tsw+) plants in Agrobacterium transient expression assay (Fig. 2).

NSs-RB NSs- ControlFig. 2. Symptoms on leaves of Brendon pepper variety (containing Tsw resistance gene) after Agrobacterium transient expression assay with NSs proteins of TSWV-WT (HR) and TSWV-RB (no HR).

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To determine which nucleotide or aa changes in NSs led to RB and how other functions altered, further (mutational analysis) investigation/analysis is needed. In the search for resistance to TSWV-RB strain, 89 Capsicum items were tested [Capsicum annuum (8), C. chinense (50), C. frutescens (8), C. chacoense (2), C. baccatum var. baccatum (4), C. baccatum var. pendulum (11) and C. praetermis-sum (6)]. Eighty-five items were susceptible and 4 C. baccatum var. pendulum items showed HR-like symptoms (Fig. 3). Further study is necessary to clear the genetic background and the possibility to use these items in resistance breeding.

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ance breaking strain of Tomato spotted wilt virus, (TSWV) on resistant pepper cultivars in Hungary. Internat. Symp. Current Trends in Plant Protection, Belgrade, Serbia 2012. pp. 239-241.

Black L. L., H. A. Hobbs, D. S. Kammerlohr (1996). Resis-tance of Capsicum chinense lines to tomato spotted wilt virus from Louisiana, USA, and inheritance of resistance. Acta Hort. 431: 393-401.

Brittlebank C. C. (1919). Tomato disease. J. Dept. Agric. Vic-torian 17: 231-235.

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de Ronde, D., P. Butterbach, D. Lohuis, M. Hedil, J. W. van Lent, R. Kormelink (2013). Tsw gene-based resistance is triggered by a functional RNA silencing suppressor pro-tein of the Tomato spotted wilt virus. Mol. Plant Pathol. 14: 405-415.

Fig. 3. HR-like symptoms on C. baccatum var. pendulum items after inoculation with TSWV-RB strain.

de Ronde, D., A. Pasquier, S. Ying, P. Butterbach, D. Lohu-is, R. Kormelink (2014). Analysis of Tomato spotted wilt virus NSs protein indicates the importance of the N-termi-nal domain for avirulence and RNA silencing suppressor. Mol. Plant Pathol. 15: 185-195.

Gáborjányi, R., G. Csilléry, I. Tóbiás, G. Jenser (1995). Toma-to spotted wilt virus: A new threat for pepper production in Hungary. IXth Eucapia Meeting, Budapest, 159-160.

German, T. L., D. E. Ullman, J. W. Moyer (1992). Tospovirus-es: Diagnosis, molecular biology, phylogeny, and vector relationships. Ann. Rev. Phytopathol. 30: 315-348.

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de Haan, P., R. Kormelink, R. R. de Oliveira, F. van Poelwijk, D. Peters, R. Goldbach (1991). Tomato spotted wilt virus L RNA encodes a putative RNA polymerase. J. Gen Virol. 72: 2207-2216.

Hanssen, I. M., M. Lapidot, B. P. Thomma (2010). Emerging viral diseases of tomato crops. Mol. Plant Microbe Inter-act. 23: 539-548.

Heinze, C., B. Letschert, D. Hristova, M. Yankulova, O. Kau-adjouor, P. Willingmann, A. Atanassov, G. Adam (2001). Variability of the N-protein and the intergenic region of the S RNA of Tomato spotted wilt tospovirus (TSWV). New Microbiol. 24: 175-187.

Jenser, G., Cs. K. Tusnádi (1989). A nyugati virágtripsz (Frankliniella occidentalis Pergande) megjelenése Mag-yarországon. (The appearence of Frankliniella occidenta-lis Pergande in Hungary). Növényvédelem 25: 389–392.

Jenser, G. (1995). The role of the Thysanoptera species in the spread of tomato spotted wilt tospovirus. Növényvédelem 31: 541–545.

Kormelink, R., P. de Haan, C. Meurs, D. Peters, R. Goldbach (1992). The nucleotide sequence of the M RNA segment of Tomato spotted wilt virus , a Bunyavirus with two am-bisense RNA segments. J. Gen. Virol. 73: 2795-2804.

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DNA and protein sequences. Brief Bioinform. 9: 299–306.Ligeti, L., G. Nagy (1972). A Lycopersicum vírus 3

dohányültetvényeink új, veszedelmes kórokozója. Do-hányipar 1: 41–43.

Margaria, P., M. Ciuffo, M. Turina (2004). Reistance breaking strain of Tomato spotted wilt virus (Tospovirus, Bunya-viridae) on resistant pepper cultivars in Almeria, Spain. Plant Pathol. 53: 795.

Prins, M., R. Goldbach (1998). The emerging problem of to-spovirus infection and nonconventional methods of con-trol. Trends Microbiol. 6: 31-35.

Roggero, P., V. Masenga, L. Tavella (2002). Field isolates of Tomato spotted wilt virus overcoming resistance in pep-per and their spread to other hosts in Italy. Plant Dis. 86: 950-954.

Salamon, P., K. Nemes, K. Salánki (2010). A paradicsom foltos hervadás vírus (Tomato spotted wilt virus, TSWV) rezisztenciatörı törzsének elsı izolálása paprikáról (Capsi-cum annuum L) Magyarországon. In: Növényvédelmi Tu-dományos Napok, pp 23.

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Thomas-Caroll, M. L., R. A. C. Jones (2003). Selection, bio-logical properties and fitness of resistance-breaking stra-ins of Tomato spotted wilt virus. Ann. Appl. Biol. 142: 235-243.

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Van de Wetering, F., R. Goldbach, D. Petrs (1996). Tomato spotted wilt tospovirus ingestion by first instar larvae of Frankliniella occidentalis is a prerequisite for transmis-sion. Phytopathol. 86: 900-905.

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Aggressiveness of Phytopathogenic Viruses: Trace Investigation and Evaluation

Antoniy Stoev

N. Poushkarov Institute for Soil Science, Agrotechnology and Plant Protection, 7 Shosse Bankya Str., 1080 Sofia, Bulgaria

ACTA MICROBIOLOGICA BULGARICA

*Corresponding author: e-mail: anton_stoev@yahoo.com

Abstract The paper deals with the particularities in the manifestation of aggressiveness by phytopathogenic

viruses and the methods of its tracing out and evaluation. Aggressiveness is a property of phytopathogens. We present some examples of viral pathogens which cause economically important diseases in the agroe-cosystems of Bulgaria. We have considered the manifestation of virus aggressiveness in association with other properties which characterize plant pathogens.Key words: phytopathogenic virus, viral pathogen, aggressiveness

РезюмеСтатията представя особеностите в проявата на агресивността на фитопатогенните вируси,

както и начините за нейното проследяване и оценяване. Дадени са примери с вирусни патогени, причиняващи стопански важни болести в агроекосистемите на България. Проявата на агресивността е разгледана във връзка с други свойства, характеризиращи растителните патогени.

Introduction The development of the pathological process

depends on the properties of pathogens and the plant immune system. The infectious virus is the virus which is determined by its ability to introduce itself into the living organism (Markov et al., 1989). The viral pathogen is in the closest link with the hospi-table cell of the plant organism. Under conditions of viral infection, the cell metabolism is directed to-wards realizing the ability of the viral genome, and eventually towards producing the pathogen (Goldin et al., 1966; Fraenkel-Konrat, 1969).

The pathogen possesses a definite phylogen-ic specialization. The infection which characterizes the beginning of the pathogenic process spreads in the frames of specific taxonomic units of the hosts (Table 1).

The pathogenic population (if it is possible to use the term of viral population) is not homoge-neous. In the population there are smaller system-atic units of strains which differ in their infective ability and the ability to cause diseases: for exam-ple, necrotic strains causing some necroses in plant

tissues, etc. This relative ability is well-known as virulence.

Viruses penetrate plant tissues via different pathways. The tobacco mosaic virus (TMV) pene-trates through micro-wounds formed after mechan-ical damage to the tissue.

Other viruses [such as: plum pox virus (PPV), barley yellow dwarf virus (BYDV), and raspberry ringspot virus (RRSV)] penetrate through the suck-ing organs of insects and nematodes using the plant sap for feeding (Esau, 1961; Trifonov, 1972; Gibbs and Harrison, 1976; http://www.eppo.int/QUARAN-TINE/data_sheets/virus/RPRSV0_ds.pdf).

Virulence and aggressiveness depend on the ability of the pathogen to overcome the protecting system of the plant organism. Aggressiveness char-acterizes the development of infection and the abil-ity of the pathogen to spread into the tissues of the host plant (Gorlenko, 1973, according to Markov et al., 1989).

Methods to follow the spreading of virus The visual following of viral spread depends

on the appearance of disease symptoms. These can

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appear on certain plant organs under conditions of infection after the expiration of the incubation pe-riod. This way is uncertain because the incubation period for the various viruses has different duration which depends on many factors. These factors are not considered in this paper.

It is possible for the infection to remain latent. This is a problem concerning the quarantine and the producing of virus-free propagation material.

The visual observation of the viral spread can be made visible under microscope. It is possible for some viruses which form specific bodies (called vi-ral inclusions) in the plant cell. These viral inclu-sions vary in form, composition and texture. The differences can help us to determine the systemat-ical belonging taxonomic affiliation? of the virus.

For TMV, these viral inclusions are visible under a light microscope (Goldin et al., 1966). Discovering the real viral particles needs the appli-cation of electronic microscope (Matthews, 1970; Protsenko and Legunova, 1960).

Additional helping method is the use of dyes for colouring the substances which are accumulated in the plant tissues as a result of the infection. As diagnosis reference point, some intrinsic deforma-tions can be used, for example, tylosis in the xylem (Kovachevski et al., 1995).

Another method for visual following of virus spreading is by biological testing. In this method, plants are used which are indicators of viral infec-tion (Matthews, 1970; Gibbs and Harrison, 1976).

These plants respond to the supposed carrier of viruses in a specific way with inoculum prepared from different tissues and organs of the carrier. The biological test is reliable for proving and tracing the infection. Its application needs time and availabil-ity of vegetation greenhouse or specialized planta-tions, which is a disadvantage of the method.

The modern methods for determination of plant viruses are based on immunization reactions between virus and antibody. Plants cannot form an-tibodies. They are produced in an animal organism injected with prepared and purified viral substance. These methods create the possibility for artificial production of viral nucleic acid and give quick and exact evidence of the presence of a virus (Starke, 1968; Clark and Adams, 1977; Wetzel et al., 1991).

Evidence of viral infection can be express-ly obtained by applying diagnostic kits out of the laboratory in crop fields and plantations (Stoev and Tomeva, 2005).

Determination of virus aggressiveness de-pends not only on the chosen method and diag-nostic reagents. The researcher has to know the defense reaction of the plant which is in contact with the viral pathogen. The plant might manifest a hypersensitivity reaction. In this case, plant cells die (Corbett and Sisler, 1964; Gibbs and Harrison, 1976; Kegler and Kleinhempel, 1987; Hartmann, 2001).

A necrotic zone can be formed which stops the development of the infection process. The hy-

Infective abilityAbility of virus to infect the plants It depends on:(a) immune reaction of the plant(b) phylogenic specialization of the pathogen, which can be:Wide – The pathogen infects plant species of different botanical families.

Narrow – The pathogen infects plant species in the frame of one botanical family.

(c) Ontogenetic specialization – The infectious ability is depending on the plant age (phase of development). Aggressiveness Ability of virus to disseminate in the plant It depends on: (a) immune reaction of the plant (b) specialization of the pathogen, which can be: Histotropic OrganotropicThe virus spreads in some tissues of the plant The virus spreads in some organs of the plant

Table 1. Infection ability and aggressiveness of phytopathogenic viruses

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persensitivity reaction can lead to perishing of the plant organs and even of the whole plant when the necrosis develops in vital parts of the plant.

All these cases require mutual taking into consideration of the experimental setting and the natural and agrotechnical conditions which exert influence on the plant health.

Features in the manifestation of aggressiveness Some viruses (ТМV) which infect the plant

through micro-wounds formed by mechanical im-pact are disseminated and spread in the whole plant (Samuel, 1934 according to Gibbs and Harrison, 1976).

For the receptive variety of tobacco, the vi-rus is accumulated in the leaves which manifest the specific symptoms of the disease. These symptoms gave the name of mosaic virus and infection of to-bacco (Kovachevski et al., 1995). In the sphere of hosts of Nicotiana genus, the infection can provoke the appearance of necrotic lesions (Gibbs and Har-rison, 1976).

ТМV keeps its infection ability in the plant‘s sap. When there is a contact between an infected plant and an uninfected one, the infection can go over to healthy plants. Under natural conditions, the wind can cause the contact. When feeding, the natural carriers of infection can be some gnawing insects, passing from the diseased plant to a healthy one.

Different processes in growing tobacco can cause the opening of micro-wounds, which contrib-ute to spreading the infection into plants of field, where the seedlings are grown, as well as in the fu-ture plantation (Kovachevski et al., 1995).

For viruses which have natural carriers (as leaf-lice, etc.), the spreading of the infection de-pends on the attractiveness of the plant hosts for the carriers.

Crop plant species have great diversity of va-rieties. They differ by many indices. Among these indices is the index of the plant reaction, which is due to the different viruses provoking the diseases.

Having in mind the indicated examples, it can be concluded that aggressiveness is a relative concept, which depends on many factors. Some of these factors are external to the pathogen itself. The human activity is one of these external factors. The creation of new hybrids and varieties should be considered, which are resistant to the attacks of different disease provoking viruses (Kegler et al., 1998).

Resistance is coded in the plant genotype. It can be considered and systematized from differ-ent view points. Theoretically, full resistance can be assumed. In this case, there is no existence of infection. The pathogen cannot be introduced and disseminated into the plant organism. The plant possesses immunity. It is obviously healthy and the tests for presence of infection give negative results (Kegler and Kleinhempel, 1987; Vlasov and Lari-na, 1982).

When the plant is tolerant, the virus can affect the plant tissues and organs of the host completely or partially. In this case, it is not compulsory for the plant to give evidence of disease. From econom-ic point of view, the tolerant property is valuable when the infection does not significantly decrease the quality and quantity of plant production. The details concerning the pathological evidence and the damaging consequences are not considered in this publication. Resistance and tolerance are dif-ferent categories when characterizing the interac-tions between the pathogen and the host (Vlasov and Larina, 1982).

In the case of infection the following differ-ent cases are possible: duration of incubation period; appearance of symptoms which can disappear at a later stage and the plant is restored and looks healthy; manifestation of symptoms which are common pnenomena concerning some viral infections in tree species; manifestation of hypersensitivity, which leads to premature perishing of the infected plant; availability of symptomswhich vary by the strength of manifestation but the plant-host does not perish.

In the above cases, depending on its aggres-siveness, the pathogen can be established in the whole organism of the plant. Its determination de-pends on the specificity of the chosen method for identification. The visual diagnosis of viral diseases is of secondary importance only.

Methods for evaluation of aggressiveness Aggressiveness of viruses can be evaluat-

ed through their organotropic and histotropic spe-cializations (Table 2). The presence of the virus in one or another organ or in the issues of the plant-host is a qualitative index (Petrov, 2014; Stoev and Kamenova, 1995).

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Table 2. Estimation of virus aggressiveness

The virus concentration in the infected tissue is an additional quantitative indicator, which char-acterizes the infection (Gibbs and Harrison, 1976). The measurement of concentration is performed by applying physical and chemical methods. The num-ber of local lesions gives an idea on the infection degree of the crude sap, which is used as inoculum (Nordam, 1973).

An indirect indicator of the virus concentra-tion is the optical density, which is measured at the end of the enzyme-linked assay of the immuno-sorbent (Clark and Adams, 1977).

The movement velocity of the virus in the plant organism is another indicator of its aggres-siveness (Samuel, 1934, according to Gibbs and Harrison, 1976). The formed lesions can serve as a reference point for orientating, which should not abolish the check-up concerning the presence of vi-rus in the rest of plant tissues (Dikova, 2009, 2011, 2014; Milusheva, 2014).

According to Van der Plank (1963), described and cited by Pariaud et al. (2009), aggressiveness is a non-specific component of the pathogen phe-nomenon. From the quantitative point of view, the strength of the disease manifestation (or the patho-logical activities) can be characterized through the size of the lesions, according to Markov et al. (1989).

The aggressiveness of a pathogen has to be considered also in relation to the whole crop fields or plantation. In the areas with crop plants, the in-fection can be general or local. For example, in the fields with cereals, having a combined homogene-ous distribution in the area (such as wheat, barley and oats), the plants can be infected in separate land parcels, looking like threshing-floor or halos (Ko-vachevski et al., 1995). In other cases, the periph-ery of the crop field can be affected, etc.

In plum (Prunus domestica) plantations cre-ated with material of saplings free of viruses, the

PPV infection can be localized in separate trees. These have to be eradicated. In these plantations, sprinklings with insecticides are carried out against leaf lice vectors of the mentioned pathogen as a protecting measure (Dragoiski et al., 1990).

In the nursery gardens, the trees infected by viruses which can be spread through the pollen, have to be eradicated. This measure ensures the production and distribution of healthy propagative material (Trifonov, 1972).

Significance of aggressiveness for the prac-tices

Cultivation of crop plants can be developed in different aspects. Obtaining food for humans and animals is of primary significance. Furthermore, plant production can be basic material for obtaining certain goods which are necessities of life or raw materials for different industrial branches.

Other important aspects of growing crop plants are: reproduction of the crop species and varieties

(production of seeds and sapling material); selection (plant breeding), creating new hy-

brids and cultivars; variety testing and cultivar maintenance; domestication of new plant species.

When all these plant-growing aspects are implemented, the plants grow and develop under conditions of a fixed background of infection. The plants interact with the pathogens available in this background.

The distribution of the infection inside the agroecosystems depends on certain conditions. It has its own characteristics. These conditions and characteristics are subjects for investigation in the field of epidemiology. The possibility of mixed infections should be taken into account (Petrov, 2015).

The ability of plants to limit the spreading of viruses is of important significance for obtain-

Qualitative estimation 1. According to the availability or the absence of pathogen (а) investigation of different plant tissues, organs and parts (б) following and measuring of the spreading velocity Quantitative estimation1. Taking into account the affected area (volume) of the infected organ 2. Taking into account the number of the affected organs (fruits, leaves, blooms etc.)

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ing generative (sexual) and vegetative non-infected posterity. The availability of seeds, tubers and bulb which are free of viruses is an important prerequi-site for obtaining a good crop yield in amount and quality.

The hypersensitivity reaction does not allow the survival of the virus in the plant organism. In this case, there is no appearance or retention of in-fected areas in the crop fields.

The knowledge on genetical determination of the relationships between the virus and its host enables the creation of new plant cultivars which possess not only valuable economic and market qualities of the production, but also high resistance to the virus agents of dangerous diseases in crop plants.

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croplate method enzyme-linked immunosorbent assay for the detection of plant viruses. J. Gen. Virol. 34: 475-483.

Corbett, M. K., H. D. Sisler (1964). Plant virology. University of Florida, Gainesville, pp. 527.

Dikova, B. (2009). Establishment of some viruses on econom-ical important essential oil-bearing and medicinal plants in Bulgaria. Biotechnol. Biotechniol. Eq. 23/SE: 80-84.

Dikova, B. (2011). Tomato spotted wilt virus on some med-ical and essential oil-bearing plants in Bulgaria. Bulg. J. Agric. Sci. 17: 306-313.

Dikova, B. (2014). Tomato spotted wilt virus and tomato mo-saic virus on some medicinal and essential plants in Bul-garia. J. Balkan Ecol. 17: 357-367.

Dragoiski, K., I. Minev, P. Mondeshka, H. Dinkova, B. Mi-hovska (1990). Work related to the plum pox virus. The Institute of Mountains Animal-breeding and Land Man-agement, XXVII (4): 10-15 (In Bulgarian).

Esau, K. (1961). Plants, viruses and insects. Harvard Univer-sity Press, Cambridge, Massachusetts, pp. 110.

Fraenkel-Konrat, H. (1969). Chemistry and biology of virus-es. Academic Press, New York and London. In Russian (1972), Mir Publisher, Moscow, pp. 336.

Gibbs, A., B. Harrison (1976). Plant virology. The Principles. Edward Arnold, London (In Russian - Mir Publisher , Moscow, 1978, pp. 432).

Goldin, M. I., A. E. Protsenko, V. L. Rizhkov (1966). About the nature of the viruses. Nauka Publisher , Moscow, pp. 226.

Hartmann, W. (2001). The importance of hypersensitivity for breeding plums and prunus resistant to plum pox virus (Sharka). Acta Horticulturae, 577: 33-37.

Kegler, H., H. Kleinhempel (1987). Virusresistenz der Pflan-zen. Akademie. Verlag, Berlin, pp. 184.

Kegler, H., E. Fuchs, M. Gruntzig, S. Swcharz (1998). Some results of 50 years of research on the resistance to plum pox virus. Acta Virol. 42: 200-215.

Kovachevski, I., M. Markov, M. Yankulova, D. Trifonov, D. Stoyanov, V. Kacharmazov (1995). Virus and Virus-like Diseases of Crop Plants. Publish ScieSet-Agri (PSSA), Sofia, pp. 408.

Markov, M., F. Straka, I. Balinov, V. Boyadzhiev, N. Velche-va, E. Videnova, M. Vitanov, N. Genchev, E. Genche-va-Chetkarova, T. Zaharieva, H. Karzhin, Y. Lyubenov, P. Mihaylova, P. Nachev, M. Tsalev, B. Koleva (1989). Ter-minological Dictionary of Plant Protection. A-P. Publisher ZEMIZDAT, Sofia, No. 86. (In Bulgarian).

Matthews, R. E. F. (1973). Plant Virology. Academic Press Publisher, New York and London, (In Russian - Mir Pub-lisher, Moscow, p. 600).

Milusheva, S. (2014). Diagnosis, epidemiology and control of viral diseases on cultural fruit plants. Brief Presentation of Scientific Papers Concerning Habilitation Procedure, p. 10.

Nordam, D. (1973). Identification of Plant Viruses. Centre for Agricultural Publishing and Documentation, Wageningen, pp. 217.

Pariaud, B., V. Ravigne, F. Halkett, H. Goyeau, J. Carlier, C. Lannou (2009). Aggressiveness and its role in the adapta-tion of plant pathogens. Plant Pathol. 58: 409-424.

Petrov, N. (2014). Tracking of organ and tissue tropism of plum pox virus (PPV) and prunus necrotic ring spot virus (PNRV) in plums and apricots. J. Agric. Balkans 17: 433-445.

Petrov, N. (2015). Mixed viral infections in tomato as precon-dition for economic loss. Agric. Sci.Technol. 7: 134-228.

Protsenko, A. E., R. M. Legunova (1960). Electronic Micros-copy on Viruses. - Publisher of Academy of Sciences of USSR, Moscow, pp. 96, (In Russian).

Starke, G. (1968). Virologische Praxis. VEB Gustav Fischer Verlag, Jena , (In Russian – Kolos Publisher, Moscow, p. 352, 1979).

Stoev, A., I. Kamenova (1995). Studies on the relationship between plum cultivar zhulta butilkovidna and plum pox virus. Plant Sci. 32: 144-145.

Stoev, A., E. Tomeva (2005). Actual problems in the plant protection concerning the system for biological protection and reaction in the case of extreme situation (in Bulgari-an). Proceedings of the First National Research Confer-ence on Emergency Management and Protection of the Population. BAS, Sofia, November 2005, pp. 440-448.

Trifonov, D. (1972). Viral Diseases on Fruit Trees (in Bulgar-ian). Zemizdat, Sofia, p. 192.

Vlasov, Y. I., E. I. Larina (1982). Agricultural Virology (in Russian). Kolos Publisher, Moscow, p. 240.

Wetzel, T., Candrese T., Ravelonandro M. and J. Dunez. (1991) A polomerase chain reaction assay adapted to plum pox virus. J. Virol. Methods 33: 355-365.

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ACTA MICROBIOLOGICA BULGARICA

Cucumber Mosaic Virus on Aromatic and Medicinal Plants of Lamiaceae and Asteraceae Families

Bistra Dikova

Institute of Soil Science, Agrotechnologies and Plant Protection, 1080 Sofia, 7, “Shosse Bankya” Str., Bulgaria

AbstractThe occurrence of Cucumber mosaic virus (CMV) and the related disease symptoms on aromatic

and medicinal plants of Lamiaceae and Asteraceae families were studied in 2004-2015. The best essential oil plant species belong to these families. Virus identification was done by the serological method DAS ELISA and indicator method of test plants for CMV. The results for CMV detection on fifteen most popular species of the Lamiaceae and eight of the Asteraceae families were presented. CMV was found on ten of all aromatic and medicinal plant species in over fifty percent of the analyzed plants of each of them. These species were: Agastache foeniculum, Melissa officinalis, Ocimum basilicum, Origanum heracleoticum, Salvia officinalis, Salvia sclarea, Satureja hortensis, Thymus officinalis (Lamiaceae), Inula helenium, Ser-ratula coronata (Asteraceae). The control of CMV caused viroses during the cultivation process is crucial for obtaining high yield of prime quality.Key words: Cucumber mosaic virus on species of Lamiaceae and Asteraceae.

РезюмеВ периода 2004-2015 г. бе проучено разпространението на Cucumber mosaic virus (CMV) и на

причиняваните от него симптоми на заболявания при ароматни и медицински растения от семейства Lamiaceae и Asteraceae, към които спадат най-перспективните за добив на етерично масло растител-ни видове. Идентифицирането на вируса бе извършено чрез серологичен метод DAS-ELISA и чрез индикаторен метод на тестови за СМV растения. Представени са резултати от установяване на СМV по растения от 15 широко застъпени за отглеждане видове, принадлежащи към Lamiaceae и 8 вида – към Asteraceae. При 10 от видовете, СМV присъства в над 50% от анализираните от всеки вид растения. Тези видове са: Agastache foeniculum, Melissa officinalis, Ocimum basilicum, Origanum heracleoticum, Salvia officinalis, Salvia sclarea, Satureja hortensis, Thymus officinalis (Lamiaceae), Inula helenium, Serratula coronata (Asteraceae). Висок и качествен добив би се получил, ако в процеса на отглеждане се обърне специално внимание върху контрола на причиняваната от СМV вироза.Introduction

The aromatic and medicinal (curative) plants of the Lamiaceae and Asteraceae families account for the largest share of essential oil production in the world. The world production of essential oils at the beginning of the 21st century was 11785.8 tons at the value of USD 211.634 million. The species of Lamiaceae family produced 10396.2 tons of essen-tial oils, valued at USD 129.195 million (Yankulov, 2000). The species of the following families are the richest in essential oil in the temperate climatic zone, which includes Bulgaria: Lamiaceae – 187,

Asteraceae – 177, Apiaceae – 170, Rosaceae – 58, Brassicaceae – 35 and Cupressaceae – 35 (Yanku-lov, 2000).

Cucumber mosaic virus (CMV) is among the five most common viruses in the world that cause diseases on vegetables, flowers, some field and me-dicinal cultures. According to Zitter and Murphy (2009), more than 1200 species of over 100 fam-ilies of monocotyledon and dicotyledonous plants, including vegetables, ornamental, woody and semi woody plants, were CMV hosts. Plant viruses, in-cluding СМV, decrease the yield and deteriorate the quality of production (Bellardi et al., 2006a, *Corresponding author: b.dikova @ abv bg.

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2006b; Dikova et al., 2013). The СМV caused vi-rus infection has a negative effect on the quality of root extracts from Echinacea purpurea L. Moench (Bellardi et al., 2001).

Kovachevsky (1965) found СМV in Hysso-pus officinalis and Ocimum basilicum (Lamiaceae) as well as Calendula officinalis (Asteraceae) in Bulgaria. СМV was proven in Lavandula angusti-folia Mill. (Kobylko et al., 2008); Echinacea pur-purea (L). Moench. (Dikova, 2009; Dikova et al., 2013); Ocimum sanctum L., Salvia officinalis L. and Calendula officinalis (Seth and Raychaudhuri, 1972); Salvia sclarea L. (Pisi and Vicci, 1989) and Mentha sp., (Sevik, 2012).

The objective of the study was to identify the essential oil-bearing (aromatic) and medicinal (cu-rative) plants that acted as natural hosts of Cucum-ber mosaic virus (СМV) in Bulgaria.

Material and methodsEssential oil-bearing (aromatic) and medici-

nal (curative) plants, belonging to Lamiaceae and Asteraceae families, were analyzed in the period 2004-2015. Samples of single plants or tufts, taken from the trial fields of the Institute of Roses, Essen-

tial and Medical cultures (IREMC) near Kazanlak, showed symptoms of the virus disease. Each sam-ple (as well CMV test plants) was analyzed by the ELISA method (DAS – ELISA); (Clark and Adams, 1977) with a kit, purchased from the German com-pany LOEWE, Biochemica. The extinction values were measured using a spectrophotometer SUMAL PE, Karl Zeiss, Jena, Germany. All samples show-ing values two and a half times higher than the negative controls were assumed as virus positive. The negative controls were samples of symptom-less healthy plants and the positive - CMV infected indicator plants as well as the positive control from the kit.

The extinction values (optical density) of the samples were processed by statistical analysis of Student’s criterion, quoted by Lidanski (1988). The average extinction values of optical density were calculated as well as the standard deviation. The confidence intervals had a significance rate of p ≤ 0.05 according to Student’s criterion. The confi-dence intervals of the positive and negative extinc-tion values of the samples are given in Tables 1 and 2. The indicator method of Noordam (1973) was used for the identification of CMV isolates from

Board I. Symptoms of Cucumber mosaic virus (CMV) on aromatic and medicinal plants

Fig. 1. Symptoms of mosaic on Nepeta cataria, on the right – symptomless leaf

Fig. 2. Symptoms of mosaic on Ocimum basilicum plant

Fig. 3. Symptoms of mosaic and chlorosis on Satureja hortensis tuft

Fig. 4. Symptoms of severe mosaic on Echinacea purpurea leaves, on the left – symptomless leaf

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aromatic and medicinal plants. Besides the sero-logical method DAS-ELISA that allowed for the analysis of a large number of plant species, we also applied the method of test plant infection. The so called indicator method was used for the following CMV infected species: Chenopodium quinoa and Vigna unguiculata as test plants reacting with local lesions and Cucumis sativus, Nicotiana glutinosa Nicotiana tabacum as test plants reacting with sys-temic symptoms according to Franski et al. (1979) and Palukaitis and Garcia-Arenal (2003) (Board II, Fig.4 and Board III, Fig.1 and 2).

ResultsThe symptoms caused by Cucumber mosaic

virus (CMV) on the leaves of Nepeta cataria (Fig. 1), Ocimum basilicum (Fig. 2), Satureja hortensis (Fig. 3) and Echinacea purpurea (Fig. 4) were as follows: light to dark green mosaic spots or entire-ly chlorotic young leaves as on Satureja hortensis (Fig. 3; Board 1).

CMV infected a large number of plants of each species of Lamiaceae family – more than 1/3 and 1/2 of all tested plants (Table 1).

Plant species Total number of samples

Samples with CMV

% Extinction values (Optical density - OD)Positive extinction values

Negative extinction values

Agastache foeniculum (Purch.) Kuntze - blue giant hyssop

8 4 50.00 0.503 ± 0.109 0.120 ± 0.055

Hyssopus officinalis L. - hyssop

8 3 37.5 0.553 ± 0.220 0.136 ± 0.030

Lavandula angustifolia Mill. - lavender

25 9 36.00 0.666 ± 0.324 0.085 ± 0.024

Melissa officinalis L. - lemon balm

25 16 64.00% 0.278 ± 0.052 0.082 ± 0.017

Mentha piperita L - peppermint

17 7 41.1 0.467 ± 0.176 0.162 ± 0.011

Mentha spicata L - spearmint

13 6 46.15 0.418 ± 0.146 0.124 ± 0.023

Monarda fistulosa L. - bee balm

12 2 16.67 0.767 ± 0.097 0.084 ± 0.025

Nepeta cataria L. - catmint

22 8 36.36 0.472 ± 0.128 0.125 ± 0.022

Nepeta racemosa L. - Persian catmint

17 6 35.29 0.400 ± 0.059 0.124 ± 0.013

Ocimum basilicum L. - basil

18 9 50.00 1.466 ± 0.544 0.106 ± 0.035

Origanum heracleoticum (L.) Letsw. - Greek oregano

11 7 63.64 0.573 ± 0.246 0.080 ± 0.010

Salvia officinalis L. - garden sage

19 10 52.63 0.635 ± 0.206 0.161 ± 0.025

Salvia sclarea L. clary sage

38 19 50.00 0.616 ± 0.141 0.097 ± 0.010

Satureja hortensis L. summer savory

9 5 55.56 0.391 ± 0.146 0.100 ± 0.050

Thymus vulgaris L. - thyme

14 10 71.43 0.417 ± 0.131 0.090 ± 0.034

Table 1. CMV in aromatic and curative plant species of Lamiaceae family.

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Plant species Total number of samples

Samples with

TSWV

% Extinction values(Optical density, OD)

Positive extinction values

Negative extinction values

Artemisia absinthium L. - wormwood 11 3 27.27 0.858 ± 0.560 0.129 ± 0.044

Calendula officinalis L.- pot marigold 12 3 25.00 0.297 ± 0.059 0.087 ± 0.023

Echinacea purpurea (L.) Moench. - purple coneflower

53 24 45.28 1.121 ± 0.290 0.121 ± 0.011

Inula helenium L. white elecampane 8 4 50.00 0.349 ± 0.137 0.110 ± 0.076

Leuzea carthamoides (Willd.) DC - maral root

56 20 35.71 0.419 ± 0.099 0.103 ±0.01

Matricaria chamomilla L. - chamomile

9 4 44.44 0.422 ± 0.401 0.117 ± 0.047

Silybum marianum (L.) Gaerth. - milk thistle

27 11 40.74 0.420 ± 0.120 0.118 ± 0.025

Serratula coronata L. - sickle moon 22 14 64.00 0.299 ± 0.023 0.105 ±0.036

Table 2. CMV in aromatic and medicinal plant species of Asteraceae family

The high percentage of CMV infected plants was characteristic of almost all species, belonging to Lamiaceae family with one exception for Mo-narda fistulosa (Тable 1). Of all 256 plants of La-miaceae family, СМV was found in 121 (47.27 %) (Table 1). Some of the Lamiaceae species: Hysso-pus officinalis, Monarda fistulosa, Origanum vul-gare ssp. hirtum (Link.) are perspective cultures, recommended to the farmers for 2016 by IREMC. CMV was established in high percentage in the species of both Lamiaceae and Asteraceae family, except for Artemisia absinthium and Calendula of-ficinalis, where the infected plants were less than 30% (Table 2). Of a total of 198 analyzed plants of

the Asteraceae family, eighty three (41.92 %) were CMV carriers.

The rate of CMV infection was 50% or over 50% in ten of the 23 species of both families - eight plant species of Lamiaceae and two of Asterace-ae family. They belonged to the following species: Agastache foeniculum Melissa officinalis, Ocimum basilicum, Origanum heracleoticum, Salvia offic-inalis, Salvia sclarea, Satureja hortensis, Thymus

officinalis (Lamiaceae), Inula helenium and Serrat-ula coronata (Asteraceae). CMV monoinfection in aromatic and medicinal plant species is not a fre-quent phenomenon. Usually, СМV is in mixed in-fection with one or more plant viruses, common for these cultures. Thus, both CMV and Tomato spot-ted wilt virus (TSWV) monoinfections were found in plants of Serratula coronata species as well as mixed infection of both viruses (Figs. 5, 6 and 7; Board 2).

Indicator plant species Chenopodium quinoa and Vigna unguiculata reacted to CMV infection with local chlorotic, turning to necrotic, lesions and were suitable for diagnosis of CMV isolates from

aromatic and medicinal species (Table 3). The identification and maintenance of multi-

plying CMV isolates was carried out on indicator plants that responded with systemic symptoms of this virus, namely Cucumis sativus, Nicotiana ta-bacum and Nicotiana glutinosa (Table 3 and Figs. 8, 9, 10).

Moreover, the species Cucumis sativus served to differentiate the two viruses – СМV и To-

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Board III. Symptoms of CMV on test (indicator) plants

Board II. Mixed CMV and TSWV infection

Fig. 5. Leaves with chlorotic spots and mosaic on Serratula coronata plant

Fig. 6. Large bright yellow areas, caused by TSWV on Serratula coronata plant

Fig. 7. Dwarfed plant Serratula coronata with mixed infection of both viruses – CMV and TSWV, on the right - healthy S. coronata

Fig. 8. Symptoms of CMV on Cucumis sativus cv. Delikates- on the left, lack of symptoms on C. sativus cv. Delikates, inoculated with TSWV – on the right

Fig. 9. Systemic symptoms on the leaves of Nicotiana glutinosa plant

Fig. 10. Systemic symptoms on some leaves of Nicotiana tabacum cv. Samsun NN

mato spotted wilt virus (TSWV) that infected the medicinal species Serratula coronata in mixed in-fection (Board II, Figs. 7 and 8). CMV caused sys-temic mosaic symptoms (Fig.8, left), while TSWV caused no systemic symptoms on the leaves of Cu-cumis sativus (Fig. 8, right).

DiscussionThe symptoms of mosaic, spotting, chlorosis

and plant dwarfing due to stem and branch shorten-ing affected the yield of drugs in terms of quanti-ty and quality both in CMV monoinfection and in mixed infection with TSWV as well as other virus-

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Indicator (test) plant Description of symptoms on leaves

Optical density (OD) at 405 nm (extinction values) for CMV

Chenopodium quinoa Chlorotic local lesions, gradating to necrotic

*

Cucumis sativus cv. Delikates Chlorotic local lesions, chlorotic systemic spots and mosaic

2.005

Nicotiana tabacum cv. Samsun NN

Latent local infection, chlorotic systemic spots, mosaic, deformation

1.738, 0.628**

Nicotiana glutinosa Chlorotic local lesions, chlorotic systemic spots, mosaic, blisters, deformation

1.957, 2.011, 1.248,2.0970.683

Vigna unguiculata Necrotic local lesions *Capsicum annuum cv. Sivria Chlorotic systemic spots,

mosaic1.458

Lycopersicon esculenthum cv. Ideal

Chlorotic systemic spots, mosaic, narrow strand leaves

1.549

Negative control for CMV (K-) Young symptomless leaf from cucumber

0.063

Positive control for CMV (K+) Chlorotic systemic spots, mosaic

0.847

Table 3. Data on the reaction of indicator plants and some host plants to the isolate 11/09 of Cucumber mosaic virus (CMV), originating from Echinacea purpurea

*- indicator plants were not tested with antiserum to the corresponding virus**- more than one test of the indicator plants

es (Dikova, 2010). The yield of leaves, seeds and roots of Echinacea purpurea from spotted plants was twice to several times lower in comparison with symptomless plants (Dikova et al., 2013). CMV in-fection in the above aromatic and medicinal plants should be kept under control as they could become a serious source of infection for the vegetables and flowers, cultivated nearby. It is necessary to effect strict control of the CMV vectors, namely, aphids, especially those of the species Myzus persicae Sulz during the vegetative season. Spatial isolation from vegetable and flower gardens would be necessary as well.

ConclusionCucumber mosaic virus (CMV) was one of

the major viral pathogens – agents of diseases in more than half of the individual plants of aromatic and medicinal species, namely 8 species of Lami-aceae and 2 species of Asteraceae families. They were as follows: Agastache foeniculum, Melissa officinalis, Ocimum basilicum, Origanum hera-cleoticum, Salvia officinalis, Salvia sclarea, Satu-

reja hortensis, Thymus officinalis (Lamiaceae) and Inula helenium, Serratula coronata (Asteraceae). The percentage of infection was lower in 7 spe-cies of Lamiaceae (Hyssopus officinalis, Lavandu-la angustifolia, Mentha piperita, Mentha spicata, Monarda fistulosa, Nepeta cataria, and Nepeta racemosa) and 6 species of Asteraceae (Artemisia absinthium, Calendula officinalis, Echinacea pur-purea, Leuzea carthamoides Matricaria chamomil-la, Silybum marianum).

ReferencesBellardi, M. G., C. Rubies-Autonell, M. Hudaib (2001). Ef-

fect of Cucumber mosaic virus infection on the quality of Echinacea purpurea root extracts. J. Plant Pathol. 83: 69-70.

Bellardi, M. G., A. Benni, R. Bruni, A. Bianchi, G. Parrella, S. Biffi (2006a). Chromatographic (GC-MS) and virologi-cal evaluations of Lavandula hybrida “Alardi” infected by Alfalfa mosaic virus. Acta Hort. 723: 387-392.

Bellardi, M. G., A. Benni, R. Bruni, A. Bianchi (2006b). A vi-rus disease affecting Salvia officinalis L. “Maxifolia” and its effects on essential oil production and composition. Acta Hort. 723: 381-386.

Clark, M., A. Adams (1977). Characteristics of the microplate

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method of enzyme linked Immunosorbent assay for the detection of plant viruses. J. Gen. Virol., 34: 475-483.

Dikova, B. (2009). Establishment of some viruses-polyph-agues on economically important Essential oil-bearing and medicinal plants in Bulgaria. Biotechnol. Biotechnol. Eq. 23: 80-84.

Dikova, B. (2010). Virus diseases on economically important essential oil and medicinal plants in Bulgaria. Internation-al Scientific Conference Science and Society, 4, 2, 90-95, October 13-14, 2010, Kardzhali, Bulgaria.

Dikova, B. (2013). The Cucumber mosaic virus in essential oil and medicinal plants in Bulgaria. Agr. Sci. 46: 39-49.

Dikova, B., A. Djourmanski, H. Lambev (2013). Establish-ment of economically important viruses on Echinacea purpurea and their influence on the yield. International Scientific Conference Innovational Approach to the Study of Echinacea, June 25-27, 2013, Poltava, 36-45 (ISBN 978-617-633-073-8).

Franski, R. I. B., D. W. Mossop, T. Hatta (1979). Cucumber mosaic virus. Descriptions of Plant Viruses № 213, July 1979.

Kovachevcky, I. (1965). Cucumber Mosaic Virosis in Bulgar-ia, S., BAS.

Lidanski, T. (1988). Statistical methods in Biology and Agri-

culture. Zemizdat, Sofia, pp. 50-157.Kobylko, T., P. Danda, B. Haslow, N. Borodynko, H. Po-

spieszny (2008). First report of Cucumber mosaic viruson Lavandula angustifolia in Poland. Plant Dis. 92: 978.

Noordam, D. (1973). Identification of plant viruses: methods and experiments. Wageningen Pudoc, pp. 207.

Palukaitis, P., F. Garcia-Arenal (2003). Cucumber mosaic vi-rus. Descriptions of Plant Viruses, № 400 July 2003.

Pisi, A., V. Vicchi (1989). Infection by Cucumber mosaic virus (CMV) on Salvia sclarea in Italy. Informatore fito-pathologico 39: 49-51.

Seth, M. L., S. P. Raychaudhuri (1972). Further studies on a new mosaic disease of brinjal (Solanum melongena L.). Proc. Indian Sci. Acad. 39: 122-128.

Sevik, M. A. (2012). Natural occurrence of Cucumber mosaic virus infecting water mint (Mentha aquatica) in Antalya and Konya, Turkey. Acta Botanica Croatica 71: 187-193.

Zitter, T. A., J. F. Murphy (2009). Cucumber mosa-ic virus. The Plant Health Instructor DOI:10.1094/PHI-I-2009-0518-01

Yankulov, J. (2000). Fundamental (basic) aromatic plants 19. Temporary technologies for cultivation, Plovdiv, ET “MDM Zv. Markova”, pp. 533.

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Therapeutic Strategy for Survival of Mice Infected with Influenza Virus by Combination of S-Adenosyl-L-Methionine and Oseltamivir

Adriana Dimitrova1, Milka Mileva1, Dimo Krastev2, Galina Gegova1, Angel S. Galabov1 1 The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences,Acad. G. Bontchev Str., Bl.26, 1113 Sofia, Bulgaria2 Medical University of Sofia, Medical College “JordankaFilaretova”, JordankaFilaretova Str. No 3,Sofia, Bulgaria

AbstractInfluenza is a highly contagious viral infection of the respiratory system. Many studies provide com-

pelling evidence that the abnormal production of reactive oxygen species is a crucial mediator of acute lung injury in influenza A virus infection. Therefore, antioxidants are potentially useful against this ongo-ing clinical problem. Our studies showed that S-adenosyl-L-methionine (SAM) has a protective effect in a model of influenza infection in mice. This substance converts into glutathione - the main antioxidant in the body, through a multistep biochemical cycle. In the present study, we report the effect of combined treat-ment with SAM and the antiviral agent oseltamivir in infected with influenza A virus mice. SAM was given as a single daily dose of 50 and 100 mg/kg in different mice groups, starting from 5 days before infection until day 4 after infection. Oseltamivir was given in a dose of 2.5 mg/kg daily in two intakes for 5 days, starting from 4 h before infection. End-point evaluation was 14 - day survival. Survival was 70% in the treatment with oseltamivir and rose to 90% in the treatment with oseltamivir and SAM in both doses. SAM alone did not show any antiviral activity. The present findings suggest that therapy with molecules convert-ed into antioxidants in the body increases survival by modulating the host defense mechanisms and by a direct antioxidant effect against oxidative stress associated with viral infections. This study demonstrated the effectiveness of combining agents that act through different mechanisms - antiviral drug oseltamivir as a specific neuraminidase inhibitor of influenza virus, and SAM as a precursor of the most important anti-oxidant - glutathione.Key words: influenza, oxidative stress, antioxidant, glutathione, S-adenosyl-L-methionine, oseltamivir

РезюмеГрипът е остра инфекциозна вирусна инфекция на дихателните пътища. Много изследвания

предоставят убедителни доказателства, че абнормната генерация на активни форми на кислорода е фактор от изключителна важност като медиатор на острото белодробно увреждане при грипната инфекция. Затова антиоксидантите са потенциално полезни при този клиничен проблем. Нашите изследвания показват, че S-аденозил-L-метионин (SAM) има протективно действие при експериментален модел на грипна инфекция в мишки. Чрез многоетапен биохимичен цикъл SAM се превръща в глутатион - основния антиоксидант в живата. В настоящото проучване ние отчитахме ефекта на комбинираното действие на SAM и антивирусния препарат оселтамивир в инфектирани с грипен вирус тип А мишки. SAM се прилагаше под формата на единични дневни дози от 50 и 100 мг/кг, започвайки 5 дни преди заразяването и продължавайки 4 дни след него. Оселтамивир се при-лагаше в дневна доза от 2.5 мг/кг, разделена в 2 приема, в продължение на 5 дни, като се започваше 4 часа преди заразяването. Референтен пункт в оценката беше 14 дневната преживяемост на мишките, която беше 70% при индивидуалното прилагане на оселтамивир и достигаше до 90% при комбинация на оселтамивир и SAM и в двете дози. SAM не показа антивирусна активност. Получените резултати подсказват, че лечението с молекули, които се превръщат в антиоксиданти в организма, увеличава преживяемостта чрез модулиране на защитните механизми на гостоприемника. Изследването

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демонстрира ефективността на комбинирането на средства, които действат чрез различни механи-зми – антивирусния препарат оселтамивир като специфичен инхибитор на невраминидазата на грипния вирус, и SAM като прекурсор на най-важния антиоксидант - глутатион.

IntroductionInfluenza virus infection is a major public

health problem, occurring typically in the Northern Hemisphere between the months of December and April. Epidemics of influenza are characterized by an increased morbidity and mortality in the com-munity (Monto et al., 2000). Influenza viruses en-compass a major group of human and animal path-ogens belonging to enveloped, segmented, nega-tive-strand RNA viruses (Biswas et al., 1998). They have a multipartite, negative-sense, single-strand-ed RNA genome and a lipid envelope (Beigel and Bray, 2008) and are one of the rare RNA viruses to replicate in the nucleus (Boulo et al., 2007). Cur-rent pharmacological strategies to control influen-za A virus - induced lung disease are problematic owing to antiviral resistance and the requirement for strain-specific vaccination. The production of reactive oxygen species (ROS), particularly super-oxide, is an important host defense mechanism for killing invading pathogens. However, excessive su-peroxide may be detrimental following influenza A virus infection (Vlahos, 2012).

Oseltamivir is a potent and selective inhibi-tor of the neuraminidase enzyme of the influenza viruses A and B. The neuraminidase enzyme is re-sponsible for cleaving sialic acid residues on new-ly formed virions and plays an essential role in the release and spread of progeny virions (Kamps and Hoffman, 2006).

S-Adenosyl-L-methionine (SAM, also known as AdoMet and SAMe) is an important mol-ecule that is found in all living organisms. The im-portance of SAM stems from the fact that SAM is the principal biological methyl donor, the precursor of aminopropyl groups utilized in polyamine bio-synthesis and, in the liver, SAM is also a precur-sor of glutathione (GSH) through its conversion to cysteine via the transsulfuration pathway (Lu, 2000). The tripeptide glutathione is the most abun-dant thiol present in mammalian cells. GSH has im-portant functions as an antioxidant. The glutathione system is especially important for cellular defense against ROS. GSH reacts directly with radicals in nonenzymatic reactions and is the electron donor in the reduction of peroxides catalyzed by glu-tathione peroxidase (GPx) (Dringen et al., 2000). Glutathione serves as the major scavenger of reac-tive oxygen species. Certain lymphocyte functions,

such as the DNA synthetic response, are exquisitely sensitive to reactive oxygen species and, therefore, are favored by relatively high levels of glutathione. Even a moderate depletion of the intracellular glutathione pool has dramatic consequences for a variety of lymphocyte functions (Droge and Breit-kreutz, 2000).

Our previous research confirmed that using S-adenosyl-L-methionine (SAM) did not have a positive response in influenza infected mice.

The combined therapy of influenza is a ques-tion of particular interest. Garozzo et al. (2007) achieved up to 100% survival rate in mice infected with influenza virus and treated with a combination of N-acetylcysteine as a precursor of glutathione in a dose of 1000 mg/kg and oseltamivir in a dose of 1 mg/kg.

Hence, the present study focuses on the anal-ysis of the positive effect when combining oseltam-ivir as a specific neuraminidase inhibitor of the influenza virus replication with S-adenosyl-L-me-thionine as a precursor of glutathione - the most abundant antioxidant in the body. This is a new ap-proach which needs to be studied in details.

Materials and MethodsWhite male mice of the ICR line with body

weight 14–16 g, obtained from Slivnitza Animal Pharm (Bulgarian Academy of Sciences (BAS), Bulgaria), were placed in specially designed, well-ventilated acrylic cage containers, with free access to water and food, and maintained in the Ani-mal House facility of the Stephan Angeloff Institute of Microbiology, BAS. During a 3-day acclimation period (prior to experimental onset), they were ob-served for any signs of diseases and/or physical abnormalities. Animal husbandry and experiments were conducted in accordance with the guidelines of Bulgaria’s Directorate of Health Prevention and Humane Behaviour toward Animals.

For the purpose of the experiment they were anaesthetized with ether and infected intranasally with 10×LD50 of an influenza virus strain adapted for mice: A/Aichi/2/68 (H3N2). The experimental groups were designed as follows:

I. Healthy, non-infected animals; II. Mice infected with influenza virus,

non-treated;

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III. Mice infected with influenza virus and treated with oseltamivir in a dose of 2.5 mg/kg dai-ly in two intakes, per os, for 5 days, starting 4 h before infection and for the subsequent 4 days;

IV. Mice infected with influenza virus and treated with SAM in a dose of 50 mg/kg, i.p, once a day starting 5 days before infection and for the subsequent 4 days after infection;

V. Mice infected with influenza virus and treated with SAM in a dose of 100 mg/kg, i.p, once a day starting 5 days before infection and for the subsequent 4 days after infection;

VI. Mice infected with influenza virus and treated with oseltamivir in a dose of 2.5 mg/kg dai-ly in two intakes, per os, for 5 days, starting 4 h before infection and for the subsequent 4 days and SAM in a dose of 50 mg/kg, i.p, once a day start-ing 5 days before infection and for the subsequent 4 days after infection;

No Group Experimental groups Number of survivals/ total Survival

rate (%)

I Healthy, non-infected animals 10/10 100II Mice infected with influenza virus 3/10 30

III Mice infected with influenza virus and treated with oseltamivir 2.5 mg/kg per os 7/10 70

IV Mice infected with influenza virus and treated with SAM in a dose of 50 mg/kg,i.p 1/10 10

V Mice infected with influenza virus and treated with SAM in a dose of 100 mg/kg, i.p 2/10 20

VI Mice infected with influenza virus and treated with oseltamivir 1.25 mg/kg, per os, and SAM in a dose of 50 mg/kg, i.p.

9/10 90

VIIMice infected with influenza virus and treated with oseltamivir 1.25 mg/kg, per os and SAM in a dose of 100 mg/kg, i.p

9/10 90

Table 1. Results of treatment with oseltamivir and SAM on 14 days survival of influenza virus infected mice.

VII. Mice infected with influenza virus and treated with oseltamivir in a dose of 2.5 mg/kg dai-ly in two intakes, per os, for 5 days, starting 4 h before infection and for the subsequent 4 days and SAM in a dose of 100 mg/kg, i.p, once a day start-ing 5 days before infection and for the subsequent 4 days after infection.

Mice were observed daily for 14 days for sur-vival after infection.

Results and DiscussionExperimental data on the effect of the com-

bination of oseltamivir plus SAM were compared against control uninfected and untreated animals of group I, and also compared to infected with influ-enza virus and non-treated animals of group II. The effect of treatment of influenza-infected mice with oseltamivir and SAM used alone and in combina-tion is shown in Table 1.

The survival rate was estimated from the number of mice who survived till day 14 of the experiment calculated as a percentage of the total number of mice in the experimental group. The number of survivals shows the total number of mice who survived till day 14 of the experiment out of 10 in the experimental group. SAM was applied once a day intraperitoneally in doses of 50 and 100 mg/kg, starting 5 consecutive days before virus inoculation up to day 5 after infection. Oseltamivir was applied per os for 5 days after viral infection in a dose of

2.5 mg/kg daily in two intakes.For statistical analysis of the results one-way

ANOVA tests with Bonferroni’s post-test were used.

The survival rate of the animals from the non-treated control group II, infected with influenza virus in a dose of 10 LD50, was 30%. Treatment with oseltamivir in a dose of 2.5 mg/kg increased the survival rate of the infected mice from 30% to 70%. SAM alone did not have any positive effect. SAM in a dose of 50 mg/kg showed a very low survival

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rate - 10%, and in a dose of 100 mg/kg the survival rate slightly increased to 20%. The combination of oseltamivir and SAM showed significantly better protection in mice infected with influenza virus. In both groups – the combinations of oseltamivir in a dose of 2.5 mg/kg and SAM in a dose of 50 mg/kg; and oseltamivir in a dose of 2.5 mg kg and SAM in a dose of 100 mg/kg, 90% of the animals survived till day 14 of the experiment (Fig. 1).

The present study shows the positive effect of combining oseltamivir as a specific neuramin-idase inhibitor of the influenza virus replication with S-adenosyl-L-methionine as a precursor of glutathione - the most abundant antioxidant in the body. As our previous research confirmed, SAM alone does not have a positive effect in influenza infected mice. The good results of the combina-tion could be explained by modulation of host de-fense mechanisms and by direct antioxidant effect of increased glutathione against oxidative stress associated with influenza infection. Many authors indicate that glutathione plays a key role in airway function (Rahman and MacNee, 1995; Cai et al.,

2003; Fitzpatrick et al., 2011; Kettle et al., 2014). It is also important for the normal function of the T-cell mediated immunity (Droge and Breitkreutz, 2000).

Oseltamivir (Tamiflu®, F. Hoffmann-La Ro-che Ltd.) is an orally administered antiviral drug that is approved for the treatment of influenza A and B in adults and children (including full term neonates) who present with symptoms typical of influenza when influenza virus is circulating in the community, and for the prophylaxis of influenza in patients aged 1 year or older. Oseltamivir is a prodrug that is administered as a phosphate salt (oseltamivir phosphate; OP). It is then converted by hepatic carboxylesterases to the active metabo-lite oseltamivir carboxylate (OC). In humans, OP is readily absorbed and converted to OC, which is de-tectable in plasma within 30 min, and the absolute bioavailability for OC is 80%. Peak plasma con-centrations of OC are attained in about 3–4 h, and the apparent half-life is 6–10 h, with elimination primarily through renal excretion of OC (Reddy et al., 2015). When exposed to oseltamivir, the influ-

Fig. 1. Survival rate on day 14th after infection. Experimental groups are as follow:I - Healthy, non-infected animals II - Mice infected with influenza virusIII - Mice infected with influenza virus and treated with oseltamivir 2.5 mg/kg per os,IV - Mice infected with influenza virus and treated with SAM in a dose of 50 mg/kg, i.p.V - Mice infected with influenza virus and treated with SAM in a dose of 100 mg/kg, i.p.VI - Mice infected with influenza virus and treated with oseltamivir 1,25 mg/kg, per os,and SAM in a dose of 50 mg/kg, i.p.VII - Mice infected with influenza virus and treated with oseltamivir 1,25 mg/kg, per osand SAM in a dose of 100 mg/kg, i.p.*** p< 0.001 vs group I; ** p < 0.01 vs group I; +++ p< 0.001 vs group II;++p < 0.01 vs group II; +p < 0.05 vs group IIn.s. - non significant vs group I

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enza virions aggregate on the surface of the host cell, thereby limiting the extent of infection within the mucosal secretions and reducing viral infectivi-ty (Kamps and Hoffman, 2006).

SAM is synthesized in the cytosol of every cell, but the liver plays the central role in the ho-meostasis of SAM as the major site of its synthe-sis and degradation (Lu, 2000). SAM contains a high-energy sulfonium ion, which activates each of the attached carbons toward nucleophilic attack and confers on SAM the ability to participate in 3 major types of reactions: transmethylation, transsulfura-tion, and aminopropylation (Lieber and Packer, 2002). The methylation cycle involves the conver-

sion of methionine via S-adenosylmethionine and S-adenosylhomocysteine into homocysteine, fol-lowed by reconversion of homocysteine into me-thionine. This cycle has three major cellular func-tions. First, it provides SAM, necessary for polyam-ine synthesis and for the methylation of numerous essential cell constituents, such as phospholipids, methyl-accepting proteins, CpG islands in DNA, adrenergic, dopaminergic and serotoninergic mol-ecules. Second, it feeds the transsulphuration path-way that leads to the formation from homocysteine of glutathione, the main cellular antioxidant, re-quired for the detoxification of various compounds and for the scavenging of free radicals (Fig. 2).

Fig. 2. Hepatic methionine metabolism and GSH synthesis. The transsul-furation pathway converts methionine to cysteine, which is then converted to GSH via the GSH synthetic pathway (Lu, 1999).

Finally, it recycles 5-methyltetrahydrofolate into tetrahydrofolate, a necessary cofactor for the synthesis of DNA and RNA. SAM plays a central role in the methylation cycle by controlling both the remethylation of homocysteine to methionine and its catabolism through the transsulfuration path-way. A normal adult makes about 8 g of SAM per day, the majority of it in the liver, where it is also mainly consumed (Mato et al., 1997). It is the sec-ond most widely used enzyme substrate after ATP. SAM is biosynthesized during the reaction of me-thionine with ATP, which is catalyzed by SAM syn-thetase or methionine adenosyltransferase. SAM is recognized as the major methyl-donor reagent for

essential methylation reactions that occur in all liv-ing organisms (Fontecave et al., 2004).

ConclusionIn our experimental mice model of influen-

za, the virus infection with 10 LD50 causes 70% le-thality. The monotherapy with oseltamivir in a dose of 2.5 mg/kg increases the survival rate from 30 to 70%. The monotherapy with SAM in a dose of 50 mg/kg and 100 mg/kg does not increase the surviv-al rate. It is a matter of interest that the combined therapy - SAM in both doses applied i.p. combined with oseltamivir in a dose of 2.5 mg/kg daily in two intakes applied per os achieve 90% survival rate

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in mice infected with influenza virus. Our study demonstrates the advantage of combining agents acting through different mechanisms – the antivi-ral drug oseltamivir and SAM as a precursor of the main antioxidant - glutathione. Further experiments are necessary to clarify this statement.

AcknowledgmentsThe authors would like to express gratitude to

Mrs. Kirilka Todorova for their excellent technical assistance and to Dr. Petya Stoyanova for supplying and housing the experimental animals.

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protein interacts with influenza virus polymerase proteins. J. Virol. 72: 5493-5501.

Beigel, J., M. Bray (2008). Current and future antiviral therapy of severe seasonal and avian influenza. Antiviral Res. 78: 91–102.

Boulo, S., H. Akarsu, R.W. Ruigrok, F. Baudin (2007). Nuclear traffic of influenza virus proteins and ribonucleoprotein complexes. Virus Res. 124: 12–21.

Cai, J., Y. Chen, S. Seth, S. Furukawa, R. Compans, D. Jones (2003). Inhibition of influenza infection by glutathione, Free Rad. Biol. Med. 34: 928–936.

Dringen, R., J.M Gutterer, J. Hirrlinger (2000). Glutathione metabolism in brain metabolic interaction between astro-cytes and neurons in the defense against reactive oxygen species. Eur. J. Biochem. 267: 4912-4916.

Dröge. W, R. Breitkreutz (2000). Glutathione and immune function. Proc. Nutr. Soc. 59: 595-600.

Fitzpatrick A.M., W.G. Teague, L. Burwell, M.S. Brown, L.A. Brown (2011). Glutathione Oxidation is associated with airway macrophage functional impairment in children with severe asthma. Pediatric Res. 69: 154–159.

Fontecave, M., M. Atta, E. Mulliez (2004). S-adenosylmethi-onine: nothing goes to waste, TRENDS Biochem. Sci. 29: 243-249.

Garozzo, A., G. Tempera, D. Ungheri, R. Tipanaro, A. Cas-tro (2007). N-Acetylcysteine synergizes with oseltamivir in protecting mice from lethal influenza infection, Int. J. Immunopathol. Pharmacol. 20: 145-354.

Kamps, B., Ch. Hoffman (2006). Drug profiles, in: Kamps, B., et al. (Eds), Influenza Report 2006, Flying Publisher, Paris, 188-219.

Kettle, J., R. Turner, L. Gangell, T. Harwood, S. Khalilova, A. Chapman, C. Winterbourn, P. Sly, CF. AREST (2014). Ox-idation contributes to low glutathione in the airways of children with cystic fibrosis. Eur. Respir. J. 44: 122-129.

Lieber, C., L. Packer (2002). S-Adenosylmethionine: molecu-lar, biological, and clinical aspects—an introduction. Am. J. Clin. Nutr. 76: 1148S-1150S

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Mato, J., L. Alvarez, P. Ortiz, M. Pajares (1997). S-Adenosyl-methioninesynthesis: molecular mechanisms and clinical implications, Pharmacol. Ther. 73: 265-280.

Rahman, I., W. MacNee (1999). Lung glutathione and oxida-tive stress: implications in cigarette smoke-induced airway disease, Am. J. Physiol. 277: 1067-1088.

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About Pathogenesis of the Vaccine Mumps Meningitis

Hristo Odisseev

National Centre of Infectious and Parasitic Diseases, Sofia

ACTA MICROBIOLOGICA BULGARICA

AbstractMeningeal reactions are common with mumps patients. It is considered that such reactions are due

to the direct impact of the mumps virus on meningitis. With the development of the mumps vaccines, this opinion also included the vaccinated people who are considered at risk of serious post vaccinal meningitis. In this article, the author expresses the opinion that these reactions are pathogenetically conditioned and inevitable, and are due to inflammation of the choroid plexus, caused by both virulent and vaccinal strains. The inflamation leads to enhanced secretion of cerebrospinal fluid, causing an increase in the intracranial pressure and marked meningeal symptoms. On the basis of the morphological changes in the choroid plexus in experiments with monkeys, as well as on the clinical symptoms of meningeal reactions in vaccinated children - rare, in a mild form, shortlived, and without any consequences - the author believes these reac-tions to be forms of meningism, not of meningitis.Key words: mumps virus, choroid plexus, meningitis, meningisms

РезюмеМенингиалните реакции са чести при болните от паротит. Съществува мнението, че се

дължат на прякото действие на паротитния вирус върху меките мозъчни обвивки, менингите. Със създаването на паротитни ваксини това сановище се прехвърли и върху менингиалните реакции, появяващи се и при ваксинираните и считани за сериозни постваксинални усложнения. В настоящата статия, авторът изразява становището, че тези реакции са патогенетично обусловени и неизбежни и, че не се дължат нa пряко засягане на менингите от паротитния вирус, а на възпаление на плексус хороидеус предизвикано от вируса. Това възпаление повишава секрецията на ликвора, увеличава интракраниалното налягане и с това предизвиква менингиалните симптоми. Базирайки се на морфологичните промени в плексус хороидеус при експерименти с маймуни и на клиничните симптоми нa реакциите при ваксинираните деца: редки, леко протичащи, бързо преходни и без никакви последици, авторът счита, че тези реакции не са менингити, а менингизми.

*Corresponding author: odisseev@abv.bg

Aseptic meningitis was considered to be a serious and common reaction of the mumps infec-tion. This type of meningitis is explained with the direct attack on the meninges by the virus. After the development of mumps vaccine strains, this opin-ion was also addressed to them. Тhis fact has an adverse effect on the mass vaccine prophylaxis of mumps.

It is widely known that the mumps virus has a tropizm for the excretory cells of the salivary, testi-cles, pancreas as well as choroid plexus glands. The mumps virus has its own surface receptors corre-sponding to the relevant receptors on the excretory

cells of the above mentioned glands. The virus does not affect the cells with endocrine function (en-zymes expression as insulin, testosterone, amylase etc.) of the above mentioned glands since they have not similar receptors.

Once the virus has entered the body through respiratory tract, it moves to the regional lymph nodes and thereafter to the blood flow. It prolifer-ates in the blood system (in the lymphocytes), and causes viremia (Fleischer and Kroeth, 1971). This fact has been proven by isolation of the mumps virus from blood, saliva, urine and liquor. Having interacted with the relevant receptors of the cited excretory cells, mumps virions enter them, repro-duce and cause inflammation.

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The present standpoint primarily view the re-lationship of the mumps virus (virulent and vaccine strains) with choroid plexus.(Noback et al., 1996) (Fig. 1). As a single-stranded negative RNA, the virus replicates in the cytoplasm, leaving the cell nucleic acid unaffected.

Newly produced virions leave the endothelial cells of the plexus by budding, do nоt destroy the membranes of the cells but cause a benign inflam-matory process. This process increases the function of secretory granules and the volume of cytoplasm as well as the permeability of the cell membranes, and causes hyperemia, augments the capillary vol-ume and increases the count of perivascular lym-phocyte infiltrates among the braided vascular plexus. These lesions increase the secretion of ce-rebrospinal fluid (CSF) and cause disorder in CSF dynamics. As a result, the brain ventricles overflow, liquor enters the intercellular space and swells it. All these processes increase the intracranial pres-sure and cause meningeal syndrome - meningism.

The outwandering of the surplus liquor con-taining virus is a continuous process (Bernard, 2014). It escapes from the fourth ventricles into the cistern magna of the sub-arachnoid space, moves slowly and finally reaches the superior sagittal si-nus and by the Pacchioni granules leaves the cranial cavity carrying both the virus and the waste prod-ucts of excess fluid. Мeningeal symptoms disap-pear without any consequences.

During its stay in the cerebrospinal fluid and in the intra-cerebral space the virus contacts neuronal, glial and meningeal cells. However, the mumps virus does not enter and proliferate into them since they have no receptors corresponding to this causer.

Mumps vaccines are live and vaccine strains replicate the pathway of the wild mumps virus. Un-like it, however, the inflammatory processes pro-voked by them are significantly weaker, intracra-nial pressure remains largely lower and meningitis symptoms are manyfold scarcer, milder and fast transient, due to which their clinical presentation is incidental.

The degree of inflammation of choroid plex-us depends on the degree of attenuation of a strain and viral load in the blood and correlates with the intensity of the immune response.

The leading role of choroid plexus inflamma-tion was supported also by experiments in monkeys with mumps vaccine strains Leningrad-3, Sofia-6 and Jeril Lynn (Rosina et al., 1978; Rosina and Hilgenfeldt, 1985).

The role of the choroid plexus is also in line with clinical observations in vaccinated individuals manifesting meningeal symptoms a dissociation of the clinical symptoms and liquor finding: mild clin-ical signs, most often cervical rigidity, against clear liquor, outflow under pressure with puncture, with higher pleocytosis and increased protein (Odisseev, 1971; Radev et al., 1980; Kaneva et al., 1982).

The above facts give us a reason to suggest that the main cause of the occurrence of vaccine “mumps meningitis” reactions in some of the vac-cinated individuals is not the inflammation of the meninges but inflammation of the choroid plex-us and the surplus liquor secretion causes the in-creased pressure in the intracranial space, hence the mumps meningism is pathogenetically conditioned and inevitable, but acceptable. Given the above, we maintain that the term mumps chroiditis compli-ance with the concurrent parotitis, pancreatitis and orchitis. This is particularly valid for the leaflets of routinely used vaccines, in which the term men-ingitis, where it appears, is considered a serious syndrome and this gives parents a reason to deny children vaccination.We believe the term should be modified into meningism, which is the result of a mild inflammation of choroid plexus (excreting lit-tle liquor), with mild clinical signs and without any consequences.

Vaccine meningeal reactions, which are considered to be an inflammation of the menin-ges, were the object of in-depth discussions in the WHO at international and national forums world-wide and an abundance of papers. As a matter of fact, we have defended our statement in discus-sions and papers and also have expressed it to the WHO (Odisseev, 2006). In the beginning of 2007, the WHO concluded: “that in terms of safety, all mumps vaccine preparations for which relevant data are available and acceptable for use in immu-nization programes” (WHO, 2007).

The problem of mumps vaccines was termi-nated and it does not present in the further plans of the WHO. From now on they will be seamlessly in-cluded in the combination with measles-mumps-ru-bella (MMR) and measles-mumps-rubella-varicel-la (MMRV) vaccines and distributed parallel with them.

However, when used in the vaccine informa-tion leaflets, the term ”mumps meningitis - inflam-mation of the soft cerebral lining that covers the brain” ( for instance in leaflet of the vaccine Priorix applicable in Bulgaria ) has an adverse impact in the course of immunization process with mumps

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vaccines, hence we suggest that this term should be modified into the meaning proposed above – meningism.

ReferencesBernard, C. (2014). Le liquide qui nettoie notre cerveau. La

Recherche 491: 56-60.Fleischer, B., H.,V. Kroeth (1971). Lymphoid mumps virus

replication in human lymphocytis cell lines and in periph-eral blood: preference T cells. Infect. Immun. 35: 25-31.

Kaneva, J., B. Ezekieva, R. Katzarova, T. Duhovnikova (1982). Neurological complications during revaccination with mumps vaccine. Reporte pp. 6.

Noback, CB, N. L. Strominger, R. J. Demarsted (1996). Me-ninges, ventricles and cerebrospinal fluid in human ner-vous system. In: Structure and Function. Fifth edition. Williams & Wilkins. NY. pp. 67-82.

Odisseev, H. (1971). Vaccinoprophylaxis of mumps using strain Sofia-6. Tesis of PhD. pp. 216

Odisseev, H. (2006). Letter to Chairman of the Global Ad-visory Committee Vaccine Safety, WHO, in Switzerland.

Radev, M., J. Kaneva, I. Dikov (1980). About neurological complications during of vaccination with mumps vaccine 1976-1979. Report. pp. 5.

Rosina, E. E., T. Captzova, O. Andjaparidse, H. Odisseev, V. Jukovetz, H. Todorova (1978). Comparative morpholog-ic study of the neurovirulence of vaccinal mumps strаins Leningrad-3 and Sofia 6. Epidemiol. Microbiol. Inf. Dis. 15: 174-179.

Rosina, E., M. Hilgenfeldt (1985). Comparative study on the neurovirulence with different vaccine strains of mumps virus in monkeys. Acta Virol. 29: 225-230.

WHO. 2007. Position paper. In WER, 82, 7. Mumps virus vaccines, 58- 60. София, 21. 04. 2016.

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Polymerase Chain Reaction for Detection of Chlamydia abortus in Samples from Aborted Ruminants using Primers for Chlamydia psittaci

Konstantin B. Simeonov1, Ivo N. Sirakov2, Raina T. Gergova2, Kostadinka Koburova3, Tzvetan G. Todorov3

1Department of Virology, National Diagnostic and Research Veterinary Medical Institute “Prof. Dr. G. Pavlov”, 15“Pencho Slaveykov” Blvd., 1606 Sofia, Bulgaria; 2 Medical University – Sofia, 15 „Akademik Ivan Evstratiev Geshov“ Blvd., 1431 Sofia, Bulgaria;3 G-Lab, Ltd, 2 “Hristo Belchev” Str., 1000 Sofia, Bulgaria

AbstractIn this study, the CPsittF(R) and Or1(2) primers, targeting the ompA gene of Chlamydia psittaci,

were used for PCR detection of Chlamydia abortus DNA in clinical samples from aborted animals. The sensitivity and specificity of the reaction were compared with those achieved by C. abortus-specific primers CpsiA(B). The comparative analysis of the results showed that all samples that were positive for C. abor-tus in PCR, performed with primers CpsiA(B) also gave a positive result in the PCR using the CPsittF(R) primers, generating a 1041 bp specific amplification product. PCR amplification using the Or1(2) primers was uncertain and produced an amplification product of 212 bp, which was different from the expected length of 245 bp. In an attempt to improve these primers, their sequence was modified at the 11th and the 21st nucleotide. Although the sensitivity of the reaction performed with the modified primers was improved, it was still lower compared to that achieved with the original C. abortus-specific primers CpsiA(B) and prim-ers CPsittF(R). The results show that the primer pair CPsittF(R), developed for the detection of C. psittaci could be successfully used in the diagnosis of abortions, induced by C. abortus, while the primer set Or1(2) is less effective. Key words: Chlamydia abortus, Chlamydia psittaci, primers, PCR

РезюмеПраймери CPsittF(R) и Or1(2), насочени към ompA гена на Chlamydia psittaci бяха изпитани в

PCR за доказване наличието на ДНК на Chlamydia abortus в клинични проби от абортирали животни. Чувствителността и специфичността на реакцията бяха сравнени в PCR, проведена със специфичните за C. abortus праймери CpsiA(B). Сравнителния анализ показа, че всички изследвани проби с положителен резултат за C. abortus от PCR, проведена с праймери CpsiA(B), реагират положително и в PCR, проведена с праймери CPsittF(R), при което се генерира специфичен амплификационен продукт с големина 1041bp. Реакцията, проведена с праймери Or1(2) показа непостоянни резултати, като при това големината на получения продукт (212bp) не съответстваше на очакваната големина от 245bp. Тези праймери бяха модифицирани чрез замени в 11-та и 21-та база в нуклеотидната им последователност. Въпреки, че това повиши чувствителността на реакцията, тя остана по-ниска в сравнение с PCR, проведена с праймери CpsiA(B) и CPsittF(R). Резултатите показват, че праймери CPsittF(R), разработени за детекция на C. psittaci могат успешно да бъдат използвани в диагностиката на абортите, породени от C. abortus, докато праймерната двойка Or1(2) е по-малко ефективна.

ACTA MICROBIOLOGICA BULGARICA

*Corresponding author: kbsimeonov@yahoo.com

Introduction Chlamydia abortus is the main etiological

agent of abortions and stillbirths in ruminants in Europe (Longbottom et al., 2013). Similar pathol-ogy, although with a lower incidence, it also caus-es in other animals (pigs, horses) (Bocklisch et al.,

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1991; Schautteet et al., 2011). This infectious agent has also proved to have a zoonotic potential, as it can cause abortions in risk-group pregnant women being in contact with infected animals, or flu-like conditions, characterized by fever, headache and muscle ache (Rodolakis and Mohamad, 2010).

Since a number of other viruses and bacteria also have an abortogenic potential, it is important to identify C. abortus etiology in view to develop an adequate strategy for treatment and prevention of such infections, respectively, abortions. In addition to conventional diagnostic assays, such as antigen detection by immunohistochemistry, microscopic observation of intracellular inclusions in stained impression smears and serological detection of antibodies by complement fixation test (CFT) and ELISA, the molecular methods, based on nucleic acid amplification are increasingly being used for detection of C. abortus and other Chlamydiales. Due to the close genetic relationship between the members of Chlamydiaceae family, they are com-monly detected using conventional and real-time PCR as an initial screening step and, if necessary, differentiation at the species level is then done by sequencing (Kaltenboeck et al., 1993; Sheehy et al., 1996; Hartley et al., 2001) or restriction frag-ment length polymorphism (RFLP) analisys of the PCR products (Everettt and Andersen, 1999; Sait et al., 2011).

In the PCR-based diagnostics of C. abortus infections, there is a wide range of primers target-ing different regions of the chlamydial genome, e.g. the intergenic spacer region between the 16S and 23S ribosomal RNA (rRNA) genes (Madico et al., 2000), the conserved regions of the gene encoding the outer membrane protein (omp2) (Hartley et al., 2001), the helicase gene (Creelan et al., 2000), as well as the genes involved in the synthesis of the polymorphic outer membrane proteins (pmp) (Laurucao et al., 2001). However, the sensitivity of the reactions may vary depending on the primer set (Creelan et al., 2000; Greco et al., 2005; Soomro et al., 2012). Therefore, searching for new combina-tions of primers in order to optimize this molecular assay and expand the range of C. abortus strains which can be detected is needed.

Although both C. abortus and C. psittaci could infect ruminants (Cox et al., 1998; Lenzko et al., 2011), from practical point of view their spe-cies differentiation is not necessary since the ther-apeutic approaches in case of infection are one and the same. Presuming that, as well as the close ge-netic relationships between these two members of

Chlamydiaceae family, we performed this study to examine whether C. abortus could be successfully detected by PCR in clinical samples from placentae and internal organs of aborted fetuses using prim-ers, originally constructed for the diagnosis of C. psittaci.

Materials and methods. Clinical samples

Between December of 2013 and February of 2015, fetuses and placentae from 21 sheep and 7 goats, originating from 23 flocks with a high per-centage of abortion were delivered in the laboratory for diagnostic investigation for C. abortus and Cox-iella burnetii. Samples were investigated by light microscope observation on impression smears from cotyledons, stained by Giemsa method for the pres-ence of intracellular elementary bodies and PCR. The samples were tested in other laboratories both bacteriologicaly and virologicaly for the presence of other abortogenic agents. Where available, epi-zootiological data were collected for the period of the pregnancies to exclude any concomitant factors able to cause abortions (mycotoxicoses, feeding with frost-damaged feed and other technological errors).DNA extraction

DNA was extracted from placental cotyledons or tissues (abomazum and lung) from aborted fetus-es using Animal and Fungi DNA Preparation Kit (Jena Bioscience) or Tissue & Cell Genomic DNA Mini Kit (Guangzhou Geneshun Biotech., Ltd.) fol-lowing manufacturer’s instructions. Extraction effi-ciency was assessed by agarose gel electrophoresis and extracted DNA was stored at -20°C until use.PCR

DNA was amplified by conventional PCR using the CpsiА and CpsiB primers, which were designed from the 4 available C. abortus pmp gene sequences (Larocaou et al., 2001). An automatic FluoroCycler 12, HAIN, LKB, was used. The re-action mixtures were 25 µL and consisted of 12.5 µL of 2x PCR Taq Mix (Guangzhou Geneshun Biotech., Ltd), 2 µL of each primer at a concen-tration of 10pmol µL-1, 2 µL of target DNA and 6.5 µL ddH2O. The amplification program was set as follows: initial denaturation at 94 °C for 5 min, followed by 30 cycles of denaturation at 94 оС for 30 s, annealing at 50 оС for 1 min and elongation at 72 оС for 2 min; and a final elongation step at 72 оC for 10 min. As a control, which was run in parallel, we used DNA, extracted from the placenta of an aborted goat, identified as C. abortus positive by

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Table 1. Target genes and primer sequences used in the study:

*C. burnetii amplification was performed as described by Stein and Raoult, 1992 with modifications.

microscopy of stained impression smears, PCR and serology (ELISA).

To check for a possibility to detect C. abortus using C. psittaci-specific primers, three CpsiА(B) PCR positive and three negative samples were test-ed twofold by PCR using the CPsittF(R) and Or1(2) primers. The same amplification program as for CpsiА(B) PCR was used. The target genes, prim-er sequences, amplification product lengths and the corresponding references are given in Table 1.

All amplified fragments were analyzed us-ing 2% agarose gel electrophoresis and GeneRuler 100bp Plus DNA Ladder. Software

Comparison and modification of primers was done using the Basic Local Alignment Search Tool (BLAST) (National Center for Biotechnology In-formation-NCBI, http://www.ncbi.nlm.nih.gov/), software MEGA 6.06 (Tamura et al., 2013) and its integrated alignment tool (Edgar, 2004).

ResultsIn the CpsiA(B) PCR 5 of 21 sheep and one

of seven goat resulted positive. One goat was posi-tive for Coxiella burnetii infection (Q fever), how-ever coinfection with C. abortus was not detected. BLAST analysis of the CpsiA(B) primers showed that they are targeted to more than one region of the C. abortus genome as the forward primer (А) dis-plays complementarities to other 3 regions and the reverse primer (В) to another 2 regions of the se-

Gene Primer Sequence Amplicon size (bp)

Reference

pmpC. abortus

CpsiАCpsiB

5’-ATG AAA CAT CCA GTC TAC TGG-3’5’- TTG TGT AGT AAT ATT ATC AAA-3’

300 Larocaou et al., 2001

ompAC. psittaci

CPsittFCPsittR

5’-GCT ACG GGT TCC GCT CT-3’5’-TTT GTT GAT YTG AAT CGA AGC -3’

1041 Heddema et al., 2006

ompAC. psittaci

Or1Or2

5’-TTT CGA TCG TGT ATT AAA AGT T-3’5’-AGA AAA TGT CGA AGC GAT CCA-3’

245 Olsen et al., 1998

ompAC. psittaci

Or1mOr2m

5’-TTT CGA TCG TGT ATT AAA AGT T-3’ 5’-AGA AAA TGT CRA AGC GAT CCG-3’

212 This study

superoxide dismutase*Coxiel la burnetii

C.B.-1C.B.-2

5’-ACT CAA CGT ACT GGA ACC GC-3’5’-TAG CTG AAG CCA ATT CGC C-3’

257 Stein and Raoult, 1992

Fig.1. Results of PCR analysis of C. abortus DNA and Coxiella burnetii DNA, using C. abortus- and C. psittaci-specific, and Coxiella burnetii-specific primers, respectively. Lane 1, primers CpsittF(R), 1041bp; lane 2, primers Or1(2), 212bp; lane 3, primers CpsiA(B), 300 bp; lane 4, DNA molecular size marker 100bp; lane 5, negative control (DNA, tested negative with CpsiA(B) and re-tested with Cpsitt(F)R; lane 6, PCR product of placenta form goat, tested with C.B.-1(2) primers (Coxiella burnetii), 257bp; lane 7, ddH2O and C.B.-1(2) primers (Coxiella burnetii)-negative control.

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quenced C. abortus genome (GenBank, accession number (LN554882.1, CR848038.1). In addition, difference was found in the sequence complemen-tary to CpsiA in position 10, where C→T.

In the comparative study with different prim-ers in all three samples PCR, performed with CPsit-tF(R) generated products with the expected size of 1041bp. (Fig.1). In contrast, on the first run of the reaction carried out with Оr1(2) one of the samples resulted negative but, after being repeated, all sam-ples gave positive results. Nevertheless, at electro-phoresis the generated amplicons were with a size of about 200bp, which differed significantly from the theoretically expected size of 245bp, indicated in the original report (Olsen et al., 1998). In addi-tion, visualized bands showed low intensity stain-ing. In an attempt to find a possible explanation for the unsatisfactory results, the homology of the re-ported Оr1(2) primer pair with their nucleic acid se-quences was analyzed by comparison to GenBank (NCBI) data, using MEGA 6.06 and an alignment tool (Edgar, 2004). The alignment revealed the presence of two discrepancies as follow: G→A(R) in position 11 (in two sequences, KM609418.1 and KM609419.1) and A→G in position 21 (in all se-quences). Moreover, BLAST analysis revealed a difference of 34 bases in the size of the fragments encompassed by Оr1(2) primers as compared to that, reported by Olsen et al. (1998).

Presuming this we modified Оr1(2) primers, in which these mismatches were corrected. The modified primers Оr1(2)m showed greater sensi-tivity in PCR amplification (two positive from two runs) and generated a 212 bp product, which cor-responded well to the length of the targeted region of the ompA gene. Nevertheless, the electropho-retic bands in most cases showed lower intensity as compared to those generated by CpsiA(B) and CPsittF(R) PCR. PCR of samples that tested nega-tive with CpsiA(B) gave the same results when per-formed with primers CPsittF(R), Оr1(2) and Оr1(2)m.

DiscussionThe results obtained by PCR for diagnosis of

С. abortus (21.43% positives) confirmed the im-portant role this agent plays as an etiological agent of the abortions in the sheep and goats from tested flocks. These values exceeded the percent of pos-itive reagents detected by Giemsa-staining of im-pression smears (unpublished data) and showed the high diagnostic potential of PCR for diagnosis of chlamydial infections.

Considering the close genetic relationship within the Chlamydiaceae, we examined the avail-ability of primers CPsittF(R) and Or1(2), designed for the detection of C. psittaci to diagnose C. abor-tus in clinical samples by PCR. In this we consider also studies demonstrating that primers targeting C. abortus genes can be successfully used for amplifi-cation of DNA extracted from C. psittaci (Creelan and McCullough, 2000; Larocaou et al., 2001), C. caviae (Larocaou et al., 2001), as well as primers targeting sequences common for all members of order Chlamydiales (Lienard et al., 2011). In our study, the results from CpsiA(B)-PCR coincided entirely with those of PCR, conducted with primers CPsittF(R), although these primers target different genes of the chlamydial genome, pmp and ompA, respectively. This indicates that the two primer pairs amplify sequences in these genes, which are identical in both C. abortus and C. psittaci.

Some differences in terms of repeatability of the results and the size of the amplification products were observed when the same samples were test-ed in PCR, using primers Or1(2). We suggest that the mismatches we identified between the primer sequences and their complementary regions in the ompA gene of C. psittaci could serve as a possible explanation, since such mismatches are known to reduce the specificity of PCR amplification.

Although some authors report differences in the detection rates of the PCR reactions performed with different primers (Greco et al., 2005; Soomro et al., 2012), in our study we did not observe such variation in the sensitivity and all three samples that proved positive by CpsiA(B) PCR also resulted positive in CPsittF(R) and Or1(2) PCR. It is known that C. abortus is characterized by low genetic het-erogeneity (Denamur et al., 1991) and inter-strain differences can only be detected using monoclo-nal antibodies, sequencing or restriction fragment length polymorphism (RFLP) analysis of the PCR amplification products. Recent advancements in modern techniques for analysis of tandem repeats in DNA (multiple loci VNTR analysis (MLVA) and multi locus sequence typing (MLST)) have even made possible to divide C. abortus strains in differ-ent genotypes (Laroucau et al., 2009; Pannekoek et al., 2010; Siarkou et al., 2015), partly dependent on the geographical origin as well (Laroucau et al., 2009). In this study, only samples collected from Bulgaria were analyzed and no comparative anal-ysis with reference strains or isolates from other countries was carried out. Moreover, only a limited number of samples were tested. Thus, the existence

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of strains within C. abortus which cannot be detect-ed by the PCR primer pairs used for detection of C. psittaci cannot be excluded completely.

Although different annealing temperatures have been reported in the original papers as optimal for the primers, compared to this study, 50оС for CpsiA(B), 62оС for CPsittF(R) and 48оС for Or1(2), respectively, we performed reactions following one and the same regime of amplification at annealing temperature of 50оС and obtained positive results with all of the primers tested. This allows simulta-neous amplification of different genome segments of one and the same sample, when further sequenc-ing or RFLP analyses are planned. In addition, the primers used by us for detection of C. abortus also have the advantage to generate amplicons of differ-ent size, thus facilitating their electrophoretic dif-ferentiation when other abortogenic agents (most commonly Coxiella burnetii) have been examined simultaneously in multiplex PCR.

Due to the uneven results observed with Or1(2), we do not recommend these primers for routine PCR diagnosis of C. abortus in small ru-minants. In contrast, C. psittaci specific primer pair CpsittF(R) could be efficiently used for this purpose. However, additional techniques such as sequencing or RELF analysis are needed to specif-ically discriminate C. abortus from C. psittaci or other Chlamydia that may be involved in induce-ment of abortions.

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Cox, H., P. Hoyt, R. Poston, T. Snider, T. Lemarchand, K. O‘Reilly (1998). Isolation of an avian serovar of Chla-mydia psittaci from a case of bovine abortion. J. Vet. Di-agn. Invest. 10: 280-282.

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Letter to the Editor

Inhibition of Human Herpes Virus Type 2 Replication by Water Extract from Nepeta nuda L.

Petia Angelova, Anton Hinkov, Venelin Tsvetkov, Kalina Shishkova, Daniel Todorov, Stoyan Shishkov

Laboratory of Virology, Faculty of Biology, University of Sofia ‘‘Saint. Kliment Ohridski’’, Sofia, Bulgaria

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Herpes simplex virus type 2 (HSV-2), a hu-man pathogen, is a member of the large family of Herpesviridae. HSV-2 infection is usually transmit-ted sexually and can cause recurrent, painful gen-ital ulcers. In neonates the infection is associated with significant morbidity and mortality. Moreover, HSV-2 infection increases the risk of human im-munodeficiency virus (HIV) acquisition. There are about 11 licensed antiherpetic drugs for treatment of herpesvirus infections (De Clercq et al., 2006). The most commonly used ones are the nucleoside ana-logs acyclovir, its derivatives and cidofovir (Elion, 1993). Unfortunately, continuous therapy leads to a selec-tion of resistant strains. (Piret et al., 2011). This requires development of new antivirals directed against other viral targets. Moreover, the toxici-ty associated with some antivirals limits their use, and therefore less toxic and more effective drugs are needed. For these reasons special attention is focused on compounds of natural origin. Plant ex-tracts have complex chemical structures and lower cytotoxicity, hence the occurrence of strains resist-ant to their action is delayed.

The genus Nepeta (Lamiaceae) comprises about 250 species distributed in the central and southern parts of Europe, Asia and the Middle East. Nepeta species are widely used in folk medicine be-cause of their antispasmodic, expectorant, diuretic, antiseptic, antitussive, antiasthmatic and febrifuge activities. (Zargari, 1990; Newall et al., 1996; Bas-er et al., 2000).

We have studied the antiviral activity of a water extract from Nepeta nuda L. derived from in vivo propagated plants. The cytotoxicity was tested on Madin Darby Bovine Kidney (MDBK) cell line. The maximal nontoxic concentration (MNC) and the cytotoxic concentration (CC50) of the extract

were determined by a colorimetric method (MTT assays) (Mosmann, 1983). The results were mea-sured at 48 hour and 72 hour after the addition of the extract. The maximal nontoxic concentration (MNC) of the extract determined at 48 hour was ≈ 4 mg/ml, and the cytotoxic concentration (CC50) was ≈8 mg/ml. The results obtained for MNC and CC50 at 72 hour after the addition of the extract were 2 mg/ml and 4.5 mg/ml, respectively. To de-termine the antiviral activity of the extract against HSV-2 strain BA, a modification of MTT assays at low MOI was used (Takeuchi et al., 1991) (ef-fect expressed as % of protection). As a long-term experiment (results are measured 5-6 days p.i.), MNC values measured at 72 hour after the extract addition were used. The water extract from Nepe-ta nuda significantly inhibited the replication of HSV-2. The protection rate is up to 70% (IC50 is ≈ 0.75 mg/ml). We also conducted a yield reduction assay at high MOI (Souza et al. 2008). As long as this experiment is terminated at the 24th hour, MNC measured at the second day can be used (effect ex-pressed as % of inhibition). Inhibition yield produc-tion reached ≈98 % at 4.5 mg/ml. There was almost no activity at 2 mg/ml. Further we tested the direct inactivating effect of the extract against the extra-cellular form of HSV-2. The extract did not show any change in the virus titer.

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Assoc. Prof. Dr. Margarita Yankulova was born on December 30, 1930 in Blagoevgrad to a clerical family. She graduated from primary and high schools and became a student of the Agricultural Academy “Georgi Dimitrov” in Sofia, specializing in viticulture. She began her professional activity as a teacher-agriculturist in the village of Pordim near the town of Pleven. She married for Prof. Dr. Jordan Yankulov – medicinal plants scientist and breeder. Their daughter is a pharmacologist. Prof. Dr. M. Jankulova became a regular post-graduate student at the Bulgarian Academy of Sciences with Cor. Member Prof. Dr. Kovachevsky as her mentor. She defended a dissertation on “Methods for diagnosis of virus diseases on potato”. M. Yankulova worked together with Prof. Dr. Todora Ivancheva-Gabrovska on the development of methods for plant viruses’ purification and serological methods of virus detection. Shortly after the defense of her thesis, Dr. Yankulova was nominated for the Humboldt’s grant and specialized at the Institute of Plant Virology (IPV), Braunschweig, Germany. She was recommended for this scholarship by Prof. Dr. Dimitar Atanassov, Academician Christo Daskalov and Cor. Member Prof. Ivan Kovachevsky. Dr. Yankulova was trained to work with grapevine viruses and mastered new methods for plant virus diagnostics and identification in IPV, Braunschweig.

Back from Germany, Dr. Yankulova was nominated Assistant Prof. at the Plant Protection Institute (PPI). At the beginning, she worked at the PPI Department of Immunity with Prof. Dr. Dimitar Dodov as Head of Department. Later, it was divided into Immunity and Virology departments, with Prof. Dimitar Trifonov Head of the latter. Assist. Prof. Dr. Yankulova began her virological research at the Virology Department and became Associate Prof. in 1972. She studied economically important viruses on tobacco, different vegetables, grapevine and sugar beet, etc., at the PPI. She also produced

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85th Anniversary of Margarita Yankulovaantisera for a lot of these viruses in the institute’s laboratory for purification and serodiagnosis. As Associate Professor, Dr. Yankulova trained a lot of Bulgarian and foreign post-graduate students.

Assoc. Prof. Dr. Yankulova was invited to the Institute of Genetic Engineering (IGE) by its Director Acad. Atanas Atanassov to work with molecular genetics researchers. At the same time, Dr. M. Yankulova was awarded The People’s Republic of Bulgaria medal for her overall activity. M. Yankulova and T. Gabrovska were members of the Virological Council of Bulgaria. Assoc. Prof. Dr. Yankulova took part in the development of biotechnologies at IGE. She was the first to introduce the serological Enzyme Linked Immunosorbent Assay (ELISA) in Bulgaria and taught this method to many plant virologists. A construct for creating TSWV resistant tobacco cultivars was developed in a collaboration of Bulgarian and German researchers in IPV, Braunschweig. Transgenic tobacco cultivars were created with this construct at IGE, Bulgaria. Assoc. Prof. Dr. Yankulova organized and did the entire work on testing the transgenic tobacco plants in laboratory conditions and in the fields of the Experimental Station of Tobacco in the town of Gotse Delchev. The study of the transgenic tomato cultivars for TSWV resistance was done together with Assoc. Prof. Dr. Dimitrinka Christova, PPI.

Even though Assoc. Prof. Dr. Yankulova has been exceptionally diligent in her experimental work, she would always emphasize how lucky she has been to have the best teachers, principals and colleagues, such as Prof. Dr. Dimitar Atanassov, Corr. Member Prof. Ivan Kovachevsky, Prof. Dr. Todora Ivancheva-Gabrovska, Prof. DSc. Penka Kaytasova, Prof. DSc. Dimitar Trifonov, Assoc. Prof. Marko Markov and many others, as well as good laboratory assistants. Assoc. Prof. Dr. Yankulova will always be a teacher and model for all of us.

Bistra Dikova

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In memoriam

Penka Kaytazova1933 – 2014

Penka Yosheva Kaytazova was born on 08.12.1933 in the village Bejanovo, Lovech dis-trict in the family of Yosho Petkov Nedyalkov and Vojka Tzanova Nedyalkova. Her father Yo-sho Petkov worked as an accountant in the rural cooperation and with his active participation the building of the cooperation in the village was built. Her mother was a housewife. She had a little sister Teodora Yosheva Naydenova, who along with her husband Nayden Naydenov lived and worked in the farm village during their career. Primary edu-cation Penka Yosheva finished in his native village. In 1951 he graduated Pleven Girls’ School “Anas-tasia Dimitrova.” In 1952 she was a student at the Agronomy Faculty of Agricultural Academy in So-fia. He graduated as an agronomist in “Plant Pro-tection” in 1957. She was married to Angel Ivanov Kaytazov - Senior Fellow at the Plant Protection Institute (PPI). They have two daughters, Elena - physicist and Valentina - physician. They have two grandsons, two granddaughters, great-grandson and great-granddaughter. From 1957 Penka Yoshe-va Kaytazova worked as an agronomist - specialist and later as a researcher at the virology section in PPI. From 1964 to 1967 he specialized in electron microscopy, electron microscopy lab at the Institute of Histology and Embryology, Faculty of Medicine in Vienna with a scholarship of the Austrian Min-istry of Education. She was assigned as an official PhD student at the Institute of Forest Pathology and

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Plant Protection of the Higher School of crops - now University. Her thesis was on „Studies to iden-tify viruses in tobacco in Austria“ (Untersuhungen zur Differenzialdiagnose der Tabakviren in Oster-reich). With this work was first identified viruses in deciduous tobaccos in Austria. In this study it was found that serious damages on this culture were in-duced by a mixed infection of Potato virus Y and Cucumber mosaic virus. She rejected the view that Tobacco rattle virus was the causative agent of these damages. After his return to Bulgaria in 1968 under the pressure of responsible government officials in Agricultural Academy, the Higher Attestation Commission under Council of Ministers refused to legalize her PhD diploma, although she went with the permission of the Agricultural Academy to use Austrian scholarship for training and to defend a dissertation on Electron microscopy in the field of botany. Thanks to the moral support of prof. Dim-itar Dodov and the President of the AA, she trans-lated her PhD thesis and successfully defended in Bulgaria in 1970. At the same time she organized at the Institute of Plant Protection, Electron mi-croscope laboratory with full equipment, which mainly studied the morphology of viral pathogens in plants, the morphology of some viruses caus-ing diseases in insects and other ultra-microscopic studies. She developed methods for rapid diagnosis of plant viruses. Along with research work she mi-croscope and diagnose viral diseases in dozens of samples from production areas in the country.

In 1989 she defended degree of “Doctor of Agricultural Sciences” on “Morphology and di-agnosis of plant viruses.” She wrote about 60 sci-entific papers related to study the morphology of the viruses in plants. Her scientific research activ-ity was awarded with the medal “St. St. Cyril and Methodius “.

Prof. Dr. Penka Kaytasova was emphasized researcher, but also socially active person. Her so-cial activity was evidenced by the book “One life, one Golgotha” in memoriam of Prof. Dr. Dimitar Atanassov, notable scientist – phytopathologist and virologist, whose scientific work for virus agent of the disease “sharka” on plum is fundamental for the plant virology.

Antoniy Stoev