[Persistence of antibodies specific to West Nile virus in blood of reconvalescents of Volgograd region].

AIM: Determine the duration of persistence of IgM and IgG in reconvalescents of West Nile fever (WNF) 1 year after the disease in southern regions of Russia and evaluate effectiveness of PCR method for acute infection diagnostics. MATERIALS AND METHODS: Blood sera of 87 patients with WNF diagnosis was studied for the presence of West Nile virus (WNV) RNA and IgM and IgG by PCR and EIA. Samples of the first sera were collected in 2010 at days 2 - 30 after the onset of the disease, samples of the second sera--at days 5 - 23 and third--264 - 385 days later. RESULTS: During the first 2 weeks of the disease WNV RNA was detected in more than 50% of patients. In all the first sera IgM at titers of ≥ 1:800 were detected. Seroconversion of IgG titers of 4 and more times was observedin 83% (30/36) of patients. In 2011 IgG were detected in 91% of reconvalescents (79/87), IgM--in 57% (50/87), and in 25% (22/87) IgM titers were ≥ 1:800. CONCLUSION: The results obtained give evidence on the necessity of using several diagnostic criteria simultaneously for the confirmation of WNF clinical diagnosis.

[The tick-borne encephalitis virus detection efficiency in the Ixodid ticks (Acari: Ixodidae) with ELISA and real-time PCR].

According to the data of the Federal Service on Customers' Rights Protection and Human Well-being Surveillance, the enzyme-linked immunosorbent assay (ELISA) detects the tick-borne encephalitis virus (TBEV) in ticks more often than the polymerase chain reaction (PCR). The goal of this work was to compare TBEV detection efficiency in the ixodid ticks of different species with the commercial kits based on ELISA and real-time PCR. Ticks of five species were parenterally infected with 2-6 IgPFU of the European or Siberian TBEV subtypes. We formed randomized and encoded series of infected and intact ticks of different species, and in "blind" experiment analyzed the ticks on the TBEV presence with the kits based on ELISA and real-time PCR. ELISA and real-time PCR effectiveness of the TBEV detection in ticks was not affected by gender, species of ticks or presence of blood meal. The kits based on ELISA were less sensitive than those based on real-time PCR. ELISA effectiveness depended on the TBEV subtype. The presence of the false positive reactions and sensitivity of ELISA were affected by the protocols of reaction. The problem of the different TBEV prevalence in the field-collected ticks obtained with various methods remains to be studied.

[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.

[Interaction of the Siberian and Far Eastern subtypes of tick-borne encephalitis virus in mammals with mixed infection. Competition of the subtypes in acute and inapparent infection].

Long-term monitoring of natural tick-borne encephalitis virus (TBEV) populations could reveal the change of TBEV subtypes, the displacement of the Far Eastern (FE) subtype, and its substitution for the Siberian (Sib) subtype. Acute and inapparent mixed infections were studied in Syrian hamsters to understand this phenomenon. The animals were inoculated with the Sib subtype and then with the FE one of TBEV (JQ845440-YaroslavI-Aver-08 and Fj214132-Kemerovo-Phateev-1954 strains). The inapparent form developed more frequently in mixed infection. Viral progeny was genotyped by reverse transcription polymerase chain reaction and hybridization fluorescence detection using genotype-specific probes. Independent reproduction of strains in the brain gave way to competition. The FE subtype dominated in hamster youngsters with acute infection. The Sib subtype had selective benefits in asymptomatic infection (adult hamsters infected intracerebrally and subcutaneously and youngsters infected subcutaneously). The competition of the subtypes was imperfect.

[Genotyping of West Nile fever virus strains circulating in southern Russia as an epidemiological investigation method: principles and results].

AIM: Characteristic of West Nile fever (WNF) virus strains circulating in southern Russia. MATERIALS AND METHODS: WNF RNA was amplified directly from clinical samples, mosquitoes and bird tissues by PCR, nucleotides were sequenced directly and analyzed comparatively. RESULTS: Related but different genovariants of WNF lineage 1a--"Volgograd" and "Astrakhan"--circulated during WNF outbreaks of 1999 and 2000-2003 in Volgograd and Astrakhan regions. In 2005 "Volgograd" WNF variant emerged in Astrakhan region and along with "Astrakhan" variant caused a new morbidity increase. In 2004 in sera of 2 WNF patients from Rostov region WNF lineage 2 RNA was detected, this was the first WNF clinical case caused by WNF lineage 2 outside of Africa. WNF outbreak in Volgograd region in 2007 was caused by this unique WNF lineage that may preliminary be called Russian. Finally, during a major WNF outbreak in 2010 in Volgograd and Rostov regions in clinical samples only russian genovariant WNF lineage 2 RNA was detected again. CONCLUSION: After emergence of a certain WNF genovariant the virus is capable of persisting in natural foci in southern Russia. A near disappearance of one of the WNF clones by substitution or displacement with another maybe possible. Determination of genetic characteristics of WNF strains circulating in Russia is an important element of WNF epidemiological surveillance and control of this disease.

[Usage of real time polymerase chain reaction for diagnostics of different tick-borne infections].

AIM: To create and test the complex of polymerase chain reaction-based methods for detection of pathogens vectored by ticks in clinical and environmental samples. MATERIALS AND METHODS: Real time PCR methods with hybridization-fluorescent detection were developed for detection of tick-borne encephalitis virus, Borrelia burgdorferi sensu lato, Anaplasma phagocytophillum, Erlichia muris/E. chaffeensis, and B. miyamotoi. First four methods were combined in one assay in multiprime format. Efficacy of the assay was assessed by testing of blood samples from patients with tickborreliosis (166 patients), tick-born encephalitis (22 patients) and mixed infection tick-borne encephalitis + borreliosis (21 patients) from Sverdlovsk region. RESULTS: It was shown that using PCR-based assay for testing the blood samples obtained during admission, it was possible to determine the etiology of disease in 39% of patients, whereas on the basis of serological data diagnosis, as a rule, is made not earlier than on 2nd week of therapy. False-positive results of PCR diagnostics were not observed. Infections caused by Anaplasma or Erlichia were not observed. It was shown that > 50% of cases of tick borreliosis without erythema were caused by B. miyamotoi, whereas B. burgdorferi sensu lato predominated as a causative agent of erythemic form of borreliosis. CONCLUSION: Proposed complex of methods is useful for rapid diagnostics of tick-borne infections including previously unknown infection caused by B. miyamotoi.

[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.