Genetic Characteristic Of H9N2 Avian Influenza Virus Isolated From Free-Range Poultry In Eastern China,During 2014-2015

The first case of serious lower respiratory tract infection caused by H7N9 avian influenza virus emerged in China's Yangtze River Delta region,in 2013.H7N9 was a new type of recombinant virus whose internal genes derived from H9N2 avian influenza virus.H9N2 is a common low pathogenic avian influenza virus(LPAIV),since it first appeared in China in 1994,its popularity has been rising with broad host range.Further,the virus can provide internal genes for a variety of subtypes of AIV which can infect human.Previous studies showed that,people infected with H7N9 have had a history of live poultry exposure or live poultry market(LPM)exposure.Therefore,LPM is the main source leading to human infection with the H7N9.Meanwhile,there is a large amounts of Free-range Poultry(FP)which is widely distributed in Eastern China.The FP are transported to different places through live poultries trading which is one of an important sources of LPMs trade.In recent years,all government departments have stepped up the detection of AIV in LPM,but the attention to FP is relatively weak.Based on this,during 2014-2015,an investigation was conducted to study H7N9 and H9N2 carried by LPMs and FP in Eastern China,aimed to assess the prevalence of H7N9 and H9N2 in FP and LPMs and to further explore the evolutionary characteristics of the viruses in the region.We collected throat and anal swab samples from poultry of LPMs and FP in the Eastern China including Jiangxi Province,Anhui Province,Zhejiang Province and Shanghai,during 2014-2015.The method of Real-time PCR was used to identify H9N2 and H7N9 subtypes and the positive representative strains were selected for genome sequencing.The molecular characteristics,genetic evolution and its pathogenicity to mice were analysised.The main results are as follows:(1)The isolation and identification of AIV: a total of 876 samples were collected from LPMs and FP in Eastern China,28 samples were positive for H9N2,the total positive rate was 3.2%,of which the positive rate of AIV isolated from FP was 2.92% and the positive rate of AIV isolated from LPMs was 7.14%.No H7N9 subtype was detected in these samples.(2)Homology analysis: eight representative strains were selected for whole genome sequencing.According to its sequence homology,the eight isolates were divided into two different branches named group 1 and group 2.The nucleotide homology between group 1 strains was 86.3%-100%,the nucleotide homology between group 2reached 99.9%-100%.Compared with the classical representative strains,group 1strain had the highest homology with A/Chicken/Zhejiang/HJ/2007,the strain is the ancestor of G57 genotype,so group 1 strains belong to G57 genotype.group 2 strains had closest evolutionary relationships with A/Hong Kang/33982/2009 isolated from human in Hongkong.(3)Phylogenetic analyses: group 1 strains clustered in the same branch with H9N2 isolated from poultry and human in Eastern China and H7N9 isolated from poultry and human in Eastern China and Southern China.Group 2 strains clustered in the same branch with H9N2 isolated from wild birds in Jiangxi in early age,and human in Hongkong.(4)Molecular characteristics analyses: eight representative strains have characteristics of low pathogenic avian influenza virus(LPAIV),some part of which have characteristics of preferentially binding human cell receptor,while some part have characteristics of preferentially binding avian cell receptor.The eight representative strains have strong sensitive to oseltamivir(Tamiflu)or other neuraminidase inhibitors.Some strains were resistant to amantadine.(5)Pathogenicity analyses: the isolates from free range poultry can replicate in trachea and lung tissues of mice and it can cause slight infection in mice,but but it can not lead to death.The above results showed that the positive rate of H9N2 subtype in FP is lower than that of LPMs,because the strains from FP had close relationship with H9N2 and H7N9 isolated from poultry,wild birds and human in Eastern China,there might exist gene flow between FP and LPMs.Isolates of FP in Eastern China were low pathogenic avian influenza virus,but they can infect mammals,causing mild symptoms of infection.In view of the particular farming environment of free-range poultry,the AIV surveillance need to be studied further to provide risk warning for human infection of AIV.Meanwhile,we should strengthen the monitoring of free-range poultry and pay attention to improve its biosafety level.