BACKGROUND
baculoviruses are specific pathogens of the insect orders lepidoptera, diptera and hymenoptera and exhibit rod-shaped nucleocapsids embedded in a crystalline protein matrix  composed of polyhedrin in nucleopolyhedroviruses  and granulin in granuloviruses   <cit> .
the replication cycle of the baculoviruses is characterized by production of two viral phenotypes: occlusion derived viruses  and budded viruses .
these particles are genotypically identical, but they are morphologically and functionally distinct, with the bvs involved in systemic infection within host larvae  and the odvs involved in the horizontal transmission of the virus in the host population   <cit> .
the baculoviridae family consists of four genera: alphabaculovirus , betabaculovirus , gammabaculovirus  and deltabaculovirus   <cit> .
alphabaculoviruses can be further divided into groups i and ii based on dna sequence data and differences in bvs, where the envelope fusogenic protein in group i is gp <dig> and in group ii is the fusion  protein  <cit> .
so far,  <dig> complete baculovirus genomes are present in genbank, including many of the alphabaculoviruses  followed by  <dig> betabaculoviruses,  <dig> gammabaculoviruses and the culex nigripalpus deltabaculovirus  .
baculovirus genomes range in size from  <dig>   to  <dig>  kbp  with gc content below 50% and containing from  <dig>  to  <dig>  predicted orfs  <cit> .
the gene diversity in baculoviruses has been estimated to be about  <dig> genes, among which  <dig>  may play essential biological functions in the replication cycle  <cit> .
the common genomic features of the baculoviridae family include large double-stranded circular dna, bidirectionally oriented open reading frames  which are distributed on both dna strands,  <dig> genes common to all baculoviruses , promoters that regulate the temporal cascade of gene expression and viral genome replication in the host cell nucleus  <cit> .
the soybean looper, chrysodeixis includens    is a lepidopteran pest with restricted distribution in the western hemisphere, occurring from the northern united states to southern south america  <cit> .
soybean, cotton, beans, potatoes, tomatoes, tobacco, sunflower, lettuce, cauliflower, cabbage and okra are the most common crops attacked by c. includens  <cit> .
however, the polyphagous c. includens was found feeding on  <dig> plant species from  <dig> different families in brazil  <cit> .
until  <dig>  anticarsia gemmatalis was considered one of the most important pests on soybean and the baculovirus anticarsia gemmatalis mnpv was widely used as a bioinsecticide on approximately two million hectares of soybeans  <cit> .
recently, c. includens has begun to have an economic impact due to its population growth, causing significant losses in soybean production.
among other factors, this was attributed to a decline in natural enemies, which previously controlled the pest, and to development of resistance due to indiscriminate use of chemical pesticides in soybean fields  <cit> .
other forms of control are therefore required, and for this, new baculoviruses may be strong candidates for the biocontrol of this emerging pest.
pseudoplusia includens single nucleopolyhedrovirus  is a group ii alphabaculovirus pathogenic to c. includens  <cit> .
seven psinsnpv  isolates collected on cotton and soybean crops from guatemala and brazil were reported to cause fatal infections in c. includens larvae  <cit> .
evidence of significant genetic variations and different degrees of pathogenicity were observed among the isolates analyzed in our previous studies  <cit> .
other psinnpv isolates, psinnpv-usa and psinnpv-gt, have been reported, but little is known about them  <cit> .
the isolate psinsnpv-ie was obtained from c. includens larvae collected on brazilian soybean crops and was found to be one of the most virulent against c. includens among seven isolates analyzed  <cit> .
in this manuscript, we report the complete sequence and organization of the psinsnpv-ie genome and speculate on the origin of the p <dig> gene within the baculoviridae family by potentially distinct acquisition events.
the analysis of the psinsnpv genome will provide important information for a better understanding of its virulence, evolution and molecular biology.
these findings may also contribute to the development of a psinsnpv bioinsecticide for the control of c. includens.
RESULTS
nucleotide sequence and gene content of the psinsnpv genome
the psinsnpv genome was sequenced using next generation technology  on the roche  <dig> gs-flx titanium platform.
a total of  <dig>  reads were obtained with an average length of  <dig>  ±  <dig>  bp.
pre-processing yielded  <dig>  sequences, with a mean length of  <dig>  ±  <dig>  bp .
following assembly, the size of the double-stranded circular dna psinsnpv genome  was determined to be  <dig>  bp  with a gc content of  <dig>  %, which is in agreement with the average gc content of group ii alphabaculoviruses   <cit> .
in silico restriction digest analysis of the psinsnpv genome was conducted, corroborating previous physical restriction maps  <cit>  .
the orfs were sequentially numbered starting from the polyhedrin gene in a clockwise orientation.
a total of  <dig> putative orfs, including the  <dig> core genes present in all baculoviruses and two psinsnpv unique orfs , were identified, comprising  <dig> % of the psinsnpv genome with  <dig> orfs in clockwise, and  <dig> orfs in counterclockwise orientation .figure  <dig>
circular map of the psinsnpv genome.
the  <dig> predicted orfs and transcription direction are represented by arrows with displayed name.
genome position is indicated in bp, starting from the polh gene, by convention.
comparison of psinsnpv with others alphabaculoviruses
the psinsnpv genome was compared with the alphabaculoviruses chchnpv, tnsnpv, maconpv-b  and the reference baculovirus, acmnpv .
the information is summarized in table  <dig> and additional file  <dig>  as expected, the two viruses most closely related to psinsnpv are chchnpv and tnsnpv, sharing similar genome sizes and possessing high nucleotide sequence similarity  <cit> .
however, the psinsnpv genome is  <dig>  bp larger than tnsnpv and  <dig>  bp smaller than chchnpv .
global alignment and dot matrix analysis of the psinsnpv genome compared with chchnpv, tnsnpv, maconpv-b and acmnpv revealed that psinsnpv is highly similar and collinear with chchnpv and tnsnpv , but not with maconpv-b and acmnpv  .
psinsnpv shares  <dig> orfs with acmnpv,  <dig> orfs with macomnpv-b,  <dig> orfs with chchnpv and  <dig> orfs with tnsnpv .
the orfs reported as unique in chchnpv  and in tnsnpv  showed similarity to psinsnpv orfs- <dig>  − <dig>  − <dig>  − <dig> and psinsnpv orf- <dig>  − <dig>  respectively .table  <dig>
characteristics of the psinsnpv genome compared with other alphabaculoviruses
features
baculoviruses
psinsnpv
acmnpv
maconpv-b
chchnpv
tnsnpv
genome size
gc content
orfs
coding sequence
hr
sequences
bro
genes
mean % aa id with psinsnpv
homologs in psinsnpv
orfs unique to psinsnpv
genbank accession numberdot plot matrix analysis using lbdotview v.  <dig>  software.
blue dots represent the homolog regions of the psinsnpv genome compared with  chchnpv,  tnsnpv,  acmnpv and  maconpv-b genome, both in the direct orientation, and red dots compare the psinsnpvgenome in the direct orientation with npv genomes in the reverse orientation.
replication, transcription and structural genes
the baculovirus genes are categorized based on their functions during the viral cycle as follows: dna replication, rna transcription, odv and bv structural proteins or oral infectivity proteins  <cit> .
baculovirus genome replication mechanisms are still not fully understood.
several studies have been developed to try to identify the genes responsible for dna replication and translation.
the essential dna replication factors late expression factor  <dig> , lef- <dig>  lef- <dig>  dna polymerase , p <dig> , 38 k, helicase  and immediate early  <dig>  homologs are all present in the psinsnpv genome.
in addition, the psinsnpv genome contains genes homologous to proliferating cell nuclear antigen , major early-transcribed protein  <dig> , dna binding protein , alkaline exonuclease  and exon-0/ie- <dig>  which were not identified in all baculoviruses but may influence viral dna replication  <cit> .
the acmnpv transcription system is activated in two main stages.
at first, lef- <dig>  lef- <dig>  lef- <dig> and p <dig> are transcribed to encode the  <dig> subunits of the viral rna polymerase complex  <cit> .
this complex acts on gene transcription and mrna processing, including capping and polyadenylation.
then, the transcription enhancers lef- <dig> and very late factor  <dig>  are transcribed  <cit> .
all these genes were also found in the psinsnpv genome.
in addition, some supposedly non-essential genes involved in acmnpv transcription regulation are also present in the psinsnpv genome: lef- <dig>  lef- <dig>  39 k, lef- <dig> and protein kinase  <dig> .
the psinsnpv genome has  <dig> known baculovirus genes coding for structural proteins.
genes for odv and bv structural proteins include polyhedrin , orf <dig>  pk <dig>  occlusion derived virus envelope protein  <dig> , occlusion derived virus enveloped capsid protein  <dig> , p <dig>  viral protein  <dig> , few polyhedra protein/25 k , desmoplakin, 41-kda glycoprotein , telokinin-like peptide  <dig> , viral protein  <dig> , vp <dig>  p <dig>  odv-e <dig>  p87/vp <dig>  odv-ec <dig>  odv-e <dig>  p <dig>  calyx/polyhedrin enveloped protein , p <dig>  per os infectivity factor  <dig> , pif- <dig>  pif- <dig>  pif- <dig>  odv-e28/pif- <dig>  odv-e56/pif- <dig> and fusion  protein.
the six pif genes which are components of odvs and are involved in oral infectivity exhibited high sequence similarity to chchnpv and tnsnpv pifs.
the genus alphabaculovirus is divided into groups i and ii based on gene content, and in particular, the bvs fusion protein: gp <dig> and f protein, respectively.
psinsnpv, a group ii alphabaculovirus, possesses the expected f protein homolog.
nucleotide metabolism and dna repair
several group ii alphabaculoviruses and betabaculoviruses encode genes involved in nucleotide biosynthesis.
psinsnpv possesses the ribonucleotide reductase  large  and small  subunits and the dutpase protein.
these rr proteins are enzymes involved in the formation of deoxyribonucleotides from ribonucleotides  <cit> .
the dutpase protein is responsible for preventing incorporation of mutagenic dutp into dna  <cit> .
poly  polymerase  and poly adp-ribose glycohydrolase  are enzymes involved in synthesis of adp riboses that activate and recruit dna repair enzymes  <cit> .
although it has been reported that all group ii genomes encode parg homologs  <cit> , the psinsnpv genome is notable for its absence.
the cpd photolyase, encoded by the dna photolyase  gene, acts at cyclobutane pyrimidine dimers to repair ultraviolet  -induced dna damage.
the phr gene was identified in chchnpv  <cit> , tnsnpv  <cit> , plusia acuta npv   <cit>  and thysanoplusia orichalcea npv-b <dig>   <cit> .
studies suggest that the phr gene is conserved in group ii alphabaculoviruses that infect lepidopteran insects in the plusiinae subfamily of the noctuidae family  <cit> .
however, the phr gene was also identified in baculoviruses that infect insects of other subfamilies, such as spodoptera litura gv    <cit> , clanis bilineata npv    <cit> , apocheima cinerarium npv    <cit>  and ampelophaga rubiginosa npv     <cit> .
psinsnpv belongs to a group where the phr gene is conserved and, as expected, its genome encodes a cpd photolyase protein .
the complete nucleotide sequence of the psinsnpv phr gene is of  <dig>  bp with gc % =  <dig> %.
the deduced phr amino acid sequence of psinsnpv, chchnpv- phr <dig>  −phr <dig>  and tnsnpv were aligned, revealing that the psinsnpv photolyase possesses high identity to tnsnpv and chchnpv- phr <dig> .
previous studies showed that chchnpv - phr <dig> is not active when tested in an escherichia coli photolyase deficient strain  <cit> .
the active copy  is distinct in its possession of two conserved tryptophan residues, which may be involved in an electron transfer mechanism  <cit> .
in the psinsnpv photolyase protein, the tryptophan residues are replaced by histidine and tyrosine in positions  <dig> and  <dig> aa, respectively .
therefore, both tryptophans are absent in the psinsnpv photolyase, suggesting that this protein might not be active.
the partial phr amino acid sequences of psinnpv –gt <dig> ; −gt <dig> , psinnpv - usa  and psinsnpv-ia to -ig isolates described in the literature  <cit>  were aligned, where psinnpv-gt <dig> and psinnpv – usa isolates showed high similarity to the psinsnpv-ia to -ig isolates.
interestingly, in contrast to other psinnpv isolates reported so far, psinnpv - gt <dig> possesses a tryptophan residue at position  <dig>  which is thought to be essential for enzyme activity .
further studies are needed to confirm and investigate the activity of the psinsnpv - gt <dig> photolyase.
auxiliary genes
the auxiliary genes viral ubiquitin , viral cathepsin , chitinase , 37-kda glycoprotein , conotoxin , superoxide dismutase  <dig> , fibroblast growth factors , phosphotyrosine phosphatase , ecdysone glucose transferase , actin rearrangement infectivity factor  <dig> , inhibitor of apoptosis  <dig> , iap- <dig> and p35/p <dig> were found in the psinsnpv genome.
the auxiliary genes are non-essential for dna replication, translation or viral particle formation.
however, these genes confer selective advantages to viruses as has been observed in homologs of psinsnpv auxiliary genes described in the literature.
the activity of the v-cath and chia genes is notable in p. includens larvae infected with psinsnpv, where the encoded proteins cause degradation and liquefaction of the host cadaver  <cit> .
the fgf, ptp and egt genes were reported to be involved in host hyperactive behaviors, increasing larval motility and preventing the molt to extend insect life, respectively  <cit> .
the ptp gene was previously reported to be only present in group i npvs  <cit> , however this gene is present in the psinsnpv genome.
homologous regions  are absent from the psinsnpv genome
homologous regions  are repeated sequences with an imperfect palindromic core that are distributed in the genome as singletons or arranged in tandem.
these repeat sequences are present in baculovirus genomes and other closely related invertebrate viruses  <cit> .
these regions act as enhancers of early gene transcription in npvs and may serve as origins of replication in npvs and gvs  <cit> .
homologous regions are a common feature found in genomes of the four genera of the baculoviridae family.
however, no typical baculoviral hrs were found in the psinsnpv genome, which is also the case in buzura supressaria npv   <cit> , chchnpv, tnsnpv and agrotis segetum gv   <cit> .
psinsnpv bro genes
the psinsnpv genome sequence contains two baculovirus repeated orfs , named according to their order in the genome: bro-a  and bro-b .
the bro genes commonly occur in alpha-, beta- and gammabaculoviruses, varying in number of copies and length among the viruses  <cit> .
these genes were first reported from baculoviruses, but bro gene homologs were subsequently identified in other insect dsdna viruses, such as entomopoxvirus and entomoiridovirus  <cit> .
bro proteins exhibit a highly conserved n-terminal dna binding domain  in the first 100– <dig> aa and a variable c-terminal domain   <cit> .
the functions of bro proteins are not clear, but were proposed to be involved in host dna replication and/or transcriptional regulation and as a viral replication enhancer in the late phase  <cit> .
although a deletion of 425 bp   is present in the psinsnpv bro-b gene compared with the chchnpv bro-b gene, the genes share 72% identity.
the psinsnpv bro-a gene showed higher similarity to the lymantria xylina mnpv bro-m gene and the mamestra brassicae mnpv bro-a gene with  <dig> and 53% identity, respectively.
in contrast to the psinsnpv bro-b protein with one bro-n domain, psinsnpv bro-a protein contains two bro-n domains  at amino acid position  <dig> to  <dig> and  <dig> to  <dig>  in addition, the psinsnpv bro-a protein contains a domain of unknown function duf <dig>  in amino acid position  <dig> to  <dig>  although psinsnpv, chchnpv and tnsnpv are closely related, their bro genes do not show high similarity.
orfs unique to psinsnpv
two putative orfs, psin <dig> and psin <dig>  were found to be unique to the psinsnpv genome.
these orfs do not show significant similarity to other previously described baculovirus orfs and exhibit signature sequences that describe domains predicted by interproscan  <dig>  <cit> .
psin <dig> is predicted to encode a  <dig> amino acid  protein with molecular weight of  <dig>  kda and shows low homology to a signal transducer and activator of transcription protein in the avian species, pseudopodoces humilis  .
psin <dig> is predicted to encode a  <dig> aa,  <dig>  kda protein, but shows no significant similarity to any genes in genbank databases .
the taag late promoter motif, combined with a tata early promoter , was identified about 100 bp upstream of both the psin <dig> and psin <dig> start codons .
these promoters are thought to be transcribed both by the host rna polymerase ii and viral rna polymerases  <cit> , suggesting that these genes could be expressed both early and late in infection.
a search was made for protein families, domains and functional sites found in transmembrane domains in the predicted psin <dig> and psin <dig> proteins.
using tmhmm server v  <dig> , transmembrane helices from amino acid position  <dig> to  <dig> and  <dig> to  <dig> in psin <dig> and  <dig> to  <dig> in psin <dig> hypothetical proteins were predicted .
two p <dig> homologs in psinsnpv
two p <dig>  gene homologs were identified in the psinsnpv genome.
the function of the p <dig> gene is not well understood, but studies have shown that deletion of the acmnpv p <dig> gene produced no differences in phenotype from wild-type acmnpv in cells and in larvae  <cit> .
however, a combined deletion of p <dig>  p <dig> and p <dig> genes in acmnpv resulted in polyhedra containing few or no virions  <cit> .
for this reason, p <dig> is thought to be required for optimal virion occlusion in the obs.
one or more copies of the p <dig> gene are present in all group i and ii alphabaculoviruses, except spodoptera littoralis multiple nucleopolyhedrovirus, splinpv .
the viruses with more than one p <dig> copy belong to group ii alphabaculoviruses .
however, in group i alphabaculovirus genome sequences available at genbank, choristoneura fumiferana  mnpv, choristoneura occidentalis  npv and choristoneura rosaceana  npv contain two p <dig> homologs .table  <dig>
amino acid sequences used in phylogenetic analysis of the p <dig> amino acid sequence
position
alphabaculoviruses
abbreviation
genbank
1
a
2
b
3
c
group i
group ii
ap <dig> orf number adjacent to p <dig> gene.
bp <dig> orf number adjacent to iap- <dig> gene.
cp <dig> orf number adjacent to ptp − <dig> and − <dig> genes.
the p <dig> gene is conserved in position, adjacent to the p <dig> gene in all the alphabaculoviruses containing a single copy.
the psinsnpv p26a/orf- <dig> and p26b/orf- <dig> are positioned adjacent to the p <dig> gene and adjacent to the iap- <dig> gene, respectively.
the copy adjacent to the iap- <dig> gene is also positionally conserved in all group ii alphabaculoviruses containing two p <dig> copies.
however, the second p <dig> copy in group i alphabaculoviruses is positioned adjacent to the ptp <dig> and ptp <dig> genes.
the phylogenetic tree obtained by bayesian phylogenetic inference  using the p <dig> copies found in all alphabaculovirus genomes so far sequenced showed four clearly defined clades  nested within the larger clades i and ii .
the clade ia contains p <dig> copies from group i alphabaculoviruses and clade ii from group ii alphabaculoviruses.
clade ib contains p <dig> copies also from group ii alphabaculoviruses, except for a monophyletic group with cfmnpv_orf <dig>  chocmnpv_orf <dig> and chronpv_orf <dig> from group i alphabaculoviruses.
clades i  and ii correlate with the position of the genes in the genome, where clade i contains the p <dig> copies adjacent to the p <dig> gene , except for cfmnpv_orf <dig>  chocmnpv_orf <dig> and chronpv_orf <dig>  which are adjacent to ptp <dig> and ptp <dig> , and clade ii contains the p <dig> copies that are adjacent to iap- <dig> .
this clustering pattern suggests the occurrence of three independent acquisition events of the p <dig> gene by baculoviruses.
the first acquisition event occurred in position  <dig> of the common ancestral genome of all baculoviruses containing this gene.
the second acquisition event generated the p <dig> second copy in position  <dig> of the group ii alphabaculovirus genome with two p <dig> homologs.
finally, the third acquisition event occurred in cfmnpv, chocnpv and chronpv , inserting the second p <dig> copy in position  <dig> figure  <dig>
bayesian phylogram based on p <dig> deduced amino acid sequences.
the clustering pattern suggests the occurrence of three independent p <dig> gene acquisition events that are marked by circles.
the arrow indicates the node where occurred the signal peptide acquisition.
numbers at branches indicate the bayesian posterior probabilities .
the tree was rooted by midpoint approach.
the scale bar indicates the number of substitutions per site.
the acquisition of baculovirus genes may be the results of duplication and horizontal gene transfer by transposable element and/or homologous recombination.
the second acquisition event probably occurred by horizontal gene transfer.
in this case, the gene duplication hypothesis can be refuted, since the similarity between p <dig> copies in the same virus is low .
furthermore, clades i and ii are clearly separated, indicating that the p <dig> copies did not originate from a common, recent ancestor.
in the third acquisition event, there is high similarity between the second p <dig> copy of the group i alphabaculoviruses and first p <dig> copy of the group ii alphabaculoviruses, grouping these genes in the same clade .
therefore, the second p <dig> copy of the group i alphabaculoviruses may have been acquired from a group ii alphabaculovirus.
the isoelectric point  and molecular weight  of the deduced p <dig> protein from alphabaculoviruses with two p <dig> copies were calculated, and are presented in the context of their genomic positioning .
p <dig> protein from position  <dig> showed an average pi of  <dig>  ±  <dig>  and average mw of  <dig>  ±  <dig> ; position  <dig> p <dig> proteins have an average pi of  <dig>  ±  <dig>  and average mw of  <dig>  ±  <dig> ; and position  <dig> p <dig> proteins have an average pi of  <dig>  ±  <dig>  and an average mw of  <dig>  ±  <dig>  the mean scores were examined using the student’s t-test and the pi average showed a significant difference  between the three positions.
the isoelectric point difference suggests that the p <dig> sequence in position  <dig> exhibits more basic amino acids than in those in positions  <dig> and  <dig>
the presence and location of signal peptide cleavage sites in p26a and p26b amino acid sequences from psinsnpv were analysed using signalp v. <dig> .
the p26a protein showed a signal peptide cleavage site at amino acid residues  <dig> to  <dig> and the cleavage site  between amino acids  <dig> and  <dig> .
however, the signal peptide cleavage site was absent in the p26b protein.
signal peptides direct the proteins to their proper cellular and extracellular locations.
the export of proteins occurs via the secretory pathway, where proteins labeled by an n-terminal signal sequence are translocated across the cytoplasmic membrane, whereafter the n-terminal signal peptide is usually cleaved by an extracellular signal peptidase.
the signal peptide cleavage sites were predicted for other p <dig> proteins of alphabaculoviruses with complete sequenced genomes and the results are shown in table  <dig>  the presence or absence of signal peptides in p <dig> proteins correlates with the clustering obtained in the phylogenetic analysis, where only p <dig> proteins from clade ib possess signal peptides.
however, four sequences belonging to this clade, lesenpv_orf <dig>  ecobnpv_orf <dig>  orlenpv_orf <dig> and chchnpv_orf <dig>  do not possess a signal peptide.
the presence or absence of the signal peptide may have led to the differential results found in predicted molecular weight and isoelectric points between the p <dig> proteins analyzed.
the presence of a signal peptide in the first p <dig> copy of group ii alphabaculoviruses and in the second p <dig> copy of group i alphabaculoviruses suggests that this domain was acquired from a common ancestor of these viruses.
although the function of p <dig> is not well understood, the signal peptide may lead to differences in activity of the clade ib proteins compared to the p <dig> from other clades, which warrants further investigation.
CONCLUSIONS
in summary, the complete psinsnpv-ie genome sequence is apparently different from other baculoviruses sequenced so far.
the genome does not contain the typical baculovirus hrs and contains two orfs with predicted transmembrane domains that are unique to psinsnpv.
the psinsnpv genome, however, exhibits high sequence similarities and co-linearity to the closely related chchnpv and tnsnpv.
the psinsnpv genome contains two p <dig> copies and a phylogenetic analysis of p <dig> sequences of alphabaculoviruses showed three potential acquisition events of these genes within the baculoviridae family.
one of the clades comprises p <dig> protein with a signal peptide, indicating a possible distinct function from other classes of p <dig> protein.
however, further investigations are needed for a better understanding of this protein in baculoviruses.
this research reports the first completely sequenced genome of a strain of psinsnpv, a currently little known baculovirus.
it is anticipated that this data will both promote advances in investigations of its molecular biology and gene function and accelerate its development as a biocontrol agent.

 baculoviruses are specific pathogens of the insect orders lepidoptera, diptera and hymenoptera and exhibit rod-shaped nucleocapsids embedded in a crystalline protein matrix composed of polyhedrin in nucleopolyhedroviruses and granulin in granuloviruses. the replication cycle of the baculoviruses is characterized by production of two viral phenotypes: occlusion derived viruses and budded viruses. these particles are genotypically identical, but they are morphologically and functionally distinct, with the bvs involved in systemic infection within host larvae and the odvs involved in the horizontal transmission of the virus in the host population. the baculoviridae family consists of four genera: alphabaculovirus, betabaculovirus, gammabaculovirus and deltabaculovirus. alphabaculoviruses can be further divided into groups i and ii based on dna sequence data and differences in bvs, where the envelope fusogenic protein in group i is gp and in group ii is the fusion protein. so far, complete baculovirus genomes are present in genbank, including many of the alphabaculoviruses followed by betabaculoviruses, gammabaculoviruses and the culex nigripalpus deltabaculovirus. baculovirus genomes range in size from to kbp with gc content below 50% and containing from to predicted orfs. the gene diversity in baculoviruses has been estimated to be about genes, among which may play essential biological functions in the replication cycle. the common genomic features of the baculoviridae family include large double-stranded circular dna, bidirectionally oriented open reading frames which are distributed on both dna strands, genes common to all baculoviruses, promoters that regulate the temporal cascade of gene expression and viral genome replication in the host cell nucleus. the soybean looper, chrysodeixis includens is a lepidopteran pest with restricted distribution in the western hemisphere, occurring from the northern united states to southern south america. soybean, cotton, beans, potatoes, tomatoes, tobacco, sunflower, lettuce, cauliflower, cabbage and okra are the most common crops attacked by c. includens. however, the polyphagous c. includens was found feeding on plant species from different families in brazil. until anticarsia gemmatalis was considered one of the most important pests on soybean and the baculovirus anticarsia gemmatalis mnpv was widely used as a bioinsecticide on approximately two million hectares of soybeans. recently, c. includens has begun to have an economic impact due to its population growth, causing significant losses in soybean production. among other factors, this was attributed to a decline in natural enemies, which previously controlled the pest, and to development of resistance due to indiscriminate use of chemical pesticides in soybean fields. other forms of control are therefore required, and for this, new baculoviruses may be strong candidates for the biocontrol of this emerging pest. pseudoplusia includens single nucleopolyhedrovirus is a group ii alphabaculovirus pathogenic to c. includens. seven psinsnpv isolates collected on cotton and soybean crops from guatemala and brazil were reported to cause fatal infections in c. includens larvae. evidence of significant genetic variations and different degrees of pathogenicity were observed among the isolates analyzed in our previous studies. other psinnpv isolates, psinnpv-usa and psinnpv-gt, have been reported, but little is known about them. the isolate psinsnpv-ie was obtained from c. includens larvae collected on brazilian soybean crops and was found to be one of the most virulent against c. includens among seven isolates analyzed. in this manuscript, we report the complete sequence and organization of the psinsnpv-ie genome and speculate on the origin of the p gene within the baculoviridae family by potentially distinct acquisition events. the analysis of the psinsnpv genome will provide important information for a better understanding of its virulence, evolution and molecular biology. these findings may also contribute to the development of a psinsnpv bioinsecticide for the control of c. includens.  nucleotide sequence and gene content of the psinsnpv genome the psinsnpv genome was sequenced using next generation technology on the roche gs-flx titanium platform. a total of reads were obtained with an average length of  ±   bp. pre-processing yielded sequences, with a mean length of  ±   bp. following assembly, the size of the double-stranded circular dna psinsnpv genome was determined to be  bp with a gc content of %, which is in agreement with the average gc content of group ii alphabaculoviruses. in silico restriction digest analysis of the psinsnpv genome was conducted, corroborating previous physical restriction maps. the orfs were sequentially numbered starting from the polyhedrin gene in a clockwise orientation. a total of putative orfs, including the core genes present in all baculoviruses and two psinsnpv unique orfs, were identified, comprising % of the psinsnpv genome with orfs in clockwise, and orfs in counterclockwise orientation.figure circular map of the psinsnpv genome. the predicted orfs and transcription direction are represented by arrows with displayed name. genome position is indicated in bp, starting from the polh gene, by convention. comparison of psinsnpv with others alphabaculoviruses the psinsnpv genome was compared with the alphabaculoviruses chchnpv, tnsnpv, maconpv-b and the reference baculovirus, acmnpv. the information is summarized in table  and additional file as expected, the two viruses most closely related to psinsnpv are chchnpv and tnsnpv, sharing similar genome sizes and possessing high nucleotide sequence similarity. however, the psinsnpv genome is  bp larger than tnsnpv and  bp smaller than chchnpv. global alignment and dot matrix analysis of the psinsnpv genome compared with chchnpv, tnsnpv, maconpv-b and acmnpv revealed that psinsnpv is highly similar and collinear with chchnpv and tnsnpv, but not with maconpv-b and acmnpv. psinsnpv shares orfs with acmnpv, orfs with macomnpv-b, orfs with chchnpv and orfs with tnsnpv. the orfs reported as unique in chchnpv and in tnsnpv showed similarity to psinsnpv orfs- − − − and psinsnpv orf- − respectively.table characteristics of the psinsnpv genome compared with other alphabaculoviruses features baculoviruses psinsnpv acmnpv maconpv-b chchnpv tnsnpv genome size gc content orfs coding sequence hr sequences bro genes mean % aa id with psinsnpv homologs in psinsnpv orfs unique to psinsnpv genbank accession numberdot plot matrix analysis using lbdotview v. software. blue dots represent the homolog regions of the psinsnpv genome compared with chchnpv, tnsnpv, acmnpv and maconpv-b genome, both in the direct orientation, and red dots compare the psinsnpvgenome in the direct orientation with npv genomes in the reverse orientation. replication, transcription and structural genes the baculovirus genes are categorized based on their functions during the viral cycle as follows: dna replication, rna transcription, odv and bv structural proteins or oral infectivity proteins. baculovirus genome replication mechanisms are still not fully understood. several studies have been developed to try to identify the genes responsible for dna replication and translation. the essential dna replication factors late expression factor, lef- lef- dna polymerase, p, 38 k, helicase and immediate early homologs are all present in the psinsnpv genome. in addition, the psinsnpv genome contains genes homologous to proliferating cell nuclear antigen, major early-transcribed protein, dna binding protein, alkaline exonuclease and exon-0/ie- which were not identified in all baculoviruses but may influence viral dna replication. the acmnpv transcription system is activated in two main stages. at first, lef- lef- lef- and p are transcribed to encode the subunits of the viral rna polymerase complex. this complex acts on gene transcription and mrna processing, including capping and polyadenylation. then, the transcription enhancers lef- and very late factor are transcribed. all these genes were also found in the psinsnpv genome. in addition, some supposedly non-essential genes involved in acmnpv transcription regulation are also present in the psinsnpv genome: lef- lef- 39 k, lef- and protein kinase. the psinsnpv genome has known baculovirus genes coding for structural proteins. genes for odv and bv structural proteins include polyhedrin, orf pk occlusion derived virus envelope protein, occlusion derived virus enveloped capsid protein, p viral protein, few polyhedra protein/25 k, desmoplakin, 41-kda glycoprotein, telokinin-like peptide, viral protein, vp p odv-e p87/vp odv-ec odv-e p calyx/polyhedrin enveloped protein, p per os infectivity factor, pif- pif- pif- odv-e28/pif- odv-e56/pif- and fusion protein. the six pif genes which are components of odvs and are involved in oral infectivity exhibited high sequence similarity to chchnpv and tnsnpv pifs. the genus alphabaculovirus is divided into groups i and ii based on gene content, and in particular, the bvs fusion protein: gp and f protein, respectively. psinsnpv, a group ii alphabaculovirus, possesses the expected f protein homolog. nucleotide metabolism and dna repair several group ii alphabaculoviruses and betabaculoviruses encode genes involved in nucleotide biosynthesis. psinsnpv possesses the ribonucleotide reductase large and small subunits and the dutpase protein. these rr proteins are enzymes involved in the formation of deoxyribonucleotides from ribonucleotides. the dutpase protein is responsible for preventing incorporation of mutagenic dutp into dna. poly polymerase and poly adp-ribose glycohydrolase are enzymes involved in synthesis of adp riboses that activate and recruit dna repair enzymes. although it has been reported that all group ii genomes encode parg homologs, the psinsnpv genome is notable for its absence. the cpd photolyase, encoded by the dna photolyase gene, acts at cyclobutane pyrimidine dimers to repair ultraviolet -induced dna damage. the phr gene was identified in chchnpv, tnsnpv, plusia acuta npv and thysanoplusia orichalcea npv-b. studies suggest that the phr gene is conserved in group ii alphabaculoviruses that infect lepidopteran insects in the plusiinae subfamily of the noctuidae family. however, the phr gene was also identified in baculoviruses that infect insects of other subfamilies, such as spodoptera litura gv, clanis bilineata npv, apocheima cinerarium npv and ampelophaga rubiginosa npv. psinsnpv belongs to a group where the phr gene is conserved and, as expected, its genome encodes a cpd photolyase protein. the complete nucleotide sequence of the psinsnpv phr gene is of  bp with gc % =  %. the deduced phr amino acid sequence of psinsnpv, chchnpv- phr −phr and tnsnpv were aligned, revealing that the psinsnpv photolyase possesses high identity to tnsnpv and chchnpv- phr. previous studies showed that chchnpv - phr is not active when tested in an escherichia coli photolyase deficient strain. the active copy is distinct in its possession of two conserved tryptophan residues, which may be involved in an electron transfer mechanism. in the psinsnpv photolyase protein, the tryptophan residues are replaced by histidine and tyrosine in positions and aa, respectively. therefore, both tryptophans are absent in the psinsnpv photolyase, suggesting that this protein might not be active. the partial phr amino acid sequences of psinnpv –gt; −gt, psinnpv - usa and psinsnpv-ia to -ig isolates described in the literature were aligned, where psinnpv-gt and psinnpv – usa isolates showed high similarity to the psinsnpv-ia to -ig isolates. interestingly, in contrast to other psinnpv isolates reported so far, psinnpv - gt possesses a tryptophan residue at position which is thought to be essential for enzyme activity. further studies are needed to confirm and investigate the activity of the psinsnpv - gt photolyase. auxiliary genes the auxiliary genes viral ubiquitin, viral cathepsin, chitinase, 37-kda glycoprotein, conotoxin, superoxide dismutase, fibroblast growth factors, phosphotyrosine phosphatase, ecdysone glucose transferase, actin rearrangement infectivity factor, inhibitor of apoptosis, iap- and p35/p were found in the psinsnpv genome. the auxiliary genes are non-essential for dna replication, translation or viral particle formation. however, these genes confer selective advantages to viruses as has been observed in homologs of psinsnpv auxiliary genes described in the literature. the activity of the v-cath and chia genes is notable in p. includens larvae infected with psinsnpv, where the encoded proteins cause degradation and liquefaction of the host cadaver. the fgf, ptp and egt genes were reported to be involved in host hyperactive behaviors, increasing larval motility and preventing the molt to extend insect life, respectively. the ptp gene was previously reported to be only present in group i npvs, however this gene is present in the psinsnpv genome. homologous regions are absent from the psinsnpv genome homologous regions are repeated sequences with an imperfect palindromic core that are distributed in the genome as singletons or arranged in tandem. these repeat sequences are present in baculovirus genomes and other closely related invertebrate viruses. these regions act as enhancers of early gene transcription in npvs and may serve as origins of replication in npvs and gvs. homologous regions are a common feature found in genomes of the four genera of the baculoviridae family. however, no typical baculoviral hrs were found in the psinsnpv genome, which is also the case in buzura supressaria npv, chchnpv, tnsnpv and agrotis segetum gv. psinsnpv bro genes the psinsnpv genome sequence contains two baculovirus repeated orfs, named according to their order in the genome: bro-a and bro-b. the bro genes commonly occur in alpha-, beta- and gammabaculoviruses, varying in number of copies and length among the viruses. these genes were first reported from baculoviruses, but bro gene homologs were subsequently identified in other insect dsdna viruses, such as entomopoxvirus and entomoiridovirus. bro proteins exhibit a highly conserved n-terminal dna binding domain in the first 100– aa and a variable c-terminal domain. the functions of bro proteins are not clear, but were proposed to be involved in host dna replication and/or transcriptional regulation and as a viral replication enhancer in the late phase. although a deletion of 425 bp is present in the psinsnpv bro-b gene compared with the chchnpv bro-b gene, the genes share 72% identity. the psinsnpv bro-a gene showed higher similarity to the lymantria xylina mnpv bro-m gene and the mamestra brassicae mnpv bro-a gene with and 53% identity, respectively. in contrast to the psinsnpv bro-b protein with one bro-n domain, psinsnpv bro-a protein contains two bro-n domains at amino acid position to and to in addition, the psinsnpv bro-a protein contains a domain of unknown function duf in amino acid position to although psinsnpv, chchnpv and tnsnpv are closely related, their bro genes do not show high similarity. orfs unique to psinsnpv two putative orfs, psin and psin were found to be unique to the psinsnpv genome. these orfs do not show significant similarity to other previously described baculovirus orfs and exhibit signature sequences that describe domains predicted by interproscan. psin is predicted to encode a amino acid protein with molecular weight of  kda and shows low homology to a signal transducer and activator of transcription protein in the avian species, pseudopodoces humilis. psin is predicted to encode a aa,  kda protein, but shows no significant similarity to any genes in genbank databases. the taag late promoter motif, combined with a tata early promoter, was identified about 100 bp upstream of both the psin and psin start codons. these promoters are thought to be transcribed both by the host rna polymerase ii and viral rna polymerases, suggesting that these genes could be expressed both early and late in infection. a search was made for protein families, domains and functional sites found in transmembrane domains in the predicted psin and psin proteins. using tmhmm server v, transmembrane helices from amino acid position to and to in psin and to in psin hypothetical proteins were predicted. two p homologs in psinsnpv two p gene homologs were identified in the psinsnpv genome. the function of the p gene is not well understood, but studies have shown that deletion of the acmnpv p gene produced no differences in phenotype from wild-type acmnpv in cells and in larvae. however, a combined deletion of p p and p genes in acmnpv resulted in polyhedra containing few or no virions. for this reason, p is thought to be required for optimal virion occlusion in the obs. one or more copies of the p gene are present in all group i and ii alphabaculoviruses, except spodoptera littoralis multiple nucleopolyhedrovirus, splinpv. the viruses with more than one p copy belong to group ii alphabaculoviruses. however, in group i alphabaculovirus genome sequences available at genbank, choristoneura fumiferana mnpv, choristoneura occidentalis npv and choristoneura rosaceana npv contain two p homologs.table amino acid sequences used in phylogenetic analysis of the p amino acid sequence position alphabaculoviruses abbreviation genbank 1 a 2 b 3 c group i group ii ap orf number adjacent to p gene. bp orf number adjacent to iap- gene. cp orf number adjacent to ptp − and − genes. the p gene is conserved in position, adjacent to the p gene in all the alphabaculoviruses containing a single copy. the psinsnpv p26a/orf- and p26b/orf- are positioned adjacent to the p gene and adjacent to the iap- gene, respectively. the copy adjacent to the iap- gene is also positionally conserved in all group ii alphabaculoviruses containing two p copies. however, the second p copy in group i alphabaculoviruses is positioned adjacent to the ptp and ptp genes. the phylogenetic tree obtained by bayesian phylogenetic inference using the p copies found in all alphabaculovirus genomes so far sequenced showed four clearly defined clades nested within the larger clades i and ii. the clade ia contains p copies from group i alphabaculoviruses and clade ii from group ii alphabaculoviruses. clade ib contains p copies also from group ii alphabaculoviruses, except for a monophyletic group with cfmnpv_orf chocmnpv_orf and chronpv_orf from group i alphabaculoviruses. clades i and ii correlate with the position of the genes in the genome, where clade i contains the p copies adjacent to the p gene, except for cfmnpv_orf chocmnpv_orf and chronpv_orf which are adjacent to ptp and ptp, and clade ii contains the p copies that are adjacent to iap-. this clustering pattern suggests the occurrence of three independent acquisition events of the p gene by baculoviruses. the first acquisition event occurred in position of the common ancestral genome of all baculoviruses containing this gene. the second acquisition event generated the p second copy in position of the group ii alphabaculovirus genome with two p homologs. finally, the third acquisition event occurred in cfmnpv, chocnpv and chronpv, inserting the second p copy in position figure bayesian phylogram based on p deduced amino acid sequences. the clustering pattern suggests the occurrence of three independent p gene acquisition events that are marked by circles. the arrow indicates the node where occurred the signal peptide acquisition. numbers at branches indicate the bayesian posterior probabilities. the tree was rooted by midpoint approach. the scale bar indicates the number of substitutions per site. the acquisition of baculovirus genes may be the results of duplication and horizontal gene transfer by transposable element and/or homologous recombination. the second acquisition event probably occurred by horizontal gene transfer. in this case, the gene duplication hypothesis can be refuted, since the similarity between p copies in the same virus is low. furthermore, clades i and ii are clearly separated, indicating that the p copies did not originate from a common, recent ancestor. in the third acquisition event, there is high similarity between the second p copy of the group i alphabaculoviruses and first p copy of the group ii alphabaculoviruses, grouping these genes in the same clade. therefore, the second p copy of the group i alphabaculoviruses may have been acquired from a group ii alphabaculovirus. the isoelectric point and molecular weight of the deduced p protein from alphabaculoviruses with two p copies were calculated, and are presented in the context of their genomic positioning. p protein from position showed an average pi of  ±  and average mw of  ± ; position p proteins have an average pi of  ±  and average mw of  ± ; and position p proteins have an average pi of  ±  and an average mw of  ±  the mean scores were examined using the student’s t-test and the pi average showed a significant difference between the three positions. the isoelectric point difference suggests that the p sequence in position exhibits more basic amino acids than in those in positions and the presence and location of signal peptide cleavage sites in p26a and p26b amino acid sequences from psinsnpv were analysed using signalp v.. the p26a protein showed a signal peptide cleavage site at amino acid residues to and the cleavage site between amino acids and. however, the signal peptide cleavage site was absent in the p26b protein. signal peptides direct the proteins to their proper cellular and extracellular locations. the export of proteins occurs via the secretory pathway, where proteins labeled by an n-terminal signal sequence are translocated across the cytoplasmic membrane, whereafter the n-terminal signal peptide is usually cleaved by an extracellular signal peptidase. the signal peptide cleavage sites were predicted for other p proteins of alphabaculoviruses with complete sequenced genomes and the results are shown in table  the presence or absence of signal peptides in p proteins correlates with the clustering obtained in the phylogenetic analysis, where only p proteins from clade ib possess signal peptides. however, four sequences belonging to this clade, lesenpv_orf ecobnpv_orf orlenpv_orf and chchnpv_orf do not possess a signal peptide. the presence or absence of the signal peptide may have led to the differential results found in predicted molecular weight and isoelectric points between the p proteins analyzed. the presence of a signal peptide in the first p copy of group ii alphabaculoviruses and in the second p copy of group i alphabaculoviruses suggests that this domain was acquired from a common ancestor of these viruses. although the function of p is not well understood, the signal peptide may lead to differences in activity of the clade ib proteins compared to the p from other clades, which warrants further investigation.  in summary, the complete psinsnpv-ie genome sequence is apparently different from other baculoviruses sequenced so far. the genome does not contain the typical baculovirus hrs and contains two orfs with predicted transmembrane domains that are unique to psinsnpv. the psinsnpv genome, however, exhibits high sequence similarities and co-linearity to the closely related chchnpv and tnsnpv. the psinsnpv genome contains two p copies and a phylogenetic analysis of p sequences of alphabaculoviruses showed three potential acquisition events of these genes within the baculoviridae family. one of the clades comprises p protein with a signal peptide, indicating a possible distinct function from other classes of p protein. however, further investigations are needed for a better understanding of this protein in baculoviruses. this research reports the first completely sequenced genome of a strain of psinsnpv, a currently little known baculovirus. it is anticipated that this data will both promote advances in investigations of its molecular biology and gene function and accelerate its development as a biocontrol agent. 
pseudoplusia includens single nucleopolyhedrovirus is a baculovirus recently identified in our laboratory, with high pathogenicity to the soybean looper, chrysodeixis includens. the circular double-stranded dna genome is  bp in length, with a gc content of % and contains open reading frames. the p phylogenetic tree suggests that this gene was obtained from three independent acquisition events within the baculoviridae family. p is thought to be required for optimal virion occlusion in the occlusion bodies, but its function is not well characterized. the presence of a signal peptide only in the psinsnpv p26a/orf- homolog indicates distinct function between the two p proteins. in brazil, the c. includens caterpillar is an emerging pest and has caused significant losses in soybean and cotton crops. the complete genome sequence of psinsnpv will provide a valuable resource, contributing to studies on its molecular biology and functional genomics, and will promote the development of this virus as an effective bioinsecticide. psinsnpv possesses the conserved baculovirus core genes, genes found in other baculoviruses and unique orfs. the psinsnpv genome was determined and the phylogeny of the p gene within the family baculoviridae was investigated. the homologous repeats are absent from the psinsnpv genome, which is also the case in chchnpv and tnsnpv. two p gene homologs were found in the psinsnpv genome. two baculovirus repeat orfs homologs, bro-a and bro-b, were identified and compared with chrysodeixis chalcites nucleopolyhedrovirus and trichoplusia ni single nucleopolyhedrovirus bro genes and showed high similarity, suggesting that these genes may be derived from an ancestor common to these viruses.
 pseudoplusia includens single nucleopolyhedrovirus is a baculovirus recently identified in our laboratory, with high pathogenicity to the soybean looper, chrysodeixis includens. in brazil, the c. includens caterpillar is an emerging pest and has caused significant losses in soybean and cotton crops. the psinsnpv genome was determined and the phylogeny of the p gene within the family baculoviridae was investigated.  the complete genome of psinsnpv was sequenced, annotated and compared with other alphabaculoviruses, displaying a genome apparently different from other baculoviruses so far sequenced. the circular double-stranded dna genome is  bp in length, with a gc content of % and contains open reading frames. psinsnpv possesses the conserved baculovirus core genes, genes found in other baculoviruses and unique orfs. two baculovirus repeat orfs homologs, bro-a and bro-b, were identified and compared with chrysodeixis chalcites nucleopolyhedrovirus and trichoplusia ni single nucleopolyhedrovirus bro genes and showed high similarity, suggesting that these genes may be derived from an ancestor common to these viruses. the homologous repeats are absent from the psinsnpv genome, which is also the case in chchnpv and tnsnpv. two p gene homologs were found in the psinsnpv genome. p is thought to be required for optimal virion occlusion in the occlusion bodies, but its function is not well characterized. the p phylogenetic tree suggests that this gene was obtained from three independent acquisition events within the baculoviridae family. the presence of a signal peptide only in the psinsnpv p26a/orf- homolog indicates distinct function between the two p proteins.  psinsnpv has a genomic sequence apparently different from other baculoviruses sequenced so far. the complete genome sequence of psinsnpv will provide a valuable resource, contributing to studies on its molecular biology and functional genomics, and will promote the development of this virus as an effective bioinsecticide. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. issue-copyright-statement© the author 2015 