| Porcine epidemic diarrhea(PED), with the clinical symptoms of vomiting, diarrhea and dehydration, is an intestinal infectious disease caused by porcine epidemic diarrhea virus(PEDV). Since 2010, PED has been reported in many provinces of China and resulted in huge economic losses to pig industry. Currently, PED has become one of important swine infectious disease to restrict the healthy development of pig industry. Henan is a main swine-raising province in China. PED has caused a heavy frustration and lasting effect to the pig industry of Henan since 2010. It is very important for understanding epidemiological characteristics and making control measures for PED to investigate its phylogenetic characteristics and immunological detection methods.S(spike) protein of PEDV is a structure protein on the surface of viral particle, with the main epitopes, and it can be used for the diagnosis of PEDV and the study of molecular epidemiological; ORF3 is an solely auxiliary protein of PEDV, which is associated with the virulence of PEDV and can be used to differ virulent from attenuated strains of PEDV. In this study, phylogenetic analysis based on the ORF3 and S gene was carried out, which is of great significance for clarifying the epidemic and genetic characteristics of PEDV in Henan; N(nucleocapsid) protein is one of the major structural proteins of PEDV. The high level of antibody against N protein can be detected in the sera of porcine at the early stage of PEDV infection. Therefore, N protein can be used as target protein for the early diagnosis of PEDV; As an enteric virus of porcine, PEDV causes mainly the mucosal immune response. The level of Ig A can reflect the status of virus infection and immunity protective efficacy of vaccine. It is of great significance to develop the methods of detecting Ig A of PEDV for the evaluation of PEDV vaccine. By preparing the S and N recombinant protein, we developed the ELISA methods for detecting the Ig A antibody based on p S protein and chromatography test strip based on N protein of PEDV. The contents and results for this study are as follows:1. The samples were collected from 116 pig farms with different scales from the regions of Xuchang, Xinzheng, Pingdingshan, Zhengzhou, Xinxiang, Hebi, Kaifeng, Zhumadian, Zhoukou, Luoyang, and Sanmenxia in Henan Province, and these samples were used to detect PEDV antigen by RT-PCR. The results showed that the positive rate of PEDV in the feces and milk samples was 60.8 ﹪ and 56.7 ﹪, respectively; The genetic analysis was conducted based on the ORF3 gene and partial S gene of 14 PEDV positive samples. The results showed that the similarity of nucleotide and amino acid sequence of ORF3 gene between 14 samples were 96.7-100.0 ﹪ and 94.0-100.0 ﹪, respectively, and the nucleotide and amino acid sequence of ORF3 gene had 96.9-99.8% and 94.5-99.3 ﹪ similarity compared to the reference strains, respectively; Phylogenetic tree based on the nucleotide sequences of ORF3 and part of S gene displayed closer genetic distance between 14 sample strains and with the domestic strains CH/ZMDZY/11, BJ-2011-1, JS-HZ2012, GD-B, CH/FJZZ-9/2012, CH/FJND-3/2011, CH/S, but it showed a distant relationship with the SM98, LZC and vaccine strain CV777; The amino acid sequences deduced from the S gene analysis showed that there were some changes of amino acids in the epitopes of 7-146 aa and 271-278. It is speculated that these changes of amino acids may be associated with changes in the antigenicity of PEDV and consequently result in vaccination failure.2. We studied the pathological changes in the intestine of the piglet infected with CH/HNQX-3/14 strain by HE staining, immunohistochemical and morphological methods, and demonstrated preliminary the importance of the intestinal villious M cells during PEDV infection; CH/HNQX-3/14 genome had four obvious deletions in the S gene(nt 20,813-20,824, nt 21,058-21,063, nt 21,127-21,132 and nt 22,252-22,257) compared with CV777 strain. The genomic sequence of CH/HNQX-3/14 had the highest similarity with CH/ZMDZY/11(99.1 ﹪); Recombinant analysis showed that there were four recombination sites with high probability in the genome of CH/HNQX-3/14, which located in ORF1a(nt 2,769), S(nt 20,691 and nt 22,176) and N(nt 27,252) gene, respectively. The nucleotide sequences in the regions after the breakpoint(nt 2,769) and before the breakpoint(nt 27,252) were mainly from CV777, and from mainly DR13(vaccine strain from Korea) in the regions between the breakpoint nt 20,691 and nt 22,176. It is speculated that CH/HNQX-3/14 may be a mosaic strain evolved from three putative parental-like strains(CV777, DR13 and CH/ZMDZY/11) through recombination.3. The partial S1 gene of CH/HNQX-3/14 which contained three neutralization epitopes of S1 protein(499-638 aa, 748-755 aa and 756-771 aa) was cloned. The recombinant plasmid p ET30a(+)-p S1 was constructed and expressed efficiently in BL21 with soluble form by optimizing the expression conditions, which was able to react with PEDV positive serum of porcine and His monoclonal antibody. ELISA was developed for detecting the Ig A antibody against PEDV using the purified p S1 protein as coating antigen, which had good specificity, sensitivity and repeatability. Compared to PEDV Ig A kit from Korea BIONOTE, the coincidence rates of serum and milks samples reached 93.6 % and 93.9 ﹪, respectively. Therefore, the ELISA in this study can be applied to clinical detection of Ig A antibody against PEDV and vaccine evaluation.4. The plasmid p ET28a(+)-N was constructed and transformed into BL21 for expressing N protein of PEDV. The recombinant N protein was soluble by optimizing the expression conditions. It was purified by Ni-NTA affinity chromatography and gel filtration chromatography and labeled with colloidal gold, which was fixed on the bonding pads as the probe for detecting Ig G antibody against PEDV. Then, staphylococcus protein A(SPA)(1 mg/m L) and His monoclonal antibody(2.9 mg/m L) were coated on the nitrocellulose membrane as the test line and control line, respectively. The immunochromatography test strip was prepared and assembled, which showed no cross reacting with the positive serum of TGEV, CSFV, PRRSV, PRV and FMDV. There was the coincidence rate of 94.0 ﹪ compared with the ELISA kit. The strip provides new technical means for rapid diagnosis of PEDV. |
PDF Full Text Request