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Warwick HRI, Wellesbourne, Warwick CV35 9EF, UK
Correspondence
Sally Hilton
sally.hilton{at}warwick.ac.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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The GenBank/EMBL/DDBJ accession number for the AdorNPV genome sequence reported in this paper is EU591746.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). There are two genera of baculovirus, the Nucleopolyhedrovirus (NPV) and the Granulovirus (GV) based on OB morphology (Theilmann et al., 2005). The NPVs are further classed into group I and II NPVs (Bulach et al., 1999; Zanotto et al., 1993). The majority of the sequenced NPVs infect insects of the Lepidopteran order, but NPVs that infect dipteran or hymenopteran insect species like the mosquito-infecting Culex nigripalpus NPV, or the NPVs from three Neodiprion species, have been sequenced (Afonso et al., 2001; Garcia-Maruniak et al., 2004; Lauzon et al., 2004; Duffy et al., 2006). Baculoviruses are used worldwide as biological control agents of agricultural and forest pests. The summer fruit tortrix moth (Adoxophyes orana) is a pest of apples and pears in most of Europe and Japan. During the past 25 years, there has been increased interest in the use of a naturally occurring GV as a control agent for the summer fruit tortrix (Cross et al., 1999). Recently, a mixture of baculoviruses was recovered from larvae in the UK (Hilton & Winstanley, 2008). These were separated and found to comprise an NPV and a GV. The GV (Adoxophyes orana GV) has been characterized and completely sequenced (Wormleaton et al., 2003; Hilton & Winstanley, 2008). Here, we present the sequence of the isolated single-nucleocapsid type NPV (SNPV), Adoxophyes orana NPV (AdorNPV). Previously, an SNPV from Adoxophyes honmai (AdhoNPV) in Japan, which infects the tea tree tortrix, has been characterized and sequenced (Ishii et al., 2003; Nakai et al., 2003). Using an LD90 dose, AdhoNPV is slow-killing, with host larvae including A. orana dying in the final (fifth) instar (Ishii et al., 2003). Here, we show AdorNPV to be fast-killing with A. orana larvae dying predominantly in the first and second instars. To determine the genetic basis behind the diversity in speed of kill of these different NPVs, we sequenced the genome of AdorNPV and compared it to the previously published AdhoNPV sequence and other sequenced NPVs. The fast-killing AdorNPV may provide an improved biocontrol agent, either alone or in combination with AdorGV or AdhoNPV. |
METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). The original virus also contained a GV, which was separated and removed by using glycerol and sucrose gradients following methods described previously (Crook & Payne, 1980). A cloned genotype of AdorNPV was obtained through three successive rounds of in vivo cloning, using the limiting dilution method described by Smith & Crook (1988a). Absence of the GV was confirmed by PCR using AdorGV-specific primers. The virus OBs were purified using sucrose gradients and the DNA was extracted and purified following methods described previously (Smith & Crook, 1988b). A Helber 0.02 mm depth counting chamber was used to count the NPV OBs under dark-field illumination. Duplicate samples for each of two dilutions of the virus were counted and the concentration of the sample calculated.
Insects.
A laboratory stock of A. orana was reared at 25 °C with a 16 : 8 h light : dark cycle on a semi-synthetic diet as described in Hilton & Winstanley (2008).
Determination of virus-caused mortality in neonate and fifth instar larvae.
The droplet-feeding method was used to inoculate neonate larvae (Hughes & Wood, 1981; Hughes et al., 1986) and described in Hilton & Winstanley (2008). The volume ingested by neonates had previously been found to be 5.35±0.945 nl (Hilton & Winstanley, 2008). Fifty larvae were dosed at each of five virus dilutions (18, 54, 162, 486 and 1458 OBs per larva) and 50 larvae dosed with a water/phenol red solution as a control. For fifth instar larvae, 50 larvae were dosed at each of five virus dilutions (1x103, 5x103, 2.4x104, 1.25x105 and 6.25x105) and 50 larvae dosed with water as a control by using the method described in Hilton & Winstanley (2008). The larvae from all bioassays were monitored every 24 h for symptoms of virus infection, mortality and instar. All bioassays were performed in triplicate. LD50 and LD80 values were calculated from the medians of the three replicate assays and fitted using binomial regression analysis with a probit link function using GenStat 10th edition.
Calculation of median survival time (ST50).
ST50 refers to the time required for 50 % of the larvae to die post-inoculation (p.i.). LD80 doses were used to inoculate duplicate groups of 60 neonate and 60 fifth instar larvae and the mortalities were recorded daily. The doses were 144 OBs for neonate larvae and 1.44x105 OBs for fifth instar larvae. The experimental set up was such that all of the larvae received a prescribed dose within a short time span and were then maintained individually on their diet. ST50 calculations were based on the total number of larvae responding to virus infection, rather than the total number of larvae used in each test (Allaway & Payne, 1984). For each instar, the point in time at which 50 % of the total mortality response occurred was interpolated from the sigmoid-response curve (Bliss, 1937). The mean ST50 was calculated from the duplicate assays carried out for each instar.
DNA cloning and sequencing.
Shotgun library construction and sequencing was performed by Lark Technologies as follows: an aliquot of AdorNPV DNA was nebulized to produce randomly sheared fragments of DNA. The sheared DNA fragments were blunt-ended with the Klenow fragment of Escherichia coli DNA polymerase and polynucleotide kinase. The blunt-ended fragments were run on a low-melting-point agarose gel for size selection and the fragments in the size range of 1–3 kb were collected and purified. The purified DNA fragments were used to create a shotgun library by ligating the blunt-ended DNA fragments into the SmaI site of the pUC19 vector. The ligations were used to transform competent XL1-blue MRF' cells. The cells were plated on LB agar plus ampicillin containing X-Gal and IPTG. White colonies were picked into ten 96-well blocks containing LB plus ampicillin and then cultured overnight. Plasmid DNA was isolated from the shotgun library using the Eppendorf Vacuum Manifold Plasmid Preparation. DNA sequencing was performed using BigDye Terminator Cycle Sequencing Ready Reaction kit, with AmpliTaq DNA Polymerase, FS (Applied Biosystems) and the reactions were analysed on ABI 3100 or 3700 instruments.
DNA sequence analysis.
Double-stranded DNA sequences were assembled using the SeqMan II sequence analysis package (Lasergene software version 4.03; DNASTAR). The coding regions were predicted using the package GeneQuest II (DNASTAR) by locating translation start and stop codons of open reading frames (ORFs) of 50 or more amino acids. Database searches using the PSI-BLAST program were used to identify proteins sharing similarity. Percentage pairwise identities were calculated using the GAP program of Wisconsin Package version 10.0, Genetics Computer Group (GCG), Madison, Wisconsin (Devereux et al., 1984), with default settings. Multiple alignments were produced as in Wormleaton & Winstanley (2001).
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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. LDs increased, as expected, as the instar increased. The LD50 for AdorNPV in neonates (56 OBs) was similar to that of AdorGV (31 OBs) (Hilton & Winstanley, 2008). Whereas, the LD50 for AdorNPV in fifth instar (2.3x104 OBs) was nearly two orders of magnitude lower than that for AdorGV (1.4x106 OBs) (Hilton & Winstanley, 2008). Neonate larvae had an ST50 of 8.8 days using an LD80 dose (144 OBs). However, it was apparent during the dose-response assays that neonate larvae infected with a high dose (
1458 OBs) died faster, predominantly as neonates, and larvae infected with a low dose (
162 OBs) died more slowly, predominantly as second instars (Fig. 1). Fifth instar larvae had an ST50 of 7.0 days using an LD80 dose (1.44x105 OBs). It was interesting to note that most of the infected fifth instar larvae (62.0 %), which died earlier (at 5 and 6 days p.i.), died as larvae–pupae intermediates, as if pupation had been attempted. After 6 days p.i., only 3.0 % of larvae died as larvae–pupae intermediates. Larvae infected with AdorGV also showed this pathology upon death (Hilton & Winstanley, 2008).
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, Supplementary Table S1 available in JGV Online) and they were numbered from polyhedrin in a clockwise direction. The AdorNPV ORFs had minimal intergenic distances and no preferred orientation or clustering according to expression or function.
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). This observation has been confirmed in other annotated, AT-rich, viral genomes such as AdorGV (Wormleaton et al., 2003).
Whereas most AdorNPV ORFs had an AT composition (average 65 %) very close to the average AT composition of the AdorNPV genome (65 %), polyhedrin and p6.9 had an AT composition that was significantly lower at 52 and 45 %, respectively. This was also found to be the case in the AdorGV genome where it was noted that for some proteins, such as polyhedrin/granulin, it might be impossible for the virus to maintain its preferred nucleotide composition and codon usage and still encode a particular peptide (Wormleaton et al., 2003). AdhoNPV also displays the same high AT composition and purine (AG) bias. Therefore, three baculoviruses that infect A. orana all have a high AT content and purine bias, which may suggest that the tRNA pool is limited in the host.
Promoter analysis
Regions 120 bp upstream of putative ORFs were screened for promoter elements. Early promoters consisting of a TATA-box motif with a cap-site CAKT 25–35 bp downstream were found in 14 ORFs compared with 17 ORFs in AdhoNPV (Supplementary Table S1). Only eight genes shared by AdorNPV and AdhoNPV contained early promoters. A baculovirus late promoter element (DTAAG) was found in 70 ORFs compared with 75 in AdhoNPV. In total, 76 ORFs were identified as having some type of early or late promoter elements in AdorNPV. Of these 76 ORFs, eight had both early and late promoter elements. The upstream 120 bp of 45 ORFs did not yield a match to the promoter elements that we searched for. Of these 45 genes, several were homologues of previously characterized, expressed baculovirus proteins. For example, AdorNPV ie-1, a known immediate-early gene in other baculoviruses, had no recognizable promoter element. This was also the case for AdhoNPV ie-1 (Nakai et al., 2003). It is possible that the AdorNPV and AdhoNPV ie-1 gene, is transcribed as a spliced gene from a distal promoter element. The promoter elements for AdorNPV and AdhoNPV ORFs are shown in Supplementary Table S1. The most significant differences are the lack of a late promoter for p74 in AdhoNPV and the lack of a late promoter for lef-10 and pkip-1 in AdorNPV. All other baculovirus homologues of these genes contain a late promoter.
Comparison of AdorNPV gene content with that of other baculoviruses
The 62 genes conserved in other lepidopteran baculoviruses (Jehle et al., 2006) were present in AdorNPV. Fifty-six additional ORFs had homologues in the AdhoNPV genome. The average amino acid sequence identity between AdorNPV and AdhoNPV, Mamestra configurata NPV-A (MacoNPV-A) (Q.J. Li et al., 2002), Spodoptera exigua MNPV (SeMNPV) (IJkel et al., 1999), Lymantria dispar MNPV (LdMNPV) (Kuzio et al., 1999) and Autographa californica MNPV (AcMNPV) (Ayres et al., 1994) were 93.9, 45.4, 45.3, 44.2 and 41.8 %, respectively. The most conserved ORFs are polyhedrin/granulin (ORF1), ubiquitin (ORF16), sod (ORF101), lef-9 (ORF33) and lef-8 (ORF46) (Supplementary Table S1). Three ORFS were unique to AdorNPV (ORF15, ORF82 and ORF111).
AdorNPV structural genes
In line with all other sequenced group II NPVs (and GVs), AdorNPV does not encode the envelope glycoprotein gp64, the major envelope fusion protein of AcMNPV, Bombyx mori MNPV (BmNPV) (Gomi et al., 1999), Orgyia pseudotsugata (Ahrens et al., 1997) and Epiphyas postvittana NPV (EppoNPV) (Monsma et al., 1996; Hyink et al., 2002). This protein appears to be unique to group I NPVs (Pearson et al., 2000; IJkel et al., 2000). In LdMNPV, the functionally analogous envelope fusion protein is the product of the Ld130 gene, now called the F protein. AdorNPV encodes an F protein homologue, ORF114, which shows 48 % aa identity to Ld130. Ld130 homologues are present in all lepidopteran and dipteran baculoviruses that have been completely sequenced, including those that contain gp64. The role of the Ld130 homologue in the latter species is unclear, but it is likely they have lost their envelope fusion properties (Pearson et al., 2000, 2001).
AdorNPV does not contain a protein tyrosine phosphatase gene (ptp). It also lacks a gp37 (spindle-like protein), which apart from AdhoNPV is found in all lepidopteran NPVs. All other structural proteins listed by Hayakawa et al. (1999) are present.
Genes involved in DNA replication and transcription
There are 19 lef genes in AcMNPV that have been implicated in DNA replication and transcription (Rapp et al., 1998). Early baculovirus genes are transcribed by the host cell RNA polymerase II, but these are often transactivated by genes such as ie-0, ie-1, ie-2 and pe38 (Friesen, 1997). Of these genes, ie-0 and ie-1 are present in AdorNPV. Both ie-2 and pe38 appear to be group I NPV-specific genes. Six genes are reported to be essential for baculovirus DNA replication: lef-1, lef-2, lef-3, dnapol, helicase and ie-1 (Lu et al., 1997). Homologues of all are present in AdorNPV. They are moderately well conserved, with the exception of lef-3 and ie-1 (Supplementary Table S1). AdorNPV does not have a lef-7 homologue, which appears to be a group I NPV-specific gene found to stimulate transient DNA replication in AcMNPV and BmNPV (Morris et al., 1994; Gomi et al., 1997).
AdorNPV lacks genes for enzymic functions in nucleotide metabolism, such as the large (rr1) and small (rr2) subunits of ribonucleotide reductase and deoxyuridyltriphosphate (dUTPase), which are found in several baculoviruses. These enzymes are involved in nucleotide metabolism and catalyse the reduction of host cell rNTPs to dNTPs (Lange & Jehle, 2003). The three sequenced Adoxophyes viruses (AdorGV, AdhoNPV and AdorNPV) all lack these genes involved in nucleotide metabolism. They also all maintain a high AT content and purine bias. This may reflect the nucleotide composition of the host, as without the genes involved in nucleotide metabolism, these viruses are restricted to the nucleotide composition of their hosts.
Many genes required for late gene transcription have been described, including lef 4–6, 8–11, 39K, p47 and vlf-1 (Lu & Miller, 1997). All of these are found in AdorNPV. Generally, these genes are more conserved than the early transcription activators (IJkel et al., 1999). Phylogenetic analysis based on gene sequences of the baculovirus sequences available to date was performed using a concatenated alignment of the lef-8 and lef-9 genes. These analyses aligned AdorNPV very closely to AdhoNPV in a group II NPV clade (data not shown). The relationship between the closely related AdhoNPV and other baculoviruses is presented comprehensively in Nakai et al. (2003) by using maximum-parsimony of the combined sequences of 30 common genes, break-point distance analysis and gene content phylogenetic analysis.
Genes with auxiliary functions
Auxiliary genes are genes that are not essential for viral replication, but provide it with some selective advantage (O'Reilly, 1997). The AdorNPV genome does not contain a chitinase gene but does contain a cathepsin gene (ORF49). Chitinase and cathepsin genes have been identified in many other baculoviruses that have been completely sequenced to date. It appears that baculoviruses encode these enzymes to aid the breakdown of insect tissues at the end of infection, releasing OBs into the environment to aid their horizontal spread. Deletion of either the cathepsin or chitinase genes resulted in the failure of AcMNPV to cause liquefaction of the host (Slack et al., 1995; Hawtin et al., 1997). This indicated that these proteins function together to promote degradation of the host tissues at the end of the infection process (Hawtin et al., 1997). AdorNPV-infected larvae do not liquefy at the end of infection, presumably due to the absence of chitinase, and the cuticle retains its integrity despite melanization occurring.
AdorNPV contains a truncated ecdysteroid UDP-glucosyltransferase (EGT) protein compared with AdhoNPV and other baculoviruses (Fig. 3). The AdorNPV EGT protein is 363 aa compared with 512 aa in AdhoNPV. EGT is an enzyme that catalyses the conjugation of ecdysteroids with sugar, which prevents metamorphosis of the host (O'Reilly, 1995). Ten conserved regions have been identified among EGT proteins (Hu et al., 1997). AdorNPV EGT lacks domains 7–10 due to the truncation of the protein. There are seven conserved amino acids for all UDP-glucosyltransferases (Hu et al., 1997). Only one of these seven is present in the AdorNPV EGT, which strongly suggests that the AdorNPV is a dysfunctional EGT. Larval–larval moulting is usually prevented by infection with a baculovirus expressing a functional EGT, which prolongs the period the infected larvae feed, increasing the yield of progeny virus during infection (O'Reilly et al., 1998). However, deletion or inactivation of the egt gene often reduces ST of NPV-infected larvae by a significant extent, possibly due to stresses involved in the moult of an infected larva or by the infection of the malpighian tubules (Chen et al., 2000; Eldridge et al., 1992; Flipsen et al., 1995; O'Reilly & Miller, 1991; Slavicek et al., 1999; Treacy et al., 1997). The interaction regarding EGT in Adoxophyes viruses and hosts is unusual. AdorGV contains a functional EGT protein (Wormleaton & Winstanley, 2001), yet A. orana larvae infected as neonates continue to moult until final instar, which is extended by on average of 25 days. In AdhoNPV-infected A. honmai larvae there is a similar scenario except the fifth instar is extended by just a few days (Ishii et al., 2003). In AdorNPV-infected A. orana the situation is reversed; the EGT is most likely dysfunctional, yet larvae die in the first or second instar. Although it is unclear how EGT is functioning in these larval–host interactions, it is likely that the lack of a functional EGT in AdorNPV is responsible for the increased speed of kill of AdorNPV compared with AdhoNPV.
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Inhibitors of apoptosis
Baculoviruses possess two families of genes that suppress apoptosis, the P35/P49 family and the inhibitor of apoptosis (IAP) family. The IAP-3 protein of CpGV was the first member of the baculovirus IAP family of proteins to be identified (Crook et al., 1993). It has been shown to block apoptosis in diverse systems and to substitute for the p35 gene in blocking AcMNPV-induced apoptosis in Sf21 cells (Crook et al., 1993). p35 genes have only been identified in AcMNPV, BmNPV, Spodoptera litura MNPV and Maruca vitrata MNPV (Ayres et al., 1994; Gomi et al., 1999; Pang et al., 2001). Whereas, all baculoviruses have been found to contain IAP homologues. IAP homologues generally contain two baculovirus IAP repeats (BIR), which are associated with binding to apoptosis-inducing proteins, and a C-terminal zinc finger-like (RING) Cys/His motif (Crook et al., 1993; Birnbaum et al., 1994; Vucic et al., 1997). These features have enabled the iap genes to be divided into five groups, iap-1 to iap-5 (Luque et al., 2001) Two members of the IAP gene family were observed in AdorNPV, iap-2 (ORF 50) and iap-3 (ORF 85). AdhoNPV contains a further iap gene, iap-4. However, this gene does not contain any BIRs. The significance of the extra iap gene in AdhoNPV relative to AdorNPV is unclear.
Baculovirus repeated ORFs (bro genes)
Bro genes are present, between 1 and 16 copies, in all lepidopteran and dipteran NPVs sequenced to date and in some of the GVs. They comprise a highly repetitive and conserved gene family, which is widespread among insect DNA viruses (Bideshi et al., 2003). The function of these genes is unclear but they have been shown to bind to DNA (Zemskov et al., 2000). These genes have also been found to be associated with regions of viral genome rearrangement (Li et al., 2002a, 2005). AdorNPV contains three bro genes and are named bro-a to bro-c based on their order in the genome (Supplementary Table S1, Fig. 2
). Most BROs contain a core sequence of 41 aa at the N-terminal half and several different domains throughout the sequence. The bro gene family has been divided into four groups based on the similarity of those domains (Kuzio et al., 1999). AdorNPV bro-a (ORF32) and bro-c (ORF88) belong to group II, whereas bro-b (ORF81) belongs to group I. AdhoNPV contains an additional bro gene (Adho ORF85) next to the AdorNPV bro-a homologue, which encodes a small truncated protein (113 aa) lacking most of the 41 aa core domain but containing the conserved N-terminal region of the group I bro genes (Nakai et al., 2003).
Homologous regions (hrs)
Many baculovirus genomes have several hrs dispersed throughout the genome. An individual NPV hr typically comprises direct repeats usually centred around a palindrome. The hrs may serve as origins of replication in NPVs and GVs (Kool et al., 1995; Hilton & Winstanley, 2007) and as enhancers of transcription in NPVs (Guarino & Summers, 1986; Guarino et al., 1986). Although no single homologous repeat region is essential for DNA replication of AcMNPV (Carstens & Wu, 2007). A second type of replication origin, a non-hr ori, has been identified in several NPVs (Habib & Hasnain, 2000; Heldens et al., 1997; Kool et al., 1994). These complex structures are composed of multiple direct and inverted repeats within a region spanning up to 4000 bp and are present only once per genome.
The AdorNPV genome contains four small homologous repeat regions ranging from 137 to 399 bp. The first two are directly either side of the p74 gene. The p74 region appears to be an area in many baculovirus genomes where hrs are located. The hr regions are in the same relative position as the four AdhoNPV hr regions, although the AdorNPV hrs do not overlap with p74 as they do in AdhoNPV. They also consist of the same two domains but differ in their number of repeats and their orientation, with AdorNPV having generally fewer domains (Fig. 4). Domain A contains a 16 bp imperfect palindrome flanking a 16 bp sequence. This may form a stem–loop structure (Fig. 4). Domain B shares significant similarity with the SeMNPV 36 bp DR2 repeat (Broer et al., 1998). This was also found in AdhoNPV domain B (Nakai et al., 2003).
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Comparison of AdorNPV to AdhoNPV
The AdorNPV genome is 1496 bp smaller than the AdhoNPV genome and contains four fewer ORFs. AdorNPV contains three ORFs that are absent in AdhoNPV, whereas AdhoNPV contains seven ORFs absent in AdorNPV including an iap-4 gene and a bro gene as mentioned previously. AdorNPV and AdhoNPV share four ORFs not present in other baculoviruses (ORFs 7, 9, 109 and 112). These ORFs had no significant homologues identified by a BLAST search, although ORF9 did have 48 % identity with a 38 aa region from ORF43 of EppoNPV. These shared genes range in size from 238 to 307 aa in AdorNPV and all contain recognizable promoters. It is possible that these genes are host range genes involved specifically in the NPV infection of the Adoxophyes species.
There are 118 ORFs in common between AdorNPV and AdhoNPV. Several of these ORFs are of different lengths as shown in Fig. 5. The most notable being egt, also lef-12, p43, bro-c (ORF88) and lef-2 amongst other genes with unassigned functions. The gene order between the 118 shared genes of AdorNPV and AdhoNPV is identical. The hrs are in a similar position but with different numbers and orientation of repeat units. The differences in gene content, ORF length and hr composition are likely to account for the different speed of kill and pathology exhibited by AdorNPV and AdhoNPV.
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In conclusion, AdorNPV shows a high degree of collinearity and sequence similarity with AdhoNPV. However, the two viruses from different geographical locations are biologically distinct in their speed of kill and phenotype characteristics.
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ACKNOWLEDGEMENTS |
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Received 8 April 2008;
accepted 25 June 2008.
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