Isolation And Identification Of Porcine Epidemic Diarrhea Virus And Its Genomic Sequencing And Analysis

Porcine epidemic diarrhea virus(PEDV)is an enveloped,single-stranded positive-sense RNA virus,it is the causative agent of an acute,highly contagious,and severe enteric disease that leads to high mortality rates in suckling piglets.The replication of PEDV in intestinal epithelial cells leads to villous atrophy,malabsorption and severe diarrhea.PEDV infection of host cell is mediated by viral spike glycoprotein(S),which attaches to target cells and undergoes membrane fusion into cells.Proliferation of PEDV in vitro requires the presence of trypsin.The activity of trypsin proteolytic protein results in the cleavage of S protein into S1 and S2 subunits,which enhances the fusion between the virus and cells,increases the infectivity of the virus,and replicates and then releases in infected cells.In this study,the specimen,which was suspected of infected of PEDV,was collected on a pig farm in Harbin,Heilongjiang Province,China.The small intestine samples were used to prepare tissue sections.There appeared villous atrophy of the intestine villi,vacuolar degeneration of epithelial cells,hyperemia of the lamina propria,and infiltration of inflammatory cells.RT-PCR analysis confirmed that no contamination of intestinal sample with porcine rotavirus virus and transmissible gastroenteritis of swine virus.Porcine small intestinal epithelial cell line(IPEC-J2)was used for the blind adaption culture of PEDV.IPEC-J2 cells were isolated from the jejunum of neonatal piglets to obtain undifferentiated columnar epithelial cells,which could better simulate the intestinal environment.Typical cell lesions appear at 5th generation.Indirect immunofluorescence and electron microscopy demonstrated successful isolation of PEDV HLJ isolates.According to the sequence information of PEDV strains uploaded on GenBank,9 pairs of overlapping primers were designed to amplify the whole PEDV sequence using primer design software.At the same time,the virus 5' UTR sequence was amplified according to the method of the 5' RACE kit.The bioinformatics analysis of PEDV HLJ strain showed that the full length of the 27,953 nt,5' and 3' noncoding regions of the genome were relatively conserved,and the sizes of ORF1 b,M,N,and E were the same as those of the reference PEDV stain,while the lengths of the ORF1 a,S,and ORF3 genes were different.For the PEDV HLJ isolates,phylogenetic analysis indicated it belonged to the group I population,and had closer relationship with domestic strain of SC1402 in Guangdong Province,the JS-2/2015 strain in Shanghai,and the attenuated DR13 in South Korea from 2014 to 2015,and they shared 99.9%,99.8% and 99.9% of homology,respectively.Regarding of group II population,PEDV HLJ strain was quite far from the phylogenetic location of the refenrecne PEDV-LS strain,and they had lower homology of 96.8% By analyzing the structure of protein S,it was found that the amino acid sequence of protein S had densley populated hydrophobic regions and had a high antigenic index.There are 28 potential glycosylation sites and a typical transmembrane helix located between amino acids 1324-1346.Integrated comparison with three methods of amino acid hydrophilicity,antigenic index and surface accessibility,it was predicated that S protein had 22 dominant B-cell antigenic epitopes.This study isolated and identified the PEDV HLJ strain successfully,and further analyzed its molecular biology characteristics,genetic evolutionary relationships and major protein antigenicity.This will help to understand the biological characteristics of domestic epidemic PEDV strains and their genetic evolution,and provide the theoretical and material basis for further study the pathogenesis mechanism,and prevention of PED.