Demonstration of the protective effects of fluorescent proteins in baculoviruses exposed to ultraviolet light inactivation.

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) recombinants, namely AcRFP produced by fusion of the red fluorescent protein (RFP) gene with the polyhedrin gene, and a recombinant (pAcUW21-23GFP) carrying the green fluorescent protein (GFP) in its viral envelope, were evaluated for their resistance to inactivation by ultraviolet light. AcRFP recombinants produced incomplete polyhedra with low infectivity for Trichoplusia ni larvae, whereas AcuW21-23GFP produced normal polyhedra with high infectivity. Electron microscopy of AcRFP CL14 showed the incorporation of very few viral particles into polyhedrin matrix protein material. The LC50 for AcuW21-23GFP was 0.10 occlusion bodies/mm2, whereas the LC50 values for several AcRFP recombinants ranged from 20 to 329 occlusion bodies/mm2. When both the RFP and GFP recombinants were exposed to ultraviolet light (UV-B 280-320 nm), the results support the conclusion that these fluorescent proteins afford some protection against its damaging effects.

Establishment and characterization of insect cell lines from 10 lepidopteran species.

Cell lines from selected lepidopteran species were established for the overall purpose of use in baculovirus production. A total of 36 new cell lines from 10 lepidopteran species were generated, including cell lines from a pyralid, the European corn borer, Ostrinia nubilalis, a plutellid, the diamondback moth, Plutella xylostella, as well as eight noctuids: the black cutworm, Agrotis ipsilon, the celery looper, Anagrapha falcifera, the velvetbean caterpillar, Anticarsia gemmatalis, the corn earworm, Helicoverpa zea, the tobacco budworm, Heliothis virescens, the beet armyworm, Spodoptera exigua, the fall armyworm, Spodoptera frugiperda, and the cabbage looper, Trichoplusia ni. Tissues used for cell line establishment included fat bodies, ovaries, testes, or whole embryos/larvae/pupae. All the cell lines were subcultured numerous times, characterized by isoenzyme analysis and/or deoxyribonucleic acid amplification fingerprinting using polymerase chain reaction, and stored in liquid nitrogen. Many of the cell lines were adapted to grow in serum-free medium, with cell lines from A. ipsilon and H. virescens being adapted to suspension culture using shaker flasks. The potential use for these cell lines in baculovirus production is discussed.

Production of selected baculoviruses in newly established lepidopteran cell lines.

One key to the in vitro mass production of baculoviruses is the development of insect cell lines capable of producing high levels of extracellular virus (ECV) and/or occlusion bodies (OBs). For this study, 34 newly established cell lines from 10 lepidopteran species were screened for their ability to produce ECV and OBs from a variety of baculoviruses. The selected baculoviruses included: the alfalfa looper virus (AcMNPV); the celery looper virus (AfMNPV); the velvetbean caterpillar virus (AgMNPV), the bollworm virus (HzSNPV), the diamondback moth virus (PxMNPV), and the beet armyworm virus (SeMNPV). ECV titers were determined using TCID50 assays (50% tissue culture infectivity dose), with the presence or absence of OBs being noted. For AcMNPV, 28 new cell lines were tested, with eight producing AcMNPV ECV titers of 1.1-47.3 x 10(6) TCID50/ml and 11 producing OBs. For AgMNPV, six new cell lines were tested, with all producing AgMNPV ECV titers of 3.5-62.3 x 10(6) TCID50/ml and generating OBs. For HzSNPV, four new cell lines were tested with three lines producing HzSNPV ECV titers of 1.4-5.0 x 10(6) TCID50/ml, but none generating OBs. For PxMNPV, 10 new cell lines were tested with seven generating PxMNPV ECV titers of 4.7-232.6 x 10(6) TCID50/ml and eight producing OBs. Lastly, using qualitative or semiquantitative methods, homologous cell lines were tested for AfMNPV and SeMNPV production, all of which produced OBs. Overall, many of the cell lines tested were found to produce OBs and generate moderate to high levels of ECVs of one or more baculoviruses.

Establishment and characterization of an Ostrinia nubilalis cell line, and its response to ecdysone agonists.

A cell line derived from embryonic tissues of the European corn borer, Ostrinia nubilalis (UMC-OnE), was established in EX-CELL 401 medium containing 10% fetal bovine serum. The cells grew in suspension, and were mainly spherical in shape. The cell doubling times at the 17th and 79th passages were 56 and 36 h, respectively. DNA amplification fingerprinting showed that the DNA profile of the OnE cell line was different from that of the southwestern corn borer, Diatraea grandiosella (UMC-DgE), and that of the cotton bollworm, Helicoverpa zea (BCIRL-HZ-AM1). The OnE cell line was responsive to treatments of 20-hydroxyecdysone and the ecdysone agonists, methoxyfenozide (RH-2485) and tebufenozide (RH-5992). These compounds caused similar effects on the cells, which included cell clumping and decreased cell proliferation. The clumps were observed on the third day of incubation, and became larger after 7 d of incubation. After 168 h of incubation, methoxyfenozide and tebufenozide were 35 and 11 times more effective, respectively, in inhibiting proliferation of the OnE cells than was 20-hydroxyecdysone.

In vitro host range studies with a new baculovirus isolate from the diamondback moth Plutella xylostella (L.) (Plutellidae: Lepidoptera).

The in vitro host range of a newly isolated baculovirus from the diamondback moth Plutella xylostella was tested against six lepidopteran cell liness. Two baculoviruses with wide host ranges from the alfalfa looper Autographa californica (A. californica multiple nucleopolvhedrovirus, AcMNPV) and the celery looper Anagrapha falcifera (AfMNPV) were also included in this study for comparative purposes. PxMNPV replicated in all six cell lines and produced occlusion bodies, with HV-AM1 and TN-CL1 cells producing the highest viral titers and greatest number of occlusion bodies. There was no significant replication of AcMNPV and AfMNPV in the HZ-FB33 cell line and thus no production of occlusion bodies. The restriction endonuclease profiles of the three baculoviruses showed similarities but could be readily distinguished from each other. Either HV-AM1 or TN-CL1 would be suitable cell lines for the in vitro production of PxMNPV.

Expression of the green fluorescent protein carried by Autographa californica multiple nucleopolyhedrovirus in insect cell lines.

A recombinant AcMNPV containing the green fluorescent protein (gfp) gene under the polyhedrin promoter (polh) was used to investigate the expression of the gfp gene as well as the production of recombinant extracellular virus in 14 continuous insect cell lines, including Heliothis virescens (BCIRL-HV-AM1), Helicoverpa zea (BCIRL-HZ-AM1), Anticarsia gemmatalis (BCIRL-AG-AM1), Trichoplusia ni (TN-CL1), Spodoptera frugiperda (IPLB-SF21), Spodoptera exigua (BCIRL/AMCY-Se-E1 and BCIRL/AMCY-Se-E5), Bombyx mori (BMN), Sf9 (a clone of IPLB-SF21), and five cell line clones of BCIRL-HV-AM1. The susceptibility of the cell lines to the recombinant virus (AcMNPV.GFP) was ascertained by calculating the mean percentage number of green light-emitting cells as well as by TCID50 titration of extracellular virus with fluorescence as a sign of infection. Of the 14 cell lines tested, all were permissive with varying degrees to AcMNPV.GFP, except BCIRL-HV-AMCL2 and BCIRL-HZ-AM1, both grown in serum-containing medium, and BMN, grown in serum-free medium, which were nonpermissive to the virus. Except for BCIRL/AMCY-Se-E1, IPLB-SF21, and four of the five BCIRL-HV-AM1 clones, all the other cell lines (BCIRL-HV-AM1, BCIRL-AG-AM1, TN-CL1, Se-E5, and Sf9) expressed detectable levels of GFP by 48 h postinoculation. The BCIRL/AMCY-Se-E1 and IPLB-SF21 cells, grown in serum-free medium (Ex-Cell 401), expressed detectable levels of GFP at 72 h postinoculation. By contrast, in BCIRL/AMCY-Se-E1 in serum-containing medium (Ex-Cell 401 + 10% FBS [fetal bovine serum]), GFP was detected at 48 h postinoculation. Furthermore, TN-CL1 cells produced the largest mean percentage number of fluorescent (76.6%) cells in both serum-containing and serum-free medium (64.8%) at 120 h postinoculation. All the BCIRL-HV-AM1 clones showed no GFP expression until 96 h postinoculation, and only then about 1% of the cell population fluoresced. The mean extracellular virus (ECV) production at 120 h postinoculation was highest in BCIRL/AMCY-Se-E5 cells grown in Ex-Cell 401 + 10% FBS (37.8 x 10% TCID50/ml) followed by BCIRL-HV-AM1 in TC199-MK (33.4 x 10(6) TCID50/ml). Only the BCIRL-HV-AMCL3 clone produced any substantial level of ECV at 120 h postinoculation (16.9 x 10(6) TCID50/ml). However, there was no significant correlation between ECV production and the mean percentage number of fluorescent cells. This study provides further information on the susceptibility of 14 insect cell lines to a recombinant AcMNPV containing the green fluorescent protein gene. This information might avail researchers with information to facilitate decisions as to what other cell lines are available for in vitro studies of the gfp gene.

Infectivity studies of a new baculovirus isolate for the control of the diamondback moth (Plutellidae: Lepidoptera).

This study describes a new baculovirus isolate recovered from infected larvae of the diamondback moth, Plutella xylostella (L.), and identified as a multiple nucleopolyhedrovirus (MNPV). The plaque purified isolate designated as PxMNPVCL3 was found to be pathogenic to P. xylostella, Heliothis virescens (F.), Trichoplusia ni (Hübner), H. subflexa (Guenée), Helicoverpa zea (Boddie), Spodoptera exigua (Hübner), and S. frugiperda (J. E. Smith) larvae in decreasing order of susceptibility. The LC50 for diamondback moth, the most susceptible, was 6 occlusion bodies (OB)/cm2, whereas the most resistant species, namely S. frugiperda, was 577 OB/cm2. PxMNPVCL3 was more pathogenic to diamondback moth by 3-4 log cycles as compared with 2 broad-spectrum baculoviruses, namely Autographa california (alfalfa looper) MNPV and Anagrapha falcifera (celery looper) MNPV. The 3 baculoviruses were compared with each other and characterized by restriction endonuclease (REN) analysis, hybridization, and neutralization tests. Fragmentation profiles generated by REN showed that the 3 baculoviruses shared some fragments in common. Hybridization studies employing digoxigenin labeled PxMNPVCL3 DNA as a probe revealed the close but distinct relationship of these 3 viruses. Neutralization tests confirmed the hybridization studies, namely that the 3 viruses although genetically similar are distinguishable from each other.

The establishment of heliothine cell lines and their susceptibility to two baculoviruses.

A total of eight cell lines were established from Helicoverpa armigera (3) and H. punctigera (5) embryos and ovaries. Cell lines were established and grown in TC100 and/or TC199-MK containing 10% fetal bovine serum. The serum-free medlium ExCell 400 was also used, with and without 10% supplemental fetal bovine serum, but failed to generate cell lines from fat bodies, embryos, or ovarian tissues. Cell lines consisted of heterogenous cell types ranging from oval to fibroblast-like. This is the first report on the successful establishment of cell lines from H. punctigera. Cell lines from the two species were distinguishable from each other by DAF-PCR, and noticeable differences in minor bands were observed among cell lines from the same species. All of the established cell lines from both species were susceptible to HzSNPV but did not replicate more virus than that of a H. zea cell line (BCIRL-HZ-AM1-A11). However, an H. punctigera cell line (HP1) replicated AcMNPV to the highest titer (1.0 X 10(8) 50% tissue culture infective dose/ml), and only one of the H. armigerm cell lines (HA1) was susceptible to this virus.

In vitro and in vivo host range of Anticarsia gemmatalis multiple nuclear polyhedrosis virus.

A clone of the wild type (wt) Anticarsia gemmatalis multiple nuclear polyhedrosis virus AgMNPV, derived from a geographical isolate (Hondrina, Brazil) and designated AgMNPV-CL4-3A1, was used to determine the host range of this virus in six established lepidopteran cell lines: Anticarsia gemmatalis (BCIRL-AG-AM1), Helicoverpa zea (BCIRL-HZ-AM1), Heliothis virescens (BCIRL-HV-AM1), Helicoverpa armigera (BCIRL-HA-AM1), Trichoplusia ni (TN-CL1), Bombyx mori (BMN), and a coleopteran cell line Anthonomus grandis (BRL-AG-1). In addition, the in vivo host range of this clone was also assayed in larvae of Helicoverpa zea, Heliothis virescens, Trichoplusia ni, and the homologous species Anticarsia gemmatalis by probit analysis. On the basis of temporal studies of TCID50 values, BCIRL-HV-AM1 cells gave the highest extracellular virus (ECV) titer (9.7 x 10(6) TCID50/ml) followed by BCIRL-HA-AM1 cells (8.3 x 10(5) TCID50/ml) and BCIRL-AG-AM1 cells (3.2 x 10(5) TCID50/ml). In addition, a low ECV titer of 1.37 x 10(3) TCID50/ml was detected from TN-CL1 cells 96 h postinoculation, while BRL-AG-1, BMN, and BCIRL-HZ-AM1 cells were nonpermissive to AgMNPV-CL4-3A1 on the basis of TCID50 results. AgMNPV-CL4-3A1 and the wild type AgMNPV had similar restriction profiles that were different from wild type AcMNPV. The LC50 values were 96.9, 564.6, 733.3, and 1.1 x 10(4) occlusion bodies/cm2 of diet for A. gemmatalis, Helicoverpa zea, Heliothis virescens, and T. ni, respectively.

Identification of insect cell lines by DNA amplification fingerprinting (DAF).

Fingerprint profiles were generated from twenty insect cell lines spanning the Orders, Lepidoptera, Diptera, Coleoptera and Homoptera employing DNA amplification fingerprinting (DAF) with arbitrarily selected primers. The fingerprint pattern is a stable characteristic of the cell line because high and low passages generated the same profile. In addition, insect hosts and homologous cell lines generated similar profiles. All cell lines could be distinguished from each other with the following exceptions: Plutella xylostella (BCIRL-PX2-HNU3) and Mamestra brassicae (IZD-MB-0503) produced identical patterns to Trichoplusia ni (TN-CL1). Also Spodoptera exigua (UCR-SE-1C) produced the same profile as Spodoptera frugiperda (SF9) and the parent cell line IPLB-SF21. All these cases of identical patterns are believed to be due to cross contamination or mislabelling of cultures. DAF will serve as an additional, valuable and reliable technique for the identification of insect cell lines.

Expression of beta-galactosidase and luciferase in insect cell lines infected with a recombinant AcMNPV.

An AcMNPV recombinant (Ac-gal-luc) carrying the beta-galactosidase and luciferase genes under the control of the p10 and polyhedrin promoters, respectively, was used to study expression in nine insect cell lines. All AcMNPV-permissive cell lines expressed both reporter genes with the coleopteran cell line, Anthonomus grandis (AGE), producing the highest concentrations of beta-galactosidase (5.0 x 10(6) pg/ml) and luciferase (2.67 x 10(3) pg/ml). Both enzymes were detected as early as 12 h postinoculation in lysate samples of the AGE cell line. Helicoverpa armigera (HA), a nonpermissive cell line, expressed beta-galactosidase at 72 h postinoculation at a concentration of 3.5 x 10(3) pg/ml. However, expression of luciferase was not detected. Expression of luciferase and beta-galactosidase was also not detected in the nonpermissive Helicoverpa zea (HZ) cell line.

Replication of Autographa californica baculovirus (AcMNPV) in a coleopteran cell line.

A coleopteran cell line (AGE) derived from the cotton boll weevil Anthonomus grandis supported replication of Autographa californica multiple nuclear polyhedrosis virus (AcMNPV). The titer of extracellular virus (ECV) and the number of occlusion bodies (OB) produced in AGE cells were approximately equal to those produced by a Trichoplusia ni cell line (TN-CL1), and the OB produced by both cell lines were equally infectious for T. ni larvae. The identity of the AGE cell line was established by chromosome and isoenzyme analyses.

Replication of Heliothis zea nuclear polyhedrosis virus in cloned cell lines.

Clones from two Heliothis zea (Lepidoptera:Noctuidae) ovarian cell lines, BCIRL-Hz-AM1 (AM1) and BCIRL-Hz-AM3 (AM3), were generated and their ability to produce H. zea nuclear polyhedrosis virus (HzSNPV) was compared. Titers of extracellular virus (ECV) ranged from 5.5 (AM3-F9) to 44.9 x 10(5) PFU/ml (AM1-A4), with the parental cell lines AM3 and AM1 producing 14.8 and 26.4 x 10(5) PFU/ml, respectively. Concentrations of polyhedral inclusion bodies (PIB) produced by the cloned lines ranged from 0.7 (AM3-F9) to 59.6 x 10(6) PIB/ml (AM1-B3), while the parental cell lines generated 6.5 (AM3) and 12.9 x 10(6) PIB/ml (AM1). The percentage of cells from the cloned lines that produced PIB ranged from 39 to 86.4% for AM3-F9 and AM1-A7, respectively, with the parental lines exhibiting 49.1% (AM3) and 75.3% (AM1) cells with PIB. The number of PIB per cell also differed markedly between cell lines, varying from 18.3 (AM3-F9) to 184.4 (AM1-B3) PIB/cell. The parental lines produced 57.3 (AM3) and 75.9 (AM1) PIB/cell. Thus, significant differences were seen in virus production (ECV, PIB) between parental cell lines, as well as between parental cell lines and their clones. In addition, cell lines were characterized with regard to their growth rates and isoenzyme patterns.

Digestive proteinases of larvae of the corn earworm, Heliothis zea: characterization, distribution, and dietary relationships.

Proteinases and peptidases from the intestinal tract of fifth-instar larvae of Heliothis (= Helicoverpa) zea (Boddie) (Lepidoptera:Noctuidae) were characterized based on their substrate specificity, tissue of origin, and pH optimum. Activity corresponding to trypsin, chymotrypsin, carboxypeptidases A and B, and leucine aminopeptidase was detected in regurgitated fluids, midgut contents, and midgut wall. High levels of proteinase activity were detected in whole midgut homogenates, with much lower levels being observed in foregut and salivary gland homogenates. In addition, enzyme levels were determined from midgut lumen contents, midgut wall homogenates, and regurgitated fluids. Proteinase activities were highest in the regurgitated fluids and midgut lumen contents, with the exception of leucine aminopeptidase activity, which was found primarily in the midgut wall. Larvae fed their natural diet of soybean leaves had digestive proteinase levels that were similar to those of larvae fed artificial diet. No major differences in midgut proteinase activity were detected between larvae reared under axenic or xenic conditions, indicating that the larvae are capable of digesting proteins in the absence of gut microorganisms. The effect of pH on the activity of each proteinase was studied. The pH optima for the major proteinases were determined to be pH 8.0-8.5 for trypsin, when tosyl-L-arginine methyl ester was used as the substrate; and pH 7.5-8.0 for chymotrypsin, when benzoyl-L-tyrosine ethyl ester was used as the substrate.

Restriction endonuclease cleavage patterns of commercial and serially passaged isolates of Heliothis baculovirus.

The restriction endonuclease patterns of three Baculovirus isolates (Br, Vh, El) from Heliothis zea having different passages and production histories were compared. Digestion of the ds DNA genomes of the three isolates with EcoRI, HindIII, and XhoI showed no major difference in the cleavage patterns, although submolar fragments were detected. One of the commercial isolates (Vh) was serially passaged 20 times through larvae of H. zea and a substitute host, H. virescens. EcoRI cleavage profiles of the ds DNA showed the absence of a band in the 3-megadalton region that was present in the original isolates. In addition, the cleavage pattern of the DNA from Vh passed in H. zea showed additional submolar fragments in the 15- and 6-megadalton regions. Differences also were observed in the HindIII and XhoI restriction patterns. No significant differences in virulence between the original isolate and passage isolates were detected after 20 serial passages in larvae of either H. zea or H. virescens.

In vitro host range of five baculoviruses in lepidopteran cell lines.

The in vitro host range of five nuclear polyhedrosis viruses (NPV) was assessed in five lepidopteran cell lines from three genera. Multiple-enveloped baculoviruses of Autographa californica (ACMNPV), Trichoplusia ni (TNMNPV), and Galleria mellonella (GMMNPV) replicated in cells of T. ni, Spodoptera frugiperda, and Heliothis virescens to a titer of approximately 10(7) TCID50/ml. The multiple-enveloped baculovirus of S. frugiperda (SFMNPV) replicated only in S. frugiperda cells. The single-enveloped baculovirus of H. zea (HZSNPV) replicated in cells of H. zea and H. virescens but not in cells of H. armigera, T. ni, or S. frugiperda. Low levels of replication of ACMNPV, TNMNPV, and SFMNPV in cultures of H.zea, H. virescens, and T. ni, respectively, could not be detected by using a sensitive tritiated thymidine technique. However, two characteristically labelled peaks at densities of 1.145 and 1.245 g/ml were obtained in H. virescens cells inoculated with ACMNPV.60 min postinoculation ACMNPV particles were observed both entering and inside S. frugiperda cells but were not observed in H. zea or H. armigera cells. None of the five baculoviruses replicated in H. armigera cells.

Glycoproteins of nuclear polyhedrosis viruses.

The glycopeptides and glycoproteins of two nuclear polyhedrosis viruses (NPVs) grown in two lepidopteran insect cell lines were studied using Sephadex elution chromatography and polyacrylamide gel electrophoresis. No quantitative or qualitative differences were observed in the glycopeptide and glycoprotein patterns between the viruses grown in either cell line. However, both viruses synthesized a glycoprotein which was not observed in either insect cell line and may be virus-directed.

Biological studies of a baculovirus in a mammalian cell line.

In two of five experiments, replication (TCID50) was observed following inoculation of Chinese hamster cells with Autographa californica nuclear polyhedrosis virus (NPV). Radiolabeling experiments employing tritiated thymidine consistently resulted in a labeled entity which banded in sucrose gradients at a density of 1.24-1.25 g/ml, similar to that observed for A. californica NPV in the permissive Trichoplusia ni cell line. The labeled entity is believed to be A. californica NPV, based on infectivity and serological data. The infectious center assay revealed that at an MOI of 3 only 0.38% of Chinese hamster cells was infected compared with 28% for cabbage looper cells. However, 4.2% of Chinese hamster cells was infected at an MOI of 41.

Baculovirus replication in a mosquito (dipteran) cell line.

The baculovirus from the lepidopteran host Autographa californica (alfalfa looper) was shown to replicate in a dipteran cell line without the production of characteristic polyhedral inclusion bodies. The low level of replication could not be detected by 50% tissue culture infective dose titrations, but was apparent by [3H]thymidine labeling of the viral genome. Immunoprecipitation of the radioactive product confirmed baculovirus production.