| Porcine epidemic diarrhea (PED) is a diarrhea disease of swine caused by porcine epidemic diarrhea virus (PEDV), which was characterized by acute enteritis and lethal watery diarrhea followed by dehydration leading to death with a high mortality rate in piglets, the morbidity and mortality rate of newborn piglets is very high, up to100%, the prevalence of PED have caused severe economic losses to China’s swine industry, during the year of2010, the mortality rate have significantly increased, even in the immunized farms. To investigate the genetic characteristics of the prevalent strains, therefore, the author has sequenced and analyzed the full-length sequence of S, ORF3and M gene of13pandemic strains from12provinces, spike protein mainly responsible for the induction of neutralizing antibodies, specific receptor binding and cell membrane fusion. ORF3encodes a nonstructural protein, it’s a transmembrane protein and functions as an ion channel and related to the virulence of PEDV. The M protein plays a central role in PEDV assembly, and it can direct the incorporation of the S protein and the nucleocapsid protein into to budding particle, and neutralizing antibodies can be induced by M protein.According to the reported genome sequence of strain CV777(Accession No. AF353511.1), five pairs of PCR primers were designed to amplify the full-length of S gene, ORF3gene and M gene, the amplicons were cloned, sequenced and analysised. The results showed that we have successfully obtained the full-length sequences of the three genes of thirteen field isolates, and the percentage of identity at the nucleotide level of S gene, ORF3gene and M gene among the thirteen strains were93.2%-99.9%,89.9%-100%and97.7%-100%, respectively. When compared with the vaccine strain CV777, the nucleotide homology of S, ORF3and M gene were93.8%-96.8%,90.8%-97.2%and98.1%-99.1%, respectively, the homologies of the amino acid sequence were92.3%-95.8%,86.8%-100%and98.2%-99.6%, to our surprise, the ORF3gene of strain CH3and CH4shared high amino acid sequence homology (100%and99.6%, respectively) with Korean vaccine strain attenuated DR13, and all of the three strains have138amino acid deletion, so these two strains may be originated from DR13; Strain CHI, CH8, CH17and CH18shared similar sequence identity, including53point mutation,5amino acid insertion (at position59-63and position141of the S protein),2amino acid insertion (at position164-165of the S protein) when compared with strain CV777, they have high pathogenicity to piglets, so we defined them as variant strains. Based on our results, both classical and variant stains were detected in the field, implying a diverse distribution profile for PEDV on pig farms in China. In addition, there are seven and three point mutations in ORF3and M gene of the prevalent strains, respectively, but the effect of these mutations to the biological characteristics of these strains remains to be studied.The sequence insertions and mutations found in the variant strains may have imparted a stronger pathogenicity to the new PEDV variants that influenced the effectiveness of the CV777-based vaccine, our study of the full-length S gene revealed a more comprehensive distribution profile that reflects the current PEDV status in pig farms in China, including the presence of two strains (CH3and CH4) similar to South Korea vaccine strain DR13, further study will be done to confirm the origin of these two strains. M gene was found to be conserved in our study, so M gene-based nucleotide detection method and M protein-based antibody detection method can be developed to the diagnosis of PED.The results of this study revealed the molecular epidemiological characteristics of PEDV, and provided the theory basis for the clinical prevention and control of the disease. |
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