Polyhedrosis Virus Of Autographa Californica Research Articles

Effect of Viral Concentration and Body Weight on Mortality of Larvae of Trichoplusia ni (Lepidoptera: Noctuidae) Exposed to Wild-Type or Recombinant Strains of the Nuclear Polyhedrosis Virus of Autographa californica (Lepidoptera: Noctuidae)

A parental, wild-type strain of the nuclear polyhedrosis virus of Autographa californica (Speyer) (AcMNPV) and 2 recombinant strains, engineered to express a scorpion toxin (AcAaIT) or a juvenile hormone esterase (AcJHE-KK), were used to infect larvae of cabbage looper, Trichoplusia in (Hubner). Initial mortality of 1st, 4th, and 5th instars, regardless of the polyhedral inclusion body (PIB) concentration, was not detected earlier than the 2nd d after exposure. At equivalent concentrations of virus, the recombinant AcAaIT was the fastest acting strain followed by the recombinant AcJHE-KK and then the wild-type AcC6. Each doubling of the viral concentration of AcC6 decreased the LT 50 by 5%. Therefore, an 8- to 16-fold increase in the concentration of virus of the wild-type AcC6 was needed to attain LT 50 s equivalent to that of the recombinants AcAaIT and AcJHE-KK. Six weight classes (ranging from 0.3 to 95 mg per larva) of T. ni larvae also were exposed to AcC6 or the recombinant strain AcAaIT. The recombinant AcAaIT always killed cabbage loopers more quickly than the wild AcC6 strain regardless of larval size. Differences in LT 50 between strains AcC6 and AcAaIT were significant for all weight classes except for larvae weighing 45-60 mg per larva. The greatest difference in the LT 50 of the strains was for 1st instars weighing 0.3-0.5 mg per larva and the least difference was for 3rd instars weighing 45-50 mg per larva.

Rate of Larval Lysis and Yield and Activity of Inclusion Bodies Harvested fromTrichoplusia niLarvae Fed a Wild or Recombinant Strain of the Nuclear Polyhedrosis Virus ofAutographa californica

Rate of Larval Lysis and Yield and Activity of Inclusion Bodies Harvested fromTrichoplusia niLarvae Fed a Wild or Recombinant Strain of the Nuclear Polyhedrosis Virus ofAutographa californica

Development of a recombinant baculovirus expressing a modified juvenile hormone esterase with potential for insect control

AbstractBaculovirus insecticides are receiving renewed attention as insect pest control agents following the development of fast‐acting recombinant baculoviruses. Here we report on the construction and biological activity of a recombinant baculovirus derived from the nuclear polyhedrosis virus of Autographa californica which expresses a modified form of juvenile hormone esterase (JHE). The serine at the catalytic site of the JHE has been mutated to a glycine residue so that the protein does not degrade JH. The recombinant baculovirus expressing this modified form of JHE, named AcJHE‐SG, has enhanced activity against lepidopteran larvae. Lethal times of the recombinant are 20 to 30% lower than for the wild type virus, and a 66% reduction in feeding damage caused by infected larvae is observed. This result is comparable to the best recombinant baculovirus developed to date, AcAaIT, which expresses an insect‐selective scorpion toxin. The potential of these recombinant viruses for commercialization as insecticides is discussed. Bioassays of AcJHE‐SG in conjunction with anti‐JH agents indicate that the virus is not killing by an anti‐JH mechanism. Larvae apparently die from contraction‐paralysis, or disruption of the normal sequence of events at the molt. © 1995 Wiley‐Liss, Inc.

The structural and functional organization of the Autographa californica nuclear polyhedrosis virus genome.

Baculoviruses are used as biological control agents of insect pests in agriculture and forestry. The multiple-nucleocapsid nuclear polyhedrosis virus of Autographa californica (AcMNPV) is the prototype baculovirus. Recently, this virus has become widely used as vector for the high-level expression of foreign genes in insect cells. An updated physical map of restriction sites as well as the location of open reading frames (ORFs) and transcripts are presented. Most characteristic is the dispersal of "early", "late", and "very late" genes over the genome and the presence of nested sets of 3' and 5' coterminal transcripts.

Characterization of baculovirus-expressed hemagglutinin and fusion glycoproteins of the attenuated measles virus strain AIK-C

With measles virus cDNA of the avirulent vaccine strain AIK-C, two cDNAs of H or F genes were amplified by the polymerase chain reaction. The amplified cDNAs were inserted respectively to the baculovirus transfer vector pAcYM1 derived from the nuclear polyhedrosis virus of Autographa californica (AcNPV). After co-transfection of the transfer vectors with AcNPV DNA to Spodoptera frugiperda cells, recombinant baculoviruses were screened by plaque assay, and the viruses containing H-cDNA or F-cDNA were named H-AIK or F-AIK, respectively. By Western blot analyses, the band around 80 kDa and some smaller bands were appeared in the H-AIK infected S. frugiperda cells, and the band around 40 kDa was detected in the F-AIK infected cells. Immunofluorescence studies on unfixed S. frugiperda cells infected with H- or F-AIK recombinants showed that both antigens were transported to the cell surface. When green monkey red blood cells were added to the recombinant infected cells, H-AIK infected cells showed haemadsorption, and cells infected with F-AIK lysed the red blood cells. The recombinant proteins elicited the neutralizing antibodies against measles virus.

Insect virus: assays for viral replication and persistence in mammalian cells

Viral pesticidal agents must be evaluated for potential health hazards prior to utilization. Assessment of the likelihood of replication in humans has included in vitro exposure of human cells to the potential pesticidal agent. Previous in vitro evaluation strategies have lacked positive controls. Thus, negative results, interpreted as no effect of the virus on human cells, could reflect basic deficiencies in the testing protocols. We designed a testing scheme for viral pesticides and used it to test the nuclear polyhedrosis virus of Autographa californica. Tests were aimed at evaluating potential replication or gene expression in primate cells. Parallel tests were run utilizing identical protocols with primate viruses known to produce the biological effect being evaluated. Thus protocols described were tested with positive viral controls.

Expression, characterization, and purification of a phosphorylated rabies nucleoprotein synthesized in insect cells by baculovirus vectors

A baculovirus expression vector (AcNPV3) derived from the nuclear polyhedrosis virus of Autographa californica (AcNPV) was prepared containing the complete coding region of the nucleoprotein (N) gene of rabies virus (Gif-sur-Yvette clone of the CVS strain). The gene was placed under the control of the AcNPV polyhedrin promoter and was expressed to high levels (66 mg N protein/liter of 2 × 10 9 cells) by the derived recombinant virus using a Spodoptera frugiperda cell line. Using available antisera, it was established that the antigenic characteristics of the N protein were similar by comparison with those of the native N protein of rabies virus. Characterization of the expressed protein established that, like the N protein of mammalian cell-grown CVS virus, the N protein was phosphorylated. The expressed rabies N protein induced antibodies in mice that reacted strongly with the rabies viral protein. The expressed nucleoprotein was recovered from the insect cells by differential centrifugation followed by ion exchange chromatography. The expressed rabies N protein represents a source of authentic protein suitable for virus diagnosis as well as structural studies.

Insecticidal Activity of a Bacterial Crystal Protein Expressed by a Recombinant Baculovirus in Insect Cells

Baculoviruses are insect pathogens with a relatively slow speed of action, and this has limited their use as control agents of insect pests. Introduction into baculoviruses of genes which code for proteins interfering specifically with insect metabolism or metamorphosis, such as toxins, hormones, and enzymes, may enhance the pathogenicity of these viruses. The complete insecticidal crystal protein gene cryIA(b) of Bacillus thuringiensis subsp. aizawai 7.21 was engineered into the nuclear polyhedrosis virus of Autographa californica (AcNPV) in place of the polyhedrin gene. In infected Spodoptera frugiperda cells, the cryIA(b) gene was expressed at a high level without interference with AcNPV production. The crystal protein was found in the cytoplasm of S. frugiperda cells, mainly as large crystals with an ultrastructure similar to that of B. thuringiensis crystals. Infected-cell extracts inhibited feeding of the large cabbage white Pieris brassicae. The toxicity of the crystal protein expressed by AcNPV recombinants was comparable with that of the crystal protein expressed by a corresponding Escherichia coli recombinant.

Immunogenic and protective properties of rabies virus glycoprotein expressed by baculovirus vectors

The gene encoding the glycoprotein of rabies virus (G protein, CVS strain) has been cloned and inserted into the baculovirus transfer vector pAcYM1 derived from the nuclear polyhedrosis virus of Autographa californica (AcNPV). The gene was placed under the control of the AcNPV polyhedrin promoter and expressed to high levels by the derived recombinant virus using a Spodoptera frugiperda cell line. It has been established that the antigenic characteristics of the protein were conserved by comparison with those of the native glycoprotein of rabies virions. The immunogenicity of the expressed product was also demonstrated. Intraperitoneal or intramuscular injection of G antigen conferred protection to mice and was associated with the induction of high titers of neutralizing antibodies. The availability of large quantities of antigenically and immunogenically reactive rabies G protein may make feasible crystallographic studies and the safe preparation of a low cost subunit vaccine for the disease.

Insect virus: assays for toxic effects and transformation potential in mammalian cells.

The nuclear polyhedrosis virus of Autographa californica (AcNPV) was evaluated by using in vitro test systems for toxicity and transforming potential in mammalian cells. Mass cell cultures of CV-1 and WI38 cells appeared unaffected by AcNPV at a multiplicity of infection of 5. Human foreskin cells grew more slowly after inoculation but eventually produced healthy monolayers. The sensitivities of the inhibition of reproductive survivability assays were greater and demonstrated slight AcNPV toxicity to CV-1, WI38, and human foreskin cells. Toxicity was not ameliorated when gradient-purified or psoralen-inactivated virus was used, suggesting that the toxic component of the preparation is part of the virion or copurifies with it. AcNPV was not toxic to and did not transform BALB/c 3T3 cells or primary cell cultures derived from Syrian hamster embryo cells (SHE). Unlike the BALB/c 3T3 transformation assay, the SHE assay detected no spontaneous transformants. The SHE transformation assay can employ simian adenovirus 7 as a positive control. SHE are transformed by numerous viruses and so are useful in assessment protocols. This study suggests that in vitro assessment of viral pesticide toxicity should employ the inhibition of reproductive survivability assay and that transformation assessment is best done with the SHE-simian adenovirus 7 procedure.

Glycosylation is not required for the fusion activity of the G protein of vesicular stomatitis virus in insect cells

The gene encoding the complete glycoprotein of vesicular stomatitis virus (VSV, Indiana serotype G protein) with potential asparagine-linked glycans at amino acid residues 179 and 338 was inserted into a baculovirus transfer vector pAcYM1, derived from the nuclear polyhedrosis virus of Autographa californica (AcNPV). The gene was placed under the control of the AcNPV polyhedrin promotor and expressed by the derived recombinant viruses to high levels in Spodoptera frugiperda cell lines. The principal product was the glycosylated version of the G protein, although some alternative (including probable degradation) forms of the protein were also observed. Similar recombinant viruses were prepared with deletion of one, the other, or both glycosylation sites of the VSV G protein. All forms expressed VSV G protein derivatives and mediated cell fusion and the production of syncytia at low pH. The fusogenic properties of the VSV G protein expressed on the surface of insect cells was prevented using anti-VSV sera, or by elevating the pH above 6.2. A reduction of the pH to 5.5, or 5.0, accelerated the rate of syncytia formation.

Semipermissive Replication of a Nuclear Polyhedrosis Virus of Autographa californica in a Gypsy Moth Cell Line

Several gypsy moth cell lines have been previously described as nonpermissive for the multiple-embedded nuclear polyhedrosis virus of Autographa californica (AcMNPV). In this report, we demonstrate the semipermissive infection of a gypsy moth cell line, IPLB-LD-652Y, with AcMNPV. IPLB-LD-652Y cells infected with AcMNPV produced classic cytopathic effects but failed to yield infectious progeny virus. Results of experiments employing DNA-DNA dot hybridization suggested that AcMNPV DNA synthesis was initiated from 8 to 12 h postinfection (p.i.), continued at a maximum rate from 12 to 20 h p.i., and declined from 20 to 36 h p.i. The rate of AcMNPV DNA synthesis approximated that observed in the permissive TN-368 cell line. AcMNPV-infected IPLB-LD-652Y cells, pulse-labeled with [(35)S]methionine at various time intervals p.i. and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, revealed four virus-induced proteins, one novel to the semipermissive system and three early alpha proteins, synthesized from 1 to 20 h p.i. Thereafter, both host and viral protein synthesis was completely suppressed. These results suggest that AcMNPV adsorbed, penetrated, and initiated limited macromolecular synthesis in the semipermissive gypsy moth cell line. However, the infection cycle was restricted during the early phase of AcMNPV replication.

In Vivo Treatment of a Nuclear Polyhedrosis Virus of Autographa californica (Lepidoptera: Noctuidae) with Chemical Mutagens: Determination of Changes in Virulence in Four Lepidopteran Hosts

Chemical treatment of a nuclear polyhedrosis virus of Autographa californica (Speyer) was carried out initially in Trichoplusia ni (Hubner), Heliothis zea (Boddie), H. virescens (F.), and Spodoptera frugiperda (J. E. Smith), and subsequent effects of treatment on virulence of the virus were assessed in each host. No significant effects were noted in any of the four insect species after treatment with 5-bromodeoxyuridine or nitrosoguanidine. Almost a 100-fold increase in virulence was observed, however, when the virus was treated in T. ni with 3-methylcholanthrene and assayed in S. frugiperda . This isolate also maintained a high degree of pathogenicity for T. ni . Significant changes were also observed in the LT50 of several virus samples, but none of these changes persisted after several serial passages. These observations support use of such low-passage virus isolates, which demonstrate a significant increase in virulence, on crops affected with a particular insect pest.

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.

Morphogenesis of nuclear polyhedrosis virus from Autographa californica in a cell line from Mamestra brassicae (cabbage moth). Further aspects on baculovirus assembly.

Cultures of cell line IZD-MB 0503 from Mamestra brassicae were inoculated with nonoccluded nuclear polyhedrosis virus of Autographa californica (AcNOV). At 1 h postinoculation (p.i.) nucleocapsids were found in the cytoplasm near nuclear pores and within the nucleoplasm. Formation of virogenic stroma was observed at 7 h p.i. The first short empty capsids were seen at 10 h postinoculation (p.i.), followed by partially and completely filled nucleocapsids (11-12 h p.i.) with most capsids filled at 12 h and later. This suggests that nucleocapsid components, such as capsid and DNA-core, assemble in successive stages rather than simultaneously. Viral progeny, being released from the cells by budding, were observed at 13 h p.i.

DNA Sequence Homology between Autographa californica and Orgyia pseudotsugata Nuclear Polyhedrosis Viruses

SUMMARY To investigate the DNA sequence homology between the multicapsid nuclear polyhedrosis virus of Autographa californica and the multicapsid and single capsid nuclear polyhedrosis virus of Orgyia pseudotsugata, the stringency of hybridization conditions was varied. Thirteen to 25 % homology between the multicapsid viruses of A. californica and O. pseudotsugata was detected in 20, 30 and 40% formamide, indicating that fairly stable duplexes were being formed. Under the most stringent conditions in 50% formamide little homology was detected. In contrast to the multicapsid viruses, the single capsid virus of O. pseudotsugata demonstrated about 10% sequence homology in 20% formamide, but these duplexes were unstable and the percentage homology decreased markedly in the higher formamide concentrations. Examination of the specific regions of homology by hybridizing labelled DNA to Southern blots of the virus DNAs revealed that the regions of homology between these viruses were not limited to one region of the genome but were found on a number of restriction fragments.

The effect of inoculum concentration on the development of the nuclear polyhedrosis virus of Autographa californica in TN-368 cells

A multiplicity of infection (m.o.i.) of 25 or 50 mean tissue culture-infective doses (TCID 50) of Autographa californica NPV per cell of a TN-368 cell line initially infected >90% of attached cells whereas an m.o.i. of 1 or 5 TCID 50/cell initially infected <50% of the cells. An immunoperoxidase technique first detected nucleocapsid antigens at 6–12 hr postinfection (PI) and polyhedral protein antigen 12–18 hr PI, which was followed 4–6 hr later by polyhedra formation. At a m.o.i. of 50, the extracellular virus titer (nonoccluded progeny virus) increased between 6 and 12 hr PI while at m.o.i. of 25, 5, and 1, the titer increased at 12–18 hr PI. Antisera to nucleocapsids and polyhedral protein were specific and also failed to react with viral envelope antigens.

Stimulation of DNA Synthesis in Human Lymphocytes by an Insect Virus Antigen 12

Synthesis of DNA in lymphocytes isolated from adult human peripheral blood was detected by measuring incorporation of tritiated thymidine after cell cultures were exposed to polyhedra of a nuclear polyhedrosis virus of Autographa californica . Similar results obtained with lymphocytes isolated from human umbilical cord blood suggests that this mitogenic activity is nonspecific. No DNA synthesis was evident when lymphocyte cultures were treated with enveloped or unenveloped nucleocapsids. Observation of lymphocytes cultured over a 3D-day period, in the presence of baculovirus, provided no evidence of permanently transformed cells.

The influence of the condition of cells and medium on production of polyhedra of Autographa californica nuclear polyhedrosis virus in vitro

A culture of an established cell line of Trichoplusia ni (TN-368) was infected with the nuclear polyhedrosis virus of Autographa californica (ACNPV) at different stages of growth. After 50 hr of growth (early log phase), production of polyhedral inclusion bodies (PIBs) declined rapidly as cell number increased. Medium taken from cultures in the later stages of growth suppressed PIB production in early log-phase cells. Cultures infected with ACNPV and inoculated into fresh medium at high cell concentrations produced relatively fewer PIBs than those inoculated at lower concentrations. The depressive effect of utilized medium is suggested to be the result of exhaustion of a precursor rather than accumulation of an inhibitor.