Abstract
Glossina pallidipes salivary gland hypertrophy virus (GpSGHV; family Hytrosaviridae) can establish asymptomatic and symptomatic infection in its tsetse fly host. Here, we present a comprehensive annotation of the genome of an Ethiopian GpSGHV isolate (GpSGHV-Eth) compared with the reference Ugandan GpSGHV isolate (GpSGHV-Uga; GenBank accession number EF568108). GpSGHV-Eth has higher salivary gland hypertrophy syndrome prevalence than GpSGHV-Uga. We show that the GpSGHV-Eth genome has 190 291 nt, a low G+C content (27.9 %) and encodes 174 putative ORFs. Using proteogenomic and transcriptome mapping, 141 and 86 ORFs were mapped by transcripts and peptides, respectively. Furthermore, of the 174 ORFs, 132 had putative transcriptional signals [TATA-like box and poly(A) signals]. Sixty ORFs had both TATA-like box promoter and poly(A) signals, and mapped by both transcripts and peptides, implying that these ORFs encode functional proteins. Of the 60 ORFs, 10 ORFs are homologues to baculovirus and nudivirus core genes, including three per os infectivity factors and four RNA polymerase subunits (LEF4, 5, 8 and 9). Whereas GpSGHV-Eth and GpSGHV-Uga are 98.1 % similar at the nucleotide level, 37 ORFs in the GpSGHV-Eth genome had nucleotide insertions (n = 17) and deletions (n = 20) compared with their homologues in GpSGHV-Uga. Furthermore, compared with the GpSGHV-Uga genome, 11 and 24 GpSGHV ORFs were deleted and novel, respectively. Further, 13 GpSGHV-Eth ORFs were non-canonical; they had either CTG or TTG start codons instead of ATG. Taken together, these data suggest that GpSGHV-Eth and GpSGHV-Uga represent two different lineages of the same virus. Genetic differences combined with host and environmental factors possibly explain the differential GpSGHV pathogenesis observed in different G. pallidipes colonies.
Introduction
Tsetse flies (Diptera; Glossinidae) transmit African trypanosomoses, a set of neglected tropical zoonotic diseases with devastating socio-economic impacts in sub-Saharan Africa (Jordan, 1986; Steelman, 1976). Due to a lack of effective vaccines and increasing drug resistance and counterfeits (i.e. substandard drugs that mimic authentic drugs) (Barrett et al., 2011), vector (tsetse fly) control is of critical importance and represents a sustainable trypanosomoses control method (Schofield & Kabayo, 2008). An effective vector control strategy is the application of the sterile insect technique (SIT) as a component of an area-wide integrated pest management approach (Vreysen et al., 2013).
A prerequisite for the application of SIT programmes is mass production of healthy and productive colonies of the target tsetse species. A SIT programme was initiated to eradicate Glossina pallidipes from the Rift Valley of Ethiopia (Feldmann et al., 2005). For this programme, a G. pallidipes colony was established at the UN Food and Agriculture Organization/International Atomic Energy Agency's Insect Pest Control Laboratory (IPCL), Seibersdorf, Austria, using pupae collected from the target area. However, this colony completely collapsed within 2 years of its establishment (Abd-Alla et al., 2010) due to infection by Glossina pallidipes salivary gland hypertrophy virus (GpSGHV; family Hytrosaviridae) (Abd-Alla et al., 2007). Up to 85 % of the flies in this colony had salivary gland hypertrophy (SGH) syndrome – a phenomenon that was first described in wild G. pallidipes populations (Whitnall, 1934) and later observed in other Glossina species (Kariithi et al., 2013c).
GpSGHV can establish a chronic non-debilitating asymptomatic infection and an acute symptomatic infection in the adult insect host (Boucias et al., 2013). Whereas asymptomatic virus infection has no apparent host fitness cost, symptomatic infection, which is characterized by severe and extensive SGH symptoms, causes reproductive dysfunction and colony collapse (Abd-Alla et al., 2007; Jaenson, 1978a, b). Overt SGH occurs in G. pallidipes, although GpSGHV infection is largely asymptomatic in other Glossina species (Kariithi et al., 2013a). Expression of overt SGH in G. pallidipes can be successfully suppressed by oral administration of antiviral drugs (Abd-Alla et al., 2014), and by modification of fly feeding and colony handling protocols (Abd-Alla et al., 2013). Asymptomatic GpSGHV infection potentially represents viral latency (Kariithi et al., 2013c) and the occurrence of SGH symptoms is an exception rather than a rule.
GpSGHV pathogenesis and SGH prevalence differ from one G. pallidipes colony to another. For instance, despite a high prevalence of asymptomatic infection in the IPCL G. pallidipes colony (originating from Uganda), SGH prevalence averages at < 10 % and the colony has been stable for almost three decades (Abd-Alla et al., 2010). This prevalence markedly differs from the 85 % SGH prevalence in another IPCL G. pallidipes colony (originating from Ethiopia in 2001), despite the two colonies being maintained under the same insectary conditions (Abd-Alla et al., 2010, 2011), suggesting that the different pathogenesis could be the result of two different strains of the same virus.
Here, we hypothesize that (viral and host) genetic factors contribute to the expression of overt SGH symptoms. We tested this hypothesis by complete genome sequence annotation of GpSGHV isolated from an Ethiopian G. pallidipes colony and compared it with the reference GpSGHV genome (GenBank accession number EF568108), which originated from Uganda (Abd-Alla et al., 2008). We enhanced the GpSGHV genome annotations by mapping onto the virus genomic sequence transcripts and peptides obtained from RNA sequencing (RNA-Seq) and MS data, respectively, which were analysed from hypertrophied salivary gland (HSG) extracts.
Results and Discussion
The current study was conceived from the observed differential pathogenesis of GpSGHV in different G. pallidipes colonies. We used a combination of proteogenomic and transcriptomic mapping approaches (Nesvizhskii, 2014) to complement the annotation of ORFs in the GpSGHV-Eth genome. In addition to using peptides identified from flies infected by GpSGHV-Eth, we also included data obtained from previous proteomics of the GpSGHV-Uga (Kariithi et al., 2010, 2011, 2013b, 2016). Moreover, we compared the GpSGHV-Eth genome to the GpSGHV-Uga genome, which was annotated in silico (Abd-Alla et al., 2008).
General features of the GpSGHV-Eth genome
The sequencing results revealed that the GpSGHV-Eth genome had a size of 190 291 nt and a low (27.9 %) G+C content (the GpSGHV-Uga genome has a size of 190 032 nt; 28.0 % G+C content). From this genomic sequence, a total of 319 ORFs (methionine-initiated ORFs; ≥ 50 aa) were predicted, of which 174 ORFs had minimal overlaps and were therefore considered to encode putative viral proteins (Table 1). The 174 putative ORFs were evenly distributed on both strands (51.15 % forward; 48.85 % reverse), mostly arranged in unidirectional clusters (Fig. 1). The blast analyses revealed that the majority of the putative GpSGHV-Eth ORFs were nearly identical to the homologous GpSGHV-Uga ORFs with only minor differences at the amino acid level (Table S1, available in the online Supplementary Material). Overall, the GpSGHV-Eth and GpSGHV-Uga genomes were 98.1 % identical at the nucleotide level (Fig. 2). Analysis of the nucleotide sequences upstream of the first methionine residues revealed that 89.7 % (n = 156) of the GpSGHV-Eth ORFs contained a putative TATA-like box promoter element. Furthermore, 83.9 % (n = 146) contained the canonical polyadenylation [poly(A)] signal, whilst 35.8 % (n = 62) had putative baculovirus upstream late (T/G/A)TAAG transcription initiation motifs (Table 1).
Table 1. Annotation of 174 ORFs potentially expressed in the GpSGHV-Eth genome.
Homologies of the GpSGHV-Eth ORFs to other known virus and/or cellular genes and to the corresponding ORFs in GpSGHV-Uga are shown in columns 2–7. Of the 174 putative ORFs, protein products of 86 ORFs were detected by LC-MS/MS. Out of the 86 proteins, 10, 15, 20 and 12 were classified as envelope, nucleocapsid, tegument and virion proteins, respectively, based on the detection of their homologues in highly purified GpSGHV-Uga virions by LC-MS/MS (Kariithi et al., 2013b). The remaining 29 of the 86 proteins without specific localization were designated infected cell-specific viral proteins (ICSVPs). The annotation of the structural features of the ORFs, the presence of putative transcriptional signals, the peptide and/or transcript mapping on the viral genome, and the expression levels of the ORFs are also indicated. ORFs under positive selection pressure are indicated in bold (see discussion in main text and details in Table S4). The gene expression levels were normalized with fragments per kilobase of exon per million mapped reads (FKPM). The mean number of reads representing a given nucleotide in the reconstructed sequence is shown as mean coverage. ORFs marked ‘Yes’ on transcript (T) mapping were those mapped by exonic transcripts, i.e. read alignments were completely (100%) contained within the respective exons defined by the database used in RNA-seq quantification.
| Best blast match (description of homologies) | Transcription signals | Transcript (T) and peptide (P) mapping | Transcript expression levels | ||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| ORF (position and orientation) | Homology to viral and/or cellular proteins (localization) | Best match | Identity (%) | GenBank accession no. | E value | Score | Functional and/or structural annotation | TATA-like box (position) | Poly(A) signal (position) | G/T/ATAAG late motif (position: T of TAAG) | T | P | Mean coverage | Raw read count | FPKM |
| SGHV-Eth001 (1 → 2088) | PIF-0 (P74) Spodoptera lituranucleopolyhedrovirus (envelope protein) | SGHV-Uga001 | 99.71 | YP_001686949.1 | 0.0 | 1442 | TM; SP; isoleucine-rich activation motif | TATATT (190205) | AATAAT (2106) | TGAATAAATAAG (190286) | Yes | Yes | 1126.4 | 23287 | 16093 |
| SGHV-Eth002 (3071 ← 2091) | (Tegument protein) | SGHV-Uga002 | 100.00 | YP_001686950.1 | 0.0 | 671 | TATAAT (3100) | AATAAA (2046) | Not found | Yes | Yes | 310.8 | 3019 | 4441 | |
| SGHV-Eth003 (3785 ← 3237) | SGHV-Uga003 | 100.00 | YP_001686951.1 | 2.00e–130 | 375 | TATAAA (3839) | AATAAA (3238) | TATAAAATTAAG (3831) | No | No | 126.2 | 686 | 1803 | ||
| SGHV-Eth004 (5440 ← 4382) | ODV-E66 protein Euproctis pseudoconspersanucleopolyhedrovirus | SGHV-Uga005 | 99.72 | YP_001686953.1 | 0.0 | 724 | TM helix; INM-SM; chondroitin AC/alginate lyase | TATAAT (5453) | AATTATT (4349) | ATGTATAATAAG (5448) | Yes | No | 515.6 | 5406 | 7366 |
| SGHV-Eth005 (6580 ← 5495) | Lecithin-cholesterol acyltransferase Pseudomonas sp. (ICSVP) | SGHV-Uga006 | 98.90 | YP_001686954.1 | 0.0 | 719 | Lecithin : cholesterol/phospholipid : diacylglycerol acyltransferase | Not found | AATAA (5450) | TACACAAATAAG (6590) | Yes | Yes | 77.4 | 832 | 1105 |
| SGHV-Eth006 (6718 → 7779) | d-3-Phosphoglycerate dehydrogenase Clostridium ultunense(tegument protein) | SGHV-Uga007 | 98.87 | YP_001686955.1 | 0.0 | 691 | TATAAA (6646) | AATAAA (7800) | ATAAATCTTAAG (6655) | Yes | Yes | 48.5 | 510 | 693 | |
| SGHV-Eth007 (8621 ← 7809) | (Virion protein) | SGHV-Uga008 | 97.79 | YP_001686956.1 | 0.0 | 529 | TATATT (8712) | AATAAA (7798) | GTTGCTGATAAG (8698) | Yes | Yes | 35.3 | 284 | 504 | |
| SGHV-Eth008 (8634 → 10868) | MAL7P1.132 Plasmodium falciparum3D7 (ICSVP) | SGHV-Uga009 | 92.73 | YP_001686957.1 | 0.0 | 1347 | SWI/SNF-related matrix-associated actin-dependent regulator of chromatin | TATAAT (8583) | AATAAA (10880) | Not found | Yes | Yes | 719.3 | 15916 | 10276 |
| SGHV-Eth009 (14184 ← 10894) | ORF MSV156 Melanoplus sanguinipes entomopoxvirus (tegument protein) (desmoplakin) | SGHV-Uga010 | 94.04 | YP_001686958.1 | 0.0 | 1999 | Coiled coils; proline-rich; BP-NLS; PPase-tensin | TATATT (14259) | AATAAA (10879) | AAATTGTATAAG (14242) | Yes | Yes | 591.2 | 19264 | 8446 |
| SGHV-Eth010 (14759 ← 14184) | ORF AMV179 Amsacta mooreientomopoxvirus (ICSVP) | SGHV-Uga011 | 98.44 | YP_001686959.1 | 2.00e–129 | 374 | TATAAA (14813) | AATAA (14043) | Not found | Yes | Yes | 160.8 | 917 | 2297 | |
| SGHV-Eth011 (15876 ← 14818) | SGHV-Uga012 | 99.15 | YP_001686960.1 | 0.0 | 691 | TATATT (15984) | AATATAA (14815) | Not found | Yes | No | 13.5 | 141 | 192 | ||
| SGHV-Eth012 (16760 ← 16554) | No hits | TM helix | Not found | Not found | Not found | No | No | ||||||||
| SGHV-Eth013 (16935 → 17501) | UDP-glucose-6 deshydrogenase Pseudobutyrivibrio ruminis | SGHV-Uga016 | 100.00 | YP_001686964.1 | 4.00e–83 | 253 | TM | TATATAA (16760) | AATAAA (17504) | AGATAAGATAAG (16878) | Yes | No | 487.7 | 2738 | 6968 |
| SGHV-Eth014 (18701 ← 17514) | Putative ubiquitin ligase Feldmannia sp. virus | SGHV-Uga017 | 89.39 | YP_001686965.1 | 0.0 | 694 | Zinc finger; RING/U-box fold | TATAAA (18795) | AATAAA (17452) | Not found | Yes | No | 1565.5 | 18414 | 22366 |
| SGHV-Eth015 (19087 ← 18923) | No hits | TM helix | TATAT (19214) | Not found | Not found | No | No | 28.6 | 47 | 411 | |||||
| SGHV-Eth016 (20108 ← 19947) | No hits | NAD-specific glutamate dehydrogenase domain | TATATA (20173) | Not found | Not found | No | No | 130.8 | 210 | 1870 | |||||
| SGHV-Eth017 (20613 ← 20314) | Uncharacterized conserved protein (COG3868) | No hits | TATAAA (20703) | AATAAA (20306) | Not found | No | No | 49.5 | 147 | 707 | |||||
| SGHV-Eth018 (22022 ← 20721) | Putative ubiquitin ligase Feldmannia sp. virus | SGHV-Uga019 | 90.97 | YP_001686967.1 | 0.0 | 805 | Zinc finger; RING/U-box fold | TATAAA (22101) | AATAAA (20699) | Not found | Yes | No | 258.7 | 3335 | 3696 |
| SGHV-Eth019 (22943 ← 22677) | No hits | TM helix | TATAAA (23032) | Not found | Not found | No | No | ||||||||
| SGHV-Eth020 (23443 → 23970) | SGHV-Uga020 | 99.43 | YP_001686968.1 | 2.00e–123 | 357 | TATAAA (23375) | AATAAA (24150) | Not found | Yes | No | 52.3 | 273 | 746 | ||
| SGHV-Eth021 (25248 ← 23971) | SGHV-Uga021 | 99.06 | YP_001686969.1 | 0.0 | 856 | TATATAA (25289) | AATAAT (23921) | Not found | Yes | No | 83.4 | 1055 | 1191 | ||
| SGHV-Eth022 (25256 → 26284) | ORF AMV156 Amsacta mooreientomopoxvirus | SGHV-Uga022 | 98.83 | YP_001686970.1 | 0.0 | 693 | TATATT (25147) | AATAAA (26335) | Not found | Yes | No | 9.1 | 93 | 130 | |
| SGHV-Eth023 (26341 → 27468) | ORF AMV253Amsacta mooreientomopoxvirus (possible surface protein) | SGHV-Uga023 | 99.73 | YP_001686971.1 | 0.0 | 742 | AARP2CN (NUC121) domain SM00785 | TATATA (26295) | AAATAA (27589) | Not found | Yes | No | 4.4 | 49 | 63 |
| SGHV-Eth024 (29032 ← 27479) | ORF098 Orgyia pseudotsugatanucleopolyhedrovirus | SGHV-Uga024 | 96.14 | YP_001686972.1 | 0.0 | 990 | TM helix | TATAAT (29058) | AATTAA (27442?) | Not found | No | No | 329.8 | 5075 | 4712 |
| SGHV-Eth025 (29124 → 29873) | ORF55 Leucania separatanucleopolyhedrovirus | SGHV-Uga025 | 98.00 | YP_001686973.1 | 2.00e–176 | 497 | TATAAT (29055) | AATAAA (29885) | Not found | Yes | No | 16.8 | 125 | 240 | |
| SGHV-Eth026 (30249 → 31292) | (Possible surface protein) | SGHV-Uga026 | 99.71 | YP_001686974.1 | 0.0 | 698 | TATAAA (30196) | AATAAA (31310) | Not found | Yes | No | 120 | 1240 | 1714 | |
| SGHV-Eth027 (31318 → 32688) | ORF010 Pieris rapaegranulosis virus type II chitinase (virion protein) | SGHV-Uga027 | 99.33 | YP_001686975.1 | 0.0 | 900 | SP; chitinase family 18 | TATATA (31194) | AATAAA (32703) | CATATTAATAAG (31296) | Yes | Yes | 2892 | 39257 | 41317 |
| SGHV-Eth028 (33059 ← 32772) | No hits | Rifin_STEVOR, Rif/Stevor family | TATATA (33106) | AATAAA (32733) | Not found | No | No | 88.2 | 251 | 1258 | |||||
| SGHV-Eth029 (33186 → 33776) | ORF 091R invertebrate iridovirus 25 GIY-YIG nuclease-like protein | SGHV-Uga028 | 99.49 | YP_001686976.1 | 5.00e–137 | 393 | Coiled coils; GIY-YIG superfamily family domain | TATATA (33101) | AATAAA (33876) | Not found | Yes | No | 9.9 | 58 | 142 |
| SGHV-Eth030 (33807 → 34487) | NADH ubiquinone oxidoreductase | SGHV-Uga029 | 98.68 | YP_001686977.1 | 1.00e–156 | 446 | Staphylococcal AgrD protein | TATAAA (33765) | AATAAA (34486) | ACTGTTTGTAAG (33791) | Yes | No | 34.4 | 232 | 492 |
| SGHV-Eth031 (34895 ← 34497) | (Virion protein) | SGHV-Uga030 | 100.00 | YP_001686978.1 | 2.00e–93 | 277 | DNA breaking-rejoining enzyme | TATAT (34947) | AATAAA (34451) | Not found | Yes | Yes | 5736.1 | 22660 | 81948 |
| SGHV-Eth032 (35834 ← 34980) | (Nucleocapsid protein) | SGHV-Uga031 | 100.00 | YP_001686979.1 | 0.0 | 575 | Leucine/isoleucine-rich; HDAC domain | TATAAA (35848) | AATAAT (34938) | Not found | Yes | Yes | 211.8 | 1793 | 3026 |
| SGHV-Eth033 (36661 ← 35885) | LEF-9 ORF24 Gryllus bimaculatusnudivirus (ICSVP) | SGHV-Uga032 | 99.61 | YP_001686980.1 | 0.0 | 526 | Coiled coils | TATATA (36797) | AATAAA (35844) | Not found | Yes | Yes | 87 | 669 | 1242 |
| SGHV-Eth034 (38003 ← 36960) | LEF-9 ORF24 Gryllus bimaculatusnudivirus (ICSVP) | SGHV-Uga033 | 99.71 | YP_001686981.1 | 0.0 | 705 | TATAAA (38029) | AATAAA (36870) | Not found | Yes | Yes | 621.7 | 6427 | 8883 | |
| SGHV-Eth035 (39105 ← 38056) | ORF AMV260 Amsacta moorei entomopoxvirus (virion protein) | SGHV-Uga034 | 98.57 | YP_001686982.1 | 0.0 | 659 | TATATA (39172) | AATAAA (38046) | TATCAAAATAAG (39124) | Yes | Yes | 601.5 | 6253 | 8593 | |
| SGHV-Eth036 (39378 ← 39118) | ORF MSV238 Melanoplus sanguinipesentomopoxvirus (thymidylate synthase) (ICSVP) | SGHV-Uga035 | 100.00 | YP_001686983.1 | 1.00e–54 | 175 | TM helix; SP | TATAAA (39484) | AATAAA (39058) | Not found | Yes | Yes | 944.8 | 2442 | 13501 |
| SGHV-Eth037 (40006 ← 39662) | ORF067 white spot syndrome virus dihydrofolate reductase-thymidylate synthase (envelope protein) | SGHV-Uga036 | 100.00 | YP_001686984.1 | 5.00e–77 | 234 | SP; TM | TATAAA (40048) | AATAAT (39648) | Not found | Yes | Yes | 288.4 | 985 | 4120 |
| SGHV-Eth038 (40289 ← 40053) | SGHV-Uga037 | 88.24 | YP_001686985.1 | 1.00e–21 | 90.1 | TATATTT (40363) | AATAAA (40038) | Not found | Yes | No | 17866.9 | 41925 | 255255 | ||
| SGHV-Eth039 (44292 ← 40702) | Maltodextrin glycosyltransferase (tegument protein) | SGHV-Uga038 | 97.70 | YP_001686986.1 | 0.0 | 2246 | Coiled coils; SP; RGD motif | Not found | AAATAA (40644) | TTTAATAATAAG (44304) | Yes | Yes | 5406.5 | 192225 | 77240 |
| SGHV-Eth040 (45360 ← 44359) | HSP90-like ATPase (envelope protein) | SGHV-Uga039 | 89.60 | YP_001686987.1 | 4.00e–166 | 478 | TM; SP; threonine-rich | TATAAT (45383) | AATAAT (44309) | CTTTATAATAAG (45378) | Yes | Yes | 14559.5 | 144442 | 208006 |
| SGHV-Eth041 (45680 → 48382) | LEF-8 Spodoptera lituranucleopolyhedrovirus (ICSVP) | SGHV-Uga040 | 99.78 | YP_001686988.1 | 0.0 | 1849 | TATATT (45572) | AATAAA (48402) | Not found | Yes | Yes | 135 | 3614 | 1929 | |
| SGHV-Eth042 (49675 ← 48437) | Casein kinase isoform I-d ORF MSV214 Melanoplus sanguinipesentomopoxvirus (envelope protein) | SGHV-Uga041 | 98.79 | YP_001686989.1 | 0.0 | 831 | PD-(D/E) XK nuclease fold | TATAAT (49779) | AATAAA (48413) | Not found | No | Yes | 46.3 | 568 | 661 |
| SGHV-Eth043 (50093 ← 49722) | (ICSVP) | SGHV-Uga042 | 100.00 | YP_001686990.1 | 2.00e–83 | 251 | Coiled coils; TM helix | TATAAA (50226) | AATAAA (49586) | AATGTTAATAAG (50102) | Yes | Yes | 2189.8 | 8066 | 31287 |
| SGHV-Eth044 (50562 ← 50131) | (Tegument protein) | SGHV-Uga043 | 99.31 | YP_001686991.1 | 4.00e–97 | 288 | SP; glutamine-rich region | TATATA (50672) | AATATT (50088) | Not found | Yes | Yes | 1213.6 | 5191 | 17339 |
| SGHV-Eth045 (51813 ← 50731) | (Nucleocapsid protein) | SGHV-Uga044 | 98.34 | YP_001686992.1 | 0.0 | 672 | Coiled coils; TM; t-SNAREs; SF3 helicase | TATAAT (51848) | AATAAT (50690) | Not found | Yes | Yes | 267.3 | 2866 | 3819 |
| SGHV-Eth046 (56956 ← 51773) | LEF-3 ORF51 Maruca vitrata multiple nucleopolyhedrovirus (envelope protein) | SGHV-Uga045 | 99.31 | YP_001686993.1 | 0.0 | 3511 | PPase-tensin | TATAAA (57002) | AATATA (51736) | TTAGCCCATAAG (56975) | Yes | Yes | 72.6 | 3726 | 1037 |
| SGHV-Eth047 (57195 ← 57001) | No hits | GTPase-activator protein for Ras-like GTPase; TM helix | TATATA (57313) | AATAAA (56936) | CTTATTTATAAG (57210) | No | No | ||||||||
| SGHV-Eth048 (57226 → 58824) | Glutathione S-transferase (tegument protein) | SGHV-Uga046 | 99.81 | YP_001686994.1 | 0.0 | 1098 | PPase (inorganic pyrophosphatase) | TATAAA (57164) | AATAAT (58999) | CTTATAAATAAG (57215) | Yes | Yes | 395.2 | 6257 | 5646 |
| SGHV-Eth049 (58843 → 60060) | Cellular protein CBG22662 Coenorhabditis briggsae (tegument protein) | SGHV-Uga047 | 98.52 | YP_001686995.1 | 0.0 | 758 | NUDIX hydrolase domain-like; coiled coils; pre-mRNA splicing factor 9-like protein | TATATA (58756) | AATAAA (60131) | AATAATAATAAG (58837) | Yes | Yes | 180.8 | 2181 | 2584 |
| SGHV-Eth050 (60099 → 60881) | Budded virus envelope protein 31 Helicoverpa armigeranucleopolyhedrovirus NNg1 | SGHV-Uga048 | 97.72 | YP_001686996.1 | 2.00e–180 | 508 | NUDIX superfamily hydrolases motif | TATAAT (60052) | AAATAT (60883) | ATTTTTAATAAG (60076) | No | No | 63.7 | 494 | 910 |
| SGHV-Eth051 (60833 → 62080) | (Tegument protein) | SGHV-Uga049 | 90 | YP_001686997.1 | 6.00e–49 | 168 | TATATT (60755) | AAATAT (62114) | TATATTAATAAG (60763) | Yes | Yes | 5246 | 64822 | 74947 | |
| SGHV-Uga050 | 98.97 | YP_001686998.1 | 0.0 | 587 | |||||||||||
| SGHV-Eth052 (64069 ← 62096) | LEF-4 ORF96 Gryllus bimaculatus nudivirus (ICSVP) | SGHV-Uga051 | 98.94 | YP_001686999.1 | 0.0 | 1237 | TM helix | TATAAA (64108) | AATAAA (62097) | Not found | Yes | Yes | 10.7 | 208 | 152 |
| SGHV-Eth053 (65113 ← 64196) | ORF MSV152 putative core protein (nucleocapsid protein) | SGHV-Uga052 | 100.00 | YP_001687000.1 | 0.0 | 603 | TM helix | TATAA (65130) | AATAAA (64126) | TGAAGATATAAG (65128) | Yes | Yes | 46.3 | 421 | 662 |
| SGHV-Eth054 (66208 ← 65129) | PIF-2 Apis melliferafilamentous virus (envelope protein) | SGHV-Uga053 | 98.89 | YP_001687001.1 | 0.0 | 731 | SP | TATAT (66295) | AATAAA (65003) | Not found | Yes | Yes | 1920.3 | 20534 | 27435 |
| SGHV-Eth055 (67392 ← 66361) | ORF AMV054 Amsacta mooreientomopoxvirus putative RNA polymerase TF (ICSVP) | SGHV-Uga054 | 99.13 | YP_001687002.1 | 0.0 | 665 | TATAAA (67507) | AATAAA (66362) | Not found | Yes | Yes | 12.6 | 129 | 180 | |
| SGHV-Eth056 (69283 ← 67388) | ORF AMV253 Amsacta mooreientomopoxvirus (possible surface protein) | SGHV-Uga055 | 99.37 | YP_001687003.1 | 0.0 | 1257 | TATAAA (69337) | AAATAA (67369) | Not found | Yes | No | 2 | 37 | 0 | |
| SGHV-Eth057 (69349 → 69993) | SGHV-Uga056 | 99.07 | YP_001687004.1 | 5.00e–150 | 427 | TATAAT (69292) | AAATAA (70097) | Not found | Yes | No | 0.8 | 5 | 0 | ||
| SGHV-Eth058 (70035 → 70988) | Rhoptry protein Plasmodium yoleistrain 17XNL | SGHV-Uga057 | 99.69 | YP_001687005.1 | 0.0 | 617 | TATATA (69943) | AATTAAA (71059) | ATTTAAAATAAG (70031) | Yes | No | 2.1 | 19 | 0 | |
| SGHV-Eth059 (70997 → 71203) | No hits | NifE/NifN | TATAAT (70977) | AATAA (71224) | TAATTTAATAAG (70992) | Yes | No | 4721.1 | 9676 | 67449 | |||||
| SGHV-Eth060 (71212 → 71409) | (ICSVP) | No hits | Amino acid transporter | TATAAT (71189) | AATAAA (71408) | Not found | Yes | Yes | 1220.8 | 2393 | 17439 | ||||
| SGHV-Eth061 (71482 → 71676) | (ICSVP) | No hits | Domain of unknown function (DUF3557) | TATAAT (71368) | AATAAA (71704) | Not found | Yes | Yes | 5.3 | 10 | 74 | ||||
| SGHV-Eth062 (71936 ← 71775) | No hits | TATAAA (72040) | Not found | Not found | No | No | |||||||||
| SGHV-Eth063 (72216 → 72488) | No hits | Not found | Not found | Not found | No | No | 16.3 | 44 | 233 | ||||||
| SGHV-Eth064 (73411 → 73713) | Signalling mucin HKR1-like Xenopus (Silurana) tropicalis | No hits | 37.00 | XP_006821602.1 | 9.00e–07 | 53.9 | TATAAT (73344) | AATAAA (73558) | Not found | No | No | 6.6 | 20 | 95 | |
| SGHV-Eth065 (74476 ← 74321) | No hits | SplB; DNA repair; TM helix | TATAAA (74514) | Not found | Not found | No | No | ||||||||
| SGHV-Eth066 (75576 ← 74602) | RpoD-like protein P. falciparum | SGHV-Uga059 | 98.15 | YP_001687007.1 | 0.0 | 619 | TATAAT (75627) | AATAAA (74591) | Not found | Yes | No | 3.8 | 37 | 55 | |
| SGHV-Eth067 (76209 ← 75586) | (ICSVP) | SGHV-Uga060 | 100.00 | YP_001687008.1 | 5.00e–148 | 422 | TATATA (76248) | AATAAT (75447) | Not found | Yes | Yes | 367.9 | 2273 | 5256 | |
| SGHV-Eth068 (77752 ← 76268) | Acanthamoeba polyphaga mimivirus (nucleocapsid protein) | SGHV-Uga061 | 99.60 | YP_001687009.1 | 0.0 | 1007 | TM helix | Not found | AATAAA (76136) | Not found | Yes | Yes | 106.8 | 1571 | 1527 |
| SGHV-Eth069 (77713 → 90453) | ORF147 Trichoplusia ni ascovirus-2c (nucleocapsid protein) | SGHV-Uga062 | 91.59 | YP_001687010.1 | 0.0 | 7128 | SP; BP-NLS; GBD-FH3; ezrA; NUMOD3 motifs; spectrin repeats; t-SNAREs | TATAAA (77660) | AATATAA (90504) | TATAATAATAAG (77704) | Yes | Yes | 69 | 8707 | 986 |
| SGHV-Eth070 (91852 ← 90461) | SGHV-Uga063 | 100.00 | YP_001687011.1 | 0.0 | 924 | TM helix | TATAAT (91903) | Not found | Not found | Yes | No | 54.6 | 735 | 781 | |
| SGHV-Eth071 (93816 ← 92032) | ORF AMV130 Amsacta mooreientomopoxvirus ABC transporter protein (tegument protein) | SGHV-Uga064 | 99.83 | YP_001687012.1 | 0.0 | 1192 | Coiled coils; PUM repeats; zinc finger domain | TATAAT (93944) | AATAAA (91991) | TCTCTAAATAAG (93826) | Yes | Yes | 123.3 | 2178 | 1761 |
| SGHV-Eth072 (98093 ← 93831) | ORF AMV039 Amsacta moorei entomopoxvirus ATPase/DNA helicase protein | SGHV-Uga065 | 99.01 | YP_001687013.1 | 0.0 | 2825 | TATATA (98104) | Not found | AATATATATAAG (98098) | No | No | 12.4 | 524 | 177 | |
| SGHV-Eth073 (98139 → 98453) | SGHV-Uga066 | 99.06 | YP_001687014.1 | 2.00e–67 | 209 | α-Helix–β-strand–α-helix | TATATA (98097) | AATAAA (98459) | Not found | Yes | No | 831.6 | 2594 | 11883 | |
| SGHV-Eth074 (99279 ← 98503) | Helicase (tegument protein) | SGHV-Uga067 | 98.46 | YP_001687015.1 | 2.00e–180 | 508 | TM helix | TATATA (99305) | AATAAA (98504) | ATAATAAGTAAG (99290) | Yes | Yes | 281.5 | 2165 | 4021 |
| SGHV-Eth075 (99625 ← 99302) | Riboflavin uptake protein, chain A (ECF transporter) (envelope protein) | SGHV-Uga068 | 100.00 | YP_001687016.1 | 2.00e–70 | 217 | TM helix; SP | TATAAT (99746) | AATAAT (99299) | TTAATAAATAAG (99672) | Yes | Yes | 222.8 | 715 | 3184 |
| SGHV-Eth076 (100496 ← 99687) | Ca2+- and Zn2+-binding protein (tegument protein) | SGHV-Uga069 | 100.00 | YP_001687017.1 | 0.0 | 542 | TATATT (100581) | AATAAA (99644) | TATATAAATAAG (100500) | Yes | Yes | 1340.2 | 10748 | 19147 | |
| SGHV-Eth077 (101994 ← 100687) | ORF AMV156 Amsacta moorei entomopoxvirus (nucleocapsid protein) | SGHV-Uga070 | 98.39 | YP_001687018.1 | 0.0 | 858 | TATATA (102020) | Not found | Not found | Yes | Yes | 10.5 | 136 | 150 | |
| SGHV-Eth078 (102058 → 103881) | Probable capsid protein 3 Acanthamoeba polyphaga mimivirus (tegument protein) | SGHV-Uga071 | 99.34 | YP_001687019.1 | 0.0 | 1225 | NUDIX hydrolase domain-like | TATATA (102001) | AATAAA (103927) | ACTTATTATAAG (102040) | Yes | Yes | 29.1 | 525 | 415 |
| SGHV-Eth079 (104676 ← 103870) | FAD-dependent thiol oxidase African swine fever virus Malawi LIL 20/1 (envelope protein) | SGHV-Uga072 | 99.63 | YP_001687020.1 | 0.0 | 540 | TM; ERV/ALR thiol oxidase domain (mitochondrial) | TATAAA (104760) | AATAAA (103871) | Not found | Yes | Yes | 879.1 | 7024 | 12559 |
| SGHV-Eth080 (104928 ← 104692) | SGHV-Uga073 | 98.73 | YP_001687021.1 | 2.00e–46 | 154 | TATAAA (104965) | Not found | Not found | Yes | No | 1023.6 | 2402 | 14624 | ||
| SGHV-Eth081 (104975 → 107110) | Helicase 2-like protein Spodoptera frugiperdagranulosis virus | SGHV-Uga074 | 99.16 | YP_001687022.1 | 0.0 | 1439 | P-loop NTPase fold (Walker A/B motifs) | TATAAA (104771) | AATAAA (107120) | Not found | Yes | No | 32.5 | 687 | 464 |
| SGHV-Eth082 (107969 ← 107157) | SGHV-Uga075 | 99.26 | YP_001687023.1 | 0.0 | 526 | TATAAA (108021) | Not found | Not found | Yes | No | 65.6 | 538 | 937 | ||
| SGHV-Eth083 (107993 → 108625) | PIF-3 Euproctis pseudoconspersa single nucleopolyhedrovirus (virion protein) | SGHV-Uga076 | 100.00 | YP_001687024.1 | 3.00e–148 | 423 | TM helix | Not found | AATAAA (108645) | Not found | Yes | Yes | 478.9 | 3002 | 6843 |
| SGHV-Eth084 (108695 → 111871) | ORF AMV130 Amsacta moorei entomopoxvirus ABC transporter protein (ICSVP) | SGHV-Uga077 | 99.72 | YP_001687025.1 | 0.0 | 2117 | DNA polymerase (B); exonuclease/H-like domains | TATATA (108633) | AATAAA (111979) | CAAACAAATAAG (108691) | Yes | Yes | 6.6 | 208 | 94 |
| SGHV-Eth085 (111886 → 112041) | No hits | Ycf48-like protein (provisional); TM helix | Not found | AATAAA (112241) | Not found | No | No | ||||||||
| SGHV-Eth086 (112845 ← 112138) | ORF004 Oryctes rhinocerosnudivirus Ac81-like protein (ICSVP) | SGHV-Uga078 | 98.31 | YP_001687026.1 | 2.00e–166 | 471 | TM | TATATT (113012) | AATAAA (112133) | ATAATAAATAAG (112856) | Yes | Yes | 325.7 | 2283 | 4653 |
| SGHV-Eth087 (112896 → 115754) | ORF9 human herpesvirus 8 type M DNA polymerase | SGHV-Uga079 | 99.58 | YP_001687027.1 | 0.0 | 1955 | DNA/RNA polymerases (α/β multidomains) | TATAAA (112820) | AATAAA (115763) | Not found | Yes | No | 188.2 | 5327 | 2689 |
| SGHV-Eth088 (116378 ← 115776) | (ICSVP) | SGHV-Uga080 | 99.50 | YP_001687028.1 | 1.00e–141 | 405 | TATAAA (116401) | AATAAA (115777) | Not found | Yes | Yes | 140.7 | 840 | 2010 | |
| SGHV-Eth089 (116899 ← 116387) | SGHV-Uga081 | 99.42 | YP_001687029.1 | 5.00e–117 | 340 | TM helix | TATAAA (117011) | AATAAA (116385) | ATTAACTATAAG (116909) | Yes | No | 28.4 | 144 | 405 | |
| SGHV-Eth090 (117392 ← 116916) | (Virion protein) | SGHV-Uga082 | 100.00 | YP_001687030.1 | 3.00e–110 | 322 | TM | TATAAT (117445) | AATAAA (116776) | ATAGCAAATAAG (117397) | Yes | Yes | 112.9 | 533 | 1612 |
| SGHV-Eth091 (119479 ← 117398) | ORF AMV214 Amsacta mooreientomopoxvirus (nucleocapsid protein) | SGHV-Uga083 | 99.42 | YP_001687031.1 | 0.0 | 1402 | BP-NLS | TATAAT (119523) | AATAAA (117389) | Not found | Yes | Yes | 116.5 | 2401 | 1664 |
| SGHV-Eth092 (119571 → 120227) | (Virion protein) | SGHV-Uga084 | 99.09 | YP_001687032.1 | 3.00e–149 | 426 | TATATT (119537) | AATAAA (120246) | TATAAATGTAAG (119556) | Yes | Yes | 13.9 | 90 | 198 | |
| SGHV-Eth093 (120447 → 121211) | Signalling protein (tegument protein) | SGHV-Uga085 | 96.47 | YP_001687033.1 | 0.0 | 509 | α-Helix–β-strand–α-helix | Not found | AATAAA (121218) | Not found | Yes | Yes | 644 | 4877 | 9199 |
| SGHV-Eth094 (121304 → 123079) | ORF398 megavirus chiliensis ankyrin repeat protein (tegument protein) | SGHV-Uga086 | 100.00 | YP_001687034.1 | 0.0 | 1201 | α-Helix–β-strand–α-helix | TATAAT (121236) | AATAAA (123078) | TAGATATATAAG (121292) | Yes | Yes | 1031.3 | 18134 | 14733 |
| SGHV-Eth095 (123392 ← 123105) | LEF-5 ORF99 Culex nigripalpusnucleopolyhedrovirus | SGHV-Uga087 | 96.88 | YP_001687035.1 | 7.00e–62 | 194 | TFIIS-type zinc finger; RNA polymerase subunit RPO30 | TATAAT (123456) | AATAAA (123074) | Not found | Yes | No | 93.1 | 265 | 1328 |
| SGHV-Eth096 (123514 → 125466) | ORF AMV258 Amsacta mooreientomopoxvirus vaccinia G1L metalloprotease (envelope protein) | SGHV-Uga088 | 98.16 | YP_001687036.1 | 0.0 | 1226 | TM helix | TATAAT (123434) | Not found | TGTTGTTATAAG (123503) | Yes | Yes | 180.6 | 3492 | 2580 |
| SGHV-Eth097 (125484 → 126023) | ORF AMV134 Amsacta moorei entomopoxvirus leucine-rich repeat gene family protein | SGHV-Uga089 | 97.78 | YP_001687037.1 | 2.00e–117 | 342 | Coiled coils | TATATA (125431) | AATAAA (126050) | Not found | Yes | No | 14.4 | 77 | 206 |
| SGHV-Eth098 (126211 ← 126026) | No hits | TATAAT (126291) | AATAAA (125962) | Not found | No | No | |||||||||
| SGHV-Eth099 (126398 → 126631) | SGHV-Uga090 | 100.00 | YP_001687038.1 | 2.00e–43 | 146 | TM helix | TATAAA (126646) | AATAAA (127611) | Not found | Yes | No | ||||
| SGHV-Eth100 (126731 → 127534) | Putative capsid protein 3 Cafeteria roenbergensisvirus BV_PW1 (virion protein) | SGHV-Uga091 | 99.25 | YP_001687039.1 | 0.0 | 541 | TM; PS; RNA-dependent RNA polymerase | TATAAA (127546) | AATAAA (127828) | GCAATTATTAAG (126556) | Yes | Yes | 33.1 | 264 | 474 |
| SGHV-Eth101 (127557 → 127805) | (ICSVP) | SGHV-Uga092 | 100.00 | YP_001687040.1 | 1.00e–50 | 165 | TATATA (127252) | AATAAT (126196) | Not found | Yes | Yes | 5496.9 | 13552 | 78533 | |
| SGHV-Eth102 (127816 → 128802) | Hydrolase (IIe RE) (tegument protein) | SGHV-Uga093 | 100.00 | YP_001687041.1 | 0.0 | 665 | TM | Not found | AATAAA (128797) | Not found | Yes | Yes | 1188.1 | 11611 | 16975 |
| SGHV-Eth103 (128827 → 129645) | (Tegument protein) | SGHV-Uga094 | 98.90 | YP_001687042.1 | 0.0 | 541 | TM; α-helix–β-strand–α-helix | Not found | AATAAT (129731) | Not found | Yes | Yes | 278.7 | 2260 | 3982 |
| SGHV-Eth104 (129659 → 130129) | (Virion protein) | SGHV-Uga095 | 98.09 | YP_001687043.1 | 2.00e–103 | 305 | TATATA (129559) | AATAAA (130132) | TAAAGAAGTAAG (129590) | Yes | Yes | 33.1 | 154 | 472 | |
| SGHV-Eth105 (130169 → 131311) | Metal-binding protein (tegument protein) | SGHV-Uga096 | 98.16 | YP_001687044.1 | 0.0 | 747 | TM helix | TATAAA (130096) | AATAAA (131321) | TCATTAGATAAG (130156) | Yes | Yes | 796.3 | 9012 | 11377 |
| SGHV-Eth106 (131301 → 132482) | Vesicle-associated membrane protein (tegument protein) | SGHV-Uga097 | 98.98 | YP_001687045.1 | 0.0 | 800 | TM helix; RNA polymerase | TATATT (131262) | AATAAA (132580) | Not found | Yes | Yes | 1843.2 | 21571 | 26333 |
| SGHV-Eth107 (132505 → 132849) | (Nucleocapsid protein) | SGHV-Uga098 | 96.52 | YP_001687046.1 | 9.00e–73 | 223 | TM | Not found | AATAAA (132874) | Not found | Yes | Yes | 1015.7 | 3469 | 14509 |
| SGHV-Eth108 (133544 ← 133065) | SGHV-Uga099 | 98.75 | YP_001687047.1 | 3.00e–109 | 320 | PD-(D/E) XK nuclease fold | TATATA (133638) | AATAAT (133030) | TATAACTATAAG (133555) | Yes | No | 399.8 | 1900 | 5712 | |
| SGHV-Eth109 (133566 → 133856) | Hypothetical conserved protein PRK06126 (ICSVP) | No hits | Not found | Not found | Not found | Yes | Yes | 6160.8 | 17750 | 88015 | |||||
| SGHV-Eth110 (133970 → 134227) | (ICSVP) | No hits | Serpentine type 7TM GPCR chemoreceptor Srx; TM helix | Not found | AATAAA (134356) | Not found | Yes | Yes | 2063.8 | 5272 | 29485 | ||||
| SGHV-Eth111 (134308 → 134625) | (Nucleocapsid protein) | SGHV-Uga101 | 100.00 | YP_001687049.1 | 3.00e–67 | 209 | TM helix | TATAAT (134265) | AATAAA (134673) | Not found | Yes | Yes | 473.3 | 1493 | 6775 |
| SGHV-Eth112 (134681 → 136636) | PIF-1 Neodiprion abietisnucleopolyhedrovirus (envelope protein) | SGHV-Uga102 | 99.54 | YP_001687050.1 | 0.0 | 1332 | TM helix; SP; EGF-like domain | TATATA (134644) | AATAAA (136700) | TTATATAATAAG (134651) | Yes | Yes | 608.1 | 11776 | 8687 |
| SGHV-Eth113 (137858 ← 136731) | Viral capsid-associated protein 1054 Neodipron abietisnucleopolyhedrovirus | SGHV-Uga103 | 97.87 | YP_001687051.1 | 0.0 | 743 | TATAAA (137935) | AATAAA (136732) | Not found | Yes | No | 29.6 | 331 | 423 | |
| SGHV-Eth114 (137907 → 139886) | (Nucleocapsid protein) | SGHV-Uga104 | 99.39 | YP_001687052.1 | 0.0 | 1335 | TM; coiled coil region | TATAAA (137859) | AATAAA (139896) | Not found | Yes | Yes | 31.9 | 625 | 455 |
| SGHV-Eth115 (140947 ← 140072) | (Virion protein) | SGHV-Uga105 | 98.97 | YP_001687053.1 | 0.0 | 562 | Coiled coils | TATATT (141031) | AATAAA (139965) | TAATATATTAAG (140963) | Yes | Yes | 110.1 | 955 | 1573 |
| SGHV-Eth116 (142410 ← 140953) | ORF MSV156 Melanoplus sanguinipes entomopoxvirus (nucleocapsid protein) | SGHV-Uga106 | 98.29 | YP_001687054.1 | 0.0 | 919 | Coiled coils; serine/threonine/glutamine-rich stretches | TATAAT (142466) | AATAAA (140839) | Not found | Yes | Yes | 522.8 | 75.47 | 74.69 |
| SGHV-Eth117 (143935 ← 142373) | Cell division protein 48 lymphocystis disease virus (China isolate) (nucleocapsid protein) | SGHV-Uga107 | 97.89 | YP_001687055.1 | 0.0 | 1046 | SP; AAA+-ATPase; PAN | TATAAA (143960) | AAATAA (142334) | Not found | Yes | Yes | 806.7 | 12484 | 11525 |
| SGHV-Eth118 (145550 ← 143916) | Cell division protein 48 lymphocystis disease virus (China isolate) (nucleocapsid protein) | SGHV-Uga108 | 99.08 | YP_001687056.1 | 0.0 | 1096 | SP; P-loop/AAA+-ATPase; PAN | TATACA (145574) | Not found | Not found | Yes | Yes | 30 | 486 | 429 |
| SGHV-Eth119 (145628 → 145804) | No hits | Phage shock protein B; TM helix | TATAAA (145571) | AATAAA (145900) | Not found | No | No | ||||||||
| SGHV-Eth120 (146791 ← 145937) | (Virion protein) | SGHV-Uga109 | 99.30 | YP_001687057.1 | 0.0 | 567 | TM helix; CBS domain | Not found | AATAAA (145853) | Not found | Yes | Yes | 601.6 | 5093 | 8595 |
| SGHV-Eth121 (147433 ← 146831) | Spodoptera lituragranulosis virus matrix metalloproteinase-38 (MP-NASE-like protein) (tegument protein) | SGHV-Uga110 | 99.50 | YP_001687058.1 | 9.00e–145 | 413 | TM helix; SP; helix–turn–helix domain | TATAAT (147490) | AATAAA (146701) | TTGTATTTTAAG(147463) | Yes | Yes | 2331.8 | 13921 | 33312 |
| SGHV-Eth122 (148189 ← 147533) | MP-NASE-like protein (ICSVP) | SGHV-Uga111 | 98.63 | YP_001687059.1 | 7.00e–153 | 435 | TM; SP; zinc protease | TATATT (148280) | Not found | CTGTTTAATAAG (148194) | Yes | Yes | 6661.1 | 43330 | 95164 |
| SGHV-Eth123 (148826 ← 148302) | Regulatory protein (tegument protein) | SGHV-Uga112 | 94.86 | YP_001687060.1 | 2.00e–110 | 323 | TM helix; SP | Not found | Not found | Not found | No | Yes | 2444.4 | 12706 | 34922 |
| SGHV-Eth124 (149662 ← 148820) | Cellular protein PY00593 Plasmodium yoelli strain 17XNL (nucleocapsid protein) | SGHV-Uga113 | 98.93 | YP_001687061.1 | 0.0 | 553 | TATAT (149806) | Not found | TTTAATAGTAAG (149788) | Yes | Yes | 1162 | 9699 | 16602 | |
| SGHV-Eth125 (149925 ← 149668) | No hits | Vps52/Sac2 family | TAATAT (150036?) | AATAAT (149666?) | Not found | No | No | ||||||||
| SGHV-Eth126 (149956 → 151275) | ORF MSV016 Melanoplus sanguinipesentomopoxvirus leucine-rich repeat gene family protein | SGHV-Uga114 | 99.77 | YP_001687062.1 | 0.0 | 868 | Leucine-rich repeat protein-like protein | TATATT (149803) | AATAAA (151306) | TGTTCTTATAAG (149937) | Yes | No | 8.9 | 116 | 127 |
| SGHV-Eth127 (151332 → 152561) | SGHV-Uga115 | 99.27 | YP_001687063.1 | 0.0 | 830 | F-box/RNI superfamily | TATATA (151281) | AATAAA (152653) | Not found | No | No | 82.5 | 1004 | 1178 | |
| SGHV-Eth128 (152582 → 153655) | ORF AMV134 Amsacta mooreileucine-rich repeat gene family protein | SGHV-Uga116 | 98.88 | YP_001687064.1 | 0.0 | 696 | RNase inhibitor | TATATA (152445) | AATAAA (153722) | Not found | Yes | No | 21.4 | 227 | 305 |
| SGHV-Eth129 (154378 ← 153662) | ISKNV ORF 099L RING finger protein Phaeocystis globosa virus | SGHV-Uga117 | 97.91 | YP_001687065.1 | 1.00e–158 | 451 | E3-ubiquitin-protein ligase TRAIP | TATAAA (154406) | Not found | Not found | Yes | No | 578.3 | 4105 | 8261 |
| SGHV-Eth130 (154650 ← 154405) | ORF103L scale drop disease virus | SGHV-Uga118 | 98.78 | YP_001687066.1 | 5.00e–52 | 168 | Coiled coils; E3-ubiquitin-protein ligase TRAIP | TATAAA (154742) | AATAAA (154264) | Not found | Yes | No | 906.3 | 2207 | 12945 |
| SGHV-Eth131 (155345 ← 154695) | SGHV-Uga119 | 100.00 | YP_001687067.1 | 6.00e–152 | 432 | RNase inhibitor | Not found | Not found | Not found | Yes | No | 159.3 | 1026 | 2274 | |
| SGHV-Eth132 (155374 → 156426) | SGHV-Uga120 | 97.72 | YP_001687068.1 | 0.0 | 696 | TATAT (155238) | AAATAA (156489) | TAATGTTTTAAG (155291) | Yes | No | 114.2 | 1190 | 1631 | ||
| SGHV-Eth133 (156715 → 157473) | SGHV-Uga121 | 100.00 | YP_001687069.1 | 0.0 | 523 | TM | TATAAT (156598) | AAATAA (157541) | ATTATTTTTAAG (156610) | Yes | No | 52.1 | 391 | 743 | |
| SGHV-Eth134 (57669 → 158016) | SGHV-Uga122 | 100.00 | YP_001687070.1 | 8.00e–79 | 239 | TM helix | TATAAA (157605) | AATAAA (158193) | Not found | Yes | No | 170.1 | 586 | 2430 | |
| SGHV-Eth135 (158076 → 158225) | No hits | CRISPR/Cas system-associated RAMP superfamily protein Csb3 | TATAAT (158058) | AATAAA (158236) | Not found | Yes | No | 11.5 | 17 | 164 | |||||
| SGHV-Eth136 (158840 → 159307) | chmu148 Choristoneura murinana alphabaculovirus | SGHV-Uga123 | 94.87 | YP_001687071.1 | 1.00e–102 | 303 | Coiled coils; zinc finger (C2H2) domain | TATATA (158794) | Not found | Not found | Yes | No | 34.5 | 160 | 493 |
| SGHV-Eth137 (160866 → 162386) | chmu148 Choristoneura murinanaalphabaculovirus | SGHV-Uga124 | 34.95 | YP_001687072.1 | 4.00e–86 | 289 | Coiled coils | TATAAT (160695) | AATAAA (162437) | Not found | Yes | No | 0.6 | 9 | 0 |
| SGHV-Eth138 (162549 ← 162373) | No hits | Protein trafficking PGA2 | TATAAA (162638) | AATAAA (162259) | Not found | No | No | ||||||||
| SGHV-Eth139 (162679 → 164472) | ORF067 Ecotropis obliqua nucleopolyhedrovirus Cg30 protein | SGHV-Uga125 | 83.82 | YP_001687073.1 | 6.00e–85 | 273 | Coiled coils | TATAAC (162635) | AATAAA (164478) | GTTAATCTTAAG (162668) | Yes | No | 22.3 | 396 | 319 |
| SGHV-Eth140 (164571 → 164813) | ORF149 Anticarsia gemmatalis nucleopolyhedrovirus Pe38-like protein | SGHV-Uga126 | 98.72 | YP_001687074.1 | 2.00e–43 | 147 | Coiled coils | TATATG (164516) | AATAAA (164819) | AATAATTATAAG (164563) | Yes | No | 67.2 | 162 | 962 |
| SGHV-Eth141 (165783 → 166046) | SGHV-Uga127 | 88.89 | YP_001687075.1 | 8.00e–46 | 152 | TATAAA (165725) | AATAAA (166059) | CTTATAGTTAAG (165757) | Yes | No | 265.7 | 694 | 3793 | ||
| SGHV-Eth142 (166616 ← 166461) | No hits | Zinc finger RING of BARD1-type protein domain | TATAAT (166659) | AATAAA (166462) | Not found | Yes | No | 147.9 | 228 | 2109 | |||||
| SGHV-Eth143 (166602 → 166784) | SGHV-Uga128 | 87.30 | YP_001687076.1 | 2.00e–22 | 91.7 | Zinc finger (C2H2) domain | TATATT (166532) | AATAAA (166913) | Not found | No | No | 1894.6 | 3433 | 27069 | |
| SGHV-Eth144 (167032 → 167292) | (ICSVP) | SGHV-Uga129 | 100.00 | YP_001687077.1 | 7.00e–43 | 145 | Coiled coils | TATAAA (167007) | Not found | GTTAACAATAAG (167026) | Yes | Yes | 4.1 | 11 | 61 |
| SGHV-Eth145 (167445 → 167969) | SGHV-Uga130 | 93.71 | YP_001687078.1 | 2.00e–111 | 326 | TATATA (167300) | AATAAA (168020) | CCGAGTTGTAAG (167348) | Yes | No | 730.3 | 3796 | 10433 | ||
| SGHV-Eth146 (168084 → 170510) | ORF MSV156 Melanoplus sanguinipesentomopoxvirus | SGHV-Uga131 | 86.66 | YP_001687079.1 | 0.0 | 1415 | Coiled coils | TATAAA (168036) | Not found | Not found | Yes | No | 85.5 | 2055 | 1222 |
| SGHV-Eth147 (170635 → 170832) | SGHV-Uga132 | 100.00 | YP_001687080.1 | 1.00e–39 | 135 | TATATA (170587) | AATAAA (170859) | Not found | No | No | 2125.9 | 4168 | 30375 | ||
| SGHV-Eth148 (171023 → 172279) | SGHV-Uga133 | 99.28 | YP_001687081.1 | 0.0 | 865 | EGF-like domain signature 1 | TATAAT (170966) | Not found | Not found | Yes | No | 552.5 | 6876 | 7893 | |
| SGHV-Eth149 (172423 → 172719) | Tail length tape-measure protein Oenococcusphage phi9805 (ICSVP) | SGHV-Uga134 | 97.98 | YP_001687082.1 | 2.00e–61 | 194 | Coiled coils | TATAAA (172395) | Not found | Not found | Yes | Yes | 418.9 | 1232 | 5986 |
| SGHV-Eth150 (172742 → 172969) | (ICSVP) | SGHV-Uga135 | 100.00 | YP_001687083.1 | 2.00e–45 | 151 | Coiled coils; helix hairpin bin | TATAAG (172718) | AATAAA (172990) | TAGTTTTATAAG (172720) | No | Yes | 337.6 | 762 | 4822 |
| SGHV-Eth151 (173210 ← 173010) | No hits | TATATT (173349) | AATAAA (173004) | TTTTCGTTTAAG (173320) | Yes | No | 1359.2 | 2705 | 19419 | ||||||
| SGHV-Eth152 (173287 → 173448) | (ICSVP) | SGHV-Uga136 | 100.00 | YP_001687084.1 | 1.00e–29 | 109 | Coiled coils | TATAAA (173247) | AATAAA (173447) | GATATAAATAAG (173253) | No | Yes | 286.2 | 459 | 4088 |
| SGHV-Eth153 (173515 → 173784) | (ICSVP) | SGHV-Uga137 | 93.33 | YP_001687085.1 | 3.00e–53 | 172 | Coiled coils | TATAAA (173457) | AATAAA (173783) | CATATAAATAAG (173463) | No | Yes | 33.8 | 90 | 481 |
| SGHV-Eth154 (173800 → 174159) | (ICSVP) | SGHV-Uga138 | 100.00 | YP_001687086.1 | 6.00e–80 | 242 | Coiled coils | TATATA (173747) | AATAAA (174209) | Not found | Yes | Yes | 21 | 75 | 301 |
| SGHV-Eth155 (174442 → 174636) | (ICSVP) | SGHV-Uga139 | 100.00 | YP_001687087.1 | 1.00e–37 | 130 | Coiled coils | TATAAA (174313) | AATAAA (174635) | AGTAGTGGTAAG (174381) | No | Yes | 482.5 | 932 | 6897 |
| SGHV-Eth156 (174647 → 175870) | ORF148 Choristoneura murinanaalphabavculovirus (virion protein) | SGHV-Uga140 | 99.51 | YP_001687088.1 | 0.0 | 840 | Coiled coils | TATAAA (174520) | AATAAA (175908) | GAAATAAATAAG (174641) | Yes | Yes | 341.4 | 4137 | 4877 |
| SGHV-Eth157 (176052 ← 175789) | LEF-8 Penaeus monodon nudivirus | SGHV-Uga141 | 96.59 | YP_001687089.1 | 3.00e–51 | 167 | TM | TATATT (176190) | AATAAA (175620) | Not found | Yes | No | 124.1 | 324 | 1771 |
| SGHV-Eth158 (176252 → 177322) | SGHV-Uga142 | 99.72 | YP_001687090.1 | 0.0 | 730 | TATAAA (176200) | AATAAA (177377) | TTATAATTTAAG (176230) | Yes | No | 53.1 | 563 | 759 | ||
| SGHV-Eth159 (177366 → 178655) | SGHV-Uga143 | 98.60 | YP_001687091.1 | 0.0 | 866 | Coiled coils | TATAAA (177321) | Not found | Not found | Yes | No | 10.2 | 130 | 145 | |
| SGHV-Eth160 (179750 ← 179379) | ORF AMV193 protein phosphatase 1 Amsacta mooreientomopoxvirus | SGHV-Uga144 | 100.00 | YP_001687092.1 | 5.00e–80 | 243 | PP1 (regulatory subunits 15A/B) | TATAAA (179853) | AATAAA (179387) | Not found | Yes | No | 371.7 | 1369 | 5310 |
| SGHV-Eth161 (180040 → 180726) | ORF179 shrimp white spot syndrome virus | SGHV-Uga145 | 70.74 | YP_001687093.1 | 6.00e–96 | 291 | TATAAA (180029) | AATAAA (180756) | Not found | Yes | No | 5.9 | 40 | 84 | |
| SGHV-Eth162 (180745 → 181377) | (Virion protein) | SGHV-Uga146 | 98.58 | YP_001687094.1 | 2.00e–139 | 400 | RNA-dependent RNA polymerase | TATAAT (180690) | AATAAA (181376) | TATAATAATAAG (180733) | No | Yes | 6.2 | 39 | 89 |
| SGHV-Eth163 (181828 → 182496) | ORF179 shrimp white spot syndrome virus | SGHV-Uga148 | 70.23 | YP_001687096.1 | 1.00e–90 | 279 | TATAGA (181701) | AAATAA (182525) | Not found | No | No | 5.9 | 39 | 84 | |
| SGHV-Eth164 (182922 → 183491) | ORF179 shrimp white spot syndrome virus | SGHV-Uga148 | 64.58 | YP_001687096.1 | 7.00e–69 | 222 | Not found | Not found | Not found | No | No | 5.9 | 36 | 83 | |
| SGHV-Eth165 (185006 ← 184023) | (ICSVP) | SGHV-Uga150 | 97.57 | YP_001687098.1 | 0.0 | 653 | TATATT (185048) | AATAAA (184021) | Not found | Yes | Yes | 362.8 | 3545 | 5183 | |
| SGHV-Eth166 (185128 → 185373) | SGHV-Uga151 | 98.78 | YP_001687099.1 | 3.00e–49 | 161 | TATAAA (185081) | AATAAA (185414) | Not found | No | No | 324.9 | 791 | 4640 | ||
| SGHV-Eth167 (185476 → 185844) | Bcr–Abl-like protein | SGHV-Uga152 | 100.00 | YP_001687100.1 | 1.00e–82 | 249 | Coiled coils; Bcr–Abl (α-1/2) | TATAAA (185461) | AATAAA (185942) | Not found | Yes | No | 109.8 | 401 | 1568 |
| SGHV-Eth168 (185847 → 186077) | SGHV-Uga153 | 100.00 | YP_001687101.1 | 2.00e–49 | 161 | TATATA (185823) | AATAAA (186080) | Not found | Yes | No | 132.5 | 303 | 1893 | ||
| SGHV-Eth169 (186134 → 187147) | ORF026 Wiseanairidescent virus (nucleocapsid protein) | SGHV-Uga154 | 99.11 | YP_001687102.1 | 0.0 | 668 | Glutamine-rich region | TATAAA (186098) | Not found | Not found | Yes | Yes | 37.2 | 374 | 532 |
| SGHV-Eth170 (187212 → 187409) | SGHV-Uga155 | 98.48 | YP_001687103.1 | 1.00e–35 | 125 | TATAAA (187151) | AATAAA (187408) | Not found | Yes | No | 52.2 | 108 | 787 | ||
| SGHV-Eth171 (187737 ← 187570) | SGHV-Uga156 | 96.49 | YP_001687104.1 | 5.00e–26 | 100 | TATAAA (187796) | AATAAA (187433) | Not found | Yes | No | 282.3 | 470 | 4037 | ||
| SGHV-Eth172 (188119 ← 187892) | SGHV-Uga157 | 100.00 | YP_001687105.1 | 9.00e–46 | 152 | Not found | AATAAA (187792) | Not found | Yes | No | 9.8 | 22 | 139 | ||
| SGHV-Eth173 (188916 ← 188248) | SGHV-Uga158 | 100.00 | YP_001687106.1 | 6.00e–154 | 439 | TATATA (188947) | AATAAA (188066) | Not found | Yes | No | 17.7 | 117 | 252 | ||
| SGHV-Eth174 (189097 → 190281) | ORF AMV253 Amsacta mooreientomopoxvirus (possible surface protein) | SGHV-Uga160 | 99.49 | YP_001687108.1 | 0.0 | 789 | F-box protein 7-like domain | TATATA (189034) | AATAAA (190280) | Not found | Yes | No | 33.9 | 397 | 483 |
TM, Transmembrane domain; SP, signal peptide; INM-SM; inner nuclear membrane sorting motif; SWI/SNF, gene family that affects mating-type switching (SWI) and sucrose fermentation [sucrose non-fermenting (SNF)] pathways; BP-NLS, bipartite nuclear localization signal/sequence; AARP2, asparagine and aspartate-rich protein 2 domain; AgrD, pro-peptide precursor of the auto-inducing peptide (AIP) of the accessory gene regulatory protein (Agr); HDAC, histone deacetylase; RGD, arginyl-glycyl-aspartic acid; PD-(D/E) XK, conserved domain of nuclease superfamily involved in various aspects of nucleic acid metabolism; ABC, ATP-binding cassette; GPCR, G-protein-coupled receptor; EGF, epidermal growth factor; EzrA, septation FtsZ ring formation regulator; NUMOD3, nuclease-associated modular domain 3; PUM, pumilio homology domain; ERV/ALR domain, Erv1 (essential for respiratory and vegetative growth 1) and ALR (augmenter of liver regeneration) domain; TFIIS-type zinc finger; zinc finger motif found in transcription factor IIs; Bcr–Abl, an oncogene arising from the fusion of the breakpoint cluster (bcr) gene with chromosomal Abelson murine leukaemia (c-abl) proto-oncogene; GADD, growth arrest and DNA damage protein; Rifin, repetitive interspersed family; Stevor, subtelomic variant ORF; NifE/NifN, bifunctional nitrogenase fixation proteins E/N.
Fig. 1.
Linear representation of the GpSGHV-Eth genome: The linearization is presented starting with the ATG initiating codon of p74 (SGHV-Eth001). The arrows indicate the positions and orientations of the transcription potential of each ORF. In total, 112 ORFs in the GpSGHV-Eth genome were identical in length (amino acid residues) to the homologous ORFs in the GpSGHV-Uga genome; 37 ORFs had insertions and/or deletions, 11 ORFs were deleted and 24 ORFs were novel in the GpSGHV-Eth genome compared with the GpSGHV-Uga genome. The ORFs are colour-coded to show their homology to the corresponding ORFs in the GpSGHV-Uga genome. The abbreviations used are explained in the footnote to Table 1. The figure is drawn to scale.
Fig. 2.
Genome synteny visualization of the relationship between GpSGHV-Eth and GpSGHV-Uga. The positions of the genomes are shown between the thick black lines, which represent the dsDNA viral genomes. (a) The dot-plot was generated from the whole-genome DNA homology alignments between GpSGHV-Eth and GpSGHV-Uga genomic sequences. The red and blue colours indicate the sequence direction (reverse and forward). (b, c) Syntenic maps show the overall collinearity of GpSGHV-Eth and GpSGHV-Uga compared with Musca domestica salivary gland hypertrophy virus (MdSGHV). The red lines (b, c) indicate identity levels between the viruses, whilst the blue lines (c) indicate instances of inversions. The bands represented in the thick black lines do not necessarily indicate the ORFs, but rather the conserved genomic regions.
Proteogenomic mapping of the GpSGHV-Eth genome
We authenticated the assignment of the GpSGHV-Eth ORFs by mapping transcripts and peptides onto the virus genomic sequence. After quality filtering of the transcriptome data, ∼40 million paired reads remained, of which ∼12.3 million mapped to the host (tsetse fly) and were therefore discarded from further analysis. The remaining reads mapped onto the viral genome and were used to construct 545 putative transcripts (represented by transcript isoforms). Of these, 431 transcripts were predicted to contain protein-coding regions and were used in the functional gene mapping; they mapped onto 141 of the 174 predicted GpSGHV-Eth ORFs (Table 1).
Similarly, filtering out reverse hits, contaminants and host-specific peptides from the liquid chromatography (LC)-MS/MS data resulted in 1314 unique viral peptides. These peptides mapped to 86 of the 174 predicted GpSGHV-Eth ORFs (Table 1). Of the 86 ORFs, 68 ORFs had ATG as the start codon, whilst 13 ORFs had either CTG or TTG as the start codon (see Table S2). Additionally, some of these ORFs contained mapped peptides on sequences upstream of the first methionine residues (Table S2). Non-AUG codons are mostly exhibited by viral mRNAs encoding regulatory proteins (Corcelette et al., 2000). Non-canonical translation initiation is a rare event that occurs in addition to initiation at a downstream AUG (Gupta et al., 1994), possibly resulting in translation of multiple related proteins from a single ORF. Five of the 13 GpSGHV-Eth ORFs with non-AUG start codons were homologues to known regulatory proteins, i.e. SGHV-Eth008 (MAL7P1.132), SGHV-Eth033 (lef-9), SGHV-Eth036 (ts), SGHV-Eth117 (cd-48) and SGHV-Eth122 (mp-nase).
Overall, 45.4 % (n = 79) of the 174 GpSGHV-Eth ORFs had both peptides and transcripts mapped onto their sequences, whilst only seven ORFs had peptide mapping only. Taking into account the proteomic datasets from GpSGHV-Uga (Kariithi et al., 2010, 2011, 2013b, 2016) and the strong homologies between the two virus isolates (Table S1), additional homologous ORFs could be functionally annotated in the GpSGHV-Eth genome (Table 1). Furthermore, of these 79 ORFs, 68 had appropriately upstream positioned TATAA-like box and/or (G/T/A) TAAG transcriptional signals of early and late baculovirus genes, respectively (Chen et al., 2013), whilst 70 ORFs had poly(A) signals. A total of 60 ORFs contained both the TATA-like box and poly(A) signals, and had mapped transcripts and peptides. Only one ORF (SGHV-Eth109) remained without any transcriptional signals. It is worth mentioning that all the ORFs harbouring the TAAG promoter motif were of the (G/T/A)TAAG type characteristic of baculovirus late promoters (Chen et al., 2013; Rohrmann, 2013). This motif was present upstream of 80 % of the 45 ORFs encoding proteins identified in purified virus particles and thus assumed to correspond to late genes (see Table 1). The experimental validation by 5′–3′ RACE sequencing of whether these motifs correspond to functional transcription and polyadenylation signals is currently ongoing.
Functional elements in the GpSGHV-Eth genome
The peptides from the experimentally derived proteomics data helped us to identify potential functional ORFs in the GpSGHV-Eth genome and to improve the genome annotation. In particular, the GpSGHV-Eth genome contained homologues to 12 out of the 31 so-called core genes of baculoviruses and nudiviruses that are involved in five main processes of baculovirus infection, i.e. (i) replication, (ii) transcription, (iii) packaging, assembly and release, (iv) cell cycle arrest and/or interactions with host proteins, and (v) oral infectivity (Miele et al., 2011). Of the 12 GpSGHV-Eth ORFs homologous to the core genes, nine contained both TATA-like box and poly(A) signals, and had both mapped transcripts and peptides, implying that they were functional in GpSGHV. The remaining three ORFs lacked peptide mapping only (Table 1). The homologues to the core genes in GpSGHV-Eth included one of the four baculovirus core genes involved in DNA repair and recombination, i.e. SGHV-Eth081, a homologue to helicase 2-like protein (Spodoptera frugiperda granulosis virus) (Wang et al., 2007). Other homologues were five genes involved in transcription, i.e. SGHV-Eth052, SGHV-Eth095, SGHV-Eth041 and SGHV-Eth033/34, which are homologues to the transcription factors LEF-4, LEF-5, LEF-8 and LEF-9, respectively. Homologues to desmoplakin (Ac66) and to Ac81, which are involved in egress of baculovirus virions and in virus–host interactions at late infection stages (Miele et al., 2011), respectively, were identified in GpSGHV (SGHV-Eth009 and SGHV-Eth086, respectively). Finally, five of the baculovirus per os infectivity factors (PIFs), i.e. SGHV-Eth001, SGHV-Eth054, SGHV-Eth112, SGHV-Eth083 and SGHV-Eth004, which are homologues to baculovirus P74 ( = PIF-0), PIF-1, PIF-2, PIF-3 and ODV-e66 (Slack & Arif, 2007), respectively, were detected (Table 1). These homologies to the core genes suggest that hytrosaviruses share with baculoviruses and nudiviruses similar modes of entry and transcription of their late genes, which strongly supports the hypothesis that they are derived from a common ancestor (Jehle et al., 2013). We also detected homologues to other viral genes that are potentially functional in GpSGHV, notably SGHV-Eth087 (homologue to herpesvirus DNA polymerase), SGHV-Eth095 (homologue to LEF-5 of Culex nigripalpus nucleopolyhedrovirus) and SGHV-Eth053 (homologue to a putative core protein encoded by ORF152 of Melanoplus sanguinipes entomopoxvirus) (Tables 1 and S2).
The expressed proteins provide experimental evidence that the viral genes are transcribed and translated to produce functional proteins. Nevertheless, some of the putative ORFs remained without peptide and/or transcript mapping. The lack of equivalence between the transcripts and peptides could be due to several reasons. The most important reasons are that not all mRNAs are actively translated at any particular time, and that the protein content is dependent on both synthesis of new proteins and degradation of existing proteins. Further, the intrinsic MS/MS peptide properties (e.g. abundance, ionization efficiency, solubility, etc.), incorrect assignment of peptides harbouring multiple coding mutations (Dresang et al., 2011) and post-translational modifications may result in under-representation of the full protein repertoires. It is, however, worth mentioning that most of the ORFs without peptide or transcript mapping are of small size ( < 100 aa residues) and at least some of them could correspond to non-functional ORFs.
Genetic heterogeneity of the GpSGHV-Eth genome
Various insertions and deletion events were detected in the genome of GpSGHV-Eth (Tables 2 and S3). A 111 nt deletion was observed in the 89 508–89 648 nt region in 30 % of reads (Fig. 3a). This deletion was not detected in GpSGHV-Uga. The nucleotide variation in this region was within ORF SGHV-Eth069, which is homologous to the structural protein ORF147 of Trichoplusia ni ascovirus 2c (Cui et al., 2007) (Table 1). Notably, compared with its SGHV-Uga062 homologue, ORF SGHV-Eth069 contains indels (Table S3); overall, the latter is 126 nt shorter than the former.
Fig. 3.
Repeat status and genetic heterogeneity in the GpSGHV-Eth genome. (a) Approximately 30 and 70 % of the 89 508–89 648 nt region were covered by short repeats of 30 and 26 nt, respectively. (b) Two repeat sequences, 27 nt each, occur two and three times in 65 and 35 %, respectively, of the reads in the 183 153–183 354 nt region.
Similarly, two repeat sequences, 27 nt each, occur two and three times, in 65 and 35 %, respectively, of the reads in the 183 153–183 354 nt region (Fig. 3b). The nucleotide variations in this region occur in ORF SGHV-Eth164, which was annotated as a trophozoite antigen-like protein and homologous to shrimp white spot syndrome virus ORF94 (Table 1). Compared with its homologous ORF SGHV-Uga148, SGHV-Eth164 has two deletion events encompassing a total of 57 nt (Table S3). However, this nucleotide region has more repeat sequences in GpSGHV-Uga than GpSGHV-Eth, making SGHV-Uga148 longer than SGHV-Eth164 by a total of 255 nt. It is, however, doubtful whether this ORF is expressed as it contains no transcriptional signals and remains without any peptide or transcript mapping (Table 1).
Likewise, 75 and 25 % of the reads in the 165 386 nt region contained two and one C repeats, respectively. This nucleotide region occurs between ORFs SGHV-Eth140 and SGHV-Eth141. Whereas the 967 nt between these two ORFs did not have any predicted ORF in the GpSGHV-Eth genome, the corresponding region in the GpSGHV-Uga genome (containing 1052 nt between ORFs SGHV-Uga126 and SGHV-Uga127) contained a predicted ORF (159 nt) which was not annotated during the sequencing of the GpSGHV-Uga genome (Abd-Alla et al., 2008).
Such nucleotide deletions potentially result in a change at the ORF position or destroy the ORF if they are located in non-coding regions or coding regions, respectively. The apparent genetic heterogeneity observed within the GpSGHV-Eth genome could be attributed to the fact that the virus sample was isolated from HSGs collected from many symptomatically infected G. pallidipes individuals. This notwithstanding, deletions and/or insertions are known to result in several virus strains, e.g. for baculoviruses (Barrera et al., 2013; López-Ferber et al., 2003; Virto et al., 2014). Furthermore, such variation may play important roles in virus pathogenesis (Bernal et al., 2013; Clavijo et al., 2010; Simón et al., 2004, 2005). The high error rate observed in high-throughput sequencing is well known and the observed single nucleotide polymorphisms (SNPs) could be due simply to read errors. To test this, the probability of obtaining the number of reads observed, assuming that read errors are random, was calculated by the log-likelihood ratio test (Sokal & Rohlf, 2012) and is presented in Table 2. All of the observed SNPs are highly significant.
Table 2. Sequence polymorphisms in the GpSGHV-Eth genome: the table shows the positions with nucleotide polymorphisms in the genome detected from pyrosequencing reads.
| Genome position | A* | C* | G* | T* | Nucleotide sequence | Maximum† | Second‡ | Percentage§ | P|| |
|---|---|---|---|---|---|---|---|---|---|
| 22760 | 2 | 2 | 2735 | 91 | ACTTGGAAATAAG/TTCGCAATAAGTT | 2735 | 91 | 3.33 | 1e–37 |
| 31111 | 5 | 2112 | 1 | 94 | TTGTCGAATTTAC/TTTTTCTATTGCA | 2112 | 94 | 4.45 | 2e–37 |
| 32379 | 51 | 1 | 2208 | 0 | TCATTCAAAACAG/ACAATCGGCGCAA | 2208 | 51 | 2.31 | 2e–24 |
| 62381 | 82 | 1 | 1533 | 1 | AGCAAATCTTTCG/ACCACCACTAAAA | 1533 | 82 | 5.35 | 4e–36 |
| 72054 | 72 | 3 | 1893 | 4 | GGACAAACTTTCG/AGATAAGGCTACC | 1893 | 72 | 3.80 | 4e–26 |
| 88953 | 11 | 3174 | 2 | 308 | AGATGAATTATCC/TAAAGAGCAAGAA | 3174 | 308 | 9.70 | 8e–128 |
| 101549 | 270 | 4 | 2228 | 1 | GAAACTTCACTTG/ATAGCAATATTTA | 2228 | 270 | 12.12 | 5e–120 |
| 101553 | 13 | 2219 | 2 | 106 | CTTCACTTGTAGC/TAATATTTATTTG | 2219 | 106 | 4.78 | 3e–36 |
| 130384 | 2 | 2140 | 0 | 100 | AAAAAAATTACGC/TTGTGGACTAACA | 2140 | 100 | 4.67 | 2e–46 |
| 137360 | 57 | 2701 | 0 | 1 | CCTTTATATTATC/ATCCATTGAATAG | 2701 | 57 | 2.11 | 2e–27 |
| 160377 | 0 | 182 | 2 | 2349 | ACTGATCACACAT/CGTGTAGGGTGCC | 2349 | 182 | 7.75 | 4e–85 |
| 163075 | 114 | 2 | 2400 | 3 | GGATCATCCATTG/ACAACAAATTGGA | 2400 | 114 | 4.75 | 5e–47 |
| 164395 | 11 | 3178 | 1 | 89 | AAGCAAGAGATTC/TGTCATTTGAAAG | 3178 | 89 | 2.80 | 2e–31 |
| 164892 | 2487 | 4 | 211 | 0 | GCTGAGACATTCA/GTTCGCATTATTC | 2487 | 211 | 8.48 | 4e–96 |
| 179546 | 2 | 1777 | 1 | 110 | CAAAGTCCCAAGC/TAATTATTTTATG | 1777 | 110 | 6.19 | 8e–48 |
| 183486 | 4 | 2981 | 4 | 220 | ATAAAAAAGACGC/TAAATATGAATGA | 2981 | 220 | 7.38 | 5e–92 |
| 185137 | 147 | 2603 | 1 | 4 | GACATGGAAGAGC/AGATATGAATTAA | 2603 | 147 | 5.65 | 1e–62 |
Number of reads of each of the four bases at the stated position. Only reads matching exactly the flanking sequences in the column ‘Nucleotide sequence’ are included in the count.
Count of the dominant residue.
Count of the second most frequently observed base.
Percentage of all reads with the second most frequent base.
‖‖ Probability that the second most frequent base occurs at this frequency or greater due to random sequence reading error calculated by the log-likelihood ratio test.
Repetitive regions in the GpSGHV-Eth genome
We detected several repetitive regions with head-to-tail tandem repeat sequences (TRSs) and one inverted repeat sequence in the GpSGHV-Eth genome (Fig. 4). These repeat elements were found to be distributed throughout the GpSGHV-Eth genome, representing ∼3 % of the genomic sequence. The lengths of the repetitive regions ranged from 171 to 556 nt and consisted of 78 TRS minifragments. Most of these TRS segments were highly homologous to each other and clustered in two genomic regions (86 000–88 000 and 179 000–183 000 nt). The sizes of the TRS varied from 52 to 246 nt and the number of TRSs per repetitive region varied from 2.7 to 14.5. Within the same repetitive region, the identity of TRS was >80 %, but the sequence identity amongst the different repetitive region varied from 21.2 to 96.2 %. TRS9, 11 and 13, and 10, 12 and 14 shared >80 % sequence identities amongst each other. Occurrence of repeat sequences at multiple locations along the genomes has been reported in baculoviruses (Cochran & Faulkner, 1983), nudiviruses (Wang et al., 2011) and whispovirus (Syed Musthaq et al., 2006). Potentially, the repetitive elements may serve as regulators of viral gene expression (Schnitzler et al., 1987).
Fig. 4.
Loci of the GpSGHV-Eth genome with repeating elements. (a) The arrows represent the repeat core elements in the indicated directions. The name, type, size, copy number and genomic location of the repeat are indicated on the right. DR, direct repeat; IR, indirect repeat. Parameters: length >50 nt, n>2. (b) Alignment of the consensus nucleotide sequences of the direct repeats R9, R10, R11, R12, R13 and R14.
Comparison between the GpSGHV-Eth and GpSGHV-Uga genomes
We observed a strong collinearity between GpSGHV-Eth and GpSGHV-Uga genomic sequences (Fig. 2), which was corroborated by blast searches in that only 24 of the 174 GpSGHV-Eth ORFs remained without any hits to GpSGHV-Uga ORFs (Table 1). The major differences between the genomes of the two viruses are depicted in Figs 1 and 5. Compared with the GpSGHV-Uga genome, the GpSGHV-Eth genome had an insertion of ∼500 nt in the 20 000 nt region, which is immediately followed by a deletion of ∼600 nt. Similarly, a short insertion was observed in the 72 000 nt region and a long deletion (∼400 nt) was observed in the 87 000 nt region. Several other insertions of a combined length of almost 1250 nt were observed in the 150 000–161 000 nt region, followed by several deletions in the 162 000–1 90 000 nt region (see summary in Fig. 5). Combined, these insertions and deletions make the GpSGHV-Eth genome 249 nt longer and more ORF-dense than the GpSGHV-Uga genome. Taken together, our analyses of the two virus genomes led us to omit 10 small ORFs (four in the 16 000–18 000 nt region and six dispersed along the sequence) reported in the GpSGHV-Uga genome. On the same note, we included 24 new small ORFs (five in the 16 000–22 000 nt region and 19 dispersed along the sequence) in the GpSGHV-Eth genome (Fig. 1).
Fig. 5.
Inserted and deleted nucleotides in the GpSGHV-Eth genome compared with the GpSGHV-Uga genome. Positive and negative numbers indicate insertions and deletions, respectively, in the two virus genomes.
We then determined which specific ORFs contained deletions and/or insertions. We found that, compared with GpSGHV-Uga, a total of 37 ORFs in the GpSGHV-Eth ORFs contained several nucleotide insertions and/or deletions, of which 17 and 20 ORFs had insertions and deletions, respectively (Table S1; compare with Fig. 1). As can be observed in Fig. 1, the ORFs with insertions and deletions appeared to occur in clusters. For instance, the 5000–25 000 nt region contains three ORFs with insertions (SGHV-Eth005, SGHV-Eth013 and SGHV-Eth014) and three ORFs with deletions (SGHV-Eth008, SGHV-Eth009 and SGHV-Eth018). Interestingly, this region also contained four ORFs that lack homologues in GpSGHV-Eth (SGHV-Uga013, SGHV-Uga14, SGHV-Uga015 and SGHV-Uga018) and five novel ORFs (SGHV-Eth012, SGHV-Eth015, SGHV-Eth016 and SGHV-Eth019) (Fig. 1). Another notable region is the 70 000-90 000 nt region, which contains two ORFs with insertions (SGHV-Eth064 and SGHV-Eth068), a high-molecular-mass ORF with deletions (SGHV-Eth069) and seven novel ORFs (SGHV-Eth059–SGHV-Eth065). Similarly, the 150 000–170 000 nt region contains three ORFs with insertions, six ORFs with deletions and three novel ORFs (Fig. 1). The 162 000–190 000 nt region contains six ORFs with insertions, nine ORFs with deletions, three novel ORFs and two missing ORFs (Fig. 1). Finally, except for ORF SGHV-Eth073 (with deletion) and a small novel ORF (SGHV-Eth085), the remaining 24 ORFs in the 90 000–123 000 nt region did not have any variations when comparing the two virus genomes.
Putative novel ORFs in the GpSGHV-Eth genome
A total of 24 ORFs in GpSGHV-Eth had no homologues to any known gene, including to GpSGHV-Uga (Table 1), implying that they are novel. However, notable motifs/domains were detected in some of the novel ORFs. For instance, ORF SGHV-Eth016 contained an NAD-specific glutamate dehydrogenase (GluDH) motif, whilst ORF SGHV-Eth028 contained the subtelomic variant ORF and repetitive interspersed family (Rif/Stevor) domain. Stevor and Rif family proteins are implicated in the regulation of antigenic variations and gene duplication events in some pathogens (Joannin et al., 2008; Niang et al., 2009), but their role in viruses is not well studied. ORF SGHV-Eth110 contained a viral small hydrophobic protein (v-SHP) domain, which is a retention signal for intracellular microvesicles (McCarthy & Theilmann, 2008) potentially involved in virus docking. ORF SGHV-Eth155 contained a repeat-associated mysterious proteins (RAMPs) domain – a protospacer sequence targeted by Cas nucleases to cleave viral genomes (Heler et al., 2015). Other notable domains detected in the putative novel ORFs are shown in Table 1. Out of the 24 novel ORFs, 14 contained both putative transcriptional signals [TATA-like box and poly(A) signals]. Four of these ORFs (SGHV-Eth060, SGHV-Eth061, SGHV-Eth109 and SGHV-Eth110) showed both transcripts and peptides mapping onto their sequences, implying that these ORFs are functional.
Selection pressures acting on GpSGHV ORFs
We estimated numbers and rates of synonymous and non-synonymous substitutions in the ORF sequences of GpSGHV-Eth in comparison with the homologous ORFs in GpSGHV-Uga (Table S4). Mutation and selection have different effects on silent (d S) and amino acid replacement (d N) substitutions. The d S/d N ratios amongst sites provide insights into the functional constraints at different amino acid sites and are useful in detection of sites under positive selection (Nielsen & Yang, 1998). A d S/d N < 1.0 or d N/d S>1.0 is considered to be a convincing indicator of genes that are under purifying or positive selection pressure, respectively (Kreitman & Akashi, 1995). Based on this criterion, a total of 21 ORFs were considered to be under positive selection pressure. Of the 21 ORFs under positive selection, 11 ORFs had significant homologies to known viral genes. These include three homologues to baculovirus genes (SGHV-Eth136, SGHV-Eth139 and SGHV-Eth140), four homologues to entomopoxvirus genes (SGHV-Eth009, SGHV-Eth035, SGHV-Eth096 and SGHV-Eth116), and homologues to nudivirus (SGHV-Eth052), ascovirus (SGHV-Eth069), mimivirus (SGHV-Eth68) and nimavirus (SGHV-Eth164) genes (Table 1). Two ORFs (SGHV-Eth049 and SGHV-Eth064) were homologous to known cellular genes, whilst the GpSGHV envelope and tegument proteins encoded by ORFs SGHV-Eth040 and SGHV-Eth123 were annotated to be HSP90-like ATPase and regulatory proteins, respectively (Table 1). The remaining six ORFs (i.e. SGHV-Eth051, SGHV-Eth108, SGHV-Eth141, SGHV-Eth153, SGHV-Eth165 and SGHV-Eth171) remained without homologies to known genes. It is thought that genes encoding virulence factors undergo intensified episodes of positive selection to maintain or improve the advantage of pathogens over their hosts (adaptive evolution) (van der Ende et al., 1998; Wolf et al., 2006). In this case, some of the notable GpSGHV genes under positive selection pressure include SGHV-Eth139 and SGHV-Eth140 (homologues to baculovirus cg30 and pe38 involved in maximum production of occlusion bodies and apoptosis, respectively), SGHV-Eth052 (homologue to the nudivirus very late gene transcription factor lef-4), and SGHV-Eth069 (homologue to the ascovirus 2c ORF147) (Table 1). Negative selection occurs in conservative viral genes that are maintained under structural and/or functional constraints to avoid reproductive and other fitness disadvantages (Doi, 1991; Wilson et al., 1977). Knowledge of viral genes under positive selection pressure is critical in the identification of genes involved in virulence/pathogenesis without prior knowledge of the exact mechanism(s) that govern virulence and pathogenesis. Our analysis may clarify the driving force of Hytrosaviridae evolution.
Differential pathogenesis of GpSGHV-Eth and GpSGHV-Uga
The differential pathogenesis of GpSGHV in different tsetse colonies could be attributed to the nucleotide variations described above or to the susceptibility of the host itself to virus infection. It has been demonstrated that insect host-encoded factors have an impact on virus pathogenesis, e.g. in the baculoviruses (Asser-Kaiser et al., 2010, 2011). It should be noted that whereas the Ugandan G. pallidipes fly population has been maintained at the IPCL for many generations ( ≥ 30 years), the Ethiopian G. pallidipes population is quite recently colonized. Consequently, the prolonged exposure of the Ugandan G. pallidipes population to GpSGHV infections could have resulted in establishment of a virus–host equilibrium and co-existence. Alternatively, colony handling and feeding regimes practised in different mass production facilities could influence expression of SGH, as recently reported by Abd-Alla et al. (2013).
Conclusions
Based on our data, we draw the following conclusions. (i) With a size of 190 291 nt and encoding 174 putative ORFs, the GpSGHV-Eth genome is 98.1 % identical to the reference genome of GpSGHV-Uga. (ii) The combined proteogenomic and transcriptomic mapping significantly improved the annotation of the GpSGHV-Eth genome, allowing the detection of 141 transcripts and 86 proteins. Of the 86 ORFs that were proteogenomically mapped on the virus genome, 13 ORFs appear to contain non-canonical start codons; four of the 13 ORFs had peptides mapping upstream of the first methionine residues. (iii) Of the 132 ORFs that contained transcriptional signals [TATA-like box promoter elements and poly(A) signals], 60 ORFs had both mapped transcripts and peptides, implying that these ORFs are translated into functional viral proteins. Compared with the GpSGHV-Uga genome, where only 47 ORFs could be annotated, 114 ORFs could be annotated in the GpSGHV-Eth genome, including 61 structural proteins. (iv) Amongst the potentially functional elements in the GpSGHV-Eth genome were homologues to the core proteins of the baculoviruses, nudiviruses and herpesvirus that are important in various virus replication cycles. (v) Over 10 % (n = 21) of the GpSGHV genes are potentially under positive selection pressure, implying that these genes may have evolved new functions in GpSGHV-Eth compared with the reference GpSGHV-Uga. (vi) As the characteristics of virulence are often due to the synergistic effects of various genomic loci, the cohesive activities of the genetic heterogeneity presented here potentially result in the differential pathogenesis of the two GpSGHV isolates. In addition, it is possible that G. pallidipes colonies maintained under different insectary conditions may vary in their susceptibility to GpSGHV infections and may therefore influence the occurrence of overt SGH symptoms. Our data provide a foundation for future investigations of the Hytrosaviridae family of insect viruses.
Methods
Isolation, purification, extraction and sequencing of viral genomic DNA
G. pallidipes flies were obtained from a tsetse production facility at the National Institute for Control and Eradication of Tsetse Fly and Trypanosomosis (NICETT), Addis Ababa, Ethiopia. The virus sample was purified from HSGs (dissected from naturally infected 4-week-old males) and the viral DNA sequenced as described by Abd-Alla et al. (2008), with slight modifications. Briefly, after extraction from purified virus suspension, viral DNA was released by Sarkosyl/proteinase K treatment (Qiagen), followed by phenol/chloroform extraction. Based on the genomic sequence of the reference GpSGHV genome, 136 primer pairs were designed and used to amplify 1500 bp amplicons covering the entire virus genomic sequence (Abd-Alla et al., 2007). Each of the PCR products was then purified using a High Pure PCR Purification kit (Roche Biochemicals). The respective PCR primers were used to sequence the PCR products from both ends by the Sanger method (MWG-Biotech). In certain cases, PCR products were cloned into pGEM-T/pGEM T-Easy vector systems (Promega), and then sequenced using T7 and SP6 universal primers according to standard protocols. To cover the sequence of the AT-rich regions of the virus genomic sequence, 10 μg intact DNA was subjected to pyrophosphate-based sequencing (pyrosequencing) (454 Life Sciences) according to Margulies et al. (2005). Repeat regions were resolved by DNA sequencing at Macrogen using the HiSeq 2000 platform (Illumina). Sequencing reads were trimmed by Trimmomatic tools version 0.36 (Bolger et al., 2014), and assembled by SeqMan (Lasergene version 7.0; Dnastar) and Vector NTI version 9.0 (Invitrogen) packages. Sequence assembly was validated using a set of routines as described by Parker & Parker (2008).
Protein extraction, fractionation and MS/MS data acquisition
Preparation of G. pallidipes salivary gland proteins and MS analyses were performed as described by Kariithi et al. (2013b). Briefly, proteins were extracted from HSG extracts, fractionated using 12 % SDS-PAGE and subjected to in-gel trypsin digestion. The resultant tryptic peptides were analysed by LC coupled to electrospray ionization and high-accuracy LTQ-Orbitrap XL tandem MS (LC-MS/MS). The MS/MS spectra were searched against Glossina hytrosavirus and tsetse fly protein databases downloaded from UniProt (version May 2014), a database containing sequences of common contaminants and a decoy database created by reversing the database sequences. The MS searches were performed using MaxQuant version 1.3.0.5 (Cox & Mann, 2008) with the Andromeda search engine (Cox et al., 2011). The default false discovery rate of ≤ 0.01 was used at protein and peptide levels during the searches. Any peptide hits to the decoy sequences and hits with modified peptides only were excluded from further analyses.
Deep sequencing of RNAs, assembly and annotation of viral transcriptome
Total RNA was extracted from HSGs (obtained from 4-week-old males as described above) using TRIzol reagent (Invitrogen) and treated with DNase I to remove residual DNA contaminants. RNA samples were purified using a RNeasy Plus Mini kit (Qiagen). Then, RNA libraries were constructed using a TruSeq RNA Sample Prep kit version 2 (Illumina), and the pooled libraries sequenced using the Illumina HiSeq2000 platform in high-throughput mode. To map the RNA-Seq reads onto the GpSGHV genomic sequence, residual Illumina sequencing adapters, low-quality read pairs with average Phred +33 quality scores < 30 and low-quality bases ( < 30) on the ends of the reads were removed using EA-Utils software (Aronesty, 2013). To ascertain from which part of the GpSGHV genome the sequencing reads occurred, the paired reads were aligned to the virus genome sequence using Bowtie 2 (Langmead et al., 2009). Estimation of the virus gene expression levels from the RNA-Seq data was done using EDGE-pro (Magoc et al., 2013).
Prediction, validation and annotation of virus ORFs
The composition and features of the virus genome were analysed as described by Abd-Alla et al. (2008), with modifications. The modifications were that, in addition to selection of ORFs containing ≥ 50 aa and with minimal overlaps ( < 100 nt), the coding regions of the ORFs predicted in the GpSGHV genome were delineated by mapping RNA-Seq transcripts and LC-MS/MS peptides onto the virus genome (Armengaud, 2009). For the transcript mapping, the transcripts were mapped on to the virus genome sequence translated into all six reading frames using BioEdit local blast alignment. For proteogenomic mapping, the validated unique peptides derived from the LC-MS/MS spectral matches were mapped back to the virus genome (translated in all six reading frames) using the Proteogenomic Mapping Pipeline (PMP) (Sanders et al., 2011). Briefly, three files were inputted into the PMP: a fasta file containing validated unique MS/MS peptides, a fasta file of the GpSGHV genome and a text file containing the National Center for Biotechnology Information (NCBI) genetic code (genetic_code_table). The output file was used to analyse the expressed protein sequence tags created after the peptides had mapped to the virus nucleotide sequence (Sanders et al., 2011). Gene ontology annotation of the predicted ORFs was performed using Blast2GO version 3.0.4 (Conesa et al., 2005). Protein domain analyses were performed using various databases, including Pfam (Finn et al., 2008), InterPro (Mitchell et al., 2015) and the NCBI conserved domain database (Marchler-Bauer et al., 2015).
Comparative analysis and genomic synteny of viral genomes
After the annotation, the genomic sequence of GpSGHV-Eth was compared with that of the reference GpSGHV-Uga. For this, the predicted GpSGHV-Eth ORFs were used as query sequences to blast against the nr (non-redundant) NCBI protein database. The Artemis comparison tool (Carver et al., 2005) was used to analyse the gene synteny of GpSGHV-Eth compared with GpSGHV-Uga and the closely related hytrosavirus of the house fly, Musca domestica salivary gland hypertrophy virus (Garcia-Maruniak et al., 2009). The occurrence of insertions, deletions and SNPs in the protein-coding regions of the GpSGHV-Eth genomic sequences compared with those of GpSGHV-Uga was investigated by aligning the nucleotide sequences of the homologous ORFs using MegAlign (Dnastar). The pair-wise alignments were visualized within MegAlign and descriptions of the nucleotide variations were based on a previously described nomenclature system (den Dunnen & Antonarakis, 2001).
Analysis of positive and negative selection pressures
The occurrence of selection pressures in the protein-coding regions of the GpSGHV-Eth genome in comparison with the GpSGHV-Uga genome was assessed by analysing the rates of non-synonymous substitution per synonymous site (d N) versus synonymous substitution per synonymous site (d S). For this, codon-aligned nucleotide sequence sets of GpSGHV-Eth and GpSGHV-Uga ORFs were analysed using snap version 2.1.1 (Korber, 2000). snap computes the nucleotide substitution differences (codon by codon) between pairs of homologous sequences based on the Nei–Gojobori method (Nei & Gojobori, 1986). Evidence for the occurrence of negative or positive selection pressures is indicated when d S/d N>1.0 or d N/d S>1.0, respectively.
Nucleotide/protein sequence databases and accession numbers
The accession numbers for the ORFs presented in this paper are from the GenBank, UniProt, or PIR databases. The GpSGHV-Eth genome sequence has been deposited in GenBank.
Acknowledgements
This work was funded by the Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, IAEA, Austria. The authors acknowledge Ms Irene Meki, Mr Moges Hidoto, and the staff members of NICETT and IPCL for technical assistance.
Supplementary Data
Supplementary Data
References
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