Study On The Identification And Biological Characteristics Of Porcine Epidemic Diarrhea Virus Isolate

Porcine epidemic diarrhea(PED)is an actue diarrhea intestinal infectious disease in swine caused by Porcine epidemic diarrhea virus(PEDV).In this study,in clinical samples of piglets with diarrhea were collected during 2013-2016 were tested for diarrhea-related virus.The genome,pathogenicity,and invasion mechanisms of acquired epidemic strains PEDV were studed.A total of 898 fecal and intestine tissue simples from clinically infected piglets were collected from pig farms with diarrhea in Shandong,Henan,Beijing,Jiangxi and etc.provinces during 2013-2016.The viral pathogens which may cause diarrhea in piglets was detected by RT-PCR.The results showed that the positive rates of PEDV,TGEV,PoRV and PDCoV were 90.42%(812/898),3.89%(35/898),1.23%(11/898),2.89%(26/898),respectively.The data porved that the PEDV was the main pathogen causing piglets diarrhea in piglet farms.Several PEDV epidemic strains were isolated from clinical samples using Vero cells,one of them was named SDSX16.The isolate SDSX16 was serially propageated in cell cluture.At the 10th passage,the syncytial lesions colud be observed and at the 100th passage the viral titer reach 107TCID50.3-day-old piglets were vaccinated with the 10th passage of SDSX16 virus via the oral route.The results showed that the SDSX16-infected group showed depression,watery feces,and decreased body temperature at 2 days psot-inocalation(dpi).At 3 dpi,the piglets began to die.The mesenteric lymphadenopathy,intestinal dilatation,and intestinal fluid filled with yellow liquids were observed by necropsy.Intestinal epithelial cells shed with necrosis were observed by histopathology.Immunohistochemistry showed that PEDV was mainly concentrated in small intestinal villus epithelial cells.This results indicate that the PEDV isolate SDSX16 is highly pathogenic for newborn piglets,and can cause diarrhea,dehydration and death in piglets.The full genome sequence of the PEDV SDSX16 isolate was obtained by RT-PCR.The entire genome length was 28036 nt(GenBank accession:MH117940).Compared with the PEDV CV777 standard strain,the 5’UTR and S genes of the SDSX16 genome contained base insertions,deletions,and point mutations and the S gene had a large variation.Amino acid alignment result showed that four amino acids(QGVN)were inserted at position 59-62 of the S gene coding region,one amino acid(N)was inserted at position 139-141,and one amino acid(G)was deleted at position 159-161.The nucleotide and amino acid homology of the SDSX16 S gene with the reported 28 reference strains(including the CV777 strain)were 93.6%-96.7%and 92.2%-97.8%,respectively.Based on the amino acid evolution analysis of S gene,the SDSX16 strain belonged to Subgroup Ⅱb subpopulation and had a distant relationship with the standard strain CV777.The heptad repeat regions(HR)of PEDV S2 gene was predicted by biological software.Six polypeptides(HR1M,HR1L,HR2,HR2M,HR2L,HR2P)were designed to express using the GST fusion expression system.It was comfirmed that HR2M,HR2L,and HR2P could inhibit PEDV infection in vitro,and HR2P had the highest inhibitory effect.Further studies had shown that antibodies from mice immunized with HR2P peptide could block PEDV infection in vitro.In order to explore possible receptors for PEDV,we constructed hDC-SIGN and hL-SIGN BHK-21 stable cell lines using the lentivirus system,in vitro infection assay confirmed that hDC-SIGN and hL-SIGN could promote PEDV infection in host cells;Adding of 50 μg/mL mannose and 20 mg/mL anti-hDC-SIGN antibody could inhibit PEDV infection,whereas adding 100 μg/mL D-galactose had no inhibitory effect.The results indicate that hDC-SIGN and hL-SIGN can act as receptors to mediate PEDV invasion cells.In summary,porcine epidemic diarrhea virus(PEDV)is the major pathogen of diarrhea piglets in China.The PEDV isolate SDSX16 has high virulence in suckling piglets,and its S gene shows a characteristic variation,and it has a distant relationship with the CV777 strain.In vitro cell infection assay confirmed that HR2M,HR2L,and HR2P could competitively inhibite PEDV invasion cells,and HR2P antibody could block PEDV infection.Expressing hDC-SIGN and hL-SIGN could induce PEDV invasion cells.