A few-polyhedra mutant and wild-type nucleopolyhedrovirus remain as a stable polymorphism during serial coinfection in Trichoplusia ni

Abstract

Few-polyhedra (FP) mutants of nucleopolyhedroviruses (NPVs) are a well-known phenomenon during serial passage of virus in cell culture. Under these circumstances such mutants produce low yields of occlusion bodies (OBs) and poorly occlude virions, but they are selected for through advantageous rates of budded virus replication. Spontaneous insertion of transposable elements originating from host cell DNA into the viral fp25 gene has been shown to be a common cause of the phenotype. A model of NPV population genetics predicts that mutants with these characteristics might persist within stable polymorphisms in viral populations during serial passage of virus in vivo. However, this hypothesis was previously untested, and FP mutants have not been recovered from field isolates of NPVs. We isolated and characterized an FP mutant that arose during routine passage of Autographa californica multinucleocapsid NPV (AcMNPV) in cell culture and identified a transposable element within the fp25 gene. We tracked the fates of coinfecting wild-type and FP mutant AcMNPV strains through serial passage in fifth-instar Trichoplusia ni larvae. The levels of both strains remained stable during successive rounds of infection. We applied the data obtained to a model of NPV population genetics in order to derive the frequency distribution of the multiplicity of cell infection in infected insects and estimated that 4.3 baculovirus genomes per OB-producing cell would account for this equilibrium.

FIG. 1.

Electron micrographs of AcMNPV OBs. TN368 cells were infected with virus, and OBs were harvested 48 h postinfection. Many occluded virions are present in the wild-type (L1) OB. For the FP mutant (fp-1), there are few signs of occluded virions; this OB is typical of those observed. OBs harvested after infection of cells with the revertant virus (rev) appeared to be wild type. Bars = 0.5 μm. The arrows indicate the calyx (C) and a virion (V)).

FIG. 2.

Mortality bioassay for AcMNPV strains in T. ni. Larvae were infected 2 days after hatching by placing individual insects on equal areas of diet containing various concentrations; each portion was inoculated with a 50-μl aliquot of a purified OB suspension. There was not a significant difference between the lethal concentrations of L1 and rev (log-linear analysis; P = 1.00; df = 1). However, fp-1 displayed significantly reduced infectivity compared to the infectivity of L1 (P = 7.22E-06; df = 1).

FIG. 3.

Replication rates of AcMNPV strains. The titers of BVs harvested from infected fifth-instar T. ni larvae were measured for 3 days postinfection (pi) and compared. The error bars indicate standard errors (n = 3). (A) fp-1 reached a titer that was 1.6 times that of L1. The difference was significant (P = 0.01, as determined by a t test). This was considered the replication advantage of the FP mutant. (B) There was not a significant difference between the titers of the wild-type strain (L1) and the revertant strain (rev) (P = 0.55, as determined by a t test).

FIG. 4.

Percentages of L1 in serial rounds of co-occlusion. (A) Example (round 2) of a Southern blot used to track the fate of the L1 and fp-1 genotypes during serial rounds of infection. Each lane contained the total virus DNA recovered from OBs from a single insect. The upper band was derived from fp-1, whereas the lower band was derived from L1. Lines 1 to 10 were independent series of infection. (B) Means ± standard errors for the percentages of the L1 genotype for the 10 independent lines. Also shown are the best-fit model prediction with an fp-1 replicative advantage of 1.6 (W=1.6) and the outcome of that model for no replicative advantage (W=1).