Study On Isolation, Identification And Biological Characteristics Of Porcine Deltacoronavirus HB-BD Strain

Porcine deltacoronavirus(PDCoV)is a novel enteropathogenic coronavirus in pigs that can cause enteric disease with clinical signs including diarrhea,vomiting,dehydration and mortality in neonatal piglets.PDCoV was initially reported in pigs in Hong Kong in 2012 and the disease subsequently broke out in the United States in 2014.At present,PDCoV has been detected in many countries in the world.And it has been reported that PDCoV was detecred in pig farms in some provinces in China.The virus has become a new pathogen that seriously affects the healthy development of the swine industry.In this study,to isolate and identify PDCoV from the faecal/intestinal contents of diarrhea piglets,the samples of PDCoV positive detected by RT-PCR was inoculated to ST cells.The cell cultures were confirmed by the cytopathic effect(CPE),RT-PCR,determination of virus titer and indirect immunofluorescence assay(IFA).The results revealed that HB-BD strain was successfully isolated.The complete genome sequence of PDCoV HB-BD was deduced by RT-PCR.Sequencing results showed that the complete genome sequence was 25,420 nucleotides(nt)in length excluding the 3' poly(A)tail.The gene order of its genomic structure was 5'UTR-ORF1-S-E-M-NSP6-N-NSP7-3'UTR,and the nucleotides numbers of these parts were 540,18803,3480,252,654,285,1029,603 and 392,respectively.The complete genome sequence of PDCoV HB-BD shared 97.5%-99.5% nucleotide identity with the other PDCoV reference strains and had the highest nucleotide identity(99.5%)with PDCoV/NH.Phylogenetic analysis based on the complete genome sequence revealed that all PDCoV strains were separated into the genus deltacoronavirus,further analysis demonstrated that the PDCoV isolate,HB-BD,was more closely related to other Chinese PDCoV isolates than to those isolated from the United States,South Korea and Thailand.According to optimizing the reaction conditions,the purified recombinant PDCoV S1 protein was used as coating antigen to develop an indirect enzyme-linked immunosorbent assay(ELISA)for detecting IgG antibody against PDCoV in serum to PDCoV.Using a checkerboard ELISA,the optimal antigen concentration and serum sample dilution were set at 1?g/ml and 1:200,respectively.The optimal blocking buffer was 5% fetal bovine serum in PBST,and the best blocking time was 2 h at 37°C.The optimal dilution of secondary antibody was defined as 1:20000.After the abovementioned conditions were determined,for the optimal incubation time of serum samples and secondary antibody,they were both determined to be 1 h,the TMB substrate was at 37? for 15 min.The specificity test showed that no cross-reaction was detected for the developed ELISA with other coronavirus or other common pig pathogens and both the Both the intra-assay and inter-assay coefficients of variation(CV)were < 10%.The cut-off value was 0.369 determined by 30 negetive samples.ROC curve analysis showed that compared with western blot,the sensitivity and specificity of the method were 100% and 92.3%,respectively.The above test results show that the establisged ELISA based the recombinant S1 protein could be used for detecting the PDCo V antibodies.Using the established indirect ElISA method,542 serum samples collected from Hebei province from Januanr 2017 to December 2017 were tested and the positive rate was 18.6%.Given that there are currently no effective treatments or vaccines available to control PDCoV.In order to investigate the interference effect of RNA interference(RNAi)technology on PDCoV replication in vitro,two shRNA expression plasmids(pGenesil-M and pGenesil-N)targeting the M and N genes of PDCoV,respectively,were constructed and transfected into ST cells conducting genetic interference experiments.TCID50 was detected between the transfection group and the control group,as well as the CPE was observed and the mRNA level of N detected by real-time PCR.The results showed that the two shRNAs(pGenesil-M and pGenesil-N)could inhibit the replication of PDCoV on ST cells,and the pGenesil-N expressing plasmid had higher inhibition than the pGenesil-M expressing plasmid.In summary,this study isolated and identified PDCoV HB-BD strains,as well as amplify the sequence the whole genome of HB-BD strain,and established an indirect ELISA method based on S1 protein to detect serum samples from some regions of Hebei Province in 2017.Besides,RNAi had been studied for the inhibition of PDCoV replication in vitro.This study provides a theoretical basis for the study of PDCo V genetic evolution,pathogenesis and other mechanisms,and is also of great significance in epidemiological investigations,diagnosis of diseases,development of vaccines,prevention and treatment of PDCoV.