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BACKGROUND: In terms of serological properties and immunization, the wild type of HBsAg HBV and its G145R mutant behave as different antigens. This testifies to serious structural changes, which presumably could have a significant impact on the morphogenesis of virions and subviral particles. Nevertheless, morphological and ultrastructural investigations of HBV with G145R mutation have not been carried yet. OBJECTIVES: Research of structural and morphological organization of HBV in the presence of the G145R escape mutation. METHODS: Studies of sera, purified viruses and recombinant HBsAg were carried out by transmission electron microscopy by the method of negative staining and indirect reaction of immunelabeling using monoclonal antibodies of different specificity. Specimens of wild type HBV and HBV with S143L mutation obtained in an identical manner were used as the control. RESULTS: The presence of typical virus particles of HBV was shown in the specimens of wild strain and HBV with S143L mutation. Specimens of HBV with G145R mutation were characterized by expressed morphological heterogeneity. In the initial serum and in the specimen of purified virus containing G145R mutant, large oval particles 60-70 nm and up to 200 nm in size, respectively, were found. The presence of antigen structures of HBV in all heterogeneous forms was confirmed. It was shown that forming of subviral particles in the process of expression of the recombinant HBsAg with G145R mutation depends on conditions of expression and purification of the protein. They can vary from well-formed circular and oval particles to practically unstructured fine-grained masses. CONCLUSION: Direct data on the impact of G145R escape-mutation in S-gene, in contrast to S143L mutation, on the morphogenesis of virions and subviral particles of HBV were obtained.

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Comparative study of widely distributed on Russian market commercial test-systems for HBsAg detection by enzyme immunoassay was performed. Panel of serum samples containing mutant forms of HBsAg was developed for the study. It showed that only 2 out of 7 studied test-systems are able to detect mutant forms of HBsAg with the same sensitivity as "wild-type" forms of HBsAg. Test-systems based only on monoclonal antibodies did not detect HBsAg with G145R substitution in concentration 5 ng/ml. The study confirms the relevance of problem of HBsAg-mutants detection for hepatitis B immunodiagnostics.

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Mice monoclonal antibodies against lypopolysaccharides (LPS) of Brucella abortus has been obtained and characterized. The antibodies detected LPS of B. abortus, B. melitensis and B. suis with high sensivity and specificity and did not react with LPS of Yersinia enterocolitica O:3, Y. enterocolitica O:9, Salmonella typhimurium, and Francisella tularensis. It has been shown that interaction of monoclonal antibodies and LPS of Brucella species can be critically dependent from buffer system. Obtained monoclonal antibodies allowed to develop highly sensitive assay which was able to detect antigens of Brucella species in concentrations 0.05 - 0.1 ng/ml. The assay can be used for detection and identification of Brucella species.

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Algorithm of serologic screening for HBsAg-mutants in hepatitis B virus (HBV) carriers with high level of HBsAg was developed which is based on the detection of defects of interactions of serum HBsAg with monoclonal anti-HBs realizing as a decrease of ELISA sensitivity in 10 times or more during serial 10-fold dilutions. During 1st stage commercial test-systems based on monoclonal antibodies was used to select serum samples with discrepancy of test results. During 2nd stage HBsAg contained in selected sera was analyzed by the panel of monoclonal and polyclonal anti-HBs conjugates using decrease in ELISA sensitivity as a criterion. Serum samples from 2510 chronic carriers of HBV with high level of HBsAg were studied. 19 samples with discrepant results were found. Subsequent characterization of HBsAg with panel of 11 monoclonal and 1 polyclonal conjugates allowed to distinguish groups of sera with specific serologic "portraits". Atypical features of HBsAg were confirmed by genotyping 9 of 19 samples. Analysis of primary nucleotide sequence revealed serologically meaningful mutations in S-gene of HBV in all 9 isolates: 3 of them contained substitution mutation G145R, 5--S143L, and one--T143M. Distribution of mutations in HBsAg corresponded with specific serologic "portraits". Prevalence of HBsAg mutations in HBV carriers with high level of HBsAg was assessed for the first time: prevalence of G145R, S143L/T143M mutations, and all serologically atypical variants was 0.12%, 0.24%, and 0.76% respectively. Developed algorithm was proposed for epidemiologic monitoring of HBsAg-mutants of HBVand control of diagnostic test-systems.

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