Open Access

Comparative genomic analysis of the family Iridoviridae: re-annotating and defining the core set of iridovirus genes

  • Heather E Eaton1,
  • Julie Metcalf1,
  • Emily Penny1,
  • Vasily Tcherepanov2,
  • Chris Upton2 and
  • Craig R Brunetti1Email author
Virology Journal20074:11

DOI: 10.1186/1743-422X-4-11

Received: 27 November 2006

Accepted: 19 January 2007

Published: 19 January 2007

Abstract

Background

Members of the family Iridoviridae can cause severe diseases resulting in significant economic and environmental losses. Very little is known about how iridoviruses cause disease in their host. In the present study, we describe the re-analysis of the Iridoviridae family of complex DNA viruses using a variety of comparative genomic tools to yield a greater consensus among the annotated sequences of its members.

Results

A series of genomic sequence comparisons were made among, and between the Ranavirus and Megalocytivirus genera in order to identify novel conserved ORFs. Of these two genera, the Megalocytivirus genomes required the greatest number of altered annotations. Prior to our re-analysis, the Megalocytivirus species orange-spotted grouper iridovirus and rock bream iridovirus shared 99% sequence identity, but only 82 out of 118 potential ORFs were annotated; in contrast, we predict that these species share an identical complement of genes. These annotation changes allowed the redefinition of the group of core genes shared by all iridoviruses. Seven new core genes were identified, bringing the total number to 26.

Conclusion

Our re-analysis of genomes within the Iridoviridae family provides a unifying framework to understand the biology of these viruses. Further re-defining the core set of iridovirus genes will continue to lead us to a better understanding of the phylogenetic relationships between individual iridoviruses as well as giving us a much deeper understanding of iridovirus replication. In addition, this analysis will provide a better framework for characterizing and annotating currently unclassified iridoviruses.

Background

Iridoviruses are large DNA viruses (~120–200 nm in diameter) that replicate in the cytoplasm of infected cells. Iridovirus genomes are circularly permuted and terminally redundant, and range in size from 105 to 212 kbp [1, 2]. The family Iridoviridae is currently subdivided into five genera:Chloriridovirus, Iridovirus, Lymphocystivirus, Megalocytivirus, and Ranavirus [3].

Iridoviruses have been found to infect invertebrates and poikilothermic vertebrates, including amphibians, reptiles, and fish [4]. Iridovirus infections produce symptoms that range from subclinical to very severe, which may also result in significant mortality [59]. The high pathogenicity associated with some members of the iridovirus family has had a significant impact on modern aquaculture, fish farming, and wildlife conservation. For example, systemic iridovirus infections have been found in economically important freshwater and marine fish species worldwide. In addition, iridovirus infections have been implicated in amphibian population declines, representing a set of emerging infectious diseases whose spread has been accelerated by human activities [1014].

Despite the economic and ecological significance of iridoviruses, very little is currently known about their molecular biology. One approach towards gaining a deeper understanding of iridoviral pathogenesis is to investigate the core set of essential genes conserved among all members of the family. The genomes of twelve iridoviruses, including at least one from each genus, have been completely sequenced (Table 1). According to the previously published annotations, these genomes contained only 19 core genes associated with a variety of viral activities: transcriptional regulation, DNA metabolism, protein modification, and viral structure. Definition of this core set of genes also highlights those genes that are conserved across some, but not all, genera, and unique genes found within a single species. These non-core genes may be involved in specific virus-host interactions, enhancement of virus replication, and augmented pathogenesis in certain species.
Table 1

Iridovirus Genomes

Virus

Abbreviation

Genus

Genome Size (bp)

# ORFs*

GenBank accession #

Ref

Frog virus 3

FV3

Ranavirus

105903

97

AY548484

[27]

Tiger frog virus

TFV

Ranavirus

105057

103

AF389451

[1]

Ambystoma tigrinum virus

ATV

Ranavirus

106332

92

AY150217

[30]

Grouper iridovirus

GIV

Ranavirus

139793

139

AY666015

[21]

Singapore grouper iridovirus

SGIV

Ranavirus

140131

139

AY521625

[22]

Lymphocystis disease virus 1

LCDV-1

Lymphocystivirus

102653

108

L63545

[34]

Lymphocystis disease virus China

LCDV-China

Lymphocystivirus

186250

178

AY380826

[24]

Infectious spleen and kidney necrosis virus

ISKNV

Megalocytivirus

111362

117

AF371960

[20]

Rock bream iridovirus

RBIV

Megalocytivirus

112080

116

AY532606

[19]

Orange-spotted grouper iridovirus

OSGIV

Megalocytivirus

112636

116

AY894343

[18]

Invertebrate iridescent virus 6

IIV-6

Iridovirus

212482

211

AF303741

[2]

Invertebrate iridescent virus 3

IIV-3

Chloriridovirus

191100

126

DQ643392

[26]

*The number of ORFs in each viral genome reflects the results of the analysis done in this paper

Despite the growing number of sequenced iridovirus genomes, no systematic comparative genomic analysis of the family has yet been performed. Thus, annotation of these genomes has been performed without standardization and has so far been guided primarily by the position of start/stop codons rather than the presence of homologous sequences. As a result, some long overlapping potential ORFs have been automatically designated as coding sequences, and smaller homologous ORFs overlooked. In this paper, we have taken a comparative genomics approach to re-examine the annotation of all twelve iridovirus genomes, using the Viral Orthologous Clusters (VOCs) [15] and Viral Genome Organizer (VGO) [16] software. These re-annotated genomes were then analysed further, both to define the core set of iridovirus genes more accurately, and to provide a deeper understanding into the phylogenetic relationship between individual iridovirus species.

Results & discussion

Re-annotation of Iridovirus genomes

One objective of this project was to demonstrate the application of comparative genomics to annotating viral genomes, particularly those that have been poorly characterized experimentally. In an earlier study, we utilized comparative genomics to identify previously unannotated small viral ORFs in the Poxviridae [17]. Here, we focused our analysis on the Iridoviridae family, which represents a challenge in genome annotation since there is little experimental evidence available to confirm gene expression. Another problem is that iridovirus promoter elements have not been well characterized, and thus cannot be used as a reliable criterion for assigning ORFs. These combined factors made previous iridovirus gene annotation a somewhat arbitrary process, resulting in closely related iridovirus species with dramatic differences in their genomic annotations. Therefore, we decided to analyse all members of this family using a standardized comparative genomics approach, using the fact that ORFs that are conserved in more than one divergent species are likely to be functional genes.

Analysis was begun with the Megalocytivirus genus, which contains three sequenced genomes: infectious spleen and kidney necrosis virus (ISKNV), rock bream iridovirus (RBIV), and orange-spotted grouper iridovirus (OSGIV). These three viruses display a co-linear arrangement of genes with an overall DNA sequence identity of greater than 90%. In the analysis of this genus, differences in gene content were examined in detail. Dotplots were used to determine presence of orthologous DNA and a variety of BLAST searches and the VGO genome visualization software were used to determine the reason (frameshifts, extra stop codons) behind the apparent absence of some ORFs.

Using this approach, a substantial number of ORFs were either added to, or deleted from members of the Megalocytivirus genus (Table 2). OSGIV and RBIV share 99% DNA sequence identity, and thus are probably different strains of the same virus; however, previous annotation described only 82 out of 118 total annotated ORFs shared by the two genomes [18, 19]. After our re-analysis, the RBIV and OSGIV genomes had an identical complement of annotated genes. Furthermore, this re-annotated ISKNV genome contained 110 ORFs orthologous with both RBIV and OSGIV (compared to 71 in the old annotation.) (Table 2) [18, 20].
Table 2

Re-annotation of the Megalocytivirus genus

ISKNVa

Start

Stopc

aad

RBIVa

Start

Stopc

aad

OSGIVa

Start

Stopc

aad

1L

1270

134

378

1L

1270

134

378

1L

1270

134

378

2R

1394

2044

216

2R b

1394

1597/1781

67

2R

1394

1789

131

-

-

-

-

3R

1841

2056

71

3R

1849

2064

71

3L

2634

2077

185

4L

2605

2102

167

4L

2613

2110

167

4L

2890

2681

69

5L

2800

2624

58

5L

2808

2632

58

5L

3648

2893

251

6L

3541

2876

221

6L

3548

2883

221

6L

5155

3794

453

7L

5147

3786

453

7L

5154

3793

453

7L

6631

5174

485

8L

6621

5164

485

8L

6628

5171

485

8R

6669

8246

525

9R

6692

8239

515

9R

6699

8246

515

9R

8342

8503

53

10R

8335

8496

53

10R

8342

8503

53

10L

9054

8662

130

11L

9047

8655

130

11L/12L b

9055

8849/8662

130

11L

9311

9051

86

11.5L

9304

9044

86

13L

9312

9052

86

12R

9330

9659

110

12R

9323

9655

110

14R

9331

9663

110

13R

9669

11054

461

13R

9662

11059

465

15R

9670

11067

465

14R

11309

12268

319

14R

11314

12288

324

16R

11322

12296

324

15R

12278

13069

263

15R

12298

13089

263

17R

12302

13093

263

16L

13716

13129

195

16L

13733

13146

195

18L

13738

13151

195

17L

14095

13718

125

17L

14086

13748

112

19L

14088

13753

111

17.5R

14089

14325

78

18R

14171

14410

79

20R

14094

14351

85

18.5L b

14563

14233

109

19L

14648

14472

58

21L

14607

14431

58

19R

14579

17425

948

20R

14664

17510

948

22R

14623

17469

948

20L

17642

17454

62

21L

17756

17574

60

23L

17715

17533

60

21L

17900

17778

40

21.5L

18014

17892

40

24L

17973

17851

40

22L

19489

17990

499

22L

19714

18104

536

25L

19715

18063

550

23R

19562

22132

856

23R

19787

22204

805

26R

19788

22922

1044

24R

22300

23238

312

26R

23035

23973

312

27R

23207

24145

312

25R

23354

23779

141

27R

23997

24380

127

28R

24169

24696

175

26L

24145

23822

107

27.5L

24697

24377

106

29L

25013

24693

106

27L

25063

24167

298

28L

25615

24719

298

30L

25931

25035

298

28L

28559

25080

1159

29L

29138

25632

1168

31L

29454

25948

1168

29L

28814

28593

73

29.5L b

29362

29087/29145

91

32L

29682

29461

73

31.5L

29414

28884

176

30.5L

29957

29430

175

33.5L

30277

29750

175

32R

29447

30061

204

31R

29990

30622

210

34R

30310

30942

210

33L

31079

30138

313

32L

31654

30713

313

35L

31935

31033

300

34R

31144

34278

1044

33R

31700

34861

1053

36R

32018

35176

1052

35L

35508

34360

382

34L

36067

34934

377

37L

36382

35249

377

36R

35546

36601

351

35.5R

36061

37113

350

38R

36376

37431

351

37L

37950

36598

450

37L

38219

37110

369

39L

38777

37428

449

38L

39395

37959

478

38L

39974

38469

501

40L

40225

38786

479

39R

39439

40311

290

39.5R b

40012

40506/40857

164

41R

40290

41168

292

40L

41443

40304

379

41L

41995

40850

381

42L

42306

41161

381

41L

42788

41445

447

42L

43346

41997

449

43L

43657

42308

449

42R

42803

43396

197

43R

43361

43959

198

44R

43672

44271

199

43L

43842

43480

120

43.5L

44405

43975

142

45L

44717

44355

120

44L

44645

43845

266

44L

45208

44408

266

46L

45524

44724

266

45L

45564

44650

304

45L

46127

45213

304

47L

46443

45529

304

46L

46241

45558

227

46L

46804

46121

227

48L

47120

46437

227

47R

46401

46664

87

47R

47150

46887

87

49R

47280

47543

87

48R

46661

47005

114

47.5R b

47224

47433/47588

69

50R

47540

47893

117

49R

47021

47191

56

48.5R

47604

47774

56

51R

47909

48079

56

50L

47678

47250

142

49L

48270

47842

142

52L

48575

48147

142

51R

47733

47864

43

-

-

-

-

-

-

-

-

52L

48403

47951

150

50L

48999

48547

150

53L

49306

48854

150

53R

48405

48620

71

51R

49001

49195

64

54R

49308

49502

64

54L

49559

48633

308

52L

50173

49229

314

55L

50480

49536

314

55L

50508

49582

308

53L

51137

50196

313

56L

51444

50503

313

56L

51166

50519

215

54L

51795

51148

215

57L

52102

51455

215

57L

51433

51173

86

55L

52062

51802

86

58L

52369

52109

86

59L

52414

51749

221

56L

52839

52327

170

59L

53146

52634

170

61L

53162

52359

267

57L

53709

52903

268

60L

54016

53210

268

62L

56785

53159

1208

58L

57467

53706

1253

61L/62L b

55131

54013/53893

372

63L

59875

57227

882

59L

60567

57919

882

63L

60876

58228

882

64L

61393

59918

491

60L b

62102

60855/60635

415

64L

62416

60944

490

65L

61900

61439

153

61L

62611

62144

155

65L

62928

62458

156

66L

63025

61982

347

62L

63744

62662

360

66L

64061

62979

360

67L

63855

63271

194

63.5L

64446

63865

193

67.5L

64763

64182

193

68L

65329

63896

477

64L

65917

64484

477

69L

66234

64801

477

69L

66001

65336

221

65L b

66661

66215/65929

148

70L

66977

66246

243

70L

66331

66101

76

-

-

-

-

-

-

-

-

71L

68042

66432

536

68L

68529

67120

469

71L

68973

67417

518

72R

68173

69177

334

-

-

-

-

-

-

-

-

73R

69203

69622

139

69R b

68717

69191/69135

157

72R

69078

69497

139

74R

69669

70682

337

70R

69184

70203

339

73R

69546

70568

340

75L

71043

70777

88

71L

70573

70304

89

74L

70938

70669

89

76L

74017

71045

990

72L

73541

70575

988

75L

73912

70940

990

77R

74035

75369

444

73R

73559

74893

444

76R

73930

75264

444

78R

75366

75830

154

75R

74890

75354

154

77R

75261

75725

154

79L

76053

75832

73

76L

75580

75356

74

78L

75951

75727

74

-

-

-

-

77R

75664

76137

157

79R

76039

76512

157

80L

76368

76165

67

-

-

-

-

-

-

-

-

81R

76367

76864

165

78R

76150

76647

165

80R

76525

77022

165

82L

78007

76901

368

79L

77802

76696

368

81L

78177

77071

368

83R

78152

78418

88

80R

77827

78225

132

82R

78202

78600

132

84L

79881

78526

451

81L

79556

78252

434

83L

79931

78627

434

85R

79884

80486

200

82R

79643

80173

176

84R

80018

80548

176

86R

80483

80947

154

82.5R

80170

80637

155

84.5L

80545

81012

155

87R

80940

81710

256

83R

80603

81400

265

85R

80978

81775

265

88R

81717

83720

667

84R

81503

83425

640

86R/87R b

82234

83805/82279

523

90.5L

84663

83701

320

85L

84457

83630

275

88.5L

84811

83786

341

93L

85786

84860

308

86L

85504

84578

308

90L

85918

84992

308

94L

86296

85796

166

87L

86014

85514

166

91L

86428

85928

166

95L

87481

86321

386

88L

87202

86039

387

92L

87616

86453

387

96L

88298

87489

269

89.5L b

87601

87218/87210

127

93L

88361

87624

245

97.5L

88723

88232

163

90L

88443

87952

163

94L

88857

88366

163

99L

89097

88774

107

91L

88814

88491

107

95L

89229

88906

107

100L

89689

89144

181

92L

89515

88868

215

96L

90024

89377

215

101L

90251

89736

171

93.5L

89999

89470

175

97L

90508

89993

171

102R

90311

91753

480

94R

90068

91513

481

98R

90577

92022

481

103R

91760

92161

133

95.5R

91477

91935

152

99R

92029

92442

137

104R

92215

92991

258

96R

91994

92773

259

100R

92501

93280

259

105R

92993

93358

121

97R

92775

93146

123

101R

93282

93653

123

-

-

-

-

98R

93240

94127

295

102R

93747

94634

295

106L

94501

93482

339

99L

95042

94221

273

103L

95548

94676

290

108.5L

95093

94494

199

100.5L

95699

95068

209

104.5L

96217

95574

213

109L

97950

95185

921

101L

98557

95792

921

106L

99060

96298

920

110R

97997

98152

51

101.5L

98609

98764

51

107R

99113

99268

51

111L

99039

98149

296

102L

99657

98761

298

108L b

99885

99265/99849

206

112R

99059

99802

247

103R b

99677

100426/100422

249

109R

100183

100926

247

113R

99937

100290

117

104.5R

100493

100909

138

110R

100998

101414

138

114L

103159

100334

941

106L b

103615

102539/100953

358

111L

104041

101594

815

115R

103203

104213

336

108.5R

104050

104781

243

112R

104476

105486

336

116R

104219

105667

482

110R

105060

106493

477

113R

105547

106701

384

117L

106395

105721

224

111L

107221

106547

224

114L

107652

106978

224

118L

108093

106723

456

112L

108913

107549

454

115L

109299

107986

437

119R

108105

108392

95

113R

108931

109104

57

116R

109369

109656

95

120R

108424

108933

169

114R

109248

109637

129

117R

109687

110193

168

121L

109584

108934

216

115L

110419

109756

220

118L

110849

110214

211

122R

109594

110313

239

116R

110429

111148

239

119R

110859

111578

239

123R

110391

110576

61

117R

111226

111420

64

120R

111656

111850

64

124L

111351

110665

228

118L

112037

111576

153

121L

112625

111939

228

aORFs that have been added or altered are highlighted in bold. If a previously annotated ORF is not listed in the table, it has been deleted.

bPotentially frameshifted ORF

cWhere an ORF has a potential sequencing error resulting in a frameshift mutation, 2 stop codons are provided in the format X/Y. The first number represents the actual physical stop in the reported sequence. The second number is the proposed stop if a sequencing error occurred.

dLength of ORF in amino acids

In the process of re-examining these genomes, we annotated a number of genes containing apparent frameshift mutations between species. In RBIV we annotated ten genes with potential frameshift mutations, while OSGIV had four such genes (Table 2). All of the genes containing potential frameshift mutations had orthologs in the other two members of the Megalocytivirus genus (Table 2). In some cases, these mutations may be the result of natural mutations within the viruses; however, it is also possible that these apparent frameshift mutations are actually sequencing errors. For both RBIV and OSGIV, PCR primers based on the ISKNV sequence were used to amplify genomic fragments, which were subsequently sequenced [18, 19]. It is possible that errors were introduced during the PCR process, leading to apparent frameshifts in the reported sequence. It is interesting to note that the genomic sequence of ISKNV (sequenced using subcloned fragments rather than PCR products) [20], had significantly fewer annotation changes made during our re-analysis. Though we have not experimentally proven that the frameshift mutations in OSGIV and RBIV are the result of sequencing errors, it would be useful to focus future sequencing efforts on these regions, to determine if the reported sequences are indeed correct.

After re-annotating the Megalocytivirus genus, we applied the same comparative genomic analysis to the Ranavirus genus. The genus contains five sequenced members divided into two groups, each with a high degree of sequence conservation and a co-linear arrangement of genes. The first group is comprised of frog virus 3 (FV3), tiger frog virus (TFV), and Ambystoma tigrinum virus (ATV). The second group contains Singapore grouper iridovirus (SGIV) and grouper iridovirus (GIV).

The first step in the re-annotation of the Ranavirus genus was a comparative genomic analysis of FV3, TFV, and ATV. This resulted in an increase in the number of conserved annotated genes from 76 to 87 (Table 3). Subsequent re-analysis of the second Ranavirus group, containing SGIV and GIV, resulted in an increase from 131 to 138 conserved annotated ORFs (Table 4). It should be noted that two of the newly annotated ORFs, SGIV 0.5L and GIV 120.5L, appear to "wrap around", beginning at one end of the genome with the remainder of the ORF located at the opposite end [21, 22]. These apparent "split ORFs" are actually the result of the circularly permutated iridovirus genome being represented as a linear genomic sequence, when the arbitrarily chosen start point happens to fall in the middle of an ORF [23].
Table 3

Re-annotation of FV3, TFV, and ATV of the Ranavirus genus

FV3a

Start

Stopc

aad

TFVa

Start

Stopc

aad

ATVa

Start

Stopc

aad

1R

272

1042

256

105R

103809

104576

256

91R

104836

105606

256

2L

2611

1649

320

2L b

1028

315/11

237

1L

981

70

303

2.5L

3488

2649

279

2.5L

1943

1065

292

2L

1858

1019

279

3R

3418

4734

438

4R

1937

3151

404

3R

1892

3106

404

4R

4775

4957

60

5R

3190

3372

60

4R

3149

3331

60

5R

5390

6004

204

6R

3816

4418

200

-

-

-

-

6R

6007

6234

75

-

-

-

-

-

-

-

-

-

-

-

-

6.5R

4411

4578

55

-

-

-

-

7.5L

7025

7411

128

7L

5452

5024

142

5L

4416

3994

140

8R

7503

11384

1293

8R

5531

9415

1294

6R

4495

8379

1294

9L

14599

11753

948

9L

12599

9753

948

7L

11725

8879

948

10R

14615

15028

137

10R

12615

13028

137

8R

11741

12154

137

11R

15378

15590

70

11R

13380

13592

79

88L

102924

102712

70

12L

16549

15656

297

12L

14551

13658

297

87R

101753

102646

297

-

-

-

-

13L

14947

14747

66

86R

101169

101363

64

13R

17090

17296

68

14.5R b

15041

15184/15247

47

85.5L

101128

100871

85

14R

17311

17670

119

15R

15261

15620

119

84L

100856

100482

124

15R

17766

18734

322

16R

15716

16663

315

83R

100400

99474

308

16R

19014

19841

275

17R

16838

17665

275

82.5L

98809

98438

123

17L

21590

20082

502

18L

19414

17906

502

81R

96410

97918

502

18L

21864

21628

78

18.5L

19687

19451

78

80.5R

96137

96373

78

19R

21916

24471

851

19R

19686

22271

861

80L

96083

94086

665

20R

24519

24965

148

20R

22319

22774

151

79L

94038

93589

149

21L

25861

25202

219

21L

23657

23998

219

78R

92383

93042

219

22R

25991

28912

973

22R

23789

26716

975

77L

92253

89326

975

23R

29290

30438

382

23R

27093

28241

382

53R

58082

59230

382

24R

30821

31918

365

24R

28636

29733

365

54R

59613

60710

365

25R

32112

32900

262

25R

29930

30709

259

55R

62328

63335

335

-

-

-

-

26R

30778

30936

52

56R

63402

63500

32

26R b

32967

33197

76

27R

31033

31812

259

57R

63659

64438

259

-

-

-

-

28L

32190

32002

62

-

-

-

-

27R

33728

36640

970

29R

32345

35257

970

58R

64968

67880

970

28R

36689

37177

162

30R

35306

35794

162

59R

67929

68417

162

29L

37652

37356

98

31L

36122

35823

99

-

-

-

-

30R

37854

38006

50

-

-

-

-

-

-

-

-

31R

38068

38487

139

32R

36565

36984

139

60R

68786

69205

139

32R

38537

40426

629

33R

37098

39047

649

61R

69255

71471

738

33R

40509

40700

63

34R

39133

39324

63

62R

71555

71746

63

34R

40844

41164

106

35.5R

39467

39787

106

62.5R

71894

72235

113

35L

41717

41256

153

37L

40308

39772

178

63L

72576

72220

118

36L

42353

41256

365

38.5L b

40938

40543/40367

131

-

-

-

-

-

-

-

-

39R

41112

41246

44

-

-

-

-

37R

42749

43378

209

40R

41296

41952

218

64R

74110

74739

209

38R

43519

45216

565

41R

42091

43788

565

65R

74878

76575

565

39R

45322

45672

116

42R

43899

44249

116

66R

76682

76948

88

40R

45761

46309

182

43R

44335

44883

182

67R

77048

77671

207

-

-

-

-

44R

44973

45239

88

68R

77899

78039

46

41R

46691

50188

1165

45R

45270

48767

1165

69R

78111

81608

1165

43.5L b

50940

51455/5684

171

46L

50362

49133

409

70L

82913

82152

253

45L

52348

51938

136

47L

50899

50489

136

71L

83450

83040

136

46L

52968

52723

81

48L

51411

50953

152

72L

84331

83504

275

47L

53509

53093

138

49L

51953

51537

138

73L

84874

84458

138

48L

53763

53512

83

50L

52207

51956

83

74L

85187

84804

127

49L

54621

53872

249

51L

52899

52315

194

75L

86776

85235

513

50L

55459

54770

229

52L

53876

53136

246

-

-

-

-

51R

55539

57224

561

53R

53956

55641

561

76R

86858

88543

561

52L

58548

57481

355

54L

56965

55898

355

52R

57441

57602

53

53R

58886

60454

522

55R

57301

58869

522

51L

57102

55522

526

54L

60899

60669

76

-

-

-

-

-

-

-

-

55L

62232

60937

431

56L

60615

59320

431

50R

53770

55065

431

-

-

-

-

57L

60772

60623

49

49R

53613

53762

49

56R

62320

62757

145

58R

60809

61213

134

48L

53576

53172

134

57R

62871

64367

498

59R

61254

62750

498

47L

53130

51634

498

-

-

-

-

60L

62888

62757

43

-

-

-

-

58.5R

64819

65373

184

61R

63264

63818

184

46L

50770

50216

184

59L

67014

65956

352

62L

65445

64387

352

45R

48676

49734

352

60R

67176

70217

1013

63R

65605

68646

1013

44L

48512

45471

1013

61L

70408

70226

60

-

-

-

-

-

-

-

-

-

-

-

-

64R

69029

69151

40

-

-

-

-

62L

74516

70851

1221

65L

72940

69281

1219

43R

41447

45112

1221

62.5R

74515

74778

87

66.5R

72927

73202

91

42.5L

41460

41185

91

63R

74895

75389

164

68R

73319

73813

164

42L

41068

40631

145

64R

75529

75816

95

69.5R

73946

74209

87

40L

40492

40205

95

65L

76373

76209

54

-

-

-

-

-

-

-

-

66L

76921

76370

183

70.5L

75301

74685

204

38.5R

39094

36681

195

67L

78139

76976

387

71L

76525

75362

387

38R

37876

39039

387

68R

78422

78709

95

72R

76785

76982

65

37bL

37592

37416

58

-

-

-

-

73L

77175

77020

51

36R

36736

36891

51

69R

78845

79111

88

74R

77244

77507

88

35L

36677

36411

88

70R

79129

79503

124

75R

77507

77902

131

34.5L

36392

36018

124

71R

79543

79776

77

76R

77942

78175

77

34L

35978

35742

78

72L

80549

79833

238

77L

78948

78232

238

32R

34970

35293

107

73L

81971

80997

324

78L

80299

79325

324

31R

33319

34311

330

74L

83258

82146

370

79L

81498

80506

330

30R

31947

33128

393

75L

83544

83290

84

80L

81809

81555

84

29R

31637

31891

84

76R

83607

83828

73

81R

81872

82093

73

28L

31574

31353

73

77L

84172

83825

115

82L

82437

82090

115

27R

31009

31356

115

78L

85395

84757

212

83L

83568

82894

224

-

-

-

-

79R

85531

87249

572

84R

83668

85386

572

26L

30729

28999

576

80L

88987

87872

371

85L

86988

85873

371

25R

27224

28345

373

81R

89043

89321

92

86R

87046

87324

92

24L

27168

26890

92

82R

89450

89923

157

87R

87454

87927

157

23L

26762

26289

157

-

-

-

-

88R

88138

88512

124

22L

25564

25277

95

83R

90373

91017

214

89R

88857

89501

214

21L

24912

24268

214

84R

91389

92126

245

90R

89903

90640

245

20L

23923

23141

260

85R

92201

92788

195

91.5R

90715

91302

195

19L

23066

22479

195

86L

93363

93178

61

92L

91943

91650

97

18R

21742

22119

125

87L

95533

93716

605

93L

94096

92279

605

17R

19571

21397

608

88R

95566

96018

150

94R

94129

94581

150

16L

19538

19086

150

89R

96086

97252

388

95R

94649

95845

398

15L

19018

17744

424

90R

97345

98736

463

96R

95938

97329

463

14L

17651

16260

463

91R

98860

100047

395

97R

97453

98640

395

13L

16136

14949

395

92R

100398

100637

79

98R

98927

99232

101

-

-

-

-

93L

100986

100819

55

99L

99593

99426

55

12R

14091

14246

51

94L

101563

101096

155

100R

100169

99702

155

11L

13512

13979

155

95R

101656

102747

363

101R

100180

101352

390

10L

13419

12325

364

96R

103549

104220

223

103R

102169

102840

223

89R

103279

103965

228

97R

104303

104716

137

104R

102923

103372

149

90R

104031

104444

137

aORFs that have been added or altered are highlighted in bold. If a previously annotated ORF is not listed in the table, it has been deleted.

bPotentially frameshifted ORF

cWhere an ORF has a potential sequencing error resulting in a frameshift mutation, 2 stop codons are provided in the format X/Y. The first number represents the actual physical stop in the reported sequence. The second number is the proposed stop if a sequencing error occurred.

dLength of ORF in amino acids

Table 4

Re-annotation of SGIV and GIV of the Ranavirus genus

SGIVa

Start

Stopc

aad

GIVa

Start

Stopc

aad

14L

12773

12348

141

1.3L

2020

1595

141

15L

13000

12821

59

1.5L

2247

2068

59

16L

14289

13048

413

2L

3536

2295

413

18R

14317

15174

285

3R

3564

4421

285

19R

15196

16224

342

4R

4443

5399

318

20L

17246

16278

322

5L

6421

5453

322

21L

17725

17306

139

6L

6900

6475

141

22L

18277

17777

166

7L

7486

6950

178

24L

18774

18319

151

8L

7983

7528

151

25L

20488

18956

510

9L

9682

8165

505

26R

20567

22267

556

10R

9761

11461

566

28L

23363

22350

337

11L

12559

11546

337

29L

24445

23447

332

12L

13659

12643

338

30L

25635

24610

341

13L

14850

13816

344

31L

27160

26144

338

14L

16384

15362

340

32.5L

28666

27609

352

15L

17904

16846

352

33L

29760

28726

344

16L

19010

17964

348

34L

30161

29823

112

16.5L

19411

19073

112

35L

31388

30261

375

17L

20638

19511

375

36L

32515

31526

329

18L

21835

20771

354

37L

33696

32668

342

19L

23016

21988

342

38L

34236

33724

170

20L

23556

23044

170

39L

37417

34262

1051

21L

26738

23583

1051

41L

37978

37547

143

22L

27296

26865

143

42R

38058

38285

75

-

-

-

-

43R

38285

40288

667

23R

27608

29605

665

45L

41090

40362

242

24L

30407

29679

242

46L

41866

41120

248

25L

31204

30467

245

47L

43063

41909

384

26L

32401

31247

384

48L

43489

43214

91

27L

32824

32549

91

49L

44002

43535

155

28L

33336

32857

159

50L

44695

44033

220

29L

34029

33367

220

51L

45563

44868

231

30L

34896

34201

231

52L

37997

35097

968

31L

37997

35097

966

54R

48777

49424

215

32R

38100

38747

215

55R

49447

50169

240

33R

38770

39492

240

56R

50198

50938

246

34R

39521

40261

246

57L

54510

51004

1168

35L

43833

40327

1168

59L

55000

54560

146

35.5L

44323

43391

146

60R

54967

57879

970

36R

44348

47200

950

61R

57914

58528

204

37R

47235

47849

204

62R

58593

59363

256

38R

47914

48708

264

64R

59415

61133

572

39R

48760

50478

572

65R

61268

61510

80

39.5R

50614

50856

80

66R

61603

61845

80

39.7R

50949

51191

80

67L

62482

61907

191

40L

51829

51254

191

68L

63334

62516

272

41L

52681

51863

272

69L

64967

63321

548

42L

54314

52668

548

70R

64994

65452

152

43R

54341

54799

152

71R

65483

66307

274

44R

54830

55654

274

72R

66404

67795

463

45R

55751

57142

463

73L

71185

67874

1103

46L

60532

57221

1103

74R

68472

68738

88

47R

57819

58085

88

75R

71239

71775

178

48R

60586

61122

178

76L

72715

71858

285

49L

62064

61207

285

77L

73747

72839

302

50L

63096

62188

302

-

-

-

-

51L

62944

62282

220

78L+81L a

76809

76246/73855

984

52L

66156

63202

984

82L

77592

76924

222

53L

66939

66271

222

83R

77672

79009

445

54R

67019

68356

445

84L

80193

79066

375

55L

69540

68413

375

85R

80251

80529

92

56R

69598

69876

92

86R

80591

81055

154

57R

69938

70402

154

87R

81385

82032

215

58R

70728

71375

215

88L

84187

82667

506

59L

73355

71835

506

89L

85420

84248

390

60L

74588

73416

390

90L

86627

85506

373

61L

75794

74673

373

91L

87886

86750

378

62L

77051

75915

378

92L

89216

88086

376

63L

78373

77240

377

93L

90497

89280

405

64L

79654

78437

405

95R

90635

91111

158

64.5L

80265

79792

157

96R

91148

91618

156

65R

80301

80771

156

97L

92774

91626

382

66L

81926

80778

382

98R

92428

93231

267

67R

81580

82383

267

99R

93244

93492

82

67.5R

82380

82646

88

101R

93753

94694

313

68R

82906

83847

313

102L

95007

94774

77

69L

84161

83928

77

103R

95092

95385

97

70R

84246

84539

97

104L

99252

95446

1268

71L

88406

84600

1268

105R

95498

95731

77

72R

84652

84885

77

107R

99308

100453

381

73R

88462

89088

208

111R

100766

101533

255

74R

89401

90168

255

112R

101588

102655

355

75R

90223

91326

367

114L

103050

102712

112

77.5L

91721

91383

112

115R

103122

103580

152

78R

91793

92251

152

116R

103700

104476

258

79R

92371

93147

258

117L

104733

104575

52

79.5L

93401

93241

52

118R

104795

105754

319

80R

93463

94422

319

119R

105799

106050

83

80.5R

94467

94718

83

120L

106525

106103

140

81L

95162

94779

127

121R

106615

106869

84

81.5R

95291

95547

84

122L

107599

106967

210

82L

96275

95643

210

123L

108740

107652

362

83L

97416

96328

362

124R

108863

109399

178

83.5L

98976

97684

130

125R

109474

110028

184

84R

98151

98705

184

126R

110101

110658

185

85R

98692

99330

212

127R

110731

111252

173

86R

99403

99924

173

128R

112041

115070

1009

87R

100788

103817

1009

129L

115490

115308

60

87.5L

104245

103884

119

131R

115749

116303

184

88R

104499

105053

184

132R

116321

117148

275

89R

105071

105898

275

134L

118498

117527

323

90L

107244

106273

323

135L

118885

118547

112

90.5L

107631

107258

123

136R

118946

119260

104

91R

107692

108006

104

137R

119282

120667

461

92R

108028

109413

461

138L

120907

120713

64

92.3L

109653

109457

64

139R

121013

121324

103

92.5R

109757

110068

103

140R+141R b

121397

122311/124558

 

93R

110141

113554

1137

143L

124882

124643

79

94L

113878

113639

79

144R

124963

125421

152

95R

113959

114417

152

145R

125480

125977

165

96R

114476

114973

165

146L

127052

126078

324

97L

116050

115076

324

147L

128221

127187

344

98L

117220

116186

344

148R

128324

128803

159

99R

117323

117802

159

149R

128843

129220

125

99.5R

117842

118219

125

150L

130827

129301

508

100L

119826

118300

508

151L

131435

130848

195

101L

120434

119847

195

152R

131534

132772

412

102R

120533

121771

412

153L

132661

132089

190

103L

121660

121088

190

154R

132788

133081

97

103.5R

121787

122080

97

155R

133172

134899

575

104R

122172

123896

574

156L

135860

135048

270

105L

124852

124043

269

157R

135948

136472

174

106R

124940

125464

174

158L

136944

136528

138

106.5L

125936

125520

138

159R

137020

137511

163

106.7R

126012

126503

163

160L

137996

137508

162

107L

126988

126500

162

161.5L

138598

138309

95

107.5L

127561

127296

87

162L

139822

138674

382

108L

128797

127649

382

0.5L

1029

0/140141-140020

391

109L

130138

128963

391

1L

1971

1057

304

110L

131080

130166

304

3R

2018

3163

381

111R

131127

132272

381

4L

4332

3235

365

113L

133442

132345

365

5L

5542

4400

380

114L

134652

133510

380

6R

5570

6349

259

115R

134680

135453

257

7L

7339

6416

307

116L

136425

135520

301

8L

7886

7194

230

117L

136972

136280

230

9L

8444

7980

154

118L

137530

137066

154

10L

8888

8517

123

118.5L

137974

137603

123

11L

9132

8944

62

119L

138218

138030

62

12L

12293

9219

1024

120.5L

138307/139793

1/1540

1008

aORFs that have been added or altered are highlighted in bold. If a previously annotated ORF is not listed in the table, it has been deleted.

bPotentially frameshifted ORF

cWhere an ORF has a potential sequencing error resulting in a frameshift mutation, 2 stop codons are provided in the format X/Y. The first number represents the actual physical stop in the reported sequence. The second number is the proposed stop if a sequencing error occurred.

dLength of ORF in amino acids

As seen above, our comparative genomic approach was able to identify previously unannotated ORFs, homologous ORFs with potential frameshifts, and ORFs split between the two ends of a circular genome. Although this approach proved extremely successful for the Ranavirus and Megalocytivirus genera, we were unable to use it for the Chloriridovirus, Iridovirus, and Lymphocystivirus genera. This is due to the lack of co-linearity and the highly divergent sets of genes that exist between the members of these genera, as well as the low number of available genome sequences. However, we did modify the annotations of lymphocystis disease virus-China (LCDV-China) and invertebrate iridescent virus-6 (IIV-6). The previous annotations of these genomes of both species had contained a large number of overlapping ORFs [2, 24], which we decided to exclude on several grounds. First, LCDV-China and IIV-6 are the only iridoviruses, out of the twelve so far sequenced, in which overlapping ORFs have been annotated. In addition, the original sequencing paper for IIV-6 [2] and a follow-up paper by the same group [25] did not include a number of the overlapping ORFs reported in the database sequence, presumably due to their small size and lack of similarity with other viral and cellular genes. Finally, there is no experimental or bioinformatics evidence to suggest that any of these ORFs encode proteins. Therefore, to improve the overall consistency of the Iridoviridae family annotations, we removed the small overlapping ORF annotations from the LCDV-China and IIV-6 genomic sequences (Table 5, Additional File 1 &2).
Table 5

Overlapping ORFs deleted from the Iridovirus and Lymphocystivirus genera

Virus

Deleted

LCDV-C

4L, 8R, 17R, 20L, 21L, 26L, 28L, 30L, 31L, 32L, 35R, 36R, 44R, 46L, 48L, 52R, 55L, 68L, 74R, 76R, 78R, 79R, 81R, 88R, 92R, 94R, 98R, 102R, 103R, 113L, 120R, 130L, 132L, 134L, 138R, 141R, 144L, 152L, 155L, 156L, 163L, 167R, 174L, 183L, 188L, 192L, 193L, 194L, 195L, 198R, 199L, 200R, 204R, 207L, 210L, 213L, 223R, 225R, 232R, 233L, 236L, 238R, 240L

IIV-6

1R, 2R, 3R, 4R, 5R, 7R, 8R, 11L, 13R, 14R, 15R, 16L, 17R, 18R, 20L, 21R, 23L, 24L, 25R, 26R, 27L, 28L, 31R, 33L, 35L, 36R, 38R, 39R, 40R, 46R, 47R, 48R, 51R 52R, 53R, 54R, 55R, 57L, 58L, 59R, 63R, 64L, 66L, 68L, 70R, 72R, 73R, 74R, 76L, 78R, 79L, 80L, 81L, 86R, 87R, 88L, 89L, 90R, 91R, 92R, 93R, 97L, 99L, 102R, 103R, 105R, 108R, 109R, 112R, 114L, 119R, 124L, 125L, 128L, 129R, 131L, 133R, 134L, 144R, 147L, 150R, 151R, 152R, 153L, 154R, 158R, 163L, 164R, 166L, 167L, 168R, 171R, 173R, 174R, 177L, 178L, 180L, 181L, 182L, 183L, 185L, 186L, 187R, 188L, 189L, 190R, 191L, 194R, 199L, 202L, 204L, 207L, 108L, 210L, 214L, 215R, 217L, 220L, 222R, 223L, 230L, 231R, 233L, 237R, 239R, 243R, 245R, 248R, 252L, 256R, 257R, 258R, 260R, 262R, 263L, 264R, 265L, 266L, 267R, 269R, 270R, 271R, 275R, 276L, 277R, 278L, 279R, 280R, 281R, 282R, 283L, 286L, 288R, 290R, 291R, 292L, 294R, 296R, 297L, 298R, 299R, 303R, 304R, 305L, 310R, 311R, 314L, 316R, 318R, 319L, 320L, 321R, 323L, 324L, 326L, 327R, 328L, 330L, 331R, 333L, 334R, 336R, 338L, 339L, 341R, 344R, 345R, 351R, 352R, 353L, 354L, 355R, 356L, 360R, 362R, 363L, 364L, 365L, 367L, 370R, 371R, 372R, 377R, 379L, 381L, 382R, 383L, 386R, 387R, 390R, 392R, 394R, 397L, 398R, 399R, 402L, 403L, 405L, 406R, 407R, 408R, 409R, 410L, 412L, 416R, 417L, 418R, 419L, 421L, 424R, 425R, 427R, 429R, 430R, 431L, 432R, 433R, 434L, 435R, 440R, 442L, 444R, 445L, 446L, 447L, 448L, 449L, 450L, 452R, 455L, 456R, 459L, 461R, 462R, 464R, 465R

Defining the conserved genes in Iridoviruses

As a result of this re-annotation of the Iridoviridae family, species within each genus now have a much greater consensus among their annotated ORFs. Prior to re-annotation, only 19 ORFs appeared to be conserved across all iridovirus species (Table 6). Although a previous report has suggested that 27 core genes exist within the Iridoviridae family [26], those core genes reported are found in most, but not all published iridoviridal species. In light of our previous results, we re-examined this core set of genes using the VOCs software. We identified seven novel core genes (Table 7), increasing the total number to 26 (Table 6 &7). This increase in the number of core genes was primarily due to the five new genes annotated during the re-analysis of RBIV (Table 7 bold highlighted genes). As expected most of the core genes are predicted to have essential functions, required for transcription, replication, and virus formation. Interestingly, three core genes, the orthologs of FV3 12L, 41R, and 94L, have no predicted functions. As previously stated Delhon et al. [26] identified 27 core genes, one more than we identified after our re-analysis. Delhon et al. [26] report the orthologs of FV3 20L represent a core [26]. However, our analysis shows that orthologs of FV3 20L exist in all genera except the Megalocytivirus (Figure 1) suggesting that FV3 20L is not a core gene. Future research to determine the functions of these genes, which are also likely to be essential, will provide important data for understanding the replication cycle of iridoviruses.
Table 6

Iridoviridae Core Genes

 

Gene Namea

FV3

TFV

ATV

SGIV

GIV

LCDV-1

LCDV-C

ISKNV

RBIV

OSGIV

IIV-6

MIV

1.

Putative replication factor and/or DNA binding-packing

1R

105R

91R

116R

79R

162L

181R

61L

57L

60L

282R

79L

2.

DNA-dep RNA pol-II Largest subunit

8R

8R

6R

104L

71L

16L

191R

28L

29L

31L

176R, 343L

90L

3.

Putative NTPase I

9L

9L

7L

60R

36R

132L

075L

63L

59L

63L

22L

87L

4.

ATPase-like protein

15R

16R

83R

134L

90L

54R

114L

122R

116R

119R

75L

88R

5.

Helicase family

21L

21L

78R

54R

32R

6L

7L

56L

54L

57L

67R

4R

6.

D5 family NTPase involved in DNA replication

22R

22R

77L

52L

31L

128L

80L

109L

101L

106L

184R

121R

7.

Putative tyrosin kinase/lipopolysaccharide modifying enzyme

27R

29R

58R

78L+81L b

52L

195R

173R

61L, 114L

57L, 106Lb

60L, 111L

179R, 439L

35R

8.

NIF-NLI interacting factor

37R

40R

64R

61R

37R

82L

148L

5L

6L

6L

355R

104R

9.

Unknown

41R

45R

69R

57L

35L

163R

235R

76L

72L

75L

295L

16R

10.

Myristilated membrane protein

53R

55R

51L

88L

59L

67L

158R

7L

8L

8L

118L, 458R

6R

11.

DNA pol Family B exonuclease

60R

63R

44L

128R

87R

135R

203L

19R

20R

22R

37L

120L

12.

DNA-dep RNA pol-II second largest subunit

62L

65L

43R

73L

46L

25L

25R

34R

33R

36R

428L

9R

13.

Ribonucleotide reductase small subunit

67L

71L

38R

47L

26L

27R

41L

24R

26R

27R

376L

48L

14.

Ribonuclease III

80L

85L

25R

84L

55L

137R

187R

87R

83R

85R

142R

101R

15.

Proliferating cell nuclear antigen

84R

90R

20L

68L

41L

3L

197L

112R

103R b

109R

436L

60L

16.

Major capsid protein

90R

96R

14L

72R

45R

147L

43L

6L

7L

7L

274R

14L

17.

Putative XPPG-RAD2-type nuclease

95R

101R

10L

97L

66L

191R

169R

27L

28L

30L

369L

76L

18.

Serine-threonine protein kinase

19R

19R

80L

39L

21L

10L

45R

55L

53L

56L

380R

10L

19.

Serine-threonine protein kinase

57R

59R

47L

150L

100L

143L

178L

13R

13R

15R

98R

98L

The Iridoviridae core genes are shown.

aORFs that have been added or altered are highlighted in bold

bPotentially frameshifted ORF

Table 7

Additional Iridoviridae Core Genes Identified After Genome Re-analysis

 

Newly Characterized Gene Namea

FV3

TFV

ATV

SGIV

GIV

LCDV-1

LCDV-C

ISKNV

RBIV

OSGIV

IIV-6

MIV

1.

Myristilated membrane protein

2L

2L b

1L

19R

4R

160L

38R

90.5L

85L

88.5L

337L

47R

2.

Unknown

12L

12L

87R

118R

80R

108L

100L

96L

89.5L b

93L

287R

56L

3.

Transcription elongation factor TFIIS

81R

86R

24L

85R

56R

171R

115R

29L

29.5L b

32L

349L

55R

4.

Deoxynucleoside kinase

85R

91.5R

19L

67L

40L

136R

027R

32R

31R

34R

143R

29R

5.

Erv1/Alr family

88R

94R

16L

70R

43R

106L

142L

43L

43.5L

45L

347L

96R

6.

Immediate early protein ICP-46

91R

97R

13L

162L

108L

47L

162R

115R

108.5R

112R

393L

39R

7.

Hypothetical protein-Clostridium tetani

94L

100R

11L

98R

67R

19R

153L

86R

82.5R

84.5L

307L

33L

The Iridoviridae core genes are shown.

aORFs that have been added or altered are highlighted in bold

bPotentially frameshifted ORF

https://static-content.springer.com/image/art%3A10.1186%2F1743-422X-4-11/MediaObjects/12985_2006_Article_224_Fig1_HTML.jpg
Figure 1

Conserved Iridovirus Genes. Every Iridoviridae gene that has an ortholog in at least 2 Iridoviridae genera are shown. Orthologs share the same row on the table. The genes within each genus are color-coded for easier identification. As long as at least one member of the genus contains an ortholog, the entire genus is highlighted. Where multiple ORFs are listed for a particular gene name, the ORFs represent multiple orthologs of the gene in that viral species. The remainder of the figure showing just the genes conserved between the Iridovirus and Chloriridovirus genera are included in Additional File 3.

Identifying genes conserved between some, but not all, iridovirus species can give us important information when investigating evolutionary relationships within the family. A number of past phylogenetic analyses of Iridoviridae have used phylogenic trees constructed from aligned protein sequences [1, 1820, 22, 24, 27]. However, there are potential problems with phylogenic analysis based on comparisons of single genes. This type of analysis is rarely consistent due to horizontal gene transfer [28] and variable rates of evolution [29]. Therefore, we decided to take a whole genome comparative phylogenetic analysis to understand the relationship between iridoviruses. Our approach was to identify all the genes conserved between different genera to gain a better understanding of the relationships within the iridovirus family. This approach yields an indication of how similar in gene content 2 genomes are. Our whole-genome comparative analysis, grouped orthologous genes between genera (Figures 1 &2 and Additional File 3), and was consistent with phylogenic trees constructed from single protein sequences. Based on gene conservation, the Ranavirus and Lymphocystivirus genera appear to be most closely related to one another (Figure 2). In addition, the Iridovirus and Chloriridovirus genera are also closely related to one another based on presence of orthologous genes (Figure 2). In contrast, the Megalocytivirus genus and the Iridovirus/Chloriridovirus genera are equally divergent from each other as well as all other Iridoviridae family members (Figure 2).
https://static-content.springer.com/image/art%3A10.1186%2F1743-422X-4-11/MediaObjects/12985_2006_Article_224_Fig2_HTML.jpg
Figure 2

Phylogenetic relationships between the five iridovirus genera based on gene content. Individual viral species were compared within a genus to identify the number of orthologous genes. Orthologous genes between viral genera were then determined. The numbers on each line identify the number of orthologous genes shared between viral species or genera including the 26 core genes. The Iridovirus and Chloriridovirus genera have a high degree of gene conservation and a combined genera box (Iridovirus/Chloriridovirus) was used to compare orthologous genes between genera. In addition, two subgroups of the Ranavirus genus are shown. Each subgroup contains a virtually identical complement of genes. However, a comparison between the FV3/TFV/ATV subgroup with the SGIV/GIV subgroup revealed 72 orthologous genes.

As the list of sequenced iridovirus genomes grows, the non-co-linearity between many of these genomes becomes more apparent. The Megalocytivirus and Ranavirus, but not the Chloriridovirus, Iridovirus, and Lymphocystivirus genera, show a co-linear arrangement of genes within each genus. However, comparisons of genomic sequences from different genera suggest no co-linearity. This trend may be the result of the high recombination rates [30] seen in some iridovirus members [31]. For example, within the Ranavirus genus, ATV has two inversions relative to the FV3 and TFV sequences [30], reducing the co-linearity of these genomes to some degree. Figure 3A shows how two recombination events could convert FV3 to the ATV arrangement of genes. In contrast, a comparison between the more distantly related members within the Ranavirus genus (such as FV3 and GIV) demonstrate a much more dramatic loss of co-linearity. No long stretches of co-linear genes exist between these sequences, although small sections of co-linearity remain as seen through a dotplot analysis between FV3 and GIV (Figure 3B). The dotplot shows small regions of co-linearity scattered throughout the genome of FV3 and GIV as seen by short diagonal lines on the dotplot (Figure 3B). A schematic representation of the co-linearity between FV3 and GIV demonstrates that co-linearity occurs in small clusters of genes often only 2–4 genes in length (Figure 3C).
https://static-content.springer.com/image/art%3A10.1186%2F1743-422X-4-11/MediaObjects/12985_2006_Article_224_Fig3_HTML.jpg
Figure 3

Co-linearity found within the Ranavirus genus. (A) FV3 and ATV, both members of the Ranavirus genus possess almost complete co-linearity of orthologous genes as visualized by a dotplot. However, 2 inversions have occurred. The FV3 genes 10–52 and 77–88 have switched genomic locations as shown, potentially through two recombination events. The inversion has also resulted in the loss of the ortholog of FV3 9L in ATV. (B) There is a limited amount of co-linearity found between FV3/TFV/ATV and SGIV/GIV. The co-linearity has been visualized using a dotplot analysis between FV3 (horizontal sequence) and GIV (vertical sequence). Genes are colored either red or blue representing right- or left-ward transcription respectively. (C) The co-linearity between FV3 and GIV is generally composed of stretches of 2 or 3 co-linear orthologous genes. Orthologous genes, in a co-linear arrangement are schematically shown as blocks of the same color on either FV3 or GIV genomic sequence.

Conclusion

The Iridoviridae family can cause severe diseases resulting in significant economic and environmental losses. Very little is known about how iridoviruses cause disease in their host. Our re-analysis of genomes within the Iridoviridae family provides a unifying framework to understand the biology of these viruses. For example, the re-analysis of the Iridoviridae family has increased the consistency of annotated sequences from viruses within the same genus. In addition, the re-analysis has helped create a much greater consensus among Iridoviridae family members and enhanced our understanding of this virus family as a whole. The updated annotations that we have produced for the iridovirus sequences can be found in the additional files to this paper; in addition, the databases and tools to analyse Iridoviridae genomes are available to all researchers [32]. This database will contain genomes from the original GenBank files and also the edited genomes described in this paper. Further re-defining the core set of iridovirus genes will continue to lead us to a better understanding of the phylogenetic relationships between individual iridoviruses as well as giving us a much deeper understanding of iridovirus replication. In addition, this analysis will provide a better framework for characterizing and annotating currently unclassified iridoviruses.

Methods

Re-annotation of the iridoviridae

Annotated sequences for the twelve completely sequenced iridovirus genomes (Table 1) were obtained from GenBank files and imported into the Viral Orthologous Clusters (VOCs) database [15]. Species from the same genus were examined using VOCs to identify all of the orthologous genes. The analysis then focused on the differences found between genomes within the same genus. For those genomes that contained co-linear arrangements of genes (those in the Ranavirus and Megalocytivirus genera), we compared those regions containing annotated ORFs. If more than two sequenced genomes were available for a given genus, and the ORF was present in at least two of the genomes, then we set out to determine if that ORF was also present in the remainder of the genomes. By this method, we were able to re-annotate small segments of each genome without needing to re-analyse the entire genome. The Viral Genome Organizer (VGO) software [16] was used to visualize the annotated ORFs, as well as the start and stop codons found within each genome.

Analysis of orthologous genes

We used a combination of BLAST searches and queries using the VOCs software [32] to define orthologous genes between Iridoviridae genera. VOCs is a JAVA client-server that accesses a sequence query language (SQL) database containing iridovirus genomes. This SQL database permits complex queries to be assembled in an easy to use graphical user interface. VOCs initially groups orthologous genes into families based on BLASTP scores, these can be manually checked and altered if necessary.

Dotplot analysis

Dotplots of FV3 and GIV were done using JDotter [33]. JDotter provides an interactive input window that links JDotter to the VOCs database. The sequences for the FV3 and GIV were obtained through the VOCs database.

Declarations

Acknowledgements

We would like to thank Daniel Rock for sharing information about mosquito iridescent virus prior to publication and Cristalle Watson for critically reviewing the manuscript. This work was supported by Discovery Grants (Natural Science and Engineering Research Council (NSERC) of Canada) to C.R.B. and C.U. H.E.E. is the recipient of an NSERC postgraduate scholarship.

Authors’ Affiliations

(1)
Department of Biology, Trent University
(2)
Department of Biochemistry and Microbiology, University of Victoria

References

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© Eaton et al; licensee BioMed Central Ltd. 2007

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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