[Biological properties and phylogenetic relationships of tick-borne encephalitis virus (Flaviviridae, Flavivirus) isolates of siberian subtype isolated in the south of East siberia in modern period].

INTRODUCTION: Tick-borne encephalitis virus (TBEV) is medically most important representative of the same-name serogroup of genus Flavivirus (Flaviviridae). In the view of various researchers there are 3 to 5 TBEV subtypes, of them siberian being the most prevalent. The aim of the work is to compare the biological properties and to reveal phylogenetic relationships of large group of modern (2006-2019) TBEV isolates of siberian subtype from natural foci in southern East Siberia. MATERIAL AND METHODS: Ixodid ticks (Ixodidae) and small mammals (Mammalia) from tick-borne encephalitis (TBE) natural foci in Irkutsk Region, Republic of Buryatia and Republic of Tuva, as well as specimens from TBE patients, were examined for TBEV markers using enzyme-linked immunosorbent assay (ELISA) and reverse transcription polymerase chain reaction (RT-PCR). Virus was isolated from suspensions with positive result, and its pathogenicity for white mice (Mus) (WM) was studied by different inoculation ways. Analysis of the nucleotide sequences of E gene was performed for isolates at 1st passage. Phylogenetic tree was constructed using MEGA X program. RESULTS: The phylogenetic analysis has shown that TBEV of siberian subtype that circulates in natural foci of the studied territory belong to two genetic lines. These lines are «Vasilchenko¼ and «Zausaev¼ with a strong predominance of the first. The differences in biological properties between the two groups of strains have been demonstrated. Most of the strains from both groups showed high virulence for WM both after intracerebral and subcutaneous inoculation. Only four strains demonstrated the reduced ability to overcome the blood-brain barrier. However, the analysis of the E protein coding sequences revealed evident correlation between phylogenetic clustering and geographical origin of isolates, but not with TBE host or pathogenicity for WM. CONCLUSION: Further search for TBE genome regions associated with pathogenicity require the analysis of complete genome sequences of representative group of strains with different biological properties.

Analysis of effectivity of the tickborne encephalitis virus detection methods in ixodid ticks.

Tick-borne encephalitis (TBE) is transmissible viral disease widely common in temperate zone of Eurasia. ELISA and PCR are used for express identification of the vector's infection, but the results of the two methods often do not agree. Aim of the work is comparative analysis for TBE virus of Ixodid ticks from nature using complex of methods, including ELISA, PCR, and isolation of the virus in laboratory mice. 18608 Ixodid ticks were collected during 2013-2019 in TBE natural foci of the Baikal Region. The ticks suspensions were examined individually, using ELISA (n=17610) and PCR (n=2999). Suckling mice were inoculated with the suspensions positive in the both tests. The TBEV antigen was found in 1.2 % of ticks in average. All ticks positive in ELISA were examined in PCR (Group 1). Randomly selected part of negative-ELISA samples were examined in PCR too (Group 2). The PCR results were positive in 68.9±3.13 % of the Group 1, with average Ct index 24.6±0.38. Positive results of PCR in Group 2 accounted for just 2.7±0.31 % with average Ct index 31.0±0.70. The average Ct margin of the Groups 1 and 2 is statistically significant (p < 0.001; df = 118). Isolation of strains was significantly more successful in Group 1 (21.7±2.77 %), than in Group 2 (8.2±5.26 %; p < 0.05; df = 50). ELISA is more useful for examining large amounts of ticks. To get a more complex picture about epidemically dangerous part of the vectors in TBE natural foci, the results of the two express-methods is better to sum. The isolation of the virus is useful to carry out of the samples positive in ELISA and PCR concurrently.

[PLAGUE IN MANCHURIA (1910-1911) AND EBOLA VIRUS DISEASE IN WEST AFRICA (2014-2015): COMMON PREREQUISITES FOR THE DEVELOPMENT OF EPIDEMICS].

The paper gives the results of a comparative analysis of the prerequisites for the emergence and spread of epidemics of particularly dangerous infections, by using plague in Manchuria (1910-1911) and Ebola virus disease in West Africa (2014-2015) as examples. Analysis of literature and archival data and online information could reveal a number of common factors and conditions, which substantially contributed to the epidemics. Organization of anti-epidemic (preventive) measures in cases of the threatening epidemic spread, of particularly dangerous diseases must be based on the minimization, of the influence of the specific factors and conditions, which facilitate disease transmission in a given area in a given time.

Tick-borne encephalitis virus isolates from natural foci of the Irkutsk region: clarification of the genotype landscape.

The Irkutsk region is the unique territory where all known subtypes of tick-borne encephalitis virus (TBEV) circulate. In the last years, the phenomenon of changes in TBEV subtypes (substitution of the Far-Eastern subtype by the Siberian one) was noted in some regions of the Russian Federation. The results of individual investigation of 11522 Ixodes persulcatus ticks and brain specimens from 81 small mammals collected in natural foci of the Irkutsk region during 2006-2014 are presented in the article. More than 60 TBEV strains have been isolated and studied by virological methods; E gene fragments (1193 b.p.) of 68 isolates have been typed. The majority of the strains (irrespective of subtype) were of high virulence for laboratory mice (LM) in case of both intracerebral and subcutaneous inoculation of virus. All isolates from warm-blooded small mammals and humans were of high virulence for LM, but placed in the same clusters of the phylogenetic tree with ticks collected in the same area. Tick-borne strains of different virulence also did not form separate clusters on the tree. Phylogenetic analysis showed that modern TBEV genotypic landscape of the studied territory is changing toward absolute predominance of the Siberian subtype (94.1%). This subtype is represented by two groups with prototype strains "Zausaev" and "Vasilchenko". The "Vasilchenko" group of strains is spread on the whole territory under study; the strains of "Zausaev" group were isolated previously in the Irkutsk suburbs. The European subtype of TBEV circulates in natural foci of Pribaikalie permanently (at least 5% of the random sampling); the strains are of high virulence for LM. The Far-Eastern TBEV subtype was not found within the group of isolates collected in 20062014. The phylogenetic relationship of the strains under study had a higher correlation with the place of isolation than with the year or source.

[POLYMORPHISM IN THE PHENOTYPIC STRUCTURE OF A POPULATION OF TAIGA TICK AND ITS EPIDEMIOLOGICAL SIGNIFICANCE].

The paper presents the results of 10-year (2005-2014) observations of an Ixodespersulcatus Schulze population. The purpose of this investigation was to trace long-term changes in the structure of the taiga tick population from the proportion of specimens with external skeletal anomalies and to assess a relationship between the pattern of imago phenotypic variation and the virus percentage of a carrier. There were a total of reports of the external skeletal structure of 1123 females gathered from plants to a flag in an area at 43 km from the Baikal Road connecting Irkutsk and the settlement of Listvyanka (Irkutsk Region). The proportion of specimens with anomalies averaged 37.8 +/- 1.88%. Four-to-seven varying anomalies were annually recorded. There was a preponderance of scutum impairment (an average of 17.0 +/- 3.08% of all females) that was a conglomerate of prominences and indentations along the entire clypeus surface and that was denoted P9. The nature of a change in the proportion of ticks with two anomalies (average monthly registration rate, 2.5 +/- 0.66%) is exhibited by three-year high-frequency oscillations whereas the specimens with P9 anomalies fail to show so clear cycling. The percentage of virus-containing taiga ticks was individually determined estimating the level of tick-borne encephalitis virus antigen by an enzyme immunoassay. A total of 4022 ticks were examined. The male and female data were pooled. There was a positive correlation between the change in the proportion of females with P9 anomaly and the infection of ticks in the examined population (Spearman's correlation coefficient, 0.88; P < 0.01). This supports the earlier observation of the greater epidemiological significance of the imago of a taiga tick with external skeletal anomalies particularly with considerably marked ones.

[Monitoring of enterovirus circulation in Irkutsk region].

AIM: Monitoring of circulation of enteroviruses (EVI) in Irkutsk Region and study of regional specter of circulating enteroviruses. MATERIALS AND METHODS: 1419 samples from patients with suspected EVI, contact in foci ofenterovirus infection, acute intestine infections and 964 samples of sewage water were studied in total. In 2011 isolation of viral agents from 97 samples positive on enterovirus by RT-PCR from patients with preliminary EVI diagnosis and 5 samples of sewage water of Irkutsk city was carried out. Transplantable line of human rhabdomyosarcoma RD cell culture was used for isolation of enteroviruses. Infection of cells and 2 serial passages of the studied material were carried out. The isolates were typed in neutralization reaction (NR) with a set of 32 diagnostic type-specific immune sera against viral poliomyelitis I-III; Coxsackie B1-6; Coxsackie A2, A4, A7, A9, A10; ECHO 68 - 71; ECHO 2, 4, 7, 8, 9, 12, 16, 20, 25, 26, 27, 29, 31, 33. RESULTS: In 2011 circulation of enterovirus serotypes that were previously absent on the territory of the region was established: ECHO 68, ECHO 70, ECHO 71. These strains were isolated from patients, circulation of ECHO 70 serotype was established also in samples of sewage water. The analysis of enterovirus landscape carried out showed the possibility of complication of epidemic situation on the territory of the region due to change of serovariants of causative agents of non-polioenterovirus infections and detection ofepidemically significant enteroviruses - ECHO 68, 70 and 71 serotypes. CONCLUSION: Determination of specter ofenterovirus serotypes, detection of serotypes that had not previously circulated in Irkutsk Region allows to prognose epidemic situation on morbidity of enterovirus infections and timely develop and make decisions for ensuring epidemiologic welfare of the population.

[Epidemiological situation and prophylaxis of zoonotic and natural-focal infectious diseases in Siberia and the Far East].

Analysis of zoonotic and natural-focal infectious disease morbidity in 2009 - 2011 in Siberia and the Far East is presented, and a complex of measures aimed at their prophylaxis is proposed. Analysis is carried out based on the data received by Reference Center of Monitoring of Natural-Focal Infection Causative Agents and Regional Center of Monitoring of I-II Pathogenicity Group Causative Agents at the Irkutsk Research Institute of Plague Control from departments and Centers of Hygiene and Epidemiology of Siberian, Far Eastern, 3 subjects of Urals Federal District and 5 Stations of Plague Control of Federal Service for Control in the Sphere of Protection of Consumers' Rights and Well-Being of Humans. In the morbidity structure in this region "tick-borne" infections were established to predominate--69.4%, among bacterial--yersiniosis dominates. Deterioration of epizootic situation on rabies is observed in the Republics of Tuva and Buryatia.

[Polytypic strains in the genofund of tick-borne encephalitis virus].

Eighteen polytypic tick-borne encephalitis virus (TBEV) strains containing the fragments of E and NS1 protein genes of Siberian and Far Eastern, occasionally Siberian and European subtypes were isolated in the European and Asian parts of the tick-borne encephalitis (TBE) area. They were identified using real-time polymerase chain reaction, hybridization-fluorescence detection with genotype-specific probes, restriction fragment length polymorphism analysis, and E protein sequencing. The polytypic strains were isolated from individual Ixodes persulcatus ticks, their pools, from the blood of patients and the brain of dead patients. The isolation rates of the polytypic strains in the sympathry area of different TBEV subtypes ranged from 4.4% (the Irkutsk Region) to 15.1% (the Yaroslavl Region). In addition to 2 polytypic strains, a strain similar to the TBEV 886-84 strain was isolated. The TBEV subtypes entering into the composition of the polytypic strains show nongenetic interactions, such as neutral replication or competition. The polytypic strains are stable during passages in the cultured pig embryo kidney epithelial cells and on cloning. Mouse brain passage promotes dissociation of polytypic strains. The conditions for the formation of polytypic strains and their role in the etiology of TBE are discussed.

[Evolution of tick-borne encephalitis and a problem of evolution of its causative agent].

The evolution of tick-borne encephalitis (TBE) is marked by the expanded nosological area, the transformation of landscapes, the formation of anthropurgic foci, the change of environmental systems, the increase of mortality rate mainly among urban dwellers, as well as pathomorphism. The evolution of natural TBE virus (TBEV) populations was studied in Eastern and Western Siberia, Middle Urals, and the European part of the nosological area. The paper first describes the types of evolutionary transformations of viral populations under the conditions of a varying environmental and epidemiological situation. These include: 1) the change of TBEV subtypes over 50-60 years; substitution of the Far-Eastern subtype for its Siberian subtype (the Sverdlovsk and Kemerovo regions); 2) the steady-state circulation of one Siberian subtype with mutanttypes being accumulated (the Vologda region); 3) co-existence of the Far-Eastern and Siberian subtypes with the common vector Ixodes persulcatus (the Yaroslavl and Irkutsk regions, etc.); 4) original mixed TBEV strains including the gene sites of proteins E and NSI of two subtypes. There is new evidence that the Siberian subtype is able to induce focal TBE forms, leading to death.

[Dynamics of expression of Marburg and Ebola virus antigens in infected Vero cells ]. / Dinamika ékspressii antigenov virusov Marburg i Ebola v infitsirovannykh kletakh Vero.

Time course of Marburg and Ebola virus antigens expression in Vero cells was studied by indirect immunofluorescence test. The maximum accumulation of virus specific antigens in Vero cells infected with a high dose was observed after 48-54 h of incubation. It is essential for laboratory diagnosis that virus specific antigens can present as incorporations of different shape and size, starting from small hardly discernible granules (immediately after the virus adsorption) to large lumps, cords, accumulations, and diffuse fluorescence.

[Ebola virus reproduction in cell cultures]. / Reproduktsiia virusa Ebola v kletochnykh kul'turakh.

Ebola-Zaire virus production in Vero and BGM cells was studied. The CPE developed in both cell cultures. The cell monolayer destruction by 80-90% was seen at a low multiplicity of infection in 7-8 days after virus inoculation. An overlay composition was developed for virus titration using plaque assay. The plaque production was shown to be directly proportional to the virus dose. The curve of Ebola virus production in Vero cell culture fluid was determined. At a multiplicity of infection of 0.01 PFU/cell, the maximum virus titer of 10(6.4) PFU/ml was reached in 7 days postinfection. Specific antisera were generated by inoculation of guinea pigs. Indirect immunofluorescent assay was used for testing of virus-specific antigen and antibody.