BACKGROUND
the family baculoviridae consists of viruses that contain circular dna genomes ranging in size from approximately  <dig> to  <dig> kbp.
this family of viruses has only been shown to be pathogenic to arthropods particularly insects  <cit>  and have a very variable g+c content that ranges from 28% to  <dig> %  <cit> .
the virions have a complex construction and consist of an envelope and a nucleocapsid.
baculoviruses are divided into nucleopolyhedrovirus  and granulovirus  generally.
based on phylogenetic analysis, baculoviruses can be classified into five major groups including gvs, group i and group ii npvs, a group of the dipteran viruses, culex nigripalpus npv  <cit>  and a group of the hymenopteran viruses  <cit> .
npvs are mainly found in lepidoptera and other insects such as hymenoptera, diptera, coleoptera, thysanura and trichoptera.
the virions contain either single or multiple nucleocapsids.
all hymenopteran npvs virions contain single nucleocapsid.
gvs have been found only in lepidoptera  <cit> .
previous reports demonstrated that there are  <dig> conserved genes present in all baculoviruses and  <dig> conserved genes present in all lepidopteran baculoviruses  <cit> , and a total of  <dig> genes are present in all lepidopteran npvs <cit> .
to date, the complete genomes of  <dig> npvs and  <dig> gvs are published or available in genbank.
<dig> npv genomes have been published from autographa californica npv   <cit>  to neodiprion abietis npv   <cit> .
<dig> gv genomes published include xestia c-nigrum gv  <cit> , plutella xylostella gv  <cit> , cydia pomonella gv  <cit> , adoxophyes orana gv <cit> , cryptophlebia leucotreta gv  <cit>  and choristoneura occidentalis gv <cit> .
the chinese tussah silkworm , antheraea pernyi , is an insect that can spin silk cocoons and eats the leaves of oak trees.
as an important economic insect, antheraea pernyi is commercially cultivated mainly in middle and northeastern china.
it is also used as a source of food and for cosmetics.
antheraea pernyi npv  can infect antheraea pernyi resulting in nuclear polyhedrosis that can potentially result in an outbreak of infectious disease.
the average incidence rate of this disease reaches approximately 30% and occasionally reaches above 70%.
the yield of cocoons can be decreased by 30% when anpenpv occurs.
this viral disease has brought about huge economic losses to sericulturist in china.
anpenpv belongs to the lepidopteran family of npv.
the physical map for anpenpv genome has been constructed and the genome was estimated at approximately  <dig> kbp and  <dig> kbp in size, respectively  <cit> .
our group and others have reported the sequences of several anpenpv genes including lef- <dig>  lef- <dig>  polyhedrin <cit> , lef- <dig>  cathepsin, chitinase <cit> , genes ie- <dig>  pe <dig> ,truncated egt, lef- <dig> , odv-e <dig> , lef- <dig>  and gp <dig> <cit> .
a total of approximately  <dig> kbp of the genome of anpenpv has been sequenced and released in genbank.
in this paper, we report the complete sequence and organization of the anpenpv genome.
the phylogeny of anpenpv in comparison with previously published baculovirus genomes is also addressed.
sequence and analysis of the anpenpv genome suggest significance for the epidemiological studies in the research community.
RESULTS
sequencing, assembly, and analysis of the anpenpv genome
a total of  <dig> recombinant plasmids containing cloned viral dna fragments  were obtained.
the size of most of the inserted fragments ranged from  <dig>  to  <dig>  kbp.
both ends of the inserted fragments were sequenced.
the complete sequence of anpenpv genome was assembled from the sequenced restriction fragments and pcr amplified products.
approximately over  <dig> times coverage of the genome was achieved.
the entire anpenpv dsdna genome was sequenced and assembled into a contiguous sequence of  <dig>  bp with a g+c content of  <dig> % .
of the previously published sequences of anpenpv, only gp <dig>  showed sequence differences .
the reason may be the variation of different virus isolates.
the cytosine of the complement of stop codon of polyhedrin was designated as the first nucleotide of the genome.
compared with other baculovirus genomes, the size of anpenpv genome was larger than those of nelenpv, nesenpv, cuninpv, epponpv, adhonpv, plxygv, adorgv, phopgv and crlegv.
the g+c content of the opmnpv and ldmnpv genomes was higher than that of the anpenpv genome.
only three hrs were identified in anpenpv genome, but twenty-four perfect or imperfect drs were found in the intergenic spacer regions.
in addition,  <dig> drs were also found in the intragenic spacer regions of anpenpv genome.
the number of drs in anpenpv is by far the most in any baculovirus genome.
the results of the comparisons between anpenpv and other baculovirus genomes are listed in additional files .
identification of anpenpv orfs
a total of  <dig> orfs, with no or minimal overlap and encoding putative proteins of more than  <dig> amino acid residues, were identified .
an exception regarding the size of the orf was made for anpe <dig> which encoded a protein with  <dig> amino acid residues and showed high homology with the ctl- <dig> gene of other baculoviruses.
the anpenpv orfs demonstrated no preference in terms of orientation  forward and 52%  reverse) or clustering based on putative function or expression.
of the  <dig> identified orfs,  <dig> had homologues in at least one other baculovirus and  <dig> were unique to anpenpv.
orf encoding nucleotides accounted for  <dig>  bp, hrs for  <dig>  bp and the remaining  <dig>  bp represented intergenic regions.
the g+c content of the coding regions was 55%, but the g+c content of intergenic regions was only  <dig> %, which was significantly lower than that of coding regions.
the results of orfs identified in anpenpv were listed in additional file  <dig> and plotted in figure  <dig>  as a comparison, the cfmnpv and opmnpv orfs were shown along with anpenpv orfs in figure  <dig>  codon usage for the  <dig> orfs is shown in additional files .
classification of genes in anpenpv and other baculoviruses
the classification of genes encoded in different baculovirus genomes may help to determine essential genes for virus survival and to understand features including host range, virulence and morphology.
the genes found in all baculoviruses are more likely to be essential, whereas auxiliary genes found in only some baculoviruses may give viruses a selective advantage in nature  <cit> .
we classified  <dig> orfs into six groups: 1) orfs found in all baculoviruses; 2) orfs found in all lepidopteran baculoviruses; 3) orfs found in all lepidopteran npvs; 4) orfs found in all group i npvs; 5) orfs found in partial group i npvs; 6) orfs that are unique to anpenpv.
the detailed results were shown in additional files  and figure  <dig>
in first group, the  <dig> genes present in all baculovirus genomes reported previously were also found in anpenpv genome .
comparison of these  <dig> genes between anpenpv and the baculovirus type species acmnpv dedicated that their organization was consistent with group i npvs.
the  <dig> genes are more likely to be essential for all baculoviruses survival.
lauzon et al.
identified  <dig> orfs present in all lepidopteran baculoviruses.
all of these orfs were found in anpenpv .
the  <dig> conserved genes are more likely to be essential for all lepidopteran baculoviruses survival.
in addition to the genes present in all baculovirus and all lepidopteran baculoviruses described above,  <dig> orfs in anpenpv were identified in all lepidopteran npvs .
some of these orfs were also present in one or more gvs.
of these  <dig> genes,  <dig> were found exclusively in all lepidopteran npvs , anpe <dig> , anpe <dig> , anpe <dig> , anpe <dig> , anpe <dig> ).
de jong et al.
identified  <dig> orfs exclusive to all lepidopteran npvs including ac <dig>  ac <dig> and ac <dig> <cit> .
however, due to the absence of ac <dig> in chchnpv <cit> , ac <dig> and ac <dig> in adhonpv <cit> , these  <dig> orfs are now excluded from this class of orfs.
including these  <dig> genes, there were still  <dig>  lepidopteran npv specific orfs.
in the work of de jong et al.,  <dig> baculovirus orfs were also identified in all lepidopteran npvs <cit> , however these  <dig> are not the same as those identified here .
in this class of genes, anpe <dig> had only a partial egt , which matched to 5' end of egt gene of other lepidopteran npvs by blastp, and the amino acid identity ranged from 43%  to 70%  for group i npvs.
egt is a auxiliary gene in lepidopteran npvs, which is not essential for replication but may facilitate a selective advantage in nature  <cit> .
another  <dig> orfs in anpenpv were found in all group i npvs.
of the  <dig> orfs,  <dig> were found exclusively in all group i npvs by de jong et al.
and hyink et al.
<cit> , all of which were also found in anpenpv including anpe <dig>  anpe <dig> , anpe <dig>  anpe <dig> , anpe <dig> , anpe <dig> , anpe <dig> , anpe <dig> , anpe <dig> , anpe <dig> , anpe <dig> and anpe <dig>  .
<dig> orfs of anpenpv  had no recognizable baculovirus homologs .
a homologous region between the anpe <dig> and microtuble associated protein of drosophila melanogaster was found by blast search.
the microtuble associated protein is required for the integrity of mitotic spindle.
additionally, a region of anpe <dig> showed homology to a carbamate kinase gene of streptococcus ratti.
in addition, another  <dig> orfs  were considered in the class of orfs found in some group i npvs.
these genes may be auxiliary genes and give viruses a selective advantage in nature.
in these orfs, anpe <dig> and anpe <dig> were determined as bro, a baculovirus repeated orf, which are found in almost all baculoviruses.
cfdeforf <dig> and hycuorf <dig> were identified as genes unique to cfdefnpv and hycunpv, respectively, but alignment of anpe <dig> to cfdef <dig> and anpe <dig> to hycu <dig> revealed high conservation .
anpe <dig> was a homologue of a gene only found in hycunpv and opmnpv .
anpe <dig> and anpe <dig> were homologs of genes observed only in acmnpv and ronpv.
alignment of the deduced amino acid sequence of anpe <dig>  to ac <dig>  and ro <dig>  showed high conservation in their amino acid sequences.
at the same time, anpe <dig>  ac <dig>  and ro <dig> shared a  <dig> residue-long nk  motif.
the percentage identity within these regions  suggested that they are functionally important regions.
additionally, multiple alignment of anpe <dig>  to ac <dig> and ro <dig> revealed that anpe <dig> matched to two regions of ac <dig> and ro <dig> in the 5' and 3'ends .
on the other hand, anpe  <dig> also matched to  <dig> k gene of some group ii npvs and gvs in the two terminal regions.
this implied that the function region of  <dig> k may be in the two regions.
similarly, anpe <dig> was homologous to eukaryotic trex gene, which was observed only in cfmnpv, cfdefnpv and agmnpv.
the trex gene encodes a 3' to 5' exonuclease  <cit> .
also, multiple alignment of anpe  <dig> to cf <dig>  and cfdef <dig>  appeared to be highly conservative in their amino acid sequences.
furthermore, anpe <dig> was 87%  identical at the amino acid level to v-trex gene of agmnpv.
anpe <dig>  cf <dig>  cfdef <dig> and the v-trex gene of agmnpv had a  <dig> residue-long exoiii motif .
the percentage identity  within these regions indicated that this region was also functionally important.
this kind of genes may give anpenpv a selective advantage in nature.
functional genes
baculoviruses contain a lot of genes involved in essential and selective functions, such as viral replication, transcription, inhibition of apoptosis, protein structure, assembly and auxiliary.
besides, there were some conserved genes, of which functions were not clearly identified.
all of the conserved function genes, previously identified in all baculoviruses and all lepidopteran-specific baculoviruses  <cit> , were also found in anpenpv.
several variable function genes identified in some baculoviruses and some conserved baculovirus orfs of unknown function were also found in anpenpv.
the detailed functions of these genes are listed in additional files .
whereas, several variable dna replication genes, including helicase- <dig>  dna-ligase, dutpase , rnase reductase- <dig>  and rnase reductase- <dig> , were not found in anpenpv.
five genes have so far been implicated as inhibitors of apoptosis, including iap- <dig>  iap- <dig>  iap- <dig>  iap- <dig> and p <dig>  however, only iap-1and iap- <dig> were identified in anpenpv and considered as genes present in all group i npvs.
baculovirus genomes typically encode auxiliary genes that are non-essential for replication but provide a selective advantage to the virus  <cit> .
anpenpv contained some auxiliary genes including ctl- <dig> and ctl- <dig> , which are small disulfide-rich ion channel antagonists.
ctl <dig> was found in acmnpv, opmnpv, ldmnpv, cfmnpv and hycunpv, but ctl <dig> was only found in opmnpv, hycunpv and ldmnpv.
numerous structural genes were also found in anpenpv, which composed the structure of virus.
three genes in anpenpv, pif- <dig>  pif- <dig> and p <dig> , were identified as structural genes as well as per os infectivity factor involved in baculovirus infection to midgut epithelial cells of the host.
other structural genes identified in some baculoviruses but not in anpenpv included homologues to vef- <dig>  vef- <dig>  vef- <dig>  pk <dig>  vefs have been found to dramatically increase the infectivity of baculoviruses in non-natural lepidopteran hosts  <cit>  and only found in gvs and group ii npv.
hrs repeats and the genes around them
most baculoviruses contain hr regions.
an individual npv hr is typically comprised of direct repeats usually centered around a perfect or imperfect palindrome.
hrs have been implicated as origins of dna replication  <cit>  and as enhancers of rna polymerase ii-mediated transcription  <cit> .
gv hrs are different from npv hrs except for plxygv, whose hrs appear more similar to those from npvs.
gv hrs usually lack a palindrome and are homologous to all other hrs within the same genome, but there are no two gvs that have similar hr regions.
gv hrs are characterized to be repeat regions that are variable and at-rich  <cit> .
three hrs have been identified in the anpenpv genome.
the number of repeats per hr ranged from two in hr <dig> to nine in hr <dig>  the multiple alignment of the anpenpv hrs  showed a high degree of conservation within the hrs and the hr palindrome consensus sequence, agcdatgcgtcagcgcygayybtgcttttcragtayarccawtctwgaaaamcgt.
this palindrome consensus is also found in hycunpv, cfmnpv, opmpv, bmnpv, acmnpv, cfdefnpv and agmnpv .
there was no interval between each repeat unit of hr <dig> and hr <dig> in the anpenpv genome.
this was different from that of some other npv hrs, in which an interval between each repeat unit is found.
the repeats of hr <dig> were nearly the same.
the even repeats of hr <dig> hr1b,hr1d,hr1f,hr1h, hr1j were the same too.
the odd repeats appeared to be formed by even repeats in which a and t was alternately replaced with g .
the average g+c content of the hrs was  <dig> %, significantly lower than the  <dig> % for the complete anpenpv genome.
the relative location of hrs in anpenpv genome was also characterized.
the genes that flanked the hrs were mainly similar between anpenpv and the other seven group i npvs .
anpenpv hr <dig> was in the same relative genomic location as epponpv hr <dig>  opmnpv hr <dig> and cfdefnpv hr <dig> between gene p <dig> and me <dig>  anpenpv hr <dig> was in the same relative genomic location as cfmnpv hr <dig>  epponpv hr <dig>  opmnpv hr <dig>  acmnpv hr  <dig>  romnpv hr <dig> and cfdefnpv hr <dig> between gene pif- <dig> and chitinase.
anpenpv hr <dig> was in the same relative genomic location as cfmnpv hr <dig>  epponpv hr <dig>  bmnpv hr <dig>  acmnpv hr <dig>  romnpv hr <dig> and cfdefnpv hr <dig> near gene op <dig> and fgf.
it proved that the group i npvs showed conserved genomic locations for certain hrs.
furthermore, the unique gene anpe <dig> for anpenpv and unique gene anpe <dig> for anpenpv and opmnpv existed around hr <dig> and hr <dig>  respectively .
instead of a putative hr between odv-e <dig> and ie- <dig> that is present in hycunpv, cfmnpv and epponpv, this was replaced in anpenpv by the unique gene, anpe <dig> .
furthermore, instead of the apoptosis inhibiting gene iap- <dig> located between the sod and fgf genes in some other group i npvs, the iap- <dig> homolog was putatively replaced by unique gene anpe <dig> and ctl- <dig> gene in anpenpv .
also, the relative direction of transcription for fgf and sod around hr <dig> in anpenpv was different from that of other group i npvs, except for that of hycunpv .
only in anpenpv, hycunpv and opmnpv was the odv-e <dig> and ie- <dig> transcription in the same relative orientation .
this was consistent with the description by de jong et al.
that a high degree of variability existed around the hrs and by ahrens et al.
that major rearrangements, insertions and deletions exist around the hrs in acmnpv and opmnpv <cit> .
in particular, the v-trex gene homolog  was inserted between pif- <dig> and chitinase around hr <dig> in anpenpv.
besides anpenpv, the v-trex gene was inserted only into cfmnpv and cfdefnpv at the same location  around cf-hr <dig> and cfdef-hr <dig> respectively.
using blastp, we found that the anpe <dig> had high amino acid identity  to trex genes from some eukaryotes, such as canis familiaris, rattus norvegicus, anopheles gambiae str.
pest,bos taurus, mus musculus, homo sapiens, drosophila melanogaster, and drosophila pseudoobscura.
by multi-aligning the deduced amino acid sequences of these genes, two conserved regions were found as following: -x- - -d- -e- -t- -i-p at the n terminus, and f-i-x-p-x-c-l-v-a- -n-g-x- -d-f- - - , respectively.
phylogenetic tree analysis  by clustal x supported the assumption that the v-trex was transferred horizontally, possibly from an insect host, which was similar with the assumption by slack et al.
and lauzon et al.
<cit> .
the v-trex gene product has the potential to be involved in virus recombination or uv-light tolerance, which could contribute to the high degree of variability existed around the hrs and may give anpenpv a tolerance advantage in nature.
similarly, anpe <dig> , anpe <dig>  and anpe <dig>  showed high amino acid sequence similarity with the homologues from insect hosts, respectively , implying evolutionary conservation of them, suggesting that baculovirus maybe acquire genes that were inserted into the regions around hrs from insect hosts.
only iap was studied in detail supporting the argument  <cit> .
drs repeats
direct repeats  as described by garcia-maruniak et al.
<cit>  are suggested to be a putative non-hr, ori-like regions containing an unusual at-rich sequences and perfect or imperfect short palindromes in intergenic spacer or within orfs.
these drs are not repeated elsewhere in the genome.
with the exception of nelenpv, the majority of npvs have hrs and many of these hrs have homologous palindromes core.
the nelenpv has only nine drs shared a closer similarity to the organization of repeat regions in gvs than to those in npvs and showed little similarity to typical npv hrs, which are characterized by the presence of perfect or imperfect palindrome and at rich  <cit> .
in comparison nesenpv not only has the hrs, but also has four drs <cit> .
similar to nesenpv,  <dig> perfect or imperfect drs were identified by tandem repeats finder software in addition to the hrs in anpenpv .
these drs contained from  <dig> to  <dig> repeats.
except for dr <dig>  they were found to be at-rich, especially dr <dig>  dr <dig>  dr <dig>  dr <dig>  dr <dig>  dr <dig>  dr <dig>  dr <dig>  dr <dig>  dr <dig>  dr <dig>  dr <dig>  dr <dig> and dr <dig>  palindromes ranging in size from  <dig> to  <dig> bp were found in several of the drs.
all of the drs were located inside intergenic spacer regions of the anpenpv genome.
the existence of twenty-four drs in the anpenpv intergenic spacer regions may reflect a different genome replication strategy for the npvs, and these drs may be a new type putative non-hr, ori-like region as is found in grlegv, cpgv, and adorgv.
in addition, nine tandem repeats were also identified in orfs of anpenpv genome with a high gc content .
at the same time, a specific gt-repeat was located from  <dig> to122259nt.
the sequences contained continuous gt residues with dinucleotide  repeats.
the gt-repeat was only found in opmnpv <cit> .
similar sequences of gt repeats have been characterized in other organisms, e.g., drosophila virilis <cit>  and caenorhabditis elegans  <cit> .
comparison of anpenpv and other baculoviruses
the gene order of anpenpv was compared with those of other group i npvs , two group ii npvs  and a gv  by gene parity plots  <cit> .
anpenpv shared  <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig> and  <dig> orfs with hycunpv, epponpv, cfmnpv, opmnpv, acmnpv, ronpv, bmnpv and cfdefnpv, respectively .
among the group ii npvs, anpenpv shared the highest number of orfs with maconpv a  and the least with adohnpv .
the organization of the anpenpv genome was collinear to that of hycunpv, epponpv, cfmnpv, and opmnpv .
in particular, anpenpv showed a high similarity to hycunpv and they shared a gene  unique to them.
the gene order in two and one regions of the anpenpv genome were inverted with respect to the corresponding regions of acmnpv, ronpv and bmnpv; cfdefnpv respectively .
collinearity was not found between anpenpv and maconpv a, hearnpv and xcgv.
similarity between the  <dig> core baculovirus genes between anpenpv and other group i npv was obviously higher than the similarities between anpenpv and other groups .
cuninpv was particularly dissimilar, of which similarities were  <dig> %– <dig> % range and average of  <dig> %.
phylogeny of anpenpv
based on concatenated  <dig> conserved genes, a phylogenetic tree was estimated for the  <dig> baculoviruses.
sequences were aligned using clustal w, with a gap penalty of  <dig>  an extend gap penalty of  <dig>  and a delay of divergence of 30%.
bootstrap values for  <dig>  replicates were given.
the baculovirus phylogenetic tree is shown in figure  <dig>  from the tree, anpenpv appeared as a group i npv and most closely related to hycunpv, opmnpv, cfmnpv, cfdefnpv, epponpv, and to a lesser extent to bmnpv, ronpv and acmnpv.
ikeda et al.
suggested that the close relationship among hycunpv, cfmnpv and opmnpv may be due to a geographical overlap among their hosts <cit> .
also, the relationship between anpenpv and the other npvs, especially hycunpv, may be due to the same reason.
hyphantria cunea was firstly introduced into dandong, liaoning province of china in  <dig>  which is the main distribution area for antheraea pernyi and has attacked many kinds of trees including oak tree  <cit> .
the anpenpv virus strain used in this study originated from liaoning province.
antheraea pernyi also has a narrow distribution in japan where hycunpv had been isolated since  <dig> <cit> .
CONCLUSIONS
in summary, we found that anpenpv is a member of group i npv and is most closely related to hycunpv opmnpv, epponpv, cfmnpv, cfdefnpv and to a lesser extent to bmnpv, ronpv and acmnpv based on the results of multi-alignment, phylogenetic analysis and gene parity plots.
analysis of the genome organization of anpenpv and other group i npvs demonstrated that there are one gene rearrange region in anpenpv with cfdefnpv, and two gene rearrange regions in anpenpv with bmnpv, ronpv and acmnpv.
despite its close relation to opmnpv, cfmnpv, hycunpv, epponpv and cfdefnpv, the anpenpv genome has several distinct features including the presence of  <dig> genes unique to anpenpv and  <dig> genes previously identified as new genes unique to cfdefnpv and hycunpv, respectively, and that, unlike other baculoviruses there are fewer hrs , more direct repeats  and a gt-repeat previously found only in opmnpv.
at the same time, analysis of the genes around the hrs of anpenpv supported the argument that an ancestral baculovirus acquired genes from ancestral insect hosts that were inserted into the regions around the hrs.
there are still  <dig> conserved genes present in all baculoviruses and  <dig> genes present in all lepidopteran baculoviruses.
besides genes present in all baculoviruses and lepidopteran baculoviruses,  <dig> orfs in anpenpv were identified in all lepidopteran npvs.
of the  <dig> orfs,  <dig> were found exclusively in all lepidopteran npvs , therefore the number of genes common to all lepidopteran npvs is  <dig>  however these  <dig> genes are not the same as those identified before.
in addition,  <dig> orfs are now exclusive to and present in all group i npvs.
only two bro genes  and two inhibitors of apoptosis  were found in anpenpv.
so far, two ctls,ctl <dig> and ctl <dig>  are found only in the genomes of anpenpv, hycunpv, opmnpv and ldmnpv.
these differences identified between anpenpv and other baculovirus genomes might provide insight into their evolution, host specificity and pathogenicity.
the sequence and analysis of anpenpv complete genome can provide a continuing resource to better follow the evolution and phylogeny of baculovirus.

 the family baculoviridae consists of viruses that contain circular dna genomes ranging in size from approximately to kbp. this family of viruses has only been shown to be pathogenic to arthropods particularly insects and have a very variable g+c content that ranges from 28% to %. the virions have a complex construction and consist of an envelope and a nucleocapsid. baculoviruses are divided into nucleopolyhedrovirus and granulovirus generally. based on phylogenetic analysis, baculoviruses can be classified into five major groups including gvs, group i and group ii npvs, a group of the dipteran viruses, culex nigripalpus npv and a group of the hymenopteran viruses. npvs are mainly found in lepidoptera and other insects such as hymenoptera, diptera, coleoptera, thysanura and trichoptera. the virions contain either single or multiple nucleocapsids. all hymenopteran npvs virions contain single nucleocapsid. gvs have been found only in lepidoptera. previous reports demonstrated that there are conserved genes present in all baculoviruses and conserved genes present in all lepidopteran baculoviruses, and a total of genes are present in all lepidopteran npvs. to date, the complete genomes of npvs and gvs are published or available in genbank. npv genomes have been published from autographa californica npv to neodiprion abietis npv. gv genomes published include xestia c-nigrum gv, plutella xylostella gv, cydia pomonella gv, adoxophyes orana gv, cryptophlebia leucotreta gv and choristoneura occidentalis gv. the chinese tussah silkworm, antheraea pernyi, is an insect that can spin silk cocoons and eats the leaves of oak trees. as an important economic insect, antheraea pernyi is commercially cultivated mainly in middle and northeastern china. it is also used as a source of food and for cosmetics. antheraea pernyi npv can infect antheraea pernyi resulting in nuclear polyhedrosis that can potentially result in an outbreak of infectious disease. the average incidence rate of this disease reaches approximately 30% and occasionally reaches above 70%. the yield of cocoons can be decreased by 30% when anpenpv occurs. this viral disease has brought about huge economic losses to sericulturist in china. anpenpv belongs to the lepidopteran family of npv. the physical map for anpenpv genome has been constructed and the genome was estimated at approximately kbp and kbp in size, respectively. our group and others have reported the sequences of several anpenpv genes including lef- lef- polyhedrin, lef- cathepsin, chitinase, genes ie- pe,truncated egt, lef-, odv-e, lef- and gp. a total of approximately kbp of the genome of anpenpv has been sequenced and released in genbank. in this paper, we report the complete sequence and organization of the anpenpv genome. the phylogeny of anpenpv in comparison with previously published baculovirus genomes is also addressed. sequence and analysis of the anpenpv genome suggest significance for the epidemiological studies in the research community.  sequencing, assembly, and analysis of the anpenpv genome a total of recombinant plasmids containing cloned viral dna fragments were obtained. the size of most of the inserted fragments ranged from to kbp. both ends of the inserted fragments were sequenced. the complete sequence of anpenpv genome was assembled from the sequenced restriction fragments and pcr amplified products. approximately over times coverage of the genome was achieved. the entire anpenpv dsdna genome was sequenced and assembled into a contiguous sequence of bp with a g+c content of %. of the previously published sequences of anpenpv, only gp showed sequence differences. the reason may be the variation of different virus isolates. the cytosine of the complement of stop codon of polyhedrin was designated as the first nucleotide of the genome. compared with other baculovirus genomes, the size of anpenpv genome was larger than those of nelenpv, nesenpv, cuninpv, epponpv, adhonpv, plxygv, adorgv, phopgv and crlegv. the g+c content of the opmnpv and ldmnpv genomes was higher than that of the anpenpv genome. only three hrs were identified in anpenpv genome, but twenty-four perfect or imperfect drs were found in the intergenic spacer regions. in addition, drs were also found in the intragenic spacer regions of anpenpv genome. the number of drs in anpenpv is by far the most in any baculovirus genome. the results of the comparisons between anpenpv and other baculovirus genomes are listed in additional files. identification of anpenpv orfs a total of orfs, with no or minimal overlap and encoding putative proteins of more than amino acid residues, were identified. an exception regarding the size of the orf was made for anpe which encoded a protein with amino acid residues and showed high homology with the ctl- gene of other baculoviruses. the anpenpv orfs demonstrated no preference in terms of orientation forward and 52% reverse) or clustering based on putative function or expression. of the identified orfs, had homologues in at least one other baculovirus and were unique to anpenpv. orf encoding nucleotides accounted for bp, hrs for bp and the remaining bp represented intergenic regions. the g+c content of the coding regions was 55%, but the g+c content of intergenic regions was only %, which was significantly lower than that of coding regions. the results of orfs identified in anpenpv were listed in additional file and plotted in figure as a comparison, the cfmnpv and opmnpv orfs were shown along with anpenpv orfs in figure codon usage for the orfs is shown in additional files. classification of genes in anpenpv and other baculoviruses the classification of genes encoded in different baculovirus genomes may help to determine essential genes for virus survival and to understand features including host range, virulence and morphology. the genes found in all baculoviruses are more likely to be essential, whereas auxiliary genes found in only some baculoviruses may give viruses a selective advantage in nature. we classified orfs into six groups: 1) orfs found in all baculoviruses; 2) orfs found in all lepidopteran baculoviruses; 3) orfs found in all lepidopteran npvs; 4) orfs found in all group i npvs; 5) orfs found in partial group i npvs; 6) orfs that are unique to anpenpv. the detailed results were shown in additional files and figure in first group, the genes present in all baculovirus genomes reported previously were also found in anpenpv genome. comparison of these genes between anpenpv and the baculovirus type species acmnpv dedicated that their organization was consistent with group i npvs. the genes are more likely to be essential for all baculoviruses survival. lauzon et al. identified orfs present in all lepidopteran baculoviruses. all of these orfs were found in anpenpv. the conserved genes are more likely to be essential for all lepidopteran baculoviruses survival. in addition to the genes present in all baculovirus and all lepidopteran baculoviruses described above, orfs in anpenpv were identified in all lepidopteran npvs. some of these orfs were also present in one or more gvs. of these genes, were found exclusively in all lepidopteran npvs, anpe, anpe, anpe, anpe, anpe ). de jong et al. identified orfs exclusive to all lepidopteran npvs including ac ac and ac. however, due to the absence of ac in chchnpv, ac and ac in adhonpv, these orfs are now excluded from this class of orfs. including these genes, there were still lepidopteran npv specific orfs. in the work of de jong et al., baculovirus orfs were also identified in all lepidopteran npvs, however these are not the same as those identified here. in this class of genes, anpe had only a partial egt, which matched to 5' end of egt gene of other lepidopteran npvs by blastp, and the amino acid identity ranged from 43% to 70% for group i npvs. egt is a auxiliary gene in lepidopteran npvs, which is not essential for replication but may facilitate a selective advantage in nature. another orfs in anpenpv were found in all group i npvs. of the orfs, were found exclusively in all group i npvs by de jong et al. and hyink et al., all of which were also found in anpenpv including anpe anpe, anpe anpe, anpe, anpe, anpe, anpe, anpe, anpe, anpe and anpe. orfs of anpenpv had no recognizable baculovirus homologs. a homologous region between the anpe and microtuble associated protein of drosophila melanogaster was found by blast search. the microtuble associated protein is required for the integrity of mitotic spindle. additionally, a region of anpe showed homology to a carbamate kinase gene of streptococcus ratti. in addition, another orfs were considered in the class of orfs found in some group i npvs. these genes may be auxiliary genes and give viruses a selective advantage in nature. in these orfs, anpe and anpe were determined as bro, a baculovirus repeated orf, which are found in almost all baculoviruses. cfdeforf and hycuorf were identified as genes unique to cfdefnpv and hycunpv, respectively, but alignment of anpe to cfdef and anpe to hycu revealed high conservation. anpe was a homologue of a gene only found in hycunpv and opmnpv. anpe and anpe were homologs of genes observed only in acmnpv and ronpv. alignment of the deduced amino acid sequence of anpe to ac and ro showed high conservation in their amino acid sequences. at the same time, anpe ac and ro shared a residue-long nk motif. the percentage identity within these regions suggested that they are functionally important regions. additionally, multiple alignment of anpe to ac and ro revealed that anpe matched to two regions of ac and ro in the 5' and 3'ends. on the other hand, anpe also matched to k gene of some group ii npvs and gvs in the two terminal regions. this implied that the function region of k may be in the two regions. similarly, anpe was homologous to eukaryotic trex gene, which was observed only in cfmnpv, cfdefnpv and agmnpv. the trex gene encodes a 3' to 5' exonuclease. also, multiple alignment of anpe to cf and cfdef appeared to be highly conservative in their amino acid sequences. furthermore, anpe was 87% identical at the amino acid level to v-trex gene of agmnpv. anpe cf cfdef and the v-trex gene of agmnpv had a residue-long exoiii motif. the percentage identity within these regions indicated that this region was also functionally important. this kind of genes may give anpenpv a selective advantage in nature. functional genes baculoviruses contain a lot of genes involved in essential and selective functions, such as viral replication, transcription, inhibition of apoptosis, protein structure, assembly and auxiliary. besides, there were some conserved genes, of which functions were not clearly identified. all of the conserved function genes, previously identified in all baculoviruses and all lepidopteran-specific baculoviruses, were also found in anpenpv. several variable function genes identified in some baculoviruses and some conserved baculovirus orfs of unknown function were also found in anpenpv. the detailed functions of these genes are listed in additional files. whereas, several variable dna replication genes, including helicase- dna-ligase, dutpase, rnase reductase- and rnase reductase-, were not found in anpenpv. five genes have so far been implicated as inhibitors of apoptosis, including iap- iap- iap- iap- and p however, only iap-1and iap- were identified in anpenpv and considered as genes present in all group i npvs. baculovirus genomes typically encode auxiliary genes that are non-essential for replication but provide a selective advantage to the virus. anpenpv contained some auxiliary genes including ctl- and ctl-, which are small disulfide-rich ion channel antagonists. ctl was found in acmnpv, opmnpv, ldmnpv, cfmnpv and hycunpv, but ctl was only found in opmnpv, hycunpv and ldmnpv. numerous structural genes were also found in anpenpv, which composed the structure of virus. three genes in anpenpv, pif- pif- and p, were identified as structural genes as well as per os infectivity factor involved in baculovirus infection to midgut epithelial cells of the host. other structural genes identified in some baculoviruses but not in anpenpv included homologues to vef- vef- vef- pk vefs have been found to dramatically increase the infectivity of baculoviruses in non-natural lepidopteran hosts and only found in gvs and group ii npv. hrs repeats and the genes around them most baculoviruses contain hr regions. an individual npv hr is typically comprised of direct repeats usually centered around a perfect or imperfect palindrome. hrs have been implicated as origins of dna replication and as enhancers of rna polymerase ii-mediated transcription. gv hrs are different from npv hrs except for plxygv, whose hrs appear more similar to those from npvs. gv hrs usually lack a palindrome and are homologous to all other hrs within the same genome, but there are no two gvs that have similar hr regions. gv hrs are characterized to be repeat regions that are variable and at-rich. three hrs have been identified in the anpenpv genome. the number of repeats per hr ranged from two in hr to nine in hr the multiple alignment of the anpenpv hrs showed a high degree of conservation within the hrs and the hr palindrome consensus sequence, agcdatgcgtcagcgcygayybtgcttttcragtayarccawtctwgaaaamcgt. this palindrome consensus is also found in hycunpv, cfmnpv, opmpv, bmnpv, acmnpv, cfdefnpv and agmnpv. there was no interval between each repeat unit of hr and hr in the anpenpv genome. this was different from that of some other npv hrs, in which an interval between each repeat unit is found. the repeats of hr were nearly the same. the even repeats of hr hr1b,hr1d,hr1f,hr1h, hr1j were the same too. the odd repeats appeared to be formed by even repeats in which a and t was alternately replaced with g. the average g+c content of the hrs was %, significantly lower than the % for the complete anpenpv genome. the relative location of hrs in anpenpv genome was also characterized. the genes that flanked the hrs were mainly similar between anpenpv and the other seven group i npvs. anpenpv hr was in the same relative genomic location as epponpv hr opmnpv hr and cfdefnpv hr between gene p and me anpenpv hr was in the same relative genomic location as cfmnpv hr epponpv hr opmnpv hr acmnpv hr romnpv hr and cfdefnpv hr between gene pif- and chitinase. anpenpv hr was in the same relative genomic location as cfmnpv hr epponpv hr bmnpv hr acmnpv hr romnpv hr and cfdefnpv hr near gene op and fgf. it proved that the group i npvs showed conserved genomic locations for certain hrs. furthermore, the unique gene anpe for anpenpv and unique gene anpe for anpenpv and opmnpv existed around hr and hr respectively. instead of a putative hr between odv-e and ie- that is present in hycunpv, cfmnpv and epponpv, this was replaced in anpenpv by the unique gene, anpe. furthermore, instead of the apoptosis inhibiting gene iap- located between the sod and fgf genes in some other group i npvs, the iap- homolog was putatively replaced by unique gene anpe and ctl- gene in anpenpv. also, the relative direction of transcription for fgf and sod around hr in anpenpv was different from that of other group i npvs, except for that of hycunpv. only in anpenpv, hycunpv and opmnpv was the odv-e and ie- transcription in the same relative orientation. this was consistent with the description by de jong et al. that a high degree of variability existed around the hrs and by ahrens et al. that major rearrangements, insertions and deletions exist around the hrs in acmnpv and opmnpv. in particular, the v-trex gene homolog was inserted between pif- and chitinase around hr in anpenpv. besides anpenpv, the v-trex gene was inserted only into cfmnpv and cfdefnpv at the same location around cf-hr and cfdef-hr respectively. using blastp, we found that the anpe had high amino acid identity to trex genes from some eukaryotes, such as canis familiaris, rattus norvegicus, anopheles gambiae str. pest,bos taurus, mus musculus, homo sapiens, drosophila melanogaster, and drosophila pseudoobscura. by multi-aligning the deduced amino acid sequences of these genes, two conserved regions were found as following: -x- - -d- -e- -t- -i-p at the n terminus, and f-i-x-p-x-c-l-v-a- -n-g-x- -d-f- - -, respectively. phylogenetic tree analysis by clustal x supported the assumption that the v-trex was transferred horizontally, possibly from an insect host, which was similar with the assumption by slack et al. and lauzon et al.. the v-trex gene product has the potential to be involved in virus recombination or uv-light tolerance, which could contribute to the high degree of variability existed around the hrs and may give anpenpv a tolerance advantage in nature. similarly, anpe, anpe and anpe showed high amino acid sequence similarity with the homologues from insect hosts, respectively, implying evolutionary conservation of them, suggesting that baculovirus maybe acquire genes that were inserted into the regions around hrs from insect hosts. only iap was studied in detail supporting the argument. drs repeats direct repeats as described by garcia-maruniak et al. are suggested to be a putative non-hr, ori-like regions containing an unusual at-rich sequences and perfect or imperfect short palindromes in intergenic spacer or within orfs. these drs are not repeated elsewhere in the genome. with the exception of nelenpv, the majority of npvs have hrs and many of these hrs have homologous palindromes core. the nelenpv has only nine drs shared a closer similarity to the organization of repeat regions in gvs than to those in npvs and showed little similarity to typical npv hrs, which are characterized by the presence of perfect or imperfect palindrome and at rich. in comparison nesenpv not only has the hrs, but also has four drs. similar to nesenpv, perfect or imperfect drs were identified by tandem repeats finder software in addition to the hrs in anpenpv. these drs contained from to repeats. except for dr they were found to be at-rich, especially dr dr dr dr dr dr dr dr dr dr dr dr dr and dr palindromes ranging in size from to bp were found in several of the drs. all of the drs were located inside intergenic spacer regions of the anpenpv genome. the existence of twenty-four drs in the anpenpv intergenic spacer regions may reflect a different genome replication strategy for the npvs, and these drs may be a new type putative non-hr, ori-like region as is found in grlegv, cpgv, and adorgv. in addition, nine tandem repeats were also identified in orfs of anpenpv genome with a high gc content. at the same time, a specific gt-repeat was located from to122259nt. the sequences contained continuous gt residues with dinucleotide repeats. the gt-repeat was only found in opmnpv. similar sequences of gt repeats have been characterized in other organisms, e.g., drosophila virilis and caenorhabditis elegans. comparison of anpenpv and other baculoviruses the gene order of anpenpv was compared with those of other group i npvs, two group ii npvs and a gv by gene parity plots. anpenpv shared and orfs with hycunpv, epponpv, cfmnpv, opmnpv, acmnpv, ronpv, bmnpv and cfdefnpv, respectively. among the group ii npvs, anpenpv shared the highest number of orfs with maconpv a and the least with adohnpv. the organization of the anpenpv genome was collinear to that of hycunpv, epponpv, cfmnpv, and opmnpv. in particular, anpenpv showed a high similarity to hycunpv and they shared a gene unique to them. the gene order in two and one regions of the anpenpv genome were inverted with respect to the corresponding regions of acmnpv, ronpv and bmnpv; cfdefnpv respectively. collinearity was not found between anpenpv and maconpv a, hearnpv and xcgv. similarity between the core baculovirus genes between anpenpv and other group i npv was obviously higher than the similarities between anpenpv and other groups. cuninpv was particularly dissimilar, of which similarities were %– % range and average of %. phylogeny of anpenpv based on concatenated conserved genes, a phylogenetic tree was estimated for the baculoviruses. sequences were aligned using clustal w, with a gap penalty of an extend gap penalty of and a delay of divergence of 30%. bootstrap values for replicates were given. the baculovirus phylogenetic tree is shown in figure from the tree, anpenpv appeared as a group i npv and most closely related to hycunpv, opmnpv, cfmnpv, cfdefnpv, epponpv, and to a lesser extent to bmnpv, ronpv and acmnpv. ikeda et al. suggested that the close relationship among hycunpv, cfmnpv and opmnpv may be due to a geographical overlap among their hosts. also, the relationship between anpenpv and the other npvs, especially hycunpv, may be due to the same reason. hyphantria cunea was firstly introduced into dandong, liaoning province of china in which is the main distribution area for antheraea pernyi and has attacked many kinds of trees including oak tree. the anpenpv virus strain used in this study originated from liaoning province. antheraea pernyi also has a narrow distribution in japan where hycunpv had been isolated since.  in summary, we found that anpenpv is a member of group i npv and is most closely related to hycunpv opmnpv, epponpv, cfmnpv, cfdefnpv and to a lesser extent to bmnpv, ronpv and acmnpv based on the results of multi-alignment, phylogenetic analysis and gene parity plots. analysis of the genome organization of anpenpv and other group i npvs demonstrated that there are one gene rearrange region in anpenpv with cfdefnpv, and two gene rearrange regions in anpenpv with bmnpv, ronpv and acmnpv. despite its close relation to opmnpv, cfmnpv, hycunpv, epponpv and cfdefnpv, the anpenpv genome has several distinct features including the presence of genes unique to anpenpv and genes previously identified as new genes unique to cfdefnpv and hycunpv, respectively, and that, unlike other baculoviruses there are fewer hrs, more direct repeats and a gt-repeat previously found only in opmnpv. at the same time, analysis of the genes around the hrs of anpenpv supported the argument that an ancestral baculovirus acquired genes from ancestral insect hosts that were inserted into the regions around the hrs. there are still conserved genes present in all baculoviruses and genes present in all lepidopteran baculoviruses. besides genes present in all baculoviruses and lepidopteran baculoviruses, orfs in anpenpv were identified in all lepidopteran npvs. of the orfs, were found exclusively in all lepidopteran npvs, therefore the number of genes common to all lepidopteran npvs is however these genes are not the same as those identified before. in addition, orfs are now exclusive to and present in all group i npvs. only two bro genes and two inhibitors of apoptosis were found in anpenpv. so far, two ctls,ctl and ctl are found only in the genomes of anpenpv, hycunpv, opmnpv and ldmnpv. these differences identified between anpenpv and other baculovirus genomes might provide insight into their evolution, host specificity and pathogenicity. the sequence and analysis of anpenpv complete genome can provide a continuing resource to better follow the evolution and phylogeny of baculovirus. 
however, perfect or imperfect direct repeats with a high degree of at content were found within the intergenic spacer regions that may function as non-hr, ori-like regions found in grlegv, cpgv and adorgv. drs were also found in intragenic spacer regions of anpenpv. furthermore, there are still and conserved genes present in all baculoviruses and all lepidopteran baculoviruses respectively. open reading frames that putatively encode proteins of or more amino acid residues with minimal overlap were determined. in addition, the total number of genes common to all lepidopteran npvs is sill however the genes are somewhat different from the genes identified before because of some new sequenced npvs. only genes were found exclusively in all lepidopteran npvs and genes were found exclusively in all group i npvs. anpenpv encodes v-trex, which was observed only in cfmnpv and cfdefnpv in group i npvs. anpenpv belongs to group i npvs and is most similar to hycunpv, epponpv, opmnpv and cfmnpv based on gene content, genome arrangement, and amino acid identity. the g+c content of the genome, %, was higher than that of most of the sequenced baculoviruses. the completion and reporting of baculovirus genomes is extremely important as it advances our understanding of gene function and evolution.
 the completion and reporting of baculovirus genomes is extremely important as it advances our understanding of gene function and evolution. due to the large number of viral genomes now sequenced it is very important that authors present significantly detailed analyses to advance the understanding of the viral genomes. however, there is no report of the antheraea pernyi nucleopolyhedrovirus genome.  the genome of anpenpv, which infects chinese tussah silkworm, was sequenced and analyzed. the genome was bp in size. the g+c content of the genome, %, was higher than that of most of the sequenced baculoviruses. open reading frames that putatively encode proteins of or more amino acid residues with minimal overlap were determined. of the orfs, appeared to be homologous to other baculovirus genes, and were unique to anpenpv. furthermore, there are still and conserved genes present in all baculoviruses and all lepidopteran baculoviruses respectively. in addition, the total number of genes common to all lepidopteran npvs is sill however the genes are somewhat different from the genes identified before because of some new sequenced npvs. only genes were found exclusively in all lepidopteran npvs and genes were found exclusively in all group i npvs. anpenpv encodes v-trex, which was observed only in cfmnpv and cfdefnpv in group i npvs. this gene potentially originated by horizontal gene transfer from an ancestral host. in addition, anpenpv encodes two conotoxin-like gene homologues, ctl and ctl which were observed only in hycunpv, opmnpv and ldmnpv. unlike other baculoviruses, only typical homologous regions were identified containing 2~ repeats of a bp-long palindromic core. however, perfect or imperfect direct repeats with a high degree of at content were found within the intergenic spacer regions that may function as non-hr, ori-like regions found in grlegv, cpgv and adorgv. drs were also found in intragenic spacer regions of anpenpv.  anpenpv belongs to group i npvs and is most similar to hycunpv, epponpv, opmnpv and cfmnpv based on gene content, genome arrangement, and amino acid identity. in addition, analysis of genes that flank hrs supported the argument that these regions are involved in the transfer of sequences between the virus and host. 