Identification Of Anti-PEDV ISGs And Mechanism Of 2'-5' Oligoadenylate Synthetases Like Against PEDV Replication

Porcine epidemic diarrhea(PED)is a severe infectious disease with diarrhea,vomit,dehydration,high morbidity and mortality in piglets caused by Porcine epidemic diarrhea virus(PEDV).PEDV is an enveloped virus with a single-stranded RNA,and the genomic structure is 5' UTR-ORF1a/b-S-E-ORF3-M-N-3'UTR.During the process of clinical epidemic and infecting cell,the genome is constantly mutated.Among them,the spike protein(S)has a good immunogenicity.Interferon stimulated genes(ISGs)are the main antiviral effectors of interferon and play a major role in the innate immune defense against microbial infections,especially viral infections.The 2'-5' oligoadenylate synthetase-like(OASL)can be induced by interferon and crucial in interferon-mediated antiviral innate immunity.This study is divided into three parts.1 Screening of ISGs against PEDV replication based on transcriptome analysisThe growth curves of PEDV 85-7 parental strain and C40 strain were analyzed.It was found that the replication titer of C40 strain on interferon-deficient Vero cells was significantly higher than that of the parental strain.However,the replication titer of C40 strain was significantly lower than that of the parental strain in the same type of MARC-145(interferon-normal)cells.This phenomenon was associated with interferon signaling pathways.Then,we found that C40 strain could induce the production of type I interferon in MARC-145 cells,compared with 85-7 parental strain.Transcriptome analysis was used to compare the differences of two PEDV-induced ISGs,and qPCR verified transcriptome data.We constructed 12 recombinant plasmids,including N1-OASL,N1-RSAD2,N1-USP18,N1-ISG15,N1-TRIM16,N1-STAT2,N1-ATF1,N1-IFIT2,N1-IFIT3,N1-IFIT5,N1-IFI16 and N1-IFI35 using pEGFP-NI plasmid.After over-expression of ISGs for 24 h,MARC-145 cells were infected with PEDV,then the plaque assay was used to detect the amount of virus in the supernatant,and the level of viral replication in the cells was detected by qPCR.The results showed that the virus titer decreased by about 60%after overexpression of OASL,RSAD2 and STAT2;the virus titer decreased by about 50%after overexpression of ISG15,TRIM16 and IFIT2.In conclusion,we confirmed that OASL,RSAD2,STAT2,ISG15,TRIM16 and IFIT2 are resistant to PEDV replication,and suggested that these ISGs are important causes of the difference between PEDV C40 and 85-7 strain2 Study the mechanism of OASL protein inhibiting PEDV replicationTo further study the antiviral mechanism of OASL,PEDV can induce OASL expression in a time-dependent manner after infection with MARC-145 cells.Transient overexpression of OASL significantly inhibited the proliferation of PEDV on MARC-145 cells,and the transient silencing of OASL by RNA interference technology promoted the proliferation of PEDV,demonstrating that OASL has an inhibitory effect on PEDV replication.By co-expressing the OASL and siRNase L,it was found that silencing of the RNase L did not affect the antiviral effect of OASL,demonstrating that the antiviral effect of OASL does not depend on the classical OAS/RNase L pathway.Subsequent studies found that overexpression of OASL can significantly increase the transcription and expression of IFN-?,while silencing OASL can down-regulate the transcription and expression of IFN-?.In addition,the overexpression of OASL on Vero(interferon-deficient)cells did not play an anti-PEDV role of OASL.Moreover,it was proved by Co-IP test that OASL interacts directly with RIG-1,but not with MDA5.In addition,co-expression of OASL and RIG-1 can promote the transcription level of IFN-?.This suggests that OASL can act as an anti-PEDV ISG and exert an antiviral effect through the RIG-1 mediated type I interferon pathway,rather than the classical OAS/RNase L pathway,which helps to further elucidate the mechanism of host innate immunity against PEDV infection.3 Prokaryotic expression of PEDV antigen epitopes and antibody preparationA hypothetical neutralizing linear epitope S582 of PEDV S1 protein was predicted using biological software.The COE,894 and CSS were designed with reference to the neutralization epitope of PEDV CV777,which predict that these flanking fragments contain neutralizing epitopes.Then PCR amplify the four target genes in the PEDV 85-7 parent strain and the 311-1 epidemic strain,and construct a recombinant plasmid containing the target genes.According to the standard immunization procedures,New Zealand white rabbits were immunized with S582 protein for 4 times,and rabbit polyclonal antibody was obtained by Protein A/G purification method.The immune serum titer reached 1:128,000,and virus neutralization assays showed that this antibody was unable to neutralize PEDV,indicating that the S582 sequence is not a PEDV linear neutralizing epitope.