High degree of genetic diversity of non-polio enteroviruses identified in Georgia by environmental and clinical surveillance, 2002-2005.

Enterovirus surveillance data are useful for establishing temporal and geographical patterns of circulation and for virus characterization to determine phylogenetic relationships between strains. Almost no information is available on circulating enteroviruses in Georgia and the surrounding region. To describe enterovirus circulation in Georgia, determine relationships with previously characterized strains and assess the role of environmental and clinical enterovirus surveillance, this study analysed a total of 112 non-polio enterovirus isolates identified during 2002-2005 from sewage and human stool samples. Viruses were isolated in cell culture using standard methods and typed by partial sequencing of the VP1 gene. A total of 20 different non-polio enterovirus serotypes were identified over the 4-year period. The most commonly detected enteroviruses included echovirus (E) 6 (21 isolates; 18.8 %), E20, E3 and E7 (11 isolates each; 9.8 %), E11, coxsackievirus (CV) B4 and CVB5 (seven isolates each; 6.3 %), and E13, E19 and E30 (six isolates each; 5.4 %). Phylogenetic analysis showed that many serotypes were represented by more than one genetic lineage. The present study showed a very high degree of enterovirus diversity in Georgia and demonstrated the added value of environmental enterovirus surveillance, particularly in settings with limited clinical surveillance. Several serotypes would not have been detected without having both clinical and environmental surveillance in place. Several serotypes detected in Georgia were among those rarely reported in the USA and Europe (e.g. E3, E20 and E19). As the emergence of new genetic lineages of enterovirus in a particular area is often associated with large-scale outbreaks, continued monitoring of enterovirus strains by both environmental and clinical surveillance and genetic characterization should be encouraged.

[Polyclonal immunoenzyme system for determining hantaviral antigens: assessment of specificity using monoclonal antibodies and the polymerase chain reaction]. / Poliklonal'naia immunfermentnaia sistema dlia opredeleniia antigenov khantavirusov: otsenka spetsifichnosti s pomoshch'iu monoklonal'nykh antitel i polimeraznoi tsepnoi reaktsii.

Genus-specific monoclonal antibodies to nucleocapsid protein of Pumala virus and polymerase chain reaction were used to assess the specificity of a previously described polyclonal (based on human IgG to Pumala hantavirus) enzyme immunoassay (EIA) system for detecting hantavirus antigens. Parallel testing of crude lung suspensions from wild rodents trapped in different regions of Russia for hantavirus antigens and RNA showed 100% coincidence of the results of three tests. These data indicate a high specificity of polyclonal EIA system and its efficacy for screening of natural samples.

[Genetic differentiation of hantaviruses using the polymerase chain reaction and sequencing]. / Geneticheskaia differentsiatsiia khantavirusov s pomoshch'iu polimeraznoi tsepnoi reaktsii i sekvenirovaniia.

Thirty-two hantavirus strains and 8 samples of lung tissue from rodents collected in different regions of Russia have been examined by molecular biological methods. Two methodological approaches have been employed for the study of genetic relationships between the viruses: nested PCR assay and common RT-PCR with subsequent direct sequencing of 200 and 365 base pair of G2 protein encoding regions of M-segment, respectively, and the resultant sequences were compared with those of the prototype hantavirus. The study revealed a mosaic pattern of distribution of different hantavirus genotypes on the territory of Russia.

[Hybridomas producing monoclonal antibodies to Crimean hemorrhagic fever virus]. / Gibridomy, produtsiruiushchie monoklonal'nye antitela k virusu Krymskoi gemorrhagicheskoi likhoradki.

A series of hybridomas producing monoclonal antibodies to Crimean hemorrhagic fever virus was obtained and their cultural properties were characterized. HEMA-12 and HEMA-24 secreted monoclonal IgG2b antibodies, HEMA-101 secreted monoclonal IgG1 antibodies, HEMA-31, HEMA-9 and HEMA-11 secreted monoclonal IgG2 antibodies. According to the results of the indirect immunofluorescence test, the titer of specific immunoglobulins in the culture fluid was 1:16-1:32, but sometimes reached 1:64-1:128. The titer of antibodies in ascitic fluid amounted to dilutions of 10(4)-10(5). Hybridomas were cloned by the method of ultimate dilutions. The injection of 5-15 million HEMA cells into the abdominal cavity of BALB/c mice induced the formation of ascitic tumors in the animals within 7-11 days and the accumulation of ascitic fluid in a volume of 1-4 ml. Hybridomas, found to be capable of passage from mouse to mouse, underwent 5-16 passages during the term of observation.

[Characteristics of the monoclonal antibodies induced by the Crimean hemorrhagic fever virus]. / Kharakteristika monoklonal'nykh antitel, indutsirovannykh virusom krymskoi gemorragicheskoi likhoradki.

Twelve lines of hybridomas secreting monoclonal antibodies (MCA) have been generated by fusion of spleen lymphocytes of BALB/c mice immunized with crude Crimean hemorrhagic fever virus (CHF). The hybridomas multiply actively in vitro (over 50 passages) and as ascitic tumors in the abdominal cavity of BALB/c mice. MCA were characterized by indirect immunofluorescence (IF), complement fixation (CF), biological neutralization test (NT), agar gel diffuse precipitation tests, and by the type of immunoglobulins. In the indirect IF, all kinds of MCA reacted with CHF virus, in CF only GEMA 12 and GEMA 31 which also precipitated the virus antigen in AGDPT. The CF-positive MCA belonged to the IgG2a and IgG2b subclasses, but 2 more MCA species of the same immunoglobulin type did not react in CF. No neutralizing properties have been found with MCA. All MCA reacted similarly in indirect IF and CF both with CHF and Congo viruses. Among other members of the CHF-Congo antigenic group, only GEMA 4 MCA interacted with Hazara virus. MCA generated in this study are intended for use in identification of CHF and Congo viruses in diagnostic tests.

[Monoclonal antibodies to flavivirus antigens induced by a vaccinal strain of yellow fever]. / Monoklonal'nye antitela k flavivirusnym antigenam, indutsirovannye vaktsinnym shtammom zheltoi likhoradki.

Monoclonal antibodies (MABs) YEL-2 induced by the vaccine FNS Dakar yellow fever (YF) virus were characterized for their capacity to enter into serological reactions and to react with heterologous flaviviruses. YEL-2 MABs belong to the IgG2a class of immunoglobulins, possess the antihemagglutination properties, are active in indirect IF test but do not activate complement and have no neutralizing properties. The inability to enter into CFT in the presence of antihemagglutinating properties suggests that YEL-2 MABs are directed for the structural E glycoprotein. YEL-2 MABs reacted similarly with the vaccine 17D strain and the FNS Dakar strain by which they had been induced. In addition to YF virus, YEL-2 MABs reacted with Tyuleniy, Rosio, Ilhéus, Uganda S, Karshi, and Sokuluk viruses, the reaction with Tyuleniy virus reaching the same titer as with the homologous virus but was of one-way nature. No reaction of YEL-2 MABs was observed with the viruses of the tick-borne encephalitis complex, Japanese encephalitis, West Nile, Dengue 2 and 4 viruses. These results specify the antigenic classification of flaviviruses.

[Monoclonal antibodies to the yellow fever virus]. / Monoklonal'nye antitela k virusu zheltoi likhoradki.

A highly technological Yel-2 hybrid cell clone was isolated. The clone produces monoclonal antibodies to protein E of the yellow fever virus (Dakar strain). The Yel-2 hybridoma was cloned by the method of limiting dilutions. By the data of indirect immunofluorescence the titre of the specific antibodies reached 1:128 in the culture fluid and 1:10240 in the ascitic fluid. The Yel-2 antibodies had no cross reactions with forest-spring encephalitis, Dengue 2 and Dengue 4 viruses. In the hemagglutination inhibition test the activity of the Yel-2 ascitic preparations was observed at a dilution of 1:50000 while the complement fixation test did not reveal it. Administration of the Yel-2 cells into the abdominal cavity of mice BALB/c induced development of ascitic tumors in 97 per cent of pristane-sensitized mice and in 90 per cent of nonsensitized mice. The hybridoma was successfully transplanted from mouse to mouse (more than 20 passages during the observation period) and maintained high constant levels of specific antibody secretion. The high ascite forming capacity of the Yel-2 hybridoma provided decreasing of the cell dose administered to the mice from 10(7) to 10(4) cells per mouse.