Isolation of a factor, from the capsule of a granulosis virus, synergistic for a nuclear-polyhedrosis virus of the armyworm

When the capsules of a granulosis virus are fed together with the polyhedra of a nuclear-polyhedrosis virus to larvae of the armyworm, Pseudaletia unipuncta, the former enhances the infectivity of the latter virus, a synergistic interaction. The enhancement of infectivity depends upon the concentration of the polyhedra and the capsules. The factor responsible for the synergistic activity in the capsule can be dissolved in alkaline solution, separated from the virus particles by centrifugation, and further purified by Sephadex G-200 gel filtration with 4 m urea. The fraction obtained from Sephadex filtration and containing the synergistic factor can be separated into two components by disc-electrophoresis with 8 m urea. Both components possess synergistic activity. The ID₅₀ of the synergistic factor corresponds to 0.0015 OD₂₈₀. Its optimum pH is 8.5. Synergism is most evident when the factor is fed to larvae together with the polyhedra or is fed 24 hr prior to the ingestion of the polyhedra. The factor appears to be a simple or a conjugated protein of the capsule.     

The effect of inoculum concentration on the development of the nuclear polyhedrosis virus of Autographa californica in TN-368 cells

A multiplicity of infection (m.o.i.) of 25 or 50 mean tissue culture-infective doses (TCID₅₀) of Autographa californica NPV per cell of a TN-368 cell line initially infected >90% of attached cells whereas an m.o.i. of 1 or 5 TCID₅₀/cell initially infected <50% of the cells. An immunoperoxidase technique first detected nucleocapsid antigens at 6–12 hr postinfection (PI) and polyhedral protein antigen 12–18 hr PI, which was followed 4–6 hr later by polyhedra formation. At a m.o.i. of 50, the extracellular virus titer (nonoccluded progeny virus) increased between 6 and 12 hr PI while at m.o.i. of 25, 5, and 1, the titer increased at 12–18 hr PI. Antisera to nucleocapsids and polyhedral protein were specific and also failed to react with viral envelope antigens.     

Effects of Substituting Granulin or a Granulin-Polyhedrin Chimera for Polyhedrin on Virion Occlusion and Polyhedral Morphology in Autographa californica Multinucleocapsid Nuclear Polyhedrosis Virus

Substitution of granulin from the Trichoplusia ni granulosis virus (TnGV) for polyhedrin of the Autographa californica multinucleocapsid nuclear polyhedrosis virus (AcMNPV) yielded a few very large (2 to 5 μm) cuboidal inclusions in the cytoplasm and nucleus of infected cells. These polyhedra lacked the beveled edges characteristic of wild-type AcMNPV polyhedra, contained fractures, and occluded few virions. Placing a nuclear localization signal (KRKK) in granulin directed more granulin to the nucleus and resulted in more structurally uniform cuboidal inclusions in which no virions were observed. A granulin-polyhedrin chimera produced tetrahedral occlusions with more virions than granulin inclusions but many fewer than wild-type polyhedra. Despite the unusual structure of the granulin and granulin-polyhedrin inclusions, they interacted with AcMNPV p10 fibrillar structures and electron-dense spacers that are precursors of the polyhedral calyx. The change in inclusion shape obtained with the granulin-polyhedrin chimera demonstrates that the primary amino acid sequence affects occlusion body shape, but the large cuboidal inclusions formed by granulin indicate that the amino acid sequence is not the only determinant. The failure of granulin or the granulin-polyhedrin chimera to properly occlude AcMNPV virions suggests that specific interactions occur between polyhedrin and other viral proteins which facilitate normal virion occlusion and occlusion body assembly and shape in baculoviruses.     

Comparative biochemical studies of polyhedral proteins of nuclear polyhedrosis viruses]

Using disc polyacrylamide gel electrophoresis, the molecular weights of polyhedral proteins of nuclear polyhedrosis viruses (NPV) of Porthetria dispar, Mamestra brassicae, and Aporia crataegi were found to be 28000 +/- 3000. It was shown that NPV polyhedra of Bombyx mori, Galleria mellonella, P. dispar, and M. brassicae contain a protease. During dissolution of the polyhedra at pH 10,5 this protease specifically cleaves the matrix protein into 2--5 fragments. The amino acid compositions of NPV polyhedral proteins of P. dispar, M. brassicae, A. crataegi, Hyphantria cunae were shown to be very similar. It was found that tyrosine is a C-terminal amino acid of NPV polyhedral proteins of P. dispar, M. brassicae, and A. crataegi.     

Electron microscopical studies and restriction analysis ofHelicoverpa armigera nucleo polyhedrosis virus

Nuclear polyhedrosis virus ofHelicoverpa armigera (HaNPV) from Madurai (south India) was isolated, purified and subjected to electron microscopical studies. The results indicate that the isolate is a multi embedded form of NPV. Further, STEM observations on polyhedra and polyhedral envelope of the HaMNPV are presented. The restriction pattern and genome size of HaMNPV Madurai isolate are compared with HaNPV isolates reported earlier. A dendrogram is presented to show the relatedness among the isolates.     

Purification,electron microscopy and serology of virus isolated fromEuonymus europaea

“Euonymus mosaic virus”, purified from cucumber cotyledons by the differential and density-gradient centrifugation, shows typical nucleoprotein absorption spectrum. Electron microscopy reveals isometric virus particles of about 37 nm diameter. No reaction of purified “Euonymus mosaic virus” was observed with antisera against a raspberry ringspot virus, tobacco ringspot virus, cherry leaf roll virus, strawberry latent ringspot virus, tomato ringspot virus, elm mosaic virus, arabis mosaic virus, tomato bushy stunt virus and watermelon mosaic virus.     

An electron microscope study of effects of various fixatives and thin-section enzyme treatments on a nuclear polyhedrosis virus

A comparison of fixatives and embedding media used in thin sectioning of polyhedra and isolated virions of the Pseudoplusia includens nuclear polyhedrosis virus demonstrated that best results are obtained with glutaraldehyde-OsO₄ fixation and epoxy embedding. Fixation was obtained with formaldehyde, acrolein-formaldehyde, or OsO₄ alone but the crystalline array of the polyhedral protein was not preserved. Glycol methacrylate embedding medium resulted in images of poor quality. Treatment of thin sections of polyhedra and virions with enzymes showed that the polyhedral membrane is resistant to digestion with proteases but the interiors of polyhedra were removed with pepsin, pronase, subtilisin, and a mixture of deoxyribonuclease and trypsin. Enzyme treatment of thin sections of virions resulted in removal of the nucleocapsid by all proteases tested except trypsin. A mixture of deoxyribonuclease and trypsin digested the nucleocapsid. The envelope of the virion resisted enzyme treatment.     

Detection of Autographa californica and Heliothis zea baculovirus proteins by enzyme-linked immunosorbent assay (ELISA)

The structural proteins of Autographa californica (AcMNPV) and Heliothis zea (HzSNPV) nuclear polyhedrosis viruses were detected by indirect enzyme-linked immunosorbent assay (ELISA). The immunoassay detected less than 1 ng of AcMNPV protein. The extent of immunological relatedness between AcMNPV-occluded virus and AcMNPV polyhedral protein, AcMNPV-nonoccluded virus, Estigmene acrea granulosis virus, Amsacta moorei entomopoxvirus Heliothis zea NPV, and Lymantria dispar NPV was determined. No immunological relatedless was detected between HzSNPV, AcMNPV, and a persistent rod-shaped virus isolated from the Heliothis zea cell line (IMC-Hz-1). The polyhedral proteins of HzSNPV and AcMNPV were found to be immunologically identical.     

Purification of polyhedra of a cytoplasmic-polyhedrosis virus from soil

Polydedra of a cytoplasmic-polyhedrosis virus of the silkworm, Bombyx mori, were purified from soil by multistep procedures consisting of desorption of the polyhedra with sodium pyrophosphate, aqueous two-phase separation with the dextran-polyethylene glycol system, and CsCl density-gradient centrifugation. The overall recovery of polyhedra was 7% and there was a 170-fold increase in the number of polyhedra per gram minerals.     

Characterization of a Nuclear Polyhedrosis Virus Isolated from Diseased Gonometa podocarpi (Lepidoptera:Lasiocampidae)

Gonometa podocarpi is an important pest of several species of pine in East Africa, and large numbers of trees in plantations in Kenya were partially or completely defoliated by the larval stage of this insect. After the infestation in the Mt. Elgon region, large numbers of dead and moribund larvae were found on the ground. Examination of extracts of these larvae demonstrated the presence of an occluded virus. Electron microscopy of purified sectioned polyhedra demonstrated the presence of virus particles containing from 1 to 12 nucleocapsids. Purification of virus particles from polyhedra was accomplished by using alkali solubilization and sucrose gradient centrifugation. Virus particles contained 15 proteins as determined by polyacrylamide gel electrophoresis. Detergent solubilization of the virus particles released polyhedra containing one major structural protein. Electron microscopy of purified virus particles and nucleocapsids demonstrated them to be similar in structure to previously recorded nuclear polyhedrosis viruses. The viral deoxyribonucleic acid was extracted and spread for electron microscopy and was determined to have a size of approximately 80 × 10⁶ daltons.     

The infectivity of a nuclear polyhedrosis virus of the velvetbean caterpillar for eight noctuid hosts

Eight species of noctuid larvae were tested for susceptibility to a nuclear polyhedrosis virus of the velvetbean caterpillar, Anticarsia gemmatalis. Velvetbean caterpillar larvae were highly susceptible to crude preparations of polyhedral inclusion bodies (PIBs; LD₅₀ = 4.7 PIBs/larva), but preparations of purified polyhedra were much less effective against these larvae (LD₅₀ = 319.7 PIBs/larva). Of seven other noctuid species tested, only Heliothis virescens was as susceptible to the virus as A. gemmatalis. High dosages were required to kill Heliothis zea, Trichoplusia ni, Pseudoplusia includens, and Spodoptera ornithogalli. Plathypena scabra and Spodoptera frugiperda were not susceptible.     

Characterization of a rearrangement in viral DNA: mapping of the circular simian virus 40-like DNA containing a triplication of a specific one-third of the viral genome

Serial passage of the non-defective form of a simian virus 40-like virus (DAR) isolated from human brain results in the appearance of three distinct classes of supercoiled DNAs: R_I resistant, R_I sensitive (one cleavage site) and R_I “supersensitive” (three cleavage sites). The R_I cleavage product of the “super sensitive” form is one-third the physical size of simian virus 40 DNA (10.4 S) and reassociates about three times more rapidly than “standard” viral DNA. To identify the portions of the DAR genome present in the 10.4 S segment, the plus strand of each of the 11 fragments of ³²P-labeled simian virus 40 DNA, produced by cleavage with the Hemophilus influenzae restriction endonuclease, was hybridized in solution with the sheared R_I cleavage product of the “supersensitive” class of viral DNA. Reaction was observed with fragments located in two distinct regions of the simian virus 40 genome: (1) Hin-A and C; (2) Hin-G, J, F and K.Further studies indicated that simian virus 40 complementary RNA transcribed in vitro with Escherichia coli RNA polymerase from one strand of simian virus 40 DNA reacts with both strands of the denatured 10.4 S cleavage product when hybridization is monitored with hydroxyapatite. Treatment of the 10.4 S DNA-simian virus 40 cRNA hybrid with the single-strand spcific nuclease, S₁, converted approximately 50% of the radioactive counts to an acid-soluble product. These results indicate that the 10.4 S product contains a transposition of sequences originally present on one of the DAR DNA strands to the other strand. Examination of heteroduplexes formed between the 10.4 S segment and unique linear forms of DAR DNA produced with the R · Eco RI restriction endonuclease have confirmed these observations. Thus it appears that a molecular rearrangement(s) has resulted in the recombination and inversion of viral DNA sequences from two separate loci on the parental DAR genome. This 1.1 × 10⁶ dalton segment is reiterated three times in a supercoiled molecule equivalent in physical size to parental DAR DNA.     

Triplication of a unique genetic segment in a simian virus 40-like virus of human origin and evolution of new viral genomes

The non-defective (heavy) virions from a simian virus 40-like virus (DAR virus) isolated from human brain have been serially passaged at high input multi-plicities in primary monkey kidney cells. The ³²P-labeled, progeny DAR-viral genomes have been purified and tested for sensitivity to the R_I restriction endouclease from Escherichia coli (Eco RI3 restriction nuclease). The parental DAR-viral genomes share many physical properties with “standard” simian virus 40 DNA and are cleaved once by the Eco RI restriction nuclease. After the fourth serial passage, three populations of genomes could be distinguished: Eco RI resistant, Eco RI sensitive (one cleavage site) and Eco RI “supersensitive” (three, symmetrically-located, cleavage sites). The Eco RI cleavage product of the “supersensitive” form is one-third the physical size (10.4 S) of simian virus 40 DNA and reassociates about three times more rapidly than sheared, denatured simian virus 40 DNA. From the fourth to the eighth serial passages, the genomes containing this specific triplication of viral DNA sequences were selected for and became the predominant viral DNA species.     

Activation of latent virus infections in larvae of Adoxophyes orana (Lepidoptera: Tortricidae) and Barathra brassicae (Lepidoptera: Noctuidae) by foreign polyhedra

The number of larvae containing polyhedra increased when larvae of Adoxophyes orana and Barathra brassicae were fed on polyhedra of nuclear polyhedrosis virus (NPV) of the reciprocal species. Comparison of restriction endonuclease EcoRI cleavage patterns of DNA isolated from polyhedra used as inocula and from polyhedra obtained after cross-inoculation showed that cross infection did not occur. The observations indicate that latent viruses were activated in both insects. Activation of the A. orana latent NPV with polyhedra of a cytoplasmic polyhedrosis virus (CPV) of B. brassicae, and cross-inoculation with an extract prepared from healthy larvae indicated that an activating agent does not have to be a component of nuclear polyhedra.     

Serial passage of Heliothis zea singly embedded nuclear polyhedrosis virus in a homologous cell line

This paper describes the replication and serial passage of Heliothis zea nuclear polyhedrosis virus (NPV) in a H. zea cell line. It was demonstrated that long-term serial passages of the H. zea NPV in homologous host cell culture decreased both the total number of polyhedral inclusion bodies (PIBs) produced and the infectivity of the supernatant as measured by TCID₅₀. The growth curve indicated that infectious material was released from cells 24 hr postinfection (p.i.) and approached a maximal titer 3 days p.i. The kinetics of H. zea NPV decay at 4°, 27°, and 37°C were determined. Infectivity was not detected after 3 weeks at 37°C, but approximately 10^3.5 TCID₅₀/ml activity was still present after 3 and 8 weeks storage at 27° and 4°C, respectively. Electron microscopy confirmed the presence of single embedded virions in the inoculated cells.     

Infectivity of the components of a nuclear polyhedrosis virus of the silkworm, Bombyx mori

The polyhedra of the nuclear polyhedrosis virus (NPV) of Bombyx mori were dissolved under controlled dissolution with weak alkali. Five components were separated by centrifugation of the dissolved solution in a sucrose gradient. The assay of the separated components in B. mori pupae showed that two types of rods were almost equally, highly infectious, whereas the small spherical particles were much less infectious than the rods by an order of 4 log units. The infectious unit of this virus appeared to be the virus rod without the outer membrane, and the small spherical particle did not represent any infectious functional unit but was a degradation product of the viral internal substance and the outer membrane may not be essential for the virus infection in the pupal hemocoel.     

Analysis of the proteins in thymocyte plasma membrane and smooth endoplasmic reticulum by sodium dodecylsulfate-gel electrophoresis

We have purified the plasma membranes and membranes of endoplasmic reticulum from calf and rabbit thymocytes and from calf mediastinal lymph node lymphocytes. We disrupted the cells by the “nitrogen cavitation method” and prepared a microsomal isolate by differential centrifugation. We fractionated this by isopycnic ultracentrifugation in dextran gradients into membrane vesicles, PM₁ and PM₂, most likely derived from plasma membrane and a fraction, ER, most likely originating from endoplasmic reticulum. More than 80% of the microsomal 5′-nucleotidase and acid p-nitrophenylphosphatase concentrates in the PM₁ and PM₂ fractions; alkaline p-nitrophenylphosphatase, another presumptive PM marker, is concentrated in the PM₁ fraction. These data are confirmed by the lacroperoxidase radioiodination of intact rabbit thymocytes followed by subcellular fractionation. The specific content of phospholipids (822 nmoles/mg protein) and cholesterol (1032 nmoles/mg protein) is highest in PM₁ and PM₂ plasma membrane fractions. NADH-oxidoreductase, our endoplasmic reticulum marker, is clearly enriched in gradient pellet.The membrane proteins were separated by electrophoretic molecular sieving in sodium dodecylsulfate-polyacrylamide gel electrophoresis, containing dithiothreitol (sodium dodecylsulfate-polyacrylamide gel electrophoresis). We numbered the 10 major protein components of the “microsomal fraction” (apparent molecular weights between 280000 and 15000) from 1–10 according to their decreasing molecular weights. Of these proteins, those with higher molecular weight, predominantly glycoproteins, appear in the PM₁ fraction, while the endoplasmic reticulum fraction contains mainly low molecular weight components.     

Infectivity of nuclear polyhedrosis virus extracted with digestive juices

Autographa californica NPV, which had been obtained by dissolving polyhedra in the digestive juice of Estigmene acrea larvae, was infectious to a Trichoplusia ni cell line (TN-368). Virions thus botained were infective, and as few as 0.0025–0.005 polyhedral equivalents could infect newly transferred tissue culture cells. Activity decreased after 8 min of digestion.     

Acylamines enhance the infection of a baculovirus of the armyworm, Pseudaletia unipuncta (Noctuidae,Lepidoptera)

The acylamines (cationic detergents) enhance the infection of a nuclear polyhedrosis virus (NPV) in larvae of the armyworm, Pseudaletia unipuncta. Those with 6 carbons (hexylamine) or 12 carbons (dodecylamine) in the acyl group are more effective in enhancing the NPV than those with 4, 8, or 10 carbons. Hexylamine and dodecylamine increase the rate of infection beyond the 50% level (ID₅₀) by 10 to 100 times when based on the concentration of the virus polyhedra (inclusion bodies).     

An RNA virus in Autographa californica nuclear polyhedrosis virus preparations: Incidence and influence on baculovirus activity

A small RNA virus infectious to Trichoplusia ni larvae (TRV) was observed as a contaminant of several Autographa californica nuclear polyhedrosis virus preparations (AcMNPV). The extent of contamination in various AcMNPV preparations was studied by means of serial enrichment passages through T. ni larvae and enzyme-linked immunosorbent assay (ELISA). TRV could not be detected by ELISA in the original preparation of AcMNPV polyhedra prepared in 1968 even after five enrichment passages. Antibody inactivation offers a possible prophylactic method against TRV but temperature inactivation (55°C) does not. Although TRV reduced larval weight, it had little or no effect on bioassays of AcMNPV to T. ni and Heliothis virescens.