Nonoccluded Virus Research Articles

The isolation and characterization of the MP and FP plaque variants of Galleria mellonella nuclear polyhedrosis virus

Two plaque morphologies were detected in assays of Galleria mellonella nuclear polyhedrosis virus preparations which had been serially passaged in the TN-368 cell line. The FP (few polyhedra) plaques were larger (0.7-mm average diameter) and the infected cells contained less than 10 polyhedra each. The MP (many polyhedra) plaques were smaller (0.5-mm average diameter), and the infected cells contained greater than 10 each. Only the FP variant could be purified by plaque picking and remained pure through 10 serial injection passages in last-instar Galleria mellonella larvae, while the MP variant always produced FP plaques after serial passage in cell cultures or by injection of larvae. Bioassays of MP and FP polyhedra demonstrated a 350-fold reduction in virulence for FP polyhedra. Electron micrographs of these polyhedra showed that both variants were MNPV type viruses, although the FP polyhedra were predominantly devoid of nucleocapsids. Electron microscopic observations of MP- and FP-infected cell cultures revealed an apparent deficiency in de novo membrane synthesis in FP infections. SDS-polyacrylamide gel electrophoresis of cell-released nonoccluded virus proteins demonstrated several differences in protein composition between the two variants. The evidence indicates that the FP variant of G. mellonella nuclear polyhedrosis virus is a stable genotypic variant which arises from spontaneous mutation of the MP variant.

Cell culture studies with the IMC-Hz-1 nonoccluded virus

Studies were conducted on an adventitious agent (Hz-1V) isolated from the IMC-Hz-1 cell line. It appeared identical to the virus first obtained by Granados et al (R. R. Granados, T. Nguyen, and B. Cato, 1978, Intervirology, 10, 309–317) from a persistent infection of this cell line. Restriction endonuclease digestion of Hz-1V DNA indicated the agent was different from the S nuclear polybedrosis virus (HzSNPV) of the host species, Heliothis zea, from which the IMC-Hz-1 cell line was derived by Hink and Ignoffo (W. F. Hink and C. M. Ignoffo, 1970, Exp. Cell Res. 60, 307–309) . Hz-1V caused extensive cytopathic effects (CPE) in cultures of TN-368 cells and was moderately infectious to several other insect cell lines. The lytic nature of the infection permitted development of a plaque assay employing TN-368 cells. Following acute infections of TN-368 cells, persistently infected cultures were readily established by the surviving cells. Attempts to induce persistent infections of clones 10 and 13 of TN-368 were, however, unsuccessful. Infectious Hz-1V was shed continuously into the medium throughout subculturings of persistently infected cells. The infectious titer dropped sharply during initial subculturings, but subsequently reached a basal level at about the 25th passage. These cultures were resistant to superinfection by homologous Hz-1V, but remained as susceptible as normal TN-368 cells to heterologous Autographa californica M NPV (Ac MNPV).

Increased virulence of Autographa californica nuclear polyhedrosis virus by mutagenesis

A mutant of the Autographa californica nuclear polyhedrosis virus (AcMNPV) with increased virulence in Trichoplusia ni larvae was isolated following replication of a random virus clone in the presence of 2-aminopurine. The LT 50 of the mutant, designated HOB, was significantly shorter than those of either the wild isolate or parental clone of AcMNPV. Also, fifth-instar larvae infected with this mutant gained significantly less weight and consistently produced more virus occlusion bodies than larvae infected with the wild isolate or parental clone. No alterations in the in vitro replication of nonoccluded virions, occluded virus structural proteins, or DNA restriction endonuclease patterns were observed with the HOB mutant.

A pathogenic nonoccluded virus in hemocytes of the navel orangeworm, Amyelois transitella (Pyralidae: Lepidoptera)

A small, nonoccluded virus was isolated from the granular hemocytes (=adipocytes) of moribund larvae of the navel orangeworm, Amyelois transitella, collected from almonds in northern California. The virus was readily transmitted perorally in laboratory tests and was highly pathogenic to neonate larvae; however, third- and fourth-stage larvae usually acquired attenuated infections that retarded growth and delayed mortality. Large paracrystalline viral arrays filled the cytoplasm of granular hemocytes of diseased larvae. Attempts to transmit the virus perorally to larvae of nine other species of moths were unsuccessful. The virus is isometric, about 25 nm in diameter, and contains single-stranded RNA. It has been designated “chronic stunt virus” (CSV) and tentatively assigned to the picorna-like viruses.

Quantitative Effects of Heat on Baculovirus Virions Released from Polyhedra (Nuclear-Polyhedrosis Virus- Galleria mellonella )

The Galleria mellonella -nuclear polyhedrosis virus ( Baculovirus ) system was used to investigate the effects of heat on virions released from polyhedra. These virions were found to be 3.5 times more thermostable than nonoccluded virions. The inactivation threshold was 42°C e and 359 degree-minutes caused 50 per cent inactivation. The significance of these differences is discussed.

Isolation and replication of an occlusion body-deficient mutant of the Autographa californica nuclear polyhedrosis virus

A spontaneous mutant of the Autographa californica nuclear polyhedrosis virus which was deficient in viral occlusion body formation was studied in Trichoplusia ni tissue culture cells. Virus multiplication could be divided into four phases based on growth curves and the sequential appearance of 25 viral-induced proteins identified by pulse labeling and polyacrylamide gel electrophoresis. During the latent phase, five viral-induced proteins with molecular weights of 68, 47, 35, and 30 thousand (K) were detected. The synthesis of 17 additional viral-induced proteins with molecular weights ranging from 64 to 14K was detected during the nonoccluded virus (NOV) phase, 10 to 16 hr postinoculation (pi). During the transition from NOV to occluded virus formation, 16 to 34 hr pi, the synthesis of occlusion body matrix protein (33K) and two additional proteins (15.6 and 13K) was detected. During the occlusion phase of replication, after 34 hr pi, NOV synthesis ceased; however, the synthesis of the 25 viral-induced proteins continued. Posttranslational cleavage was not detected in pulse-chase experiments. The occlusion body-deficient mutant induced lower levels of occlusion body matrix protein synthesis than the wild type virus. The decrease in amount of occluded virions was accompanied by a corresponding increase in the number of NOV particles produced.

Comparative infectivity of nonoccluded virions, polyhedra, and virions released from polyhedra for larvae of Galleria mellonella

Comparative infectivity of nonoccluded virions, polyhedra, and virions released from polyhedra for larvae of Galleria mellonella

Serum neutralization of nucleopolyhedrosis viruses ( Baculovirus subgroup A) pathogenic for Orgyia pseudotsugata

The preparation of antisera to intracellular nonoccluded virions and an in vivo neutralization test procedure (constant serum and virus in dilutions) are described. Results of homologous neutralization tests showed that rabbit antisera to two multicapsid viruses pathogenic for Orgyia pseudotsugata had higher neutralization indices than antiserum to a unicapsid Baculovrus from O. pseudotsugata. Based on reciprocal tests, the three viruses are antigenically distinguishable. Blood serum of rats which had been exposed by inhalation to 25 projected acre doses of a technical-grade Baculovirus preparation demonstrated no viral neutralizing activity. Since the neutralization test used in this study does not require availability of susceptible cell lines and is sensitive and accurate, it could find application in quality control programs and in field monitoring of Baculovirus strains.

Protein synthesis in cells infected by Autographa californica nuclear polyhedrosis virus (Ac-NPV): The effect of cytosine arabinoside

Twenty-two virion polypeptides (VP) were detected reproducibly when the occluded form (OV) of [ 35S]methionine-labeled Ac-NPV, purified from polyhedral inclusion bodies (PIB), was analyzed by polyacrylamide gel electrophoresis and autoradiography. Ten of the VPs and the polyhedral protein (PP) were phosphorylated and 6 VPs were glycoproteins. Purified, nonoccluded virus (NOV) revealed 25 polypeptides. During a single cycle of virus replication, the synthesis of 33 infected cell polypeptides (1CP) was detected in different relative proportions at different times. They were arbitrarily designated as early (0–12hr), middle (12–18 hr), and late 18–24 hr) polypeptides. Most of the middle and late proteins seemed to be viral structural proteins. Rapid post -translat ional cleavage of IPCs was not observed; however, pulse-chase experiments revealed post-translational modifications of at least four polypeptides. Inhibition of DNA synthesis at the time of infection did not prevent the synthesis of ICPs, VPs, or the formation of PIBs. Progeny PIB from both control and cysosine arabinoside (Ara C)-treated cells that were infected with radiochemically pure [ 3H]thymidine-labeled NOV contained parental virus DNA. Electron micrographs of thin sections showed that, whereas PIBs from control cultures contained complete virions, those from Ara (C-treated cells contained both full and empty virus.

Comparison of nuclear polyhedrosis virus replication in five lepidopteran cell lines

Comparative studies were performed on the replication of the Autographa californica nuclear polyhedrosis virus in cell lines from Estigmene acrea, BTI-EAA; Lymantria dispar, IPLB-LD64BA; Mamestra brassicae, IZD-MB0503; Spodoptera frugiperda, IPLB-SF1254; and Trichoplusia ni, TN-368. Significant differences were observed in the amount of virus obtained from the cell lines, with M. brassicae and T. ni producing more polyhedra than the other lines. These two cell lines also produced nonoccluded virus most rapidly, followed by S. frugiperda, E. acrea, and L. dispar. Sensitivities of the cell lines to infection by the virus, as determined by plaque formation, followed the same pattern, with M. brassicae being most sensitive and L. dispar least so. The T. ni cell line produced polyhedra which were more pathogenic to T. ni larvae than those from the other cells. These differences have important implications in the application of cell cultures in the development of microbial insecticides.

A Partial Genetic Map of the Baculovirus, Autographa californica Nuclear Polyhedrosis Virus, based on Recombination Studies with ts Mutants

SUMMARY Recombination experiments involving a series of two-factor crosses have been done with ts mutants of the baculovirus, Autographa californica nuclear poly-hedrosis virus (NVP). Six mutants which fail to produce non-occluded virus at 33 °C (NOV mutants) have been ordered on a linear genetic map.

Titration of a cytoplasmic polyhedrosis virus by a tissue microculture assay: some applications.

A simple tissue microculture technique was developed for the titration of a cytoplasmic polyhedrosis virus (CPV) from Euxoa scandens. The procedure was similar to the 50% tissue culture infectious dose assay, but a single infected cell, detected by the presence of cytoplasmic polyhedra, was scored rather than the degeneration of cell monolayers. The filtration of CPV suspensions resulted in decreased virus titers under certain conditions. This microculture assay was used to determine the effect of cell disruption methods on virus yields. Sonication of infected cells was more efficient than freeze-thawing for the recovery of nonoccluded virus.

The proteins of nonoccluded Autographa californica nuclear polyhedrosis virus produced in an established cell line of Spodoptera frugiperda

Nonoccluded virions have been isolated from the extracellular fluid of cultured Spodoptera frugiperda cells that have been infected with Autographa californica nuclear polyhedrosis virus. By sucrose density gradient centrifugation, infectious virus particles have been obtained that exhibit densities of about 1.19 g/cm 3. Using staining and autoradiographic techniques, about 35 polypeptides, including some high molecular weight proteins, have been detected by analysis of the virion proteins on sodium dodecyl sulfate-polyacrylamide slab gels (SDS-PAGE). Two major proteins of MW 65,000 and 42,000 have been found.

Characterization of Autographa californica Nuclear Polyhedrosis Virus Deoxyribonucleic Acid

SUMMARY The DNA of Autographa californica nuclear polyhedrosis virus (AcNPV) was isolated from extracellular non-occluded virions. The guanosine plus cytosine content of AcNPV DNA was estimated from its buoyant density in CsCl and its Tm value to be about 43 mol %. In ethidium bromide-CsCl gradients double-stranded covalently closed DNA molecules (form I) were detected, while relaxed circular (form II) and linear (form III) DNA molecules were demonstrated by velocity sedimentation in sucrose. The mol. wt. of AcNPV DNA was calculated to be about 78 × 106 from the sedimentation value of form III DNA. Digestion of AcNPV DNA with restriction endonucleases EcoRI and BamI resulted in 21 and 7 fragments, respectively, after analysis on agarose gels. The mol. wt. of the fragments and their molar ratio were determined. By this method the mol. wt. of AcNPV DNA was calculated to be about 83 × 106.

Isolation, Complementation, and Initial Characterization of Temperature-Sensitive Mutants of the Baculovirus Autographa californica Nuclear Polyhedrosis Virus

Sixteen temperature-sensitive mutants of Autographa californica nuclear polyhedrosis virus were isolated. Several interesting phenotypes were observed. A large proportion of the mutants were unable to form polyhedral occlusion bodies (polyhedra) at the nonpermissive temperature (32.5 degrees C). At 32.5 degrees C, one mutant formed plaques in which the cells lacked polyhedra. Another mutant type was defective in the production of progeny extracellular nonoccluded virus and produced a "plaque" consisting of only a single cell containing polyhedra at 32.5 degrees C. One mutant was defective in plaque formation, progeny nonoccluded virus formation, and polyhedra formation at 32.5 degrees C. Several mutants produced nonoccluded virus but failed to produce plaques or polyhedra at 32.5 degrees C. Other phenotypes were also distinguished. Complementation analyses, performed by either measuring the increase in extracellular nonoccluded virus formation or by observing polyhedra formation in mixed infections at 32.5 degrees C, indicated the presence of 15 complementation groups. A high frequency of recombination was observed. Four of the mutants were found to be host dependent in their temperature sensitivity for polyhedra formation.

Genetic Analysis of a Baculovirus, Autographa californica Nuclear Polyhedrosis Virus I. Isolation of Temperature-Sensitive Mutants and Assortment into Complementation Groups

Temperature-sensitive (ts) mutants were isolated from the baculovirus Autographa californica (alfalfa looper) MNPV, grown in Spodoptera frugiperda (fall armyworm) cells in the presence of N-methyl-N'-nitro-N-nitrosoguanidine. Of 567 plaque isolates screened, 27 were temperature sensitive (ts), representing a mutation frequency of 4.8%. Ten ts mutants were studied in detail: six failed to yield nonoccluded virus at 33 degrees C (NOV mutants), whereas the other four produced nonoccluded virus but were restricted in formation of polyhedra at 33 degrees C (Poly mutants). One of the six NOV mutants failed to synthesize viral DNA. Reversion and leak frequencies were determined, and the mutants were assorted into complementation groups based on the yield of polyhedrin synthesis in cells coinfected with pairs of mutants at 33 degrees C, as measured by radioimmunoassay. For NOV mutants, complementation indexes were also based on virus yield and were consistent with those based on polyhedrin synthesis. Nine mutants were assorted into five complementation groups. One mutant remained unclassified.

Isolation of genotypic variants of Autographa californica nuclear polyhedrosis virus

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, SalI, and Bam HI 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 genotype 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.

Immunoperoxidase Detection of Baculovirus Antigens in Insect Cells

The sequence of events in the infection of TN-368-10 and TN-368-13 cells by Autographa californica nuclear polyhedrosis virus (AcMNPV) was investigated by using the indirect immunoperoxidase technique. Antisera raised against enveloped nucleocapsids detected homologous antigens at 6 to 8 h post infection which was about 2 h before the appearance of both intracellular and extracellular infectious virus. Similar tests using polyhedrin antiserum showed that polyhedrin is first synthesized at 12 h post infection, 2 to 4 h after the appearance of infectious nonoccluded virus. The immunoperoxidase technique was also applied to four other invertebrate cell lines after inoculation with AcMNPV. The most significant result was that 90% of AcMNPV-inoculated Bombyx mori 5 cells produced enveloped nucleocapsid antigens and infectious virus but only 1% or less of the cells produced polyhedrin. This disparity emphasizes the need for assays for NPV infection that are independent of polyhedron production.

In Vitro Infectivity of the Autographa californica Nuclear Polyhedrosis Virus Under Various Conditions

SUMMARY The stability of the in vitro infectivity of the non-occluded virions (NOV) of the Autographa californica nuclear polyhedrosis virus (Ac-NPV) was tested. Exposure of Ac-NPV NOV to solutions ranging from pH 7.5 to 11.4 with molarities of 0.1 to 0.001 showed that infectivity was most stable in 0.001 m, pH 7.5, tris(hydroxymethyl)aminomethane (tris)-HCl buffer. Infectious NOV could be concentrated by precipitation with 50% saturated ammonium sulphate or 9% (w/w) polyethylene glycol 4000. Treatment of concentrated preparations with 1 m-urea did not affect infectivity or the centrifugal properties of the NOV. NOV were partially purified by centrifugation through a sucrose underlay.

Functional differences between occluded and nonoccluded viruses of a nuclear polyhedrosis of the silkworm, Bombyx mori

Comparative infectivity and virus neutralization studies on occluded and nonoccluded viruses of Bombyx mori nuclear polyhedrosis revealed that the infectious unit causing peroral infection differed from that causing hemocoelic infection. There were functional differences between the occluded (mainly virons with envelopes) and the nonoccluded virus (mainly virions without envelopes) preparations. The peroral infection was largely due to the virion with an envelope (peroral infectious unit), and the hemocoelic infection was due largely to the virion without an envelope (hemocoelic infectious unit). The apparent change of the virions with envelope to those without envelopes was detected as a slight increase in hemocoelic infectivity when the occluded virus was diluted and incubated at 4°C for more than 6 days.