Establishment Of Multiple RT-PCR Assay For Four Porcine Enteroviruses And Isolation And Identification Of Porcine Epidemic Diarrhea Virus

Porcine epidemic diarrhea virus（PEDV）,porcine transmissible gastroenteritis virus（TGEV）,porcine deltacoronavirus（PDCo V）and porcine rotavirus（Po RV）are the four main pathogens that cause viral diarrhea in pigs at present,and mixed infections often occur,which are very harmful to the pig industry in China.These four diarrheal diseases are characterized by vomiting,anorexia,diarrhea and dehydration,which are difficult to distinguish only by clinical symptoms and need to be diagnosed differently with the help of laboratory methods.In this study,a quadruple RT-PCR assay for PEDV,TGEV,PDCo V and Po RV was established,and based on this,the isolation and identification of PEDV and sequence analysis of virulent strains were performed as follows:1.Specific primers were designed for the M protein gene of PEDV,N protein gene of TGEV,N protein gene of PDCo V and VP7 protein gene of Ro RV,and then the corresponding plasmid standards were constructed.After the optimization of three parameters,namely primer concentration,annealing temperature and cycle number,the optimal primer concentration was determined to be 0.375 pmol/μL,the optimal annealing temperature was50.9℃and the optimal cycle number was 30 cycles for this quadruple RT-PCR assay in this study.Sensitivity,specificity,and reproducibility tests were then performed to validate the method.The results of the sensitivity test showed that the lowest detection limits for PEDV-M,TGEV-N,PDCo V-N and Po RV-VP7 were 1.75×10~2 Copies/μL,1.5×10~3 Copies/μL,1.6×10~2 Copies/μL and 1.6×10~2 Copies/μL,respectively;the results of the specificity test showed that The specificity test results showed that only the four target viruses in this study could be detected,but not CSFV,PRRSV,PCV and PRV,which are common viruses in swine;The repeatability test results showed that three different concentrations of 10~8,10~6 and 10~4Copies/μL of recombinant plasmids were selected as templates,and other conditions remained unchanged,and five replicate tests were conducted respectively,and clear and uniform bands could be amplified in all five tests.Fifty-two clinical samples sent for testing were tested by the established quadruple RT-PCR method,and the results showed that PDEV,TGEV,PDCo V and Po RV were 19（37%）,3（6%）,5（10%）and 13（25%）,respectively,of which 2（4%）were mixed infections of PEDV and Po RV 2（4%）were mixed infections of PEDV and TGEV,and2（4%）were mixed infections of PEDV and TGEV.Among them,2（4%）were mixed with PEDV and Po RV,2（4%）were mixed with PEDV and TGEV,and 1（2%）was mixed with PEDV and PDCo V 5 samples were randomly selected and sequenced for the corresponding viral gene fragments.2.PEDV-positive tissue samples detected by applying the quadruple RT-PCR method described above were selected and used for PEDV isolation.The supernatant of the processed disease material was inoculated into Vero cells and validated using RT-PCR and indirect immunofluorescence（IFA）assay,resulting in the successful isolation of a PEDV strain named QH-202105.the isolate was passaged and cultured to the 10th generation,RNA was extracted and the whole genome was amplified and sent to the company for sequencing,and combined with the reference sequence downloaded from Gen Bank.Nucleotide homology matching and phylogenetic tree construction were performed on the whole genome of the isolate QH-202105.The results of nucleotide homology analysis showed that the isolate had the highest nucleotide homology with the US isolate USA/Colorado/2013 at 99.1%;The lowest homology was 96.5%with CHM2013 strain;Homology with the mutant strain AJ1102 was high at 98.2%;The homology with the classical strain CV777 was low at 96.6%.The results of the genetic development tree showed that the isolate belonged to the G2 gene group,which was a different branch from the classical strain CV777 in the G1 gene group,and was closely related to the variant AJ1102 in the G2 gene group.A genetic development tree was also constructed for the S protein gene of strain QH-202105,and the degree of variation in the amino acid sequence of the S protein of strain QH-202105 was further analyzed and the effect of the predicted amino acid mutation on the function of the strain was determined.The results of the genetic development tree showed that the isolate belonged to the G2b subgroup,which was distantly related to the classical strain CV777 and belonged to the variant strain.the results of the S protein amino acid sequence analysis showed that the S gene of the isolate in this study was 4,161 bp in length and encoded 1,387 amino acids.Further amino acid sequence variation analysis was performed with the classic reference strains such as USA lowa107 2013,AJ1102,CHM2013,CV777,DR13 and OH851.The isolate QH-202105 had more than 60 mutations in the N terminus of the S gene,two amino acid insertions（including4 amino acid insertions in QGVN at amino acids 59-62,insertion of amino acid N at position145）and two amino acid deletions（deletion of amino acids 115-118,deletion of amino acids167-168）,as well as only the isolate at amino acid 32 mutation（F→L）,at position 143（N→D）,at position 235（P→L）,and at position 239（N→D）.Further comparison with the mutant strain AJ1102 revealed three amino acid mutations at amino acids 505（T→I）,514（N→I）,and 526（A→S）in one of the key antigen-neutralizing epitope regions of the S protein（aa 499-638）.PROVEAN predictions revealed that amino acid comparison of the S protein with the mutant strain AJ1102 revealed that the mutation of strain QH-202105 was determined to be"Deleterious"because of the mutation of amino acid from G to C at 1,361.In conclusion,the results of this study are intended to provide a reference for efficient detection of viral diarrhea in pigs and epidemiological investigation of PEDV variants and vaccine development.