Rapid Formation of Few Polyhedra Mutants of Lymantria dispar Multinucleocapsid Nuclear Polyhedrosis Virus during Serial Passage in Cell Culture

Abstract Four genotypic variants of Lymantria dispar multi-nucleocapsid nuclear polyhedrosis virus (LdMNPV) were used to investigate the generation of few polyhedra (FP) mutants during viral propagation in the L. dispar 652Y cell line. Titers of budded virus, the percentage of infected cells producing polyhedra, the amount of polyhedra produced per cell, the proportion of virus exhibiting an FP phenotype, virion occlusion characteristics, and changes in genomic DNA digestion profiles were investigated through five serial passages. LdMNPV FP mutants arose and became predominate very quickly during serial passage. Greater than 92% of the virus present after the second passage exhibited an FP phenotype in three of the four LdMNPV isolates studied. The amount of budded virus produced by the isolates was from approximately 150- to 250-fold greater at the third passage compared to the amount produced at the first passage. The percentage of infected cells producing polyhedra decreased by an average of 35.6% during serial passage. The number of polyhedra produced per cell decreased between 2.3- and 5.6 fold from the first to the third passage. Restriction endonuclease digestion profiles of the genomic DNA of the FP mutants were analyzed to determine whether genotypic changes occurred that could be correlated with the appearance of the FP phenotype. The genomic DNA digestion profiles of FP mutants in three of the four isolates matched the profiles of the original wild-type isolates. A DNA insertion of 0.9 kb was found in the fourth isolate; however, it was shown not to be the basis for the FP phenotype. These results indicate that FP mutants arise rapidly during serial propagation of LdMNPV in cell culture. In contrast to other baculoviruses, the LdMNPV FP mutants studied in this investigation appear 60 lack DNA insertions of readily detectable length that could be correlated with the appearance of the FP phenotype.