Y. Tanada

Enhanced infection of a nuclear-polyhedrosis virus in larvae of the armyworm, Pseudaletia unipuncta, by a factor in the capsule of a granulosis virus

Abstract Capsules of a granulosis virus (GV) enhanced the infectivity of a nuclear-polyhedrosis virus (NPV) for larvae of the armyworm, Pseudaletia unipuncta. When mixtures of the capsules and polyhedra were fed to armyworm larvae, there was a direct increase in the number of larvae infected with the NPV as the concentration of the capsules increased. A capsule factor, possibly a protein, rather than the virus particle, occluded within the capsule, was responsible for the enhancement of infection of the NPV. The capsule factor retained its enhancing activity when dissolved with NaOH, Na2CO3, urea, and Σ-dithioerythritol. Fractionation of dissolved capsule protein on DEAE cellulose column revealed that the enhancing factor was not present in the major protein fraction, but appeared to be a minor component. The capsule factor enhanced the infection of the NPV even when the larvae were treated under gnotobiotic condition. When the NPV was introduced into the hemocoel, the perorally administered capsules did not enhance infection.

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.

Phospholipid, an enhancing component in the synergistic factor of a granulosis virus of the armyworm, Pseudaletia unipuncta

The synergistic factor (SF) in the capsule of a granulosis virus (Hawaiian strain) of the armyworm, Pseudaletia unipuncta, contained polypeptides and phospholipids. Its molecular weight estimated by SDS-polyacrylamide gel electrophoresis was 126,000 ± 8,700. The capsule proteins were digested by a proteinase released from the capsule under alkaline conditions, and by trypsin added to the proteinase-free capsules. Neither enzyme affected the synergistic factor or its activity. The synergistic factor was slowly depolymerized by 2% sodium dodecyl sulfate and was more rapidly depolymerized when phospholipase C (phosphatidylcholine cholinephosphohydrolase) was also added. Phospholipase C alone did not decompose the synergistic factor, but it did destroy the capacity of the synergistic factor to enhance the nuclear polyhedrosis virus. In contrast, phospholipase A2 (phosphatidyl 2-acylhydrolase) had no effect on the synergistic factor. The different reactions of the two phospholipases on the synergistic factor suggested that the hydrophilic group of the phospholipid was exposed to the action of phospholipase C and was associated with the synergistic activity. This interpretation was supported by the detection of a phospholipid in the SF by thin-layer chromatography.

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.

Multiplication of a granulosis virus in larval midgut cells of Trichoplusia ni and possible pathways of invasion into the hemocoel.

The ultrastructure of the midgut epithelium of the fourth-instar cabbage looper larva of Trichoplusia ni has been described. The epithelium consists of columnar and goblet cells. A granulosis virus of T. ni infects and multiplies in columnar midgut cells and in fat cells. The goblet cells appear not to be infected. Complete virus particles with developmental (or outer) membranes have been found, 24 hours after virus ingestion, in some of the intercellular spaces between midgut cells. There is evidence of virus multiplication in the nuclei of columnar cells as early as 24 hours after virus ingestion. A few virus particles occur in the cytoplasm near the bases of the microvilli, but most are found in the basal portion of the cytoplasm of the infected cells. Some particles also appear in the intercellular spaces at the bases of the columnar cells and in the basement membrane. The infected midgut cells contain “naked” and complete virus particles, but very rarely virus inclusion bodies (capsules). Occasionally hexagonal inclusions occur in virus-infected cells, but they do not seem to have virus particles in them. Based on these observations, we have postulated two pathways by which the ingested granulosis virus may invade into the hemococl of the cabbage looper larva.

Biochemical properties of viral envelopes of insect baculoviruses and their role in infectivity

Abstract The enveloped virions of a nuclear polyhedrosis virus (NPV) and those of a granulosis virus (GV) of the armyworm, Pseudaletia unipuncta , were isolated and purified from their inclusion bodies. The enveloped virion of NPV contained a large amount of phosphatidyl choline which was not detected in that of GV. The total electric charges distributed on the surface of the envelopes of NPV and GV were negative in neutral and alkaline solutions. Although there was little difference in charges between NPV and GV, the charge was less negative in NPV than in GV. When the negative charges were neutralized by cationic detergents, the NPV infectivity was enhanced.

A strain of nuclear-polyhedrosis virus causing extensive cellular hypertrophy☆

Abstract A newly discovered strain of a nuclear-polyhedrosis virus causes extensive hypertrophy to the tracheal cells of the armyworm, Pseudaletia unipuncta. This extreme hypertrophy, the absence of infection in the fat cells, and the long period of lethal infection clearly differentiate it from the typical strain of a nuclear-polyhedrosis virus which has been described previously. It also differs from the typical strain in causing the following cytopathological changes: an increase in the number and an alteration in the appearance of the mitochondria in the infected cell, the development of innumerable folds in the cell and nuclear membranes, and the formation of electron-transparent areas in the cytoplasm. At an early stage of infection when the cellular hypertrophy is marked, there is at first no virus rods. The rods appear later, and the polyhedra still later. The polyhedra and virus rods of the new strain resemble those of the typical strain. Its infectivity is also enhanced by a granulosis virus in a synergistic association as in the case of the typical strain. When armyworm larvae are fed equal concentrations of the new and the typical strains, very few of them, if any, develop the signs and symptoms of the new strain. The larval specimens, from which the new strain was obtained, had been collected in a field in Hawaii during 1958 and 1960, and showed the typical signs and symptoms of nuclear polyhedrosis at the time of collection. Accordingly, the larval specimens may have been infected originally by two strains of a nuclear-polyhedrosis virus. Three larvae collected in alfalfa fields at Davis, California, also exhibited signs and symptoms of the new strain.

A nonsynergistic strain of a granulosis virus of the armyworm, Pseudaletia unipuncta

Abstract A granulosis virus isolated in Hawaii from the larva of the armyworm, Pseudaletia unipuncta , acts as a synergist and enhances the nuclear-polyhedrosis virus in infecting the armyworm larva. On the other hand, a granulosis virus obtained from Oregon (probably collected in California) is not capable of enhancing the nuclear-polyhedrosis virus. This difference in the synergistic property is not directly associated with the virulence of the viruses from the two separate sources. The virus from Hawaii at a concentration below its infectious dose does not enhance the nuclear-polyhedrosis virus. The viruses from the two sources also differ in the lengths of the virus rod enclosed within a developmental membrane and of the capsule. On the basis of differences in their synergistic property and in their sizes, the granulosis viruses from the two sources are considered as separate strains.

The formation and circulation, in Galleria, of hyphal bodies of entomophtoraceous fungi☆

Abstract The hyphal bodies of Entomophthora coronata and Entomophthora apiculata are formed in vivo and in vitro. The number of hyphal bodies produced in stationary culture was less than that produced in shake cultures. The accumulation of waste products and the exhaustion of nutrients apparently did not play a significant role in hyphal-body formation. The hyphal bodies of the two Entomophthora species were not circulated in the blood of the larva of Galleria mellonella, whereas those of Metarrhizium anisopliae and Beauveria bassiana were distributed throughout the larva in the blood of the body cavity. The factors that are apparently involved in the circulation of hyphal bodies in the insect are (1) the size of the hyphal body, (2) the optimal site for the growth of the fungus, and (3) the behavior of the fungus in a specific insect host. Both species of Entomophthora, in general, invaded and multiplied in the solid tissues and organs of the host. On the other hand, M. anisopliae and B. bassiana developed in the hemolymph and did not invade the solid organs and tissues until the host died. M. anisopliae produced chlamydospores in the larvae of Galleria mellonella.

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.