Isolation, identification and genomic analysis of HP-PRRSV obtained from one province in east China | 我国华东某省HP-PRRSV的分离鉴定及全基因组序列分析

Chinese. 从我国华东地区某省3个不同市县的疑似猪繁殖与呼吸综合征(PRRS)发病猪场送检的病料中分离PRRSV毒株，并对其全基因组进行测序，进而对当前PRRSV流行毒株的分子特征和近几年我国PRRSV毒株的演化特点进行分析。采集养殖场病死猪的淋巴结和肺脏病料，处理后进行PRRSV的RT-PCR检测。对PRRSV检测呈阳性的病料进行病原分离和免疫荧光试验（IFA）鉴定，测定IFA鉴定呈阳性的PRRSV分离株的TCID50。参照NCBI基因数据库已提交的PRRSV VR-2332、CH-1a、HB-2及JXA1等毒株序列设计6对引物，对所得分离毒株分别进行全基因组序列扩增、测序，并将测序结果与LV、VR-2332、JXA1等国内外16个PRRSV毒株进行序列同源性和进化分析。试验共分离鉴定获得3株PRRSV毒株，分别命名为SDCXA/2008、SDWF、SDLY。全基因组Blast比对结果表明，这3个分离株均为北美洲型毒株。序列分析发现，3个分离株的全基因组序列与欧洲株的同源性极低（61.6%～61.8%），与2006年前分离的美洲型毒株的同源性较低（86.6%～97.1%），与2006后分离的美洲型毒株同源性较高（98.3%～99.7%）。Nsp2基因在3个易变区中变异最大， ORF5次之，ORF3相对最小；推导氨基酸序列中变异最大的亦为Nsp2。3株PRRSV毒株Nsp2基因推导氨基酸的第481和532～560位处共存在30个氨基酸缺失，与以HUN4、JXA1等为代表的国内高致病性分离株序列的同源性较高，结合进化分析将其均划为与JXA1类似的高致病性猪蓝耳病毒株（HP-PRRSV）。分离到3株HP-PRRSV毒株，其遗传特征相对稳定，但同时也发生了一定的变异，说明PRRSV还在不断地变异和演化。PRRSV的分布无明显地域性。

English. PRRSV strains from pig IFArms suspected to have broken out PRRS in 3 different regions of one province in middle east China were isolated. The genome of the obtained PRRSV isolates was determined and then molecular characteristics of dominant strains was analyzed, the evolutionary law of PRRSVs isolated in China recent years according detaild sequences analysis was observed. Samples of lymph nodes and lungs from dead or sick pigs were collected, treated samples by PRRSV RT-PCR were determined, then the strains from positive samples were isolated. IFA was conducted after the isolation and TCID50 of IFA positive isolates was measured. 6 pairs of primers were designed based on PRRSV VR-2332, CH-1a, HB2 and JXA1 and other PRRSV strains available on NCBI Genebank. The genome of obtained PRRSV isolates were amplified and determined by these primers. Detailed sequences analysis and evolutionary relationship research among 3 obtained isolates and LV, VR-2332, JXA1 and other 13 domestic and foreign representative strains or isolates were carried out. 3 PRRSVstrains were obtained and were named as SDCXA/2008, SDWF and SDLY respectively. The Blast result of complete genome indicated that all 3 isolates were Amercian-type strains. According to the sequences analysis, the genome sequences of 3 isolates showed a very low identity of 61.6%-61.8% with Europe-type PRRSV, a relatively low identity of 86.6%-97.1% with American-type strains isolated before 2006, a high identity of 98.3%-99.7% with American-type strains isolated after 2006. Nsp2 gene was the most variable labile gene compared with ORF5 and ORF3 gene. ORF5 gene was more mutable than ORF3 gene. The deduced aa of Nsp2 gene was most variable too. The sequences analysis showed that all 3 isolates were high pathogenic PRRSV like representative HP-PRRSV strains of JXA1 and HUN4 having 30 discontinuous aa deletions at the position of 481 and 532-560 aa site in Nsp2 deduced aa sequence. The sequences analysis with and phylogenetic tree was combined and 3 obtained HP-PRRSV strains were clossified into JXA1-like HP-PRRSV group. 3 HP-PRRSV strains were obtained, the genetic characteristics of 3 strains showed to be comparatively stable, but some mutations appeared in the genomes-suggested that the PRRSVs would continually evolve with variation in future. The result also showed that there were no apparent relations between the distribution features of PRRSVs and the places.