Persistence of an occlusion-negative recombinant nucleopolyhedrovirus in Trichoplusia ni indicates high multiplicity of cellular infection

Abstract

We use data from the serial passage of co-occluded recombinant Autographa californica nuclear polyhedrosis virus (AcMNPV) to estimate the viral multiplicity of infection of cells within infected insects. Co-occlusion, the incorporation of wild-type and mutant virus genomes in the same occlusion body, has been proposed as a strategy to deliver genetically modified viruses as insecticides in a way that contains their spread in the environment. It may also serve as a means whereby naturally occurring mutant forms of NPVs can be maintained in a stable polymorphism. Here, a recombinant strain of AcMNPV was constructed with a deletion of its polyhedrin gene, rendering it incapable of producing occlusion bodies (i.e., occlusion negative). This was co-occluded with wild-type AcMNPV and used to infect fifth-instar Trichoplusia ni larvae. The fate of both genotypes was monitored over several rounds of insect infection. Levels of the occlusion-negative virus genome declined slowly over successive rounds of infection. We applied these data to a model of NPV population genetics to derive an estimate of 4.3 +/- 0.3 viral genomes per occlusion body-producing cell.

FIG. 1

Replication rates of AcMNPV strains. The titer of BV was measured during a single round of infection for L1 and occ^–1 over 4 days postinfection and compared. Both in cell culture (A) and in insects (B), neither virus was seen to have an advantageous rate of replication. The SE is indicated by bars (n = 3).

FIG. 2

Percentage of L1 through serial rounds of co-occlusion. (A) Example (round 3) of a Southern blot used to track the fate of the L1 and occ^–1 genotypes over serial rounds of infection. Each lane represents the total virus DNA recovered from OBs from a single insect. The upper band derives from L1, whereas the lower band derives from occ^–1. Lines 1 to 10 were independent series of infection. (B) Percentage of the L1 genotype over the course of six rounds of serial infection for 10 independent lines of insects and the best-fitting model prediction if a Poisson distribution of MOI is assumed. In this series, all insects were infected with 5,000 OBs each.

FIG. 3

Percentage of L1 after reduction of infectious dose. The percentage of the L1 genotype was measured over three serial rounds of infection at a dose of 5,000 OBs per insect (the same data as shown in rounds 1 to 3 of Fig. 2) for 10 independent lines of insects. Subsequently, all insects were infected with 50 OBs each (rounds 4 to 6), and the 10 independent lines of insects were continued. Also shown is the mean (SE [error bars], n = 10) for the 10 lines of infection.