Comparative chemical studies of the polyhedral proteins of the nuclear polyhedrosis viruses of Bombyx mori and Galleria mellonella

Amino and carboxyl terminal groups, amino acid composition, and peptide maps of polyhedral proteins of the nuclear polyhedrosis viruses (NPV) of Bombyx mori and Galleria mellonella were investigated. It is shown that both the proteins have a tyrosine residue as their carboxyl terminal group and no amino terminal group. Amino acid compositions of the proteins are similar. The proteins are found to have 242 residues. From the amino acid composition, a molecular weight of 28,000 was calculated. The tryptic peptide maps of both the proteins differed only in a few peptides.It is inferred that the polyhedral proteins of B. mori and G. mellonella NPV have a closely similar primary structure.     

Serological relationship of nuclear polyhedrosis viruses: I. Hemagglutination by polyhedral inclusion body protein from the nuclear polyhedrosis virus of Porthetria (= Lymantria) dispar

Polyhedral inclusion body protein (PIBP), harvested from the nuclear polyhedrosis virus (NPV) infecting the gypsy moth, Porthetria dispar, illicits the hemagglutination of chicken erythrocytes. Antisera to PIBP, polyhedral inclusion bodies (PIB), and virions (RODS) from the NPV's infecting P. dispar and the European Pine Sawfly, Neodiprion sertifer, inhibits hemagglutination when utilized to neutralize the PIBP from P. dispar. The crossreactivity of antisera to viral components from N. sertifer, a hymenopteran insect, with viral antigens from P. dispar, a lepidopteran insect, demonstrates a serological relationship exists between two viruses which have widely separated host ranges.     

Comparative studies on the nuclear polyhedrosis viruses of Lymantria monacha and L. dispar

Immunodiffusion and tube precipitation tests, polyacrylamide gel electrophoresis of virus polypeptides, and cross-transmission experiments suggest that two nuclear polyhedrosis viruses, one from Lymantria monacha and one from L. dispar, are partially related to each other, but not identical. The virus particle proteins seem to be more specific than the polyhedron proteins.     

Biochemical comparison of polyhedral protein from five nuclear polyhedrosis viruses infecting plusiine larvae (Lepidoptera: Noctuidae)

Polyhedral protein preparations from five nuclear polyhedrosis viruses isolated from four closely related host insects of the noctuid subfamily Plusiinae were characterized by sodium dodecyl sulfate-polyacrylamide-gel electrophoresis (SDS-PAGE), high voltage paper electrophoresis, and amino acid analysis. The viruses were Autographa california multiple-embedded virion type (MEV), Pseudoplusia includens singly embedded virion type (SEV), Rachiplusia ou MEV, Trichoplusia ni MEV, and T. ni SEV. Each was produced in its own host; A. californica MEV was also produced in T. ni larvae to determine possible host influence on polyhedral protein chemistry. Each test revealed minor, reproducible differences among most isolates. In SDS-PAGE, the major protein component ranged from 26,700 to 28,300 MW among the isolates. Differences were confined to minor protein bands or to band intensity. Peptide maps showed differences among most isolates in numbers of acidic and basic peptide spots, but all had an identical number of neutral spots. Migration patterns also differed among most isolates. The amino acid compositions of the six polyhedral inclusions were very similar, with aspartic and glutamic acids being the predominant residues. The greatest differences were found between the MEV and SEV groups, with lesser differences within each group. In all analyses, A. californica MEV produced in A. californica was indistinguishable from virus produced in T. ni.     

Serological relationships of five nuclear polyhedrosis viruses from lepidopterous species

The serological relationships of five nuclear polyhedrosis viruses (NPV) were investigated using the immunodiffusion technique with intragel absorption. Reciprocal tests demonstrated that virion fractions from Autographa californica multiple embedded virus (MEV), Heliothis armigera MEV, and H. zea single embedded virus (SEV) are not related to each other or to virions from Trichoplusia ni SEV and Pseudoplusia includens SEV. Virion fractions of T. ni and P. includens NPV were shown to be closely related, sharing several antigens. Matrix fractions possessed a common group antigen and one or two antigens specific for the individual NPV with the exception that T. ni and P. includens NPV shared one of these antigens. The specific antigens of the matrix fraction were also shared with the homologous virion fraction.     

The production of nuclear polyhedrosis viruses in large-volume cell cultures

Methods were tested for growing cell lines from the fall armyworm, Spodoptera frugiperda, in roller bottle cultures. The effects of inoculum size, medium volume, and serum level were tested for effect on the cell yield. A protocol is described which gives yields of 3–5 × 10⁸ cells per bottle. Several protocols were then tested for producing the NPV of Autographa californica in this culture system and the results are described.     

Conservation of genome organization in two multicapsid nuclear polyhedrosis viruses.

The genome of the multicapsid nuclear polyhedrosis virus of Orgyia pseudotsugata was mapped by examining overlapping HindIII fragments from cosmid clones which had been constructed from partial HindIII digests of viral DNA. Five OpMNPV cosmid clones containing fragments encompassing the entire OpMNPV genome were hydridized to blots of DNA from the multicapsid nuclear polyhedrosis virus of Autographa californica. The hybridization pattern indicated that the genomes of these viruses are similarly organized.     

Interaction of vertebrate viruses and insect nuclear polyhedrosis viruses with transplantable diploid embryonal drosophila cells

When the Drosophila cells were infected with the mixo- and arboviruses, in case of influenza A/WSN virus a rise in the titre and slight cytopathogenic effect with the subsequent decrease in the titre was observed. Since the decrease in the virus titer was not observed when actinomycin D was added, it was supposed that interferonlike inhibitor may be produced by the infected cells. Vacuolization and increase in the size of the infected cells were caused by all the nuclear polyhedrosis viruses tested. The number of the infected cells depended on the virus type and multiplicity of the infection.     

Selection of nuclear polyhedrosis viruses as biological control agents ofSpodoptera exigua [Lep.: Noctuidae]

The virulence of 5 nuclear polyhedrosis viruses infectious for larvae of beet armyworm,Spodoptera exigua, was studied and their potential as biological control agents of this accidentally introduced pest in Dutch greenhouse crops is discussed. Three of the virus isolates were collected from deceased beet armyworm larvae found in Dutch greenhouses. Based on restriction endonuclease patterns of their DNA they appeared to be closely related toMamestra brassicae nuclear polyhedrosis virus (MbMNPV) and therefore were named MbMNPV-NL80, MbMNPV-NL82 and MbMNPV-NL83. These isolates were not related toAutographa californica MNPV (AcMNPV) or toSpodoptera exigua MNPV (SeMNPV), both originating from the USA. Comparison of the oiological activity of these 5 isolates showed that the SeMNPV was more virulent against beet armyworm than the other isolates. There was no significant difference in virulence between MbMNPV-NL80, NL82, NL83 and AcMNPV forS. exigua. The LD-50 values of the 5 isolates for 2nd instar larvae were 3, 26, 14, 17 and 18 polyhedra, respectively. Despite compensating qualities of the other MNPVs, such as a broader host range and potential production in alternate hosts or cell-lines, SeMNPV is considered to be the most suitable candidate as biological control agent of beet armyworm.      

Biochemical comparison of virion proteins from five nuclear polyhedrosis viruses infecting plusiine larvae (Lepidoptera: Noctuidae)

The virions of six isolates of five nuclear polyhedrosis viruses (NPV) infecting plusiine larvae (Lepidoptera: Noctuidae) were reproducibly separated by sucrose density gradient centrifugation and fractionation. Purity of the preparations was established by electron microscopy. Virion proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE); each produced 12 distinct polypeptides ranging from 10,300 to 82,900 mw. Qualitative and quantitative differences were found between most of the polypeptide patterns. The singly embedded viron (SEV)-type isolates had two major components with mw in the range of 32,900–35,200; multiply embedded virion (MEV)-type isolates had a major component of ca. 12,500 mw. SEV isolates showed almost no within-group differences, while minor differences were found among the MEV banding patterns in both intensity and presence of certain bands. Capsids and envelopes from MEV had two to four polypeptides with mw between 10,800 and 26,900. The presence of more than one polypeptide and electron microscopy of sample composition suggested that the capsid and envelope are composed of several distinct proteins.     

Characterization of the polyhedral envelope of the nuclear polyhedrosis virus of Heliothis virescens

The polyhedral envelope of the nuclear polyhedrosis virus of Heliothis virescens was separated from the matrix proteins and nucleocapsids by alkaline dissolution and differential centrifugation. Spectrophotometric analyses and histochemical staining demonstrated that the envelope was composed of carbohydrates. The envelope contained 60.9% (by weight) hexose and 29.1% pentose. Further examination revealed significant amounts of uronic acids (8.4%) and hexosamines (1.6%).     

SDS-PAGE comparative studies on the polyhedral and viral polypeptides of the nuclear polyhedrosis viruses of Mamestra brassicae, Autographa californica, and Lymantria dispar

After solubilization of polyhedra of Autographa californica, Lymantria dispar, and Mamestra brassicae nuclear polyhedrosis viruses, PAGE showed at least eight distinct polyhedral polypeptide bands. Whereas the molecular weights of the major polypeptide were similar for the three NPVs (28.0–30.0 kdalton), characteristic differences between the species were found for the minor polypeptides having molecular weights in the range from 12.4 to 62.0 kdalton. It is assumed that these polypeptides are not generated by polyhedral alkaline protease since they are detected after protease inactivation. The data demonstrate that different baculoviruses can be distinguished from each other by SDS-PAGE of their polyhedral polypeptides.     

Interference between two nuclear polyhedrosis viruses of the armyworm,Pseudaletia unipuncta [Lep.: Noctuidae]

The larva ofPseudaletia unipuncta (Haworth) is susceptible to 2 nuclear polyhedrosis viruses (NPV), the typical (TNPV) and the hypertrophy (HNPV) strains. The interference between the 2 viruses was studied primarily in the tracheal and fat tissues. When both viruses were microfed simultaneously, no larva had mixed virus infections and most were infected with TNPV. In order to obtain 50% infection by each virus the inoculum of HNPV had to be increased by 1000 polyhedra/ml at the LD₅₀ value of TNPV. Mixed infection resulted when TNPV was inoculated 1 and especially 2 days after HNPV inoculation. In mixed infection, each virus attacked certain areas of the tracheae instead of random cells. These results indicated an interference or antagonism between the 2 viruses. The possible cause for the interference is discussed.

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Résumé La larve dePseudaletia unipuncta (Haworth) est sensible à 2 virus à polyèdres nucléaires (NPV), la souche typique (TNPV) et la souche causant une hypertrophie (HNPV). L'interférence entre ces 2 virus a été étudiée essentiellement dans les trachées et le corps adipeux. Si les deux virus sont inoculés simultanément par micro ingestion aucune larve ne présente d'infection mixte et la plupart d'entre elles est infectée par le TNPV. Pour obtenir 50% d'infection par chacun des virus, l'inoculum de HNPV doit être accru de 1000 polyèdres par ml par rapport à l'inoculum provoquant la DL 50 du TNPV. Une infection mixte est enregistrée quand le TNPV est inoculé 1 jour et de préférence 2 jours après l'introduction du HNPV. Dans ce cas d'infection mixte chaque virus attaque certaines régions des trachées et non pas des cellules réparties au hasard. Ces résultats prouvent une interférence ou un antagonisme entre les 2 virus, dont la cause est discutée.

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Portion of a dissertation submitted by the senior author for the degree of doctor of philosophy from the University of California, Berkeley. Part of the study was supported by a NSF grant provided to the junior author.     

Characterization of DNA from three nuclear polyhedrosis viruses pathogenic for Choristoneura sp

Three nuclear polyhedrosis viruses isolated from larvae of the insect genus Choristoneura showed polyhedrins of 28–30,000 daltons, genome sizes of 78–82 × 10⁶ daltons, and guanine plus cytosine contents of 47.9–49.4%. It was demonstrated by comparison of restriction endonuclease fragment patterns that two of the viruses are closely related genetically.     

Efficacy of two nuclear polyhedrosis viruses againstOstrinia nubilalis [Lep.: Pyralidae] in the laboratory and field

Nuclear polyhedrosis viruses isolated from the alfalfa looper,Autographa californica (Speyer) (AcMNPV), and a mint looper,Rachiplusia ou (Guenée) (RoMNPV), were assayed against the European corn borer,Ostrinia nubilalis Hübner), and applied to field corn for suppression of this insect. Fourteen days post-treatment, LC₅₀ values were 46.50 abd 0.95×10⁵ PIB/ml diet for AcMNPV and RoMNPV, respectively. Both viruses caused significant reductions in number of larvae per plant and in plant damage when the material was applied with a backpack sprayer. Data indicate that RoMNPV kills in less time than AcMNPV. When the viruses were applied with a commercial highboy applicator, the centimeters of damage to cornstalks was reduced by 65% with RoMNPV during the 1st generation and by 32.9% in the 2nd generation.     

Two tissue culture media for production of lepidopteran cells and nuclear polyhedrosis viruses

Two media supporting the growth of several established lepidopteran cell lines in monolayer and suspension culture are described. The medium designated $\text{BML-TC}\text{10}$ was developed specifically as an inexpensive medium for production of cells of Spodoptera frugiperda and the homologous nuclear polyhedrosis virus (NPV) of this species. Simultaneously, a second medium was formulated in which the amino acid requirements were provided by enzymatic protein hydrolysates, one of which was termed $\text{BML-TC}\text{7A}$. Several cell lines could be adapted easily to this medium. $\text{BML-TC}\text{10}$ supported growth of S. frugiperda cells and production of the NPV's of S. frugiperda and Autographa californica. $\text{BML-TC}\text{7A}$ supported the growth of cells of S. frugiperda. Carpocapsa pomonella, Heliothis zea, and Trichoplusia ni. Cells of the latter produced the polyhedra of T. ni and A. californica NPV's in this medium.