Investigation of genetic heterogeneity in wild isolates of Spodoptera frugiperda nuclear polyhedrosis virus by restriction endonuclease analysis of plaque-purified variants

doi:10.1016/0042-6822(81)90624-3

Virology

Volume 112, Issue 1, 15 July 1981, Pages 190-197

https://doi.org/10.1016/0042-6822(81)90624-3 Get rights and content

Abstract

Spodoptera frugiperda nuclear polyhedrosis virus (SfMNPV was obtained from four different sources and each isolate was cultured in IPLB-SF21 cells. Viral DNA from each isolate was analyzed with EcoR1, Xho1, and BamH1 restriction endonucleases. All four isolates could be distinguished on the basis of minor differences in EcoR1 patterns. Submolar DNA restriction fragments were seen in all isolates indicating that each isolate consisted of a mixture of different genotypes. To investigate this, two of the isolates were plaque purified. Of the total of 13 clones isolated, the DNA from 5 of these had distinctly different EcoR1 restriction endonuclease patterns. The five cloned variants were further analyzed with Xho1, BamH1, and Sac1. Three of the five clones had fragments which comigrated with submolar fragments seen in the wild isolates. Thus, the submolar fragments observed in digests of the DNA from two wild isolates of SfMNPV were due to a mixture of genetic variants.

References (23)

J. Hamm
Comparative histopathology of a granulosis and a nuclear polyhedrosis of Spodoptera frugiperda
J. Invertebr. Pathol.
(1968)
K.A. Harrap et al.
The properties of three baculoviruses from closely related hosts
Virology
(1977)
M. Jurkovíčová et al.
Characterization of the nuclear polyhedrosis virus DNA of Adoxophyes orana and of Barathra brassicae
Virology
(1979)
D.C. Kelly
The DNA contained by nuclear polyhedrosis viruses isolated from four Spodoptera sp. (Lepidoptera, Nocuidae): genome size and homology assessed by DNA reassociation kinetics
Virology
(1977)
S.T. Tjia et al.
Infection of Spodoptera frugiperda cells with Autographa californica nuclear polyhedrosis virus
Virology
(1979)
S. Burgess
Molecular weights of Lepidopteran baculovirus DNAs: derivation by electron microscopy
J. Gen. Virol.
(1977)
G. Crozier et al.
Sélection de types viraux dans les infections doubles à Baculovirus chez les larves de lépidoptère
C. R. Acad. Sci. Paris
(1980)
W.A.L. David
The status of viruses pathogenic for insects and mites
Annu. Rev. Entomol.
(1975)
R.B. Helling et al.
Analysis of endonuclease R-Eco-R1 fragments of DNA from lambdoid bacteriophages and other viruses by agarose-gel electrophoresis
J. Virol.
(1974)
D.L. Knudson et al.
Replication of a nuclear polyhedrosis virus in a continuous cell culture of Spodoptera frugiperda: Purification, assay of infectivity, and growth characteristics of the virus
J. Virol.
(1974)

H.H. Lee et al.

Isolation of genotypic variants of Autographa californica nuclear polyhedrosis virus

J. Virol.

(1978)

Cited by (54)

In vivo production of recombinant proteins using occluded recombinant AcMNPV-derived baculovirus vectors
2017, Journal of Virological Methods
Citation Excerpt :
Wild-type baculovirus isolates contain several genomes as the result of the infective strategy for a successful use of host resources (Fisher and Viney, 1998; Hodgson et al., 2004; Meijer and Leuchtmann, 1999; Read and Taylor, 2001; Taylor et al., 1997). This favors the maintenance of diversity during insect–insect transmission (Clavijo et al., 2010; Cory et al., 2005; Hodgson et al., 2001; Knell and Summers, 1981; Lee and Miller, 1978). Therefore, to generate a baculovirus donor vector Polh+, carrying a unique and known polyhedrin sequence into its genome, is highly relevant for industrial and commercial purposes.
Trichoplusia ni insect larvae infected with vectors derived from the Autographa californica multiple nucleopolyhedrovirus (AcMNPV), are an excellent alternative to insect cells cultured in conventional bioreactors to produce recombinant proteins because productivity and cost-efficiency reasons. However, there is still a lot of work to do to reduce the manual procedures commonly required in this production platform that limit its scalability. To increase the scalability of this platform technology, a current bottleneck to be circumvented in the future is the need of injection for the inoculation of larvae with polyhedrin negative baculovirus vectors (Polh-) because of the lack of oral infectivity of these viruses, which are commonly used for production in insect cell cultures. In this work we have developed a straightforward alternative to obtain orally infective vectors derived from AcMNPV and expressing recombinant proteins that can be administered to the insect larvae (Trichoplusia ni) by feeding, formulated in the insect diet. The approach developed was based on the use of a recombinant polyhedrin protein expressed by a recombinant vector (Polh+), able to co-occlude any recombinant Polh- baculovirus vector expressing a recombinant protein. A second alternative was developed by the generation of a dual vector co-expressing the recombinant polyhedrin protein and the foreign gene of interest to obtain the occluded viruses. Additionally, by the incorporation of a reporter gene into the helper Polh+ vector, it was possible the follow-up visualization of the co-occluded viruses infection in insect larvae and will help to homogenize infection conditions. By using these methodologies, the production of recombinant proteins in per os infected larvae, without manual infection procedures, was very similar in yield to that obtained by manual injection of recombinant Polh- AcMNPV-based vectors expressing the same proteins. However, further analyses will be required for a detailed comparison of production yields reached by injection vs oral infections for different recombinant proteins. In conclusion, these results open the possibility of future industrial scaling-up production of recombinant proteins in insect larvae by reducing manual operations.
Insect pathology
2012, Insect Pathology
Development of a quantitative real-time PCR for determination of genotype frequencies for studies in baculovirus population biology
2008, Journal of Virological Methods
Two bacmid-derived Autographa californica Multiple-capsid Nucleopolyhedrovirus genotypes – that differ only in a short tag sequence for differential PCR recognition – were generated. By electron microscopy, these genotypes were found to have identical polyhedra morphology. Mixtures of quantified polyhedra were made and used to validate a SYBR Green I-based quantitative real-time PCR (qPCR) to determine genotype frequencies in mixed genotype populations. The PCR could accurately quantify genotype ratios over a range of 8 orders of magnitude. Only a small correction of the genotype ratio was necessary to obtain a valid result. Low levels of aspecific background (a fluorescent signal when the template corresponding with the primer set used is not present) were measured in these validation experiments and in a typical laboratory setup. A small fitness difference between the genotypes generated was observed in a median lethal dose bioassay. The bacmid-derived virus genotypes generated and the qPCR assays are valuable tools for studying the population biology of baculoviruses.
Genetic and phenotypic variability in Spodoptera exigua nucleopolyhedrovirus isolates from greenhouse soils in southern Spain
2006, Biological Control
Genotypic and phenotypic variation of SeMNPV was examined in seven isolates of SeMNPV originating from occlusion body (OB) populations in the soil of greenhouses in Spain. Semi-quantitative PCR indicated that some of the isolates were composed of a single dominant genotype, whereas other isolates were composed of two or three genotypes in equal proportions. The coexistence of genotypes could be explained by trade-offs among the three phenotypic traits analyzed, namely pathogenicity (LD₅₀), speed of kill (mean time to death), and OB yields, so that increases in one trait were accompanied by decreases in another. Mixed genotype isolates tended to behave differently to single genotype isolates. Two of the genotypic mixtures were significantly more pathogenic (lower LD₅₀ values) for Spodoptera exigua (Hübner) larvae than the single genotypes that they comprised. OB yield/insect was greater for single genotype compared to mixed genotype isolates, despite genotype-specific differences in mean times to death. Total OB production/insect was positively correlated with time to death. Two out of three of the mixed genotype isolates had lower OB yield/mg insect weight at death compared to single genotype isolates. Each genotypic combination appeared to interact to produce a unique phenotype. This suggests the existence of trade-offs between traits leading to the coexistence of distinct genotypes and genotypic mixtures with similar transmissibility.
Baculoviruses - Re-emerging biopesticides
2006, Biotechnology Advances
Biological control of agricultural pests has gained importance in recent years due to increased pressure to reduce the use of agrochemicals and their residues in the environment and food. Viruses of a few families are known to infect insects but only those belonging to the highly specialized family Baculoviridae have been used as biopesticides. They are safe to people and wildlife, their specificity is very narrow. Their application as bioinsecticides was limited until recently because of their slow killing action and technical difficulties for in vitro commercial production. Two approaches for the wider application of baculoviruses as biopesticides will be implemented in future. In countries where use of genetically modified organisms is restricted, the improvements will be mainly at the level of diagnostics, in vitro production and changes in biopesticide formulations. In the second approach, the killing activity of baculoviruses may be augmented by genetic modifications of the baculovirus genome with genes of another natural pathogen. It is expected that the baculoviruses improved by genetic modifications will be gradually introduced in countries which have fewer concerns towards genetically modified organisms.
Genotypic and phenotypic diversity of a baculovirus population within an individual insect host
2005, Journal of Invertebrate Pathology
It is becoming increasingly apparent that many pathogen populations, including those of insects, show high levels of genotypic variation. Baculoviruses are known to be highly variable, with isolates collected from the same species in different geographical locations frequently showing genetic variation and differences in their biology. More recent studies at smaller scales have also shown that virus DNA profiles from individual larvae can show polymorphisms within and between populations of the same species. Here, we investigate the genotypic and phenotypic variation of an insect baculovirus infection within a single insect host. Twenty four genotypically distinct nucleopolyhedrovirus (NPV) variants were isolated from an individual pine beauty moth, Panolis flammea, caterpillar by in vivo cloning techniques. No variant appeared to be dominant in the population. The PaflNPV variants have been mapped using three restriction endonucleases and shown to contain three hypervariable regions containing insertions of 70–750 bp. Comparison of seven of these variants in an alternative host, Mamestra brassicae, demonstrated that the variants differed significantly in both pathogenicity and speed of kill. The generation and maintenance of pathogen heterogeneity are discussed.

View all citing articles on Scopus

View full text

Article preview

Virology

Abstract

References (23)

Comparative histopathology of a granulosis and a nuclear polyhedrosis of Spodoptera frugiperda

J. Invertebr. Pathol.

The properties of three baculoviruses from closely related hosts

Virology

Characterization of the nuclear polyhedrosis virus DNA of Adoxophyes orana and of Barathra brassicae

Virology

The DNA contained by nuclear polyhedrosis viruses isolated from four Spodoptera sp. (Lepidoptera, Nocuidae): genome size and homology assessed by DNA reassociation kinetics

Virology

Infection of Spodoptera frugiperda cells with Autographa californica nuclear polyhedrosis virus

Virology

Molecular weights of Lepidopteran baculovirus DNAs: derivation by electron microscopy

J. Gen. Virol.

Sélection de types viraux dans les infections doubles à Baculovirus chez les larves de lépidoptère

C. R. Acad. Sci. Paris

The status of viruses pathogenic for insects and mites

Annu. Rev. Entomol.

Analysis of endonuclease R-Eco-R1 fragments of DNA from lambdoid bacteriophages and other viruses by agarose-gel electrophoresis

J. Virol.

Replication of a nuclear polyhedrosis virus in a continuous cell culture of Spodoptera frugiperda: Purification, assay of infectivity, and growth characteristics of the virus

J. Virol.

Isolation of genotypic variants of Autographa californica nuclear polyhedrosis virus

J. Virol.

Cited by (54)

In vivo production of recombinant proteins using occluded recombinant AcMNPV-derived baculovirus vectors

Insect pathology

Development of a quantitative real-time PCR for determination of genotype frequencies for studies in baculovirus population biology

Genetic and phenotypic variability in Spodoptera exigua nucleopolyhedrovirus isolates from greenhouse soils in southern Spain

Baculoviruses - Re-emerging biopesticides

Genotypic and phenotypic diversity of a baculovirus population within an individual insect host