Epidemiological Investigation Of PEDV And Preliminary Development Of Rapid Detection Methods

Porcine epidemic diarrhea (PED) caused by Porcine epidemic diarrhea virus (PEDV) was a devastating enteric disease with acute diarrhea, dehydration and significant mortality in swine. Thereby, it is one of the most important disease causing heavy economic losses in China. In 2010, an outbreak of PED occurred in China and caused significant high morality 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 three parts as described below.1. Epidemiology of PED in China314 specimen samples were collected in 5 provinces of china during 2012 to 2015 and were detected by reverse transcription-polymerase chain reaction(RT-PCR) to investigate the epidemiology of PED. The results showed the positive rate reached to 24.2%(76/314). PED positive rate of Guangdong、Shanghai、Jiangsu、Henan and Jilin were 94.7%、27.6%、17.8%、 31.6% and 0% respectively. Some representative samples were selected, and S, M, ORF3 gene of which were amplified, cloned and sequenced. Nucleotide sequence alignment revealed that Chinese epidemic strains shared the highest identity with South Korean and American epidemic strains, but lowest identity with European strains and CV777. Amino acid sequence analysis based on structural proteins showed a significant diversity in S1 protein. Amino acid sequence of M and ORF3 were highly conserved. ORF3 gene was the only accessory gene of PEDV, and the nucleotide sequence alignment showed a deletion (about 49 bp) in ORF3 of HN13 and YM.2. Preparation of monoclonal antibodies against PEDVBalb/c mice were immunized with ultracentrifugation-purified virions. After boost immunization the spleen cells of immunized mice were fused with SP2/0 myeloma cells. Detction by IFA, nine hybridoma cells which can secrete monoclonal antibodies against PEDV were generated. These hybridoma cells were named as PEDV-1C12、PEDV-2A9、PEDV-2C11、 PEDV-2E5、PEDV-2F1、PEDV-3E9、PEDV-5B12、PEDV-6D1、PEDV-6E10. The immunoglobulin subtypes of monoclonal antibodies were identified with a commercial capture-ELISA kit. The results showed that seven monoclonal antibodies belonged to IgG subgroup and two monoclonal antibodies belonged to IgM. The IFA titers of ascites were: 5.12×104、1.08×104、5.12×104、2.16×104、1.08X104、2.048×105、3.2×103、1.024×105、1.024×105. The results of Western-blot assay showed that eight antibodies were specific to the PEDV N protein.3. Development of a test strip with monoclonal antibodies of PEDVIn this part, nanometer colloidal gold was prepared by reducing gold chloride with sodium citrate, than 30nm colloidal gold was acquired. The colloidal gold strip was developed according to the principle of double antibodies sandwich. Gold-labeled antibody PEDV-2C11 was dispensed onto the conjugate pad, the PEDV-1C12 monoclonal antibody was dispensed on the bottom of the NC membrane as the test line, while the rabbit anti-mouse IgG was dispensed at the upper position as the control line. When the samples contain PEDV antigen, two red bands will be visible, otherwise, just a single band will be shown on the control line. The test strip could detect PEDV specifically, and its sensitivity was 310 TCIDso PEDV. The results prove that the strip can be used in clinical detection.