[Identification by enzyme immunoassay of escape mutants S143L and G145R of hepatitis B virus (Hepadnaviridae: Orthohepadnavirus: Hepatitis B virus)].

INTRODUCTION: The achievement of the goal of the World Health Organization to eliminate viral hepatitis B by 2030 seems to be problematic partly due to the presence of escape mutants of its etiological agent, hepatitis B virus (HBV) (<i>Hepadnaviridae: Orthohepadnavirus: Hepatitis B virus</i>), that are spreading mainly in the risk groups. Specific routine diagnostic assays aimed at identification of HBV escape mutants do not exist.The study aimed the evaluation of the serological fingerprinting method adapted for routine detection of escape mutations in 143 and 145 aa positions of HBV surface antigen (HBsAg). MATERIAL AND METHODS: HBV DNA from 56 samples of HBsAg-positive blood sera obtained from donors, chronic HBsAg carriers and oncohematology patients has been sequenced. After the identification of mutations in HBsAg, the samples were tested in the enzyme-linked immunosorbent assay (ELISA) kit «Hepastrip-mutant-3K¼. RESULTS AND DISCUSSION: Escape mutations were detected mainly in patients with hematologic malignancies. Substitutions in 143 and 145 aa were found in 10.81% and in 8.11% of such patients, respectively. The G145R mutation was recognized using ELISA kit in almost all cases. The kit specifically recognized the S143L substitution in contrast to the S143T variant. The presence of neighbor mutation D144E can be assumed due to it special serological fingerprint. CONCLUSION: ELISA-based detection of escape mutations S143L, D144E and G145R can be used for routine diagnostics, especially in the risk groups. The diagnostic parameters of the kit can be refined in additional studies. This immunoassay and methodology are applicable for the development and quality control of vaccines against escape mutants.

[Detection of Epstein-Barr virus genome in oral cavity squamous cell carcinoma samples of russian patients.]

INTRODUCTION: Oral cavity squamous cell carcinoma (OC-SCC) is the most common and aggressive malignancy of the oral cavity. Recent studies have revealed infections with human papilloma virus (HPV) as an additional risk factor for oral squamous cell carcinoma development, while distinguished role of Epstein-Barr virus (EBV) remains still uncertain. However, the evidence for association between virus infection and risk of oral squamous cell carcinoma is controversially and varies significantly by geographic regions and race. PURPOSE: The aim of the present study was to elucidate the prevalence of HPV and EBV in OC-SCC samples of Russian patients from Moscow region. MATERIAL AND METHODS: We investigated fresh-frozen tumor tissue fragments obtained from 11 patients with OC-SCC. DNA was extracted and the viral genome was examined by quantitative PCR assays with highrisk type-specific HPV and EBV specific markers followed by sequencing-based analysis. RESULTS: No HPV infection in analyzed OC-SCC samples was observed, while EBV was identified in 70.0% (7/10) of patients. Further based on Q-PCR amplification of the EBV targets including BamHI-W, EBNA1 and C-terminal fragment of LMP1 gene, EBV infection and measurement of virus load in the tumor samples was assessed. Sequencing LMP1-positive products revealed that the most samples (5/6) contained variants LMP1 with Cao deletion characterized by an increased transforming potential. CONCLUSION: These data suggest that prevalence of EBV infections is common and may influence cancer development, although detected LMP1 variants of EBV are not necessarily associated with the pathogenesis of OC-SCC. Further studies are necessary to determine the potential role of EBV and its possible importance as an infection factor in OC-SCC.

[Antiviral effect of «Kagocel¼ substance in vitro on influenza viruses H1N1, H1N1pdm09 and H3N2.]

INTRODUCTION: Active circulation of pandemic influenza and new variants of influenza H3N2 strains requires monitoring of antiviral efficacy of drugs permitted for influenza therapy in the Russian Federation. PURPOSE: Assessment of antiviral efficacy of «Kagocel¼ substance against influenza viruses H1N1, H1N1pdm09 and H3N2 in vitro. MATERIAL AND METHODS: Cytotoxic effect of «Kagocel¼ substance on MDCK cells had been determined by stained with MTS. Antiviral efficacy of «Kagocel¼ substance against influenza infection has been studied in vitro in the culture of MDCK cells infected with influenza virus strains: A/Puerto Rico/8/34 (H1N1), А/California/7/2009 (H1N1)pdm09, А/Hong Kong/1/68 (H3N2) and А/ Hong Kong/4801/2014 (H3N2). The antiviral activity of «Kagocel¼ substance was tested by its effect on the infectious titer of the influenza viruses and on its impact on the expression level of viral antigens in the enzyme immunoassay test system. RESULTS: «Kagocel¼ substance had low toxicity for MDCK cells. «Kagocel¼ inhibited the infection titer of influenza virus strains A/Puerto Rico/8/34 (H1N1), А/California/7/2009 (H1N1)pdm09, А/Hong Kong/1/68 (H3N2) and А/ Hong Kong /4801/2014 (H3N2) in the MDCK cell culture with equal efficacy. Study of the impact of «Kagocel¼ substance on the expression level of viral antigens by ELISA also revealed its antiviral efficacy for all tested strains. Dose dependence was observed from concentration of substance and from infective dose of virus. DISCUSSION: Effective suppression of the reproduction of influenza virus strains A(H1N1), A(Н1N1)pdm09 and A(H3N2) in the different sublines of MDCK cells with «Kagocel¼ was shown by the different methods. These results give the possibility to suggest that along with the ability to induce interferons, «Kagocel¼ can impact on the reproduction of influenza virus, but the further research is needed. CONCLUSION: «Kagocel¼ substance effectively inhibits the reproduction of influenza virus strains A(H1N1), A(Н1N1)pdm09 and A(H3N2) in vitro. At the same time, the selectivity index is quite high.

Pretreatment with Antiviral Preparation Kagocel Normalizes the Content of Bone Marrow Multipotent Stromal Cells and TNFα in Blood Serum of CBA Mice Disturbed by Administration of S. typhimurium Antigen Complex In Vivo and Maintains High Concentration of IL-10 and Th1 Cytokines.

Pretreatment with the active substance of antiviral preparation Kagocel, inductor of type I endogenous IFN, in a daily therapeutic dose (30 µg/mouse) 3 h prior to administration of S. typhimurium antigens to CBA mice reduced the number of bone marrow multipotent stromal cell (significantly increased by 3.2 times on the next day after antigen injection) to the initial level. Thus, activation of the stromal tissue induced by administration of the bacterial antigen was blocked. In addition, preliminary administration of Kagocel modulated the cytokine profile of the blood serum affected by S. typhimurium antigens: reduced 1.6-fold elevated concentration a proinflammatory cytokine TNFα to the control level (in 4 h after antigen injection) and maintained this level in 20 h after antigen administration. Kagocel also maintained the concentration of anti-inflammatory cytokine IL-10 at the level surpassing the normal by 1.6 times and high concentrations of Th1 cytokines (IL-2, IFNγ, and IL-12). These results suggest that Kagocel can reduce the immune response to bacterial antigens (similar to type I IFN [7]), which can contribute to its therapeutic and preventive effects in addition to its well documented antiviral activity and then this preparation can be used for the therapy of diseases accompanied by excessive or chronic inflammation.

MORPHOLOGICAL ANALYSIS OF HEPATITIS B VIRUS WITH ESCAPE MUTATIONS IN S-gene G145R AND S143L.

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.

Effects of Kagocel® on the Counts of Multipotent Stromal Cells, Expression of Cytokine Genes in Primary Cultures of Bone Marrow Stromal Cells, and Serum Cytokine Concentrations in CBA Mice.

The efficiency of cloning of bone marrow multipotent stromal cells (ECF-MSC) from CBA mice and the MSC counts in the femoral bone increased 24 h after a single in vivo (but not in vitro) injection of kagocel (active substance of antiviral drug Kagocel (®) ) 1.4 times (in response to 50-80 µg) and 4.6 times (in response to 250 µg). The maximum increase of ECF-MSC in response to 50 µg per mouse was detected just 1 h after Kagocel injection to intact mice and to mice previously receiving the drug for 3 days (2 and 1.7 times, respectively). The increase of ECF-MSC was 3-fold less intense in response to oral Kagocel in a dose of 250 µg/mouse vs. intraperitoneal Kagocel, ECF-MSC corresponding to its level in response to oral Poly (I:C). In vivo Kagocel led to emergence of proinflammatory cytokine IFN-γ, IL-1ß, and IL-8 mRNA in primary cultures of bone marrow stromal cells. Serum concentrations of IL-2, IL-5, IL-10, GM-CSF, IFN-γ, TNF-α, IL-4, and IL-12 increased 1.5 and 2 times just 1 h after Kagocel injection in doses of 30-50 and 250 µg, respectively, to intact mice and to animals previously treated with the drug for 3 days. The cytokine concentrations normalized after 3 h and increased again after 24 h, though did not reach the levels recorded 1 h after the drug injection. These data indicated that the therapeutic and preventive effects of Kagocel, together with its previously demonstrated stimulation of α- and ß-interferon production during several days, could be due to the capacity of this drug to increase the bone marrow ECF-MSC, serum cytokine concentrations, and induce the expression of proinflammatory cytokine genes in the bone marrow stromal cells 1 h after its injection.

[Role of macrophage migration inhibitory factor in systemic inflammatory response and development of sepsis].

I.I Mechnikov was the first who characterized phagocytosis as cellular defense mechanism. Infiltration of infectious process focus with phagocytes and subsequent activation of these cells is a fundamental defense reaction of the organism. However inflammation may be destructively dangerous if inflammatory response prolongates and/or generalization of the process leading to death of the host develops. The main trigger mechanisms in the pathogenesis of systemic inflammatory response and sepsis are release of bacterial endo- and exotoxins as well as hyperproduction of proinflammatory cytokines. Macrophage migration inhibitory factor (MIF) has exceptional multifunctionality and significant potential for activation of inflammatory system by various mechanisms acting as proinflammatory cytokine, hormone-contaregulator of immunosuppressive effect of corticosteroids and regulator of glucose metabolism. Data about the role of MIF as a crucially dangerous factor in pathogenesis of systemic inflammatory response obtained on experimental models of sepsis as well as about the efficacy of anti-MIF therapy were discussed, specific molecular mechanisms were analyzed. Prognostic value of high blood concentration of MIF during septic complications in clinic situations was assessed. In general, existing data on key role of MIF in sepsis pathogenesis show that MIF is one of the most promising targets for development of new strategies of immunotherapy for this life-threatening pathology.

[Assessment of sensitivity of commercial test-systems for HBsAg immunodetection according to their ability to detect HBsAg-mutants of hepatitis B virus].

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.

[Highly sensitive enzyme-linked assay based on monoclonal antibodies for detection of Brucella antigens].

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.

[Algorithm of serologic screening and assessment of prevalence of serologically meaningful mutations of HBsAg in hepatitis B virus carriers].

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.