Study On The Molecular Mechanisms Of Persistent Replication Of Bovine Viral Diarrhea Virus And Construction Of Site-directed Mutation Of Bovine Viral Diarrhea Virus

Bovine viral diarrhea virus(BVDV) is the main animal pathogen resulting in bovine viral diarrhea-mucosal disease(BVD-MD) of artiodactyl domestic animals, such as porcine, cattle, sheep and deer, and other wild animal. BVDV spreads widely around the world and greatly threatens the healthy development of animal husbandry and the quality improvement of biological products originated from cattle, such as serum and frozen semen. However, at present there is no effective drug or preventive measure against BVDV infection, and one of the main reasons is that the molecular mechanisms of persistent infection are not fully understood. However, many researches show that the signaling pathway of autophagy and apoptosis directly affects viral persistent replication, and microRNA(miRNA) also regulates the signaling pathway of autophagy and apoptosis and further affects viral persistent replication. Therefore, our study thoroughly explores the molecular mechanisms of the interactions among BVDV, miRNA, antophagy and apoptosis, which will provide the theoretic foundation for illustrating the molecular mechanisms of persistent infection. Otherwise, construction of BVDV mutated strain using the technology of site-directed mutagenesis and reverse genetics will provide theoretical and technical support for developing the effective method against BVDV, and provide a new method and theory for resistance breeding of domestic animals.Objective: In this study, BVDV standard strain NADL served as the research object, investigating the molecular mechanisms of BVDV regulate autophagy and apoptosis, and the effects of autophagy change on BVDV replication, and exploring the construction of BVDV site-directed mutation using reverse genetics method.(1) to explore the molecular mechanisms of the effects of BVDV NADL infection on autophagy;(2) to investigate the effects of autophagy changes on BVDV replication;(3) to investigate the effects of miR-29 b on BVDV NADL infection-related autophagy and BVDV replication;(4) to investigate the effects of miR-29 b on apoptosis;(5) to construct the site-directed mutation of BVDV using reverse genetics. This research will provide theoretical basis for illustrating the molecular mechanisms of persistent infection, and develop a new method for prevention and control of BVDV, and resistance breeding of domestic animals.Methods: At different time intervals after treatment with BVDV NADL in GFP-LC3 transfected MDBK cells, the expression levels of autophagy-related proteins and autophagy activity using real-time quantitative PCR, Western blot, laser confocal microscopy and transmission electron microscopy; and using the same methods to detect autophagy activity in MDBK cells treated with red fluorescent labeled BVDV NADL glycoprotein Erns-, E1- and E2-overexpressed lentivirus.(2) After treatment with autophagy inhibitors, promoter and RNA interference technology, autophagy activity and the replication levels of BVDV NADL after autophagy change were detected.(3) Prediction and identification of the target genes of miR-29 b in autophagy pathway, ATG14 and ATG9A; the expression of target genes, autophagy activity and BVDV replaiction levels were detected in pre-miR-29b-lv and BVDV NADL infected MDBK cells; Designing the experiment of ATG14 and ATG9 A overexpression, and further verificating the effects of miR-29 b on BVDV NADL-infeced autophagy and its replication through downregulating of ATG14 and ATG9 A expression.(4) Prediction and identification of the target genes of miR-29 b in apoptosis pathway, NAIF1 and caspase-7; After transfection miR-29 b mimics and inhibitors into MDBK cells, the rate of apoptosis and the expression levels of caspase-7 and NAIF1 were detected. Designing the experiment of NAIF1 and caspase-7 overexpression, and further verificating the effects of miR-29 b on apoptosis through inhibiting NAIF1 and caspase-7 expression.(5) T7 RNA polymerase-overexpressed lentivirus was constructed and used to infect MDBK cells. After stable screening with puromycin, the expression level of T7 RNAP and T7 RNA polymerase activity were detect. The stability of MDBK-T7 RNAP cells was identified using the same methods at passage 40. E2- and NS4B-mutated plasmids were constructed from pACNR/NADL plasmid, and rescuing site-directed mutation after transfecting E2- and NS4B-mutated plasmids into MDBK-T7 RNAP cells. Meanwhile, the change of site-directed mutation replication was detected, and their genetic stability was detected after passage.Results:(1) BVDV NADL infection and overexpression of Erns and E2 significantly increased the number of autophagosome and autolysosome, GFP-LC3 puncta and the expression levels of Beclin1 and ATG14.(2) Treatment with 3-MA, wortmannin and RNA interference inhibited autophagy activity and BVDV NADL replication. On the contrary, rapamycin significantly promoted autophagy activity and increased BVDV NADL replication. After 18 h post-infection, the transcription level of 5’UTR was decreased, and the transcription level was increased after treatment with chloroquine.(3) miR-29 b specifically targeted the 3 ’UTR region of ATG14 and ATG9 A, and inhibited their expression. Meanwhile, the high expression of miR-29 b significantly decreased BVDV NADL infection-induced autophagy and its replication. Overexpression of ATG14 and ATG9 A, BVDV NADL replication and BVDV NADL infection-induced autophagy were increased.(4) miR-29 b reduced apoptosis of MDBK cells. miR-29 b specifically targeted the 3 ’UTR region of NAIF1 and caspase-7, and inhibited their expression. Apoptosis was significantly increased after NAIF1 and caspase-7 overexpression.(5) MDBK-T7 RNAP cells with strong T7 RNA polymerase activity and high genetic stability were obtained. BVDV sitedirected mutation, E2 M and NS4 BM, were successfully constructed. Compared with its original parent, the replication and proliferation ability of BVDV E2 M and NS4 BM strain were reduced.Conclusion:(1) BVDV infection and overexpression of Erns and E2 significantly increased the activity of autophagy. Our study first expound that the effects of BVDV infection on autophagy and its molecular mechanisms.(2) The replication of BVDV NADL was inhibited in autphagy downregulated MDBK cells, and autophagy during early stages contributed to BVDV replication. Our study elaborated the effects of autophagy change on BVDV replication and further provided a theoretical foundation for clarifying the molecular mechanisms of persistent infection.(3) miR-29 b directly targeted autophagy-related protein ATG14 and ATG9 A and inhibited their expression, and further inhibited BVDV NADL infection-induced autophagy and its replication, which expounded the molecular mechanisms of miR-29 b affected BVDV replication through affecting autophagic activity.(4) miR-29 b directly targeted apoptosis related protein caspase-7 and NAIF1 and inhibited their expression, and further downregulated apoptosis. Our study clarified the molecular mechanisms of miR-29 b affected apoptosis and BVDV replication.(5) BVDV site-directed mutation E2 M and NS4 BM with lower growth capacity and good genetic stability were constructed, which provides technical support for the establishment of an effective site-directed and vaccine candidate BVDV mutation.