| Porcine enterovirus G(EV-G)infection was an infectious gastrointestinal disease of pigs.Pigs in different ages were all susceptible to the virus,and there were always mutiple enterovirus serotypes in the herd.The clinical symptoms were characterized by diarrhea,muscle relaxation paralysis and reproductive disorders,often complicated with other diarrhea related diseases.The genome of EV-G was prone to mutation,and it differed greatly between different countries and regions.At present,there were some reports of pigs infected with EV-G abroad,while the epidemiological data of EV-G in China are limited,It is necessary to understand the prevalence of EV-G in Guangxi and the characteristics of whole genome structure and genetic variation of EV-G,for promoting the healthy development of the pig industry in Guangxi.1.Epidemiological investigation of porcine enterovirus G infection in pigs in GuangxiEpidemiological investigation of EV-G infection in pigs in Guangxi region was conducted from 2017 to 2018 by RT-PCR in this study.It was found that among the 222 clinical diarrhea samples,the total positive rate of EV-G was6.76%and the positive rate of pig farms was 16.98%,the positive rates of EV-G samples from 2017 to 2018 were 4.55%and 10.00%,respectively.Most of the EV-G positive samples detected in this study were mixed with other porcine diarrhea virus,and the mixed infection rate of EV-G was 6.31%,among them,the mixed infection of EV-G with porcine rotavirus was the highest(1.80%).2.Establishment and preliminary application of real-time fluorescence quantitative PCR for detection of porcine enterovirus GA real-time fluorescence quantitative PCR method was successfully established in this study that could rapidly detect EV-G,with good specificity,repeatability and sensitivity.And the minimum detection limit was 2.92×10copies/μL,the Cq value of the standard curve showed a good linear relationship with the plasmid standard template in the range of 2.92×10~8 copies/μL-2.92×10~2copies/μL,the variation coefficient of intra-group and inter-group repeatability test was low.The real-time fluorescent quantitative PCR method was used to test the tissue samples and intestinal samples of EV-G challenge piglets(7d),the results showed that the copy number of EV-G virus in the cecum and stomach of challenge piglets was high.3.Complete genome sequencing and analysis of two isolated strains of porcine enterovirus GTwo complete genomes of Guangxi EV-G isolates CH/GXBH/2018 and CH/GXQZ/2017 were sequenced in this study.The full-length genomes of the two isolates were 7359 nt and 7364 nt,respectively,containing 5’UTR with lengths of 816 nt and 821 nt.They containing the same size of ORF and 3’UTRs,with the ORF length of 6471 nt,encoding 2157 amino acids,and 3’UTR length of 72 nt.The nucleotide homology between the two isolated strains reached99.7%,the nucleotide homology with the reference strains was 70.1%to 83.8%.Genetic and evolutionary data analysis showed that the Guangxi isolates were belong to the G1 subtype,they closely related to Ch-ah-f1 strain from Jiangsu province,China and 13-03212 strain from the United States.CH/GXBH/2018had 13 consecutive A base insertions,and CH/GXQZ/2017 had 6 consecutive A base insertion between 676 nt and 688 nt in the 5’UTR region;CH/GXBH/2018and CH/GXQZ/2017 both had 36 base deletions between 2493 nt and 2528 nt in the VP3.A total of 149 mutations occurred in the VP1 gene nucleotides of the two EV-G isolates in Guangxi,and 144 nucleotide mutation sites were the same.By comparing the amino acid sequence of EV-G VP1 with that of the reference strains,it showed that the VP1 amino acid of the two EV-G isolates in Guangxi region had variation,especially at positions of 111-115.Compared with the reference strains,the VP1 structural protein antigen index of the isolated strains in Guangxi significantly changed in the 170-180 aa and 220-230 aa regions.In this study,we conducted an epidemiological survey on the prevalence of EV-G in Guangxi from 2017 to 2018,and we had established an effective diagnosis method.At the same time,the genetic evolution of EV-G strains in Guangxi region has been analyzed,which enriched the whole genome bioinformation database of EV-G,it laid the foundation for further research on molecular biological characteristics of EV-G and understand the genetic characteristics as well as the epidemic situation of EV-G in Guangxi,China. |
