Detection Of Two Kinds Of Porcine Diarrhea Coronavirus In Some Provinces And Cities And Exploration On Inhibition Of PDCoV Replication By IFIT1

Previous studies have shown that porcine deltacoronavirus(PDCoV)and porcine epidemic diarrhea virus(PEDV)are two common pathogens in pig farms.They can infect pigs of all ages,and mainly infects newborn piglets,causing vomiting,diarrhea,dehydration and even death.In 2010,PEDV broke out in pig farms in southern China,with the piglet mortality rates as high as 80%to 100%.Although the infection rate of PDCoV in China is generally lower than that of PEDV according to the current survey results,and there has not been a pandemic.It is worth mentioning that PDCoV has caused a large-scale epidemic of piglet diarrhea in the United States in early 2014,and it is likely that PDCoV highly pathogenic strains will endanger the development of the pig industry in the future.There are 266 pig intestinal tissues and feces samples from 10 provinces or cities including Jiangxi,Anhui,Jiangsu,Guangxi,Hubei,Zhejiang,Shandong,Fujian,Hebei and Shanghai during February 2016 to March 2017 were collected for RT-PCR detection.The results showed that the PDCoV-positive rate was 19.9%in total samples and 22.8%in diarrhea samples,PEDV-positive rate was 41.0%in total samples and 47.4%in diarrhea samples.The co-infection rate of PDCoV and PEDV was 12.1%in total samples and 14.2%in diarrhea samples.Our results of this study indicate that PDCoV and PEDV infection are still prevalent in the surveyed pig farms,and the rate of infection and incidence of PEDV are higher than PDCoV.RNA-seq technology is an effective means to study the transcriptome levels during the process of viral infection of host cells.This study was the first to use RNA-seq technology combined with bioinformatics to study the transcripts changes of swine testicular cells(ST cells)after infected with PDCoV.Total 8139 differential transcripts were identified.Among them,4236 were significantly up-regulated and 3903 significantly down-regulated.GO and KEGG enrichment analysis showed that the differentially expressed genes were mainly concentrated in the function of protein binding,organelle and metabolic processes and mainly involved in metabolic,endocytosis and viral carcinogenesis pathway.The RNA-seq results were further verified by RT-qPCR,and the results showed that RNA-seq were reliable.From the RNA-seq analysis report,we screened a differentially expressed ifit1(IFN-induced proteins with tetratricopeptide repeats-1)gene for further study.In this study,the recombinant His-IFIT1 protein was successfully expressed in bacterial inclusion bodies by constructing the E.coli expression system of His-IFIT1.Following purified using Ni-NTA,IFIT1 protein were obtained.And the specific polyclonal antibodies were prepared by immunizing rabbit with purified His-IFIT1 protein.The IFIT1 protein was detected in PDCoV-infected ST cells and recombinant His-IFIT1 protein using polyclonal anti-IFIT1 protein antibodies in western blot,but not in indirect immunofluorescence assay.We demonstrated that the transcription and translation levels of ifitl gene were time-and dose-dependent with the infection of PDCoV.Further study showed that the mRNA level,protein level and virus titer of PDCoV were significantly inhibited after transient overexpression of IFIT1 in ST cells.These data confirmed that porcine IFIT1 as a host antiviral protein can significantly inhibit the proliferation of PDCoV.