Epidemiological Investigation,Virus Isolation,Identification And Gene Sequence Analysis Of H9 Subtype Avian Influenza In Hunan Province In 2021

In order to understand the epidemic situation,strains and gene sequences of H9subtype avian influenza in Hunan Province in 2021,samples were collected and detected by H9 real-time fluorescence quantitative PCR in markets and farms in 14cities and prefectures of Hunan Province in 2021.HA gene sequences and 9 NA gene sequences were obtained by chicken embryo inoculation,virus isolation,virus inactivation,target gene amplification and sequencing.The results are as follows:1.The epidemiological investigation results of H9 subtype avian influenza show that:（1）The overall positive rate of H9 subtype avian influenza in the market was12.5%（427/3465）,and the field positive rate was 80.68%（71/88）;The positive rates of H9 subtype avian influenza in chicken,waterfowl,pigeon and environmental swabs were 11.79%（265/2247）,9.31%（65/698）,9.52%（2/21）and 19.03%（94/494）,respectively;According to the statistics of regional results,the positive rate of H9 AIV subtype in Shaoyang market is high,which is 20%.There were no positive samples to be detected in Yongzhou market.（2）The overall positive rate of H9subtype avian influenza in farms was 1.19%（47/3946）and 19.01%（23/121）.The positive rates of H9 subtype in chicken and duck samples were 1.34%（41/3055）and0.67%（6/891）respectively;According to the statistics of time results,the positive rate was higher in winter and spring,and the positive rate was the highest in November,which was 7.78%（7/90）;According to the regional distribution statistics,the positive rate of H9 subtype avian influenza in farms in Yiyang City is the highest,with a positive rate of 3.45%.No positive samples were detected in Zhangjiajie City.2.The isolation of H9 AIV subtype strain and sequence of target gene show that:（1）The HA gene sequence:（1）The cleavage site of HA protein amino acid sequence is ³³³PSRSSR³³⁸,which is consistent with the molecular characteristics of low pathogennic avian influenza;Q mutation at amino acid 226 was L,and the isolate showed human receptor binding characteristics.G mutation at amino acid 228 was not found to be S,which showed avian receptor binding characteristics;There was no glycosylation site at amino acid position 145,indicating that the current isolate tended to bind to mammals on the basis of amphiphilic;（2）The genetic evolution analysis of HA gene:Six HA gene fragments were located in H9 4.2.5 branch:the nucleotide homology of HA gene between isolates is 98%～99.9%,and 86%～88%compared with the classical strain Y280.The results show that there is a large variation from the original classical strain.（2）The NA gene sequence:（1）The results of NA protein amino acid sequence showed that the isolates were N2 subtype,except CK.YY.01.21、CK.YY.02.21 and CK.YY.In addition to 03.21,the other six Na proteins were mutated from n at amino acid 402 to D,resulting in the deletion of 402～404glycosylation site;Compared with reference strain and vaccine strain（except vaccine strain SZ）,E/K/G368N and D369G mutations occurred,resulting in the emergence of ³⁶⁸NGS³⁷⁰glycosylation site;The active center of NA protein was¹⁴⁰LKNEHSNGTTHDRTPH-RT¹⁵⁷.The K mutation was E/D at amino acid 143 and the amino acid I mutation was T at amino acid 153;To compared with the classical strain Y280,the red blood cell adsorption sites at positions 431～433 of the isolated strain were highly conservative,which was ⁴³¹PEQ⁴³³.Compared with the vaccine strains HL,SZ and WJ57,the N mutation of amino acid at position 402 was s/D,and the red blood cell adsorption sites at positions 400～403 changed to a certain extent;（2）The genetic evolution analysis of NA gene showed that the NA genes of 9 isolates were located in Y280 like subgroup.The results showed that the prevalence of H9 AIV subtype in Hunan Province had the characteristics of wide coverage,obvious seasonality and"concave"trend,and the host was mainly chickens;The isolates showed a tendency to infect mammals,including humans.The epidemic strain of H9 AIV subtype was H9N2 subtype.Therefore,in the front line of market and breeding,we should pay attention to and strengthen the prevention and control of H9 subtype avian influenza,so as to reduce the impact on poultry industry and public health.