欧洲型猪繁殖与呼吸综合征病毒GD2022株的分离鉴定及遗传进化分析

Isolation,Identification and Genetic Evolutionary Analysis of European Type Porcine Reproductive and Respiratory Syndrome Virus GD2022 Strain

为了解广东省欧洲型猪繁殖与呼吸综合征病毒(PRRSV-1)流行情况,将RT-PCR检测PRRSV-1阳性的病料(肺脏和淋巴结)接种猪肺泡巨噬细胞(PAMs)进行病原分离鉴定,以分离毒株核酸作为模板,对分离的PRRSV-1进行全基因克隆和测序,并与国内外PRRSV-1和PRRSV-2参考毒株序列进行同源性及遗传进化分析。结果显示,成功分离1株PRRSV-1,命名为GD2022株,其接种PAMs可引起典型的细胞病变,从出现病变的F5细胞中提取核酸,用PCR分段扩增出PRRSV-1 GD2022株10个基因组片段,分别连接到pCE2 TA/Blunt-Zero载体进行测序,拼接后获得PRRSV-1 GD2022株全基因组长15058个核苷酸(不包含polyA尾)。同源性分析显示,该分离株全基因组序列与PRRSV-1参考毒株(LV株)和PRRSV-2参考毒株(VR2333株)核苷酸序列同源性分别为87%和59.7%。遗传进化树分析显示,全基因序列和ORF5基因与国内外PRRSV-1毒株为同一进化分支,且与疫苗毒分支较远,推断分离株为1株PRRSV-1,属于基因亚型1。氨基酸序列比对分析显示,ORF3和ORF4重叠区域缺失3个核苷酸,导致GP3蛋白和GP4蛋白分别在第245和第65氨基酸位点缺失了1个氨基酸,且与LV株比对,GD2022毒株GP5的氨基酸序列在中和抗原表位第33氨基酸位点(D→A)出现点突变,在高变区第56氨基酸位点(D→A)和第63氨基酸位点(G→D)出现两处突变。重组分析显示,GD2022毒株未发生重组事件。成功分离鉴定1株PRRSV-1毒株,为国内PRRSV-1生物学特性研究提供了材料,也为了解广东省内PRRSV-1流行情况及变异特点提供参考依据。

To investigate the prevalence of European type porcine reproductive and respiratory syndrome virus(PRRSV-1)in Guangdong province,PRRSV-1 positive samples(lung and lymph nodes)were inoculated into porcine alveolar macrophages(PAMs)for pathogen isolation and identification.The nucleic acid of the isolated strains was used as a template.The full gene of the isolated PRRSV-1 was cloned and sequenced.Then the homology and genetic evolution were analyzed with the sequences of PRRSV-1 and PRRSV-2 reference strains from different countries.The results showed that one PRRSV-1 strain,named GD2022 strain,was successfully isolated,which developed typical cytopathic effect when inoculated into PAMs.Nucleic acid was extracted from the 5th-passage infected cells with cytopathic effect,and 10 genomic segments of PRRSV-1 GD2022 strain were amplified by PCR to be cloned to the pCE2 TA/Blunt-Zero vector for sequencing.The complete genome of PRRSV GD2022 strain was 15058 nucleotides in length(excluding polyA tail).The complete genome sequence of the isolate shared 87%and 59.7%nucleotide sequence identity with the PRRSV-1 reference strain(LV strain)and PRRSV-2 reference strain(VR2333 strain),respectively.The full gene sequence and ORF5 gene were in the same evolutionary branch with the domestic and external PRRSV-1 strains,and were far from the vaccine virus branch.The isolated strain belonging to subgenotype 1 was inferred to be PRRSV-1 strain.Amino acid sequence alignment analysis showed that ORF3 and ORF4 overlapped with 3 nucleotides,resulting in GP3 and GP4 protein 245 and 65 amino acid deletion of 1 amino acid comparing with LV strain.The GP5 amino acid sequence of GD2022 strain showed a site mutation at 33 amino acid site(D→A)in the neutralizing epitope,and two mutations at 56 amino acid site(D→A)and 63 amino acid site(G→D)in the hypervariable region.Recombination analysis showed that no recombination event occurred in the GD2022 strain.A PRRSV-1 strain was successfully isolated and identified,which provides a material for the study of biological characteristics of PRRSV-1 in China as well as providing a reference for understanding the epidemic and variation characteristics of PRRSV-1 in Guangdong province.