Alkaline protease activity associated with iridescent virus type 6

Abstract An alkaline protease activity has been found to be associated with Iridovirus type 6. The enzyme showed a pH optimum of 10–10.5 with azocoll and hemoglobin but was essentially inactive on casein. From inhibitor studies the enzyme behaved like a serine protease. An M _r value of about 11500 was determined by SDS-PAGE.     

A cysteine protease encoded by the baculovirus Bombyx mori nuclear polyhedrosis virus.

Sequence analysis of the BamHI F fragment of the genome of Bombyx mori nuclear polyhedrosis virus (BmNPV) revealed an open reading frame whose deduced amino acid sequence had homology to those of cysteine proteases of the papain superfamily. The putative cysteine protease sequence (BmNPV-CP) was 323 amino acids long and showed 35% identity to a cysteine proteinase precursor from Trypanosoma brucei. Of 36 residues conserved among cathepsins B, H, L, and S and papain, 31 were identical in BmNPV-CP. In order to determine the activity and function of the putative cysteine protease, a BmNPV mutant (BmCysPD) was constructed by homologous recombination of the protease gene with a beta-galactosidase gene cassette. BmCysPD-infected BmN cell extracts were significantly reduced in acid protease activity compared with wild-type virus-infected cell extracts. The cysteine protease inhibitor E-64 [trans-epoxysuccinylleucylamido-(4-guanidino)butane] inhibited wild-type virus-expressed protease activity. Deletion of the cysteine protease gene had no significant effect on viral growth or polyhedron production in BmN cells, indicating that the cysteine protease was not essential for viral replication in vitro. However, B. mori larvae infected with BmCysPD showed symptoms different from those of wild-type BmNPV-infected larvae, e.g., less degradation of the body, including fat body cells, white body surface color due presumably to undegraded epidermal cells, and an increase in the number of polyhedra released into the hemolymph. This is the first report of (i) a virus-encoded protease with activity on general substrates and (ii) evidence that a virus-encoded protease may play a role in degradation of infected larvae to facilitate horizontal transmission of the virus.     

Alkaline protease associated with the matrix protein of a virus infecting the cabbage looper.

We report here a convenient and inexpensive method of attaching enzymes to solid supports which contain diols. Dextran coated porous glass, Sepharose and glass coated with a glyceryl silane were oxidized with NaIO₄. Trypsin, carboxypeptidase A, and carboxypeptidase B were bound to the oxidized supports by treatment with NaBH₄. The pH dependence of the coupling reaction and loss of lysine in bound trypsin indicate that the immobilization occurs via reductive alkylation. The bound enzymes display good catalytic activity against synthetic substrates and proteins.     

Effect of alkaline protease on the antigenic nature of wiseana nuclear polyhedrosis virus polyhedron protein

Polyhedron protein from Wiseana spp. nuclear polyhedrosis virus was found to be degraded by an alkali protease when polyhedra are dissolved in alkali. The protease activity did not occur at high pH (0.1 M NaOH) and was inactivated by heating polyhedra to 70°C for 3 h. The products from the protease degradation of Wiseana spp. nuclear polyhedrosis virus polyhedron protein retain the antigenicity of undegraded polyhedron protein as measured by the direct solid-phase radioimmunoassay and immunoadsorption. Degradation products below 27,000 daltons could not be detected by the sandwich radioimmunoassay, indicating that they are probably monovalent.     

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.     

In vivo and in vitro host range of Autographa californica nuclear polyhedrosis virus and Spodoptera frugiperda nuclear polyhedrosis virus

Baculoviruses from Autographa californica (AcNPV-E2) and Spodoptera frugiperda (SfNPV-2) were titered in five insect cell lines: IAL-PID2, IAL-SFD1, IPLB-SF-21AE, TN-368, and IAL-TND1. AcNPV-E2 replicated in all the cell lines while SfNPV-2 did not replicate in the lines TN-368 and IAL-TND1. Further in vivo studies of SfNPV-2 showed the virus was not infectious when fed to Trichoplusia ni larvae per os or when injected into the hemocoel. These data suggest that the barrier to SfNPV-2 infectivity in T. ni is at the cellular level, as opposed to the midgut.     

Electron microscope study on the replication of Autographa nuclear polyhedrosis virus and Spodoptera nuclear polyhedrosis virus in Spodoptera exigua

A comparative study of Spodoptera nuclear polyhedrosis virus (NPV) and Autographa NPV replication in Spodoptera exigua revealed some cytopathologic differences. Infection with Spodoptera NPV was accompanied by electron-dense intranuclear granules. Autographa infection within the midgut led to secretion within the lumens of the goblet cells of paracrystalline arrays of small, round particles, 9.5 nm in diameter. Autographa virus was also observed in various stages of possible replication within the cytoplasm.     

Bioassay of a purified nuclear polyhedrosis virus against Heliothis armigera

A precise bioassay method, which is not limited by lack of field applicability, as are peroral administration techniques, is described. Purified nuclear polyhedrosis virus (NPV) suspensions were assayed against third and fourth instar Heliothis armigera larvae to provide standards for additive and field testing. Third instar larvae proved to be approximately one hundred times more susceptible to the NPV disease than fourth instar larvae. The minimum time to mortality was 4 days.     

Host range expansion by recombination of the baculoviruses Bombyx mori nuclear polyhedrosis virus and Autographa californica nuclear polyhedrosis virus.

The mechanisms of host specificity of nuclear polyhedrosis viruses (NPVs) (Baculoviridae) were analyzed after coinfection of Bombyx mori NPV (BmNPV) and one of four distinct groups of Spodoptera litura NPV (SlNPV), including an Autographa californica NPV (AcNPV) variant (S. Maeda, Y. Mukohara, and A. Kondo, J. Gen. Virol. 71:2631-2639, 1990), into various lepidopteran cell lines. Replication of BmNPV in nonpermissive cells (TN-386, SF-21, and CLS-79) was induced by coinfection with AcNPV but not with the other three SlNPV groups. These induced progeny NPVs were plaque purified in BmN cells, which are susceptible to only BmNPV, and characterized. Most of these isolates did not replicate in the cell lines in which they were produced, indicating the existence of a helper function of AcNPV for BmNPV replication in nonpermissive cells. Some of these isolates, however, were able to replicate in cell lines nonpermissive to BmNPV, indicating the appearance of a new virus with wider host specificity. DNA restriction endonuclease analysis showed that the isolates exhibiting wider host range were recombinant viruses between the parents, AcNPV and BmNPV, resulting from various types of crossovers of relatively large areas of their genomes. Expansion of host range was also observed in larvae.     

Susceptibility of Heliothis armiger to a commercial nuclear polyhedrosis virus

The susceptibility of Heliothis armiger larvae of different ages to a commercial nuclear polyhedrosis virus (NPV), Elcar, was determined by bioassay. The median lethal dosage (LD₅₀) increased 150-fold during the first week of larval life at 25°C, i.e., during development to early fourth instar, but daily feeding rate and thus potential virus acquisition also increased. A linear relationship was determined between log LD₅₀ and larval length, indicating that larval length constitutes a useful index for estimating the susceptibility of larval populations. Median lethal times (LT₅₀s) were similar for larvae tested at ages of 0 to 7 days and ranged from 3.6 to 8.0 days at 30°C. The amount of virus produced in a single, infected neonate was equivalent to 1.4 × 10⁶ LD₅₀s for neonates, a 900,000-fold increase on the dose supplied. The data support the practice of directing the NPV against neonates, but, on the basis of larval susceptibility alone, the age of larvae at treatment may not always be critical.     

An enzootic nuclear polyhedrosis virus of pink shrimp: Ultrastructure,prevalence, and enhancement

A nuclear polyhedrosis virus exists in pink shrimp, Penaeus duorarum, from waters of the northern Gulf of Mexico. This virus is rod-shaped, 269 nm long, and possesses an outer envelope surrounding its nucleocapsid. The nucleocapsid is 50 nm in diameter. The virus occurs in nuclei of host hepatopancreatic and midgut cells, and is both free in the nucleus and occluded within pyramidal-shaped polyhedral inclusion bodies (PIB's). Histochemically and ultrastructurally, the shrimp PIB's appear to be ribonucleoprotein and in fine structure bear close resemblance to polyhedral inclusion bodies of Baculovirus species from insects. However, the lattice line-to-line spacing is greater than that usually reported for insect PIB's. Crowding and chemical stress of shrimp in aquaria may enhance and increase the virus infection and prevalence. In limited experiments, shrimp fed heavily infected hepatopancreatic tissues had much higher mortality than controls fed only fish. The virus appears to be enzootic in pink shrimp in nature. Cytopathological changes in infected cells of shrimp appear similar to those in insects infected with certain species of Baculovirus. The name Baculovirus penaei n.sp. is proposed for the shrimp virus.     

Food consumption by cabbage loopers infected with nuclear polyhedrosis virus

The effects of age, temperature, and dose on artificial medium consumption by healthy and nuclear polyhedrosis virus-infected cabbage looper larvae were measured using gravimetric methods. Instar in which lethal infection occurred was more closely related to subsequent food consumption than was larval age in days. Larval cabbage loopers, lethally infected in either the first or second instar, consumed 2% or less of their potential consumption. Larvae infected in the third instar consumed ca. 5% of their subsequent potential. In the fourth instar, this amount increased to ca. 10%. If infection occurred in the fifth instar, no significant amount of feeding was prevented. Increasing the virus dosage significantly decreased consumption and length of feeding period over the range of dosages tested. The relationship between consumption patterns of diseased and healthy insects remained constant over a 20–35°C temperature range.     

Field evaluation of a nuclear polyhedrosis virus of Neodiprion taedae linearis

Aerial applications of a nuclear polyhedrosis virus were evaluated for control of Neodiprion taedae linearis. Based on colony size, cocoon counts, and defoliation, rates of 5 × 10⁷, 1 × 10⁷, and 5 × 10⁶ PIB/ml of spray concentration were most effective in reducing sawfly populations. Sex ratio of cocoons recovered from the treated plots indicated a greater percentage of females succumbed to the virus than did males.     

Comparison of bioassay and enzyme-linked immunosorbent assay for quantification of Spodoptera frugiperda nuclear polyhedrosis virus in soil

Standard curves with known amounts of Spodoptera frugiperda nuclear polyhedrosis virus (NPV) in soil were established with a bioassay and with an enzyme-linked immunosorbent assay (ELISA). The bioassay detected as few as 4 × 10⁴ polyhedral inclusion bodies (PIB)/g sandy soil and <10 PIB/g soils with large amounts of silt or clay. The ELISA detected as few as 360 PIB/g in all three soil types, and absorbance values were inversely related to the amount of clay. Results of the bioassay and ELISA were significantly (P < 0.01) correlated for natural NPV from field samples of silt (R = 0.961) and sandy soil (R = 0.723). Soil samples from Louisiana pastures and corn fields contain up to 7.6 × 10⁴ PIB/g, and 2× 10⁴ PIB/g are commonly present.