| Porcine epidemic diarrhea (PED) is an acute infectious disease with severe diarrhea, vomiting and dehydration clinical features caused by porcine epidemic diarrhea virus (PEDV). Porcine epidemic diarrhea virus and (TGEV) belong to the coronavirus family of the genus Coronavirus. Pigs of various ages may be vulnerable, but piglets are the most serious victims, whose morbidity and mortality can reach one hundred percent. Since its outbreak in China in2011, the disease had become epidemic diseases in the world, had become one of the major diseases causing piglet death in farms of China, seriously loss in economic interests of farmers and the development of livestock farming. Therefore, the annual epidemiological surveillance, analysis and immunization of PED are essential tasks; especially monitoring the molecular epidemiological characteristics of PEDV by molecular biology can let us understand the source and the degree of variation of PEDV strain, and the genetic relationship between the various strains, providing a theoretical basis for disease prevention and control. For this, this paper carried out the following contents:(1) We detected the whole genome sequence of epidemic strains SDZB_CH2014By RT-PCR, molecular cloning and other molecular biology techniques, using CLUSTAL1.83and MAGE5software to compare sequence of SDZB_CH2014with sixty-four reference strains sequences from China, United States,South Korea, Canada and other countries, and we determined the genetic relationship between the various strains, and drew a phylogenetic tree. The results showed that:length of the strain were28034bp, and the vaccine strain CV777, DR13, SM98homology were96.7%,97.3%,96.5%, evolutionary tree were divided into two branches, namely G1and G2type. G1were mainly vaccine strains, and G2contain ed the most prevalent strains of PEDV, including different provinces of China2011-2014epidemic strains and the strains that come from United States, South Korea, Canada and other country, which indicated G2group was the major regnant strains of PEDV in the world today. Analysis of a single gene phylogenetic tree showed that the sequences ORF3gene, E gene, M gene, N gene were more conservative compared to the S gene of easy-to-mutation.(2) S genes of thirty-nine strains from13provinces and cities in2013-2014were cloned and sequenced, and we used the biological software to compare the sequences of S genes in thirty-nine strains with the sequences of S’ genes in the134reference strains from China, United States, South Korea,Japan and other countries to determine the genetic relationship between the various strains, and we drew a phylogenetic tree. The results showed that:S gene phylogenetic tree of thirty-nine PEDV wild strains and134reference strains were divided into two evolutionary branches, namely G1and G2type. G1were mainly vaccine strains, G2contained the most prevalent strains of PEDV, indicating G2group was the major regnant strains of PEDV in the world today. PEDV epidemic strains were not regional in China.(3) In order to better select antigenic fragments of S gene of PEDV, we used biological software to analysis the antigenic sites of S gene of PEDV, linking three segments of different antigenic sites to conduct prokaryotic expression, then carried out Western-Blotting detection. The results showed that the fusion protein finally obtained has immunological activity, indicating that existence of antigenic fragments in the three fragments. As how many fragments with antigenicity there are needs further experimental validation. |
