Gypsy Moth Cell Line Research Articles

Ectopic Transgene Expression in Butterfly Imaginal wing Discs using Vaccinia Virus

and larval wing tissue, we examined the use of vaccinia virus. It has been reported that vaccinia virus, a vertebrate poxvirus, is able to infect gypsy moth cell lines in vitro (9). The vaccinia virus enters the gypsy moth cells and carries out both early and late viral protein synthesis as well as viral DNA replication (9). However, processing of viral late proteins does not occur, and therefore infectious virions are not produced. In this study, we set out to determine whether vaccinia virus could be used as a gene expression vector to ectopically express transgenes on the wings of butterflies in vivo.

Effects of Long- and Short-Term Passage of Insect Cells in Different Culture Media on Baculovirus Replication

Two insect cell lines that had been maintained in both serum-free (SFM) and serum-containing (SCM) media for over 5 years were each tested for their ability to replicate baculovirus. The gypsy moth cell line, IPLB-LdEIta (Ld), produced similar (not statistically different) amounts of gypsy moth nucleopolyhedrovirus (LdMNPV) occlusion bodies (OBs) in the two media (serum-free Ex-Cell 400 and TC-100 with 9% (v/v) fetal bovine serum, SCM1) but produced more of the Autographa californica nucleopolyhedrovirus (AcMNPV) OBs in SFM than in SCM1. When Ld cells normally grown in SCM1 were switched to SFM, production of OBs from both viruses improved and, after three passages, reached higher levels of AcMNPV production than in cells normally maintained in that medium. Alternatively, cells switched from SFM to SCM1 initially produced as much (in the case of LdMNPV) or higher (in the case of AcMNPV) levels of virus OBs than cells normally maintained in SCM1 but productivity dropped off over subsequent passages such that after five passages in SCM1, cells produced substantially fewer OBs of both viruses. A fall armyworm cell line (IPLB-SF21AE; Sf) showed slightly different effects from long- and short-term passage in SFM (Ex-Cell 400) or SCM2 (TMN-FH). Cells maintained in SFM produced about 20 times more AcMNPV OBs than cells maintained long-term in SCM. Sf cells switched from SFM to SCM maintained the level of production of that seen in SFM at the first passage, but quickly dropped off OB production levels to that normally seen in SCM. Alternatively, SCM-maintained Sf cells produced higher levels at the first passage in SFM and, within five passages in SFM, reached levels found in cells maintained for long term in this medium. Under the conditions in which these two cell lines were infected, the highest levels of AcMNPV OB production in Ld cells were about five times that of Sf cells. In a separate series of experiments, cells normally grown in SFM were passaged over five times in Ex-Cell 400 to which serum was added; both cell lines produced as much virus as that in SFM. These results suggest that it is not the serum per se but rather some other components which differ between the SFM and the SCM formulations that are responsible for the varied virus production obtained in these studies. The results of these studies suggest that a maintenance and virus production protocol can be developed with Ld cells which could improve overall efficiency of virus production. These studies also suggest that long-term maintenance of cells in SFM was not detrimental to their ability to produce baculoviruses.

Responses of insect cells to baculovirus infection: protein synthesis shutdown and apoptosis.

Protein synthesis is globally shut down at late times postinfection in the baculovirus Autographa californica M nuclear polyhedrosis virus (AcMNPV)-infected gypsy moth cell line Ld652Y. A single gene, hrf-1, from another baculovirus, Lymantria dispar M nucleopolyhedrovirus, is able to preclude protein synthesis shutdown and ensure production of AcMNPV progeny in Ld652Y cells (S. M. Thiem, X. Du, M. E. Quentin, and M. M. Berner, J. Virol. 70:2221-2229, 1996; X. Du and S. M. Thiem, Virology 227:420-430, 1997). AcMNPV contains a potent antiapoptotic gene, p35, and protein synthesis arrest was reported in apoptotic insect cells induced by infection with AcMNPV lacking p35. In exploring the function of host range factor 1 (HRF-1) and the possible connection between protein synthesis shutdown and apoptosis, a series of recombinant AcMNPVs with different complements of p35 and hrf-1 were employed in apoptosis and protein synthesis assays. We found that the apoptotic suppressor AcMNPV P35 was translated prior to protein synthesis shutdown and functioned to prevent apoptosis. HRF-1 prevented protein synthesis shutdown even when the cells were undergoing apoptosis, but HRF-1 could not functionally substitute for P35. The DNA synthesis inhibitor aphidicolin could block both apoptosis and protein synthesis shutdown in Ld652Y cells infected with p35 mutant AcMNPVs but not the protein synthesis shutdown in wild-type AcMNPV-infected Ld652Y cells. These data suggest that protein synthesis shutdown and apoptosis are separate responses of Ld652Y cells to AcMNPV infection and that P35 is involved in inducing a protein synthesis shutdown response in the absence of late viral gene expression in Ld652Y cells. A model was developed for these responses of Ld652Y cells to AcMNPV infection.

Ichnovirus Infection of an Established Gypsy Moth Cell Line

In the present study, a lepidopteran cell line (Ld-652Y, from the gypsy moth, Lymantria dispar) exposed to Hyposoter fugitivus polydnavirus (HfPV) was found to display a variety of cytopathic effects. These included a transient inhibition of cell proliferation, rounding up, aggregation and apoptosis. In addition, unusual paracrystalline structures appeared within the lumen of the rough endoplasmic reticulum; similar structures were observed in the spherulocytes of parasitized Malacosoma disstria. Following Coomassie Blue staining, two new cell-associated polypeptides were detected; one of these, an 8 kDa polypeptide, could also be observed following exposure of LD-652Y cells to media taken from infected cultures or to cell-free haemolymph from parasitized M. disstria. After a period of 2-4 weeks, the L. dispar cell cultures were observed to largely recover from the effects of exposure to virus, and resumed proliferation; "transformed' cell populations tended to form aggregates, and adhered less tightly to the substrate. Viral DNA was stably maintained in all recovered cell lines, possibly in chromosomally integrated form.

Selection and Screening of Clonal Isolates of the Abington Strain of Gypsy Moth Nuclear Polyhedrosis Virus

Clonal isolates of the Abington strain of the Lymantria dispar multiple-embedded nuclear polyhedrosis virus (LdMNPV) were selected by plaque purification. After an initial screening for productivity of 35 clones in a continuous gypsy moth cell line (IPLB-LdFB), 17 clones were selected for further evaluation in vivo and in vitro. An eighteenth clone, LdMNPV-Ab-a624, was also included in these experiments as our "type" isolate. Based on these tests, extensive variability can be seen in the Abington isolate of the gypsy moth virus. While relatively little in vitro productivity differences were observed between the clones, two clones showed no activity in a larval bioassay while the mean lethal concentration for 50% of the test insect (LC50's) for the other clones ranged from 2.2 × 104 to greater than 6.5 × 105 occlusion bodies per diet cup, with the a624 clone having a LC50 of 1.2 × 105. The LT50's (time for half the larvae to die) varied from 9.8 to 18.7 days in these clones with a624 having a LT50 of 13.5 days. While certain advantages exist for the use of clones in biopesticide production schemes, the results of this study indicate careful screening of isolates should be made to ensure an acceptable product.

Saturable attachment sites for polyhedron-derived baculovirus on insect cells and evidence for entry via direct membrane fusion.

This research provides the first evidence for specific receptor binding of polyhedron-derived baculovirus (PDV) to host cells and to lepidopteran brush border membrane vesicles (BBMV) and demonstration of entry via a nonendocytotic pathway involving direct membrane fusion. The technique of fluorescence-activated cell sorting analysis was used to investigate the specificity of binding between the PDV phenotype of Lymantria dispar nuclear polyhedrosis virus (LdNPV) and host membranes. Fluorescein isothiocyanate-labeled PDV was found to bind in a saturable manner to the gypsy moth cell line IPLB-LdEIta and to L. dispar BBMV. The IPLB-LdEIta cell line was found to possess approximately 10(6) PDV-specific receptor sites per cell. Excess levels of unlabeled PDV were highly efficient in competing with fluorescein isothiocyanate-labeled PDV for limited receptor sites, further supporting the specificity of the interaction. Major reductions in virus binding (as high as 70%) after protease treatment of cells indicated that a protein receptor is involved. A fluorescence dequenching assay of membrane fusion with octadecyl rhodamine B (R18)-labeled PDV was used to identify PDV fusion to host cells and BBMV. Direct membrane fusion of PDV occurred at 27 degrees C to both target membranes as well as at 4 degrees C at approximately 55% of the levels achieved at 27 degrees C. Viral fusion to BBMV occurred throughout the pH range of 4 to 11, with dramatically increased fusion levels (threefold) under the alkaline conditions normal for lepidopteran larval midguts. Treatment of cells with chloroquine, a lysosomotropic agent, did not significantly affect PDV fusion to cells or infectivity in tissue culture assays.

Viral and host cellular transcription in Autographa californica nuclear polyhedrosis virus-infected gypsy moth cell lines.

Infection of two gypsy moth cell lines (IPLB-Ld652Y and IPLB-LdFB) by the Autographa californica multiple-enveloped nuclear polyhedrosis virus (AcMNPV) is characterized by extremely attenuated viral protein synthesis followed by a total arrest of all viral and cellular protein production. In this study, AcMNPV- and host cell-specific transcription were examined. Overall levels of viral RNAs in infected gypsy moth cells were, at most measured times, comparable to RNA levels from an infected cell line (TN-368) permissive for AcMNPV replication. Northern blot (RNA) analyses using viral and host gene-specific probes revealed predominantly normal-length virus- and cell-specific transcripts postinfection. Transport of viral RNAs from the nucleus to the cytoplasm and transcript stability in infected gypsy moth cells also appeared normal compared with similar parameters for AcMNPV-infected TN-368 cells. Host cellular and viral mRNAs extracted from gypsy moth and TN-368 cells at various times postinfection and translated in vitro yielded similar spectra of host and viral proteins. Treatment of infected gypsy moth cells with the DNA synthesis inhibitor aphidicolin eliminated the total protein synthesis shutoff in infected IPLB-LdFB cells but had no effect on protein synthesis inhibition in infected IPLB-Ld652Y cells. The apparent selective block in the translation of viral transcripts early in infection and the absence of normal translation or transcription of host cellular genes at later times is discussed.

Production of a cellular macromolecular synthesis inhibition factor(s) in gypsy moth cells infected with the Autographa californica nuclear polyhedrosis virus

The gypsy moth cell lines IPLB-Ld652Y and IPLB-LdFB have been shown to be semipermissive for replication of the Autographa californica multiple-embedded nuclear polyhedrosis virus (AcMNPV). We report here that AcMNPV infection of these cell lines results in the production, by the infected cells, of a proteinaceous viral derived factor(s) which is secreted into the tissue culture medium (IPL-52B). Uninfected IPLB-Ld652Y and IPLB-LdFB cells, when incubated with media from AcMNPV-infected cells, exhibit markedly reduced levels of cell growth, mitosis, DNA, and protein synthesis. The factor(s), which has been designated the macromolecular synthesis inhibition factor (MSIF), is produced and secreted from infected cells between 1 and 30 hr postinfection and is produced in the absence of viral gene activity in infected cells. A preliminary characterization of the MSIF revealed the presence of a heat-labile, proteinaceous, pH sensitive molecule(s) whose activity was neutralized by three different monoclonal antibodies directed against the AcMNPV 64-kDa envelope glycoprotein. Production of the MSIF(s) did not require any new viral or cellular gene activity and was inhibited by treatment of infected cells with the lysosomal protease inhibitor, leupeptin. It is postulated that the MSIF is the AcMNPV 64-kDa glycoprotein or some component or complex of this protein, which is removed from the inoculum virus, and secreted, by the infected cells, into the tissue culture medium.

Changes in macromolecular synthesis of gypsy moth cell line IPLB-Ld652Y induced by Autographa californica nuclear polyhedrosis virus infection

The aberrant replication of the Autographa californica multiple-enveloped nuclear polyhedrosis virus (AcMNPV) in the Lymantria dispar cell line IPLB-Ld652Y was used as a model system for the investigation of factors regulating baculovirus host specificity. A previous study of this system indicates that viral gene expression in infected cells is extremely attenuated and subsequently all cellular and viral protein synthesis is inhibited. In the present study, infection of IPLB-Ld652Y cells with AcMNPV photochemically inactivated in situ resulted in a rapid reduction in cell mitotic indices and cell growth, as well as inducing a series of distinct morphological changes in these cells. At the molecular level, infection with inactivated virus, followed by pulse labelling with [3H]thymidine, resulted in a rapid [0 to 2 h post-infection (p.i.)] and permanent inhibition of host cellular DNA synthesis. Assays of cellular DNA polymerases in isolated IPLB-Ld652Y nuclei confirmed the reduction in cellular DNA synthesis observed in intact cells and indicated an initial (0 to 2 h p.i.) reduction in the activity of aphidicolin-sensitive DNA polymerases. Activity of all cellular DNA polymerases was inhibited at later times p.i. Host cell protein synthesis was completely inhibited after 48 h p.i. Treatment of inactivated virus and virus-infected cells with various chemical and physical factors (i.e. pH and temperature) or lysosomotropic agents revealed that virus entry into cells and fusion of endocytic vesicles (containing virus) with lysosomes were essential for suppression of cellular macromolecular synthesis. The possible involvement of structural components of the AcMNPV virion in these effects is discussed.

Replication of the entomopoxvirus from Amsacta moorei in serum-free cultures of a gypsy moth cell line

The entomopoxvirus isolated from Amsacta moorei (Lepidoptera: Arctiidae) replicated to the mature occluded stage during six serial passages in the serum-free cultured cells of a continuous cell line (IPLB-LD-652) from the gypsy moth, Lymantria dispar (Lepidoptera: Lymantriidae). Replication of the virus in cells grown in serum-free RIL-14 (Table 1) medium was similar to replications of the same virus observed in the same and other susceptible cell lines in various serum-containing media. Virus developmental stages are described from cells grown in serum-free medium after virus passages two and six, and from cells in vivo from gypsy moth larval fat body infected by intrahemocoelic inoculation with extracellular virus from infected cultures of the same cell line.

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.

Gypsy moth cell lines divergent in viral susceptibility. I. Culture and identification.

A series of cell lines unique in insect virus susceptibility pattern have been isolated from the ovaries of the gypsy moth (Lymantria dispar: Lepidoptera: Lymantriidae) on a synthetic medium with mammalian and avian serum supplementation. Growth curves showed the poorest growth occurring on peptone-based media with somewhat better growth on amino-acid-based media. The best growth was obtained with combined media. Serological study distinguished the present cell lines from one another and from cell lines derived from other insect species grown routinely in the same laboratory. Baculovirus susceptibility among the new lines varied from no response to a specific complete replication response upon challenge by the homologous (gypsy moth) nuclear polyhedrosis virus.