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1 Laboratório de Virologia Molecular, Núcleo Integrado de Biotecnologia, Universidade de Mogi das Cruzes, Mogi das Cruzes, SP, Brazil
2 Embrapa Milho e Sorgo, Sete Lagoas, MG, Brazil
3 Laboratório de Evolução Molecular e Bionformática, Departamento de Microbiologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, SP, Brazil
Correspondence
José Luiz Caldas Wolff
caldaswolff{at}pq.cnpq.br
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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The GenBank accession number of the sequence reported in this paper is EU258200.
Supplementary Tables are available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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; Blissard & Rohrmann, 1990). This family of virus is divided into two genera: Nucleopolyhedrovirus (NPV) and Granulovirus (GV) (Blissard et al., 2000). Phylogenetic analyses of baculovirus genes confirmed the division of the two genera and indicated that NPVs could possibly be separated into two groups, named group I and II (Zanotto et al., 1993). Baculoviruses have enveloped rod-shaped virions containing a circular supercoiled double-stranded DNA molecule ranging in size from 81 756 bp for Neodiprion lecontei NPV (Lauzon et al., 2004) to 178 733 bp for Xestia c-nigrun GV (Hayakawa et al., 1999). Their genomes comprise from 90 to 181 genes that are closely packaged, sometimes even partially overlapped, in both strands of the DNA molecule.
The first baculovirus to be completely sequenced was the Autographa californica NPV (AcNPV; Ayres et al., 1994). Three years later, the genome of another NPV, the Orgyia pseudotsugata NPV (OpMNPV), was published (Ahrens et al., 1997). Today, genomes from over 40 baculoviruses have been determined. Most of these genomes are from NPVs and GVs that infect Lepidoptera. However, in 2001 the genome of a NPV that infects Culex nigripalpus (Diptera) was determined (Afonso et al., 2001). More recently, genomes of NPVs that infect hymenopteran species were also sequenced (Garcia-Maruniak et al., 2004; Lauzon et al., 2004; Duffy et al., 2006). Analysis of the available genomes has shown that 29 genes are conserved in all baculoviruses sequenced so far (Herniou et al., 2003; Garcia-Maruniak et al., 2004; Lauzon et al., 2005). This group of core genes represent a relatively small part of the over 800 different orthologous gene groups found in all baculovirus genomes up to the present date (Jehle et al., 2006). The phylogenetic investigation of these genomes revealed that gene acquisition and loss are important factors in the evolution of baculoviruses (Herniou et al., 2001, 2003; Oliveira et al., 2006). Moreover, genomic analysis suggests that NPVs infecting members of distinct invertebrate Orders are sufficiently different to arguably merit classification into separate virus genera (Jehle et al., 2006).
Spodoptera frugiperda is a severe pest of corn and other crops. In Brazil, where infestations can result in losses of up to 34 % in corn yield, the control of S. frugiperda is often difficult and requires the undesirable use of repeated chemical control. For that reason, the application of alternative methods of control, such as baculoviruses, has become a priority. Isolates of S. frugiperda NPV (SfMNPV) have been identified from S. frugiperda larvae collected from various regions of the Americas (Maruniak et al., 1984; Shapiro et al., 1991; Barreto et al., 2005; Loh et al., 1981, 1982; Knell & Summers, 1981). Several studies have shown that SfMNPV isolates often display genetic and biological differences (Loh et al., 1982; Knell & Summers, 1981; Berretta et al., 1998). Recently, the analysis of 22 SfMNPV geographical isolates collected in Brazil identified an isolate, named SfMNPV-19, which had a lower LC50 (Barreto et al., 2005). This geographical isolate was used in this investigation.
Sequencing studies with SfMNPV started with the polyhedrin gene, one of the first baculovirus genes to be sequenced (Gonzalez et al., 1989). Six years later, the gp41 gene sequence was determined and its expression pattern analysed (Liu & Maruniak, 1995). In 2003, the sequence of a 5122 bp region that encompasses the gp37, ptp-2 and egt genes and three other ORFs was determined in a study that showed that SfMNPV genome expresses a functional ecdysteroid UDP-glucosyltransferase (EGT) protein (Tumilasci et al., 2003). The sequencing of this genomic region revealed that genes located in the proximity of the SfMNPV egt are highly conserved and collinear with genes from Spodoptera exigua NPV (SeMNPV) and Mamestra configurata NPV (MacoMNPV) (Tumilasci et al., 2003). In the past few years, the sequences from the p74 gene (GenBank accession no. AY174684
[GenBank]
), the immediate early gene (ie-0) (AY846365
[GenBank]
) and the pif gene (Simon et al., 2005b) have also became available. Finally, 38 ORFs located in termini of cloned restriction fragments were partially sequenced in a study with a Nicaraguan isolate (Simon et al., 2005a). The distribution of these putative genes along the genome and the level of sequence similarity observed confirmed that the SfMNPV genome is indeed highly similar to SeMNPV and MacoMNPV (Simon et al., 2005a). As we were completing the sequence of SfMNPV-19, the sequence of another SfMNPV isolate, named isolate 3AP2, was deposited in the database (GenBank accession no. EF035042
[GenBank]
). The sequence of this genome was included in our analysis. Herein we report the complete genome sequence of SfMNPV-19, an SfMNPV isolate that has good potential to be used as a biological control agent due to its high infectivity. The analysis of the genome is presented as well as a comparison with closely and distantly related members of the Baculoviridae.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). The DNA was digested with the restriction enzymes HindIII and BamHI, which did not appear to yield submolar bands.
Construction of a genomic library representing the entire genome.
Purified viral DNA was digested with the restriction enzymes BglII, EcoRV, HincII, HindIII, KpnI, PstI and SalI and digested simultaneously with BamHI and BglII. DNA fragments were cloned into pGEM-3Z (Promega) following standard procedures (Sambrook et al., 1989). A total of 66 unique clones, covering approximately 90 % of the SfMNPV-19 genome, were chosen as templates for DNA sequencing. The selection of the unique clones was done in accordance with the sequences identified on the edges of the cloned fragments. The fact that most genes in the genome of SfMNPV-19 were collinear to those in the closely related SeMNPV and Agrotis segetum NPV (AgseMNPV-A) helped to find possible gaps in the genomic library. Missing genomic segments were amplified by PCR using oligonucleotides derived from the cloned flanks of each gap.
Sequence determination and analysis.
DNA templates were sequenced initially using universal primers and oligonucleotides of the PCR reactions. The internal sequences were determined by primer-walking using specific primers. Sanger reactions were performed by cycle sequencing using Eppendorf thermocycler machines with the ABI PRISM Big Dye Terminator sequencing ready reaction kit v. 3 (Applied Biosystems). Electrophoreses were done on an ABI 377 DNA sequencer (Applied Biosystems). Aligner version 1.3.4 (CodonCode) was used for contig assembly using default Phrap and Phred parameters. The complete genome was checked manually in order to detect possible errors. Alternative oligonucleotides were used for resequencing regions that repeatedly displayed ambiguity and/or poor quality.
Sequence analysis and genome annotation.
The release 8 of Artemis (Rutherford et al., 2000) (The Sanger Institute; http://www.sanger.ac.uk/Software/Artemis/) and ORF Finder (http://www.ncbi.nlm.nih.gov/projects/gorf/) were used to find and analyse open reading frames of over 50 aa, except for those that were completely overlapped by other ORFs. Identity values of ORFs were obtained using BLASTP (Altschul et al., 1990), also available at the NCBI (http://www.ncbi.nlm.nih.gov/blast/). Regions of the genome that differed (presence or absence of ORFs as well as any major differences in size) from the closely related genomes were checked in detail to verify possible sequencing or assembly errors. The complete genome was compared with other baculovirus genomes using the Artemis comparative tool (ACT) release 5 (Carver et al., 2005) (The Sanger Institute; http://www.sanger.ac.uk/Software/ACT/). The Dotter program (Sonnhammer & Durbin, 1995) was used to locate homologous regions (hrs). Palindromes present in the hrs were identified using the program PALINDROME from the EMBOSS package (http://emboss.bioinformatics.nl/cgi-bin/emboss/palindrome). Annotation of the genome was done with Artemis (Rutherford et al., 2000).
Phylogenomics.
Since the reciprocal value of bit score (1/S') obtained with the TBLASTX program (Altschul et al., 1990) available in the NCBI BLAST distribution (http://www.ncbi.nlm.nih.gov/blast/download.shtml) is a direct measure of evolutionary distance (Oliveira et al., 2006), pairwise distance matrices of median values of 1/S' from complete genomes were clustered using the weighted neighbour-joining (weighbor) (Bruno et al., 2000), neighbour-joining, unweighted pair group mean average (UPGMA) and Fitsch and Kitsch methods from the PHYLIP package (Felsenstein, 1989). Pairwise distance matrices were calculated with the BlastPhen pipeline (Oliveira et al., 2006) to establish the relationship of SfMNPV-19 to 42 other sequenced baculoviruses. For each pair of genomes, median values from bit scores S' (Altschul et al., 1990; Pertsemlidis & Fondon, 2001) were calculated for all local high-score pairs (HSP), generated by the TBLASTX program.
Gene content and genomic organization.
We generated a binary gene-content matrix, expanded from Herniou et al. (2001, 2003) and Oliveira et al. (2006) to analyse the gene content of the baculoviruses. This matrix scored the presence (1) or absence (0) of 862 known baculovirus genes from 43 baculovirus genomes. The most parsimonious reconstruction (MPR) of each gene gain or loss event was estimated along the branches of a complete genome-based phenogram for the Baculoviridae obtained with the BlastPhen algorithm (Oliveira et al., 2006). We also included a synthetic outgroup (i.e. a ‘basal’ root in which all genes are absent), in order better to define gain and loss of genes among baculoviruses and other cellular and viral organisms (including prokaryotes, eukaryotes, DNA and RNA viruses) (Herniou et al., 2001, 2003). Complete genome alignments were built in order to study genome structure and conservation (i.e. synteny) among the baculoviruses. Syntenic maps for genomes from members of group II were built with the Artemis comparative tool, by plotting the high similarity scoring pairs along pairs of genomes that were obtained with TBLASTX (using default settings) using the proteins encoded in all six reading frames.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). In agreement with the convention for baculoviruses (Vlak & Smith, 1982), the polyhedrin gene was designated ORF number 1 and the putative coding regions were numbered sequentially in the clockwise orientation. The adenine of the start codon of the polyhedrin gene was assigned base number 1. Seventy-four ORFs (52.5 %) were in the clockwise direction and 67 (47.5 %) in the opposite direction (Fig. 1).
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; Garcia-Maruniak et al., 2004) were also found in the SfMNPV-19 genome. The phenogram shown in Fig. 2 summarizes the relationship of SfMNPV-19 to 42 other complete genomes of baculoviruses. The topology of the genome phenogram was congruent with trees obtained from individual genes (Zanotto et al., 1993) or trees for concatemers of the 29 baculovirus conserved genes (Garcia-Maruniak et al., 2004) and was obtained with all the clustering methods used. Nevertheless, we preferred to consider the weighbor tree, since this method allows for more precise branch length estimates (Bruno et al., 2000). The phenogram had the SfMNPV-19 and the SfMNPV-3AP2 clustering with the NPVs that infect S. exigua (SeMNPV), A. segetum (AgseMNPV-A) and, to a lesser extent, to the two NPVs that infect M. configurata (MacoNPV-A and B) (Fig. 2). Moreover, as observed in previous gene-based phylogenies (Zanotto et al., 1993; Bulach et al., 1999; Herniou et al., 2003; Garcia-Maruniak et al., 2004; Jehle et al., 2006) and genome-based phylogenies (Oliveira et al., 2006), branch lengths leading to group II NPV in Fig. 2 were longer than those for group I NPV. As a consequence, the monophyly of group II appeared to be supported by a limited set of shared traits.
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) to 44 mol% in SeMNPV (IJkel et al., 1999). The variation of the G+C content along the SfMNPV-19 genome is shown in Fig. 1
. Computer-generated cleavage patterns for HindIII and BamHI were in agreement with the restriction pattern generated in the laboratory (data not shown). Nevertheless, the analysis of 6596 bases (approx. 5 % of the total genome) whose sequence was derived from two or more different cloned fragments revealed that a total of only eight bases (0.12 %) had nucleotide polymorphism, which was within an acceptable sequencing error level (99.88 % unambiguous nucleotides) and suggested that our assembly reflects good depiction of a stable and homogeneous SfMNPV genome. Moreover, the comparison of gene content with that of closely related NPVs did not show discrepancies that could be explained by genetic diversity in the DNA source.
Homologous repeat sequences
Canonical homologous regions (hrs) are made of direct repeat sequences with imperfect palindromes. The baculovirus hrs have been implicated in important functions such as origins of DNA replication (Leisy & Rohrmann, 1993; Leisy et al., 1995; Kool et al., 1995) and transcriptional enhancement (Guarino et al., 1986; Theilmann & Stewart, 1992). A total of eight hrs were found in the SfMNPV-19 genome (Supplementary Table S1, available with the online version of the paper). The main feature of the hrs was imperfect palindromic repeats of 44 bp, which were similar to palindromic repeats found in the hrs of SeMNPV and AgseMNPV (Broer et al., 1998; Jakubowska et al., 2006). The SfMNPV-19 hrs harboured from one up to four of these imperfect palindromes. The consensus sequence of the 18 palindromes found in the hrs was: CCATGTTTGCTTTCGGCGAAGTGTTTCGCTGAAAGCAAACATCA.
A non-hr origin of replication has been identified in SeMNPV (Heldens et al., 1997). Sequences that resemble the SeMNPV non-hr origins of replication (ORI) were found in the intergenic region between ORFs 83 and 84 of SfMNPV-19.
Comparison of SfMPV-19 with other SfMNPV isolates
The first physical map of an SfMNPV isolate was done by Loh et al. (1981). A more detailed physical map was presented in 1984 in a study that analysed seven plaque-purified variants (Maruniak et al., 1984). The computer-generated digestion the SfMNPV-19 genome revealed a profile that differed from those of the previous investigations in terms of number and size of fragments generated (data not shown). Moreover, the genome of SfMNPV-19 was 10.9 kbp larger than the estimated size of the SfMNPV-2 clone (Maruniak et al., 1984). More recently, the physical and partial genetic map of a Nicaraguan SfMNPV isolate was constructed and the data obtained showed a genome of approximately 129.3 kbp (Simon et al., 2005a). Comparison with SfMNPV-19 revealed differences in terms of number and position of restriction sites along the genomes. Finally, the sequence of SfMNPV isolate 3AP2 (GenBank accession no. EF035042
[GenBank]
) became available as we were completing the genome of SfMNPV-19. Alignment of the genomic sequence from isolates SfMNPV-19 and 3AP2 with CLUSTAL W confirmed that the two viruses were variants of the same species (data not shown). Major differences were limited to regions that had deletions and insertions. The largest of these were observed around the egt gene. In the 3AP2 isolate, there was a 1427 nt deletion that removed the amino terminal end of the putative EGT protein and the carboxy terminal end of the ORF that corresponds to SfMNPV-19 ORF 26 (data not shown).
Another evident difference was observed in the putative dutpase gene. In the SfMNPV-19 genome, ORF 53 encodes a putative dUTPase protein that is 21 aa shorter than that encoded by isolate 3AP2, due to differences in the 5' end of the genes. A comparison with the putative dUTPase proteins encoded by SeMNPV and AgseMNPV-A showed that the longer version of the gene was shared by the most closely related members of the SfMNPV cluster (data not shown). It is noteworthy that the non-coding region located immediately upstream to the SfMNPV-19 dutpase gene was poorly conserved, suggestive of a mutation hot spot. Other differences were also observed in the hr5 region that was longer in isolate 3AP2 by one repeat. Moreover, these two genomes differed in a series of point mutations and short deletions or insertions that added or removed codons. Computer-generated restriction profiles for SfMNNPV isolates 19 and 3AP2 were distinct, since the former had 19 HindIII sites and the latter had only 17 (data not shown).
Comparison of SfMNPV-19 genome with those of SeMNPV and AgseNPV-A
SfMNPV-19, SeMNPV, AgseNPV-A, MacoNPV-A and MacoNPV-B formed a closely related cluster in our phylogenetic tree (Fig. 2). Within this group, SfMNPV-19, SeMNPV and AgseNPV-A were very similar in terms of gene content, genomic organization and conservation. For instance, SfMNPV-19 had 125 and 129 orthologues in the genomes of SeMNPV and AgseMNPV-A, respectively.
The average value for the identities between genes from SfMNPV-19 and SeMNPV was 67 % (SD=16 %). However, several structural proteins (polyhedrin, P45/P48, ODV-E25, ODV-EC27, GP41, ODV-E18, VP39 capsid, F protein, P74, PIF-2 and PIF-3) had identity values above 83 %. These results were in agreement with those obtained for other closely related baculoviruses (Gomi et al., 1999; Li et al., 2002; Oliveira et al., 2006). The conservation of structural proteins may indicate that functional constraints impose strong stabilizing (i.e. negative) selection on these genes. Similarly, the proteins that make the DNA-dependent RNA polymerase (LEF-4, LEF-8, LEF-9 and P47) and two proteins involved in viral DNA replication (DNA polymerase and helicase) were also highly conserved. It is also noteworthy that several non-essential proteins with auxiliary functions (cathepsin, chitinase, superoxide dismutase, EGT and 38K hydrolase) displayed levels of sequence identity above 83 %, suggesting that their genes may be under strong negative selection.
SfMNPV-19 lacked 14 ORFs present in SeMNPV (Supplementary Table S1, available with the online version of this paper). In general, the SfMNPV-19 genome had different genes in place, suggesting that SfMNPV-related viruses present considerable genomic plasticity via gene replacement. However, ORFs Se39 and 49 (Supplementary Table S1) were missing and were not replaced in SfMNPV-19. Nine of the unique SeMNPV ORFs were found in two ORF clusters, one from Se20 to 24 and another from Se83 to 86. The SfMNPV-19 putative lef-7 gene (Sf20) was homologous with two SeMNPV ORFs, Se17 and Se18 (Supplementary Table S1). One bro gene (Sf68) was identified in SfMNPV-19 (Supplementary Table S1), whereas the SeMNPV genome did not have a single bro gene (IJkel et al., 1999). Likewise, the SfMNPV-19 genome contained 24 fewer ORFs than the AgseMNPV-A genome (Supplementary Table S1). Three of these (Agse75, Agse76 and Agse128) are putative vef genes, which encode virus-enhancing factors (Jakubowska et al., 2006). The AgseNPV-A genome has four copies of the bro genes, named bro-a to bro-d (Jakubowska et al., 2006). Of these, only bro-b had a homologue in the SfMNPV-19 genome (Supplementary Table S1). The genomes of both SfMNPV-19 and SeMNPV had two copies of the odv-e66 gene (Sf56 and Sf114), as has been observed previously (Simon et al., 2005a). However, AgseMNPV-A had only one complete copy of odv-e66 (Agse125) and this gene had 49 % sequence identity to its SfMNPV-19 equivalent (Supplementary Table S1). The odv-66 gene encodes a structural protein involved in the formation of occluded virus envelope (Hong et al., 1997). Two copies of the p26 gene were found in SfMNPV-19 (Sf85 and Sf129) and in the genomes of SeMNPV and AgseMNPV-A (Supplementary Table S1).
Interestingly, the genomes of both SeMNPV and AgseMNPV-A carry two ribonucleotide reductase genes, rr1 and rr2b (IJkel et al., 1999; Jakubowska et al., 2006). The rr1 gene is usually linked to the presence of either rr2a or rr2b (Herniou et al., 2003). However, only the rr1 gene was found in SfMNPV-19 and it had 34 and 33 % identity to the rr1 genes of SeMNPV and AgseMNPV-A, respectively. This value is considerably lower than the identities observed for other closely related viruses that carry ribonucleotide reductase genes (Li et al., 2002; Jakubowska et al., 2006). For instance, the identity level for the rr1 gene of AgseMNPV-A and those of SeMNPV and MacoNPV-B are 58 and 60 %, respectively (Jakubowska et al., 2006). It is possible that the loss of one of the two copies of rr2 caused rr1 to experience elevated rates of substitution.
ORFs with no homology to baculovirus genes
Most unique genes identified in SfMNPV-19 were located in regions that also had unique genes in the other viruses of the cluster, suggesting that these regions experience increased genetic variation. A total of 11 unique ORFs were identified in the genome of SfMNPV-19 (Sf5, Sf6, Sf7, Sf10, Sf31, Sf42, Sf43, Sf46, Sf54, Sf84 and Sf95). The size of these ORFs varied considerably, ranging from 52 to 484 aa, and seven of them were larger than 100 aa (Supplementary Table S1). ORFs Sf31 (121 putative aa), Sf42 (395 putative aa), Sf43 (263 putative aa) and Sf95 (166 putative aa) had known baculovirus promoter motifs upstream from their putative initiation codon and polyadenylation signals at their 3' end (data not shown). Sf5 was 88 aa long and displayed some homology to the putative HOAR protein gene from MacoNPV-A (with a BLAST e-value of 0.059). The SfMNPV-19 hoar gene (Sf6) encoded a putative protein that was at least 130 aa shorter than the HOAR proteins from other baculoviruses. As these two ORFs were conserved in both SfMNPV-19 and the 3AP2 isolate, it seems that the hoar gene was split into two in SfMNPV. It also came to our attention that the region between the hoar and the odv-e56 (Sf08) putative genes was highly variable among the members of group II NPV. In SeMNPV, AgseMNPV-A and Trichoplusia ni SNPV (TnSNPV), the genomic region between the hoar and the odv-e56 is occupied either by unique ORFs or by poorly conserved ORFs (IJkel et al., 1999; Willis et al., 2005; Jakubowska et al., 2006).
Genomic organization, gene content analysis and evolution in group II NPVs
The tree for 43 complete genomes of baculoviruses isolated from Lepidoptera (39), Hymenoptera (3) and Diptera (1) (Fig. 2) was similar to others obtained previously for single genes (Zanotto et al., 1993), for a set of conserved orthologues (Herniou et al., 2001, 2003) and for complete genomes (Garcia-Maruniak et al., 2004; Oliveira et al., 2006). As expected, SfMNPV was placed within group II NPV (Fig. 2). The subdivisions of group II into two (Zanotto et al., 1993) or three subgroups (Bulach et al., 1999) has been proposed based on the analysis of the polyhedrin and the DNA polymerase genes. These subdivisions proved to be insufficient given the genomic information now available. The syntenic maps generated in this study showed extensive collinearity and gene content conservation between the genomes of SfMNPV, SeMNPV, AgseMNPV-A and, to a lesser extent, to both MacoNPV-A and B (Fig. 3a). Similar results were observed in syntenic maps for members of two other individual clusters within group II [one including the viruses of the Heliothis and the other including Chrysodeixis chalcites NPV (ChchNPV) and TnSNPV, data not shown]. On the other hand, the inclusion of one member of each of these three clusters and the other seven viruses of the group always resulted in extensive lack of collinearity and gene conservation (Fig. 3b). This lack of conservation of gene order and content between members of the different lineages of group II contrasted markedly with group I NPV (Oliveira et al., 2006) and invited further analyses.
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). The NPV had 22 synapomorphies and the Lepidopteran baculovirus (NPV and GV together) had 18 synapomorphies. These critical differences in the number of shared MPR are not detected in phylogenetic analyses that include only the core set of shared baculovirus genes (Herniou et al., 2003; Garcia-Maruniak et al., 2004; Jehle et al., 2006; Oliveira et al., 2006). Our current analyses indicated, for instance, that the LdMNPV had 52 apomorphies (i.e. genes unique to that lineage), which questions its inclusion in any of the current NPV groups. A similar argument could be extended to the Leucania separata NPV (LeseNPV), with 51 apomorphies, to the SpltNPV, with 49 apomorphies, and possibly to the three Heliothis NPVs, with 29 synapomorphies (Fig. 2). These analyses led to the conclusion that, within group II, there were at least ten deep lineages that were as divergent from each other as from group I (Fig. 2).
Given the complex pattern of gene gain and loss, it became interesting to ask how the gene gain and loss process correlated along the genome tree that had branch lengths estimated as median values of 1/S' (i.e. reciprocal of the median values of the bit-scores on pairwise genome comparisons with TBLASTX) (Oliveira et al., 2006). Interestingly, more than 60 % of the data had a power-law relation expressing the positive dependence of the number of gene gain (g) on branch length (b) in the weighbor tree (g=2537.11b10.870, r2=0.54), whereas loss events (l) were less frequent and had less dependence (12 %) on branch lengths (l=29.96b10.409, r2=0.12). For the gain process, the signature of the power-law relation was also obtained by a good linear fitting of a log-log plot (logg=1.063logb+3.871, r2=0.61). In this regard, it is necessary to point out that BlastPhen comparisons considered only traits shared among pairs of genomes, and not gain and loss events used to estimate MPRs. Therefore, the dependency of values of MPRs on the estimated branch lengths did not entail circular reasoning, since both sets of values were obtained from fairly independent methods of measurement. Nevertheless, it needs to be acknowledged that our method could inflate distances among closely related viruses, due to the larger amount of high-score pairs and polymorphisms found in non-coding regions during the pairwise TBLASTX search. This effect tends to disappear as genomes lose similarity in their non-coding regions (i.e. among less related genomes). However, we do not find that our main results are wrong because (i) the tree topology in Fig. 2 is congruent with that obtained previously from the conserved core set of genes and (ii) there was no change in the topology when three viruses closely related to AcMNPV and SfMNPV-19 (i.e. PlxyMNPV, RoMNPV and SfMNPV-3AP2) were taken out from the MPR/branch length comparison and the power-law signature was altered by only 6.7 %, which is almost exactly the amount of data (6.9 %) taken out of the analyses.
In summary, an implication of our findings is that, to a considerable extent, the process of gene gain in baculoviruses appears to be temporally organized (since branch lengths increase in time) following a power-law, whereas genes loss events appear to be less frequent and not correlated with time. Nevertheless, most baculovirus lineages we studied are apparently increasing in genome complexity with time, since gene losses are less frequent and more irregular. Similar accretion processes of auxiliary gene functions in large DNA viruses were observed for pox viruses (McLysaght et al., 2003) and herpesviruses (Montague & Hutchison, 2000). These findings posit renewed challenge to our understanding of the evolution of baculovirus genome organization and taxonomy.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 10 November 2007;
accepted 25 January 2008.
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