Abstract
The bacmid system first established for Autographa californica nucleopolyhedrovirus (AcNPV) is a powerful tool for generating recombinant viral DNA in E. coli, which allows one to produce knockout viruses (even viruses missing essential genes) independent of viral replication in host cells. We constructed a bacmid system for the type strain (T3) of Bombyx mori NPV (BT3Bac) and analyzed the functional necessity of a delayed-early gene he65 using the polyhedrin-gene-rescued BmNPV/T3 bacmid (BT3Bac+polh). An apparent difference in the viral replication profile judging from numbers of polyhedra-forming cells and amounts of viral DNA in the culture medium was not observed between BmNPV/T3 and BT3Bac+polh. On the other hand, the accumulation of viral DNA was delayed by about 12 hrs in he65-knockout BT3Bac (BT3Bac+polh-he65KO) compared to those in BT3Bac+polh or BmNPV/T3, indicating that he65 is not essential for BmNPV replication but critical for an optimal replication in BmN cells. The BmNPV/T3 bacmid system constructed here is useful for the functional analysis of BmNPV genes.
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