Study On The Function Of CyPB During ORFV Infection

The infection of Orf virus (ORFV) causes enormous economic losses to the world’ssheep industry. As a zoonosis, this disease is a serious threat to human health, and hasalready become a significant hazard which has an important impact on our country andthe global public health security. Previous researches of ORFV are mainly about etiology,however, viral infection is the process of interaction between pathogen and host. Virusinfection is capable of replicating at different phases through different ways and interactswith the various components of the host cell, which can then cause some differences inthe gene expression in the host cell and change the mediated cellular signal pathway,consequently, it results in cell physiological dysfunction and leads to the disease.Therefore, it is difficult to understand the mechanism of viral infection just based on thestudy of etiology. From this study, we start from the interaction of the virus with the hostcell. We have finished gene library constructed from ORFV early infection in host cell byusing suppression subtractive hybridization. After plasmid PCR, reverse dot blothybridization screening, sequencing, we obtain81differential expressed genes in ourresearch. Online biologically applied software was used for predicting gene function, theresults indicate that functions of differential expressed genes are divided into structuralmolecule activity, binding, transcription regulator activity, transporter activity, translationregulator activity, regulation of enzyme activity, catalytic activity. The biologicalprocesses where these genes are involved include metabolic process, transportationprocess, localization, immune process, cellular process, development process, response tostimulus and the apoptotic process.According to the prediction of the function of differential expressed genes, wechoose the cyclophilin B (CyPB) as study object, which involved in immuneprocess.Many diseases like tumors, immune regulation, and blood vessel formation areassociated with abnormal expression and distribution of CyPB. CyPB plays an importantrole in the infection of vaccinia virus, swine fever virus, human immunodeficiency virus,hepatitis C virus, Japanese encephalitis virus, bovine vesicular stomatitis virus, influenza virus etc. However, there is no report about CyPB in ORFV infection. In this study, westudy the function of CyPB during ORFV infection. First, we analyze the changes insequence of CyPB in ORFV infection, there is no mutation of CyPB gene found in thehost cells after ORFV infection. By using Real-Time PCR and Western Blot to monitorCyPB expression in mRNA and protein levels respectively within24h（2h,4h,8h,12h,18h,24h）after ORFV infection we found that the level of CyPB geneexpression increases rapidly in the early viral infection. The number of mRNA of CyPBin ORFV infected cells is106times more than that in normal cells at4h after infection. Inthis research, we construct PCDNA3.1/His A+CyPB eukaryotic expression plasmid, andobtain1CyPB over-expressing MDBK cells by G418selection, and inhibit CyPBexpression in MDBK cells using siRNA. After the virus infection, the results from viraltiter and Real-Time PCR assays demonstrate that CyPB plays a key role in the viralreplication: the over-expressed CyPB accelerates viral DNA replication. Virus titer reacha peak ahead of12hours. After the inhibition of CyPB expression, the viral replication issignificantly blocked. The MTT assay reveals that CsA (an inhibitors of CyPB) at losedose can effectively inhibit the cytotoxicity caused by ORFV infection.