Production of baculovirus defective interfering particles during serial passage is delayed by removing transposon target sites in fp25k.

Accumulation of baculovirus defective interfering particle (DIP) and few polyhedra (FP) mutants is a major limitation to continuous large-scale baculovirus production in insect-cell culture. Although overcoming these mutations would result in a cheaper platform for producing baculovirus biopesticides, little is known regarding the mechanism of FP and DIP formation. This issue was addressed by comparing DIP production of wild-type (WT) Autographa californica multiple nucleopolyhedrovirus (AcMNPV) with that of a recombinant AcMNPV (denoted Ac-FPm) containing a modified fp25k gene with altered transposon insertion sites that prevented transposon-mediated production of the FP phenotype. In addition to a reduction in the incidence of the FP phenotype, DIP formation was delayed on passaging of Ac-FPm compared with WT AcMNPV. Specifically, the yield of DIP DNA in Ac-FPm was significantly lower than in WT AcMNPV up to passage 16, thereby demonstrating that modifying the transposon insertion sites increases the genomic stability of AcMNPV. A critical component of this investigation was the optimization of a systematic method based on the use of pulsed-field gel electrophoresis (PFGE) to characterize extracellular virus DNA. Specifically, PFGE was used to detect defective genomes, determine defective genome sizes and quantify the amount of defective genome within a heterogeneous genome population of passaged virus.

Removal of transposon target sites from the Autographa californica multiple nucleopolyhedrovirus fp25k gene delays, but does not prevent, accumulation of the few polyhedra phenotype.

Low-cost, large-scale production of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) using continuous insect cell culture is seriously hindered by the accumulation of AcMNPV mutants. Specifically, few-polyhedra (FP) mutants, with a reduced yield of occluded virus (polyhedra) and decreased infectivity, usually accumulate upon passaging in cell culture. FP mutations result from transposon insertions in the baculovirus fp25k gene, leading to significantly reduced levels of FP25K protein synthesis. This study evaluated the effects of removing the transposon insertion sites from the wild-type baculovirus fp25k gene; the mutated virus was denoted Ac-FPm. Specifically, this study involved a detailed comparison of wild-type (WT) AcMNPV and Ac-FPm with regard to the proportion of cells having polyhedra, number of polyhedra per cell, the fraction of empty polyhedra, number of occlusion-derived viruses per polyhedron, number of nucleocapsids in the nuclei, FP25K protein synthesis and genetic analysis of the fp25k gene. Removal of TTAA transposon insertion sites from the fp25k gene stabilized FP25K protein synthesis and delayed the appearance of the FP phenotype from passage 5 to passage 10. Electron micrographs revealed that more virus particles were found inside the nuclei of cells infected with Ac-FPm than in the nuclei of cells infected with WT AcMNPV (at passage 10). Abnormalities, however, were observed in envelopment of nucleocapsids and virus particle occlusion within Ac-FPm polyhedra. Thus, the FP phenotype appeared in spite of continued FP25K protein synthesis, suggesting that mechanisms other than fp25k gene disruption can lead to the FP phenotype.