Isolation And Identification Of Porcine Rotavirus GZAS2020 Strain And Analysis Of Its Genomic Diversit

Porcine rotavirus（PoRV）belongs to the family Reoviridae and is a segmented virus of the rotavirus genus.It contains six structural proteins（VP1～VP4,VP6,VP7）and six non-structural proteins（NSP1～NSP6）.PoRV is widely distributed in the world,and there are A,B,C,E and H serogroups.Porcine Group A rotavirus（PoRVA）is the most prevalent in the world,with prevalence rates of 3.3%～67.3%.The most widely prevalent genotype is G5P[7].PoRV can infect pigs at different growth stages,especially young piglets.The infection rate can be as high as 90%～100%.The sick pigs have symptoms such as loss of appetite,vomiting,diarrhea,and feces are watery or paste,causing huge economic losses to the animal husbandry.At present,there are no specific therapeutic drugs for PoRV infection.The only vaccines on sale in clinical practice is porcine transmissible gastroenteritis,porcine epidemic diarrhea,and porcine rotavirus（G5）triple live vaccine（attenuated Chinese strain+attenuated CV777 strain+NX strain）.The vaccine can prevent the infection of PoRV G5 type,but the PoRV genotype is variable.PoRV infection still occurs in pig farms that had been immunized with PoRV G5 vaccine,as well as the prevalence of G3,G9,G10 and other genotypes clinically,resulting in poor immunization effect of PoRV G5 vaccine.Guizhou Province lacks the complete genome sequence of PoRV epidemic strains,and Gen Bank also lacks the complete genome sequence of PoRV G5 vaccine strains.This study aims to isolate a PoRV wild virus strain from samples from diseased pig farms in Guizhou Province,and obtain the complete genome sequences of the wild virus strain and the NX vaccine strain.Then,differential analysis will be conducted to understand the genetic evolution relationship of each genome segment of the PoRV wild virus strain and the NX vaccine strain in Guizhou Province,as well as their matching degree in genotype and antigenic epitopes,providing reference for the screening of candidate vaccine strains of porcine rotavirus.1.Isolation and identification of PoRV GZAS2020 strainIn order to obtain a wild strain of PoRV from Guizhou Province,the PoRV positive samples were inoculated into Vero cells for virus identification on cell cultures with stable CPE.The results of nucleic acid amplification and sequencing results showed that part of the VP7 gene sequence of group A porcine rotavirus;IFA test showed specific green fluorescence in the infected cells;Electron microscope observation showed microtubule like structure and lattice like cytoplasmic inclusion bodies,about70 nm virion without capsule;The virus titer of the 10th generation virus solution is10^-5.67 TCID₅₀/100μL.The above results showed that a porcine group A rotavirus was successfully isolated and named PoRV GZAS2020 strain.2.Cloning of the whole genome of PoRV GZAS2020 strain and NX vaccine strainIn order to obtain the complete genome sequences of the PoRV GZAS2020 wild strain and the NX vaccine strain,this study referred to the PoRV genotype sequences of Group A in Gen Bank,designed 11 pairs of annexing primers,and cloned and sequenced the genome segments of the GZAS2020 and NX vaccine strains,respectively.Clone sequencing results showed that:（1）The full-length genome of GZAS2020 was 18 514 bp.The genomic segments of VP1～VP4 and VP6～VP7 were 3 302 bp,2 717 bp,2 591 bp,2 368 bp,1 356 bp and1 062 bp,respectively.The ORFs of each gene were 3 267 bp,2 673 bp,2 508 bp,2 337 bp,1 194 bp and 981 bp,respectively.The genomic segments of NSP1～NSP5were 1 569 bp,1 059 bp,1 076 bp,750 bp and 664 bp,respectively.The ORFs of each gene were 1 482 bp,954 bp,942 bp,528 bp and 594 bp,respectively.（2）The full-length genome of NX vaccine strain was 18 504 bp.The genomic segments of VP1～VP4 and VP6～VP7 were 3 302 bp,2 717 bp,2 591 bp,2 362 bp,1 356 bp and 1 061 bp,respectively.The ORFs of each gene were 3 267 bp,2 673 bp,2 508 bp,2 337 bp,1 194 bp and 981 bp,respectively.The genomic segments of NSP1～NSP5 were 1 567 bp,1 059 bp,1 075 bp,750 bp and 664 bp,respectively.The ORFs of each gene were 1 482 bp,954 bp,942 bp,528 bp and 594 bp,respectively.3.Differential analysis of the genomes of PoRV GZAS2020 strain and NX vaccine strainIn order to explore the genetic evolution relationship between various genomic segments of PoRV GZAS2020 wild strain and NX vaccine strain in Guizhou Province,as well as their matching degree at genotype and antigen levels,the experiment conducted differential analysis on GZAS2020 and NX strains using various software and online websites.The analysis results show that:（1）The nucleotide homology of VP1～VP4,VP6,VP7,NSP1～NSP5 genomic segments of GZAS2020 strain and NX vaccine strain was 87.4%,92.7%,86.2%,74.5%,89.5%,78.1%,82.5%,88.4%,87.6%,87.9%,96.5%,respectively.The amino acid homology was 98.0%,99.3%,93.9%,78.5%,97.2%,84.4%,82.8%,92.8%,94.9%,94.9%,97%,respectively.There were significant differences in nucleotides and amino acids between GZAS2020 strain and NX vaccine strain in VP4 and VP7 genome segments.There were significant differences between the genome segments of GZAS2020 strain and the reference strains of different genotypes,and the difference of the same genotype was small.（2）The genotype of GZAS2020 strain was G5-P[13]-I5-R1-C1-M1-A8-N1-T1-E1-H1,and the genotype of NX vaccine strain was G9-P[7]-I5-R1-C1-M1-A8-N1-T1-E1-H1.The VP4 and VP7 genomic segments of the GZAS2020 strain were in different evolutionary branches from the corresponding genomic segments of the NX vaccine strain,and the remaining genomic segments were in the same evolutionary branch.GZAS2020 strain only had recombination signals in VP4 and NSP4 genome segments,and the recombination sites were located at 1 541 bp～2 161 bp and 321 bp～500 bp,respectively.（3）There were 160 and 23 amino acid differences in VP4 protein between GZAS2020 strain and NX vaccine strain and CMP178 strain,respectively.There were6 and 1 amino acid site differences in VP6 protein between GZAS2020 strain and NX vaccine strain and VNM/30378 strain,respectively.There were 151 and 20 amino acid differences in VP7 protein of GZAS2020 strain,NX strain and JN-2 strain,respectively.Among them,there were 18 and 1 amino acid differences in the neutralization epitopes of VP7,respectively.The amino acids of the main virulence genes of VP4 and the main antigen genes of VP4 and VP7 were significantly different between GZAS2020 strain and NX vaccine strain,but the amino acids of the main antigen genes of VP6 were relatively conserved.（4）The epitopes of VP4 protein of GZAS2020 strain and NX vaccine strain were quite different.GZAS2020 strain had 14 epitopes,2 more than NX vaccine strain.Only the aa 41～56 and aa 62～76 of GZAS2020 strain had some of the same amino acid composition as the aa 22～72 epitope region of NX vaccine strain,and the rest were different.The GZAS2020 strain and the NX vaccine strain had little difference in the epitopes on the VP6 protein.The GZAS2020 strain had six epitopes,one less than the NX vaccine strain,of which three epitopes were identical and two epitopes were partially identical.The antigenic epitopes of VP7 protein of GZAS2020 strain and NX vaccine strain were quite different.Although there were six antigenic epitopes,only one antigenic epitope region had the same amino acid composition.The above results showed that the PoRV GZAS2020 wild strain isolated and identified in this study was significantly different from the NX vaccine strain in the genomic segments of the main antigen genes VP4 and VP7,and the amino acids and linear epitopes of the encoded proteins were significantly different,and the antigen matching degree was low.The differences of other genomic segments between GZAS2020 strain and NX vaccine strain were small,which were located in the same evolutionary branch.The homology and recombination analysis of GZAS2020 strain and NX vaccine strain showed that the isolated strain may be a gene reassortment strain from multiple host sources.It is recommended to screen and study vaccines with high antigenic genotype matching based on understanding the genotype of the current epidemic strain.Further epidemiological investigation of PoRV and isolation and identification of PoRV epidemic strains with different genotypes will be beneficial for the screening and research of candidate vaccine strains.