Mixed genotype transmission bodies and virions contribute to the maintenance of diversity in an insect virus

Abstract

An insect nucleopolyhedrovirus naturally survives as a mixture of at least nine genotypes. Infection by multiple genotypes results in the production of virus occlusion bodies (OBs) with greater pathogenicity than those of any genotype alone. We tested the hypothesis that each OB contains a genotypically diverse population of virions. Few insects died following inoculation with an experimental two-genotype mixture at a dose of one OB per insect, but a high proportion of multiple infections were observed (50%), which differed significantly from the frequencies predicted by a non-associated transmission model in which genotypes are segregated into distinct OBs. By contrast, insects that consumed multiple OBs experienced higher mortality and infection frequencies did not differ significantly from those of the non-associated model. Inoculation with genotypically complex wild-type OBs indicated that genotypes tend to be transmitted in association, rather than as independent entities, irrespective of dose. To examine the hypothesis that virions may themselves be genotypically heterogeneous, cell culture plaques derived from individual virions were analysed to reveal that one-third of virions was of mixed genotype, irrespective of the genotypic composition of the OBs. We conclude that co-occlusion of genotypically distinct virions in each OB is an adaptive mechanism that favours the maintenance of virus diversity during insect-to-insect transmission.

Figure 1.

Semiquantitative PCR of the inocula prepared for the bioassays ((wt) is SfNIC-wt, B + C is 50B + 50C and SfNIC-lab). Amplification of SfNIC-wt DNA extracted from OBs confirmed the proportion of genotypes with per os transmission factor genes (75%) and those lacking these genes (25%) that survive in the natural population by complementation with complete genotypes in co-infected cells. Amplification of 50B + 50C DNA from OBs confirmed the presence of each genotype in the expected proportion. The SfNIC-lab inoculum was amplified with all pairs of primers to calculate the relative proportions of genotypes present. Letters A–I represent their corresponding genotypes (SfNIC-A–SfNIC-I). Relative density values are shown above or below the corresponding amplicon. Reactions were performed in triplicate and pooled prior to densitometric analysis. Molecular markers were M1 1 kb DNA ladder (Stratgene, La Jolla, CA, USA) and M2 was 100 bp DNA ladder (Invitrogen Corp., Carlsbad, CA, USA).