Artem Tikunov

Evolutionary time-scale of primate bocaviruses

Human bocavirus (HBoV) is associated with acute gastroenteritis in humans, occurring mostly in young children and elderly people. Four bocavirus genotypes (HBoV1-HBoV4) have been found so far. Since there were no data on the contribution of HBoV to gastroenteritis in Russia, 1781 fecal samples collected from infants hospitalized with acute gastroenteritis in Novosibirsk, Russia during one year were tested for the presence of nucleic acids from HBoV and three major gastrointestinal viruses (rotavirus A, norovirus II, and astrovirus). HBoV was detected only in 1.9% of the samples: HBoV1 was detected in 0.6% and HBoV2, in 1.3%. Complete genome sequencing of three Novosibirsk isolates was performed. An evolutionary analysis of these sequences and the available sequences of human and great apes bocaviruses demonstrated that the current HBoV genotypes diverged comparatively recently, about 60-300years ago. The independent evolution of bocaviruses from chimpanzees and gorillas commenced at the same time period. This suggests that these isolates of great apes bocaviruses belong to separate genotypes within the species of human bocavirus, which is actually the primate bocavirus. The rate of mutation accumulation in the genome of primate bocaviruses has been estimated as approximately 9×10(-4)substitutions/site/year. It has been demonstrated that HBoV1 diverged from the ancestor common with chimpanzee bocavirus approximately 60-80years ago, while HBoV4 separated from great apes bocaviruses about 200-300years ago. The hypothesis postulating independent evolution of HBoV1 and HBoV4 genotypes from primate bocaviruses has been proposed.

Molecular epidemiology of noroviruses associated with sporadic gastroenteritis in children in Novosibirsk, Russia, 2003-2012.

Noroviruses (NoVs) are an important cause of acute gastroenteritis worldwide. To monitor the molecular epidemiology of NoVs genogroup II (GII) in Novosibirsk, Russia, a total of 10,198 stool samples from young children hospitalized with acute gastroenteritis and two asymptomatic comparison groups were collected from 2003 to 2012. All samples were screened for the presence of NoV GII, rotavirus, and astrovirus by RT‐PCR. The prevalence of NoV in gastroenteritis cases was 13.1%, varying from 7.1% to 21.3% in different seasons. Rotavirus and/or astrovirus were detectable in 25% of the NoV‐positive samples. NoV was detected throughout the year with a seasonal increase during winter months. Based on sequence analysis of regions D and/or C within the VP1 gene, 892 identified NoV strains were divided into nine genotypes—GII.3 (51%), GII.4 (44%), GII.6 (2%), as well as GII.1, GII.2, GII.5, GII.7, GII.16, and GII.21 (totally, 3%). The prevalence of NoV in the comparison groups was considerably lower (∼2.5%); only GII.4 (n = 6), GII.21 (n = 2) and GII.1 (n = 1) genotypes were revealed. Based on phylogenetic analysis of the ORF1/ORF2 junction region sequences, GII.P21/GII.3 recombinant and GII.P4/GII.4 were prevalent genotypes (totally, 93%) and their ratio changed every season. The median age of children with NoV infection was 6.6 months (range, <1–35 months), but it was different depending on NoV genotype. Children infected with the NoV GII.3 were younger (median 6.2 months) than GII.4‐positive patients (median 9.1 months). This is the first long‐term systematic study of NoV molecular epidemiology in Russia. J. Med. Virol. 87:740–753, 2015.

High evolutionary rate of human astrovirus.

Human astrovirus is one of the etiological agents of acute gastroenteritis in humans, mostly in young children and elderly people. Complete genome sequencing of four human astrovirus strains isolated in Novosibirsk, Russia was performed. Analysis of these sequences and the sequences available in GenBank database has detected numerous potential recombination breakpoints. For the first time the rate of human astrovirus evolution was estimated based on the genome fragments without recombination breakpoints; the determined rate is typical of the RNA viruses with high evolutionary rate, amounting to approximately 3.7 × 10(-3) nucleotide substitutions per site per year, and for the synonymous changes, 2.8 × 10(-3) nucleotide substitutions per site per year.

A study of the human bocavirus replicative genome structures.

The complete genomes of two human bocavirus 4 (HBoV4) isolates recovered in 2011 in Novosibirsk, Russia have been determined. A set of primers was designed based on the determined and previously published HBoV sequences; this primer pair was able to detect all possible HBoV replicative intermediates. This primer set was used to assay all HBoV genotypes and detected only those structures that correspond to an episomal form of this viral genome. Also, for the first time, head-to-tail nucleotide sequences have been determined for HBoV4. Secondary structures of the terminal noncoding regions (NCRs) of episomal forms have been computed for all HBoV genotypes, as well as for the canine bocavirus. Conserved secondary structures in episomal NCRs, which are likely to play an important part in the replication of bocaviruses, were found. NCR heterogeneity in the genomes of individual HBoV isolates has been shown for the first time.

Genetic variability of Rickettsia spp. in Ixodes persulcatus/Ixodes trianguliceps sympatric areas from Western Siberia, Russia: Identification of a new Candidatus Rickettsia species

Rickettsia spp. are the causative agents of a number of diseases in humans. These bacteria are transmitted by arthropods, including ixodid ticks. DNA of several Rickettsia spp. was identified in Ixodes persulcatus ticks, however, the association of Ixodes trianguliceps ticks with Rickettsia spp. is unknown. In our study, blood samples of small mammals (n=108), unfed adult I. persulcatus ticks (n=136), and I. persulcatus (n=12) and I. trianguliceps (n=34) ticks feeding on voles were collected in two I. persulcatus/I. trianguliceps sympatric areas in Western Siberia. Using nested PCR, ticks and blood samples were studied for the presence of Rickettsia spp. Three distinct Rickettsia species were found in ticks, but no Rickettsia species were found in the blood of examined voles. Candidatus Rickettsia tarasevichiae DNA was detected in 89.7% of unfed I. persulcatus, 91.7% of engorged I. persulcatus and 14.7% of I. trianguliceps ticks. Rickettsia helvetica DNA was detected in 5.9% of I. trianguliceps ticks. In addition, a new Rickettsia genetic variant was found in 32.4% of I. trianguliceps ticks. Sequence analysis of the 16S rRNA, gltA, ompA, оmpB and sca4 genes was performed and, in accordance with genetic criteria, a new Rickettsia genetic variant was classified as a new Candidatus Rickettsia species. We propose to name this species Candidatus Rickettsia uralica, according to the territory where this species was initially identified. Candidatus Rickettsia uralica was found to belong to the spotted fever group. The data obtained in this study leads us to propose that Candidatus Rickettsia uralica is associated with I. trianguliceps ticks.

Recombination in the evolution of human bocavirus

Whole genome sequencing of Novosibirsk human bocavirus (HBoV) isolates has detected an isolate that emerged via recombination between HBoV3 and HBoV4 genotypes. The recombination site is located between regions with abnormally low and abnormally high GC contents in the genome. This site is a bocavirus recombination hotspot and coincides with one of two parvovirus recombination hotspots. The Novosibirsk recombinant isolate, which is similar to a previously studied isolate from Thailand, utilizes the strategy of borrowing ORF3, which encodes structural proteins, of a rare genotype HBoV4. The role of recombination in HBoV evolution is discussed.

Recombination analysis based on the HAstV-2 and HAstV-4 complete genomes.

Complete genome sequences of previously unstudied human astrovirus subgenotypes - HAstV-2a and HAstV-2c - and two isolates of a rare genotype HAstV-4 have been determined. These isolates were recovered from fecal samples of young children hospitalized with acute intestinal infections in Novosibirsk (Russia). Three of the four sequenced isolates (HAstV-2a, HAstV-2c, and HAstV-4) are recombinants. It has been shown that all known HAstV-2 genomes have emerged via recombination; the HAstV-1 and HAstV-4 genotypes contain both recombinant and non-recombinant isolates; and all HAstV-3, HAstV-5, and HAstV-6 whole-genome sequences display no reliable signs of recombination. The average mutation accumulation rate has been determined based on an extended ORF2 fragment and amounts to 1.0×10(-3) substitutions per site per year. The evolutionary chronology of current HAstV genotypes has been reconstructed.

Detection and genetic characterization of a wide range of infectious agents in Ixodes pavlovskyi ticks in Western Siberia, Russia

BackgroundThe Ixodes pavlovskyi tick species, a member of the I. persulcatus/I. ricinus group, was discovered in the middle of the 20th century in the Russian Far East. Limited data have been reported on the detection of infectious agents in this tick species. The aim of this study was to investigate the prevalence and genetic variability of a wide range of infectious agents in I. pavlovskyi ticks collected in their traditional and recently invaded habitats, the Altai Mountains and Novosibirsk Province, respectively, which are both located within the Western Siberian part of the I. pavlovskyi distribution area.ResultsThis study reports the novel discovery of Borrelia bavariensis, Rickettsia helvetica, R. heilongjiangensis, R. raoultii, “Candidatus Rickettsia tarasevichiae”, Anaplasma phagocytophilum, Ehrlichia muris, “Candidatus Neoehrlichia mikurensis” and Babesia microti in I. pavlovskyi ticks. In addition, we confirmed the previous identification of B. afzelii, B. garinii and B. miyamotoi, as well as tick-borne encephalitis and Kemerovo viruses in this tick species. The prevalence and some genetic characteristics of all of the tested agents were compared with those found in I. persulcatus ticks that were collected at the same time in the same locations, where these tick species occur in sympatry. It was shown that the prevalence and genotypes of many of the identified pathogens did not significantly differ between I. pavlovskyi and I. persulcatus ticks. However, I. pavlovskyi ticks were significantly more often infected by B. garinii and less often by B. bavariensis, B. afzelii, “Ca. R. tarasevichiae”, and E. muris than I. persulcatus ticks in both studied regions. Moreover, new genetic variants of B. burgdorferi (sensu lato) and Rickettsia spp. as well as tick-borne encephalitis and Kemerovo viruses were found in both I. pavlovskyi and I. persulcatus ticks.ConclusionAlmost all pathogens that were previously detected in I. persulcatus ticks were identified in I. pavlovskyi ticks; however, the distribution of species belonging to the B. burgdorferi (sensu lato) complex, the genus Rickettsia, and the family Anaplasmataceae was different between the two tick species. Several new genetic variants of viral and bacterial agents were identified in I. pavlovskyi and I. persulcatus ticks.

First genetic characterization of rotavirus C in Russia.

Rotaviruses C (RVC) cause sporadic cases and outbreaks of diarrhea in humans and animals worldwide. The aim of this study was to monitor RVC during a surveillance study of sporadic cases of viral gastroenteritis in the Novosibirsk and Omsk regions of Russia from 2006 to 2011. A total of 2144 stool samples from children and adults hospitalized with acute gastroenteritis were tested for RVC by RT-PCR. Sixteen RVC-positive stool samples were detected at a rate of 0.6% (13/2037) in children and 2.8% (3/107) in adults. The low detection rate suggested that RVC infection was an uncommon cause of hospitalization in Russia. The complete VP7, VP4, VP6, and NSP4 gene sequences were determined. It was found that RVCs with at least two different genome backgrounds circulated in Siberia. VP4, VP6, and NSP4 gene sequences of most Russian RVC strains clustered with South Asian strains, while the VP7 gene showed a closer relationship to European strains. Meanwhile, only VP4 and NSP4 sequences of the strain Omsk08-386 clustered with South Asian strains, while its VP6 and VP7 sequences clustered with European strains. This is the first genetic characterization of Russian RVC strains and the first report on the prevalence of RVC in the Asian part of Russia.

Erratum to: Genetic diversity of Ixodes pavlovskyi and I. persulcatus (Acari: Ixodidae) from the sympatric zone in the south of Western Siberia and Kazakhstan

The most epidemiologically significant tick species in Siberia involved in transmission of a large number of pathogens causing human infectious diseases is Ixodes persulcatus. Ixodes pavlovskyi, being more active, also poses epidemiological threats. These tick species share morphology, activity seasons and geographic distribution range. In this paper, we characterize the geographic and genetic structures of I. persulcatus and I. pavlovskyi populations inhabiting the southern part of Western Siberia (Russia and Kazakhstan)—the western part of I. pavlovskyi distribution range. The data are based on six distinct Ixodes tick populations. Analysis of the concatenated mitochondrial marker sequences (16S rRNA and COI) and the nuclear sequence (ITS2) showed genetic polymorphisms in both I. persulcatus and I. pavlovskyi ticks inhabiting the sympatric zone. We could not determine the phylogeographic structure of I. pavlovskyi populations whereas for I. persulcatus significant within-region variance was shown. Notably, the abundance of I. persulcatus ticks negatively correlates with nucleotide and haplotype diversity in the concatenated sequence of mitochondrial gene (16S rRNA and COI) fragments. This is the first description of the genetic polymorphism of I. persulcatus and I. pavlovskyi ticks coexisting in a sympatric zone based on analysis of mitochondrial and nuclear markers.