Seroplrevalence of <i>Anaplasma phagocytophilum</i> and <i>Ehrlichia</i> sp. among people affected by tick bites

Background. In spring and summer, the population of the Baikal region regularly comes into contact with the pathogens transmitted through the bites of ixodid ticks. In the Center for Diagnosis and Prevention of Tick-Borne Infections (Irkutsk, Russian Federation), we annually detect anaplasmas of the Anaplasma phagocytophilum species, as well as Ehrlichia chaffeensis/E. muris in both ixodid ticks and blood samples from people who have been bitten by ticks. At the same time, there are no data in open sources on the incidence of human granulocytic anaplasmosis and human monocytic ehrlichiosis in the Baikal region. Currently, there is very little information on the studies of intensity of the immune response to anaplasmas and ehrlichia in people living in the surveyed area, although this information is critical for assessing the frequency of contacts and the risk of infection of people in a territory endemic for tick-borne infections.   The aim. To update information on the presence and prevalence of specific immunoglobulins M and G to A. phagocytophilum and Ehrlichia sp. among the population of the Irkutsk Region affected by tick bites.   Materials and methods. In total, 204 samples of blood serum from the residents of the Irkutsk Region who were registered to be bitten by ticks were analyzed for the presence of IgM and IgG to human monocytic ehrlichiosis and human granulocytic anaplasmosis agents.   Results. IgG to A. phagocytophilum were found in 9 samples, IgG to E. chaffeensis/E. muris – in 1 sample; no IgM to both pathogens were found in any sample.   Conclusions. The results obtained indicate regular infection of the population with anaplasmas and ehrlichia which is a testifies to the existence of active natural foci of human monocytic ehrlichiosis and human granulocytic anaplasmosis in the Baikal region. To clarify the real epidemic role of these infections, a detailed study of the immune status is required both among healthy individuals and among patients with symptoms of an infectious disease

Features of Reproduction of Tick-Borne Encephalitis Virus in a New Cell Line of the Siberian Bat Myotis sibiricus (Kastschenko, 1905)

Background. Tick-borne encephalitis virus (TBEV) in nature exists due to the constant circulation between vertebrate animals and tick viruses. To study the characteristics of reproduction of TBEV in the cells of vertebrate hosts of various species, it is necessary to simulate infection in the cell lines of both natural and accidental hosts of TBEV.Aim. To study the possibility of reproduction of TBEV in the cell line of the kidney of Siberian bat Myotis sibiricus (Kastschenko, 1905) – an accidental host of the virus.Materials and methods. The cell line of Siberian bat M. sibiricus was established by serial passages of primary culture of kidney cells. The SPEV line porcine kidney cells were used as reference. Cells were infected with a strain of TBEV of the Siberian subtype 92M and both cells and cell culture fluids were sampled every 2 hours during first 24 hours post infection. In addition, the samples were collected daily up to 5 days post infection. Evaluation of the amount of intracellular RNA of TBEV of positive polarity (+RNA) was performed  using quantitative real-time PCR. The concentration of infectious TBEV was evaluated using the method of titration TBEV on plaque forming units.Results. The continuous cell line, designated as MdbK, was established from the kidney cell suspension of M. sibiricus and was used for experiments after 20 serial passages. MdbK cells were able to support the replication of TBEV RNA and production of infectious virus was also possible. The concentration of intracellular RNA had reached 9.1 lg copies/μl by day 3 post infection, whereas highest titer of infectious TBEV in cell culture fluid had comprised 5,5 PFU/ml and was detected by day 4. The concentration of intracellular RNA and the virus infectivity in MdbK cell line was significantly lower than in convenient SPEV line porcine kidney cell.Conclusions. The results suggest the low fitness of TBEV to the intracellular environment of an accidental host

Characteristics of tickborne infections in the underexplored areas of the Trans-Baikal Territory

Background. Infections transmitted to humans by the bites of ixodid ticks remain an urgent public health problem. In this work we explored the natural foci of tickborne infections located in the valley of the Chikoy River, which is a part of the buffer zone of the Baikal natural territory.The aim. To characterize the modern diversity and prevalence of tick-borne pathogens in the ecosystems of the valley of the Chikoy River (Trans-Baikal Territory, Russian Federation).Materials and methods. Thirteen sampling sites were located in typical biotopes throughout the Chikoy valley. In total 48  adult Ixodes persulcatus ticks, 1  female Haemaphysalis concinna tick and 38 specimens of small mammals were studied. All samples were tested for infection with seven tick-borne pathogens using multiplex real-time PCR.Results. No pathogens were detected in the H. concinna specimen. No R. sibirica and R. heilongjiangensis were detected both in ticks and in rodents. Among I. persulcatus, tick-borne encephalitis virus (TBEV), and the prevalence of Borrelia burgdorferi s. l. comprised 39.5 %, A. phagocytophilum – 16.7 %, B. miyamotoi – 8.3 % and Ehrlichia sp. – 2.1 %. Among infected ticks 6.2 % were co-infected with B. burgdorferi s. l. and A. phagocytophilum. Four rodent hosts of ticks and infections were identified: Myodes rufocanus (44.7  %), Apodemus peninsulae (39  %), Microtus oeconomus (13.2  %) and M.  rutilus (2.6  %). Mean prevalence of B.  burgdorferi  s.  l. in  rodents comprised 39.5 %, B. miyamotoi – 28.9 %, Ehrlichia sp. – 21.1 % and A. phagocytophilum – 18.4 %. TBEV was detected in 5.3 % of rodents.Conclusion. At least five tick-borne pathogens circulate in the Chikoi River valley, i. e. TBEV, B. burgdorferi sensu lato, A. phagocytophilum, B. miyamotoi and Ehrlichia sp. The wide spread of I. persulcatus and abundance of competent rodent hosts of infections and ticks indicates that natural foci of tick-borne diseases are widely distributed in the Chikoi River valley

Optimization of a Quantitative Real-Time RT-PCR Technique for Evaluation of Concentration of Genomic +RNA of Tick-Borne Encephalitis Virus

Background. The specific detection of genomic/template +RNA and replicative –RNA of tick-born encephalitis virus (TBEV) is necessary to study the mechanisms of viral replication in the cells of reservoir and accidental hosts. However, the current approaches of quantitative reverse transcription – polymerase chain reaction (qRT-PCR) are rather focused on the detection of total viral RNA load in the sample. Thus, the significant optimization is necessary both for RT-PCR and for RNA copy number standard preparation.Aims. To develop the set of standard samples of synthetic +RNA of TBEV and to optimize qRT-PCR for quantification of genomic +RNA of the virus.Materials and methods. Fragment of the genomic +RNA of TBEV was synthesized using pTZ57R-T\A plasmid vector with embedded T7 promoter and T7 RNA polymerase. The DNA contamination was removed using RNase-free DNase I treatment followed by additional RNA purification step. Reverse transcription was performed using specific antisense primer 11154R 5`- AGCGGGTGTTTTTCCG-3` and qPCR detection was used according to the modified procedure of M. Schwaiger and P. Cassinotti (2003).Results. As a result of the amplification of standard samples, the concentration of positive polarity ТBEV RNA, carried out in five independent repetitions on different days, the correlation coefficient R2 between the quantification cycle and the concentration of the standard sample was 0.99, and the efficiency of PCR was 100 %. The coefficient of variation in assessing the inter-test accuracy of determination averaged 2.8 %.Conclusions. Optimized qRT-PCR procedure and set of +RNA standards allow to determine the concentration of genomic +RNA of TBEV in routine laboratory practice

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

Findings of <i>Amblyomma americanum</i> L., 1758 in the Territory of Eastern Siberia (Russia)

In June, 2008, in the Irkutsk suburban zone, a man was beaten by a tick of an unknown in this area species. According to its morphological characteristics this tick was classified as Amblyomma americanum L., 1758 species, which is the endemic one in the North, Central, and South America. In this respect, the discovery of the unknown previously in this region and in the Russian Federation species of the ixodic tick is not only of a great scientific importance, but presents specific practical interest, as the ticks of this species are the carriers of a wide range of infectious diseases. Probable scenarios of importation of the new species of ticks in the territory of the Russian Federation are under discussion now and the risks, related to this problem, are being assessed

GENETIC IDENTIFICATION AND PHYLOGENETIC RELATIONSHIPS OF DERMACENTOR SP. TICKS IN IRKUTSK REGION

Here we present the results of the analysis of mt16S ribosomal RNA gene of two species of ticks from Irkutsk region - Dermacentor nuttalli and D. silvarum.. The ticks from are closely related to each other with 99-100 % identity of nucleotide sequence of mt16S rRNA gene. The phylogenetic analysis has shown that both species are closely related and. formed the separate clade together with specimens of D. nuttalli and. D. silvarum. from China

GENETIC ANALYSIS OF HOKKAIDO HANTAVIRUS AMONG MYODES RUFOCANUS IN THE BAIKAL LAKE AREA

Hokkaido hantavirus (HOKV) identified originally in the grey red-backed vole (Myodes rufocanus) in Hokkaido, Japan. Subsequent studies showed different genetic lineages of HOKV in Sakhalin, Buryatia and Far Eastern regions of Russia and in China. Tissuesfrom 68 arvicolid rodents, captured in regions south and west of Baikal Lake, were initially tested for hantaviral antigen by ELISA, and tissues from antigen-positive rodents were analyzed for hantavirus RNA by RT-PCr. Taxonomic identification of host species was based on phylogenetic analysis of partial cytochrome b gene sequences. Hantavirus Land S-segment sequences were detected in two antigen-positive M. rufocanus, from the Tunka region of Buryatia Republic (south side) and the Olhon region of Irkutsk Oblast (west side). Sequence analysis showed that the newfound hantavirus strains, designated Baikal and Siberia, represented genetic variants of HOKV Previously unknown genetic variant designated Siberia was identified in M. rufocanus captured in Olhon region. Second genetic variant from Tunka region, designated Baikal, was closely related to previously described hantavirus strain from the same region. Alignment and comparison of the nucleotide and amino acid sequences showed intra-strain differences of 18,4 % and 5,3 % for the L segment and 17,4 % and 3,5 % for the S segment, respectively. Sequence divergence from geographically distant HOKV strains were 17,4-21,5 % and 3,9-6,8 % for the L segment and 15,2-17,0 % and 3,3-4,0 % for the S segment, respectively. Phylogenetic analysis, based on a 346-nucleotide region of the L segment, revealed four lineages represented by previously reported variants from Japan and Sakhalin (strains Kitahiyama128L/2008, Tobetsu35L/2010 and Sakhalin99L/1998), Shkotovo in Far-Eastern Russia (strain Khekhtsir37L/2002) and the new variants from Baikal Lake. Analysis of the N protein, coding by the S segment, identified specific amino acid signatures for YJRV of Lys5, Arg26, Val/Ile68, Val/Ala9 He262, Pro283. Conclusions: HOKV is widespread across the geographic range of its arvicolid rodent reservoir host

Development of DNA aptamer selection approach based on membrane ultrafiltration of aptamer/target complex

Background. Aptamers are small single-stranded DNA or RNA molecules that have an affinity for a specific target molecule. The main method of aptamers construction is the technology of systematic evolution of ligands with exponential enrichment (SELEX). However, the exact approach depends on the nature of target molecules, and is selected and optimized by each researcher independently. The article describes the technique of production of aptamers to the tick-borne encephalitis virus (TBEV) using membrane ultrafiltration with a molecular weight cut-off of 100 kDa. As a result, the pool of aptamers with observable affinity for TBEV is successfully selected and enriched.The aim. To develop the technique suitable for selection of specific DNA aptamers to a live, crude TBEV suspension directly in cell culture supernatant.Materials and methods. The selection of aptamers was carried out using a modified SELEX DNA aptamer technology in combination with semipermeable membrane ultrafiltration using Vivaspin 6 (Sartorius, Germany) concentrators of molecular weight cut-off of 100 kDa. Enrichment of a specific pool of aptamers was performed using real time polymerase chain reaction. Aptamers were sequenced with automated Sanger sequencing method. The direct virucidal effect of the aptamers was determined by the decrease in the titer of the infectious virus after incubation with the aptamer.Results. The pool of aptamers to TBEV was selected and enriched. This aptamer pool expressed affinity both to the infectious TBEV and to the TBEV antigen. Sixteen aptamers were sequenced from this pool and four of them were synthesized and tested for antiviral activity against TBEV. No antiviral activity was observed.Conclusions. The technique developed that can be successfully used to select aptamers to a live virus culture for the viruses comparable in size to TBEV or larger

Influence of substrate wood species on the formation of antiviral properties of <I>Inonotus rheades</I> Pers. P. Karst. (1882) mycelium extracts regarding tick-borne encephalitis virus

Background. The tick-borne encephalitis virus (TBEV) is one of the most dangerous and epidemiologically significant vector-borne pathogens. There is a need for effective antiviral agents for the treatment and prevention of this infection. Previously we found that the mycelium of Inonotus rheades grown on birch wood contains water-soluble substances with strong virulicidal properties against TBEV. It is necessary to check whether the mycelium of I. rheades can synthesize virulicidal substances from wood of other species.The aim: to study the antiviral properties of extracts of I. rheades mycelium grown on coniferous wood, both in the presence and in the absence of blue light during cultivation.Materials and methods. The mycelium of I.  rheades was grown on birch, pine, and fir wood. The direct virulicidal effect of the extract was evaluated by the decrease in the titer of the infectious virus incubated in the presence of the extract. The ability of the extract to inhibit the reproduction of the virus in infected cells was studied by  the  calculation of 50  % effective concentration (EC50). The toxicity of extracts for cells was evaluated based on the calculation of 50 % cytotoxic concentration.Results. Mycelium extracts grown on conifers under blue light do not cause a statistically significant decrease in the concentration of infectious TBEV (p = 0.2563). However, the BP10 extract (pine, blue light) inhibits TBEV reproduction in infected cells (EC50 = 0.28 ± 0.06 mg/mL). Toxicity for SPEV cell culture is low. In the extracts of conifers grown in the dark, no antiviral effect was found at all.Conclusions. The component composition and mechanism of the antiviral action of I. rheades extracts are determined by the species of the wood substrate. The most promising sources of new drugs in relation to TBEV appear to be extracts of I. rheades mycelium grown on birch and pine