Isolation And Identification Of BVDV-2 GS2018 Strain And Effects On The Proliferation And Transcription Of Bovine PBLCs

Bovine viral diarrhea virus(BVDV)is an important pathogen causing bovine viral diarrhea/mucosal disease(BVD-MD),and it is also the common source of contaminant in bovine serum and related biological products.BVDV could infect a range of domestic and wild ruminants.Commonly result in reproductive disorders,immunosuppression and persistent infection.Owing to the numerous subtypes and genetic diversity,BVDV has currently caused the enormous economic losses to the cattle industry.BVDV has evolved various strategies to evade the immune response of host,which can establish persistent infection(PI)in calves after the fetal infection and continuously shed infectious virus throughout their lives.Therefore,exploring the immune response mechanism caused by BVDV is of great significance for elucidating the interaction mechanisms of host-BVDV and early diagnosis of BVDV in cattle herds.In this study,the obvious cytopathic effect(CPE)were found in Madin-Darby bovine kidney cells(MDBK)cell,which cultured with fetal bovine serum suspected contaminated with BVDV.Based on the plaque formation method obtained the purified virus particles and then measured the titer was up to1×10^6.2/0.1 m L.Then the virus was identified by transmission electron microscopy(TEM),indirect immunofluorescence assay,molecular identification,complete genome sequencing and genetic evolution analysis respectively,and the CP BVDV-2a GS2018 strain was obtained with a size of 12235nt.However,there is no nucleic acid fragment insertion in the NS2/3 region,which is obviously different with most CP BVDV-2,suggesting the complexity of BVDV induced cytopathic.The results of cell proliferation and apoptosis assay showed that,GS2018 could inhibit the proliferation of PBLCs and increase PBLCs apoptosis,which increased apoptosis rate by 10%(p<0.01)at 48 h after infection with GS2018.In addition,transcriptomic sequencing analysis was performed on bovine PBLCs infected with GS2018 at 12 hpi.Gene expression profiling demonstrated that 1052 genes were differentially expressed in GS2018 infected PBLCs compared with the control group.Of these genes,485 genes were up-regulated and 567 were down-regulated.19 differential expressed genes(DEGs)were selected for validation using quantitative real-time PCR,which were consistent with the expression tendency of RNA-Seq.Gene ontology enrichment and KEGG pathway analysis showed that the DEGs associated with viral infection were significantly enriched in 16 pathways,including cytokine-cytokine receptor interaction,NOD-like receptor,IL17,PI3K-Akt,MAPK and TNF signaling pathway etc.Besides,the result of flow cytometry showed that the Th17 cells differentiation was induced by GS2018,particularly at 24 hpi,with the number of Th17 cells were increased by 5%(p<0.01).In addition,as well as q PCR and Western blot showed that,the production of IL17A in PBLCs was continuously increased after GS2018 infection.Further study was conducted on the effects of IL17A and IL22 on the GS2018 replication.The IL17A and IL22 were cloned from the bovine PBLCs and inserted into the eukaryotic expression vector p EGFP-N1 and then transfected into PK15 cells respectively.At 12 h post transfection,the fluorescence was observed and then inoculated with GS2018 at MOI of 1.The expression levels of IL17A,IL22 and the copy number of GS2018 were detected by q PCR and Western blot at 24 h,48 h,and 72 h after infection.The results showed that overexpression of IL17A could significantly inhibit virus replication(p<0.05),while overexpression of IL22 has no significant effect on viral replication.In brief,the production of IL17A induced by GS2018 at an early stage of infection,which could inhibit viral replication at the later stage.Taken together,the CP BVDV-2a GS2018 strain with no exogenous fragment insertion in the NS2/3region was isolated and identified in this study.The GS2018 could inhibit the proliferation and promote apoptosis of PBLCs.Meanwhile,induce Th17 cells differentiation and stimulate the production of IL17A and thereby inhibit the replication of BVDV.These results will establish a solid foundation for clarifying the molecular mechanism of immune response underlying BVDV-host interactions.