1株欧亚类禽猪流感病毒的分离鉴定及遗传进化分析

Isolation,Identification and Genetic Evolution Analysis of a Eurasian Avian-like Swine Influenza Virus

【目的】了解广东地区猪流感病毒(Swine influenza virus,SIV)的流行情况并探究其分子生物学特征。【方法】采集广东某猪场疑似猪流感病毒感染猪的鼻拭子和肺脏组织样品进行病毒分离鉴定、遗传进化和关键氨基酸位点分析。【结果】样品经实时荧光定量RT-PCR检测为猪流感病毒核酸阳性;在红细胞凝集试验中,该病毒对鸡红细胞有凝集作用,血凝效价为1∶128;8个基因片段序列结果经BLAST比对和进化树分析显示,HA、NA基因属于欧亚类禽猪流感病毒(H1N1)分支,PA、PB 1、PB 2、NP和M基因属pdm/09分支,NS基因属于北美三源重组分支,因此,本试验分离株属于G4基因型欧亚类禽猪流感病毒,将其命名为A/swine/Guangdong/CJM2/2022(H1N1)。关键氨基酸位点分析显示,分离株HA蛋白裂解位点序列为PSIQSR/GL,具有典型低致病性流感病毒的分子特征。HA基因在受体结合位点处的190、225、226位氨基酸分别为D、E、Q,表明其既具有结合人型唾液酸受体的潜能又具有结合禽型唾液酸受体的潜能。NA基因关键氨基酸残基均未发生突变,提示分离株对奥司他韦和扎那米韦等神经氨酸酶抑制剂的敏感性较高,而M基因3处氨基酸位点突变为V27A、A30T、D44A,提示对金刚烷胺类药物耐药性增加。【结论】本研究分离鉴定的毒株为G4基因型欧亚类禽猪流感病毒,关键氨基酸位点分析提示该毒株具有适应在哺乳动物中复制和毒力增强的特征。本研究结果为广东地区猪流感的防控提供了参考数据。

【Objective】The experiment was aimed to understand the prevalence of Swine influenza virus(SIV)in Guangdong and explore its molecular biological characteristics.【Method】Nasal swab and lung tissue samples of pigs suspected to be infected with Swine influenza virus were collected from a pig farm in Guangdong for virus isolation and identification,genetic evolution and key amino acid site analysis.【Result】The samples were tested positive for Swine influenza virus nucleic acid by Real-time quantitative RT-PCR.In the red blood cell agglutination test,the virus exhibited agglutination with chicken red blood cells,with a hemagglutination titer of 1∶128.The results of sequencing analysis of eight gene segments were analyzed by BLAST comparison and construction of evolutionary trees,and HA and NA genes belonged to the Eurasian avian-like Swine influenza virus(H1N1)branch,PA,PB 1,PB 2,NP and M genes belonged to the pdm/09 branch,and NS gene belonged to the North American triple reassortant branch,therefore,the isolate in this study belonged to the G4 genotype Eurasian avian-like Swine influenza virus,and was named A/swine/Guangdong/CJM2/2022(H1N1).Key amino acid site analysis showed that the cleavage site sequence of HA protein of the isolate was PSIQSR/GL,which had the molecular characteristics of a typical low pathogenicity influenza virus.The amino acids at position 190,225 and 226 of the receptor binding site of HA gene were D,E and Q,respectively,which suggested that it had the potential ability to bind to the human salivary acid receptor and it also had the potential to bind avian sialic acid receptors.None of the amino acid residues of NA gene were mutated,suggesting that the isolate was sensitive to neuraminidase inhibitors such as oseltamivir and zanamivir.However,the mutations at three amino acid sites in M gene were V27A,A30T and D44A,suggesting an increased resistance to amantadine-based drugs.【Conclusion】The strain isolated and identified in this study was a G4 Eurasian avian-like Swine influenza virus.The analysis of key amino acid sites suggested that this strain had the characteristics of adapting to replication and enhancing virulence in mammals.The results of this study provided reference data for the prevention and control of swine influenza in Guangdong.