Joint-Laboratory of Invertebrate Virology, Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei 430071, Peoples Republic of China1
Laboratory of Virology, Wageningen University, Binnenhaven 11, 6709 PD Wageningen, The Netherlands2
Greenomics, Plant Research International, PO Box 16, 6700 AA Wageningen, The Netherlands3
Author for correspondence: Zhihong Hu. Fax +86 27 87641072. e-mail huzh{at}pentium.whiov.ac.cn
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Abstract |
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Introduction |
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Baculoviruses are frequently used as bio-insecticides of phytophagous insects, mainly belonging to the orders Lepidoptera, Hymenoptera and Diptera (Moscardi, 1999 ; Federici, 1999
). The SNPV of the bollworm Helicoverpa armigera (HaSNPV) has been extensively used to control this insect in cotton and vegetable crops in China (Zhang, 1994
). In 1999, about 100000 hectares of cotton had been treated with a commercial virus preparation based on HaSNPV. Recently, recombinant HaSNPVs with improved insecticidal properties have been engineered (Chen et al., 2000b
) and field tested (S. Sun, X. Chen, Z. Zhang, H. Wang, F. J. J. A. Bianchi, H. Peng, J. M. Vlak & Z. H. Hu, unpublished). However, the genetics of HaSNPV have only been partly described.
The nucleotide sequences of five MNPVs, Autographa californica (Ac) MNPV (Ayres et al., 1994 ), Bombyx mori (Bm) NPV (Gomi et al., 1999
), Orgyia pseudotsugata (Op) MNPV (Ahrens et al., 1997
), Lymantria dispar (Ld) MNPV (Kuzio et al., 1999
) and Spodoptera exigua (Se) MNPV (IJkel et al., 1999
), and one granulovirus, Xestia c-nigrum (Xc) GV (Hayakawa et al., 1999
), have been determined. The size of these genomes ranges from 128413 bp for BmNPV to 178733 bp for XcGV. This size difference is predominantly due to the presence of gene duplications including the so-called baculovirus repeat ORF or bro genes (Gomi et al., 1999
). However, no SNPV genome has been sequenced to date and it is therefore of interest to see whether the sequence of HaSNPV would reveal some unique features contributing to, among others, the SNPV phenotype and to the specificity of this virus for heliothine insects.
A physical map of HaSNPV has been previously constructed and the size was estimated to be about 130 kb (Chen et al., 2000a ). Analysis of approximately 45 kb of random sequence from the HaSNPV genome resulted in the identification of 53 ORFs with homologies to ORFs of other baculoviruses. Partial alignment of the HaSNPV genome with other baculovirus genomes using GeneParityPlot (Hu et al., 1998
) revealed a close relationship of HaSNPV and SeMNPV in terms of genomic organization (Chen et al., 2000a
). A few genes, notably polyhedrin (Chen et al., 1997b
), ecdysteroid UDP-glucosyltransferase (egt) (Chen et al., 1997a
), DNA polymerase (Bulach et al., 1999
) and late expression factor 2' (lef-2) (Chen et al., 1999
), have been characterized in some detail. Phylogenetic analysis of these genes also revealed a close ancestral relationship between HaSNPV, SeMNPV and LdMNPV at the gene level.
In this paper we describe the complete nucleotide sequence and organization of the HaSNPV genome. This baculovirus is characterized by the absence of extensive gene duplications and by the presence of a limited number of homologous repeat (hr) regions, the structure of which is distinctly different from the hr sequences of other baculoviruses. Finally, a genomic comparison is made with the complete sequences of AcMNPV, SeMNPV, LdMNPV and XcGV using GeneParityPlot (Hu et al., 1998 ).
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Methods |
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HaSNPV DNA isolation, cloning and sequence determination.
The HaSNPV G4 strain (Sun et al., 1998 ) was sequenced to a sixfold genomic coverage using a shotgun approach. The viral DNA was caesium chloride-purified (King & Possee, 1992
) and sheared by nebulization into fragments with an average size of 1200 bp. Blunt repair of the ends was performed with Pfu DNA polymerase (Stratagene), according to the manufacturers directions. DNA fragments were size-fractionated by gel electrophoresis and cloned into the EcoRV site of pBluescriptSK (Stratagene). After transformation into E. coli XL2-Blue competent cells (Stratagene), 1000 recombinant colonies were picked randomly. DNA templates for sequencing were isolated using QIAprep Turbo kits (Qiagen) on a QIAGEN BioRobot 9600. Sequencing was performed using the ABI PRISM Big Dye Terminator Cycle Sequencing Ready reaction kit with FS AmpliTaq DNA polymerase (Perkin Elmer) and analysed on an ABI 3700 DNA Analyser.
Shotgun sequences were base-called by the PHRED basecaller and assembled with the PHRAP assembler (Ewing & Green, 1998 ; Ewing et al., 1998
). Using the PREGAP4 interface, PHRAP-assembled data were stored in the GAP4 assembly database (Bonfield et al., 1995
). The GAP4 interface and its features were then used for editing and sequence finishing. Consensus calculations with a quality cut-off value of 40 were performed from within GAP4 using a probabilistic consensus algorithm based on expected error rates output by PHRED. Sequencing the PCR products bridging the ends of existing contiguous fragments filled the remaining gaps in the sequence.
DNA sequence analysis.
Genomic DNA composition, structure, repeats and restriction enzyme pattern were analysed with the University of Wisconsin Genetics Computer Group programs (Devereux et al., 1984 ) and DNASTAR (Lasergene). ORFs encoding more than 50 amino acids (150 bp) were considered to be protein-encoding and hence designated putative genes. The maximal alignment of 115 ORFs (out of 135) was checked with known baculovirus gene homologues extracted from GenBank; ORFs with an overlap of hr region were excluded from the alignment analysis. The overlap between any two ORFs with known baculovirus homologues was set to a maximum of 25 amino acids; otherwise the largest ORF was selected.
DNA and protein comparisons with entries in the sequence databases were performed with FASTA and BLAST programs (Pearson, 1990 ; Altschul et al., 1990
). Multiple sequence alignments were performed with the GCG PileUp and Gap computer programs version 10.0 (Genetics Computer Group, Madison, WI, USA) with gap creation and extension penalties set to 9 and 2, respectively (Devereux et al., 1984
). Percentage identity indicates the percentage of identical residues between two complete sequences. The GENESCAN program was used for gene predictions (http://ccr-081.mit.edu/GENESCAN.html). The DOTTER program (http://www.cgr.ki.se/cgr/groups/sonnhammer/Dotter.html) was used to identify and classify repeat families and miniature inverted repeat transposable elements (MITEs). GeneParityPlot analysis was performed on the HaSNPV genome versus the genomes of AcMNPV, SeMNPV, LdMNPV and XcGV as described previously (Hu et al., 1998
).
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Results and Discussion |
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Homologous repeat (hr) regions
Regions with homologous repeats were first found in AcMNPV (Cochran & Faulkner, 1983 ) and appear to be present in all baculoviruses. They occur at multiple locations along the genome and may serve as origins of DNA replication (Kool et al., 1995
) and as enhancers of transcription (Guarino & Summers, 1986
; Guarino et al., 1986
). Hr regions are characterized by the presence of multiple, often imperfect, tandemly repeated palindromic sequences (AcMNPV). Five hr regions were previously identified on the genome of HaSNPV by direct sequencing and Southern blot hybridization (Chen et al., 2000a
). No further hr regions were detected in the complete sequence (Fig. 1
; Table 1
). These five hr regions were found dispersed along the HaSNPV genome around map positions 17.5 (hr1), 37.7 (hr2), 40.2 (hr3), 70.8 (hr4) and 83.6 (hr5) and are located in AT-rich intergenic regions. Their sizes vary from 750 (hr3) to 2800 nt (hr5). It is interesting to note that hr2 and hr3 are separated by two bro-related genes (Fig. 1
). This configuration might have been the result of an insertion of two bro genes into what originally may have been a single hr. Assuming that hr2 and hr3 have been a single hr, the hr regions of HaSNPV are remarkably similar is size (21002800 nt).
Using a dot matrix analysis, the HaSNPV sequence was compared to itself and its complementary strand. Two types of repeats were identified, type A and type B, with imperfect 40 and 107 bp long repeats, respectively, or truncated versions thereof (Fig. 2). The type A and type B repeats are found in each of the hr regions. There is no sequence homology with other known baculovirus hr regions. The type B repeats contain short internal stretches of palindromic and direct repeats. Not only is the sequence of the HaSNPV hr regions different from those of other baculovirus hr regions, but their structure is also rather unique. The function of the type A and type B repeats remains to be determined.
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Of the 135 HaSNPV ORFs identified, 100 have homologues in AcMNPV and a further 15 have homologues in other baculoviruses (Tables 1 and 2
). HaSNPV shares the largest number of homologues (103) with SeMNPV, underscoring the close relationship between these two viruses as evidenced from gene phylogeny analyses involving polyhedrin, egt, lef-2 (Chen et al., 1997a
, b
, 1999
) and DNA polymerase (Bulach et al., 1999
). Polyhedrin is the most conserved ORF of the six NPVs, with a mean amino acid identity of 83% to other NPV polyhedrin genes; the identity to GV granulin is much less (51% for XcGV). Ubiquitin (ubi), which is involved in the targetting of proteins for degradation, is the next most conserved gene among the seven sequenced baculoviruses, with 75% amino acid identity, followed by superoxide dismutase (sod) with 70% amino acid identity. The mean ORF amino acid identity between HaSNPV and the group II baculoviruses SeMNPV and LdMNPV is similar (46%) and higher than to group I baculoviruses (41%). This is in support of the distinct phylogenetic relationship between group I and group II NPVs (Zanotto et al., 1993
; Bulach et al., 1999
).
Structural virion genes
The HaSNPV genome contains the known genes encoding the common virion structural proteins of NPVs (Table 2). In contrast to SeMNPV, where odv-e66 is duplicated (IJkel et al., 1999
), HaSNPV does not contain duplicate genes for virion structural proteins. However, HaSNPV apparently lacks a homologue of the BV envelope surface glycoprotein gene gp64 (Ac128). The product of this gene, GP64, is acquired by virions during budding through the plasma membrane and is involved in the association with cell receptors upon invasion and fusion in endosomes (Oomens & Blissard, 1999
). However, an ORF has been identified in HaSNPV (Ha133) with an average amino acid identity with Ld130 (43%) and Se8 (40%). The latter viruses also lack a gp64 homologue and it has been suggested that Ld130 and Se8 are the functional homologues of AcMNPV gp64 (Kuzio et al., 1999
; IJkel et al., 1999
). Recently, direct evidence was obtained that the products of Ld130 and Se8 are the major constituents of the BV envelope and are responsible for the fusogenic activity of SeMNPV (Pearson et al., 2000
; IJkel et al., 2000
).
DNA replication and late gene expression
There are 19 lef genes in AcMNPV that have been implied in DNA replication and late gene expression (Kool et al., 1995 ; Lu & Miller, 1995
). They were all required for late and very late gene expression. Of these, six (lef-1, lef-2, lef-3, dnapol, helicase, ie-1) are essential for DNA replication, whereas others are involved in transcription (ie-2, lef-4, lef-5, lef-8, lef-9) (Guarino et al., 1998
) or in inhibition of apoptosis (such as p35 and iap genes) (Clem & Miller, 1994
). In silico analysis indicated that the genome of HaSNPV contains homologues of 16 of the above AcMNPV lef genes and lacks ie-2, p35 and lef-12 (Table 4
). The latter genes are also absent in LdMNPV, SeMNPV and XcGV, suggesting that they occur only in the group I NPVs. HaSNPV also has a homologue (Ha8) to the first exon of a spliced transcript from Ac141 (ie-0). This transcript also includes Ac147 located 4 kb downstream of ie-0 (Chisholm & Henner, 1988
). In contrast, this exon encoded by Se138 is not functional in SeMNPV (Van Strien et al., 2000
).
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Similar to SeMNPV, LdMNPV and XcGV, HaSNPV also lacks a p35 homologue (Table 4). Instead, two members of the iap (Crook et al., 1993
) gene family were observed in HaSNPV, iap-2 (Ha62) and iap-3 (Ha103). Homologues of iap subclasses (14) have been found in AcMNPV (Ac27, iap-1 and Ac71, iap-2), OpMNPV (Op41, iap-1; Op74, iap-2; Op35, iap-3 and ORF106, iap-4), SeMNPV (Se88, iap-2 and Se110, iap-3), LdMNPV (Ld79, iap-2 and Ld139, iap-3) and XcGV (Xc137, iap-3). The HaSNPV iap-3 gene has high homology to the CpGV iap gene, which could functionally complement an AcMNPV p35 deletion mutant (Crook et al., 1993
). OpMNPV iap-3 can also complement AcMNPV p35 null mutants (Birnbaum et al., 1994
). The function of the iap-1, iap-2 and iap-4 genes is unknown. Through partial DNA sequence analysis, three iap gene homologues (iap-1, iap-2 and iap-3) were found in Buzura suppressaria SNPV (Hu et al., 1998
).
HaSNPV lack genes for enzymatic functions in nucleotide metabolism, such as ribonucleotide reductase (rr) and deoxyuridyltriphosphatase (dUTPase). The products of rr and dUTPase allow the virus to convert rNTPs into dNTPs to the benefit of virus DNA replication. RR reduces NDPs into dNDPs and dUTPase converts dUTP into dUMP, thereby excluding dUTP from incorporation into DNA and providing dUMP as a precursor for dTTP. dUTPase and rr are present in SeMNPV (IJkel et al., 1999 ), OpMNPV (Ahrens et al., 1997
) and LdMNPV (Kuzio et al., 1999
) but are absent from AcMNPV and BmNPV and also from XcGV. The latter virus contained a DNA ligase (Xc141), which appeared to be absent from NPVs except LdMNPV.
Genes with auxiliary functions
Baculovirus auxiliary genes are not essential for virus replication per se but are important, for example, for interaction with the insect host (OReilly, 1997 ). HaSNPV has a very similar set of auxiliary genes as SeMNPV, encoding for example chitinase (chitA, Ha41), cathepsin (v-cath, Ha56) and egt (Ha126) (IJkel et al., 1999
). These genes are quite well conserved, with 66, 47 and 49% amino acid identity, respectively, whereas the fibroblast growth factor (fgf, Ha113) is poorly conserved among baculoviruses with 28% amino acid identity.
HaSNPV lacks a gene for protein tyrosine/serine phosphatase (ptp) with dual-specificity (dsPTP) (Tilakaratne et al., 1991 ; Kim & Weaver, 1993
). This protein specifically removes phosphates from both tyrosine and serine/threonine residues (Wishart et al., 1995
). The absence of a ptp gene homologue in HaSNPV is striking, since such a gene is present in all NPV genomes sequenced to date and is thought to be involved in the regulation of the phosphorylation status of viral and host proteins during infection.
Duplicated bro genes
A common characteristic of baculovirus genomes is the presence of a group of related genes, the so-named bro genes. Five homologues of AcMNPV ORF2 (Ac2) are present in BmNPV (Gomi et al., 1999 ). In LdMNPV, SeMNPV and XcGV sixteen, one and five bro-related genes are found, respectively (Kuzio et al., 1999
; IJkel et al., 1999
; Hayakawa et al., 1999
). In OpMNPV, a truncated version and two smaller bro-related ORFs are present (Ahrens et al., 1997
). Three bro-related genes were identified in HaSNPV, named bro-a (Ha59), bro-b (Ha60) and bro-c (Ha105). Ha59 is most closely related to Ld150 (bro-m), belonging to the group II bro family (Kuzio et al., 1999
), with 50% amino acid identity. Ha60 also belongs to the group II bro genes and shares the largest homology to Ld140 (bro-l) and Xc159 (bro-g), but has an N-terminal duplication of 183 amino acids. It thus seems unlikely that the Ha59 and Ha60 bro genes have a common recent ancestor and therefore might have been spliced in tandem into an hr sequence (hr3 and hr4). Ha105 and Xc60 are 66% identical and related to the group III bro genes (Kuzio et al., 1999
).
HaSNPV ORFs with homologues in a few other baculoviruses
HaSNPV possesses 22 ORFs that have no homologues in AcMNPV, BmNPV, OpMNPV, SeMNPV, LdMNPV or XcGV (Table 2). Of these, Ha6 is identical to Hz480 from Helicoverpa zea SNPV (HzSNPV) (Le et al., 1997
). In HaSNPV, a homologue of the Leucania separata NPV (LsNPV) p13 gene (Ha97) is found. This homologue is, in contrast to the SeMNPV homologue, not C-terminally extended (Wang et al., 1995
; IJkel et al., 1999
). The two leucine-zipper-like structures present in LsNPV P13 (Wang et al., 1995
) are also conserved in Ha97. The function of this ORF in LsNPV as well as in SeMNPV (Se59) and XcGV (Xc48) is unknown.
Three HaSNPV ORFs have a homologue in only one other baculovirus. None of these genes has yet been assigned functions. Ha19 has a gene homologue in LdMNPV (Ld26) with an amino acid identity of 40%. This ORF, however, is rather small, encoding an 11 kDa protein. Ha57, encoding a putative 21 kDa product, has a homologue in XcGV (Xc83) with an amino acid identity of 33%. An Se68 homologue is identified in HaSNPV as Ha83 encoding a putative protein of 18·8 kDa but with a low amino acid identity of 26%. All ORFs, however, have baculovirus early and/or late transcription motifs and may therefore be functional.
HaSNPV ORF100 (Ha100) was found to encode a putative poly(ADP-ribose) glycohydrolase (parg). The homology with Drosophila melanogaster (24% identity) and Homo sapiens (23% identity) genes was found in the C-terminal portion of the putative protein. Homologues of Ha100 were also found in LdMNPV (Ld141) and SeMNPV (Se52), so that their presence appears to be limited to group II NPVs. In eukaryotes this enzyme is involved in the breakdown of polyribose and recruitment of this compound for nuclear functions such as DNA replication and repair (DAmours et al., 1999 ). The function of this enzyme in baculovirus group II morphogenesis or pathology is not known, but it is possible that it is involved in similar capacity during the NPV infection process. The baculovirus parg gene is much longer than the eukaryotic counterpart and thus may have additional activities.
A few HaSNPV ORFs (Ha14, Ha6, Ha911, Ha1315, Ha41, Ha69, Ha71 and Ha126; Table 2) have a high degree of amino acid identity (>90%) to sequences available from HzSNPV (Ma et al., 1993
; Cowan et al., 1994
; Le et al., 1997
). This suggests that the overall homology between HaSNPV and HzSNPV is very high and that they are most likely variants of the same virus species. Sequencing of the HzSNPV genome would reveal whether this assumption is correct.
Unique HaSNPV ORFs
To date, 20 ORFs in the HaSNPV genome are unique to this virus and also do not exhibit significant homology to any other sequences in the GenBank. Most of these ORFs are either very small, encoding putative proteins of up to 100 amino acids (Ha5, Ha7, Ha17, Ha18, Ha40, Ha45, Ha54, Ha104 and Ha112), or contain no common baculovirus transcription initiation sites for early or late gene expression (Ha102, Ha108 and Ha109). Eight ORFs (Ha29, Ha34, Ha99, Ha107, Ha122, Ha125, Ha134 and Ha135) are larger than 100 amino acids and have early and late baculovirus promoter motifs. Ha34 and Ha107 are of interest as they encode putative proteins of 41·1 and 51·2 kDa, respectively. The possible functions of these ORFs are being investigated. For convenience, the ORFs present in the other baculovirus sequences, AcMNPV, BmNPV, OpMNPV, LdMNPV and SeMNPV, are listed in Table 4.
The HaSNPV genome organization
The genomic organization, i.e. the order of genes, of HaSNPV has been studied in a comparative manner using GeneParityPlot analysis (Hu et al., 1998 ). As the gene order between AcMNPV, BmNPV and OpMNPV is basically identical, except for a small number of rearrangements (Ahrens et al., 1997
; Hu et al., 1998
; Gomi et al., 1999
), AcMNPV was taken as a representative example of this group in the analysis (Fig. 3A
). A comparison was made between the recently sequenced MNPVs, SeMNPV (IJkel et al., 1999
) and LdMNPV (Kuzio et al., 1999
), and XcGV (Hayakawa et al., 1999
) (Fig. 3B
D
). To obtain maximum alignment in the GeneParityPlot analysis, the order of genes had to be reversed for the calculation. By convention, the orientation of a circular baculovirus genome is determined by the relative position of two genes, polyhedrin at map unit 0 and p10 approximately at map unit 90 (Vlak & Smith, 1982
). In the initial GeneParityPlot analysis, the orientation of the HaSNPV genome appeared to be reversed for more than 50% of the ORFs compared with AcMNPV and LdMNPV in order to obtain maximum alignment compared with the physical map constructed previously (Chen et al., 2000a
). A similar situation exists for SeMNPV (IJkel et al., 1999
). The gene organization of HaSNPV is most conserved in the central region of the linearized baculovirus genomes and confirms the supposition of Heldens et al. (1998)
. The left region of the linearized HaSNPV genome displays a considerable number of gene inversions and translocations as deduced from the GeneParityPlot analyses. The right region showed a high degree of gene scrambling (Fig. 3A
D
). From these analyses it is concluded that the organization of HaSNPV is highly characteristic and distinct from those of AcMNPV, SeMNPV, LdMNPV and XcGV.
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Comparison of the cluster organization of HaSNPV with that of other baculoviruses (Fig. 4) suggests that the genomic organization of HaSNPV is more closely related to that of SeMNPV and LdMNPV than to that of group I NPVs (AcMNPV, BmNPV and OpMNPV) or XcGV. This is in agreement with the phylogenetic analysis of single genes such as egt, lef-2, dnapol and rr (Chen et al., 1997a
, b
, 1999
; Bulach et al., 1999
). When the order of gene clusters is taken to represent the baculovirus genome organization, the common structure of group II baculoviruses becomes apparent (Fig. 4A
). Within each group, the structural difference is relatively small and predominantly determined by inversions of gene clusters as well as inversions of individual genes (e.g. polyhedrin). Comparison of the two groups showed extensive genomic translocations in addition to cluster inversions. When the inverted genes remained functional, they could be translocated to other genomic regions. These jumping genes can be used as phylogenetic markers to follow baculovirus evolution in retrospect. A common genome structure for group I and group II viruses can be derived, showing a major inversion of a genomic segment containing the cluster c-b-a-m-n (Fig. 4B
).
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Acknowledgments |
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Footnotes |
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References |
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Altschul, S. F., Gish, W., Miller, W., Meyers, E. W. & Lipman, D. J.(1990). Basic local alignment search tool. Journal of Molecular Biology 215, 403-410.[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.[Medline]
Birnbaum, M. J., Clem, R. J. & Miller, L. K.(1994). An apoptosis-inhibiting gene from a nuclear polyhedrosis virus encoding a polypeptide with Cys/His sequence motifs. Journal of Virology 68, 2521-2528.[Abstract]
Bonfield, J. K., Smith, K. F. & Staden, R.(1995). A new DNA sequence assembly program. Nucleic Acids Research 24, 4992-4999.
Bulach, D. M., Kumar, C. A., Zaia, A., Liang, B. F. & Tribe, D. E.(1999). Group II nucleopolyhedrovirus subgroups revealed by phylogenetic analysis of polyhedrin and DNA polymerase gene sequences. Journal of Invertebrate Pathology 73, 59-73.[Medline]
Chen, X., Hu, Z., Jehle, J. A., Zhang, Y. & Vlak, J. M.(1997a). Analysis of the ecdysteroid UDP-glucosyltransferase gene of Heliothis armigera single-nucleocapsid baculovirus. Virus Genes 15, 219-225.[Medline]
Chen, X., Hu, Z. H. & Vlak, J. M.(1997b). Nucleotide sequence analysis of the polyhedrin gene of Heliothis armigera single nucleocapsid nuclear polyhedrosis virus. Virologica Sinica 12, 346-353.
Chen, X., IJkel, W. F. J., Dominy, C., Zanotto, P. de A., Hashimoto, Y., Faktor, O., Hayakawa, T., Wang, C. H., Krell, P. J., Hu, Z. & Vlak, J. M.(1999). Identification, sequence and phylogeny of the lef-2 gene of Helicoverpa armigera single-nucleocapsid baculovirus. Virus Research 65, 21-32.[Medline]
Chen, X., Li, M., Sun, X., Arif, B. M., Hu, Z. H. & Vlak, J. M. (2000a). Genomic organization of Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus. Archives of Virology 145, (in press).
Chen, X., Sun, X., Hu, Z. H., Li, M., OReilly, D. R., Zuidema, D. & Vlak, J. M.(2000b). Genetic engineering of Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus as an improved bioinsecticide. Journal of Invertebrate Pathology 76, 140-146.[Medline]
Chisholm, G. E. & Henner, D. J.(1988). Multiple early transcripts and splicing of the Autographa californica nuclear polyhedrosis virus IE-1 gene. Journal of Virology 62, 3193-3200.[Medline]
Clem, R. J. & Miller, L. K.(1994). Control of programmed cell death by the baculovirus genes p35 and iap. Molecular Cell Biology 14, 5212-5222.[Abstract]
Cochran, M. A. & Faulkner, P.(1983). Location of homologous DNA sequences interspersed at five regions in the baculovirus AcMNPV genome. Journal of Virology 45, 961-970.
Cowan, P., Bulach, D., Goodge, K., Robertson, A. & Tribe, D. E.(1994). Nucleotide sequence of the polyhedrin gene region of Helicoverpa zea single nucleocapsid nuclear polyhedrosis virus: placement of the virus in lepidopteran nuclear polyhedrosis group II. Journal of General Virology 75, 3211-3218.[Abstract]
Crook, N. E., Clem, R. J. & Miller, L. K.(1993). An apoptosis-inhibiting baculovirus gene with a zinc finger-like motif. Journal of Virology 67, 2168-2174.[Abstract]
DAmours, D., Desnoyers, S., dSilva, I. & Poirier, G. G.(1999). Poly (ADP-ribosyl)ation reactions in the regulation of nuclear functions. Biochemical Journal 342, 249-268.[Medline]
Devereux, J., Haeberli, P. & Smithies, O.(1984). A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Research 12, 387-395.[Abstract]
Domingo, E., Holland, J. J., Biebricher, C. & Eigen, M.(1995). Quasispecies: the concept and the world. In Molecular Basis of Evolution , pp. 171-180. Edited by A. Gibbs, C. Calisher& F. Garcia-Arenal. Cambridge:Cambridge University Press.
Ewing, B. & Green, P.(1998). Basecalling of automated sequencer traces using PHRED. II. Error probabilities. Genome Research 8, 186-194.
Ewing, B., Hillier, L., Wendl, M. C. & Green, P.(1998). Basecalling of automated sequencer traces using PHRED. I. Accuracy assessment. Genome Research 8, 175-185.
Federici, B. A.(1999). Naturally occurring baculoviruses for insect pest control. Methods in Biotechnology 5, 301-320.
Figueiredo, E., Muñoz, D., Escribano, A., Mexia, A., Vlak, J. M. & Caballero, P.(1999). Biochemical identification and comparative insecticidal activity of nucleopolyhedrovirus isolates pathogenic for Heliothis armigera (Lep., Noctuidae) larvae. Journal of Applied Entomology 123, 165-169.
Gettig, R. R. & McCarthy, W. J.(1982). Genotypic variation among wild isolates of Heliothis spp nuclear polyhedrosis viruses from different geographical regions. Virology 117, 245-252.
Gomi, S., Majima, K. & Maeda, S.(1999). Sequence analysis of the genome of Bombyx mori nucleopolyhedrovirus. Journal of General Virology 80, 1323-1337.[Abstract]
Guarino, L. A. & Summers, M. D.(1986). Interspersed homologous DNA of Autographa californica nuclear polyhedrosis virus enhances delayed-early gene expression. Journal of Virology 60, 215-223.
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. Journal of Virology 60, 224-229.
Guarino, L. A., Xu, B., Jin, J. P. & Dong, W.(1998). A virus-encoded RNA polymerase purified from baculovirus-infected cells. Journal of Virology 72, 7985-7991.
Hayakawa, T., Ko, R., Okano, K., Seong, S., Goto, C. & Maeda, S.(1999). Sequence analysis of the Xestia c-nigrum granulovirus genome. Virology 262, 277-297.[Medline]
Heldens, J. G. M., Yi, L., Zuidema, D., Goldbach, R. W. & Vlak, J. M.(1998). A highly conserved genomic region in baculoviruses: sequence and transcriptional analysis of an 11·3 kbp DNA fragment (46·555·1 mu) from the Spodoptera exigua multicapsid nucleopolyhedrovirus. Virus Research 55, 187-198.[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. Journal of General Virology 79, 2841-2851.[Abstract]
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. Journal of General Virology 80, 3289-3304.
IJkel, W. F. J., Westenberg, M., Goldbach, R. W., Blissard, G. W., Vlak, J. M. & Zuidema, D.(2000). A novel baculovirus envelope fusion protein with a proprotein convertase cleavage site. Virology 275, 30-41.[Medline]
Kim, D. & Weaver, R. F.(1993). Transcription mapping and functional analysis of the protein tyrosine/serine phosphatase (PTPase) gene of the Autographa californica nuclear polyhedrosis virus. Virology 195, 587-595.[Medline]
King, L. A. & Possee, R. D. (1992). The Baculovirus Expression System: A Laboratory Guide, pp. 180194. London: Chapman and Hall.
Kool, M., Ahrens, C. H., Vlak, J. M. & Rohrmann, G. F.(1995). Replication of baculovirus DNA. Journal of General Virology 76, 2103-2118.[Medline]
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.[Medline]
Le, T. H., Wu, T., Robertson, A., Bulach, D., Cowan, P., Goodge, K. & Tribe, D.(1997). Genetically variable triplet repeats in a RING-finger ORF of Helicoverpa species baculoviruses. Virus Research 49, 67-77.[Medline]
Lu, A. & Miller, L. K.(1995). The roles of eighteen baculovirus late expression factor genes in transcription and DNA replication. Journal of Virology 69, 975-982.[Abstract]
Ma, S.-W., Corsaro, B. G., Klebba, P. E. & Fraser, M. J.(1993). Cloning and sequence analysis of a p40 structural protein of Helicoverpa zea nuclear polyhedrosis virus. Virology 192, 224-233.[Medline]
Mikhailov, V. S., Mikhailova, A. L., Iwanaga, M., Gomi, S. & Maeda, S.(1998). Bombyx mori nucleopolyhedrovirus encodes a DNA-binding protein capable of destabilizing duplex DNA. Journal of Virology 72, 3107-3116.
Moscardi, F.(1999). Assessment of the application of baculoviruses for control of Lepidoptera. Annual Reviews of Entomology 44, 257-289.
Muñoz, D., Castillejo, J. I. & Caballero, P.(1998). Naturally occurring deletion mutants are parasitic genotypes in a wild-type nucleopolyhedrovirus population of Spodoptera exigua. Applied and Environmental Microbiology 64, 4372-4377.
Murphy, F. A., Fauquet, C. M., Bishop, D. H. L., Ghabrial, S. A., Jarvis, A. W., Martelli, G. P., Mayo, M. A. & Summers, M. D. (editors) (1995). Virus Taxonomy. Sixth Report of the International Committee on Taxonomy of Viruses. New York: SpringerVerlag.
Okano, K., Mikhailov, V. S. & Maeda, S.(1999). Colocalization of baculovirus IE-1 and two DNA-binding proteins, DBP and LEF-3, to viral replication factories. Journal of Virology 73, 110-119.
Oomens, A. G. P. & Blissard, G. W.(1999). Requirement for gp64 to drive efficient budding of Autographa californica multicapsid nucleopolyhedrovirus. Virology 254, 297-314.[Medline]
OReilly, D. R.(1997). Auxiliary genes of baculoviruses. In The Baculoviruses , pp. 267-295. Edited by L. K. Miller. New York:Plenum.
Pearson, W. R.(1990). Rapid and sensitive sequence comparison with FASTP and FASTA. Methods in Enzymology 183, 63-98.[Medline]
Pearson, M. N., Groten, C. & Rohrmann, G. F.(2000). Identification of the Lymantria dispar nucleopolyhedrovirus envelope fusion protein provides evidence for a phylogenetic division of the Baculoviridae. Journal of Virology 74, 6126-6131.
Smith, I. R. & Crook, N. E.(1988). In vivo isolation of baculovirus genotypes. Virology 166, 240-244.[Medline]
Sun, X. L., Zhang, G. Y., Zhang, Z. X., Hu, Z. H., Vlak, J. M. & Arif, B. M.(1998). In vivo cloning of Helicoverpa armigera single nucleocapsid nuclear polyhedrosis virus genotypes. Virologica Sinica 13, 83-88.
Tilakaratne, N., Hardin, S. E. & Weaver, R. F.(1991). Nucleotide sequence and transcript mapping of the HindIII F region of the Autographa californica nuclear polyhedrosis virus. Journal of General Virology 72, 285-291.[Abstract]
Todd, J. W., Passarelli, A. L. & Miller, L. K.(1996). Factors regulating baculovirus late and very late gene expression in transient-expression assays. Journal of Virology 70, 2307-2317.[Abstract]
Van Strien, E. A., IJkel, W. F. J., Gerrits, H., Vlak, J. M. & Zuidema, D.(2000). Characteristics of the transactivator gene ie-1 of Spodoptera exigua multiple-nucleocapsid nucleopolyhedrovirus. Archives of Virology 145, 2115-2133.[Medline]
Vlak, J. M. & Smith, G. E.(1982). Orientation of the genome of Autographa californica nuclear polyhedrosis virus: a proposal. Journal of Virology 41, 1118-1121.
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. Archives of Virology 140, 2283-2291.[Medline]
Wishart, M. J., Denu, J. M., Williams, J. A. & Dixon, J. E.(1995). A single mutation converts a novel phosphotyrosine binding domain into a dual-specificity phosphatase. Journal of Biological Chemistry 270, 26782-26785.
Wu, Y. & Carstens, E. H.(1998). A baculovirus single-stranded DNA binding protein, LEF-3, mediates the nuclear localization of the putative helicase P143. Virology 247, 32-40.[Medline]
Zanotto, P. M. de A., Kessing, B. D. & Maruniak, J. E.(1993). Phylogenetic interrelationships among baculoviruses: evolutionary rates and host associations. Journal of Invertebrate Pathology 62, 147-164.[Medline]
Zhang, G.(1994). Research, development and application of Heliothis viral pesticide in China. Resources and Environment in the Yangtze Valley 3, 1-6.
Zhang, G., Zhang, Y., Ge, L. & Shan, Z.(1981). The production and application of the nuclear polyhedrosis virus of Heliothis armigera (Hübner) in biological control. Acta Phytophylacica Sinica 8, 235-240.
Received 9 June 2000;
accepted 15 September 2000.