Rescue Of The Linearized Genome Of Bombyx Mori Bidensovirus, And Characterization Of A Recombinant Virus

Bombyx mori bidensovirus(BmBDV), the first bidensovirus identified in insects, belongs to the family Bidnaviridae, which was established by the International Committee on Taxonomy of Viruses in 2012. Bombyx mori bidensovirus(BmBDV) can specifically infect the columnar cells of the midgut epithelium of silkworms, and it causes chronic densonucleosis disease。The genome structure of this virus was similar to parvovirus, but the replication of this virus DNA could use themechanism similar to that of adenovirus. Bm BDV has the characteristics of adenoviruses and parvoviruses. Although BmBDV has the potential to be used as a tool in biological pest control and as an expression vector, virus rescue has been a bottleneck in the application of this virus. In this study, we constructed a full-length genomic clone of BmBDV and showed that its terminal structure was restored. A recombinant BmBDV that expressed the green fluorescence protein(GFP) gene was constructed. Then, BmN cells, which are an ovarian cell line, were co-transfected with the linearized genome using continuous culture and expanded cell culture methods. To observe the pathological changes and expression of exogenous green fluorescent protein GFP gene in con-transfected BmN cells with optical microscope and inverted fluorescence microscope. Using DNA methylation analysis detection method to determine whether the BmBDV genome DNA could be synthesized in BmN cells after co-transfection; Using RT-PCR detection method to confirm whether the BmBDV genome could be transcribed in BmN cells; Using western blot detection method to confirm whether the BmBDV genome or exogenetic GFP gene could be expressed. First instar silkworm(strain QingSong) larvae were fed with a virus-infected cell suspension to determine whether the BmBDV virions are produced successfully in BmN cells.We constructed a genomic clone in which the terminal structure could be reinstated through diagnostic restriction endonuclease digestion. To construct BmBDV genome full length clone pUC-VD1/p,pUC-VD2/p, we created the specific PmaCI recognition sequence CACGTG by introducing three nucleotides, CAC, at the end of the CTS. We constructed a recombinant plasmid pVD2-mcp-gfp,in which a GFP gene expression cassette p5-gfp-sv40 was inserted into the Sac I and SpeI sites of the mcp gene.The linear genomic terminal region was exposed by digestion, so that a large number of double-stranded, linearized viral genomes could be obtained,then liposome-entrapped the linear genomic were co-transfected in to BmN cells.The co-transfected BmN cells were continuous cultured 15 d,to study the co-transfection and rescue of the genome of Bombyx mori bidensovirus. BmN cells were co-transfected respectively with linearized genome-containing plasmids and linearized recombinant plasmids could be observed that the shape of the cell is changed,the cells began to become round and began to break up by optical microscope. The direct visualization of green fluorescence allowed us to detect recombinant plasmids that were expressed in BmN cells by inverted fluorescence microscopy.Using DNA methylation analysis detection method to determine Cell precipitation,PCR data illustrated that there were newly synthesized progeny virus DNA in co-transfected BmN cells. NS1 mRNA was detected by RT-PCR, illustrated that the BmBDV genome could be transcribed in BmN cells. Western blot data also indicated that the BmBDV structural protein VP was expressed in BmN cells and GFP was also expressed in BmN cells after co-transfection. The VP protein is the most important structural protein of BmBDV. The expression of VP indicates that BmBDV is likely to be packaged in BmN cells. GFP was used as a marker for exogenous genes expression, GFP protein expression provide the possibility for Bm BDV developing as a viral vector.First instar silkworm(strain QingSong) larvae were fed with a virus-infected cell suspension and served as the experimental group,then the larvae started showing symptoms, and midgut tissues that were removed from silkworm larvae that were invaded by Bm BDV were yellow and thin, and green fluorescence could be observed. These results indicate that an infectious viral genome was rescued.