Isolation,identification,and genome sequence analysis of a strain of G9P[23]por-cine group A rotavirus from Guizhou
To understand the genomic characteristics and genetic evolution relationship of G9 por-cine rotavirus(PoRV)epidemic strain in Guizhou Province,the pathogen of intestinal contents and fecal samples of diarrhea piglets was detected,and the positive samples of G9 PoRV were isolated and cultured by Vero cells,and the genetic variation of the isolated strain was analyzed.The results showed that the positive samples were inoculated into Vero cells after pre-treatment with trypsin,and stable CPE appeared after blind transmission to the 15th generation.After the cells were rounded in the early stage,they contracted and fell off,and the cytoplasm boundary was unclear in the later stage,which led to the net was pulled.The specific primers were used to identify the cell culture by RT-PCR,and the target band of 341 bp was successfully amplified.Indirect immunofluo-rescence identification showed specific green fluorescence reaction,and electron microscope observation showed that there were lattice-arranged encapsulated virus particles with a size of a-bout 70 nm.A strain of PoRV was successfully isolated and named GZZY2021.The growth curve showed that the virus content of GZZY2021 strain was the lowest in 0-12 h,and the virus prolif-eration rate was the fastest in 12-30 h,and the virus proliferation rate was gentle in 30-48 h,and the virus titer reached the peak at 48 h(1 0-5.60/0.1 mL),and then entered a stable period.Genome cloning and sequencing analysis showed that the whole genome of GZZY2021 isolate was 18 506 bp,and the homology of the most similar sequences of 11 genes was more than 95.00％.The genes VP1,VP2,VP4,VP7,NSP1,NSP3 and NSP4 have the highest homology with pig-derived strains,while VP3 had the highest homology with giant panda-derived strains.The VP6,NSP2 and NSP5 genes have the highest homology with human strains,suggesting that the isolated strain is a ternary recombinant strain,and the complete genotype is G9-P[23]-I1-R1-C1-M1-A8-N1-T1-E1-H1.Compared with reference strains of the same type,VP4 and VP7 genes have five unique amino acid sites and three unique amino acid sites variations respectively.There are 22 amino acid sites and 4 amino acid sites which are different from the neutralizing epitopes of NX vaccine strain.In summary,a G9P[23]PoRV strain was successfully isolated using Vero cells,which can not only provide research data for continuously monitoring the epidemic trend of PoRV in Guizhou Prov-ince,but also lay the foundation for the screening of candidate vaccine strains as well as the in-depth study of the disease.
group A porcine rotavirusisolation and identificationsequence analysis
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