Laboratory of Virology, Wageningen University, Binnenhaven 11, 6709 PD Wageningen, The Netherlands1
Joint Lab of Invertebrate Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei 430071, Peoples Republic of China2
Dupont Agricultural Products, StineHaskell Research Center, E.I. du Pont de Nemours and Co., PO Box 30, Newark, DE 19714-0030, USA3
Author for correspondence: Just Vlak. Fax +31 317 484820. e-mail just.vlak{at}medew.viro.wau.nl
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Abstract |
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Introduction |
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Genome sequence analysis has recently begun to reveal the distinctive features of baculoviruses and the extent of their diversity. At present, the nucleotide sequences have been determined of six 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
), Spodoptera exigua (Se) MNPV (IJkel et al., 1999
) and Spodoptera litura MNPV (Pang et al., 2001
), one SNPV, Helicoverpa armigera (Ha) SNPV (Chen et al., 2001
), and three granuloviruses, Xestia c-nigrum (Xc) GV (Hayakawa et al., 1999
), Plutella xylostella (Px) GV (Hashimoto et al., 2000
) and Cydia pomonella GV (Luque et al., 2001
). The genome sizes range from 100999 bp for PxGV to 178733 bp for XcGV. Sixty-three ORFs are conserved in all baculovirus genomes sequenced to date (Chen et al., 2001
; Hayakawa et al., 2000
; Herniou et al., 2001
).
A number of NPVs have been isolated worldwide from insect species belonging to the genus Helicoverpa (Lepidoptera: Noctuidae), which includes agricultural pest insects such as Helicoverpa zea, Helicoverpa virescens, H. armigera and Helicoverpa punctigera (Gettig & McCarthy, 1982 ). These species are major global pests that attack at least 30 different food and fibre crops and are, in many instances, resistant to chemical insecticides (Ignoffo, 1973
; Cunningham, 1998
). These Helicoverpa sp. NPVs can be divided morphologically into two groups, MNPVs and SNPVs (Blissard et al., 2000
). HzSNPV was registered as one of the first commercial baculovirus pesticides (Virion-H, Biocontrol-VHZ, Elcar) in the 1970s and has been used extensively to control the cotton bollworm in the USA (Shieh, 1989
; Cunningham, 1998
). HaSNPV, isolated in 1975 in Hubei province, Peoples Republic of China, has been used successfully in China for over 20 years to control H. armigera in cotton and vegetable crops in an area of about 100000 hectares (Zhang, 1994
).
Previous studies have indicated that HzSNPV and HaSNPV are more closely related to each other than to Helicoverpa sp. MNPVs (Getting & McCarthy, 1982 ; Sun et al., 1998
). The restriction enzyme patterns and even the physical maps of HzSNPV and HaSNPV are quite similar (Knell & Summers, 1984
; Chen et al., 2000
). A few genes present in these two viruses, notably polyhedrin (Chen et al., 1997b
), ecdysteroid UDP glucosyltransferase (egt) (Chen et al., 1997a
), hoar (Le et al., 1997
) and DNA polymerase (Bulach et al., 1999
), have a very high degree of amino acid identity. Recently, sequence analysis of the HaSNPV genome has indicated that known HaSNPV ORFs have over 97% amino acid identity on average to reported partial HzSNPV sequences in databases (Chen et al., 2001
). This suggests that the overall homology between HaSNPV and HzSNPV is probably very high and that these two viruses may be variants of the same virus species. This is then in line with the observation that these two viruses share the same heliothine host range, although they differ in biological activity against individual heliothine species (Hughes et al., 1983
). Sequencing of the HzSNPV genome would therefore substantiate these hypotheses further.
In this paper, we describe the complete nucleotide sequence and organization of the HzSNPV genome and make a comparison with the HaSNPV genome sequence, published recently (Chen et al., 2001 ). These two baculoviruses appeared to be almost identical, most ORFs sharing over 97% amino acid identity. The major difference occurs in the sequence and organization of the homologous repeat (hr) regions. Furthermore, HzSNPV and HaSNPV are characterized by the presence of different baculovirus repeat ORF (bro) genes. Genomic comparison of HzSNPV and HaSNPV gives full support to the view that these two viruses are variants of the same virus species. As such, this is the first report of a full sequence comparison of two variants of the same baculovirus.
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Methods |
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A plaque-purified isolate (F16) of HzSNPV strain Elcar (Ignoffo, 1965 ) was obtained from the USDA at Brownsville, TX, USA, and polyhedra were propagated in H. virescens larvae. Polyhedra were isolated from infected H. virescens larvae by sucrose-gradient centrifugation (OReilly et al., 1994
).
HzSNPV DNA isolation, cloning and sequence determination.
HzSNPV genomic DNA was isolated from purified polyhedra by solubilization in an alkaline solution and viral DNA was prepared as described in OReilly et al. (1994) . Size-selected viral DNA (24 kb) was cloned into the SmaI site of M13mp18 (Novagen) following nebulization of 50 µg HzSNPV genomic DNA in a Nebulizer 4207 (Inhalation Plastics Inc.). Fragments were treated with T4 DNA polymerase and the large fragment of T4 DNA polymerase (Klenow) according to the manufacturers protocol to repair the ends prior to cloning. Ligation products were transformed into E. coli JM101 cells and ssDNA for sequencing was prepared from over 2000 clones by using standard protocols (Sambrook et al., 1989
).
Sequencing reactions were 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 Model 377 automated DNA sequencer. The combined sequence generated from these clones represented 6-fold genomic coverage. Additional sequences for confirmation of ambiguous regions and for filling in of gaps in the assembled sequence were obtained from sequencing of PCR products by using customized oligonucleotide primers.
DNA sequence analysis.
Assembly of the complete sequence of the HzSNPV genome was accomplished as described previously (Chen et al., 2001 ). ORFs encoding more than 50 amino acids (150 bp) were considered to be protein-encoding and hence designated putative genes. One hundred and thirty-five ORFs (out of 139) were checked for maximal alignment with known baculovirus gene homologues extracted from GenBank. 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 by computational analysis as described previously (Chen et al., 2001
).
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Results and Discussion |
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Forty-five of the 139 HzSNPV ORFs (32%) exhibited 100% identity in amino acid sequence to the corresponding HaSNPV ORFs, of which 21 (15% of 139 ORFs) were even identical in nucleotide sequence (Table 2). Sixty-eight HzSNPV ORFs had amino acids replaced in the corresponding HaSNPV ORFs, 28 of which had only a single amino acid change. Due to deletion/insertion or to stop codon changes, a total of 25 HzSNPV ORFs (15·8%) exhibited lower identity or had different sizes in HaSNPV (Table 2
).
bro genes
The most divergent ORFs in HzSNPV relative to HaSNPV are the bro genes, bro-a (Hz60) and bro-b (Hz61) (Fig. 2). bro-a and bro-b had relatively low degrees of amino acid identity (39 and 71%) to HaSNPV bro-a (Ha59) and bro-b (Ha60), respectively. Hz-bro-a is 33 amino acids smaller than Ha-bro-a, whereas Ha-bro-b has an N-terminal extension of 183 amino acids compared with Hz-bro-b (Fig. 2
). These additional 183 amino acids of Ha-bro-b have significant identity to Ha-bro-a (43·9%), Hz-bro-a (48%), Hz-bro-b (59%) and the C-terminal portion of Ha-bro-b (52%). Hz-bro-c (Hz108) has a very high degree of identity to Ha105 (99%).
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Unique genes
So far, only four ORFs (Hz26, Hz42, Hz62 and Hz79) are unique to HzSNPV. All are quite small, with sizes ranging from 50 to 68 amino acids. Hz26 and Hz79 are located downstream of homologous repeat regions (hr1 and hr3) and overlap the respective downstream ORFs, Hz27 (67 nt) and Hz80 (34 nt). The HaSNPV genome did not contain Hz26 and Hz62 homologues, due respectively to three insertions with a total of 120 nucleotides and a 27 bp insertion in the corresponding regions of HaSNPV. No Hz79 (58 aa) homologue was found in HaSNPV due to a 17 bp deletion. The small ORFs (Hz26, Hz62 and Hz79) are probably not functional, but this has to be tested experimentally, e.g. by deletion mutagenesis. The most notable unique ORF is Hz42 (68 aa), which is located between the chitinase gene (Hz41) and Hz43 in the HzSNPV genome. The corresponding region (total 334 bp), including the entire ORF (Hz42) and the up-/downstream sequence, is not present in HaSNPV. Hz42 has a baculovirus late promoter motif, TAAG (-34 to -30 nt), and an early conserved motif, AATCGTGT (-134 to -128 nt), upstream of the start codon ATG. It is possible that Hz42 might have a unique function for HzSNPV.
Homologous repeat regions (hrs)
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 serve as putative origins of DNA replication (Kool et al., 1995
) and as enhancers of transcription (Guarino & Summers, 1986
; Guarino et al., 1986
). In AcMNPV, hrs are characterized by the presence of multiple, often imperfect, tandemly repeated palindromic sequences. The HzSNPV genome contains five hrs and they have the same genomic location and organization as the HaSNPV hrs. The latter are unique compared with other baculovirus hrs as they include two types of repeat, type A and type B, respectively containing imperfect 40 and 107 bp repeats, or truncated versions thereof (Chen et al., 2001
). The type A and B repeats are also found in each of the hrs in HzSNPV.
Sequence alignment between HzSNPV and HaSNPV hrs indicated that these homologous regions have a significantly lower nucleotide identity (95%), and that numerous insertions/deletions have occurred (Table 3). The most divergent region is hr1, with 92% sequence identity, which also contains more than 10 insertions/deletions. The biggest deletion in HzSNPV hr1 compared with HaSNPV is a 271 bp segment that contains both type A and type B repeats, while a 171 bp insertion contains only a type B repeat. The other deletions/insertions, ranging from 1 to 59 bp, did not contain, and in most cases were too short to contain, an entire repeat. This situation also occurred in the other hrs. hr2, with the lowest identity of 90·5%, contains a few deletions/insertions of different sizes. No major deletion/insertion was found in hr3, the smallest hr, but the sequence identity is only 94·4%. As indicated previously (Chen et al., 2001
), hr2 and hr3 might originally have been a single hr that was split by two bro genes. The hr2hr3 region therefore stands out as the most divergent region in the Hz/HaSNPV genome (Fig. 2
). hr4 has 95% sequence identity and contains a single 132 bp deletion, with other small deletions/insertions ranging from 3 to 12 bp. The most stable hr is hr5, with 98·6% sequence identity and a single 290 bp deletion that contains type A and type B repeats.
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Sequence divergence between HzSNPV and HaSNPV
Sequence divergence between homologous genes and the corresponding intergenic regions from HzSNPV and HaSNPV has been investigated in detail and a summary is presented in Table 4 and Figs 1
and 3
. The hypervariable hr regions and bro-a and bro-b mentioned earlier are not included in this analysis.
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The mean frequency of nucleotide substitution in the coding regions of HzSNPV/HaSNPV is 6·8 . For individual genes, the substitution frequency ranges from 0 (21 ORFs mentioned earlier) to 45·8
for Hz40. Seven nucleotide substitutions, causing five amino acid differences in a 150 bp stretch of coding sequence, were found in this ORF, which is so far unique to HaSNPV and HzSNPV. On average, the identity of sequences of proteins involved in DNA replication and regulation of gene expression and that of the structural proteins is 99·6%, while for auxiliary proteins it is 99·4%, which is higher than the mean identity of all ORFs (98·3%). The 63 proteins conserved among all baculoviruses (Herniou et al., 2001
) have lower divergence in these two related Helicoverpa baculoviruses.
The extent of synonymous substitution also varied among ORFs, which is indicated by the different frequencies of synonymous and non-synonymous divergence in HzSNPV genes (Fig. 3). The extents of amino acid and nucleotide sequence divergence were correlated, but the relationship between them is interesting. In genes encoding highly conserved proteins, for example the 63 conserved baculovirus ORFs, amino acid sequence identity exceeds nucleotide identity, apparently because only silent (synonymous) substitutions are permitted in these genes. However, in less conserved genes or ORFs, nucleotide similarity exceeds amino acid similarity. This presumably reflects the degeneracy of the genetic code: for example, a single codon with nucleotide substitutions at positions 2 and 3 (the former causing an amino acid replacement, the latter probably silent) exhibits 33% nucleotide sequence identity but 0% amino acid sequence identity. This picture is the same as that observed in rodent genes (363 genes compared between mouse and rat; Wolf & Sharp, 1993
), but markedly different from that seen in a study of bacterial sequence divergence (67 genes compared between Escherichia coli and Salmonella typhimurium; Sharp, 1991
). From the bacterial data, protein sequences are invariably more similar than nucleotide sequences over a range of 76 to 100% amino acid sequence identity. This difference is probably related to a much higher mean ratio of synonymous to non-synonymous divergence in bacterial genes.
Conclusions
The complete HzSNPV genome sequence has been analysed and compared with other baculovirus genomes, especially that of HaSNPV (Chen et al., 2001 ). One hundred and thirty-nine ORFs with more than 50 amino acids and five hrs were identified. The genome of HzSNPV is very similar to that of HaSNPV, but distinct from other baculovirus genomes in both gene content and gene arrangement. Except for four unique ORFs and two bro genes, 133 HzSNPV ORFs have very high amino acid sequence identity (mean 98·9%) to the corresponding ORFs in HaSNPV. Gene translocation was not observed, but nucleotide substitutions and deletions/insertions with sizes ranging from a few base pairs to 334 bp were found. Sequence alignment of the HzSNPV and HaSNPV genomes indicated that the hrs are the most variable regions, while the central region, i.e. the region flanking the helicase gene, is the most conserved (Heldens et al., 1996). Genes common to all baculoviruses have a much lower degree of divergence than other ORFs. The two bro genes, bro-a and bro-b, might have different sources.
We conclude that HzSNPV and HaSNPV are two variants of the same virus species, on the basis of genome sequence features and other structural and biological properties, including the morphotype (SNPV), their common heliothine host and their more or less equal virulence to insect hosts (Hamm, 1982 ; Hughes et al., 1983
; Kelly et al., 1980
; Williams & Payne, 1984
; Sun et al., 1998
). H. virescens SNPV and H. punctigera SNPV might also belong to the same virus species, on the basis of their similar physical maps (Getting & McCarthy, 1982
). Different genotypic variants of HzSNPV and HaSNPV have been reported. Polymorphism in the HaSNPV isolate (G4) was at approximately 100 nucleotide locations (0·07%) (Chen et al., 2001
). This indicates that the substitution ratio between HaSNPV isolates (genotypes) is much lower compared with the HzSNPV genome. Taking into account the hypervariable hrs, the bro genes and four unique ORFs, it is clear that HzSNPV and HaSNPV are related less closely to each other than is observed among genotypes in isolates of HaSNPV. We hypothesize that HaSNPV and HzSNPV are adapted/adapting to their respective host insects, H. armigera and H. zea. The same situation probably exists with the baculovirus type species, AcMNPV, with Anagrapha falcifera NPV, Galleria mellonella NPV, Rachiplusia ou MNPV, Spodoptera exempta MNPV and Trichoplusia ni MNPV being genotypic variants that have diverged into strains with distinctive genotypic profiles. The sequence information presented in this paper calls for a revision of baculovirus nomenclature (Blissard et al., 2000
) to provide a better reflection of the genetic information.
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Acknowledgments |
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Footnotes |
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Received 17 May 2001;
accepted 15 October 2001.