Characterization of the genome of the nucleopolyhedrosis bundle virus pathogenic for Orgyia pseudotsugata

282

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

mentioning

confidence: 99%

Isolation of genotypic variants of Autographa californica nuclear polyhedrosis virus

Lee¹,

Miller²

1978

282

“…Nuclear polyhedrosis viruses (NPVs) are members of the baculovirus group and possess double-stranded, circular, supercoiled DNA genomes with molecular weights between 70 x 106 and 100 x 106 (2,3,8,14,15,17). Kinetics of NPV DNA reassociation indicate that the viral genome contains predominantly nonrepeated sequences (9,14,15); this sequence complexity is reflected in the restriction endonuclease (REN) fragment patterns of NPV DNA (12). REN analysis of the DNA genomes has been suggested as a basis for identifying NPVs (12) and has been utilized to distinguish and characterize closely related genotypic variants of Autographa californica NPV (AcNPV) (10,13).…”

mentioning

confidence: 99%

Physical Map of the DNA Genome of Autographa californica Nuclear Polyhedrosis Virus

Miller

Dawes

1979

A physical map of the 88 x 10' dalton, circular DNA genome of Autographa californica nuclear polyhedrosis virus was constructed. The complete order of BamHI and XmaI restriction enzyme sites was determined. The EcoRI and HindIII fragments were partially ordered, and their general locations, relative to the BamHI and XmaI maps, were determined. Alterations in the restriction endonuclease fragment patterns of natural genotypic variants of A. californica nuclear polyhedrosis virus, including Trichoplusia ni MEV nuclear polyhedrosis virus, were located on the physical map. Alterations were found throughout the A. californica nuclear polyhedrosis virus DNA genome.

“…1.715 g/ml for Orgyia pseudotsugate MNPV (19). In this study, Hz-lV DNA had a slightly higher density than those of H.zea SNPV, Spodoptera frugiperda MNPV, and AcMNPV.…”

Section: Discussionmentioning

confidence: 42%

Characterization of the DNA of a Nonoccluded Baculovirus, Hz-1V

Huang

Hedberg

Kawanishi

1982