贵州省高致病性猪繁殖与呼吸综合征病毒的分离鉴定及遗传变异分析  

Isolation, identification and genetic variation of highly pathogenic porcine reproductive and respiratory syndrome virus in Guizhou Province

作　　者：郭倩妤 石远菊 汤德元[1] 曾智勇[1] 王彬[1] 黄涛[1] 杨伟[1] 叶丽[1] 廖少山 张森 GUO Qianyu;SHI Yuanju;TANG Deyuan;ZENG Zhiyong;WANG Bin;HUANG Tao;YANG Wei;YE Li;LIAO Shaoshan;ZHANG Sen(College of Animal Science,Guizhou University,Guiyang 550025,China)

机构地区：[1]贵州大学动物科学学院

出　　处：《畜牧与兽医》2019年第7期67-74,共8页Animal Husbandry & Veterinary Medicine

基　　金：贵州省教育厅创新群体重大研究项目(黔教合KY字[2017]027);贵州省科技支撑计划项目(黔科合支撑[2017]2583)

摘　　要：利用已建立的猪呼吸和繁殖障碍类病毒性疫病多重PCR检测方法对2017年以来贵州9个不同地区采集的150份病料进行检测,对猪繁殖与呼吸综合征病毒(PRRSV)检测为阳性的病料采用细胞接毒培养技术、病毒的形态结构观察和间接免疫荧光试验等分离鉴定PRRSV,并对其ORF5和Nsp2基因的核苷酸和氨基酸序列进行差异性分析。结果表明:分别接种Marc-145细胞并连传5代,获得5株PRRSV,分别命名为GZ-Fq、GZ-Bj、GZ-Kl、GZ-Hz和GZ-Zy;病毒粒子在电镜下呈球形或卵圆形,直径约30~80nm;5株毒株与近年来国内各地流行的高致病性PRRSV(HP-PRRSV)同源性较高,分别为96.2%~100%和97.1%~98.6%,其中,ORF5基因的第13位氨基酸均为精氨酸(R),第137位氨基酸均为丝氨酸(S),Nsp2基因的氨基酸均存在第481位和532-560位氨基酸2处不连续的缺失位点;其ORF5基因及Nsp2基因均与2008年之后流行的HP-PRRSV亲缘关系较近。研究表明,贵州省PRRSV发生了变异,与近年来国内各个地区的HP-PRRSV序列的变异位点一致,属于美洲型PRRSV的变异株。This study used the multiplex PCR assay for porcine respiratory and reproductive disorders in the laboratory to examine 150 samples collected from 9 different regions in Guizhou Province since 2017. The cells used for PRRSV were positive. Five highly pathogenic porcine reproductive and respiratory syndrome viruses were isolated and identified by toxic culture technique, virus morphological structure observation and indirect immunofluorescence assay, and were named GZ-Fq, GZ-Bj, GZ-Kl, GZ-Hz, respectively. The GZ-Zy strain and the nucleotide and amino acid sequences of the ORF5 and Nsp2 genes of the five strains of PRRSV were differentially analyzed. The results of isolation and identification showed that the Marc-145 cells had been inoculated separately for 5 generations, and RT-PCR amplified the specific bands of PRRSV. Under electron microscope, the virions were spherical or oval with a diameter of about 30-80 nm. The 5 strains were highly homologous with HP-PRRSV prevalent in recent years, at 96.2%-100% and 97.1%-98.6%, respectively. Among the strains, the 13^th amino acid of the ORF5 gene was arginine(R), the amino acid at the site of 137 was serine(S), and the amino acids of the Nsp2 gene were located at the sites of 481^th and 532-560 amino acid 2 non-contiguous deletion;the ORF5 and Nsp2 genes have both been prevalent since 2008. The highly pathogenic reproduction of the genes was closely related to the respiratory syndrome. This indicated that the highly pathogenic reproductive and respiratory syndrome virus in pigs in Guizhou Province has been mutated, which was consistent with the mutation sites of highly pathogenic reproductive and respiratory syndrome virus sequences in other regions in China in recent years;to the viruses were of a mutant strain of the American-type PRRSV.

关 键 词：HP-PRRSV 分离鉴定 ORF5基因 NSP2基因 遗传变异 

分 类 号：S855.3[农业科学—临床兽医学]