Isolation And Identification Of BVDV And Preliminary Study On The Effect Of RhoA Protein On Its Infection Of MDBK Cells

Bovine viral diarrhea virus(B VDV),a member of the Flaviviridae family and the genus Plague virus,is a single-stranded positive-stranded RNA virus that causes bovine viral diarrhea-mucosal disease(BVD-MD or BVD).BVDV-infected cattle may develop diarrhea,fever,leukopenia,oral and gastrointestinal mucosal erosion and necrosis,and abortion or deformed fetuses in pregnant cows.BVDV infection in early gestation may lead to the production of persistently infected(PI)cattle,and PI cattle are detoxified outward for life.BVD has become endemic worldwide,causing serious economic losses to the livestock industry.The World Organization for Animal Health(WOAH)has classified it as a mandatory quarantine disease for import and export animal trade,and China has classified it as a Class III animal disease.Therefore,it is important to establish a rapid diagnostic test for BVDV and control virus transmission.The positive samples with high viral load detected were subjected to virus isolation and genetic evolutionary analysis to determine the genotype of the isolates and to provide some reference for the molecular epidemiological situation of BVDV in Ningxia region.Through the established detection method and virus isolation results,it is extremely important to explore the epidemiological status of BVDV in Ningxia region and to explore novel anti-BVDV infection methods for the healthy development of Ningxia livestock industry.The main contents of this study include:(1)Establishment and application of a one-step BVDV and BRSV single/dual PCR assayBased on the existing full-length genomic strains of BRSV and BVDV in the GenBank database,the sequences were matched using MAFFT to select the conserved BVDV 5’-UTR region and the BRSV M gene,and the primers and probes for BRSV and BVDV were designed to establish a one-step BVDV and BRSV duplex PCR assay and a one-step BVDV and BRSV duplex PCR assay.The one-step BVDV and BRSV duplex PCR assay and the one-step BVDV and BRSV duplex TaqMan real-time fluorescence quantitative PCR assay were also developed,and the one-step BVDV/BRSV single PCR assay and the one-step BVDV/BRSV single TaqMan real-time fluorescence quantitative PCR assay were also developed.The established assays were used to amplify BRSV,BVDV,IBRV,bovine mycoplasma,bovine coronavirus,bovine microvirus,standard strains of Staphylococcus aureus and enterococci of bovine origin,and none of the pathogens were specifically amplified except for BRSV and BVDV.The minimum detection limits of the one-step BVDV and BRSV single PCR assays were 103 copies/μL and 10 copies/μL;the minimum detection limits of the one-step BVDV and BRSV single TaqMan real-time quantitative fluorescence PCR assays were 102 copies/μL and 10 copies/μL;the minimum detection limits of the one-step BVDV/BRSV The minimum detection limits of the one-step BVDV/BRSV duplex PCR assay were 104 copies/μL and 10 copies/μL;the minimum detection limit of the one-step BVDV/BRSV duplex TaqMan real-time fluorescence quantitative PCR assay was 10 copies/μL,which was 1000 times more sensitive than the duplex PCR assay and 10 times more sensitive than the single BVDV fluorescence quantitative assay.The sensitivity of BVDV is 1000 times higher than that of the duplex PCR method and 10 times higher than that of the single BVDV fluorescence assay.The established assays were used to amplify BRSV,BVDV,IBRV,bovine mycoplasma,bovine coronavirus,bovine microvirus,standard strains of Staphylococcus aureus,and enterococci of bovine origin,and none of the pathogens were specifically amplified except for BVDV and BRSV.The minimum detection limits of the one-step BVDV and BRSV single PCR assays were 103 copies/μL and 10 copies/μL,and the minimum detection limits of the one-step BVDV and BRSV single TaqMan real-time quantitative fluorescence PCR assays were 102 copies/μL and 10 copies/μL,compared with the PCR assays.The one-step BVDV/BRSV duplex PCR assay has the lowest detection limit of 104 copies/μL and 10 copies/μL,and the one-step BVDV/BRSV duplex TaqMan real-time quantitative fluorescence PCR assay has the lowest detection limit of 10 copies/μL,which is 1000 times more sensitive than the duplex PCR assay.The sensitivity of BVDV was 1000-fold higher than that of the duplex PCR method,and 10-fold higher than that of the single BVDV fluorescence quantification method.One-step BVDV/BRSV duplex PCR assay and one-step BVDV/BRSV duplex TaqMan realtime fluorescence PCR assay were used to detect 197 samples from Ningxia area,and the detection rates of BVDV and BRSV by one-step BVDV/BRSV duplex PCR assay were 19.28%and 68.52%,respectively.The detection rates of BVDV and BRSV by the one-step BVDV/BRSV duplex TaqMan real-time fluorescence PCR assay were 30.96%and 73.6%for BVDV and BRSV,respectively,and the mixed infection rate was 13.2%.The detection rate was higher.(2)BVDV isolation and identification and genetic evolution analysisThe detected positive samples were processed and inoculated in bovine kidney cells(MDBK)for virus isolation.The tests of qRT-PCR identification,indirect immunofluorescence(IFA)and electron microscopy observation were performed respectively,and the virus Npro gene was specifically amplified and sequenced and compared with each other.The results showed that the strain was identified as BVDV positive by qRT-PCR,indirect immunofluorescence(IFA)and electron microscopy,and did not cause significant cytopathogenic effects,suggesting that the strain was non-cytopathogenic(NCP).Sequencing of the Npro gene of this strain,followed by sequence matching and genetic evolutionary analysis,revealed that the isolate was BVDV subtype Ⅱ a,which was most closely related to MN5273 54.1,and the sequence identity of the two Npro strains was 99%-100%,and was named BVDV-2a-NX02.(3)Study on the regulatory effect of RhoA and its derived peptides on bovine viral diarrhea virus-infected cellsPreliminarily,we verified that BVDV infection of MDBK cells could cause up-regulation of RhoA expression;after interfering with cellular RhoA expression using siRNA,there was a certain promotion effect on BVDV infection;after overexpressing RhoA by plasmid transfection,there was a certain inhibition effect on virus amplification;after using RhoA short peptide to infect cells after incubation with BVDV,the infectivity of the virus could be reduced.In conclusion,this study successfully established one-step BVDV and BRSV duplex PCR assay and one-step BVDV and BRSV duplex fluorescence quantitative PCR assay,both of which are highly sensitive,specific and reproducible,and have good clinical application value;BVDV virus was isolated from Ningxia area,and genetic evolution analysis was performed by Npro gene,and the virulent strain was found to be 2a subtype;it was found that BVDV infection of MDBK cells could upregulate RhoA expression,and BVDV infection of MDBK cells could be affected by knocking down RhoA or overexpressing RhoA using siRNA,while RhoA peptide could significantly inhibit BVDV infection of MDBK cells after co-incubation with BVDV.