Genetic Structure of a Spodoptera frugiperda Nucleopolyhedrovirus Population: High Prevalence of Deletion Genotypes

Abstract

A Nicaraguan field isolate (SfNIC) of Spodoptera frugiperda nucleopolyhedrovirus was purified by plaque assay on Sf9 cells. Nine distinct genotypes, A to I, were identified by their restriction endonuclease profiles. Variant SfNIC-B was selected as the standard because its restriction profile corresponded to that of the wild-type isolate. Physical maps were generated for each of the variants. The differences between variants and the SfNIC-B standard were confined to the region between map units 9 and 32.5. This region included PstI-G, PstI-F, PstI-L, PstI-K and EcoRI-L fragments. Eight genotypes presented a deletion in their genome compared with SfNIC-B. Occlusion body-derived virions of SfNIC-C, -D and -G accounted for 41% of plaque-purified clones. These variants were not infectious per os but retained infectivity by injection into S. frugiperda larvae. Median 50% lethal concentration values for the other cloned genotypes were significantly higher than that of the wild type. The variants also differed in their speed of kill. Noninfectious variants SfNIC-C and -D lacked the pif and pif-2 genes. Infectivity was restored to these variants by plasmid rescue with a plasmid comprising both pif and pif-2. Transcription of an SfNIC-G gene was detected by reverse transcription-PCR in insects, but no fatal disease developed. Transcription was not detected in SfNIC-C or -D-inoculated larvae. We conclude that the SfNIC population presents high levels of genetic diversity, localized to a 17-kb region containing pif and pif-2, and that interactions among complete and deleted genotypic variants will likely influence the capacity of this virus to control insect pests.The family Baculoviridae comprises a large group of viruses that are pathogenic to invertebrates and are characterized by enveloped rod-shaped virions with large (85 to 166 kb) circular double-stranded DNA genomes (2). These viruses have been reported from numerous insect species, particularly Lepidoptera (28). Due to their restricted host range (16) and high pathogenicity to a number of insect pest species, they have received considerable attention as microbial insecticides (31).Baculoviruses produce two kinds of particles, one occluded and the other nonoccluded. Usually, both forms of virus are produced in a sequential manner in the same cell. When a susceptible insect consumes foliage contaminated with occlusion bodies (OBs), occlusion derived virions are released from the OB by alkali disruption of the OB protein. The occlusionderived virions infect midgut cells, resulting in a primary infection. Subsequently, nonoccluded budded virus is produced, which buds out of the infected cell to allow cell-to-cell spread of the infection throughout the host. Budded virus particles consist of a nucleocapsid containing a single genome, enveloped by a membrane in which some virus-encoded proteins are anchored. Recent work indicates that each cell within the insect is infected by an average of four to five budded viruses (3). Later, the OB is produced, which stays inside t...

Mixtures of Complete andpif1- andpif2-Deficient Genotypes Are Required for Increased Potency of an Insect Nucleopolyhedrovirus

“…OBs of a Nicaraguan isolate (SfNIC) of SfMNPV were amplified in fourth-instar S. frugiperda (8). The complete genotype, SfNIC-B, was obtained from plaque-purified material described by Simón et al (40). SfNIC-B OBs were solubilized with thioglycolate buffer (0.25 M thioglycolic acid, 0.25 M sodium carbonate [pH 10.5]).…”

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

“…Genetic diversity is common in NPV populations (Graham et al 2004;Cory et al 2005;Erlandson et al 2006;Ogembo et al 2007), and provides clear advantages for virus transmission under varying ecological conditions (Hodgson et al 2001(Hodgson et al , 2004Cooper et al 2003;Simó n et al 2004Simó n et al , 2006Hitchman et al 2007). It seems reasonable to accept that a system of multiple occlusion is adaptive in facilitating continuous transmission of high variability of the virus population when the pathogen density is low, leading to an improved likelihood of virus survival.…”

“…An artificial 'wild-type' population was produced by mixing purified OBs of [75B þ 25C] with the OBs of the other six genotypes in proportions similar to those found in the natural SfNIC isolate (Simó n et al 2004). The overall composition of the mixture, in terms of percentages of OBs of each genotype, was 80 per cent [75B þ 25C], 0.5 per cent SfNIC-A, 3 per cent SfNIC-E, 9 per cent SfNIC-F, 0.6 per cent SfNIC-G, 0.2 per cent SfNIC-H and 5 per cent SfNIC-I.…”

Quality Control of Baculoviral Bioinsecticide Production

“…Most of baculoviruses applied as bioinsecticides derived from homogenous populations cloned or partially cloned by different procedures (Wang et al, 2003;Simón et al, 2004). This is a remarkable aspect since it allows establishing genotypic characteristic patterns that can be detected by different approaches.…”

“…This is a remarkable aspect since it allows establishing genotypic characteristic patterns that can be detected by different approaches. Among them, the visualization of RFLPs (Restriction Fragment Lenght Polymorphism) in agarose gel electrophoresis stained by different dyes and UV exposition is usually a good indicator of genome integrity, revealing the gain or loss of DNA (Simón et al, 2004;Eberle et al, 2009;Rowley et al, 2010). In fact, this is a classic approach to characterize genotypic variants of a viral species.…”