Genomic Analysis Of Bovine Viral Diarrhea Virus And Effects Of LDLR Knockout On Its Infection Of MDBK Cells

Background and Aims:Bovine viral diarrhea(BVD)is an important disease of cattle caused by bovine viral diarrhea virus(BVDV).There are numerous subtypes of bovine viral diarrhea virus,and also new subtypes have been continued to emerge,which make it more difficult to prevent and control BVDV in clinic.However,the use of receptors and induced immune responses by BVDV into the cell remains unclear,so it is impossible to develop specific antiviral drugs.Through the analysis of BVDV genome sequence and its infection mechanism,this study can provide theoretical support for prevention and control of BVDV,and it lays the foundation for the design of new anti-BVDV therapeutic targets.Methods:The clinically isolated BVDV viruses BJ1201,BJ1305 and BJ1308 were extensively propagated on Madin-darby bovine kidney(MDBK)epithelial cells,viral RNA was extracted and subjected to whole genome sequencing;the bioinformatics method was used to analyze the characteristics of whole genomes in clinical isolates and compare with the typical strains of BVDV in the NCBI database for in-depth analysis of their genetic evolutionary relationship.MDBK cells were infected with the BVDV standard strain NADL and the clinical isolate BJ1305,and the total RNA and protein were extracted at different time points respectively.Also,the effect of RIG-1 pathway on BVDV replication was evaluated using poly I:C that can activate RIG-1 protein.Low-density lipoprotein receptors(LDLR),CD46,RIG-I,TLR-3/TLR-7,interferon regulatory factor 3(IRF-3)and IFN-?/?were quantified at the gene level using quantitative Real-time PCR;and the expression levels of LDLR,CD46,RIG-I,and BVDV E2 protein were detected by Western blot.To further investigate the role of LDLR in the invasion of MDBK cells by BVDV,we used the CRISPR/Cas9 gene editing technique to knock out the LDLR gene in MDBK cells.Guide RNA was ligated into the pCRISPR-sg5 vector to construct the pCRISPR-LDLR-sg5 plasmid,and finally electroporated into MDBK cells together with the pCRISPR-W9 and pCAG-Pbase knockout systems;LDLR-/-MDBK cells were identified by sequencing and Western blot.Finally,LDLR-/-MDBK cells were used for BVDV infection,and the effects of LDLR deletion on BVDV-infected host were evaluated by immunofluorescence and western blot analysis.Results:According to the genetic phylogenetic trees constructed using the genome-wide,5'-UTR and Npro gene sequence,three BVDV clinical isolates were identified as BVDV-1d subtypes.BVDV standard strains NADL and clinical isolate BJ1305 can significantly up-regulate the expression of membrane receptors LDLR and CD46 in MDBK cells at 24 h,and can significantly activate RIG-? and inhibit the mRNA expression levels of TLR-3/TLR-7 and IRF-3;also,poly ?:C can inhibit the replication of CP BVDV after activating RIG-?,but it has no effect on the replication of NCP.Cytopathogenic(CP)BVDV can induce the mRNA expression levels of IFN-?(IFN-?),whereas Noncytopathogenic(NCP)BVDV significantly inhibit the mRNA expression levels of IFN-?(IFN-?/IFN-?)for escaping host immunity.The LDLR of MDBK cells was successfully knocked out.LDLR-deleted MDBK cells showed weak viral protein expression at 24 h and 36 h after challenge,while wild-type cells had obvious viral protein blots at 24 h and 36 h after challenge.Conclusion:The three BVDV clinically isolates belong to the subtype BVDV-1d.LDLR and CD46 are cell membrane surface receptors facilitating the BVDV into MDBK cells,and LDLR is the main receptor for BVDV entry into cells.BVDV can be recognized by RIG-? pattern recognition receptors,and can down-regulate the mRNA expression levels of TLR-3/TLR-7 and IRF3;CP BVDV can promote mRNA expression levels of IFN-?,NCP BVDV could inhibit mRNA expression levels of IFN-? for escaping the host immune.