Characterization of a local isolate of Bombyx mori nuclear polyhedrosis virus

Polyhedral bodies of Bombyx mori nuclear polyhedrosis virus, BmNPV (BGL) isolated from infected silkworms around Bangalore were propagated either in the cultured B. mori cell line, BmN or through infection of larvae. Electron microscopic (EM) observations of the polyhedra revealed an average length of 2 mu m and a height of 0.5 mu m. The purified polyhedra derived virions (PDV) showed several bands in sucrose gradient centrifugation, indicating the multiple nucleocapsid nature of BmNPV. Electron microscopic studies of PDV revealed a cylindrical, rod-shaped nucleocapsid with an average length of 300 nm and a diameter of 35 nm. The genomic DNA from the PDV was characterized by extensive restriction analysis and the genome size was estimated to be 132 kb. The restriction pattern of BmNPV (BGL) resembled that of the prototype strain BmNPV-T3. Distinct differences due to polymorphic sites for restriction enzyme HindIII were apparent between BmNPV (BGL) and the virus isolated from a different part of Karnataka (Dharwad area), BmNPV (DHR)

Baculovirus mediated high-level expression of luciferase in silkworm cells and larvae

Bombyx mori nuclear polyhedrosis virus (BmNPV)-based baculovirus expression system exploits silkworm larvae as an economical alternative to large-scale cell cultures for production of biomolecules. To generate recombinant BmNPV at high efficiency, we have achieved high efficiency transfection of B. mori cells, BmN, through lipofection. Optimal conditions for lipofection were standardized by quantification of the transient expression level of firefly luciferase (luc) reporter gene under control of an immediate early gene promoter of BmNPV Lipofection was 50-fold and 100-fold more efficient than the calcium phosphate method for transfecting BmN and Sf9 cells, respectively. Lipofection enabled us to generate a recombinant BmNPV (vBmluc), harboring luc under control of the strong polyhedrin promoter On infection with vBmluc, luciferase was expressed at very high levels, 170 mu g/10(6) BmN cells or 13 mg/larva. Expression of luciferase in vBmluc-infected larvae was visualized by luminescence emission instantaneously following luciferin injection generating ''glowing silkworms''