Study On Genetic Variation Of HA Gene Of H9 Subtype Avian Influenza Virus In Some Poultry Farms In China

Avian influenza(AI)is a malignant and lethal infectious disease caused by influenza A virus.It has brought huge economic losses to the poultry industry in China and the world.H9 subtype AIV belongs to the low pathogenic influenza virus.It has been widely spread in China and has been delayed and brought great economic losses.At present,it has been confirmed that H9 subtype avian influenza not only infected poultry,but has gradually begun to have the ability to infect mammals.In addition,avian influenza viruses are prone to mutation and cause antigen drift,and have the possibility of recombination between different subtypes of avian influenza viruses,resulting in frequent mutations of antigens,these bring tremendous pressure to its prevention and control.To clarify the molecular epidemiology and the evolution of HA gene epidemic variation of H9 subtype avian influenza virus in China,H9 subtype AIV were isolatied and identificated from 2014 to 2018,HA gene were sequenced and then analyzed by Clustal W,DNA Star and MEGA software.The results showed that total of 532 samples were isolated.Obtained 113 strains of H9 subtype avian influenza virus,including 111 strains of chicken-derived virus,1strain of Duck-Derived virus and 1 strain of bird-derived virus.and which distributed in 15 provinces of China.Shandong and Guangdong had the largest number of H9 positive strains;the highest positive rate was found in the five northern provinces,with the positive rate as high as 27.7%,followed by South China and Yunnan-Guichuan,but the positive rate in other areas was not optimistic,all above10%;H9 subtypes were also isolated in chickens without any symptoms of avian influenza,suggesting that the current epidemic situation of H9 subtypes is severe,and the animal population carrying and transmitting the virus far exceeded the estimate.The homology and phylogenetic tree analysis of HA gene showed that 113 strains of virus that isolated all belong to Y280 subtype and belong to h9.4.2 branch,had obvious genetic differences with SS strain,F strain and 696 strain that commonly used vaccine strains,and the genetic distance was large.At the same time,the homology of HA gene of H9 subtype avian influenza was only 95% in the same region,suggesting that even in the same region,the HA gene had undergone great variation,with the big degree of variation.Two strains of A-CK-YN-KMTQ50-2014 and A-CK-SC-DYYAzw01-2015 were isolated from Sichuan and Yunnan provinces,with only 91% homology with the vaccine strain.It suggests that the current H9 subtype vaccine may be difficult to control the prevalent H9 strain.The analysis of HA protein showed that the amino acids encoded by HA gene of the strains that isolated in the experiment also had a great variation.The amino acid main variation locations were 13,66,88,145,167,168,199,424 and 477,and there has a relatively conservative among 220-423 amino acids,which variation was very small.A-CK-GD-HYys-2016 strain inserted an amino acid at 424 in HA sequence.There is a highly variable region at the end of the sequence,which varies frequently.The similarity of the total amino acid sequence is between 88.7 and 97.9%.The analysis of HA cleavage site showed that the amino acid composition of HA cleavage site could be divided into two modes.Except for A/CK/SD/GMkh/2015 is PYRSSR?GLF,the other isolates were PSRSSR?GLF.There has only one basic amino acid(R),this was consistent with the characteristics of low pathogenicity avian influenza virus.Potential glycosylation sites of HA gene of H9 subtype strain were found at 29-31,141-143,218-220,298-300,305-307,492-494 and 551-553 amino acid sites.However,some strains showed new glycosylation sites occurred ordeletion of glycosylation sites,suggesting that the structure and antigenicity of HA protein had been gradually changed.At present,it has been reported that the dominant strain of H9 subtype avian influenza has changed to Y280 or Y280-like subtype,and the existed vaccines can not produce complete immune protection.Our sequencing comparison of isolates from 2014 to 2018 also shows this point.It is urgent to develop new vaccine strains.