To develop an advanced baculovirus insecticide with additional advantages, such as higher toxicity and recovering to wild-type baculovirus, a novel recombinant baculovirus, NeuroBactrus was constructed. Bacillus thuringiensis crystal protein gene (cry1-5) and an insect-specific neurotoxin gene (AaIT) were introduced into Autographa californica nucleopolyhedrovirus genome by fusion of polyhedrin-cry1-5-polyhedrin under the control of poyhedrin gene promoter, and by fusion of orf603 partial genes and AaIT under the control of early promoter of ORF3006 from Cotesia plutellae bracovirus. About 150 kDa of Polyhedrin-Cry1-5-Polyhedrin fusion protein expressed by NeuroBactrus was occluded into the polyhedra, and activated as about 65 kDa of crystal protein when treated with trypsin. RT-PCR analysis indicated that transcription of AaIT gene occurs by 2 h postinfection (p.i.) and increased at 16 h p.i.. NeuroBactrus showed high toxicity against Plutella xylostella larvae and significant reduction in median lethal time (LT50) against Spodoptera exigua larvae compared to those of wild-type AcNPV. Re-recombinants derived from NeuroBactrus, NBt-Del5 (deleted cry1-5), NBt-DelA (deleted AaIT) and NBt-Del5A (deleted cry1-5 and AaIT; wild-type baculovirus) were generated in serial passages in vitro. This result showed that the NeuroBactrus could be transferred to wild-type baculovirus along with serial passages by the homologous recombination between two polyhedrin genes and two partial orf603 genes.

To develop an improved baculovirus insecticide with additional advantages, a novel recombinant baculovirus, AcB5B-AaIT was constructed. B. thuringiensis crystal protein gene (cry1-5) and insect-specific neurotoxin gene (AaIT) were introduced into Autographa californica nucleopolyhedrovirus genome by fusion of polyhedrin-cry1-5-polyhedrin under the control of polyhedrin (polh) gene promoter, and AaIT under the control of early promoter of ORF3004 from Cotesia plutellae bracovirus, respectively. About 150 kDa of Polyhedrin-Cry1-5-Polyhedrin fusion protein expressed by AcB5B-AaIT was occluded into the polyhedra produced by the recombinant virus, and activated as about 65 kDa of crystal protein when treated with gut-juice of Bombyx mori. The AcB5B-AaIT showed about 50% reduced LT50 value compared to that of the recombinant virus, Ap1Ac, expressing Cry1Ac against Plutella xylostella larvae. In addition, Spodoptera exigua larvae fed the recombinant polyhedra of AcB5B-AaIT showed about 4 fold higher refusing diet effect compared S. exigua larvae fed the recombinant polyhedra of the recombinant virus, Ap1C, expressing Cry1C. AcB5B-AaIT could be transferred to wild-type baculovirus along with serial passage by the homologous recombination between two polyhedrin genes contained in polh-cry1-5-polh fusion protein gene. These results suggested that the novel recombinant baculovirus, AcB5B-AaIT, could be applied as advanced viral insecticide.