Evolutionary Analysis And Immunological Evaluation Of H9N2 Subtype Avian Influenza Virus HA And NA Gene In Parts Of China From 2014 To 2017

Avian influenza belong to a class infectious diseases.The H9N2 subtype avian influenza virus was one of the most prevalent and harmful avian infectious diseases in poultry,and mainly infected chickens,breedersandcommercial broilers.It was first reported in China form 1994,and subsequent widespread outbreaks have been recorded in China,causing significant losses to the poultry industry.Influenza virus genomes were susceptible to mutation and recombination.H9N2 subtype of avian influenza virus could provide internal genes for H5N1,H7N9,H10N8 and H5N6 subtype avian influenza virus in humans,potentially threatening human health and safety due to the susceptibility of influenza virus to mutation and recombination.Since 1994,at least 13 cases of human H9N2infection had been reported in our country,indicating that H9N2 subtype influenza also had important public health significance.In order to continuously monitor the epidemic variation of the H9N2 subtype avian influenza virus,the 30 purifiedH9N2 avian influenza viruses isolated in South,Central and Southwest China from 2014 to 2017 of HA and NA genes sequence were analysised and took the genetic phylogenetic tree.Thus,it could master the genetic variation direction epidemic regularity of the H9N2 AIV in morbidity.According to the HA sequence genetic system phylogenetic tree,6 strains representing different strains in the source,year and region were selected and then single factor serum was prepared.And the results were determined for analysis of antigenic changes in the virus by HI test of serological cross-reactivity and the cross neutralization test of specific pathogen free?SPF?chicken embryos.The inoculated vaccine was prepared by selecting the candidate vaccine strain CK/HN/15347/2015 and CK/SD/WHKY/2014.Finally,the immune protection experiment was carried out to evaluate the immuneprotective effect of the two strains.Phylogenetic anylasis shows:30 strains of H9N2 subtype avian influenza virus were derivedfrom CK/Bei-like,H9.4.2.5 branch of H9.4.2 gene represented by A/Duck/HongKong/Y280/97 strain,HA cleavage site PSRSSR/GLF,there is only one basic amino acid at the cleavage site,showing typical character of low pathogenic avian influenza.There were V/Adifference in the HA gene receptor binding site at position 198,and the 226th amino acid in all 30 isolates was glutamine?Q?.Typical of human influenza receptor binding properties present.NA gene had a 9 bp deletion so the viruses had three amino-acid deletions at 63⁶5 in NA stem.Serum cross HI test data and chicken embryo cross neutralization test results showes.The HA titer and EID₅₀0 results of CK/SD/WHKY/2014 and CK/HN/15347/2015 were significantly higher than those of other strains,and there was a good breeding ability in the6 strains of H9N2 subtype avian influenza virus.Serum cross-HI test results of six strains from the antigen difference is divided into two antigen groups,belong to an antigen group of cross-border is better,but there was a certain cross between different populations,indicating that the isolation of the virus despite the antigenicity.There were differences in variation,still able to produce a certain cross-protection,and the same virus and serum between the titer was the highest.Inactivated vaccines were preparedwith CK/SD/WHKY/2014 strains and CK/HN/15347/2015 strains and used to inoculate immune SPF chickens.For the vaccine antigen itself,after 21 days of immunization could stimulate the body to produce high titer of antibodies,the resulting HI antibody titer average between 9.0log2⁹.4log2,which indicated that the strains had a good immunogenicity,could be very effective resistance against the parent of the drug attack,the protection rate can reach 100%,showed that two strains could be used as H9N2 subtype avian influenza inactivation Candidate for the vaccine.