Accumulation of few-polyhedra mutants upon serial passage of Anticarsia gemmatalis multiple nucleopolyhedrovirus in cell culture

Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) has been widely used to control the velvetbean caterpillar, Anticarsia gemmatalis, in Brazil. To date, AgMNPV has been produced by larval infection and, due to in vivo production limitations and the continuing high demand for the biopesticide, attempts should be made to develop in vitro production of this virus. In order to investigate the effects caused by serial passage of AgMNPV in cell culture, we carried out a total of ten passages and analyzed the morphological and the genomic changes of the virus. After six passages, the many-polyhedra (MP) phenotype started to switch to the few-polyhedra (FP) phenotype which rapidly accumulated in the virus population. Ultrastructural analysis showed typical signs of FP mutant formation such as decrease in the number of polyhedra per cell, polyhedra aberrant morphology and low numbers of virions occluded in the protein matrix. Also enhanced BV production was observed from the fifth passage indicating that FP mutants were becoming predominant in comparison to the wild type virus. Restriction endonuclease analysis of the viral DNA revealed that lower and higher passages had similar profiles indicating that there were no large insertions or deletions or rearrangements in their genomes and indicating the generation of FP mutants instead of defective interfering viruses.