Viral DNA was subjected to whole genome shotgun sequencing using purified virus pooled from the calyx fluid of ~400 female wasps from an outbred population. As judged by sizing on agarose gels, the GiBV viral genome was expected to contain 13 segments with a genome size of ~250 kbp 41. However, assembly of our preliminary sequence data indicate an aggregate genome size of ~490 kbp and ~24 different segments. Many segments are of similar sizes and would have co-migrated on agarose gels. A high frequency of single nucleotide polymorphism (SNP) (~1/70 bp) and insertions and deletions (indels) in the DNA of the viral population that was sampled complicated the closure phase of the sequencing project. Nevertheless, 19 of the 24 preliminary viral genome segment sequences were of sufficient quality to allow development of segment-specific PCR primers (data not shown). These primers were used to determine the proviral genome segment composition of BAC clones that hybridized with ³²P-labeled GiBV viral DNA. Priority was given to closing sequence and physical gaps in 8 viral genome segments that were encoded by two overlapping BAC clones (see below). A consensus sequence was generated for each viral genome segment (see Materials and Methods), and the resulting sequences, which varied in length from 10 to 26 kbp, were deposited in GenBank (EF051505–EF051512). Individual sequence reads were deposited in the NCBI Trace Archive (1472627677-1472629890).

Radioactive probes derived from total GiBV viral DNA hybridized at varying intensity to 127 clones from a BAC library of 9,216 clones made from the larvae of G. indiensis. Nineteen viral genome segment-specific PCRs were used to genotype 60 BAC clones to determine the proviral genome segment composition. These BAC clones segregated into 7 sets that contained non-overlapping profiles of viral genome segments (Table 1). Each set contained 1 to 7 proviral genome segments, and in total 17 of the 19 proviral genome segments were identified. Additionally, a sub-set of 30 BAC clones were fingerprinted using EcoRI and the resulting restriction enzyme patterns were used to place the BAC clones into overlapping contigs. This method of clustering was consistent with the results of the segment-specific PCRs (data not shown).

Two overlapping BAC clones that appeared to code for a cluster of 7 proviral genome segments were selected for sequencing. BAC clones 18I8 and 20D14 were 120,708 kbp and 116,222 kbp in length, respectively, and overlapped by 14,273 bp. The region of sequence overlap contained 53 SNPs, indicating the BAC clones were derived from different individuals from a population of G. indiensis. A contiguous DNA sequence was generated by fusing positions 1–109,055 of clone 18I8 with positions 2,560–116,222 of clone 20D14, resulting in a region spanning 222,657 bp. The annotated DNA sequence was deposited in GenBank (AC191960).

The coordinates of the 7 proviral genome segment sequences in this region were determined by aligning viral genome segment sequences to it. A search of the BAC sequences against the entire assembly of viral genome segment shotgun sequence data led to the identification and closure of an extra viral genome segment sequence. This assembly was not of high enough quality for primer design during the BAC clone screening phase. Thus, a cluster of 8 proviral genome segments labeled 1p to 8p separated by 7 inter-segmental regions (isg1 to isg7) that vary in length from 122 bp to 8.4 kbp occupies ~163 kbp of DNA which we call GiBV proviral locus 1. Interestingly, the 34 kbp and 25 kbp region of DNA that flank locus 1 contain a 6–7 kbp section of DNA (L1R1 and L1R2) consisting primarily of non-coding tandem DNA sequence repeats (Figure 1, Table 2).

A variety of nucleotide compositional differences exist between the flanking regions I-IV, inter-segmental regions, and proviral genome segments. The latter sequences and L1R1/L1R2 have the highest average G+C content (37%), followed by the flanking regions (32%) while the inter-segmental regions have the lowest G+C content (26%). The difference in G+C content between coding and non-coding DNA is greater in flanking regions I-IV (44% vs. 28%) than in proviral genome segment sequences (41% vs. 34%) (Table 2). Relative dinucleotide frequencies which correct for background G+C composition were calculated for each region > 500 bp in length, except L1R1 and L1R2 as tandemly repetitive sequences have highly biased dinucleotide frequencies. Neighbor-joining clustering of the distances derived from these data (Figure 2) revealed that all of the proviral genome segments cluster together and have a highly similar dinucleotide composition, which is distinct from flanking DNA. Regions I and IV clustered together and the most distantly from proviral genome segments, whereas regions II and III and the inter-segmental regions clustered between the proviral genome segments and regions I and IV.

Visual examination of the DNA sequence at the junctions between GiBV proviral genome segments and inter-segmental regions led to the identification of a 6 bp direct sequence repeat (AGCTTT), which is perfectly conserved at 14 of the 16 junctions and has one nucleotide substitution at the remaining 2 junctions. Because this repeat is encoded on the top DNA strand for 3 proviral genome segments (1p, 3, p, and 5p) and the bottom DNA strand for the remaining 5 proviral genome segments (2p, 4p, 6p, 7p, and 8p), the 5' and 3' boundaries of a proviral genome segment were defined as the first and second copy of the AGCTTT repeat relative to the sequence depicted in Figure 1. The 16 junction sequences were separated into 5' and 3' boundaries and searched using MEME, a motif discovery tool. An extended sequence motif centered on the AGCTTT repeat was identified in each group of sequences. The 5' and 3' motifs are different to each other and the 5' motif is more conserved than the 3' motif. Conservation of both motifs was greater and longer on the segmental side of the excision site than on the inter-segmental side (Figure 3).

MEME analysis of the 8 GiBV viral genome segment sequences revealed the presence of a single copy of the AGCTTT repeat surrounded by a recombined motif from the 5' and 3' motifs (Figure 3). By comparing proviral and viral genome segment sequences, it was determined that the two nucleotide polymorphisms present in the AGCTTT repeat of the proviral genome segment sequences appeared in the single copy of the repeat in viral genome segment sequences. Specifically, the 5' repeat of segment 5p has a substitution at the fifth position while the 3' repeat of segment 3p has a substitution at the first position and both changes occur in the corresponding viral segment.

MEME was also used to search the complete CcBV and MdBV viral genomes, and the 5 available viral genome segments of CiBV. A sequence motif highly similar to the recombined GiBV segment motif was found in all 30 CcBV viral genome segments and 13 out of 15 MdBV viral genome segments (Figure 3). No similar motif was found in CiBV, although described CiBV exision sites show conservation of varying degrees to the AGCTTT repeat 3340.

Two previously described GiBV cDNAs (p325 and p494) expressed in infected gypsy moth larvae 41 which encode hypothetical proteins map to multiple genes in proviral locus 1. These cDNAs provide direct evidence for the presence of 1 and 2 introns in the p325 and p494 gene families, respectively; p494 maps to 2 genes in proviral genome segment 2p, while p325 maps to 1 gene of proviral genome segment 3p, 4p, and 5p. The shortest and longest intron was 83 bp and 591 bp in length, respectively. Four variations of ab initio gene modeling programs were tested for their ability to recover the correct intron-exon structure of these 5 genes. A combination of Softberry's FGENESH trained on the honey bee (Apis mellifera) and the Beijing Genome Institute's BGF trained on the silkmoth (Bombyx mori) were most accurate and these programs, in addition to protein alignments generated with the AAT package 43 were used to predict protein-coding gene models (see Materials and Methods).

A total of 62 protein-coding genes were predicted to be encoded within the 8 proviral genome segments (Table 2 and 3). As judged by sequence similarity using BLASTP, 47 genes have homologs in CcBV, but only 3 genes, all of which are members of hypothetical family 4, have homologs in MdBV. A TBLASTN analysis of the predicted GiBV proteins against the MdBV and CcBV genomes showed no additional similarity to MdBV. However, of the 15 proteins which did not show BLASTP similarity to CcBV, 5 showed similarity to translated CcBV sequences, suggesting homologs of these genes may exist in CcBV but were not previously predicted. The 10 remaining GiBV genes which do not have homologs in CcBV encode novel hypothetical proteins.

Only 10 of the 62 predicted GiBV genes in the locus were assigned a potential function, namely C-type lectins and proteins containing a cystatin or ribonuclease T2 domain (Table 3). Surprisingly, 50 genes were predicted to access the secretory pathway as they contained a signal peptide at the N-terminus. Of these 6 genes were predicted to have trans-membrane domains, and 6 genes were predicted to have potential glycosylphosphatidylinisotol anchors. Only 3 proviral genome segment genes were not predicted to contain introns and the remaining genes contain either 1 or 2 introns. A protein domain-based clustering pipeline placed 43 of the 62 proteins into 14 gene families (see Methods and Table 3). The distribution of members of these gene families was generally not restricted to specific proviral genome segments–8 gene families, including all families with 4 members or more, were located on at least 2 non-adjoining proviral genome segments.

Regions L1R1 and L1R2 and the inter-segmental regions were not predicted to contain protein-coding genes, nor did these sequences produce any significant matches when tested against the GenBank non-redundant protein database using BLASTX (E = e-10). On the other hand, regions I to IV were predicted to encode 9 genes and potential function was assigned to 6 of them (Table 3). These genes had a top blast hit to genes from Apis mellifera (BLASTP, E < e-45), including the 5'-nucleotidase, trans-2-enoyl-CoA reductase, hyaluronidase, N-myristoyltransferase, and 1 hypothetical protein. By contrast, none of the genes encoded by the proviral genome segments had any sequence similarity to A. mellifera (BLASTP, E = e-10), other than proteins with conserved domains encoded in a large number of genomes (e.g., the C-type lectin domain). Four of 6 genes are encoded on A. mellifera chromosome 14, although only the honey bee hyaluronidase and N-myristoyltransferase genes were located in close proximity to each other.

Proviral genome segments in locus 1 share 99.5–99.9% sequence identity with their homologous viral genome segment sequence. The distribution of 2,159 SNPs in the 8 GiBV viral genome segment sequences relative to the corresponding proviral genome segment sequence is shown in Table 4. Viral genome segment 2 showed a low frequency of polymorphisms, averaging ~5 SNPs/kbp, while the remaining segments had an average SNP density of ~16 SNPs/kbp. The majority of genome segments showed no significant correlation between sequence coverage and SNP density (Table 4), with the exception of segment 1, which showed a slight correlation (R² = 0.25, p < 0.05). All SNPs were placed in one of three classes: non-coding, synonymous, and non-synonymous. As expected, there was a significantly higher SNP density in synonymous sites than non-synonymous sites (χ²_{1 df} = 37.3, p < 0.01). However, there was also a higher SNP density in synonymous sites relative to non-coding sites (χ²_{1 df} = 38.2, p < 0.01), and no difference in SNP density between non-coding and non-synonymous sites (χ²_{1 df} = 1.8, p > 0.05).

The number of SNPs per gene ranged from 0 to 68, and dN/dS ratios were calculated for the 39 out of 62 genes that contained 5 or more SNPs (Table 3). Most of these genes appear to be under purifying selection and 32 of 39 genes had dN/dS ratio < 0.8 with a majority of the ratios falling in the range of 0.40–0.59 (Figure 4). Three genes appear to be evolving neutrally (dN/dS = 0.8–1.2) and code for 2 hypothetical proteins and 1 member of gene family 3. Four genes had a dN/dS > 1.9, including 1 member each of gene families 1, 10, and 11 (the ribonuclease T2 domain) and an EP1-like protein. No correlation was found between dN/dS ratios and specific genome segments or gene families–most segments and gene families contained genes under different degrees of selection.

Prior to this study, it was believed that bracovirus proviral genome segments were closely linked in a tandem array in the wasp genome with short stretches of intervening DNA separating them 12333435. Our study indicates that this is not the case for GiBV. While some GiBV proviral genome segment sequences are clustered in tandem arrays others occur in isolation as singletons. This conclusion is supported by the segregation of BAC clones coding for 18 of ~24 proviral genome segments into 7 non-overlapping sets of clones via viral genome segment-specific PCRs (Table 1), and preliminary BAC shotgun sequence data support the typing data (not shown). Furthermore, although we describe a tandem array of proviral genome segments in this paper at GiBV proviral locus 1, the array codes for only 8 proviral genome segment sequences and this cluster is flanked by at least 34 kbp and 25 kbp of DNA (Figure 1) that is not packaged into GiBV virions. It remains to be determined whether the 7 loci encoding GiBV proviral segment sequences are linked on the same chromosome as a macrolocus but with longer stretches of intervening DNA between them, or whether they are dispersed across more than one chromosome. Although the former scenario remains compatible with a study of C. congregata where probes from 3 different viral genome segments bound to the same location on C. congregata chromosome 5 35, the structural organization of BV proviral genome segment sequences appears to be more complex than previously hypothesized.

It is reasonable to propose that the inter-segmental regions in GiBV proviral locus 1 should be classified as part of the GiBV proviral genome. However, to what extent the proviral genome extends into flanking DNA is less easily determined. BV viral genome segments are thought to be excised from the amplified products of one or more large precursor molecules, and there is no evidence for post-excision amplification of segments 343637. Thus copy number studies of regions immediately flanking GiBV proviral locus 1 and other loci containing proviral genome segment sequences at the time of viral genome segment formation could be used as a surrogate marker for identifying potential components of the GiBV proviral genome.

Due to the limited transcriptional data available for BVs, there is substantial disagreement on the structural complexity of BV genes, particularly with regards to the percentage of PDV genes that contain introns. While Espagne et al 24 predicted that 69% of CcBV proteins contain introns, Webb et al 28 re-annotated the CcBV genome and predicted only 6.8% of CcBV genes contain introns–a ten-fold difference in intron content. In GiBV proviral locus 1, using a combination of Hymenoptera- and Lepidoptera-trained gene prediction programs (see Methods), we predicted that 81% of the 63 genes contain introns. Sequence data from 2 cDNAs derived from genes in proviral locus 1 suggests that the 7 introns predicted for 5 members of the 2 gene families are real and not artifacts of improper gene modeling. However, this number is probably not reflective of the entire GiBV genome, as PTP and ankyrin genes usually do not contain introns 444546 and generally comprise a large percentage of BV genes (21% and 41% of predicted CcBV and MdBV genes, respectively), but are not present in GiBV proviral locus 1. Regardless, the accuracy of most predicted gene models awaits experimental verification. While the presence of introns may be unusual for virus genes, some DNA viruses which replicate in the host cell nucleus encode genes with introns (e.g., adenoviruses 47).

GiBV genes in proviral locus 1 predicted to contain introns have an extremely simple intron-exon structure compared to often complex higher eukaryotic genes, and generally contain a single short exon followed by a long exon encoding the remainder of the protein. Remarkably, 80% of the genes at this locus, including the p494 and p325 gene families which are transcribed in infected gypsy moth larvae, are predicted to encode a secretion signal peptide within the first exon. Secretion of some proteins may compensate for differences in the abundance of segment sequences in virions. Since it is unclear whether the entirety of the GiBV genome is packaged into a single virion 41, secretion of a large number of proteins may be necessary for properly delivery of these proteins. Attempts to functionally annotate the 62 predicted genes in the 8 GiBV proviral genome segment sequences identified the presence of a C-type lectin 48, CrV1-like proteins 49, and a number of conserved hypothetical proteins encoded by other PDV genomes 19242650. Most of the genes in locus 1 were predicted to have homologs in CcBV, while only gene family 4 showed homology to a gene on MdBV segment B. Although the function of this gene family is unknown, it is the only gene family in GiBV proviral locus 1 for which none of the members are predicted to contain signal peptides.

The placement of 43 GiBV genes into 14 gene families suggests that extensive duplication of genes has occurred within proviral locus 1. Typically, gene duplications are thought to result in relaxation of the selection on the duplicated gene, allowing it to acquire a new function. However, the majority of genes in proviral locus 1, even multiple members of the same gene family, appear to be under purifying selection (Figure 4). This implies that members of gene families are, for the most part, not free to acquire entirely new functions but may play different roles within the constraints of their gene family, such as differential targeting as seen in some inhibitors of NF-κBs 45 or differential expression as seen in some PTPs 46. Alternatively, conserved function across duplicated genes may be important for increasing the level of expression of functional classes of genes 2651. Despite the large proportion of genes under purifying selection, 7 genes appear to be evolving neutrally or under diversifying selection, potentially allowing a limited set of genes to acquire new functions or adapt to changes in host defenses.

The inter-segmental regions which separate the proviral genome segments are not predicted to contain protein coding genes. However, regions that map outside of proviral locus 1 are predicted to contain 9 genes, and potential function has been assigned to some of them, e.g., N-myristoyltransferase, ecto-5'-nucleotidase, and hyaluronidase (Figure 1). It is interesting to note that viral proteins are often modified with a lipid tail, hyaluronidase is a component of venom 52 that hydrolyzes complex carbohydrate structures allowing tissue diffusion, and ecto-5'-nucleotidase is involved in the extracellular formation of adenosine, a regulator of innate immune responses 5354. It is unclear whether these regions constitute part of the GiBV proviral genome but there is a striking difference in the structural complexity of these predicted gene models and those present in proviral locus 1. Nevertheless, it is tempting to speculate that proteins encoded in the flanking regions, perhaps as components of ovarian fluids, and genes that are located close to other proviral segment loci, may play a role in GiBV biology. Also notable is a sex-linked wasp gene coding for a homolog of female-lethal(2) [fl(2)d] that is present in region I. In Drosophila fl(2)d plays a critical role in alternative splicing regulation of genes involved in sex determination (including Sex-lethal and transformer), dosage compensation, oogenesis, and differentiation, as well as non sex-specific functions, and is expressed throughout larval and adult life 55565758. Since excision of proviral genome segments from the wasp chromosome and encapsidation into virion particles occurs only in females, it is possible that regulation of this sex-linked process is related, at least in part, to expression of fl(2)d.

The presence of a near perfect AGCTTT direct DNA sequence repeat was discovered at the boundaries of proviral genome segment sequences and flanking sequences (Figure 3). As the viral genome segment sequences contain a single copy of this repeat, it appears to define the site of proviral genome segment excision. This suggests an excision mechanism via conservative site specific recombination as described for formation of other PDV genome segments 123339. The presence of two SNPs within this repeat at the junction of proviral segment sequences and the ability to follow these nucleotide differences from the proviral to the viral genome segments suggests that the site of proviral genome segment excision and circularization must be located between the first and fifth position within the AGCTTT repeat (Figure 3). A study of excision sites in CiBV similarly concluded that GCT was the preferred site of excision 40.

An extended but different sequence motif around the excision site was identified at the 5' and 3' proviral genome segment junction sequences using MEME and the recombined sequence motif is found on viral genome segment sequences (Figure 3). While sequence conservation exists on both sides of excision sites, a higher level of conservation is seen in the side of the motif which is retained in circularized segment, and in particular at the 5' junction. The asymmetry of the 5' and 3' sequence motifs suggests that there is directionality to the recognition of excision sites. Since recombined sites have a different motif we predict they are no longer substrates for the excision enzymes. Excision and circularization of segments from a large precursor molecule could occur via release of single segments or a smaller molecule containing multiple segments. In the latter case the segments flanking the site of circularization would no longer be available for excision. For example, if a molecule encompassing 1p through 3p in proviral locus 1 were excised, only 2p would remain a substrate for subsequent excision and circularization (Figure 1). Such a pathway could contribute to differences in the abundance of packaged viral genome segments but it portrays a complex scenario. Assuming that sequence coverage of a viral genome segment in our shotgun sequencing approach correlates with the abundance of the segment it is interesting to note that the GiBV viral genome segments encoded in proviral locus 1 appear to be present in about the same levels (Table 4), suggesting that generation of intermediate excision products is not a common occurrence. The sequencing data also suggest that intermediates or by-products of excision, if they occur, are excluded from the packaging process, perhaps by the presence of inter-segmental DNA.

We found that the predicted site of excision/circularization and the recombined extended motif present in GiBV viral genome segments is also present in CcBV and MdBV viral genome segments (Figure 3). Conservation of the GCT portion of the excision repeat sequence exists in the CiBV viral genome segment sequences that are available 40, although more CiBV sequences will be required to determine how closely the CiBV extended motif mirrors that of GiBV, CcBV, and MdBV. As C. congregata, G. indiensis, and M. demolitor are all members of Microgasterinae, the most derived clade of bracovirus-bearing braconids, and C. inanitus is a member of Cheloninae, the most basal clade of the bracovirus-bearing wasps 5960, it is possible that the predicted excision motif is one of the very few sequence features that is highly conserved across bracoviruses, and provides additional support for the hypothesis that bracoviruses have a single evolutionary origin 2060. This observation also predicts conservation of the enzyme(s) involved in BV proviral genome segment excision and circularization.

Analysis of SNP data derived from sequencing the GiBV viral genome from an outbred population of female wasps revealed that non-coding sites in the 8 viral genome segments derived from locus 1 had a significantly lower SNP density than synonymous sites within coding DNA. As we presume synonymous sites to be evolving neutrally, this result suggest that there is likely to be selective pressure on non-coding DNA. The lack of difference between rates of change at non-coding and non-synonymous sites suggests that in these segment sequences, non-coding DNA may be as highly conserved as coding DNA. Such areas could encode non-coding RNAs or contain sequence motifs vital to DNA replication, gene expression or segment packaging. Limited experimental evidence support the idea that PDV non-coding DNA is functional–studies of CsIV segment B found 2 sequences of 0.6 and 1.2 kbp which are transcribed but do not encode proteins 6162.

Several differences between the cluster of 8 GiBV proviral segment sequences which are excised and packed into virus particles and flanking DNA suggest that proviral segment sequences are not simply host genetic elements evolved for the export of wasp parasitism genes. For example, the proviral segments exhibit similar nucleotide compositions to each other but their G+C composition and dinucleotide frequencies differ from those of inter-segmental regions and flanking regions I-IV (Table 2 and Figure 2). Given the estimated age of the integration of bracoviruses into the wasp genome, ~74 million years, and using substitution rates estimated from Drosophila 63, one would predict that a sufficient period of time has passed for the process of ameliorization, i.e., the adjustment over time of the nucleotide composition of the integrated DNA to that of the resident genome 64, to have occurred. The different nucleotide composition of the proviral segment sequences may be maintained or its ameliorization may be slowed by the purifying selection found to be acting on both non-coding and coding DNA. However, as differences in nucleotide composition can be caused by different origins of DNA 64 or by the widespread purifying selection itself 65, the origins of the compositional differences between proviral and flanking DNA remain to be determined. Additionally, it is possible that inter-segmental and flanking regions, rather than the proviral segment sequences, differ from the remainder of the wasp genome.

Outbred populations of Glyptapanteles indiensis, solitary endoparasitoids of gypsy moths (Lymantria dispar), were maintained at the USDA-ARS-Beneficial Insects Introduction Research Unit, Newark, Delaware, as part of a biocontrol program. The colony was founded in May 1998 from a shipment of 168 moths collected from 4 localities in India. In May 2002, the colony was outcrossed with 242 moths collected from the same localities. The mean colony size was 400 with an average sex ratio of 7 females:13 males. Host larvae were fed on a high wheat-germ diet. Both wasp and host larvae were maintained at 26°C, 58% relative humidity, and a light-dark (L:D) cycle of 16L:8D hr according to established protocol 66. G. indiensis parasitize late first instar gypsy moth larvae. Cocoons formed from parasitized hosts were stored at 24°C until adult parasitoid emergence and then separated by sex. G. indiensis larvae were dissected from parasitized host 10 days post parasitization, briefly rinsed in phosphate buffered saline (PBS), flash frozen in liquid nitrogen and stored frozen at -80°C.

Virions were purified from G. indiensis females using established protocols 67. Briefly, female wasps were anaesthetized in 75% ethanol and rinsed in PBS. Ovaries were dissected from the females in a drop of PBS and ruptured, draining the calyx fluid. Pooled calyx fluid was subsequently filtered through a 0.45 μm filter to remove eggs and cellular debris 68. Viral DNA was extracted according to established protocol 41. Briefly, viral DNA was isolated from the calyx fluid using a proteinase K/SDS buffer, DNA was extracted with phenol, precipitated with ethanol, and recovered by centrifugation.

A BAC library of G. indiensis with a 120 kb average insert size was constructed by Amplicon Express 69, using a partial BamHI digest inserted into an MboI site of a pECBAC1 vector. A nylon filter arrayed with 9,216 BAC clones was created from the library. In order to identify BAC clones containing proviral DNA, GiBV viral DNA was radioactively labeled with ³²P-labeled α-dCTP (NEN/Perkin-Elmer) using the Redi-prime II DNA labeling kit (Amersham Biosciences). Labeled DNA was then purified using a QIAquick PCR purification kit (Qiagen). The filter was pre-hybridized at 65° for at least 3 hours with Rapid-hyb Buffer (Amersham Biosciences) and 500 μg of salmon testes DNA (denatured at 100°C, Sigma-Aldritch). The probe was added and allowed to hybridize overnight at 65°C. The filter was then washed 2 times for 60 minutes each at 65°C with a 0.1 × SSC/0.1% SDS solution, wrapped in plastic wrap, and autoradiographed using Kodac BioMax MS film.

BAC clones were grown in 5 mL LB with 12.5 μg/ml chloramphenicol overnight at 37°C and shaking at 200 rpm. BAC DNA was extracted using the Sigma Phaseprep BAC DNA Kit (Sigma-Aldritch) without the endotoxin removal step. BAC DNA was digested with EcoRI (Invitrogen) in a 1:150 dilution of RNase cocktail (Sigma Phaseprep Kit) at 37°C for 2 hours. Digested DNA was run overnight on a 1.2% agarose gel, stained with Vistra Green and imaged using a FluorImager SI (Amersham Biosciences). Gel images were processed using Image 70, and contigs were assembled using FingerPrintContig 71 using the default e-value of e-10.

Approximately 7.5 μg of GiBV DNA was sheared and DNA fragments in the size range of 3.5–4.5 kbp purified after separation by agarose gel electrophoresis. The fragments were blunt ended and, after addition of BstXI adaptors, cloned into the BstXI site of pHOS2. Shotgun libraries were made from the 2 BAC clones as described for GiBV DNA. Celera Assembler 72 and TIGR Assembler 73 were used to assemble random sequence data from the viral whole genome shotgun and BAC clones, respectively. Gap closure was assisted by a closure editor tool called Cloe that also permits the manual inspection and editing of sequence data. A variety of methods were used to close gaps including re-sequencing the ends of random clones, transposon assisted sequencing (GPS, New England Biolabs™) or "micro-library" construction of single or pooled templates, and conversion of physical gaps to sequence gaps using "POMP" (pipette optimal multiplex PCR) 74 and or/a "Genome Walker" kit (Invitrogen™).

Primers were developed to be specific to 19 GiBV viral genome segment sequences. Primers were designed to be 22–26 nt in length, have a Tm of 62–65°C, a GC clamp, and a maximum identity to the remainder of the unclosed GiBV genome of 70%. Designed primers were tested for potential secondary structure using NetPrimer 75. PCR was performed in a 10 μl solution which included 0.1 μl template DNA, 0.3 μl 50 mM MgCl₂, 1 μl 10 × PCR buffer, 0.2 μ10 mM dNTPs, 7.9 μl H₂O, 0.1 μl Platinum Taq (Invitrogen), 0.2 μl F primer (20 pm/μl), and 0.2 μl RC primer (20 pm/μl). PCR protocol was 94° for 2 min; 35 cycles of 94° for 30 sec, 58° for 30 sec, 72° for 45 sec; followed by 72° for 7 min.

As shotgun sequencing of the GiBV DNA was carried out using a sample pooled from a population of ~400 wasps, a large number of SNPs and indels were present in the sequence assembly. Because individual sequence reads could not be associated with individual wasps, a conical consensus sequence was generated for each viral genome segment using the SliceTools package 76. At a given position in a conical consensus, all bases with a cumulative quality value within 50% of the highest cumulative quality value are assigned to that position.

Gene models were generated with a variety of software: Softberry's FGENESH 77 using both the honey bee (Apis mellifera) and fruit fly (Drosophila melanogaster) training sets, the Beijing Genome Institute's BGF 78 trained on the silkmoth (Bombyx mori), and GENSCAN 79 using the vertebrate training set. Predicted gene models were compared to gene models generated using cDNA from 2 gene families for their ability to predict correct intron-exon structure. Most of the gene finders accurately predicted the 2 intron structure of the p494 genes, with the exception of GENSCAN which predicted an extra exon. The single intron in p325 genes were significantly more difficult to predict – only FGENESH (A. mellifera) and BGF properly predicted these genes. FGENESH (D. melanogaster) and GENSCAN both mis-predicted the majority of intron-exon boundaries and showed a tendency to combine multiple genes into single genes with a large number of introns. Based on these results, a combination of FGENESH (A. mellifera) and BGF was used for gene prediction, in addition to the AAT package 43 which allows spliced alignment of proteins to genomic DNA thereby revealing potential exon-intron boundaries. Gene models from FGENESH were generally accepted except when multiple other sources of information contradicted those models. SignalP 8081, TM-HMM 82, and GPI-SOM 83, were used to predict signal peptides, transmembrane domains, and glycosylphosphatidylinisotol anchors, respectively. Predicted genes were clustered into gene families using previously described methods 84, which utilize Pfam 85 and TIGRFAM 86 domains and calculate novel shared domains within the genome. Predicted GiBV proviral segment genes were analyzed for potential homology to genes in CcBV and MdBV and CsIV using BLASTP (CcBV only) and TBLASTN (CcBV and MdBV), with a cutoff of E = e-10.

Relative dinucleotide frequencies 87, were calculated for each region > 500 bp in length except the flanking repeats, as they are expected to have highly biased dinucleotide frequencies. A Euclidean distance matrix between the regions was constructed from these frequencies. Regions were then clustered using the Neighbor-joining algorithm in PAUP* 88 and the resulting tree was visualized using PHY·FI 89.

Boundaries between the proviral segments and inter-segmental regions, and the inter-segmental regions themselves were analyzed for motifs using MEME 90. In the first analysis a 103 bp DNA sequence (50 bp upstream to 50 bp downstream of the GCT excision motif) was extracted from each segmental boundary. The boundaries of proviral segments 1p, 3p, and 5 bp were reverse complemented so that orientation of the excision motif was the same for all sequences. All 16 sequences were analyzed together, and then split into 8 5' (upstream) and 8 3' (downstream) motifs relative to the directionality of the excision motif. Next, an analysis was conducted using the entire length of the 7 inter-segmental regions. Analyses used a minimal and maximal motif length of 5 and 100 bp, respectively. MEME was also used to search 30 CcBV [Genbank :AJ632304–AJ632333], 15 MdBV [Genbank:AY887894, AY875680–AY875690, AY848690, AY842013, DQ000240], and 5 CiBV viral genome segments [Genbank :AJ627175, AJ278677, AJ319654, Z58828, Z31378] for common motifs. All motifs were visualized using WebLogo 9192.

Ambiguous consensus sequences were generated from the viral genome sequence by recalling contigs so that all high quality (quality value > = 30) base calls in the reads were represented in the new consensus as ambiguity codes. This ensured all variants of a given circle were encoded within a single consensus sequence, while preventing low quality sequencing error from introducing artificial polymorphisms. Then, the ambiguous viral genome segment consensus sequences were globally aligned to their corresponding proviral genome segment sequences using nucmer from the MUMmer package 93. This alignment was parsed to determine the positions of all polymorphisms relative to the reference proviral sequence, including both substitutions and indels. Substitutions were found by mismatches in the alignment between the viral consensus sequence and proviral reference sequence. The distribution of polymorphisms was analyzed using the gene-snps tool from the AMOS package 94. The tool examines each polymorphism to determine if it occurs within an exon, and if so, whether the change is synonymous or non-synonymous. Additionally, the tool estimates dN/dS for each gene using the unweighted pathway method 95. The final analysis the tool performs is a test of independence between SNP density and sequence coverage (i.e. if more sequences covering any given position means that position is more likely to contain a polymorphism). To do so, it computes the Pearson's correlation of the polymorphism rate and depth of coverage using a sliding window of size 500 bp offset by 250 bp across each circle. Statistical significance of correlation coefficients were evaluated using a 2-tailed t test, where degrees of freedom equals the number of SNPs minus two. Differences between the relative number of substitutions of non-coding, synonymous, and non-synonymous sites were evaluated using Pearson's χ² test.