Research Of Cell-adaption Of Porcine Epidemic Diarrhea Virus

Coronaviruses are viruses of Coronaviridae, Nidovirales on the system. They are enveloped positive-stranded RNA viruses that replicate in the cytoplasm. Coronaviruses gained particular notoriety when the severe acute respiratory syndrome (SARS) outbreak shook the world in2003. The infections of another coronavirus, middle east respiratory syndrome coronavirus (MERS-CoV), can cause considerable morbidity and mortality(-50%), and coronavirus gained a lot of attention one more. Porcine Epidemic Diarrhea Virus (PEDV) belongs to the alphacoronavirus genus within the Coronavirinae subfamily of the Coronaviridae family, and has only one serotype. An outbreak of PEDV has swept China with substantial economic losses in the swine industry from2010. The PEDV RNA genome is composed of six open reading frames (ORFs), and the fixed order of genes is ORFla/b-S-ORF3-E-M-N. The first ORF (ORFla/b) comprises two-thirds of the genome and encodes the replicase proteins, and the ORF3encode accessory protein. The structural genes encode the spike protein(S), the membrane protein (M), the envelope protein and the nucleocapsid protein. The spike glycoprotein(S) mediates virus entry and is a primary determinant of cell tropism and pathogenesis. There are also some neutralizing epitopes in the S1domain of spike protein. Porcine aminopeptidase N(pAPN) is a functional receptor for the PEDV coronavirus. pAPN is a150-kDa glycosylated transmembrane protein that is highly expressed in small intestinal mucosa. The S protein of coronavirus is highly glycosylated as it contains21-35N-glycosylation sites. S proteins assemble into trimers on the virion surface to form the distinctive "corona", or crown-like appearance. The spike protein ectodomain consists of the S1and S2domains. The S1domain contains the receptor binding domain and is responsible for recognition and binding to the host cell receptor. The S2domain, responsible for fusion, contains the putative fusion peptide and the heptad repeat HR1and HR2. The mechanism of coronavirus entry cell is mediated by the viral spike protein. The S protein is classified as a class I fusion protein in2003by Bosch. Judith M White has identified three classes of viral membrane fusion proteins based on structural criteria and summarized four distinct mechanisms by which viral fusion proteins can be triggered to undergo fusion-inducing conformational changes. It is shown that all characterized viral fusion proteins convert from a fusion-competent state to a membrane-embedded homotrimeric prehairpin, and then to a trimer-of-hairpins that brings the fusion peptide and the transmembrane domain into close proximity thereby facilitating the union of viral and target membranes. Coronavirus also mediate fusion by the same overall strategy. The culture of PEDV is very hard. Currently, cell-adapted PEDV was only seven strains, and the molecular basis of these strains adapted to Vero cells is still unknown. Some studies suggest that mutations in the S gene may be critical molecular basis of PEDV adapting to cells. Our research group collaborated with Utrecht University in2013, and we constructed reverse genetics system of PEDV based on RNA Targeted recombination. We obtained recombinant virus contained chimeric S gene by substitution of parental S gene for attenuated S gene using the reverse genetics system, finally we will find the key motifs or sites of PEDV adapting to Vero cells, and completely solve the problem of PEDV in cell culture.