Susceptibility of Newly Established Cell Lines from ... - J-Stage

Journal of Insect Biotechnology and Sericology 77, 25-34 (2008)

Susceptibility of Newly Established Cell Lines from Helicoverpa armigera to Homologous and Heterologous Nucleopolyhedroviruses Javier Gordon Ogembo1, Sudawan Chaeychomsri2, Barbara L. Caoili1,3, Motoko Ikeda4 and Michihiro Kobayashi1,＊ 1

Laboratory of Biodynamics and 4 Laboratory of Sericulture and Entomoresources, Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya 464-8601, Japan, 2 Central Laboratory and Greenhouse Complex, Kasetsart University, Nakhon Pathom 73140, Thailand, and 3 Crop Protection Cluster, College of Agriculture, University of the Philippines Los Baños, College, Laguna 4031, Philippines (Received July 31, 2007; Accepted August 20, 2007)

A total of five new cell lines were established from Helicoverpa armigera; including KU-HaEmb1 (HaEmb1) and KU-HaEmb2 (HaEmb2) from embryos, KU-HaPO1 (HaPO1) and KU-HaPO2 (HaPO2) from pupal ovaries, and KU-HaAO1 (HaAO1) from adult ovaries. These cell lines were examined for their permissiveness for homologous H. armigera nucleopolyhedrovirus (HearNPV) and seven heterologous NPVs. Upon infection with HearNPV, the cell line HaAO1 yielded the highest productivity in terms of amounts of polyhedra, budded virus (BV), polyhedrin and viral DNA among the six cell lines, including the five newly established cell lines and the cell line from Helicoverpa zea (BCIRL-Hz-AM1), which is commonly used for in vitro studies and production of HearNPV. The cell lines HaPO1, HaPO2 and HaEmb2 were also permissive for HearNPV but they yielded lower productivity than that of the cell line HaAO1. Only the cell line HaEmb1 was nonpermissive for HearNPV infection among the cell lines established. Examination with seven heterologous NPVs showed that all the new cell lines continued to proliferate without any cytopathic effects (CPE), following infection with five NPVs from Bombyx mori, Hyphantria cunea, Lymantria dispar, Orgyia pseudotsugata and Spodoptera litura. Upon infection with NPVs from Autographa californica (AcMNPV) and Spodoptera exigua (SeMNPV), all the new cell lines, with the exception of HaAO1, manifested CPE to varying degrees, synthesizing significant amount of viral DNA. These cell lines, with the exception of AcMNPV-infected HaPO1 that produced significant amount of BVs, yielded negligible or very low level of BVs and polyhedrin following infection with AcMNPV and SeMNPV, indicating that they were semipermissive for these two NPVs. These results indicate that the newly established H. armigera cell line HaAO1 provide a promising candidate for in vitro production of HearNPV biopesticide, as well as a useful system for the analysis of molecular mechanisms of NPV-cell interactions. Key words: Helicoverpa armigera, African bollworm, cell line establishment, baculovirus, nucleopolyhedrovirus, HearNPV, permissiveness

INTRODUCTION Helicoverpa spp. (Lepidoptera: Noctuidae) larvae are serious polyphagous pests for a variety of important agricultural and ornamental plants causing extensive economic losses worldwide (Zalucki et al., 1986, 1994; King, 1994). The larvae of Helicoverpa spp, which include three main agricultural pests, H. armigera, H. zea and H. punctigera (King, 1994), have been reported to develop resistance to chemical pesticides, reducing quality and quantity of products and profitability of farmers by increasing the cost of pesticides. A number of nucleopolyhedroviruses (NPVs) have been isolated from these insect species in different geographical regions. These NPVs have been genetically characterized to be closely related, causing natural epizootics against Helicoverpa spp. (Gettig and McCarthy, 1982; Chen et al., 2001; Zhang et al., 2005; Ogembo et al., 2007), and offer an attractive strategy for *To whom correspondence should be addressed. Fax: +81-52-789-4036. Tel: +81-52-789-4038. Email: [email protected]

the control of Helicoverpa spp. complex with minimal adverse environmental effects due to their high pathogenicity and virulence, restricted host range, and absence of toxic residues in grains, fruits, vegetables and flowers (Moore et al., 2004). The NPVs isolated from H. armigera (HearNPV) and H. zea (HzNPV) have been developed and widely used as commercial biopesticides to control host insects (HunterFujita et al., 1998). These viruses are being produced in vivo and registered under different trade names (Viron-H, Biocontrol-VHZ, Elcar™, Gemstar LC, Vivus) in various countries (Hunter-Fujita et al., 1998). However, in vivo production systems lack the capacity to support wide scale demand for the biopesticides. They also have limitations in terms of quality control of virus products necessary for practical commercialization of a biopesticide. These limitations have necessitated the development of efficient in vitro systems for the propagation of these viruses. In an effort to develop HearNPV as an effective biopesticide, we have cloned NPVs from uncloned populations isolated from H. armigera larvae collected in different geographical regions of Africa and have biologically cha-

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racterized these HearNPV clones, employing the cell line Hz-AM1 derived from H. zea (Ogembo et al., 2007). The cell line Hz-AM1 has been used widely to examine possible factors affecting the yields and the potency of HearNPV. These factors included medium, supplemented serum, cell density at infection, multiplicity of infection, viral strain and passage effect (Chakraborty et al., 1996, 1999; Chakraborty and Reid, 1999; Lua and Reid, 2000; Lua et al., 2002; Ogembo et al., 2007). These studies revealed that one of the most serious limitations on the optimal production of HearNPV in the cell line Hz-AM1 was rapid formation of few polyhedra mutants during serial passage, producing polyhedra and budded viruses (BVs) of low virulence (Chakraborty and Reid, 1999). The type and degree of the passage effect are dependent on the cell lines, the virus species and the combination of cell lines and virus species (Krell, 1996). Homologous cell lines are desirable for the production of HearNPV, since heterologous NPV infection of cell lines or individual insects decreases the productivity, and yields less virulent progeny viruses compared with those from the homologous infection (Tompkins et al., 1981, 1988). Several cell lines have been established from H. armigera (McIntosh and Ignoffo, 1981; McIntosh et al., 1999; Sudeep et al., 2002), but replication profiles of HearNPV in the homologous cell line have not been well-characterized. In this study, we established five H. armigera cell lines and tested for their permissiveness for HearNPV and seven heterologous NPVs, including Autographa californica multicapsid (M) NPV (AcMNPV), Bombyx mori NPV (BmNPV), Hyphantria cunea MNPV (HycuMNPV), Lymantria dispar MNPV (LdMNPV), Orgyia pseudotsugata MNPV (OpMNPV), Spodoptera exigua MNPV (SeMNPV) and Spodoptera litura MNPV (SpltMNPV). Based on the data on BV yields, polyhedra formation, viral DNA replication, capsid protein synthesis and polyhedrin production, we show that four of the five newly established H. armigera cell lines, with the exception of the cell line HaEmb1 from embryos, are permissive for HearNPV, and that the cell line HaAO1 from adult ovaries has superior characteristics in terms of the virus productivity. It is also shown that these established cell lines are semipermissive for AcMNPV and SeMNPV, and nonpermissive for BmNPV, HycuMNPV, LdMNPV, OpMNPV and SpltMNPV.

MATERIALS AND METHODS Establishment and maintenance of cell lines Primary cell cultures were initiated from embryos, and pupal and adult ovaries of H. armigera from the laboratory colony of Central Laboratory and Greenhouse Complex, Kasetsart University, Thailand. Tissue dissections

were carried out as described previously (Goodman et al., 2001). In brief, eggs, pupae and adults used for initiating cell cultures were surface sterilized by submersion in 70% ethanol for 5 min and dried under a stream of clean air in a laminar flow cabinet. Ovaries were aseptically removed from pupae or newly emerged moths, minced and dissociated in collagenase. For embryonic tissues, the explants were obtained from the eggs that were crushed and dissociated in collagenase. The primary cultures were maintained in TC100 medium (JRH Biosciences) supplemented with 10% fetal bovine serum (FBS) and 3% hemolymph from H. armigera larvae in the 12.5 cm2 culture flasks (Falcon) and incubated at 27°C. Initially, half of the medium was replaced with fresh medium weekly. Viruses Clonal isolates of eight different NPVs were used in these experiments. These NPVs included HearNPV NNg1 from the African bollworm, H. armigera, (HearNPV; Ogembo et al., 2007); AcMNPV E2 from the fall armyworm, A. californica, (AcMNPV; Smith and Summers, 1978); BmNPV N9 from the silkworm, B. mori, (BmNPV; Nagamine et al., 1989); HycuMNPV N9 from the fall webworm, H. cunea, (HycuMNPV; Kamiya et al., 2003); LdMNPV from the gypsy moth, L. dispar, (Slavicek et al., 1996); OpMNPV from the Douglass-fir tussock moth, O. pseudotsugata, (Leisy et al., 1986); SeMNPV G3 from the beet armyworm, S. exigua, (SeMNPV; Wu et al., 2000); and SpltMNPV P7 from the common cutworm, S. litura, (SpltMNPV; Laviña et al., 2001). Cell lines and cell culture Five newly established H. armigera cell lines, KUHaEmb1 (HaEmb1) and KU-HaEmb2 (HaEmb2) from embryos, KU-HaPO1 (HaPO1) and KU-HaPO2 (HaPO2) from pupal ovaries, and KU-HaAO1 (HaAO1) from adult ovaries, were used in these experiments. The cell line BCIRL-Hz-AM1 (Hz-AM1; McIntosh and Ignoffo, 1981) was employed as the reference cell line. The cell lines Sf9 from Spodoptera frugiperda and Se301 from S. exigua (Hara et al., 1995) were used for the plaque assays of AcMNPV and SeMNPV, respectively. The newly established H. armigera cell lines as well as the cell lines HzAM1 and Sf9 were cultured in TC100 medium and the cell line Se301 was maintained in IPL41 medium (Gibco BRL, Grand Island, NY). These culture media were supplemented with 10% FBS and the cells were maintained at 28°C. Virus infection, sample preparation for budded virus titration, and slot-blot hybridization and immunoblot analyses Samples for budded virus (BV) titration, slot-blot

Susceptibility of new Helicoverpa armigera cell lines to NPVs

hybridization analysis of viral DNA and immunoblot analysis of polyhedrin and VP39 proteins were prepared from the cell culture in a single flask at each time postinfection (pi), as described previously (Laviña et al., 2001). In brief, 1 × 106 of cells from respective cell lines were seeded in 12.5 cm2 culture flasks, infected with each of the eight different NPVs at a multiplicity of infection (MOI) of 5 plaque-forming units (PFU) per cell, and cultured in 2 ml of TC100 medium. At 24 h intervals, 500 μl aliquots of the culture medium were harvested and stored at −80°C for BV titration. The infected cells were scraped into the culture media by a rubber policeman and 100 μl aliquots of the cell suspensions were transferred into microcentrifuge tubes for slot-blot hybridization analysis. The remaining cell suspensions were pelleted at 3000 rpm for 5 min at 4°C and the resulting pellets were lysed in 100 μl of sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% (w/v) SDS, 5% (v/v) 2-mercaptoethanol, 10% glycerol) for SDS-polyacrylamide gel electrophoresis (PAGE), followed by immunoblot analysis. Viral DNA isolation and slot-blot hybridization analysis Slot-blot hybridization analysis of viral DNAs from HearNPV, AcMNPV and SeMNPV were carried out as described previously (Ikeda and Kobayashi, 1999; Laviña et al., 2001). The aliquots of virus-infected cells were pelleted at 3000 rpm for 5 min and suspended in 100 μl of pure water. The suspended cells were treated with 81 μl of heat supersaturated sodium iodide and boiled for 10 min before chilling on ice for 5 min. The chilled cells were blotted onto the Hybond-N+ nylon membranes (Amersham Biosciences) and hybridized with probes for viral DNA detection. The probes used for the detection of viral DNAs were prepared from a portion of viral DNA polymerase gene for both HearNPV and SeMNPV and GP64 gene for AcMNPV. The probes were amplified by PCR using the specific primers and genomic DNAs from HearNPV, AcMNPV and SeMNPV as templates. The specific paired primers used for PCR amplification were; 5’-ACGAT ACGATTTGAGTCCGG-3’ and 5’-CGCATAAAC AACGGCATGCT-3’ for HearNPV, 5’-CAGTGGG TCAAAGGC-3’ and 5’-GACACGCGAACACTG-3’ for AcMNPV and 5’-CTTCGTCGTCGTCGTCGTCTG-3’ and 5’-GATTACATTTGGGACACGCGC-3’ for SeMNPV. The primers were labelled by Fluorescein Gene Images (Amersham Biosciences), and the hybridized fluorescein-labelled probes were detected by Gene Images CDP-Star detection module (Amersham Biosciences). Immunoblot analysis Proteins from the infected cells were resolved on SDS-

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polyacrylamide gels, transferred onto a nitrocellulose membrane (Advantec Toyo, Tokyo, Japan) or an Immobilon transfer membrane (Millipore, Bedford, MA) and processed for immunoblot analysis as described previously (Shirata et al., 1999; Laviña et al., 2001). The polyhedrin and the major capsid protein VP39 were probed with antiBmNPV polyhedrin antibody and anti-AcMNPV/BmNPV VP39 (anti-Ac/BmVP39) antibody, respectively, as the primary antibodies and goat anti-rabbit IgG antibody conjugated with horseradish peroxidase (Zymed Laboratories, San Francisco, CA) as the secondary antibody. Positive signals for polyhedrin and VP39 proteins were visualized by Konica Immunostain HRP-1000 (Konica, Osaka, Japan) and ECL Western blotting detection reagents (Amersham Biosciences), respectively. The antibodies against BmNPV polyhedrin and Ac/BmVP39 protein were raised in rabbits, using purified BmNPV polyhedrin and a partial amino acid sequence of VP39 protein that was common for AcMNPV and BmNPV (262-QPNRLQIRNVLKFEGDT-278), respectively (Shirata et al., 1999; Katou et al., 2006). Dr. Western (Oriental Yeast, Tokyo, Japan) and BenchMark Prestained protein ladder (Invitrogen) were used as the molecular size marker. Budded virus titration BVs in the culture medium of infected cells were titrated by the plaque assay as described previously (Shirata et al., 1999; Laviña et al., 2001) in the cell lines Hz-AM1, Sf9 and Se301 for HearNPV, AcMNPV and SeMNPV, respectively.

RESULTS Characteristics of the established cell lines Most cultured tissues of H. armigera began to attach to the flask after 1 day of incubation. The primary cultures reached confluence after a month. The first subculture was done when cells in the culture flask became confluent. Subcultures were carried out every 5 days by detaching the cells from the bottom of the flask, and the floated cells were transferred to a new flask. Once the cells had been cultured for 6 months, the concentration of H. armigera hemolymph was gradually reduced and deleted. Cell growth was slow at the initial stages, but improved with increase in the number of cell passages. All the cell lines attached strongly onto the bottom of the flask except for the cell line HaAO1, in which the seeded cells attached strongly but proliferated cells attached loosely, some of which floated in the medium. The HaPO2 cells aggregated at the initial stages, but they grew singly as the number of passages increased. All the cell lines were heterogeneous in nature, comprised of spherical, spindle, tadpole-shaped and irregular cells. They also varied in sizes (Fig. 1).

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Cytopathology and polyhedra formation in the newly established H. armigera cell lines infected with HearNPV The five new H. armigera cell lines, together with HzAM1, were infected with HearNPV at an MOI of 5 PFU/ cell and examined for their cytopathic effects (CPE) and polyhedra formation at 12 h intervals until 96 h pi (Fig. 1; Table 1). Upon infection with HearNPV, all the newly established cell lines, except for the cell line HaEmb1 from embryos, showed CPE with polyhedra formation, indicating that four of the five established cell lines were permissive for HearNPV. Among the permissive cell lines, the cell line HaAO1 from adult ovaries was most suscep-

tible to HearNPV infection, in which polyhedra were first observed at 24 h pi and the cells containing polyhedra increased rapidly to about 58 and 89% of total cells at 36 and 60 h pi, respectively. At 96 h pi, some of the infected HaAO1 cells fractured (Fig. 1). In the other established cell lines, HaPO1, HaPO2 and HaEmb2, polyhedra were not observed at 24 h pi, but were clearly visible by 36 h pi, indicating that the polyhedra formation was delayed by about 12 h in these cell lines compared with that in the cell line HaAO1. Also, ultimate proportion of cells with polyhedra to the total number of cells in the cell lines HaPO1, HaPO2 and HaEmb2 was lower than that in the cell line HaAO1, in-

Fig. 1. Cytopathology and polyhedra formation in the five newly established H. armigera cell lines and the cell line Hz-AM1 following infection with HearNPV. The cell lines were infected with viruses at an MOI of 5 PFU per cell and examined at 12 h intervals under a microscope. The bar indicates 100 µm. Table 1. Replication of HearNPV in the newly established H. armigera cell lines1 Cell line HaAO1 HaEmb1 HaEmb2 HaPO1 HaPO2 Hz-AM1 1

0 − − − − − −

24 + − − −+ −+ +

36 +++ − + + + ++

Hours postinfection 48 60 +++ ++++ − − + + ++/ap +++/ap ++ ++ ++ +++

72 ++++ − + +++/ap ++ +++

96 ++++ − ++ +++/ap +++ ++++

Cytopathic effects (CPE), polyhedra formation and budded virus yields were examined at intervals until 96 h postinfection − No CPE apparent under a light microscopy. −+ CPE observed but no clear polyhedra formation observed in the infected cells. + Complete replication with formation of polyhedra in 0 - 24% of the total cells. ++ Complete replication with formation of polyhedra in 25 - 49% of the total cells. +++ Complete replication with formation of polyhedra in 50 - 74% of the total cells. ++++ Complete replication with formation of polyhedra in > 75% of the total cells. ap Apoptosis observed in the infected cells.


dicating that the proportion of cells refractory to HearNPV infection was higher in the cell lines HaPO1, HaPO2 and HaEmb2 than in the cell line HaAO1. The cell line HaPO1 showed signs of apoptosis at 48 h pi and underwent severe apoptosis by 96 h pi (Fig. 1). The cell line HaEmb1 infected with HearNPV continued to proliferate and reached confluence without any appreciable CPE. In the cell line Hz-AM1 infected with HearNPV, polyhedra formation was first detected at 24 h pi, as in the cell line HaAO1. However, the proportion of cells with polyhedra was much lower than that in the cell line HaAO1, showing that 58 and 38% of the infected cells at 36 h pi had polyhedra in the cell lines HaAO1 and Hz-AM1, respectively. Examination under a microscope also showed that individual cells from the cell lines HaAO1, HaPO1 and HaPO2 produced more polyhedra compared with those from the cell lines HaEmb2 and Hz-AM1 (Fig. 1). BV yields in the newly established H. armigera cell lines infected with HearNPV BV yields in the culture medium of infected cells were determined by the plaque assay (Fig. 2). The BV titers in the cell lines HaAO1, Hz-AM1, HaPO1, HaPO2 and HaEmb2 increased sharply between 0 and 48 h pi and reached a plateau of 6.6 × 106, 6.3 × 106, 5.9 × 105, 5.5 × 105, and 3.4 × 103 PFU/ml of culture medium, respectively, at 72 h pi. The cell line HaEmb1 showed no detectable increase in BV titer throughout the experiment.

Fig. 2. Budded virus yields in the five newly established H. armigera cell lines and the cell line Hz-AM1 following infection with HearNPV. Cells from respective cell lines were seeded in culture flasks and infected with HearNPV. The infected cells were washed twice with TC100 medium after a 60 min adsorption period at room temperature and cultured in 2 ml of fresh TC100 medium at 28°C. Culture media were harvested at 0, 24, 48, 72 and 96 h postinfection, and examined for virus titers by the plaque assay in the cell line Hz-AM1. The vertical bars indicate the standard deviations of the means from three independent determinations.

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Production of viral DNA and polyhedrin in the newly established H. armigera cell lines infected with HearNPV Viral DNA production in HearNPV-infected cells was examined by slot-blot hybridization analysis (Fig. 3). The amount of viral DNA accumulated in the infected cells varied depending on the cell lines throughout the experiment. The viral DNA was first detected at 24 h pi in all the established cell lines, although only a very low or an undetectable amount of viral DNA was detected in the cell lines HaEmb1, HaEmb2 and HaPO2. At 24 h pi, the cell line HaAO1 produced the highest amount of viral DNA, which was comparable to that in the cell line HzAM1, and the cell line HaPO1 had less viral DNA compared with those in the cell lines HaAO1 and Hz-AM1. The viral DNA in the cell lines HaEmb2 and HaPO2 increased continuously from 24 to 96 h pi. The cell line HaEmb1 produced a significant amount of viral DNA at 48 h pi, without any detectable production of BVs and polyhedra. The viral DNA in the HearNPV-infected HaEmb1 decreased strikingly at 72 h pi. Polyhedrin production of HearNPV-infected cells was examined by immunoblot analysis using an antibody against BmNPV polyhedrin (Fig. 4). Following infection with HearNPV, four of the five established H. armigera cell lines, excluding the cell line HaEmb1, produced a significant amount of polyhedrin. In those cell lines, polyhedrin was first detected at 24 or 48 h pi, and increased with time pi until 96 h pi. The amount of polyhedrin produced was highest in the cell line HaAO1, followed by the cell line HaPO1. A significant but much smaller amount of polyhedrin was produced in the cell lines HaEmb2 and HaPO2. In the cell line Hz-AM1, the amount of polyhedrin produced was very low at 24 h pi but increased gradually to a level comparable to that of the cell line HaAO1 at 96 h pi. Permissiveness of newly established cell lines for heterologous NPVs The five newly established cell lines, as well as the cell line Hz-AM1, were infected with seven different heterologous NPVs, AcMNPV, BmNPV, HycuMNPV, LdMNPV, OpMNPV, SeMNPV and SpltMNPV at an MOI of 5, and examined for cytopathology (Table 2). AcMNPV and SeMNPV manifested CPE in four of the five newly established cell lines. Upon infection with AcMNPV, CPE was observed clearly at 24 h pi in all the new cell lines, except in the cell line HaAO1, and became more severe with time pi. These AcMNPV-infected cell lines, however, produced neither polyhedra nor polyhedrin even at 96 h pi (data not shown). In addition, the plaque assay showed that the BV titer in the medium from the infected culture increased significantly only in the cell line HaPO1

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(Fig. 5A). Examination by slot-blot hybridization analysis showed that AcMNPV synthesized viral DNA in all the five new cell lines (Fig. 5B), although the viral DNA remained at a very low level in the cell line HaAO1 that supported HearNPV replication to the highest titer. The highest amount of viral DNA was detected in the cell line HaPO1, followed by the cell lines HaEmb1, HaPO2 and HaEmb2 in decreasing order. The cell line Hz-AM1 produced viral DNA similar in amount to that in the cell line HaPO1 at 72 and 96 h pi, although the amount of viral DNA in the cell line Hz-AM1 at 24 and 48 h pi was significantly lower than that in the cell line HaPO1. To determine if the viral DNA replication was followed by the synthesis of viral structural proteins in those cell

lines infected with AcMNPV, the major capsid protein VP39 was examined by immunoblot analysis at different time pi. The cell lines HaPO1, HaPO2 and HaEmb2, as well as Hz-AM1, produced VP39 protein following infection with AcMNPV, yet the amount of VP39 in HaEmb2 was extremely low (Fig. 5C), while no detectable VP39 was observed in the cell lines HaEmb1 and HaAO1 under the conditions used (Fig. 5C). Upon infection with SeMNPV, the cell lines HaEmb1, HaEmb2, HaPO1 and HaPO2 manifested mild CPE at

Fig. 3. Slot-blot hybridization analysis of viral DNA in the five newly established H. armigera cell lines and the cell line Hz-AM1 following infection with HearNPV. Viral DNAs were isolated from the infected cells at 0, 24, 48, 72 and 96 h postinfection in heat-supersaturated NaI and blotted onto a Hybond-N+ nylon membrane. The viral DNAs were hybridized with the probe prepared from a portion of HearNPV DNA polymerase gene and labeled by Gene Images random prime labeling module (Amersham), and visualized by Gene Images CDP-Star detection module. For the viral infection, see the legend to Fig. 2.

Fig. 4. Immunoblot analysis of polyhedrin protein in the five newly established H. armigera cell lines and the cell line HzAM1 following infection with HearNPV. At 24, 48, 72 and 96 h postinfection, polypeptides corresponding to 8 x 104 cells at infection were resolved on a 12% SDS-polyacrylamide gel and processed for immunoblot analysis using the antibody against BmNPV polyhedrin as the primary antibody. The polyhedrin reacted with goat anti-rabbit IgG antibody conjugated with horseradish peroxidase was visualized by Konica Immunostain HRP-1000. For the viral infection, see the legend to Fig. 2.

Table 2. Replication of heterologous NPVs in the newly established H. armigera cell lines1 Virus Autographa californica MNPV Spodoptera exigua MNPV Lymantria dispar MNPV Spodoptera litura MNPV Hyphantria cunea MNPV Bombyx mori NPV Orgyia pseudotsugata MNPV Helicoverpa armigera NPV 1

HaAO1 − − − − − − − ++++

HaEmb1 −+ −+ − − − − − −

Cell line HaEmb2 HaPO1 −+ −+ −+ + − − − − − − − − − − ++ +++/ap

HaPO2 −+ ++ − − − − − +++

Cytopathic effects (CPE), polyhedra formation and budded virus yields were examined at 96 h post infection. − No CPE apparent by visual inspection under a light microscopy. −+ CPE observed but no clear polyhedra formation observed in the infected cells. + Complete replication with formation of polyhedra in 0 - 24% of the total cells. ++ Complete replication with formation of polyhedra in 25 - 49% of the total cells. +++ Complete replication with formation of polyhedra in 50 - 74% of the total cells. ++++ Complete replication with formation of polyhedra in > 75% of the total cells. ap Apoptosis observed in the infected cells.

Hz-AM1 − −+/ap − − − − − ++++


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Fig. 5. Budded virus yields, and accumulations of viral DNA and major capsid protein VP39 in the five newly established H. armigera cell lines and the cell line Hz-AM1 following infection with AcMNPV. Cells from respective cell lines were infected with AcMNPV and sampled for the analyses at 0, 24, 48, 72 and 96 h postinfection. The budded viruses were titrated by the plaque assay in Sf9 cells (A). Viral DNA was analyzed by the slot-blot hybridization analysis, using a fluorescein-labeled probe prepared from a portion of AcMNPV gp64 gene (B). The capsid protein VP39 was analyzed by the immunoblot analysis using the antibody against AcMNPV/BmNPV VP39 protein and the VP39 protein reacted with goat anti-rabbit IgG antibody conjugated with horseradish peroxidase was visualized by ECL Western blotting detection reagents (C). For the immunoblot analysis of VP39 protein, polypeptides equivalent to 1.6 × 105 cells at infection were analyzed in each lane. In panel A, the vertical bars indicate standard deviations of the means from three independent determinations.

48 h pi, but no appreciable CPE was observed in the cell line HaAO1 that represented the highest permissiveness for HearNPV infection. The CPE in the SeMNPV-infected H. armigera cells became more severe with time pi, without any appreciable polyhedra formation in the infected cells (data not shown). A significant, but low, increase in BV titer was observed only in the cell line HaPO2 (Fig. 6A). Slot-blot hybridization analysis showed that viral DNA was synthesized in all the newly established cell lines upon infection with SeMNPV, although the amount of viral DNA synthesized was significantly lower than that in the SeMNPV-infected Hz-AM1 cells (Fig. 6B). A negligible or a very low amount of polyhedrin was detected only in the cell lines HaPO1 and HaPO2 at 96 h pi (Fig. 6C). The cell line Hz-AM1 infected with SeMNPV showed a hallmark of apoptosis that was detected at 48 h

pi and clearly observed at 72 h pi (data not shown). Neither polyhedra nor polyhedrin was produced in the SeMNPV-infected Hz-AM1 cells throughout the experiment.

DISCUSSION In this study, we established a total of five H. armigera cell lines, two from embryos (HaEmb1 and HaEmb2), two from pupal ovaries (HaPO1 and HaPO2) and one from adult ovaries (HaAO1). These established cell lines were examined for their permissiveness for homologous HearNPV and seven heterologous NPVs. Based on BV yield and polyhedrin production as well as polyhedra formation, we found that four of the five newly established cell lines, with the exception of HaEmb1, were permissive

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Fig. 6. Budded virus yields, and accumulations of viral DNA and polyhedrin protein in the five newly established H. armigera cell lines and the cell line Hz-AM1 following infection with SeMNPV. Cells from respective cell lines were infected with SeMNPV and sampled for the analyses at 0, 24, 48, 72 and 96 h postinfection. The budded viruses were titrated by the plaque assay in Se301 cells (A). Viral DNA was analyzed by the slot-blot hybridization analysis using a fluorescin-labeled probe prepared from a portion of SeMNPV DNA polymerase gene (B). Polyhedrin was analyzed by the immunoblot analysis using the antibody against BmNPV polyhedrin and visualized by Konica Immunostain HRP-1000 (C). For the immunoblot analysis of polyhedrin, polypeptides equivalent to 1.6 × 105 cells at infection were analyzed in each lane. In panel A, the vertical bars indicate standard deviations of the means from three independent determinations.

for HearNPV infection. Comparative analyses of parameters relevant to viral replication that included BV yield, polyhedra formation, and productions of viral DNA and polyhedrin showed that the cell line HaAO1 from adult ovaries supported the highest productivity of HearNPV among the newly established cell lines. Our results also showed that the overall productivity of HearNPV in the cell line HaAO1 is better than or comparable to that in the cell line Hz-AM1 that was derived from H. zea and had been used routinely for the production and the study of HearNPV. Specifically, the cell line HaAO1 was superior to the cell line Hz-AM1 in expediting the production of large amounts of polyhedrin and polyhedra following infection with HearNPV. In addition, observations under a microscope showed that individual HearNPV-infected cells of the cell line HaAO1 produced more polyhedra than those of other cell lines, suggesting that cell lines with higher productivity of polyhedra could be obtained through cloning of the cells from the cell line HaAO1. Thus, the present study provides a promising novel H.

armigera cell line for the mass production of HearNPV in vitro. All the remaining established cell lines permissive for HearNPV were inferior to the cell line Hz-AM1 in terms of HearNPV productivity. The cell lines HaPO1, HaPO2 and HaEmb2 yielded significantly less BVs compared with that of the cell line Hz-AM1, accounting for the BV yields at 72 h pi in the cell lines HaPO1, HaPO2 and HaEmb2 to be about 10.7, 11.5 and 184 times lower, respectively, than that in the cell line Hz-AM1. Also, the polyhedrin produced in these H. armigera cell lines was significantly lower than that in the cell line Hz-AM1. Visual observations of the infected cultures under a microscope indicated that at least two factors were involved in the low productivity of HearNPV in these H. armigera cell lines. First, the low proportion of infected cells is a possible factor that restricts the overall productivity of HearNPV in those cell lines. This is evidenced by the fact that only a low proportion of cells in the cultures produced polyhedra even at the late stages of infection. The


phenomenon that a high proportion of cells are refractive to viral infection has commonly been observed in other established cell lines from different insect species (Goodwin et al., 1978; McIntosh and Ignoffo, 1981; McIntosh et al., 1983; Rice et al., 1989; Lynn and Shapiro, 1998). Cloning of the cells is a possible way to obtain cell lines with higher virus productivity. Second, in the case of the cell line HaPO1, the apoptosis induced by the HearNPV infection is another possible factor that restricts the productivity of HearNPV. This is evidenced by the activated caspase 3-like protease and the occurrence of characteristic fragmentation of cellular DNA, as well as the apoptotic body formation (data not shown). Examination with seven heterologous NPVs showed that all the newly established H. armigera cell lines were refractive to the infection of five different NPVs that included BmNPV, HycuMNPV, LdMNPV, OpMNPV and SpltMNPV, neither manifesting appreciable CPE nor yielding progeny BVs. In contrast, four of the five new cell lines exhibited CPE upon infection with AcMNPV and SeMNPV. However, BVs and polyhedrin yielded in the cell lines that manifested CPE upon infection with AcMNPV and SeMNPV remained at a negligible or a very low level, yet a considerable amount of viral DNA was synthesized. One exception was found in the cell line HaPO1 infected with AcMNPV, which yielded a significant, but low, amount of BVs, producing no detectable polyhedrin and polyhedra (Fig. 5). Thus, based on the criteria proposed (McClintock et al., 1986; McIntosh et al., 2005), it is reasonable to conclude that the majority of the newly established H. armigera cell lines are categorized into the cell line semipermissive for AcMNPV and SeMNPV. Whether newly established H. armigera cell lines are permissive for NPVs from other insect species within the same subfamily, such as H. zea, H. virescens and H. punctigera, remains to be explored. Among the five newly established cell lines, only the cell line HaEmb1 derived from the embryos did not support the productive infection of homologous HearNPV. In the cell line HaEmb1, a considerable amount of viral DNA was produced while neither BVs nor polyhedrin was yielded upon infection with HearNPV. It is not known if the HearNPV infection of the cell line HaEmb1 was abortive at a stage either prior or posterior to the expression of viral structural proteins, since the anti-Ac/BmVP39 antibody used in this study did not react with the VP39 protein encoded by HearNPV genome. Finally, it is worthwhile to describe that the cell line Hz-AM1 used as the reference cell line in this study exhibited three different types of response against NPV infection. This cell line supported productive infection for HearNPV, while it was non-productive for AcMNPV, in which syntheses of viral DNA and major capsid protein

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VP39 occurred without any substantial yields of BVs and polyhedrin. In addition, the cell line Hz-AM1 underwent severe apoptosis upon infection with SeMNPV. Thus, the cell line Hz-AM1 provides an interesting system for the analysis of molecular mechanisms of NPV-cell interactions.

ACKNOWLEDGMENTS We thank Dr. T. Yaginuma and Dr. T. Niimi of the Laboratory of Sericulture and Entomoresources, Nagoya University, Japan, for their helpful discussion during this study. J.G.O. was a recipient of a scholarship from the Ministry of Education, Science, Sports and Culture of Japan (Monbukagakusho). B.L.C. was supported by Postdoctoral Fellowship for Foreign Researchers from the Japan Society for the Promotion of Science (JSPS). This work was supported by JSPS-NRCT Joint Research Program between Japan and Thailand and, in part, by grantsin-aid (16380040, 19208006) from the JSPS.

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