Cases of Q fever detected in residents of the novosibirsk region hospitalized with suspection of infections transmitted by ticks.

Coxiella burnetii is the causative agent of Q fever (coxiellosis), which, in addition to acute manifestations, often occurs in a latent form, is prone to chronic course and, in the absence of antibiotic therapy, has a high risk of disability or death. As a result of the presence of a wide range of clinical manifestations specific to other infectious diseases, the use of laboratory test methods (LTM) is required to make a diagnosis. The presence of Q fever anthropurgic foci in the Novosibirsk region was described in the 90s of the last century, but due attention to its laboratory diagnostics is not paid in this region. The aim of the study was to identify genetic and serological markers of the causative agent, C. burnetii, in patients of the Novosibirsk region who were admitted for treatment with fever with suspected tick-borne infections (TBIs). DNA marker of the causative agent of Q fever was detected in blood samples by real time PCR in 9 out of 325 patients. In three patients, the presence of C. burnetii DNA was confirmed by sequencing of the IS1111 and htpB gene fragments. In ELISA tests, antibodies against the causative agent of coxiellosis were detected in the blood sera of 4 patients with positive results of PCR analysis. Contact with tick was registered in 7 out of 9 patients who had C. burnetii DNA and lacked markers of other TBIs. Six people were infected in the Novosibirsk region, two suffered from tick's bite in Altai, and one case was from the Republic of Kyrgyzstan. Thus, a complex approach using both PCR analysis and ELISA provided the identification of markers of the Q fever causative agent in patients admitted with suspected TBIs, thereby differentiating it from other infections. Contact with ticks in most cases suggests that infection with C. burnetii had a transmissible pathway.

[Detection of potential sites of recombination in the Tick-borne encephalitis virus by the methods of comparative genomics].

The results of the bioinformatic search for the potential sites of the recombination in the genome-wide structures of the tick-borne encephalitis virus (TBEV) through a series of software techniques were presented in this work. The genomes of the 55 TBEV strains were assayed, 21 of them showed the presence of the recombination sites. Recombinant strains belonged to the Far Eastern (19 strains) and European (2 strains) genotypes. 22 sites of the recombination attributed were identified to five types based on position, strain, and regional characteristics. The parental strains were identified based on the genotypic and geographical parameters, which do not contradict the possibility of the formation of the recombinants. Nearly two-thirds of the sites are located in the regions of NS4a and NS4b genes, which are the "hot spots" of the recombination, most of them being concentrated in the gene NS4. It was shown that the recombination processes did not occur at the level of the genotypes (European genotype) or certain groups within the genotype (Far East) and were typical of the peripheral populations.

[Genotypes 4 and 5 of the tick-borne encephalitis virus: features of the genome structure and possible scenario for its formation].

On the basis of the comparison of complete genome structures of 32 strains and gene E fragments (160 ndt) of 643 strains and RNA isolates of tick-borne encephalitis (TBE) virus, we confirmed our previously expressed assumption (Zlobin V.I. et al, 2001) of existence, along with the three major genotypes, of genotypes 4 (strain 178-79) and 5 (strain 886-84). "Mosaic" structure of the polyprotein in the two strains was established. It manifests itself in particular in the sequences of 14 positions (C-3, E-206, NS1-54, NS-285, NS2A-100, NS2A-127, NS2A-174, NS2A-175, NS2A-225, NS3-376, NS4B-28, NS4B-96, NS5-18, NS5-671) containing the amino acids strictly conserved for each of the three major genotypes and is consistent with a uniform pattern of distribution of nucleotide substitutions that are specific for genotypes 1, 2 and 3. Possible scenario of the origin of TBE genotypes 4 and 5 was suggested.

[Detection of recombinations in tick-borne encephalitis virus by using computer analysis of viral genomes].

Computer programs were used to search for tick-borne encephalitis (TBE) virus recombinants among the isolates whose complete nucleotide sequences are deposited in the GenBank database. The application of RDP, Chimaera, Maximum chi2, and TOPAL programs has revealed recombinant sites in a number of sequences, which indicates that TBE virus has recombinations and that the programs are suitable for their detection.

Genotyping and characterization of the geographical distribution of tick-borne encephalitis virus variants with a set of molecular probes.

A panel of deoxyoligonucleotide probes for studying the genetic variability and genotyping of Tick-borne encephalitis virus (TBEV) strains by molecular hybridization of nucleic acids (MHNA) was created. This panel allows to estimate the genetic structure of individual TBEV strains, as the targets for probes are both variable and genotype (subtype)-specific sequences of all TBEV genes. With the help of this panel using the method of molecular hybridization of nucleic acids 268 archived TBEV isolates were investigated and the distribution of its genotypes and subgenotypes of genotype 3 was made more precise in the territory of Eurasia. The conclusion made earlier has confirmed that five genotypes of TBEV co-circulate in Eastern Siberia. It is generally recognized that the Far Eastern (TBE-FE), European (TBE-Eu), and Siberian (TBE-Sib) genotypes are widespread and epidemiologically important. The fourth genotype is presented by only one isolate, TBE178-79, originated from Irkutsk region, Russia. The fifth genotype includes 10 isolates, 1 of them, TBE886-84, was found earlier and recognized as unique [Zlobin et al. (2001b): Vopr Virusol 1:12-16 (Russian)].

Genetic diversity and geographical distribution of the Siberian subtype of the tick-borne encephalitis virus.

The tick-borne encephalitis virus (TBEV), a member of the Flaviviridae family, is currently subdivided into three main subtypes-the European (TBEV-Eu), the Far-Eastern (TBEV-FE), and the Siberian (TBEV-Sib). The TBEV-Sib is the most common subtype and found in all regions where TBEV was detected, except for Central and Western Europe. Currently, four genetic lineages have been described within TBEV-Sib. In this study, detailed analysis of TBEV-Sib genetic diversity, geographic distribution, phylogeography and divergence time of different TBEV-Sib genetic lineages based on E gene fragments, complete genome sequences, and all currently available data in the GenBank database was performed. As a result, a novel Bosnia lineage within the TBEV-Sib was identified. It was demonstrated that the Zausaev lineage is the most widely distributed among the TBEV-Sib lineages, and was detected in all studied regions except the Far East. The Vasilchenko lineage was found from Western Siberia to the Far East. The Baltic lineage is presented from Europe to Western Siberia. The Obskaya lineage was found only in Western Siberia. TBEV strains from a newly described Bosnia lineage were detected in Bosnia, the Crimean peninsula, Kyrgyzstan and Kazakhstan. The greatest divergence of the TBEV-Sib genetic variants was observed in Western Siberia. Within the TBEV-Sib, the Obskaya lineage diverged from the common ancestor the earliest, after that the Bosnia lineage was separated, then the Baltic lineage, and the Zausaev and Vasilchenko lineages diverged most recently.

[The genetic variability and genotyping of tick-borne encephalitis virus with deoxyoligonucleotide probes].

A panel of genotype-specific molecular probes has been designed, which is used to indicate and differentiate tickborne encephalitis (TBE) virus. It assesses the individual genetic structure of each strain since the targets for the probes are the variable sequences of all 10 virus genes, which are specific for each of three genotypes. The molecular nucleic acid hybridization by means of the panel was used to study 273 TBE virus strains. Isolated from a Eurasian area; along with the representatives of three genotypes, the virus strains, the genomic structures of which do not fit in the established concept on three genotypes, circulate in Eastern Siberia.

DEFINITION AND COMPARATIVE ANALYSIS OF THE GENOMIC STRUCTURE OF SIBERIAN STRAINS OF TICK-BORNE ENCEPHALITIS VIRUS OF THE EUROPEAN SUBTYPE.

Tick-borne encephalitis virus (TBEV) is classified into three subtypes: Far Eastern (TBEV-FE), European (TBEV-EU) and Siberian (TBEV-SIB). In Russia, these are also called genotypes 1, 2 and 3, respectively. Geographically, TBEV-EU dominates in Central and Northern Europe, but its representatives are also found to the east - along the southern part of the forest zone of extratropical Eurasia - up to Eastern Siberia and South Korea. However, the strains isolated outside Europe remain poorly investigated. In the proposed study, eight full genomes of the Siberian isolates of TBEV-EU were determined and 13 complete genomes were compared. The analysis of 152 full-genome TBEV sequences showed that the TBEV-EU has a higher degree of stability of the genome-coding region in the entire Eurasian area (3.1% of differences) compared to TBEV-FE (6.6%) and TBEV-SIB (7.8%). At the same time, the maximum differences are observed not between European and Siberian strains, as one could expect, but between the representatives from Europe - TBEV strains Mandl-2009 from Norway and Hypr from the Czech Republic. The studied strains from Siberia form the compact genetic cluster of 42 TBEV-EU strains and are divided into two subclusters - West Siberian and East Siberian variants. These variants differ in the combinations of amino acid substitutions in all proteins except NS2B. The West Siberian variant mostly circulates in the territory of Altai, and the closest relative of its representatives is Absettarov strain from the European part of Russia. The strains similar to the East Siberian variant of the European subtype were recorded in the Altai (strain 84.2, 2007) and in Belarus (N256, about 1940).

A comparative analysis on the physicochemical properties of tick-borne encephalitis virus envelope protein residues that affect its antigenic properties.

This work is dedicated to the study of the variability of the main antigenic envelope protein E among different strains of tick-borne encephalitis virus at the level of physical and chemical properties of the amino acid residues. E protein variants were extracted from then NCBI database. Four amino acid residues properties in the polypeptide sequences were investigated: the average volume of the amino acid residue in the protein tertiary structure, the number of amino acid residue hydrogen bond donors, the charge of amino acid residue lateral radical and the dipole moment of the amino acid residue. These physico-chemical properties are involved in antigen-antibody interactions. As a result, 103 different variants of the antigenic determinants of the tick-borne encephalitis virus E protein were found, significantly different by physical and chemical properties of the amino acid residues in their structure. This means that some strains among the natural variants of tick-borne encephalitis virus can potentially escape the immune response induced by the standard vaccine.

Comparative analysis of complete genome sequences of European subtype tick-borne encephalitis virus strains isolated from Ixodes persulcatus ticks, long-tailed ground squirrel (Spermophilus undulatus), and human blood in the Asian part of Russia.

Tick-borne encephalitis virus (TBEV) is divided into three subtypes: European (TBEV-Eu), Siberian (TBEV-Sib), and Far Eastern (TBEV-FE) subtypes. The geographical range of TBEV-Eu dominates in Europe, but this subtype is present focally across the whole non-tropical forested Eurasian belt, through Russia to South Korea. However, the TBEV-Eu strains isolated outside Europe remain poorly characterized. In this study, full-genome sequences of eight TBEV-Eu isolates were determined. These strains were isolated from Ixodes persulcatus ticks, long-tailed ground squirrel (Spermophilus undulatus), and human blood in the natural foci of Western and Eastern Siberia, Russia. A phylogenetic analysis of all available TBEV-Eu genomic sequences revealed that strains from Siberia were closely related to other strains from Europe and South Korea. The closest relation was identified between the Siberian strains and strains from Zmeinogorsk (Western Siberia, Russia) and strain Absettarov (Karelia, Russia), and were most divergent from strains from the Czech Republic and Norway. TBEV-Eu strains isolated in Eastern Siberia were more closely related phylogenetically to strains from South Korea, but strains from Western Siberia grouped together with the strains from Europe, suggesting two genetic TBEV-Eu lineages present in Siberia.

[Detection of RNA in vaccine " EnceVir" against tick-borne encephalitis virus].

Fractionation of RNA isolated from the inactivated purified concentrated vaccine "EnceVir" ("Virion", Tomsk, Russia) allowed the authors to reveal predominantly double-stranded RNA and trace quantities of single-stranded RNA sizing mainly less than 200 n. The fragments of tick-borne encephalitis virus (TBEV) were detected among the total RNA by the reverse transcription test, followed by nested polymerase chain reaction with the primers specific to the E gene of TBEV. The findings may suggest that immunity may be induced through activation of Th1 and Th2 after immunization with such an inactivated vaccine for effective elimination of intracellular infection and intracellular virions, respectively.

Tick-borne encephalitis virus strains of Western Siberia.

Tick-borne encephalitis virus (TBEV) strains were isolated from ticks in Western Siberia for 12 years. Molecular hybridization of the 46 viral RNA with the TBEV cDNA and oligonucleotide probes revealed differences between the Siberian and Far Eastern strains. A comparison of the viral E gene fragment nucleotide sequence showed 89-98% homology between Siberian TBEV strains, whereas their similarity with strains from other populations was less than 83%. However, the viral E and NS1 glycoprotein antigenic structures appeared to be conservative because of the degenerate genetic code. This was shown by enzyme-linked immunosorbent assay with the corresponding monoclonal antibodies (MAb). The single exception was the MAb 17C3 against nonstructural glycoprotein NS1, which could distinguish Siberian from Far Eastern strains. Moreover, the neurovirulence differed between strains from the two natural populations. Lower neuroinvasiveness of the Siberian strains in comparison with Far Eastern Sofyin strain might be caused by both E and NS1 glycoprotein mutations.

[Newly isolated mink parvovirus strain]. / Novyi izoliat parvovirusa norok.

A new mink enteritis virus (MEV) strain, called Cherepanovo, was isolated in the Novosibirsk region (West Siberia). The level of this strain's accumulation in cell culture is lower than that of commercial MEV strains Rodniki or Beregovoi. Identification of Cherepanovo strain by hemagglutination inhibition test, protein electrophoresis in denaturing polyacrylamide gel, and PCR showed its similarity to the previously described MEV strains. Comparative analysis of this strain's 5' and 3' nucleotide sequences of VP2 gene showed its more than 98% homology with other feline parvoviruses. Nucleotide residues 2920, 2971, and 4245 in the Cherepanovo strain VP2 gene differed from those of all previously described MEV strains. It is noteworthy that transition at the position 4245 led to Phe-->Cys replacement in amino acid residue.

[Genetic analysis of tick-borne encephalitis virus strains from West Siberia]. / Geneticheski'i analiz shtammov virusa kleshchevogo éntsefalita Zapadno'i Sibiri.

Tick-borne encephalitis (TBE) virus strains were isolated in West Siberia in the forest-steppe region near the Ob river in 1981-1992. Hybridization of genome RNA of 46 TBE strains with [32P]cDNA of TBE Sofyin strain revealed essential differences in the genomes of West-Siberian and Far-Eastern Sofyin strains of TBE virus. Nucleotide sequences of 6 TBE strains (1348-1503 n.) have been determined. A 89-98% homology of Siberian TBE strains has been shown, while the similarity of the respective fragment of E gene for West Siberian and Sofyin strains was no more than 81%. No significant changes in E gene of TBE strains have been detected over a 12-year period.

The correlation between tick (Ixodes persulcatus Sch.) questing behaviour and synganglion neuronal responses to odours.

We examined the behavioural and electrophysiological responses of taiga ticks (Ixodes persulcatus) to several olfactory stimuli: Osmopherone® (5-a-androst-16-en-3-ol), Osmopherine® (butanoic and 3-methylbutanoic acids), DEET® (N,N-diethyl-meta-toluamide), ethanol (96%), and water (control stimulus). To study individual tick behavioural reactions to these stimuli, we used a Y-shaped glass maze (n=50). To study the electrophysiological reactions of the ticks' synganglia to these olfactory stimuli, we recorded the shifts of total potential (TP) of pre-oesophageal neurons in response to odour stimulation of Haller's organ (n=25). We found that Osmopherine® attracted ticks and frequently evoked negative shifts of TP, whereas the response to Osmopherone® did not differ from the reaction to water. DEET® and ethanol acted as tick repellents and generally evoked positive shifts of TP. We also tested each tick for its questing height (QH) on a glass rod that was at an incline of 75°, and we tested for the presence of pathogens i.e., DNA of Borrelia burgdorferi sp. s.l. and RNA of tick-borne encephalitis virus (TBEV). The degree of response to Osmopherine® positively correlated with the QH. The ticks with the highest values of QH showed a greater prevalence of the tick-borne pathogen Borrelia sp. s.l. compared with the ticks that did not reach the maximum QH. The present results show a correlation between the electrophysiological reaction of the synganglia of ticks and their behavioural responses to different odours.

The brain 5-HT1A receptor gene expression in hibernation.

Hibernation is a unique physiological state characterized by profound reversible sleep-like state, depression in body temperature and metabolism. The serotonin 5-hydroxytryptamine(1A) (5-HT(1A)) receptor gene sequence in typical seasonal hibernator, ground squirrel (Spermophilus undulatus), was specified. It was found that the fragment encoding the fifth transmembrane domain showed 93.6% of homology with the analogous fragment of the mouse and rat genes and displayed 88.5% homology with the human 5-HT(1A) receptor gene. Using primers designed on the basis of obtained sequence, the expression of 5-HT(1A) receptor gene in the brain regions in active, entering into hibernation, hibernating and coming out of hibernation ground squirrels was investigated. Significant structure-specific changes were revealed in the 5-HT(1A) messenger RNA (mRNA) level in entry into hibernation and in arousal. An increase in the 5-HT(1A) gene expression was found in the hippocampus during the prehibernation period and in ground squirrels coming out of hibernation, thus confirming the idea of the hippocampus trigger role in the hibernation. Significant decrease in 5-HT(1A) receptor mRNA level in the midbrain was found in animals coming out of hibernation. There was no considerable changes in 5-HT(1A) receptor mRNA level in different stages of sleep-wake cycle in the frontal cortex. Despite drastically decreased body temperature in hibernating animals (about 37 degrees C in active and 4-5 degrees C in hibernation), 5-HT(1A) receptor mRNA level in all examined brain regions remained relatively high, suggesting the essential role of this 5-HT receptor subtype in the regulation of hibernation and associated hypothermia.