Molecular Mechanism Of Bovine Viral Diarrhea Virus Infection And Replication

Bovine viral diarrhea (BVD) is an important viral disease and listed as a reportable disease of cattleby the World Organization for Animal Health (OIE). BVDV is the causative agent of Bovine viraldiarrhea which causes a series of clinical symptoms of acute or chronic mucosal disease, persistentinfection and immunosuppression. At present, the BVDV prevalence rate is rising in a worldwide andthe host range has expanded, which brings more challenges to prevent and control BVDV infection.Autophagy is induced by virus infection. It may not only contribute as an intrinsic host defensemechanism against invading viruses but may also mediate antigen presentation, which plays crucial rolein the control of intracellular pathogen. However, many viruses have a number of mechanisms to blockautophagy or even manipulate autophagy for their own benefit. In addition, the interactions betweenBVDV and cellular protein can destroy the normal function of cell and lead to cell damage, which is akey element of causing disease. However, the studies on BVDV-autophagy and BVDV-host interactionsare limited. In order to elucidate the BVDV replication mechanism, the following results were obtained:1. In recent years, three subgentypes: BVDV-1b, BVDV-1d and BVDV-1q are circulating inWestern China. In order to investigate possible infection in dariy cattle of Ningxia and yaks of Qinghai,all samples were tested for viral nucleic acids by RT-PCR. The results showed the prevalence was26.8%and24%in dariy cattle and yak, respectively. The phylogenetic reconstructions demonstratedthat these samples are BVDV-1, specifically belonging to subgenotypes BVDV-1b, BVDV-1d andBVDV-1q, through the nucleotide analysis of5′UTR and Nproregions. Thirteen viruses were isolatedfrom the antigen-positive samples, and all of them were ncp biotype. Virus particles can be observedunder electron microscopy and direct immunofluorescence.2. Preparation of high affinity antibodies with Core and Nproproteins. The prokaryotic expressionplasmids pET-30a-Core and pET-30a-Nprowere constructed and transformed into E.coli BL21. Highlypurified Core and Nproproteins were obtained by an affinity chromatography and electroelution afterinduction by IPTG. The western-blot assay indicated that recombinant proteins had good antigenicity.Then, polyclonal antibodies against Core and Nproproteins were prepared. IFA and western-blotexperiments demonstrated that polyclonal antibodies can identify the natural proteins and can be used infollow-up studies.3. Induction of autophagy can enhance BVDV replication. To determine the role of autophagy inthe infection of BVDV, we performed on MDBK cell infected by BVDV strain Oregon C24V. A largenumber of autophagosome-like double-memberane vesicles in the cytoplasm were observed undertransmission electron microscopy. BVDV infection resulted in conversion of LC3-Ⅰ/Ⅱ, degradation ofp62and the accumulation of GFP-LC3dot. Inhibition of autophagy using wortmannin and smallinterfering RNAs targeting Beclin-1can reduce BVDV replication and expression of Npro.4. Interaction of cellular PIAS4with BVDV core protein is beneficial to BVDV replication. Usinga yeast two-hybrid screen, the interacting partners of the Core protein were screened in PBMC cDNAlibrary and PIAS4as a novel interacting partner was selected. The interaction between Core and PIAS4 was confirmed by co-immunoprecipitaion and confocal assays. Moreover, Knockdown of PIAS4bysmall interfering RNA resulted in reduction of the virus titer as well as Core protein, whileoverexpression of PIAS4promoted BVDV growth and expression of Core protein.According to the research, the key host factors that regulate the BVDV replication have beendiscovered, which provide clues for further study on virus-autophagy interaction and mechanism inresistance to host immunity.