The Genetic Vairation Analysis And Diagnostic Methods Development Of Porcine Epidemic Diarrhea Virus

Porcine epidemic diarrhea caused by Porcine epidemic diarrhea virus is a devastating enteric disease with acute diarrhea, dehydration and significant mortality in swine. Thereby, it is one of the most important diseases causing heavy economic losses in China. In1976, it was firstly reported in our country. In2011, an outbreak of PED occurred in China and caused significant higher mortality in piglets. To investigate the molecular epidemiological features and dynamic trends of PEDV and committed to better prevention and control of the disease, we conducted this study mainly including four parts as described below.First,115clinical samples were collected from different farms in five provinces in the occurrence of diarrhea epidemic. These samples were tested by RT-PCR method, and the results showed a81.7%PEDV positive rate. We selected some representative samples, and their complete genome and S, M, N, ORF3gene were amplified, cloned and sequenced. Nucleotide sequence alignment based on the whole genome revealed that ZJCZ4shared the highest identity of99.1%with isolate CHGD-01, and96.9%with CV777. Amino acid sequence analysis based on structural proteins showed a significant diversity in S protein, and the most mutations were in SI domain. Compared with CV777, a discontinuous5-amino-acid insertion (59QGVN62and140N) and two continuity amino acid deletions (161GK162or163NI164) exists in the SI domain of epidemic strains. Besides, there are eight mutations in two neutralising epitopes (499-638aa and764-771aa) and six mutations in potential N-glycosylation point. Amino acid sequences of M and N protein are highly conserved, the homologies are96.5%-100%and95.3%-98.1%respectively. The results of phylogenetic analyses of indicated a close relationship between epidemic strains and reference isolates(CHGD-01and CH/FJND-3/2011), thus constituting a new cluster.The majority of sample isolates are closely related to Korea strains.ORF3gene is the only accessory gene of PEDV, and the nucleotide sequence alignment showed a truncation (about49bp) in ORF3of HEB-1and SD-5. These two strains are closely related to attenuated DR13, and we speculated they are attenuated PEDVs. According to the truncation in the ORF3, two pairs of primers were designed. The procedure of the RT-PCR was perfected. And we collected a large number of clinical samples to carry out the specificity and sensitivity test. The RT-PCR was shown to specifically amplify a300bp fragment from the wild-type PEDVs or a250bp fragment from the attenuated PEDVs. This method showed no cross-amplification between PEDVs and other porcine virus. And the sensitivity of detection of viral RNA extract was to100TCIDso/0.1mL. The test of clinical samples show a65.4%positive rate in wild-type PEDV infection.The RT-PCR could be used as an effective tool for differentiating diagnosis of the PEDVs in epidemiological investigations.Since the nucleocapsid protein is highly conserved, it is a candidate antigen for diagnosis. N protein of PEDV was expressed successfully, about81kDa. The Western blot shows the recombinant N protein can interact with the positive PEDV serum specifically.We selected20clinical pig serums, the origin and background of which were clear. The ELISA plates were coated with purified N protein,and was for detection of these20clinical pig serum samples. And the results are in line with the rate of85%, compared with RT-PCR method. It indicats that nucleocapsid protein may be a useful antigen for sera-diagnosis of PEDV. To better understand the antigenicity of N protein, BALB/c mice were immunized with purified recombinant N protein of PEDV emulsified in Freund’s adjuvant. Five hybridoma cell lines that secreted monoclonal antibodies against recombinant N protein were established. Five McAbs were characterised by indirected ELISA, with ascetic titres of105. The results of IF A showed that McAb reacted with PEDV. Also we used McAb N-l as detecting antibody to established a double-antibody sandwich ELISA. And the results revealed, N-l interact with PEDV from clinical samples, and sensitivity is up to100TCID50. The ELISA had no cross-reaction with other swine disease virus, indicating that the McAbs have high level of specificity and sensitivity, and can be used as a powerful tool to detect PED antigen.