猪流行性腹泻病毒CH/GSMQ/2022株的分离鉴定与致病性

Isolation,identification and pathogenicity of porcine epidemic diarrhea virus strain CH/GSMQ/2022

采集了我国甘肃省民勤县某养殖场疑似猪流行性腹泻发病猪的粪便和肠道内容物,通过RT-PCR方法对疑似阳性病料进行检测后,利用Vero细胞对其进行体外分离培养,对成功分离的病毒进行实验室鉴定,对其全基因组序列进行遗传进化分析,并通过动物回归试验对其致病力进行评价。结果显示,该猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV)阳性病料处理液接种Vero细胞盲传至第4代时可观察到典型的合胞体病变现象,第6代病毒滴度达到10^(-4.75)TCID_(50)/mL;电镜观察可以看到直径约为100 nm、圆形、表面有明显囊膜和纤突的PEDV样病毒粒子;全基因组测序分析表明该毒株全长28085 bp,与经典毒株CV777代表的G1亚型毒株亲缘关系较远(同源性为96.6%),与G2b型毒株BJ-2011-1、IA1、USA/Colorado/2013和WELL同源性较高(为98.6%),表明该毒株属于G2b型流行毒株。动物回归试验结果显示,5日龄的仔猪在攻毒后12 h即出现呕吐、急性水样腹泻、消瘦和精神沉郁等临床症状,于24 h内症状加重而死亡,剖检后可以观察到感染仔猪的胃部肿胀,小肠高度臌气、肠壁薄而透明,内部还有未消化完的乳汁凝块。结果表明,本试验成功分离鉴定到1株PEDV G2b型流行毒株,并对其全基因组序列和致病性进行了分析,为今后针对PEDV基因功能、致病机制的研究及疫苗的开发提供了研究材料。

Feces and intestinal contents of pigs suspected with porcine epidemic diarrhea virus were collected from a farm in Minqin County,Gansu Province,China.After the suspected positive samples were detected by RT-PCR,Vero cells were used to isolate and culture them in vitro.The successfully isolated virus was identified in the laboratory,and its whole genome sequence was analyzed for genetic evolution.The pathogenicity was evaluated by animal regression test.The results showed that typical syncytial lesions could be observed when the PEDV-positive treatment solution was inoculated with Vero cells in the 4th generation,and the virus titer in the 6th generation reached 10^(-4.75)TCID_(50)/mL.PEDV-like virions with a diameter of about 100 nm and a round shape with obvious capsular membranes and spikes were observed by electron microscopy.Whole genome sequencing analysis showed that the total length of this strain was 28085 bp,which was far from the G1 subtype represented by the classical strain CV777(96.6%),and had a high homology with the G2bstrains BC-2011-1,IA1,USA/Colorado/2013and WELL(98.6%).This indicated that the strain belonged to the G2bepidemic strain.The animal regression test showed that the 5-day-old piglets developed vomiting,acute watery diarrhea,emaciation and mental depression within12hafter the attack,and the symptoms worsened and died within 24h.After autopsy,the infected piglets could be observed with stomach swelling,high intestinal heave,thin and transparent intestinal wall,and undigested milk clots inside.In summary,a PEDV G2bepidemic strain was successfully isolated and identified in this study,and its whole genome sequence and pathogenicity were analyzed,providing research materials for future studies on PEDV gene function,pathogenic mechanism and vaccine development.