| H9N2 subtype avian influenza virus（AIV）,a global epidemic of low pathogenic virus,can infect domestic poultry,wild birds,even some mammalians.Waterfowl not only serve as a natural host of AIV,but also play an important role of AIV transmission from migratory birds to poultry.The mutations and recombination easily happens among AIV genome,and leading to the change of virulence,avidity and antigenicity.H9N2 AIVs also were known to served as a donor of other subtype influenza virus by providing their internal genes.According to these,it is necessary to keep tracking the variation of the virus and to screening the vaccine candidate strains for further prevention.In this study,from October 2015 to March 2017,6 strains of H9N2 subtype AIV were isolated from 469 swab samples collected from a live poultry market in Foshan,Guangdong Province.The whole genome of 6 isolates were obtained through RT-PCR and sequenced.Afterward,the viral genome and amino acids sequences`characteristics were analyzed which showed that the cleavage sites amino acid sequences of HA protein of 6 newly found strains belong to low pathogenic strains（Two strains were PARSSR↓GL and 4 strains were PSRSSR↓GL）.Mutant Q226L on receptor binding site of HA protein was found in 4 isolates which suggested that such viruses had potential ability of binding the humanα2,6-sialic acid receptor.Amino acid deletions in NA stem region were found in all of the 6 isolates.The mutation of S31N locus in M2 protein,The PB2,M gene is derived from the G1 subline.The PB1,PA,NP,NS gene was derived from the F98 subline.The mutation of P42S site in the NS1 protein of 6 strains of virus resulted in the production of interferon in antagonistic host.The analysis showed that the internal genes of the 6 strains were similar to those of the human influenza H7N9,H10N8,H5N6 subtype,suggesting that there might be a gene recombination between the isolated virus and the human virus mentioned above.The biological characteristics of A/Goose/Guangdong/A11/2016（H9N2）（GS/A11）strain were also determined in this study.The results showed that the average mean death time（MDT）of chicken embryo was 120 h,and that Chicken embryo half infection(EID50)of GS/A11 was 10-8.632/0.1 mL.In order to understand the pathogenicity of GS/A11 on goose,6-week-old geese without significant clinical signs and influenza antibody negative,were challenged i.v.with 106EID50 viral dosage.No obvious clinical symptoms could be observed on the animals.According to the virus shedding detection,the GS/A11 could be shed through larynx trachea and cloacal cavity.The histopathological and viral distribution of the trachea of the Goose was observed.The results showed that the mucosal structure was destroyed,the epithelial cells were denatured and necrotized,and a small number of inflammatory cells were infiltrated.The positive signal of virus was found in the epithelial cells of goose-infected trachea mucosa by immunohistochemistry.In order to understand the immunogenicity of goose GS/A11 strain to goose,GS/A11strain was inactivated and produced as oil emulsion vaccine before immunizing the geese.The results showed that 2 weeks after the first immunization,the geese began to produce antibodies,and the average antibody level reached a peak value of 8.1 log2 at the 4th week after the second immunization.The GS/A11 immunized goose serum and the H9 standard serum which against A/Chicken/Shanghai/10/01（H9N2）（SH10）,a commonly used vaccine candidate,were enrolled for the cross protection tested against those 6 newly isolated H9N2strains,respectively.The results of HI cross test showed that the antigenicity of 6 isolates was different from that of vaccine candidate representative strains,while the GS/A11 can provide a more broadly cross reaction among these newly found strains.The HA protein of goose GS/A11 strain was expressed in prokaryotic and purified.The method of HA protein expression was optimized.Western blot was enrolled to verified weather the HA protein were express correctly.The results showed that there was a clear protein imprinting at 56 k Da,which indicated that H9 HA protein was successfully expressed in prokaryotic expression.It provides a basis for laboratory study of downstream tests related to H9N2subtype AIV HA protein（preparation of monoclonal antibodies subunit vaccines and recombinant live vector vaccines etc.）.The study provides a basis for exploring the key points of H9N2 AIV’s recent epidemic evolution,clarifying its role in the process of avian influenza transmission and mutation and the selection of vaccine candidate strains. |
