猪流产胎儿中猪繁殖与呼吸综合征病毒的检测及GP5抗原分析

Detection of Porcine Reproductive and Respiratory Syndrome Virus in Aborted Fetus from Sows and Antigen Analysis of GP5

河南某规模化猪场,按照每年4次高致病性猪繁殖与呼吸综合征弱毒疫苗免疫接种,2016年11月,妊娠后期母猪陆续产死胎。为确诊病因,采集死产胎儿肺脏组织,采用RT-PCR方法,扩增PRRSV NSP2基因部分片段(根据产物片段大小,能区分高致病性与经典毒株)和ORF5全长基因,结果显示,NSP2片段扩增大小约为418bp,表明检测毒株是高致病性PRRSV毒株;ORF5全长基因扩增结果,与预期大小片段一致。为分析猪繁殖与呼吸综合征疫苗免疫失败的原因,利用DNA Star软件,将检测毒株ORF5基因序列与其他17种PRRSV毒株ORF5基因序列构建遗传进化树,并对检测毒株与其他9种高致病性PRRSV毒株ORF5基因推导的氨基酸序列进行分析、比较及GP5抗原性分析。结果显示,ORF5序列分析表明,检测毒株与2015年福建分离毒株FJYR、河南分离毒株TJbd14-2同源性最高,分别为97.2%和97.0%,与美洲型经典毒株VR-2332同源性为87.7%,与欧洲型毒株Lelystad virus、NMEU09-1亲缘关系最远,分别为62.8%和62.6%;根据ORF5DNA序列推导氨基酸序列分析表明,与其他9种高致病性PRRSV毒株相比,从病料中检测毒株的GP5共有13个位置发生变异;GP5抗原指数分析表明,第30-40、51-63位抗原性,相比其他9种高致病性PRRSV毒株显著降低,据此推测这是导致免疫失败的根本原因,导致该猪场母猪流产的病原为一种变异的美洲型高致病性猪繁殖与呼吸综合征毒株。

There were some sows in late gestation which produced dead fetus in November 2016, at a large scale pig farm in Henan. However, all of pigs in the pig farm were inoculated with the highly pathogenic PRRSV attenuated vaccine four times in a year.In order to clarify the agent of the disease,lungs were col- lected from stillbirth for RT-PCR amplification of NSP2 gene fragment distinguishing the highly pathogen- ic and classical strains of PRRSV according to the fragment size and whole ORF5 genes.The results of RT- PCR showed that the NSP2 fragment of the detected PRRSV strain is about 418 bp,indicating that the de- tected strain is the highly pathogenic PRRSV strain; that the size of the full-length ORF5 gene amplifica- tion was consistent with the expected length.To explore the cause of the failure of PRRSV attenuated vac- cine immunization, phylogenetic trees were constructed using DNA Star by sequences of 18 different PRRSV ORF5 genes ; at the same time,the deduced amino acid sequences between the detected strain and the other 17 PRRSV strains were analysed and compared.Sequence analysis of ORF5 genes showed that the detected PRRSV strain shares the highest homology with the FJYR isolate （97.2%） from Fujian,China, and the TJbd14-2 isolate（97.0%） from Henan, China in 2015; 87.7% with America classic strain VR- 2332; respectively 62.8% and 62.6% with Europe strain Lelystad virus and NMEU09-1.Analysis of the de- duced amino acid sequences according to ORF5 genes between the detected PRRSV strain and nine highly pathogenic PRRSV strains showed that 13 amino acid positions in the detected PRRSV strain were muta- ted.Analysis of GP5 antigen index showed that compared with the other 9 highly pathogenic PRRSV strains, antigenicity between 30-40 amino acid positions, and 51-63 amino acid positions in GP5 of the detec- ted PRRSV strain significantly decreased .Therefore, we speculated antigenicity variation may cause the immune failure of the highly pathogenic attenuated vaccine.The etiology of the abortion of the pig farm is a highly pathogenic,variant PRRSV strain belonging to American type PRRSV.