Esther M. Omi

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

Abstract 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 ID 50 of the synergistic factor corresponds to 0.0015 OD 280 . Its optimum p H 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.

Isolation and characterization of a synergistic enzyme from the capsule of a granulosis virus of the armyworm, Pseudaletia unipuncta

Abstract A synergistic factor than enhances the infection of a nuclear polyhedrosis virus in the armyworm, Pseudaletia unipuncta, was isolated from the occlusion body (capsule) of a granulosis virus of the armyworm. Disc electrophoresis indicated that the purified factor was a single homogeneous compound. Chemical identification and amino acid analysis showed that it was a simple protein, with a molecular weight of 152,000–163,000. Proteolytic enzymes did not markedly reduce the activity of the factor. It could be stored at −20°C or lyophilized. The synergistic factor displayed properties of an enzyme. It enhanced the hydrolysis of p-nitrophenyl esters of fatty acids with an optimum of pH 9.0. The relative hydrolytic activity increased with increase in number of carbon atoms in the fatty-acid chain from 2 to 8 and gradually decreased with the number of carbon atoms from 10 to 18. Copper sulfate markedly and mercuric chloride completely prevented enhancement of the hydrolysis of butyrate. When the synergistic factor was fed to larvae with mercuric chloride, it did not enhance the nuclear polyhedrosis virus.

Site of action of a synergistic factor of a granulosis virus of the armyworm, Pseudaletia unipuncta

Abstract A synergistic factor (SF), which is present in the capsule matrix protein of a granulosis virus of the armyworm, Pseudaletia unipuncta , enhances baculovirus infection in armyworm larvae. The site of action of the SF was investigated. The oral inoculation of SF did not enhance the infectious hemolymph virions which had been inoculated into the hemocoel. The SF also did not enhance the infection of purified enveloped virions when both virus and SF were inoculated into the hemocoel, but enhancement occurred when they were inoculated orally. Thus, the activity of the SF was confined to the midgut lumen. Observations with ferritin-conjugated antibody indicated that the site of action of SF was the cell membrane of the microvillus. There were more ferritin particles attached to midgut cell membranes of larvae inoculated orally with SF than to those of control larvae inoculated with buffer.

Lethal effect of purified spore and crystalline endotoxin preparations of Bacillus thuringiensis on several lepidopterous insects

Abstract Purified preparations of crystals and spores of Bacillus thuringiensis were tested, mainly by inoculation per os, for toxicity against larvae of Colias eurytheme, Trichoplusia ni , and Pseudaletia unipuncta . When the larvae were raised on a diet containing antibiotics, crystal solution surprisingly showed little lethal effect. A mixture of crystal solution and spores was more effective than the crystal solution alone. Death also resulted from ingestion of crystal-free spores. Examination of cadavers showed systemic infection in all cases; when the larvae had been inoculated with spores, large numbers of rods of the B. thuringiensis type were present although spores were never observed. B. thuringiensis was isolated from over 70% of the cadavers in this group. The results indicate that, in addition to the B. thuringiensis endotoxin, the spore may play an important role in killing the insect larva.

Persistence of insect viruses in field populations of alfalfa insects.

Abstract The persistence of viruses of five insects was observed in alfalfa fields. The insects were Autographa californica, Colias eurytheme, Pseudaletia unipuncta, Spodoptera exigua, and Trichoplusia ni. The isolated viruses were the granulosis (GV), the cytoplasmic-polyhedrosis (CPV), and the nuclear-polyhedrosis (NPV) viruses. The viruses persisted in the soil, on the alfalfa foliage, and in alternate hosts. In the soil, the viruses persisted even during the winter months when no foliage remained on the plants. Alfalfa sprouts harboring virus-infected larvae of C. eurytheme and S. exigua produced virus infections in larvae of these insects, but those with larvae of A. californica and P. unipuncta did not cause virus infection. The GVs and CPVs isolated from these insects were transmitted to nearly all of the other four species, but the NPVs appeared to be host specific.

Binding sites on the midgut cell membrane for the synergistic factor of a granulosis virus of the armyworm (Pseudaletia unipuncta)

Abstract The midgut membrane fraction from armyworm larvae ( Pseudaletia unipuncta ) has specific binding sites for the synergistic factor (SF), a protein in the capsule matrix of a granulosis virus (GV), that enhances the infection of nuclear polyhedrosis viruses (NPVs). The GVs and NPVs are insect baculoviruses. Optimum binding of the SF was obtained after a 30 min incubation of the membrane fraction at pH 6.0. The number of binding sites is 4.20 × 10 −13 mol/50μg membrane protein and the equilibrium dissociation constant is 1.57 × 10 −9 M. Inactivation of the binding sites by trypsin and heat treatment suggests that some portion of the binding site is proteinaceous. Pre-treatment of the midgut membrane fraction with Concanavalin A and castor bean lectin also inhibits specific binding. These results support the hypothesis that the SF acts as a binding molecule for the attachment of enveloped virions of baculoviruses to the cell plasma membrane.

Eclipse period of baculovirus infection in larvae of the armyworm, Pseudaletia unipuncta

Two strains of a nuclear polyhedrosis virus (baculovirus) infect larvae of the armyworm, Pseudaletia unipuncta. The hypertrophy strain (HNPV) produces a gradient of infected epithelial cells along the tracheae indicating the movement of infectious material to adjacent cells. Cytopathology of the eclipse period up to the appearance of the virogenic stroma has been separated into three phases during which the chromatin disappears and is replaced by dense interconnected strands of fibrils and dense punctate bodies. Cellular hypertrophy occurs in phase 1 and the virogenic stroma appears in phase 3. The typical strain (TNPV) does not produce structures comparable to those of HNPV infection.

The movement and invasion of an insect baculovirus in tracheal cells of the armyworm, Pseudaletia unipuncta

Abstract The hypertrophy nuclear polyhedrosis virus of the armyworm, Pseudaletia unipuncta , causes a unique gradient of infected cells to form on the trachea. The movement and invasion of the virus apparently were not through adjacent intercellular membranes. The enveloped viruses emerged from the initially infected cell into an area between the cell plasma membrane and basal lamina, and then entered the uninfected tracheal cell either by lateral attachment and fusion of the viral envelope and the plasma membrane or by viropexis. The two methods of viral invasion into the cell suggest the presence of at least two phenotypically different enveloped viruses. Viropexis was initiated with an alignment of the peplomer spikes with regularly spaced, short radial striations on the inner coat of the plasma membrane. At a late state in viropexis, the viral envelope fused with the vacuole membrane, and an opening developed below the site of membrane fusion through which the nucleocapsid might enter the cytoplasm. Some nucleocapsids in membrane-lined vesicles resulting from viropexis appeared to be in a state of dissolution. Naked nucleocapsids were found along the nuclear envelope and within the nucleoplasm. No uncoating of the nucleocapsids was observed at the nucleopores, but uncoating seemed to occur in the nucleoplasm. Nucleocapsids were also found in the cytoplasm of nonsusceptible fat body cells, in which virus replication was not observed.

Unique virus morphogenesis and cytopathology of a baculovirus (hypertrophy strain) in larva of the armyworm, Pseudaletia unipuncta

Abstract Nuclear polyhedrosis in tracheal cells caused by the hypertrophy strain of a nuclear polyhedrosis virus (HNPV) has many morphological and developmental features that distinguish it from that caused by the typical strain (TNPV). The most obvious difference is the morphogenic sequence due to the relatively slow virogenic development in HNPV-infected cells, in which are found extensive membranous profiles similar to viral envelopes, electron dense granules, large fibrous bodies, and microtubules. These structures also occur in TNPV-infected cells but are far less abundant and conspicuous. Fibrous bodies found in the cytoplasm and nucleus appear to be morphologically identical. Cellular distortion in a hypertrophied tracheal cell is seen as tearing of mestracheon folds between cells, separation of septate desmosomes, and attenuation in the cellular sheath.

Epizootiology of virus diseases in three lepidopterous insect species of alfalfa

The incidence of virus infections in three lepidopterous insect species was studied from 1965 to 1968 in alfalfa fields in California. The insects were the alfalfa caterpillar,Colias eurytheme; the beet armyworm,Spodoptera exigua; and the alfalfa looper,Autographa californica. InC. eurytheme, the major virus was a nuclear polyhedrosis virus (NPV); inS. exigua, a granulosis virus (GV) and an NPV; inA. californica, a GV. Virus epizootics did not develop in very high densities ofC. eurytheme. Virus epizootics occurred in low host densities of the three insect species, especially in populations ofA. californica. The virus acted as a density-dependent factor in the regulation of the populations ofS. exigua andA. californica. Temperature, humidity and rainfall had no marked effect on the incidence of virus infections.