摘要
目的 对活禽市场环境中检出的3份H5N6亚型禽流感病毒(avian influenza virus,AIV)标本进行测序鉴定和基因特征分析,为预防人感染H5N6亚型AIV提供依据.方法 通过实时荧光定量PCR确定病毒型别,采用二代测序方法对病毒血凝素(hemagglutinin,HA)和神经氨酸酶(neuraminidase,NA)基因序列进行测序.使用NCBI中BLAST模块进行比对,用Mega7.0软件构建进化树并进行基因特征分析.结果 3份H5亚型阳性标本经测序鉴定为H5N6亚型AIV,属于Clade2.3.4.4支系.病毒的HA蛋白存在6个连续碱性氨基酸(LRERRRKRGL,其中R,K为碱性氨基酸);病毒HA蛋白的受体结合位点238～240位氨基酸是QRG(氨基酸排序以H3-HA为准),受体特征为禽源;NA蛋白第69～73位点氨基酸未出现茎缺失.结论 环境中3份H5亚型AIV核酸阳性病毒鉴定为新型H5N6亚型AIV,属于高致病AIV,但具有不容易感染人的受体结合特征.活禽市场中持续存在H5N6病毒,对人类健康和公共卫生存在威胁,需要继续加强监测.
Abstract
Objective To sequence,identify H5N6 subtype avian influenza virus(AIV)from three environment samples from live poultry market and analyze the genetic characteristics of,so as to provide a basis for the prevention of human infection with H5N6 subtype AIV.Methods The real-time fluorescence quantitative PCR amplification was used to determine the type of AIVs.The next generation sequencing method was used to sequence virus hemagglutinin(HA)gene and neuraminidase(NA)gene.The sequences were aligned by NCBI BLAST module,and the MEGA7.0 software was used to construct the phylogenetic tree and analyze the genetic characteristics.Results The three AIV strains were identified as H5N6 subtype,belonging to clade 2.3.4.4 by sequencing.There were six consecutive basic amino acids(LRERRRKRGL,where R and K are basic amino acids)in the HA protein.The amino acids of in the receptor binding sites of 238-240 in the HA protein of the virus were QRG(amino acid sequence of H3-HA as reference).The receptor characteristics were avian origin.There was no stem deletion of amino acids at sites 69-73 of NA protein.Conclusions A novel H5N6 subtype of AIV was identified in three environment samples that were positive of nucleic acid of H5 subtype AIV.The virus stains were high pathogenic AIV but without receptor binding characteristics feasible to infect human.The persistence of H5N6 virus in live poultry markets poses a threat to human health and public health.It is necessary to continue to strengthen the AIV surveillance.
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