Pathogenicity And Biosafety Of Recombinant Viruses Of Bombyx Mori Bidensovirus

Bombyx mori bidensovirus(BmBDV)belongs to the genus bidensovirus of the bidensoviridae.BmBDV mainly infect the midgut cells of silkworms,causing the silkworm to suffer from the densonucleosis-like symptoms.The genome structure of BmBDV is similar to parvovirus and its replication pattern is similar to the adenovirus.Therefore,BmBDV has the potential to be developed as a viral vector that combines the characteristics of parvovirus and adenovirus.BmBDV can be created as viral vectors with specific functions by recombining foreign genes.For example,Developed a new type of safe and effective bio-pesticides through genetic transformation.The scorpion toxin BmK ITa1 isolated from the Buthus martensii Karsch is an inhibitory anti-insect toxin composed of 65 amino acid residues and has a lethal effect rely on its insect-specific selective neural paralysis.It is unclear whether the introduction of the scorpion toxin BmK ITa1 gene into BmBDV genomes can enhance the toxicity of BmBDV or not.The main contents of this study includes:(1)the construction of the BmBDV viral vector recombinant with BmK ITa1 gene;(2)the infectivity of the recombinant BmBDV with BmK ITa1 gene in insect cells and silkworm;(3)the infectivity of the recombinant BmK ITa1 virus in mammalian cell;(4)the homology of the BmBDV genes and other insect virus genes.The results of the study are as follows:(1)The construction of the recombinant BmBDV viral vector with BmK ITa1 gene.We first constructed the BmK ITa1 gene expression cassette.The BmK ITa1gene(198 bp)with restriction enzyme sites at both ends synthesized by company was inserted between the baculovirus immediate early gene promoter ie1 and the sv40 tailing signal sequence to construct the plasmid pT-ie1-BmK ITa1-sv40 that possess the scorpion toxin expression cassette.Then,the scorpion toxin BmK ITa1 expression cassette was inserted into the BmBDV minor structural protein mcp gene to construct a recombinant scorpion toxin viral vector pVD2-mcp/BmK ITa1.(2)The infectivity of the recombinant BmBDV virus with BmK ITa1 gene ininsect cells and silkworm.The recombinant virus pVD2-mcp/BmK ITa1 was linearized and co-transfected into Hi5 insect cells with the proviral genome(VD1,VD2).Compared with the control group transfected with pUC119,the cells appeared the phenomenon of becoming round and floating.96 hours post-transfection,the cells continued to undergo cell disruption,which was consistent with the infection of the original virus genome(linearized VD1,VD2).The transcription of BmK ITa1 gene was detected in diseased cells using reverse transcription PCR by specific primer;BmK ITa1 protein and viral VP protein were detected by western blot using His and VP monoclonal antibody.The diseased Hi5 cell lysate was fed to susceptible BmBDV silkworm larvae by coating mulberry leaf.After 4 days of re-infection,the larvae showed signs of anorexia,spasm and paralysis.This results shown that the recombinant exogenous BmK ITa1 gene expressed and enhanced the virulence of BmBDV in the silkworm midgut.(3)The infectivity of the recombinant BmK ITa1 virus in mammalian cells.Using the linearized pVD2-mcp/BmK ITa1 and the original virus genome(VD1,VD2),co-transfected into the mouse fibroblast cells L929.The cell growed normally post-transfection,and no obvious difference has been observed contrast with the blank control at 10 days after transfection.The transcription of scorpion toxin gene has not been detected by reverse transcription PCR and the expression of viral VP protein has not been detected by western blot,indicating that no virus particles were produced in L929 cells after transfection.prove that the recombinant scorpion toxin BmBDV vector can not infect mammals cells initially.(4)The homology of the BmBDV virus genes and other insect virus genes.Using Blast to search the sequence-like proteins of BmBDV,phylogenetic analysis shows that the ns1 and vp genes of VD1 are homologous to the densovirus ns1 and vp genes.DNA polymerase polB is homologous to the polymerase of eukaryotic self-synthetic DNA transposons(Polinton transposons).the ns1 and vp genes are in the same single-strand.It is speculated that the VD1 originated from amonosemantic densovirus.DNA polymerase genes were obtained from the Polinton transposon by gene transfer.the genome of original virus experienced the monosemantic to ambisence evolution mechanism at the same time;The ns3 gene of VD2 was homologous to the granulosis virus and ns3 gene and the minor structural protein mcp gene was derived from the double-stranded RNA reovirus.It is speculated that VD2 originated from a primitive Ambidensovirus.In conclusion,this studies proved that the recombinant BmK ITa1 gene accelerates the death of silkworm larvae that is enhances the pathogenicity of BmBDV;Recombinant BmBDV can not infect the mouse fibroblast cells L929,suggest that the recombinant BmBDV possesses mammalian biosafety.The evolutionary analysis has found that VD1 originated from the monosemantic densovirus,VD2 originated from the Ambidensovirus.This study is of great significance for the further understand the characteristics of BmBDV and its application to viral vectors.