Open Access

Genomic characterization of two novel pathogenic avipoxviruses isolated from pacific shearwaters (Ardenna spp.)

BMC Genomics201718:298

https://doi.org/10.1186/s12864-017-3680-z

Received: 18 December 2016

Accepted: 5 April 2017

Published: 13 April 2017

Abstract

Background

Over the past 20 years, many marine seabird populations have been gradually declining and the factors driving this ongoing deterioration are not always well understood. Avipoxvirus infections have been found in a wide range of bird species worldwide, however, very little is known about the disease ecology of avian poxviruses in seabirds. Here we present two novel avipoxviruses from pacific shearwaters (Ardenna spp), one from a Flesh-footed Shearwater (A. carneipes) (SWPV-1) and the other from a Wedge-tailed Shearwater (A. pacificus) (SWPV-2).

Results

Epidermal pox lesions, liver, and blood samples were examined from A. carneipes and A. pacificus of breeding colonies in eastern Australia. After histopathological confirmation of the disease, PCR screening was conducted for avipoxvirus, circovirus, reticuloendotheliosis virus, and fungal agents. Two samples that were PCR positive for poxvirus were further assessed by next generation sequencing, which yielded complete Shearwaterpox virus (SWPV) genomes from A. pacificus and A. carneipes, both showing the highest degree of similarity with Canarypox virus (98% and 67%, respectively). The novel SWPV-1 complete genome from A. carneipes is missing 43 genes compared to CNPV and contains 4 predicted genes which are not found in any other poxvirus, whilst, SWPV-2 complete genome was deemed to be missing 18 genes compared to CNPV and a further 15 genes significantly fragmented as to probably cause them to be non-functional.

Conclusion

These are the first avipoxvirus complete genome sequences that infect marine seabirds. In the comparison of SWPV-1 and −2 to existing avipoxvirus sequences, our results indicate that the SWPV complete genome from A. carneipes (SWPV-1) described here is not closely related to any other avipoxvirus genome isolated from avian or other natural host species, and that it likely should be considered a separate species.

Keywords

Avipoxvirus Poxvirus Next generation sequencing dermatitis Ardenna Shearwater

Background

The Avipoxvirus genus includes a divergent group of viruses that cause diseases in more than 278 species of wild and domestic birds in terrestrial and marine environments worldwide [1, 2]. Relatively little is known about the origins, worldwide host distribution and genetic diversity of avipoxviruses [3]. In affected birds, avipoxviruses typically cause proliferative ‘wart-like’ growths that are most commonly restricted to the eyes, beak or unfeathered skin of the body (so-called ‘dry’ pox), but infections can also develop in the upper alimentary and respiratory tracts (‘wet’ or ‘diptheritic’ pox) [2]. The incubation period and magnitude of avipoxvirus infection is variable, and is rarely fatal although secondary bacterial or fungal infections are common and cause increased mortality [2]. Such conditions in naïve populations can reach a much higher prevalence with substantial fatality [4, 5].

Avipoxviruses belong to the subfamily Chordopoxvirinae (ChPV) of the Poxviridae family, which are relatively large double-stranded DNA (dsDNA) viruses that replicate in the cytoplasm of infected cells [6]. Although poxviruses have evolved to infect a wide range of host species, to date only six avipoxvirus genomes have been published; a pathogenic American strain of Fowlpox virus (FPVUS) [7], an attenuated European strain of Fowlpox virus (FP9) [8], a virulent Canarypox virus (CNPV) [9], a pathogenic South African strain of Pigeonpox virus (FeP2), a Penguinpox virus (PEPV) [3], and a pathogenic Hungarian strain of Turkeypox virus (TKPV) [10]. Although these genome sequences demonstrate that avipoxviruses have diverged considerably from the other chordopoxviruses (ChPVs), approximately 80 genes have been found to be conserved amongst all ChPVs and to comprise the minimum essential poxvirus genome [11]. These genes tend to be present in the central core of the linear genome with the remainder presumed to be immunomodulatory and host specific genes located towards the terminal regions of the genome [3]. With the exception of TKPV (188 kb), avipoxvirus genomes (266–360 kb) tend to be bigger than those of other ChPVs due in part to multiple families of genes.

Over the past two decades, the status of the world’s bird populations have deteriorated with seabirds declining faster than any other group of birds [12]. On Lord Howe Island in eastern Australia, the Flesh-footed Shearwater Ardenna carneipes has been declining for many years and is therefore listed as Vulnerable in the state of New South Wales [13]. The ongoing threat of plastic pollution, and toxicity from the elevated concentration of trace elements such as mercury could be confounding drivers of this declining species [14]. Infectious diseases, including those caused by avipoxviruses, have also been identified as an important risk factor in the conservation of small and endangered populations, particularly in island species [1518]. The impact of the introduction of avipoxviruses has been severe for the avifauna of various archipelagos [19]. The emergence of distinctive avipoxvirus with a high prevalence (88%) in Hawaiian Laysan Albatross (Phoebastria immutabilis) enabled one of the first detailed studies of the epidemiology and population-level impact of the disease in the seabirds [20]. However, relatively little is known about the general prevalence or effects of poxviruses in seabird species, including for shearwaters (Ardenna or Puffinus spp.). Therefore, the aim of the present study was to identify and characterize pathogens associated with clinical disease in breeding colonies of Flesh-footed Shearwater and Wedge-tailed Shearwater sourced from Lord Howe Island in 2015.

Results

Identification of fungal pathogens

In the sample from A. pacificus (15–1526, and 15–1527), there were multifocal areas of inflammation and exudation associated with serocellular surface crust that contained abundant branching fungal hyphae and aggregations of bacteria (Fig. 1c). A PCR screening was conducted for the presence of fungal pathogen using the ITS region to amplify a segment of approximately 550 bp. Two samples (out of 6) were positive for fungal pathogens, and direct Sanger sequencing of the purified gel bands resulted in a 550 bp sequence after trimming off primer sequences (data not shown). These sequences were further verified using high-throughput NGS, and generated con tigs of 3,430 bp (15–1526; GenBank accession KX857213) and 5,188 bp (15–1527; GenBank accession KX857212). A BLASTn search for the bird coinfected with fungal pathogen (15–1526) returned multiple hits to various fungal species, all with very similar scores; however, the best match (88%) was to the Phaeosphaeria nodorum (GenBank Accession EU053989.1, and value ≤ e-153), a major necrotrophic fungal pathogen of wheat [21]. Similar search model for the fungal pathogen of bird 15–1527, demonstrated a highest hit (96%) to the Metarhizium anisopliae var. anisopliae (GenBank Accession AY884128.1, and value ≤ e-173), an entomopathogenic fungus [22].
Fig. 1

Pathological evidence of characteristic pox and fungal lesions. a Grossly well circumscribed, popular, crusting pox lesions across the featherless skins (white arrows). b Feather skin demonstrating diffuse proliferation of the epidermis and follicular infundibula with keratinocytes containing eosinophilic intracytoplasmic inclusions (Bollinger bodies) and serocellular surface crust (double head arrow). c Inflammatory exudates associated with serocellular surface crust that contained abundant branching fungal hyphae and aggregations of bacteria

Identification of virus

Samples from six shearwater chicks of two different species, A. carneipes and A. pacificus, with evidence of gross well circumscribed, popular, crusting lesions across the feather skins (Fig. 1a), were biopsied, with blood and liver samples also collected. Histological examinations of the skin demonstrated focal to diffuse full thickness necrosis of the epidermis and a thick serocellular surface crust. A marked heterophilic rich inflammatory cellular response and exudation was present alongside abundant macrophages and perifollicular fibroplasia. In some areas there was focal proliferation of the adjacent epidermis associated with ballooning degeneration of keratinocytes with eosinophilic intracytoplasmic inclusions (Fig. 1b). A PCR screening was conducted for the presence of poxvirus, circovirus and reticuloendotheliosis virus, which are likely to cause this type of skin lesions. Two birds (A. pacificus 151526 and A. carneipes 15–1528) were positive by PCR targeting the 4b gene that encodes a core protein of ChPV, however, there were no evidence of either circovirus or reticuloendotheliosis for any of the samples used in this study. Direct Sanger sequencing of the purified gel bands resulted in a 578 bp sequence after trimming off primer sequences (data not shown). A BLASTn search with these sequences returned multiple hits to the 4b core gene from a variety of poxviruses, all with very similar scores; however, the best match was to the Canarypox virus 4b core protein gene ((bird 15–1526; similarity with AY318871 was 99% and identity score ≤ e-162), and bird 15–1528; similarity with LK021654 was 99% and identity score ≤ e-157)).

Genome sequence and annotation of viruses

The Shearwaterpox virus complete genomes were assembled using CLC Genomics workbench 9.5.2 under La Trobe University Genomics Platform. The assembled complete genomes of SWPV-1 and −2 were 326,929 and 351,108 nt, respectively. The SWPV-1 and −2 complete genomes were annotated as described in the methods using CNPV as a reference genome (Additional file 1: Table S1 and Additional file 2: Table S2). We took a conservative approach to the annotation in order to minimize the inclusion of ORFs that were unlikely to represent functional genes. Table 1 lists the 310 and 312 genes annotated for SWPV-1 and −2, respectively. For the most part, these two new complete genomes are collinear to CNPV although there are a number of rearrangements of blocks of 1–6 genes in addition to insertions and deletions with respect to CNPV (Table 1). Comparison of the predicted proteins of SWPV-2 to orthologs in CNPV reveal the vast majority are >98% identical (aa), with more than 80 being completely conserved. In contrast, the orthologs of SWPV-1 only have an average aa identity of 67% to CNPV. However, with the lower average identity, greater genetic distance, comes a much greater range of variation in the level of identity and a significant number of predicted proteins are 80 – 90% identical (aa) to CNPV orthologs.
Table 1

Shearwaterpox virus (SWPV) genome annotations and comparative analysis of ORFs relative to CNPV genomes

SWPV1 synteny

SWPV2 synteny

CNPV synteny

CNPV BLAST hits

SWPV1 % identity

SWPV2 % identity

SWPV1 AA size

SWPV2 AA size

Reference AA size

notes

  

CNPV001

CNPV001 hypothetical protein

    

72

 
 

SWPV2-001

CNPV002

CNPV002 hypothetical protein

 

92.941

 

171

171

 

SWPV1-001

SWPV2-002

CNPV003

CNPV003 C-type lectin-like protein

32.044

85.99

181

208

204

 

SWPV1-002

 

CNPV004

CNPV004 ankyrin repeat protein

56.458

 

468

 

514

 

SWPV1-003

SWPV2-003

CNPV005

CNPV005 conserved hypothetical protein

87.387

99.55

220

222

222

 
 

SWPV2-004

CNPV006

CNPV006 hypothetical protein

 

88.71

 

134

182

SWPV2: C-terminus fragment, not likely translated

  

CNPV007

CNPV007 ankyrin repeat protein

    

674

 

SWPV1-004

SWPV2-005

CNPV008

CNPV008 C-type lectin-like protein

50

98.225

174

169

169

 
 

SWPV2-006

CNPV009

CNPV009 ankyrin repeat protein

 

99.564

 

688

688

 
  

CNPV010

CNPV010 ankyrin repeat protein

    

734

 
 

SWPV2-007

CNPV011

CNPV011 ankyrin repeat protein

 

99.147

 

586

586

 
 

SWPV2-008

CNPV012

CNPV012 hypothetical protein

 

100

 

189

189

 
 

SWPV2-009

CNPV013

CNPV013 hypothetical protein

 

98.81

 

168

168

 
 

SWPV2-010

CNPV014

CNPV014 immunoglobulin-like domain protein

 

99.184

 

490

490

 
 

SWPV2-011

CNPV015

CNPV015 ankyrin repeat protein

 

97.538

 

528

528

 

SWPV1-005

  

CNPV035 C-type lectin-like protein

35.556

 

138

 

134

 

SWPV1-006

  

CNPV318 ankyrin repeat protein

58.932

 

487

 

514

 

SWPV1-007

SWPV2-012

CNPV016

CNPV016 C-type lectin-like protein

52.128

98.81

117

168

168

 

SWPV1-008

SWPV2-013

CNPV017

CNPV017 ankyrin repeat protein

64.471

97.912

425

479

486

 

SWPV1-009

  

CNPV295 ankyrin repeat protein

56.41

 

277

 

396

 
 

SWPV2-014

CNPV018

CNPV018 IL-10-like protein

 

90.805

 

190

191

 
 

SWPV2-015

CNPV019

CNPV019 ankyrin repeat protein

 

99.083

 

436

436

 

SWPV1-010

SWPV2-016

CNPV020

CNPV020 ankyrin repeat protein

56.311

99.761

412

419

419

 

SWPV1-011

  

CNPV320 Ig-like domain protein

31.656

 

483

 

469

 

SWPV1-012

SWPV2-017

CNPV021

CNPV021 ankyrin repeat protein

62.313

99.626

528

535

535

 

SWPV1-013

SWPV2-018

CNPV022

CNPV022 putative serpin

65.642

98.324

356

358

358

 

SWPV1-014

  

PEPV260 ankyrin repeat protein

53.158

 

190

 

192

 

SWPV1-015

  

CNPV011 ankyrin repeat protein

34

 

530

 

586

 
 

SWPV2-019

CNPV023

CNPV023 vaccinia C4L/C10L-like protein

 

98.595

 

424

427

 
 

SWPV2-020

CNPV024

CNPV024 hypothetical protein

 

96.629

 

178

178

 

SWPV1-016

SWPV2-021

CNPV025

CNPV025 alpha-SNAP-like protein

57.491

98.667

304

300

300

 

SWPV1-017

SWPV2-022

CNPV026

CNPV026 ankyrin repeat protein

54.271

98.953

397

382

382

 

SWPV1-018

SWPV2-023

CNPV027

CNPV027 ankyrin repeat protein

59.375

98.722

646

626

626

 

SWPV1-019

SWPV2-024

CNPV028

CNPV028 ankyrin repeat protein

57.618

99.164

408

365

362

 

SWPV1-020

SWPV2-025

CNPV029

CNPV029 C-type lectin-like protein

50.35

99.296

142

142

142

 

SWPV1-021

SWPV2-026

CNPV030

CNPV030 ankyrin repeat protein

63.72

98.529

345

340

340

 

SWPV1-022

SWPV2-027

CNPV031

CNPV031 hypothetical protein

60.331

97.479

120

119

119

 

SWPV1-023

  

CNPV013 conserved hypothetical protein

44.048

 

168

 

168

 

SWPV1-024

SWPV2-028

CNPV032

CNPV032 Ig-like domain putative IFN-gamma binding protein

51.837

92.149

242

242

242

 

SWPV1-025

SWPV2-029

CNPV033

CNPV033 Ig-like domain protein

48.095

93.496

238

246

246

 
 

SWPV2-030

CNPV034

CNPV034 ankyrin repeat protein

 

99.848

 

659

659

 
 

SWPV2-031

CNPV035

CNPV035 C-type lectin-like protein

 

94.776

 

133

134

 

SWPV1-026

SWPV2-032

CNPV036

CNPV036 conserved hypothetical protein

48.235

98.947

88

95

95

 

SWPV1-027

SWPV2-033

CNPV037

CNPV037 conserved hypothetical protein

63.068

99.441

178

179

179

 

SWPV1-028

SWPV2-034

CNPV038

CNPV038 vaccinia C4L/C10L-like protein

54.523

99.516

411

413

413

 

SWPV1-029

SWPV2-035

CNPV039

CNPV039 G protein-coupled receptor-like protein

67.284

97.859

323

327

327

 

SWPV1-030

SWPV2-036

CNPV040

CNPV040 ankyrin repeat protein

57.36

93.401

589

591

591

SWPV2: High SNP Density

SWPV1-031

SWPV2-037

CNPV041

CNPV041 ankyrin repeat protein

66.284

98.605

432

430

430

 

SWPV1-032

SWPV2-038

CNPV042

CNPV042 ankyrin repeat protein

72.712

99.339

608

605

605

 

SWPV1-033

SWPV2-039

CNPV043

CNPV043 conserved hypothetical protein

74.627

99.005

202

201

201

 

SWPV1-034

SWPV2-040

CNPV044

CNPV044 ankyrin repeat protein

67.316

99.583

470

480

480

 

SWPV1-035

SWPV2-041

CNPV045

CNPV045 G protein-coupled receptor-like protein

65.231

100

331

332

332

 

SWPV1-036

SWPV2-042

CNPV046

CNPV046 ankyrin repeat protein

68.08

98.667

452

450

450

 

SWPV1-037

SWPV2-043

CNPV047

CNPV047 conserved hypothetical protein

65.6

99.194

125

124

124

 

SWPV1-038

SWPV2-044

CNPV048

CNPV048 alkaline phosphodiesterase-like protein

68.238

98.502

804

801

801

 

SWPV1-039

SWPV2-045

CNPV049

CNPV049 hypothetical protein

72.667

100

148

150

150

 

SWPV1-040

SWPV2-046

CNPV050

CNPV050 ankyrin repeat protein

67.422

98.864

352

352

352

 

SWPV1-041

SWPV2-047

CNPV051

CNPV051 DNase II-like protein

63.683

96.75

398

408

401

 

SWPV1-042

SWPV2-048

CNPV052

CNPV052 C-type lectin-like protein

50

100

182

171

171

 

SWPV1-043

  

FWPV ankyrin repeat protein

45

 

329

 

406

 

SWPV1-044

SWPV2-049

CNPV053

CNPV053 conserved hypothetical protein

68.148

100

135

146

146

 

SWPV1-045

SWPV2-050

CNPV054

CNPV054 conserved hypothetical protein

62.59

99.286

141

140

140

 

SWPV1-046

SWPV2-051

CNPV055

CNPV055 conserved hypothetical protein

74.534

100

162

163

163

 

SWPV1-047

SWPV2-052

CNPV056

CNPV056 dUTPase

80.986

98.621

155

145

145

 

SWPV1-048

SWPV2-053

CNPV057

CNPV057 putative serpin

63.107

99.02

301

306

306

 

SWPV1-049

SWPV2-054

CNPV058

CNPV058 bcl-2 like protein

51.744

98.857

174

180

175

 

SWPV1-050

SWPV2-055

CNPV059

CNPV059 putative serpin

71.302

99.704

338

338

338

 

SWPV1-051

SWPV2-056

CNPV060

CNPV060 conserved hypothetical protein

46.939

95.098

236

206

316

SWPV2: Large internal deletion, Translated but not likely functional

SWPV1-052

SWPV2-057

CNPV061

CNPV061 DNA ligase

80.995

98.761

567

565

565

 

SWPV1-053

SWPV2-058

CNPV062

CNPV062 putative serpin

70.94

100

349

350

350

 

SWPV1-054

SWPV2-059

CNPV063

CNPV063 hydroxysteroid dehydrogenase-like protein

71.348

99.441

359

358

358

 

SWPV1-055

SWPV2-060

CNPV064

CNPV064 TGF-beta-like protein

56.897

98.587

272

283

282

 

SWPV1-056

SWPV2-061

CNPV065

CNPV065 semaphorin-like protein

69.735

99.485

573

583

583

 

SWPV1-057

SWPV2-062

CNPV066

CNPV066 hypothetical protein

37.349

98.519

139

399

405

SWPV1: Low BLAST hits, possible unique ORF

 

SWPV2-063

CNPV067

CNPV067 hypothetical protein

 

100

 

57

57

 

SWPV1-058

  

no significant BLAST hits

    

239

SWPV1: Possible Unique ORF

SWPV1-059

SWPV2-064

CNPV068

CNPV068 GNS1/SUR4-like protein

84.825

99.611

257

257

257

 

SWPV1-060

SWPV2-065

CNPV069

CNPV069 late transcription factor VLTF-2

87.5

100

154

155

155

 

SWPV1-061

SWPV2-066

CNPV070

CNPV070 putative rifampicin resistance protein, IMV assembly

88.065

100

553

551

551

 

SWPV1-062

SWPV2-067

CNPV071

CNPV071 mRNA capping enzyme small subunit

89.273

100

289

289

289

 
 

SWPV2-068

CNPV072

CNPV072 CC chemokine-like protein

 

96.262

 

132

312

SWPV2: N-terminus fragment

SWPV1-063

SWPV2-069

CNPV073

CNPV073 hypothetical protein

45.263

100

110

109

109

 

SWPV1-064

SWPV2-070

CNPV074

CNPV074 NPH-I, transcription termination factor

92.756

99.685

635

635

635

 

SWPV1-065

SWPV2-071

CNPV075

CNPV075 mutT motif putative gene expression regulator

79.295

100

226

228

230

 

SWPV1-066

SWPV2-072

CNPV076

CNPV076 mutT motif

84.549

99.569

233

232

232

 
  

CNPV077

CNPV077 hypothetical protein

    

78

 

SWPV1-067

  

CNPV011 ankyrin repeat protein

29.806

 

435

 

586

 

SWPV1-068

SWPV2-073

CNPV078

CNPV078 RNA polymerase subunit RPO18

82.39

100

161

160

160

 
 

SWPV2-074

CNPV079

CNPV079 Ig-like domain protein

 

94.161

 

274

272

 

SWPV1-069

SWPV2-075

CNPV080

CNPV080 early transcription factor small subunit VETFS

96.682

100

633

633

633

 
 

SWPV2-076

CNPV081

CNPV081 Ig-like domain protein

 

97.006

 

334

333

 

SWPV1-070

SWPV2-077

CNPV082

CNPV082 NTPase, DNA replication

88.818

99.748

790

794

794

 

SWPV1-071

SWPV2-078

CNPV083

CNPV083 CC chemokine-like protein

60.352

91.855

223

221

221

 

SWPV1-072

  

CNPV215 CC chemokine-like protein

30.994

 

195

 

204

 

SWPV1-073

SWPV2-079

CNPV084

CNPV084 uracil DNA glycosylase

86.364

97.706

220

218

218

 

SWPV1-074

SWPV2-080

CNPV085

CNPV085 putative RNA phosphatase

67.895

74.312

245

303

403

SWPV2: High SNP Density

SWPV1-075

  

CNPV216 conserved hypothetical protein

39.225

 

398

 

404

 

SWPV1-076

SWPV2-081

CNPV086

CNPV086 TNFR-like protein

67.327

71.569

103

112

117

 
 

SWPV2-082

CNPV087

CNPV087 putative glutathione peroxidase

 

98.473

 

131

198

SWPV2: C-terminus fragment, not likely translated

SWPV1-077

  

CNPV227 N1R/p28-like protein

74.638

 

256

 

359

 

SWPV1-078

SWPV2-083

CNPV088

CNPV088 conserved hypothetical protein

55.769

97

104

100

100

 

SWPV1-079

SWPV2-084

CNPV089

CNPV089 conserved hypothetical protein

64.935

100

164

159

159

 

SWPV1-080

SWPV2-085

CNPV090

CNPV090 conserved hypothetical protein

62.393

100

124

127

127

 

SWPV1-081

SWPV2-086

CNPV091

CNPV091 HT motif protein

64.634

100

77

83

83

 

SWPV1-082

SWPV2-087

CNPV092

CNPV092 conserved hypothetical protein

64.901

97.945

140

146

146

 

SWPV1-083

SWPV2-088

CNPV093

CNPV093 virion protein

60.37

99.625

270

267

267

 

SWPV1-084

SWPV2-089

CNPV094

CNPV094 T10-like protein

75

98.909

282

275

275

 

SWPV1-085

SWPV2-090

CNPV095

CNPV095 conserved hypothetical protein

71.111

100

47

45

45

 

SWPV1-086

SWPV2-091

CNPV096

CNPV096 ubiquitin

100

100

77

85

85

 

SWPV1-087

SWPV2-092

CNPV097

CNPV097 conserved hypothetical protein

70.031

99.705

298

339

339

 

SWPV1-088

SWPV2-093

CNPV098

CNPV098 hypothetical protein

67.442

98.75

61

80

80

 

SWPV1-089

SWPV2-094

CNPV099

CNPV099 beta-NGF-like protein

62.162

97.949

186

195

195

 

SWPV1-090

SWPV2-095

CNPV100

CNPV100 putative interleukin binding protein

51.176

98.225

211

168

169

 
 

SWPV2-096

CNPV101

CNPV101 hypothetical protein

 

98.824

 

85

85

 

SWPV1-091

SWPV2-097

CNPV102

CNPV102 conserved hypothetical protein

54.167

99.048

102

105

105

 

SWPV1-092

SWPV2-098

CNPV103

CNPV103 N1R/p28-like protein

62.304

98.947

188

190

190

 

SWPV1-093

SWPV2-099

CNPV104

CNPV104 putative glutaredoxin 2, virion morphogenesis

86.4

99.2

125

125

125

 

SWPV1-094

SWPV2-100

CNPV105

CNPV105 conserved hypothetical protein

77.35

98.718

234

234

234

 

SWPV1-095

SWPV2-101

CNPV106

CNPV106 putative elongation factor

76.829

98.039

103

102

102

 
 

SWPV2-102

CNPV107

CNPV107 hypothetical protein

 

100

 

77

77

 

SWPV1-096

  

PEPV083 transforming growth factor B

64

 

444

 

336

 

SWPV1-097

SWPV2-103

CNPV108

CNPV108 putative metalloprotease, virion morphogenesis

85.489

100

633

632

632

 

SWPV1-098

SWPV2-104

CNPV109

CNPV109 NPH-II, RNA helicase

86.05

99.706

681

681

681

 

SWPV1-099

SWPV2-105

CNPV110

CNPV110 virion core proteinase

87.441

99.763

421

422

422

 

SWPV1-100

SWPV2-106

CNPV111

CNPV111 DNA-binding protein

80.612

99.488

391

391

391

 

SWPV1-101

SWPV2-107

CNPV112

CNPV112 putative IMV membrane protein

81.481

100

81

81

81

 

SWPV1-102

SWPV2-108

CNPV113

CNPV113 thymidine kinase

75.978

99.441

181

179

179

 

SWPV1-103

SWPV2-109

CNPV114

CNPV114 HT motif protein

69.62

100

79

82

82

 

SWPV1-104

SWPV2-110

CNPV115

CNPV115 DNA-binding phosphoprotein

71.429

82.353

282

289

289

SWPV2: High SNP density

SWPV1-105

SWPV2-111

CNPV116

CNPV116 unnamed protein product

73.913

98.551

66

69

69

 

SWPV1-106

SWPV2-112

CNPV117

CNPV117 DNA-binding virion protein

88.854

99.677

314

310

310

 

SWPV1-107

SWPV2-113

CNPV118

CNPV118 conserved hypothetical protein

75.762

99.387

656

652

653

 

SWPV1-108

SWPV2-114

CNPV119

CNPV119 virion core protein

83.969

100

131

131

131

 

SWPV1-109

SWPV2-115

CNPV120

CNPV120 putative IMV redox protein, virus assembly

80.851

100

94

93

93

 

SWPV1-110

SWPV2-116

CNPV121

CNPV121 DNA polymerase

89.17

99.899

988

988

988

 

SWPV1-111

 

CNPV122

CNPV122 putative membrane protein

83.088

 

273

 

274

 

SWPV1-112

SWPV2-117

CNPV123

CNPV123 conserved hypothetical protein

82.312

85.336

571

502

571

SWPV2: High SNP density

SWPV1-113

SWPV2-118

CNPV124

CNPV124 variola B22R-like protein

67

98.957

1906

1916

1918

 

SWPV1-114

SWPV2-119

CNPV125

CNPV125 variola B22R-like protein

71.669

99.66

1742

1767

1767

 

SWPV1-115

SWPV2-120

CNPV126

CNPV126 variola B22R-like protein

64.456

98.847

1902

1839

1951

SWPV2: N-terminus fragment

 

SWPV2-121

 

CNPV126 variola B22R-like protein

 

96

 

153

1951

SWPV2: C-terminus fragment, not likely translated

SWPV1-116

SWPV2-122

CNPV127

CNPV127 RNA polymerase subunit RPO30

96.154

100

182

182

182

 

SWPV1-117

SWPV2-123

CNPV128

CNPV128 conserved hypothetical protein

77.072

98.752

742

721

721

SWPV2: High SNP Density

SWPV1-118

SWPV2-124

CNPV129

CNPV129 poly(A) polymerase large subunit PAPL

83.898

99.788

472

472

472

 

SWPV1-119

SWPV2-125

CNPV130

CNPV130 DNA-binding virion core protein

76.471

100

114

119

119

 

SWPV1-120

SWPV2-126

CNPV131

CNPV131 conserved hypothetical protein

64.115

99.517

212

207

207

 

SWPV1-121

SWPV2-127

CNPV132

CNPV132 conserved hypothetical protein

81.081

99.324

151

148

148

 

SWPV1-122

SWPV2-128

CNPV133

CNPV133 conserved hypothetical protein

73.737

100

90

99

99

 

SWPV1-123

SWPV2-129

CNPV134

CNPV134 variola B22R-like protein

65.517

99.001

1774

1801

1801

 

SWPV1-124

SWPV2-130

CNPV135

CNPV135 putative palmitylated EEV envelope lipase

89.418

99.735

378

378

378

 

SWPV1-125

SWPV2-131

CNPV136

CNPV136 putative EEV maturation protein

75.602

99.68

622

625

625

 

SWPV1-126

SWPV2-132

CNPV137

CNPV137 conserved hypothetical protein

62.26

98.925

467

462

465

 

SWPV1-127

SWPV2-133

CNPV138

CNPV138 putative serine/threonine protein kinase, virus assembly

83.632

100

445

444

444

 

SWPV1-128

SWPV2-134

CNPV139

CNPV139 conserved hypothetical protein

81.69

100

213

213

213

 

SWPV1-129

SWPV2-135

CNPV140

CNPV140 conserved hypothetical protein

78.788

100

65

66

66

 

SWPV1-130

SWPV2-136

CNPV141

CNPV141 HAL3-like domain protein

88.333

100

182

184

184

 

SWPV1-131

  

no significant BLAST hits

28

 

101

 

571

SWPV1: Possible Unique ORF

SWPV1-132

SWPV2-137

CNPV142

CNPV142 N1R/p28-like protein

48.266

98.442

314

321

321

 

SWPV1-133

SWPV2-138

CNPV143

CNPV143 ankyrin repeat protein

54.103

98.361

634

671

671

 

SWPV1-134

SWPV2-139

CNPV144

CNPV144 ankyrin repeat protein

59.011

99.281

562

556

556

 

SWPV1-135

SWPV2-140

CNPV145

CNPV145 conserved hypothetical protein

75.814

100

439

440

440

 

SWPV1-136

SWPV2-141

CNPV146

CNPV146 RNA polymerase subunit RPO7

88.525

100

66

62

62

 

SWPV1-137

SWPV2-142

CNPV147

CNPV147 conserved hypothetical protein

80.851

100

188

188

188

 

SWPV1-138

SWPV2-143

CNPV148

CNPV148 virion core protein

86.533

100

347

348

348

 
 

SWPV2-144

CNPV149

CNPV149 putative thioredoxin binding protein

 

99.673

 

306

306

 
  

CNPV150

CNPV150 ankyrin repeat protein

    

351

 
 

SWPV2-145

CNPV151

CNPV151 ankyrin repeat protein

 

99.029

 

412

412

 
 

SWPV2-146

CNPV152

CNPV152 hypothetical protein

 

98

 

149

187

SWPV2: C-terminus fragment, not likely translated

 

SWPV2-147

CNPV153

CNPV153 Rep-like protein

 

99.359

 

312

312

 

SWPV1-139

  

CNPV159 N1R/p28-like protein

78.488

 

333

 

337

 

SWPV1-140

  

FWPV121 CC chemokine-like protein

46

 

93

 

121

 

SWPV1-141

SWPV2-148

CNPV154

CNPV154 variola B22R-like protein

90.067

98.286

1939

875

1928

SWPV2: N-terminus fragment/SWPV1: Low SNP Density

SWPV1-142

SWPV2-149

CNPV155

CNPV155 variola B22R-like protein

82.427

99.454

1810

1831

1830

 
 

SWPV2-150

CNPV156

CNPV156 hypothetical protein

 

96.287

 

834

832

 
 

SWPV2-151

CNPV157

CNPV157 TGF-beta-like protein

 

87.679

 

343

349

 
  

CNPV158

CNPV158 TGF-beta-like protein

    

172

 
  

CNPV159

CNPV159 N1R/p28-like protein

    

337

 
  

CNPV160

CNPV160 N1R/p28-like protein

    

396

 
 

SWPV2-152

CNPV161

CNPV161 TGF-beta-like protein

 

99.441

 

358

358

 
 

SWPV2-153

CNPV162

CNPV162 TGF-beta-like protein

 

97.987

 

149

149

 
  

CNPV163

CNPV163 hypothetical protein

    

92

 
  

CNPV164

CNPV164 hypothetical protein

    

98

 
 

SWPV2-154

CNPV165

CNPV165 N1R/p28-like protein

 

98.75

 

320

346

SWPV2: C-terminus fragment, not likely translated

SWPV1-143

SWPV2-155

CNPV166

CNPV166 Ig-like domain protein

96.812

95.652

345

345

345

SWPV1: Low SNP Density

SWPV1-144

SWPV2-156

CNPV167

CNPV167 Ig-like domain protein

94.767

88.372

172

168

171

SWPV1: Low SNP Density

 

SWPV2-157

CNPV168

CNPV168 N1R/p28-like protein

 

96

 

350

358

 

SWPV1-145

 

CNPV169

CNPV169 N1R/p28-like protein

83.578

 

337

 

332

SWPV1: CNPV-168/169 Fusion

SWPV1-146

SWPV2-158

CNPV170

CNPV170 thymidylate kinase

100

100

121

212

212

SWPV1: N-terminus fragment

SWPV1-147

SWPV2-159

CNPV171

CNPV171 late transcription factor VLTF-1

96.923

100

260

260

260

 

SWPV1-148

SWPV2-160

CNPV172

CNPV172 putative myristylated protein

83.125

99.403

336

335

335

 

SWPV1-149

SWPV2-161

CNPV173

CNPV173 putative myristylated IMV envelope protein

91.358

98.354

243

243

243

 

SWPV1-150

SWPV2-162

CNPV174

CNPV174 conserved hypothetical protein

47.917

100

96

96

96

 

SWPV1-151

SWPV2-163

CNPV175

CNPV175 conserved hypothetical protein

84.158

100

303

303

303

 

SWPV1-152

SWPV2-164

CNPV176

CNPV176 DNA-binding virion core protein

87.747

100

253

252

252

 

SWPV1-153

SWPV2-165

CNPV177

CNPV177 conserved hypothetical protein

84.733

100

131

130

130

 

SWPV1-154

SWPV2-166

CNPV178

CNPV178 putative IMV membrane protein

85.135

100

148

148

148

 

SWPV1-155

SWPV2-167

CNPV179

CNPV179 poly(A) polymerase small subunit PAPS

88.667

100

300

302

302

 

SWPV1-156

SWPV2-168

CNPV180

CNPV180 RNA polymerase subunit RPO22

87.634

99.462

186

186

186

 

SWPV1-157

SWPV2-169

CNPV181

CNPV181 conserved hypothetical protein

82.353

100

136

136

136

 

SWPV1-158

SWPV2-170

CNPV182

CNPV182 RNA polymerase subunit RPO147

93.866

99.922

1288

1288

1288

 

SWPV1-159

SWPV2-171

CNPV183

CNPV183 putative protein-tyrosine phosphatase, virus assembly

85.542

100

166

166

166

 

SWPV1-160

SWPV2-172

CNPV184

CNPV184 conserved hypothetical protein

91.534

100

190

189

189

 

SWPV1-161

SWPV2-173

CNPV185

CNPV185 ankyrin repeat protein

32.632

96.341

337

328

328

 

SWPV1-162

SWPV2-174

CNPV186

CNPV186 IMV envelope protein

100

100

329

330

330

 

SWPV1-163

SWPV2-175

CNPV187

CNPV187 RNA polymerase associated protein RAP94

91.114

99.75

799

799

799

 

SWPV1-164

SWPV2-176

CNPV188

CNPV188 late transcription factor VLTF-4

70.115

92.941

170

170

170

 

SWPV1-165

SWPV2-177

CNPV189

CNPV189 DNA topoisomerase

88.608

99.684

316

316

316

 

SWPV1-166

SWPV2-178

CNPV190

CNPV190 conserved hypothetical protein

77.124

99.346

153

153

153

 

SWPV1-167

SWPV2-179

CNPV191

CNPV191 conserved hypothetical protein

70.874

99.029

103

103

103

 

SWPV1-168

SWPV2-180

CNPV192

CNPV192 mRNA capping enzyme large subunit

88.221

99.764

848

846

846

 

SWPV1-169

SWPV2-181

CNPV193

CNPV193 HT motif protein

72.619

100

104

106

106

 

SWPV1-170

SWPV2-182

CNPV194

CNPV194 virion protein

71.223

100

139

140

140

 

SWPV1-171

SWPV2-183

CNPV195

CNPV195 hypothetical protein

51.2

98.611

139

144

144

 

SWPV1-172

SWPV2-184

CNPV196

CNPV196 conserved hypothetical protein

62.963

100

189

190

190

 

SWPV1-173

SWPV2-185

CNPV197

CNPV197 N1R/p28-like protein

61.679

97.818

279

275

275

 

SWPV1-174

SWPV2-186

CNPV198

CNPV198 C-type lectin-like protein

55.844

99.359

159

156

156

 

SWPV1-175

SWPV2-187

CNPV199

CNPV199 deoxycytidine kinase-like protein

79.111

100

222

225

225

 

SWPV1-176

SWPV2-188

CNPV200

CNPV200 Rep-like protein

72.903

97.59

152

166

166

 

SWPV1-177

SWPV2-189

CNPV201

CNPV201 conserved hypothetical protein

60

97.661

197

167

192

 

SWPV1-178

SWPV2-190

CNPV202

CNPV202 N1R/p28-like protein

69.203

99.638

275

276

276

 

SWPV1-179

SWPV2-191

CNPV203

CNPV203 N1R/p28-like protein

64.935

99.738

380

382

382

 

SWPV1-180

SWPV2-192

CNPV204

CNPV204 conserved hypothetical protein

53.226

100

53

61

61

 

SWPV1-181

SWPV2-193

CNPV205

CNPV205 N1R/p28-like protein

71.885

99.371

317

318

318

 

SWPV1-182

SWPV2-194

CNPV206

CNPV206 putative photolyase

84.989

99.364

464

472

472

 

SWPV1-183

  

CNPV081 Ig-like domain protein

53.988

 

332

 

333

 

SWPV1-184

SWPV2-195

CNPV207

CNPV207 N1R/p28-like protein

64.535

98.235

193

173

183

 

SWPV1-185

SWPV2-196

CNPV208

CNPV208 conserved hypothetical protein

52.239

97.5

172

200

200

 

SWPV1-186

SWPV2-197

CNPV209

CNPV209 N1R/p28-like protein

65.686

100

311

310

310

 

SWPV1-187

SWPV2-198

CNPV210

CNPV210 N1R/p28-like protein

74.419

99.237

130

131

131

 

SWPV1-188

SWPV2-199

CNPV211

CNPV211 conserved hypothetical protein

49.02

98.148

54

54

54

 

SWPV1-189

SWPV2-200

CNPV212

CNPV212 N1R/p28-like protein

76.136

98.295

175

176

176

 

SWPV1-190

  

no significant BLAST hits

  

70

  

SWPV1: Possible Unique ORF

SWPV1-191

SWPV2-201

CNPV213

CNPV213 deoxycytidine kinase-like protein

58.768

99.539

216

216

217

 
 

SWPV2-202

CNPV214

CNPV214 vaccinia C4L/C10L-like protein

 

99.438

 

356

356

 

SWPV1-192

  

CNPV012 conserved hypothetical protein

37.41

 

165

 

189

 

SWPV1-193

  

CNPV223 ankyrin repeat protein

31.579

 

674

 

847

 

SWPV1-194

SWPV2-203

CNPV215

CNPV215 CC chemokine-like protein

49.751

96.078

202

204

204

 
 

SWPV2-204

CNPV216

CNPV216 conserved hypothetical protein

 

98.762

 

401

404

 
 

SWPV2-205

CNPV217

CNPV217 N1R/p28-like protein

 

95.152

 

330

330

 

SWPV1-195

  

CNPV223 ankyrin repeat protein

38.474

 

729

 

847

SWPV1: N-terminus fragment

SWPV1-196

SWPV2-206

CNPV218

CNPV218 N1R/p28-like protein

66.667

99.522

318

223

437

SWPV2: N-terminus fragment

SWPV1-197

  

CNPV228 N1R/p28-like protein

53

 

161

 

371

SWPV1: N-terminus fragment

SWPV1-198

  

CNPV160 N1R/p28-like protein

79.293

 

367

 

396

SWPV1: Fragment/CNPV-220/221 Fusion

SWPV1-199

  

CNPV160 N1R/p28-like protein

66.582

 

360

 

396

SWPV1: Paralog to SWPV1-198?

SWPV1-200

  

CNPV161 TGF-beta-like protein

36.882

 

256

 

358

 

SWPV1-201

  

CNPV162 TGF-beta-like protein

50

 

141

 

149

 

SWPV1-202

  

no significant BLAST hits

  

98

  

SWPV1: Possible Unique ORF

 

SWPV2-207

CNPV219

CNPV219 N1R/p28-like protein

 

99.713

 

349

349

 
 

SWPV2-208

CNPV220

CNPV220 N1R/p28-like protein

 

80.263

 

85

178

SWPV2: N-terminus fragment

 

SWPV2-209

CNPV221

CNPV221 N1R/p28-like protein

 

94.231

 

213

281

SWPV2: N-terminus fragment

 

SWPV2-210

CNPV222

CNPV222 N1R/p28-like protein

 

99.649

 

285

285

 
 

SWPV2-211

CNPV223

CNPV223 ankyrin repeat protein

 

98.819

 

847

847

 

SWPV1-203

SWPV2-212

CNPV224

CNPV224 hypothetical protein

50.382

100

126

239

239

 
 

SWPV2-213

CNPV225

CNPV225 N1R/p28-like protein

 

74.038

 

94

159

SWPV2: N-terminus fragment

 

SWPV2-214

CNPV226

CNPV226 N1R/p28-like protein

 

96.825

 

126

134

 
  

CNPV227

CNPV227 N1R/p28-like protein

    

359

 
  

CNPV228

CNPV228 N1R/p28-like protein

    

371

 

SWPV1-204

SWPV2-215

CNPV229

CNPV229 ankyrin repeat protein

44.498

97.926

423

434

434

 
 

SWPV2-216

CNPV230

CNPV230 hypothetical protein

 

98.462

 

65

65

 

SWPV1-205

SWPV2-217

CNPV231

CNPV231 MyD116-like domain protein

72.222

98.101

100

158

158

SWPV1: large in-frame deletions

SWPV1-206

SWPV2-218

CNPV232

CNPV232 CC chemokine-like protein

59.024

93.137

205

204

204

 

SWPV1-207

SWPV2-219

CNPV233

CNPV233 ankyrin repeat protein

56.936

99.788

476

471

471

 
 

SWPV2-220

CNPV234

CNPV234 ankyrin repeat protein

 

100

 

508

508

SWPV2: High SNP Density

SWPV1-208

  

PEPV008 vaccinia C4L/C10L-like protein

55

 

420

 

411

 
 

SWPV2-221

CNPV235

CNPV235 conserved hypothetical protein

 

88.426

 

432

432

 

SWPV1-209

SWPV2-222

CNPV236

CNPV236 ribonucleotide reductase small subunit

83.282

95.666

324

323

323

 
 

SWPV2-223

CNPV237

CNPV237 ankyrin repeat protein

 

97.732

 

441

441

 

SWPV1-210

  

CNPV234 ankyrin repeat protein

30.545

 

559

 

508

 

SWPV1-211

SWPV2-224

CNPV238

CNPV238 late transcription factor VLTF-3

95.111

100

225

225

225

 

SWPV1-212

SWPV2-225

CNPV239

CNPV239 virion redox protein

80.282

100

72

75

75

 

SWPV1-213

SWPV2-226

CNPV240

CNPV240 virion core protein P4b

88.788

99.848

660

659

659

 

SWPV1-214

SWPV2-227

CNPV241

CNPV241 immunodominant virion protein

47.368

99.07

242

215

215

 

SWPV1-215

SWPV2-228

CNPV242

CNPV242 RNA polymerase subunit RPO19

88.166

98.817

169

169

169

 

SWPV1-216

SWPV2-229

CNPV243

CNPV243 conserved hypothetical protein

81.501

98.928

373

373

373

 

SWPV1-217

SWPV2-230

CNPV244

CNPV244 early transcription factor large subunit VETFL

95.91

100

709

709

709

 

SWPV1-218

SWPV2-231

CNPV245

CNPV245 intermediate transcription factor VITF-3

90.667

99.667

300

300

300

 

SWPV1-219

SWPV2-232

CNPV246

CNPV246 putative IMV membrane protein

80

98.667

76

75

75

 

SWPV1-220

SWPV2-233

CNPV247

CNPV247 virion core protein P4a

81.494

99.664

897

893

893

 

SWPV1-221

SWPV2-234

CNPV248

CNPV248 conserved hypothetical protein

78.723

100

281

279

279

 

SWPV1-222

SWPV2-235

CNPV249

CNPV249 virion protein

74.269

99.405

167

168

168

 

SWPV1-223

SWPV2-236

CNPV250

CNPV250 conserved hypothetical protein

36.082

94.595

73

56

99

SWPV2: N-terminus fragment

SWPV1-224

SWPV2-237

CNPV251

CNPV251 putative IMV membrane protein

69.565

100

69

69

69

 

SWPV1-225

SWPV2-238

CNPV252

CNPV252 putative IMV membrane protein

68.478

98.913

92

92

92

 

SWPV1-226

SWPV2-239

CNPV253

CNPV253 putative IMV membrane virulence factor

73.585

98.113

53

53

53

 

SWPV1-227

SWPV2-240

CNPV254

CNPV254 conserved hypothetical protein

75

98.958

96

96

96

 

SWPV1-228

SWPV2-241

CNPV255

CNPV255 predicted myristylated protein

84.282

99.728

368

368

368

 

SWPV1-229

SWPV2-242

CNPV256

CNPV256 putative phosphorylated IMV membrane protein

81.006

100

188

192

192

 

SWPV1-230

SWPV2-243

CNPV257

CNPV257 DNA helicase, transcriptional elongation

87.229

99.784

462

462

462

 

SWPV1-231

SWPV2-244

CNPV258

CNPV258 conserved hypothetical protein

77.647

100

86

89

89

 

SWPV1-232

SWPV2-245

CNPV259

CNPV259 DNA polymerase processivity factor

81.86

100

432

112

434

 

SWPV1-233

SWPV2-246

CNPV260

CNPV260 conserved hypothetical protein

91.071

99.77

112

434

112

 

SWPV1-234

SWPV2-247

CNPV261

CNPV261 Holliday junction resolvase protein

80.405

100

151

152

152

 

SWPV1-235

SWPV2-248

CNPV262

CNPV262 intermediate transcription factor VITF-3

86.126

100

383

383

383

 

SWPV1-236

SWPV2-249

CNPV263

CNPV263 RNA polymerase subunit RPO132

94.301

100

1158

1157

1157

 

SWPV1-237

SWPV2-250

CNPV264

CNPV264 A type inclusion-like protein

81.015

99.502

602

601

603

 

SWPV1-238

SWPV2-251

CNPV265

CNPV265 A type inclusion-like/fusion protein

67.015

99.789

471

475

475

 

SWPV1-239

SWPV2-252

CNPV266

CNPV266 conserved hypothetical protein

89.286

99.286

140

140

140

 

SWPV1-240

SWPV2-253

CNPV267

CNPV267 RNA polymerase subunit RPO35

77.558

99.016

303

305

305

 

SWPV1-241

SWPV2-254

CNPV268

CNPV268 conserved hypothetical protein

73.529

100

72

75

75

 

SWPV1-242

SWPV2-255

CNPV269

CNPV269 conserved hypothetical protein

70.796

100

113

113

113

 

SWPV1-243

SWPV2-256

CNPV270

CNPV270 conserved hypothetical protein

70.588

100

119

120

120

 

SWPV1-244

SWPV2-257

CNPV271

CNPV271 DNA packaging protein

89.963

99.648

272

284

284

 

SWPV1-245

SWPV2-258

CNPV272

CNPV272 C-type lectin-like EEV protein

76.136

99.448

182

181

181

 

SWPV1-246

  

CNPV012 conserved hypothetical protein

30.147

 

172

 

189

 

SWPV1-247

SWPV2-259

CNPV273

CNPV273 conserved hypothetical protein

62.816

99.635

276

274

274

 

SWPV1-248

SWPV2-260

CNPV274

CNPV274 putative tyrosine protein kinase

63.197

99.628

286

269

269

 

SWPV1-249

SWPV2-261

CNPV275

CNPV275 putative serpin

72.271

99.408

340

338

338

 

SWPV1-250

SWPV2-262

CNPV276

CNPV276 conserved hypothetical protein

56.667

100

227

252

252

 

SWPV1-251

SWPV2-263

CNPV277

CNPV277 G protein-coupled receptor-like protein

90

99.677

310

310

310

 

SWPV1-252

SWPV2-264

CNPV278

CNPV278 conserved hypothetical protein

89.691

98.958

97

96

96

 

SWPV1-253

SWPV2-265

CNPV279

CNPV279 beta-NGF-like protein

63.415

100

167

169

169

 

SWPV1-254

SWPV2-266

CNPV280

CNPV280 HT motif protein

67.692

99.231

134

130

130

 

SWPV1-255

SWPV2-267

CNPV281

CNPV281 conserved hypothetical protein

71.728

99.533

192

214

214

 

SWPV1-256

SWPV2-268

CNPV282

CNPV282 HT motif protein

71.552

100

118

120

120

 

SWPV1-257

SWPV2-269

CNPV283

CNPV283 CC chemokine-like protein

63.208

100

110

111

111

 

SWPV1-258

SWPV2-270

CNPV284

CNPV284 putative interleukin binding protein

37.405

90.769

192

193

195

 

SWPV1-259

SWPV2-271

CNPV285

CNPV285 EGF-like protein

62.992

99.206

123

126

126

 

SWPV1-260

SWPV2-272

CNPV286

CNPV286 putative serine/threonine protein kinase

76.744

99.672

303

305

305

 

SWPV1-261

SWPV2-273

CNPV287

CNPV287 conserved hypothetical protein

73.248

98.758

165

160

161

 

SWPV1-262

SWPV2-274

CNPV288

CNPV288 C-type lectin-like protein

52.414

88.435

163

147

147

 

SWPV1-263

SWPV2-275

CNPV289

CNPV289 putative interleukin binding protein

58.993

99.281

132

139

139

 

SWPV1-264

SWPV2-276

CNPV290

CNPV290 conserved hypothetical protein

84

83.784

75

75

75

 

SWPV1-265

SWPV2-277

CNPV291

CNPV291 ankyrin repeat protein

48.067

98.99

613

594

594

 

SWPV1-266

SWPV2-278

CNPV292

CNPV292 hypothetical protein

37.209

100

101

74

74

 

SWPV1-267

SWPV2-279

CNPV293

CNPV293 ankyrin repeat protein

55.634

99.648

305

284

284

 

SWPV1-268

SWPV2-280

CNPV294

CNPV294 ankyrin repeat protein

68.447

99.07

424

430

430

 

SWPV1-269

  

PIPV223 host range protein

51

 

138

 

143

 

SWPV1-270

  

FWPV217 hypothetical protein

50

 

330

 

328

 

SWPV1-271

SWPV2-281

CNPV295

CNPV295 ankyrin repeat protein

57.736

100

264

396

396

 

SWPV1-272

SWPV2-282

CNPV296

CNPV296 ankyrin repeat protein

67.195

99.127

438

458

458

 

SWPV1-273

SWPV2-283

CNPV297

CNPV297 ankyrin repeat protein

54.972

99.457

717

737

737

 

SWPV1-274

SWPV2-284

CNPV298

CNPV298 ankyrin repeat protein

64.591

99.825

573

571

571

 

SWPV1-275

SWPV2-285

CNPV299

CNPV299 putative serine/threonine protein kinase

67.893

99.333

303

300

300

 

SWPV1-276

SWPV2-286

CNPV300

CNPV300 ankyrin repeat protein

75.82

98.77

253

244

244

 

SWPV1-277

  

CNPV219 N1R/p28-like protein

28.467

 

142

 

349

 

SWPV1-278

  

CNPV228 N1R/p28-like protein

43.038

 

87

 

371

 

SWPV1-279

  

TKPV163 ankyrin repeat protein

40

 

432

 

434

 

SWPV1-280

SWPV2-287

CNPV301

CNPV301 ankyrin repeat protein

59.546

99.241

510

527

527

 

SWPV1-281

SWPV2-288

CNPV302

CNPV302 conserved hypothetical protein

45.026

100

175

193

193

 

SWPV1-282

SWPV2-289

CNPV303

CNPV303 ankyrin repeat protein

68.938

99.4

499

500

500

 

SWPV1-283

SWPV2-290

CNPV304

CNPV304 ankyrin repeat protein

62.105

99.785

476

466

466

 

SWPV1-284

SWPV2-291

CNPV305

CNPV305 N1R/p28-like protein

54.545

100

261

262

262

 

SWPV1-285

SWPV2-292

CNPV306

CNPV306 hypothetical protein

30.769

98.611

73

72

72

 

SWPV1-286

SWPV2-293

CNPV307

CNPV307 C-type lectin-like protein

55.828

100

165

154

154

 

SWPV1-287

SWPV2-294

CNPV308

CNPV308 ankyrin repeat protein

58.757

99.44

359

357

357

 

SWPV1-288

SWPV2-295

CNPV309

CNPV309 ankyrin repeat protein

69.388

100

195

196

196

 

SWPV1-289

SWPV2-296

CNPV310

CNPV310 ankyrin repeat protein

47.359

99.255

540

537

537

 

SWPV1-290

SWPV2-297

CNPV311

CNPV311 EFc-like protein

54.4

99.194

125

124

124

 

SWPV1-291

SWPV2-298

CNPV312

CNPV312 conserved hypothetical protein

53.704

98.795

168

166

166

 

SWPV1-292

SWPV2-299

CNPV313

CNPV313 Ig-like domain protein

69.43

98.165

213

218

218

 

SWPV1-293

SWPV2-300

CNPV314

CNPV314 ankyrin repeat protein

71.552

99.829

580

629

584

 

SWPV1-294

  

CNPV011 ankyrin repeat protein

32

 

513

 

586

 

SWPV1-295

SWPV2-301

CNPV315

CNPV315 G protein-coupled receptor-like protein

59.17

99.365

315

315

315

 

SWPV1-296

  

CNPV014 Ig-like domain protein

59.624

 

230

 

490

 

SWPV1-297

  

CNPV014 Ig-like domain protein

59.641

 

240

 

490

 

SWPV1-298

  

CNPV015 ankyrin repeat protein

45.455

 

74

 

528

 

SWPV1-299

  

CNPV150 ankyrin repeat protein

36.364

 

84

 

351

 

SWPV1-300

SWPV2-302

CNPV316

CNPV316 ankyrin repeat protein

35.294

99.632

162

544

544

 
 

SWPV2-303

CNPV317

CNPV317 hypothetical protein

 

100

 

55

55

 
 

SWPV2-304

CNPV318

CNPV318 ankyrin repeat protein

 

98.054

 

514

514

 
 

SWPV2-305

CNPV319

CNPV319 ankyrin repeat protein

 

97.638

 

637

739

SWPV2: C-terminus fragment, not likely translated

SWPV1-301

  

PIPV253 EFc-like protein

69

 

124

 

124

 

SWPV1-302

  

CNPV015 ankyrin repeat protein

45.276

 

520

 

528

 

SWPV1-303

  

CNPV223 ankyrin repeat protein

40

 

480

 

847

 

SWPV1-304

SWPV2-306

CNPV320

CNPV320 Ig-like domain protein

76.858

99.787

468

469

469

 
 

SWPV2-307

CNPV321

CNPV321 EFc-like protein

 

99.194

 

124

124

 
 

SWPV2-308

CNPV322

CNPV322 ankyrin repeat protein

 

98.408

 

689

690

 

SWPV1-305

  

CNPV035 C-type lectin-like protein

35.556

 

138

 

134

 

SWPV1-306

  

CNPV008 C-type lectin-like protein

50

 

174

 

169

 

SWPV1-307

SWPV2-309

CNPV323

CNPV323 conserved hypothetical protein

75.61

93.651

84

186

182

 

SWPV1-308

SWPV2-310

CNPV324

CNPV324 conserved hypothetical protein

87.387

99.55

220

222

222

 

SWPV1-309

 

CNPV325

CNPV325 ankyrin repeat protein

56.458

 

468

 

514

 

SWPV1-310

SWPV2-311

CNPV326

CNPV326 C-type lectin-like protein

32.044

85.99

181

208

204

 
 

SWPV2-312

CNPV327

CNPV327 hypothetical protein

 

92.941

 

171

171

 
  

CNPV328

CNPV328 hypothetical protein

    

72

 
This difference in similarity between the new viruses and CNPV is easily visualized in complete genome dotplots (Fig. 2a and b). Significantly more indels are present in the SWPV-1 vs CNPV dotplot (Fig. 2a). However, when the phylogenetic relationships of these viruses were examined together with the other available complete genomes, SWPV-1 was still part of the CNPV clade (Fig. 3a). From this alignment, CNPV is 99.2%, 78.7%, 69.4%, 69.5%, 68.8% and 66.5% identical (nt) to SWPV-2, SWPV-1, FeP2, PEPV, FWPV and TKPV, respectively. A greater selection of viruses was included in the phylogenetic tree by using other fragments of incompletely sequenced avipoxvirus genomes. For example, Vultur gryphus poxvirus (VGPV), Flamingopox virus (FGPV) and Hawaiian goose poxvirus (HGPV) are all more similar to SWPV-2 and CNPV than SWPV-1 (Fig. 3b), this confirms that other poxviruses are as closely related to CNPV as SWPV-2. By also building phylogenetic trees with partial nucleotide sequences from the p4b gene (Fig. 4) and DNA polymerase gene (Fig. 5), we discovered that several other viruses are within the SWPV-1, SWPV-2 and CNPV clade. This includes a poxvirus isolated from Houbara Bustards (Chlamydotis undulata) in captive-breeding programs in Morocco [23], but named CNPV-morocco, and avipoxviruses isolated from American crow (Corvus brachyrhynchos) and American robin (Turdus migratorius) [24], which is almost identical to CPNV-1 within this relatively small fragment of the genome.
Fig. 2

Dotplots of Shearwaterpox viruses (SWPV-1 and 2) vs CNPV genomes. Horizontal sequence: SWPV-1 (a) and SWPV-2 (b), vertical sequence CNPV. Red and blue boxes represent genes transcribed to the right and left of the genome, respectively

Fig. 3

Phylogenetic relationship between Shearwaterpox viruses (SWPV-1 and 2) and other avipoxviruses. a Phylogenetic tree of 173 kbp core region (large gaps removed) from available complete avipoxvirus genomes. b Phylogenetic tree highlighting viruses closely related to CNPV. The sequences were aligned with ClustalO and MEGA7 was used to create a maximum likelihood tree based on the Tamura-Nei method and tested by bootstrapping with 1000 replicates. The abbreviations and GenBank accession details for poxviruses strains were used: Canarypox virus (CNPV; AY318871), Pigeonpox virus (FeP2; KJ801920), Penguinpox virus (PEPV; KJ859677) Fowlpox virus (FWPV; AF198100), Shearwaterpox virus 1 (SWPV-1; KX857216), Shearwaterpox virus 2 (SWPV-2; KX857215), Turkeypox virus (TKPV; NC_028238), Vultur Gryphus poxvirus (VGPV; AY246559), Flamingopox virus (FGPV; HQ875129 and KM974726), Hawaiian goose poxvirus (HGPV; AY255628)

Fig. 4

Maximum likelihood phylogenetic tree from partial DNA sequences of p4b gene of avipoxviruses. Novel Shearwaterpox viruses (SWPV-1 and SWPV-2) are highlighted by gray background

Fig. 5

Maximum likelihood phylogenetic tree from partial DNA sequences of DNA polymerase gene of avipoxviruses. Novel Shearwaterpox viruses (SWPV-1 and SWPV-2) are highlighted by gray background

Features of SWPV-2

As noted above, and displayed in the Dotplot (Fig. 2b), SWPV-2 is very similar to CNPV with almost 98% nt identity. However, a 1% difference still gives approximately 10 mutations in an average sized gene any of which could have drastic effects if an early STOP codon is introduced to the gene sequence. Similarly, small changes to promoter regions can significantly alter gene expressions that are impossible to predict in these viruses. With this annotation strategy, 18 CNPV genes were deemed to be missing from the SWPV-2 complete genome and a further 15 genes significantly fragmented as to probably cause them to be non-functional (Table 1). No novel genes were predicted in SWPV-2, and no rearrangement of genes compared to CNPV was observed.

Features of SWPV-1

As expected from the much lower percent nt identity, SWPV-1 was found to be considerably more different to CNPV than SWPV-2 when compared at the level of genes present or absent. (Table 1). 43 CNPV genes are absent from SWPV-1 and a further 6 are significantly fragmented. There are 4 predicted genes in SWPV-1 that are not present in any other poxvirus, nor do they match any sequences in the NR protein database using BLASTP. However, they are all relatively short ORFs and it is possible that they are not functional genes. Additionally, SWPV-1 encodes nine polypeptides that do not match CNPV proteins, but do match proteins from other avipoxviruses (penguinpox, turkeypox, pigeonpox and fowlpox). This could be due to recombination among ancestral viruses, but could also result from the loss of the corresponding ortholog in CNPV leaving another virus to provide the “best match”.

As might be expected given the greater distance between SWPV-1 and CPNV than between SWPV-2 and CNPV, there are more instances of minor rearrangements that created a loss of synteny (Table 1). However, since most of these involve the families of repeated genes, it is also possible that divergence of these sequences has led to the inability to distinguish between the orthologous and paralogous genes.

Evidence of recombination among avipoxviruses

When we reviewed a graph of nt identity between the 2 new complete genomes and CNPV using BBB (not shown), there were several relatively short syntenic regions where 1) SWPV-1 matched CNPV significantly better than the majority of the genome, and 2) SWPV-2 matched CNPV significantly worse than the majority of the genome. To examine these regions in more detail, the Visual Summary feature of BBB was used to display individual SNPs for these genome comparisons (Fig. 6a and b). This analysis revealed that SWPV-1 and SWPV-2 were unique in these regions and confirmed that the genome sequences of SWPV-1and SWPV-2 were not contaminated during their assembly. However, when these regions were used as query sequences in BLASTN searches of all poxvirus sequences the best match remained CNPV suggesting that these sequences originated from avipoxvirus genomes that are not represented in the public databases.
Fig. 6

Region of recombination in Shearwaterpox viruses (SWPV) detected in A. carneipes and A. pacificus. Nucleotide differences to CNPV are shown in blue (SNPs), green/red (indels). Figure 6a. Region of recombination in SWPV-2. On the middle track, SWPV-2 has very few differences to CNPV except for highly divergent block in the middle of this region. Figure 6b. Region of recombination in SWPV-1. On the bottom track, SWPV-1 is very different to CNPV except for highly similar block between nt 193,000 and 195,500

Discussion

This paper describes the detection and characterization of two novel avipoxvirus complete genome sequences in a naturally occurring infections of avian pox in a naïve population of shearwaters. The DNA sequences of SWPV-1 and SWPV-2 are significantly different than each other but nevertheless had closest similarity with Canarypox virus (67% and 98%, respectively). Furthermore, the genetic distance and novel genome structure of SWPV-1 from A. carneipes considered to be missing 43 genes likened to CNPV and contained 4 predicted genes which are not found in any other poxvirus and is overall sufficiently genetically different to be considered a separate virus species. Whilst, the SWPV-2 complete genome was missing 18 genes compared to CNPV, with a further 15 genes significantly fragmented as to probably cause them to be non-functional. Furthermore, the phylogenetic distribution of SWPV-1 indicates that shearwaters and perhaps other long-lived, vagile marine birds could be important hosts for avipoxvirus dispersal around the globe. The natural hosts of these avipoxviruses maybe this population of shearwaters, other migratory birds that use Lord Howe Island for breeding or resident avian host reservoir species. Species such as the Lord Howe White-eye (Zosterops tephropleura) and Lord Howe Golden Whistler (Pachcephala petoralis contempta) are candidate passerine birds that might provide such function.

Examining the phylogenetic relationship between the Shearwaterpox viruses and other avipoxviruses, it is evident that the SWPV-2 is most closely related to Canarypox virus. The SWPV-1 and SWPV-2 complete genomes both contain several genes that are more closely related to CNPV throughout their entire genome. As shown in Fig. 3 it is reasonable to postulate that these viruses originated from a common ancestor that diverged from a CNPV-like progenitor related to fowlpox, penguinpox and pigeonpox viruses. Finer resolution of the phylogenetic relationship using partial nucleotide sequences of p4b and DNA polymerase genes of avipoxviruses revealed that SWPV isolated from seabirds also clustered in global clade B consisting of avipoxviruses originating from Canary Morocco, Canarypox and poxviruses from American crow and American robin. Given their genetic diversity, it is perhaps not surprising that Shearwater species can be exposed to multiple avipoxviral infections. Studies such as those by Barnett et al. [25] suggest that the species specificity of poxviruses is variable. Some genera, such as Suipoxvirus are highly restricted to individual vertebrate hosts, swinepox for instance, whereas others, such as avipoxviruses demonstrate some evidence of cross-species infection within a predator–prey system [24]. This suggests that the avipoxviruses can infect a diverse range of bird species if they are within a close enough proximity to each other [26]. Thus far, there were no clear patterns regarding species-specificity in the Shearwaterpox viruses described here.

While overt and systemic lesions and fatal disease can occur, avian pox tends to be a self-limiting localized infection of apterial skin with full recovery possible. Many bird species experience life-long immunity if the immune system is not weakened and or the birds are not infected by different strains [27, 28]. As shown in our example, secondary infections can occur and these may contribute to morbidity and mortality [2931]. Similar to the example in shearwaters, Shivaprasad et al. [30] reported evidence of poxvirus infection and secondary fungal pathogens in canaries (Serinus canaria). Stressful conditions, poor nutrition, overt environmental contamination and other underlying causes of immunosuppression and ill health may contribute to the pathogenesis of such lesions. This was the primary reason we tested for avian circovirus and other potential pathogens.

Avian pox has not been previously reported in shearwaters (Ardenna spp.) from Lord Howe Island, nor has it been documented for any other bird species in this region. So it is difficult to attribute the causality of this unique event in these species. The value of complete genome characterization and analysis is highlighted since a phylogenetic relationship based on single gene studies such as the polymerase gene may have falsely implicated Canarypox virus as a potential exotic introduced emerging disease from domesticated birds. Although we cannot trace the actual source of infection in the shearwater chicks, it is more likely that the infection in the birds resulted from parental feeding or arthropod mediated transmission from other island bird species [32]. While, the reservoir host of these novel Shearwaterpox viruses is unknown, mosquitoes are suspected to play a part in transmission within the island. Avipoxvirus infection appears to be relatively rare in seabirds, but it has been reported in several species when they occur on human-inhabited islands that harbor mosquito vectors [33]. According to the Lord Howe Island Board, ship rats, mice, cats, humans and other invasive pest species such as owls are implicated in the extinction of at least five endemic birds, two reptiles, 49 flowering plants, 12 vegetation communities and numerous threatened invertebrates [34]. These rodents and invasive pests have also been highlighted for the potential reservoir of poxvirus infections [3, 35]. Transmission of avipoxvirus by prey–predator and other migratory seabirds likely plays a prominent role; however, the mode of avipoxvirus transmission on Lord Howe Island is not completely understood. Studies by Gyuranecz et al. [24], for example, postulated that raptors may acquire poxvirus infection from their avian prey. This suggests that the poxvirus in shearwaters is likely to be transmitted from other island species such as other migratory seabirds and/or prey–predator, although, it is difficult to be certain without further studies.

Interestingly, these new shearwaterpox virus complete genomes also provide evidence that supports the hypothesis that recombination may play an important role in the evolution of avipoxviruses. A number of genes in SWPV-1 appear to be rearranged compared to CNPV and blocks of unusual similarity scores were seen in both SWPVs. Software that is designed to look for gross recombination between two viruses, such as two strains of HIV, fails to detect this level of recombination and it is left to the investigator to observe such small events by eye after visualizing the distribution of SNPs between viruses. Such relatively small exchanges of DNA may still exert important influences on virus evolution, and has been predicted to have been a driver in the evolution of smallpox [36].

Conclusions

These are the first avipoxvirus complete genome sequences that infect marine bird species. The novel complete genome sequences of SWPV-1 and −2 have greatly enhanced the genomic information for the Avipoxvirus genus, which will contribute to our understanding of the avipoxvirus more generally, and track the evolution of poxvirus infection in such a non-model avian species. Together with the sequence similarities observed between SWPV and other avipoxviruses, this study concluded that the SWPV complete genome from A. carneipes (SWPV-1) described here is not closely related to any other avipoxvirus complete genome isolated from avian or other natural host species, and that it likely should be considered a separate species. Further investigations of Shearwaterpox viruses genetic and pathogenesis will provide a unique approach to better assess the risk associated to poxvirus transmission within and between marine bird species.

Methods

Source of sampling

A total of six samples were collected from two different species of shearwater, five were from Flesh-footed Shearwater (ID: 15-1527-31), and other one was from Wedge-tailed Shearwater (ID: 15–1526). Of size birds, two were recoded to have evidence of gross well circumscribed lesions in the beak (Fig. 1a) and ankle, and others had feather defects (fault lines across the vanes of feathers). Samples were collected from fledglings (approximately 80–90 days of age) of both species on Lord Howe Island, New South Wales (32.53̊S, 159.08̊E) located approximately 500 km off the east coast of Australia during April-May 2015. Samples were collected with the permission of the Lord Howe Island Board (permit no. LHIB 02/14) under the approval of the University of Tasmania and Charles Sturt University Animal Ethics Committees (permit no. A0010874, A0011586, and 09/046). Samples from one individual of each shearwater species were collected including skin lesions, liver and skin biopsies, as well as blood for identifying the causative agents. Depending on the samples, either 25 mg of skin tissue were cut out and chopped into small pieces or 50–100 μL of blood were aseptically transferred into clean 1.5 mL microcentrifuge tube (Eppendorf), and genomic DNA was isolated using the Qiagen blood and tissue mini kit (Qiagen, Germany). The extracted DNA has been stored at −20 °C for further testing. Histopathological examination of the skin was performed.

Archived viral and fungal pathogen testing

Initially, the extracted DNA was screened for detecting novel circoviruses [37, 38] and reticuloendotheliosis virus [39]. For poxvirus screening, the primers PoxP1 (5′-CAGCAGGTGCTAAACAACAA-3′) and PoxP2 (5′-CGGTAGCTTAACGCCGAATA-3′) were synthesized from published literature and used to amplify a segment of approximately 578 bp from the 4b core protein gene for all ChPV species [40]. Optimized PCR reactions mixture contained 3 μL of extracted genomic DNA, 25 pmol of each primer (GeneWorks, Australia), 1.5 mM MgCl2, 1.25 mM of each dNTP, 1xGoTaq® Green Flexi Reaction Buffer, 1 U of Go Taq DNA polymerase (Promega Corporation, USA) and DEPC distilled H2O (Invitrogen, USA) was added to a final volume of 25 μL. The PCR amplification was carried out in an iCycler thermal cycler (Bio-Rad) under the following conditions: denaturation at 94 °C for 2 min followed by 35 cycles of 94 °C for 1 min, 60 °C for 1 min and 72 °C for 1 min, and a final extension step of 2 min at 72 °C.

The internal transcribed spacer (ITS) region was chosen for screening and identification of fungal pathogens [41]. A set of fungus-specific primers ITS1 (5′- TCCGTAGGTGAACCTGCGG -3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC -3′) were designed and used to amplify a segment of approximately 550 bp from the fungal ITS gene [42]. The PCR was standardized to amplify ITS genes, and the 25-μL reaction mixture contained 3 μL of extracted genomic DNA, 25 pmol of each primer (GeneWorks, Australia), 1.5 mM MgCl2, 1.25 mM of each dNTP, 1xGoTaq® Green Flexi Reaction Buffer, 1 U of Go Taq DNA polymerase (Promega Corporation, USA). The PCR reaction involved initial denaturation at 95 °C for 5 min, followed by 30 cycles of denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, and extension at 72 °C for 1 min, and with a final step of one cycle extension at 72 °C for 10 min.

Amplified PCR products, together with a standard molecular mass marker (Sigma), were separated by electrophoresis in 2.0% agarose gel and stained with GelRed (Biotium, CA). Selected bands were excised and purified using the Wizard® SV Gel and PCR Clean-Up System (Promega, USA) according to the manufacturer’s instructions. Purified amplicons were sequenced with PCR primers by the Australian Genome Research Facility Ltd (Sydney) using an AB 3730xl unit (Applied Biosystems). For each amplicon, sequences were obtained at least twice in each direction for each isolate. The sequences were trimmed for primers and aligned to construct contigs (minimum overlap of 35 bp, minimum match percentage of 95%) using Geneious Pro (version 10.0.2).

High throughput sequencing

Next-generation sequencing (NGS) was used to sequence the poxvirus genomes. Virion enrichment was performed by centrifugation for 2 min at 800 × g to remove tissue debris, and the supernatants were subsequently filtered through 5 μm centrifuge filters (Millipore) [43]. The filtrates were nuclease treated to remove unprotected nucleic acids using 8 μL RNase Cocktail Enzyme Mix (Invitrogen). Viral nucleic acids were subsequently extracted using QIAamp DNA mini (Qiagen). The genomic libraries were prepared with an insert size of 150 paired-end. DNA sequencing (NGS) was performed on a HiSeq4000 sequencing platform (Illumina) by Novogene, China.

Bioinformatics

Assembly of the viral genome was conducted according to the established pipeline [44] in CLC Genomics workbench 9.5.2 under La Trobe University Genomics Platform. Briefly, the preliminary quality evaluation for each raw read was generated using quality control (QC) report. The raw data were preprocessed to remove ambiguous base calls, and bases or entire reads of poor quality using default parameters. The datasets were trimmed to pass the quality control based on PHRED score or per base sequence quality score. Trimmed sequence reads were mapped against closely available host genome (Albatross) to remove possible remaining host DNA contamination, and post-filtered reads were mapped against reference Canarypox virus complete genome sequence. Consensus sequences were used to generate the complete poxvirus genome. Avipoxvirus complete genome sequences were aligned using MAFFT [45]. Then the poxvirus specific bioinformatics analyses were performed using the Viral Bioinformatics Resource Centre (virology.uvic.ca) [46], and the further analyses were conducted using the following tools: Viral Orthologous Clusters Database for sequence management (VOCs) [11]; Base-By-Base for genome/gene/protein alignments [47, 48]; Viral Genome Organizer for genome organization comparisons (VGO) [11], and Genome Annotation Transfer Utility for annotation (GATU) [49].

Open reading frames (ORFs) longer than 60 amino acids with minimal overlapping (overlaps cannot exceed 25% of one of the genes) to other ORFs were captured using the CLC Genomics Workbench (CLC) ORF analysis tool as well as GATU [49], and other protein coding sequence and annotation software described in Geneious (version 10.0.2, Biomatters, New Zealand). These ORFs were subsequently extracted into a FASTA file, and similarity searches including nucleotide (BLASTN) and protein (BLASTP) were performed on annotated ORFs as potential genes if they shared significant sequence similarity to known viral or cellular genes (BLAST E value ≤ e-5) or contained a putative conserved domain as predicted by BLASTp [50]. The final SWPV annotation was further examined with other poxvirus ortholog alignments to determine the correct methionine start site, correct stop codons, signs of truncation, and validity of overlaps.

Phylogenetic analysis

Phylogenetic analyses were performed using full poxvirus genome sequences for Shearwater species determined in this study with related avipoxvirus genome sequences available in GenBank database. A selection of partial sequences from seven completely sequenced avipoxvirus genomes and fragments of incompletely sequenced avipoxvirus genomes from Vultur Gryphus poxvirus (VGPV), flamingopox virus (FGPV) and Hawaiian goose poxvirus (HGPV) were also used for phylogenetic analysis. To investigate closer evolutionary relationship among avipoxviruses, partial nucleotide sequences of p4b and DNA polymerase genes were selected. The avipoxvirus sequences were aligned using ClustalO, and then manually edited in Base-by-Base. MEGA7 was used to create a maximum likelihood tree based on the Tamura-Nei method and tested by bootstrapping with 1000 replicates. An additional analysis was performed using complete genome nucleotide sequences of Canarypox virus (CNPV; AY318871), Pigeonpox virus (FeP2; KJ801920), Fowlpox virus (FPV; AF198100), Turkeypox virus (TKPV; NC_028238), Shearwaterpox virus strain-1 (SWPV-1; KX857216), and Shearwaterpox virus strain-2 (SWPV-2; KX857215), which were aligned with MAFTT in Base-By-Base for genome/gene/protein alignments [48]. The program jModelTest 2.1.3 favoured a general-time-reversible model with gamma distribution rate variation and a proportion of invariable sites (GTR + I + G4) for the ML analysis [51].

Abbreviations

ChPV: 

Chordopoxvirinae

CNPV: 

Canarypox virus

dsDNA: 

double-stranded

FGPV: 

Flamingopox virus

FP9: 

European strain of Fowlpox virus

FPVUS: 

South African strain of Fowlpox virus

GATU: 

Genome Annotation Transfer Utility

HGPV: 

Hawaiian goose poxvirus

ITS: 

internal transcribed spacer

ML: 

Maximum likelihood

NGS: 

Next-generation sequencing

ORF: 

open reading frame

PCR: 

polymerase chain reaction

PEPV: 

Penguinpox virus

QC: 

Quality control

SWPV-1: 

Shearwaterpox virus 1

SWPV-2: 

Shearwaterpox virus 2

TKPV: 

Turkeypox virus

Declarations

Acknowledgments

The authors are extremely grateful to La Trobe University School of Life Science Publication Booster Award for their financial support to SS. Additional funding for this project was generously provided by the Detached Foundation, Trading Consultants Ltd, and L. Bryce. Assistance in the field was provided by the Lord Howe Island community and numerous dedicated volunteers, particularly A. Fidler, P. Lewis, A. Lombal, K. Richards, and V. Wellington. The authors thank Chad Smithson for help with genome assembly.

Funding

Funding for this project was generously provided by the Detached Foundation, Trading Consultants Ltd, and L. Bryce. However, none of these have grant numbers assigned since all are donations from private philanthropists. Additional financial support was provided to SS through the La Trobe University School of Life Science Publication Booster Award. CU and JI were funded by the Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant. The funding bodies had no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.

Availability of data and materials

The complete genome sequences of the Shearwaterpox virus 1 from a Flesh-footed Shearwater (Ardenna carneipes) and Shearwaterpox virus 2 from a Wedge-tailed Shearwater (Ardenna pacificus) have been deposited in the NCBI database under GenBank accession numbers: [SWPV-1, GenBank: KX857216] and [SWPV-2, GenBank: KX857215].

Authors’ contributions

Conceived and designed the experiments: SS, SRR. Performed the experiments: SS, SRR. Analyzed the data: SS, CU, JI, SRR. Contributed reagents/materials/analysis tools: SS, SD, JLL, IH, KH, CU, JI, SRR. SS, JLL, CU, JI, SRR wrote the initial manuscript. All authors read, edited and approved the final manuscript.

Competing interests

The authors declare that they have no competing interests.

Consent for publication

Not applicable.

Ethics approval

Samples were collected with the permission of the Lord Howe Island Board (permit no. LHIB 02/14) under the approval of the University of Tasmania and Charles Sturt University Animal Ethics Committees (permit no. A13836, A0010874, A0011586, and 09/046).

Publisher’s Note

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Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Department of Physiology, Anatomy and Microbiology, School of Life Sciences, La Trobe University
(2)
School of Animal and Veterinary Sciences, Charles Sturt University
(3)
Institute for Marine and Antarctic Studies, University of Tasmania
(4)
Lord Howe Island Museum
(5)
Department of Biochemistry and Microbiology, University of Victoria

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