| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 Invasive Insect Biocontrol and Behavior Laboratory, USDA Agricultural Research Service, Plant Sciences Institute, 10300 Baltimore Avenue, Beltsville, MD 20705, USA
2 Division of Plant Sciences (Entomology), University of Missouri, Columbia, MO 65211, USA
3 Biological Control of Insects Research Laboratory, USDA Agricultural Research Service, 1503 S. Providence Road, Columbia, MO 65203, USA
Correspondence
Robert L. Harrison
Robert.L.Harrison{at}ars.usda.gov
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are EF035042 (SfMNPV-3AP2 genome), EU095337–EU095342 (SfMNPV field isolates) and EU095942–EU095946 (SfMNPV plaque isolates).
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
) consist of rod-shaped, arthropod-specific viruses with double-stranded, circular DNA genomes ranging from 80 to 180 kb. During infection, baculoviruses produce both secreted, extracellular virions (the budded virus, or BV) and intracellular virions that are packaged into a protein crystalline matrix to form viral occlusions (occluded virus, or OV). Baculoviruses isolated from Lepidoptera (moths and butterflies) are currently divided into two genera, Granulovirus and Nucleopolyhedrovirus (Jehle et al., 2006a). Viruses of the genus Granulovirus produce ovoid occlusions that contain a single virion, whilst those of the genus Nucleopolyhedrovirus produce larger cuboidal occlusions containing many virions. The virions of both genera are enveloped, but nucleopolyhedroviruses (NPVs) produce virions containing either a single nucleocapsid (SNPV) or multiple nucleocapsids (MNPV) per envelope. The lepidopteran NPVs are divided into two groups, I and II, on the basis of phylogenies inferred from gene sequences, content and order (Herniou et al., 2003). NPVs are used extensively as gene expression vectors (Kost et al., 2005) and as environmentally safe, species-specific, narrow-spectrum insecticides (Moscardi, 1999).
Species of the lepidopteran genus Spodoptera are important pests of many crops. Of these species, the fall armyworm (Spodoptera frugiperda) is a significant pest of maize, sorghum, rice, wheat, vegetable crops and pastures in the Americas (Sparks, 1979). Several NPV isolates have been isolated and characterized from fall armyworm populations in North, Central and South America, and these isolates appear to be variants of the same virus (Berretta et al., 1998; Escribano et al., 1999; Loh et al., 1982; Shapiro et al., 1991). S. frugiperda MNPV (SfMNPV) has been evaluated in field trials as a potential biopesticide to control S. frugiperda on maize (Armenta et al., 2003; Cisneros et al., 2002; Moscardi, 1999; Williams et al., 1999). Applications of SfMNPV cause significant levels of mortality among S. frugiperda larvae in maize plots without affecting populations of natural enemies (Armenta et al., 2003; Williams et al., 1999). In addition, SfMNPV has been a model for studies into the ecology of NPVs (reviewed by Fuxa, 2004). Partial sequences reported previously from different SfMNPV isolates indicate that it is closely related to Spodoptera exigua MNPV (SeMNPV) (Simón et al., 2005a, b; Tumilasci et al., 2003).
In this study, the complete genomic sequence of a new clone of SfMNPV was determined. This clone, named SfMNPV-3AP2, was originally derived from a virus population isolated from infected S. frugiperda larvae collected in Missouri, USA, and was selected for genome sequencing because of its rapid speed of kill in bioassays. We report an analysis of the gene organization and content of SfMNPV-3AP2 and its relationship to other SfMNPV isolates and other NPVs.
|
METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
). The viral isolates prepared from the six original cadavers were numbered 1–6. To produce plaque-purified viral isolates, DNA isolated from the polyhedra of each field-collected larva was packaged with Lipofectin (Invitrogen) and used to transfect Sf21 cells maintained as monolayers at 28 °C in Excel 401 medium (JRH Biosciences) supplemented with 10 % fetal bovine serum (Integen) (O'Reilly et al., 1992b). Plaque assays were set up with cell culture medium from the transfections (O'Reilly et al., 1992b). One plaque was picked from each of the original six transfections, subjected to two additional rounds of plaque purification and amplified. For bioassays of the plaque-purified isolates, 5 µl BV was injected into S. frugiperda larvae and polyhedra were purified from killed larvae as above.
Bioassay analysis of viruses.
Neonate S. frugiperda larvae were infected per os by the droplet feeding method developed by Hughes et al. (1986) with five doses of SfMNPV polyhedral inclusion bodies (PIBs) ranging from 1x105 to 1x108 PIBs ml–1. Larvae were placed on fresh food, maintained at 28±1 °C at a photoperiod of 14 : 10 h (light : dark) and monitored two or three times daily for 7 days. The LC50 (concentration of occluded virus required to kill 50 % of the test larvae) for each virus was calculated using POLO-PC (Robertson & Preisler, 1992). The LT50 (mean time to kill 50 % of the test larvae, h) for all viruses was determined using the ViStat 2.1 program (Hughes, 1990). The L1 strain of Autographa californica MNPV (AcMNPV) was also bioassayed for comparison with the SfMNPV isolates (Lee & Miller, 1978). The diet (#9781B) and S. frugiperda eggs were purchased from Bio-Serv. All bioassays were repeated twice.
DNA cloning and sequencing.
Ethanol-precipitated SfMNPV-3AP2 DNA was pelleted by microcentrifugation and resuspended in distilled deionized H2O. After resuspension, the DNA was sheared with a GeneMachines Hydroshear device (Genomic Solutions) following the manufacturer's instructions. Fragments ranging in size from 0.8 to 2 kb were gel-purified and cloned into the pCR-Blunt II-TOPO plasmid vector (Invitrogen). After transformation into competent Escherichia coli TOP10 cells, cloned products were plated on Luria–Bertani agar plates containing kanamycin (50 µg ml–1) and spread with X-Gal (Fisher Scientific). White colonies from the transformation were picked into 25 µl 10 mM Tris/HCl (pH 8.0)/0.1 mM EDTA/0.5 % Triton X-100 and lysed by incubation at 100 °C for 5 min.
Cloned SfMNPV-3AP2 sequence fragments were amplified from colony lysates (2 µl per lysate) by PCR with plasmid vector-specific primers M13F (5'-TTGTAAAACGACGGCCAGT-3') and M13R (5'-GGAAACAGCTATGACCATG-3'). To eliminate excess primers and deoxynucleotides, the resulting PCR products were precipitated by incubation with an equal volume of 20 % PEG/2.5 M NaCl for 30 min at 37 °C. PCR products were pelleted by centrifugation and washed twice with 80 % ethanol.
PCR products were sequenced using nested plasmid vector-specific primers T7 (5'-GTAATACGACTCACTATAGGG-3') and SP6 (5'-GCTATTTAGGTGACACTATAG-3'). Reactions were set up using the Applied Biosystems BigDye Terminator Cycle Sequencing kit with AmpliTaq DNA polymerase and electrophoresed on an Applied Biosystems 3100 DNA sequencer.
DNA sequence analysis.
DNA sequence data was compiled and analysed using the software of the Lasergene suite (DNASTAR). Gaps and ambiguities in the genome sequence were resolved by amplifying the corresponding regions of the sequence from viral DNA by PCR (40 pg DNA per reaction) with custom-designed primers and sequencing the PCR products. A complete sequence of the SfMNPV-3AP2 genome was obtained with 16.2-fold coverage.
Open reading frames (ORFs) greater than 50 codons in length that did not overlap larger ORFs by more than 75 nt and were not present in a homologous repeat (hr) region were selected for further characterization. ORFs with homologues in other baculovirus genomes were also characterized. Predicted amino acid sequence identities were obtained from the results of protein database searches using the standard protein–protein BLAST algorithm (http://www.ncbi.nlm.nih.gov/BLAST/).
For phylogenetic inference, amino acid sequences derived from selected genes were aligned by CLUSTAL W (Thompson et al., 1994) using GONNET matrices with a gap penalty of 10 and a gap extension penalty of 0.1 for pairwise alignments and 0.2 for multiple alignments. Sequence alignments for different genes were concatenated using BioEdit (Hall, 1999). The concatenated amino acid alignments were used to construct phylograms with MEGA version 3.1 (Kumar et al., 2004) using minimum-evolution (ME) and maximum-parsimony (MP) methods. ME and MP trees were sought by using a close-neighbour-interchange heuristic search, starting with either one initial neighbour-joining tree (ME) or ten initial trees generated by random addition of sequences (MP). For ME trees, Poisson correction distances were estimated with a gamma shape parameter of 2.25. In both cases, the reliability of the trees was tested with bootstrap resampling using 1000 replicates.
Pairwise nucleotide sequence alignments were carried out using the Martinez/Needleman–Wunsch method of MEGALIGN (DNASTAR) with a gap penalty of 1.10 and a gap length penalty of 0.33.
|
RESULTS AND DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
). LC50 values ranged from 3.2x105 to 1.6x106 PIBs ml–1 for the non-plaque-purified virus. Plaque-purified isolates SfMNPV-4AP2 and SfMNPV-5AP1 did not exhibit per os insecticidal activity against larvae. The other plaque isolates exhibited a somewhat higher LC50 range (1.6x106–4.1x106 PIBs ml–1). The LC50 of AcMNPV-L1 against S. frugiperda was significantly higher than that of the SfMNPV isolates, ranging from 1.3x107 to 2.9x107 PIBs ml–1.
|
). LT50 values for plaque-purified isolates ranged from 34.4 to 49.7 h post-infection (p.i.), whilst those of the non-plaque purified isolates ranged from 72.3 to 90.5 h p.i. AcMNPV-L1 infections resulted in survival times of 73.4 to 83.0 h p.i., in the same range as those obtained with non-plaque-purified SfMNPV.
|
). The genome sequence had a G+C content of 40.2 %, which is higher than that of Adoxophyes honmai NPV (AdhoNPV; Nakai et al., 2003), Chrysodeixis chalcites NPV (ChChNPV; van Oers et al., 2005), Helicoverpa zea SNPV (HzSNPV; Chen et al., 2001) and Helicoverpa armigera SNPV (HearSNPV; Chen et al., 2002), but comparable or slightly less than that of other group II NPVs, including SeMNPV (IJkel et al., 1999) and Spodoptera litura MNPV (SpltMNPV; Pang et al., 2001). The first nucleotide position for the SfMNPV-3AP2 genome sequence was set to the adenine of the polyhedrin ORF initiation codon. Of the ORFs in the sequence that equalled or exceeded 50 codons in length, 144 had minimal (<75 bp) overlap with larger ORFs or shared significant sequence identity with previously characterized baculovirus ORFs (Table 3, Fig. 1). Of these ORFs, 77 were in the polyhedrin-sense orientation and 67 were in the anti-polyhedrin-sense orientation. Ninety-two ORFs were preceded by baculovirus early and late gene promoter motifs (Table 3).
|
|
). Eight hrs consisting of imperfect palindromic repeats were found in the SfMNPV-3AP2 sequence (Table 3
, Fig. 1). The repeats were 44 bp in size and the number of repeats in each hr varied from one (hr2, hr3 and hr6) to seven (hr1). SfMNPV-3AP2 hr1, hr4, hr5, hr7 and hr8 occupied the same positions on the genome with respect to surrounding ORFs as SeMNPV hr1, hr2, hr3, hr5 and hr6, respectively (Table 3). In addition, SfMNPV-3AP2 hr4, hr5 and hr7 were located in the same relative positions as Agrotis segetum NPV (AgseNPV) hr2, hr3 and hr4, respectively. The hr consensus sequence of SfMNPV-3AP2 exhibited sequence identities of 75 and 68 % with hr sequences of SeMNPV and AgseNPV, respectively.
Relationships with other NPVs
The results from BLAST searches with the SfMNPV-3AP2 ORF predicted amino acid sequences indicated that SfMNPV-3AP2 was closely related to SeMNPV, AgseMNPV and the NPVs characterized from Mamestra configurata. To examine the relationship of SfMNPV-3AP2 with other group II NPVs, phylogenetic trees were inferred from two sets of aligned sequences: (i) the concatenated aligned sequences for 28 baculovirus genes from completely sequenced group II NPVs as well as AcMNPV-C6 (Ayres et al., 1994) and Cydia pomonella granulovirus (CpGV; Luque et al., 2001) (Fig. 2a), and (ii) the concatenated aligned partial amino acid sequences of polyhedrin and late expression factors 8 and 9 (polh, lef-8 and lef-9; Fig. 2b) for SfMNPV-3AP2, other completely sequenced group II NPVs, a selection of group II NPV sequences produced by Jehle et al. (2006b), AcMNPV-C6 and CpGV. The first dataset included all of the genes in common among all baculoviruses sequenced as of 2006 (Jehle et al., 2006a) except for p6.9, which was not identified in the annotation for the Clanis bilineata NPV (ClbiNPV) genome sequence (GenBank accession no. NC_008293) at the time the analysis was carried out.
|
) and other Agrotis species NPVs (Fig. 2b). This clade in turn was part of a group that included M. configurata NPV (MacoNPV; Fig. 2a) and Mamestra brassicae MNPV (MbMNPV; Fig. 2b). These relationships enjoyed strong bootstrap support in both ME and MP phylograms, especially those constructed with aligned core genes (Fig. 2a). Sequences from SpltMNPV did not group with SeMNPV and SfMNPV-3AP2, but instead were either distantly related to other group II NPVs (Fig. 2a) or formed a separate clade containing only sequences from other isolates of SpltMNPV, isolates of Spodoptera littoralis NPV (SpliNPV) and a sequence from Spodoptera terricola NPV (SpteNPV) (Fig. 2b).
Gene content and order
All of the 62 genes common among lepidopteran NPV genome sequences (Jehle et al., 2006a) were found in the SfMNPV-3AP2 genome. One of these conserved ORFs, sf130/ac29, was almost entirely contained within a larger ORF, sf129 (Fig. 1, Table 3). Sixty-four additional ORFs had homologues in the SeMNPV genome, including a homologue of se121, which was also present only in AgseNPV (Jakubowska et al., 2006). ORFs sf55 and sf70 only had homologues in AgseNPV and the MacoNPV isolates. The ORF sf133 had homologues only in the MacoNPV isolates and Xestia c-nigrum granulovirus (XecnGV; Hayakawa et al., 1999), whilst sf135 had homologues only in AgseNPV, the MacoNPV isolates, HzSNPV and HearSNPV. ORF sf23 had homologues only in the MacoNPV isolates and SpltMNPV. Twelve SfMNPV-3AP2 ORFs (sf5, sf6, sf7, sf8, sf11, sf32, sf43, sf44, sf47, sf85, sf96 and sf129) had no homologues in other baculovirus sequences. Four of these unique ORFs were preceded by promoter motifs and six were larger than 100 codons (Table 3). The SfMNPV-3AP2 genome, in turn, lacked 14 ORFs that were present in SeMNPV, including se5, se20–se24, se33, se39, se45, se49 and se83–se86.
The gene order and organization of the SfMNPV-3AP2 genome was compared with that of other NPVs by gene-parity plot analysis (Hu et al., 1998). The SfMNPV-3AP2 genome possessed a strong degree of collinearity with the genomes of SeMNPV and AgseNPV (Fig. 3a and b). Comparison with AcMNPV-C6 and SpltMNPV revealed that the order of several ORFs in these NPVs was conserved with that of SfMNPV-3AP2, but the orientation of a large proportion of these ORFs was inverted relative to the polyhedrin gene (Fig. 3c and d). Similar ORF orientation with respect to AcMNPV has been observed with MacoNPV-A and -B (Li et al., 2002a, b), but not with Trichoplusia ni SNPV (TnSNPV; Willis et al., 2005) and ChchNPV (van Oers et al., 2005).
|
). Although gene expression, nucleic acid binding activity, nucleosome association, and protein localization and trafficking have been characterized for some Bombyx mori NPV bro genes and proteins (Kang et al., 1999, 2006; Zemskov et al., 2000), the functions of NPV bro genes are unclear. SfMNPV-3AP2 bro shared the greatest degree of amino acid sequence identity with MacoNPV-B bro-d, MacoNPV-A bro-e and AgseNPV bro-b. The SfMNPV-3AP2 bro amino acid sequence also exhibited a low degree of similarity to the SeMNPV bro-like ORF, se13.
As reported for SeMNPV (IJkel et al., 1999) and MacoNPV-A and -B (Li et al., 2002a, b), SfMNPV-3AP2 had two copies of both the odv-e66 and the p26 genes. The two odv-e66 genes (sf57 and sf115) and the two p26 genes (sf86 and sf131) only shared 30.8 and 28.2 % amino acid sequence identity, respectively. The odv-e66 gene encodes an OV envelope protein (Hong et al., 1994), whilst the function of p26 is unknown.
The SfMNPV-3AP2 odv-e56 gene was split into two ORFs, sf9 and sf9a, due to the occurrence of a stop codon after codon 160 of sf9. This stop codon was detected in 13 sequences from 13 different clones containing this region of the SfMNPV-3AP2 genome. The sf9 predicted amino acid sequence aligns with residues 1–154 of SeMNPV odv-e56, whilst the sf9a sequence aligns with residues 277–370 of the same gene. The odv-e56 gene product localizes to the OV envelope (Braunagel et al., 1996) and has undergone positive selection among group I NPVs (Harrison & Bonning, 2004), but the function of this gene is otherwise unknown. Conversely, the gene encoding late expression factor-7 (lef-7) was split into two ORFs in SeMNPV (se17 and se18) but was intact in SfMNPV-3AP2 (sf18). The SeMNPV lef-7 ORFs aligned with residues 44–207 and 213–323 of sf18. Like odv-e56, lef-7 has undergone positive selection in group I NPVs (Harrison & Bonning, 2004). The lef-7 gene is required for optimal levels of AcMNPV late gene expression and hr-directed plasmid DNA replication in transient assays in S. frugiperda (Sf21) cells (Lu & Miller, 1995). This gene is also required for optimal AcMNPV viral replication in S. frugiperda and S. exigua cell lines but not in a T. ni cell line (Chen & Thiem, 1997).
Comparison with other SfMNPV isolates
The nucleotide sequence of SfMNPV-3AP2 was strongly conserved with partial sequences reported from other SfMNPV isolates (Table 4). In general, nucleotide sequence identities were at least 99.5 % with few gaps required for an optimal alignment. Complete ORFs contained within these sequences showed a similarly high degree of predicted amino acid sequence conservation with corresponding sequences from SfMNPV-3AP2 ORFs. The most divergent sequence was from the p74 region of an unidentified SfMNPV isolate, which exhibited a relatively low nucleotide sequence identity of 97.4 % with 11 gaps over 2398 bp.
|
) revealed that the SfMNPV-3AP2 genome contained a deletion of 1426 bp. The 5' end point of this deletion occurred after the first position of codon 190 of the egt ORF (sf26). Whilst the egt genes of the Brazilian SfMNPV isolate and SeMNPV are 525 and 523 codons, respectively, the SfMNPV-3AP2 egt ORF was truncated, consisting of 207 codons. The baculovirus egt gene encodes an ecdysteroid UDP-glucosyltransferase. This enzyme transfers glucose or galactose from a nucleotide-sugar donor substrate to a specific hydroxyl group on ecdysteroid hormones (O'Reilly et al., 1991, 1992a). In insects, ecdysteroid hormones regulate development. Expression of egt during infection delays or blocks larval development, which in turn prolongs the period that infected larvae feed (O'Reilly & Miller, 1989; O'Reilly et al., 1998; Wilson et al., 2000). This activity is thought to increase the yield of progeny virus generated during infection (O'Reilly et al., 1998). The C-terminal truncation of the SfMNPV-3AP2 egt ORF removed domains containing residues thought to be required for glucosyltransferase activity (O'Reilly, 1995). The 3' end point of the 1426 bp deletion occurred between codons 77 and 78 of the Brazilian SfMNPV sf27 ORF. The SfMNPV-3AP2 sf27 ORF was initiated from an ATG codon that was downstream and in frame with the Brazilian SfMNPV sf27. Homologues of sf27 occur in SeMNPV, AgseMNPV, MacoNPV-A, MacoNPV-B, TnSNPV and ChChNPV. The expression and function of this ORF have not been characterized.
Structure of the egt region in field and plaque isolates of SfMNPV
Deletion or inactivation of the egt gene often reduces survival time of NPV-infected larvae to a significant extent (Chen et al., 2000; Eldridge et al., 1992; Flipsen et al., 1995; O'Reilly & Miller, 1991; Slavicek et al., 1999; Treacy et al., 1997). The deletion of most of the egt ORF in SfMNPV-3AP2 provides a potential explanation for its rapid speed of kill in bioassays. If so, one would expect that the non-plaque-purified SfMNPV isolates would contain an intact egt gene. The other fast-killing SfMNPV plaque isolates might also carry deletions in egt.
Restriction endonuclease mapping, PCR analysis and sequencing of the egt region in these viruses revealed that the egt sequences missing from SfMNPV-3AP2 were present in viral DNA from the non-plaque-purified SfMNPV isolates (Fig. 4). PCR using primers that flanked the deletion end points in SfMNPV-3AP2 amplified a sequence that contained the 1426 nt sequence present in the Brazilian isolate and missing from SfMNPV-3AP2 in the six field isolates of SfMNPV, with differences at 6 nt positions in this region among the six field isolates and the Brazilian isolate. PCR of DNA from field isolate SfMNPV-3 only amplified a sequence containing the intact egt and sf27 ORFs, and did not produce an amplification product corresponding to the deletion genotype of SfMNPV-3AP2 (data not shown).
|
). The deletions present in the other plaque isolates were larger than the deletion in SfMNPV-3AP2 and resulted in the removal of several neighbouring ORFs. SfMNPV-4AP2 contained the largest deletion in this area, with one end point occurring near the 3' end of lef-7 and the other end point occurring near the 3' end of pif-2. As pif-2 is required for oral infectivity (Ohkawa et al., 2005; Pijlman et al., 2003), the removal of most of this ORF from SfMNPV-4AP2 probably explains its lack of virulence towards S. frugiperda larvae. A complex rearrangement event or series of events appears to have occurred in SfMNPV-5AP1. One end point of the rearrangement occurred at nt 18622. Sequences including parts of sf27 and sf28 occurred at this point, but in an opposite orientation. Next to this sequence was a sequence derived from sf96 (nt 90488–90789), followed by the other end point of the rearrangement (at nt 27718, within sf29). The deletion and rearrangement end points for all plaque isolates were identical in DNA from tissue-culture-derived BV and larval-derived polyhedra. The end points of the deletion in SfMNPV-3AP2 were unchanged after two passages in Sf21 cells.
Although genotypes with deletions in the egt gene occur spontaneously in tissue culture stocks of AcMNPV (Kumar & Miller, 1987; O'Reilly et al., 1990), there is no direct evidence that egt mutations provide a growth advantage in tissue culture. It is possible that the deletion genotypes represented by the plaque isolates described in this study are variant genotypes that occur naturally in Missouri field isolates of SfMNPV. In a Nicaraguan isolate of SfMNPV (SfMNPV-NIC), nine distinct genotypes are present in the original field isolate (López-Ferber et al., 2003; Simón et al., 2004, 2005c). Eight of these variants carry deletions in the same region as the Missouri SfMNPV plaque isolates, extending from sf19 to sf36. None of the variants with deletions contain the egt gene. Three variants are not infectious per os to S. frugiperda larvae and two variants kill S. frugiperda larvae faster than the presumptive non-deleted genotype (Simón et al., 2004). The SfMNPV-NIC field isolate is more virulent than the non-deleted genotype in bioassays, and mixtures of the various genotypes are more virulent towards S. frugiperda larvae in bioassays than the non-deleted genotype alone, suggesting that the deletion genotypes contribute to the virulence of the field isolate (López-Ferber et al., 2003; Simón et al., 2005c, 2006). The non-deleted genotype of SfMNPV-NIC was represented in only 15 % of plaque isolates derived from SfMNPV-infected larvae, although it was the prevalent genotype as assessed by restriction digestion of viral DNA from larval-derived polyhedra (Simón et al., 2004). A high prevalence of alternative genotypes also may be present in the Missouri field isolates when assessed by plaque assay.
Conclusions
Although an extensive degree of collinearity was detected between SfMNPV-3AP2 and SeMNPV, the level of sequence divergence and differences in ORF content indicated that SfMNPV-3AP2 was a distinct virus and not a variant of SeMNPV. Comparison of the SfMNPV-3AP2 genome sequence with partial sequences from other SfMNPV isolates revealed that SfMNPV-3AP2 was a variant of other isolates previously described from geographically distant populations of S. frugiperda. The occurrence of variant genotypes containing deletions in the region of the egt gene appears to be a common feature of SfMNPV populations.
SfMNPV offers considerable promise as a safe, ecologically friendly means of controlling infestations of S. frugiperda where they occur. The isolation and genomic sequence of a naturally occurring, fast-killing isolate of SfMNPV may lead to further advances in the development of this NPV as an insecticide and to a greater understanding of baculovirus genetics and molecular biology in general.
|
ACKNOWLEDGEMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
Ayres, M. D., Howard, S. C., Kuzio, J., López-Ferber, M. & Possee, R. D. (1994). The complete DNA sequence of Autographa californica nuclear polyhedrosis virus. Virology 202, 586–605.[CrossRef][Medline]
Berretta, M. F., Rios, M. L. & Sciocco de Cap, A. (1998). Characterization of a nuclear polyhedrosis virus of Spodoptera frugiperda from Argentina. J Invertebr Pathol 71, 280–282.[CrossRef][Medline]
Bideshi, D. K., Renault, S., Stasiak, K., Federici, B. A. & Bigot, Y. (2003). Phylogenetic analysis and possible function of bro-like genes, a multigene family widespread among large double-stranded DNA viruses of invertebrates and bacteria. J Gen Virol 84, 2531–2544.
Braunagel, S. C., Elton, D. M., Ma, H. & Summers, M. D. (1996). Identification and analysis of an Autographa californica nuclear polyhedrosis virus structural protein of the occlusion-derived virus envelope: ODV-E56. Virology 217, 97–110.[CrossRef][Medline]
Chen, C. J. & Thiem, S. M. (1997). Differential infectivity of two Autographa californica nucleopolyhedrovirus mutants on three permissive cell lines is the result of lef-7 deletion. Virology 227, 88–95.[CrossRef][Medline]
Chen, X., Sun, X., Hu, Z., Li, M., O'Reilly, D. R., Zuidema, D. & Vlak, J. M. (2000). Genetic engineering of Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus as an improved pesticide. J Invertebr Pathol 76, 140–146.[CrossRef][Medline]
Chen, X., Ijkel, W. F., Tarchini, R., Sun, X., Sandbrink, H., Wang, H., Peters, S., Zuidema, D., Lankhorst, R. K. & other authors (2001). The sequence of the Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus genome. J Gen Virol 82, 241–257.
Chen, X., Zhang, W. J., Wong, J., Chun, G., Lu, A., McCutchen, B. F., Presnail, J. K., Herrmann, R., Dolan, M. & 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.
Cisneros, J., Perez, J. A., Penagos, D. I., Ruiz, J., Goulson, D., Caballero, P., Cave, R. D. & Williams, T. (2002). Formulation of a nucleopolyhedrovirus with boric acid for control of Spodoptera frugiperda (Lepidoptera: Noctuidae) in maize. Biol Control 23, 87–95.[CrossRef]
Eldridge, R., O'Reilly, D. R., Hammock, B. D. & Miller, L. K. (1992). Insecticidal properties of genetically engineered baculoviruses expressing an insect juvenile hormone esterase gene. Appl Environ Microbiol 58, 1583–1591.
Escribano, A., Williams, T., Goulson, D., Cave, R. D., Chapman, J. W. & Caballero, P. (1999). Selection of a nucleopolyhedrovirus for control of Spodoptera frugiperda (Lepidoptera: Noctuidae): structural, genetic, and biological comparison of four isolates from the Americas. J Econ Entomol 92, 1079–1085.[Medline]
Finney, D. J. (1971). Probit Analysis. Cambridge: Cambridge University Press.
Flipsen, J. T., Mans, R. M., Kleefsman, A. W., Knebel-Morsdorf, D. & Vlak, J. M. (1995). Deletion of the baculovirus ecdysteroid UDP-glucosyltransferase gene induces early degeneration of Malpighian tubules in infected insects. J Virol 69, 4529–4532.
Fuxa, J. R. (2004). Ecology of insect nucleopolyhedroviruses. Agric Ecosyst Environ 103, 27–43.[CrossRef]
Gonzalez, M. A., Smith, G. E. & Summers, M. D. (1989). Insertion of the SfMNPV polyhedrin gene into an AcMNPV polyhedrin deletion mutant during viral infection. Virology 170, 160–175.[CrossRef][Medline]
Hall, T. A. (1999). BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41, 95–98.
Harrison, R. L. & Bonning, B. C. (2004). Application of maximum-likelihood models to selection pressure analysis of group I nucleopolyhedrovirus genes. J Gen Virol 85, 197–210.
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., Olszewski, J. A., Cory, J. S. & O'Reilly, D. R. (2003). The genome sequence and evolution of baculoviruses. Annu Rev Entomol 48, 211–234.[CrossRef][Medline]
Hong, T., Braunagel, S. C. & Summers, M. D. (1994). Transcription, translation, and cellular localization of PDV-E66: a structural protein of the PDV envelope of Autographa californica nuclear polyhedrosis virus. Virology 204, 210–222.[CrossRef][Medline]
Hu, Z. H., Arif, B. M., Jin, F., Martens, J. W., 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]
Hughes, P. R. (1990).. ViStat: Statistical Package for the Analysis of Baculovirus Bioassay Data. Ithaca, NY: Boyce Thompson Institute at Cornell University.
Hughes, P. R., van Beek, N. A. M. & Wood, H. A. (1986). A modified droplet feeding method for rapid assay of Bacillus thuringiensis and baculoviruses in noctuid larvae. J Invertebr Pathol 48, 187–192.[CrossRef]
IJkel, W. F., van Strien, E. A., Heldens, J. G., 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.
Jakubowska, A. K., Peters, S. A., Ziemnicka, J., Vlak, J. M. & van Oers, M. M. (2006). Genome sequence of an enhancin gene-rich nucleopolyhedrovirus (NPV) from Agrotis segetum: collinearity with Spodoptera exigua multiple NPV. J Gen Virol 87, 537–551.
Jehle, J. A., Blissard, G. W., Bonning, B. C., Cory, J. S., Herniou, E. A., Rohrmann, G. F., Theilmann, D. A., Thiem, S. M. & Vlak, J. M. (2006a). On the classification and nomenclature of baculoviruses: a proposal for revision. Arch Virol 151, 1257–1266.[CrossRef][Medline]
Jehle, J. A., Lange, M., Wang, H., Hu, Z., Wang, Y. & Hauschild, R. (2006b). Molecular identification and phylogenetic analysis of baculoviruses from Lepidoptera. Virology 346, 180–193.[CrossRef][Medline]
Kang, W., Suzuki, M., Zemskov, E., Okano, K. & Maeda, S. (1999). Characterization of baculovirus repeated open reading frames (bro) in Bombyx mori nucleopolyhedrovirus. J Virol 73, 10339–10345.
Kang, W., Kurihara, M. & Matsumoto, S. (2006). The BRO proteins of Bombyx mori nucleopolyhedrovirus are nucleocytoplasmic shuttling proteins that utilize the CRM1-mediated nuclear export pathway. Virology 350, 184–191.[CrossRef][Medline]
Kost, T. A., Condreay, J. P. & Jarvis, D. L. (2005). Baculovirus as versatile vectors for protein expression in insect and mammalian cells. Nat Biotechnol 23, 567–575.[CrossRef][Medline]
Kumar, S. & Miller, L. K. (1987). Effects of serial passage of Autographa californica nuclear polyhedrosis virus in cell culture. Virus Res 7, 335–349.[CrossRef][Medline]
Kumar, S., Tamura, K. & Nei, M. (2004). MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform 5, 150–163.
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]
Lee, H. H. & Miller, L. K. (1978). Isolation of genotypic variants of Autographa californica nuclear polyhedrosis virus. J Virol 27, 754–767.
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, 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.
Liu, J. C. & Maruniak, J. E. (1995). Nucleotide sequence and transcriptional analysis of the gp41 gene of Spodoptera frugiperda nuclear polyhedrosis virus. J Gen Virol 76, 1443–1450.
Loh, L. C., Hamm, J. J., Kawanishi, C. & Huang, E. S. (1982). Analysis of the Spodoptera frugiperda nuclear polyhedrosis virus genome by restriction endonucleases and electron microscopy. J Virol 44, 747–751.
López-Ferber, M., Simón, O., Williams, T. & Caballero, P. (2003). Defective or effective? Mutualistic interactions between virus genotypes. Proc Biol Sci 270, 2249–2255.
Lu, A. & Miller, L. K. (1995). Differential requirements for baculovirus late expression factor genes in two cell lines. J Virol 69, 6265–6272.
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.
Moscardi, F. (1999). Assessment of the applications of baculoviruses for control of Lepidoptera. Annu Rev Entomol 44, 257–289.[CrossRef][Medline]
Nakai, M., Goto, C., Kang, W., Shikata, M., Luque, T. & Kunimi, Y. (2003). Genome sequence and organization of a nucleopolyhedrovirus isolated from the smaller tea tortrix, Adoxophyes honmai. Virology 316, 171–183.[CrossRef][Medline]
Ohkawa, T., Washburn, J. O., Sitapara, R., Sid, E. & Volkman, L. E. (2005). Specific binding of Autographa californica M nucleopolyhedrovirus occlusion-derived virus to midgut cells of Heliothis virescens larvae is mediated by products of pif genes Ac119 and Ac022 but not by Ac115. J Virol 79, 15258–15264.
O'Reilly, D. R. (1995). Baculovirus-encoded ecdysteroid UDP-glucosyltransferases. Insect Biochem Mol Biol 25, 541–550.[CrossRef]
O'Reilly, D. R. & Miller, L. K. (1989). A baculovirus blocks insect molting by producing ecdysteroid UDP-glucosyl transferase. Science 245, 1110–1112.
O'Reilly, D. R. & Miller, L. K. (1991). Improvement of a baculovirus pesticide by deletion of the egt gene. Biotechnology (NY) 9, 1086–1089.[CrossRef]
O'Reilly, D. R., Passarelli, A. L., Goldman, I. F. & Miller, L. K. (1990). Characterization of the DA26 gene in a hypervariable region of the Autographa californica nuclear polyhedrosis virus genome. J Gen Virol 71, 1029–1037.
O'Reilly, D. R., Howarth, O. W., Rees, H. H. & Miller, L. K. (1991). Structure of the ecdysone glucoside formed by a baculovirus ecdysteroid UDP-glucosyltransferase. Insect Biochem 21, 795–801.[CrossRef]
O'Reilly, D. R., Brown, M. R. & Miller, L. K. (1992a). Alteration of ecdysteroid metabolism due to baculovirus infection of the fall armyworm Spodoptera frugiperda: host ecdysteroids are conjugated with galactose. Insect Biochem Mol Biol 22, 313–320.[CrossRef]
O'Reilly, D. R., Miller, L. K. & Luckow, V. A. (1992b). Baculovirus Expression Vectors. New York: W. H. Freeman and Company.
O'Reilly, D. R., Hails, R. S. & Kelly, T. J. (1998). The impact of host developmental status on baculovirus replication. J Invertebr Pathol 72, 269–275.[CrossRef][Medline]
Pang, Y., Yu, J., Wang, L., Hu, X., Bao, W., Li, G., Chen, C., Han, H., Hu, S. & Yang, H. (2001). Sequence analysis of the Spodoptera litura multicapsid nucleopolyhedrovirus genome. Virology 287, 391–404.[CrossRef][Medline]
Pijlman, G. P., Pruijssers, A. J. & Vlak, J. M. (2003). Identification of pif-2, a third conserved baculovirus gene required for per os infection of insects. J Gen Virol 84, 2041–2049.
Possee, R. D. & Rohrmann, G. F. (1997). Baculovirus genome organization and evolution. In The Baculoviruses, pp. 109–140. Edited by L. K. Miller. New York: Plenum.
Robertson, J. L. & Preisler, H. K. (1992). Pesticide Bioassays with Arthropods. Boca Raton, FL: CRC Press.
Shapiro, D. I., Fuxa, J. R., Braymer, H. D. & Pashley, D. P. (1991). DNA restriction polymorphism in wild isolates of Spodoptera frugiperda nuclear polyhedrosis virus. J Invertebr Pathol 58, 96–105.[Medline]
Simón, O., Williams, T., López-Ferber, M. & Caballero, P. (2004). Genetic structure of a Spodoptera frugiperda nucleopolyhedrovirus population: high prevalence of deletion genotypes. Appl Environ Microbiol 70, 5579–5588.
Simón, O., Chevenet, F., Williams, T., Caballero, P. & López-Ferber, M. (2005a). Physical and partial genetic map of Spodoptera frugiperda nucleopolyhedrovirus (SfMNPV) genome. Virus Genes 30, 403–417.[CrossRef][Medline]
Simón, O., Gutiérrez, S., Williams, T., Caballero, P. & López-Ferber, M. (2005b). Nucleotide sequence and transcriptional analysis of the pif gene of Spodoptera frugiperda nucleopolyhedrovirus (SfMNPV). Virus Res 108, 213–220.[CrossRef][Medline]
Simón, O., Williams, T., López-Ferber, M. & Caballero, P. (2005c). Functional importance of deletion mutant genotypes in an insect nucleopolyhedrovirus population. Appl Environ Microbiol 71, 4254–4262.
Simón, O., Williams, T., Caballero, P. & López-Ferber, M. (2006). Dynamics of deletion genotypes in an experimental insect virus population. Proc Biol Sci 273, 783–790.
Slavicek, J. M., Popham, H. J. R. & Riegel, C. I. (1999). Deletion of the Lymantria dispar multicapsid nucleopolyhedrovirus ecdysteroid UDP-glucosyl transferase gene enhances viral killing speed in the last instar of the gypsy moth. Biol Control 16, 91–103.[CrossRef]
Sparks, A. N. (1979). A review of the biology of the fall armyworm. Fla Entomol 62, 82–87.[CrossRef]
Theilmann, D. A., Blissard, G. W., Bonning, B., Jehle, J. A., O'Reilly, D. R., Rohrmann, G. F., Thiem, S. & Vlak, J. M. (2005). Baculoviridae. In Virus Taxonomy. Classification and Nomenclature of Viruses. 8th Report of the International Committee on the Taxonomy of Viruses, pp. 177–185. Edited by C. X. Fauquet, M. A. Mayo, J. Maniloff, U. Desselberger & L. A. Ball. Amsterdam: Elsevier.
Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 4673–4680.
Treacy, M. F., All, J. N. & Ghidiu, G. M. (1997). Effect of ecdysteroid UDP-glucosyltransferase gene deletion on efficacy of a baculovirus against Heliothis virescens and Trichoplusia ni (Lepidoptera: Noctuidae). J Econ Entomol 90, 1207–1214.[Medline]
Tumilasci, V. F., Leal, E., Zanotto, P. M., Luque, T. & Wolff, J. L. (2003). Sequence analysis of a 5.1 kbp region of the Spodoptera frugiperda multicapsid nucleopolyhedrovirus genome that comprises a functional ecdysteroid UDP-glucosyltransferase (egt) gene. Virus Genes 27, 137–144.[CrossRef][Medline]
van Oers, M. M., Abma-Henkens, M. H., Herniou, E. A., de Groot, J. C., 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.
Williams, T., Goulson, D., Caballero, P., Cisneros, J., Martinez, A. M., Chapman, J. W., Roman, D. X. & Cave, R. D. (1999). Evaluation of a baculovirus bioinsecticide for small-scale maize growers in Latin America. Biol Control 14, 67–75.[CrossRef]
Willis, L. G., Seipp, 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]
Wilson, K. R., O'Reilly, D. R., Hails, R. S. & Cory, J. S. (2000). Age-related effects of the Autographa californica multiple nucleopolyhedrovirus egt gene in the cabbage looper (Trichoplusia ni). Biol Control 19, 57–63.[CrossRef]
Zemskov, E. A., Kang, W. & Maeda, S. (2000). Evidence for nucleic acid binding ability and nucleosome association of Bombyx mori nucleopolyhedrovirus BRO proteins. J Virol 74, 6784–6789.
Zhang, C. X., Ma, X. C. & Guo, Z. J. (2005). Comparison of the complete genome sequence between C1 and G4 isolates of the Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus. Virology 333, 190–199.[CrossRef][Medline]
Received 6 November 2007;
accepted 22 November 2007.
This article has been cited by other articles:
|
|
 |
|
  O. Simon, T. Williams, R. D. Possee, M. Lopez-Ferber, and P. Caballero Stability of a Spodoptera frugiperda Nucleopolyhedrovirus Deletion Recombinant during Serial Passage in Insects Appl. Envir. Microbiol., February 1, 2010; 76(3): 803 - 809. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 O. Simon, T. Williams, A. C. Asensio, S. Ros, A. Gaya, P. Caballero, and R. D. Possee Sf29 Gene of Spodoptera frugiperda Multiple Nucleopolyhedrovirus Is a Viral Factor That Determines the Number of Virions in Occlusion Bodies J. Virol., August 15, 2008; 82(16): 7897 - 7904. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
