Isolation,Identification,Genetic Evolution Analysis And Study Of Pathogenicity In Mice Of Avian Influenza Virus And Swine Influenza Virus

In order to master the regular patterns of the pandemic and genetic variation of avian influenza virus and swine influenza virus in Hubei province,we isolated and identified the influenza virus pathogens,and on this basis,researched the genetic evolution of virus sequences.Genetic and phylogenetic analysis of influenza virus was conducted.We investigate the pathogenicity of A/swine/Hubei/221/2016(H1N1)and A/Hunan/42443/2015.A statistical analysis of the distribution,genetic evolution and prevalence of swine influenza virus in China was conducted.Provide theoretical basis for effective prevention and control of the outbreak of influenza.The main research contents are as follows:1.Isolation,identification and genetic evolution analysis of AIVTwo strains of avian influenza virus were isolated from the diseased chicken flocks.Blast analysis of the sequencing results showed that two strains were H9N2 subtype avian influenza viruses.The phylogenetic trees of whole genome sequence of the H9N2 isolated strains were constructed.The result showed HA and NA gene of two H9N2 isolated strains belonged to Y280-like lineage,which widely spread in our country.PB1,PA,NP and NS gene belonged to SHF98-like lineage.PB2 and M gene belonged to G1-like lineage.Molecular analysis of whole genome sequence found that the amino acid sequence of HA genes of the H9N2 isolated strains had the cleavage sites-RSSR?GLF-,which represents the molecular basis of low pathogenic avian influenza virus strain.Compared with the prevalent strain Y280,the two H9N2 isolated strains had 313(NCS)potential glycosylation site in HA protein.The position L234 Q of HA gene of the H9N2 isolated strains were mutated,indicating that there was an affinity of avian influenza viruses for human-type receptors.The position N31 S of M2 protein of the H9N2 isolated strains were mutated,indicating that there was a resistance to amantadine drugs.2.Isolation,identification and genetic evolution analysis of one H1N1 isolated strain and pathogenicity of one H1N1 isolated strain in miceOne strain of swine influenza virus was isolated from the diseased swine flocks.RT-PCR was used to amplify whole genome sequence from one isolated strain,Blast analysis of the sequencing results showed that the isolated strain was H1N1 subtype SIV.The phylogenetic trees of whole genome sequence of the H1N1 isolated strain were constructed.The virus isolated in 2016(A/swine/Hubei/221/2016)was highly identical toa human isolated H1N1 in 2015(A/Hunan/42443/2015).Thus,swines might be the source of human H1N1 infections.The H1N1 swine influenza virus isolate and A/Hunan/42443/2015 belonged to eurasian avian like-H1N1.PB2,PB1,PA and NP gene of A/swine/Hubei/221/2016 belonged to pandemic H1N1/2009.HA,NA,M and NS gene belonged to eurasian avian like H1N1.The pattern of genetic reassortment of A/Hunan/42443/2016 was the same as A/swine/Hubei/221/2016.Analysis of the epidemic trend of swine influenza virus in China revealed Eurasian avian like H1N1 swine influenza virus was an important constituent part of swine influenza virus.In addition,the Eurasian avian like H1N1 SIV with PB2,PB1,PA and NP gene belonged to pandemic H1N1/2009 appeared more and more frequently in recent years.The virus isolated in 2016(A/swine/Hubei/221/2016)was highly identical to A/Hunan/42443/2015.To investigate the pathogenicity of H1N1 isolated strain,pathogenic test was carried out on BALB/c mice with A/Hunan/42443/2015 served as a control group.A/Hunan/42443/2015 replicated higher than A/swine/Hubei/221/2016 in MDCK,Nptr and A549 cells.All the mice inoculated with A/Hunan/42443/2015 were resulting in 100% lethality.Mice infected with A/swine/Hubei/221/2016 caused a lethality of 25%.A/swine/Hubei/221/2016 and A/Hunan/42443/2015 replicated systemically in mice,which could be detected from the tested organs,including the lung,trachea and nasal turbinate.In summary,this study carried out the isolation and identification of avian influenza virus and swine influenza virus.We analysed the genetic evolutionary relationship of genome gene of two H9N2 AIV isolates and one H1N1 SIV isolate.We investigated the pathogenicity of the H1N1 isolate in mice.Analysis of the epidemic trend of swine influenza virus in China was carried out.The study can help us to master the genetic characteristics and variation of H9N2 AIV and H1N1 SIV from molecular level,and laid an important foundation for the prevention and control technology of influenza and as well as vaccine development.