Full-length (72K) and truncated (61K) CrylVD mosquitocidal proteins of Bacillus thuringiensis (Bt) were expressed in Spodoptera frugiperda cells and larvae of Trichoplusia ni using a baculovirus vector to investigate the role of CrylVD peptides in toxicity as well as to evaluate further the baculovirus/lepidopteran system for expressing Bt proteins. The crylVD genes were inserted into the Autographa californica multinucleocapsid nuclear polyhedrosis virus (AcMNPV) under control of the polyhedrin promoter by recombination in S. frugiperda cells between a transfer vector carrying the Bt genes and vDA26Z, a recombinant AcMNPV carrying the Escherichia coli fl-galactosidase gene under control of the DA26 promoter. Recombinant AcMNPVs carrying the genes were detected as blue occlusion body-negative plaques in monolayers of S. frugiperda cells grown in the presence of X-Gal. Infection of S. frugiperda cells and T. ni larvae with plaque-purified recombinant virus, expressing either the full-length or truncated CrylVD protein, resulted in the synthesis of proteins of the expected size, as confirmed by immunoblot analyses, and their crystallization into cuboidal inclusions in the cytoplasm. Infected cells and purified inclusions from the virus (AcCrylVD) expressing the full-length protein were highly toxic to mosquito larvae, but similar preparations from the virus (AcCrylVD-C) expressing the truncated protein with a 9-6K deletion at the N terminus were non-toxic. Proteolysis with trypsin of CrylVD proteins produced by Bt and the recombinant AcMNPVs yielded peptides corresponding in size, showing that synthesis of mosquitocidal Bt proteins in lepidopteran cells occurred. The lack of toxicity of the truncated CryIVD protein, which like the toxic fulllength protein yielded a 34K protein on proteolysis that has been implicated in toxicity, indicates that by itself this protein is non-toxic. These results demonstrate the utility of the baculovirus system for expression of mosquitocidal Bt proteins and for investigation of their mode of action.

A typical apoptosis of BTI-Tn-5B1-4 (Hi5) cells induced by Heliothis armigera single capsid nucleopolyhedrovirus (HaSNPV) infection was completely suppressed by coinfection with Trichoplusia ni multicapsid nucleopolyhedrovirus polyhedron-negative recombinant (TnMNPV-SVING) (OCCN) at a low multiplicity of infection (6

When Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) is grown in insect cell culture, defective viruses are generated. These viruses lack about 25 kbp of sequence information and are no longer infectious for insects. This makes the engineering of SeMNPV for improved insecticidal activity or as expression vectors difficult to achieve. Recombinants of Autographa californica MNPV have been generated in insects after lipofection with viral DNA and a transfer vector into the haemocoel. In the present study a novel procedure to isolate SeMNPV recombinants was adopted by alternate cloning between insect larvae and cultured cells. The S. exigua cell line Se301 was used to select the putative recombinants by following a green fluorescent protein marker inserted in the p10 locus of SeMNPV. Polyhedra from individual plaques were fed to larvae to select for biological activity. In this way an SeMNPV recombinant (SeXD1) was obtained with the speed of kill improved by about 25%. This recombinant lacked 10593 bp of sequence information, located between 13

The complete Spodoptera litura multicapsid nucleopolyhedrovirus (SpltMNPV) genome contained 139,342 bp with a G1C content of 42.7%, and 141 putative open reading frames (ORFs) or genes of 150 nucleotides or greater that showed minimal overlap. Ninety-six ORFs had homologues in Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV), 16 had homologues in other baculoviruses, and 29 were unique to SpltMNPV. The homologues of ubiquitin and gp37 are fused in SpltMNPV. The genome lacked a homologue of the major budded virus glycoprotein gene gp64, but it contained a homologue of ORF130 of Lymantria dispar multicapsid nucleopolyhedrovirus (LdMNPV). There were two homologues of AcMNPV ORF2 (bro gene), and a DnaJ protein gene (SpltORF39) in which the N-terminus showed homologies with the J domain of DnaJ family proteins. Seventeen homologous regions (hrs) were identified, each containing 2

Direct evidence of in vivo apoptosis of Spodoptera litura larvae was demonstrated by haemocoel inoculation with wild-type Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) budded virus (BV). In sharp contrast to natural infection, cadavers did not melt, liquefy and melanize. Typical morphological changes of apoptosis in insect haemocytes post-infection, including blebbing of the cell surface, chromatin margination and condensation, vacuolization of the cytoplasm and formation of apoptotic bodies, were observed by light and electron microscopy. Total DNAs extracted from virus-infected haemocytes showed DNA ladders. Cleavage of chromatin DNA by endogenous endonucleases were detected in the cells of most tissues cells, including epithelial cells and fat body cells, using terminal dUTP nick end labelling assays. Virogenic stroma and viral nucleocapsids could be seen in the nuclei of a few haemocytes. Yields of BV and OV (occluded virus) produced from the infected S. litura larvae were much lower than from the infected S. exigua larvae. These data suggest that host apoptotic responses to virus infection reduce AcMNPV spread at the level of the organism and that apoptosis could be host-range limiting factor for baculovirus infections.