一株猪高致病性蓝耳病病毒的分离鉴定与遗传变异分析

Isolation，Identification and its Genetic Variation Analysis of a Highly Pathogentic Porcine Reproductive and Respiratory Syndrome Virus

关键词:  猪高致病性蓝耳病病毒  分离；Marc-145细胞；基因组；进化分析

Key words:  PRRSV  Isolation  Marc-145 cell line  Genome Evolutionary analysis  

摘要:

为研究猪蓝耳病病毒（PRRSV）的遗传进化规律，于2018年从新疆某猪场采集的病猪血液中分离了一株PRRSV，命名为XJ-b。对临床样品采用RT-PCR扩增鉴定，将阳性样品过滤处理之后接种在Marc-145细胞系进行培养，将盲传3代之后出现病变（CPE）的Marc-145样品进行间接免疫荧光鉴定。通过噬斑纯化后，利用RT-PCR对分离株的全基因组进行扩增，使用SeqMan软件对测序结果进行拼接，并根据NCBI上已公布的PRRSV全基因组及Nsp2基因核苷酸序列进行遗传进化与同源性对比分析。结果表明，临床病料RT-PCR检测呈PRRSV核酸阳性，处理后的病料接种至Marc-145细胞72h之后产生CPE，间接免疫荧光鉴定为PRRSV抗原阳性。序列拼接显示分离株全基因组开放阅读框大小为15119bp。全基因组和Nsp2基因核苷酸序列分析和同源性比对显示该分离毒株属于中国的高致病性PRRSV亚群。本研究结果为进一步分析近年来PRRSV在新疆地区的毒株差异，为PRRSV在新疆地区的流行提供了科学参考，同时也为我国边远地区PRRS的防控与净化具有一定的指导意义。

Abstract:

In order to study the genetic evolution of Porcine reproductive and respiratory syndrome virus, a PRRSV strain was successfully isolated from the blood of suspected PRRSV infected pigs in Xinjiang Uygur Autonomous Region in 2018, and named as XJ-b. In this study, the Marc-145 cell line was used to isolate and culture the clinical material. The isolate was subjected to examinations by RT-PCR, plaque test, IFA identification. After plaque purification, the complete genome of the isolate was amplified by RT-PCR, spliced by SeqMan software, and genetically evolved with the PRRSV whole genome and Nsp2 gene nucleotide sequences published on the NCBI. The results showed that clinical material was positive of nucleic acid of PRRSV. The CPE of Marc-145 cells were appeared after 72h of post-infection. Antigenicity positive were proved by IFA tests. Sequence splicing showed that the genome-wide open reading frame size of the isolate was 15119bp. The isolated strain appertained to the highly pathogenic PRRSV subgroup of China according to its whole genome and Nsp2 nucleotide sequence. The results of this study are to further explore the genetic variation of PRRSV, provide a scientific reference for the spread of PRRSV in Xinjiang and also lay a foundation for the prevention and control of PRRS in remote areas of China.