Study On The Infection Of Porcine Epidemic Diarrhea Virus,the Response Of Host Antibody Repertoire And The Regulation Of M~6A Modification

Porcine epidemic diarrhea virus(PEDV)is an enveloped,single-stranded,positive-sense RNA virus that belongs to the order Nidovirales,family coronaviridae and genus Alphacoronavirus.In this study,we focused on the receptor usage of PEDV entry and host response and regulation against the PEDV infection,which aims to comprehensively understand the relationship between PEDV and host as well as provide theoretical basis for clinical prevention and treatment of PEDV.A monkey cell line Vero(ATCC CCL-81)is commonly used for PEDV propagation in vitro.However,it is still controversial whether the porcine aminopeptidase N(pAPN)counterpart on Vero cells(vAPN)confers PEDV entry.We found that endogenous expression of vAPN was undetectable in the mRNA and the protein levels in Vero cells.We cloned the partial vAPN gene(3340-bp)containing exons 1 to 9 from cellular DNA and subsequently generated two APN-knockout Vero cell lines by CRISPR/Cas9 approach.PEDV infection of two APN-knockout Vero cells had the same efficiency as the Vero cells with or without neuraminidase treatment.A Vero cells stably expressing pAPN did not increase PEDV production.SiRNA-knockdown of pAPN in porcine jejunum epithelial cells had no effects on PEDV infection.The results suggest that there exists an additional cellular receptor on Vero or porcine jejunal cells independent of APN for PEDV entry.Next,in order to study the host response to PEDV infection,we applied multiplex PCR and high-throughput sequencing to analyze the functional antibody repertoire in peripheral blood mononuclear cells(PBMCs)from normal three-week-old pigs,and evaluated the dynamics of antibody repertoire stimulated by infection with PEDV.In normal conditions,several V gene segments were preferentially used,with IGHV1-4 and IGHV1S2 the two most frequently used genes.However,in PEDV-inoculated pigs there was increased usage of IGHV1-6 and IGHV1-12 gene segments and shorter-length HCDR3s.In particular,some high-frequency HCDR3 sequences appeared in the antibody repertoire in response to PEDV infection.Finally,we studied the regulatory role of N6-methyladenosine(m6A)modification in PEDV infection.We determined that m6A modifications of PEDV are mostly located in the N gene at the 3'-end of the genome by MeRIP-seq.Depletion of m6A demethylases significantly increased PEDV replication and gene expression whereas knockdown of m6A methyltransferases slightly decreased PEDV infection.Interestingly,m6A binding proteins YTHDF2 and YTHDF3 significantly inhibited PEDV replication whereas YTHDF1 had the opposite effect.These results suggested that the host m6A-related proteins are involved in the regulation of PEDV infection.In addition,when the major m6A sites in the N gene were mutated by reverse genetic systems,the resultant recombinant PEDVs and PEDV replicons had a significant increase in replication and gene expression,suggesting that addition of m6A to PEDV RNA inhibits viral replication.This study illustrates that both host and viral m6A machinery regulate PEDV replication.