| Porcine deltacoronavirus(PDCoV)is a positive-stranded RNA virus with an envelope,belonging to the family Coronaviridae and D-type coronavirus.PDCoV can cause swine diarrhea and piglet death at all ages.It is similar to the clinical symptoms of porcine epidemic diarrhea virus infection,and it is difficult to distinguish by clinical symptoms.PDCoV has a wide range of tissue tropism.PDCoV can be detected in various organs,and the infection mechanism is complex.Exploring its infection mechanism is of great significance for the prevention and control of PDCoV.Long noncoding RNA(lncRNA)is a non-coding RNA(ncRNA)that is more than 200 nucleotides in length and does not translate proteins.The transcripts produced by 4-9%of the mammalian genome sequence is lncRNAs.Studies have shown that lncRNA plays an important role in many life activities,so it has become the research focus of genetics.The research on lncRNA has been developed rapidly,numerous types of lncRNA have been discovered,but the function of most lncRNAs is still unclear.Most of the current research on IncRNA has focused on cancer and cardiovascular disease,but little is known about the regulatory role played by lncRNA in viral infection.In order to explore the function of IncRNA in PDCoV infection,this study first used RTqPCR to verify the differential expression profile of lncRNA in PDCoV infected pig-derived cells ST.The verification results of five randomly selected lncRN As(LNC-001155,LNC000737,LNC-000520,LNC-000781,ALDBSSCT0000010704,etc.)that significantly upregulated the expression showed that the expression dynamics were consistent with the RNAseq data.Based on this,lncRNA-000781,which was significantly up-regulated,was selected as the research object,and the effect of different infection doses and time of PDCoV on the expression of lncRNA-000781 and the effect of lncRNA-000781 expression on host cells on PDCoV replication were analyzed.The results showed that the expression of lncRNA000781 was positively correlated with the infection dose and time of PDCoV in ST cells,and the overexpression of lncRNA-000781 in ST cells could also significantly inhibit PDCoV replication.The open reading frame on lncRNA-000781 was predicted and verified by ORF Finfer.It was found that lncRNA-000781 ORF3 and ORF6 can encode small peptides,and it was further found that lncRNA-000781 ORF3 was almost completely co-localized with the cell mitochondria by confocal laser microscope,and the specific mechanism needs further research.Antibodies specific for viral proteins are important tools for studying the mechanism of viral infection.In order to further study the replication mechanism of PDCoV,we prepared specific antibodies for the non-structural proteins of PDCoV Nsp5,Nsp8 and Nsp10.Using the cDNA of PDCoV as a template,we successfully cloned the Nsp5,Nsp8,and Nsp10 genes and inserted them into the prokaryotic expression vector pET-2 8a to achieve protein expression in E.coli BL21(DE3).Western blot results showed that Nsp5,Nsp8 and Nsp10 were expressed in soluble form in E.coli.Polyclonal antibodies were prepared by immunizing BALB/C mice with purified Nsp5,Nsp8,and Nsp10 proteins as antigens.Western blot results showed that mouse-derived Nsp5,Nsp8,and Nsp10 antibodies could interact with PDCoV non-structural proteins Nsp5,Nsp8 and Nsp10 reacted specifically,indicating that the prepared mouse Nsp5,Nsp8 and Nsp10 antibodies had good reactivity and specificity.In summary,this study demonstrated that lncRNA-000781 inhibits PDCoV replication,and found that ORF3 and ORF6 of lncRNA-000781 have coding potential.Murine polyclonal antibodies to Nsp5,Nsp8,and Nsp10 of PDCoV were prepared.It lays the foundation for further research on the function of lncRNA-000781 in PDCoV replication. |
