Epideminological Investigation,Isolation And Antiviral Research In Vitro Of Porcine Deltacoronavirus

The concept of porcine deltacoronavirus(PDCoV)was first proposed in 2012 and it was a new high intestinal-infected porcine coronavirus that could cause acute diarrhea,vomiting,dehydration and even death in piglets.The PDCoV genome is a single-stranded positive-stranded RNA with 25.4 kb.PDCoV has been popular in many countries around the world,bringing new challenges to the world's pig industry.It has been confirmed that PDCoV has undergone recombination and evolution in the global epidemic.Therefore,continue monitoring the genome of PDCoV by collecting diarrhea samples from multiple regions and discover new anti-PDCoV drugs are of great significance for preventting diarrhea diseases.In this study,pig diarrhea samples from various provinces in China were collected,and virus isolation and identification of PDCoV-positive samples were conducted.The materials and foundations were accumulated to fully understand the prevalence of PDCoV in China.In addition,we also found that LiCl and Diammonium Glycyrrhizinate(DG)inhibite the replication of the PDCoV in LLC-PK1 cells.The specific results are as follows:1.Epidemiological investigation and genetic evolution analysis of PDCoV.From March 2018 to March 2019,we collected 645 samples of diarrhea from Shandong,Anhui,Henan,Jiangsu and other provinces.The RT-PCR results showed that the positive rate of PDCoV was 9.3%.The PEDV co-infection rate was 20%,and the PKV co-infection rate was 21.7%.We successfully amplified 18 genomes and 47 S gene sequences.Through genetic analysis,it can be seen that the sequences in this study belong to the Chinese clade.At the same time,it was found that the genotypes of the strains in different regions had genetic changes at different times,indicating that PDCoV was widely spread in China.2.Isolation and identification of PDCoV.We successfully isolated one PDCoV strain of SD2018/10 from single-infected PDCoV positive samples on LLC-PK1 cells,it was cultured on LLC-PK1 cells and selected through plague selection,and then adapted to LLC-PK1 cells.The strain was identificated by PCR,WB and IFA.The growth characteristic of the isolated strain were also investigated:LLC-PK1 cells-were inoculated with MOI=0.05,then the CPE appeared.and the virus titer(TCID50)could reach about 10^8.0/mL.3.Investigation of the antiviral properties of lithium chloride and diammonium glycyrrhizinate on PDCoV.We found that LiCl and DG could inhibit the replication of the PDCoV strain SD2018/10 on LLC-PK1 cells in a dose-dependent manner.By investigating the effective stage of the two drugs on the virus,it was founded that although both drugs mainly play an important role in the early stage of viral infection,LiCl does not affect the stage of viral attachment and DG could inhibit the attachment of the virus.At the same time,we also found that the inhibition efficiency of LiCl was higher than that of DG at the maximum non-toxic concentration;and both drugs could inhibit the early apoptosis of LLC-PK1 cells induced by PDCoV.