Genetic Analysis And Immunogenicity Of H9N2 Subtype Avian Influenza Virus Isolated From Chickens With Vaccine Antibodies

H9N2 avian influenza(AI)is a low-pathogenic avian influenza caused by influenza A virus.It is an important infectious disease in poultry that is dangerous and widespread.Avian influenza virus(AIV)was first isolated in North American turkey in 1966.In 1992,after the virus was first isolated in China,AIV broke out in several provinces.AIV was mixed with a variety of pathogens to infect chickens,and cause serious economic losses in the poultry industry.In 1998,China began to widely use the commercial H9N2 subtype avian influenza vaccine,effectively controlling the development of the epidemic in chicken farms.However,due to virus antigen drift and gene recombination of H9N2 subtype avian influenza virus during 2010 to 2013,the original vaccine cannot be fully protected against the infection of H9N2 subtype avian influenza viruses.H9N2 subtype avian influenza viruses can be isolated in the immunized chickens occasionally,resulting in widespread transmission of H9N2 subtype AIV in China.In this study,15 strains of H9N2 subtypes avian influenza virus were isolated and identified in chickens with the vaccine antibodies titer of 16-128 in several chicken flocks in 2015 and 2016,where the chickens were routinely immunized with H9N2 subtypes avian influenza vaccine.Gene mutations and immunogenicity of the 15 isolated strains were explored by using molecular biology techniques,animal experimentsin this study.1.Isolation and identification of H9N2 subtype AIV in chickens with of vaccine antibodyIn this study,the tissues of trachea and lungs,larynx and cloacal swabs from the chickens with vaccine antibody were collected at several chicken farms during 2015 and 2016,and were used to isolate the virus.Hemagglutination test and hemagglutination inhibition test were used to identify the virus.5 strains of H9N2 subtype AIV were isolated in 2015,and 10 strains of H9N2 subtype AIV were isolated in 2016.Virus purification and virulence assays were performed using chicken embryo final concentration dilution method.The egg infectious doses(EID50)of isolates were from 10-6 20 to 10-8 25.The isolates could not cause death of chicken embryos,and the ELD50 of chicken embryos could not be calculated.2.Genome analysis of the isolated strainIn order to explore the genetic molecular characteristics of the H9N2 isoforms isolated by the vaccine antibody,the HA genes of the 15 isolates and whole genome of the 10 representative strains were sequenced and analyzed.The phylogenetic tree analysis of HA gene showed that 15 isolates belonged to the H9.4.2.5 lineage represented by Ck/GX/55/05.In the HA phylogenetic tree,most of the strains isolated in 2016 could be divided into the A branch,and the isolates of 2015 and part isolates of the 2016 were divided into B branch.The vaccine strains Ck/GD/SS/94 and Ck/SD/6/96 belong to the lineage h9.4.2.3 and the vaccine strain Ck/SH/F/98 lies in the lineage h9.4.2.1.The HA protein cleavage site of the isolates was PSRSSR?GL,except that the protein cleavage site of the isolate Ck/JS/TM151/16 was HSRSSR?GL.The glycosylation sites of the Ck/FJ/SDLH35/15 and Ck/SD/WF39/16 was NGT and NNT respectively,and the number of glycosylation sites was 8,while the remaining isolated strains had 7 glycosylation sites.The Ck/JS/JT141/16 increased NGT glycosylation sites in 145-147.The isolated strains increased NCS glycosylation sites glycosylation sites in 313-315 by compared with vaccine strains.In isolates,the glycosylation sites of NA protein at positions 264-266 and 402-404 were not appeared,although the 264-266 glycosylation sites of NA protein appeared in H9N2 influenza viruses published prior to 2008,and the 402-404 glycosylation sites of NA protein were found after 2008.The S31N mutation of M2 gene occurred in all isolates,which results in the amantadine resistance of the strain.The PA gene of the isolates have A515T mutations,which may induce lower virulence to the duck.3.Immunogenicity research of the isolatedNine representative strains on different phylogenetic tree of HA genes were selected:Ck/FJ/SDLH35/15.Ck/JS/TM123/15,Ck/JS/WJ179/15,Ck/AH/AH196/15,Ck/JS/JT154/16,Ck/SD/WF39/16,Ck/JS/JT141/16,Ck/JS/TM210/16,Ck/JS/TM224/16.Cross-hemagglutination inhibition test in the nine isolates and vaccine strain Ck/SH/F/98 virus were performed.The results showed that the antigen distance of between most isolates and Ck/SH/F/98 strain was far,except of the 2 isolates(JS/TM123/15 and AH/AH196/15).Based on above results,the isolates could be divided into 4 antigen regions.First group were SDLH35/15 and JT154/16,second groups were AH196/15 and TM210/16,third groups were WJ179/15,TM123/15,JT141/16and TM224/16,and fourth groups were vaccine strains Ck/SH/F/98 and WF39/16.To identify the immunogenicity of the siolates each other,four isolates were selected in different antigen religen for animal experiment.The research results provided that the protective rates of Ck/FJ/SDLH35/15Ck/AH/AH196/15 and Ck/SD/WF39/16 vaccines for their own viruses were 100%,but the protection rate of Ck/JS/TM224/16 was 83.3%.In addition,the protection rates of Ck/FJ/SDLH35/15 vaccine for Ck/AH/AH196/15 were 83.3%,the lowest protection rates of Ck/FJ/SDLH35/15 vaccine for Ck/JS/TM224/16 and Ck/SD/WF39/16 were 33.3%;the lowest protection rates of Ck/AH/AH196/15 vaccine for Ck/JS/TM224/16 was 16.6%,the protection rates of Ck/JS/TM224/16 vaccine for Ck/FJ/SDLH35/15 and Ck/AH/AH196/15 was 100%;and the protection rates of Ck/SD/WF39/16 vaccine for Ck/AH/AH196/15 and Ck/FJ/SDLH35/15 were 100%;while the lowest protection rate of Ck/SD/WF39/16 vaccine for Ck/JS/TM224/16 was 33.3%.All results indicate that the H9N2 avian influenza virus gene and antigenicity under pressure by the vaccine antibody have been evolved,and the evolved may caused by a lack of a polymerase corrector function of the RNA virus.However,the phenomenon warns us to know the vaccine dialectically that The protection efficiency of Ck/JS/TM224/16 isolates was decreased.