Researches On The Molecular Mechanism Of Inhibition Of Host Type ? Interferon Response By BPV VP1 And BCoV N Proteins

Bovine parvovirus(BPV)and bovine coronavirus(BCoV)are the main pathogens causing bovine diarrhea in cattle,both of which can cause digestive tract diseases of calves with similar clinical symptoms,which bring great trouble to clinical diagnosis,especially differential diagnosis.Simple or mixed infection of these pathogens will reduce the immunity of cattle and even cause serious economic losses to the cattle industry.Therefore,in-depth study of the pathogenic mechanism of BPV and BCoV and rapid and reliable detection methods are very necessary.At present,the research on BPV and BCoV is mainly focused on the isolation of virus strains,epidemiological investigation and the establishment of detection methods,but the molecular mechanism of BPV and BCoV has not been reported.In this study,the exogenous recombinant expression plasmid of BPV virus protein was constructed according to the genome sequence of BPV Haden strain(Gen Bank No:DQ335247).BPV virus protein was screened by real-time fluorescence quantification(qRT-PCR).It was found that BPV VP1 protein significantly promoted the proliferation of BPV in vitro(P<0.001),and BPV VP1 significantly inhibited the production of IFN-?induced by vesicular stomatitis virus(VSV).Further studies showed that BPV VP1 could inhibit the transcriptional expression of downstream factors of JAK/STAT pathway induced by VSV and the production of interferon?(IFN-?)induced by key proteins of exogenous JAK/STAT pathway and the transcription of key proteins of IFN-?pathway.The results showed that BPV VP1 was involved in the inhibition of JAK/STAT signaling pathway activation and IFN-?production.In the study of BCoV,the regulatory role of BCoV nucleocapsid phosphoprotein(N)in IFN-?production and RIG-I-like receptors(RLRs)pathway was preliminarily discussed based on the study of coronavirus homologous viruses.It was found that BCoV N was dose-dependent(P<0.01)inhibit the production of IFN-?induced by VSV,and BCoV N can inhibit the production of IFN-?induced by key proteins of RLR pathway and the transcriptional level of key proteins.In addition,the results showed that BCoV N protein was involved in the inhibition of RLR signal pathway activation and IFN-?production.In this study,using Taq Man qRT-PCR technique,using BCoV N protein and BPV VP1protein as monitoring genes,a dual Taq Man qRT-PCR detection method for BCoV and BPV was established.The results showed that the established double Taq Man qRT-PCR detection method of BCoV and BPV could only specifically amplify BPV and BCoV,and the R~2of the standard curve was more than 0.996,indicating a good linear relationship.The repeatability test results showed that the coefficients of variation within and between groups were less than 1.63%,indicating good repeatability.The sensitivity test results show that the detection limit of BCoV is200 copies/?L,that of BPV is 20 copies/?L,and that of ordinary PCR is 2.0×10~6copies/?L,indicating that the dual Taq Man qRT-PCR method established in this study is more accurate than conventional PCR.In summary,this study confirmed that BPV VP1 protein is involved in inhibiting the activation of JAK/STAT signal pathway and the production of,BCoV N protein by down-regulating the expression of key proteins in RLRs signal pathway.It suppresses the production of IFN-?and the activation of RLRs signal pathway by down-regulating the expression of IFN-?.In this study,a double Taq Man qRT-PCR detection method of BCoV and BPV was successfully established,which provides a technical means for rapid and efficient detection and differential diagnosis of bovine BCoV and BPV.