title: Production of baculovirus defective interfering particles during serial passage is delayed by removing transposon target sites in fp25k.
abstract: 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.
authors: Department of Chemical and Biochemical Engineering, University of Iowa, Iowa City, IA, USA.
authors: Lopamudra
authors: L
authors: Giri
authors: Department of Microbiology, University of Iowa, Iowa City, IA, USA.
authors: Michael G
authors: MG
authors: Feiss
authors: Department of Entomology, Iowa State University, Ames, IA, USA.
authors: Bryony C
authors: BC
authors: Bonning
authors: Department of Chemical and Biochemical Engineering, University of Iowa, Iowa City, IA, USA.
authors: David W
authors: DW
authors: Murhammer
mesh_terms: D000818:Animals; D004251:DNA Transposable Elements; D003673:Defective Viruses / Q000235:genetics* / Q000302:isolation & purification; D007915:Lepidoptera / Q000821:virology; D017924:Nucleopolyhedroviruses / Q000235:genetics* / Q000254:growth & development* / Q000648:ultrastructure; D017384:Sequence Deletion; D012692:Serial Passage; D014774:Virulence
chemical_list: D004251:DNA Transposable Elements