Effect of genetic modification of the fp25k gene on few polyhedra and defective interfering particle mutations in baculovirus

Use of chemical pesticides has several undesirable consequences, including the development of insecticide resistance, reduction of soil fertility and adverse health effects on non-target organisms. To minimize the use of chemical pesticides, baculoviruses can be used as potential bio-insecticides since they do not kill beneficial insects and are environmentally benign. However, low cost, large-scale production of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) using continuous insect cell culture is seriously hindered by undesirable mutations in the baculovirus genome. Few polyhedra (FP) and defective interfering particle (DIP) mutants are commonly responsible for the reduction in occluded virus yield and decreased infectivity, which is unacceptable for bio-insecticides. The FP25K protein is generally missing from the FP mutants, which commonly results from the insertion of transposons into the baculovirus fp25k gene. The current research tested the hypothesis that FP mutant accumulation can be reduced by removing the transposon target sites from the fp25k gene of the wild type AcMNPV genome. It was demonstrated that removal of the transposon target sites from the wild type baculovirus fp25k gene (named as Acfp25km(TTAA)) stabilized the FP25K protein expression and delayed the incidence of the FP phenotype from passage 5 to passage 10. Electron micrographs of infected cells revealed that more virus particles were found inside the nucleus in the Acfp25km(TTAA) compared to WT AcMNPV (at passage 10), but abnormalities were observed in envelopment of neucleocapsids and occlusion of virus particles in polyhedra. A significant loss in FP25K protein expression was observed in cells infected with WT AcMNPV by passage 12, whereas the Acfp25km(TTAA) was stable in FP25K protein expression through 30 passages. Hence, even if the correlation exists between FP phenotype and insertion mutation in fp25k gene, mutations in other genes also contribute to FP phenotype accumulation. Additionally, a simple and novel assay based on restriction enzyme digestion of viral DNA and pulse field gel electrophoresis was developed to detect and quantify the DIP mutants. Genomic analysis of passaged viral DNA exhibited a delay in DIP formation for the stabilized virus and thereby suggesting a relationship between the FP and DIP mutations.