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

  • Heather E Eaton1,

    Affiliated with

    • Julie Metcalf1,

      Affiliated with

      • Emily Penny1,

        Affiliated with

        • Vasily Tcherepanov2,

          Affiliated with

          • Chris Upton2 and

            Affiliated with

            • Craig R Brunetti1Email author

              Affiliated with

              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 familyIridoviridae 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 theIridoviridae 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 theRanavirus andMegalocytivirus genera in order to identify novel conserved ORFs. Of these two genera, theMegalocytivirus 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 theIridoviridae 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 familyIridoviridae is currently subdivided into five genera: Chloriridovirus, Iridovirus, Lymphocystivirus, Megalocytivirus, andRanavirus [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 thePoxviridae [17]. Here, we focused our analysis on theIridoviridae 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 theMegalocytivirus 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 theMegalocytivirus 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 theMegalocytivirus 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 theMegalocytivirus 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 theMegalocytivirus genus, we applied the same comparative genomic analysis to theRanavirus 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), andAmbystoma tigrinum virus (ATV). The second group contains Singapore grouper iridovirus (SGIV) and grouper iridovirus (GIV).

              The first step in the re-annotation of theRanavirus 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 secondRanavirus 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 theRanavirus 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 theRanavirus 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 theRanavirus andMegalocytivirus genera, we were unable to use it for theChloriridovirus, Iridovirus, andLymphocystivirus 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 theIridoviridae 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 theIridovirus andLymphocystivirus 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 theIridoviridae 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 theIridoviridae 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,106L b

              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

              TheIridoviridae core genes are shown.

              aORFs that have been added or altered are highlighted in bold

              bPotentially frameshifted ORF

              Table 7

              AdditionalIridoviridae 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

              TheIridoviridae core genes are shown.

              aORFs that have been added or altered are highlighted in bold

              bPotentially frameshifted ORF

              http://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 2Iridoviridae 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 ofIridoviridae 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, theRanavirus andLymphocystivirus genera appear to be most closely related to one another (Figure 2). In addition, theIridovirus andChloriridovirus genera are also closely related to one another based on presence of orthologous genes (Figure 2). In contrast, theMegalocytivirus genus and theIridovirus/Chloriridovirus genera are equally divergent from each other as well as all otherIridoviridae family members (Figure 2).
              http://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. TheIridovirus andChloriridovirus 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 theRanavirus 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. TheMegalocytivirus and Ranavirus, but not theChloriridovirus, Iridovirus, andLymphocystivirus 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 theRanavirus 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 theRanavirus 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).
              http://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 theRanavirus genus. (A) FV3 and ATV, both members of theRanavirus 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

              TheIridoviridae 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 theIridoviridae family provides a unifying framework to understand the biology of these viruses. For example, the re-analysis of theIridoviridae 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 amongIridoviridae 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 analyseIridoviridae 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 theRanavirus andMegalocytivirus 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 betweenIridoviridae 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

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              Copyright

              © Eaton et al. 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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