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The complete genomes of three viruses assembled from shotgun libraries of marine RNA virus communities Culley, Alexander I; Lang, Andrew S; Suttle, Curtis A Jul 6, 2007

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ralssBioMed CentVirology JournalOpen AcceResearchThe complete genomes of three viruses assembled from shotgun libraries of marine RNA virus communitiesAlexander I Culley1, Andrew S Lang2 and Curtis A Suttle*1,3Address: 1University of British Columbia, Department of Botany, 3529-6270 University Blvd, Vancouver, B.C. V6T 1Z4, Canada, 2Department of Biology, Memorial University of Newfoundland, St. John's, NL A1B 3X9, Canada and 3University of British Columbia, Department of Earth and Ocean Sciences, Department of Microbiology and Immunology, 1461-6270 University Blvd, Vancouver, BC, V6T 1Z4, CanadaEmail: Alexander I Culley - culley@interchange.ubc.ca; Andrew S Lang - aslang@mun.ca; Curtis A Suttle* - csuttle@eos.ubc.ca* Corresponding author    AbstractBackground: RNA viruses have been isolated that infect marine organisms ranging from bacteriato whales, but little is known about the composition and population structure of the in situ marineRNA virus community. In a recent study, the majority of three genomes of previously unknownpositive-sense single-stranded (ss) RNA viruses were assembled from reverse-transcribed whole-genome shotgun libraries. The present contribution comparatively analyzes these genomes withrespect to representative viruses from established viral taxa.Results: Two of the genomes (JP-A and JP-B), appear to be polycistronic viruses in the proposedorder Picornavirales that fall into a well-supported clade of marine picorna-like viruses, thecharacterized members of which all infect marine protists. A temporal and geographic surveyindicates that the JP genomes are persistent and widespread in British Columbia waters. The thirdgenome, SOG, encodes a putative RNA-dependent RNA polymerase (RdRp) that is related to theRdRp of viruses in the family Tombusviridae, but the remaining SOG sequence has no significantsimilarity to any sequences in the NCBI database.Conclusion: The complete genomes of these viruses permitted analyses that resulted in a morecomprehensive comparison of these pathogens with established taxa. For example, in concordancewith phylogenies based on the RdRp, our results support a close homology between JP-A and JP-B and RsRNAV. In contrast, although classification of the SOG genome based on the RdRp placesSOG within the Tombusviridae, SOG lacks a capsid and movement protein conserved within thisfamily and SOG is thus likely more distantly related to the Tombusivridae than the RdRp phylogeneyindicates.BackgroundRNA viruses of every classification have been isolatedfrom the ocean; nevertheless, the marine RNA virus com-munity remains largely uncharacterized. Although therethe organisms in the sea; therefore it is unlikely thatviruses infecting these organisms make up a significantfraction of the natural RNA virioplankton. Marine RNAphages appear to be rare [2] and thus it is more likely thatPublished: 6 July 2007Virology Journal 2007, 4:69 doi:10.1186/1743-422X-4-69Received: 10 May 2007Accepted: 6 July 2007This article is available from: http://www.virologyj.com/content/4/1/69© 2007 Culley et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Page 1 of 9(page number not for citation purposes)are several examples of RNA viruses that infect marine ani-mals [1] these organisms represent a very small portion ofthe dominant RNA viruses infect the diverse and abun-dant marine protists. For example, RNA viruses haveVirology Journal 2007, 4:69 http://www.virologyj.com/content/4/1/69recently been isolated that infect a number of marine pro-tists including a diatom [3], a dinoflagellate [4], araphidophyte [5], a prasinophyte [6] and a thrausto-chytrid [7].Picorna-like viruses are a "superfamily" of positive-sensesingle-stranded RNA (ssRNA) viruses that have similargenome features and several conserved protein domains[8]. Previously, we investigated the diversity of marinepicorna-like viruses by analysis of RNA-dependent RNApolymerase (RdRp) sequences amplified from marinevirus communities and demonstrated that picorna-likeviruses are present and persistent in a diversity of marineenvironments [9]. Furthermore, phylogenetic analysesshowed that none of the environmental sequences fellwithin established virus families.In a recent study, reverse-transcribed whole-genome shot-gun libraries were used to characterize two marine RNAvirus communities [10]. Positive-sense ssRNA viruses thatare distant relatives of known RNA viruses dominated thelibraries. One RNA virus library (JP) was characterized bya diverse, monophyletic clade of picorna-like viruses, butthe second library (SOG) was dominated by viruses dis-tantly related to members of the family Tombusviridae andthe genus Umbravirus. Moreover, in both libraries, a highpercentage of sequence fragments were part of only a fewcontiguous segments of sequence (contigs). Specifically,in the SOG sample 59% of the sequence fragmentsformed a single contig. Similarly, 66% of JP sequence frag-ments contributed to only four contigs that representedtwo viral genomes. Using a RT-PCR-based approach toincrease the amount of sequence for each dominant con-tig resulted in the assembly of three complete viralgenomes. This contribution analyzes these genomes fromthree previously unknown marine RNA viruses and inves-tigates their similarities and differences with respect torepresentative genotypes from established viral taxa.Results and DiscussionJericho Pier siteThe two assembled genomes (JP-A and JP-B) from the Jeri-cho Pier sampling site (Figure 1) are single molecules oflinear ssRNA.The JP-A genome is positive-sense, 9212 nt in length witha 632 nt 5' untranslated region (UTR) followed by 2 pre-dicted open reading frames (ORFs) of 5067 nt (ORF 1, ntposition 633 to 5699) and 3044 nt (ORF 2, nt position5848 to 8799) separated by an intergenic region (IGR) of149 nt (Figure 2A). ORF 2 is followed by a 3' UTR of 413nt (nt position 8800 to 9212) and a polyadenylate [poly(A)] tail. The base composition of JP-A is 27.1% A, 19.4%percentage similar to other polycistronic picorna-likeviruses (Table 1).Comparison to known viral sequences shows that the pro-tein sequence predicted to be encoded by ORF 1 of JP-Acontains conserved sequence motifs characteristic of atype III viral Helicase (aa residues 430 to 545), a 3C-likecysteine protease (aa residues 1077 to 1103) and a type IRdRp (aa residues 1350 to 1591) [11] (Figure 1A).BLASTp [12] searches of the NCBI database with the pre-dicted ORF 1 protein sequence showed significantsequence similarities (E value < 0.001) to nonstructuralprotein motifs of several viruses, including members ofthe families Dicistroviridae (Drosophila C virus), Marna-viridae (HaRNAV), Comoviridae (Cowpea mosaic virus)and the unassigned genus Iflavirus (Kakugo virus). The topmatches for ORF 1 were to RsRNAV [E value = 3 × 10-119,identities = 302/908 (33%)], a newly sequenced, unclas-sified positive-sense ssRNA virus that infects the widelydistributed diatom Rhizosolenia setigera [3], HaRNAV [Evalue = 2 × 10-32, identities = 156/624 (25%)] and Dro-sophila C virus [E value = 1 × 10-29, identities = 148/603(24%)], a positive-sense ssRNA virus that infects fruit flies.Comparison of the protein sequence predicted to beencoded by ORF 2 of JP-A to known viral sequences showsthat it has significant similarities to the structural proteinsof viruses from the families Dicistroviridae (Drosophila Cvirus), Marnaviridae (HaRNAV), and the genus Iflavirus(Varroa destructor virus 1). The sequences that are mostsimilar to ORF 2 of JP-A were the structural proteinregions of RsRNAV [E value = 6 × 10-78, identities = 212/632 (33%)], HaRNAV [E value = 6 × 10-68, identities =187/607 (30%)] and SssRNAV [E value = 2 × 10-49, iden-tities = 241/962 (25%)].The JP-B RNA genome is also likely from a positive-sensessRNA virus. The 8839 nt genome consists of a 5' UTR of774 nt followed by two predicted ORFs of 4842 nt (ORF1, nt position 775 to 5616) and 2589 nt (ORF 2, nt posi-tion 5914 to 8502) separated by an IGR of 298 nt (nt posi-tion 5617 to 5913) (Figure 2B). The 3' UTR is 337 nt longand followed by a poly (A) tail. The base composition ofthe genome is A, 30.8%; C, 17.9%; G, 19.7%; U, 31.6%.Like JP-A, this % G+C value of 38% is comparable to the% G+C observed in other polycistronic picorna-likeviruses (Table 1).The position of core sequence motifs conserved amongpositive-sense ssRNA viruses and BLAST searches of theNCBI database with the translated JP-B genome suggestthat nonstructural proteins are encoded by ORF1, and thestructural proteins are encoded by ORF2. We identifiedconserved sequence motifs in ORF 1 characteristic of aPage 2 of 9(page number not for citation purposes)C, 22.0% G, and 31.6% U; this results in a G+C of 41%, a type III viral Helicase (aa residues 328 to 441), a 3C-likecysteine protease (aa residues 882 to 909) and a type IVirology Journal 2007, 4:69 http://www.virologyj.com/content/4/1/69RdRp (aa residues 1143 to 1408) [11] (Figure 2B).BLASTp [12] searches of the GenBank database showedthat ORF 1 has significant similarities (E value < 0.001) tononstructural genes from positive-sense ssRNA virusesfrom a variety of families, including the Comoviridae(Peach rosette mosaic virus), Dicistroviridae (Taura syn-drome virus), Marnaviridae (HaRNAV), Sequiviridae (Ricetungro spherical virus) and Picornaviridae (Avian enceph-alomyelitis virus). The top scoring sequences [E value = 2× 10-69, identities = 232/854 (27%)] were to a RdRpsequence from RsRNAV and a partial picorna-like virusRdRp from an unidentified virus [E value = 2 × 10-40, iden-tities = 85/150 (56%)] amplified from the same JP stationduring an earlier study [9]. Significant similarities to ORF2 include the structural genes of viruses from the familiesDicistroviridae (Rhopalosiphum padi virus), Marnaviridae(HaRNAV) and Picornaviridae (Human parechovirus 2), aswell as the unclassified genus Iflavirus (Ectropis obliquapicorna-like virus). The top scoring sequences were to thecapsid protein precursor regions of RsRNAV [E value = 9 ×10-88, identities = 244/799 (30%)] and HaRNAV [E valueThe JP-A and JP-B genomes appear to have a polycistronicgenome organization similar to that found in viruses inthe family Dicistroviridae. Several of these viruses containinternal ribosome entry sites (IRES) [13-16] that positionthe ribosome on the genome, actuating translation initia-tion even in the absence of known canonical initiationfactors [13]. For example, TSV, a marine dicistrovirus, hasan IRES located in the IGR that directs the synthesis of thestructural proteins [15]. Computational searches did notidentify the secondary structure elements characteristic ofdicistrovirus IGR-IRESs in the JP genomes [16,17], how-ever, JP-A and JP-B genomes have extensive predicted sec-ondary structure in the 5' UTRs and IGRs [18,19],suggestive of an IRES function. Moreover, start codons ina favorable Kozak context, i.e. conserved sequencesupstream of the start codon that are thought to play a rolein initiation of translation [20], were not found in the JPgenomes. However to unequivocally demonstrate IRESelements in the JP genomes, they must be confirmedexperimentally in polycistronic constructs. Nevertheless,it seems reasonable that JP-A and JP-B use similar mecha-nisms to initiate translation of the ORF 2 genes as areknown to be employed by several dicistroviruses.We used RT-PCR to assess the distribution and persistenceof the JP-A and JP-B viruses in situ. Amplification with spe-cific primers that target each of these viruses occurred insamples from throughout the Strait of Georgia, the Westcoast of Vancouver Island, and in every season and tidalstate at Jericho pier (Figure 1, Table 2). These results sug-gest that JP-A and JP-B are ubiquitous in the coastal watersof British Columbia.It has long been recognized that several other groups ofsmall, positive-sense ssRNA viruses share many character-istics with viruses in the family Picornaviridae. Recently,Christian et al. [8] proposed creating an order (the Picor-navirales) of virus families (Picornaviridae, Dicistroviridae,Marnaviridae, Sequiviridae and Comoviridae) and unas-signed genera (Iflavirus, Cheravirus, and Sadwavirus) thathave picornavirus-like characteristics. Viruses in the pro-posed order have genomes with a protein covalentlyattached to the 5' end, a 3' poly (A) tail, a conserved orderof non-structural proteins (Helicase-VpG-Proteinase-RdRp), regions of high sequence similarity in the helicase,proteinase and RdRp, post translational protein process-ing during replication, an icosahedral capsid with aunique "pseudo-T3" symmetry, and only infect eukaryo-tes.Although the capsid morphology, presence of a 5' termi-nal protein and replication strategy and hosts areunknown, signature genomic features and phylogeneticMap of southwestern British Columbia, Canada showing locations wher  samples were collectedFigure 1Map of southwestern British Columbia, Canada showing locations where samples were collected.Sites in coastal BC waters where the JP-A and JP-B genomes were detected are indicated and labelled. Both JP-A and JP-B were detected in samples from 5 of the 9 stations that were screened. The SOG station was not assayed for JP-A or JP-B. See Table 2 for additional information about the stations.Page 3 of 9(page number not for citation purposes)= 8 × 10-60, identities = 180/736(24%)] and SssRNAV [Evalue = 1 × 10-40. identities = 156/588 (26%)].analyses suggest that the JP viruses fall within the pro-posed order Picornavirales. Both JP genomes encode theVirology Journal 2007, 4:69 http://www.virologyj.com/content/4/1/69conserved core aa motifs and have the non-structural geneorder characteristic of viruses in the proposed Picornavi-rales. Furthermore, both JP genomes have a poly (A) tailRdRp domains [11] (Figure 3), as well as concatenated(putative) Hel+RdRp+VP3 capsid-like protein sequences(Figure 4), of the JP genomes and representative membersAnalysis of genomes for putative open reading framesFigure 2Analysis of genomes for putative open reading frames.In the ORF maps created with DNA Strider [28], for each reading frame, potential start codons (AUG) are shown with a half-height line and stop codons (UGA, UAA, and UAG) are shown by full-height lines. Recognizable conserved RNA virus protein domains (Hel = helicase, Pro = Protease, RdRp = RNA-dependent RNA polymerase) and other genomic features (UTR = untranslated region, IGR = intergenic region) are noted below each genome. See text for more detail. A. Map of the JP-A genome. B. Map of the JP-B genome. C. Map of the SOG genome.Page 4 of 9(page number not for citation purposes)and G+C content commensurate with these other viruses.Bayesian trees [21] based on alignments of conservedof the proposed Picornavirales, resolves established taxaaccording to previous taxonomic divisions. These analysesVirology Journal 2007, 4:69 http://www.virologyj.com/content/4/1/69also provide strong support for a clade comprised ofviruses (HaRNAV, RsRNAV and SssRNAV) that infectmarine protists and the JP-A and JP-B viruses. Within thisclade, RsRNAV, JP-A and JP-B have the most characteris-tics in common. For example, they have the same order ofstructural and non-structural genes, they are polycistronicand the phylogenetic analyses indicate they are moreclosely related (Figures 3 and 4). Whether JP-A and JP-Binfect host organisms related to Rhizosolenia setigeraremains unclear, although because of the inclusion of theJP genomes within this clade and the fact that protists arethe most abundant eukaryotes in the sea, we suggest thatboth JP viruses likely have a protist host.Strait of Georgia siteThe SOG genome was assembled from the Strait of Geor-gia metagenomic library, and subsequently completed asdescribed in Methods. The genome has features character-istic of a positive-sense ssRNA virus. The genome is 4449nt long and comprised of a 5' UTR of 334 bp followed bythree putative ORFs (nt position 335–1228, nt position1385–2860 and nt position 2903–4228) and is termi-nated with a 3' UTR of 221 nt. A poly (A) tail was notdetected. Another putative ORF located at nt position 49to 783 is in an alternative reading frame relative to theORFs discussed above (Figure 2C). The G+C content ofthe SOG genome is 52%.We identified the eight conserved motifs of the RdRp [11]in the SOG genome (aa residues pos 451 to 700) (Figure2C). tBLASTx [12] searches with the remainder of thefive environmental metagenomes that have been depos-ited). BLASTp searches with the putative RdRp sequenceresulted in significant similarities (E value < 0.001) toRdRp sequences from positive-sense ssRNA viruses fromthe family Tombusviridae and the unassigned genusUmbravirus. The sequence with the most similarity to SOGwas from Olive latent virus 1 [E value = 3 × 10-66, identi-ties = 180/508 (35%)]. This virus belongs to the genusNecrovirus in the family Tombusviridae that has a hostrange restricted to higher plants [22]. SOG is also signifi-cantly similar to the Carrot mottle mimic virus sequence[E value = 6 × 10-66, identities = 178/492 (36%)], a mem-ber of the unclassified genus Umbravirus whose knownmembers infect only flowering plants [23].Although the SOG putative RdRp sequence has similarityto the RdRp of viruses from the family Tombusviridae andgenus Umbravirus, the remaining SOG sequence has nodetectable similarity to any other known sequence. ABayesian maximum likelihood tree based on alignmentsof the SOG RdRp with the available Umbravirus sequencesand representative members of the Tombusviridae indi-cates that the SOG genome forms a well supported clade(Bayesian clade support value of 100) with the singlemember of the genus Avenavirus, OCSV (Figure 5). Addi-tionally, the presence of an amber stop codon (nt position1230–1232) at the end of ORF 1 of the SOG genome (Fig-ure 2C), resembles the in-frame termination codon char-acteristic of the replicase gene of viruses in 7 of the 8genera of the Tombusviridae [24]. This division of the rep-licase of the Tombusviridae by a termination codon isTable 1: Comparison of base composition between polycistronic picorna-like virusesGenome* A C G U % G+CJP-A 27.1 19.4 22.0 31.6 41JP-B 30.8 17.9 19.7 31.6 38ABPV 35.7 15.4 20.1 28.9 36ALPV 31.3 19.4 19.2 30.2 39BQCV 29.2 18.5 21.6 30.6 40CrPV 32.6 18.4 20.9 28.1 39DCV 29.9 16.3 20.4 33.4 37HiPV 29.2 18.7 20.9 31.2 39KBV 33.8 17.5 20.2 28.6 38PSIV 31.3 17.0 19.4 32.3 36RhPV 30.0 18.6 20.2 31.2 39RsRNAV 31.2 16.7 19.5 32.5 36SINV-1 32.9 18.3 20.5 28.2 39SssRNAV 24.2 26.1 23.6 26.0 50TSV 28.0 20.2 23.0 28.8 43TrV 28.7 16.1 19.8 35.4 36Average 30.4 18.4 20.7 30.5 39* See Additional file 2 for the complete virus namesPage 5 of 9(page number not for citation purposes)genome sequence showed no significant matches (E value< 0.001) to sequences in the NCBI database (including thethought to be part of a translational read though geneexpression strategy [24]. Other similarities to the Tombus-Virology Journal 2007, 4:69 http://www.virologyj.com/content/4/1/69viridae include a similar genome size, the absence of anobvious helicase motif and the 5' proximal relative posi-tion of the RdRp within the genome [22]. However,unlike viruses in the Tombusviridae, there is no recogniza-ble sequence for conserved movement or capsid proteinsin the SOG genome. The absence of a recognizable move-ment protein could indicate the SOG virus does not infecta higher plant. Our inability to identify structural genesmay indicate that, like the umbraviruses, the SOG virusdoes not encode capsid proteins. However, it is also pos-sible that movement or structural proteins encoded in theSOG genome have no sequence similarity to those cur-rently in the NCBI database.ConclusionOur analyses suggest that a persistent, widespread andpossibly dominant population of novel polycistronicpicorna-like viruses is an important component of theRNA virioplankton in coastal waters. Nevertheless, asexemplified by the SOG genome from the Strait of Geor-gia site, other marine RNA virus assemblages appear tocontain viruses whose detectable sequence similarity withestablished groups of viruses is limited to only the mostconserved genes (i.e. RdRp). The novelty of JP-A, JP-B andSOG, as revealed by sequence analyses and genome char-acterization, suggests that most of the diversity in thegenomes of these marine RNA viruses that we propose toinfect single-celled eukaryotes may be more similar to theancestral RNA viruses that gave rise to those that infecthigher organisms.MethodsStation descriptionsThe shotgun libraries were constructed from seawatersamples collected from two stations, JP (Jericho Pier), asite in English Bay adjacent to the city of Vancouver, Brit-ish Columbia and SOG (Strait of Georgia), located in thecentral Strait of Georgia next to Powell River, B.C. (Figure1).The locations of the stations where one or both of the JPgenomes were detected are shown in Figure 2. Details foreach station are listed in Table 2. In summary, sampleswere collected from sites throughout the Strait of Georgia,including repeated sampling from the JP site during differ-ent seasons, and from the West coast of Vancouver Islandin Barkley Sound.Virus concentration methodConcentrated virus communities were produced asdescribed by Suttle et al. [25]. Twenty to sixty litres of sea-water from each station were filtered through glass fibreTable 2: JP genome survey sample sites and results of assaysStation Name Station location (B.C., Canada)Date (mm/dd/yy) Location (Lat., Long.) Depth (m) Temp (°C) Salinity (ppt) JP-A PCR JP-B PCRJP Jericho Pier 04/28/00 49.27, -123.20 S 9 26 + +JP Jericho Pier 06/15/00 49.27, -123.20 S 14 12 + +JP Jericho Pier 06/29/00 49.27, -123.20 S 17 12 + +JP Jericho Pier 07/06/00 49.27, -123.20 S 16 13 + +JP Jericho Pier 07/13/00 49.27, -123.20 S 18 8 - -JP Jericho Pier 07/27/00 49.27, -123.20 S 18 11 + +JP Jericho Pier 08/17/00 49.27, -123.20 S 18 18 + +JP Jericho Pier 09/14/00 49.27, -123.20 S 15 19 + +JP Jericho Pier 09/21/00 49.27, -123.20 S 15 16 - +JP Jericho Pier 09/28/00 49.27, -123.20 S 14 21 + +JP Jericho Pier 11/23/00 49.27, -123.20 S 8 27 + +JP Jericho Pier 02/15/01 49.27, -123.20 S 7 27 + +JP Jericho Pier 06/14/01 49.27, -123.20 S 15 13 + +SEC Sechelt Inlet 07/06/03 49.69, -123.84 4 13 26 - +TEA Teakearne Inlet 07/07/03 50.19, -124.85 5 13 28 + -QUA Quadra Island 07/07/03 50.19, -125.14 3 13 28 - -ARR Arrow Pass 07/09/03 50.72, -126.67 2 10 31 + +IEC Imperial Eagle Channel06/20/99 48.87, -125.21 7 n.a. n.a. + -TRE Trevor Channel 06/28/99 48.97, -125.16 S n.a. n.a. + +BAM Bamfield Inlet 07/06/99 48.81, -125.16 S n.a. n.a. + +NUM Numukamis Bay 07/12/99 48.90, -125.01 8 n.a. n.a. + +A "+" indicates amplification and "-" indicates no amplification occurred. "n.a." indicates the data is not available and "S" means the sample was taken from the surface.Page 6 of 9(page number not for citation purposes)marine RNA virus community remains uncharacterized.Furthermore, these results raise the hypothesis that the(nominal pore size 1.2 μm) and then 0.45 μm pore-sizeDurapore polyvinylidene fluoride (PVDF) membranesVirology Journal 2007, 4:69 http://www.virologyj.com/content/4/1/69(Millipore, Cambridge, Canada), to remove particulateslarger than most viruses. This filtrate was subsequentlyconcentrated approximately 200 fold through a Tangen-tial Flow Filter cartridge (Millipore) with a 30 kDa molec-ular cut-off, essentially concentrating the 2 to 450 nm sizefraction of seawater. Remaining bacteria were removed byfiltering the concentrate two times through a 0.22 μmDurapore PVDF membrane (Millipore). Virus-sized parti-cles in each VC were pelleted via ultracentrifugation (5 hat 113 000 × g at 4°C). Pellets were resuspended overnightat 4°C in sterile 50 μM Tris chloride (pH 7.8).Whole genome library constructionA detailed description of the whole genome shotgunlibrary construction protocol can be found in Culley et al.[10]. Briefly, before extraction, concentrated viral lysateswere treated with RNase (Roche, Mississauga, Canada)and then extracted with a QIAamp Minelute Virus Spin Kit(Qiagen, Mississauga, Canada) according to the manufac-turer's instructions. An aliquot of each extract was used ina PCR reaction with universal 16S primers to ensure sam-ples were free of bacteria. To isolate the RNA fraction,samples were treated with DNase 1 (Invitrogen, Burling-ton, Canada) and used as templates for reverse transcrip-tion with random hexamer primers. Double-stranded (ds)gen) using nick translational replacement of genomicRNA [26]. After degradation of overhanging ends with T4DNA polymerase (Invitrogen), adapters were attached tothe blunted products with T4 DNA ligase (Invitrogen).Subsequently, excess reagents were removed and cDNAproducts were separated by size with a Sephacryl column(Invitrogen). To increase the amount of product for clon-ing, size fractions greater than 600 bp were amplified withprimers targeting the adapters. Products from each PCRreaction were purified and cloned with the TOPO TACloning system (Invitrogen). Clones were screened forinserts by PCR with vector-specific primers. Insert PCRproducts greater than 600 bp were purified and sequencedat the University of British Columbia's Nucleic Acid andProtein Service Facility (Vancouver, Canada). Sequencefragments were assembled into overlapping segmentsusing Sequencher v 4.5 (Gene Codes, Ann Arbor, U.S.A.)based on a minimum match % of 98 and a minimum bpoverlap of 20. Sequences were compared against the NCBIdatabase with tBLASTx [12]. A sequence was consideredsignificantly similar if BLAST E values were < 0.001. Thedetails for viruses used in phylogenetic analyses are listedin additional file 2. Virus protein sequences were alignedusing CLUSTAL X v 1.83 with the Gonnet series proteinmatrix [27]. Alignments were transformed into likelihoodBayesian maximum likelihood trees of aligned concatenated helicase, RdRp and VP3-like capsid amino acid sequences from the JP-A and JP-B gen me  and other picor a-like virusesFigure 4Bayesian maximum likelihood trees of aligned con-catenated helicase, RdRp and VP3-like capsid amino acid sequences from the JP-A and JP-B genomes and other picorna-like viruses. Bayesian clade credibility val-ues are shown for relevant nodes in boldface followed by bootstrap values based on neighbour-joining analysis. The Bayesian scale bar indicates a distance of 0.1. See Additional file 2 for complete virus names and accession numbers.Bayesian maximum likelihood trees of aligned RdRp amino acid equences from the JP-A and JP-B genomes and repre-sentative embers of the propos d ord r Picorn viralesFigure 3Bayesian maximum likelihood trees of aligned RdRp amino acid sequences from the JP-A and JP-B genomes and representative members of the pro-posed order Picornavirales. Bayesian clade credibility val-ues are shown for relevant nodes in boldface followed by bootstrap values based on neighbour-joining analysis. The Bayesian scale bar indicates a distance of 0.1. See Additional file 2 for complete virus names and accession numbers.Page 7 of 9(page number not for citation purposes)cDNA fragments were synthesized from single strandedDNA with Superscript III reverse transcriptase (Invitro-distances with Mr. Bayes v3.1.1 [21] and 250,000 genera-tions. Neighbor-joining trees were constructed with PAUPVirology Journal 2007, 4:69 http://www.virologyj.com/content/4/1/69v4.0 [28], and bootstrap values calculated based on per-centages of 10,000 replicates.5' and 3' RACEThe 5' and 3' ends of the environmental viral genomeswere cloned using the 5' and 3' RACE systems (Invitrogen)according to manufacturer's instructions. The 3' RACEwith the SOG genome required the addition of a poly (A)tract with poly (A) polymerase (Invitrogen) according tomanufacturer directions before cDNA synthesis. cDNAwas synthesized directly from extracted viral RNA fromthe appropriate library. Three clones of each 5' and 3' endwere sequenced.PCRClosing gaps in the assemblyPCR with primers targeting specific regions of the two JPenvironmental genomes were used to verify the genomeassembly, increase sequencing coverage and reconfirm thepresence of notable genome features. The template forthese reactions was the amplified and purified PCR prod-The standard PCR conditions were reactions with 1 U ofPlatinum Taq DNA polymerase (Invitrogen) in 1× Plati-num Taq buffer, 1.5 mM MgCl2, 0.2 mM of each dNTP,and 0.2 μM of each primer (see Additional file 1), in afinal volume of 50 μl. Thermocycler conditions were, acti-vation of the enzyme at 94°C for 1 min 15 s, followed by30 cycles of denaturation at 94°C for 45 s, annealing at50°C for 45s and extension at 72°C for 1 minute. Thereaction was terminated after a final extension stage of 5min at 72°C. PCR products were purified with a PCR Min-elute cleanup kit (Qiagen) and sequenced directly withboth primers.Environmental screeningTo assess the temporal and geographic distribution of theJP genomes, extracted RNA from viral concentrates werescreened with Superscript III One-step RT-PCR Systemwith Platinum Taq DNA Polymerase (Invitrogen) withprimers JP-A 5 and 6 and JP-B 6 and 7 (see Additional file1). The template for the reactions was DNase 1 treatedviral RNA, extracted with a QIAamp Minelute Virus SpinKit (Qiagen) according to the manufacturer's instructions.Each reaction consisted of RNA template, 1× reaction mix,0.2 μM of each primer, 1 μl RT/Platinum Taq mix in a vol-ume of 50 μl. Reactions were incubated 30 min at 50°C,then immediately heated to 94°C for 45 s, followed by 35cycles of denaturation at 94°C for 15 s, annealing at 50°Cfor 30 s and extension at 68°C for 1 min. After a finalextension step at 68°C for 5 min, RT-PCR products wereanalyzed by agarose gel electrophoresis. Products weresequenced to verify the correct target had been amplified.Competing interestsThe author(s) declare that they have no competing inter-ests.Authors' contributionsAC contributed to the design of the study, performed thelab work, analyzed the data and drafted the manuscript.AL contributed to the design of the study, analyzed thedata and helped prepare the manuscript. CS was involvedin the conceptualization and design of the research and inmanuscript preparation. AC, AL and CS have read andapproved this manuscript.Additional materialAdditional file 1PCR primers used to complete the three genome sequences. The table pro-vides detailed information about the primers used to complete the three viral genome sequences.Click here for file[http://www.biomedcentral.com/content/supplementary/1743-Bayesian maximum likelihood trees of aligned RdRp amino acid equences from the SOG g nome and members of the family To bu vi idae and unassig ed genus UmbravirusFigure 5Bayesian maximum likelihood trees of aligned RdRp amino acid sequences from the SOG genome and members of the family Tombusviridae and unas-signed genus Umbravirus. Bayesian clade credibility val-ues are shown for relevant nodes in boldface followed by bootstrap values based on neighbour-joining analysis. The Bayesian scale bar indicates a distance of 0.1. See Additional file 2 for complete virus names and accession numbers.Page 8 of 9(page number not for citation purposes)uct from the JP and SOG shotgun libraries. Additional file1 lists the sequence and genome position of primers used.422X-4-69-S1.doc]Publish with BioMed Central   and  every scientist can read your work free of charge"BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime."Sir Paul Nurse, Cancer Research UKYour research papers will be:available free of charge to the entire biomedical communitypeer reviewed and published immediately upon acceptancecited in PubMed and archived on PubMed Central Virology Journal 2007, 4:69 http://www.virologyj.com/content/4/1/69AcknowledgementsWe would like to thank Professor Nakashima for evaluating the IGRs of the JP genomes for the presence of dicistrovirus IRES elements and Debbie Adams from the Nucleic Acid Protein Service Unit at the University of Brit-ish Columbia for her generosity. Sequences have been deposited in Gen-Bank with accession numbers EF198240, EF198241 and EF198242. This work was supported by grants from the Natural Science and Engineering Research Council of Canada.References1. Smith A: Aquatic virus cycles.  In Viral Ecology Edited by: Hurst C.San Diego: Academic Press; 2000:447-491. 2. Weinbauer M: Ecology of prokaryotic viruses.  FEMS Microbiol Rev2004, 28:127-181.3. Nagasaki K, Tomaru Y, Katanozaka N, Shirai Y, Nishida K, Itakura S,Yamaguchi M: Isolation and characterization of a novel single-stranded RNA virus infecting the bloom-forming diatomRhizosolenia setigera.  Appl Environ Microbiol 2004, 70:704-711.4. Tomaru Y, Katanozaka N, Nishida K, Shirai Y, Tarutani K, YamaguchiM, Nagasaki K: Isolation and characterization of two distincttypes of HcRNAV, a single-stranded RNA virus infecting thebivalve-killing microalga Heterocapsa circularisquama.  AquatMicrob Ecol 2004, 34:207-218.5. 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Suttle CA, Chan AM, Cottrell MT: Use of ultrafiltration to isolateviruses from seawater which are pathogens of marine phyto-plankton.  Appl Environ Microbiol 1991, 57:721-726.26. Okayama H, Berg P: High-efficiency cloning of full-lengthcDNA.  Mol Cell Biol 1982, 2:161-170.27. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG: TheCLUSTAL_X windows interface: flexible strategies for mul-tiple sequence alignment aided by quality analysis tools.Nucleic Acids Res 1997, 24:4876-4882.28. Swofford DL: PAUP*. Phylogenetic analysis using parsimony(*and other methods). Version 4.  Sunderland, MA: Sinauer Asso-ciates; 2003. 29. Marck C: "DNA Strider": a "C" program for the fast analysisof DNA and protein sequences on the Apple Macintosh fam-ily of computers.  Nucleic Acids Res 1988, 16:1829-1836.Additional file 2Virus sequence details. 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