Development Of Delection Technique And Genetic Variation Of Porcine Epidemic Diarrhea Virus

Since late2010, porcine viral diarrhea appeared an outbreak in many areas of China.In order to investigate the etiology of newborn piglet diarrhea, a novel multiplexRT-PCR was developed, which could detect PEDV, TGEV, GARV, and PKV. Theresults show that viral diarrhea outbreaks were caused by multiple pathogens infection,and PEDV was main pathogen. In addition, RT-nested PCR and indirect ELISA weredeveloped to detect PEDV and anti-PEDV antibody, respectively, which provided atool for clinical epidemiology of PED and surveillance of antibody level. We alsoamplified, cloned, sequenced and analyzed complete M and ORF3genes of15PEDVstrains isolated from central china from2010to2011, and explored the reasons ofPED outbreaks again in China.1. Establishment and Clinical Application of a Multiplex RT-PCR for Rapid Detectionof PEDV, TGEV, GARV, and PKVIn order to investigate the etiology of newborn piglet diarrhea in central China, anovel multiplex RT-PCR was developed, which could detect PEDV, TGEV, GARV,and PKV. A total of190fecal and intestinal samples from piglets with acutegastroenteritis were collected in central China during October2010and August2012,which were tested by the established multiplex RT-PCR. The positive rates of PEDV,TGEV, GARV, and PKV are62.11%,0.53%,7.37%, and82.11%respectively. Thecoinfection rates of PEDV/PKV, PEDV/GARV, GARV/PKV, and PEDV/GARV/PKVare47.89%,4.74%,7.37%and4.74%respectively, however there isn’t coinfection ofTGEV with other virus. In addition, single infection of PEDV and PKV was detectedin27samples (14.25%) and50samples (30%) with this method respectively. Theresults show that viral gastroenteritis of piglets in central China is closely related tocoinfection of diarrhea-related virus，of which PEDV is the leading cause. The role ofPKV needs further confirmation and research.2. Establishment and Application of RT-nested PCR Assay for Detection of PEDVTo detect PEDV accurately and sensitively, two pairs of specific primers of the Mgene were designed for the RT-nested PCR. The lowest sensitivity value of the RT-nested PCR as proved by the sensitivity test was50fg RNA of the standard template,and the lowest sensitivity value of the conventional RT-PCR was50pg RNA of thestandard template. As demonstrating by the specificity test, the RT-nested PCRprimers designed in the present study could not be amplified from the PRRSV, CSFV, TGEV, and GARV RNA template. The RT-nested PCR is rapid, sensitive, and acost-effective detection method for the detection of trace PEDV template.3. Prokaryotic expression of PEDV Spike protein and development of indirect ELISAfor detection of its antibodyA spike (S) protein-based ELISA was developed to detect anti-PEDV antibody. The794bp fragment of PEDV S gene was amplified by RT-PCR and inserted into theexpression vector pET-32a，the recombinant plasmid was named pET-32a-S. A fusionprotein was expressed in BL21(DE3) that transformed by pET-32a-S and induced byIPTG. The molecular weight of the recombinant protein was about46.9kD bySDS-PAGE, and the immunoreaction activity of the recombinant protein wasconfirmed by Western-blot, in which positive serum against PEDV was used. Anindirect ELISA for detecting antibody against PEDV was developed by usingexpressed protein S as coating antigen. Results showed that the optimal coatingconcentration for S protein is0.04μg/well, the serum dilution fold is1:100, the HRP-SPA dilution fold is1:2000, and the positive criterion for this ELISA assay isDD450≥0.24. On testing279field samples, the positive rate was97.42%(227/233) ofsows sera, and71.74%(33/46) of nursery and fattening pigs sera. The resultsindicated that this assay was specific, sensitive and reproducible, which could suitableas a rapid serology detection method for monitoring anti-PEDV antibody andepidemiologic survey of PED．4.Genetic variation analysis of reemerging PEDV prevailing in central China from2010to2011To investigate and analyze the reason of this outbreak,15porcine epidemicdiarrhea viruses (PEDV) were collected from different areas of central China fromOctober2010to December2011. The M and ORF3genes were amplified by reversetranscriptase polymerase chain reaction, cloned, sequenced, and analyzed. Sequenceanalyses showed that the nucleotides and amino acids were changed at some sites inthe M and ORF3genes of the15PEDV strains compared with those genes ofreference strains. Based on the phylogenetic analyses of M and ORF3proteins, PEDVin central China and reference strains could be separated into three groups: G1, G2and G3. The15PEDV strains belonged to G3group and showed a close relationshipwith Korean strains (2007), Thai strains (2007–2008), Vietnam strains (2010) andpartial other Chinese strains (2010–2011), but differed genetically from Europeanstrains (Br1/87) and the vaccine strain (CV777vs.) being used in China. Furthermore, all15PEDV strains from central China and some other isolates in China from2003to2007(LJB-03、JS-2004-2、DX、QH and LZC) belonged to different subgroups.Therefore, PEDV exhibits rapid variation and genetic evolution, and the currentlyprevailing PEDV strains in central China are a new genotype.