Bombyx mori cypovirus (BmCPV) is one of the major viral pathogen for silkworm, and the genome of BmCPV is composed of 10 dsRNA segments. As construction system of recombinant BmCPV (rBmCPV) is scanty, researchers achieved little progress in studying gene function of BmCPV in recent decades. Here, 10 recombinant plasmids with a full-length cDNA of viral genome segments S1-S10 containing T7 promoter were constructed. After cotransfecting the BmN cells with the mixture of 10 in vitro-transcribed RNAs, pathological changes were observed. Real-time PCR and Western blot showed viral gene vp1 and structural proteins were expressed. It is found the genome of the rBmCPV is composed of 10 dsRNA segments similar to those of wild-type BmCPV. Moreover, viral particles and polyhedron with virions can be generated in the cotransfected cells and the injected silkworm midguts. These findings confirmed the formation of infective rBmCPV. Additionally, we found viable rBmCPV was generated by cotransfecting the mixture of in vitro-transcribed S1-S9 RNAs into the cultured cells, confirming polh was not essential for BmCPV replication. Moreover, an infectious rBmCPV expressing the DsRed protein was constructed based on this system. Further investigation showed S2 and S7 segments are indispensible for viral proliferation. Our findings demonstrated the construction system of rBmCPV can be utilized for exploring viral replication and pathogenesis, and investigated method for constructing rBmCPV will certainly facilitate developing novel biopesticides and expressing exogenous gene in the midgut of silkworm.
Keywords: Bombyx mori; Cypovirus; Exogenous gene; In vitro-transcribed RNAs; Recombinant virus.