生物学杂志

生物学杂志 ›› 2022, Vol. 39 ›› Issue (3): 12-.doi: 10.3969/j.issn. 2095-1736.2022.03.012

• 病毒学专题 • 上一篇    下一篇

猪流行性腹泻病毒变异毒株的分离鉴定及遗传演化分析

  

  1. 1. 山东农业大学 动物科技学院， 泰安 271018；
    2. 山东省农业科学院家禽研究所 山东省家禽疫病诊断与免疫重点实验室， 济南 250000;
    3. 山东省农业科学院畜牧兽医研究所 山东省畜禽疫病防治与繁育重点实验室， 济南 250000
  • 出版日期:2022-06-18 发布日期:2022-06-17
  • 通讯作者: 孙淑红，教授，主要从事动物病毒学研究，E-mail: sunshuhong@sdau.edu.cn；吴家强，研究员，主要从事动物病毒防控技术研究，E-mail: wujiaqiang2000@sina.com
  • 作者简介:辛忠昊，硕士研究生，主要从事动物病毒学研究，E-mail: 18366592378@163.com；郭效珍，博士研究生，主要从事动物传染病研究，E-mail: gxz0821@163.com；辛忠昊和郭效珍为共同第一作者
  • 基金资助:
    山东省自然科学基金青年项目（ZR2021QC054）； 山东省重大科技创新工程项目（2020CXGC010801）； 山东省现代农业产业技术体系项目（SDAIT-08-01）； 山东省农业重大应用技术创新项目（SD2019XM007）； 高层次人才工程项目；山东省农业创新工程项目

Isolation and identification of variant strains of porcine epidemic diarrhea virus and analysis of genetic evolution

  1. 1. Shandong Agricultural University College of Animal Science and Technology, Taian 271018, China;
    2. Poultry Research Institute, Shandong Academy of Agricultural Sciences, Key Laboratory of Poultry Disease
    Diagnosis and Immunity in Shandong Province, Jinan 250000, China; 3. Institute of Animal Husbandry and Veterinary
    Medicine, Shandong Academy of Agricultural Sciences, Key Laboratory of Animal and Poultry Disease Control
    and Breeding, Shandong Province, Jinan 250000, China
  • Online:2022-06-18 Published:2022-06-17

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摘要： 猪流行性腹泻（PED）是由猪流行性腹泻病毒（PEDV）引起猪的一种急性高度接触性肠道传染病。为了解山东地区PEDV的遗传进化趋势，对2020—2021年收集的疑似猪流行性腹泻的临床病料进行RT-PCR检测。随后将处理好的病料上清液接种于Vero细胞进行PEDV病毒分离，并利用间接免疫荧光（IFA）进行验证，成功分离到一株PEDV毒株，命名为QH-202105。将分离株传代培养至第10代，提取RNA后进行扩增并全基因组测序，结合NCBI下载的参考序列对分离株的全基因组和S基因进行核苷酸同源性比对、系统发育树构建分析，结果显示，分离株与美国分离株USA/Colorado/2013核苷酸同源性最高，为99.1％，与CHM2013株同源性最低，为96.5％。经S基因氨基酸序列分析显示，分离株QH-202105在S基因的N端有60多处突变，两处氨基酸插入（包括第59~62位氨基酸的QGVN 4个氨基酸插入，第145位氨基酸N的插入）和两处氨基酸缺失（第115~118氨基酸的缺失，第167~168氨基酸的缺失）。进化树分析结果显示，分离毒株QH-202105为G2基因群，与CH/HNAY/2015和CH/ZJCX-1/2012亲缘关系最近，属同一进化分支。在此基础上，应用PROVEAN对QH-202105的S蛋白突变氨基酸进行功能预测，结果显示，在1361处（G→C）被判定为“Deleterious”，可以推测此处的突变对病毒的侵袭力可能存在间接影响。研究结果可为我国PEDV变异株的流行情况及防控提供参考。

关键词: 猪流行性腹泻病毒, 流行毒株, 分离鉴定, 遗传变异

Abstract: Porcine epidemic diarrhea (PED) is an acute, highly contact intestinal infectious disease of pigs caused by porcine epidemic diarrhea virus (PEDV). To understand the genetic evolutionary trend of PEDV in Shandong, RT-PCR was performed on clinical material collected from 2020-2021 with suspected epidemic diarrhea. Subsequently, the supernatant of the treated disease material was inoculated into Vero cells for PEDV virus isolation and verified using indirect immunofluorescence (IFA), and a PEDV strain, named QH-202105, was successfully isolated. the isolate was passaged and cultured to the 10th generation, its RNA was extracted, amplified and whole genome was sequenced, and the whole genome and S gene of the isolate were combined with the reference sequence downloaded from NCBI. The results showed that the isolate had the highest nucleotide homology with USA/Colorado/2013 at 99.1% and the lowest homology with CHM2013 at 96.5%. Amino acid sequence analysis of the S gene showed that the isolate QH-202105 had more than 60 mutations in the N terminus of the S gene, two amino acid insertions (including four amino acid insertions in QGVN at amino acids 59-62 and an insertion in amino acid N at position 145) and two amino acid deletions (deletion in amino acids 115-118 and deletion in amino acids 167-168). The results of evolutionary tree analysis showed that the isolated strain QH-202105 was a G2 gene group, which was the closest relative to CH/HNAY/2015 and CH/ZJCX-1/2012 and belonged to the same evolutionary branch. Based on this result, PROVEAN was applied to predict the function of the mutated amino acid in the S protein of QH-202105, and the result showed that the mutation at 1361 (G→C) was determined to be “Deleterious”, and it could be assumed that the mutation here might have an indirect effect on the invasiveness of the virus.The results of the study could provide reference data for the prevalence and prevention and control of PEDV variants in China.

Key words: porcine epidemic diarrhea virus, epidemic strain, isolation and identification, genetic variation

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