Effect of Serial Passage on Genetic Homogeneity of a Plaque Variant of Lymantria dispar Nuclear Polyhedrosis Virus (Hamden LDP-67)

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Abstract
The wild-type Hamden gypsy moth baculovirus preparation (LDP-67) was subjected to plaque purification in IPLB-LD-652Y cells, resulting in three distinguishable classes of variants, based on the number of polyhedra produced per cell in vitro. Representatives were designated Ld-S (several polyhedra per cell), Ld-F (few polyhedra per cell) and Ld-V (variable numbers of polyhedra per cell). These representative phenotypic variants were compared to the wild-type by restriction enzyme analysis and by bioassay in Lymantria dispar larvae. With all enzymes tested the variants demonstrated restriction site polymorphism. The genome size of the three variants was estimated at 159 to 163 kb. In bioassay trials two of the variants, Ld-V and Ld-S, exhibited LD50 values approximately 3.9 times lower than the LD50 of wild-type virus. The Ld-F variant was not infective in feeding trials. The stability of Ld-S was tested by high multiplicity passage (HMP) in IPLB-LD-652Y cells. By the twentieth undiluted passage (P-20), the in vitro cytopathic effect of this variant was significantly altered. DNA isolated from the P-20 stock exhibited several differences in the restriction endonuclease profile relative to the early passage virus. Genomic alterations were more clearly visualized after plaque purification of the Ld-S (P-20) stock. Four of the five plaques chosen contained what appeared to be two distinct viral populations. One of these populations, which could be isolated by repeated plaque purification, had a restriction enzyme profile identical to the pre-HMP Ld-S variant. The second population, characterized by high Mr BglII bands not present in the Ld-S profile, could never be plaque-purified of submolar amounts of the first population. The possibility that this population represents a defective interfering virus is discussed. The source of additional DNA in the P-20 variants was investigated by probing blots of these variants with labelled total cellular DNA. No specific hybridization to any of the fragments was obtained, suggesting that the P-20 variants arose by rearrangements involving solely viral DNA.