Isolation of genotypic variants of Autographa californica nuclear polyhedrosis virus

Abstract

A nuclear polyhedrosis virus (MNPV) isolated from a lepidopteran (Noctuidae) insect, Autographa californica, was cloned by successive plaque purification using virions containing only one nucleocapsid per envelope as inoculum. The ability to clone the virus by this method was demonstrated by the isolation of nondefective, genotypic variants of the virus with similar but not identical restriction endonuclease fragment patterns. Five distinct variants were identified by genotypic analysis with HindIII, EcoRI, Sall, and BamHI restriction endonucleases. The characteristic genotype of each variant was maintained upon passage in insect larvae. The isolation of these virus variants demonstrates (i) the heterogeneity of the uncloned virus preparation and (ii) the ability to clone MNPVs by plaque purification of media-derived nonoccluded virions. The A. californica MNPV is being considered for commercial use as a pesticide in the United States, and the cloning of the virus, in view of the heterogeneity detected, may be advisable. The cloning and genotypic analyses are also significant with regard to understanding the genetic nature of multiply embedded NPVs (those NPVs containing more than one nucleocapsid per envelope in the occluded form of the virus) and indicate that further genetic analysis of these viruses is possible. clone this virus is essential for ensuring a uniform virus for pesticide purposes and for the 754 on September 29, 2020 by guest http://jvi.asm.org/ Downloaded from VARIANTS OF A. CALIFORNICA NPV 755 genetic characterization of the virus. MATERIALS AND METHODS Virus source. The MNPV of A. californica was originally isolated by Vail et al. (24). In the third passage from the original isolation, M. R. Bell (U.S. Department of Agriculture, Agricultural Research Station, Phoenix, Ariz.) provided the virus in the form of infected T. ni (an alternate host) larvae. All passages were through T. ni larvae, and the virus had not been plaque purified. Hemolymph from the infected larvae was extracted, diluted in TC-100 basal media (Microbiological Associates), and stored by the method of Vaughn (25). The larval carcasses were frozen at-200C. Cell line. The Spodoptera frugiperda continuous cell line (IPLB-SF-21) was obtained from D. L. Knudson (Yale University, New Haven, Conn.). The cells were propagated at 27°C in TC-100 basal media supplemented with 0.26% tryptose broth (Difco), 8% fetal calf serum (Gibco), 100 U of penicillin per ml, and 100 ,ug of streptomycin per ml. Virus inoculum. A stock of A. californica MNPV for tissue culture use (passage 1) was prepared by infecting a monolayer of S. frugiperda cells with hemolymph from A. californica MNPV-infected T. ni larvae provided by M. R. Bell. The TC-100 medium was removed from S. frugiperda cell monolayers in petri dishes (100 by 20 mm), and 0.2 ml of the hemolymph, diluted 1 to 5 in TC-100 basal media, was applied to cell monolayers. The inoculated cells were incubated at 27°C with intermittent tilting of the dishes for uniform virus distribution. After 1 h of

Divergent transcription of early 35- and 94-kilodalton protein genes encoded by the HindIII K genome fragment of the baculovirus Autographa californica nuclear polyhedrosis virus

“…Cells and virus infection. Spodoptera frigiperda IPLB-SF-21 cells (34) were inoculated with extracellular AcNPV L-1 (19) at a multiplicity of 20 PFU per cell as described previously (6). To block protein synthesis, growth medium containing 100 ,ug of cycloheximide per ml was added to cell monolayers (107 cells per 100-mm plate) 30 min prior to infection.…”

Optimized production of recombinant bluetongue core-like particles produced by the baculovirus expression system

“…After 4-6 d postinfection, the infected culture was harvested at low-speed centrifugation (100g) for 10 min. The cell pellets were discarded, and the supernatant stored as a stock which was then titrated by plaque assay by a modification of Lee and Miller's (1978) procedure. The virus stock was kept at 4°C for 6 months.…”

Mixed infections and the competitive fitness of faster‐acting genetically modified viruses

“…As natural baculovirus isolates are typically composed of numerous genotypes (Lee and Miller 1978;Smith and Crook 1988;Cory et al 2005), co-infections appear to be the norm in the field under natural conditions. Moreover, different baculovirus genotypes can be co-occluded in the same polyhedron if the host is infected with multiple genotypes (Hamblin et al 1990;Bull et al 2001).…”