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1 Natural Resources Canada, Canadian Forest Service, Laurentian Forestry Centre, 1055 du PEPS, PO Box 10380, Stn Ste-Foy, Quebec City, QC G1V 4C7, Canada
2 Department of Microbiology and Immunology, Sir Charles Tupper Medical Building, Dalhousie University, Halifax, NS B3H 4H7, Canada
Correspondence
Michel Cusson
michel.cusson{at}nrcan.gc.ca
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The GenBank/EMBL/DDBJ accession numbers for the sequences determined in this work are DQ790660–DQ790663.
Present address: Natural Resources Canada, Canadian Forest Service, Great Lakes Forestry Centre, 1219 Queen St East, PO Box 490, Sault Ste Marie, ON P6A 2E5, Canada. 
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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As PDVs do not replicate within their lepidopteran hosts, sustained viral gene expression during parasitism requires that viral genome segments, or at least some of them, persist long enough to allow the successful completion of wasp larval development. Although some of these genome segments do persist as episomes for various periods of time (Stoltz et al., 1986
; Theilmann & Summers, 1986), recent findings suggest that selected genome segments may also undergo integration into the lepidopteran host genomic DNA. Using Southern blot hybridization, circumstantial evidence has been provided for the integration of both ichnovirus (Kim et al., 1996; Volkoff et al., 2001) and bracovirus (McKelvey et al., 1996; Gundersen-Rindal & Dougherty, 2000) genome segments into the chromosomes of insect cells in culture. More recently, the sequences of viral DNA–host DNA junctions have been reported for genome segment F of the Glyptapanteles indiensis bracovirus (GiBV), which becomes integrated into the chromosomal DNA of GiBV-infected gypsy moth cells (Gundersen-Rindal & Lynn, 2003). No direct evidence has yet been provided for in vivo PDV genome segment integration into caterpillar chromosomes, although Southern blot analysis of fat-body DNA obtained from Manduca sexta larvae infected with the Cotesia congregata bracovirus (CcBV) yielded a hybridization profile suggestive of CcBV genome segment integration (Le et al., 2003). Similar results have been obtained in the case of Malacosoma disstria larvae parasitized by Hyposoter fugitivus (N. Keirstead & D. Stoltz, unpublished data).
In an effort to provide direct evidence for the integration of ichnovirus genome segments into lepidopteran chromosomal DNA, and to begin exploring the functional significance of such events, we undertook an investigation of Tranosema rostrale ichnovirus (TrIV) DNA persistence in CF-124T cells following inoculation with TrIV. These cells, derived from Choristoneura fumiferana, one of the natural hosts of T. rostrale, show transient but significant cytopathic effects as a consequence of TrIV infection. Following recovery from infection, however, the cells respond differently to trypsinization (Béliveau et al., 2003), suggestive of a transformation event. Here, we provide direct evidence that TrIV genome segment F, a 7990 bp circular DNA characterized previously (Volkoff et al., 2002), is integrated into the chromosomal DNA of CF-124T cells. In addition, we provide evidence that one of the two rep genes (TrFrep1) carried by this genome segment displays long-term transcription.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Calyx fluid (CF) was extracted from T. rostrale ovaries and filtered as described previously (Béliveau et al., 2003). Choristoneura fumiferana larvae were either parasitized (as fifth instars) or injected with CF (as sixth instars) 24 h after the moult. Inoculation of CF-124T cells with filtered CF and all post-inoculation (p.i.) procedures were carried out as described previously (Béliveau et al., 2003).
RNA isolation and Northern blotting.
Total RNA isolation from Choristoneura fumiferana larvae and CF-124T cells was carried out using Trizol reagent (Invitrogen). Agarose gel electrophoresis and Northern blot analysis were performed as described previously (Béliveau et al., 2003).
DNA isolation and Southern blotting.
TrIV DNA was isolated as described by Béliveau et al. (2000). For CF-124T DNA extraction, cells were resuspended in Grace's medium supplemented with 0.25 % tryptose broth (Difco) and 10 % heat-inactivated fetal bovine serum, pelleted by centrifugation (500 g, 5 min) and lysed in sodium sarcosinate (4 %) by repeated pipetting. Approximately 1 ml lysis solution was used per 5x106–10x106 cells; lysates were stored at –80 °C until ready for extraction. Prior to extraction, lysates were thawed and incubated at 37 °C for 30 min in the presence of proteinase K (250 µg ml–1) and RNase A (50 µg ml–1). Cellular DNA was phenol extracted, ethanol precipitated and resuspended in 30 µl water.
TrIV DNA was fractionated on agarose gels and blotted as described previously (Béliveau et al., 2000). Both untreated and restriction-enzyme-digested DNA from CF-124T cells infected with TrIV were used in Southern blotting. For digested samples, DNA (10 µg) was incubated overnight at 37 °C with either EcoRI or XbaI in a total volume of 500 µl. Digests were precipitated at –20 °C and resuspended in 10 µl water. Undigested (5 µg) or digested (10 µg) DNA was applied to a 1 % agarose gel. After completion of electrophoresis, nucleic acids were treated for 2 min with UV irradiation (302 nm) using a UVP Transilluminator and depurinated in 0.2 M HCl for 20 min. Nucleic acids were denatured in 0.5 M NaOH/1.5 M NaCl for 15 min and neutralized in 0.5 M Tris/HCl (pH 8)/1.5 M NaCl for 15 min. Transfer of DNA to Hybond-N membrane (GE Healthcare) was carried out in 20x SSC, using a VacuGene XL apparatus (GE Healthcare) according to the manufacturer's instructions.
Radioactive probes and hybridization.
To detect TrFrep1 transcripts on Northern blots or TrIV genome segment F on Southern blots, we labelled a 747 bp SspI restriction fragment from genome segment F with 32P; this fragment encompasses almost the entire TrFrep1 open reading frame (Volkoff et al., 2002). Probes used to detect TrV4 and TrV1/TrV2 and hybridization conditions for Northern and Southern blots were the same as those used in previous studies (Béliveau et al., 2000, 2003).
Construction and screening of a TrIV-infected CF-124T DNA library.
DNA from CF-124T cells maintained in culture for 215 days following inoculation with TrIV was used to construct a phage 
genomic library. Purified DNA (60 µg) was digested with 0.6 U Sau3AI for 30 min at 37 °C and ligated to Lambda DASH II/BamHI vector arms (Stratagene). Packaging of ligation reactions (2.5 µl) was done using the Gigapack III packaging extract (Stratagene); recombinant phages were selected and amplified on Escherichia coli XL1-Blue MRA (P2).
Screening of the library was done by PCR (Israel, 1993). Primers specific for TrFrep1 (forward: 5'-TCGCGGATCCATGCGCATTATCATC-3', reverse: 5'-CACGTTCCAGGTATAACTTG-3') were used to amplify a 535 bp fragment in dilutions of the library containing from 6x102 to 6x106 p.f.u. to estimate the frequency of the template and determine the optimal titre for the primary screening. PCR conditions were as follows: a denaturation step of 5 min at 94 °C, followed by 36 cycles of 1 min at 94 °C, 1 min at 45 °C and 1 min at 72 °C. Reactions were completed by an extension step of 8 min at 72 °C and products were fractionated by agarose gel electrophoresis. The primary screen was done on 1.22x106 p.f.u. from the library. Phages (0.5 ml) were incubated with 0.5 ml fresh overnight culture of E. coli XL1-Blue MRA (P2) at room temperature for 20 min. After the addition of 20 ml L broth (Helms et al., 1987), infected cells were distributed among 64 wells of a 96-well plate (100 µl per well), in an 8x8 grid, and grown for 5–6 h at 37 °C on a rotary shaker set at 225 r.p.m. to allow amplification of phage sublibraries. The wells of each row and column from the grid were pooled (25 µl from each well) and subjected to PCR and agarose gel electrophoresis as above to identify TrFrep1-positive sublibraries. Second and third rounds of PCR screening at 30 p.f.u. and 2–3 p.f.u. per well, respectively, were performed as described previously (Israel, 1993), except that agarose gels of resolved PCR products were not subjected to hybridization with a radioactive probe. Phages positive for the TrFrep1 sequence were plaque purified and phage DNA was isolated as described previously (Helms et al., 1987).
Subcloning and sequencing of cell–virus DNA junctions and CF-124T target sites.
DNA from positive phages was analysed by restriction enzyme digestion, followed by agarose gel electrophoresis. Restriction fragments were purified from the agarose (Kurien et al., 2001) and cloned into the pBluescript KS– vector (Stratagene) for sequencing with M13 forward and reverse primers. Sequences were aligned with that of TrIV genome segment F using the BLAST algorithm. Subcloned fragments that had genome segment F-related sequence on one side and unrelated DNA (assumed to be CF-124T genomic DNA) on the other side were sequenced completely in order to identify the junction site.
In addition, we sequenced the site of insertion targeted by TrIV genome segment F in the DNA of uninfected CF-124T cells. PCR primers were designed based on CF-124T DNA sequences flanking the integrated portion of TrIV genome segment F in phage clone G1. A forward primer (5'-AGATGGGCCAGAATGAATAATCC-3') designed from nt –328 to –350 upstream of the left junction site, and a reverse primer (5'- TACCTTGAAGCTATATACTAGGT-3') designed from nt 254 to 276 downstream of the right junction site, were used for PCR. PCR was conducted using the following conditions: 1 cycle of 2 min at 94 °C, 35 cycles of 30 s at 94 °C, 30 s at 55 °C and 2 min at 72 °C, and a final extension step of 8 min at 72 °C. PCR products were resolved on agarose gels, subcloned and sequenced as above.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, 2003; Cusson et al., 2001). In a previous study (Béliveau et al., 2003), the abundance of TrV4 transcripts was examined by Northern blot analysis and shown to drop to undetectable levels 2 days after the moult to the sixth instar. Although somewhat less pronounced, a decrease in the accumulation of TrV1/TrV2 mRNA could also be observed concurrent with the last larval moult of parasitized insects, using a probe that can hybridize to both transcripts (Béliveau et al., 2000, 2003). By comparison, the levels of two TrFrep1-related transcripts (900 and 2100 nt) remained at more stable levels throughout the 6 day post-parasitization period (Fig. 1a).
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). As inferred previously for the 900 nt transcript (Volkoff et al., 2002), these results confirmed that expression of the TrFrep1-related transcripts originated from the virus and not from the developing T. rostrale larva. Furthermore, the TrFrep1-related transcripts persisted longer after injection than those of TrV1/TrV2 (Béliveau et al., 2000, 2003) or TrV4 (Béliveau et al., 2003).
Expression of TrIV genes in CF-124T cells
To determine whether the differential abundance of TrV1/TrV2, TrV4 and TrFrep1-related transcripts observed in vivo could be duplicated in vitro, we inoculated CF-124T cells with filtered CF extracted from T. rostrale females, followed by isolation of total RNA from cell samples taken at nine different times after the initial inoculation (between 1 and 92 days p.i.), as well as from uninoculated cells. TrV4 and TrV1/TrV2 transcripts of the expected sizes (900 and 650 nt, respectively) accumulated to high levels within the first 24 h p.i., but neither transcript could be detected beyond day 4 p.i. (Fig. 2). In contrast, the presence of the TrFrep1-related transcripts was observed over a longer period of time, with the 2100 nt transcript being detected until at least day 22 p.i. and the 900 nt transcript until day 92 p.i. (Fig. 2). Thus, the in vitro system used here provides clear evidence for a much more prolonged persistence of TrFrep1-related transcripts, with mRNA presence extending beyond the time limits imposed by the duration of parasitoid development (14 days in its host; Doucet & Cusson, 1996).
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), electrophoretic profiles of genome segments typically include slow-migrating, nicked-circular DNA molecules, as well as faster-migrating, supercoiled species of the same molecule. The autoradiograms shown in Fig. 3 suggested that both relaxed and supercoiled forms of all three genome segments could be detected on day 4 p.i., along with other bands representing cross-hybridizing genome segments (compare with signals of the same mobility in lane V). One such signal, however, was not detected on the viral DNA blot, possibly owing to a lower signal intensity (Fig. 3, TrFrep1 panel, third arrow from top).
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, TrFrep1 panel). In contrast, no hybridization signal corresponding to either of the other two genome segments could be detected beyond the first sampling date (4 days p.i.; Fig. 3, lower two panels). Thus, the results of this experiment supported the hypothesis that the sustained accumulation of TrFrep1-related transcripts could be linked, at least in part, to the persistence of TrIV genome segment F (or a very large part thereof) in CF-124T cells.
Restriction analysis of genome segment F in TrIV-infected CF-124T cells: evidence for integration
The persistence of genome segment F for at least 100 days in TrIV-infected cells, combined with the relatively low mobility of the hybridization signal (i.e. co-migration with cellular DNA), suggested that this molecule might have undergone integration into the chromosomes of CF-124T cells. To determine whether genome segment F persisted as an episome or as a molecule integrated into host DNA, we first performed a comparative restriction analysis of DNA isolated from TrIV and from TrIV-inoculated CF-124T cells maintained in culture for >100 days. DNAs were digested with either EcoRI or XbaI and probed with a 750 bp SspI fragment spanning most of the TrFrep1 coding sequence. Southern blot analysis of digested TrIV DNA revealed sharp bands of the expected sizes. As TrIV genome segment F displays two EcoRI sites, including one in the middle of the sequence specific to the probe (the TrFrep1 coding sequence), we observed two hybridizing fragments of 3547 and 4443 bp (Fig. 4). In comparison, digestion of viral DNA with XbaI, for which genome segment F contains only one site at nt 5160, yielded a single band of approximately 8 kb, as expected. Southern blot analysis of DNA from TrIV-infected CF-124T cells, following digestion with the same restriction endonucleases, generated a different pattern of bands: the 4443 bp EcoRI fragment was no longer detectable and the remaining signals in each digest were diffuse (Fig. 4). Thus, after its delivery into cells, TrIV genome segment F becomes modified in such a way that its normal restriction enzyme pattern is lost, perhaps by integration into multiple sites (as suggested by the diffuseness of the hybridization signal) in the cell genomic DNA.
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vector. The library was screened using a modification of the PCR screening method developed by Israel (1993). Three phage clones containing the TrFrep1 gene (therefore potentially containing portions of genome segment F) were plaque purified and their respective inserts mapped with restriction enzymes (Fig. 5). Clone G1 had a 16 kb insert containing DNA junctions at both ends of the integrated portion of genome segment F, flanked downstream and upstream by 1 and 7 kb, respectively, of CF-124T genomic DNA (Fig. 5a). Two other clones, C3 and C5, had identical restriction endonuclease digestion patterns (Fig. 5b). The 12.5 kb insert of phage C3/C5 contained 3.5 kb of TrIV genome segment F DNA and 9 kb of cell genomic DNA. The portion of genome segment F found in clone G1 also differed slightly from that cloned directly from TrIV, as it displayed an additional XbaI site (compare Fig. 5a with Fig. 3 in Volkoff et al., 2002). Thus, the library screening and restriction analyses provided further evidence that TrIV genome segment F undergoes integration into CF-124T cell DNA and that both left and right putative DNA junctions had been cloned.
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). In both G1 and C3/C5 clones, the first nucleotide of genome segment F from the left junction was identified as the thymidine at nt 2227 (see Figs 4b and 6a and the map of TrIV genome segment F in Volkoff et al., 2002). Note that the cytidine at nt 2226 could also be the first nucleotide of genome segment F in phage C3/C5. The limit of TrIV genome segment F on the right junction was determined to be at nt 2193. Therefore, 33 nt were missing from the integrated form of genome segment F relative to the published sequence of the circular episome (Volkoff et al., 2002; Fig. 6a). Host DNA sequences at the left junctions of the G1 and C3/C5 clones were observed to be unrelated (Fig. 6a), indicating that TrIV genome segment F DNA can integrate at different sites within CF-124T cell DNA.
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250 bp downstream and 350 bp upstream of the integrated portion of TrIV genome segment F in clone G1, which we used to PCR amplify a 600 bp product from DNA extracted from control CF-124T cells. Sequencing of this PCR fragment (GenBank accession no. DQ790661) and its alignment with the CF-124T sequences on the left and right sides of the integrated portion of genome segment F from clone G1 revealed 100 % identity, although it seems possible that the target host sequence lost one base pair through the integration process (compare sequences in Fig. 6a and b). We also discovered that both strands of the target sequence contained a few nucleotides (2 and 4 nt) that were identical with nucleotides in the region of TrIV genome segment F that undergoes integration (Fig. 6b). |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) alone provided strong circumstantial evidence for the integration of TrIV genome segment F into CF-124T cell DNA following viral infection. First, a persistent TrFrep1-specific signal was seen to be associated with high-molecular-mass DNA on Southern blots, whilst no such signal was observed using probes specific for two other genome segments (Fig. 3). Secondly, a Southern blot comparison of TrIV DNA with that of TrIV-infected CF-124T cells, digested with EcoRI or XbaI, provided evidence for the absence of a virus-specific EcoRI restriction fragment in the cellular DNA sample (Fig. 4), suggestive of an integration event taking place in that region of the genome segment (i.e. within the 4443 bp fragment region). Finally, on the same blots, the hybridization signals obtained with cellular DNA were far more diffuse than those observed with viral DNA, suggesting that integration events had taken place at multiple sites within the host genome. Previous to this study, only circumstantial evidence of integration involving ichnovirus DNA had been reported (Kim et al., 1996; Volkoff et al., 2001). On the other hand, proof of bracovirus DNA integration in vitro exists (Gundersen-Rindal & Lynn, 2003).
Definitive evidence for an integration event relies on the cloning and sequencing of identifiable TrIV–CF-124T DNA junction sites (Figs 5 and 6), which we have now provided. As one phage clone (G1) contained both right and left junctions, we were able to show that >99 % of genome segment F was present within TrIV-transformed CF-124T chromosomal DNA; whether the missing 33 bp in the integrated form of this genome segment are lost before or after the integration event remains to be determined. In addition, sequencing of the left junctions of clones G1 and C3/C5 provided evidence that integration could take place at different sites within the host genomic DNA, as suggested by Southern blot analysis (Fig. 4). Although the two sites reported here were apparently unrelated, several additional clones will need to be analysed to assess the extent of variability among CF-124T genomic DNA insertion points. Based on the analysis of clone G1, none of the detectable open reading frames of TrIV genome segment F was disrupted by the integration process, which in theory permits continued transcription of TrFrep1 after integration (Fig. 2). It remains possible, however, that integration occurs at more than one site within genome segment F, a hypothesis that could be evaluated by analysis of other positive clones. Finally, sequencing of a large portion of the integrated form of TrIV genome segment F, in clone G1, revealed at least one difference in its sequence (resulting in an additional XhoI restriction site; see Fig. 5) relative to the published sequence of the circular genome segment (Volkoff et al., 2002). This observation is not surprising given the documented occurrence of polymorphisms in polydnavirus genomes (Stoltz & Xu, 1990) and the fact that the original viral inoculum was obtained from several wasps. Polymorphism of a genome segment bearing rep genes has been reported previously for Hyposoter didymator ichnovirus genome segment E (Volkoff et al., 2002).
The present study provides relatively few clues as to how TrIV genome segment F becomes integrated into CF-124T cellular DNA. As reported previously for a GiBV genome segment that undergoes integration into the DNA of Lymantria dispar cells in culture (Gundersen-Rindal & Lynn, 2003), TrIV genome segment F does not appear to encode enzymes such as transposases, integrases or reverse transcriptases that could participate in the process, nor do the other sequenced TrIV genome segments (unpublished data). It therefore seems likely that enzymes required for integration originate from the host. Examination of the sequences at the junction sites revealed little in terms of how these could facilitate linearization and/or integration. There was, however, an (ATTCT)2 repeat within the 33 nt region that was lost during the integration process (Fig. 6a); (ATTCT)n repeats have recently been associated with duplex unpairing under high superhelical energy (Potaman et al., 2003), a process that could favour a DNA break within that region of the genome segment. Similarly, we observed two poly(A)15–19 tracks within TrIV genome segment F, symmetrically located at 110 bp from the point of integration (data not shown). Similar A, T or AT runs have been observed in GiBV genome segment F near the point of integration within lepidopteran genomic DNA (Gundersen-Rindal & Lynn, 2003). Such AT-rich regions may favour DNA unpairing. A notable feature of the sequence of the integrated GiBV genome segment is the presence of a CATG palindrome repeat at the junction site, which the authors postulate may be involved in the integration mechanism (Gundersen-Rindal & Lynn, 2003); no such palindromic repeat was detected in TrIV genome segment F at the DNA junction site. Finally, analysis of clone G1 revealed the presence of a small region of sequence identity (5 bp) between host target DNA and TrIV genome segment F DNA at the point of integration (Fig. 6); however, this feature is unlikely to be relevant to the integration process given that the CF-124T DNA sequence at the left junction of clone C3/C5 differs from that in clone G1. Ultimately, elucidation of a mechanism of integration will, in this case, require in vitro transfection experiments using engineered truncations of TrIV genome segment F. A comparison of the host–virus DNA junctions in the lepidopteran cells with those in wasp cells may also yield new insights into the mechanism of integration; within the wasp host, such junctions have been identified for other PDV genome segments and found to display direct repeats (Gruber et al., 1996; Savary et al., 1997; Rattanadechakul & Webb, 2003; Wyder et al., 2002).
It remains to be determined whether the integration phenomenon described here, which we induced by infecting cells grown in culture, also takes place in larvae that have become parasitized under natural conditions. It has, thus far, been difficult to obtain unequivocal evidence to the effect that ichnovirus and/or bracovirus genome segments undergo integration within the chromosomal DNA of parasitized caterpillars, perhaps because of a lower infected : uninfected cell ratio achieved in vivo compared with that obtained in vitro, in which environment cells are presumably exposed to a much larger inoculum of virus. None the less, as we have shown, the expression of TrFrep1 in parasitized larvae appears to be sustained for a longer period than that of other TrIV genes borne by genome segments that appear not to undergo integration in vitro (Fig. 1), an observation suggesting that integration of genome segment F may also occur in vivo. In an independent study, Southern blot analysis of DNA obtained from CcBV-parasitized Manduca sexta larvae revealed hybridization signals suggestive of in vivo integration events (Le et al., 2003). Given that some PDV genome segments appear not to persist as episomes in host cells (Fig. 3), integration of selected genome segments could extend the persistence of viral genes whose products are essential throughout parasitoid development within the host. It is interesting to note that in the ichnovirus (this study) and bracovirus (Gundersen-Rindal & Lynn, 2003) systems where integration into lepidopteran genomic DNA has been clearly demonstrated, the genome segments that were integrated harboured genes belonging to the numerically most important gene families in each taxon (i.e. the rep and PTP gene families; Webb et al., 2006). Although the exact functions of these genes are not yet known, their high level of representation in ichnovirus and bracovirus genomes, respectively, suggests that they play key roles in some aspect of PDV virulence. The observation that integration of PDV genome segments into lepidopteran genomic DNA implicates viral DNAs carrying rep and PTP genes may be viewed as additional support for the hypothesis that these integration events have a functional significance, warranting a more in-depth examination of this phenomenon in vivo.
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ACKNOWLEDGEMENTS |
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Received 21 June 2006;
accepted 22 September 2006.
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J. Y. Choi, S.-J. Kwon, J. Y. Roh, T.-J. Yang, M. S. Li, B.-S. Park, Y. Kim, S.-D. Woo, B. R. Jin, and Y. H. Je Analysis of promoter activity of selected Cotesia plutellae bracovirus genes J. Gen. Virol., May 1, 2009; 90(5): 1262 - 1269. [Abstract] [Full Text] [PDF]
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