Study Of Glycosylation Sites On Hemagglutinin Of Pandemic H1N1/2009Influenza Virus And Pathogenicity Of H1N2Swine Influenza Virus

The two glycosylation sites (Asn142and Asn177) were observed in the HA of most human seasonal influenza A/H1N1viruses, while none in pH1N1viruses. The mutants which gained potential glycosylation sites Asn142and Asn177on HA respectively were generated from A/Mexico/4486/2009(H1N1). We investigated the effect of the two glycosylation sites on viral virulence and pathogenicity in mice using recombinant H1N1/2009, leading to the development of the glycosylation sites affect the viral pathogenicity.Currently, three predominant subtypes of influenza virus are prevalent in swine worldwide:H1N1, H3N2, and H1N2, including classical swine H1N1, avian-like H1N1, human-like H3N2, reassortant H3N2and various genotype H1N2viruses. The details about genetic characterization and reassortment events of the Chinese H1N2swine virus were not investigated systematically. In2009, two H1N2influenza viruses were isolated from trachea swabs of pigs in Hubei in China. In this study, we included these two isolates, and18other H1N2swine isolates collected from influenza database, and made an extensive analysis of long-term evolutionary patterns in Chinese H1N2swine isolates. Some reassortant viruses were discovered and their genetic parents were identified. Additionally, we also expanded upon a mouse model to study the pathogenesis and inflammatory responses of H1N2isolates, laying the foundation of prevention and control of H1N2swine influenza. The main researches were as follows:1Glycosylation on hemagglutinin affects the virulence and pathogenicity of pandemic H1N1/2009influenza A virus in miceThe H1N1/144and H1N1/177mutants which gained potential glycosylation sites Asn142and Asnl77on HA respectively were generated from A/Mexico/4486/2009(H1N1) by site-directed mutagenesis and reverse genetics, the same as the H1N1/144+177gained both glycosylation sites Asn142and Asn177. The biological characteristics and antigenicity of the mutants were compared with wild-type H1N1/2009. The virulence and pathogenicity of recombinants were also detected in mice.(1) Our results showed that HA antigenicity and viral affinity for receptor may change with introduction of the glycosylation sites. The ability of monoclonal antibodies to neutralize the virus was largely attenuated, and the time required for the elution from chicken erythrocytes were longer than H1N1/WT.(2) Compared with wild-type pH1N1, the mutant H1N1/144and H1N1/144+177can efficiently replicate in chicken embryos, and the mutant H1N1/177displayed an equivalent virus titer with H1N1/WT. The mutants displayed increased virulence and pathogenicity in chicken embryos and mice.(3) The H1N1/144and H1N1/144+177showed significantly higher virus titers than H1N1/177and H1N1/WT in mice lung. The mutants exhibited more serious virulence and pathogenicity than H1N1/WT in infected mice. The mutants and H1N1/WT did not cause any death in mice. The different proinflammatory cytokines were significantly up-regulation in prophase of infection. The H1N1/177and H1N1/144+177induced relatively higher IL-1β mRNA than did H1N1/WT. The expression of MCP-1, TNF-a, and IFN-] genes were significantly higher in mice infected with the H1N1/144+177. The production of IL-10induced by the HIN1/144and H1N1/177was higher than in other viruses. All mutants’infections produced lesions typical of influenza A virus infections: bronchiolitis with accompanying necrosis of respiratory epithelium and associated neutrophilic to histiocytic alveolitis. The mutant H1N1/144+177induced the most serious alveolar inflammation and pathogenicity in infected mice.2Genetic characterization of H1N2swine influenza virus isolated in China and its pathogenesis and inflammatory responses in miceIn2009, two H1N2swine influenza viruses, were isolated from trachea swabs of pigs that had a respiratory disease on a farm in Hubei in China.(1) In this study, we included these two isolates, A/swine/Hubei/04/2009(HB/04) and A/swine/Hubei/05/2009(HB/05), and18other H1N2swine isolates collected from influenza database, and made an extensive analysis of long-term evolutionary patterns in Chinese H1N2swine isolates. Six different genotypes, two reassortants between triple reassortant (TR) H3N2and classical H1N1swine virus, three reassortants between TR H1N2and Eurasian avian-like H1N1swine virus, and one reassortant between H1N1and H3N2human virus, were observed in these twenty swine H1N2isolates. The TR H1N2swine virus is the predominant genotype and the two Hubei H1N2isolates were located in this cluster.(2) The biological characterization of HB/04and HB/05. The logEID50/200μL of HB/04and HB/05were6.4and5.2respectively. HB/05(211) exhibited a higher viral titre (HA) on chicken embryos than HB/04(29). The MDT of HB/04and HB/05on chicken embryos were76.8h and72h. Furthermore, the log TCID50/100μL of HB/04and HB/05were both7.6.(3) We also used a mouse model to examine the pathogenesis and inflammatory responses of the two isolates. The isolates replicated efficiently in the lung. Viral shedding in the rectum of HB/04and HB/05was detected5dpi. The HB/04and HB/05viruses caused58.3%and75%survival in mice respectively. HB/04and HB/05induced significantly higher inflammatory cytokines than the PBS-mock infection including IL-1β, IL-10, TNF-α and IFN-y. The expressions of IL-1β and IL-6on2and5dpi were higher than on7and9dpi. Levels of IL-12and IFN-y were higher on7and9dpi than on2and5dpi. The production of IL-10was highest on5dpi, and TNF-a induced by the HB/04and HB/05was similar on all the detected days. Both H1N2infections produced lesions typical of influenza A virus infections:bronchiolitis with accompanying necrosis of respiratory epithelium and alveolitis. The lesions in the lung were severe on6and9dpi with HB/04and HB/05viruses.