Complete Genome Sequence of the Grouper Iridovirus and Comparison of Genomic Organization with Those of Other Iridoviruses

Abstract

The complete DNA sequence of grouper iridovirus (GIV) was determined using a whole-genome shotgun approach on virion DNA. The circular form genome was 139,793 bp in length with a 49% G+C content. It contained 120 predicted open reading frames (ORFs) with coding capacities ranging from 62 to 1,268 amino acids. A total of 21% (25 of 120) of GIV ORFs are conserved in the other five sequenced iridovirus genomes, including DNA replication, transcription, nucleotide metabolism, protein modification, viral structure, and virus-host interaction genes. The whole-genome nucleotide pairwise comparison showed that GIV virus was partially colinear with counterparts of previously sequenced ranaviruses (ATV and TFV). Besides, sequence analysis revealed that GIV possesses several unique features which are different from those of other complete sequenced iridovirus genomes: (i) GIV is the first ranavirus-like virus which has been sequenced completely and which infects fish other than amphibians, (ii) GIV is the only vertebrate iridovirus without CpG sequence methylation and lacking DNA methyltransferase, (iii) GIV contains a purine nucleoside phosphorylase gene which is not found in other iridoviruses or in any other viruses, (iv) GIV contains 17 sets of repeat sequence, with basic unit sizes ranging from 9 to 63 bp, dispersed throughout the whole genome. These distinctive features of GIV further extend our understanding of molecular events taking place between ranavirus and its hosts and the iridovirus evolution.

Iridoviruses are large icosahedral cytoplasmic deoxyriboviruses with viral particle sizes ranging from 120 to 350 nm in diameter. The family Iridoviridae can be subdivided into at least four genera, including Iridovirus, Chloriridovirus, Ranavirus, and Lymphocystivirus (59). Members of the family are notable for their variability in infecting a diverse range of vertebrate (Lymphocystivirus, Ranavirus) and invertebrate (Chloriridovirus, Iridovirus) hosts, causing diseases that range in severity from subclinical to lethal (58). The iridovirus genome is both circularly permutated and terminally redundant and is a double-stranded DNA genome ranging from 103 to 212 kbp in length (30). Moreover, the genome of the iridoviruses that infect vertebrates is highly methylated at the cytosine residues in CpG sequences by a virus-encoded methyltransferase (53, 60).

Vertebrate iridoviruses are found in fish, amphibians, and reptiles. Many of them are confirmed to be pathogens that cause systemic diseases in aquaculture. Iridovirus infection of fish and frogs is a serious problem in modern aquaculture, fish farming, and wildlife conservation because of its epidemic morbidity and ability to cause mortality. Systemic iridoviral infections have been previously described for a variety of freshwater and marine food fish species (18, 22, 32, 37), tropical freshwater ornamental fish (40), frogs (20), and salamanders (30). Since the discovery of the first fish iridovirus, LCDV (lymphocystis disease virus), in 1962 (56), many new iridovirus-like pathogens have been reported from over 140 different species of fish worldwide (15). Intriguingly, most of these fish iridoviruses have shown to be more closely related to frog virus 3, the typical species of the genus Ranavirus, than to Lymphocystivirus. Although the genus Ranavirus was originally thought to infect only amphibians, the ranavirus-like pathogens can infect fish and reptiles as well (35). This has prompted speculations that viruses belonging to genus Ranavirus are capable of infecting cross-species organisms and that one class of poikilothermic vertebrates might serve as a reservoir, alternate host, or amplifying host for viruses infecting another class of lower vertebrates (35). These observations raise several questions and research interests.

(i) The taxonomic position of newly isolated iridoviruses.

In previous studies, identification of a newly isolated virus as an iridovirus was performed on the basis of characteristic virion morphology or of the use of a phylogenic algorithm employing a few common genes or of a single gene such as the major capsid protein gene (37, 47, 54). However, there has been considerable debate on whether a phylogenetic tree constructed on the basis of any single gene could accurately represent the evolution of a species, due to the possibility of horizontal gene transfer and degradation of the phylogenetic signal because of saturation for amino acid substitutions (10). Besides, these methods cannot provide enough information for clear identification and determination of isolated viruses. There is no common or universal phylogenetic tree to support the taxonomic position of a virus because of the differences in the rates of evolution of different genes from the same virus species (38). Developing tools and criteria for the identification and classification of virus taxonomy would be a big step toward understanding the evolution process of iridoviruses.

(ii) The essence of iridovirus.

The recent advances in genome sequencing technology have made it possible to scan the whole genome to define the genes that are homologous between iridoviruses. Comparison of different iridovirus genomes has revealed that many genes have been conserved during evolution, and this comparison would enable understanding of relationship between the phenotype and genotype. Besides, the genomic comparison would also allow the identification of genes which define iridoviruses and their host range.

Recently, five iridovirus genomes have been completely sequenced, including those of Lymphocystis disease virus 1 (LCDV-1; genus Lymphocystivirus) (51), Chilo iridescent virus (CIV; genus Iridovirus) (29), Tiger frog virus (TFV; genus Ranavirus) (24), Infectious spleen and kidney necrosis virus (ISKNV; unclassified genus) (23) and Ambystoma tigrinum virus (ATV; genus Ranavirus) (30). Considering the disease problems caused by viruses in fish aquaculture and their impact on fish farming economy and to facilitate the understanding of the molecular mechanism of infection, we describe here another complete genome sequencing and analyses of an iridovirus, the grouper iridovirus (GIV), which was isolated from diseased yellow grouper, Epinephelus awoara (34, 37).

MATERIALS AND METHODS

Isolation of virus and its DNA.

The GIV used in this study was isolated from spleen tissue of a diseased yellow grouper (Epinephelus awoara). The tissue was ground with a mortar and pestle. Leibovitz's L15 medium containing no fetal bovine serum was added to homogenates and centrifuged at 6,000 × g for 10 min. The supernatant fluid was filtered through a 0.2-μm-pore-size membrane. A total of 10 μl of the filtrate was inoculated into a 25-cm² Nunc culture flask containing a 50% confluent monolayer of grouper kidney (GK) cells prepared 24 h earlier, and the mixture was incubated at 28°C until a cytopathic effect was observed. The supernatant fluid from virus-infected cells was filtered through a 0.2-μm-pore-size membrane and used for virus replication and purification. The virus was propagated in GK cells and purified by ultracentrifugation as described previously (34). For extraction of viral genomic DNA, purified GIV virions were soaked and incubated with genomic DNA extraction buffer (10 mM Tris-HCl [pH 8.0], 1 mM EDTA, 100 mM NaCl) containing 0.2% (wt/vol) sodium dodecyl sulfate and 200 μg of proteinase K/ml at 55°C for 2 h. Subsequently, viral DNA was extracted with phenol-chloroform and precipitated with alcohol. Viral DNA pellets were dissolved in TE (10 mM Tris-HCl [pH 8.0], 1 mM EDTA).

GIV DNA cloning and sequence determination.

The GIV genome was sequenced to 3,273 sequences for 11-fold of coverage by a shotgun approach, and the gaps were filled by a chromosome walking method. The viral DNA was sheared (HydroShear; GeneMachines, San Carlos, Calif.) by nebulization into fragments with average sizes of 2,000 and 5,000 bp. DNA fragments were size fractionated by gel electrophoresis and cloned into the EcoRV site of pBluescript SK(+/-) (Stratagene). After transformation into Escherichia coli DH10B competent cells (Gibco-BRL), 1,446 recombinant colonies with a fragment size of 5 kbp and 576 recombinant colonies with a fragment size of 2 kbp were picked randomly. DNA templates for sequencing were isolated using Multiscreen kits (Millipore, Bedford, Mass.). Sequencing was performed using an ABI Prism Big Dye Terminator Cycle Sequencing Ready Reaction kit with FS AmpliTaq DNA polymerase (Perkin-Elmer, Palo Alto, Calif.) and analyzed on an ABI 3730 DNA analyzer. Shotgun sequences were base called by using the PHRED basecaller and assembled with the PHRAP assembler. PHRAP-assembled data were stored in a Sun workstation assembly database by using a Sun workstation interface. The Sun workstation interface and its features were then used for editing and completing the sequence. The Sun workstation was used with a probabilistic consensus algorithm based on expected error rates for output by PHRED to perform the consensus calculation with a quality cutoff value of 40. Primers were designed for chromosome walking through the gaps of the existing contiguous fragments.

DNA sequence analysis.

Open reading frames (ORFs) encoding more than 30 amino acids (90 bp), starting with the ATG codon and ending with the termination codon, were considered protein encoding and hence were designated putative genes. The maximal 570 ORFs were predicted and analyzed using Artemis 4.0 software (42). The amino acid sequence comparisons of all 570 ORFs were carried out using the PSI-BLAST program of the National Center for Biotechnology Information, and comparisons with the Swiss-Prot sequence databases and the Conserved Domain Database (CDD) (1, 36) were also performed. The predicted ORF homologues to any previously identified ORFs in the sequence database were incorporated in our analysis list. The percentages of identity were determined on the basis of the percentages of identical residues between two complete sequences. In addition, ORFs with significant homologies to ORFs in the protein sequence database and ORFs larger than 19 kDa without any significant homology to ORFs in the sequence database were also included. The molecular weight selection criterion was selected to coincide with our previously determined viral particle sodium dodecyl sulfate-polyacrylamide gel electrophoresis pattern (37). DNA repeats were identified by using a DNA tandem-repeat finder (6). The transmembrane domains of the predicted ORFs were predicted using the TMHMM server, v. 2.0 (33, 45). Dot plotting comparisons were performed by using the pairwise program of prFLAG (http://bioinformatics.itri.org.tw/prflag/prflag.php) from the Biomedical Engineering Center, Industrial Technology Research Center, Hsinchu, Taiwan.

Nucleotide sequence accession number.

The complete GIV sequence can be obtained from GenBank (accession no. AY666015).

RESULTS AND DISCUSSION

The features of GIV genome.

The assembled GIV genome was 139,793 bp in length, which was similar to our earlier prediction based on the restriction enzyme fragment analysis. Four restriction enzyme cutting maps are presented in Fig. 1. The genome of GIV was shown to be circularly permutated, possibly assembled into a circular form, which is a major characteristic feature of iridovirus genomes (24). The genome size of GIV is larger than those of ATV (106,332 bp) (30), TFV (105,057 bp) (24), LCDV (102,653 bp) (51), and ISKNV (111,362 bp) (23) but much smaller than that of CIV (212,482 bp) (29). It seems that the invertebrate iridovirus needs a larger genome than the vertebrate iridovirus. The percentage of G+C content of GIV (49%) is comparable to those of the genomes of ATV (54%), TFV (55%), and ISKNV (55%) but much higher than those of CIV (29%) and LCDV (29%) (Table 1). Artemis analysis detected a total of 570 ORFs as methionine-initiated ORFs comprising more than 30 amino acids. Of these ORFs, 120 present in GIV genome were homologous to ORFs in the Swiss-Prot database or were larger than 19 kDa without having homology to ORFs in the sequence database (Fig. 1). The ORFs are distributed evenly along the genome in clockwise (right, 47%) and anticlockwise (left, 53%) directions. The first ORF, 001L, is referred to as the first ORF present in the A fragment of XhoI-digested GIV genome (Fig. 1). The locations, orientations, sizes, and PSI-BLAST results for the putative ORFs are listed in Table 2. Predicted ORFs, with an average length of 987 nucleotides (329 amino acids), represented 83% coding density.

FIG. 1.

Circular representation of the GIV genome. The putative 120 ORFs are shown as blank arrows in the first circle (outer ring). The second to fifth rings are the sites of EcoRI, HindIII, XhoI, and NotI restriction enzymes, respectively (the fragments are shown by alphabetic letters in descending order). The repeat sequences (R1 to R17) are indicated in the sixth ring (the innermost ring).

TABLE 1.

Summary of genomic sequence data for six viral species representing four genera within the family Iridoviridae

Characteristic	Data for indicated genus and species
	Iridovirus CIV	Ranavirus			Lymphocystivirus LCDV	Unassigned (SKNV)
		ATV	TFV	GIV
Genome size (bp)	212,482	106,332	105,057	139,793	102,653	111,362
GC (%)	29	54	55	49	29	55
No. of putative ORFs	468	96	105	120	195	124
ORF size (no. of amino acids)	40-2,432	32-1,294	40-1,294	62-1,268	40-1,199	40-1,208
Accession no.	AF303741	AY150217	NC003407	AY666015	NC001824	NC001824

TABLE 2.

Characterization of predicted open reading frames of Grouper Indovirus (GIV)^a

ORFb	Nucleotide position	Lengthc			Conserved domain or signature	CDD accession no.d	Best match homolog
		nt	a.a.	MW			Predicted function and/or similarity	% Identity	Accession no.e
001L	1:513	513	170	18,976				47	AAP33240
002L	2295:3536	1,242	413	46,218				40	AAP33180
003R	3564:4421	858	285	32,309				37	AAP33179
004R	4443:5399	957	318	34,343	DUF230, Poxvirus proteins of unknown function	pfam03003	Myristylated membrane protein	70	AAP33178
005L	5453:6421	969	322	35,362				40	AAP33250
006L	6475:6900	426	141	16,629				59	AAP33251
007L	6950:7486	537	178	19,419	Inositol-1,4,5-triphosphate 5-phosphatase	KOG0566	Neurofilament triplet Hl-like protein	41	AAP33252
008L	7528:7983	456	151	17,269				27	AAP33254
009L	8165:9682	1,518	505	55,796	SAP, Putative DNA-binding motif	smart00513		43	AAP33256
010R	9761:11461	1,701	566	63,213				39	AAP33257
011Lf	11546:12559	1,014	337	36,667
012L	12643:13659	1,017	338	37,611	IG, Immunoglobulin	smart00409	MGC52801 protein	30	AAH41253
013Lf	13816:14850	1,035	344	38,125
014Lf	15362:16384	1,023	340	37,766
015Lf	16846:17904	1,059	352	39,680
016Lf	17964:19010	1,047	348	38,155
017L	19511:20638	1,128	375	42,281	Neural cell adhesion molecule L1	KOG3513		25	AAH62581
018L	20771:21835	1,065	354	39,733			Drosophila melanogaster CG15570-PA	55	NP_572154
019L	21988:23016	1,029	342	37,096	Neural cell adhesion molecule L1	KOG3513
020L	23044:23556	513	170	19,029				46	AAP33260
021L	23583:26738	3,156	1,051	118,626				37	AAP33261
022L	26865:27296	432	143	15,072			Drosophila melanogaster CG15570-PA	42	NP_572154
023R	27608:29605	1,998	665	73,550				40	AAP33262
024Lf	29679:30407	729	242	22,813
025L	30467:31204	738	245	23,473			COG5295: Autotransporter adhesin	39	ZP_00122019
026L	31247:32401	1,155	384	43,712	NrdF, Ribonucleotide reductase, β subunit	COG0208		73	AAP33216
027L	32549:32824	276	91	10,505	CARD, caspase recruitment domain	pfam00619	CARD-like caspase	42	AAP33218
					TFIIF-interacting CTD phosphatase	KOG0323
028L	32857:33336	480	159	17,483	dUTPase	KOG3370	dUTPase	66	AAH19979
029L	33367:34029	663	220	23,582	TNFR domain	cd00185	Tumor necrosis factor receptor superfamily, member a	29	NP_571915
030L	34201:34896	696	231	25,959	TNFR domain	cd00185	Tumor necrosis factor receptor superfamily member 14 precursor	33	XP_345617
031L	35097:37997	2,901	966	109,744	Predicted ATPase	COG3378	D5 family NTPase	71	AAP33258
					Poxvirus D5 protein-like	pfam03288
032R	38100:38747	648	215	25,128				70	AAP33259
033R	38770:39492	723	240	22,715			ATV nucleotide position 39,711-40,067	50
034R	39521:40261	741	246	23,009			Collagen-like protein	51	NP_078660
035L	40327:43833	1,168	389	131,512			LCDV ORF 2-like protein	54	AAK37740
036R	44348:47200	2,853	950	107,211	DNA repair protein, SNF2 family	KOG0390	Helicase	61	AAP33184
					helicase_C, helicase conserved C-terminal domain	pfam00271
037R	47235:47849	615	204	23,319	CPDc, catalytic domain of Ctd-like phosphatase	smart00577		50	AAP33244
					TFIIF-interacting CTD phosphatase	KOG1605
038R	47914:48708	795	264	27,641	IGF-like growth factor	smart00078		38	NP_705854
					Amphiphysin	KOG3771
039R	48760:50478	1,719	572	63,753	NrdA, ribonucleotide reductase, α subunit	COG0209	Ribonucleoside-diphosphate reductase, α subunit-like protein	71	NP_571996
040L	51254:51829	576	191	21,640	Mitochondrial thymidine kinase 2-deoxyguanosine kinase	KOG4235	Deoxyribonucleoside kinase-thymidine kinase	47	AAP33196
041L	51863:52681	819	272	29,652			ISKNV ORF112R	27	NP_612334
042L	52668:54314	1,647	548	61,930				37	AAP33194
043R	54341:54799	459	152	17,035	Ervl/Alr family	pfam04777	Thiol oxidoreductase	53	AAP33193
044R	54830:55654	825	274	31,589				32	AAP33192
045R	55751:57142	1,392	463	50,520	Capsid iridovirus	pfam04451	Major capsid protein	72	AAO32315
046L	57221:60532	3,312	1,103	123,289	RNA polymerase II, second largest subunit	KOG0214	DNA-directed RNA polymerase II β subunit	66	NP_572001
047R	57819:58085	267	88	9,173				73	AAP33222
048R	60586:61122	537	178	19,947			Sensory box histidine kinase-response regulator	26	NP_793671
049L	61207:62064	858	285	30,332	Purine nucleoside phosphorylase	KOG3984	Purine nucleoside phosphorylase	50	NP_000261
050Lf	62188:63096	909	302	34,096
051Lf	62282:62944	663	220	25,860
052L	63202:66156	2,955	984	110,839	CAP10, putative lipopolysaccharide-modifying enzyme	smart00672	Putative tyrosine protein kinase	44	NP_571995
053Lf	66271:66939	669	222	24,364
054R	67019:68356	1,338	445	50,545				41	AAP33203
055L	68413:69540	1,128	375	41,572	Rnc, double-stranded RNA-specific ribonuclease	COG0571	RNase III	47	NP_572005
056R	69598:69876	279	92	10,570	RPB9, DNA-directed RNA polymerase, subunit M-Transcription elongation factor TFIIS	COG1594	Putative transcription elongation factor SII	55	NP_572006
					C2C2 zinc finger	smart00440
057R	69938:70402	465	154	17,127			Immediate-early protein ICP-18	46	P03298
058Rf	70728:71375	648	215	25,146
059L	71835:73355	1,521	506	53,963			Myristylated membrane protein A	57	AAK54492
060L	73416:74588	1,173	390	45,521				23	AAP33232
061L	74673:75794	1,122	373	43,483				21	AAP33232
062Lf	75915:77051	1,137	378	44,664
063L	77240:78373	1,134	377	44,411				20	AAP33232
064L	78437:79654	1,218	405	47,514				22	AAP33232
065R	80301:80771	471	156	16,872	TNFR domain	cd00185	Tumor necrosis factor receptor superfamily, member 11b	35	NP_037002
066L	80778:81926	1,149	382	43,338	XPG, Xeroderma pigmentosum G	cd00128	Putative DNA repair protein RAD2	54	NP_572012
067R	81580:82383	804	267	30,517			RRV 8.141-kb PstI fragment	57	AAK53746
068Rf	82906:83847	942	313	35,127
069L	83928:84161	234	77	8,547	Ubiquitin-60s ribosomal protein L40 fusion protein	KOG0003	Ubiquitin GP37 fusion protein	81	NP_258300
070R	84246:84539	294	97	11,059				38	AAP33269
071L	84600:88406	3,807	1,268	139,162	RNA polymerase III, large subunit	KOG0261	DNA-dependent RNA polymerase α subunit	61	AAP33183
					RNA polymerase II, large subunit	KOG0260
072R	84652:84885	234	77	8,106			CIV 344R	45	AAK82205
073R	88462:89088	327	208	21,540			Ati or CPXV158 protein	29	NP_619942
074R	89401:90168	768	255	29,136				36	AAP33270
075R	90223:91326	1,104	367	35,962	Collagens (type XV)	KOG3546	Procollagen, type VI, α 3	38	NP_034065
076L	90579:90971	393	130	12,179			B antigen	38	CAA42442
077L	90975:91304	330	109	10,453			Levansucrase	38	CAD48195
078R	91793:92251	459	152	17,210	BCL	smart00337	Bak protein	33	AAF89533
079R	92371:93147	777	258	30,130			Putative replication factor	58	AAP33272
080R	93463:94422	960	319	35,749				61	AAP33268
081L	94779:95162	384	127	14,315				47	AAP33204
082Lf	95643:96275	633	210	24,255
083L	96328:97416	1,089	362	41,459				31	AAL13097
084Rf	98151:98705	555	184	21,078
085Rf	98692:99330	639	212	23,960
086Rf	99403:99924	522	173	19,877
087R	100788:103817	3,030	1,009	114,730	DNA polymerase family B	pfam00136	DNA polymerase-like protein	69	NP_572000
088Rf	104499:105053	555	184	19,822
089R	105071:105898	828	275	31,303				46	AAP33263
090L	106273:107244	972	323	36,485	AAA, ATPases associated with a variety of cellular activities	smart00382	ATPase-like protein	68	NP_571992
091R	107692:108006	315	104	11,634	LITAF, LPS-induced tumor necrosis factor α factor	smart00714		60	AAP33206
092R	108028:109413	1,386	461	49,680				36	AAP33207
093R	110141:113554	3,414	1,137	119,286			Trypanosoma cruzi R27-2 protein	37	T30296
094L	113639:113878	240	79	8,967				49	AAP33212
095R	113959:114417	459	152	16,748	Fibroblast growth factor	KOG3885	Fibroblast growth factor 22	35	NP_570107
096R	114476:114973	498	165	18,060	FGF, acidic and basic fibroblast growth factor family	cd00058	Fibroblast growth factor 10	31	NP_032028
097L	115076:116050	975	324	36,684	Predicted E3 ubiquitin ligase	KOG1814	NTPase-helicase	43	AAP33208
098L	116186:117220	1,035	344	39,379				25	AAP33224
099R	117323:117802	480	159	17,613				45	AAP33210
100L	118300:119826	1,527	508	57,187			Phosphotransferase	36	AAP33226
101L	119847:120434	588	195	22,222				35	AAP33227
102R	120533:121771	1,239	412	46,549	SSL2, DNA or RNA helicases of superfamily II	COG1061	Putative helicase	44	CAB37349
103L	121088:121660	573	190	21,910			FV3 40K protein	30	S49999
104R	122172:123896	1,725	574	64,611	Semaphorins	KOG3611	Sema domain, immunoglobulin domain (1g), and GPI membrane anchor	27	NP_003603
105Lf	124043:124852	810	269	31,085
106R	124940:125464	525	174	19,604				59	AAP33225
107L	126500:126988	489	162	18,861	RNA polymerase, 25-kDa subunit	KOG3218		41	AAP33238
108L	127649:128797	1,149	382	44,105				49	AAP33190
109L	128963:130138	1,176	391	46,396				21	AAP33232
110Lf	130166:131080	915	304	34,638
111R	131127:132272	1,146	381	43,194	3-β hydroxysteroid dehydrogenase-isomerase family	pfam01073	3-β-hydroxy-δ5-C27-steroid oxidoreductase-like protein	53	NP_571998
112R	132248:132442	195	64	7,326	Neuroblastoma-amplified protein	KOG1797
113L	132345:133442	1,098	365	41,746				20	AAP33232
114L	133510:134652	1,143	380	40,361	Transmembrane amino acid transporter protein	pfam01490	N system amino acid transporter NAT-1	26	AF61849
115R	134680:135453	774	257	28,943				67	AAP33234
116L	135520:136425	906	301	29,638			COG5295: autotransporter adhesin	25	ZP_00122019
117L	136280:136972	693	230	22,247			Collagen-like protein	37	NP_078660.1
118L	137066:137530	465	154	16,433			ATV nucleotide position 71,894-72,235	28
119L	138030:138218	189	62	6,633				61	AAP33242
120L	138307:139776	1,470	489	56,353				45	AAP33240

^aNucleotides in the GIV virus genome were numbered sequentially, beginning with the nucleotide in the XhoI-A fragment.

^bThe direction of the transcripts are indicated by R (right) or L (left).

^cnt, nucleotides; a.a., amino acids; MW, molecular weight.

^dAccession numbers obtained from the Conserved Domain Database by use of PSI-BLAST software.

^eGenBank accession numbers of homologous proteins.

^fORFs unique to GIV.

Repeat sequences of GIV.

Repeat sequences have been observed in ATV, ISKNV, LCDV, and CIV genomes (23, 29, 30, 51). Although no repeat sequences are reported for the TFV genome (24), such extensive repeat regions were identified in TFV as well. Besides iridoviruses, the poxviruses, herpesviruses, baculoviruses, adenoviruses, and retroviruses also possess repeat sequences (30). These repeat sequences could be associated with important regulatory functions during viral replication. Seventeen sets of repeat sequences (R1 to R17) were found throughout the GIV genome (Fig. 1). The repeat sequences were of a basic unit length ranging from 9 to 63 bp, with copy numbers of 1.9 to 36.5. When compared to individual consensus repeat units, the repeat sequences matched from 82 to 100%. Repeat sequence R14 was 1,533 bp long and is the longest repeat sequence among the complete sequenced iridovirus genomes reported so far. R14 was composed of 25 sets of 42-bp-long complete direct-repeat units and 23 sets of 21-bp half-direct-repeat units. The function of this repeat sequence needs to be further characterized. Besides, R2, R3, R13, and R16 of GIV were located in the noncoding regions and 13 were within the putative ORFs (001L, 007L, 012L, 018L, 025L, 034R, 038R, 073R, 075R, 093R, and 116L). Two putative Goldberg-Hogness boxes, each comprising an AT-rich sequence (TATTTTA), were observed in the repeat sequences R1 and R16. A similar TATTTTA box was also found in upstream of the FV3 immediate-early gene ICR 169 and FV3 late gene ICR 534 and was demonstrated to be transcription dependent (61). Besides the TATTTTA box, repeat sequence R1 also contained a CCAAT box, which is similar to the upstream sequence result observed for the FV3 immediate-early gene ICR 489 (62). The CCAAT box was shown to bind a cellular transcriptional factor (62). In the coding region, repeat sequences R9 and R10 were found within ORF 038R; repeat sequence R9 was located in a putative amphiphysin domain (CDD accession no. KOG3771). Apart from repeat sequence R9, none of the repeat sequences was located within or formed any possible functional domain. In addition, we did not find any homology between the repeat sequences in GIV genome and those of the other five complete sequenced iridovirus genomes. However, repeat sequence homology was observed between ATV and TFV, suggesting that ATV may be closer to TFV than to GIV in taxonomic position.

Protein coding content.

Among the 570 ORFs identified by Artemis analysis, 97 of them had significant homology to ORFs in the protein database and 2 (019L and 112R) had homology to ORFs encoding a significant protein domain; however, no identifiable functional protein was found. Two ORFs (033R and 118L) had no corresponding homolog in the database, but they were found in the ATV genome (ATV nucleotide positions 39,711 to 40,067 and positions 71,894 to 72,235). A total of 19 ORFs considered to be unique to the GIV genes had no corresponding homologues in protein database. In total, 120 GIV ORFs ranging from 62 to 1,268 amino acids in length are listed in the Table 2. Fifty-six ORFs showed 40 to 81% identity to ORFs encoding known proteins from other viruses or organisms or encoding an identifiable functional domain, and 41 showed partial homology (20 to 39% identity) to ORFs encoding a known protein or an identifiable functional domain.

GIV ORFs were, for the most part, nonoverlapping, which is consistent with the results seen with other iridoviruses (30). However, 12 overlapping ORFs were observed in the GIV genome (Fig. 1 and Table 2). ORF 047R had homology with ORFs in ATV, CIV, and TFV, and it was located within the β subunit of RNA polymerase II (046L). ORF 067R had 57% sequence identity with RRV (Regina ranavirus) and overlapped with ORF 066L, which was similar to the results seen with DNA repair protein RAD2. ORF 072R, which shared 45% sequence identity with CIV 344R, was located within 071L (in similarity to the results seen with the DNA-dependent RNA polymerase α subunit). ORF 076L (similar to an ORF encoding B antigen) and ORF 077L (similar to an ORF encoding levansucrase) were located within the 075R, which shared 38% sequence identity with an ORF encoding procollagen, type VI. ORF 103L (similar to an ORF encoding FV3 40K protein) was located within 102R (similar to an ORF encoding helicase).

GIV ORF analysis. (i) Replication.

The GIV genome contains four homologues of the iridovirus genes associated with DNA replication machinery. Like those of the other five iridoviruses, the GIV genome encoded D5 family NTPase (031L), helicase (036R), DNA repair protein RAD2 (066L), and DNA polymerase (087R) (Table 3). The function prediction analysis of ORF 031L revealed that it encoded not only an ATPase domain but also a poxvirus D5 protein-like domain in its N terminus. This D5 protein is necessary for viral DNA replication and is a nucleic acid-independent nucleoside triphosphatase. ORF 036R encoded a protein containing not only a DNA repair protein, the SNF2 family domain, but also a helicase superfamily C-terminal domain. This helicase domain is found in a wide variety of helicases and helicase-related proteins. All helicases share the ability to unwind nucleic acid duplexes; however, the exact function of ORF 036R needs further investigation. ORF 066L encoded a DNA repair-like protein, RAD2, and the corresponding ORFs can also be found in the other five iridoviruses. In the ATV, LCDV, ISKNV, and CIV genomes, however, the start site of the RAD2 ORFs is different from that of TFV RAD2 gene. The TFV RAD2 (TFV 101R) had a point mutation which shifted the RAD2 start site upstream (30). Interestingly, alignment of the GIV RAD2 gene (066L) with the TFV RAD2 gene revealed that amino acid position 24 of ORF 066L matches amino acid position 28 of TFV 101R. When the start sites of the RAD2 genes of GIV and TFV are aligned, it is observed that GIV and TFV are in the same frame except for ORF 066L, which is short four amino acids. The product of ORF 087R shows significant homology to a DNA polymerase of cellular and viral origin, belonging to the family B DNA polymerase. These conserved DNA replication machinery-dependent genes may represent an advantage for viral genome replication that might ultimately lead to persistence of infection and a broad host range for viral infection. These conserved genes might have been acquired from a host or from infection of a common host by ancient iridoviruses (41). The acquired cellular genes could have remained as essential genes for virus replication or could represent nonessential genes which were lost or replaced by other genes in the virus during evolution (51).

TABLE 3.

ORF sequence homologies between GIV and other iridovirusesa^a

ORF group and putative function	GIV ORFs	ATV corresponding ORFs	TFV corresponding ORFs	LCDV corresponding ORFs	ISKNV corresponding ORFs	CIV corresponding ORFs
ORFs conserved in all six sequenced iridovirus genomes
Replication	031L	77L	022R	6	109L	184R
	036R	7L	009L	4	063L	022L
	066L	10L	101R	34	027L	369L
	087R	44L	063R	5	019R	037L
Transcription	037R	64R	040R	64	005L	355R
	046L	43R	065L	3	034R	428L
	055L	25R	085L	44	087R	142R
	056R	24L	086R	105	029L	349L
	071L	6R	008R	1	028L	176R
Nucleotide metabolism	026L	38R	071L	26	024R	376L
	040L	19L	Position 90,715-91,302	60	032R	143R
Protetin modification	043R	16L	094R	79	043L	347L
Structure proteins	004R	1L	002L	29	Position 83,701-84,663	337L
	045R	14L	096R	91	006L	274L
	059L	51L	055R	20	007L	118L
Virus-host interaction	052L	58R	029R	8	114L	179R
Other	021L	80L	019R	14	055L	380R
	032R	78R	021L	70	056L	067R
	035L	69R	045R	2	076L	295L
	067R	11R	100L	71	086L	307L
	079R	91R	105R	43	061L	282R
	080R	87R	012L	39	096L	287R
	090L	83L	016R	46	122R	075L
	100L	47L	059R	17	013R	380R
	108L	13L	097R	27	115R	393L
ORFs conserved in the ranavirus genomes
Transcription	091R	29R	080L
Virus-host interaction	007L	72L	048L
	027L	40L	Position 73,946-74,209
Other	005L	70L	046L
	008L	73L	049L
	023R	81R	018L
	044R	15L	095R
	054R	26L	084R
	057R	23L	087L
	060L	53R	023R
	061L	53R	023R
	064L	53R	023R
	070R	88L	011R
	083L	45R	062L
	092R	30R	079L
	094L	35L	074R
	098L	45R	062L
	106R	46L	061R
	109L	53R	023R
	113L	53R	023R
	119L	62R	034R
ORFs showing significant homologies to other iridoviruse genomes
Transcription	009L	75L	051L	58
	102R	50R	056L			161L
	107L	59R	030R	75
Nucleotide metabolism	028L	42L	068R			438L
	039R	65R	041R	12		085L
Protein modification	097L	31R	078L	36
Structure proteins	033R	Position 39,711-40,067		37
	034R			37
	075R			37
	117L			37
ORF group and putative function	GIV ORFs	ATV corresponding ORFs	TFV corresponding ORFs	LCDV corresponding ORFs	ISKNV corresponding ORFs	CIV corresponding ORFs
Virus-host interaction	038R			7		261R
	096R	37R
	111R	52R	054L	31
Other	001L	61R
	002L	3R	004R			229L
	003R	2L		25
	006L	71L	047L	88
	010R	76R	053R	9
	020L	79L	020R	84		117L
	041L			45	112R
	042L	17R	093L	11
	047R	43L	Position 69,879-70,073			430R
	048R			55
	063L	53R
	072R					344R
	074R	89R	103R	48
	081L	27R	082L	102
	089R	82R
	099R	33L		74
	101L	48L	058R	83
	114L				001L
	115R	55R	025R	49	118L
	118L	Position 71,894-72,235
	120L	61R

^aThe comparison analysis was performed in a bidirectional manner, meaning that every ORF in each individual genome was used to query all the other genomes.

(ii) Transcription.

The genes involved in virus transcription processes included two genes encoding subunits of a DNA-dependent RNA polymerase (046L and 071L) and ORFs encoding a SAF-A/B, Acinus, and PIAS (SAP) domain (009L), a putative Ctd-like phosphatase (037R), RNase III (055L), transcription elongation factor SII (056R), lipopolysaccharide-induced tumor necrosis factor alpha (TNF-α) factor (LITAF) (091R), a putative helicase (102R), and a 25-kDa subunit RNA polymerase (107L). ORF 46L and ORF 071L showed significant homologies to a RNA polymerase II β and α subunit, respectively. Both of them are conserved in six iridoviruses as well as in DNA virus genomes. The C terminus of ORF 009L contained a SAP domain with 91.4% sequence similarity to the SAP domain consensus sequence (smart00513). This motif can be observed in ATV 75L, TFV 051L, and LCDV 58, with 43, 45, and 43% sequence identity, respectively, to the GIV 009L motif. The SAP motif contained several positions enriched with positively charged amino acids that might make contact with the backbone of the DNA and may play a role in sequence- or structure-specific DNA binding involving DNA transcription or repair (2). ORF 037R not only matched 93.3% of the amino acid sequence of catalytic domain of Ctd-like (CPDc) but also matched a TFIIF-interacting CTD phosphatase domain. However, the function of ORF 037R remains to be studied.

ORF 055L encoded an RNase III, which is an important enzyme in the processing of cellular and viral precursor RNAs to produce mature rRNAs, mRNAs, and tRNAs (65). ORF 056R contained a DNA-directed RNA polymerase subunit M and a C2C2 zinc finger domain in its C terminus. This ORF was conserved in all six iridovirus genomes (Table 3) and may be a subunit of DNA-dependent RNA polymerase. ORF 091R encoded a protein with a LITAF domain (smart00714) in its C terminus, and it also can be found both in ATV 29R and TFV 080L, with 60 and 58% sequence identity, respectively. This domain is a LITAF and had been suggested to be a transcription factor which regulates the TNF-α gene expression (50). The product of ORF 102R showed 88.8% sequence similarity to sequences of SSL2, DNA, or RNA helicases of superfamily II, and this ORF is also observed in ATV 50R, TFV 056L, and CIV 161L, with 44, 44, and 29% of sequence identity to GIV 102R, respectively. The amino acid sequence of the C terminus of ORF 107L was homologous to that of an RNA polymerase 25-kDa subunit (KOG3218), and this domain can also be found in ATV 59R and TFV 30R. Both ATV 59R and TFV 30R showed 41% amino acid sequence identity to GIV 107L. Although LCDV 75 shares 37% sequence identity over 115 amino acids with GIV 107L, we could not find this conserved domain in LCDV 75.

(iii) Nucleotide metabolism.

GIV-encoded enzymes involved in nucleic acid metabolism include the two subunits of the ribonucleotide reductase (026L and 039R), dUTPase (028L), deoxyribonucleoside kinase-thymidine kinase (040L), and purine nucleoside phosphorylase (PNP) (049L). The product of ORF 026L showed 97.1% sequence similarity to a ribonucleotide reductase β subunit conserved domain (COG0208) in the CDD database. ORF 039R showed significant homology to a ribonucleotide reductase α subunit. Both ribonucleotide reductase α and β subunits are conserved in GIV, ATV, TFV, LCDV, and CIV genomes (Table 3). However, the ribonucleotide reductase α subunit was not observed in the ISKNV genome (Table 3) (23). Ribonucleotide reductase reduced ribonucelotides into deoxyribonucleotides as immediate precursors of DNA (31). The lack of the ribonucleotide reductase α subunit in ISKNV means that this enzyme may not be functional or that its function may be replaced by other genes. Similarly, not only ISKNV but also some large DNA viruses, such as betaherpesviruses and poxviruses, do not possess a ribonucleotide reductase (52) or show a lack of homology to one of the ribonucleotide reductase subunits, such as that of Rhesus cytomegalovirus (21). Whether the ribonucleotide reductase in GIV or in other iridoviruses is virus replication dependent or not is worth exploring. ORF 028L showed 66% sequence identity to a mouse dUTPase over 159 amino acids. This enzyme cleaves the alpha-beta phosphodiester bonds of dUTP to form pyrophosphate and dUMP, preventing the incorporation of uracil into DNA and providing the substrate for thymine synthesis. dUTP has been shown to be essential for the replication of DNA viruses (4). However, it was not observed in the ISKNV and LCDV genomes (Table 3). ORF 040L showed 85.7% sequence similarity to a mitochondrial thymidine kinase 2-deoxyguanosine kinase domain (KOG4235). Deoxyribonucleoside kinase-thymidine kinase is an enzyme that catalyzes the transfer of the gamma phosphoryl group of ATP to thymidine to generate dMTP in the deoxyribonucleoside salvage pathway. It is a key enzyme in viral DNA replication and is conserved in all six sequenced iridovirus genomes (7) (Table 3). The product of ORF 049L had over 50% sequence identity to a human PNP enzyme. PNP enzyme is a key enzyme of purine salvage pathway, which can catalyze the reversible phosphorylysis of (2′-deoxy) purine ribonucleotides to free bases and (2′-deoxy) ribose-1-phosphates (14). Despite its distribution in nature, a unique viral PNP gene has just been reported for the GIV genome (55). PNP is not found in any iridoviruses or any existing virus, according to comparisons of ORF 049L by use of the protein database. This suggests that GIV might acquire this gene from its host during evolution. However, such an assumption requires more data to support it.

(iv) Protein modification.

ORF 043R encoded a modification enzyme, namely, thiol oxidoreductase. ORF 43R contained a C-X-X-C motif with a homology to an Erv1/Alr family domain. Erv1/Alr family proteins are encoded by all eukaryotes and many cytoplasmic DNA viruses. The conserved domain of the Erv1/Alr family consisted of 100 amino acids and contained a C-X-X-C motif, suggesting that Erv1/Alr family proteins might function as thiol oxidoreductases and facilitate the disulfide bond formation of viral proteins in the host cytoplasm (44). Two ORFs (069L and 097L) are involved in the ubiquitin protein degradation process. ORF 069L encoded a protein with 77 amino acids and has 81% sequence identity with a Spodoptera litura nucleopolyhedrovirus (SpltMNPV) ubiquitin-60s ribosomal protein L40 fusion protein. The function of ubiquitin in cells is to signal the degradation of protein by the 26S proteosome (25). ORF 097L was homologous to a predicted E3 ubiquitin ligase domain (KOG1814) and showed 50.5% amino acid sequence similarity to this domain. The ubiquitin-mediated proteolysis is part of the regulated turnover of proteins required for controlling cell cycle progression. The role of E3 ubiquitin involved a three-step proteolysis mechanism. The E3 ubiquitin ligase-coupled E2 ubiquitin enzyme binds to substrates and assembles a multiubiquitin chain on the substrates. The E3 ubiquitin ligase transfers the activated ubiquitin from the E2 to the substrate lysine residue (28). It was found to be a host immunity modulator in bovine herpesvirus 4, Kaposi's sarcoma-associated herpesvirus, and swine-pox virus (11, 19). This ligase domain is also observed in ATV 31R, TFV 078L, and LCDV 36, and they share 43, 44, and 28% sequence identity, respectively, to GIV 097L. The E3 ubiquitin ligase domain can also be found in ISKNV 120R and CIV 095L. However, the sequence alignment similarities of ISKNV 120R and CIV 095L with this E3 ubiquitin ligase were as low as 10 and 12%, respectively, and no significant similarity were found in comparisons to GIV 097L.

(v) Structure proteins.

GIV encoded three putative viral structural proteins, ORFs 004R, 045R, and 059L, and these three viral structural proteins are conserved through all six sequenced iridovirus genomes. ORF 004R encoded a putative myristylated membrane protein with a poxvirus conserved region (pfam03003) and a transmembrane domain in its C terminus. The TMHMM transmembrane prediction software showed that the poxvirus conserved region might be located outside the cell membrane. The corresponding ORF of GIV 004R was not described for ISKNV, but it could be found within nucleotides 83,701 to 84,663 of the ISKNV genome. ORF 059L also showed sequence homology to a putative myristylated membrane protein, but we could not find any conserved domain or identifiable functional motif in this ORF. Besides, ORF 059L contained two transmembrane domains at amino acids 189 to 211 and 216 to 238, in the middle of this ORF. The functions of both putative membrane proteins GIV 004R and 059L are yet to be investigated. ORF 045R encodes a major capsid protein gene which is 463 amino acids long and is of the same peptide size as those of ATV and TFV. Moreover, ORF 045R shares 72% amino acid sequence identity with ORFs of both ATV and TFV. ORFs 033R, 034R, 075R, and 117L all contained glycine-rich fragments and showed homology to a collagen-like protein, LCDV 37. Besides the glycine-rich domain, ORF 075R contained a collagen type XV domain. The glycine-rich domain in the collagen-like protein was also found in the structural proteins of Ectocarpus siliculosus virus 1 (EsV-1) (12), herpesvirus saimiri (HSV) (17), and bacteriophage PRD1 (5). The glycine-rich domains in these structural proteins may play only a supplementary role in protein function (64).

(vi) Virus-host interaction.

The product of ORF 007L showed just 9.4% weakly significant sequence homology to a inositol-1,4,5-triphosphate 5-phosphatase, INP51/INP52/INP53 family domain (KOG0566). This ORF can be observed in the ATV genome (ATV 72L, with 41% sequence identity to GIV 007L) and TFV genome (TFV 048L, with 43% sequence identity to GIV 007L) (Table 3), but its function is yet to be defined. Both ORF 017L and 019L showed weak sequence homologies to a neural cell adhesion molecule L1 domain, but neither of them had sequence homology to any functional protein in database. Both ORF 017L and 019L contained two transmembrane domains in their N and C termini, respectively. The neural cell adhesion molecule L1 domain of ORFs 017L and 019L were located between these two transmembrane domains and were predicted to be located outside the cell membrane. However, the functions of these two ORFs are unknown. The N terminus of ORF 038R was homologous to an insulin-like growth factor (IGF) domain and an amphiphysin domain which follows the IGF domain. ORF 038R shows 38% sequence identity to LCDV 7. Viral proteins containing an IGF domain might be involved in virus-host interactions (51), and the amphiphysin has been implicated in synaptic vesicle endocytosis (57).

ORFs 095R and 096R show 35 and 31% sequence identity, respectively, to rat and mouse fibroblast growth factors (FGF). ORF 096R has a weak homology with ATV 37R, but 095R has no sequence similarity to ATV 37R. Besides the GIV genome, an FGF-like gene is also encoded in the nuclear polyhedrosis virus genome (3). However, the function of FGF superfamily-like proteins in the virus genome is still unknown. ORF 111R encoded a protein with homology to 3-beta-hydroxy-delta 5-C27-steroid oxidoreductase, and the corresponding ORF can also be found in TFV 054L, with 53% sequence identity to GIV 111R. This enzyme can be found in the ATV 52R, but it is truncated to 53 amino acids and shares no sequence homology with GIV 111R. This protein has been suggested to be involved in modulating the host response to poxvirus infection (43). However, the exact function of this gene in virus has yet to be defined. The identifiable domain or predicted function of these ORFs suggested that they might play a role in the signal transduction process of virus and cell interaction.

ORF 012L encoded a protein with an immunoglobulin domain in its N terminus and a transmembrane domain in its C terminus. The TMHMM prediction revealed that the immunoglobulin domain of this ORF was located outside the cell membrane. ORF 104R has 27% sequence identity to a human semaphorin K1. Semaphorin-like sequences have been identified in the genomes of poxviruses and alcelaphine herpesvirus-1 (13). Viral semaphorins have been proposed to be a part of a defense mechanism used by certain viruses to suppress the immune response of the host (63). Many viruses encode proteins that can function as immune modulators, for example, by interfering with antigen presentation, acting as cytokines or cytokine antagonists, inhibiting apoptosis, or interrupting the complement cascade (46). Many of these immunity-modulating genes have been described as nonconserved genes, revealing the possibility of coevolution of viruses and hosts (46).

ORF 027L encoded a caspase recruitment domain (CARD) (pfam00619) possessing protein. CARD can be observed in the ATV 40L and within nucleotides 73946 to 74209 of the TFV genome, with 42 and 39% sequence identities to GIV 027L, respectively. CARD-containing proteins are suggested to be involved in apoptotic signaling. Although the function of ORF 027L remains to be investigated, it might regulate apoptosis in virus-infected cells (30). The products of ORF 029L, 030L, and 065R were homologous to a TNF receptor (TNFR) domain, and these three predicted ORFs shared 26 to 30% amino acid sequence identity with each other. ORF 030L contained a transmembrane domain in its C terminus, and its TNFR domain was predicted to be located outside the cell membrane. Although ORF 029 also contained a transmembrane domain in its C terminus, the TMHMM prediction result could not reveal whether the TNFR domain is inside or outside of the membrane. ORF 065R did not contain any transmembrane domain in its amino acid sequence. A TNFR domain-containing ORF was also described for the LCDV genome (LCDV 40), but no sequence similarity could be found between LCDV 40 and GIV 029L, 030L, and 065R. The TNFR-like viral factor of LCDV has been considered to be a “viroceptor.” It could bind to the fish cytokine TNF and neutralize its effects (16).

ORF 078R encoded a protein with a BCL domain containing BH1 and BH2 regions and contained a transmembrane domain in its N terminus. Bcl-2 homologues were found in gamma herpesviruses, avipoxvirus (fowlpox), asf virus (African swine fever), alpha herpesvirus and the most-studied E1B of adenoviruses. Although advances in understanding the Bcl-2 family protein have been made in recent years, the precise biochemical mechanisms by which any Bcl-2 family protein inhibits cell death are still unknown and are the subject of a lively debate (39). ORFs 029L, 030L, 065R, 078R, and 104R all possessed putative apoptosis modulator domains and might be involved in the regulation of cell apoptosis; such observations might be coincident with our previous observations of a high virus infection titer for GIV, 10⁸ 50% tissue culture infective doses/ml (34), and the phenomenon of a lack of infected GK cell apoptosis.

ORF 52L contained a putative lipopolysaccharide modifying enzyme domain (CAP10) in its C terminus, and this ORF was conserved in all six iridovirus genomes. The protein CAP-10 was demonstrated to be required for capsule formation, and its deletion abolished fungus virulence (8). However, whether ORF 52L plays a role in virus virulence or not needs further investigation.

(vii) Other.

ORF 047R encoded a putative protein with 88 amino acids and shared sequence identities of 73 and 46% with ATV 43L and CIV 430R, respectively. ORF 47R is located within the RNA polymerase II β subunit (046L), as ATV 43L is located within the RNA polymerase II β subunit of the ATV genome (43R). Although this ORF was not mentioned with respect to the TFV genome (24), it can be seen in within nucleotides 69,879 to 70,073 bp of the TFV genome and it is also located within the RNA polymerase II β subunit (65L) of the TFV genome. This TFV ORF has 75% sequence identity with GIV 047R. The reason for the continuing presence of this ORF in these iridoviruses and its function in different iridoviruses remain to be investigated further. ORF 057R encoded a protein with 46% sequence identity to FV 3 immediate-early protein ICP-18, and it can be found in the ATV genome (23L) and TFV genome (087L), with 44 and 45% sequence identities to GIV 057R, respectively. ORF 090L was homologous to an AAA ATPase domain in its N terminus, with 97.4% sequence similarity. The AAA ATPases are enzymes containing a P-loop NTPase domain and function as molecular chaperones and ATPase subunits of protease-, helicase-, or nucleic-acid-stimulated ATPase (27). However, the exact function of ORF 090L needs to be further investigated.

ORF 100L encodes a phosphotransferase; however, the predicted function of this ORF was determined only on the basis of sequence homology with the sequence database; no identifiable kinase domains could be found. The product of ORF 114L had a homology to a mouse N-system amino acids transporter, NAT-1, and it shares 21% of sequence identity with ISKNV 001L. The amino acid sequence of ORF 114L could be aligned to 87.7% of the putative transmembrane amino acid transporter sequence in the CDD. This ORF encoded an 11-transmembrane domain protein predicted by TMHMM, and the transcription product was observed in GIV-infected GK cells by Northern blotting (data not shown). However, its function is due for elucidation. In addition to these ORFs with identifiable functions, there are another seven ORFs of the GIV genome which are conserved in all six sequenced iridoviruses, including ORFs 021L, 032R, 035L, 067R, 079R, 080R, and 108L (Table 3). The sequence identity between these seven GIV ORFs and those of the other five iridoviruses ranged from a highest identity of 70% over 215 amino acids (GIV 032R compared to ATV 78R) to a lowest identity of 20% over 282 amino acids (GIV 108L compared to ISKNV 115R). All of the ORFs of these seven iridoviruses were without significant identifiable functional domains. Their functions remain to be characterized. The conservation of these unknown ORFs may reveal that these conserved iridovirus common genes might be the essential genes of the iridovirus. ORFs 060L, 061L, 063L, 064L, 109L, and 113L all show about 20% of sequence identity with ATV 53R and TFV 023R. Although their functions are yet to be investigated, these ORFs seem to be redundant gene copies in the GIV genome and share a level of sequence identity from 36 to 21% to each other.

The genes conserved in other five iridoviruses but not observed in the GIV genome.

ATV 22L showed homology to thymidylate synthase, and this enzyme is conserved in other two genomes (TFV 088R and CIV 225R) but has not yet been found in a GIV genome. Thymidylate synthase catalyzes the methylation of dUTP to yield the nucleotide precursor dTMP. This is an important step in the de novo pathway of biosynthesis of pyrimidine. Most viruses do not contain it, and they depend mostly on the host enzyme machinery for the replication of their genome to enable a reduction of genome size in these viruses (64). However, lack of such an important enzyme in DNA viruses (GIV and ISKNV) larger than ATV and TFV is surprising. ATV 57R encodes a homology of eIF-2α, and the corresponding ORF can also be found in the TFV genome (TFV 027R). Previous studies suggested that this gene is conserved throughout the genus Ranavirus (30), but the corresponding ORF was not found in the GIV genome. Vertebrate iridoviruses were found to be highly methylated at the cytosine residues in CpG sequences, and so a conserved DNA methyltransferase enzyme can be found in four vertebrate iridovirus genomes (ATV 21L, TFV 089R, ISKNV 046L, and LCDV 51) and the sequence identities ranged from 95 to 49%. But this enzyme is not found in the GIV genome, and restriction enzyme analysis revealed that the GIV genome was not methylated at all (data not shown). The DNA methylation was previously suggested to play a role in the viral restriction modification system (51). In eukaryote cells, the DNA methylation is involved in the regulation of specific gene expression. More sequence data would be required to answer questions such as these: is there such a viral restriction modification system that exists in other DNA methyltransferase containing vertebrate iridoviruses, and why it is absent in the GIV? Does GIV regulate its gene expression with a different regulatory mechanism other than the host gene expression regulatory mechanisms? Did GIV lose this enzyme during evolution, or was this virus a mutant virus? ATV 20 had homology to a proliferating cell nuclear antigen, and this ORF was conserved in other vertebrate iridoviruses (TFV 090R, ISKNV 112R, and LCDV 45). However, the function of this ORF is yet to be investigated.

The relationship of GIV to other five iridoviruses.

Among the 120 GIV ORFs, 25 ORFs (21%) are conserved in the other five iridoviruses (Table 3). The GIV genome shared 68.4, 54.6, 37.5, 27.5, and 23.3% of BLAST significant ORFs with the ATV, TFV, LCDV, CIV, and ISKNV genomes, respectively (Table 4). In addition to 25 iridovirus-conserved ORFs, 21 GIV ORFs showed significant sequence homologies with ORFs in the ATV and TFV genomes, the genomes of the Ranavirus genus (Table 3). The sequence analysis revealed that these 21 corresponding ORFs in ATV showed very high sequence identities, generally more than 90%, to the corresponding ORFs in TFV. These genes occupied 17.5% of the whole putative gene encoding content of GIV and are conserved in all complete sequenced ranavirus genomes. They could be classified as Ranavirus-specific genes. However, such an inference might be changed as new complete sequenced ranavirus genomes become available. Classification of the ranavirus- or iridovirus-specific genes requires more iridovirus genome sequences or individual viral ORF studies.

TABLE 4.

Common gene content in iridovirus genomes

Virus	No. or % of genesa
	GIV	ATV	TFV	LCDV	ISKNV	CIV
GIV	120	68.4	54.6	37.5	23.3	27.5
ATV	67	98b	85.7	38.8	23.5	34.7
TFV	59	84	108b	38.9	20.4	24.1
LCDV	45	38	42	195	16.8	15.4
ISKNV	28	23	22	21	125b	16
CIV	33	34	26	30	20	468

^aThe number of genes shared between genomes (lower left triangle) or the percentage of genes shared between genomes (the total number divided by the number of genes in the smallest genome; upper right triangle) and the numbers of genes per genome (bold).

^bIn the ATV, TFV, and ISKNV genomes, only 96, 105, and 124 ORFs, respectively, have been identified (23, 24, 30). However, the database search performed for the present study identified a total of 98, 108, and 125 ORFs in ATV, TFV, and ISKNV, respectively.

Besides the high percentage of ORFs shared between GIV, ATV, and TFV, we also can find the partial colinearity by whole-genome nucleotide dot plotting (Fig. 2). In comparisons made using GIV itself, the dot plot graph revealed the −45° diagonal line with intragenic repeat sequence “dots” on it (Fig. 2A). The intragenic repeat sequence dots indicate short palindrome repeat sequences. Most of these palindrome sequences were located in the noncoding regions except those palindrome sequences located within the ORFs 002L, 006L, 024R, 025L, 026L, 033R, 034R, 053L, 063L, 065L, 090L, and 116L. Sequence analysis reveals that most of these sequences contain short imperfect palindromes and form hairpins with a variety of sequence sizes. The functions of these coding region-hairpin palindromes need further investigations. Furthermore, no consensus promoter regions were found within the noncoding palindrome sequences. The lacking of consensus promoter regions of these palindrome regions might suggest that GIV genes are regulated differentially within viral replication life cycle and that the GIV genes might be regulated by unique transcription factors or promoter regions. Besides the intragenic repeat sequence dots on the −45° line, the dot plot also revealed the repeat sequence dots out of the line (Fig. 2A). Most of these repeat sequences were located within GIV ORFs and contributed to the formation of redundant genes in the GIV genome. Although we could not find perfect sequence colinearity when comparing GIV to ATV and GIV to TFV, the dot plot revealed short parallel 45° or −45° lines (Fig. 2B and C). These lines indicated most of the iridovirus replication, transcription, nucleotide metabolism, and structural conserved ORFs.

FIG. 2.

prFLAG analysis of the GIV genome (horizontal axis) versus the ATV and TFV genomes (vertical axis). (A) The GIV genome; (B) the ATV genome; (C) the TFV genome.

Moreover, the short parallel lines also indicated the conserved ranavirus gene clusters, including gene clusters of ORF 020L-021R, 031L-032R, 037R-039R, and 066L-067R (Table 5). However, we could not find any sequence colinearity when comparing GIV to LCDV, ISKNV, and CIV (data not shown). In addition to the gene cluster revealing by dot plotting, comparing GIV genomic structure to those of ATV and TFV also revealed conserved gene clusters between these three ranaviruses (Table 5). Although the ORFs of gene cluster VIII are found in all six sequenced iridoviruses, they are not found to be clustered in LCDV, ISKNV, and CIV. Besides, gene cluster I and X were composed of ranavirus conserved ORFs, and so it can be considered that these gene clusters may be specific for the Ranavirus genus. However, this assumption needs more sequenced iridovirus genomes to support it.

TABLE 5.

Gene clusters of ranaviruses

Gene cluster	ORFs of ranavirus genome
	GIV	ATV	TFV
I	005L, 006L, 007L, 008L	70L, 71L, 72L, 73L	046L, 047L, 048L, 049L
II	020L, 021L	79L, 80L	020R, 019R
III	031L, 032R	77L, 78R	022R, 021L
IV	037R, 039R	64R, 65R	040R, 041R
V	042L, 043R, 044R, 045R	17R, 16L, 15L, 14L	093L, 094R, 095R, 096R
VI	046L, 047R	43R, 43L	065L, position 69, 879-70, 073
VII	054R, 055L, 056R, 057R	26L, 25R, 24L, 23L	084R, 085L, 086R, 087L
VIII	066L, 067R	10L, 11R	101R, 100L
IX	083L, 087R	45R, 44L	062L, 063R
X	091R, 092R	29R, 30R	080L, 079L
XI	100L, 101L, 102R	47L, 48L, 50R	059R, 058R, 056L

The ATV gene order of gene clusters V, VII, and IX and gene clusters II, III, VIII, X, and XI of TFV is inverse to the corresponding gene order of GIV. GIV ORFs of these gene clusters shared 20 to 70% of amino acid sequence identity with those of ATV and TFV. Although these three viruses are categorized into the same genus, Ranavirus, the comparison result suggested that ATV is closer to TFV than to GIV (30). The results of a similar genomic comparison of GIV to ISKNV, LCDV, and CIV genomes did not reveal such gene order conservation. It suggests that a greater extent of gene order is conserved in GIV, ATV, and TFV than in ISKNV, LCDV, and CIV.

The advances in high-throughput genomic sequencing technologies make the whole-genome comparison possible. Sequencing genes, which are homologous between different organisms, makes it obvious that many genes have been conserved over hundreds of millions of years of independent evolution. Such understanding makes the gene order of whole genomes a tool to distinguish the relations between different organisms (48). Generally the gene order is well preserved at close phylogenetic distance (49). When the species are not closely related, the degree of gene order conservation is usually low, and consequently it can be proposed that conservation of gene order is easily lost during evolution (26). The pairwise nucleotide alignment and genomic comparison suggest that the phylogenetic distance between GIV and ATV or between GIV and TFV could be greater than the distance between ATV and TFV (30). Repeat sequences and gene cluster inversions of GIV, ATV, and TFV genomes may suggest the higher recombination and genomic rearrangements of ranaviruses. High recombination rates have been described for the FV3 virus (9). The higher recombination rate may reflect the following facts about GIV. (i) GIV has more redundant genes. ORFs 060L, 061L, 063L, 064L, 109L, and 113L are similar to ATV 53R and TFV 23R. ORFs 017L and 019L are similar to neural cell adhesion molecule L1. ORFs 029L, 030L, and 065R are similar to TNFR. (ii) Unique genes exist in the GIV genome. Recombination may result in the insertion of new genes from host cells or other coinfected viruses such as PNP gene (ORF 049L), Bcl-2 like gene (ORF 078R), and host immunity interfering genes (ORF 104R). (iii) Conserved genus Ranavirus genes have been deleted. GIV genome lacks several conserved genes in Ranavirus, including DNA methyltransferase, thymidylate synthase, proliferating cell nuclear antigen, and eIF-2α. These facts may reflect the host range of GIV and the adaptation of GIV during host infection.

The similarity of G+C content and protein-coding content, the high percentage of ORF sharing, the high-level homology of ORF sequences, and the similar gene orders suggest that the GIV genome we describe here belongs to the genus Ranavirus along with the ATV and TFV. Syntenic conservation between GIV, ATV, and TFV provides us not only a more macroscopic analysis of the GIV genome's taxonomic position than analysis on the basis of one to a few individual genes but also facilitates our prediction of the approximate location of the relevant genes in different iridoviruses. However, to achieve a more precise prediction or understanding of iridovirus genome organization, more iridovirus genome information would be required.