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The highly pathogenic avian influenza H5N1 viruses have become widespread and evolved into several clades. In our previous studies, the antigenic sites of the H5 hemagglutinin (HA) were characterized by selection and sequencing of escape mutants. In the present studies we analyzed the antigenic epitopes recognized by monoclonal antibodies against avian influenza A/Duck/Novosibirsk/56/05 (H5N1) virus isolated in western Siberia and belonging to subclade 2.2 of the H5N1 viruses. The analysis revealed several antigenically relevant positions of amino acid residues in the globular head of the HA not encountered earlier in the escape mutants of the H5 subtype. The newly recognized positions (113, 117, 118, 120, and 123, mature H5 numbering) are concentrated in an area adjacent to the region described in earlier studies as corresponding to site B in H3 HA, but extending far beyond this area. The amino acid positions recognized by the monoclonal antibodies against A/Duck/Novosibirsk/56/05 (H5N1) virus differ from the positions recognized by the monoclonal antibodies against H5N2 influenza viruses. The data suggest that the evolution of the HA of H5 avian influenza viruses is associated not only with the changes of antigenic epitopes recognized by antibodies, but also with a change in the dominance of the immunogenicity of different sites in the HA.

BackgroundThe presence of human cytomegalovirus (HCMV) in male genital tract suggests its vertical transmission with spermatozoa and the development of a potentially dangerous fetal infection. The objective of the present study was to evaluate the possibility of intracellular HCMV localization in male germ cells and to examine the effect of the virus on human spermatogenesis.MethodsSemen samples from 91 infertile and 47 fertile men were analyzed. HCMV was detected by real time PCR, rapid culture method and PCR in situ. Human testis organotypic culture and quantitative karyological analysis were used to investigate viral effects on spermatogenesis. Localization of HCMV in immature germ cells and spermatozoa was studied by immunostaining with monoclonal antibodies and ultrastructural analysis of infected organotypic culture.ResultsViral DNA was detected in 12.3% samples of motile spermatozoa, while infectious activity only in 2.9% infertile and fertile men without statistically significant intergroup difference. According to PCR in situ, the mean percentage of infected cell in both groups was 1.5% (0.25%-15%), which can serve as a criterion for evaluating the risk of HCMV transmission. In HCMV-infected organotypic culture viral antigens were identified in spermatides on day 4, in spermatogonia and spermatocytes on day 8, and in spermatozoa on day 14. Empty and full capsides and virions were visualized in germ cells by electron microscopy. The number of cells before introduction in culture was taken for 100%. On day 14 infected culture contained 36.8% spermatogonia, 18.7% spermatocytes, 27.6% round spermatides and 42.5% elongated spermatides; in comparison with 82.2%, 51.5%, 70.4% and 65.7% in uninfected culture, respectively (all p < 0.05). There were no changes in the number and viability of spermatozoa.ConclusionsHCMV was detected in male germ cells, both in sperm samples and in testis organotypic culture. The virus may infect immature germ cells which develop to mature HCMV-carrying spermatozoa. A considerable decrease in the number of immature germ cells indicates that HCMV produces a direct gametotoxic effect and can contribute to male infertility.

Hepatitis C virus (HCV) is one of the major causes of chronic liver disease and leads to cirrhosis and hepatocarcinoma. Despite extensive research, there is still no vaccine against HCV. In order to induce an immune response in DBA/2J mice against HCV, we obtained modified mouse mesenchymal stem cells (mMSCs) simultaneously expressing five nonstructural HCV proteins (NS3-NS5B). The innate immune response to mMSCs was higher than to DNA immunization, with plasmid encoding the same proteins, and to naïve unmodified MSCs. mMSCs triggered strong phagocytic activity, enhanced lymphocyte proliferation, and production of type I and II interferons. The adaptive immune response to mMSCs was also more pronounced than in the case of DNA immunization, as exemplified by a fourfold stronger stimulation of lymphocyte proliferation in response to HCV, a 2.6-fold higher rate of biosynthesis, and a 30-fold higher rate of secretion of IFN-γ, as well as by a 40-fold stronger production of IgG2a antibodies to viral proteins. The immunostimulatory effect of mMSCs was associated with pronounced IL-6 secretion and reduction in the population of myeloid derived suppressor cells (MDSCs). Thus, this is the first example that suggests the feasibility of using mMSCs for the development of an effective anti-HCV vaccine.