Pathological response of the parasitoid, Glyptapanteles militaris, to nuclear polyhedrosis virus-infected armyworm hosts

The development of the embryonic and larval stages of the internal gregarious parsitoid, Glyptapanteles (=Apanteles) militaris, is adversely affected by the hypertrophy strain of a nuclear polyhedrosis virus in the armyworm, Pseudaletia unipuncta. The initial effects are cessation of parasitoid growth and general tissue disruption, followed by the melanization of parasitoid tissues. Melanization spreads from the parasitoids' caudal vesicle throughout the body, culminating in eventual encapsulation in virus-infected hosts. Parasitoids in armyworm hosts infected with the typical strain of nuclear polyhedrosis virus exhibited no abnormal development.     

Strategies to increase the use of entomopathogens

Synchronized cultures of the TN-368 insect cell line were infected with a nuclear polyhedrosis virus from the alfalfa looper, Autographa californica, during different phases of the cell cycle. Cultures exposed to virus during the middle and late S phase have higher percentages of infected cells than cultures inoculated with virus in the G₂ phase. The amount of virus produced from each infected cell (polyhedra and plaque forming units) is not significantly different between cultures infected at all phases of the cell cycle.     

Infectivity of a nuclear polyhedrosis virus from the alfalfa looper,Autographa californica,after passage through alternate hosts

A nuclear polyhedrosis virus isolated from the alfalfa looper, Autographa californica, was found to infect several species of caterpillars including the cabbage looper, Trichoplusia ni; the beet armyworm, Spodoptera exigua; and the saltmarsh caterpillar, Estigmene acrea. Studies were therefore conducted to determine the quantitative effects of passage through the alternate hosts, S. exigua and E. acrea, on the infectivity of this virus to newly hatched first-instar cabbage looper larvae. When 11 preparations of polyhedra obtained from a like number of primary passages through the original or alternate hosts were assayed and the mortality at 7-, 10-, and 14-day intervals were subjected to probit analysis, the LD₅₀s for the three intervals differed but those for the preparations at any given interval did not. Therefore, any of the three hosts could be used to propagate the virus, and whichever proves the easiest to rear and provides the highest yields of polyhedra can be selected.     

Replication and infectivity of the nuclear polyhedrosis virus of the alfalfa looper, Autographa californica, produced in cells grown in vitro

A virus isolated from the alfalfa looper, Autographa californica, replicated successfully and rapidly in a suspended ovarian cell line of the cabbage looper, Trichoplusia ni. Polyhedra were observed in the nucleus of cells within 20 hr after inoculation. The cytopathological changes typical of nuclear polyhedrosis infections were observed, and an average of 64 polyhedra/cell were produced. These polyhedra were quantitatively as infectious to cabbage looper larvae as those produced in vivo. In addition, they were infective to Heliothis virescens, Pectinophora gossypiella, Spodoptera exigua, A. californica, and Anagrapha falcifera.     

A plaque assay for titration of alfalfa looper nuclear polyhedrosis virus in a cabbage looper (TN-368) cell line

This is the first report of plaque formation by a pathogenic insect virus. Trichoplusia ni (TN-368) cells overlaid with medium containing 0.6% methyl cellulose continued to multiply, developed into monolayers, and produced plaques after infection with alfalfa looper nuclear polyhedrosis virus. Viral polyhedral inclusion bodies were first observed 24 hr after exposure of cells to virus, and plaques continued to increase in size for 72 hr. Two different types of plaques were observed: one in which all cells had many polyhedra in their nuclei, and another in which few cells had inclusion bodies. When virus from either plaque was injected into T. ni larvae, they died of typical nuclear polyhedrosis virus disease. The assay was reproducible, and plaque numbers were related to virus concentration.     

Orientation of the Genome of Autographa californica Nuclear Polyhedrosis Virus: a Proposal

The nuclear polyhedrosis virus of the alfalfa looper Autographa californica contains a double-stranded, circular DNA genome. Fourteen scientists agreed to accept an orientation of this circular genome with respect to a physical map of the restriction endonuclease cleavage sites.     

Activity of the nuclear-polyhedrosis virus of the cabbage looper evaluated at programmed temperature regimens

Each larval instar of the cabbage looper, Trichoplusia ni, was treated in the laboratory with a peroral, standardized lethal dose of the nuclear polyhedrosis virus disease and maintained until death at one of three programmed regimens of temperature. The resulting experimental data were tested in the field and found to provide a reliable method of predicting the course of epizootics.     

Replication and passage of alfalfa looper nuclear polyhedrosis virus plaque variants in cloned cell cultures and larval stages of four host species

As alfalfa looper nuclear polyhedrosis virus (NPV) was serially passed through TN-368 cell cultures, the percentage of the cell population that developed infection with the MP variant decreased, while cells infected with FP variant increased. The MP variant was more virulent to Trichoplusia ni than the FP variant. Cells from the TN-368 cell line and strains derived from single cells were infected with isolated MP plaques. The viral progeny remained homogenous after one or occasionally two passages in cultured TN-368 cells. However, further serial passes in TN-368 cells or one pass in cell strains resulted in loss of the homogeneity, and the FP variant was detected. Four species of Lepidopterous larvae were also infected with the MP variant. Both variants were detected in viral progeny from diseased larvae.     

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.     

Hybridization Selection and In Vitro Translation of Autographa californica Nuclear Polyhedrosis Virus mRNA

We isolated polyadenylated RNA from the cytoplasm of cells infected with Autographa californica nuclear polyhedrosis virus late after infection (21 h postinfection). At that time intracellular protein synthesis was directed almost exclusively toward infected cell-specific proteins. The polyadenylic acid-containing RNA sequences in the cytoplasm at 21 h postinfection were radiolabeled in vitro and hybridized to A. californica nuclear polyhedrosis virus DNA restriction fragments. The polyadenylic acid-containing RNA was derived from regions representing the entire viral genome. Translation in a reticulocyte cell-free protein-synthesizing system of cytoplasmic RNA selected by hybridization to viral DNA and polyadenylic acid-containing RNA produced almost identical polypeptide patterns, suggesting that late after infection almost all of the cytoplasmic polyadenylic acid-containing RNA present in infected cells was of viral origin. Polyhedrin protein (molecular weight, 33,000) and a number of virion structural proteins were among the translation products which were identified by immunoprecipitation and by comparing molecular weights. In addition, some tentative nonstructural infected cell-specific proteins were also detected. Using the hybridization selection technique, we determined that sequences complementary to the message coding for polyhedrin were located on EcoRI fragment I of A. californica nuclear polyhedrosis virus DNA, whereas sequences coding for a putative nonstructural protein (molecular weight, 39,000) were on EcoRI fragment J.     

Ultrastructure of fat body cells of the velvetbean caterpillar infected with a nuclear polyhedrosis virus

During a study of the ultrastructure of a nuclear polyhedrosis virus of the velvetbean caterpillar, Anticarsia gemmatalis, various types of nuclear and cytoplasmic inclusions were found in fat body tissue heavily infected with the virus. Virogenic stroma was present in the nuclei of most infected cells. Bundles of fibrous material were observed in the nuclei and cytoplasm of cells containing polyhedral bodies. Other nuclear inclusions included concentric multilayered material, vacuoles, and membrane structures.     

Aspects of the cross transmission to Galleria mellonella of a Baculovirus from the alfalfa looper, Autographa californica

The nuclear polyhedrosis virus originally isolated from the alfalfa looper, Autographa californica, was successfully transmitted to the greater wax moth, Galleria mellonella. Both the many polyhedra per nucleus (MP) and the few polyhedra per nucleus (FP) plaque variants of this virus were found to be infective when injected intracoelomically. When polyhedra of each plaque variant were fed to G. mellonella larvae, a difference in response was observed; the MP plaque variant was estimated to be 30 times more infective than the FP variant.     

Metabolic changes in diseased insects. IV. Radioautographic studies on protein changes in nuclear polyhedrosis, densonucleosis, and Tipula iridescent virus infections

Changes in glutamic acid, leucine, arginine, and tyrosine in Lepidoptera and Hymenoptera tissues infected with nuclear polyhedrosis (NPV), densonucleosis (DNV), or Tipula iridescent (TIV) viruses were studied by radioautography with a view to determining the effect of the viruses on protein metabolism.     

Interactions between Vairimorpha necatrix,Vairimorpha sp., and a nuclear polyhedrosis virus from Rachiplusia ou in Agrotis ipsilon larvae

Vairimorpha sp. and V. necatrix were assayed in combination with one another, and independently, with a nuclear polyhedrosis virus (RoMNPV) from a mint looper, Rachiplusia ou, against neonate and third-stage black cutworm larvae, Agrotis ipsilon. Initially the effect of Vairimorpha sp. was subadditive, additive, or slightly inhibiting to the V. necatrix in the dual microsporidian assays; later, V. necatrix antagonized the effect of the Vairimorpha sp. In combination with Vairimorpha sp. gradients, V. necatrix significantly (P < 0.05) reduced, in most instances, the LT₅₀ values in both neonate and third-stage larval assays. RoMNPV assayed against neonate and third instars, in combination with either Vairimorpha sp. or V. necatrix gradients, usually significantly reduced the LT₅₀ values (P < 0.05). RoMNPV, when combined with either Vairimorpha species, had varying effects on its pathology. In all assays, the particular relationship that was expressed seemed to be a function of the concentration of each pathogen, which may indicate that the two microorganisms compete for entry and/or infective sites within the larval host. Larval size, which was significantly reduced (P < 0.05) by both microsporidia, would be involved in such competition because it would limit the tissue mass available for infection. Histological examinations of larvae with dual infections revealed pathogens in the tissues that they normally infect. Vairimorpha sp. primarily infected epithelial, fat body, and Malpighian tubule tissue and, occasionally, muscle tissue. Viral polyhedral inclusion bodies were found in the same fat body tissues as the Vairimorpha spores.     

Aspects of substrate utilization and energy requirement during molluscan phagocytosis.

When certain ingredients were eliminated from a medium used to culture a cabbage looper cell line that can support replication of Autographa californica nuclear polyhedrosis virus, cells grew successfully and could be serially transferred a minimum of 44 times. Also, they maintained their ability to support replication of the Autographa californica virus, and the polyhedra produced were as infectious as those from cells grown on the original medium. The cost of the least expensive medium that would support cell growth was 2.8 times less than the cost of normal growth medium.     

Factors affecting the yield of virus in a cloned cell line of Trichoplusia ni infected with a nuclear polyhedrosis virus

The TN-368 tissue culture line of the cabbage looper, Trichoplusia ni, has been cloned. The doubling times of three clones at 27°C were 27.6 ± 3.4 hr, 21.9 ± 1.7 hr, and 27.4 ± 5.9 hr and that of the uncloned culture was 15.8 ± 1.5 hr. Growth of cells in all cultures was arrested after infection with a nuclear polyhedrosis virus of T. ni. There was little difference in the yield of polyhedra from cultures of uncloned or cloned cells infected at a multiplicity of infection (m.o.i) = 4. Yields of polyhedra were about the same when a m.o.i. was in the range of 0.01–4.0, but the yield tripled in the range m.o.i. = 20–30. At higher multiplicities, up to m.o.i. = 500 the yield of polyhedra progressively fell. It is concluded that the observed variation in numbers of polyhedra borne by individual cells in culture is not due to genetic variability among cells, nor can it be accounted for as a consequence of differing m.o.i. by virus. It is postulated that variation in polyhedra yield among cells in culture may be due to such factors as (1) strain differences in the virus, (2) the stage in the cell cycle at which a particular cell is present when infected.     

Relative susceptibility of several noctuid species to a nuclear polyhedrosis virus from Heliothis armiger

Several alternate hosts were tested for their relative susceptibility to an isolate of Galleria mellonella nuclear polyhedrosis virus. Neonate Trichoplusia ni, Heliothis zea, and Manduca sexta were all susceptible to per oral administration of purified polyhedra. Of the three alternate species tested, T. ni was the most susceptible, and exhibited the most variable mortality response over the dose range tested, while M. sexta was the least susceptible. We believe this represents the first report of a lethal virus infection in a sphingid species, and useful parameters for the successful inoculation of alternate hosts are discussed.     

A baculovirus of the European skipper, Thymelicus lineola (Lepidoptera: Hesperiidae)

The nuclear polyhedrosis virus (NPV) of the European skipper, Thymelicus lineola, observed for the first time in Quebec in 1974, is highly pathogenic for its host. The infected larvae fill with pure polyhedra and die within 4 to 10 days. All tissues were infected except nerve cells and silk glands, but all nuclei of infected cells were filled with polyhedra. Biochemical analyses revealed that important metabolic disturbances occurred in infected larvae, such as serious modifications in the activity of certain enzymes. Polyhedra measured from 350 to 1330 nm in diameter and contained up to about 80 single-enveloped virions which measured to 270 × 58 nm. Abnormally short and abnormally long particles were also observed. Safety tests on mammals, fish, and beneficial insects revealed that this virus had no effect on these organisms, thus, it was recommended for the control of T. lineola outbreaks.     

Enhancement in activity of homologous and heterologous baculoviruses infectious to beet armyworm (Lepidoptera: Noctuidae) by an optical brightener

The nuclear polyhedrosis virus (NPV) from the beet armyworm, Spodoptera exigua (Hübner) (SeMNPV), was the most active virus tested against the beet armyworm (LC50 = 4.1 PIBs/mm2), followed by nuclear polyhedrosis viruses from the alfalfa looper, Autographa californica (Speyer) (AcMNPV; LC50 = 92.6 PIBs/mm2), and the celery looper, Anagrapha falcifera (Kirby) (AfMNPV; LC50 = 195.7 PIBs/mm2). In the case of the nuclear polyhedrosis virus from the bollworm, Helicoverpa armigera (Hübner), LC50s could only be obtained for five/six replicates, whereas LC50s could only be obtained for two/six replicates for the nuclear polyhedrosis virus from the wax moth, Galleria mellonella (L.) (GmMNPV). When an optical brightener Tinopal LPW was added to virus suspensions, LC50 values were reduced by 130-fold for both SeMNPV and AcMNPV and by 300-fold for AfMNPV. The addition of Tinopal LPW greatly increased the activities of HaMNPV and GmMNPV. In terms of speed of kill, Tinopal LPW reduced the LT50s for all nuclear polyhedrosis viruses by 30-40%.     

Changes in growth and virulence of a nuclear polyhedrosis virus from Choristoneura fumiferana after passage in Trichoplusia ni and Galleria mellonella

An isolate of nuclear polyhedrosis virus from Choristoneura fumiferana was fed to neonate larvae of Trichopulsia ni and Galleria mellonella. It caused infection and mortality in both of these species. After passage in the alternate hosts, the isolate became increasingly virulent for these hosts. The passaged virus retained its infectivity for Choristoneura but diseased larvae did not wilt and at death they were found to contain only a few polyhedra indicating the virus had been changed.