Multiple QRT-PCR Detection Method Of Porcine Important Diarrhea Virus And Molecular Epidemiology Of PEDV And PDCoV

Porcine enteric coronaviruses(PECoVs)and porcine rotaviruses(RVs)are common viruses that cause diarrhea in pigs.PECoVs include porcine epidemic diarrhea virus(PEDV),transmissible gastroenteritis virus(TGEV),swine acute diarrhea syndrome coronavirus(SADS-CoV)and porcine deltacoronavirus(PDCoV),all of which cause similar symptoms of porcine enteric diarrhea.Rotavirus group A(RVA),rotavirus group B(RVB),rotavirus group C(RVC)and rotavirus group H(RVH)are four Po RVs known to be prevalent in pigs.Mixed infections often occur between PECoVs or Po RVs,so the establishment of rapid detection methods for eight porcine diarrhea viruses is of great importance for virus prevention and control.PEDV and PDCoV are the most common PECoVs,and conducting molecular epidemiological investigations to understand the direction of their genetic variation is of great importance for vaccine development and selection.1.Establishment and application of multiplex qRT-PCR detection method for porcine enteric coronavirusesPorcine infections with PECoVs have similar clinical symptoms,including vomiting,diarrhea and dehydration.In this study,we designed primers and probes based on PEDV N,TGEV M,PDCoV M and SADS-CoV N genes to develop a qRT-PCR method for rapid detection of these four PECoVs.The method has a LOD of 121 copies/μL for the four viruses and a coefficient of variation of less than 2% for both intra-assay and inter-assay tests,with high specificity,sensitivity and reproducibility.The method was used to detect 3236 porcine diarrhea samples collected from several pig farms in Guangxi between 2020-2022,and the results showed the positive rates of PEDV,TGEV,PDCoV,and SADS-CoV were 18.26%,0.46%,13.16%,and0.15%,respectively,with more than 99% agreement between the detection results of the previously reported qRT-PCR method.These results suggest that the multiplex qRT-PCR method provides an efficient and accurate technical tool for the detection of PECoVs.2.Establishment and application of multiple qRT-PCR detection method for porcine rotavirusRVA,RVB,RVC and RVH are the currently prevalent Po RVs in pigs,and coinfection between viruses often happens.In this study,primers and probes were designed based on the VP6 genes of RVA,RVB,RVC and RVH and the multiplex qRT-PCR assay was established.The method has a LOD of15 copies/μL for the Po RVs and a coefficient of variation of less than 2% for both intra-assay and inter-assay tests,thus showing high specificity,sensitivity and repeatability.The method was used to detect 1447 porcine diarrhea samples in Guangxi,with positive rates of 42.09%,27.50%,44.23% and13.13% for RVA,RVB,RVC and RVH,respectively,and mixed infections were more common than single virus infections.The method was shown to achieve more than 99% compliance with the reference method.The results indicated that the multiplex qRT-PCR method provides an efficient and accurate technical means for detecting porcine RVs.This study reports for the first time the prevalence of porcine RVH in China,and we need to pay more attention to the impact of Po RVs.3.Molecular epidemiology of porcine epidemic diarrhea virus in Guangxi from 2020 to 2022In this study,PEDV-positive samples were selected from diarrhea samples in various regions of Guangxi during 2020-2022 for amplification,cloning and sequencing,and the S1,M and N genes of 52 PEDV strains were finally obtained.Sequence analysis showed that the similarity of nucleotides and their encoded amino acids in the S1,M and N genes of 52 PEDV strains was 91.8%-99.9% and 89.1%-100%,96.0%-100% and 94.2%-100%,95%-100% and 94.7%-100%,respectively.There was a large amount of variation in the amino acids encoded by the S1 gene,while the amino acids encoded by the M and N genes were conserved.Genetic evolutionary tree showed that most of the 52 PEDV strains belonged to cluster GII,while six strains were in cluster S-INDEL.There were also potential homologous recombination events among the Guangxi strains.This study indicates that there is genetic diversity in Guangxi strains,and detection and molecular epidemiological investigation should be strengthened.4.Molecular epidemiological study of porcine deltacoronavirus in Guangxi from 2020 to 2022In this study,PDCoV-positive samples were selected from diarrhea samples in various regions of Guangxi during 2020-2022 for amplification,cloning and sequencing,and the S,M and N genes of 32 PDCoV strains were finally obtained.The similarity of nucleotides of S,M and N genes and their encoded amino acids between the 32 PDCoV strains and the reference strain were above 94.4%,indicating high similarity of S,M and N genes of PDCoV from different sources.A large number of amino acid loci were mutated in the amino acids encoded by the S gene,while the amino acids encoded by the M and N genes were more conserved.Genetic evolutionary tree showed that all32 PDCoV strains were in Group Ⅱ of the Chinese strain cluster.This study shows the genetic variation characteristics of the prevalent PDCoV strains in Guangxi,and provides a basis for scientific prevention and control of PDCoV.