Serological Survey Of Influenza Virus Infection In Non-avian Wildlife From Xinjiang And Study Of Influenza Virus-like Particle

Influenza A viruses are highly contagious.The known natural reservoirs of influenza viruses are the aquatic birds of the world with mounting evidence for a role of bats. Although in a relatively stable host–pathogen interaction in hosts of influenza A viruses often accompanies interspecies transmission as the virus adapts to a new host. For example, influenza A virus subtypes have been isolated from marine mammals, indicating that marine mammals can serve as hosts to avian-origin influenza subtypes.As wild birds are reservoirs for the majority of influenza A viruses, avian migration patterns may potentially contribute to the spread of AIV. Xinjiang Uyghur Autonomous Region(shortened to Xinjiang here) is an important stop-over site along the East Africa–West Asia Flyway migration route. This region also harbors a wide range of other non-avian wildlife that are potentially at risk for AIV infection due to cohabitation with migrating birds, especially in national parks away from residential areas. While these data suggest widespread circulation of AIVs in birds in Xinjiang, little is known concerning the extent of AIV infection in non-avian wildlife in this region. To better understand the frequency of AIV exposure among wildlife in Xinjiang, we collected and surveyed sera from a panel of mammalian species for the presence of antibodies against H5, H7, and H9 AIV subtypes by hemagglutination inhibition(HI) tests and competitive ELISAs.The evolutionary changes in the new hosts result from a number of processes including mutation, reassortment, and recombination, and the vaccine is the effective way with regard to prevent influenza viruses. As a new form of vaccine candidate, the non-infectious nature of VLPs and their lack of viral genomic material are attractive safety features. Influenza-virus-like particles have been developed as vaccine candidate for influenza virus. In this study, we expressed the proteins of influenza based on baculovirus expression vector system(BEVS). Base on this platform, we hope to generation chimeric VLPs for protection against influenza.Objective: Subjects were avian influenza virus in this study, and serological data of avian influenza have been investigated in some non-avian wild animals in Xinjiang, and we expressed the proteins of influenza based on BEVS. Base on this platform, we hope to provide data reference for the study of influenza reserve vaccine.(1) To understand the current situation of non-avian wildlife from Xinjiang,(2) To explore the production of influenza VLPs with the silkworm as a bioreactor,(3) To explore the preparation of a new influenza vaccine by Bac-to-Bac baculovirus expression system,(4) Evaluation of immunogenicity of chimeric influenza virus like particles.Method:(1) Anti-avian influenza virus(AIV) antibodies were conducted to detect in Gazella subgutturosa, Canis lupus, Capreolus pygargus, Sus scrofa, Cervus elaphus, Capra ibex, Ovis ammon, Bos grunniens and Pseudois nayaur from Xinjiang, China. Two hundred and forty six sera collected from 2009 to 2013 were assayed for antibodies against H5, H7 and H9 AIVs using hemagglutination inhibition(HI) tests and a pan-influenza competitive ELISA.(2) The target gene HA-M1-NA and the wild-type BmNPV gene were co-transfected into B.mori cells and injected directly into silkworm larvae. And the recombinant BmNPV(HA-M1-NA)was identification by HI, immune-fluorescence assay, Western blot and fluorescent microscopy. Mice immunized with VLPs has been tested immune responses from challenge of influenza virus.(3) The recombinant influenza VLPs based on BEVS was identification by immune-fluorescence assay, Western blot and fluorescent microscopy. Mice immunized with VLPs has been tested immune responses from challenge of influenza virus. Mice immunized with VLPs has been tested by statistics of CD4+ T/CD8+ T cell and level of cytokine from immunized mice.(4) Influenza chimeric VLPs were expressed by the SF9 cells transfected with AcMNPV-HA-fliC-NA-M1, and the chimeric VLPs was identification by immune-fluorescence assay, Western blot and fluorescent microscopy. Mice immunized with VLPs has been tested IgG antibodies and level of cytokine and challenge of influenza virus.Result:(1) Across all tested wildlife species, 4.47% harbored anti-AIV antibodies by HI assay. Seroprevalence for each AIV subtype across all species evaluated was 0% for H5 AIV, 0.81% for H7 AIV, and 3.66% for H9 AIV. H7-reactive antibodies were found in Canis lupus(9.09%) and Ovis ammon(4.55%). H9-reactive antibodies were found in Gazella subgutturosa(4.55%), Canis lupus(27.27%), Pseudois nayaur(23.08%), and Ovis ammon(4.55%).(2) The recombinant BmNPV(HA-M1-NA)was obtained. This influenza VLPs with function activity were structurally intact. Mice immunized with VLPs has been stimulated immune responses and showed partial protection to heterogenous challenge.(3) The results showed influenza VLPs were correctly expressed by the SF9 cells transfected with AcMNPV(HA-M1-NA), which elicited the high levels of IL-2、IL-4、IL-10、IFN-γ and antibody response to influenza virus.(4) The results showed influenza chimeric VLPs were correctly expressed by the SF9 cells transfected with AcMNPV-HA- fliC-NA-M1, which elicited the high levels of IL-2、IL-4、IL-10、IFN-γ and IgG antibodies response to influenza virus. Mice immunized with cVLPs has been stimulated immune responses and showed partial protection to heterogenous challenge.Conclusion:(1) The pan-influenza competitive ELISA results closely corresponded to the cumulative prevalence of AIV exposure as measured by subtype-specific HI assays, suggesting that H7 and H9 AIV subtypes predominate in the wildlife species evaluated. These data document evidence of prior infection with H7 and H9 AIVs among non-avian wildlife in Xinjiang.(2) The results provide reference for development of influenza VLPs vaccine by silkworm expression system.(3) The results suggested that influenza VLPs based on BEVS is a new vaccine candidate for protection against influenza.(4) This approach of influenza chimeric VLPs has greater potential for universal vaccine development against influenza viruses.