| In 1994,H9N2 subtype Avian Influenza Virus(AIV)was isolated from chickens in China for the first time.Since then,the H9N2 subtype AIV have gradually become one of the main epidemic AIV subtype,and had spread to most regions of China.In recent years,the H9N2 subtype AIV which not only seriously harm the poultry industry,but also can breaks through the barrier of the hosts,to achieve cross-host communication,infect mammals and people,posed a threat to public health security.Therefore,it is a great significance to study the genetic evolution analysis and biological characteristics of H9N2 subtype AIV isolated from part of China in recent years.The study of HA gene phylogenic Analysis H9N2 subtype AIV which analized based on the 75 isolated in part of China(Shandong,Henan,Hunan,Yunnan,Guangxi,Guangdong)between 2015 and 2017,14 representative isolates were selected for genome-wide genetic evolution analysis and biological characteristics.And 7 strains antigenicity were analyzed,4 strains viruses were used to study the patheogenicity of 4-week-old SPF chickens and 4-week-old SPF grade BALB/c mice.HA gene evolution analysis showed that 13 isolated out of the 14 isolated belong to BJ/94-like branches(h9.4.2),the other one isolates belonged to G1-like branches(h9.4.1).In the BJ/94-like(h9.4.2)the HA gene within the branch has been evolved into two different sub-branches,one group was represented isolated by A/chicken/Guangxi/55/2005(h9.4.2.5)and the other was represented isolated A/chicken/Guangdong/FZH/2011(h9.4.2.6)branches,the main advantage is the sub-branch of A/chicken/Guangxi/55/2005(h9.4.2.5).NA gene genetic evolution analysis showed that 13 belong to BJ/94-like branches,1 belonged to G1-like branches;the genetic evolution of its internal analysis showed that all isolated internal genes located which represent different evolutionary branches.The genetic evolution analysis of the HA gene sequences of 75 isolates combined with the representative strains and the classical representative strains in Genbank showed that our current H9N2 subtype AIV still BJ/94-like branch(h9.4.2)as the mainstream branch.The analysis of the key sites of the amino acid sequences showed: All isolates had no more consecutive basic amino acids present in the vicinity of the cleavage site of the HA protein,which indicates that the isolates belong to the low pathogenic avian influenza virus,and the mutations of some parts of its sugar glycosylation sites and thereceptor binding sites,Inparticular,all strains located at BJ/94-like branches(h9.4.2)have mutated from Q to L at 234,which is characterized by binding to the α2,6 receptor and thus has a potential risk of infection human.NA gene analysis HB binding sites found located in the BJ/1/94-like branch of the virus at K368 N are mutations located in the G1-like branch of a virus QA/GD/15945/15 compared with its representative strains in 367,369,401,402,403(K367N,D369 E,D401N,I402 N,R403W).All the strains on BJ/94-like branches had the phenomenon of neck loss.The results of sequence analysis of M2 protein showed that the site of S31 N one strain of G1-like branches one strain QA/GD/15945/15 and BJ/94-like branches one strain CK/SD/16583/16 did not have reduce the characteristics sensitivity to amantadine drug and the other BJ/94-like branches had lower characteristics sensitivity to amantadine drugs;the strains no appeared E627 K and no D701 N mutations were detected in PB2 proteins.The result of four strains H9N2 subtype AIV pathegenicity of different animals: After infection of 4-week-old SPF chickens with eye drops was carried out at a dose of 107EID50/200 ul virus,chickens can shed viruses through the throat and cloaca,and chickens shed more viruses in respiratory tract than that in digestive tract,at the 3d,there is a peak of detoxification;CK/SD/17213/17 had largest amount in throat swab sheeding,and higher titers of virus in chicken multiorgan.Infection of 4-week-old BALB/c mice with infested doses of 106EID50/50 ul virus resulted in a decrease in BALB/c mice weight significantly,it will not cause death in BJ/94-like isolates.But the strain CK/SD/17213/17 was replicated in multiple organs of mice,with the highest viral titers in the lungs.The strain QA/GD/15945/15,which was isolated from G1-like,could increase the body weight of mice slightly after three days of infection,but it did not cause the mice to die.Antigenic analysis of the subtype H9N2 subtype AIV suggested: there were differences between BJ/94-like branches and G1-like strains of antigen branches(0.42≤R≤0.66),and there were different of diversity between the small branches of BJ/94-like branches(0.57≤R≤1.18).This shows that H9N2 subtype AIV in the last three years of evolution of the antigenic differences more and more obvious in part of China,to the prevention and control of the disease increased difficulties.The antiserum of CK/HN/15347/15,CK/YN/16565/16,CK/SD/16757/16 had more HI titer against others viruses,on the country,the serum of others viruses antiserum had lower HI titer against,therefore that CK/HN/15347/15,CK/YN/16565/16 and CK/SD/16757/16 could be further investigated for vaccine candidate strains... |
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