Establishment Of Detection Methods For Porcine Deltacoronavirus And Isolation And Identification Of JS-LYG-2014 Strain

Porcine Deltacoronavirus(PDCoV)is a new coronavirus that has been identified in United States since 2014.The pathogen is enveloped,single-stranded,positive-sense RNA virus in the family deltacoronavirus genus.PDCoV belongs to intestinal pathogen and caused watery diarrhea,vomiting,and dehydration.The piglets were susceptible,but the mortality rate(30%~40%)was lower than that of PEDV infection(About 100%).In addition,PDCoV is often mixed infection with PEDV,TGEV and other pathogens,so that the detection,prevention and control are difficult.At present,there are at least 7 countries reported PDCoV infection including China,United States,South Korea,et al,resulting in losses in swine industry and threatening the safety of the global pig industry gradually.In our study,two specific RT-PCR detection methods were established targeting PDCoV M gene and N gene and can implement rapid and effective detection of clinical samples;The indirect ELISA for detecting antibody was established by using the PDCoV N protein as coated antigen.A strain of PDCoV was isolated from positive samples in ST cell,named JS-LYG-2014.The whole genome sequence was determined and analysed by 16 pairs of primers.Pathogenicity test of cell culture isolate was impletented to 0 day old piglets;This experiment provided an effective tool for the detection of PDCoV antigen and antibody in clinical samples,and maded the foundation for the studies of PDCoVs including molecular epidemiology,pathogenicity and pathogenic mechanism.The specific contents are as follows:1 RT-PCR assays were first reported in China for detection of PDCoV with two pairs of primers targeting M gene and N gene designed according to PDCoV sequence available in GenBank.The test results showed that the RT-PCR assays were specific for PDCoV detection,but had no cross-reaction with closely related coronavirus or other animal viruses,and were sensitive for PDCoV test which the detection limit of the M gene and N gene RT-PCR assay were approximately 104 copies/?L,103 copies/?L respectively.In addition,clinical samples infected PDCoV were approximately 2.85%(14/492)and 4.75%(25/526)collected from 2014 to 2015 from pig farms in Eastern China by M gene RT-PCR and N gene RT-PCR.Samples from diarrhea pigs infected PDCoV were approximately 18.60%(8/43)and 25.76%(17/66)respectively.The PCR assays indicated that PDCoV existed in pig populations in china and could be applied as a diagnostic technique for the detection of PDCoV.2 A pair of primers used for prokaryotic expression was designed according to PDCoV sequence available in GenBank.The N gene was amplified by RT-PCR and induced expression connecting the pCold 1 vector.The recombinant N protein was confirmed by western blot.The purified N protein was used as the coated antigen,and an indirect ELISA antibody detection method of PDCoV recombinant N protein was established by optimizing the reaction conditions.The ELISA we developed was specific for PDCoV detection,which had no cross-reaction with other common seven positive sera.Field samples(n=351)were detected collected from 2012 to 2016 from different areas.The data showed that the developed ELISA was a useful tool to detect antibodies against PDCoV.3 A PDCoV was isolated from pig samples with diarrhea.16 pairs of primers were designed for amplifying the PDCoV whole genome.The sequence alignment,homology and phylogenetic analysis were determined compared with other 19 strains of PDCoV reference strain sequences.The pathogenicity test of 0 day old piglets were challenged using cell culture isolate.The results showed that the total length of JS-LYG-2014 was 25370 bp,and the whole genome structure was 5'UTR-ORF1a-ORF1b-S-E-M-NS6-N-NS7-3'UTR.Among the major structural proteins,the S gene was the most variable,and the amino acid homology was 97.4%?99.6%.The E,M and N genes of different strains were highly homologous.The phylogenetic analysis showed that JS-LYG-2014 and PDCoV-CHJXNI2-2015 had the highest homology with the majority of Chinese strains.The inoculated pigs had severe diarrhea with thin and transparent intestinal wall and the intestinal villus became atrophy.This study suggested that we should strengthen the monitoring of PDCoV and provided a basis for the further study of pathogenicity of PDCoV.