Kama virus (KAMV) is an atypical representative of the seabird tick-borne flaviviruses.

According to modern classification, tick-borne flaviviruses have been divided into a mammalian tick-borne virus group and a seabird tick-borne virus group (STBVG). The STBVG includes the Tyuleniy virus, Meaban virus, Saumarez Reef virus, and the recently discovered Kama virus (KAMV). The latter was isolated from Ixodes lividus, an obligate parasitic tick of the sand martin (Riparia riparia), in 1989 in the central part of the Russian Plain. In 2014, based on molecular genetic analysis, it was shown that KAMV is a new virus belonging to STBVG, genus Flavivirus, fam. Flaviviridae. Very little is known about the Kama virus concerning its range, vectors, and reservoir hosts. GenBank contains a single sequence of the complete genome of this virus. In the present study, the complete genome sequences of two strains, isolated in 1983 in the Omsk region (Western Siberia) from gamasid mites in the nests of rooks (Corvus frugilegus), have been determined. Phylogenetic analyses of their genomes showed a close relationship both with each other (approx. 98.9% nucleotide identity) and with KAMV isolated in European Russia (approx. 98.4% nucleotide identity). The ecological features of KAMV that are due to the species of the vector (gamasid mites) and its hosts (colonial birds of the mainland of Eurasia) indicate that KAMV is an atypical representative STBVG.

Molecular variability and genetic structure of Omsk hemorrhagic fever virus, based on analysis of the complete genome sequences.

Omsk hemorrhagic fever virus (OHFV) is the etiological agent of Omsk hemorrhagic fever, a disease described in the 1940s in Western Siberia. However, until now, it has been represented in GenBank by just four complete genome sequences, which do not reflect the real genetic diversity of the virus in nature. In this study, we analyzed the molecular variability and genetic structure of OHFV based on 20 complete genome sequences, fifteen of which were obtained for the first time. All these sequences belong to virus strains isolated at different times from three regions of Western Siberia. The results suggest that the genetic diversity of OHFV is significantly wider than previously thought and is represented by at least three subtypes, rather than two. This broadens our understanding of the evolutionary history of OHFV. Also, it is argued that the OHFV reference strain Bogoluvovska (NC_005062) is actually a Kubrin strain and that either cross-contamination or a laboratory error was the cause of this.

Molecular features of Ixodes kazakstani: first results.

Ticks of the Ixodes ricinus group are important vectors of human pathogens in both Eurasia and North America; therefore, many studies have focused on their molecular systematics and evolutionary relationships. However, there are species that have not been characterized by molecular genetic methods so far. For the first time, we obtained nucleotide sequences of two nuclear and three mitochondrial genetic markers from four museum specimen of I. kazakstani Olenev et Sorokoumov, 1934, collected in Kyrgyzstan. The phylogenetic analysis showed that I. kazakstani undoubtedly belongs to the I. ricinus group but is not closely related to I. persulcatus, as was expected. Further studies of the genetic features of I. kazakstani, presumably an intermediate between Nearctic and Palearctic species, would elucidate the evolutionary relationships between tick species within the I. ricinus group.

Reconsidering the classification of tick-borne encephalitis virus within the Siberian subtype gives new insights into its evolutionary history.

Tick-borne encephalitis is widespread in Eurasia and transmitted by Ixodes ticks. Classification of its causative agent, tick-borne encephalitis virus (TBEV), includes three subtypes, namely Far-Eastern, European, and Siberian (TBEV-Sib), as well as a group of 886-84-like strains with uncertain taxonomic status. TBEV-Sib is subdivided into three phylogenetic lineages: Baltic, Asian, and South-Siberian. A reason to reconsider TBEV-Sib classification was the analysis of 186 nucleotide sequences of an E gene fragment submitted to GenBank during the last two years. Within the South-Siberian lineage, we have identified a distinct group with prototype strains Aina and Vasilchenko as an individual lineage named East-Siberian. The analysis of reclassified lineages has promoted a new model of the evolutionary history of TBEV-Sib lineages and TBEV-Sib as a whole. Moreover, we present arguments supporting separation of 886-84-like strains into an individual TBEV subtype, which we propose to name Baikalian (TBEV-Bkl).

Baltic lineage of tick-borne encephalitis virus: A putative evolutionary pathway.

Tick-borne encephalitis virus (TBEV) is transmitted by ixodid ticks and has three subtypes. The most genetically heterogeneous and widely distributed is the Siberian subtype which is subdivided into two main phylogenetic lineages, Asian (TBEV-SibAsia) and Baltic (TBEV-SibBaltic). According to the hypothesis of quantum evolution of TBEV (Kovalev et al., 2014b), TBEV-SibAsia originated about 370 years ago in Siberia, but the question concerning the time and place of origin of TBEV-SibBaltic is still to be solved. In the present paper, the sequences of a gene E fragment of 20 newly obtained TBEV-SibBaltic strains and 164 sequences of TBEV-SibBaltic from GenBank were analysed. The clusteron approach applied to TBEV-SibBaltic allowed the identification of three new clusterons. We revealed three clades of TBEV-SibBaltic, each characterized by a certain geographical distribution, and estimated their evolutionary ages. The oldest clade was Balt I, which presumably originated in North-West Russia and the Baltic countries about 300 years ago as a result of human activity, and then gave rise to the other clades in the Urals and West Siberia. The European subtype of TBEV and TBEV-SibBaltic may have originated simultaneously from the clusteron-founder 3A of TBEV-SibAsia, the former through the adaptation of the virus to Ixodes ricinus, the latter - to a European subpopulation of Ixodes persulcatus. The use of the clusteron approach complemented with the results of phylogenetic analysis, data on the geographical distribution of the virus, the population structure of ticks, and the historical evidence allow us to estimate evolutionary pathways of the subtypes and phylogenetic lineages of TBEV.

[UNIFICATION OF THE MOLECULAR EPIDEMIOLOGICAL RESEARCH OF THE TICK-BORNE ENCEPHALITIS].

Molecular genetic techniques and approaches in epidemiological studies were breakthrough in the understanding of the laws, ways, and mechanisms of the spread of the pathogens. However, lack of standard methods makes it difficult to compare results obtained by different scientific groups. In this work we propose to choose one fragment of the TBEV genome as a genetic marker whose sequencing would be both obligatory and sufficient for the molecular epidemiological studies. The best candidate for this purpose may be a fragment of the gene E of 454 nucleotides in length. The deduced amino acid sequence of this fragment was a basis for a new approach for the TBEV differentiation with clusteron being a structural unit (Kovalev and Mukhacheva, 2013). The clusteron approach was proved to be informative for studying the genetic structure of the TBEV-Sib population in the Middle Urals. TBE foci were shown to be unique in both quantitative and qualitative composition of the clusterons. The greatest clusteron diversity in the south of the Middle Urals, through the Trans-Siberian way, may reflect the history of the colonization, closely associated with the roads between Siberia and the European part of Russia. The age of three clusterons did not exceed 50 years, which may indicate an ongoing evolutionary process taking place in the TBEV-Sib populations. In turn, their spatial distribution indicates the crucial role of human factors in the spread of the TBEV (Kovalev & Mukhacheva, 2014). The clusteron approach provides formalization of ideas about the structure of the viral populations and could be used not only by researchers but also by epidemiological surveillance services. Unification of the studies of the TBEV on the basis of a standard genetic marker would consolidate the efforts of researchers from different regions of Russia and other countries.

Natural hybridization between Ixodes ricinus and Ixodes persulcatus ticks evidenced by molecular genetics methods.

The recently shown phenomenon of natural hybridization between Ixodes persulcatus and Ixodes pavlovskyi ticks (Kovalev et al., 2015) stimulated similar studies in the sympatric zones of other tick species. In the present paper, 265 Ixodes ricinus and I. persulcatus ticks from Estonia were subjected to a search for interspecific hybrids based on nuclear (ITS2) and mitochondrial (cox1) markers as well as morphological features. Surprisingly, only 72.1% of ticks morphologically identified as I. ricinus actually were I. ricinus both at nuclear and mitochondrial markers, while the accuracy of morphological species identification for I. persulcatus was 99.3%. Among ticks morphologically identified as I. ricinus, 24.6% turned out to be interspecific hybrids and 3.3% were I. persulcatus. Generally, about 11% of the individuals studied were shown to be interspecific hybrids with different levels of nuclear DNA introgression. The analysis of hybrid populations proved the mating pair female I. ricinus×male I. persulcatus to form hybrids more efficiently, then female I. persulcatus×male I. ricinus. The same trend can be observed for backcrosses preferentially mating with I. ricinus. Hybridization between I. ricinus and I. persulcatus proved the existing view about their reproductive isolation to be untenable. Interspecific hybridization occurring between both closely (I. persulcatus and I. pavlovskyi) and more distantly (I. ricinus and I. persulcatus) related Ixodes species could introduce novel alleles that modify vector competence, host use or the ability to exploit diverse microhabitats.

Borrelia spirochetes in Russia: Genospecies differentiation by real-time PCR.

Spirochetes of the Borrelia burgdorferi sensu lato complex are the causative agent of Lyme borreliosis which is widespread in Russia. Nowadays, three clinically important B. burgdorferi s.l. genospecies, B. afzelii, B. garinii, B. bavariensis sp. nov., can be found in Russia, as well as B. miyamotoi, which belongs to the tick-borne relapsing fever group of spirochetes. Several techniques have been developed to differentiate Borrelia genospecies. However, most of them do not allow detection of all of these genospecies simultaneously. Also, no method based on the RT-PCR TaqMan approach has been proposed to differentiate the genetically closely related species B. bavariensis and B. garinii. In the present paper, we investigated two species of ticks, I. persulcatus and I. pavlovskyi (1343 and 92 adults, respectively). Two sets of primers and probes for RT-PCR, with uvrA, glpQ and nifS genes as targets, were designed to detect four Borrelia genospecies in positive samples. The average prevalence of Borrelia sp. was about 40%, with B. afzelii as the most prevalent genospecies. Mixed infections of B. bavariensis and B. garinii were found to be extremely rare. While B. bavariensis was predominant in I. persulcatus, I. pavlovskyi ticks were infected exclusively by B. garinii. The proposed technique proved to be efficient in selection of individual Borrelia species for further genetic analysis, in particular, for multilocus sequence typing. Also, it could be applied for the differentiation of Borrelia genospecies in clinical material.

Tick-borne encephalitis virus: reference strain Sofjin and problem of its authenticity.

The Sofjin strain is one of the first isolates of tick-borne encephalitis virus and, due to its wide distribution in virus collections, it has become the reference strain. Until now, GenBank has recorded several sequences associated with the Sofjin strain that have significant differences between each other. We have sequenced the complete genome of the Sofjin strain from a virus collection and a genome fragment of the two vaccine Sofjin strains. According to phylogenetic analysis, we concluded that the GenBank sequences belong to three independent groups of Sofjin strains of the Far Eastern subtype. Their genetic differences are not a result of microevolution associated with numerous passages. Retrospective analysis of the peculiarities of origin and distribution for each of these groups showed the authenticity of one of them. For the first time, we have determined a complete genome sequence of the authentic reference TBEV strain Sofjin. Two other groups of strains named Sofjin were probably the result of cross-contamination or laboratory error. The high probability of contamination requires the introduction of a new standard for virological laboratories, the key point of which is the obligatory genetic identification of all collection strains.

Distribution of Far-Eastern tick-borne encephalitis virus subtype strains in the former Soviet Union.

European and Asian viruses within the tick-borne encephalitis flavivirus complex are known to show temporal, spatial and phylogenetic relationships that imply a clinal pattern of evolution. However, the isolation of recognized Far-Eastern tick-borne encephalitis virus (TBEV) strains in the European region of the former Soviet Union (SU), i.e. thousands of kilometres west of the region in which they are considered endemic, appears to contradict this concept. Here, we present a parsimonious explanation for this apparent anomaly based on analysis of the dates and regions in which these non-endemic strains were isolated, together with their phylogenetic relationships and the records of redistribution of animals under the All-Union programme for acclimatization of game animals within the former SU. Our evidence supports the concept that the anomalous distribution of Far-Eastern TBEV strains in Europe and Siberia arose primarily as the result of the large-scale westward redistribution of game animals for economic purposes.

Genetic analysis of mycobacterium tuberculosis strains isolated in Ural region, Russian Federation, by MIRU-VNTR genotyping.

SETTING: The Ural region in Russia is one of the areas most affected by a high incidence of tuberculosis (TB). Molecular epidemiological studies able to trace Mycobacterium tuberculosis transmission are of particular significance. OBJECTIVE: To characterize the population of M. tuberculosis strains circulating in the Ural region, to detect the predominant genotypes and to evaluate their phylogenetic relationship and epidemiological significance. DESIGN: Ninety-two M. tuberculosis clinical samples originating from the Ural region were genotyped using the MIRU-VNTR method. RESULTS: Two major phylogenetically distinct groups of isolates were identified: the W-Beijing family (54.3%) and a previously unreported cluster, named the Ural group (15.2%). Forty-seven different MIRU profiles were identified, including 38 unique (41.3%) and 54 isolates grouped into nine clusters (from 2 to 28 isolates in each cluster). Genetic diversity within the clusters was shown by additional sub-typing of M. tuberculosis isolates in nine additional QUB-VNTR loci. CONCLUSION: W-Beijing family isolates are associated with multiresistance to anti-tuberculosis drugs. It is possible that the strains of this family play a significant role in the spread of multidrug-resistant TB over the Ural region.