Characterization of the diversity of marine RNA viruses

Abstract

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 chapter, we describe two methods to assess RNA virus diversity from seawater. *Corresponding author: E-mail: aculley@hawaii.edu, phone: (808) 956-8629 Acknowledgments Publication costs for the Manual of Aquatic Viral Ecology were provided by the Gordon and Betty Moore Foundation. This document is based on work partially supported by the U.S. National Science Foundation (NSF) to the Scientific Committee for Oceanographic Research under Grant OCE-0608600. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the NSF. The writing of this chapter was supported in part by grants from NSF to the authors (OCE-04-42664 and OCE-0826650) and to the Center for Microbial Ecology Research and Education (EF-0424599). The authors acknowledge the efforts of two anonymous peer reviewers and their suggestions to improve the manuscript. ISBN 978-0-9845591-0-7, DOI 10.4319/mave.2010.978-0-9845591-0-7.193 Suggested citation format: Culley, A. I., C. A. Suttle, and G. F. Steward. 2010. Characterization of the diversity of marine RNA viruses, p. 193–201. In S. W. Wilhelm, M. G. Weinbauer, and C. A. Suttle [eds.], Manual of Aquatic Viral Ecology. ASLO. MAVE Chapter 19, 2010, 193–201 © 2010, by the American Society of Limnology and Oceanography, Inc. MANUAL of AQUATIC VIRAL ECOLOGY viral infections are a persistent force-shaping protistan community composition in the sea. Because this is a relatively new area of research, the number of methods described in the literature for investigating the diversity of marine RNA viruses is still limited. We present here two protocols that have been used successfully in published articles (Culley et al. 2006; Culley and Steward 2007; Djikeng et al. 2008, 2009), but note that this is a nascent field and there are no routine methods. We hope the protocols described will at least serve as starting point for further improvements. Protocol 1. A degenerate primer reverse transcriptionpolymerase chain reaction–based protocol to determine the diversity of picorna-like viruses 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. 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. Material and equipment Equipment—Peristaltic pump and pump heads, thermocycler, heating block, incubator, gel electrophoresis unit, electroporator, gel documentation system Supplies—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) Solutions, reagents, and media—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) 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