Vol. 104: 203–214, 2013 DISEASES OF AQUATIC ORGANISMS Published June 13 doi: 10.3354/dao02587 Dis Aquat Org Complete genome sequence and transcription profiles of the rock bream iridovirus RBIV-C1 Bao-Cun Zhang1,2, Min Zhang1, Bo-Guang Sun1, Yong Fang1,2, Zhi-Zhong Xiao1, Li Sun1,* 1Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, PR China 2Graduate University of the Chinese Academy of Sciences, Beijing 100049, PR China ABSTRACT: The family Iridoviridae consists of 5 genera of double-stranded DNA viruses, includ- ing the genus Megalocytivirus, which contains species that are important fish pathogens. In a pre- vious study, we isolated the first rock bream iridovirus from China (RBIV-C1) and identified it as a member of the genus Megalocytivirus. In this report, we determined the complete genomic sequence of RBIV-C1 and examined its in vivo expression profiles. The genome of RBIV-C1 is 112 333 bp in length, with a GC content of 55% and a coding density of 92%. RBIV-C1 contains 4584 simple sequence repeats, 89.8% of which are distributed among coding regions. A total of 119 potential open reading frames (ORFs) were identified in RBIV-C1, including the 26 core iridovirus genes; 41 ORFs encode proteins that are predicted to be associated with essential biological func- tions. RBIV-C1 exhibits the highest degree of sequence conservation and colinear arrangement of genes with orange-spotted grouper iridovirus (OSGIV) and rock bream iridovirus (RBIV). The pair- wise nucleotide identities are 99.49% between RBIV-C1 and OSGIV and 98.69% between RBIV- C1 and RBIV. Compared to OSGIV, RBIV-C1 contains 11 insertions, 13 deletions, and 103 single nucleotide mutations. Whole-genome transcription analysis showed that following experimental infection of rock bream with RBIV-C1, all but 1 of the 119 ORFs were expressed at different time points and clustered into 3 hierarchical groups based on their expression patterns. These results provide new insights into the genetic nature and gene expression features of megalocytiviruses. KEY WORDS: Iridovirus · Megalocytivirus · Comparative genome · Oplegnathus fasciatus Resale or republication not permitted without written consent of the publisher INTRODUCTION fish, particularly in Asian countries such as China, Korea, and Japan (Kurita & Nakajima 2012). The host Iridoviruses are a family of large, double-stranded range of megalocytiviruses includes a variety of mar- DNA viruses that range from 120 to 200 nm in dia - ine and freshwater fish species, notably rock bream, meter. According to the Eighth Report of the Inter - turbot, grouper, cichlids, red sea bream, gourami, national Committee on Taxonomy of Viruses, the and sea bass (Chou et al. 1998, Wang et al. 2003, Go family Iridoviridae is divided into 5 genera: Irido - et al. 2006, Whittington et al. 2010). virus, Chloriridovirus, Lymphocystivirus, Rana virus, The complete genome sequences of at least 17 iri- and Megalocytivirus, which differ in host range, par- doviruses have been reported (Table 1). There are ticle size, and genetic characteristics (Chinchar et al. great variations with respect to genome size, GC 2005, Williams et al. 2005). Viruses belonging to 3 content, and coding capacity between different genera, Lymphocystivirus, Ranavirus, and Megalo- genera of irido viruses, and the genus Megalocy- cytivirus, are capable of infecting vertebrates includ- tivirus, which includes ISKNV, RSIV, RBIV, OSGIV, ing teleost fish. Of these viruses, megalocytiviruses TRBIV, and LYCIV, is divergent from all other gen- are known to cause heavy economic losses in farmed era. However, within the genus Megalocytivirus, *Corresponding author. Email: [email protected] © Inter-Research 2013 · www.int-res.com 204 Dis Aquat Org 104: 203–214, 2013 Table 1. Iridoviruses from vertebrate and invertebrate hosts, for which the viral genomes have been completely sequenced Virus Abbreviation GenBank accession no. Reference Lymphocystis disease virus type 1 LCDV-1 NC_001824 Tidona & Darai (1997) Infectious spleen and kidney necrosis virus ISKNV NC_003494 He et al. (2001) Chilo iridescent virusa CIV AF303741 Jakob et al. (2001) Tiger frog virus TFV AF389451 He et al. (2002) Red seabream iridovirus RSIV BD143114 Kurita et al. (2002) Ambystoma tigrinum stebbensi virus ATV AY150217 Jancovich et al. (2003) Rock bream iridovirus RBIV KC244182 Do et al. (2004) Frog virus 3 FV-3 NC_005946 Tan et al. (2004) Singapore grouper iridovirus SGIV NC_006549 Song et al. (2004) Lymphocystis disease virus isolated in China LCDV-C NC_005902 Zhang et al. (2004) Orange-spotted grouper iridovirus OSGIV AY894343 Lü et al. (2005) Grouper iridovirusb GIV AY666015 Tsai et al. (2005) Large yellow croaker iridovirus LYCIV AY779031 Ao & Chen (2006) Invertebrate iridescent virus type 3 IIV-3 DQ643392 Delhon et al. (2006) Soft-shelled turtle iridovirusc STIV EU627010 Huang et al. (2009) Turbot reddish body iridovirus TRBIV GQ273492 Shi et al. (2010) Rana grylio virus RGV JQ654586 Lei et al. (2012) aListed as species Insect iridescent virus 6 (IIV-6) in the ICTV database (http://ictvonline.org) bGIV is believed to be a related strain to SGIV cSTIV is believed to be a related strain to FV3 there exist high levels of sequence conservation and was filtered through a 0.2 µm membrane and stored colinear arrangement of genes (Eaton et al. 2007). In at −80°C. Viral DNA preparation was performed as general, different species of megalocytiviruses have described by Shinmoto et al. (2009), and the obtained comparable genome sizes (110−112 kb), GC content DNA was dissolved in TE buffer (10 mM Tris-HCl, (53−55%), and numbers of open reading frames 1 mM EDTA, pH 8.0). (ORFs; 115−124). Zhang et al. (2012) reported the first rock bream iridovirus isolate from China, RBIV-C1, which had PCR amplification and DNA sequencing caused a severe epidemic outbreak. RBIV-C1 was classified as a megalocytivirus based on the se quences The complete genomic DNA of RBIV-C was se - of conserved genes and histopathological symptoms quenced using a PCR-based method. As RBIV-C of the infected fish. In the present study, we sequenced showed high homology to RBIV and OSGIV, the the complete ge nome of RBIV-C1 and analyzed its primers were designed according to the DNA se - genetic features in comparison with other known quences of RBIV and OSGIV recovered from the megalocytiviruses. In addition, we also examined the GenBank database (accession nos. AY532606 and transcription profiles of all predicted ORFs of RBIV- AY894343). The PCR products ranged from 1000 to C1 during the course of infection in rock bream. Our 3000 bp in length, and the initial PCR products were results provide further understanding regarding the designed to have at least 200 bp of overlapping se - genetic nature and gene expression patterns of quence. PCR was conducted with a Biometra Tper- megalocytiviruses. sonal thermocycler in a volume of 20 µl containing 50 mM KCl, 20 mM Tris-HCl (pH 8.4), 1.5 mM MgCl2, 0.1% Triton X-100, 100 pM of each primer, MATERIALS AND METHODS 0.2 mM of each dNTP, 1 U Taq DNA polymerase, and 100 ng template DNA. The PCR products were Viral DNA preparation cloned into the T-A cloning vector pEASY-T1 (Trans- Gen Bio tech), and the recombinant plasmids were Spleen and kidney from the moribund rock bream used for sequence determination with an ABI PRISM were homogenized in pre-cooled phosphate-buffered 3730 automated DNA sequencer in both directions saline (PBS; pH 7.4), and the homogenate was cen- with the universal primers of the plasmid (M13 For- trifuged at 2500 × g (30 min at 4°C). The supernatant ward Primer and M13 Reverse Primer). Zhang et al.: Complete genome sequence of RBIV-C1 205 Sequence analysis according to the manufacturer’s instructions. qRT- PCR was carried out in an Eppendorf Mastercycler Genomic DNA composition, structure, and homolo- using the SYBR ExScript qRT-PCR Kit (Takara) as gous regions were analyzed using DNASTAR. The described previously (Zheng & Sun 2011). Melting ORFs and the encoded amino acid sequences were curve analysis of amplification products was per- predicted using Genome Annotation Transfer Utility formed at the end of each PCR to confirm that only 1 (http:// athena. bioc. uvic. ca/ help/ tool- help/ help-books/ PCR product was amplified and detected. The genome- annotation-transfer- utility- gatu-document- expression level of each gene was analyzed using the ation/) and the National Center for Biotechnology comparative threshold cycle method (2−ΔΔCT). The Information (NCBI) ORF finder (www. ncbi. nlm. nih. reaction was performed in triplicate, and the data are gov/ gorf/ gorf.html). Protein database searches were presented in terms of mRNA levels relative to that of conducted using BLASTP at the NCBI Web site β-actin and expressed as means ± SE. (www.ncbi.nlm.nih.gov). Comparative genome ana - lysis was performed with Clustal-X 2.0 (Larkin et al. 2007) and MAFFT (Katoh & Toh 2008). DNA dot RESULTS AND DISCUSSION matrix analysis was conducted using Java Dot Plot Alignments (Jdotter; Brodie et al. 2004). Characteristics of the RBIV-C1 genome GC content and coding capacity In vivo transcriptional profile of RBIV-C1 The complete genome of RBIV-C1 is similar in size Rock bream (average 14.6 g) were sourced from a to the genomes of RSIV, RBIV, OSGIV, LYCIV, fish farm in Shandong Province, China. RBIV-C1 was TRBIV, and ISKNV (Table 2). Like most iridoviruses, suspended in PBS to 1 ×106 copies ml−1. The fish were the RBIV-C1 se quence is circularly permuted and experimentally infected with RBIV-C1 by intraperi- was assembled into a circular form. RBIV-C1 has toneal injection of each fish with 100 µl RBIV-C1 sus- 55% G+C, which are distributed uniformly over the pension. At 0, 2, 5, 8, and 11 d post-infection (dpi), genome (Fig.