Multiplex RT-PCR For Detection Of PEDV And TGEV And Analysis Of The Complete S1Gene Of11PEDV Isolates

The virus diarrhea of pigs is an acute enteric disease which is mainly caused by theporcine epidemic diarrhea virus (PEDV) and the porcine transmissible gastroenteritisvirus (TGEV). Pigs of all ages are susceptible, especially the piglets. It can adverselyaffect the feed conversion rate and lower feed efficiency, resulting in significanteconomic losses. Because PEDV and TGEV both belong to the coronavirus family,their clinical symptoms, pathologic changes and epidemiology are very similar, andare difficult to clinically differenciate from each other. The present study aims toestablish a multiplex RT-PCR for detection of porcine epidemic diarrhea viruses(PEDV) and porcine transmissible gastroenteritis viruses (TGEV). Via an optimizationof RT-PCR parameters, a multiplex RT-PCR assay for detection of PEDV and TGEVwas established and was used for testing121samples from diarrheaed pigs raised inJiangxi. The results showed that79out of121were PEDV positive, that is, thepositive rate is approximately65.3%; but we did not detect TGEV from any samplesexamed. At the same time, we also generated a ssRNA internal control fragment (IC)for monitoring the inhibitions of the multiplex RT-PCR，and thus to reduce the false-negative rate of the test, and improve the reliability of the assay.The S1gene of PEDVs harbars major neutralizing epitopes, which is the maintarget for analyzing the prevalent trend and the genetic variations of wild PEDVstrains. To determine the diversities of the wild types of PEDV circulating in Jiangxiprovince, we designed three pairs of primer and sequenced the compete S1gene of11field isolates sampled in Jiangxi.By a comparison of the complete S1gene of11wild types of PEDV withreference strains at the both nucleotide and amino acid level, we found that theidentity between11wild types of PEDV strain and reference strains of CV777vaccinestrain、Chinju99、KNU-0905and LJB/03were92.3%-92.8and90.9%-91.2%;92.7%-93.1%and90.1%-90.6%;94.7%-95.3%and92.9%-93.8%;93.5%-94.1%and92.8%-93.4%, respectively. The11wild types of PEDV strain had the highesthomology with the reference strain of KNU-0905(Korean strain).The phylogenetic tree of S1gene demonstrated that11endemic PEDV strains ofJiangxi province and30PEDV reference strains were grouped into G1and G2, whichcan be further subgrouped into G1-1, G1-2, G2-1and G2-2, respectively. All localisolates of Jiangxi province belonged to the subgroup G1-1, which had adistantlyrelationship with the CV777vaccine strain which belongs to subgroup G2-2. Analysisof nucleotide homology displayed that deletions and insertions could be found on S1gene of endemic strains of PEDV while compared to the vaccine strain or Koreanstrains. Besides, PEDV strains isolated in recent years and these11local strains had higher identity with PEDVs of South Korea than any other subgroups. These findingsprovide insight into PEDV molecular epidemiology and evolution of the Jiangxiprovince and surrounding regions as well as a useful tool for detection of PEDV andTGEV.The epitope region (COE) and the antigen epitopes (S1D5, S1D6) inducingneutralizing antibodies against PEDV of S1proteins of Jiangxi epidemic strainsisolated in2013were compared to those of S1protein of CV777. The results showedthat there is no amino acid deletion and insertion in the COEs of11Jiangxi strains, butthere are amino acid point mutations, and the number of amino acid mutations rangedfrom7to9. The antigen epitope S1D5had an amino acid point mutation in CH/JXZS-1/2013, while conserved in the rest10Jiangxi strains isolated in2013. The antigenepitope S1D6had an amino acid point mutation in CH/JXJJ-1/2013and CH/JXJJ-2/2013, while conserved in the remaining9Jiangxi strains isolated in2013.