1株猪流行性腹泻病毒CH/JSNT/2022/Jan株的分离及其S基因遗传进化分析  

Isolation of a porcine epidemic diarrhea virus strain CH/JSNT/2022/JAN and phylogenetic analysis of its S gene

作　　者：周金柱 范宝超[1] 李基棕[1] 茅爱华 王丹丹[1] 郭容利[1] 钟纯燕 李科茫 武琦 张雪寒[1] 李彬[1,4,5] ZHOU Jinzhu;FAN Baochao;LI Jizong;MAO Aihua;WANG Dandan;GUO Rongli;ZHONG Chunyan;LI Kemang;WU Qi;ZHANG Xuehan;LI Bin(Institute of Veterinary Medicine,Jiangsu Academy of Agricultural Sciences,Nanjing 210014,China;Chengdu SG-Biotech Co.,Ltd.,Chengdu 610000,China;Biological Engineering Department,Southwest Guizhou Vocational and Technical College for Nationalities,Xingyi 562400,China;College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210095,China;College of Animal Science,Tibet Agricultural and Animal Husbandry University,Linzhi 860000,China)

机构地区：[1]江苏省农业科学院兽医研究所,江苏南京210014 [2]成都史纪生物制药有限公司,四川成都610000 [3]黔南民族职业技术学院生物工程系,贵州兴义562400 [4]南京农业大学动物医学院,江苏南京210095 [5]西藏农牧学院动物科学学院,西藏林芝860000

出　　处：《畜牧与兽医》2023年第3期88-95,共8页Animal Husbandry & Veterinary Medicine

基　　金：国家重点研发计划(2022YFD1800601);国家自然科学基金(31872481)。

摘　　要：2022年1月份江苏南通一养猪场产房仔猪发生严重腹泻,发病率和死亡率均超过80%,原因未知。死亡仔猪肠道样本经RT-PCR检测猪流行性腹泻病毒(PEDV)、传染性胃肠炎病毒(TGEV)、猪德尔塔冠状病毒(PDCoV)和猪轮状病毒(PoRV),结果显示:仅PEDV检测为阳性,TGEV、PDCoV和PoRV检测均为阴性。将处理好的肠内容物接种Vero细胞,接种后48 h即观察到典型细胞融合、聚集成合胞体的PEDV典型细胞病变,且能够在Vero细胞上稳定传代,表明成功分离到病毒,并将其命名为CH/JSNT/2022/Jan株。通过电镜观察,观察到近似球形、直径约为100 nm、表面有囊膜和纤突的冠状病毒样粒子,表明分离毒株可能为PEDV。进一步对该毒株的S基因进行测序,序列相似性分析结果显示,分离毒株CH/JSNT/2022/Jan与PEDV S基因参考毒株的核苷酸序列相似性为93.5%~99.7%,氨基酸序列相似性为92.8%~99.9%;遗传进化分析结果显示,该分离株为GⅡa型,与多个2020年和2021年中国流行株处于同一进化分支,与疫苗株CV777、AJ1102和LW/L的遗传进化距离较远。研究结果表明,PEDV的田间流行株在不断变化,流行株的分离对PEDV疫苗研发和新的防控策略的制定具有重要意义。In January 2022, the piglets in the farrowing room of a pig farm suffered from severe diarrhea, with their morbidity and mortality both exceeding 80%, for unknown reason, in Nantong City, Jiangsu Province. The intestinal samples of dead piglets were detected using RT-PCR for porcine epidemic diarrhea virus(PEDV), transmissible gastroenteritis virus(TGEV), porcine delta coronavirus(PDCoV) and porcine rotavirus(PoRV), and the results showed that only the nucleic acid if PEDV was detected to be positive and that of the other viruses was negative. Then, the treated intestinal contents were inoculated into Vero cells for virus isolation. Typical cytopathic effect characterized by cells fused and aggregated into syncytia was observed 48 hours after the inoculation. The isolated PEDV strain could be stably passaged on Vero cells, indicating that it was successfully isolated and named the CH/JSNT/2022/Jan strain. The virons in round shape, about 100 nm diameter, with obvious envelop and spikes on the surface were clearly visible by negative-staining electron microscopy, and the morphology was consistent with that of a coronavirus. Then, the S gene of this strain was sequenced, and the sequence similarity analysis showed that the nucleotide similarity of the CH/JSNT/2022/Jan strain and the PEDV S gene from the NCBI database was 93. 5%-99. 7%, and the amino acid similarity was 92. 8%-99. 9%. The phylogenetic analysis indicated that the CH/JSNT/2022/Jan strain was clustered into GⅡa with the isolated strains in China in recent years in the same group and was far from the vaccine strains CV777, AJ1102 and LW/L. These results indicated that the field circulating strains of PEDV were constantly varying, and the isolation of the circulating strains was of great signification for the development of PEDV vaccines and the formulation of new prevention strategies.

关 键 词：猪流行性腹泻病毒 病毒分离 S基因 遗传进化 

分 类 号：S852.659[农业科学—基础兽医学]