南方农业学报2025，Vol.56Issue(3)：932-943,12.DOI:10.3969/j.issn.2095-1191.2025.03.023
G1P[7]型猪轮状病毒的分离鉴定及其全基因组分析
Isolation,identification and whole genome analysis of A G1P[7]porcine rotavirus strain
摘要
Abstract
[Objective]To isolate and identify G1P[7]porcine rotavirus(PoRV)and conduct a whole genome analy-sis,which could provide theoretical basis for the prevention and control of piglet diarrhea and vaccine development.[Method]Excrement samples from diarrheic piglets were provided by college of Veterinary Medicine,Nanjing Agricul-tural University.The samples were tested for porcine epidemic diarrhea virus(PEDV),transmissible gastroenteritis virus(TGEV),porcine deltacoronavirus(PDCoV),bovine viral diarrhea virus(BVDV)and PoRV by PCR.Trypsin was added to the supernatant of the PCR-positive samples,which were then inoculated into MA104 cells for virus isolation and purification.The strain was identified by PCR,electron microscopy,indirect immunofluorescence assay,and RNA-PAGE.The biological characteristics of the strain were explored by drawing the virus proliferation curve and testing the susceptibility of MA104,Vero,IPEC-J2,HT-29,HRT-18,and Caco-2 cells to the virus.Bioinformatics software was used to analyze the homology of the 11 genes of the isolated strain with various genotypes of rotavirus at home and abroad and to construct a phylogenetic tree of whole genome of the virus.[Result]The PCR results of the excrement samples showed that only PoRV was positive.One strain of PoRV was successfully isolated and named JSNJ2023.The virus could stably replicate and passage on MA104 cells.After three rounds of cloning,a suitable clone was selected.In the third round of cloning,the clone with the highest virus titer was expanded,and a high-titer monoclonal strain was finally ob-tained.The virus particles had a distinct spherical structure with a clear double-layered capsid feature,with a diameter of approximately 70 nm.The indirect immunofluorescence assay results showed that the JSNJ2023 strain could infect MA104 cells.The RNA-PAGE results indicated that strain JSNJ2023 had the characteristic 11-band electrophoretic pattern of group A rotavirus,arranged as 4:2:3:2.The virus proliferation curve showed that the strain JSNJ2023 reached the peak virus titer in MA104 cells 18 h after inoculation,and the virus titer gradually decreased with the extension of the ino-culation time.The susceptibility of the strain JSNJ2023 to cell lines was in the order of MA104,IPEC-J2,HRT-18,HT-29,Caco2 and Vero cells.The results of gene homology analysis and phylogenetic tree construction showed that the strain JSNJ2023 belonged to group A G1P[7]porcine rotavirus,with the genotype of G1-P[7]-I5.[Conclusion]The PoRV strain JSNJ2023 is successfully isolated and identified as a G1P[7]type rotavirus of group A,with the genotype of G1-P[7]-I5.The strain JSNJ2023 has a high degree of homology with human,porcine and bovine rotaviruses,and its evolu-tion may have been influenced by human,porcine,and bovine rotaviruses,resulting in gene recombination or exchange and the formation of a unique genotype R1-C1-M1-A8-N1-T7-E1-H1).关键词
猪轮状病毒(PoRV)/全基因组测序/遗传进化分析/基因重组Key words
porcine rotavirus(PoRV)/whole genome sequencing/genetic evolution analysis/gene recombination分类
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王建新,马润超,周金柱,卞贤宇,朱雪蛟,李昱辰,张雪寒,李彬..G1P[7]型猪轮状病毒的分离鉴定及其全基因组分析[J].南方农业学报,2025,56(3):932-943,12.基金项目
国家重点研发计划项目(2022YFD1800601) （2022YFD1800601）
江苏省重点研发计划(现代农业)项目(BE2023317) National Key Research and Development Program of China(2022YFD1800601) （现代农业）
Jiangsu Key Research and Development Plan(Modern Agriculture)Project(BE2023317) （Modern Agriculture）
