Complete comparative genomic analysis of two field isolates of Mamestra configurata nucleopolyhedrovirus-A

Lulin Li1,{dagger}, Qianjun Li2,{ddagger}, Leslie G. Willis1, Martin Erlandson2, David A. Theilmann1 and Cam Donly3

1 Pacific Agri-Food Research Centre, AAFC, Summerland, BC, Canada
2 Saskatoon Research Centre, AAFC-Saskatoon, SK, Canada
3 Southern Crop Protection and Food Research Centre, AAFC, London, ON, Canada

Correspondence
Cam Donly
donlyc{at}agr.gc.ca


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
A second genotype of Mamestra configurata nucleopolyhedrovirus-A (MacoNPV-A), variant 90/4 (v90/4), was identified due to its altered restriction endonuclease profile and reduced virulence for the host insect, M. configurata, relative to the archetypal genotype, MacoNPV-A variant 90/2 (v90/2). To investigate the genetic differences between these two variants, the genome of v90/4 was sequenced completely. The MacoNPV-A v90/4 genome is 153 656 bp in size, 1404 bp smaller than the v90/2 genome. Sequence alignment showed that there was 99·5 % nucleotide sequence identity between the genomes of v90/4 and v90/2. However, the v90/4 genome has 521 point mutations and numerous deletions and insertions when compared to the genome of v90/2. Gene content and organization in the genome of v90/4 is identical to that in v90/2, except for an additional bro gene that is found in the v90/2 genome. The region between hr1 and orf31 shows the greatest divergence between the two genomes. This region contains three bro genes, which are among the most variable baculovirus genes. These results, together with other published data, suggest that bro genes may influence baculovirus genome diversity and may be involved in recombination between baculovirus genomes. Many ambiguous residues found in the v90/4 sequence also reveal the presence of 214 sequence polymorphisms. Sequence analysis of cloned HindIII fragments of the original MacoNPV field isolate that the 90/4 variant was derived from indicates that v90/4 is an authentic variant and may represent approximately 25 % of the genotypes in the field isolate. These results provide evidence of extensive sequence variation among the individual genomes comprising a natural baculovirus outbreak in a continuous host population.

The GenBank/EMBL/DDBJ accession number for the sequence reported in this paper is AF539999.

Figures showing mutations in the promoter regions of lef-7 (orf16) and orf25, an alignment of the LEF-9 C-terminal amino acid sequences of v90/4 and v90/2 with those of 13 lepidopteran baculoviruses and an alignment of the 5'-end sequences of bro-b between v90/4 and v90/2 are available as supplementary material in JGV Online.

{dagger}Present address: Animal Disease Research Institute, 3851 Fallowfield Rd, Ottawa, ON, Canada, K2H 8P9.

{ddagger}Present address: Department of Medicine/Division of Geographic Medicine, University of Alabama at Birmingham, BBRB 203, 845 South 19th Street, Birmingham, AL 35294-2170, USA.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Baculoviruses are pathogenic for arthropods, mainly insects of the Lepidoptera, Hymenoptera and Diptera. These viruses have been investigated because of their potential as biological control agents of agricultural and forest pests. Baculoviruses contain circular, double-stranded DNA genomes of 80–180 kb. To date, the genomes of 26 nucleopolyhedroviruses (NPVs) have been sequenced completely.

The bertha armyworm, Mamestra configurata, is an important pest of cruciferous oilseed crops in western Canada, from which a number of NPVs have been isolated from field populations. In exploring the potential of these viruses for control of M. configurata and other pest insects, the viral isolates have been characterized with respect to their virulence in M. configurata, as well as their genomic restriction endonuclease (REN) profiles (Erlandson, 1990). Analysis of M. configurata NPV (MacoNPV) isolates has revealed significant diversity in their biological properties and genetics. Recently, we reported the complete genome analysis of two MacoNPV species (Li et al., 2002a, b). These two viruses are closely related, but have evolved divergently into two separate baculovirus species, designated MacoNPV-A and MacoNPV-B.

Very little is known about the genetic diversity of baculoviruses in field populations. Restriction fragment length polymorphisms have been reported for many species, suggesting that some level of natural genome variation is common among baculoviruses (Croizier & Ribeiro, 1992; Garcia-Maruniak et al., 1996; Hodgson et al., 2001; McIntosh et al., 1987; Muñoz et al., 1999). In addition, it has recently been reported that field isolates of the archetypal baculovirus Autographa californica multiple NPV (AcMNPV) contain additional genes to those in the previously reported genome sequence (Lu et al., 1996; Schetter et al., 1990; Yanase et al., 2000). These observations suggest that baculovirus genomes are quite dynamic and that this variability may provide selective or evolutionary advantages to the virus population. In this study, we describe the sequence of a second MacoNPV-A genome, variant 90/4 (v90/4), which was initially identified due to REN profile and biological differences in comparison to the archetypal MacoNPV-A variant, 90/2 (v90/2). This is the first study to perform a complete comparative analysis of two genomes of the same species from the same virus outbreak in a wild insect population. The genomes of v90/4 and v90/2 reveal that significant sequence variation exists between genotypes within the same virus species.


   METHODS
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INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Insects and viruses.
Larvae from a laboratory culture of M. configurata were maintained on a semi-synthetic diet (Bucher & Bracken, 1976) at 21 °C, 60 % relative humidity and an 18 : 6 light : dark photoperiod.

The v90/4 isolate was derived from a single larval cadaver that was collected near Lamont, Alberta, Canada (53° 50' N 112° 38' W), in 1990. The Lamont virus isolate was amplified by infection in vivo. Initial REN analysis indicated that this isolate contained a heterogeneous mixture of several genotypes. Haemolymph was collected from fourth-instar bertha armyworm larvae 4 days after being infected with the Lamont isolate and processed for infection of insect cell culture. Briefly, haemolymph samples were collected into 1·5 ml centrifuge tubes containing 0·5 ml Grace's tissue-culture medium (Gibco-BRL) on ice. Haemocytes were pelleted by low-speed centrifugation (1400 g for 5 min) and the supernatant was transferred to 0·45 µm SPIN-X centrifuge tube filters (COSTAR) and centrifuged for 1 min in a benchtop centrifuge (Eppendorf 5415 C). The filtrate was then used to infect Mamestra brassicae cells (IZD-MB-0503, ATCC CRL 8003) in plaque assays. A series of 10 plaques was selected and replaqued a second time. Because the production of virus progeny was not very efficient in this cell line (approx. 105 TCID50 units ml–1), plaque isolates were amplified in M. configurata larvae for further study. A single plaque isolate, v90/4, was chosen for sequencing.

The v90/2 isolate was derived from a single larval cadaver that was collected near Wilkie, Saskatchewan, Canada (52° 30' N 108° 41' W), in 1990. It was amplified by infection in vivo and cloned by using an in vivo isolation technique, as described by Smith & Crook (1988). REN analysis of the virus isolate did not reveal the presence of submolar fragments (Li et al., 1997) and, in subsequent analysis of the genome sequence data, very few (<50) nucleotide polymorphisms were detected, indicating that the isolate was genetically homogeneous.

Stocks of MacoNPV were produced by infection of fourth-instar bertha armyworm larvae by contamination of the diet with 1·4x104 PIB per cm2 of diet surface. Virus production and polyhedral inclusion body (PIB) isolation, virion purification and viral DNA extraction essentially followed previously described methods (Erlandson, 1990; Li et al., 1997).

REN analysis of viral DNA.
REN analysis of viral DNA was performed as described previously (Li et al., 1997). Briefly, purified DNA of MacoNPV-A v90/4 and v90/2 was digested with HindIII at 37 °C for 3 h, then separated on 0·7 % agarose gels in 0·5x TBE (45 mM Tris/borate, 1 mM EDTA) at 20–40 V for 15–22 h. Gels were stained with ethidium bromide and photographed.

Bioassays.
Bioassays were carried out with neonate bertha armyworm larvae by using a droplet-feeding bioassay with five virus doses and 100 larvae per dose, as described previously (Erlandson, 1990). Those larvae consuming virus inoculum during a 30 min exposure period were included in the assay and were transferred to an artificial diet and incubated at 21 °C, with fresh diet added as needed for the duration of the bioassay. Mortality was tabulated daily and mortality response data were analysed on the basis of mortality on day 14 post-infection. LD50 estimates were determined by using SAS-PROBIT (version 8, SAS Institute).

DNA sequencing and sequence analysis.
The MacoNPV-A (v90/4) genome was sequenced by using a shotgun approach, as described previously (Li et al., 2002a). In total, 1929 sequencing runs of 500–600 readable bases were assembled into 15 contigs by using Sequencher 4.0 software (Gene Codes Corporation). PCR was performed to synthesize DNA fragments bridging the gaps between contigs by using MacoNPV-A (v90/4) genomic DNA as template. PCR products were sequenced from both ends. The sequences were assembled with the initial contigs into a single, circular contig. Sequences were analysed with Wisconsin Genetics Computer Group programs (Devereux et al., 1984), GeneWorks 2.3 (IntelliGenetics) and MacVector 7.1 (Accelrys). Homology searches were carried out with GenBank/EMBL, SWISSPROT and PIR databases by using the BLAST algorithm (Altschul & Lipman, 1990). Multiple sequence alignments were performed by using CLUSTAL W (Thompson et al., 1994). MacoNPV genome sequence accession numbers are AF539999 for MacoNPV-A (v90/4), AF467808 for MacoNPV-A (v90/2) and AY126275 for MacoNPV-B.

Sequence analysis of HindIII fragments cloned from field-isolated virus.
Occluded virus of the non-plaque-purified MacoNPV Lamont field isolate, from which MacoNPV-A v90/4 was derived, was purified from infected M. configurata larvae (as described above). Occluded virus DNA was digested with HindIII, separated on 0·7 % agarose gels and selected HindIII fragments were purified from gel slices (QIAquick Gel Extraction kit; Qiagen) and cloned in vector pUC18. Eight clones for each HindIII fragment, 3051–4547 and 67365–70629, were sequenced. The DNA sequences were aligned with respect to MacoNPV-A v90/2 and v90/4 genome sequences (LaserGene, Seqman) and compared.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
LD50 and REN profile of MacoNPV-A (v90/4)
The v90/4 virus, along with a number of other MacoNPV isolates, including v90/2, was originally collected from NPV epizootics in an outbreak population of M. configurata throughout western Canada in 1990. Preliminary screening of these isolates by using REN profile analysis showed that v90/4 was very similar to v90/2, but contained some restriction fragment length polymorphisms, as shown in Fig. 1. Bioassays with v90/4 and v90/2 in neonate M. configurata demonstrated that v90/4 was less virulent, as its LD50 value was 128·4 (95 % confidence interval, 96–171) PIB per larva, tenfold higher than that of v90/2 at 11·9 (95 % confidence interval, 8·6–15·6) PIB per larva.



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Fig. 1. REN profiles of MacoNPV-A v90/4 and v90/2 isolates. DNA was isolated as described in Methods and 1 µg DNA was digested with HindIII and separated through a 0·7 % agarose gel in 0·5x TBE buffer. Names of isolates are given above each lane. M, Marker lane containing 1 kb Plus DNA ladder (Invitrogen), with the sizes of DNA fragments indicated to the left of the panel. The major differences between the two MacoNPV-A lanes, including two fragments (6602 and 4442 bp) unique to v90/2 and three fragments (5309, 2923 and 1527 bp) unique to v90/4, are marked by arrows.

 
Genome sequence comparison of v90/4 and v90/2
The genome of v90/4 is 153 656 bp, 1404 bp smaller than that of v90/2. A complete sequence alignment showed that 99·5 % of the v90/4 genome sequence is identical to that of v90/2. As shown in Fig. 2, there are 521 nucleotide changes in aligned regions when the genomes of v90/4 and v90/2 are compared. In addition, relative to v90/2, the v90/4 genome has 31 deletions and 14 insertions, comprising 1527 and 123 bp, respectively.



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Fig. 2. Comparison of genome structure between MacoNPV-A v90/4 and v90/2. The figure depicts a schematic representation of the MacoNPV-A genome, with map positions of the 169 ORFs of MacoNPV-A v90/2 (Li et al., 2002b) represented by arrows indicating transcriptional direction and relative size. Numbers above arrows represent the number of each ORF (Li et al., 2002b). Red vertical lines represent the location of point mutations in the v90/4 genome; dark blue bars (lowered) represent deletions and light blue bars (raised) represent insertions in v90/4, compared to v90/2. Yellow arrows represent ORFs with changes in amino acid sequences. Green arrows represent ORFs with identical amino acid sequences. hr sequences and their positions on the genome are indicated by empty boxes. Numbers below arrows represent the genome position relative to base 1.

 
Analysis of all variations showed that 398 point mutations, six insertions totalling 30 bp and 20 deletions totalling 1326 bp occur in predicted ORFs; 65 point mutations, three insertions (13 bp) and five deletions (102 bp) occur in intergenic regions; and 58 point mutations, two insertions (79 bp) and six deletions (98 bp) occur in homologous repeated (hr) regions. Only 27 % of the mutations cause amino acid sequence substitutions.

Although point mutations, insertions and deletions are dispersed throughout the genome, specific regions have a significantly higher density of changes (Fig. 2). The most variable region is located between hr1 and orf31 (bro-c) (v90/4, 15·0–27·1 kb; v90/2, 15·0–28·3 kb). In this 12·1 kb region (7·7 % of the genome), there are 261 of the 521 point mutations, accounting for 50 % of the total nucleotide changes. Of the 261 point mutations, 82 cause non-synonymous changes and only the chitinase and orf27 genes do not have any amino acid changes. This region also contains multiple deletions in the v90/4 genome. The largest deletion is a 1165 bp fragment that contains orf21 (bro-a) and a portion of orf20. An alignment of the v90/4 and v90/2 sequences around the junction regions of the 1165 bp deletion is shown in Fig. 3. The 1165 bp fragment contains a palindromic sequence (TCTAATTAGA) at its 5' end and another palindromic sequence (AAATATTT) at the 3' end.



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Fig. 3. Comparison of the sequences in the 1165 bp deletion/insertion region between MacoNPV-A v90/4 and v90/2. The v90/2 1165 bp fragment, missing in v90/4, contains a single bro-a gene (shown as the grey arrow) and a portion of orf20. At the ends of the 1165 bp fragment, there are two different palindromic sequences, TCTAATTAGA and AAATATTT (underlined). The identical sequences between v90/4 and v90/2 are linked by vertical lines, whilst nucleotides missing in either v90/4 or v90/2 are represented by dashes.

 
In total, 417 ORFs of 150 bp or longer, starting with an ATG, were detected in the v90/4 genome. Of these, 168 have minimal overlap with adjacent ORFs or hr regions, or showed significant homology to genes in GenBank. Gene content and arrangement are almost identical between v90/4 and v90/2 (Fig. 2). However, there is a single gene difference between the two viruses. As indicated above, bro-a is absent in v90/4. Of the 168 common ORFs, 49 ORFs show 1–12 % amino acid sequence variation and 12 ORFs vary in size (see Table 1). Among the 63 ORFs that are common to all lepidopteran baculoviruses (Chen et al., 2002; Li et al., 2002a), eight ORFs have amino acid substitutions in their encoded products. These include me53, lef-1, tlp-20, lef-8, lef-9, orf80, odv-e66 (orf144) and ie-1. Five of 12 ORFs that are unique to v90/2 and v90/4, orf5, orf10, orf18, orf23 and orf64 (Li et al., 2002b), have amino acid sequence substitutions in their putative gene products.


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Table 1. Amino acid sequence variations between v90/2 and v90/4

-, Deletion of amino acid residues; (+), conserved mutations as defined by BLAST terms.

 
Alterations in the regulation of gene expression can have significant effects on gene function; therefore, the promoter regions of all genes were analysed for variations in known regulatory motifs. Table 2 lists the nucleotide variations between v90/2 and v90/4 that occur in promoter regions located within 150 bp of an ORF. The promoter motifs in these regions are also presented. For example, the T to A substitution at –45 upstream of orf16 (lef-7) forms a TATA box in this region in v90/4 that is not found in v90/2 (see Supplementary Fig. S1, available in JGV Online). orf25 in v90/2 contains an early gene motif (–49-TATAAA, –21-CAGT); in v90/4, there is a C to T substitution at –22 that mutates the potential transcriptional start site, CAGT, into TAGT (see Supplementary Fig. S1, available in JGV Online). Pullen & Friesen (1995) showed that mutation of this base in the AcMNPV ie-1 promoter reduced gene expression dramatically.


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Table 2. Changes in promoter regions between v90/4 and v90/2

 
The hr sequences have been shown in numerous studies to be important as origins of DNA replication and as transcriptional enhancers (reviewed by Friesen, 1997; Lu et al., 1997). Comparing v90/2 and v90/4, hr1, hr3 and hr4 all show sequence variation but, interestingly, no changes were observed in hr2 (Fig. 2). Relative to v90/2, the v90/4 hr3 has a deletion of 78 bp and hr4 has an insertion of 76 bp, both of which represent a single hr repeat unit. This is similar to what was observed in hr elements of field-isolated variants of Spodoptera exigua multiple NPV (SeMNPV) (Muñoz et al., 1999). An alignment of hr4 sequences of v90/4 and v90/2 shows the significant changes that can occur in the hr elements (Fig. 4). The v90/4 hr4, in addition to the 76 bp insertion, also contains five deletions (totalling 20 bp) and 33 point mutations. The hr1 and hr3 elements have eight and 17 point mutations, respectively, relative to v90/2.



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Fig. 4. Nucleotide alignment of the hr4 sequences of v90/4 (nt 134258–135356) and v90/2 (nt 135725–136764), showing multiple point mutations and insertion/deletions. The large 76 bp insertion in v90/4 hr4 represents a single repeat unit. Individual repeat units are separated by slashes. Identical sequences between v90/4 and v90/2 are linked by vertical lines, whilst nucleotides missing in either v90/4 or v90/2 are represented by dashes.

 
Sequence polymorphism in the MacoNPV-A genome
In the process of assembling the v90/4 sequence from shotgun clones, clear alternative base readings or polymorphisms were occasionally observed for specific positions. After ruling out sequencing errors by close examination of all related electropherograms, 214 sequence polymorphisms were detected. As shown in Table 3, the majority (186) of the polymorphisms occurred in ORF regions, but they only caused 47 amino acid polymorphisms in 26 ORFs. Among the ORFs that contain polymorphisms, 14 are homologous to known genes. These include the potential structural protein genes gp37, gp41, 91k, vef and odv-e66 and the viral DNA replication- and transcription-associated genes lef-8 and ae, as well as homologues of fgf, cg30, hoar, bjdp, bro-d and bro-e.


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Table 3. Sequence polymorphisms in the MacoNPV-A (v90/4) genome

int, Intergenic (not including hr elements); :, nucleotide deletion; -, deletion of an amino acid residue; *, stop codon. Numbers between two letters in the amino acid variation columns indicate the positions where the amino acid polymorphisms occurred in the predicted gene products.

 
The MacoNPV field isolate from which v90/4 is derived was determined to be heterogeneous, based on REN analysis showing submolar ratios of some REN fragments. In an attempt to determine whether v90/4 is representative of the genotypes within the heterogeneous field isolate, selected HindIII fragments were cloned and sequenced. The sequences of HindIII fragment 3051–4547 clones that were taken directly from the field isolate fell into two groups. Two clones had sequences identical to that of MacoNPV-A v90/4 and the remaining six clones were of a genotype that differed from both v90/4 and v90/2 (Fig. 5). Similarly, two of eight HindIII fragment 67365–70629 clones were identical to MacoNPV-A v90/4 sequence, with the remaining six clones representing two additional genotypes (5 : 1). The sequence data indicate that MacoNPV-A v90/4 is an authentic genotype that was found in the heterogeneous mixture of genotypes in the original MacoNPV field isolate. MacoNPV-A v90/4 appears to represent approximately 25 % of the mixed genotype population, as measured by cloned fragment pools.



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Fig. 5. Sequence comparison of clones from the MacoNPV Lamont field isolate. The HindIII fragment representing nt 3051–4547 was isolated and cloned from genomic DNA extracted directly from the Lamont field isolate. Eight separate clones were sequenced and compared to the sequence of the plaque-purified MacoNPV v90/4 and the archetypal sequence of MacoNPV v90/2. The eight Lamont clone sequences were of two types, two clones having sequences identical to that of MacoNPV-A v90/4 (Lamont type 2 in the comparison) and six clones were of a genotype differing from both v90/4 and v90/2 (Lamont type 1 in the comparison). Green shading, no identity to either v90/4 or v90/2; red shading, identity only to v90/4; blue shading, identity only to v90/2.

 
Variations in structural protein genes
Seven genes encoding known structural proteins contain variations in amino acid sequences between v90/4 and v90/2 or have amino acid sequence polymorphisms in v90/4. Among these genes is the viral enhancing factor gene (vef). VEF is a metalloprotease that is known to enhance viral infectivity and is present in the viral occlusion bodies of granuloviruses and a few NPVs. MacoNPV-A VEF is 847 aa in size and has been shown to enhance infection of AcMNPV (Li et al., 2003). There are two amino acid substitutions in the putative VEF protein of v90/4 relative to v90/2, both occurring in the C-terminal region. At aa 758 and 779, v90/2 has an asparagine and a threonine, whereas v90/4 has an aspartic acid and an alanine, respectively (Table 1). In addition, v90/4 VEF has three amino acid polymorphisms, an asparagine to aspartic acid and two leucine to phenylalanine polymorphisms, at aa 536, 800 and 804, respectively (Table 3). As these variations occur outside the known functional domain region of VEF, it is unknown whether these amino acid residues are important for activity.

Two additional genes encoding structural proteins, p87/vp80 (orf82) and odv-e66 (orf144), have amino acid sequence substitutions in v90/4 relative to v90/2 (Table 1). P87/VP80 has a single substitution at aa 327, an alanine in v90/2 and serine in v90/4. In v90/4, ORF144 (ODV-E66) has a single substitution at aa 36, where a lysine is replaced by an asparagine, and a leucine to isoleucine polymorphism at aa 405. ORF78 is a second ODV-E66 homologue. It contains a threonine to isoleucine polymorphism and an alanine to serine polymorphism at aa 407 and 451, respectively. It is notable that all of the substitutions and polymorphisms appear at the C termini of the ODV-E66 proteins and do not occur in any of the predicted transmembrane domains or the nuclear-targeting signal (Hong et al., 1994).

In addition to VEF and ODV-E66, amino acid sequence polymorphisms also occur in the putative products of gp37, gp41 and 91k in v90/4 (Table 3), but none of the amino acid changes occur in regions that have known or predicted function. The 91k gene contains a polymorphism that causes an insertion of a single serine residue at aa 660/661 in one of the two putative translation products. The gp41 gene, which encodes an ODV-specific protein, contains a C to T polymorphism, changing a glutamine codon to a stop codon. This will result in two different proteins potentially being produced, one that is 333 aa in size and is identical to that of v90/2 and a second that is 146 aa, with the C-terminal sequence truncated. If translated, the smaller protein may have altered or antagonistic functions relative to the full-length GP41. Olszewski & Miller (1997) showed that a single base mutation in the C terminus of gp41 was responsible for a temperature-sensitive mutation that inactivated GP41 in AcMNPV-infected cells. Loss of functional GP41 resulted in the inhibition of virus production.

Variations in DNA replication and transcription regulatory genes
Among the v90/4 homologues of viral genes that are involved in DNA replication and transcription, ie-1, lef-1, lef-7, lef-8 and lef-9 contain sequence variations relative to v90/2 [Table 1, Supplementary Fig. S2 (available in JGV Online)]. In transient assays, AcMNPV IE-1 and LEF-1 are required for viral DNA replication (Kool et al., 1994), whereas LEF-7, LEF-8 and LEF-9 are required for late gene expression (Lu & Miller, 1995). LEF-1 has been characterized as a primase (Mikhailov & Rohrmann, 2002). AcMNPV LEF-8 and LEF-9 are subunits of the viral RNA polymerase II complex (Guarino et al., 1998). v90/4 LEF-9 has three amino acid substitutions relative to v90/2 LEF-9: tyrosine to histidine, isoleucine to methionine and glycine to valine at aa 301, 341 and 467, respectively (Table 1). LEF-8 has a single serine to asparagine substitution between v90/2 and v90/4 at aa 545, which is in a less conserved region of this protein. In addition, v90/4 LEF-8 has an arginine to cysteine polymorphism and an asparagine to threonine polymorphism at aa 364 and 632, respectively. LEF-1 and LEF-7 have single amino acid sequence substitutions, from arginine to lysine and aspartic acid to asparagine, at aa 77 and 45, respectively, between v90/2 and v90/4 (Table 1). In view of the importance of these genes in viral DNA replication and/or transcription, these changes could potentially affect virus replication. Kamita & Maeda (1997) reported that mutation of two adjacent nucleotides in the AcMNPV helicase gene, which causes a single amino acid change, resulted in host-range expansion (Argaud et al., 1998; Kamita & Maeda, 1997).

In v90/4 IE-1, a single aspartic acid residue is inserted at aa 26 compared to v90/2, which is within the acidic activation domain. The insertion increases the acidic nature of this domain, possibly increasing the transactivation potential of this region. In addition, AE, an exo-alkaline nuclease that is hypothesized to be involved in the processing of DNA replication intermediates (Li & Rohrmann, 2000), has a glutamine to histidine polymorphism at aa 273 in v90/4.

The me53 and cg30 genes are putative transcription regulatory genes, but their actual function during the baculovirus life cycle has yet to be determined. Homologues of me53 are conserved in all lepidopteran baculoviruses that have been sequenced to date. Both ME53 and CG30 contain RING finger and leucine zipper domains that are found in other polypeptides known to be involved in gene regulation (Knebel-Mörsdorf et al., 1993; Thiem & Miller, 1989). The ME53 homologue in v90/4 has a single alanine to threonine substitution at aa 288 relative to v90/2, which is within the conserved RING finger domain region. However, the amino acid at this site is divergent among baculoviruses, so it is unlikely that this will alter the function of the RING finger. The v90/4 CG30 has three amino acid polymorphisms: aspartic acid to asparagine, glutamic acid to aspartic acid and serine to glycine at aa 147, 154 and 158, respectively (see Table 3).

bro gene variations
There are seven and eight bro genes in the v90/4 and v90/2 genomes, respectively. As described above, bro-a is the sole ORF that exists in v90/2 but is missing in v90/4. v90/2 BRO-A shows low sequence identity to other BROs in the v90/2 and v90/4 genomes, with 27 % identity to v90/2 BRO-C being the highest. Other proteins related to v90/2 BRO-A are Spodoptera litura NPV (SpltNPV) BRO-B (27 %), Xestia c-nigrum granulovirus (XecnGV) BRO-A (25 %), XecnGV BRO-F (24 %), Helicoverpa armigera single NPV (HearSNPV) BRO-C (25 %), Bombyx mori NPV (BmNPV) BRO-C (23 %) and BmNPV BRO-B (23 %). As shown in Fig. 2, bro-a, bro-b and bro-c are located in the most highly variable region of the MacoNPV-A genome. In addition to missing bro-a, v90/4 bro-b and bro-c have 73 and 55 point mutations, respectively (see Supplementary Fig. S3, available in JGV Online). The nucleotide point mutations in these two bro genes account for 25 % of the point mutations in the whole genome.

All of the bro genes except for bro-d and bro-f contain mutations that cause amino acid substitutions (Table 1). BRO-B has 41 aa deleted between aa 65 and 105, as well as 32 amino acid substitutions; BRO-C has 13 amino acid substitutions; and BRO-E, -G and -H each have a single amino acid substitution. Polymorphisms also cause amino acid variation in BRO-E and -F (Table 3). These variation levels suggest strongly that bro genes, especially bro-a, -b and -c, are hot spots for MacoNPV-A mutations and genome variation.

Additional ORFs with amino acid sequence mutations or polymorphisms
The other known genes whose encoded protein sequences vary between v90/4 and v90/2 or have polymorphisms in v90/4 include homologues of fgf, p94, lsxe, pkip, tlp-20, sod, hoar and bjdp (Tables 1 and 2). Homologues exist in all of the sequenced lepidopteran baculovirus genomes for the predicted product of AcMNPV fgf, which is similar to fibroblast growth factors (Ayres et al., 1994). It was proposed that the expression of baculovirus FGF might facilitate the infection of tracheal cells, which serve as conduits for establishment of systemic AcMNPV infection (reviewed by Hayakawa et al., 2000). The v90/4 FGF contains five amino acid polymorphisms (Table 3).

AcMNPV p94 was hypothesized to be associated with the triggering of apoptosis induced by viral infection (Clem et al., 1994). The v90/4 P94 homologue has five amino acid substitutions relative to v90/2; of note is a methionine residue substituted for a conserved isoleucine residue at aa 492 (Table 1).

In addition to the above ORFs, 28 other ORFs that have not been characterized or have no predicted function have mutations resulting in amino acid substitutions in v90/4 relative to v90/2 (Tables 1 and 2). The most variable protein observed was ORF30, which shows 12 % sequence variation. This ORF, previously described as a unique MacoNPV ORF (Li et al., 2002b), is homologous to SpltNPV ORF106 (31 % amino acid sequence identity).


   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The MacoNPV-A viruses v90/4 and v90/2 were originally isolated from M. configurata larvae in relatively close geographical proximity during the same outbreak of this pest. The complete genome sequence of v90/4, which was found to be at least ten times less virulent for this host, has been determined and compared to that of the previously sequenced v90/2 genome. The results showed that the genome of v90/4 had 99·5 % sequence identity to that of v90/2 and nearly identical gene content and arrangement. Surprisingly, however, 49 ORFs were identified that contained nucleotide point mutations, insertions or deletions resulting in amino acid substitutions, as well as one ORF that was present in v90/2, but not in v90/4. These genetic variations underlie the biological differences between these isolates. This is the first study to determine the extent of sequence variability between baculovirus genomes isolated from the same naturally occurring field population.

Variability was not distributed evenly throughout the viral genomes, as the sequence data show that the region with the most mutations in v90/4 relative to v90/2 is located between hr1 and orf31 (bro-c) (Fig. 2). This region contains three genes, orf18, orf23 and orf30, that are unique to MacoNPV-A and it does not contain any genes that are conserved in all baculoviruses sequenced to date. Interestingly, we have shown previously that this region is also the most variable region between MacoNPV-A and the closely related species MacoNPV-B (Li et al., 2002a). Even when compared with more distantly related baculoviruses, this region appears to be more variable. For example, the gene arrangement of SeMNPV is highly collinear to that of v90/2; however, comparison of the two genomes shows that the relocation and inversion of a cluster of ORFs, as well as various insertions and deletions, have occurred in this highly variable region (Li et al., 2002b). In addition, an SeMNPV mutant with a single deletion of 25 kb encompassing orf15orf41, a region homologous to MacoNPV-A orf16orf54, can be isolated routinely in cell culture (Heldens et al., 1996). The SeMNPV deletion mutant did not cause host larval mortality or morbidity, suggesting that the 25 kb deletion contains information that is critical for virus virulence in vivo. MacoNPV homologues of SeMNPV genes in this region that vary between v90/4 and v90/2 include lef-7, he65, orf34 and orf40. In addition, lef-7, chitinase, orf25, orf50 and orf54 contain variations in their upstream promoter regions that could potentially affect gene expression.

The largest single difference between v90/4 and v90/2 is the deletion of the bro-a gene. The bro genes are a family of ORFs with sequence homology to AcMNPV orf2 that have been identified in a number of baculoviruses. Lymantria dispar multiple NPV (LdMNPV) has 16 bro-related genes (Kuzio et al., 1999). AcMNPV, Epiphyas postvittana NPV, Orgyia pseudotsugata NPV, BmNPV, HearSNPV, Helicoverpa zea single NPV (HzSNPV), SpltNPV, Culex nigripalpus NPV, Cydia pomonella granulovirus and XecnGV have between one and seven bro genes (Afonso et al., 2001; Ahrens et al., 1997; Ayres et al., 1994; Chen et al., 2001, 2002; Gomi et al., 1999; Hayakawa et al., 1999; Hyink et al., 2002; Luque et al., 2001; Pang et al., 2001). SeMNPV was previously reported to lack a bro gene, but the SeMNPV ORF13 was recently reported to be a BRO homologue of MacoNPV-A bro-g (IJkel et al., 1999; Li et al., 2002b). Only Plutella xylostella granulovirus has been found to lack a bro gene homologue (Hashimoto et al., 2000).

The BmNPV bro genes are transcribed as delayed-early genes. Functional studies suggested that BmNPV bro-a, -c and -d are essential for viral infection, but bro-a and bro-c could complement each other functionally (Kang et al., 1999). BmNPV BRO-A, -C and -D have nucleic acid-binding activities and are located in nucleoprotein complexes in the nuclei of infected cells. It has been proposed that BRO-A and -C may influence host DNA replication and/or transcription (Zemskov et al., 2000). The BRO N domain, proposed to be a DNA-binding domain, has recently been shown to be homologous to a family of proteins from other viruses, bacterial phages and bacteria (Iyer et al., 2002). Based on the apparently essential nature of some of the BmNPV bro genes, it is possible that the virulence difference that we observed between v90/4 and v90/2 may be due to the presence or absence of bro-a.

AcMNPV (C6) was originally reported as having only a single bro gene (Ayres et al., 1994). However, another bro gene was later identified in four AcMNPV variants isolated from Galleria mellonella, S. exigua, S. litura and X. c-nigrum (Yanase et al., 2000). In the variant isolated from S. litura, the second bro gene is contained within a 1·1 kb insert between AcMNPV orf30 and orf31. We have also previously reported that bro genes were found to be associated with a region of the MacoNPV-B genome (orf 53orf 58) that was possibly derived by recombination with a distantly related virus, XecnGV (Li et al., 2002a). In this study with MacoNPV-A, three bro genes (-a, -b and -c) were found to be located in the most highly variable region of the genome, with bro-b and bro-c containing 25 % of all point mutations in the v90/4 genome relative to that of v90/2. Alignments of the homologous bro genes of MacoNPV-A v90/2 and MacoNPV-B showed an average of 81 % sequence identity, which is much lower than the overall 87·6 % sequence identity between the two genomes (Li et al., 2002a). A high degree of sequence variability has also been observed among the bro genes of other viruses. The sequences of HearSNPV and HzSNPV have recently been reported and they have been shown to be variants of the same virus species. Interestingly, the most divergent ORFs between these two viruses are two bro genes (Chen et al., 2002). In BmNPV, among the five bro genes, bro-d is related most closely to AcMNPV orf2, with 80 % amino acid sequence identity. This is much lower than the average identity level of predicted proteins from these two viruses, which is 93 % (Gomi et al., 1999). These data indicate that bro genes are highly variable relative to other baculovirus genes. Furthermore, where multiple bro genes exist within a baculovirus, they are usually highly divergent amongst themselves, as has been shown in MacoNPV-A v90/2 and LdMNPV (Kuzio et al., 1999; Li et al., 2002b). It is likely that the highly variable bro genes are functionally different genes. Overall, the results of this study and those described above suggest strongly that bro genes are associated with baculovirus genome variation, but the reasons for this association are yet to be determined.

Analysis of the v90/4 genome identified 214 nucleotide polymorphisms. Sequence polymorphisms are common in genomes of humans, viruses and other organisms and contribute to important phenotypic diversities. This is the first paper to report all polymorphisms for an entire baculovirus genome. The v90/4 viral DNA used for sequencing was from a plaque-purified clone that was amplified in larvae of M. configurata. The presence of a pool of polymorphisms may be a more efficient mechanism than direct substitution, insertion or deletion in adapting to a changeable environment and may provide an evolutionary advantage by having a population of variable genomes.

The results of this study describe the extensive sequence variability that exists in natural baculovirus populations. The sequence does not provide direct answers as to why v90/4 is less virulent than v90/2, but suggests that the bro genes are potentially involved and may play a significant role in generating baculovirus diversity. In addition, the data indicate that natural baculovirus populations represent a spectrum of genotypes that may have significant ecological implications for the survival and adaptation of these viruses in the face of potentially dynamic changes in host species subpopulations, changing climatic conditions and/or availability of alternative host species.


   ACKNOWLEDGEMENTS
 
The technical assistance of Alison Paton and Keith Moore (AAFC, Saskatoon Research Centre) for insect rearing and MacoNPV bioassays, respectively, is greatly acknowledged. The support of Dr Jim Brandle (DNA Sequencing Laboratory), AAFC, Southern Crop Protection and Food Research Centre (London, ON, Canada) and the excellent technical contributions of LouAnn Verellen are gratefully acknowledged.


   REFERENCES
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Afonso, C. L., Tulman, E. R., Lu, Z., Balinsky, C. A., Moser, B. A., Becnel, J. J., Rock, D. L. & Kutish, G. F. (2001). Genome sequence of a baculovirus pathogenic for Culex nigripalpus. J Virol 75, 11157–11165.[Abstract/Free Full Text]

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]

Altschul, S. F. & Lipman, D. J. (1990). Protein database searches for multiple alignments. Proc Natl Acad Sci U S A 87, 5509–5513.[Abstract]

Argaud, O., Croizier, L., Lopez-Ferber, M. & Croizier, G. (1998). Two key mutations in the host-range specificity domain of the p143 gene of Autographa californica nucleopolyhedrovirus are required to kill Bombyx mori larvae. J Gen Virol 79, 931–935.[Abstract]

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]

Bucher, G. E. & Bracken, G. K. (1976). The bertha armyworm, Mamestra configurata (Lepidoptera: Noctuidae). Artificial diet and rearing technique. Can Entomol 108, 1327–1338.

Chen, X., IJkel, W. F. J., Tarchini, R. & 8 other authors (2001). The sequence of the Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus genome. J Gen Virol 82, 241–257.[Abstract/Free Full Text]

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.[Abstract/Free Full Text]

Clem, R. J., Robson, M. & Miller, L. K. (1994). Influence of infection route on the infectivity of baculovirus mutants lacking the apoptosis-inhibiting gene p35 and the adjacent gene p94. J Virol 68, 6759–6762.[Abstract]

Croizier, G. & Ribeiro, H. C. T. (1992). Recombination as a possible major cause of genetic heterogeneity in Anticarsia gemmatalis nuclear polyhedrosis virus wild populations. Virus Res 26, 183–196.[CrossRef]

Devereux, J., Haeberli, P. & Smithies, O. (1984). A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res 12, 387–395.[Abstract]

Erlandson, M. A. (1990). Biological and biochemical comparison of Mamestra configurata and Mamestra brassicae nuclear polyhedrosis virus isolates pathogenic for the bertha armyworm, Mamestra configurata (Lepidoptera: Noctuidae). J Invertebr Pathol 56, 47–56.

Friesen, P. (1997). Regulation of baculovirus early gene expression. In The Baculoviruses, pp. 141–170. Edited by L. K. Miller. New York: Plenum.

Garcia-Maruniak, A., Pavan, O. H. O. & Maruniak, J. E. (1996). A variable region of Anticarsia gemmatalis nuclear polyhedrosis virus contains tandemly repeated DNA sequences. Virus Res 41, 123–132.[CrossRef][Medline]

Gomi, S., Majima, K. & Maeda, S. (1999). Sequence analysis of the genome of Bombyx mori nucleopolyhedrovirus. J Gen Virol 80, 1323–1337.[Abstract]

Guarino, L. A., Xu, B., Jin, J. & Dong, W. (1998). A virus-encoded RNA polymerase purified from baculovirus-infected cells. J Virol 72, 7985–7991.[Abstract/Free Full Text]

Hashimoto, Y., Hayakawa, T., Ueno, Y., Fujita, T., Sano, Y. & Matsumoto, T. (2000). Sequence analysis of the Plutella xylostella granulovirus genome. Virology 275, 358–372.[CrossRef][Medline]

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]

Hayakawa, T., Rohrmann, G. F. & Hashimoto, Y. (2000). Patterns of genome organization and content in lepidopteran baculoviruses. Virology 278, 1–12.[CrossRef][Medline]

Heldens, J. G. M., van Strien, E. A., Feldmann, A. M., Kulcsár, P., Munoz, D., Leisy, D. J., Zuidema, D., Goldbach, R. W. & Vlak, J. M. (1996). Spodoptera exigua multicapsid nucleopolyhedrovirus deletion mutants generated in cell culture lack virulence in vivo. J Gen Virol 77, 3127–3134.[Abstract]

Hodgson, D. J., Vanbergen, A. J., Watt, A. D., Hails, R. S. & Cory, J. S. (2001). Phenotypic variation between naturally co-existing genotypes of a lepidopteran baculovirus. Evol Ecol Res 3, 687–701.

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]

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.[Abstract/Free Full Text]

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.[Abstract/Free Full Text]

Iyer, L. M., Koonin, E. V. & Aravind, L. (2002). Extensive domain shuffling in transcription regulators of DNA viruses and implications for the origin of fungal APSES transcription factors. Genome Biol 3, RESEARCH0012 (http://genomebiology.com/2002/3/3/research/0012.1).

Kamita, S. G. & Maeda, S. (1997). Sequencing of the putative DNA helicase-encoding gene of the Bombyx mori nuclear polyhedrosis virus and fine-mapping of a region involved in host range expansion. Gene 190, 173–179.[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.[Abstract/Free Full Text]

Knebel-Mörsdorf, D., Kremer, A. & Jahnel, F. (1993). Baculovirus gene ME53, which contains a putative zinc finger motif, is one of the major early-transcribed genes. J Virol 67, 753–758.[Abstract]

Kool, M., Ahrens, C. H., Goldbach, R. W., Rohrmann, G. F. & Vlak, J. M. (1994). Identification of genes involved in DNA replication of the Autographa californica baculovirus. Proc Natl Acad Sci U S A 91, 11212–11216.[Abstract/Free Full Text]

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]

Li, L. & Rohrmann, G. F. (2000). Characterization of a baculovirus alkaline nuclease. J Virol 74, 6401–6407.[Abstract/Free Full Text]

Li, S., Erlandson, M., Moody, D. & Gillott, C. (1997). A physical map of the Mamestra configurata nucleopolyhedrovirus genome and sequence analysis of the polyhedrin gene. J Gen Virol 78, 265–271.[Abstract]

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.[Abstract/Free Full Text]

Lu, A. & Miller, L. K. (1995). The roles of eighteen baculovirus late expression factor genes in transcription and DNA replication. J Virol 69, 975–982.[Abstract]

Lu, A., Craig, A., Casselman, R. & Carstens, E. B. (1996). Nucleotide sequence, insertional mutagenesis, and transcriptional mapping of a conserved region of the baculovirus Autographa californica nuclear polyhedrosis virus (map unit 64·8-66·9). Can J Microbiol 42, 1267–1273.[Medline]

Lu, A., Krell, P. J., Vlak, J. M. & Rohrmann, G. F. (1997). Baculovirus DNA replication. In The Baculoviruses, pp. 171–191. Edited by L. K. Miller. New York: Plenum.

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.[Abstract/Free Full Text]

McIntosh, A. H., Rice, W. C. & Ignoffo, C. M. (1987). Genotypic variants in wild-type populations of baculoviruses. In Biotechnology in Invertebrate Pathology and Cell Culture. Edited by K. Maramorosch. San Diego: Academic Press.

Mikhailov, V. S. & Rohrmann, G. F. (2002). Baculovirus replication factor LEF-1 is a DNA primase. J Virol 76, 2287–2297.[Abstract/Free Full Text]

Muñoz, D., Murillo, R., Krell, P. J., Vlak, J. M. & Caballero, P. (1999). Four genotypic variants of a Spodoptera exigua nucleopolyhedrovirus (Se-SP2) are distinguishable by a hypervariable genomic region. Virus Res 59, 61–74.[CrossRef][Medline]

Olszewski, J. & Miller, L. K. (1997). A role for baculovirus GP41 in budded virus production. Virology 233, 292–301.[CrossRef][Medline]

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]

Pullen, S. S. & Friesen, P. D. (1995). The CAGT motif functions as an initiator element during early transcription of the baculovirus transregulator ie-1. J Virol 69, 3575–3583.[Abstract]

Schetter, C., Oellig, C. & Doerfler, W. (1990). An insertion of insect cell DNA in the 81-map-unit segment of Autographa californica nuclear polyhedrosis virus DNA. J Virol 64, 1844–1850.[Medline]

Smith, I. R. L. & Crook, N. E. (1988). In vivo isolation of baculovirus genotypes. Virology 166, 240–244.[CrossRef][Medline]

Thiem, S. M. & Miller, L. K. (1989). Identification, sequence, and transcriptional mapping of the major capsid protein gene of the baculovirus Autographa californica nuclear polyhedrosis virus. J Virol 63, 2008–2018.[Medline]

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.[Abstract]

Yanase, T., Hashimoto, Y. & Kawarabata, T. (2000). Identification of insertion and deletion genes in Autographa californica nucleopolyhedrovirus variants isolated from Galleria mellonella, Spodoptera exigua, Spodoptera litura and Xestia c-nigrum. Virus Genes 21, 167–177.[CrossRef][Medline]

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.[Abstract/Free Full Text]

Received 30 July 2004; accepted 20 September 2004.