Porcine Epidemic Diarrhea Virus Of Henan Strains Biological Characteristics And The Establishment Of FQ-PCR Method

Porcine epidemic diarrhea (PED) is an acute, highly contagious diarrheadisease of swine caused by Porcine epidemic diarrhea virus (PEDV), which is amember of the family coronaviridae, subfamily coronavirinae, genusalphacoronavirus. Since the winter of2010, porcine epidemic diarrhea disease causedby the PEDV outbreaked in most provinces nationwide and led to the huge economiclosses in pig industries. Studies have shown that the PED pandemic virus as thevariant strain is different from the previous classic strains, but there is not acomprehensive system of research in aspects of its antigenicity, pathogenicity andrapid differential diagnosis of the disease methods. In order to study molecularvariation, cell culture characteristics and specific and rapid method for the differentialdiagnosis of the PEDV in Henan, PEDV representative positive samples was detectedthrough the RT-PCR method established in our laboratory. The genes of S1wassequenced and analysised, The culture conditions was explorated in Vero cell, STcells, and the PK-15cells to select the most suitable conditions for PEDVproliferation including the required concentration of trypsin, the time and dose ofinoculation when the virus proliferative in cells; compared with a lot of PEDV Mgene sequences in GenBank, selected the highly conserved and type-specific genesequences as a template, one pair of specific primers and a TaqMan MGB probe weredesigned. TaqMan fluorescent quantitative real-time PCR(FQ-PCR)to detect PEDVwas established after optimized the reaction conditions about optimal concentration ofthe primer, probe and enzyme and the optimal temperature of annealing through usingMatrix method, the sensitivity, specificity and reproducibility of FQ-PCR werecarried out, simultaneously the compliance with the existing PEDV RT-PCR and genesequencing methods.Results: sequences analysis showed that S1gene shared97.9%to99.8%nucleotide identities and amino acids homology97.0%to99.6%among sixteenPEDV isolates. The nucleotide homology was97.7%to99.5%and amino acidhomology was97.7%to99.0%compared with domestic landing the third group PEDV epidemic strains from2011. However，compared with domestic landing thefirst group PEDV epidemic strains in2011the nucleotide homology was89.9%to90.5%and amino acid homology was89.7%to91.1%. Compared with domesticlanding PEDV in2012the nucleotide homology was98.1%to99.7%and amino acidhomology was97.3%to99.6%. Compared with domestic landing PEDV in2013thenucleotide homology was97.2%to99.3%and amino acid homology was95.7%to99.6%. Compared with CV777that nucleotide homology was92.5%to92.9%andamino acid homology was90.5%to91.4%. Homology analysis showed that S1genesof16isolates shared the same genetic mutation, there were the same insertions anddeletions. Compared with domestic group three PEDV strains landed from2011, therewas no tendency. However, compared with CV777, there were three nucleotideinsertion between163bp to166bp, nine nucleotide insertions between173bp to174bp, three nucleotide insertions between405bp to406bp and three nucleotide deletionsbetween463bp to464bp. These insertions and deletions of nucleotides led to itscorresponding change in encoding the amino acid. Phylogenetic analysis showed thatS1genes of16PEDV strains belong to the third group. However, CV777was secondgroup.PEDV positive samples can be successfully propagated to the ninth generation inVero cells, and the cell layer showed the phenomenon typical cytopathic effects. Inthe propagation experiments, the optimal concentration of trypsin in medium is5μg/mL, optimal time of inoculation is32h after cell cultured, the dose of PEDVinoculation is100μL/mL; but there were nonpermissive for PEDV replication in STand PK-15cells.PEDV TaqMan MGB FQ-PCR methods for testing PEDV were successfullyestablished, There was a good linear relationship between the cycle threshold of thestandard curve and template concentration, and the correlation coefficient was0.999.The linear expression between the number of copies(X) and the Ct values is, Ct=-3.12×logX+41.67, the detection sensitivity of FQ-PCR method was up to101copy/μL, The specificity of the method was high, the result is negative when93samples that are known non-PEDV and negative controls were tested which includedthe classical swine fever virus (HCV), PRRS virus (PRRSV), porcine pseudorabiesvirus (PRV), porcine transmissible gastroenteritis virus (TGEV), porcine rotavirusvirus (PoRV) and other viruses of chickens, ducks, and cows. With3times repeatedamplification of different concentrations PEDV recombinant plasmids, respectively, the results were good. The positive coincidence rate was100%for detection withgene sequencing method, and88.7%with RT-PCR method.The results of this study indicate that Vero cells is suitable cell of PEDVproliferation, and trypsin concentration, time of inoculation, vaccination dose andgrowth state of the cell will influence the proliferation in cell culture conditions.Phylogenetic analysis showed that S1genes of prevalent strain PEDV exist first groupand third group, but epidemic strain of Henan mainly prevailing third group, thepathogenic and antigenic differences of these strains should be further studied.Successful establishment of sensitive, rapid, specific FQ-PCR detection method willprovide reliable technical support for rapid differential diagnosis of PEDV.