Investigation Of Porcine Viral Diarrhea In Anhui Province And Molecular Epidemiological Of PEDV

Porcine viral diarrhea causes acute gastrointestinal diseases in swine,the major pathogens of porcine viral diarrhea disease incluing CSFV,PRRSV,RV,TGEV,PEDV,PRV and PCV.The epidemic of porcine viral diarrhea disease showed a clear upward trend in recent years,resulting in tremendous losses in the swine industry.PEDV was the leading pathogen among the diarrhea virus,caused a devastating enteric disease with acute diarrhea, dehydration,and PEDV has become the main reason to reduce the rate of the slaughter pigs.The major viral pathogens was invested in Anhui aera,to further illustrate the point,molecular epidemiological research was performed using M,N and ORF3 genes.(1) In the present study,a total of 138 clinical samples from 23 farms were tested for presence of PEDV, TGEV,PoRV, CSFV and PRV. The results showed that the positive rate of PEDV was up to 58.7%, the positive rates of CSFV,PRRCV,TGEV, RV,PRV and PCV2 were 2.9%,1.44%,2.17%,1.44%,4.35% and 7.25%,respectively. Mixed infections between PEDV and PCV2 occured in many farms,mixed rate was 6%,showing the more serious clinical symptoms.(2)In order to investigate the molecular epidemiological of PEDV,the M,N and ORF3 genes of positive samples of 19 farms were cloned.Four sets of primers were designed and synthesized based on the genome of PEDV CV777(GenBank No.AF353511) to amplify the M,N and ORF3 genes.PCR products were subjected to gel electrophoesis on 2% agarose gel excised from the agarose gel and purified using an Agarose gel Recovery Kit (Tigen). The PCR products were cloned into pMD18-T vector according to the manufacture’s instructions,clones were sequenced in both direction.DNASTAR and MEGA5.0 software were uesd to analysis the homology and phylogenetic relationships of the Anhui PEDV strains.Results showed that the fragments of the M,N and ORF3 genes were 715bp,1400bp,675bp,respectively.There were many mutated sites in three gene segments,but seletions and insertions were not found.The nucleotide sequences of the M gene of Anhui PEDV had 98.3-100% identical with each other.Sequence comparison with other PEDV strains selected from GenBank revealed that their nucleotide sequences were 96.9%-100% identical to those PEDV strains.Six Anhui strains,including AHFY, AHLA, AHLX,2013AHFD,2013AHMAS,2013AHLX,have 100% nucleotide sequence identical with US strains for M gene.The results showed that M gene was conserved,suggesting M gene is the candidate gene for development of genetically engineered vaccine.The nucleotide sequences of the N gene of Anhui PEDV had 95.9-99.9% identical with each other.Sequence comparison with other PEDV strains selected from GenBank revealed that their nucleotide sequences were 94.1%-99.8% identical to those PEDV strains.Anhui and US PEDVs share many identical amino acid mutation.The nucleotide sequences of the ORF3 gene of Anhui PEDV had 96.7-100% identical with each other.Sequence comparison with other PEDV strains selected from GenBank revealed that their nucleotide sequences were 94.9%-100% identical to those PEDV strains.The homology of ORF3 gene of 2013 Anhui strains decreased for there were less identical strains during the comparison.Phylogenetic analysis revealed that Anhui isolates were genetically proximity to PEDV US strains but differ from European strains,Korea strains and Japan strains.