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1 Department of Biological Control and Quarantine, Institute of Plant Protection, Miczurina 20, Poznan 60-318, Poland
2 Laboratory of Virology, Wageningen University, Binnenhaven 11, 6709 PD Wageningen, The Netherlands
3 Greenomics, Plant Research International, PO Box 16, 6700 AA Wageningen, The Netherlands
Correspondence
Monique M. van Oers
monique.vanoers{at}wur.nl
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the sequence described in this paper is DQ123841.
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INTRODUCTION |
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). A. segetum is often part of a cutworm complex, further consisting of Agrotis ipsilon, Agrotis exclamationis, Agrotis subterranea, Peridroma saucia and other noctuid species, that destroys plants near the soil surface (Bourner & Cory, 2004). Viruses with biocontrol potential in the field, belonging to the family Baculoviridae, have been isolated from various Agrotis species (Caballero et al., 1991; Bourner et al., 1992; Boughton et al., 1999). Baculoviruses form a family of large, circular, double-stranded DNA viruses that occur widely in invertebrates, in particular insect species of the orders Lepidoptera, Diptera and Hymenoptera. To date, over 700 baculoviruses have been isolated and several of these have been investigated as bioinsecticides of phytophagous insects (Federici, 1999) and showed potential against field and forest pests all over the world.
The family Baculoviridae is divided into two genera, Nucleopolyhedrovirus (NPV) and Granulovirus (GV) (Theilmann et al., 2005). Lepidopteran NPVs show a further division into group I and group II NPVs (Bulach et al., 1999; Herniou et al., 2001). Group I NPVs appear to be much more conserved than those of group II (Hyink et al., 2002; Lange & Jehle, 2003). NPVs are designated single (S) or multiple (M) depending on the number of nucleocapsids packaged in a virion, although this feature has no taxonomic value (Volkman et al., 1995). Twenty-eight completely sequenced baculovirus genomes have been released in GenBank to date (25 from lepidopteran viruses, two from hymenopteran viruses and one from a dipteran virus), including a GV isolated from A. segetum (AgseGV) (GenBank accession no. NC_005839). Comparison of these baculovirus genomes revealed 29 core genes, shared by all baculoviruses, and 62 genes common to lepidopteran baculoviruses (Herniou et al., 2003; Lange & Jehle, 2003). Apart from those conserved genes, baculoviruses contain genes shared by a variable number of related virus species or even contain unique genes.
Two different NPVs have been isolated from the turnip moth A. segetum, an English/French and a Polish isolate, both of the multiple nucleocapsid (M) type (Allaway & Payne, 1983). Restriction-enzyme profiling and phylogenetic analysis based on three conserved baculovirus genes – polyhedrin (polh), late expression factor 8 (lef-8) and per os infectivity factor 2 (pif-2) – revealed that both isolates are relatively distant and probably represent different virus species (Jakubowska et al., 2005).
In this paper, the genome sequence of the Polish isolate of A. segetum NPV is analysed. To avoid nomenclatural confusion, we propose to name this Polish isolate AgseNPV-A, as it is the first AgseNPV isolate whose genome has been sequenced, and hence propose the name AgseNPV-B for the English/French isolate. The Polish isolate was indicated before as AgseNPV-P (Jakubowska et al., 2005). AgseNPV-A is compared in genome sequence and organization with Spodoptera exigua (Se) MNPV (IJkel et al., 1999), Autographa californica (Ac) MNPV (Ayres et al., 1994), Mamestra configurata (Maco) NPV-B (Li et al., 2002a) and the recently sequenced GV from A. segetum (AgseGV) (GenBank accession no. NC_005839). The gene order of AgseNPV-A was found to be highly collinear with that of SeMNPV, despite relatively low amino acid identity values (60 % on average).
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METHODS |
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DNA isolation.
Polyhedra were isolated from infected insects. Dead larvae were homogenized by using a glass homogenizer and filtered through four layers of cheesecloth. The filtrate was layered onto a 30 % (w/w) sucrose cushion and centrifuged for 15 min at 5300 r.p.m. at 4 °C. The pellet containing polyhedra was resuspended and the sucrose purification was repeated two times. The final pellet was washed three times in distilled water and finally resuspended in 1 ml water. Polyhedra were dissolved by incubation for 30 min at 37 °C in 0·1 M sodium carbonate (final pH, approx. 11). Large debris was removed by 5 min centrifugation at 1000 r.p.m. and the supernatant was centrifuged for 30 min at 14 000 r.p.m. to pellet the occluded virions. DNA was isolated according to Reed et al. (2003) and dialysed for 24 h at 4 °C against 0·1x TE buffer (10 mM Tris/HCl; 1 mM EDTA; pH 7·5). Quantity and quality of isolated DNA were determined spectrophotometrically and by electrophoresis in 0·7 % agarose.
Nucleotide sequence determination.
The full genome sequence was determined by shotgun cloning of 10 µg sheared DNA of 1–1·5 kbp. The DNA fragments were cloned into pBluescript II SK(+) (Stratagene) by using Escherichia coli XL2 Blue ultracompetent cells (Stratagene). End-in sequencing was performed on a 3730xL DNA analyser (Applied Biosystems) and a 3100 Genetic Analyser (Applied Biosystems); sequences were assembled with Gap4 from the Staden-Solaris-1-5-3 software package and then checked in detail manually (van Oers et al., 2005). In total, 1 517 059 nt were determined, with a mean redundancy of 9·88.
Sequence analysis.
Genes were located with GeneMark software (Borodovsky & McIninch, 1993) and ORF Finder (NCBI). All open reading frames (ORFs) with a minimal size of 150 nt (50 aa), which did not overlap for major parts with other ORFs, were analysed. In addition, the genome was checked in detail for the presence of any ORFs identified for SeMNPV (IJkel et al., 1999) or any other baculovirus in GenBank. Homology searches were performed by using BLAST (Altschul et al., 1990).
To easily compare sequence information from different baculovirus genomes, the GECCO program was exploited. GECCO is a gene content-comparison tool able to align large numbers of sequences quickly by using the standard ncbi BLAST (van Oers et al., 2005). The percentages of identity of all AgseNPV-A ORFs with their homologues in selected genomes were calculated for complete ORFs by using CLUSTAL_X (Thompson et al., 1997). To detect homologous regions, DOTPLOT analysis (dnastar) and the EMBOSS program (http://bioweb.pasteur.fr/seqanal/EMBOSS/) were applied under various stringency conditions. Pairwise gene-order analysis was performed by making gene-parity plots as described previously (Hu et al., 1998). In this analysis, both shared and non-shared genes were included.
Phylogenetic analysis.
For phylogenetic analysis of enhancin genes, protein sequences were aligned in CLUSTAL_X. Maximum-parsimony (MP) analysis was performed by using PAUP* (Swofford, 2003). Bootstrap analyses were performed to evaluate the robustness of the phylogenies using 1000 replicates. Branch lengths were calculated by using the neighbour-joining (NJ) method. The enhancin sequences used for this analysis were found by using the AgseNPV-A enhancin sequences as queries for the BLAST link at NCBI.
Cross-infectivity.
Second-instar S. exigua and A. segetum larvae were individually fed high doses (108 OBs) of AgseNPV-A and SeMNPV, respectively. The larvae were first starved for 16 h and then fed virus-contaminated diet plugs. After consumption of the entire contaminated diet plug, they were given fresh diet and reared separately until death or pupation. The larvae were incubated at 25 °C, in a relative humidity of 70 % and a 16 : 8 h day : night photoperiod. Fifty larvae of each species were tested.
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RESULTS AND DISCUSSION |
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). The computationally derived HindIII restriction map is in agreement with the experimentally constructed map from restriction-enzyme analysis, with the exception of an additional submolar band in the latter, indicating that more than one genetic variant is present in the isolate (Jakubowska et al., 2005). This is confirmed by the presence of several single-base polymorphisms in the sequence. The G+C content of the AgseNPV-A genome is 45·7 mol%, which is slightly higher than that of most sequenced baculoviruses. Only Choristoneura fumiferana (Cf) MNPV, Orgyia pseudotsugata (Op) MNPV, Lymantria dispar (Ld) MNPV and Culex nigripalpus (Cuni) NPV have G+C contents higher than that of AgseNPV-A (Ahrens et al., 1997; Kuzio et al., 1999; Afonso et al., 2001; de Jong et al., 2005).
Gene content and genome organization
Using computational analysis, 419 methionine-initiated ORFs of more than 50 aa were initially identified. The maximal acceptable overlap was set at 20 aa, with the exception of overlapping genes showing significant similarity to ORFs known in other baculoviruses. From those ORFs, 153 ORFs were assigned as AgseNPV-A ORFs, after elimination of ORFs located within larger ORFs and without similarity to baculovirus ORFs (Fig. 1, Table 1). The ORF density is comparable to that of other NPVs, when calculated as no. ORFs/genome size. According to the adopted convention (Vlak & Smith, 1982), polyhedrin was designated gene number 1 (Agse1) and the adenine of the start codon of the polyhedrin gene was assigned as the first nucleotide of the AgseNPV-A circular genome. Of the 153 AgseNPV-A ORFs, 143 have an assigned function or homologues in other baculoviruses. Ten AgseNPV-A ORFs have no homologues in baculoviruses and thus are considered unique to AgseNPV-A until homologues in other baculoviruses are found. The total length of those unique ORFs is 4379 nt (3 % of the genome). Four of these have no consensus promoter and may be non-functional. Predicted proteins are encoded by 89·8 % of the genome; the rest forms non-coding regions and homologous regions (hrs). Five hrs were identified in the AgseNPV-A genome dispersed around the genome (see below); the maximum distance between two hrs is 57 kbp.
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) were found in the AgseNPV-A genome. There are 21 pairs of overlapping genes along the genome, eight of which have overlaps longer than 20 nt. All of them showed significant similarity to known baculovirus ORFs and thus were assigned as AgseNPV-A ORFs. The maximal overlap of 139 nt exists between vp1054 and lef-10 (Agse113/114). Both genes are oriented anticlockwise and both have homologues in SeMNPV. However, a promoter motif was not found upstream of the AgseNPV-A lef-10 gene. In general, the AgseNPV-A genome is densely packed, with minimal intergenic distances. There are three gene clusters packed very tightly: ORFs 66–70, 95–100 and 109–115. Fifty-five per cent of the ORFs are directed clockwise and 45 % anticlockwise, with respect to the orientation of transcription of the polyhedrin gene (Vlak & Smith, 1982).
The AgseNPV-A genome content and organization were compared with those of four other baculoviruses: SeMNPV, AcMNPV, MacoNPV-B and AgseGV (Table 2). AgseNPV-A shares 127 ORFs with SeMNPV, 134 with MacoNPV-B, 103 with AcMNPV and 81 with AgseGV, with mean amino acid identities of 60·0, 55·9, 34·2 and 26·9 %, respectively. The highest mean amino acid identity was found between AgseNPV-A and SeMNPV, but the largest number of ORFs is shared with MacoNPV-B (134), underscoring a close relationship with MacoNPV-B as well. This is further evidenced by phylogenetic (Jakubowska et al., 2005) and gene-parity plot (Fig. 2) analyses. Seventy-eight ORFs show the highest percentage of identity with SeMNPV, 53 with MacoNPV-B, one with AcMNPV (Agse21) and one with AgseGV (Agse22).
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The identity of the 81 genes shared by AsgeNPV-A and AgseGV, despite being isolated from the same host (A. segetum), is very low. None of the 19 shared genes that do not belong to the 62 common lepidopteran baculovirus genes (Herniou et al., 2003) are exclusively present in AgseNPV and AgseGV.
To examine the genome organization, the order of homologous ORFs of AgseNPV-A, SeMNPV, MacoNPV-B and AcMNPV was compared by using gene-parity plot analysis as described previously (Hu et al., 1998) (Fig. 2
). Genes of AcMNPV were renumbered manually, starting with the polyhedrin gene as number 1. The gene arrangement of AgseNPV-A was completely collinear with that of SeMNPV. A high degree of collinearity was also observed with MacoNPV-B, with exception of a short gene cluster including 38.7K, lef-1 and cathepsin. When compared with AcMNPV, a major part of the AgseNPV-A genome (around 70 000–140 000 bp) is inversely oriented relative to the orientation of the polyhedrin gene, but the gene order in this region is similar in both viruses. The other parts of the genome differ considerably. Parity analysis of AgseNPV-A and AgseGV ORFs only provided a scattered, non-informative distribution (not shown).
Comparison of AgseNPV-A and SeMNPV ORFs
Having observed the high level of genome collinearity of AgseNPV-A and SeMNPV, we compared the gene content of both NPVs. Among the 127 homologues between AgseNPV-A and SeMNPV, the most conserved genes are polyhedrin, with amino acid identity of 92 %, and sod, ubiquitin, chitinase, vfl-1, lef-8 and lef-9, with >60 % amino acid identity. Two putative SeMNPV ORFs, Se17 and Se18, constitute one ORF in AgseNPV-A (Agse21), which was assigned as lef-7 according to homology with Ac125. The best BLAST match for Agse21 was found with Xestia c-nigrum (Xecn) GV ORF129 (38 % identity) (Hayakawa et al., 1999). Agse22 was homologous to three SeMNPV ORFs (Se22, Se23 and Se24) and assigned as a single ORF in HearNPV (Chen et al., 2002). The best BLAST match for Agse22 was found with AgseGV ORF39 (46 % identity), but its function is unknown.
There are 12 ORFs present in SeMNPV that are absent in AgseNPV-A: Se5, Se20, Se21, Se39, Se57, Se68, Se83, Se84, Se85, Se86, Se99 and Se116. All except Se99, which was assigned as p94, are genes with unknown functions (IJkel et al., 1999). The p94 gene is not essential for virus replication in cell culture, but may be involved in inhibition of apoptosis (Friesen & Miller, 1987; Clem & Miller, 1994). Having seen the different location of p94 in AcMNPV compared with AgseNPV, MacoNPV-A and B and SeMNPV, it may have been acquired by the ancestor of these viruses in an independent insertion and from a source different from the p94 of AcMNPV (as suggested for SeMNPV p94; IJkel et al., 2001). No homologue was found in AgseGV.
Between Agse64 and Agse65, a 182 nt fragment was detected with nucleotide similarity to odv-e66. This gene encodes an occlusion-derived virion (ODV) protein and is present in SeMNPV in two copies, Se57 and Se114, which are both >2 kbp long. The identity between the two SeMNPV ORFs is only 32 % (IJkel et al., 2001). An intact odv-e66 gene appears to be present in the AgseNPV-A genome in another position (Agse125) and has a length of 2036 nt, which is comparable in size with the complete Se114 ORF (2057 nt). In AgseNPV-A, the odv-e66 gene is located next to the p47 gene, as is the case in SeMNPV. The identity between Agse125 and Se114 is 52 %, which is higher than that between the two SeMNPV odv-e66 genes. It is probable that Se57 was acquired independently of Se114 from a source related more closely to LdMNPV (Kuzio et al., 1999) or Leucania separata (Ls) NPV (Wang et al., 1995). In AgseNPV-A, an Se57 homologue may have been lost during evolution, as there is a small part of this gene left in the AgseNPV-A genome between Agse64 and Agse65. There are also two copies of odv-e66 in AcMNPV and MacoNPV-B. All sequenced GVs, except for Adoxophyes orana (Ador) GV (Wormleaton et al., 2003), contain a single copy of an odv-e66 gene.
With an increasing number of complete baculovirus genome sequences, the number of unique ORFs decreases. Twenty unique ORFs were assigned to SeMNPV (IJkel et al., 1999). To the best of our knowledge, seven ORFs were still unique to SeMNPV with 28 baculovirus genomes having been sequenced: Se5, Se20, Se39, Se83, Se85, Se86 and Se121. A homologue of Se121 (Agse133) is now identified in the AgseNPV-A genome, thus reducing the number of unique SeMNPV ORFs to six. The function of Agse133 is so far unknown. Similarly, a homologue of Trichoplusia ni SNPV ORF62 (Willis et al., 2005) (Tn62) was identified in the AgseNPV-A genome. This ORF is so far present exclusively in TnSNPV and AgseNPV-A.
Unique genes in AgseNPV-A
There are ten unique ORFs in AgseNPV-A (Agse5, Agse14, Agse15, Agse42, Agse43, Agse48, Agse53, Agse61, Agse63 and Agse137) and they account for 3 % of the genome in total. Their length ranges from 50 to 421 aa. For six unique ORFs, baculovirus early or late promoter motifs were found upstream of the ATG codons, as well as putative polyadenylation signals, indicating that these may represent expressed genes. Five of these (Agse5, Agse15, Agse48, Agse53 and Agse63) are larger than 100 aa. Agse5 exhibits homology to an Arabidopsis thaliana helicase domain-containing protein, with a BLAST e value of 0·049.
bro genes
A common feature of baculovirus genomes is the presence of repeated ORFs, named bro (baculovirus repeated ORFs) genes by Kuzio et al. (1999). The highest number of bro genes was identified in LdMNPV (Kuzio et al., 1999), which contains 16 ORFs related to the AcMNPV bro gene Ac2. The role of the bro gene family has not yet been defined. The bro genes were subdivided into four groups, depending on the percentage of amino acid identity in both termini and in the central region, as well as their length (Kuzio et al., 1999). We have identified four bro genes dispersed along the genome of AgseNPV-A (Agse50, Agse77, Agse107 and Agse123) and named them bro-a to bro-d, according to the order of appearance on the linearized genome. bro-a shows the highest amino acid identity (53 %) to bro-c of Bombyx mori NPV, bro-b to bro-d of MacoNPV-B (63 %), bro-c to bro-h of MacoNPV-A (51 %) and bro-d to Ac2 (58 %). The size of each bro gene corresponds well to that of their homologues in other baculoviruses. bro-a, bro-b and bro-d belong to group I bro genes, whilst bro-c shows the highest similarity to group II bro genes according to the classification of Kuzio et al. (1999). AgseNPV-A bro-b is located directly downstream of the second enhancin gene (Agse76), like bro-d in the MacoNPV-B genome. We have not found any relatedness of the location of bro genes to that of hrs, which was suggested for the LdMNPV genome (Kuzio et al., 1999). Alignment of all AgseNPV-A bro genes revealed similarities ranging from 7 % (bro-b/bro-d against bro-c) to 53 % between bro-b and bro-d (analysis not shown).
hrs
hrs were first identified in AcMNPV (Cochran & Faulkner, 1983; Pearson et al., 1992). hrs are cis-acting putative origins of DNA replication (ori) (Ahrens & Rohrmann, 1995; Kool et al., 1995) and may also act as enhancers of transcription (Guarino & Summers, 1986; Guarino et al., 1986). AcMNPV hrs have two to eight repeats of a 72 bp long sequence, with an internal imperfect palindrome with an EcoRI restriction site in the centre. In five of the 28 baculoviruses sequenced until now [Chrysodeixis chalcites (Chch) NPV (van Oers et al., 2005), TnSNPV, AdorNPV, AgseGV and Cydia pomonella (Cp) GV (Luque et al., 2001)], canonical hrs were not identified. The remaining fully sequenced baculoviruses contain between three [Cryptophlebia leucotreta (Crle) GV] (Lange & Jehle, 2003) and 17 [Spodoptera litura (Splt) NPV] (Pang et al., 2001) hrs.
Five hrs have been identified in the AgseNPV-A genome (Fig. 3). They are dispersed along the genome in intergenic regions, around map positions 19·5 (hr1), 42·8 (hr2), 57·0 (hr3), 113·2 (hr4) and 124·8 (hr5) (see Fig. 1, Table 1). The conserved repeat size is around 40 nt. All AgseNPV-A hrs contain from one to four imperfect palindromic repeats. Thirteen nucleotides are absolutely conserved in all 16 repeats identified. The striking feature of AgseNPV-A hrs is a very high consensus sequence similarity to SeMNPV hrs. Two motifs also characteristic for SeMNPV hrs, TTTGCTTT and GAAAGCAAAC, are present in almost all AgseNPV-A hr repeats. A notable feature of the AgseNPV-A hrs also is a low G+C content of around 30 mol%, as in SeMNPV.
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). They function by enzymic hydrolysis of the peritrophic membrane, the barrier for pathogens in the insect midgut, or by increasing the fusion efficiency with midgut cells through interaction between the viral envelope and the cell plasma membrane (Wang et al., 1994; Bischoff & Slavicek, 1997). Enhancins are metalloproteases, which degrade mucins, the major proteins of the peritrophic membrane (Lepore et al., 1996; Wang et al., 1997). The first enhancin described originated from Pseudaletia unipuncta (Psun) GV and increased the infectivity of PsunNPV in NPV/GV mixed infections (Tanada & Hukuhara, 1971; Yamamoto & Tanada, 1980; Zhu et al., 1989). Enhancin protein constitutes up to 5 % of the total protein content in the granules of GVs and is localized in the granule matrix (Tanada, 1985). Recently, it was shown that LdMNPV enhancins are present in ODV envelopes in association with nucleocapsids (Slavicek & Popham, 2005).
To date, enhancins have been described in many GVs, including PsunGV, T. ni (Tni) GV, Helicoverpa armigera (Hear) GV (Roelvink et al., 1995), AgseGV (GenBank accession no. NC_005839), C. fumiferana (Cf) GV (GenBank accession no. AF319939) and XecnGV (Hayakawa et al., 1999), and in four group II NPVs, CfMNPV (de Jong et al., 2005), LdMNPV (Bischoff & Slavicek, 1997), MacoNPV-A isolates 90/2 and 90/4 (Li et al., 2002a, 2005) and MacoNPV-B (Li et al., 2002b). So far, enhancin has not been found in any group I NPV. Most of the group II NPVs and GVs carry a single copy of an enhancin gene. LdMNPV has two enhancin copies, whilst in the XecnGV genome, four enhancins were found.
AgseNPV-A encodes three enhancin genes, vef-1, vef-2 and vef-3 (Fig. 4), as Agse75, Agse76 and Agse128. For all three AgseNPV-A vefs, potential baculovirus consensus late promoter motifs were found, suggesting expression in the late stage of infection. This is compatible with their association with ODVs (Slavicek & Popham, 2005). The first two enhancin genes, vef-1 and vef-2, are located in tandem downstream of the 38K gene (Agse74). This location corresponds to the position of Se68 in the SeMNPV genome (IJkel et al., 1999). AgseNPV-A lacks an Se68 homologue (function unknown) at this position. Compared with SeMNPV, vef-3 (Agse128) is in place of Se116, also an ORF with unknown function. All three AgseNPV-A vef genes contain large ORFs of 2633, 2652 and 2588 nt, respectively. These sizes are comparable to those of other baculovirus enhancin genes, which range from 2352 nt in LdMNPV to 3015 nt in AgseGV. The identity between the predicted AgseNPV-A VEF proteins ranges from 30 to 40 % and the similarity oscillates around 60 % (Fig. 4a). Overall, AgseNPV-A enhancins have only about 20 % amino acid identity with other baculovirus enhancins. Identities of up to 40 % were only found with the MacoNPV species enhancins.
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). AgseNPV-A vef-1 and vef-2 encode this conserved zinc-binding domain as HEISH and HEMAH, respectively. AgseNPV-A vef-3 encodes an HAISF motif at a comparable position, which does not meet the requirement of an HEXXH motif. A similar case was described for two XecnGV enhancin genes (ORF150 and ORF166), with HQIGH and QKIGD motifs aligning with the HEXXH motif of other enhancins (Hayakawa et al., 1999). It is not known whether the aberrant XecnGV enhancin is active. Only a part of known metalloproteases contains this conserved motif, which can be even better defined as abXHEbbHbc, where ‘a’ is most often valine or threonine, ‘b’ an uncharged residue and ‘c’ a hydrophobic residue (Rawlings & Barrett, 1995).
Phylogenetic analysis of enhancins was performed (Fig. 4b) to address the question as to whether the origin of the AgseNPV-A enhancin genes could be found in the granulovirus AgseGV, a virus with the same host. In addition to previously presented phylogenies (Popham et al., 2001; Li et al., 2003), we also included in our analysis the CfMNPV, MacoNPV-A 90/4, MacoNPV-B and AgseGV enhancins, as well as several bacterial enhancin sequences from Bacillus spp. and Yersinia spp. This resulted in a slightly different clustering of baculovirus enhancin genes, but the previously observed tendency that enhancins of similar size fall in the same clade (Li et al., 2003) is supported by our analysis. Also in this case, AgseNPV and MacoNPVs are closely related.
We obtained two baculovirus enhancin clusters, one consisting of MacoNPVs and AgseNPV-A and a second one including all GV, LdMNPV and CfMNPV enhancins. In the second cluster, AgseGV groups with CfMNPV and LdMNPV enhancins and is apart from other GV enhancins. The bacterial enhancins are grouped together and are separated from the baculovirus enhancins. The analysis is supported by relatively high (mostly >80 %) bootstrap values for all branches. Phylogenetic analysis also showed that CfMNPV and ChfuGV, as well as AgseNPV-A and AgseGV, enhancins are not closely related, rejecting the hypothesis, at least for these viruses, that baculoviruses infecting a common host have gained enhancin genes from each other.
Cross-infectivity
The relatively high genetic identity between AgseNPV-A and SeMNPV and their similarity in genome organization may be reflected in their host range. Therefore, AgseNPV-A and SeMNPV infectivity was tested per os by using second-instar S. exigua and A. segetum larvae, respectively. SeMNPV was found to be non-infectious for A. segetum at a high dose, whereas AgseNPV-A caused mortality in 23 (46 %) tested S. exigua larvae. The identity of the progeny virus was confirmed by PCR with two sets of AgseNPV-A-specific primers (not shown).
Conclusion
In conclusion, the genome of AgseNPV-A was found to be highly collinear with that of SeMNPV in organization. So far, ten ORFs were found to be unique to AgseNPV-A and one ORF, Agse133, is so far only shared with SeMNPV (Se121). The most prominent difference between AgseNPV-A and SeMNPV genomes is the presence of three enhancin gene copies (vef) in AgseNPV-A. Sequence information of the AgseNPV genome adds to the knowledge of baculovirus genomes and, in comparison to SeMNPV and AgseGV, may lead to further insight into baculovirus–host interactions.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Ahrens, C. H. & Rohrmann, F. (1995). Replication of Orgyia pseudotsugata baculovirus DNA: lef-2 and ie-1 are essential and ie-2, p34, and Op-iap are stimulatory genes. Virology 212, 650–662.[CrossRef][Medline]
Ahrens, C. H., Russell, R. L. Q., Funk, C. J., Evans, J. T., Harwood, S. H. & Rohrmann, G. F. (1997). The sequence of the Orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis virus genome. Virology 229, 381–399.[CrossRef][Medline]
Allaway, G. P. & Payne, C. C. (1983). A biochemical and biological comparison of three European isolates of nuclear polyhedrosis viruses from Agrotis segetum. Arch Virol 75, 43–54.[CrossRef][Medline]
Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. (1990). Basic local alignment search tool. J Mol Biol 215, 403–410.[CrossRef][Medline]
Ayres, M. D., Howard, S. C., Kuzio, J., Lopez-Ferber, M. & Possee, R. D. (1994). The complete DNA sequence of Autographa californica nuclear polyhedrosis virus. Virology 202, 586–605.[CrossRef][Medline]
Bischoff, D. S. & Slavicek, J. M. (1997). Molecular analysis of an enhancin gene in the Lymantria dispar nuclear polyhedrosis virus. J Virol 71, 8133–8140.[Abstract]
Borodovsky, M. & McIninch, J. (1993). GeneMark: parallel gene recognition for both DNA strands. Comput Chem 17, 123–133.
Boughton, A. J., Harrison, R. L., Lewis, L. C. & Bonning, B. C. (1999). Characterization of a nucleopolyhedrovirus from the black cutworm, Agrotis ipsilon (Lepidoptera: Noctuidae). J Invertebr Pathol 74, 289–294.[CrossRef][Medline]
Bourner, T. C. & Cory, J. S. (2004). Host range of an NPV and a GV isolated from the common cutworm, Agrotis segetum: pathogenicity within the cutworm complex. Biol Control 31, 372–379.[CrossRef]
Bourner, T. C., Vargas-Osuna, E., Williams, T., Santiago Alvarez, C. & Cory, J. S. (1992). A comparison of the efficacy of nuclear polyhedrosis and granulosis viruses in spray and bait formulations for the control of Agrotis segetum (Lepidoptera: Noctuidae) in maize. Biocontrol Sci Technol 2, 315–326.
Bulach, D. M., Kumar, C. A., Zaia, A., Liang, B. & Tribe, D. E. (1999). Group II nucleopolyhedrovirus subgroups revealed by phylogenetic analysis of polyhedrin and DNA polymerase gene sequences. J Invertebr Pathol 73, 59–73.[CrossRef][Medline]
Caballero, P., Vargas-Osuna, E. & Santiago-Alvarez, C. (1991). Efficacy of a Spanish strain of Agrotis segetum granulosis virus (Baculoviridae) against Agrotis segetum Schiff (Lep., Noctuidae) on corn. J Appl Entomol 112, 59–64.
Chen, X., Zhang, W. J., Wong, J. & 9 other authors (2002). Comparative analysis of the complete genome sequences of Helicoverpa zea and Helicoverpa armigera single-nucleocapsid nucleopolyhedroviruses. J Gen Virol 83, 673–684.
Clem, R. J. & Miller, L. K. (1994). Control of programmed cell death by the baculovirus genes p35 and iap. Mol Cell Biol 14, 5212–5222.
Cochran, M. A. & Faulkner, P. (1983). Location of homologous DNA sequences interspersed at five regions in the baculovirus AcMNPV genome. J Virol 45, 961–970.
de Jong, J. G., Lauzon, H. A. M., Dominy, C., Poloumienko, A., Carstens, E. B., Arif, B. M. & Krell, P. J. (2005). Analysis of the Choristoneura fumiferana nucleopolyhedrovirus genome. J Gen Virol 86, 929–943.
Derksen, A. C. G. & Granados, R. R. (1988). Alteration of a lepidopteran peritrophic membrane by baculoviruses and enhancement of viral infectivity. Virology 167, 242–250.[CrossRef][Medline]
Federici, B. A. (1999). Naturally occurring baculoviruses for insect pest control. Methods Biotechnol 5, 301–320.
Friesen, P. D. & Miller, L. K. (1987). Divergent transcription of early 35- and 94-kilodalton protein genes encoded by the HindIII K genome fragment of the baculovirus Autographa californica nuclear polyhedrosis virus. J Virol 61, 2264–2272.
Guarino, L. A. & Summers, M. D. (1986). Functional mapping of a trans-activating gene required for expression of a baculovirus delayed-early gene. J Virol 57, 563–571.
Guarino, L. A., Gonzales, M. A. & Summers, M. D. (1986). Complete sequence and enhancer function of the homologous DNA regions of Autographa californica nuclear polyhedrosis virus. J Virol 60, 224–229.
Hayakawa, T., Ko, R., Okano, K., Seong, S.-I., Goto, C. & Maeda, S. (1999). Sequence analysis of the Xestia c-nigrum granulovirus genome. Virology 262, 277–297.[CrossRef][Medline]
Herniou, E. A., Luque, T., Chen, X., Vlak, J. M., Winstanley, D., Cory, J. S. & O'Reilly, D. R. (2001). Use of whole genome sequence data to infer baculovirus phylogeny. J Virol 75, 8117–8126.
Herniou, E. A., Olszewski, J., Cory, J. S. & O'Reilly, D. R. (2003). The genome sequence and evolution of baculoviruses. Annu Rev Entomol 48, 211–234.[CrossRef][Medline]
Hu, Z. H., Arif, B. M., Jin, F., Martens, J. W. M., Chen, X. W., Sun, J. S., Zuidema, D., Goldbach, R. W. & Vlak, J. M. (1998). Distinct gene arrangement in the Buzura suppressaria single-nucleocapsid nucleopolyhedrovirus genome. J Gen Virol 79, 2841–2851.[Abstract]
Hyink, O., Dellow, R. A., Olsen, M. J., Caradoc-Davies, K. M. B., Drake, K., Herniou, E. A., Cory, J. S., O'Reilly, D. R. & Ward, V. K. (2002). Whole genome analysis of the Epiphyas postvittana nucleopolyhedrovirus. J Gen Virol 83, 957–971.
Ignoffo, C. M. & Garcia, C. (1979). Susceptibility of larvae of the black cutworm to species of entomopathogenic bacteria, fungi, protozoa, and viruses. J Econ Entomol 72, 767–769.
IJkel, W. F. J., van Strien, E. A., Heldens, J. G. M., Broer, R., Zuidema, D., Goldbach, R. W. & Vlak, J. M. (1999). Sequence and organization of the Spodoptera exigua multicapsid nucleopolyhedrovirus genome. J Gen Virol 80, 3289–3304.
IJkel, W. F. J., Lebbink, R.-J., Op den Brouw, M. L., Goldbach, R. W., Vlak, J. M. & Zuidema, D. (2001). Identification of a novel occlusion derived virus-specific protein in Spodoptera exigua multicapsid nucleopolyhedrovirus. Virology 284, 170–181.[CrossRef][Medline]
Jakubowska, A., van Oers, M. M., Ziemnicka, J., Lipa, J. J. & Vlak, J. M. (2005). Molecular characterization of Agrotis segetum nucleopolyhedrovirus from Poland. J Invertebr Pathol 90, 64–68.[CrossRef][Medline]
Kool, M., Ahrens, C. H., Vlak, J. M. & Rohrmann, G. F. (1995). Replication of baculovirus DNA. J Gen Virol 76, 2103–2118.
Kuzio, J., Pearson, M. N., Harwood, S. H., Funk, C. J., Evans, J. T., Slavicek, J. M. & Rohrmann, G. F. (1999). Sequence and analysis of the genome of a baculovirus pathogenic for Lymantria dispar. Virology 253, 17–34.[CrossRef][Medline]
Lange, M. & Jehle, J. A. (2003). The genome of the Cryptophlebia leucotreta granulovirus. Virology 317, 220–236.[CrossRef][Medline]
Lepore, L. S., Roelvink, P. R. & Granados, R. R. (1996). Enhancin, the granulosis virus protein that facilitates nucleopolyhedrovirus (NPV) infections, is a metalloprotease. J Invertebr Pathol 68, 131–140.[CrossRef][Medline]
Li, L., Donly, C., Li, Q., Willis, L. G., Keddie, B. A., Erlandson, M. A. & Theilmann, D. A. (2002a). Identification and genomic analysis of a second species of nucleopolyhedrovirus isolated from Mamestra configurata. Virology 297, 226–244.[CrossRef][Medline]
Li, Q., Donly, C., Li, L., Willis, L. G., Theilmann, D. A. & Erlandson, M. (2002b). Sequence and organization of the Mamestra configurata nucleopolyhedrovirus genome. Virology 294, 106–121.[CrossRef][Medline]
Li, Q., Li, L., Moore, K., Donly, C., Theilmann, D. A. & Erlandson, M. (2003). Characterization of Mamestra configurata nucleopolyhedrovirus enhancin and its functional analysis via expression in an Autographa californica M nucleopolyhedrovirus recombinant. J Gen Virol 84, 123–132.
Li, L., Li, Q., Willis, L. G., Erlandson, M., Theilmann, D. A. & Donly, C. (2005). Complete comparative genomic analysis of two field isolates of Mamestra configurata nucleopolyhedrovirus-A. J Gen Virol 86, 91–105.
Luque, T., Finch, R., Crook, N., O'Reilly, D. R. & Winstanley, D. (2001). The complete sequence of the Cydia pomonella granulovirus genome. J Gen Virol 82, 2531–2547.
Pang, Y., Yu, J., Wang, L. & 7 other authors (2001). Sequence analysis of the Spodoptera litura multicapsid nucleopolyhedrovirus genome. Virology 287, 391–404.[CrossRef][Medline]
Pearson, M., Bjornson, R., Pearson, G. & Rohrmann, G. (1992). The Autographa californica baculovirus genome: evidence for multiple replication origins. Science 257, 1382–1384.
Popham, H. J. R., Bischoff, D. S. & Slavicek, J. M. (2001). Both Lymantria dispar nucleopolyhedrovirus enhancin genes contribute to viral potency. J Virol 75, 8639–8648.
Rawlings, N. D. & Barrett, A. J. (1995). Evolutionary families of metallopeptidases. Methods Enzymol 248, 183–228.[Medline]
Reed, C., Otvos, I. S., Reardon, R., Ragenovich, I. & Williams, H. L. (2003). Effects of long-term storage on the stability of OpMNPV DNA contained in TM Biocontrol-1. J Invertebr Pathol 84, 104–113.[CrossRef][Medline]
Roelvink, P. W., Corsaro, B. G. & Granados, R. R. (1995). Characterization of the Helicoverpa armigera and Pseudaletia unipuncta granulovirus enhancin genes. J Gen Virol 76, 2693–2705.
Slavicek, J. M. & Popham, H. J. R. (2005). The Lymantria dispar nucleopolyhedrovirus enhancins are components of occlusion-derived virus. J Virol 79, 10578–10588.
Swofford, D. L. (2003). PAUP*: Phylogenetic analysis using parsimony (*and other methods), version 4.0b10. Sunderland, MA: Sinauer Associates.
Tanada, Y. (1985). A synopsis of studies on the synergistic property of an insect baculovirus: a tribute to Edward A. Steinhaus. J Invertebr Pathol 45, 125–138.
Tanada, Y. & Hukuhara, T. (1971). Enhanced infection of a nuclear-polyhedrosis virus in larvae of the armyworm, Pseudaletia unipuncta, by a factor in the capsule of a granulosis virus. J Invertebr Pathol 17, 116–126.[CrossRef]
Theilmann, D. A., Blissard, G. W., Bonning, B., Jehle, J., O'Reilly, D. R., Rohrman, G. F., Thiem, S. & Vlak, J. M. (2005). Family Baculoviridae. In Virus Taxonomy: Eighth Report of the International Committee on Taxonomy of Viruses, pp. 177–185. Edited by C. M. Fauquet, M. A. Mayo, J. Maniloff, U. Desselberger & L. A. Ball. London: Elsevier.
Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G. (1997). The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25, 4876–4882.
van Oers, M. M., Abma-Henkens, M. H. C., Herniou, E. A., de Groot, J. C. W., Peters, S. & Vlak, J. M. (2005). Genome sequence of Chrysodeixis chalcites nucleopolyhedrovirus, a baculovirus with two DNA photolyase genes. J Gen Virol 86, 2069–2080.
Vlak, J. M. & Smith, G. E. (1982). Orientation of the genome of Autographa californica nuclear polyhedrosis virus: a proposal. J Virol 41, 1118–1121.
Volkman, L. E., Blissard, G. W., Friesen, P., Keddie, B. A., Possee, R. & Theilmann, D. A. (1995). Family Baculoviridae. In Virus Taxonomy: Sixth Report of the International Committee on Taxonomy of Viruses, pp. 104–113. Edited by F. A. Murphy, C. M. Fauquet, D. H. L. Bishop, S. A. Ghabrial, A. W. Jarvis, G. P. Martelli, M. A. Mayo & M. D. Summers. Vienna: Springer.
Wang, P., Hammer, D. A. & Granados, R. R. (1994). Interaction of Trichoplusia ni granulosis virus-encoded enhancin with the midgut epithelium and peritrophic membrane of four lepidopteran insects. J Gen Virol 75, 1961–1967.
Wang, J. W., Qi, Y. P., Huang, Y. X. & Li, S. D. (1995). Nucleotide sequence of a 1446 base pair SalI fragment and structure of a novel early gene of Leucania separata nuclear polyhedrosis virus. Arch Virol 140, 2283–2291.[CrossRef][Medline]
Wang, P., Hammer, D. A. & Granados, R. R. (1997). Binding and fusion of Autographa californica nucleopolyhedrovirus to cultured insect cells. J Gen Virol 78, 3081–3089.[Abstract]
Willis, L. G., Siepp, R., Stewart, T. M., Erlandson, M. A. & Theilmann, D. A. (2005). Sequence analysis of the complete genome of Trichoplusia ni single nucleopolyhedrovirus and the identification of a baculoviral photolyase gene. Virology 338, 209–226.[CrossRef][Medline]
Wormleaton, S., Kuzio, J. & Winstanley, D. (2003). The complete sequence of the Adoxophyes orana granulovirus genome. Virology 311, 350–365.[CrossRef][Medline]
Yamamoto, T. & Tanada, Y. (1980). Physiochemical properties and location of capsule components, in particular the synergistic factor, in the occlusion body of a granulosis virus of the armyworm, Pseudaletia unipuncta. Virology 107, 434–440.[CrossRef]
Zhu, Y., Hukuhara, T. & Tamura, K. (1989). Location of a synergistic factor in the capsule of a granulosis virus of the armyworm, Pseudaletia unipuncta. J Invertebr Pathol 54, 49–56.[CrossRef][Medline]
Received 25 August 2005;
accepted 22 November 2005.
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