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Limnology and Oceanography e-Books

Manual of Aquatic Viral Ecology

ASLO's first e-Book publication is the Manual of Aquatic Viral Ecology (MAVE), edited by Steven Wilhelm, Markus Weinbauer and Curtis Suttle. It contains 19 chapters reflecting state-of-the-art opinions on approaches to studying viruses in aquatic systems. Topics range from the enumeration of viruses to molecular techniques designed to dissect and query individual virus populations as well as communities of viruses. The content of this e-book was selected in consultation with the Scientific Committee for Oceanographic Research's working group on marine viruses, and its publication has been supported by the Gordon and Betty Moore Foundation.

Chapters in the MAVE e-Book are freely available for download. Citations of each chapter should follow the form recommended in its acknowledgments. For the entire book, a suggested citation is as follows.
S.W. Wilhelm, M.G. Weinbauer, and C.A. Suttle [eds.] 2010. Manual of Aquatic Viral Ecology. Waco, TX:American Society of Limnology and Oceanography. doi:10.4319/mave.2010.978-0-9845591-0-7

Table of Contents

Markus G. Weinbauer, Janet M. Rowe, and Steven W. Wilhelm 
Determining rates of virus production in aquatic systems by the virus reduction approach
Chapter 1, pp 1-8

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Full Citation: Weinbauer, M. G., J. M. Rowe, and S. W. Wilhelm. 2010. Determining rates of virus production in aquatic systems by the virus reduction approach, p. 1-8. In S. W. Wilhelm, M. G. Weinbauer, and C. A. Suttle [eds.], Manual of Aquatic Viral Ecology. ASLO. [DOI: 10.4319/mave.2010.978-0-9845591-0-7.1]

ABSTRACT: The reduction approach to assess virus production and the prokaryotic mortality by viral lysis stops new infection by reducing total virus abundance (and thus virus-host contacts). This allows for easy enumeration of viruses that originate from lysis of already infected cells due to the decreased abundance of free virus particles. This reoccurrence can be quantified and used to assess production and cell lysis rates. Several modifications of the method are presented and compared. The approaches have great potential for elucidating trends in virus production rates as well as for making generalized estimates of the quantitative effects of viruses on marine microbial communities.

Ruth-Anne Sandaa, Steven M. Short, and Declan C. Schroeder
Fingerprinting aquatic virus communities
Chapter 2, pp 9-18

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Full Citation: Sandaa, R.-A., S. M. Short, and D. C. Schroeder. 2010. Fingerprinting aquatic virus communities, p. 9-18. In S. W. Wilhelm, M. G. Weinbauer, and C. A. Suttle [eds.], Manual of Aquatic Viral Ecology. ASLO. [DOI 10.4319/mave.2010.978-0-9845591-0-7.9]

ABSTRACT: To circumvent the limitations of cultivation-based studies of complex microbial communities, molecular fingerprinting techniques such as pulsed field gel electrophoresis (PFGE) and denaturing gradient gel electrophoresis (DGGE) have been used to examine their richness, diversity, and dynamics. PFGE is based on the electrophoretic separation of extremely large DNA, raising the upper size limit from 50 kb (standard agarose separation) to well over 10 Mb. This technique has been used to separate aquatic virus genomes ranging in size from tens to hundreds of kilo base pairs (kb); aquatic virus genomes range from 15 to 630 kb, with the majority between 20 and 80 kb. DGGE, on the other hand, is based on the electrophoretic separation of PCR- amplified gene fragments of similar sizes, but differing in base composition or sequence. In this chapter, we provide a brief overview of each of these methods and their application to the study of aquatic viruses. We describe some of the common equipment, reagents, and procedures involved, and conclude by briefly considering some of the strengths and weaknesses of each method.

André M. Comeau and Rachel T. Noble 
Preparation and application of fluorescently labeled virus particles
Chapter 3, pp 19-29

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Full Citation: Comeau, A. M., and R. T. Noble. 2010. Preparation and application of fluorescently labeled virus particles, p. 19-29. In S. W. Wilhelm, M. G. Weinbauer, and C. A. Suttle [eds.], Manual of Aquatic Viral Ecology. ASLO. [DOI 10.4319/mave.2010.978-0-9845591-0-7.19]

ABSTRACT: Tracing the fate of individual host cells and viruses is a challenging problem for microbial ecology. The emergence of a new assay, using fluorescently labeled viruses (FLVs), offers the promise of a quick and easy method for monitoring host dynamics and virus decay. Using FLVs as probes (FLVPs) for host cells, an assay was optimized for use with SYBR Green I-stained phages and was shown to be an efficient and reliable method for detection of a strain of Vibrio sp. Various microcosm experiments were conducted that demonstrated the utility of the FLVP assay in resolving ecological interactions at the community level. The assay was also used to show that FLVPs, at high enough multiplicities of infection, can directly inhibit viral infection by "titering out" or "coating" the host's cell surface receptors. FLVs can also be used as tracers for studies of virus production and decay. The approach is mathematically similar to the isotope dilution technique, employed in the past to simultaneously measure the release and uptake of ammonium and amino acids. The method can be used to determine rates of viral degradation, production, and turnover for investigations of microbial food webs in aquatic systems.

John H. Paul and Markus Weinbauer 
Detection of lysogeny in marine environments
Chapter 4, pp 30-33

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Full Citation: Paul, J. H., and M. Weinbauer. 2010. Detection of lysogeny in marine environments, p. 30-33. In S. W. Wilhelm, M. G. Weinbauer, and C. A. Suttle [eds.], Manual of Aquatic Viral Ecology. ASLO. [DOI 10.4319/mave.2010.978-0-9845591-0-7.30]

ABSTRACT: We employed a comparative approach to review the vulnerability of the trophic interactions within aquatic systems to global threats associated with anthropogenic activities. The goal of this chapter was to identify and characterize mechanisms by which human-mediated environmental threats may modulate trophic dynamics across aquatic ecosystems. Trophic dynamics include some of the most obvious and pervasive factors influencing ecosystems and were used as a metric because of their importance and commonality across all aquatic environments. Our use of trophic dynamics proved to be insightful, illustrating that the flow of energy through aquatic food webs will be (or already has been) altered by invasive species, land use change, nutrient loading, exposure to ultraviolet radiation, overharvesting, acidification, and increasing global temperatures. The response of trophic dynamics to these threats was often similar across oceans, estuaries, lakes, and rivers. This similarity proved to be interesting given the differences in both the level of concern expressed by scientists and the predicted variability in environment- specific responses. As the trophic interactions of an ecosystem are at the root of its function and structure, examining trophic dynamics could be an informative method for evaluating the response of aquatic environments to global threats. If future analyses validate the use of trophic dynamics as a metric, it is our hope that trophic dynamics can be used by scientists and politicians to mitigate the effects of human actions.

Michael J. Allen, Bela Tiwari, Matthias E. Futschik, and Debbie Lindell 
Construction of microarrays and their application to virus analysis
Chapter 5, pp 34-56
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Full Citation: Allen, M. J., B. Tiwari, M. E. Futschik, and D. Lindell. 2010. Construction of microarrays and their application to virus analysis, p. 34-56. In S. W. Wilhelm, M. G. Weinbauer, and C. A. Suttle [eds.], Manual of Aquatic Viral Ecology. ASLO. [DOI 10.4319/mave.2010.978-0-9845591-0-7.34]

ABSTRACT: DNA microarray is the term used to describe a microscopic collection of DNA probes arrayed onto a solid surface. Microarrays take advantage of the highly selective nature of nucleic acid interactions and are commonly used for expression profiling, for comparative genomic hybridization, to aid genomic annotation, and for detection of mutations within genomes. In this virus-focused chapter, we deal primarily with the use of microarrays for expression analysis (the most popular usage) of host and virus systems during infection. We examine aspects related to array platform choice (spotted and oligonucleotide arrays), probe and array design considerations, experimental procedures and data analysis, normalization, processing, and curation. We also provide in-depth examples for the study of viral transcriptome analysis for both spotted long oligonucleotide (coccolithoviruses) and Affymetrix GeneChip (cyanophage) arrays.

Kenneth M. Stedman, Kate Porter, and Mike L. Dyall-Smith 
The isolation of viruses infecting Archaea
Chapter 6, pp 57-64

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Full Citation: Stedman, K. M., K. Porter, and M. L. Dyall-Smith. 2010. The isolation of viruses infecting Archaea, p. 57-64. In S. W. Wilhelm, M. G. Weinbauer, and C. A. Suttle [eds.], Manual of Aquatic Viral Ecology. ASLO. [DOI 10.4319/mave.2010.978-0-9845591-0-7.57]

ABSTRACT: A mere 50 viruses of Archaea have been reported to date; these have been investigated mostly by adapting methods used to isolate bacteriophages to the unique growth conditions of their archaeal hosts. The most numerous are viruses of thermophilic Archaea. These viruses have been discovered by screening enrichment cultures and novel isolates from environmental samples for their ability to form halos of growth inhibition, or by using electron microscopy to screen enrichment cultures for virus-like particles. Direct isolation without enrichment has not yet been successful for viruses of extreme thermophiles. On the other hand, most viruses of extreme halophiles, the second most numerous archaeal viruses, have been isolated directly from hypersaline environments. Detailed methods for the isolation of viruses of extremely thermoacidophilic Archaea and extremely halophilic Archaea are presented in this manuscript. These methods have been extremely effective in isolating novel viruses. However, Archaea comprise much more than extreme thermoacidophiles and extreme halophiles. Therefore a vast pool of archaeal viruses remain to be discovered, isolated, and characterized, particularly among the methanogens and marine Archaea. Some suggestions for expansion of the described methods are discussed. We hope these suggestions will provide an impetus for future work on these and other Archaeal viruses.

Susan A. Kimmance and Corina P. D. Brussaard 
Estimation of viral-induced phytoplankton mortality using the modified dilution method
Chapter 7, pp 65-73

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Full Citation: Kimmance, S. A., and C. P. D. Brussaard. 2010. Estimation of viralinduced phytoplankton mortality using the modified dilution method, p. 65-73. In S. W. Wilhelm, M. G. Weinbauer, and C. A. Suttle [eds.], Manual of Aquatic Viral Ecology. ASLO. [DOI 10.4319/mave.2010.978-0-9845591-0-7.65]

ABSTRACT: The modified dilution assay aims to partition phytoplankton mortality into virus- versus grazing-induced fractions and has previously been applied to several different environments to determine viral lysis rates of natural phytoplankton. The method involves creating a gradient of both grazing and viral lysis by dilution with different proportions of grazer- and virus-free filtrate, and assessing the subsequent impact on phytoplankton growth rates. We have conducted a critical evaluation of this method, and reviewed published data sets obtained using this approach to examine the utility of the modified dilution assay for estimating viral mortality rates. We provide modifications and improvements that have been incorporated into the method since it was first developed, and suggest recommendations for improving experimental success in less productive oligotrophic environments. Published data show that viral lysis rates vary between different algal groups and in response to environmental conditions. Results also suggest that this method has the potential to be a useful tool for estimating the impact of viruses on phytoplankton populations, but that the measurement of natural, low viral lysis rates (less than 0.1 d–-1) can challenge the application of this approach. Ultimately, however, the limitation of this method is associated with dilution of specific phytoplankton populations at low abundance.

Roberto Danovaro and Mathias Middelboe 
Separation of free virus particles from sediments in aquatic systems
Chapter 8, pp 74-81

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Full Citation: Danovaro, R., and M. Middelboe. 2010. Separation of free virus particles from sediments in aquatic systems, p. 74-81. In S. W. Wilhelm, M. G. Weinbauer, and C. A. Suttle [eds.], Manual of Aquatic Viral Ecology. ASLO. [DOI 10.4319/mave.2010.978-0-9845591-0-7.74]

ABSTRACT: The number of benthic viruses per unit of volume, at all depths (from shallow down to abyssal sediments), exceeds water column abundances by orders of magnitude. The need of methods for the determination of viral counting in aquatic sediment is becoming increasingly urgent along with the increasing evidence of the relevance of viruses in the benthic domain. The procedures used for determining viral abundances in sediments require specific modifications to release the viruses from sediment particles and to minimize the physical and chemical interferences of sedimentary matrix with the analysis. Dislodging viruses from sediment samples is the first crucial step for the analyses of viral abundance in benthic samples. Here we present the results of several tests aimed at optimizing the protocol for viral counts based on (i) the chemical treatment (type and modality of the use of surfactants), (ii) mechanical treatment (ultrasounds), (iii) cleaning of the samples (by enzymatic digestion of the extracellular DNA by means of DNases), and (iv) the limitations associated with viral recovery from the sediment (by serial washing steps). Sediment texture and composition vary considerably along horizontal and vertical gradients, and here we compare shallow sandy sediments with more silty deep-sea sediments. We found that the use of the surfactant tetrasodium pyrophosphate (final concentration 5 mM for 15 min), followed by ultrasound treatments (3 times for 1 min with 30 s intervals) and by the addition of an enzymatic cocktail composed of DNase I, nuclease P1, nuclease S1, and esonuclease 3, increased the detectability by staining with fluorochrome, thus resulting in significantly higher and more accurate viral counting, determined by epifluorescence microscopy. Our results also indicate that sediment samples processed using this optimized protocols displayed a significantly lower coefficient of variation, thus making sufficient the counting of a lower number of optical fields. Centrifugation of sediment samples after extraction procedures could underestimate viral counting, and we recommend here an accurate check of the potential loss or an alternative procedure based on sediment dilution prior to quantification by epifluorescence microscopy.

Steven M. Short, Feng Chen, and Steven W. Wilhelm 
The construction and analysis of marker gene libraries
Chapter 9, pp 82-91

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Full Citation: Short, S. M., F. Chen, and S. W. Wilhelm. 2010. The construction and analysis of marker gene libraries, p. 82-91. In S. W. Wilhelm, M. G. Weinbauer, and C. A. Suttle [eds.], Manual of Aquatic Viral Ecology. ASLO. [DOI 10.4319/mave.2010.978-0-9845591-0-7.82]

ABSTRACT: Marker genes for viruses are typically amplified from aquatic samples to determine whether specific viruses are present in the sample, or to examine the diversity of a group of related viruses. In this chapter, we will provide an overview of common methods used to amplify, clone, sequence, and analyze virus marker genes, and will focus our discussion on viruses infecting algae, bacteria, and heterotrophic flagellates. Within this chapter, we endeavor to highlight critical aspects and components of these methods. To this end, instead of providing a detailed experimental protocol for each of the steps involved in examining virus marker gene libraries, we have provided a few key considerations, recommendations, and options for each step. We conclude this chapter with a brief discussion of research on a major capsid protein (g20) of cyanomyoviruses using this work as a case study for polymerase chain reaction primer design and development. By building on the experience of numerous labs, this chapter should not only be useful to the new virus ecologist, but also serve as a valuable resource to established research groups.

Keizo Nagasaki and Gunnar Bratbak 
Isolation of viruses infecting photosynthetic and nonphotosynthetic protists
Chapter 10, pp 92-101

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Full Citation: Nagasaki, K., and G. Bratbak. 2010. Isolation of viruses infecting photosynthetic and nonphotosynthetic protists, p. 92-101. In S. W. Wilhelm, M. G. Weinbauer, and C. A. Suttle [eds.], Manual of Aquatic Viral Ecology. ASLO. [DOI 10.4319/mave.2010.978-0-9845591-0-7.92]

ABSTRACT: Viruses are the most abundant biological entities in aquatic environments and our understanding of their ecological significance has increased tremendously since the first discovery of their high abundance in natural waters. About 40 viruses infecting eukaryotic algae and 4 viruses infecting nonphotosynthetic protists have so far been isolated and characterized to different extents. The isolated viruses infecting phytoplankton (Chlorophyceae, Prasinophyceae, Haptophyceae, Dinophyceae, Pelagophyceae, Raphidophyceae, and Bacillariophyceae) and heterotrophic protists (Bicosoecophyceae, Acanthamoebidae, and Thraustochytriaceae) are all lytic. Some of the brown algal phaeoviruses, which infect host spores or gametes, have also been found in a latent form (lysogeny) in vegetative cells. Viruses infecting eukaryotic photosynthetic and nonphotosynthetic protists are highly diverse both in size (ca. 20-220 nm in diameter), genome type (double-strand deoxyribonucleic acid [dsDNA], single-strand [ss]DNA, ds-ribonucleic acid [dsRNA], ssRNA), and genome size [4.4-560 kb]). Availability of host-virus laboratory cultures is a necessary prerequisite for characterization of the viruses and for investigation of host-virus interactions. In this report we summarize and comment on the techniques used for preparation of host cultures and for screening, cloning, culturing, and maintaining viruses in the laboratory

Corina P.D. Brussaard, Jérôme P. Payet, Christian Winter, and Markus G. Weinbauer 
Quantification of aquatic viruses by flow cytometry
Chapter 11, pp 102-109

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Full Citation: Brussaard, C. P. D., J. P. Payet, C. Winter, and M. G. Weinbauer. 2010. Quantification of aquatic viruses by flow cytometry, p. 102-109. In S. W. Wilhelm, M. G. Weinbauer, and C. A. Suttle [eds.], Manual of Aquatic Viral Ecology. ASLO. [DOI 10.4319/mave.2010.978-0-9845591-0-7.102]

ABSTRACT: For many laboratories, flow cytometry is becoming the routine method for quantifying viruses in aquatic systems because of its high reproducibility, high sample throughput, and ability to distinguish several subpopulations of viruses. Comparison of viral counts between flow cytometry and epifluorescence microscopy typically shows slopes that are statistically not distinguishable from 1, thus confirming the usefulness of flow cytometry. Here we describe in detail all steps in the procedure, discuss potential problems, and offer solutions.

K. Eric Wommack, Télesphore Sime-Ngando, Danielle M. Winget, Sanchita Jamindar, and Rebekah R. Helton 
Filtration-based methods for the collection of viral concentrates from large water samples
Chapter 12, pp 110-117

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Full Citation: Wommack, K. E., T. Sime-Ngando, D. M. Winget, S. Jamindar, and R. R. Helton. 2010. Filtration-based methods for the collection of viral concentrates from large water samples, p. 110-117. In S. W. Wilhelm, M. G. Weinbauer, and C. A. Suttle [eds.], Manual of Aquatic Viral Ecology. ASLO. [DOI 10.4319/mave.2010.978-0-9845591-0-7.110]

ABSTRACT: Ecological investigations rely on data describing the biomass, diversity, and composition of living things. In the case of microbial communities, these data are primarily gathered using microscopy and molecular genetic approaches. The diminutive size of viruses means that obtaining genetic material sufficient for molecular approaches for examining the diversity and composition of aquatic viral assemblages can be challenging. Moreover, in procedures for the isolation and cultivation of novel viruses from natural waters, high-density viral inocula provide the best chance for success. To address the need for samples containing a high-density of viruses, investigators have used tangential-flow filtration (TFF) to concentrate viruses from large-volume (>20 L) water samples. This report outlines procedures for the preparation of viral concentrates from large volume water samples using TFF and discusses the effect of concentration procedures on viral recovery and downstream molecular genetic analyses.

Mathias Middelboe, Amy M. Chan, and Sif K. Bertelsen 
Isolation and life cycle characterization of lytic viruses infecting heterotrophic bacteria and cyanobacteria
Chapter 13, pp 118-133

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Full Citation: Middelboe, M., A. M. Chan, and S. K. Bertelsen. 2010. Isolation and life cycle characterization of lytic viruses infecting heterotrophic bacteria and cyanobacteria, p. 118-133. In S. W. Wilhelm, M. G. Weinbauer, and C. A. Suttle [eds.], Manual of Aquatic Viral Ecology. ASLO. [DOI 10.4319/mave.2010.978-0-9845591-0-7.118]

ABSTRACT: Basic knowledge on viruses infecting heterotrophic bacteria and cyanobacteria is key to future progress in understanding the role of viruses in aquatic systems and the influence of virus-host interactions on microbial mortality, biogeochemical cycles, and genetic exchange. Such studies require the isolation, propagation, and purification of host-virus systems. This contribution presents some of the most widely used methodological approaches for isolation and purification of bacteriophages and cyanophages, the first step in detailed studies of virus-host interactions and viral genetic composition, and discusses the applications and limitations of different isolation procedures. Most work on phage isolation has been carried out with aerobic heterotrophic bacteria and cyanobacteria, culturable both on agar plates and in enriched liquid cultures. The procedures presented here are limited to lytic viruses infecting such hosts. In addition to the isolation procedures, methods for life cycle characterization (one-step growth experiments) of bacteriophages and cyanophages are described. Finally, limitations and drawbacks of the proposed methods are assessed and discussed.

William H. Wilson and Declan Schroeder 
Sequencing and characterization of virus genomes
Chapter 14, pp 134-144

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Full Citation: Wilson, W. H., and D. Schroeder. 2010. Sequencing and characterization of virus genomes, p. 134-144. In S. W. Wilhelm, M. G. Weinbauer, and C. A. Suttle [eds.], Manual of Aquatic Viral Ecology. ASLO. [DOI 10.4319/mave.2010.978-0-9845591-0-7.134]

ABSTRACT: By unraveling the genetic code of viruses, genome sequencing offers a new era for aquatic virus ecology giving access to ecological function of viruses on an unprecedented scale. Although this chapter starts with the suggestion to that virus genome sequencing should be conducted professionally if financially feasible, we essentially try and guide the reader through some of the procedures that will direct a novice through a genome sequencing project. Arguably, the most important recommendation is to start with as high purity virus nucleic acid as possible. We use the adage, junk in equals junk out. Once sequence information is obtained, there is plenty of free, user-friendly software available to help build, annotate, and then compare sequence data. Acquiring metadata is another important aspect that is not often considered when embarking on a genome project. A new initiative by the Genomic Standards Consortium has introduced Minimum Information about a Genome Sequence (MIGS) that allows standardization of the way the data are collected to make it useful for downstream post-genomic analyses. Most viruses sequenced to date have produced surprises, and there is more to come from the other 1031 viruses still to be sequenced. This chapter focuses on sequencing purified virus isolates rather than virus metagenomes.

Curtis A. Suttle and Jed A. Fuhrman 
Enumeration of virus particles in aquatic or sediment samples by epifluorescence microscopy
Chapter 15, pp 145-153

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Full Citation: Suttle, C. A., and J. A. Fuhrman. 2010. Enumeration of virus particles in aquatic or sediment samples by epifluorescence microscopy, p. 145-153. In S. W. Wilhelm, M. G. Weinbauer, and C. A. Suttle [eds.], Manual of Aquatic Viral Ecology. ASLO. [DOI 10.4319/mave.2010.978-0-9845591-0-7.145]

ABSTRACT: Microbes and microbial processes are crucial and quantitatively important players in aquatic environments, and viruses as major agents of microbial mortality and nutrient cycling are a key component of aquatic systems. These roles have led to the need to routinely quantify viral abundance as the part of many investigations. Electron microscopy was first used to demonstrate high viral abundances in aquatic samples; by the mid-1990s, the greater accuracy and higher precision of estimates of viral abundance made by epifluorescence microscopy (EFM) were evident. Initially, DAPI (6-diamidino-2-phenylindole) was the stain used to enumerate virus particles in natural samples, but this dye was soon superseded by a new generation of much brighter fluorochromes. This article outlines detailed protocols for enumerating virus particles in aquatic or sediment samples using SYBR Green, SYBR Gold, and Yo-Pro-1. Each of these stains has advantages and disadvantages, but for natural water samples they produce indistinguishable estimates of viral abundance when the appropriate protocols are carefully followed.

Grieg F. Steward and Alexander I. Culley 
Extraction and purification of nucleic acids from viruses
Chapter 16, pp 154-165

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Full Citation: Steward, G. F., and A. I. Culley. 2010. Extraction and purification of nucleic acids from viruses, p. 154-165. In S. W. Wilhelm, M. G. Weinbauer, and C. A. Suttle [eds.], Manual of Aquatic Viral Ecology. ASLO. [DOI 10.4319/mave.2010.978-0-9845591-0-7.154]

ABSTRACT: Research on the diversity and ecology of viruses in the environment has been revolutionized by the ability to detect, fingerprint, and sequence viral genes and genomes. The starting point for these molecular assays is the release and recovery of the viral nucleic acids. The complexity of this task depends in large part on the nature of the starting material and the purity and quality of the nucleic acids one requires for downstream applications. In some cases, simply heating the sample will suffice; in other cases, a series of organic extractions and purification in a buoyant density gradient may be required to achieve adequate purity. Our goal in this chapter is to assist the reader in making informed choices from among the many options available. Toward this end, we briefly review the methods that have been used to harvest and store viruses in preparation for extraction, and the methods by which their nucleic acids may be released and purified. We discuss the general principles upon which various commercial extraction kits are based and conclude with the presentation of four step-by-step protocols. We discuss the advantages and disadvantages of these protocols, and the ways in which they may be adapted to various situations.

Janice E. Lawrence and Grieg F. Steward 
Purification of viruses by centrifugation
Chapter 17, pp 166-181

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Full Citation: Lawrence, J. E., and G. F. Steward. 2010. Purification of viruses by centrifugation, p. 166-181. In S. W. Wilhelm, M. G. Weinbauer, and C. A. Suttle [eds.], Manual of Aquatic Viral Ecology. ASLO. [DOI 10.4319/mave.2010.978-0-9845591-0-7.166]

ABSTRACT: Ultracentrifugation provides a means to concentrate, analyze, and purify viruses in solution, and therefore represents an invaluable tool for aquatic virologists. This chapter reviews the theory of ultracentrifugation and presents the technical knowledge necessary for an investigator to adapt or develop methods to meet his or her needs. Detailed protocols for the purification of viruses from culture lysates and vial assemblages from natural water samples are provided.

Hans-W. Ackermann and Mikal Heldal 
Basic electron microscopy of aquatic viruses
Chapter 18, pp 182-192

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Full Citation: Ackermann, H.-W., and M. Heldal. 2010. Basic electron microscopy of aquatic viruses, p. 182-192. In S. W. Wilhelm, M. G. Weinbauer, and C. A. Suttle [eds.], Manual of Aquatic Viral Ecology. ASLO. [DOI 10.4319/mave.2010.978-0-9845591-0-7.182]

ABSTRACT: For identification and structural studies, viruses are concentrated and purified by differential or density gradient centrifugation, stained with phosphotungstate (PT) or uranyl acetate (UA), and examined by transmission electron microscopy. Both PT and UA produce artifacts. PT yields negative staining only; UA produces both negative and positive staining and often gives excellent contrast. Fixation is normally not necessary. UA-stained preparations can be stored for years. Virus particles may be sedimented directly from water samples onto grids by means of special centrifuge tubes and subsequent staining. Positively stained particles are highly contrasted and easy to count at low magnification. Positive staining also provides information on bacteria containing viral particles and rough estimates of burst sizes in individual bacteria. Isometric, filamentous, and pleomorphic viruses are identified after negative staining.

Alexander I. Culley, Curtis A. Suttle, and Grieg F. Steward 
Characterization of the diversity of marine RNA viruses
Chapter 19, pp 193-201

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Full Citation: 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. [DOI 10.4319/mave.2010.978-0-9845591-0-7.193]

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.