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Complete nucleotide sequence and genome organization of a single-stranded RNA virus infecting the marine fungoid protist Schizochytrium sp.

¹ Department of Biology, Faculty of Science and Engineering, Konan University, 8-9-1 Okamoto, Higashinada, Kobe 658-8501, Japan
² Laboratory of Plant Pathology, Graduate School of Agriculture, Kyoto University, Kyoto, Japan
³ Harmful Algal Bloom Division, National Research Institute of Fisheries and Environment of Inland Sea, Fisheries Research Agency, Hiroshima, Japan

Correspondence
Daiske Honda
dhonda{at}konan-u.ac.jp

	ABSTRACT

TOP ABSTRACT INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 
The complete nucleotide sequence of the genomic RNA of a marine fungoid protist-infecting virus (Schizochytrium single-stranded RNA virus; SssRNAV) has been determined. The viral RNA is single-stranded with a positive sense and is 9018 nt in length [excluding the 3' poly(A) tail]. It contains two long open reading frames (ORFs), which are separated by an intergenic region of 92 nt. The 5' ORF (ORF1) is preceded by an untranslated leader sequence of 554 nt. The 3' large ORF (ORF2) and an additional ORF (ORF3) overlap ORF2 by 431 nt and are followed by an untranslated region of 70 nt [excluding the 3' poly(A) tail]. The deduced amino acid sequences of ORF1 and ORF2 products show similarity to non-structural and structural proteins of dicistroviruses, respectively. However, Northern blot analysis suggests that SssRNAV synthesizes subgenomic RNAs to translate ORF2 and ORF3, showing that the translation mechanism of downstream ORFs is distinct from that of dicistroviruses. Furthermore, although considerable similarities were detected by using a BLAST genome database search, phylogenetic analysis based on both the nucleotide and amino acid sequences of the putative RNA-dependent RNA polymerase (RdRp) and the RNA helicase suggests that SssRNAV is phylogenetically distinct from other virus families. Therefore, it is concluded that SssRNAV is not a member of any currently defined virus family and belongs to a novel, unrecognized virus group.

The GenBank/EMBL/DDBJ accession number for the SssRNAV complete genome sequence determined in this study is AB193726.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 
It was revealed by the end of the 1980s that viruses are highly abundant in marine environments (Bergh et al., 1989; Proctor & Fuhrman, 1990), and viruses and virus-like particles (VLPs) have been discovered in a variety of phytoplankton and bacteria (Suttle et al., 1990, 1991; Van Etten et al., 1991). Until today, more than 13 viruses infecting marine eukaryotic microalgae have been isolated and characterized (Brussaard, 2004). The majority are large (100–200 nm in diameter), icosahedral, double-stranded DNA (dsDNA) viruses that are included in the family Phycodnaviridae (Schroeder et al., 2002; Brussaard, 2004; Brussaard et al., 2004b; Nagasaki et al., 2005; Wilson et al., 2005) or are considered to probably belong to this family (Jacobsen et al., 1996; Gastrich et al., 1998; Sandaa et al., 2001; Tarutani et al., 2001).

In contrast, there are few reports concerning viruses infecting apochlorotic protists. Nagasaki et al. (1993, 1995) observed large VLPs in marine apochlorotic flagellates. Garza & Suttle (1995) isolated and characterized a dsDNA virus infecting Bodo sp. (Bodonidae, Kinetoplastida, Mastigophora), with characteristics similar to those of phycodnaviruses in shape, size and site of propagation. Recently, an extremely large dsDNA virus (400 nm in diameter, 1·2 Mbp) was found infecting amoebae and named mimivirus (La Scola et al., 2005) and a genomic analysis was performed (Raoult et al., 2004). Based on phylogenetic analysis of the DNA polymerase amino acid sequence, the mimivirus formed a sister group with African swine fever virus, but not with viruses belonging to the family Phycodnaviridae. In the thraustochytrids, the protists belonging to the family Thraustochytriaceae in the class Labyrinthulomycetes, Kazama & Schornstein (1972, 1973) found herpes-type VLPs in Thraustochytrium sp. that were round, enveloped, 110 nm in diameter and predicted to have a DNA genome. However, this study has not been followed by any detailed analysis because of unsuccessful isolation.

There are few reports describing RNA viruses infecting marine eukaryotic micro-organisms. At present, four RNA viruses infecting marine eukaryotic microalgae have been isolated and examined: Heterosigma akashiwo RNA virus (HaRNAV; Tai et al., 2003), Heterocapsa circularisquama RNA virus (HcRNAV; Tomaru et al., 2004), Rhizosolenia setigera RNA virus (RsRNAV; Nagasaki et al., 2004a) and Micromonas pusilla RNA virus (MpRNAV; Brussaard et al., 2004a). HaRNAV is infectious to one of the noxious bloom-forming phytoflagellates, Heterosigma akashiwo (Raphidophyceae), and contains an 8·6 kb single-stranded RNA (ssRNA) genome (Lang et al., 2004). The genomic RNA contains only one large open reading frame (ORF) (7·7 kb) encoding both structural and non-structural proteins. Molecular phylogenetic analysis of the deduced amino acid sequence of the RNA-dependent RNA polymerase (RdRp) shows that HaRNAV is a distinct species related to the family Dicistroviridae (Lang et al., 2004). The family Dicistroviridae is a newly recognized virus family (split from the family Picornaviridae) that includes a marine virus that causes a disease of shrimp, the Taura syndrome virus (TSV) (Mari et al., 2002). HcRNAV is a small, ssRNA virus infectious to the bivalve-killing dinoflagellate Heterocapsa circularisquama (Tomaru et al., 2004). The genomic RNA is 4·4 kb in length and contains two ORFs encoding replicases and a structural protein, respectively (Nagasaki et al., 2006). Field surveys with regard to the ecological relationship between HcRNAV and its host alga revealed that viral infection is one of the most significant factors controlling the dynamics of host algal blooms (Nagasaki et al., 2004b; Tomaru & Nagasaki, 2004). RsRNAV is a small, ssRNA virus infectious to the bloom-forming diatom Rhizosolenia setigera (Nagasaki et al., 2004a). The major nucleic acid extracted from RsRNAV particles is an ssRNA molecule of 11·2 kb, with smaller RNA molecules (0·6, 1·2 and 1·5 kb) occasionally observed. MpRNAV is the only algal virus with a double-stranded RNA (dsRNA) genome presently isolated. It harbours 11 segments of dsRNA in the viral genome at 25·5 kb (Brussaard et al., 2004a). Among these four RNA viruses, intensive genome analysis has been conducted only for HaRNAV.

There is a single report of RNA viruses infecting protists, which described the basic characteristics of Schizochytrium single-stranded RNA virus (SssRNAV) (Takao et al., 2005). Its host organism, Schizochytrium sp., is a marine fungoid protist belonging to the thraustochytrids, which are cosmopolitan, heterotrophic micro-organisms playing important roles as decomposers, particularly in coastal ecosystems (Raghukumar, 1996, 2002). SssRNAV particles are icosahedral, lacking a tail and approximately 25 nm in diameter. They have a single-stranded, positive-sense RNA genome of 10·2 kb in length. Based on a number of similarities, such as morphological features, cytoplasmic assembly in an infected cell, number and size of the structural proteins and partial genome sequence, SssRNAV is predicted to be related closely to viruses belonging to the family Dicistroviridae and the marine microalgal virus HaRNAV (Takao et al., 2005).

To test this prediction, we describe the complete genome sequence and the genome organization of SssRNAV and consider the phylogenetic classification of this virus. To our knowledge, this is the first report describing the genome organization of an ssRNA virus infecting a marine fungoid protist.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 
Purification of virus.
A fresh suspension of SssRNAV (1·5 ml) was inoculated into a 1·5 l vigorously growing culture of Schizochytrium sp. (strain NIBH N1-27) at an m.o.i. >1 and incubated at 20 °C for 48 h. Virus particles were purified from the culture lysate. The lysate was centrifuged at 14 000 g, 4 °C for 15 min to remove cellular debris, using an RS-20BH centrifuge and angle rotor BH-9 (Tomy Seiko Co.). Polyethylene glycol 6000 (Wako) was added to the supernatant at 10 % (w/v) and incubated at 4 °C overnight. The preparation was centrifuged at 3600 g, 4 °C for 1·5 h using a CP56GII ultracentrifuge and angle rotor P42A (Hitachi Koki Co.). Then, the resulting pellet was resuspended in 120 ml 10 mM phosphate buffer (10 mM Na₂HPO₄, 10 mM KH₂PO₄ in distilled water, pH 7·2) and centrifuged again at 100 000 g, 4 °C for 2 h using a CP56GII ultracentrifuge and swing rotor P40ST. The pellet was resuspended in 60 ml 10 mM phosphate buffer and centrifuged again at 3600 g, 4 °C for 15 min. The supernatant was centrifuged at 100 000 g, 4 °C for 2 h. This centrifugation process (3600 g, 4 °C for 15 min and 100 000 g, 4 °C for 2 h) was repeated four times to purify the virus particles. Finally, the purified virus pellet was resuspended in 500 µl distilled, deionized water.

RNA isolation.
Two hundred microlitres of extraction buffer [0·33 M glycine, 0·33 M NaCl, 3·3 mM EDTA, 3·3 % SDS and 8·3 mg bentonite ml^–1 (pH 9·59)] was added to 500 µl purified virus suspension. The aqueous phase was extracted twice with 500 µl phenol/chloroform/isoamyl alcohol (25 : 24 : 1). The nucleic acids were precipitated with ethanol, dried and suspended in 30 µl RNase-free water.

Synthesis of cDNA, cloning and sequencing.
The viral RNA was used to synthesize cDNA as template. First- and second-strand synthesis was performed by using the SuperScript Choice system for cDNA synthesis (Invitrogen) according to the manufacturer's instructions, using both oligo(dT)_12–18 primers and random hexamers. The cDNA products were ligated with EcoRI linkers and inserted into the EcoRI site of the alkaline phosphatase-treated pBlueScript II SK(–) vector (Stratagene) by using a TaKaRa DNA Ligation kit (TaKaRa Bio Inc.). Then, the resultant plasmids were transformed into Escherichia coli DH5 competent cells as described previously (Hanahan et al., 1995).

A 5' RACE (rapid amplification of cDNA ends) analysis was performed to determine the 5'-end sequence of the SssRNAV genome RNA, as described previously (Iwamoto et al., 2001). The viral RNA was reverse-transcribed by using SuperScript II (Invitrogen) with SssRNAV-specific primer R5 (5'-CAAACAAGTCTAAATCGGC-3'; Fig. 1b), derived from the results of the above experiment, at 37 °C for 1 h. After purifying the product by using a SUPREC-02 column (TaKaRa Bio Inc.), the first-strand cDNAs were polyadenylated by using terminal deoxynucleotidyltransferase (TaKaRa Bio Inc.) at 37 °C for 5 min. Then, the second-strand cDNAs were synthesized by using Ex Taq polymerase (TaKaRa Bio Inc.) with an anchor primer [Anchor sequence (5'-GGCCACGCGTCGACTAGTAC-3') + Poly(T) (Iwamoto et al., 2001)] with one round of the following cycle parameters: denaturation at 95 °C (15 s), annealing at 55 °C (1 min) and extension at 72 °C (2 min). The double-stranded cDNAs were amplified with Ex Taq polymerase (TaKaRa Bio Inc.) using primer 5'-GGCCACGCGTCGACTAGTAC-3' (Iwamoto et al., 2001) and the SssRNAV-specific primer R7 (5'-ATCAAGTGCTGTGGGTG-3'; Fig. 1b) according to the following cycle parameters: denaturation at 95 °C (40 s), annealing at 55 °C (1 min) and extension at 72 °C (2 min). Following 30 rounds of amplification, the resultant PCR products were ligated into the pGEM-T Easy vector (Promega). Then, the resultant plasmids were transformed into E. coli DH5 competent cells as mentioned above. DNA sequencing was conducted by using a DNA auto-sequencer (model 310; Applied Biosystems); fragmented sequences were assembled by using DNASIS Mac software (Hitachi Software Engineering). To verify the sequence of a region with low redundancy (2), RT-PCR was performed by using the primers R6 (5'-TCCCTAATAGCGGAAAC-3'; Fig. 1b) and F15 (5'-CAATCTGTCACCAGATC-3'; Fig. 1b) and the amplicons were sequenced as described above.

View larger version (13K): [in this window] [in a new window]   Fig. 1. (a) Schematic diagram of the SssRNAV genome. Shaded boxes represent ORF1, ORF2 and ORF3. Numbers at the beginning and end of each ORF indicate the position of the proposed initiation and termination codons. Approximate regions encoding RNA helicase (H), protease (P) and RNA-dependent RNA polymerase (RdRp) in ORF1 and viral protein (VP3) in ORF2 are indicated. (b) Positions of primers used for 5' RACE and RT-PCR experiments. (c) Products of RT-PCR and 5' RACE and cDNAs used to determine the total nucleotide sequence of SssRNAV. (d) RNA probes used for Northern blot analysis (see Methods).

Total RNA was extracted from SssRNAV-infected cells of Schizochytrium sp. NIBH N1-27 at 5 h post-infection by using an RNeasy Plant mini kit (Qiagen). Total RNA extracted similarly from uninfected cells served as a control. RNA size markers (3·9 and 0·7 kb) were prepared as follows. Briefly, the cDNA fragments corresponding to nt 5057–9018 and 8234–9018 of the SssRNAV genome RNA were ligated into pBlueScript II SK(–) vector in an antisense orientation behind the T7 promoter using appropriate restriction enzymes; then, each RNA marker was transcribed in vitro by using T7 RNA polymerase (Roche Molecular Biochemicals). The RNAs were fractionated on a formaldehyde/agarose gel (1·5 % agarose, 0·5 % formaldehyde) and transferred onto a Hybond-N+ membrane (Amersham Biosciences); they were then cross-linked on the blots with UV illumination at 1200x100 µJ cm^–2 using a Funa-UV Cross-linker (Funakoshi). The membranes were incubated in hybridization buffer [50 % formamide, 5x SSC (1x SSC: 150 mM NaCl, 15 mM sodium citrate, pH 7·0), 2 % blocking reagent (Roche Molecular Biochemicals), 0·1 % sarcosine and 0·02 % SDS] for >1 h at 68 °C. The membranes were hybridized at 68 °C for 16 h with the RNA probes specific for the SssRNAV genome sequence described below. Hybridized membranes were washed twice in 2x SSC containing 0·1 % SDS at room temperature for 5 min, then washed three further times in 0·1x SSC containing 0·1 % SDS at 68 °C for 15 min. After washing, the viral RNA was detected immunologically by using anti-digoxigenin (DIG)–alkaline phosphatase Fab fragments (Roche Molecular Biochemicals) and CDP-Star (Roche Molecular Biochemicals) according to the manufacturer's protocols. We examined the preparation by using a luminescence image analyser (LAS 1000 Plus; Fuji Photo Film).

RNA probes to detect the ORF1 (probe 1) and ORF2 (probe 2) were prepared by cloning the cDNA fragments corresponding to nt 2062–3652 and 6950–8234 of the SssRNAV genome RNA, respectively (Fig. 1d), into the pBlueScript II SK(–) vector in an antisense orientation behind the T7 promoter by using appropriate restriction enzymes. The RNA probe to detect ORF3 (probe 3) was prepared by cloning the cDNA fragment corresponding to nt 8234–9018 (Fig. 1d) into the pBlueScript II SK(–) vector in an antisense orientation behind the T3 promoter. After linearization, each riboprobe was transcribed in vitro by using the appropriate RNA polymerase in the presence of DIG–dUTP (Roche Molecular Biochemicals) according to the manufacturer's recommendations.

Computer analysis of the sequences.
Computer analyses were performed on both the nucleotide sequence and the amino acid sequence of the two non-structural protein domains: the putative RNA-dependent RNA polymerase (RdRp) and the putative RNA helicase. The potential coding region in the SssRNAV genome RNA was predicted by using DNASIS Mac software. Database searches were performed by using BLASTX (Altschul et al., 1997).

Alignments of datasets were prepared by using the following procedures. First, the amino acid sequences were aligned automatically by using CLUSTAL_X (Thompson et al., 1997) and refined manually. Next, the nucleotide sequences were aligned according to the amino acid alignment data. The positions of the third base in each codon, ambiguous bases and gaps were removed for subsequent phylogenetic analysis and compared with this list of organisms (GenBank/DDBJ accession numbers are shown): Aichi virus (AiV), AB010145; human poliovirus 1 Mahoney (PV), V01149; Norwalk virus (NV), M87661; bovine enteric calicivirus (BoCV), AJ011099; Bean pod mottle virus (BPMV), NC_003496; Cowpea severe mosaic virus (CPSMV), M83830; Parsnip yellow fleck virus (PYFV), D14066; Rice tungro spherical virus (RTSV), M95497; deformed wing virus (DWV), NC_004830; sacbrood virus (SBV), NC_002066; black queen cell virus (BQCV), NC_003784; Cricket paralysis virus (CrPV), NC_003924; Drosophila C virus (DCV), NC_001834; triatoma virus (TrV), NC_003783; Taura syndrome virus (TSV), NC_003005; HaRNAV, NC_005281; and SssRNAV, AB193726 (determined in this study).

Phylogenetic analyses.
Generally, it is considered problematic to infer basal evolutionary relationships between RNA viruses based only on single-gene phylogenies (Zanotto et al., 1996); thus, in most cases, phylogenetic trees are constructed by using concatenated sequences of genes, which are treated as an undivided long unity. However, analyses of combined sequences do not explicitly take into account the differences of tempo and mode of evolution among the different genes. Therefore, we selected the total-evaluation method, using maximum-likelihood analyses of multiple genes by using the TotalML program in the MOLPHY version 2.3 package (Adachi & Hasegawa, 1996). The total support for a particular tree can be evaluated simply by summing up the estimated log-likelihoods of each individual gene, and the total log-likelihoods for different trees can then be compared (Adachi & Hasegawa, 1996). Using this method, the program must calculate the likelihood values for given topologies, so the selection of topologies is very important for evaluation within a reasonable time. First, the constraint tree was used to search for the best topology. The consensus tree (Fig. 2) was generated from eight trees that were constructed by using neighbour-joining (NJ; Saitou & Nei, 1987) and maximum-likelihood (ML; Kishino & Hasegawa, 1989) methods for both amino acid and nucleotide sequences for each single gene: the RdRp and RNA helicase (phylogenetic trees 4–11 in Table 1). The monophyletic relationships in this consensus tree were supported by all eight trees, so that each monophyly was previously fixed in determining the topologies, i.e. the consensus tree was used as the constraint tree. The candidate topologies were selected by the approximate-likelihood (AL) criterion (Adachi, 1995). In the case of the nucleotide sequences, the best 2000 topologies each for RdRp and RNA helicase were searched exhaustively from all of the topologies included in the constraint tree (34 459 425 topologies) by using the NucML program in the MOLPHY package with the AL criterion (e option). Finally, the best tree(s) with the maximum log-likelihood value was selected by the total evaluation of two genes using the NucML (l option) and TotalML programs from 4000 topologies that contained each of the best 2000 topologies for RdRp and RNA helicase with allowed repetition (Table 1). The Ti/Tv ratio parameters in the HKY model (Hasegawa et al., 1985) from the RdRp and RNA helicase nucleotide sequences were estimated by using MODELTEST version 3.6 (Posada & Crandall, 1998). Amino acid sequences were analysed by using the ProtML and TotalML programs with the JTT model (Jones et al., 1992) as predicted by the above-mentioned nucleotide-sequence analysis.

View larger version (16K): [in this window] [in a new window]   Fig. 2. Consensus tree generated from the eight trees. The tree was constructed by using PHYLIP ver. 3.6a3 by the strict-rule consensus-tree method from eight phylogenetic trees in Table 1 (phylogenetic trees 4–11).

Assessing the confidence limits of the phylogenetic trees.
Selected topologies were assessed [the rejection limit was set at 10 % (P value >0·1)] for confidence by the Shimodaira–Hasegawa (SH) test (Shimodaira & Hasegawa, 1999) based on nucleotide and amino acid sequences by using a combination of NucML, ProtML and CONSEL version 0.1h (Shimodaira & Hasegawa, 2001).

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 
cDNA clones and sequence analysis of SssRNAV RNA
The nucleotide sequence of the viral genome RNA was determined by assembling four overlapping cDNA clones and five RT-PCR clones. The 5'-terminal nucleotide sequence was determined by comparing the sequences of seven clones (Fig. 1c). Consequently, the SssRNAV genome was shown to have a 3' poly(A) tail and was estimated to be 9018 nt in length, excluding the poly(A) tail (Fig. 1a). Thus, the genome was determined to be smaller than the previously estimated size by means of formaldehyde/agarose gel electrophoresis (10·2 kb; Takao et al., 2005), which may reflect the additional length of the 3' poly(A) tail. The base composition was A, 24·1 %; C, 26·1 %; G, 23·7 %; U, 26·1 %. The A+U content of SssRNAV was 50·2 %; this is not as high as those of other viruses within the family Dicistroviridae (56·8–64·1 %; Table 2).

There are three AUG initiation codons within the first 100 nt of ORF1. The first and second AUG codons are located at nt 555–557 and 578–580, respectively, and the sequences around them (UGUAUGC and UAUAUGC) are not in agreement with the most common initiation sequence Kozak consensus, ACCAUGG (Kozak, 1991). In contrast, the sequence around the third AUG codon (nt 627–629) coincides with the Kozak consensus (Fig. 1a). Although there are three and two AUG initiation codons within the first 150 nt of ORF2 and ORF3, respectively, no Kozak consensus is identified. Currently, we do not have experimental data to demonstrate which AUG codon works as the initiation codon for each ORF.

Alignment of the amino acid sequence for non-structural proteins
The deduced amino acid sequence of ORF1 showed high similarity to proteins involved in the replication of picorna-like viruses, especially from the family Dicistroviridae (e value >1x10^–25). Further analysis revealed that it contains the core motifs of the picornavirus 2C RNA helicase, 3C cysteine protease and 3D RNA-dependent RNA polymerase (RdRp) (Koonin & Dolja, 1993). The first conserved motif of RNA helicase, GXXGXGK (motif A), was found at aa 343–349, assuming that the first AUG (nt 555–557) is the initiation codon (Fig. 3a); the second conserved motif, QX₅DD (motif B), was not identified; the third domain, KGX₄SX₅STN (motif C), was found as KGX₅PX₅DTN at aa 437–452 (Fig. 3a). The cysteine protease motif, GXCG, was found at aa 933–936 (Fig. 3b). The conserved domains of RdRp, LKDE (motif I), SGX₃TX₃N (motif V), YGDD (motif VI) and LKR (motif VII), were found at aa 1208–1211, 1350–1359, 1405–1408 and 1459–1461, respectively (Fig. 3d).

View larger version (76K): [in this window] [in a new window]   Fig. 3. Multiple alignments of conserved regions in putative replicases of SssRNAV and picorna-like viruses. The number to the left of each sequence indicates amino acid position relative to the corresponding replicase protein, and those within sequences represent the number of omitted amino acids. Residues identical in more than three viruses are shaded. (a) Alignments of putative helicase domains, in which conserved regions (Koonin & Dolja, 1993) are marked Hel-A, -B and -C. (b) Alignments of protease domains where asterisks indicate amino acid residues that may be essential for protease activity. (c) Alignment of partial regions of viral capsid proteins of CrPV, DCV, BQCV, HaRNAV and SssRNAV. The vertical bar indicates the cleavage site for VP3 and VP4. (d) Alignments of RdRp domains where conserved regions (Koonin & Dolja, 1993) are marked I–VIII.

SDS-PAGE analysis revealed that SssRNAV has three major structural proteins (37, 34 and 32 kDa) and one minor protein (18 kDa) (Takao et al., 2005). We designated them VP1, VP2, VP3 and VP4 in decreasing order of their molecular masses. The N-terminal sequence of VP3 was revealed to be SKPLV by using Edman degradation (data not shown). This coincides with the deduced amino acid sequence from ORF2 (nt 7046–7060). This amino acid sequence is consistent with the N-terminal sequence of major capsid proteins from dicistroviruses, HaRNAV and picorna-like viruses (Fig. 3c). The N-terminal sequence of the other capsid proteins could not be determined by using Edman degradation, presumably due to an N-terminal block.

Northern blot analysis and translation strategy of ORF2 and ORF3
Northern blot analysis of the total RNA extracted from SssRNAV-infected cells using probe 1 (specific for ORF1) revealed one RNA band of the viral genome size (Fig. 4, lane 1). In contrast, two bands (5·7 and 4·9 kb) in addition to the genome-size RNA were detected in the analysis using probe 2 (specific for ORF2) (Fig. 4, lane 2). When using probe 3 (specific for ORF3), a dense band of 0·85 kb and two paler bands similar to those identified by using probe 2 were observed (Fig. 4, lane 3).

View larger version (51K): [in this window] [in a new window]   Fig. 4. Northern blot analysis for total RNA extracted from Schizochytrium sp. N1-27 strain 5 h post-infection with SssRNAV. Viral RNAs were detected by using probe 1 (lane 1), probe 2 (lane 2) or probe 3 (lane 3). Refer to Fig. 1(d) forthe region complementary to each riboprobe. Note that nohybridization was detected in the total RNA extracted from non-infected host cells by using any of the three probes (data not shown). Positions for the RNA size markers are indicated to the right.

There are three conceivable ways to translate ORF2 and ORF3 of SssRNAV: (i) a read-through translation and/or ribosomal frameshift may occur between ORF1 and ORF2 and between ORF2 and ORF3; (ii) translation of ORF2 and/or ORF3 is initiated independently by an internal ribosome entry site (IRES); or (iii) the subgenomic RNA may be synthesized during replication that includes ORF2 or ORF3. Although we cannot exclude the first possibility, the second method is used by the family Dicistroviridae (Mayo, 2002). The intergenic region (IGR) of these viruses forms stem–loop structures that act as an IRES (Sasaki & Nakashima, 2000). The ribosomes bind this structure directly and initiate translation of the downstream ORF without a universal AUG initiation codon (Sasaki & Nakashima, 2000; Wilson et al., 2000). Because we failed to determine the N-terminal sequence of VP1, we could not identify the IGR precisely; thus, its secondary structure was not determined. Although the IRESs of dicistroviruses share some similarity at the nucleotide-sequence level, no sequence showing similarity to IRESs of dicistroviruses was identified within the SssRNAV genome. In general, when the IGR contains an IRES, only one type of messenger-sense RNA containing both ORFs is produced, and the ribosomes bind directly to the IRES and translate each ORF independently (Sasaki & Nakashima, 2000; Wilson et al., 2000). The synthesis of subgenomic RNA is another tactic commonly found in many positive-sense ssRNA viruses to translate downstream ORFs (Miller & Koev, 2000). Northern blot analysis shows that smaller-sized RNAs also accumulated during SssRNAV replication (Fig. 4), suggesting the production of subgenomic RNAs. Further study is required to determine whether the detected RNAs are the subgenomic RNAs of SssRNAV.

Detection of a 0·85 kb band (Fig. 4, lane 3) strongly suggests that the protein encoded by ORF3 is also expressed from the subgenomic RNA that is synthesized during replication. Independent synthesis of the ORF3 subgenomic RNA is a remarkable feature of SssRNAV that caliciviruses do not have (Koopmans et al., 2005). No significant similarity was found in GenBank for the ORF3 product. Further study is necessary to determine the function of ORF3.

Phylogenetic analysis
The total evaluation of RdRp and RNA helicase with the ML analyses provided three topologies that contained two equally good topologies based on the nucleotide sequences and one best topology based on the amino acid sequences (Fig. 5). SssRNAV was located in a different phylogenetic position in each topology. Three of these trees showed the following monophyletic relationships, which were also supported by high bootstrap probabilities: Caliciviridae and Picornaviridae; Comoviridae and Sequiviridae; all members of the family Dicistroviridae, excluding the genus Iflavirus. However, SssRNAV did not form a monophyletic group with any of the described families. In the TotalML tree based on nucleotide sequence, SssRNAV and HaRNAV formed a monophyly; however, this relationship was supported by a low bootstrap probability (Fig. 5b).

View larger version (36K): [in this window] [in a new window]   Fig. 5. Phylogenetic trees selected by using the TotalML program. Best results based on nucleotide sequence (a, b) and amino acid sequence (c). The branch lengths were estimated by using the ML method based on concatenated nucleotide or the amino acid sequence of RdRp and helicase, respectively. Numbers at each internal branch represent the bootstrap values. Nuc, Nucleotide sequence; Prot, protein; LBP, local bootstrap probability.

These phylogenetic analyses suggest that all of the five established families are probably natural taxa, and that SssRNAV and HaRNAV have an independent lineage that evolved from a deep internal branch in the picorna-like viruses. Therefore, each of these marine RNA viruses should be recognized as a distinct family-level taxon, even if the number of members of the taxon is small. Moreover, if SssRNAV is classified in the family Dicistroviridae based only on the similarity of the genome, it is clear that the reliable taxonomic criterion will be lost.

Conclusions
In this study, we have determined and analysed the genome sequence of SssRNAV infecting the marine fungoid protist Schizochytrium sp. This is, to our knowledge, the first report describing the genome organization of ssRNA viruses infecting marine fungoid protists. The sequence analysis showed some similarity of SssRNAV to members of the family Dicistroviridae in genome size and arrangement, and also the amino acid sequence of structural and non-structural proteins (Figs 3). However, Northern blot data suggest that SssRNAV does not utilize IRES-mediated translation for downstream ORFs. Furthermore, phylogenetic analyses indicated that SssRNAV and dicistroviruses do not have a close relationship and they should be classified into distinct groups. Additionally, the A+U content of SssRNAV is not as high as those of members of the family Dicistroviridae. In comparing SssRNAV and HaRNAV, both infecting marine eukaryotic micro-organisms, their hosts are classified within the same category, the stramenopiles, which are characterized by common ultrastructure (Patterson, 1989); however, these two viruses are clearly different in genome size, genome structure and, presumably, in replication mechanism. From these results, we conclude that SssRNAV is a species distinct from those belonging to the family Dicistroviridae and other virus families, and that SssRNAV is a member of a previously unrecognized virus group.

Few reports for picorna-like viruses isolated from marine environments have been made, although there should exist a huge number of unrecognized and highly diversified RNA viruses. This is based on the analysis of the RdRp gene sequence amplified from the natural marine communities, where Culley et al. (2003) postulated that marine RNA viruses are highly diverse. Hence, for understanding the classification and evolutionary history of RNA viruses more precisely, we should note that the ocean is a fascinating field where findings of undiscovered RNA viruses are strongly expected; thus, more efforts should be thrown into it.

	ACKNOWLEDGEMENTS

 
This work was supported by the Kato Memorial Bioscience Foundation and the Asahi Glass Foundation. The authors are grateful to Dr Toro Nakahara, Dr Toshihiro Yokochi (National Institute of Advanced Industrial Science and Technology, Japan) and Ms Rinka Yokoyama (Konan University, Japan), who kindly provided the thraustochytrid strains. We wish to thank Dr Hiroyuki Mizumoto and Mr Masahiro Tatsuta (Kyoto University, Japan) for their technical assistance in the molecular biology experiments. We are also grateful to Dr Hidetoshi Shimodaira (Tokyo Institute of Technology, Japan), who kindly gave help in the phylogenetic analyses.

	REFERENCES

TOP ABSTRACT INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 
Adachi, J. (1995). Modeling of molecular evolution and maximum likelihood inference of molecular phylogeny. PhD thesis, The Graduate University for Advanced Studies, Hayama, Japan.

Adachi, J. & Hasegawa, M. (1996). MOLPHY version 2.3: programs for molecular phylogenetics based on maximum likelihood. Computer Science Monographs no. 28. Tokyo: The Institute of Statistical Mathematics.

Altschul, S. F., Madden, T. L., Schäffer, A. A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D. J. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs.Nucleic Acids Res 25, 3389–3402.[Abstract/Free Full Text]

Bergh, Ø., Børsheim, K. Y., Bratbak, G. & Heldal, M. (1989). High abundance of viruses found in aquatic environments. Nature 340, 467–468.[CrossRef][Medline]

Brussaard, C. P. D. (2004). Viral control of phytoplankton populations – a review. J Eukaryot Microbiol 51, 125–138.[CrossRef][Medline]

Brussaard, C. P. D., Noordeloos, A. A. M., Sandaa, R.-A., Heldal, M. & Bratbak, G. (2004a). Discovery of a dsRNA virus infecting the marine photosynthetic protist Micromonas pusilla. Virology 319, 280–291.[CrossRef][Medline]

Brussaard, C. P. D., Short, S. M., Frederickson, C. M. & Suttle, C. A. (2004b). Isolation and phylogenetic analysis of novel viruses infecting the phytoplankton Phaeocystis globosa (Prymnesiophyceae). Appl Environ Microbiol 70, 3700–3705.[Abstract/Free Full Text]

Culley, A. I., Lang, A. S. & Suttle, C. A. (2003). High diversity of unknown picorna-like viruses in the sea. Nature 424, 1054–1057.[CrossRef][Medline]

Edman, P. (1950). Method of determination of the amino acid sequence in peptides. Acta Chem Scand 4, 283–293.

Felsenstein, J. (1985). Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783–791.[CrossRef]

Felsenstein, J. (2002). PHYLIP: phylogeny inference package, version 3.6a3, July 2002. Department of Genome Sciences, University of Washington, Seattle, WA, USA.

Garza, D. R. & Suttle, C. A. (1995). Large double-stranded DNA viruses which cause the lysis of a marine heterotrophic nanoflagellate (Bodo sp.) occur in natural marine viral communities. Aquat Microb Ecol 9, 203–210.

Gastrich, M. D., Anderson, O. R., Benmayor, S. S. & Cosper, E. M. (1998). Ultrastructural analysis of viral infection in the brown-tide alga, Aureococcus anophagefferens (Pelagophyceae). Phycologia 37, 300–306.

Hanahan, D., Jessee, J. & Bloom, F. R. (1995). Techniques for transformation of E. coli. In DNA Cloning 1: Core Techniques (a Practical Approach), pp. 1–36. Edited by D. M. Glover & B. D. Hames. New York: Oxford University Press.

Hasegawa, M., Kishino, H. & Yano, T. (1985). Dating of the human-ape splitting by a molecular clock of mitochondrial DNA. J Mol Evol 22, 160–174.[CrossRef][Medline]

Iwamoto, T., Mise, K., Mori, K., Arimoto, M., Nakai, T. & Okuno, T. (2001). Establishment of an infectious RNA transcription system for Striped jack nervous necrosis virus, the type species of the betanodaviruses. J Gen Virol 82, 2653–2662.[Abstract/Free Full Text]

Jacobsen, A., Bratbak, G. & Heldal, M. (1996). Isolation and characterization of a virus infecting Phaeocystis pouchetii (Prymnesiophyceae). J Phycol 32, 923–927.[CrossRef]

Jones, D. T., Taylor, W. R. & Thornton, J. M. (1992). The rapid generation of mutation data matrices from protein sequences. Comput Appl Biosci 8, 275–282.[Abstract/Free Full Text]

Kazama, F. Y. & Schornstein, K. L. (1972). Herpes-type virus particles associated with a fungus. Science 177, 696–697.[Abstract/Free Full Text]

Kazama, F. Y. & Schornstein, K. L. (1973). Ultrastructure of a fungus herpes-type virus. Virology 52, 478–487.[CrossRef][Medline]

Kishino, H. & Hasegawa, M. (1989). Evaluation of the maximum likelihood estimate of the evolutionary tree topologies from DNA sequence data, and the branching order in hominoidea. J Mol Evol 29, 170–179.[CrossRef][Medline]

Koonin, E. V. & Dolja, V. V. (1993). Evolution and taxonomy of positive-strand RNA viruses: implication of comparative analysis of amino acid sequences. Crit Rev Biochem Mol Biol 28, 375–430.[Medline]

Koopmans, M. K., Green, K. Y., Ando, T. & 8 other authors (2005). Caliciviridae. In Virus Taxonomy: Eighth Report of the International Committee on Taxonomy of Viruses, pp. 843–851. Edited by C. M. Fauquet, M. A. Mayo, J. Maniloff, U. Desselberger & L. A. Ball. San Diego: Academic Press.

Kozak, M. (1991). An analysis of vertebrate mRNA sequences: intimations of translational control. J Cell Biol 115, 887–903.[Abstract/Free Full Text]

Lang, A. S., Culley, A. I. & Suttle, C. A. (2004). Genome sequence and characterization of a virus (HaRNAV) related to picorna-like viruses that infects the marine toxic bloom-forming alga Heterosigma akashiwo. Virology 320, 206–217.[CrossRef][Medline]

La Scola, B., Fournier, P.-E., Gouin, F., Motte, A. & Raoult, D. (2005). Mycoplasma pneumoniae: a rarely diagnosed agent in ventilator-acquired pneumonia. J Hosp Infect 59, 74–75.[Medline]

Mari, J., Poulos, B. T., Lightner, D. V. & Bonami, J.-R. (2002). Shrimp Taura syndrome virus: genomic characterization and similarity with members of the genus Cricket paralysis-like viruses. J Gen Virol 83, 915–926.[Abstract/Free Full Text]

Mayo, M. A. (2002). Virus Taxonomy – Houston 2002. Arch Virol 147, 1071–1076.[CrossRef][Medline]

Miller, W. A. & Koev, G. (2000). Synthesis of subgenomic RNAs by positive-strand RNA viruses. Virology 273, 1–8.[CrossRef][Medline]

Nagasaki, K., Ando, M., Imai, I., Itakura, S. & Ishida, Y. (1993). Virus-like particles in an apochlorotic flagellate in Hiroshima Bay, Japan. Mar Ecol Prog Ser 96, 307–310.

Nagasaki, K., Ando, M., Imai, I., Itakura, S. & Ishida, Y. (1995). Virus-like particles in unicellular apochlorotic microorganisms in the coastal water of Japan. Fish Sci 61, 235–239.

Nagasaki, K., Tomaru, Y., Katanozaka, N., Shirai, Y., Nishida, K., Itakura, S. & Yamaguchi, M. (2004a). Isolation and characterization of a novel single-stranded RNA virus infecting the bloom-forming diatom Rhizosolenia setigera. Appl Environ Microbiol 70, 704–711.[Abstract/Free Full Text]

Nagasaki, K., Tomaru, Y., Nakanishi, K., Hata, N., Katanozaka, N. & Yamaguchi, M. (2004b). Dynamics of Heterocapsa circularisquama (Dinophyceae) and its viruses in Ago Bay, Japan. Aquat Microb Ecol 34, 219–226.

Nagasaki, K., Shirai, Y., Tomaru, Y., Nishida, K. & Pietrokovski, S. (2005). Algal viruses with distinct intraspecies host specificities include identical intein elements. Appl Environ Microbiol 71, 3599–3607.[Abstract/Free Full Text]

Nagasaki, K., Shirai, Y., Takao, Y., Mizumoto, H., Nishida, K. & Tomaru, Y. (2006). Comparative genome sequence of single-stranded RNA viruses infecting the bivalve-killing dinoflagellate Heterocapsa circularisquama. Appl Environ Microbiol (in press).

Patterson, D. J. (1989). Stramenopiles: chromophytes from a protistan perspective. In The Chromophyte Algae: Problems and Perspectives, pp. 357–379. Edited by J. C. Green, B. S. C. Leadbeater & W. L. Diver. Oxford: Clarendon Press.

Posada, D. & Crandall, K. A. (1998). MODELTEST: testing the model of DNA substitution. Bioinformatics 9, 817–818.

Proctor, L. M. & Fuhrman, J. A. (1990). Viral mortality of marine bacteria and cyanobacteria. Nature 343, 60–62.[CrossRef]

Raghukumar, S. (1996). Morphology, taxonomy, and ecology of thraustochytrids and labyrinthulids, the marine counterparts of zoosporic fungi. In Advances in Zoosporic Fungi, pp. 35–60. Edited by R. Dayal. New Delhi: Publications Pvt Ltd.

Raghukumar, S. (2002). Ecology of the marine protists, the labyrinthulomycetes (thraustochytrids and labyrinthulids). Eur J Protistol 38, 127–145.[CrossRef]

Raoult, D., Audic, S., Robert, C., Abergel, C., Renesto, P., Ogata, H., La Scola, B., Suzan, M. & Claverie, J.-M. (2004). The 1·2-megabase genome sequence of mimivirus. Science 306, 1344–1350.[Abstract/Free Full Text]

Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425.[Abstract]

Sandaa, R.-A., Heldal, M., Castberg, T., Thyrhaug, R. & Bratbak, G. (2001). Isolation and characterization of two viruses with large genome size infecting Chrysochromulina ericina (Prymnesiophyceae) and Pyramimonas orientalis (Prasinophyceae). Virology 290, 272–280.[CrossRef][Medline]

Sasaki, J. & Nakashima, N. (2000). Methionine-independent initiation of translation in the capsid protein of an insect RNA virus. Proc Natl Acad Sci U S A 97, 1512–1515.[Abstract/Free Full Text]

Schroeder, D. C., Oke, J., Malin, G. & Wilson, W. H. (2002). Coccolithovirus (Phycodnaviridae): characterisation of a new large dsDNA algal virus that infects Emiliania huxleyi. Arch Virol 147, 1685–1698.[CrossRef][Medline]

Shimodaira, H. & Hasegawa, M. (1999). Multiple comparisons of log-likelihoods with applications to phylogenetic inference. Mol Biol Evol 16, 1114–1116.

Shimodaira, H. & Hasegawa, M. (2001). CONSEL: for assessing the confidence of phylogenetic tree selection. Bioinformatics 17, 1246–1247.[Abstract/Free Full Text]

Suttle, C. A., Chan, A. M. & Cottrell, M. T. (1990). Infection of phytoplankton by viruses and reduction of primary productivity. Nature 347, 467–469.[CrossRef]

Suttle, C. A., Chan, A. M. & Cottrell, M. T. (1991). Use of ultrafiltration to isolate viruses from seawater which are pathogens of marine phytoplankton. Appl Environ Microbiol 57, 721–726.[Abstract/Free Full Text]

Swofford, D. L. (2003). PAUP*: Phylogenetic analysis using parsimony (* and other methods), version 4.0b 10. Sunderland, MA: Sinauer Associates.

Tai, V., Lawrence, J. E., Lang, A. S., Chan, A. M., Culley, A. I. & Suttle, C. A. (2003). Characterization of HaRNAV, a single-stranded RNA virus causing lysis of Heterosigma akashiwo (Raphidophyceae). J Phycol 39, 343–352.

Takao, Y., Nagasaki, K., Mise, K., Okuno, T. & Honda, D. (2005). Isolation and characterization of a novel single-stranded RNA virus infectious to a marine fungoid protist, Schizochytrium sp. (Thraustochytriaceae, Labyrinthulea). Appl Environ Microbiol 71, 4516–4522.[Abstract/Free Full Text]

Tarutani, K., Nagasaki, K., Itakura, S. & Yamaguchi, M. (2001). Isolation of a virus infecting the novel shellfish-killing dinoflagellate Heterocapsa circularisquama. Aquat Microb Ecol 23, 103–111.

Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G. (1997). The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25, 4876–4882.[Abstract/Free Full Text]

Tomaru, Y. & Nagasaki, K. (2004). Widespread occurrence of viruses lytic to the bivalve-killing dinoflagellate Heterocapsa circularisquama in the western coast of Japan. Plankton Biol Ecol 51, 1–6.

Tomaru, Y., Katanozaka, N., Nishida, K., Shirai, Y., Tarutani, K., Yamaguchi, M. & Nagasaki, K. (2004). Isolation and characterization of two distinct types of HcRNAV, a single-stranded RNA virus infecting the bivalve-killing microalga Heterocapsa circularisquama. Aquat Microb Ecol 34, 207–218.[CrossRef]

Van Etten, J. L., Lane, L. C. & Meints, R. H. (1991). Viruses and viruslike particles of eukaryotic algae. Microbiol Rev 55, 586–620.[Abstract/Free Full Text]

Wilson, J. E., Powell, M. J., Hoover, S. E. & Sarnow, P. (2000). Naturally occurring dicistronic cricket paralysis virus RNA is regulated by two internal ribosome entry sites. Mol Cell Biol 20, 4990–4499.[Abstract/Free Full Text]

Wilson, W. H., Van Etten, J. L., Schroeder, D. S., Nagasaki, K., Brussard, C., Delaroque, N., Bratbak, G. & Suttle, C. (2005). Phycodnaviridae. In Virus Taxonomy: Eighth Report of the International Committee on Taxonomy of Viruses, pp. 163–175. Edited by C. M. Fauquet, M. A. Mayo, J. Maniloff, U. Desselberger & L. A. Ball. San Diego: Academic Press.

Zanotto, P. M. de A., Gibbs, M. J., Gould, E. A. & Holmes, E. C. (1996). A reevaluation of the higher taxonomy of viruses based on RNA polymerases. J Virol 70, 6083–6096.[Abstract]

Received 24 May 2005; accepted 4 November 2005.

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