BACKGROUND
brazil is the largest sugarcane  and bioethanol producer in the world  <cit> .
nowadays, sugarcane is grown on an area over  <dig> million hectares for both sugar and alcohol production  <cit> .
as with other cultures cultivated over large areas, pest control is of crucial importance.
the sugarcane borer diatraea saccharalis fabr.
is present in all sugarcane-producing regions of the country, and is considered the major sugarcane pest, especially in the southeast region  <cit> .
biological control based on the release of the parasitoid cotesia flavipes   has been used with success in the control of the sugarcane borer  <cit> .
however, other complementary and compatible methods, such as the application of baculoviruses, would be highly desirable.
baculoviruses are a large group of insect-specific viruses with circular double-stranded dna, whose hallmark is the presence of occlusion bodies   <cit> .
the family baculoviridae comprises four genera: two of them, alphabaculovirus and betabaculovirus, infect insects of the order lepidoptera; the other two gammabaculovirus and deltabaculovirus infect insects of the orders hymenoptera and diptera  <cit>  respectively.
to date, the genomes of  <dig> baculovirus species were completely sequenced, and  <dig> of them are betabaculoviruses.
the baculovirus from the species anticarsia gemmatalis multiple nucleopolyhedrovirus  has been used in brazil in one of the largest biocontrol programs in the world to control an insect pest  <cit> .
other successful programs with baculoviruses have been reported elsewhere in the world  <cit> .
the success of the agmnpv program is due to a combination of factors, such as: high virulence, dead larvae can be collected directly from the field to be used as inoculum, efficient application technology, etc.
nevertheless, development is needed on pest species that are not so easily exposed to the virus, as in the case of borers.
large-scale dna sequencing provides information on complete viral genomes allowing for “omic” approaches that will eventually facilitate the development of application strategies.
since brazil has high biodiversity, several baculoviruses have been found and their genomes sequenced .
with this prospect in mind, we have sequenced and analyzed the genome of a diatraea saccharalis granulovirus , the first betabaculovirus isolated from a member of the family crambidae.
the presence of a functional gp <dig> homolog  <cit>  in a betabaculovirus was a unique and remarkable finding.
RESULTS
viral infection confirmation
larvae of diatraea saccharalis with virus infection symptoms were found in sugarcane fields in the southern brazil.
we performed the structural characterization of the putative virus and a granulovirus infection was confirmed by transmission electron microscopy of obs extracted from larvae cadavers.
each elliptical granule had a single rod-shaped virion surrounded by a robust protein matrix coat , indicating the typical morphology of granuloviruses   <cit> .
since the protein matrix is formed by granulin produced in large amounts during late infection and because it is highly conserved among lepidopteran-infective baculovirus, we amplified and sequenced the granulin gene in order to obtain an initial confirmation to the viral type .
the 747 bp length of the disagv granulin had high amino acid identity with orthologs from the genus betabaculovirus .
importantly, a granulovirus was previously isolated from d. saccharalis caterpillars sampled from sugarcane crops in the southern united states.
however, there is no available sequence to establish any phylogenetic relationship to the brazilian strain described here  <cit> .fig.
<dig> ultrastructural analysis of diatraea saccharalis granulovirus .
transmission electron micrograph reveals granular occlusion bodies containing singly embedded rod-shaped nucleocapsid
disagv genome and phylogeny
the complete genome of disagv  was  <dig>  bp in length , which makes the disagv the smallest betabaculovirus sequenced to date, followed by adoxophyes orana granulovirus    <cit>  and plutella xylostella granulovirus    <cit> .
the g + c content was  <dig>  %, a typically low value found among gvs, and potentially encoded  <dig> open reading frames  with at least  <dig> predicted amino acid residues .
the current baculovirus proposed species demarcation criterion is based on pairwise nucleotide distances estimated using the kimura 2-parameter model of nucleotide substitution for three genes, granulin, lef- <dig>  and lef- <dig>  <cit> .
the pairwise distances of the viral sequences of disagv to other betabaculoviruses for both single loci and concatenated alignment are well in excess of  <dig>  substitutions/site, fulfilling all the criteria for a novel species .
in order to investigate the phylogenetic relationship of disagv to other baculoviruses, we carried out a maximum likelihood phylogenetic analysis based on the alignment of the  <dig> baculovirus core proteins from all baculovirus genomes publicly available using solely the unique species .
in agreement with both ob ultrastructural analysis and granulin gene sequencing , we found disagv as sister taxa of the cluster formed by choristoneura occidentalis granulovirus , erinnyis ello granulovirus , pieris rapae granulovirus  isolates, clostera anachoreta granulovirus , clostera anastomosis granulovirus , cydia pomonella granulovirus  and cryptophlebia leucotreta granulovirus  .fig.
<dig> circular genome map of disagv with all genes identified on the  <dig>  bp long.
arrows show the transcripcional orientation and relative size of each orf.
those are colored according to their presence into baculovirus genera: in blue the  <dig> core genes, in green only betabaculovirus-specific genes, in red the disagv unique genes, in yellow genes found in some subjects of both alpha and betabaculovirus, and homologous regions  in orange
fig.
<dig> maximum-likelihood tree for betabaculovirus and genome comparison.
a the phylogeny was based on the concatenated amino acid sequences of the  <dig> core proteins identified in all baculovirus genome completely sequenced so far .
we collapsed gammabaculoviruses  and alphaphabaculoviruses .
the cuninpv was used as root .
disagv  is a betabaculovirus and sister species of the cluster formed by cpgv-related species.
b genome comparison of the disagv genome against some related species including agsegv, chocgv, cpgv, epapgv, and erelgv.
locally collinear blocks  are numbered in the disagv genome from  <dig> to  <dig>  same colors depict same lcbs across the genomes.
rearrangement can be seen among the species
moreover, we performed a genomic comparison among some selected betabaculovirus genomes by mauve analysis.
we found nine locally collinear blocks , composed of genomic segments that appear to have the same relative position of their shared genes .
interestingly, lcb <dig> , lcb <dig>  and lcb <dig>  had an unexpected gene content composition.
lcb <dig> lacked baculovirus core genes , while lcb <dig> had a higher than expected number  and lcb <dig> had a higher than expected number of disagv-unique genes, a lower than expected number of baculovirus core genes and less than expected gv-specific genes .
disagv had a dna-ligase  and two helicase genes  probably involved in replication, repair, and recombination of dna  <cit> .
we also identified a deoxyuridine triphosphatase  gene  and the ribonucleotide reductase subunits rr <dig>  and rr2a , involved in nucleotide metabolism.
nevertheless, the role of those genes during baculovirus infection is not clear.
it was noteworthy the absence of several genes for early transcription factors, such as the ie- <dig>  ie- <dig>  and pe <dig>  there were also no similar sequences to the baculovirus repeated orfs , to the ecdysteroid udp-glucosyltransferase , to the apoptosis inhibitor p <dig>  and also to the cathepsin and chitinase genes.
we observed that the egt gene was absent only in the genomes of four other gvs, helicoverpa armigera granulovirus , pseudaletia unipuncta granulovirus , spodoptera litura granulovirus isolate k <dig>  and xestia c-nigrum granulovirus , that form a distinct phylogenic cluster.
on the other hand, the p <dig> gene was found only in the genomes of chocgv, calgv, clangv .
the absence of the cathepsin and chitinase genes may be compensated by the presence of the putative gene for matrix metalloproteinase .
whereas the loss of the cathepsin and chitinase genes is a common event among the betabaculoviruses  <cit> , the matrix metalloproteinase gene is present in all betabaculoviruses sequenced to date  <cit> .
the expression of a functional cpgv-encoded metalloproteinase into the autographa californica multiple nucleopolyhedrovirus  genome enhanced the virus virulence, promoted larval melanization, and could partially substitute for the viral cathepsin  <cit> .
disagv unique genes
homologs to  <dig> disagv orfs were not found in the genome of other baculoviruses.
taking into account the 450 bp region upstream of each unique orf, three of them presented no previously characterized promoter motifs,  <dig> contained exclusively early promoter motifs , and ten had both early and late  motifs .
two unique orfs, disa <dig> and disa <dig> showed significant blastp hits to other dsdna virus sequences publicly available.
orf disa <dig> encoded a putative  <dig> aa protein that showed 26 % amino acid identity  to a  <dig> aa length protein of a phycodnavirus  .
by motif search using the pfam database  <cit> , we found a conserved domain related to a pd-xk nuclease superfamily, a highly diverse class of proteins.
the exact biological functions, substrate specificity and molecular mechanisms of reactions for those nucleases remain unknown.
however, quite a few enzymes are thought to be related to many cell process including replication, restriction, dna repair and trna–intron splicing  <cit> .
moreover, orf disa <dig> coded for a hypothetical protein related to insect-infecting dsdna viruses including wiseana iridescent virus   and amsacta moorei entomopoxvirus ‘l’  .
phycodnaviruses are eukaryotic algae viruses and seem to share a common ancestor with other insect dsdna viruses, including iridoviruses and entomopoxviruses, which share baculovirus genes as well.
several baculovirus genes were found into the genome of those viruses, suggesting the occurrence of lateral gene transfer during co-infection in the same insect host, as probably expected to disa <dig> and disa <dig>  <cit> .fig.
<dig> maximum likelihood phylogenetic trees of both disa <dig>  and disa <dig>  based on their predicted amino acid sequence.
we used the raxml method under the lg + i + g model for disa <dig> and wag + i + f for disa <dig> with a nonparametric bootstrap to support the branches.
organisms:  organic lake phycodnaviruses , feldmannia species virus , feldmannia irregularis virus a  and prokaryotes.
b
wiseana iridescent virus , invertebrate iridovirus  <dig> , amsacta moorei entomopoxvirus ‘l’ , adoxophyes honmai entomopoxvirus ‘l’ , mythimna separata entomopoxvirus ‘l’  and choristoneura rosaceana entomopoxvirus ‘l’
g protein-coupled receptor
we also found another unique orf  encoding a polypeptide related to a putative class b secretin-like g-protein coupled receptor  of lepidopteran and an entomopoxvirus .
gpcrs are cell membrane-associated gtpases that transmit signals from the environment to the cell inside or between cells.
this allows them to react to a corresponding variety of extracellular stimuli that can be mediated by different peptides, lipids, proteins, nucleotides, nucleosides, organic odorants and photons  <cit> .
this type of receptor has been described in many animal species despite not being quite common in virus genomes.
we found a predicted signal peptide and seven trans-membrane domains in disa <dig> , which strongly suggests that it is a member of the secretin family  <cit> .
three subfamilies are recognized for this family and one of them, the b <dig> contains receptors with long extracellular n-termini as observed for both the predicted disa <dig> and the other related proteins.
it is not clear what role of this gene plays in the disagv life cycle.
nevertheless, it is noteworthy that human herpesvirus uses virally encoded gpcr to hijack cellular signaling network, which suggests a similar mechanism in disagv  <cit> .fig.
<dig>
in silico analyses of disa <dig>  a betabaculovirus-encoded g protein-coupled receptor gene.
a phylogenetic analysis of selected arthropod gpcrs.
disa <dig> sequence clustered with lepidopteran and an entomopoxvirus proteins.
we performed the raxml method under the wag + i + g + f model with a nonparametric bootstrap.
the tree is presented as a cladogram.
b schematic representation of disa <dig> and phylogenetically related proteins.
the betabaculovirus gpcr retained all the structures observed in the homologs including the signal peptide , soluble fraction , and the transmembrane domains .
plxy, plutella xylostella; psxu, papilio xuthus; papo, papilio polytes; myseev, mythimna separata entomopoxvirus ‘l’; and dapl, danaus plexippus
gp64
the most striking finding in the disagv genome was the presence of a gp <dig> homolog gene, disa <dig>  gp <dig> is the major envelope fusion protein  exclusively found in group i alphabaculoviruses   <cit> .
both group  <dig> alphabaculovirus  and betabaculovirus share an analog to the gp <dig>  called f protein, as the major budded virus  efp  <cit>  which is probably the ancestral efp in baculovirus  <cit> .
gp <dig> was acquired probably later by the ancestor of g1-α likely from an insect retrovirus-like element  <cit>  and is clearly related to the glycoprotein found in the genus thogotovirus   <cit> .
therefore, in attempt to understand both acquisition and evolution of gp <dig> into the disagv genome, we performed a phylogenetic reconstruction of the gene.
we found that disagv gp <dig> clustered with g1-α efp, suggestive of a horizontal gene transfer  from g1-α to betabaculovirus .
disa-gp <dig> clustered with dendrolimus kikuchii nucleopolyhedrovirus .
therefore, gp <dig> gene acquisition probably had adaptive value for the ancestor of disagv as it had for g1-α.
taken together, these results suggest that the common ancestor of the g1-α acquired this gene once by hgt from some unknown source, which was later transferred to disagv or some related ancestral betabaculovirus.
alternatively, the gene was firstly acquired by a disagv-related virus and later transferred to the common ancestor of g1-α.
an adaptation of disa <dig> to the g + c genome content of disagv was observed  depicting that the gene acquisition is likely not recent  <cit> .
experimental analysis has shown that the incorporation of gp <dig> into the genome of helicoverpa armigera nucleopolyhedrovirus , a g <dig> α-baculovirus, enhanced virus infectivity in vivo and in vitro  <cit> .
gp <dig> and f protein can exploit either distinct  <cit>  or similar  <cit>  receptors to entry into host cells.
therefore, gp <dig> acquisition has probably enhanced both fusion and binding virus capabilities  <cit>  and possibly replaced functionally the f protein in g1-α  <cit> .
this evolutionary replacement hypothesis is reinforced by the fact that g1-α encode a remnant f protein homolog in their genomes that is unable to compensate for gp <dig> loss, albeit probably playing a role in the virus pathogenicity  <cit> .
interestingly, despite the fact that the disagv genome encodes an f protein, large deletions were observed in several reads covering the gene, suggesting existence of genotypes with deleted segments in the sequenced population .
this feature may indicate that the function of f protein has been replaced or complemented by gp <dig> in disagv.
moreover, we also inspected the  <dig> nucleotides up-stream from the predicted disagv gp <dig> atg start codon and compare with several g <dig> α-baculovirus gp <dig> promoter regions .
during viral de novo synthesis, gp <dig> expression is regulated by transcription from both early and late promoters with negative and multiple positive regulatory elements  <cit> .
the gp <dig> promoter region size was previously described to be around 140 bp .
in the disagv, we found three required elements gata ,  <dig> tata box-like ,  <dig> cacgtg-like  sequences with mutation on the first c to a in both, and one tata-box -associated cagt .
it is noteworthy that tata-dependent activity and tata-independent activity is mediated by rna polymerase ii in opmnpv gp <dig>  <cit> , where two of the required gata and cacgtg specifically bind to host transcription factors and activate transcription from the tata-dependent gp <dig> promoter  <cit> .
the presence of these conserved regulatory expression sequences in the promoter region of disa-gp <dig> gene indicates that it must be transcribed and functional.
in a recently published work, we found that disa-gp <dig> is a functional envelope fusion protein able to replace the native acmnpv gp <dig> in a null recombinant baculovirus  <cit> .fig.
<dig> phylogeny, g + c content and promoter region analyses of the betabaculovirus-encoded gp <dig> homolog, disa <dig>  a the disagv homolog is related to dekinpv.
the maximum likelihood  tree was inferred using the predicted amino acid sequence of all the betabaculovirus gp <dig> , several publicly available group  <dig> alphabaculovirus genes , and thogotovirus genes .
we performed the raxml method under the wag + i + g model with a nonparametric bootstrap for phylogeny reconstruction.
thogotoviruses root the tree that is presented here as a cladogram.
b comparison of the g + c content average for the third position of the translational codon in the gp <dig> genes from all group  <dig> alphabaculovirus  and disagv.
the comparison suggests that disa <dig> underwent an adjustment for the low g + c content characteristic of betabaculoviruses.
c annotation of 110 bp long from the disa <dig> promoter region.
the elements and motifs shown were identified based on previously published analyses of alphabaculovirus genomes
CONCLUSIONS
after structural characterization, complete genome sequence, and phylogenetic analyses of the diatraea saccharalis-infecting virus , we found it to be a novel, distinct lineage of the betabaculovirus genus.
the genome seemed to be closely related to cpgv-related group but had the smallest genome found among other betabaculoviruses so far.
its genome encoded a gpcr-like gene and, remarkably for a functional gp <dig> gene, which had been previously reported solely for group  <dig> alphabaculovirus genomes.

 brazil is the largest sugarcane and bioethanol producer in the world. nowadays, sugarcane is grown on an area over million hectares for both sugar and alcohol production. as with other cultures cultivated over large areas, pest control is of crucial importance. the sugarcane borer diatraea saccharalis fabr. is present in all sugarcane-producing regions of the country, and is considered the major sugarcane pest, especially in the southeast region. biological control based on the release of the parasitoid cotesia flavipes has been used with success in the control of the sugarcane borer. however, other complementary and compatible methods, such as the application of baculoviruses, would be highly desirable. baculoviruses are a large group of insect-specific viruses with circular double-stranded dna, whose hallmark is the presence of occlusion bodies. the family baculoviridae comprises four genera: two of them, alphabaculovirus and betabaculovirus, infect insects of the order lepidoptera; the other two gammabaculovirus and deltabaculovirus infect insects of the orders hymenoptera and diptera respectively. to date, the genomes of baculovirus species were completely sequenced, and of them are betabaculoviruses. the baculovirus from the species anticarsia gemmatalis multiple nucleopolyhedrovirus has been used in brazil in one of the largest biocontrol programs in the world to control an insect pest. other successful programs with baculoviruses have been reported elsewhere in the world. the success of the agmnpv program is due to a combination of factors, such as: high virulence, dead larvae can be collected directly from the field to be used as inoculum, efficient application technology, etc. nevertheless, development is needed on pest species that are not so easily exposed to the virus, as in the case of borers. large-scale dna sequencing provides information on complete viral genomes allowing for “omic” approaches that will eventually facilitate the development of application strategies. since brazil has high biodiversity, several baculoviruses have been found and their genomes sequenced. with this prospect in mind, we have sequenced and analyzed the genome of a diatraea saccharalis granulovirus, the first betabaculovirus isolated from a member of the family crambidae. the presence of a functional gp homolog in a betabaculovirus was a unique and remarkable finding.  viral infection confirmation larvae of diatraea saccharalis with virus infection symptoms were found in sugarcane fields in the southern brazil. we performed the structural characterization of the putative virus and a granulovirus infection was confirmed by transmission electron microscopy of obs extracted from larvae cadavers. each elliptical granule had a single rod-shaped virion surrounded by a robust protein matrix coat, indicating the typical morphology of granuloviruses. since the protein matrix is formed by granulin produced in large amounts during late infection and because it is highly conserved among lepidopteran-infective baculovirus, we amplified and sequenced the granulin gene in order to obtain an initial confirmation to the viral type. the 747 bp length of the disagv granulin had high amino acid identity with orthologs from the genus betabaculovirus. importantly, a granulovirus was previously isolated from d. saccharalis caterpillars sampled from sugarcane crops in the southern united states. however, there is no available sequence to establish any phylogenetic relationship to the brazilian strain described here.fig. ultrastructural analysis of diatraea saccharalis granulovirus. transmission electron micrograph reveals granular occlusion bodies containing singly embedded rod-shaped nucleocapsid disagv genome and phylogeny the complete genome of disagv was  bp in length, which makes the disagv the smallest betabaculovirus sequenced to date, followed by adoxophyes orana granulovirus and plutella xylostella granulovirus. the g + c content was  %, a typically low value found among gvs, and potentially encoded open reading frames with at least predicted amino acid residues. the current baculovirus proposed species demarcation criterion is based on pairwise nucleotide distances estimated using the kimura 2-parameter model of nucleotide substitution for three genes, granulin, lef- and lef-. the pairwise distances of the viral sequences of disagv to other betabaculoviruses for both single loci and concatenated alignment are well in excess of substitutions/site, fulfilling all the criteria for a novel species. in order to investigate the phylogenetic relationship of disagv to other baculoviruses, we carried out a maximum likelihood phylogenetic analysis based on the alignment of the baculovirus core proteins from all baculovirus genomes publicly available using solely the unique species. in agreement with both ob ultrastructural analysis and granulin gene sequencing, we found disagv as sister taxa of the cluster formed by choristoneura occidentalis granulovirus, erinnyis ello granulovirus, pieris rapae granulovirus isolates, clostera anachoreta granulovirus, clostera anastomosis granulovirus, cydia pomonella granulovirus and cryptophlebia leucotreta granulovirus.fig. circular genome map of disagv with all genes identified on the  bp long. arrows show the transcripcional orientation and relative size of each orf. those are colored according to their presence into baculovirus genera: in blue the core genes, in green only betabaculovirus-specific genes, in red the disagv unique genes, in yellow genes found in some subjects of both alpha and betabaculovirus, and homologous regions in orange fig. maximum-likelihood tree for betabaculovirus and genome comparison. a the phylogeny was based on the concatenated amino acid sequences of the core proteins identified in all baculovirus genome completely sequenced so far. we collapsed gammabaculoviruses and alphaphabaculoviruses. the cuninpv was used as root. disagv is a betabaculovirus and sister species of the cluster formed by cpgv-related species. b genome comparison of the disagv genome against some related species including agsegv, chocgv, cpgv, epapgv, and erelgv. locally collinear blocks are numbered in the disagv genome from to same colors depict same lcbs across the genomes. rearrangement can be seen among the species moreover, we performed a genomic comparison among some selected betabaculovirus genomes by mauve analysis. we found nine locally collinear blocks, composed of genomic segments that appear to have the same relative position of their shared genes. interestingly, lcb, lcb and lcb had an unexpected gene content composition. lcb lacked baculovirus core genes, while lcb had a higher than expected number and lcb had a higher than expected number of disagv-unique genes, a lower than expected number of baculovirus core genes and less than expected gv-specific genes. disagv had a dna-ligase and two helicase genes probably involved in replication, repair, and recombination of dna. we also identified a deoxyuridine triphosphatase gene and the ribonucleotide reductase subunits rr and rr2a, involved in nucleotide metabolism. nevertheless, the role of those genes during baculovirus infection is not clear. it was noteworthy the absence of several genes for early transcription factors, such as the ie- ie- and pe there were also no similar sequences to the baculovirus repeated orfs, to the ecdysteroid udp-glucosyltransferase, to the apoptosis inhibitor p and also to the cathepsin and chitinase genes. we observed that the egt gene was absent only in the genomes of four other gvs, helicoverpa armigera granulovirus, pseudaletia unipuncta granulovirus, spodoptera litura granulovirus isolate k and xestia c-nigrum granulovirus, that form a distinct phylogenic cluster. on the other hand, the p gene was found only in the genomes of chocgv, calgv, clangv. the absence of the cathepsin and chitinase genes may be compensated by the presence of the putative gene for matrix metalloproteinase. whereas the loss of the cathepsin and chitinase genes is a common event among the betabaculoviruses, the matrix metalloproteinase gene is present in all betabaculoviruses sequenced to date. the expression of a functional cpgv-encoded metalloproteinase into the autographa californica multiple nucleopolyhedrovirus genome enhanced the virus virulence, promoted larval melanization, and could partially substitute for the viral cathepsin. disagv unique genes homologs to disagv orfs were not found in the genome of other baculoviruses. taking into account the 450 bp region upstream of each unique orf, three of them presented no previously characterized promoter motifs, contained exclusively early promoter motifs, and ten had both early and late motifs. two unique orfs, disa and disa showed significant blastp hits to other dsdna virus sequences publicly available. orf disa encoded a putative aa protein that showed 26 % amino acid identity to a aa length protein of a phycodnavirus. by motif search using the pfam database, we found a conserved domain related to a pd-xk nuclease superfamily, a highly diverse class of proteins. the exact biological functions, substrate specificity and molecular mechanisms of reactions for those nucleases remain unknown. however, quite a few enzymes are thought to be related to many cell process including replication, restriction, dna repair and trna–intron splicing. moreover, orf disa coded for a hypothetical protein related to insect-infecting dsdna viruses including wiseana iridescent virus and amsacta moorei entomopoxvirus ‘l’. phycodnaviruses are eukaryotic algae viruses and seem to share a common ancestor with other insect dsdna viruses, including iridoviruses and entomopoxviruses, which share baculovirus genes as well. several baculovirus genes were found into the genome of those viruses, suggesting the occurrence of lateral gene transfer during co-infection in the same insect host, as probably expected to disa and disa.fig. maximum likelihood phylogenetic trees of both disa and disa based on their predicted amino acid sequence. we used the raxml method under the lg + i + g model for disa and wag + i + f for disa with a nonparametric bootstrap to support the branches. organisms: organic lake phycodnaviruses, feldmannia species virus, feldmannia irregularis virus a and prokaryotes. b wiseana iridescent virus, invertebrate iridovirus, amsacta moorei entomopoxvirus ‘l’, adoxophyes honmai entomopoxvirus ‘l’, mythimna separata entomopoxvirus ‘l’ and choristoneura rosaceana entomopoxvirus ‘l’ g protein-coupled receptor we also found another unique orf encoding a polypeptide related to a putative class b secretin-like g-protein coupled receptor of lepidopteran and an entomopoxvirus. gpcrs are cell membrane-associated gtpases that transmit signals from the environment to the cell inside or between cells. this allows them to react to a corresponding variety of extracellular stimuli that can be mediated by different peptides, lipids, proteins, nucleotides, nucleosides, organic odorants and photons. this type of receptor has been described in many animal species despite not being quite common in virus genomes. we found a predicted signal peptide and seven trans-membrane domains in disa, which strongly suggests that it is a member of the secretin family. three subfamilies are recognized for this family and one of them, the b contains receptors with long extracellular n-termini as observed for both the predicted disa and the other related proteins. it is not clear what role of this gene plays in the disagv life cycle. nevertheless, it is noteworthy that human herpesvirus uses virally encoded gpcr to hijack cellular signaling network, which suggests a similar mechanism in disagv.fig. in silico analyses of disa a betabaculovirus-encoded g protein-coupled receptor gene. a phylogenetic analysis of selected arthropod gpcrs. disa sequence clustered with lepidopteran and an entomopoxvirus proteins. we performed the raxml method under the wag + i + g + f model with a nonparametric bootstrap. the tree is presented as a cladogram. b schematic representation of disa and phylogenetically related proteins. the betabaculovirus gpcr retained all the structures observed in the homologs including the signal peptide, soluble fraction, and the transmembrane domains. plxy, plutella xylostella; psxu, papilio xuthus; papo, papilio polytes; myseev, mythimna separata entomopoxvirus ‘l’; and dapl, danaus plexippus gp64 the most striking finding in the disagv genome was the presence of a gp homolog gene, disa gp is the major envelope fusion protein exclusively found in group i alphabaculoviruses. both group alphabaculovirus and betabaculovirus share an analog to the gp called f protein, as the major budded virus efp which is probably the ancestral efp in baculovirus. gp was acquired probably later by the ancestor of g1-α likely from an insect retrovirus-like element and is clearly related to the glycoprotein found in the genus thogotovirus. therefore, in attempt to understand both acquisition and evolution of gp into the disagv genome, we performed a phylogenetic reconstruction of the gene. we found that disagv gp clustered with g1-α efp, suggestive of a horizontal gene transfer from g1-α to betabaculovirus. disa-gp clustered with dendrolimus kikuchii nucleopolyhedrovirus. therefore, gp gene acquisition probably had adaptive value for the ancestor of disagv as it had for g1-α. taken together, these results suggest that the common ancestor of the g1-α acquired this gene once by hgt from some unknown source, which was later transferred to disagv or some related ancestral betabaculovirus. alternatively, the gene was firstly acquired by a disagv-related virus and later transferred to the common ancestor of g1-α. an adaptation of disa to the g + c genome content of disagv was observed depicting that the gene acquisition is likely not recent. experimental analysis has shown that the incorporation of gp into the genome of helicoverpa armigera nucleopolyhedrovirus, a g α-baculovirus, enhanced virus infectivity in vivo and in vitro. gp and f protein can exploit either distinct or similar receptors to entry into host cells. therefore, gp acquisition has probably enhanced both fusion and binding virus capabilities and possibly replaced functionally the f protein in g1-α. this evolutionary replacement hypothesis is reinforced by the fact that g1-α encode a remnant f protein homolog in their genomes that is unable to compensate for gp loss, albeit probably playing a role in the virus pathogenicity. interestingly, despite the fact that the disagv genome encodes an f protein, large deletions were observed in several reads covering the gene, suggesting existence of genotypes with deleted segments in the sequenced population. this feature may indicate that the function of f protein has been replaced or complemented by gp in disagv. moreover, we also inspected the nucleotides up-stream from the predicted disagv gp atg start codon and compare with several g α-baculovirus gp promoter regions. during viral de novo synthesis, gp expression is regulated by transcription from both early and late promoters with negative and multiple positive regulatory elements. the gp promoter region size was previously described to be around 140 bp. in the disagv, we found three required elements gata, tata box-like, cacgtg-like sequences with mutation on the first c to a in both, and one tata-box -associated cagt. it is noteworthy that tata-dependent activity and tata-independent activity is mediated by rna polymerase ii in opmnpv gp, where two of the required gata and cacgtg specifically bind to host transcription factors and activate transcription from the tata-dependent gp promoter. the presence of these conserved regulatory expression sequences in the promoter region of disa-gp gene indicates that it must be transcribed and functional. in a recently published work, we found that disa-gp is a functional envelope fusion protein able to replace the native acmnpv gp in a null recombinant baculovirus.fig. phylogeny, g + c content and promoter region analyses of the betabaculovirus-encoded gp homolog, disa a the disagv homolog is related to dekinpv. the maximum likelihood tree was inferred using the predicted amino acid sequence of all the betabaculovirus gp, several publicly available group alphabaculovirus genes, and thogotovirus genes. we performed the raxml method under the wag + i + g model with a nonparametric bootstrap for phylogeny reconstruction. thogotoviruses root the tree that is presented here as a cladogram. b comparison of the g + c content average for the third position of the translational codon in the gp genes from all group alphabaculovirus and disagv. the comparison suggests that disa underwent an adjustment for the low g + c content characteristic of betabaculoviruses. c annotation of 110 bp long from the disa promoter region. the elements and motifs shown were identified based on previously published analyses of alphabaculovirus genomes  after structural characterization, complete genome sequence, and phylogenetic analyses of the diatraea saccharalis-infecting virus, we found it to be a novel, distinct lineage of the betabaculovirus genus. the genome seemed to be closely related to cpgv-related group but had the smallest genome found among other betabaculoviruses so far. its genome encoded a gpcr-like gene and, remarkably for a functional gp gene, which had been previously reported solely for group alphabaculovirus genomes. 
surprisingly, we found in the disagv genome a g protein-coupled receptor related to lepidopteran and other insect virus genes and a gp homolog, which is likely a product of horizontal gene transfer from group alphabaculoviruses. the complete genome sequence of disagv was determined using the 454-pyrosequencing method. a phylogenetic analysis indicated that disagv is a member of betabaculovirus genus and that it is a sister group to a cluster formed by chocgv, erelgv, piragv isolates, clangv, calgv, cpgv, crlegv, adorgv, phopgv and epapgv. it had a g + c content of  % encoding putative open reading frames. disagv represents a distinct lineage of the genus betabaculovirus. a group of putative genes was not found in any other baculovirus genome sequenced so far. remarkably, we found a homolog of gp which was reported solely in group alphabaculovirus genomes so far. a betabaculovirus was isolated from diatraea saccharalis, one of the most important insect pests of the sugarcane and other monocot cultures in brazil.
 a betabaculovirus was isolated from diatraea saccharalis, one of the most important insect pests of the sugarcane and other monocot cultures in brazil.  the complete genome sequence of disagv was determined using the 454-pyrosequencing method. the genome was  bp long, which makes it the smallest lepidopteran-infecting baculovirus sequenced to date. it had a g + c content of  % encoding putative open reading frames. all the baculovirus core genes and a set of betabaculovirus-specific genes were found. a group of putative genes was not found in any other baculovirus genome sequenced so far. a phylogenetic analysis indicated that disagv is a member of betabaculovirus genus and that it is a sister group to a cluster formed by chocgv, erelgv, piragv isolates, clangv, calgv, cpgv, crlegv, adorgv, phopgv and epapgv. surprisingly, we found in the disagv genome a g protein-coupled receptor related to lepidopteran and other insect virus genes and a gp homolog, which is likely a product of horizontal gene transfer from group alphabaculoviruses.  disagv represents a distinct lineage of the genus betabaculovirus. it is closely related to the cpgv-related group and presents the smallest genome in size so far. remarkably, we found a homolog of gp which was reported solely in group alphabaculovirus genomes so far. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. http://dx.doi.org/ /501100003593conselho nacional de desenvolvimento científico e tecnológico481741/2008-9wolff josé l. c. http://dx.doi.org/ /501100003593conselho nacional de desenvolvimento científico e tecnológico407908/2013-7ribeiro bergmann m. http://dx.doi.org/ /501100005668fundação de apoio à pesquisa do distrito federal.583/2009ribeiro bergmann m. issue-copyright-statement© the author 2016 