Stability of a Spodoptera frugiperda Nucleopolyhedrovirus Deletion Recombinant during Serial Passage in Insects

Abstract

The stabilities of the Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV) complete genome bacmid (Sfbac) and a deletion recombinant (Sf29null) in which the Sf29 gene was replaced by a kanamycin resistance cassette were determined during sequential rounds of per os infection in insect larvae. The Sf29 gene is a viral factor that determines the number of virions in occlusion bodies (OBs). The Sf29null bacmid virus was able to recover the Sf29 gene during passage. After the third passage (P3) of Sf29null bacmid OBs, the population was observed to reach an equilibrium involving a mixture of those with a kanamycin resistance cassette and those with the Sf29 gene. The biological activity of Sf29null bacmid OBs at P3 was similar to that of Sfbac OBs. The recovered gene in the Sf29null virus was 98 to 100% homologous to the Sf29 genes of different SfMNPV genotypes. Reverse transcription-PCR analysis of uninoculated S. frugiperda larvae confirmed the expression of the SfMNPV ie-0 and Sf29 genes, indicating that the insect colony harbors a covert SfMNPV infection. Additionally, the nonessential bacterial artificial chromosome vector was spontaneously deleted from both viral genomes upon passage in insects.Nucleopolyhedroviruses (NPVs) (family Baculoviridae) infect the larvae of many important lepidopteran pests, and several have been developed as the basis for commercial biopesticides (28). Natural populations of the fall armyworm Spodoptera frugiperda (Lepidoptera) suffer S. frugiperda multiple nucleopolyhedrovirus (SfMNPV) disease, and some SfMNPV isolates have the potential to control this pest (12,16,33,44). The complete genome sequences of two isolates have been published (16,44). A core set of 31 genes is common to all baculoviruses, other genes are present in all group II NPVs, and others are unique to this virus. Homologues of the SfMNPV ORF29 (Sf29) are found only in group II NPVs. The role of the Sf29 gene in the structure and replication of this virus has been studied by using bacmid technology to produce a virus mutant (Sf29null) that lacks the gene (36). Sf29 encodes a viral factor that regulates the number of virions within the viral occlusion bodies (OBs). The DNA content of Sf29null bacmid OBs was reduced compared to that of wild-type OBs, and this difference was correlated with a reduced number of occlusion body-derived virions (ODVs) occluded in each OB of the Sf29null virus. ODVs are released from OBs in the insect gut and are responsible for establishing primary infection in midgut cells. Consequently, deletion of Sf29 results in a virus with lower infectivity so that greater numbers of OBs are required to initiate lethal infection in insects that consumed Sf29null compared to the number required for lethal infection in wild-type OBs (36).Genes have been inserted or removed from NPV genomes to produce recombinant viruses with improved insecticidal properties (26, 38), and these viruses have been subjected to field testing (7,39,40). In the present study, we aimed to determine the stability of...

Using Next Generation Sequencing to Identify and Quantify the Genetic Composition of Resistance-Breaking Commercial Isolates of Cydia pomonella Granulovirus

“…In experiments with Spodoptera frugiperda nucleopolyhedrovirus (SfMNPV), it has been demonstrated that laboratory virus genotype mixtures achieved an almost identical composition of genotypes of wild type isolates [ 53 ]. Other than effects which presumably derive from virus-virus interactions, the limiting factor of virus selection was the host, Spodoptera frugiperda , which was more susceptible to mixtures of genotypes than to single genotypes of SfMNPV [ 54 , 55 , 56 ]. Given these similar observations, the prevalence of partially resistant insect populations represents a bottleneck in the dynamics of virus isolates which attenuate to a certain virus composition.…”

Sequence comparison between three geographically distinct Spodoptera frugiperda multiple nucleopolyhedrovirus isolates: Detecting positively selected genes

“…SfMNPV-B bacmid DNA was sequenced to ensure that the nucleotide sequence originated from a single genotype and was not the result of contamination from a persistent infection present in the insect colony (Simón et al, 2010). The complete SfMMPV-B genome was cloned in a pBACe3.6 vector that includes a chloramphenicol resistance gene (Frengen et al, 1999).…”

Baculovirus Expression and Functional Analysis of Vpa2 Proteins from Bacillus thuringiensis

“…The rapid generation of these particles involves several recombination steps and can prevent the development of stable recombinant baculoviruses [41]. Loss of non-essential genomic regions has been observed to be a frequent phenomenon during virus replication in cell culture and in larvae, presumably because there is no adaptive advantage for the virus to retain such genes [42][43][44]. In the present study, variation in the REN profiles of the Bac-polh-Vpa2Ac1 and Bac-polh-Vpa2like2 recombinant viruses in comparison with the original inocula, suggested that deletions, mutations or recombination events had occurred, resulting in heterogeneous virus populations comprising of mixtures of genotypes.…”