Laboratoire de Pathologie Comparée, INRA, 30380 Saint Christol-lez-Alès, France
Correspondence
Anne-Nathalie Volkoff
volkoff{at}ensam.inra.fr
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ABSTRACT |
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MAIN TEXT |
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During replication, viral DNA is excised from the wasp genome then packaged into virions. Recent data obtained in braconids suggest that amplification of viral DNA precedes excision and circularization (Marti et al., 2003; Pasquier-Barre et al., 2002
). The mechanisms of polydnavirus replication are as yet unknown and represent a puzzling mystery for virologists. Replication is tissue-specific and development-dependent as it occurs solely in specialized cells of the female wasp oviduct and is first detected at the end of pupal development (Norton & Vinson, 1983
; Volkoff et al., 1995
). The regulation of virus replication is probably related to the calyx cell differentiation and is thus indirectly related to the biologically active derivatives of the insect hormone ecdysone, 20-hydroxyecdysone (20HE) (Marti et al., 2003
; Webb & Summers, 1992
). In addressing the question of polydnavirus replication, researchers have to date been limited by the impossibility of promoting in vitro replication and a lack of appropriate tools (Stoltz, 1993
). In this work, we describe cell lines that were established from Hyposoter didymator pupae. H. didymator is an ichneumonid wasp associated with the polydnavirus H. didymator ichnovirus (HdIV). These cell lines are, to our knowledge, the first to be derived from a polydnavirus-associated wasp and may constitute a valuable tool for future investigations on polydnavirus replication.
The cell lines were established from young H. didymator pupae (024 h after meconium release). Cocoons were washed in 20 % bleach solution, then rinsed twice with sterile water. Pupae of both sexes were recovered and washed rapidly in 5 % bleach, then rinsed twice with sterile water. The dissected pupae were stored in HdM cell culture medium until a sufficient amount of material had been collected (2025 pupae). HdM is composed of TC100 modified medium (Gibco) supplemented with 20 % foetal calf serum (Gibco), 1 % G5 complement (Gibco), 4 % Kniphofia nectar (Pronectar SA), 1·61 g putrescine l-1 (Sigma), 50 units nystatin ml-1 (Sigma) and 1·25 µg fungizone ml-1 (Gibco). Pupae were then placed in a Petri dish, rinsed with sterile PBS, and cut into small pieces with two scalpel blades. The lacerated samples were dissociated either by incubation in trypsine/EDTA solution (2·0 mg trypsin ml-1, 0·08 M EDTA) for 1 min or by incubation in collagenase solution (0·5 mg ml-1) for 2 min. Samples were washed twice by centrifugation (5 min, 1000 r.p.m.), resuspended in 3 ml HdM and placed in a 12·5 cm2 flask (Falcon) at 28 °C. The culture medium was replaced by fresh HdM or HdM0·5 (containing 0·5 % G5 complement) the following day and then periodically until establishment of the cell lines. After about 2 years, four different cell lines, named Hd-AA, -AD, -BBA and -K, were established. The AA and AD cell lines were established following incubation in trypsine/EDTA solution. AA cells were kept in HdM0·5, while AD cells were maintained in HdM. The BBA and K cell lines were established following incubation in collagenase solution. BBA cells were kept in HdM, whereas K cells were maintained in HdM0·5.
To control the wasp origin of the cell lines and discard the possible risk of contamination by the lepidopteran host Spodoptera littoralis cells, 18S rDNA sequences from the Hd cells, H. didymator pre-pupae and S. littoralis cells and eggs were PCR amplified and analysed. Primers F18SHd (5'-GACTGACGTCGGCAAGTCTGGTGCCAGCAG-3') and R18SHd (5'-CATGGAATTCGGTGCCCTTCCGTCAATTCC-3') were designed in conserved regions flanking a sequence shown as variable after alignment of sequences deposited in GenBank for lepidopteran and hymenopteran species (Fig. 1A). PCR amplification was conducted using Taq DNA polymerase (Roche) with 100 ng template DNA extracted from the lepidopteran cell lines Sf9 and Sl2b (S. littoralis haemocyte cell line; Volkoff et al., 1999
) and from the wasp cell lines AA (15th passage), BBA (19th passage), AD (19th passage) and K (20th passage) according to a standard protocol (Ausubel et al., 1995
). DNA was also extracted from wasp pre-pupae and S. littoralis eggs. According to sequences available in GenBank from other insects, a HindIII restriction site was predicted to be conserved in the amplified hymenopteran sequences, but absent in the lepidopteran ones. As predicted, all the amplification products from H. didymator cells and insects were digested by this enzyme (Fig. 1B
) but none from Spodoptera (data not shown). The wasp origin of the established cell lines was confirmed by sequencing of the PCR products. Indeed, rDNA from the four cell lines was found to be different from the S. littoralis sequence (Fig. 1C
) and identical to the wasp pupae rDNA (data not shown).
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As the HdIV genome was predicted to be integrated in the wasp chromosomes, Southern blot analysis was performed to analyse the HdIV DNA status in the four cell lines. The procedure employed for hybridization was as described by Volkoff et al. (1999). The probe consisted of [
-32P]dCTP-labelled (Nick-translation kit; Roche) total viral DNA. The results indicated that the HdIV DNA probe hybridized with the AA, AD, BBA and K cell genomic DNA, while no hybridization was observed with the lepidopteran cell samples (Fig. 3
A). No circular viral DNA, as observed in encapsidated virus (Fig. 3A
, lane HdIV), could be detected in the cell lines. The hybridization signal co-localized with high molecular mass DNA, suggesting integration of the viral DNA into the wasp cell chromosomes.
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Since 20HE is known to be related to polydnavirus replication, the AA cell line was submitted to increasing concentrations of 20HE and analysed by Southern and Northern blotting. Treatment was found to be deleterious, with observation of cell lysis, when doses were greater than 10-6 mg ml-1. At lower doses, 20HE treatment did not affect HdIV DNA status in AA cells (Fig. 3D, lanes E-6 and E-7) and did not promote expression of detectable HdIV genes (data not shown). A 42 °C heat-shock was also performed to attempt to trigger cellular pathways leading to virus replication. Cells were submitted to heat-shock by placing the culture flask in a 42 °C water-bath for 1 or 2 h. AA cells responded to heat-shock since a wasp hsp90 homologue transcript was detected in the samples using a S. frugiperda hsp90 probe (Landais et al., 2001
) (data not shown), but no effect was observed on HdIV transcription (Fig. 3B
, lanes T1 and T2) nor on viral DNA status (Fig. 3D
, lanes T1 and T2).
Thus, neither 20HE treatment, HdIV infection, nor 42 °C heat-shock applied to the AA cell line promoted virus replication, at least at a level detectable with the currently available tools, in the wasp cells in culture. It should be noted that sequencing data now available on several polydnavirus genomes indicate that encapsidated genomes encode few genes related to virus replication (Turnbull & Webb, 2002). Therefore, because the probe used consisted of the HdIV encapsidated genome, we cannot exclude the possibility that other genes (non-encapsidated viral genes, if they exist, or wasp cellular genes) that may be involved in virus replication are transcribed but not detected in the AA cells. Furthermore, as mentioned by Webb & Summers (1992)
, there are probably many factors involved in the regulation of polydnavirus replication, such as sex, developmental and tissue-specific factors, that are yet to be identified.
To conclude, the H. didymator cell lines described in this work are some of the few established from hymenoptera insects. They contain a polydnavirus genome and should be a valuable tool to study the status of polydnavirus genomes in their wasp hosts and to analyse various factors that may lead to HdIV replication. Having these cell lines available thus offers great potential to understand better the still mysterious polydnavirus organization and life-cycle.
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ACKNOWLEDGEMENTS |
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Received 11 October 2003;
accepted 11 December 2003.
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