TITLE: No Title
AUTHORS: 

[DESCRIPTION]
The diversity of ribonucleic acid (RNA) viruses in the ocean and the ongoing isolation and characterization of RNA viruses that infect important primary producers suggests that RNA viruses are active members of the marine microbial assemblage. At this point, little is known about the composition, dynamics, and ecology of the RNA virioplankton. In this protocol, we describe a method to assess RNA virus diversity from seawater.

[GUIDELINES]
This protocol is designed to detect picorna-like viruses from marine samples. It is based on a strategy first reported by Culley et al. (2003) and refined by Culley and Steward (2007). All picorna-like viruses have single-stranded positive-sense RNA genomes and are classified in the order Picornavirales (Le Gall et al. 2008). Picorna-like viruses are responsible for several significant human and animal diseases and infect a diversity of marine protists including a diatom (Nagasaki 2008; Lang et al. 2009). The procedure outlined below uses direct filtration of relatively small volumes of water and reverse transcriptionpolymerase chain reaction (RT-PCR) amplification of an RNAdependent RNA polymerase (RdRP) gene fragment using degenerate primer sets. <br /><br />With this method, viral polymerase sequences were amplified in several distinct aquatic environments, including an estuarine urban canal (Ala Wai canal, Waikiki, HI, USA), a tropical bay (Kaneohe Bay, HI, USA), and a temperate bay (Monterey Bay, CA, USA). Amplification occurred in samples from the same site in different seasons and at different depths showing that RNA viruses are widespread and consistently present. Sequencing of the amplified gene fragments revealed novel sequences that are highly divergent from any known isolates.<strong><br /><br />Equipment</strong>—Peristaltic pump and pump heads, thermocycler, heating block, incubator, gel electrophoresis unit, electroporator, gel documentation system<br /><br /><strong>Supplies</strong>—Sterile, 1.7 mL nucleic acid-free microcentrifuge tubes; 0.2 mL sterile, nucleic acid-free PCR tubes; 0.02 µm aluminum oxide filters (Anotop, Whatman); 10 mL sterile syringes; peristaltic pump tubing; sterile razor blades; MinElute Gel Extraction kit (Qiagen); MinElute PCR Purification kit (Qiagen); Masterpure Complete DNA and RNA Purification kit (Epicenter Biotechnologies); PCRTerminator End Repair kit (Lucigen); CloneSmart HCKan Blunt Cloning kit with Ecloni Supreme cells (Lucigen); Turbo DNA-free Kit (Applied Biosystems)<br /><br /><strong>Solutions, reagents, and media</strong>—0.02-filtered, sterile, nucleic acid-free TE buffer (10 mM Tris, 1 mM EDTA; pH 8); 0.1 M dithiothreitol (DTT); Superscript III Reverse Transcriptase and buffers (Invitrogen); RNase Out (Invitrogen); RNase H (Invitrogen); Platinum Taq DNA polymerase and buffers (Invitrogen) <br /><br />Primers in concentrations discussed as follows: 0.5 × TBE (45 mM Tris-borate, 1 mM EDTA; pH 8.0) electrophoresis buffer; nucleic acid–free, sterile water; 10 mM dNTP mix.<br /><br /><strong>Table 1: RT and PCR primer details</strong><br /><br />
<table>
<tbody>
<tr>
<td><strong>Name</strong></td>
<td><strong>Sequence (5'-3')</strong></td>
<td><strong>°C annealing</strong></td>
<td><strong>~Product (bp)</strong></td>
</tr>
<tr>
<td>Mpl.sc1F</td>
<td>TIGCIGGWGAYTWYARM</td>
<td>50</td>
<td>500</td>
</tr>
<tr>
<td>Mpl.sc1R</td>
<td>YTCCTTWTCRGSCATKGTA</td>
<td> </td>
<td> </td>
</tr>
<tr>
<td>Mpl.sc2F</td>
<td>ITWGCIGGIGATTWCA</td>
<td>43</td>
<td>500</td>
</tr>
<tr>
<td>Mpl.sc2R</td>
<td>CKYTTCARRAAWTCAGCATC</td>
<td> </td>
<td> </td>
</tr>
<tr>
<td>Mpl.sc3F</td>
<td>TIATIGMKGGIGAYTA</td>
<td>49</td>
<td>500</td>
</tr>
<tr>
<td>Mpl.sc3R</td>
<td>TTMARGAAIKMAGCATCTT</td>
<td> </td>
<td> </td>
</tr>
<tr>
<td>Mpl.cdhF</td>
<td>GMIGGTGAYTAYAGCGCTTWYGAY</td>
<td>44</td>
<td>500</td>
</tr>
<tr>
<td>Mpl.cdhR</td>
<td>ATACCCAATGCCTYTTIARRAA</td>
<td> </td>
<td> </td>
</tr>
<tr>
<td>RdRp1</td>
<td>GGRGAYTACASCIRWTTTGAT</td>
<td>50</td>
<td>450</td>
</tr>
<tr>
<td>RdRp2</td>
<td>MACCCAACKMCKCTTSARRAA</td>
<td> </td>
<td> </td>
</tr>
<tr>
<td>SL1</td>
<td>CAGTCCAGTTACGCTGGAGTC</td>
<td>50</td>
<td>NA</td>
</tr>
<tr>
<td>SR2</td>
<td>GGTCAGGTATGATTTAAATGGTCAGT</td>
<td> </td>
<td> </td>
</tr>
<tr>
<td>FR26RV-N</td>
<td>GCCGGAGCTCTGCAGATATCNNNNNN</td>
<td>NA</td>
<td>NA</td>
</tr>
<tr>
<td>FR40RV-T</td>
<td>GCCGGAGCTCTGCAGATATC(T)20</td>
<td>NA</td>
<td>NA</td>
</tr>
<tr>
<td>FR20RV</td>
<td>GCCGGAGCTCTGCAGATATC</td>
<td>65</td>
<td>NA</td>
</tr>
<tr>
<td colspan="4">NA, not applicable</td>
</tr>
</tbody>
</table>

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