Induction of apoptosis in an insect cell line, IPLB-Ld652Y, infected with nucleopolyhedroviruses

Hiroki Ishikawa1, Motoko Ikeda2, Kenichi Yanagimoto1,{dagger}, Cristiano A. Felipe Alves1, Yasuhiro Katou1, Barbara A. Laviña-Caoili1,{ddagger} and Michihiro Kobayashi1

1 Laboratory of Biodynamics, Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya 464-8601, Japan
2 Laboratory of Sericulture and Entomoresources, Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya 464-8601, Japan

Correspondence
Michihiro Kobayashi
michihir{at}agr.nagoya-u.ac.jp


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Ld652Y cells derived from the gypsy moth, Lymantria dispar, were infected with seven different nucleopolyhedroviruses (NPVs) including those from Autographa californica, Bombyx mori (BmNPV), Hyphantria cunea (HycuNPV), Spodoptera exigua (SeMNPV), L. dispar, Orgyia pseudotsugata (OpMNPV) and Spodoptera litura (SpltMNPV). The results showed that Ld652Y cells infected with BmNPV, HycuNPV, SeMNPV, OpMNPV and SpltMNPV underwent apoptosis, displaying apoptotic bodies, characteristic DNA fragmentation and increased caspase-3-like protease activity; HycuNPV induced the most severe apoptosis. In HycuNPV-infected Ld652Y cells, a considerable amount of viral DNA was synthesized although there was no detectable yield of budded virions and polyhedrin. Northern blot and immunoblot analyses revealed that HycuNPV inhibitor of apoptosis 3 (IAP3), which has been shown to function in Sf9 cells, was expressed in HycuNPV-infected Ld652Y cells at a level higher than or comparable with that in HycuNPV-infected SpIm cells, which produced a high titre of progeny virions without any apoptotic response. These results imply that the relative ease of apoptosis induction in NPV-infected Ld652Y cells is largely dependent on inherent cellular properties rather than functions of the respective NPVs, and indicate that the defect in progeny virion production is not merely due to the virus-induced apoptosis in HycuNPV-infected Ld652Y cells.

{dagger}Present address: Nikken Foods Co., Ltd, Haruoka 723-1, Hukuroi, Shizuoka 437-0122, Japan.

{ddagger}Present address: Insect Pathology Laboratory, Department of Entomology, College of Agriculture, University of the Philippines Los Baños College, Laguna 4031, Philippines.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Nucleopolyhedroviruses (NPVs), members of the family Baculoviridae, have been identified in more than 520 insect species spanning seven different orders (Adams & McClintock, 1991). Early studies collectively revealed that these NPVs generally display a narrow host range and their lethal infection is restricted to close relatives of the insect species from which the viruses were originally isolated. Studies with cultured cells have further demonstrated that the respective NPVs establish unique interactions with different cell lines, resulting in various types of non-productive infection (Carpenter & Bilimoria, 1983; Liu & Bilimoria, 1990; Liu & Carstens, 1993; Morris & Miller, 1993; Yanase et al., 1998; Shirata et al., 1999; Wu et al., 2000; Laviña et al., 2001), as well as productive infection.

One of the distinctive features of non-productive infection is observed in the cell line Ld652Y (Goodwin et al., 1978), derived from the gypsy moth, Lymantria dispar, which has been shown to be permissive for L. dispar multinucleocapsid NPV (LdMNPV) (Slavicek et al., 1992) and Orgyia pseudotsugata MNPV (OpMNPV) (Bradford et al., 1990). Infection of Ld652Y cells with Autographa californica MNPV (AcMNPV) results in a total shutdown of not only cellular but also viral protein synthesis at the level of translation (Guzo et al., 1991, 1992; Du & Thiem, 1997; Mazzacano et al., 1999). The suppressed protein synthesis in AcMNPV-infected Ld652Y cells is restored to the level of LdMNPV-infected Ld652Y cells when AcMNPV experimentally acquires the LdMNPV host range factor 1 (hrf-1) gene (Thiem et al., 1996). The recombinant AcMNPV with the hrf-1 gene successfully replicates and produces high titres of progeny virions in both Ld652Y cells and L. dispar larvae (Thiem et al., 1996; Chen et al., 1998), indicating that hrf-1 is directly related to the AcMNPV productive infection in Ld652Y cells.

Another distinctive feature of non-productive infection is observed in those cells undergoing virus-induced apoptosis. Baculovirus-induced apoptosis was first demonstrated in Spodoptera frugiperda Sf21 cells infected with an AcMNPV mutant lacking a functional p35 gene (Clem et al., 1991). Subsequent studies have shown that wild-type (wt) AcMNPV with the intact p35 gene also induces apoptosis in cell lines from Spodoptera littoralis and Choristoneura fumiferana (CF-203) (Chejanovsky & Gershburg, 1995; Palli et al., 1996a) and that the apoptosis induced in AcMNPV-infected CF-203 cells is blocked by prior inoculation with C. fumiferana MNPV (Palli et al., 1996a). In addition to AcMNPV, Spodoptera exigua MNPV (SeMNPV) and Heliothis armigera single nucleocapsid NPV (HaSNPV), which cause productive infection in cell lines from S. exigua and Trichoplusia ni (Hi5), have also been shown to induce apoptosis in S. littoralis and Heliothis zea cells, respectively (Yanase et al., 1998; Dai et al., 1999). In addition, it has been shown that T. ni (TN368) cells infected with the p35-defective AcMNPV mutant resist apoptosis and yield a high titre of progeny virions (Clem & Miller, 1993). These results imply that baculoviruses generally encode a factor(s) that triggers apoptosis in the infected cells, and that whether the baculovirus-infected cells undergo apoptosis relies on an intricate relationship between cellular and viral functions that are involved in the induction and suppression of apoptosis.

The molecular mechanisms underlying the induction and suppression of apoptosis in cells infected with baculoviruses are largely unknown. Careful analysis of the timing of apoptotic events in p35-defective AcMNPV-infected S. frugiperda cells has suggested that NPV-induced apoptosis could be triggered by both early and late events in virus infection (LaCount & Friesen, 1997). Previous studies have also shown that AcMNPV-induced apoptosis of Sf21 cells is triggered by IE1, the product of the immediate-early viral gene ie1 (Prikhod'ko & Miller, 1996), and suppressed by P35 and inhibitor of apoptosis (IAP) proteins, which are encoded by the genomes of certain NPVs (Clem, 1997, 2001). It has also been shown that IE1-induced apoptosis of Sf21 cells is augmented by the AcMNPV early gene product PE38 (Prikhod'ko & Miller, 1999). In addition, apoptosis induced by baculovirus infection has been shown to be associated with the activation of caspases (Bertin et al., 1996; Ahmad et al., 1997; LaCount & Friesen, 1997; Seshagiri & Miller, 1997; LaCount et al., 2000; Manji & Friesen, 2001).

We have previously suggested that Ld652Y cells exhibit apoptosis following infection with SeMNPV and Spodoptera litura MNPV (SpltMNPV) (Wu et al., 2000; Laviña et al., 2001). In the present study, we have demonstrated that Ld652Y cells readily undergo apoptosis following infection with a variety of NPVs, including Bombyx mori NPV (BmNPV), Hyphantria cunea NPV (HycuNPV), OpMNPV, SeMNPV and SpltMNPV. In addition, we have characterized the HycuNPV-induced apoptosis of Ld652Y cells, which exhibit severe apoptosis, and found that a substantial amount of the HycuNPV iap3 gene, whose product has been shown to play a role in blocking apoptosis, is expressed in HycuNPV-infected Ld652Y cells.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Viruses and cells.
Seven different clonal NPV isolates were used in these experiments: AcMNPV E2 from the fall armyworm, A. californica (Smith & Summers, 1978); BmNPV N9 from the silkworm, B. mori (Nagamine et al., 1989); HycuNPV N9 from the fall webworm, H. cunea (Shirata et al., 1999); SeMNPV S1 from the beat armyworm, S. exigua (Wu et al., 2000); LdMNPV A21-MPV from the gypsy moth, L. dispar (Slavicek et al., 1996); OpMNPV from the Douglas-fir tussock moth, O. pseudotsugata (Leisy et al., 1986); and SpltMNPV P7 from the common cutworm, S. litura (Laviña et al., 2001). An AcMNPV mutant that is defective in the p35 gene, vP35delBsu36Igus (AcMNPV{Delta}p35), obtained from Rollie J. Clem (Kansas State University, Manhattan, KS, USA) was also used.

Insect cell lines used in these experiments were IPLB-Ld652Y from the gypsy moth, L. dispar (Ld652Y; Goodwin et al., 1978) and FRI-SpIm1229 from the mulberry tiger moth, Spilosoma imparilis (SpIm; Mitsuhashi & Inoue, 1988). Ld652Y cells were grown at 28 °C in TC100 medium (Invitrogen) supplemented with 10 % foetal bovine serum (FBS), whereas SpIm cells were maintained at 28 °C in MM medium (Mitsuhashi & Maramorosch, 1964) supplemented with 3 % FBS.

DNA fragmentation assay.
Culture cells with apoptotic bodies in 25 cm2 culture flasks (Nunc 163371) were scraped into culture medium with a rubber policeman and collected by centrifugation at 3000 r.p.m. for 5 min at 4 °C. The precipitate was washed twice with PBS and stored frozen until used. DNA for the fragmentation assay was isolated as described previously (Palli et al., 1996b). Briefly, the thawed cells with apoptotic bodies were suspended in 200 µl lysis buffer (100 mM NaCl, 10 mM Tris/HCl, pH 7·9, 25 mM EDTA, 0·5 % SDS) containing 0·3 mg proteinase K ml-1, incubated at 55 °C for 12 h and digested with 1 mg RNase A ml-1 for 1 h at 37 °C. The DNA was extracted twice with an equal volume of phenol (saturated with 100 mM Tris/HCl, pH 8)/chloroform/isoamyl alcohol (24 : 1) and then once with chloroform alone. The extracted DNA was ethanol-precipitated and dissolved in TE-8 (10 mM Tris/HCl, pH 8, 1 mM EDTA).

In certain experiments, an NP-40 extraction procedure was used to yield preferentially the fragmented DNAs. In this procedure, cells washed with PBS were immediately lysed in 100 ml lysis buffer (1 % NP-40, 50 mM Tris/HCl, pH 7·5, 20 mM EDTA) and centrifuged at 4500 r.p.m. for 5 min at 4 °C in a microcentrifuge. The supernatant containing fragmented DNA was removed and incubated at 50 °C for 3 h after mixing well with 20 µl 10 % SDS and 5 µl RNase A (10 mg ml-1). After incubation, 5 µl proteinase K (15·6 mg ml-1) was added and the mixture was further incubated at 37 °C for 3 h. The DNA was ethanol-precipitated and dissolved in TE-8.

Caspase activity assay.
The caspase activity assay was performed using the caspase-3 fluorescent assay kit ApoProbe-3 (BioDynamics Laboratory), which allows quantitative detection of caspase-3-like protease activity. Monolayer cultures were infected with seven different NPVs. At different times post-infection (p.i.), cells were scraped into culture medium with a rubber policeman and collected by centrifugation at 3000 r.p.m. for 10 min at 4 °C. The cells were suspended in cell lysis buffer (included in the kit) and incubated on ice for 10 min and the cell lysates centrifuged at 12 000 r.p.m. for 3 min at 4 °C. The resultant supernatants were analysed for caspase-3-like protease activity using vAc-DEVD-AMC as the substrate. Accumulation of fluorescent product was monitored using a spectrofluorophotometer, model RF-5300PC (Shimadzu), with an excitation wavelength of 360 nm and an emission wavelength of 460 nm.

Slot-blot hybridization analysis.
Slot-blot hybridization analysis was performed as described previously (Ikeda & Kobayashi, 1999). Virus-infected cells were scraped into the culture medium at different times p.i., precipitated at 10 000 r.p.m. for 15 min at 4 °C and suspended in distilled water. The cells in distilled water were treated with heated supersaturated sodium iodide and boiled for 10 min. After chilling on ice, the mixtures were blotted on to Hybond-N+ nylon membranes and hybridized with the DNA probe labelled with fluorescein according to the protocol of the Gene Images CDP-Star detection module (Amersham Pharmacia Biotech). The probe used for the detection of viral DNA was the HycuNPV iap3 (hycu-iap3) gene, which was amplified by PCR using 5'-ACGCACACGGCGGAGTTAAC-3' and 5'-AGTAGTGCGACACGTGGGAC-3' as the paired primers and HycuNPV genomic DNA as the template.

Northern blot analysis.
Transcripts of hycu-iap3 were examined by Northern blot analysis, essentially as described previously (Ikeda et al., 2001). Monolayer cultures of Ld652Y cells (8x106) were prepared in 80 cm2 culture flasks (Nunc 147589) and infected with HycuNPV at an input m.o.i. of 10. At different times p.i., total RNA was isolated by TRIzol reagent (Invitrogen) from the virus-infected cells, resolved on a 1·2 % agarose gel (SeaKem GTG, FMC BioProducts) and blotted on to a Hybond-N+ nylon membrane (Amersham Pharmacia Biotech) under alkaline conditions. The RNA on the membrane was probed with 32P-labelled hycu-iap3 and analysed by imaging analyser (BAS 2000, Fuji Photo Film). The probe used was amplified by PCR as described for slot-blot analysis and labelled with [{alpha}-32P]dCTP (NEN Research Products) using the Rediprime II random prime labelling system (Amersham Pharmacia Biotech).

Immunoblot analysis.
Immunoblot analysis was carried out as described previously (Ikeda et al., 2001; Katou et al., 2001). Briefly, polypeptides from infected cells were resolved by SDS-PAGE and blotted on to nitrocellulose membranes (Advantec Toyo) or Immobilon transfer membranes (Millipore). Antibodies against BmNPV polyhedrin (Shirata et al., 1999), BmNPV occluded virions (Kobayashi et al., 1990) and Hycu-IAP3 were used as primary antibodies. The antibodies against BmNPV polyhedrin and BmNPV occluded virions were raised in rabbits and the antibody against Hycu-IAP3 in mice for the present study. The immunopositive polypeptides were detected using HRP-conjugated goat anti-rabbit or anti-mouse IgG (Zymed). The BmNPV structural polypeptides and polyhedrin were visualized by Konica immunostaining HRP-1000, and Hycu-IAP3 by ECL Western blotting detection reagents (Amersham Pharmacia Biotech).

Preparation of anti-Hycu-IAP3 antiserum.
For the preparation of anti-Hycu-IAP3 antiserum, a portion of the Hycu-IAP3 protein was generated using the pET-32b(+) expression vector (Novagen). A PCR product encoding 126 amino acid residues (Gly6–Thr131) of the Hycu-IAP3 protein (accession no. AB088850) was amplified using the 3·3 kbp HindIII fragment of HycuNPV DNA as the template and paired primers 5'-CGGGATCCCGGAGTTAACATGGAA-3' and 5'-CCGCTCGAGCGGGTAATAAAACCC-3' containing HindIII and XhoI restriction sites (underlined), respectively. The PCR product was subcloned into the HindIII–XhoI site of the pET-32b(+) expression vector and introduced into Escherichia coli BL21-DE3-pLysS. Bacteria were grown at 37 °C and the portion of Hycu-IAP3 protein was expressed by induction with IPTG at a final concentration of 10 mM. The Hycu-IAP3 protein produced was purified by His Trap (Amersham Pharmacia) and the His Trap-purified Hycu-IAP3 protein was resolved by SDS-PAGE. The Hycu-IAP3 protein band was cut out from the gel after Coomassie brilliant blue staining and the gel slices containing the Hycu-IAP3 protein were homogenized and injected into mice for immunization.

Budded virion titration.
HycuNPV BVs in the medium of virus-infected cells were titrated by plaque assay on SpIm cells, as described previously (Shirata et al., 1999).


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Cytopathology, DNA fragmentation and caspase activation in Ld652Y cells infected with NPVs
Monolayer cultures consisting of 1x106 Ld652Y cells were prepared and infected with each of seven different NPVs: AcMNPV, BmNPV, HycuNPV, LdMNPV, OpMNPV, SeMNPV and SpltMNPV. Microscopic examination revealed that hallmarks of cellular apoptosis were detectable by 24 h p.i. and were clearly observed at 72 h p.i. in Ld652Y cells infected with all the NPVs except LdMNPV and AcMNPV (Fig. 1A). The extent of apoptosis induction in Ld652Y cells was different among the NPV species infected. Infection with HycuNPV resulted in severe apoptosis, while less severe apoptosis was induced following infection with OpMNPV. In Ld652Y cells infected with HycuNPV at an m.o.i. of 20, more than 80 % of infected cells underwent apoptosis. Ld652Y cells infected with LdMNPV exhibited a cytopathic effect (CPE) characteristic of NPV infection, yielding a low number of polyhedra, whereas AcMNPV-infected Ld652Y cells showed appreciable CPE without any detectable production of polyhedra (Fig. 1A).



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Fig. 1. Cytopathology, DNA-laddering and caspase-3-like protease activity in Ld652Y cells infected with seven different NPVs. Monolayer cultures of Ld652Y cells were infected with NPVs from Hyphantria cunea (HycuNPV, Hycu), Spodoptera exigua (SeMNPV, Se), Spodoptera litura (SpltMNPV, Splt), Bombyx mori (BmNPV, Bm), Orgyia pseudotsugata (OpMNPV, Op), Lymantria dispar (LdMNPV, Ld) and Autographa californica (AcMNPV, Ac) at input m.o.i.s indicated in parentheses and examined for cytopathology at 72 h p.i. (A), apoptotic DNA laddering at 72 h p.i. (B) and caspase-3-like protease activity at 60 h p.i. (C). The numbers on the right in (B) indicate the sizes of marker DNA (kbp). Vertical bars in (C) represent standard deviations of averages from three determinations. Mock-infected cells (Mock) and the cells infected with the p35-defective AcMNPV mutant (v{Delta}35) are also incorporated into the figure for comparison. The caspase-3-like protease activities were expressed as units relative to the activity in mock-infected cells, which was given a value of 1.

 
To confirm that the morphological alterations observed in NPV-infected Ld652Y cells were due to apoptosis, cellular DNA of NPV-infected Ld652Y cells was isolated at 72 h p.i. by the NP-40 extraction method and analysed on an agarose gel. As shown in Fig. 1(B), an oligomeric DNA ladder, similar to that observed in AcMNPV{Delta}p35-infected Ld652Y cells, was clearly detected in the cells infected with HycuNPV, SeMNPV, SpltMNPV, BmNPV and OpMNPV. Under these experimental conditions, the amount of fragmented DNA in HycuNPV-infected Ld652Y cells was greater than that in AcMNPV{Delta}p35-infected cells. No detectable oligomeric DNA ladder was observed in cells infected with AcMNPV and LdMNPV or in mock-infected cells.

Caspase-3-like protease activity was determined in NPV-infected Ld652Y cells using the ApoProbe-3 kit. At 60 h p.i., Ld652Y cells infected with NPVs were subjected to a caspase-3-like protease activity assay. The results showed that caspase-3-like protease activity increased significantly in Ld652Y cells infected with HycuNPV, SeMNPV, SpltMNPV, BmNPV and OpMNPV, as well as with AcMNPV{Delta}p35 (Fig. 1C). The caspase-3-like protease activity was higher in cells infected with HycuNPV, SeMNPV and SpltMNPV than in the cells infected with BmNPV and OpMNPV. In mock-infected Ld652Y cells and Ld652Y cells infected with AcMNPV and LdMNPV, no significant increase in caspase-3-like protease activity was observed.

Cytopathology, DNA fragmentation and caspase activation in HycuNPV-infected Ld652Y cells
To characterize further the NPV-induced apoptosis, HycuNPV-infected Ld652Y cells, which exhibited severe apoptosis, were analysed in some detail. Ld652Y cells were infected with HycuNPV at an input m.o.i. of 1, 5, 10 or 20 and examined at intervals for apoptosis induction. Microscopic observation showed that the severity of apoptosis induced in HycuNPV-infected Ld652Y cells was dependent on the input m.o.i. between 1 and 20. The Ld652Y cells infected at 20 p.f.u. per cell showed blebbing by 24 h p.i. and the number of cells exhibiting characteristics of apoptosis increased up to 72 h p.i.

HycuNPV-infected Ld652Y cells were also examined for the oligomeric fragmentation of cellular DNA (Fig. 2). Ld652Y cells were infected with HycuNPV at an m.o.i. of 1, 5, 10 or 20, and cellular DNA was extracted from infected cells at 96 h p.i. Analysis of the DNA on agarose gels showed that oligomeric DNA laddering was observed, even in cells infected at an m.o.i. of 1 (Fig. 2A). In Ld652Y cells infected at an m.o.i. of 20, the oligomeric DNA ladder was detectable by 24 h p.i., became clearly observed at 48 h p.i. and decreased gradually from 72 to 96 h p.i. (Fig. 2B).



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Fig. 2. Apoptotic DNA laddering in Ld652Y cells infected with HycuNPV. (A) Ld652Y cells were infected with HycuNPV at an input m.o.i. of 1, 5, 10 or 20, and at 96 h p.i., cellular DNA was isolated. Twenty µg cellular DNA was resolved on a 1·5 % agarose gel and stained with ethidium bromide. (B) Ld652Y cells were infected with HycuNPV at an input m.o.i. of 10. The cellular DNA was isolated at 0, 24, 48, 72 and 96 h p.i. and analysed as in (A). The numbers on the left indicate the sizes of marker DNA (kbp). M, Mock-infected cells.

 
Caspase-3-like protease activity was examined in Ld652Y cells infected with HycuNPV at an m.o.i. of 10. The result showed a linear increase in caspase-3-like protease activity from 12 to 36 h p.i. (Fig. 3).



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Fig. 3. Changes in caspase-3-like protease activity in Ld652Y cells during infection with HycuNPV. Monolayer cultures of Ld652Y cells were infected with HycuNPV at an input m.o.i. of 10 and at indicated times p.i., virus-infected cells were processed and assayed for caspase-3-like protease activity. Vertical bars represent standard deviations of averages from three determinations. The caspase-3-like protease activities were expressed as units relative to the activity in the cells at 0 h p.i., which was given a value of 1.

 
Virus replication in HycuNPV-infected Ld652Y cells
HycuNPV infection of Ld652Y cells was characterized by examining various parameters relevant to viral multiplication, including viral DNA replication, synthesis of viral structural proteins, and polyhedrin and budded virion (BV) yield, as described previously (Ikeda et al., 2001; Laviña et al., 2001). Ld652Y cells were infected with HycuNPV at an m.o.i. of 10, and at different times p.i., BV yield in the culture medium of infected cells was examined by plaque assay on SpIm cells. The result showed that there was no detectable BV production in HycuNPV-infected Ld652Y cells, while BV titre in HycuNPV-infected SpIm cells increased to 3·8x106 and 1·2x107 p.f.u. ml-1 medium at 48 and 96 h p.i., respectively (Fig. 4).



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Fig. 4. Budded virion yields in Ld652Y and SpIm cells infected with HycuNPV. Ld652Y (A) and SpIm (B) cells were infected with HycuNPV at an m.o.i. of 10. At 0, 48 and 96 h p.i., culture medium from infected cells was subjected to plaque assay on SpIm cells. Vertical bars represent standard deviations of averages from three determinations.

 
To determine whether viral proteins were synthesized in HycuNPV-infected Ld652Y cells, immunoblot analysis was carried out using antisera against polyhedrin and occluded virions of BmNPV. The results showed that, in agreement with the observed absence of polyhedron production, polyhedrin was undetectable in HycuNPV-infected Ld652Y cells (Fig. 5A), while HycuNPV-infected SpIm cells showed a clear band of polyhedrin with an approximate molecular mass of 32 kDa at 48 h p.i. or later (Fig. 5A). Similarly, HycuNPV-infected Ld652Y cells showed no infection-specific polypeptides that were reactive with the specific antiserum against occluded virions, while several infection-specific polypeptide bands were detected in the SpIm cells infected with HycuNPV (Fig. 5B). A non-specific polypeptide with an approximate molecular mass of 27 kDa was observed exclusively in Ld652Y cells throughout the experiment.



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Fig. 5. Immunoblot analysis of polyhedrin and viral structural polypeptides in Ld652Y and SpIm cells infected with HycuNPV. Ld652Y and SpIm cells were infected with HycuNPV at an m.o.i. of 10. At 0, 12, 24, 48, 72 and 96 h p.i., polypeptides from infected cells were resolved by SDS-PAGE and processed for immunoblot analysis. Polypeptides were probed with antiserum against BmNPV polyhedrin (A) or BmNPV occluded virions (B). Immunoreactive polypeptides were visualized with Konica immunostaining HRP-1000. The molecular mass of a non-specific polypeptide observed exclusively in Ld652Y cells in (B) and indicated by the arrowhead on the left of the panel was approximately 27 kDa. Numbers on the left in panel B represent molecular masses (kDa) of marker proteins (Dr Western; Oriental Yeast, Tokyo, Japan).

 
Viral DNA accumulation was also examined by slot-blot hybridization analysis. In HycuNPV-infected Ld652Y cells, viral DNA was detectable by 12 h p.i., increased to a maximal level at 48 h p.i. and then decreased sharply to an undetectable level at 96 h p.i., presumably due to complete digestion of the genomic DNA of the apoptotic cells (Fig. 6). This pattern of viral DNA accumulation in Ld652Y cells was different from that in SpIm cells, in which viral DNA increased from 12 h p.i., peaking between 48 and 72 h p.i., and then decreased slightly at 96 h p.i. The maximal level of viral DNA in Ld652Y cells was approximately 60 % of that in SpIm cells (Fig. 6).



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Fig. 6. Slot-blot analysis of viral DNA in Ld652Y and SpIm cells infected with HycuNPV. Ld652Y and SpIm cells were infected with HycuNPV at an m.o.i. of 10. At 0, 12, 24, 48, 72 and 96 h p.i., infected cells were harvested and viral DNA was extracted in heated supersaturated sodium iodide. The viral DNAs were blotted on to a Hybond-N+ nylon membrane and hybridized with a fluorescein-labelled hycu-iap3 gene probe. The probe was detected using the Gene Images CDP-Star detection module (A) and quantified with the Lumi Imager F1 workstation (Boehringer Mannheim) by comparing the signal intensities with those of serially diluted HycuNPV DNAs of known amount (B).

 
Expression of the hycu-iap3 gene
Previous results from our laboratory have shown that the HycuNPV genome does not possess the p35 gene but encodes three iap genes. Functional analyses of the hycu-iap genes in Sf9 cells derived from S. frugiperda have also shown that the protein product from hycu-iap3 suppresses apoptosis triggered by actinomycin D and AcMNPV{Delta}p35 infection, and that the apoptosis-suppressing activity is dependent on the amount of Hycu-IAP3 protein accumulated in the cells into which the hycu-iap3 gene has been introduced (M. Ikeda and others, unpublished data). To examine whether hycu-iap3 was expressed in HycuNPV-infected Ld652Y cells, Northern blot hybridization analysis and immunoblot analysis were carried out. Consistent with the previous results in HycuNPV-infected SpIm cells, hycu-iap3 was expressed as two species of transcript with approximate molecular sizes of 1·4 and 0·9 kb, both of which first appeared at 8 h p.i. and rapidly increased up to 12 h p.i. (Fig. 7A). At 24 h p.i., the amount of 0·9 kb transcript remained at a similar level to that at 12 h p.i., while the 1·4 kb transcript had decreased markedly. Both transcripts were undetectable at 48 and 96 h p.i. Immunoblot analysis further showed that the Hycu-IAP3 protein with an approximate molecular mass of 34 kDa was first detectable at 12 h p.i. in HycuNPV-infected Ld652Y cells, increased to a maximum level at 24 h p.i., decreased slightly at 36 and 48 h p.i. and was barely detectable by 72 and 96 h p.i. (Fig. 7B). The amount of Hycu-IAP3 that accumulated in HycuNPV-infected Ld652Y cells was higher than or comparable with that in HycuNPV-infected SpIm cells, which produced a high titre of progeny virions without any apoptotic response.



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Fig. 7. Expression of the hycu-iap3 gene in Ld652Y cells infected with HycuNPV. Ld652Y and SpIm cells were infected with HycuNPV at an m.o.i. of 10, and at indicated times p.i., transcripts and polypeptides were examined by Northern blot and immunoblot analysis, respectively. (A) Northern blot analysis of hycu-iap3 transcripts. Total RNA was isolated from virus-infected cells and 20 µg total RNA was resolved on a 1·2 % agarose gel. The RNA was transferred on to Hybond-N+ membrane and probed with 32P-labelled hycu-iap3 DNA. The numbers on the left of the panel indicate the sizes of marker RNA (kb). (B) Immunoblot analysis of Hycu-IAP3 polypeptide. Polypeptides from the virus-infected cells were resolved by 12·5 % SDS-PAGE, transferred on to Immobilon transfer membrane and probed with anti-Hycu-IAP3 antiserum.

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Using various cell lines including Ld652Y, SpIm, Sf9 from S. frugiperda, BmN-4 from B. mori (Maeda, 1989), Se301 from S. exigua (Hara et al., 1995), SpLi221 from S. litura (Mitsuhashi, 1995) and CLS-79 from S. littoralis (Tsuda et al., 1984), previous studies have shown that SpltMNPV induces apoptosis only in Ld652Y cells and SeMNPV only in Ld652Y and CLS-79 cells (Wu et al., 2000; Laviña et al., 2001; Laviña-Caoili et al., 2001). In the present study, we have demonstrated that Ld652Y cells derived from the gypsy moth, L. dispar, readily undergo apoptosis following infection with a variety of NPVs. Our results showed that infection of Ld652Y cells with HycuNPV, BmNPV, SpltMNPV, SeMNPV and OpMNPV resulted in the induction of apoptosis, displaying apoptotic bodies, characteristic DNA fragmentation and increased caspase-3-like protease activity. Similarly, our results, together with previous results (Shirata et al., 1999), demonstrated that infection with BmNPV, HycuNPV and SeMNPV induced apoptosis only in Ld652Y cells among the examined cell lines of Ld652Y, BmN-4, SpIm, Se301 and Sf21. Furthermore, it has been shown that TN368 cells from T. ni (Hink, 1970) do not develop apoptosis following infection with any of the NPVs employed in the present study (data not shown). These results suggest that the relative ease of apoptosis induction in NPV-infected cells is largely dependent on inherent cellular properties rather than specific functions of the respective NPVs.

The suggestion that inherent cellular properties are closely related to the prompted apoptosis induction in NPV-infected Ld652Y cells is supported by circumstantial evidence derived from studies on virus-encoded apoptosis-suppressing factors. Previous studies in our laboratory have shown that HycuNPV does not possess the homologue of p35 but encodes three species of iap homologue (hycu-iap1, -2 and -3). Functional analyses with Sf9 cells transiently expressing Hycu-IAPs have further shown that hycu-iap3 exhibits clear apoptosis-suppressing activity, which depends on the amount of Hycu-IAP3 protein accumulated in the cells (M. Ikeda and others, unpublished data). Immunoblot analysis in the present study showed that the amount of Hycu-IAP3 protein accumulated in HycuNPV-infected Ld652Y cells was higher than or comparable with that in HycuNPV-infected SpIm cells, which showed no apoptotic response and generated a high titre of progeny virions. In addition, there was no apparent difference between HycuNPV-infected Ld652Y and SpIm cells in the time course of Hycu-IAP3 expression. It is thus probable that not only viral IAPs but also cellular IAPs or related factors play an important role in the suppression of apoptosis in insect cells triggered by NPV infection. Alternatively, it is possible that Hycu-IAP3 is non-functional or is required at higher levels for the suppression of apoptosis in this particular system relating to Ld652Y cells and NPVs. It may also be possible that the induction of apoptosis of NPV-infected Ld652Y cells is specifically associated with some unidentified apoptotic pathway that is insensitive to suppression by IAPs.

In S. frugiperda cells in which apoptosis is induced by infection with the mutant AcMNPV{Delta}p35, production of viral progeny is severely reduced due to delay or lack of viral gene expression in both cultured cells (Hershberger et al., 1992; Clem & Miller, 1993) and insect larvae (Clem & Miller, 1993). In HycuNPV-infected Ld652Y cells, on the other hand, our data demonstrated that neither BVs nor viral structural proteins and polyhedrin are yielded at detectable levels. Our results in HycuNPV-infected Ld652Y cells agree with those observed in S. littoralis and C. fumiferana cells undergoing apoptosis following infection with wt AcMNPV (Chejanovsky & Gershburg, 1995; Palli et al., 1996a). Using a caspase inhibitor, zVAD-FMK, we have also found that suppression of apoptosis in HycuNPV-infected Ld652Y cells does not result in the restoration of progeny virion production (H. Ishikawa, unpublished data), suggesting that the defects in progeny virion production are not merely due to the apoptosis induced in the infected cells. This result agrees with previous results in wt AcMNPV-infected S. littoralis cells (Gershburg et al., 1997) and HaSNPV-infected T. ni cells (Dai et al., 1999).

The mechanisms underlying apoptosis induction in Ld652Y cells observed in the present study are not known. In the HycuNPV-infected Ld652Y cells, our data demonstrated that the virus replication cycle is restricted at a step prior to viral late gene expression, suggesting that HycuNPV-induced apoptosis of Ld652Y cells is an event triggered in the early phase of viral infection. Previous studies have demonstrated that apoptosis of AcMNPV-infected S. frugiperda cells is triggered by an immediate-early viral protein, IE1 (Prikhod'ko & Miller, 1996) and suppressed by P35 and IAPs encoded by the viral genome (Clem, 1997, 2001). Recent studies from our laboratory with the transient expression assay have shown that the HycuNPV ie1 gene is sufficient to induce apoptosis in Ld652Y cells (H. Ishikawa, unpublished data).

Consistent with the previous results (Slavicek et al., 1992; Guzo et al., 1992; Du & Thiem, 1997; Mazzacano et al., 1999), our results showed that Ld652Y cells were permissive for LdMNPV, while infection of Ld652Y cells with AcMNPV resulted in no appreciable apoptotic response. Since LdMNPV and AcMNPV have been characterized in Ld652Y cells to induce productive infection (Slavicek et al., 1992) and total shut-down of protein synthesis, respectively (Guzo et al., 1992; Du & Thiem, 1997; Mazzacano et al., 1999), our results indicate that Ld652Y cells display three distinct types of cytopathic response following infection with different NPVs. Thus, Ld652Y cells provide an excellent system for understanding the molecular mechanisms of NPV–cell interactions.


   ACKNOWLEDGEMENTS
 
We thank Dr Rollie J. Clem (Division of Biology, Kansas State University, Manhattan, KS, USA) for the generous gift of the p35-defective AcMNPV and Drs T. Yaginuma and T. Niimi (Laboratory of Sericulture and Entomoresources, Graduate School of Bioagricultural Sciences, Nagoya University, Japan) for their helpful discussion during this study. This work was supported in part by grants-in-aid (Nos 10306005, 13660059, 14206007 and 14606001) from the Japan Society for the Promotion of Science.


   REFERENCES
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Adams, J. R. & McClintock, J. T. (1991). Baculoviridae. Nuclear polyhedrosis viruses. Part 1. Nuclear polyhedrosis viruses of insects. In Atlas of Invertebrate Viruses, pp. 89–204. Edited by J. R. Adams & J. R. Bonami. Boca Raton: CRC Press.

Ahmad, M., Srinivasula, S. M., Wang, L., Litwack, G., Fernandes-Alnemri, T. & Alnemri, E. S. (1997). Spodoptera frugiperda caspase-1, a novel insect death protease that cleaves the nuclear immunophilin FKBP46, is the target of the baculovirus antiapoptotic protein p35. J Biol Chem 272, 1421–1424.[Abstract/Free Full Text]

Bertin, J., Mendrysa, S. M., LaCount, D. J., Gaur, S., Krebs, J. F., Armstrong, R. C., Tomaselli, K. J. & Friesen, P. D. (1996). Apoptotic suppression by baculovirus P35 involves cleavage by and inhibition of a virus-induced CDE-3/ICE-like protease. J Virol 70, 6251–6259.[Abstract]

Blissard, G., Black, B., Crook, N., Keddie, B., Possee, R., Rohrmann, G., Theilmann, D. & Volkman, L. (2000). Family Baculoviridae. In Virus Taxonomy. Seventh Report of the International Committee on Taxonomy of Viruses, pp. 195–202. Edited by M. H. V. van Regenmortel, C. M. Fauquet, D. H. L. Bishop, E. B. Carstens, M. K. Estes, S. M. Lemon, J. Maniloff, M. A. Mayo, D. J. McGeoch, C. R. Pringle & R. B. Wickner. San Diego: Academic Press.

Bradford, M. B., Blissard, G. W. & Rohrmann, G. F. (1990). Characterization of the infection cycle of the Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus in Lymantria dispar cells. J Gen Virol 71, 2841–2846.[Abstract]

Carpenter, W. M. & Bilimoria, S. L. (1983). A semipermissive nuclear polyhedrosis virus infection: characterization of infection kinetics and morphogenesis. Virology 130, 227–231.

Chejanovsky, N. & Gershburg, E. (1995). The wild-type Autographa californica nuclear polyhedrosis virus induces apoptosis of Spodoptera littoralis cells. Virology 209, 519–525.[CrossRef][Medline]

Chen, C.-J., Quentin, M. E., Brennan, L. A., Kukel, C. & Thiem, S. M. (1998). Lymantria dispar nucleopolyhedrovirus hrf-1 expands the larval host range of Autographa californica nucleopolyhedrovirus. J Virol 72, 2526–2531.[Abstract/Free Full Text]

Clem, R. J. (1997). Regulation of programmed cell death by baculoviruses. In The Baculoviruses, pp. 237–266. Edited by L. K. Miller. New York: Plenum Press.

Clem, R. J. (2001). Baculoviruses and apoptosis: the good, the bad, and the ugly. Cell Death Differ 8, 137–143.[CrossRef][Medline]

Clem, R. J. & Miller, L. K. (1993). Apoptosis reduces both the in vitro replication and the in vivo infectivity of a baculovirus. J Virol 67, 3730–3738.[Abstract]

Clem, R. J., Fechheimer, M. & Miller, L. K. (1991). Prevention of apoptosis by a baculovirus gene during infection of insect cells. Science 254, 1388–1390.[Medline]

Dai, X., Shi, X., Pang, Y. & Su, D. (1999). Prevention of baculovirus-induced apoptosis of BTI-Tn-5B1–4 (Hi5) cells by the p35 gene of Trichoplusia ni multicapsid nucleopolyhedrovirus. J Gen Virol 80, 1841–1845.[Abstract]

Du, X. & Thiem, S. M. (1997). Responses of insect cells to baculovirus infection: protein synthesis shutdown and apoptosis. J Virol 71, 7866–7872.[Abstract]

Gershburg, E., Rivkin, H. & Chejanovsky, N. (1997). Expression of the Autographa californica nuclear polyhedrosis virus apoptotic suppressor gene p35 in nonpermissive Spodoptera littoralis cells. J Virol 71, 7593–7599.[Abstract]

Goodwin, R. H., Tompkins, G. J. & McCawley, P. (1978). Gypsy moth cell lines divergent in viral susceptibility. I. Culture and identification. In Vitro (Rockv) 14, 485–494.[Medline]

Guzo, D., Dougherty, E. M., Lynn, D. E., Braun, S. K. & Weiner, R. M. (1991). Changes in macromolecular synthesis of gypsy moth cell line IPLB-Ld652Y induced by Autographa californica nuclear polyhedrosis virus infection. J Gen Virol 72, 1021–1029.[Abstract]

Guzo, D., Rathburn, H., Guthrie, K. & Dougherty, E. (1992). Viral and host cellular transcription in Autographa californica nuclear polyhedrosis virus-infected gypsy moth cell line. J Virol 66, 2966–2972.[Abstract]

Hara, K., Funakoshi, M. & Kawarabata, T. (1995). A cloned cell line of Spodoptera exigua has a highly increased susceptibility to the Spodoptera exigua nuclear polyhedrosis virus. Can J Microbiol 41, 1111–1116.

Hershberger, P. A., Dickson, J. A. & Friesen, P. D. (1992). Site-specific mutagenesis of the 35-kilodalton protein gene encoded by Autographa californica nuclear polyhedrosis virus: cell line-specific effects on virus replication. J Virol 66, 5525–5533.[Abstract]

Hink, W. F. (1970). Established insect cell line from the cabbage looper, Trichoplusia ni. Nature 226, 466–467.

Ikeda, M. & Kobayashi, M. (1999). Cell-cycle perturbation in Sf9 cells infected with Autographa californica nucleopolyhedrovirus. Virology 258, 176–188.[CrossRef][Medline]

Ikeda, M., Katou, Y., Yamada, Y., Chaeychomsri, S. & Kobayashi, M. (2001). Characterization of Autographa californica nucleopolyhedrovirus infection in cell lines from Bombyx mori. J Insect Biotechnol Sericol 70, 49–58.

Katou, Y., Ikeda, M. & Kobayashi, M. (2001). Characterization of Bombyx mori nucleopolyhedrovirus infection of Spodoptera frugiperda cells. J Insect Biotechnol Sericol 70, 137–147.

Kobayashi, M., Kotake, M., Sugimori, H., Nagamine, T. & Kajiura, Z. (1990). Identification of virus-specific polypeptides and translatable mRNAs in the isolated pupal abdomens of the silkworm, Bombyx mori, infected with nuclear polyhedrosis virus. J Invertebr Pathol 55, 52–60.[Medline]

LaCount, D. J. & Friesen, P. D. (1997). Role of early and late replication events in induction of apoptosis by baculoviruses. J Virol 71, 1530–1537.[Abstract]

LaCount, D. J., Hanson, S. F., Schneider, C. L. & Friesen, P. D. (2000). Caspase inhibitor P35 and inhibitor of apoptosis Op-IAP block in vivo proteolytic activation of an effector caspase at different steps. J Biol Chem 275, 15657–15664.[Abstract/Free Full Text]

Laviña, B. A., Padua, L. E., Wu, F. Q., Shirata, N., Ikeda, M. & Kobayashi, M. (2001). Biological characterization of a nucleopolyhedrovirus of Spodoptera litura (Lepidoptera: Noctuidae) isolated from the Philippines. Biol Control 20, 39–47.[CrossRef]

Laviña-Caoili, B. A., Kamiya, K., Kawamura, S., Ikeda, M. & Kobayashi, M. (2001). Comparative in vitro analysis of geographic variants of nucleopolyhedrovirus of Spodoptera litura isolated from China and the Philippines. J Insect Biotechnol Sericol 70, 199–209.

Leisy, D. J., Rohrmann, G. F., Nesson, M. & Beaudreau, G. S. (1986). Nucleotide sequencing and transcriptional mapping of the Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus p10 gene. Virology 153, 157–167.[Medline]

Liu, H.-S. & Bilimoria, S. L. (1990). Infected cell specific protein and viral DNA synthesis in productive and abortive infections of Spodoptera frugiperda nuclear polyhedrosis virus. Arch Virol 115, 101–113.[Medline]

Liu, J. J. & Carstens, E. B. (1993). Infection of Spodoptera frugiperda and Choristoneura fumiferana cell lines with the baculovirus Choristoneura fumiferana nuclear polyhedrosis virus. Can J Microbiol 39, 932–940.

Maeda, S. (1989). Gene transfer vector of a baculovirus, Bombyx mori, and their use for expression of foreign genes in insect cells. In Invertebrate Cell System Applications, pp. 167–181. Edited by J. Mitsuhashi. Boca Raton: CRC Press.

Manji, G. A. & Friesen, P. D. (2001). Apoptosis in motion. An apical, p35-insensitive caspase mediates programmed cell death in insect cells. J Biol Chem 276, 16704–16710.[Abstract/Free Full Text]

Mazzacano, C. A., Du, X. & Thiem, S. M. (1999). Global protein synthesis shutdown in Autographa californica nucleopolyhedrovirus-infected Ld652Y cells is rescued by tRNA from uninfected cells. Virology 260, 222–231.[CrossRef][Medline]

Mitsuhashi, J. (1995). A continuous cell line from pupal ovaries of the common cutworm, Spodoptera litura (Lepidoptera: Noctuidae). Appl Entomol Zool 30, 75–82.

Mitsuhashi, J. & Inoue, H. (1988). Obtainment of a continuous cell line from the larval fat bodies of the mulberry tiger moth, Spilosoma imparilis (Lepidoptera: Arctiidae). Appl Entomol Zool 23, 488–490.

Mitsuhashi, J. & Maramorosch, K. (1964). Leafhopper tissue culture: embryonic, nymphal, and imaginal tissues from aseptic insects. Contrib Boyce Thompson Inst 22, 435–460.

Morris, T. D. & Miller, L. K. (1993). Characterization of productive and non-productive AcMNPV infection in selected insect cell lines. Virology 197, 339–348.[CrossRef][Medline]

Nagamine, T., Shimomura, M., Sugimori, H. & Kobayashi, M. (1989). Titration of Bombyx mori (Lepidoptera: Bombycidae) nuclear polyhedrosis virus in a Bombyx mori cell line. Appl Entomol Zool 24, 235–237.

Palli, S. R., Caputo, G. F., Sohi, S. S., Brownwright, A. J., Ladd, T. R., Cook, B. J., Primavera, M., Arif, B. M. & Retnakaran, A. (1996a). CfMNPV blocks AcMNPV-induced apoptosis in a continuous midgut cell line. Virology 222, 201–213.[CrossRef][Medline]

Palli, S. R., Sohi, S. S., Cook, B. J., Brownwright, A. J., Caputo, G. F. & Retnakaran, A. (1996b). RNA- and protein-synthesis inhibitors induce apoptosis in a midgut cell line from the spruce budworm, Choristoneura fumiferana. J Insect Physiol 42, 1061–1069.[CrossRef]

Prikhod'ko, E. A. & Miller, L. K. (1996). Induction of apoptosis by baculovirus transactivator IE1. J Virol 70, 7116–7124.[Abstract]

Prikhod'ko, E. A. & Miller, L. K. (1999). The baculovirus PE38 protein augments apoptosis induced by transactivator IE1. J Virol 73, 6691–6699.[Abstract/Free Full Text]

Seshagiri, S. & Miller, L. K. (1997). Baculovirus inhibitors of apoptosis (IAPs) block activation of Sf-caspase-1. Proc Natl Acad Sci U S A 94, 13606–13611.[Abstract/Free Full Text]

Shirata, N., Ikeda, M., Kamiya, K., Kawamura, S., Kunimi, Y. & Kobayashi, M. (1999). Replication of nucleopolyhedroviruses of Autographa californica (Lepidoptera: Noctuidae), Bombyx mori (Lepidoptera: Bombycidae), Hyphantria cunea (Lepidoptera: Arctiidae), and Spodoptera exigua (Lepidoptera: Noctuidae) in four lepidopteran cell lines. Appl Entomol Zool 34, 507–516.

Slavicek, J. M. & Podgwaite, J. & Lanner-Herrera, C. (1992). Properties of two Lymantria dispar nuclear polyhedrosis virus isolates obtained from the microbial pesticide Gypchek. J Invertebr Pathol 59, 142–148.

Slavicek, J. M., Mercer, M. J., Kelly, M. E. & Hayes-Plazolles, H. (1996). Isolation of a baculovirus variant that exhibits enhanced polyhedra production stability during serial passage in cell culture. J Invertebr Pathol 67, 153–160.[CrossRef]

Smith, G. E. & Summers, M. D. (1978). Analysis of baculovirus genomes with restriction endonucleases. Virology 89, 517–527.

Thiem, S. M., Du, X., Quentin, M. E. & Berner, M. M. (1996). Identification of a baculovirus gene that promotes Autographa californica nuclear polyhedrosis virus replication in a nonpermissive insect cell line. J Virol 70, 2221–2229.[Abstract]

Tsuda, K., Mizuki, E., Kawarabata, T. & Aizawa, K. (1984). Replication of Xestia c-nigrum (Lepidoptera: Noctuidae) nuclear polyhedrosis virus in continuous cell cultures. Appl Entomol Zool 19, 293–298.

Wu, F. Q., Laviña, B. A., Ikeda, M., Shirata, N., Cai, Y. X., Pan, S. X. & Kobayashi, M. (2000). Cloning and biological characterization of Spodoptera exigua nucleopolyhedroviruses isolated in China. J Seric Sci Jpn 69, 177–189.

Yanase, T., Yasunaga, C. & Kawarabata, T. (1998). Replication of Spodoptera exigua nucleopolyhedrovirus in permissive and non-permissive lepidopteran cell lines. Acta Virol 42, 293–298.[Medline]

Received 6 September 2002; accepted 6 November 2002.