NeuroBactrus, a Novel, Highly Effective, and Environmentally Friendly Recombinant Baculovirus Insecticide

Abstract

ABSTRACTA novel recombinant baculovirus, NeuroBactrus, was constructed to develop an improved baculovirus insecticide with additional beneficial properties, such as a higher insecticidal activity and improved recovery, compared to wild-type baculovirus. For the construction of NeuroBactrus, theBacillus thuringiensiscrystal protein gene (here termedcry1-5) was introduced into theAutographa californicanucleopolyhedrovirus (AcMNPV) genome by fusion of the polyhedrin–cry1-5–polyhedrin genes under the control of the polyhedrin promoter. In the opposite direction, an insect-specific neurotoxin gene,AaIT, fromAndroctonus australiswas introduced under the control of an early promoter fromCotesia plutellaebracovirus by fusion of a partial fragment oforf603. The polyhedrin–Cry1-5–polyhedrin fusion protein expressed by the NeuroBactrus was not only occluded into the polyhedra, but it was also activated by treatment with trypsin, resulting in an ∼65-kDa active toxin. In addition, quantitative PCR revealed that the neurotoxin was expressed from the early phase of infection. NeuroBactrus showed a high level of insecticidal activity againstPlutella xylostellalarvae and a significant reduction in the median lethal time againstSpodoptera exigualarvae compared to those of wild-type AcMNPV. Rerecombinant mutants derived from NeuroBactrus in whichAaITand/orcry1-5were deleted were generated by serial passagesin vitro. Expression of the foreign proteins (B. thuringiensistoxin and AaIT) was continuously reduced during the serial passage of the NeuroBactrus. Moreover, polyhedra collected fromS. exigualarvae infected with the serially passaged NeuroBactrus showed insecticidal activity similar to that of wild-type AcMNPV. These results suggested that NeuroBactrus could be recovered to wild-type AcMNPV through serial passaging.