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1 Laboratory for Molecular Virology, Great Lakes Forestry Centre, Sault Ste Marie, ON P6A 2E5, Canada
2 Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON N1G 2W1, Canada
Correspondence
Basil M. Arif
barif{at}nrcan.gc.ca
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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The GenBank/EMBL/DDBJ accession number for the sequence reported in this paper is DQ333351.
Comparison of the ChocGV genome with other granuloviruses is available as a supplementary table in JGV Online.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). GVs infect both agricultural and forest insect pests, making them potentially important as biological insecticides (Rashidan et al., 2004a). The sequencing of the Choristoneura occidentalis GV (ChocGV) genome brings the number of totally sequenced GVs to eight.
The western spruce budworm, Choristoneura occidentalis, and the eastern spruce budworm, Choristoneura fumiferana, were previously considered to be the same species separated geographically by the Rocky Mountains. They are now considered to be different species. C. occidentalis is the most widely distributed and destructive defoliator of coniferous trees in Western North America. The first recorded outbreak was in 1909 on Vancouver Island, Canada. The insects prefer new growth, damaging leaves, flowers, cones and affecting tree survival and regeneration (Fellin & Dewey, 1999). Damage begins in the spring when larvae mine into needles and enter the swollen buds. Webs are spun on new foliage, where insects feed by chewing needles off at their base. Trees may recover unless repeated severe defoliation occurs over 3–5 years (http://www.pfc.forestry.ca). Previous work by Arif et al. (1986) describes comparisons of granuloviruses isolated from Choristoneura spp. Baculoviruses that have been isolated from C. occidentalis and C. fumiferana are cross-infective (Fellin & Dewey, 1999). This manuscript reports the complete sequencing and analysis of the ChocGV genome and compares it with other baculovirus genomes.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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DNA sequencing and analysis.
ChocGV genomic DNA was nebulized, cloned into pUC18 and sequenced to yield 10 times coverage (Applied Biosystems; BGI Life Technology). Base calling was done using the software Phred (Ewing et al., 1998; Ewing & Green, 1998), pUC18 contaminations were removed with CrossMatch (Green, 2004), and the sequence was assembled using Phrap (Green, 1996). Gaps and regions with low quality data identified after preliminary assembly were filled in or refined by sequencing PCR amplified products.
Sequence analysis was carried out using NCBI open reading frame (ORF) finder (Wheeler et al., 2003), BLAST (Altschul et al., 1990), DNASTAR (Burland, 2000) and the Multi-purpose automated genomics project investigation environment (Magpie), available through the University of Calgary (Gaasterland & Sensen, 1996). Methionine-initiated ORFs potentially encoding 50 or more amino acids were considered for further analysis. Smaller ORFs with baculovirus homologues were also considered. DNA regions containing potential ORFs with unusually large repeats were deemed as non-coding and analysed separately. Repeated sequences were identified using REPuter (Kurtz & Schleiermacher, 1999) and Tandem Repeats Finder (Benson, 1999). ExPASy (Gasteiger et al., 2003), Prosite (Hulo et al., 2004) and SMART (Schultz et al., 1998) were used to identify protein motifs. Multiple sequence alignments and percentage amino acid identities were carried out using DNASTAR's MEGALIGN CLUSTAL W (Thompson et al., 1994) with default conditions. GeneDoc was used to shade conserved sequences in aligned proteins (Nicholas & Nicholas, 1997). Seven GV genomes and 11 NPV genomes were used to identify gene conservation in ChocGV. The genomes included were from: Cydia pomonella GV (CpGV; Luque et al., 2001), Cryptophlebia leucotreta GV (CrleGV; Lange & Jehle, 2003), Adoxophyes orana GV (AdorGV; Wormleaton et al., 2003), Phthorimaea operculella GV (PhopGV; GenBank accession no. AF499596
[GenBank]
), Plutella xylostella GV (PlxyGV; Hashimoto et al., 2000), Xestia c-nigrum GV (XecnGV; Hayakawa et al., 1999), Agrotis segetum GV (AgseGV; GenBank accession no. NC_005839
[GenBank]
), Autographa californica multicapsid NPV (AcMNPV; Ayres et al., 1994), Helicoverpa armigera NPV (HearNPV G4; Chen et al., 2001), Spodoptera litura NPV (SpltNPV; Pang et al., 2001), Spodoptera exigua MNPV (SeMNPV; IJkel et al., 1999), Lymantria dispar MNPV (LdMNPV; Kuzio et al., 1999), Orgyia pseudotsugata MNPV (OpMNPV; Ahrens et al., 1997), Choristoneura fumiferana defective NPV, (CfDEFNPV; Lauzon et al., 2005), Epiphyas postvittana NPV (EppoNPV; Hyink et al., 2002), Rachiplusia ou MNPV (RoMNPV; Harrison & Bonning, 2003), Bombyx mori NPV (BmNPV; Gomi et al., 1999) and Neodiprion lecontei NPV (NeleNPV; Lauzon et al., 2004). Promoter regions were found using Promoter Scan (Prestridge, 1995) and InterPro (Mulder et al., 2003). Gene parity plots (Hu et al., 1998) compared the gene order of ChocGV with CpGV, CrleGV, AdorGV and AcMNPV. Concatamers of 29 conserved baculovirus proteins from 27 genomes were used to determine phylogeny. A most parsimonious tree was constructed using CLUSTAL W protein alignments and PAUP 4.0b10 (Swofford, 2000) with maximum-parsimony, heuristic search, step-wise addition option and bootstrap analysis (1000 replicates). Genomes used in phylogenetic analysis included those previously listed plus the following: HearNPV C1 (Zhang et al., 2005), Helicoverpa zea SNPV (HzSNPV; Chen et al., 2002), Mamestra configurata NPV A (MacoNPV A; Q. Li et al., 2002), MacoNPV B, (L. Li et al., 2002), Adoxophyes honmai NPV (AdhoNPV; Nakai et al., 2003), Choristoneura fumiferana MNPV (CfMNPV; de Jong et al., 2005), Neodiprion sertifer NPV (NeseNPV; Garcia-Maruniak et al., 2004) and Culex nigripalpus NPV (CuniNPV; Afonso et al., 2001).
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). ChocGV contained 116 methionine-initiated ORFs, 62 (53.4 %) clockwise and 54 (46.6 %) counterclockwise, with respect to granulin that was designated ORF 1 (Table 1, Fig. 1). Choc41 (46 aa) was considered a potential ORF as it showed baculovirus homology (Table 1). There were minor overlaps between certain ORFs; the longest (139 bp) covered the 3' end of alkaline-exonuclease and the 5' end of helicase-2.
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), and no BLAST matches with other baculovirus genomes were found. The third intergenic space (3441 bp) between choc36 and 37 contained a 1.8 kb region with multiple direct repeats that was extremely AT rich (81 %). Initial computer analysis suggested the presence of a 1144 aa ORF in this region with 27 leucine zippers. Leucine zippers contain leucine residues (L) at every seventh position over at least seven helical turns (Gattiker et al., 2002) that establish amphipathy to help stabilize a long 
-helix (Landschulz et al., 1988). The leucine zipper motif is: [L(X6)L(X6)L(X6)L] (Bateman et al., 2004). This potential ORF did not have any baculovirus homologues. Sequencing errors are more common within repeat regions as frequent recombination can lead to differences in data between clones (Ahrens et al., 1997). Also, ChocGV was not plaque purified, increasing the possibility of clonal variation. Manual examination revealed variation among different clones covering the same area. Other fully sequenced GV genomes contain three smaller ORFs in this region (cp49, cp50 and cp51; Fig. 1). The presence of three ORFs between choc36 and 37 was suggested by BLASTN analysis. These potential ORFs had high identity to three ORFs from ChfuGV (GenBank accession nos AAN77189
[GenBank]
, AAN77190
[GenBank]
and AAN77191
[GenBank]
). The beginning of the region also showed low nucleotide identity to cp50/crle47 and its 3' region showed low identity to xecn47. Manual examination of the trace data showed several areas where sequencing errors were possible or where the addition or subtraction of a single base might cause a frameshift. Two of these potential changes involved areas with large strings of adenines. If bases were added to cause frameshifts, the first potential ORF between choc36 and 37 would be larger than its ChfuGV homologue (GenBank accession no. AAN77189
[GenBank]
) as it would have three leucine zippers instead of one, suggesting that duplication of repeat regions has occurred in the ChocGV genome. The second potential ChocGV ORF had three repeats of 210 bp constituting 84 % of the ORF and the third lacked repeats but had a transmembrane domain at its 5' end.
There were many factors to consider when interpreting the large space between choc36 and choc37: (i) the space had multiple repeats, (ii) it contained ambiguous bases in the trace data, (iii) ChocGV was not plaque purified and clonal variation existed in this region, (iv) no other baculovirus genomes contained homologues to the large potential protein between choc36 and 37, and (v) difficulties arose in deciding where bases should be added or deleted to cause a frameshift producing three small ORFs. Because of the combination of these factors, decisions to have one large ORF or three smaller ORFs between choc36 and 37 were both rejected. Instead, DNA regions containing ORFs with large, unusual repeats were considered non-coding and were analysed separately as was done with the genome of CrleGV (Lange & Jehle, 2003).
Intergenic spaces greater than 1.0 kb are found in other GVs. XecnGV has a 1473 bp intergenic region (Hayakawa et al., 1999) in approximately the same location as the first large intergenic space in ChocGV. CrleGV has a 1.8 kb intergenic space between crle26 and crle27 that contains a 300 bp AT-rich region, direct repeats, short palindromes and is considered to be a potential non-homologous region origin of DNA replication (non-hr ori) (Jehle, 2002; Lange & Jehle, 2003). CpGV has a repeat region of 1.13 kb with six short imperfect repeats, three large imperfect repeats and an AT-rich section also considered to be a non-hr ori. The CpGV non-hr ori region, however, is located within three ORFs (cp25–cp27) none of which have baculoviral homologues (Huang & Levin, 1999). The non-hr ori repeats in CpGV and CrleGV are not found elsewhere in the genomes and are not similar to their homologous regions (hrs) (Luque et al., 2001; Lange & Jehle, 2003). Other baculoviruses with non-hr oris include: AcMNPV (Kool et al., 1994), OpMNPV (Pearson et al., 1993), SeMNPV (Heldens et al., 1997) and Spodoptera littoralis (SpliMNPV) (Huang & Levin, 1999). These regions are complex, contain direct and inverted repeats and are between 800 and 4000 bp long (Luque et al., 2001). The ChocGV 1.8 kb repeat region between choc36 and 37 showed similarities to the non-hr ori regions in CrleGV and CpGV as it had multiple repeats not found elsewhere in the genome, was not similar to hrs and was very AT rich.
Hrs regions
Typically, baculoviruses have many hrs interspersed throughout their genomes. These regions may act as enhancers of RNA transcription, as origins of DNA replication and as sites of recombination (Hayakawa et al., 2000). Most NPV hrs contain 30 bp palindromes within direct repeats, whereas GV repeat regions are more variable and often lack palindromes (Wormleaton et al., 2003). XecnGV contains nine hrs each with three to six direct repeats lacking a palindromic core (Hayakawa et al., 1999). PlxyGV has four large repeat regions centred on a palindrome that more closely resemble NPV hrs (Hashimoto et al., 2000), while AdorGV has nine repeat regions unlike NPV hrs (Wormleaton et al., 2003). ChocGV contained five hrs/repeat regions varying in size and number of direct repeats. The first (hr-1), had three repeated sequences in a 157 bp area, hr-2, four repeated sequences within 129 bp, and hr-3, 10 repeated sequences within 288 bp. The fourth and fifth hrs/repeat regions had four repeated sequences in a 106 bp area and 162 bp area, respectively (Fig. 2a). Direct repeats averaged 27 bp with the shortest being 19 bp and the longest 39 bp. These regions were not like NPV hrs and did not have embedded palindromes. Each hr was homologous to all other ChocGV hrs but not to hrs in other baculoviruses. Despite the differences between GV and NPV hrs, and between GV hrs themselves, the relative locations of hrs in ChocGV were similar to the locations of three of the hrs in CrleGV, and to eight of 13 repeats in CpGV (Fig. 2b).
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). Choc36 had a 100 % amino acid identity to a ChfuGV ORF (GenBank accession no. AAN77188
[GenBank]
), which is listed in GenBank as a homologue to xecn152 (enhancin). Therefore, it would be expected that choc36 would also be a homologue to xecn152. However, BLASTP results showed that choc36 did not have a baculovirus match but had a high identity (9.0 e-10, 30.7 % amino acid identity) to a protein phosphatase regulatory subunit 15A in ORF 93 of Amsacta moorei entomopoxvirus (AmEPV; Bawden et al., 2000). CLUSTAL W alignments showed that ChfuGV (GenBank accession no. AAN77188
[GenBank]
) and choc36 shared 23.8 % amino acid identity with xecn152. Therefore, choc36 was accepted as a xecn152 homologue. Choc25 showed 21.4 % amino acid identity to a previously unique CrleGV ORF 9 and 34.3 % to a protein from a parvo-like virus [ORF 2 Yamanashi isolate from Bombyx mori densovirus (BmDNV-2; Bando et al., 1995)]. Other ChocGV ORFs had homologues in NPVs but not GVs. Choc20 shared 27.7 % amino acid identity with LdMNPV ORF 7 and 29.8 % with CfDEFNPV ORF 145. Choc53 shared a 24.4 % amino acid identity to LdMNPV ORF 111 and 27.8 % with MacoNPV A ORF 46.
Choc62 and choc63 had BLASTP matches to cp82 at 35.6 and 25.6 % amino acid identity, respectively, but shared only 10.3 % amino acid identity with each other. The combined size of choc62 (261 bp) and choc63 (597 bp) was similar to that of cp82 (921 bp), suggesting that the ORFs corresponded to different regions of cp82. Choc62 aligned with the 3' end of cp82 (664–921 bp), while choc63 aligned with the 5' end (89–746 bp). The sequencing data did not reveal any sequencing errors that could link the two potential ORFs as one. The function of cp82 is not known. It is conceivable that low expression of a cp82 homologue could take place in ChocGV even if a computational frameshift had occurred resulting in choc62 and choc63. This suggestion is corroborated by the fact that low gene expression of lacZ was detected in AcMNPV p10 deletion mutants containing a translational frameshift in the lacZ region (Williams et al., 1989).
Inhibitors of apoptosis
Apoptosis plays an important role in virus replication and in cellular response to infection (O'Brien, 1998). Viruses have acquired genes, the products of which evade or suppress programmed cell death. There are two types of baculovirus proteins that suppress apoptosis, P35/P49 homologues and inhibitors of apoptosis (IAPs) (Du et al., 1999). P35 acts as a direct inhibitor of proteases, while IAPs act upstream to prevent activation of the proteases (Vaux & Strasser, 1996). Functional homologues of P35 are found in some but not all NPVs, whereas IAPs are present in both NPVs and GVs. ChocGV is the first GV with a P35/P49 homologue. Choc15 shared the highest amino acid identity with SpliMNPV P49 (GenBank accession no. AJ006751
[GenBank]
) (27 %), SpltNPV ORF 55 P49 (25.7 %) and RoMNPV ORF 128 P35 (21 %). Choc15 showed a BLASTP match (1.0 e-07, 19.1 % amino acid identity) with a previously unreported P35 homologue from AmEPV (AMV010) (Bawden et al., 2000) (Fig. 3). While these values are not very high, functionality awaits further analysis. It is not yet known if choc15 functions as a P35 or as a P49 homologue as it shares conserved amino acids with both but its functionality and target of activity are yet to be determined. P35 proteins have a large loop domain known as the reactive site loop that contains the caspase recognition motif DQMDG at its apex, while P49 proteins contain the motif TVTDG (Pei et al., 2002). Choc15 and AMV010 appear to have different active site motifs than those of P35 and P49. Two motifs, 92DCSND96 and 86VYNFD90 in choc15 and AMV010, respectively, were found in the same relative location as the active motifs of P35 and P49 (Fig. 3). It has been shown that AMV010 is an active apoptotic suppressor and that mutating the aspartic acid residue (D90) within the above motif results in an inactive protein (R. Clem, personal communication). Experiments are under way to verify the activity of choc15 and to ascertain the essentiality of D96 within its putative active site motif. The molecular mass of choc15 was 42.6 kDa, which differs from P49 (49 kDa) and P35 (35 kDa) homologues. It is also interesting to note that the location of choc15 within ChocGV was similar to that of iap-3 in both CpGV and CrleGV (Fig. 2b). Five groups of iap genes have been identified in baculoviruses but not all are active suppressors of apoptosis (Ikeda et al., 2004). Homologues of iap-3, generally identified as active inhibitors of apoptosis, are found in both NPVs and GVs, but iap-5 homologues have so far been found only in GVs (Wormleaton et al., 2003). ChocGV contained two iap homologues; choc84 (iap-3) and choc95 (iap-5). Most IAP proteins contain baculovirus inhibitor repeats (BIRs) and a RING finger (Schultz et al., 1998). Both choc84 and choc95 contained two BIRs and one zinc finger ring. IAP and P35 are not homologues and are likely unrelated in ancestry and mechanism of action (Crook et al., 1993).
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were found in ChocGV (Table 2). Of the replication- and transcription-specific genes present in some but not all baculoviruses (Hayakawa et al., 2000), ChocGV did not have late expression factor 7 (lef-7) or lef-12 typically found in group I NPVs. The genome also lacked immediate-early gene 2 (ie-2), proliferating cell nuclear antigen (pcna), pe38, ribonuclease reductase 1 (rr1), rr2 and dutpase.
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). Granulin and ODV-E25 shared 90.9 and 72.2 % amino acid identity, respectively, with homologues from the seven sequenced GVs, making them two of the most highly conserved proteins in ChocGV. All three genes involved in per os infectivity were present in ChocGV, namely p74 (Faulkner et al., 1997) (choc46), per os infectivity factor-1 (pif-1) (Kikhno et al., 2002) (choc56) and pif-2 (Pijlman et al., 2003) (choc35). These three genes are conserved among all baculoviruses sequenced to date (Lauzon et al., 2004; Garcia-Maruniak et al., 2004).
Three ChocGV ORFs contained sequences corresponding to the N terminus of the polyhedron envelope protein (PEP). These ORFs were found in similar locations and shared high similarity with other GV PEPs. ChocGV PEP proteins (choc17, 18 and 19) and CpGV PEP proteins (cp20, 22 and 23) averaged 70.6 and 68.5 % amino acid identity, respectively, with the PEP proteins from CrleGV (crle20, 23, 24). PEP is found on the surface of OBs and is important for the formation of polyhedra as it stabilizes and prevents them from fusing (Bateman et al., 2004).
P10 proteins are characterized by having various common structural and functional domains, including a coiled-coil region, followed by a proline-rich area [(G/A/V/L/I) P (D/E/N) (V/L/I/P) P], a variable region and finally a basic region at the C-terminal. P10 proteins from different baculoviruses are characterized by the size differences among their shared domains and their sequences are generally poorly conserved (Van Oers & Vlak, 1997). Among the seven sequenced GV genomes, only three have P10. AdorGV and XecnGV each have one (ador13 and xecn5, respectively), while PlxyGV has three (plxy2, plxy21 and plxy50). The mean amino acid identity of choc45 with GV P10 proteins was only 14.5 %, but was 30 % with nine NPV P10s (Fig. 4a), with the highest match being op133 (46.2 %), making choc45 closer to NPV P10s than to GV P10s. ChfuGV P10 (GenBank accession no. AAN77200
[GenBank]
), which is identical to choc45, clustered within group I NPV P10s in a phylogenetic tree (Lange & Jehle, 2003), and the gene arrangement of p10 in ChfuGV was identical to that of many NPVs (Rashidan et al., 2004b).
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. Along with their P10 proteins, AdorGV and XecnGV also have PEP/P10 homologues with a P10-like motif (ador17 and xecn19, respectively). Other GVs with PEP/P10 proteins include: CpGV (cp22), PhopGV (phop20), CrleGV (crle23) and AgseGV (agse19). The P10 domains within crle23 and choc18 shared 51.1 % amino acid identity and the PEP domains 87.8 %. The close association of P10 and the polyhedron envelope has been well documented in many NPVs and it is known that the presence of P10 is essential for the formation of the polyhedron envelope. In some GVs, the functional association of PEP and P10 might have been conserved in a single protein (Lange & Jehle, 2003). Further work must be done to fully understand the role of the different P10 homologues in ChocGV.
Entomopoxviruses produce cytoplasmic fibrils made of filament-associated late proteins (FALPE), while baculoviruses have filaments in the nuclei and cytoplasm of infected cells made of P10 protein (Alaoui-Ismaili & Richardson, 1998). FALPE homologues share some of the structural features of P10 proteins (a proline-rich region and a basic C-terminal tail), but amino acid identity between FALPE and P10 proteins is low (Alaoui-Ismaili & Richardson, 1998). AMV032 (FALPE) and choc45 (P10), however, share a high amino acid identity (30.5 %) due to their identical proline-rich region, alternating proline (P) and glutamic acid (E) (Fig. 4c) not seen in other NPV or GV P10s to date (except ChfuGV). However, these EP repeats have been seen in Trypanosoma sp. procyclins (glycosyl phosphatidylinositol-anchored proteins that contain EP/GPEET amino acid repeats at the C-terminal) (Rashidan et al., 2004b; Ruepp et al., 1999). In phylogenetic analysis (data not shown), FALPE and choc45 grouped with each other, suggesting a strong evolutionary relationship.
Auxiliary genes
Auxiliary genes are not essential for replication but may give the virus a selective advantage in nature (O'Reilly, 1997). ChocGV did not appear to contain baculovirus repeat orfs (bros), iap-1, chitinase, cathepsin and some other auxiliary genes present in some GVs (Table 2). Bro genes are highly repetitive (16 present in LdMNPV) and conserved, but their function is not yet clear (Kuzio et al., 1999). Chitinases are directly involved in the degradation of insect cuticle during moulting, and cathepsin is involved with insect liquefaction (Slack et al., 1995; Hawtin et al., 1997). The absence of these two genes from ChocGV is manifested by the larvae not liquefying upon death. It is not yet clear as to why ChocGV lacks chitinase and cathepsin. ChocGV, however, had an ORF (choc9) with a coiled-coil domain, a chitin-binding domain (type2 ChtBD2), and a high amino acid identity with crle8 (72.7 %) and cp9 (66.7 %), which also have ChtBD2. Chitin-binding domains are extracellular domains that contain six cysteines needed to form three disulfide bridges (Schultz et al., 1998). Auxiliary genes present in ChocGV are listed in Table 2. Ubiquitin (ubq) was the second most conserved gene in ChocGV, having a mean of 78.8 % amino acid identity to homologues in seven sequenced GVs. All GV genomes sequenced to date have three fibroblast growth factors (fgfs). ChocGV is no exception, with choc57, 102 and 114 in the same relative locations as fgfs in other GVs. FGFs are ubiquitous in nature and are highly conserved in structure and amino acid sequence. They are found throughout the genome and play important roles in cell proliferation, differentiation and tissue repair (Ornitz & Nobuyuki, 2001).
Comparison of ChocGV with other baculoviruses
Comparative analysis of baculoviruses can provide insight into their evolutionary history. Similarities and differences in amino acid sequences and in gene order help place baculoviruses in groups sharing gene characteristics and overall genome relatedness. ChocGV and ChfuGV are almost identical in size and restriction enzyme digestion profiles. Comparison of DNA profiles from both viruses cut with EcoRI, HindIII, KpnI and BamHI reveals only minor differences in band size ranging from 0.05 to 5 kb, with the largest difference being in HindIII-P. Comparison of structural proteins also reveals only minor size differences between the two viruses (Arif et al., 1986). CLUSTAL W alignment of individual ChocGV sequences with ChfuGV sequences revealed 100 % amino acid identity for 27 of 38 ChfuGV ORFs sequenced to date (Table 1), with the mean amino acid identity for all being 97.2 %. With similar restriction patterns, biological relationships and high genomic homology, ChocGV and ChfuGV may be variants of the same viral species. The fully sequenced baculoviruses with the highest mean amino acid identity with ChocGV were CpGV (106 homologous ORFs, 52.3 % amino acid identity), CrleGV (106 ORFs, 52.1 % amino acid identity) and PhopGV (103 ORFs, 48.1 % amino acid identity) (Supplementary Table S1 available in JGV Online). The NPV with the highest mean amino acid identity to ChocGV was HearNPV G4 (65 homologous ORFs averaging 31.2 %).
Gene arrangement in baculoviruses is a reflection of their evolutionary history, with more closely related viruses sharing a higher degree of gene collinearity (Hu et al., 1998). Gene parity plots compare the positions of homologous genes in different genomes and are used to illustrate conservation between baculovirus genomes (Herniou et al., 2003). Examination of the number of ORFs in the same order as ChocGV (diagonal line in Fig. 5), showed that ChocGV shared the highest gene order with CrleGV (88.8 % collinearity), CpGV (87.9 %), AdorGV (85.3 %) and AcMNPV (24 %) (Fig. 5). The positions of iap-3 and p10 were the most variable. In ChocGV, p10 was found immediately downstream of p74, as in most NPVs. However in most GVs, p74 is distal from p10. One drawback of using parity plot analysis as compared with phylogenetic trees is that they do not give quantitative information on genome relatedness (Herniou et al., 2001).
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), dna polymerase (Bulach et al., 1999) or ecdysteroid UDP-glucosyltransferase (Barrett et al., 1995) from several genomes. Recently, however, a concatenation of shared proteins has been shown to produce more reliable trees (Herniou et al., 2001). A most parsimonious tree produced using concatamers of the 29 conserved proteins from 27 fully sequenced baculovirus genomes (Fig. 6) reflected the three major taxonomic divisions within the lepidopteran baculoviruses: group I NPV, group II NPV and GVs. ChocGV grouped in a clade with CpGV and CrleGV. This was expected, as they shared the highest mean amino acid identity to ChocGV. The Hymenopteran and Dipteran baculoviruses grouped in separate clades. A new baculovirus classification system has been proposed for these non-lepidopteran baculoviruses, as they do not group with lepidopteran NPVs or GVs (Herniou et al., 2003; Lauzon et al., 2004).
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ACKNOWLEDGEMENTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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