The structure and behavioral patterns of the human population affected by ixodid tick bites in Irkutsk Region, Eastern Siberia, Russia.

The bites of hard ticks are the major route of transmission of tick-borne infections to humans, causing thousands of cases of diseases worldwide. However, the characteristics of the human population that is exposed to tick bites are still understudied. This work is aimed at characterizing both the structure of the population directly contacting ticks and the human behavioral features associated with tick bites. We studied 25,970 individuals who sought medical help after a tick bite at the Centre for Diagnostics and Prevention of Tick-borne Infections (CDPTBI) in Irkutsk City (Russian Federation). The demographic and behavioral characteristics of the human population were analyzed using z-tests for proportions, the Mann-Whitney U test, and the Spearman rank correlation coefficient. The majority of bitten people were urban residents (70 %), and most of them were either of active ages between 30 and 74 years old (62 %), or children between 0 and 9 years old (approximately 20%). Tick bites occurred mostly in the range of 150 km around the location of the diagnostic facility (83 %). In comparison to the general population, significant differences were revealed in the representation of different age groups among bitten people. The population affected by tick bites included fewer men and women in the ages of 10-29 and over 75 years old than would be predicted based on the demographics of the general population. Vice versa, the proportions of people in the ages of 5-9 and 60-74 increased among bitten people. Among men, such activities (in order of occurrence) as "leisure and recreation", "visiting allotments", "foraging for forest food", and "fulfilling work duties" tend to be more associated with tick bites. Among women, tick bites occurred mainly during "visiting allotments", "leisure and recreation", "visiting cemeteries" and "contact with pets and plants at home". The overall vaccination rate was 12 %; however, significantly more men than women were vaccinated against tick-borne encephalitis (up to 20 % vs. approximately 7 % respectively). The structure of the tick bite - affected population suggests that it is age-specific human behavior that mainly determines the frequency of contact between people and ticks. However, in several age groups, especially among children from 5 to 9 and people aged 30-39 years old, gender-related factors could significantly change the exposure of people to tick bites.

Bat ectoparasites: chigger mites (Trombiculidae), ticks (Ixodidae and Argasidae), and bugs (Cimicidae) in the Eastern Palaearctic.

Nine species-level taxa of bat ectoparasites, three chigger mites (Trombiculidae), three hard (Ixodidae), and one soft tick (Argasidae) species, as well as two bug (Cimicidae) species from nine bat species hosts were detected in the Eastern Palaearctic. Trombiculid larvae of Leptotrombidium schlugerae, Leptotrombidium album, and Ascoschoengastia latyshevi were first recorded on bats in the temperate zone of eastern Russia. L. schlugerae was more abundant than A. latyshevi in the same study sites in Eastern Siberia, and the main hosts of both chigger species were Plecotus ognevi and Eptesicus nilssonii. Ixodid ticks Dermacentor marginatus, Ixodes simplex, and Ixodes sp. were sampled from bats in Kazakhstan, the Far East, and Eastern Siberia, respectively. Phylogenetic analysis based on Cox1, 16S rDNA, and ITS2 sequences of I. simplex showed that the specimens from the Far East grouped into a clade distributed in the Eastern Palaearctic and India. In turn, the specimen of Ixodes sp. from Eastern Siberia was most closely related to Ixodes soricis and Ixodes angustus with p-distance of 9.8-10.7% (Cox1), suggesting that this tick probably belongs to a new species. Argas vespertilionis larvae were collected from three widespread bat species in Kazakhstan. Two bug species, Cimex pipistrelli and Cimex aff. lectularius, were recorded in the Far East and Eastern Siberia, respectively. Specimens from Transbaikalia were morphologically identified as Cimex lectularius. However, they differed from the latter by 12.5-12.9% of Cox1 sequences, indicating that C. aff. lectularius may be a new species.

Co-infections with multiple pathogens in natural populations of Ixodes persulcatus ticks in Mongolia.

BACKGROUND: In Mongolia, the taiga tick Ixodes persulcatus is the major vector of tick-borne pathogens. Knowledge about co-infections of these pathogens in ticks is necessary both for understanding their persistence in nature and for diagnosing and treating tick-borne diseases. METHODS: The prevalence of seven tick-borne infections in 346 I. persulcatus collected from the Selenge and Bulgan provinces of Mongolia was evaluated using real-time PCR. Quantification of Borrelia spp. was performed using multiplex quantitative PCR targeting the 16S rRNA gene. Genetic analysis of Borrelia spp. in 11 ticks infected with Borrelia miyamotoi, including six ticks co-infected with Borrelia burgdorferi sensu lato (s.l.), was performed by high-throughput sequencing of the flaB gene fragment. RESULTS: Six ticks (1.7%) were infected with tick-borne encephalitis virus (TBEV); 171 (49.4%), with B. burgdorferi sensu lato; 17 (4.9%), with B. miyamotoi; 47 (13.6%), with Anaplasma phagocytophilum; and 56 (16.2%), with Ehrlichia sp. Neither Rickettsia sibirica nor R. heilongjiangensis were detected. Borrelia burgdorferi s.l. occurred as co-infection in 55 (32.2%) of all infected ticks. The other pathogens co-infected ticks in 58.8-70.2% of cases. No pairwise associations between co-infecting pathogens were observed, with the exception of a positive association between A. phagocytophilum and Ehrlichia sp. INFECTIONS: The spirochete loads of B. miyamotoi were significantly higher than those of B. burgdorferi s.l. (mean: 5.2 vs 4.0 log10 genome copies/tick, respectively). Ten isolates of B. miyamotoi belonged to the Siberian lineage. Borrelia burgdorferi s.l was represented by nine isolates of B. afzelii, B. bavariensis and B. garinii. CONCLUSIONS: In populations of I. persulcatus inhabiting the Selenge and Bulgan provinces of Mongolia, five vector-borne pathogens, i.e. TBEV, B. burgdorferi s.l., B. miyamotoi, A. phagocytophilum and Ehrlichia sp., persist independently from each other, with the exception of A. phagocytophilum and Ehrlichia sp. which seem to share the circulation mode. The discrepancies in B. burgdorferi s.l. and B. miyamotoi prevalence and spirochete load per tick suggest that different ecological niches are occupied by Lyme disease and relapsing fever agents. High-throughput sequencing allows genetic identification of borreliae species in co-infected ticks.

Multilocus sequence analysis of Borrelia burgdorferi sensu lato isolates from Western Siberia, Russia and Northern Mongolia.

Lyme borreliosis (LB) is the most frequently recorded tick-transmitted disease in Eurasia. Tomsk Province, Western Siberia in Russia and Selenge Aimag in Northern Mongolia are leading regions in the LB incidence rate in these countries. Spirochaetes of the Borrelia burgdorferi sensu lato (s.l.) complex isolated from Ixodes ticks from Tomsk Province (n = 56) and Ixodes persulcatus ticks from Selenge Aimag (n = 5) were genetically characterized using Multi Locus Sequence Typing (MLST), analysis of the 5S23S rRNA intergenic spacer (IGS) amplicons, and p83/100 gene sequencing. According to MLST, B. afzelii (n = 26), B. bavariensis (n = 23), B. garinii (n = 11), and B. valaisiana (n = 1) isolates were detected in Tomsk Province, while B. afzelii and B. bavariensis isolates were identified in Selenge Aimag. Of the 32 revealed sequence types (ST), 21 STs were new and 14 of the new STs belonged to B. afzelii. Several STs of B. afzelii, B. garinii and B. valaisiana identified in this study clustered with European STs found in I. ricinus ticks. Analysis of the 5S23S IGS demonstrated that the studied Borrelia strains showed RFLP pattern characteristic for the following 5S23S IGS types: VS461 (B. afselii), NT29 (B. bavariensis), 20047 (B. bavariensis and B. garinii), VS116 (B. valaisiana), and three new groups (B. afzelii and B. bavariensis). Notably, this is the first report of Asian B. bavariensis possessing a 5S23S IGS RFLP pattern identical to 20047, and analysis of the 5S23S IGS did not provide correct determination of Borrelia species occurring in Asia. Genotyping of Borrelia strains using the clpA, pepX, and p83/100 genes demonstrated the same result as genotyping based on MLST; and further investigations are required to confirm that these three genetic loci could be used for determination of bacterial species from the B. burgdorferi s.l. complex because data based on single loci may be misleading.

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.

Prevalence of Borrelia miyamotoi in Ixodes persulcatus in Irkutsk City and its neighboring territories, Russia.

Adult Ixodes persulcatus were collected in highly populated districts in Irkutsk city, Russia, and in popular recreational and professional areas in its neighboring territories. Borrelia miyamotoi infection in I. persulcatus was examined using multiplex Taqman-PCR targeting 16S rDNA, and nested PCR and sequencing analyses targeting flaB and 16S rDNA. B. miyamotoi and Lyme disease Borrelia species were detected in 13 (infection rate, 2.9%) and 77 (17.3%) out of 445 I. persulcatus ticks, respectively, collected from 4 sites around the Baikal Lake. The 16S rDNA and flaB sequences of these amplicons were closely related to those of B. miyamotoi detected and/or isolated from I. persulcatus in Japan and Far Eastern Russia, and clustered separately from those of Europe and North America. These results indicate that additional surveillance for B. miyamotoi infection is needed in order to determine how it affects human health in Irkutsk City and its neighboring territories.

The diversity and prevalence of hard ticks attacking human hosts in Eastern Siberia (Russian Federation) with first description of invasion of non-endemic tick species.

Hard ticks are the vectors of many pathogens including tick-borne encephalitis virus and the Lyme disease agent Borrelia burgdorferi sensu lato. In Eastern Siberia, Ixodes persulcatus, Dermacentor nuttalli, Dermacentor silvarum and Haemaphysalis concinna are regarded as aggressive to humans. Recently, significant changes in world tick fauna have been reported and this affects the spread of tick-borne pathogens. We studied the current species diversity, population structure and prevalence of tick-borne pathogens of hard ticks (Acari: Ixodidae) that attacked humans in Eastern Siberia (Irkutsk region, Russia). In total, 31,892 individual ticks were identified and analysed during the years 2007-2014. The majority (85.4%) of victims was bitten by I. persulcatus, 14.55% of attacks on humans were caused by D. nuttalli and D. silvarum, whereas H. concinna was documented only in 15 cases (0.05%). The seasonal activity and the age/gender structure of the tick population were studied as well. Among all the studied ticks, three unconventional species, i.e. Rhipicephalus sanguineus, Dermacentor reticulatus and Amblyomma americanum, were identified. Analysis of tick bite histories indicates at least three events of invasion of non-endemic ticks into the ecosystems of northern Eurasia with harsh continental climates. Invading ticks are able to reach the adult life stage and are aggressive to the local human population. Phylogenetic analysis of mt 16S rRNA gene fragments suggests multiple independent routes of tick migration to Eastern Siberia. Possible implications to human health and epidemiology of tick-borne infections are discussed.

New insights into flavivirus evolution, taxonomy and biogeographic history, extended by analysis of canonical and alternative coding sequences.

To generate the most diverse phylogenetic dataset for the flaviviruses to date, we determined the genomic sequences and phylogenetic relationships of 14 flaviviruses, of which 10 are primarily associated with Culex spp. mosquitoes. We analyze these data, in conjunction with a comprehensive collection of flavivirus genomes, to characterize flavivirus evolutionary and biogeographic history in unprecedented detail and breadth. Based on the presumed introduction of yellow fever virus into the Americas via the transatlantic slave trade, we extrapolated a timescale for a relevant subset of flaviviruses whose evolutionary history, shows that different Culex-spp. associated flaviviruses have been introduced from the Old World to the New World on at least five separate occasions, with 2 different sets of factors likely to have contributed to the dispersal of the different viruses. We also discuss the significance of programmed ribosomal frameshifting in a central region of the polyprotein open reading frame in some mosquito-associated flaviviruses.

Evaluation of direct antiviral activity of the Deva-5 herb formulation and extracts of five Asian plants against influenza A virus H3N8.

BACKGROUND: The herb formulation Deva-5 is used in traditional medicine to treat acute infectious diseases. Deva-5 is composed of five herbs: Gentiana decumbens L., Momordica cochinchinensis L., Hypecoum erectum L., Polygonum bistorta L., and Terminalia chebula Retz. Deva-5 and its five components were investigated for in vitro antiviral activity against avian influenza A virus subtype H3N8. METHODS: The water extracts of the herbal parts of G. decumbens, H. erectum and P. bistorta, the seeds of T. chebula and M. cochinchinensis and Deva-5 were prepared by boiling and clarified by low-speed centrifugation and filtration. To assess the antiviral properties, avian influenza virus isolate A/Teal/Tunka/7/2010(H3N8) was incubated at 37°C for 30 min in the presence and absence of the extracts of five plants and DEVA-5 in various concentrations. Subsequently, the concentration of infectious virus in each sample was determined by plaque assays. Neutralisation indexes and 90% plaque reduction concentrations were estimated for each extract, and the significance of the data was evaluated using statistical methods. RESULTS: The extracts of G. decumbens, H. erectum, P. bistorta and Deva-5 demonstrated no significant toxicity at concentrations up to 2%, whereas extracts of T. chebula and M. cochinchinensis were well-tolerated by Madin-Darby canine kidney cells at concentrations up to 1%. The extracts of H. erectum, M. cochinchinensis and T. chebula reduced the titre of A/Teal/Tunka/7/2010 (H3N8) by approximately five-fold (p ≤ 0.05). The other three extracts did not significantly reduce the infectivity of the virus. The plaque reduction neutralisation tests revealed that none of the extracts tested were able to inhibit formation of plaques by 90%. However, three extracts, H. erectum, T. chebula and M. cochinchinensis, were able to inhibit formation of plaques by more than 50% at low dilutions from 1:3 to 1:14. The T. chebula extract had a concentration-dependent inhibitory effect. CONCLUSIONS: For the first time, the consistent direct antiviral action of the extracts of H. erectum, T. chebula and M. cochinchinensis was detected. These extracts significantly reduced the infectivity of influenza A virus H3N8 in vitro when used at high concentrations (0.5-1%). However, Deva-5 itself and the remainder of its components did not exhibit significant antiviral action. The results suggest that H. erectum, T. chebula and M. cochinchinensis plants contain substances with direct antiviral activity and could be promising sources of new antiviral drugs.

Molecular identification and phylogeny of Dermacentor nuttalli (Acari: Ixodidae).

Dermacentor nuttalli is an epidemiologically important tick in Palearctic Asia which transmits several infectious diseases including tularemia, North Asian tick-borne rickettsiosis, Lyme disease and tick-borne encephalitis. The genetic specificity and phylogeny of D. nuttalli from four geographic localities in Eastern Siberia were characterized using the mitochondrial (mt) 16S ribosomal RNA (rRNA) gene and internal transcribed spacer 2 (ITS2). Low genetic diversity was observed in the populations of ticks distributed from South Siberia to North China. From 11 detected mt 16S haplotypes, one was found in all populations, whereas the others were restricted to specific localities. These results suggested that the genetic structure of D. nuttalli represents integrated populations with no geographic isolation across the distribution area. The phylogenetic reconstructions inferred from the mt 16S rRNA gene and ITS2 were in agreement and showed a distinct D. nuttalli clade within a monophyletic Eurasian lineage of Dermacentor sp.

Non-hemagglutinating flaviviruses: molecular mechanisms for the emergence of new strains via adaptation to European ticks.

Tick-borne encephalitis virus (TBEV) causes human epidemics across Eurasia. Clinical manifestations range from inapparent infections and fevers to fatal encephalitis but the factors that determine disease severity are currently undefined. TBEV is characteristically a hemagglutinating (HA) virus; the ability to agglutinate erythrocytes tentatively reflects virion receptor/fusion activity. However, for the past few years many atypical HA-deficient strains have been isolated from patients and also from the natural European host tick, Ixodes persulcatus. By analysing the sequences of HA-deficient strains we have identified 3 unique amino acid substitutions (D67G, E122G or D277A) in the envelope protein, each of which increases the net charge and hydrophobicity of the virion surface. Therefore, we genetically engineered virus mutants each containing one of these 3 substitutions; they all exhibited HA-deficiency. Unexpectedly, each genetically modified non-HA virus demonstrated increased TBEV reproduction in feeding Ixodes ricinus, not the recognised tick host for these strains. Moreover, virus transmission efficiency between infected and uninfected ticks co-feeding on mice was also intensified by each substitution. Retrospectively, the mutation D67G was identified in viruses isolated from patients with encephalitis. We propose that the emergence of atypical Siberian HA-deficient TBEV strains in Europe is linked to their molecular adaptation to local ticks. This process appears to be driven by the selection of single mutations that change the virion surface thus enhancing receptor/fusion function essential for TBEV entry into the unfamiliar tick species. As the consequence of this adaptive mutagenesis, some of these mutations also appear to enhance the ability of TBEV to cross the human blood-brain barrier, a likely explanation for fatal encephalitis. Future research will reveal if these emerging Siberian TBEV strains continue to disperse westwards across Europe by adaptation to the indigenous tick species and if they are associated with severe forms of TBE.