Biological Characteristics Of Two H5N8 Subtype Avian Influenza Viruses And Identification Of Antigenic Epitope Of HA1 Protein

Avian influenza is a highly contagious poultry disease caused by avian influenza virus?AIV?,which continues to spread throughout the world.AIV infection in poultry may cause mild clinical symptoms,respiratory disease and decreased egg production,and in severe cases,systemic disease with a mortality rate of up to 100%.In particular,highly pathogenic H5 subtype avian influenza viruses,such as H5N1,can across the interspecies barrier and spread from infected poultry to humans,leading to a serious public health crisis.With the spread of H5 avian influenza viruses,it had attracted worldwide attention.The H5N8 subtype highly pathogenic avian influenza viruses?HPAIV?was first detected in Chinese poultry in 2010 and appeared in poultry in South Korea and Japan in early 2014.By the end of 2014,the H5N8HPAIV had appeared in Europe and North America.There are no reports of human infection with H5N8HPAIV so far.However,due to the rapid geographical spread of H5N8 HPAIV and their ability to infect various species of birds,once the outbreak of HPAI H5N8 viruses will cause serious economic losses to poultry industry.Based on these,the biological characteristics of 2 strains of H5N8 AIVs?QH448 and QH450?isolated from Qinghai Lake region in 2017 were analyzed in this study.On the one hand,this study provided a theoretical basis for rapid response to possible influenza emergency;on the other hand,it lays a foundation for evaluating the virulence,pathogenicity,epidemiology and other biological characteristics of H5N8 AIVs in different regions.Firstly,genetic evolution analysis of 2 strains of H5N8 avian influenza viruses shown that the two strains belonged to Clade 2.3.4.4.It was found that the two strains of H5N8 AIVs were highly pathogenic avian influenza viruses.Through the infection and pathogenicity test in mice and the contact transmission test in guinea pigs,it was found that the two strains were highly infectious to mice,and the high-dose infection of the two strains was fatal to mice,and the two strains could not transmits between guinea pigs through direct-contact route.In addition,we prepared 5 hybridoma cell lines which can secrete monoclonal antibodies against QH448 HA1 protein,named 1H12B9,2A9D11,7E9G6,8A12D10 and 9H10F5,respectively.It was shown by western blot assay that all 5 monoclonal antibodies could react with H5 avian influenza virus.Neutralization test and hemagglutination inhibition test shown that the 5 monoclonal antibodies had no neutralization activity with H5 avian influenza virus.Indirect immunofluorescence assay shown that all5 monoclonal antibodies had high reactivity and specificity.The epitopes of 5 monoclonal antibodies were identified by using a series of overlapping QH448HA1 protein truncations.It was preliminarily determined that the antigenic epitope recognized by1H12B9 and 7E9G6 were located at HA1 ₃₃₀ATGLRNSPLREKRRKR₃₄₅.The recognition epitope of2A9D11 was located at HA1 ₃₁₀IHPLTIGECPKYVKSN₃₂₅.The epitope of 8A12D10 was located at₂₇₁KIVKKGDSTIMKSEV₂₈₅.The epitope of 9H10F5 was located at ₁₂₁LSRINHFEKILIIPKS₁₃₆.It was revealed through homology comparison that these epitopes recognized by monoclonal antibodies were highly conserved in the H5HA sequences.In summary,the biological characteristics of two strains of H5N8 avian influenza viruses were systematically analyzed in this study.In addition,the characteristics of 5 monoclonal antibodies against H5 HA1 protein were analyzed,and four conserved epitopes were identified.The conserved antigenic proteins of AIV and the conserved epitopes in virus antigenic proteins are potential immunogens to prepare cross-protective vaccine.The conserved linear epitopes selected in this study may provide important targets for the preparation of new vaccines.