Molecular detection and phylogenetic analysis of tick-borne encephalitis virus in ticks in northeastern China.

Tick-borne encephalitis virus (TBEV) is an important causative agent that causes neurological infections in humans and animals. In recent years, only few epidemiological surveys on TBEV have been conducted in China. The purpose of this study was to determine the prevalence and subtype of TBEV in ticks in northeastern (NE) China. A total of 3799 questing ticks were collected in NE China between April 2015 and June 2016. Ticks were pooled and tested for TBEV RNA using semi-nested reverse transcriptase-polymerase chain reaction. Positive pools were used to isolate the virus and amplify complete sequences, followed by sequence identity and phylogenetic analysis. TBEV RNA was detected in Ixodes persulcatus ticks at a total prevalence of 2.9% (6/143; 95% confidence interval: 1.2%-5.9%). Three TBEV strains were isolated (JL-T75, HLB-T74, and DXAL-T83) and showed 93.9%-99.1% nucleotide identities and 97.1%-99.5% amino acid identities in Far Eastern (FE) TBEV subtypes, and 82.9%-87.6% nucleotide identities and 92.9%-96.4% amino acid identities in other subtypes. For polyprotein, the JL-T75, HLB-T74, and DXAL-T83 strains showed 29, 50, and 55 amino acid residues, respectively, different from those in the TBEV vaccine (Senzhang) strain in China. Phylogenetic analysis revealed that these viruses were clustered in the FE-TBEV branch but formed distinct clades depending on the natural foci. The results of this study suggest that the FE-TBEV subtype is still endemic in I. persulcatus ticks in NE China, and the viruses in different natural foci in NE China are more likely to have genetic differences.

Exploration of binary protein-protein interactions between tick-borne flaviviruses and Ixodes ricinus.

BACKGROUND: Louping ill virus (LIV) and tick-borne encephalitis virus (TBEV) are tick-borne flaviviruses that are both transmitted by the major European tick, Ixodes ricinus. Despite the importance of I. ricinus as an arthropod vector, its capacity to acquire and subsequently transmit viruses, known as vector competence, is poorly understood. At the molecular scale, vector competence is governed in part by binary interactions established between viral and cellular proteins within infected tick cells. METHODS: To investigate virus-vector protein-protein interactions (PPIs), the entire set of open reading frames for LIV and TBEV was screened against an I. ricinus cDNA library established from three embryonic tick cell lines using yeast two-hybrid methodology (Y2H). PPIs revealed for each viral bait were retested in yeast by applying a gap repair (GR) strategy, and notably against the cognate protein of both viruses, to determine whether the PPIs were specific for a single virus or common to both. The interacting tick proteins were identified by automatic BLASTX, and in silico analyses were performed to expose the biological processes targeted by LIV and TBEV. RESULTS: For each virus, we identified 24 different PPIs involving six viral proteins and 22 unique tick proteins, with all PPIs being common to both viruses. According to our data, several viral proteins (pM, M, NS2A, NS4A, 2K and NS5) target multiple tick protein modules implicated in critical biological pathways. Of note, the NS5 and pM viral proteins establish PPI with several tumor necrosis factor (TNF) receptor-associated factor (TRAF) proteins, which are essential adaptor proteins at the nexus of multiple signal transduction pathways. CONCLUSION: We provide the first description of the TBEV/LIV-I. ricinus PPI network, and indeed of any PPI network involving a tick-borne virus and its tick vector. While further investigation will be needed to elucidate the role of each tick protein in the replication cycle of tick-borne flaviviruses, our study provides a foundation for understanding the vector competence of I. ricinus at the molecular level. Indeed, certain PPIs may represent molecular determinants of vector competence of I. ricinus for TBEV and LIV, and potentially for other tick-borne flaviviruses.

Phylogenetic analysis of tick-borne encephalitis virus strains found in an engorged tick and traveler returning from Russia.

Although travel-related tick-borne encephalitis (TBE) cases have been increasingly registered worldwide, very few published case studies are available to date. The present report describes a travel-related TBE case and provides genotypic characterization of two viral isolates. Laboratory diagnostics were based on complement fixation test and virus isolation. This report is unique because the TBE case was first confirmed by virus isolation from the engorged tick and only later from the patient's blood. Moreover, this case demonstrated a successful prophylaxis performed on day 8 post tick exposure although it is generally recommended that anti-TBEV immunoglobulins should be administered not later than on day 4 after tick bite. Sequences of E protein gene fragments were used to phylogenetically characterize the two isolates. The results demonstrated that both viral isolates belonged to clusteron 3A (Zausaev group) of the Asian lineage of the TBEV Siberian subtype. The synonymous nucleotide substitution, C351 T, was identified in E protein gene fragments of TBEV 88 and TBEV 89, which could have been induced by virus transmission. A few important take-home messages can be gleaned from the reported case. First, travelers should be aware of TBE endemic areas that they plan to visit and be proactive when exposed to Ixodes ticks. Second, medical practitioners should always consider travel history and potential tick exposure of patients. Lastly, engorged Ixodes spp. ticks removed from the patients, who have arrived from endemic areas, should be tested for TBEV even in the absence of TBE clinical signs.

Tick-borne encephalitis virus (TBEV) prevalence in field-collected ticks (Ixodes ricinus) and phylogenetic, structural and virulence analysis in a TBE high-risk endemic area in southwestern Germany.

BACKGROUND: Tick-borne encephalitis (TBE) is the most common viral CNS infection with incidences much higher than all other virus infections together in many risk areas of central and eastern Europe. The Odenwald Hill region (OWH) in southwestern Germany is classified as a TBE risk region and frequent case numbers but also more severe infections have been reported within the past decade. The objective of the present study was to survey the prevalence of tick-borne encephalitis virus (TBEV) in Ixodes ricinus and to associate TBEV genetic findings with TBE infections in the OWH. METHODS: Ticks were collected by the flagging methods supported by a crowdsourcing project implementing the interested public as collectors to cover completely and collect randomly a 3532 km2 area of the OWH TBE risk region. Prevalence of TBEV in I. ricinus was analysed by reversed transcription quantitative real-time PCR. Phylogeographic analysis was performed to classify OWH TBEV isolates within a European network of known TBEV strains. Mutational sequence analysis including 3D modelling of envelope protein pE was performed and based on a clinical database, a spatial association of TBE case frequency and severity was undertaken. RESULTS: Using the crowd sourcing approach we could analyse a total of 17,893 ticks. The prevalence of TBEV in I. ricinus in the OWH varied, depending on analysed districts from 0.12% to 0% (mean 0.04%). Calculated minimum infection rate (MIR) was one decimal power higher. All TBEV isolates belonged to the European subtype. Sequence analysis revealed a discontinuous segregation pattern of OWH isolates with two putative different lineages and a spatial association of two isolates with increased TBE case numbers as well as exceptional severe to fatal infection courses. CONCLUSIONS: TBEV prevalence within the OWH risk regions is comparatively low which is probably due to our methodological approach and may more likely reflect prevalence of natural TBEV foci. As for other European regions, TBEV genetics show a discontinuous phylogeny indicating among others an association with bird migration. Mutations within the pE gene are associated with more frequent, severe and fatal TBE infections in the OWH risk region.

Tick-Borne Encephalitis Virus: An Emerging Ancient Zoonosis?

Tick-borne encephalitis (TBE) is one of the most important viral zoonosis transmitted by the bite of infected ticks. In this study, all tick-borne encephalitis virus (TBEV) E gene sequences available in GenBank as of June 2019 with known date of isolation (n = 551) were analyzed. Simulation studies showed that a sample bias could significantly affect earlier studies, because small TBEV datasets (n = 50) produced non-overlapping intervals for evolutionary rate estimates. An apparent lack of a temporal signal in TBEV, in general, was found, precluding molecular clock analysis of all TBEV subtypes in one dataset. Within all subtypes and most of the smaller groups in these subtypes, there was evidence of many medium- and long-distance virus transfers. These multiple random events may play a key role in the virus spreading. For some groups, virus diversity within one territory was similar to diversity over the whole geographic range. This is best exemplified by the virus diversity observed in Switzerland or Czech Republic. These two countries yielded most of the known European subtype Eu3 subgroup sequences, and the diversity of viruses found within each of these small countries is comparable to that of the whole Eu3 subgroup, which is prevalent all over Central and Eastern Europe. Most of the deep tree nodes within all three established TBEV subtypes dated less than 300 years back. This could be explained by the recent emergence of most of the known TBEV diversity. Results of bioinformatics analysis presented here, together with multiple field findings, suggest that TBEV may be regarded as an emerging disease.

First detection of tick-borne encephalitis virus in Ixodes ricinus ticks and their rodent hosts in Moscow, Russia.

Here, we report the first confirmed autochthonous tick-borne encephalitis case diagnosed in Moscow in 2016 and describe the detection of tick-borne encephalitis virus (TBEV) in ticks and small mammals in a Moscow park. The paper includes data from two patients who were bitten by TBEV-infected ticks in Moscow city; one of these cases led to the development of the meningeal form of TBE. Both TBEV-infected ticks attacked patients in the same area. We collected ticks and trapped small mammals in this area in 2017. All samples were screened for the presence of pathogens causing tick-borne diseases by PCR. The TBEV-positive ticks and small mammals' tissue samples were subjected to virus isolation. The sequencing of the complete polyprotein gene of the positive samples was performed. A total of 227 questing ticks were collected. TBEV was detected in five specimens of Ixodes ricinus. We trapped 44 small mammals, mainly bank voles (Myodes glareolus) and pygmy field mice (Apodemus uralensis). Two samples of brain tissue from bank voles yielded a positive signal in RT-PCR for TBEV. We obtained six virus isolates from the ticks and brain tissue of a bank vole. Complete genome sequencing showed that the obtained isolates belong to the European subtype and have low diversity with sequence identities as high as 99.9%. GPS tracking showed that the maximum distance between the exact locations where the TBEV-positive ticks were collected was 185 m. We assume that the forest park had been free of TBEV and that the virus was recently introduced.

Characterization of tick organic anion transporting polypeptides (OATPs) upon bacterial and viral infections.

BACKGROUND: Ixodes scapularis organic anion transporting polypeptides (OATPs) play important roles in tick-rickettsial pathogen interactions. In this report, we characterized the role of these conserved molecules in ticks infected with either Lyme disease agent Borrelia burgdorferi or tick-borne Langat virus (LGTV), a pathogen closely related to tick-borne encephalitis virus (TBEV). RESULTS: Quantitative real-time polymerase chain reaction analysis revealed no significant changes in oatps gene expression upon infection with B. burgdorferi in unfed ticks. Synchronous infection of unfed nymphal ticks with LGTV in vitro revealed no significant changes in oatps gene expression. However, expression of specific oatps was significantly downregulated upon LGTV infection of tick cells in vitro. Treatment of tick cells with OATP inhibitor significantly reduced LGTV loads, kynurenine amino transferase (kat), a gene involved in the production of tryptophan metabolite xanthurenic acid (XA), levels and expression of several oatps in tick cells. Furthermore, bioinformatics characterization of OATPs from some of the medically important vectors including ticks, mosquitoes and lice revealed the presence of several glycosylation, phosphorylation and myristoylation sites. CONCLUSIONS: This study provides additional evidence on the role of arthropod OATPs in vector-intracellular pathogen interactions.

A survey on murine gammaherpesvirus 68 in ticks collected in Slovakia.

Murine gammaherpesvirus 68 (MHV-68) is a natural pathogen that infects murid rodents which serve as hosts for Dermacentor reticulatus and Ixodes ricinus ticks. For the first time, MHV-68 was detected in immature I. ricinus ticks feeding on lizards trapped in Slovakia. Later on, MHV-68 infection was detected in D. reticulatus and Haemaphysalis concinna ticks collected on vegetation, which supported the idea that ticks can acquire the virus from feeding on infected hosts. Here, we report MHV-68 infection, which was detected by nested PCR, in D. reticulatus and I. ricinus adult ticks and I. ricinus nymphs collected in five geographically isolated localities, in west, southwest, south and central Slovakia. Viral incidence in ticks was 46.7% (121/259) without considering the season, site of collection and tick species and their life stage. MHV-68 infection was detected in all five localities investigated and in both tick species. Here, for the first time, we report MHV-68 infection in I. ricinus nymphs collected from the vegetation. The finding of virus in ticks from five separated localities suggested that ticks became infected with MHV-68 via feeding on infected rodents; thus, this virus might be a newfound natural pathogen in ticks.

Saliva of Ixodes ricinus enhances TBE virus replication in dendritic cells by modulation of pro-survival Akt pathway.

It has been suggested that tick saliva facilitates transmission of tick-borne encephalitis virus (TBEV) to vertebrates. The mechanism of this facilitation has not been elucidated yet. Since dendritic cells (DCs) are among first cells attacked by the virus, we examined the amount of virus and changes induced by saliva in TBEV-infected DCs. We found that virus replication was significantly increased by saliva of Ixodes ricinus tick. Next, saliva-induced enhancement of Akt pathway activation was observed in TBEV-infected DCs. Akt mediated pathway is known for its anti-apoptotic and pro-survival effects. Accordingly, apoptosis of TBEV-infected DCs was declined and cellular viability increased in the presence of tick saliva. Saliva-induced enhancement of STAT1 and NF-κB was also observed in TBEV-infected DCs. In conclusion, we suggest that tick saliva provides pro-survival and anti-apoptotic signals to infected DCs via upregulation of Akt, which may have positive consequences for TBEV replication and transmission.

Tick-Borne Transmission of Murine Gammaherpesvirus 68.

Herpesviruses are a large group of DNA viruses infecting mainly vertebrates. Murine gammaherpesvirus 68 (MHV68) is often used as a model in studies of the pathogenesis of clinically important human gammaherpesviruses such as Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus. This rodent virus appears to be geographically widespread; however, its natural transmission cycle is unknown. Following detection of MHV68 in field-collected ticks, including isolation of the virus from tick salivary glands and ovaries, we investigated whether MHV68 is a tick-borne virus. Uninfected Ixodes ricinus ticks were shown to acquire the virus by feeding on experimentally infected laboratory mice. The virus survived tick molting, and the molted ticks transmitted the virus to uninfected laboratory mice on which they subsequently fed. MHV68 was isolated from the tick salivary glands, consistent with transmission via tick saliva. The virus survived in ticks without loss of infectivity for at least 120 days, and subsequently was transmitted vertically from one tick generation to the next, surviving more than 500 days. Furthermore, the F1 generation (derived from F0 infected females) transmitted MHV68 to uninfected mice on which they fed, with MHV68 M3 gene transcripts detected in blood, lung, and spleen tissue of mice on which F1 nymphs and F1 adults engorged. These experimental data fulfill the transmission criteria that define an arthropod-borne virus (arbovirus), the largest biological group of viruses. Currently, African swine fever virus (ASFV) is the only DNA virus recognized as an arbovirus. Like ASFV, MHV68 showed evidence of pathogenesis in ticks. Previous studies have reported MHV68 in free-living ticks and in mammals commonly infested with I. ricinus, and neutralizing antibodies to MHV68 have been detected in large mammals (e.g., deer) including humans. Further studies are needed to determine if these reports are the result of tick-borne transmission of MHV68 in nature, and whether humans are at risk of infection.

Tick-Borne Encephalitis Virus Structural Proteins Are the Primary Viral Determinants of Non-Viraemic Transmission between Ticks whereas Non-Structural Proteins Affect Cytotoxicity.

Over 50 million humans live in areas of potential exposure to tick-borne encephalitis virus (TBEV). The disease exhibits an estimated 16,000 cases recorded annually over 30 European and Asian countries. Conventionally, TBEV transmission to Ixodes spp. ticks occurs whilst feeding on viraemic animals. However, an alternative mechanism of non-viraemic transmission (NVT) between infected and uninfected ticks co-feeding on the same transmission-competent host, has also been demonstrated. Here, using laboratory-bred I. ricinus ticks, we demonstrate low and high efficiency NVT for TBEV strains Vasilchenko (Vs) and Hypr, respectively. These virus strains share high sequence similarity but are classified as two TBEV subtypes. The Vs strain is a Siberian subtype, naturally associated with I. persulcatus ticks whilst the Hypr strain is a European subtype, transmitted by I. ricinus ticks. In mammalian cell culture (porcine kidney cell line PS), Vs and Hypr induce low and high cytopathic effects (cpe), respectively. Using reverse genetics, we engineered a range of viable Vs/Hypr chimaeric strains, with substituted genes. No significant differences in replication rate were detected between wild-type and chimaeric viruses in cell culture. However, the chimaeric strain Vs[Hypr str] (Hypr structural and Vs non-structural genomic regions) demonstrated high efficiency NVT in I. ricinus whereas the counterpart Hypr[Vs str] was not transmitted by NVT, indicating that the virion structural proteins largely determine TBEV NVT transmission efficiency between ticks. In contrast, in cell culture, the extent of cpe was largely determined by the non-structural region of the TBEV genome. Chimaeras with Hypr non-structural genes were more cytotoxic for PS cells when compared with Vs genome-based chimaeras.

Immune Cell Targets of Infection at the Tick-Skin Interface during Powassan Virus Transmission.

Powassan virus (POWV) is a tick-borne flavivirus that can result in a severe neuroinvasive disease with 50% of survivors displaying long-term neurological sequelae. Human POWV cases have been documented in Canada, the United States, and Russia. Although the number of reported POWV human cases has increased in the past fifteen years, POWV remains one of the less studied human pathogenic flaviviruses. Ixodes ticks are the vectors for POWV, and the virus is transmitted to a host's skin very early during the tick feeding process. Central to the successful transmission of a tick-borne pathogen are complex interactions between the host immune response and early tick-mediated immunomodulation, all of which initially occur at the skin interface. In our prior work, we examined the cutaneous immune gene expression during the early stages of POWV-infected Ixodes scapularis feeding. The present study serves to further investigate the skin interface by identifying early cell targets of infection at the POWV-infected tick feeding site. An in vivo infection model consisting of POWV-infected ticks feeding on mice for short durations was used in this study. Skin biopsies from the tick feeding sites were harvested at various early time points, enabling us to examine the skin histopathology and detect POWV viral antigen in immune cells present at the tick feeding site. The histopathology from the present study demonstrates that neutrophil and mononuclear cell infiltrates are recruited earlier to the feeding site of a POWV-infected tick versus an uninfected tick. This is the first report demonstrating that macrophages and fibroblasts contain POWV antigens, which suggests that they are early cellular targets of infection at the tick feeding site. These data provide key insights towards defining the complex interactions between the host immune response and early tick-mediated immunomodulation.

Expression of a second open reading frame present in the genome of tick-borne encephalitis virus strain Neudoerfl is not detectable in infected cells.

A short upstream open reading frame (uORF) was recently identified in the 5' untranslated region of some tick-borne encephalitis virus (TBEV) strains. However, it is not known if the peptide encoded by TBEV uORF (TuORF) is expressed in infected cells. Here we show that TuORF forms three phylogenetically separated clades which are typical of European, Siberian, and Far-Eastern TBEV subtypes. Analysis of selection pressure acting on the TuORF area showed that it is under positive selection pressure. Theoretically, TuORF may code for a short hydrophobic peptide embedded in a biological membrane. However, expression of TuORF was detectable neither by immunoblotting in tick and mammalian cell lines infected with TBEV nor by immunofluorescence in TBEV-infected mammalian cell lines. These results support the idea that TuORF is not expressed in TBEV-infected cell or expressed in undetectably low concentrations. Therefore we can assume that TuORF has either minor or no biological role in the TBEV life cycle.

Nuclease Tudor-SN Is Involved in Tick dsRNA-Mediated RNA Interference and Feeding but Not in Defense against Flaviviral or Anaplasma phagocytophilum Rickettsial Infection.

Tudor staphylococcal nuclease (Tudor-SN) and Argonaute (Ago) are conserved components of the basic RNA interference (RNAi) machinery with a variety of functions including immune response and gene regulation. The RNAi machinery has been characterized in tick vectors of human and animal diseases but information is not available on the role of Tudor-SN in tick RNAi and other cellular processes. Our hypothesis is that tick Tudor-SN is part of the RNAi machinery and may be involved in innate immune response and other cellular processes. To address this hypothesis, Ixodes scapularis and I. ricinus ticks and/or cell lines were used to annotate and characterize the role of Tudor-SN in dsRNA-mediated RNAi, immune response to infection with the rickettsia Anaplasma phagocytophilum and the flaviviruses TBEV or LGTV and tick feeding. The results showed that Tudor-SN is conserved in ticks and involved in dsRNA-mediated RNAi and tick feeding but not in defense against infection with the examined viral and rickettsial pathogens. The effect of Tudor-SN gene knockdown on tick feeding could be due to down-regulation of genes that are required for protein processing and blood digestion through a mechanism that may involve selective degradation of dsRNAs enriched in G:U pairs that form as a result of adenosine-to-inosine RNA editing. These results demonstrated that Tudor-SN plays a role in tick RNAi pathway and feeding but no strong evidence for a role in innate immune responses to pathogen infection was found.

[Taxonomic status of the Chim virus (CHIMV) (Bunyaviridae, Nairovirus, Qalyub group) isolated from the Ixodidae and Argasidae ticks collected in the great gerbil (Rhombomys opimus Lichtenstein, 1823) (Muridae, Gerbillinae) burrows in Uzbekistan and Kazakhstan].

Full-length genome of the Chim virus (CHIMV) (strain LEIV-858Uz) was sequenced using the next-generation sequencing approach (ID GenBank: KF801656). The CHIMV/LEIV-858Uz was isolated from the Ornithodoros tartakovskyi Olenev, 1931 ticks collected in the great gerbil (Rhombomys opimus Lichtenstein, 1823) burrow in Uzbekistan near Chim town (Kashkadarinsky region) in July of 1971. Later, four more CHIMV strains were isolated from the O. tartakovskyi, O. papillipes Birula, 1895, Rhipicephalus turanicus Pomerantsev, 1936 collected in the great gerbil burrows in Kashkadarinsky, Bukhara, and Syrdarya regions of Uzbekistan, and three strains--from the Hyalomma asiaticum Schulze et Schlottke, 1930 from the great gerbil burrows in Dzheskazgan region of Kazakhstan. The virus is a potential pathogen of humans and camels. The phylogenetic analysis revealed that the CHIMV is a novel member of the Nairovirus genus (Bunyaviridae) and closely related to the Qalyub virus (QYBV), which is prototype for the group of the same name. The amino acid homology between the CHIMV and QYBV is 87% for the RdRp catalytic center (L-segment) that is coincident with both QYBV and CHIMV associated with the Ornithodoros ticks and burrow of rodents as well. The CHIMV homologies with other nairoviruses are 30-40% for the amino acid sequences of precursor polyprotein GnGc (M-segment), whereas 50%--for the nucleocapsid N (S-segment). The data obtained permit to classify the CHIMV as a member of the QYBV group in the genus of Nairovirus (Bunyaviridae).

Prevalence and phylogenetic analysis of tick-borne encephalitis virus (TBEV) in field-collected ticks (Ixodes ricinus) in southern Switzerland.

BACKGROUND: Tick-borne encephalitis is the most common tick-borne viral infection in Europe with 3,000 human cases reported each year. In Western Europe, the castor bean tick, Ixodes ricinus, is the principal vector of the tick-borne encephalitis virus (TBEV). TBEV appears to be spreading geographically and was recently detected for the first time in Canton Valais in the southern part of Switzerland. The purpose of the present study was to survey the I. ricinus tick populations of Canton Valais for TBEV. METHODS: We collected a total of 19,331 I. ricinus ticks at 45 different sites in Canton Valais between 2010 and 2013. Ticks were processed in pools and tested for TBEV using reverse transcription quantitative PCR. The NS5 gene and the envelope gene of the TBEV isolates were partially sequenced for phylogenetic analysis. RESULTS: TBEV was detected in tick populations at six of the 45 sites. These six sites were all located in a 33 km transect along the Rhône River. TBEV was detected in two sites for three of the four years of the study showing the temporal persistence of the pathogen. Prevalence of TBEV in the six positive sites ranged from 0.16% to 11.11%. Phylogenetic analysis found that all TBEV isolates from Canton Valais belonged to the European subtype. Genetic analysis found two distinct lineages of TBEV suggesting that Canton Valais experienced two independent colonization events. CONCLUSIONS: TBEV appears to be well established at certain locations in Canton Valais.

[Genetic characterization of the Zaliv Terpeniya virus (ZTV, Bunyaviridae, Phlebovirus, Uukuniemi serogroup) strains isolated from the ticks Ixodes (Ceratixodes) uriae White, 1852, obligate parasites of the Alcidae birds, in high latitudes of Northern Eurasia and the mosquitoes Culex modestus Ficalbi, 1889, in subtropics Transcaucasus].

Complete genome sequences were obtained for the LEIV-13841Ka (ID GenBank KF767463-65) and LEIV-279Az (ID GenBank KF767460-62) virus strains, which were classified as different strains of the Zaliv Terpeniya virus (ZTV). LEIV-13841Ka was isolated from the ticks Ixodes (Ceratixodes) uriae White, 1852 collected on Ariy Kamen (Commander Islands) in 1986. LEIV-279Az was isolated from the mosquitoes Culex modestus Ficalbi, 1889, collected in heron colony (Ardea Linnaeus, 1758) in Azerbaijan (1969) and was initially identified as Uukuniemi virus (UUKV). According to the results obtained LEIV-279Az is ZTV strain as well. LEIV-13841Ka and LEIV-279Az RdRp sequences have high level of homology (99%) with previously sequenced ZTV/LEIV-271Ka. The L-segment nucleotide sequences are homological with ZTV/LEIV-271Ka on the level of 94% and 98% for LEIV-13841Ka and LEIV-279Az, respectively; M-segment--89% and 88%, respectively. Such homologies for the amino acid sequences of Gn/Gc polyprotein are 98.3% and 97.7%. NP proteins of ZTV/LEIV-13841Ka and LEIV-279Az have 88.7% and 84.6% homologies with ZTV/LEIV-271Ka both for amino acid and nucleotide sequences, respectively. Thus, for the very first time we demonstrated ZTV strain isolated from mosquitoes in subtropical Transcaucasia zone. Obtained results permit to expand suggested areal of ZTV and to fill up data upon the ecology of the Uukuniemi virus group.

Clusteron structure of tick-borne encephalitis virus populations.

Tick-borne encephalitis is a natural focal transmissible zooanthroponosis. The causative agent of the disease is a tick-borne encephalitis virus (TBEV) belonging to the genus Flavivirus of the family Flaviviridae and is widespread in Eurasia. Current TBEV classification based on molecular genetic data comprises three phylogenetically separate subtypes: Far Eastern, European and Siberian (TBEV-Sib). Further differentiation of TBEV isn't developed, making it difficult to investigate the origins, distribution and evolution of the virus. In the present study we determined the nucleotide sequence of the gene E fragment for 282 TBEV-Sib isolates from Ixodes persulcatus ticks or their pools from various natural foci in Russia. Analysis of these sequences and sequences obtained from the GenBank database (more than 600), made it possible to cluster TBEV-Sib strains by identical amino acid sequences of a glycoprotein E fragment. In total, 18 groups were identified (from 3 to 285 strains in the group). It was shown that TBEV strains belonging to the same group are phylogenetically related and have a territorial attachment showing either a local or a corridor type distribution. These groups were named as clusterons showed to be the smallest unit of TBEV classification. The grouping of TBEV strains allows characterization of endemic areas both in quantitative and qualitative composition of the clusterons. The approach could be successfully used to record and monitor the TBEV populations.

[The taxonomy of the Baku virus (BAKV; Reoviridae, Orbivirus) isolated from the birds obligate parasites Argasidae ticks in Azerbaijan, Turkmenistan, and Uzbekistan].

The Baku virus (BAKV) was originally isolated from the ticks Ornithodoros capensis Neumann, 1901 (Acari: Argasidae) collected from the seagull (Larus argentatus) seating nests on the islands of the Baku archipelago, the Caspian sea. BAKV was assigned to Kemerovo group (KEMV) (Orbivirus, Reoviridae). The BAKV was frequently isolated from the ticks O. coniceps Canestrini, 1980, collected from L. argentatus and tern (Sterna hirundo) nests in Turkmenia and pigeon (Columba livia neglecta) nests in Uzbekistan. In this work, the genome of the BAKV was sequenced using the next-generation sequencing technology. The BAKV Pol protein has 48.6% identity level with the viruses of the Great Island Virus group and at average 41% with non-tick orbiviruses. The BAKV T2 protein level identity with the orbiviruses ranges from 23.7% to 64.8%. The maximum identity level of the T2 protein (64.8%) is observed for the tick-borne viruses of the GIV (KEMV) group. According to the conducted molecular-genetic and phylogenetic analysis, the BAKV is a novel species of the genus Orbivirus. It forms a phylogenetic group distinctly related to the GIV group.

[Polytypic strains in the genofund of tick-borne encephalitis virus].

Eighteen polytypic tick-borne encephalitis virus (TBEV) strains containing the fragments of E and NS1 protein genes of Siberian and Far Eastern, occasionally Siberian and European subtypes were isolated in the European and Asian parts of the tick-borne encephalitis (TBE) area. They were identified using real-time polymerase chain reaction, hybridization-fluorescence detection with genotype-specific probes, restriction fragment length polymorphism analysis, and E protein sequencing. The polytypic strains were isolated from individual Ixodes persulcatus ticks, their pools, from the blood of patients and the brain of dead patients. The isolation rates of the polytypic strains in the sympathry area of different TBEV subtypes ranged from 4.4% (the Irkutsk Region) to 15.1% (the Yaroslavl Region). In addition to 2 polytypic strains, a strain similar to the TBEV 886-84 strain was isolated. The TBEV subtypes entering into the composition of the polytypic strains show nongenetic interactions, such as neutral replication or competition. The polytypic strains are stable during passages in the cultured pig embryo kidney epithelial cells and on cloning. Mouse brain passage promotes dissociation of polytypic strains. The conditions for the formation of polytypic strains and their role in the etiology of TBE are discussed.