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1 Introduction

Marine viruses are like any other viruses defined as small infectious agents, consisting of a core of nucleic acids (RNA or DNA) in a protein coat, that replicate only in the living cells of a host. A lipid envelope may be found outside or inside of the capsid. In the strict sense, complete viral particles found outside host cells and constituting the infective form are called virions, however, throughout this chapter the term viruses will be used for both the intracellular and extracellular forms. Viruses are classified by their genome type (DNA or RNA, single or double stranded, segmented or not, circular or linear) and size (from few Kbp to 2.5 Mbp), particle structure (e.g., icosahedral or helical) and size (from ~20 to 500 nm), capsid coat protein composition, whether it is enveloped or not, the replication strategy, the latent period (i.e., the time until progeny viruses are released from the host cell), sensitivity to physicochemical factors (e.g., temperature, pH, UV), and last but not least the host they infect. There are no universal oligonucleotide primers for marine viruses due to their huge genetic diversity, but the presence of specific conservative genes aids to the detection and classification of marine viruses, e.g., primers for fragments of the conserved cyanophage structural gene g20, or for the DNA polymerase gene (polB) are used to detect large dsDNA algal viruses (Chen and Suttle 1995; Short and Suttle 2005).

Unable to replicate without a host cell, viruses are generally not considered living organisms or alive (Forterre 2010). The finding of large genome-sized viruses displaying a localized viral factory in the host’s cytoplasm where the viral genome is replicated and virions are produced (La Scola et al. 2003; Santini et al. 2013), together with the discovery of viruses infecting the viral factory (c.q. virophages meaning virus eaters; La Scola et al. 2008; Fischer and Suttle 2011), renewed the discussion as to whether viruses are alive. Both ways, viruses have the ability to pass on genetic information to upcoming generations and as such they play an important role in biodiversity and evolution. Due to their mode of replication, viruses can take some genetic material from their host in their progeny but can also add their own genetic material to the host, thereby increasing genetic diversity and biodiversity as a whole. Actually, viruses can in a way be considered part of the genetic reservoir of their host, i.e., the genomes of viruses are part of the larger pan-genome (e.g., Bacteria or Archaea) like other extra-chromosomal elements. Given their rapid reproduction rates, and often high mutation rate, viruses represent an important source of genetic innovation. Besides the enormous reservoir of uncharacterized genetic diversity (Suttle 2007), viruses have the potential to be interesting material for biotechnological use (Sánchez-Paz et al. 2014).

The research on marine viruses, their population dynamics, diversity and ecological relevance has expanded exponentially over the last years. Marine viruses are the most abundant biological entities in the seas and oceans, ranging between 1 and 100,000 × 106 L−1, whereby the higher abundances are generally found in the more coastal, eutrophic surface waters and the lowest numbers in the deep ocean. Per unit volume, viruses in marine sediments (benthic viruses) with the higher load of bacterial hosts exceed pelagic viruses by one or more orders of magnitude (Danovaro et al. 2008; Glud and Middelboe 2004; Hewson et al. 2001). The numerically dominant hosts are the marine microorganisms belonging to all three domains of life, i.e., Bacteria, Archaea, and Eukarya. With the development of sensitive nucleic acid-specific dyes in combination with epifluorescence microscopy (Noble and Fuhrman 1998), pelagic viruses could be more easily counted than by transmission electron microscopy (Fig. 5.1). Moreover, the availability of fluorescent dyes allowed counting of the total viral community and not just the infectious viruses for specific hosts in culture as determined traditionally by plaque assay (using agar plates) and most probable number (end-point dilution of liquid cultures). More recently, staining of the viral nucleic acids combined with flow cytometry allowed a more objective and faster analysis (Brussaard et al. 2000; Brussaard 2004a) opening further the research field of marine viruses to a better spatial and temporal sample coverage, as well as higher degree of replication. Besides these methods for direct detection of virus particles, there are molecular approaches helping to identify and quantify (by qPCR) marine viruses (Sandaa and Larsen 2006; Tomaru et al. 2011a, b, c). Combining techniques such as, for example, flow cytometry sorting of virus and/or infected host with molecular sequencing will further advance prospective research aiming at answering questions on viral diversity and adaptation (Labonté et al. 2015; Martínez-Martínez et al. 2014; Zeigler et al. 2011). Metagenomic analysis has even revealed new virus families, e.g., ssDNA viruses infecting a wide range of marine hosts (Labonté and Suttle 2013a, b).

Fig. 5.1
figure 1

Different techniques to detect marine viruses, i.e., epifluorescence microscopy upon staining the viral nucleic acid (left panel), transmission electron microscopy (TEM) thin section of host cell showing virions PgV infecting the phytoplankton Phaeocystis globosa (virus 150 nm diameter; mid upper panel), TEM of free bacteriophage (virus head diameter 100 nm, tail length 180 nm; mid lower panel), and flow cytometry of natural seawater sample showing several virus clusters (right panel)

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Although essential, quantification and identification by itself is not enough to answer the major research questions in marine viral ecology, e.g., who infects whom, what is the impact of viral infection on host population dynamics and subsequent species succession, and how do viruses affect ecosystem efficiency and consequently biogeochemical fluxes? One needs to be able to determine actual viral infection and lysis rates in the field. At the same time, there is need to isolate and bring into culture more (and new) virus-host model systems in order to allow optimal understanding of the mechanisms underlying successful infection and alterations in virus–host interactions due to change in environmental factors.

The outcome of viral infection has implications for ocean function and it is an exciting time of discovery (some milestones are listed in Table 5.1). This chapter describes some of the milestones in marine viral ecology (Table 5.1) and outlines the different aspects of marine viral action, i.e., the different viral infection strategies, horizontal gene transfer, viral diversity, ecological influence on host mortality, microbial biodiversity, food web structure, organic carbon and nutrient cycling, the culturing and storage of virus-host systems, and concludes with several biotechnological applications.

Table 5.1 Overview of some of the milestones obtained in marine virus ecology during the last 1–2 decades
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2 Viral Infection Strategies

Microbial host cells and their viral predators have been evolving together for billions of years. At first glance, one might think that their interactions are simpler than those of other predator or parasite-host pairs but they are actually extremely complex. The main predators of microbes typically discriminate for prey size, along rough lines of taxonomy and morphology and can eat many individual prey of diverse genomic make up throughout its individual cell life span (Sintes and Del Giorgio 2014). In contrast, viruses are single opportunity killers and highly host-specific. One virus particle has only one chance to reproduce and requires a refined adjustment to the cell machinery on which it depends for ultimate replication and virion component. Therefore, viruses are specialized parasites that are fine-tuned to the host structure and metabolism (Leggett et al. 2013). Actually, the delicate discriminatory power of viruses has been used classically for typing, for example, bacteria. Precise host cell recognition is an essential step in the life cycle of any virus. Accordingly, viruses have refined molecular recognition modules that allow discrimination of encountered cells before the infection process in triggered (Garcia-Doval and van Raaij 2013). These molecular recognition devices are among the most sensitive in nature and match in complexity, and probably in diversity as well, the vertebrate immune system (Fraser et al. 2007). Mirroring this diversity, host cells have an enormous diversity of virus potential receptors. They can be any outermost exposed structure of the cell, frequently proteins such as pillins, porins or transporters but, above all, polysaccharides or the glycosidic moiety of glycoproteins. Actually, the interaction with viruses might be behind the commonality of polysaccharides and glycoproteins in exposed structures of Bacteria, Archaea, and Eukarya.

Through the lytic cycle, viruses can reproduce inside the host cell directly upon penetration, causing destruction of the host cell to release the progeny viruses into the surrounding water. Alternatively, viruses may integrate into the host genome as a provirus and propagate together with the host. Thus, in contrast to lytic viruses, these temperate (or latent) phages establish a stable, but reversible, relationship with their host, which is termed lysogeny. For the prophage being inside the host provides protection against virion decay by environmental factors (Mojica and Brussaard 2014). Although at first glance it does not seem beneficial for the host to carry a prophage, it generally protects from infection by similar and related phages. Furthermore, prophage may provide useful genes and improve host fitness (Paul 2008). This transfer of genes via prophages is a form of horizontal gene transfer (HGT) that aids to the genetic diversity within the host species. The prophage can be released again by an inducing event triggering a lytic cycle. The environmental factors responsible for the transition from a lysogenic to a lytic cycle are not well identified. UV and pollution have been reported to induce the lytic stage, potentially to produce progeny viruses before potential cell death of the host (Paul et al. 1999). Alternatively, the triggering factor may represent more optimal growth conditions for the host (e.g., release of P-limitation; Wilson et al. 1996), whereby the shift from prophage to lytic phage is a favorable one as many more progeny viruses can be produced than under the lysogenic life style (one progeny every time the host divides). In agreement with this potential mechanism, the percentage lysogeny of total mortality of bacteria was found highest under oligotrophic conditions with low host abundance, both geographically as well as seasonally (McDaniel et al. 2002; Weinbauer et al. 2003).

Besides lytic and lysogenic cycles, the host cell can also release viruses by continuous or intermittent budding or extrusion without its immediate lysis. This replication strategy, known as chronic cycle or carrier state, is far less investigated in marine environments, yet it has been described for viruses that infect marine eukaryotic photoautotrophs (Mackinder et al. 2009; Thomas et al. 2011), but also for archaeal viruses (Geslin et al. 2003; Gorlas et al. 2012).

3 Virus Characteristics

The characterization of virus isolates typically includes a phenotypic analysis (particle morphology, symmetry and size; genome type and size; composition of structural proteins) in order to assign a taxonomical status to these isolates. Additionally, parameters related to virus life cycle, specificity pattern, environmental stability or resistance, are other important viral features. The information generated by one-step growth experiments, also referred to as “single burst experiments,” have proven essential in this respect. This classical procedure developed by Ellis and Delbrück (1939) is designed to quantitate and monitor the growth of lytic viruses and it provides two fundamental viral properties: the latent period (that is the duration between virus adsorption and extracellular release from the infected host cell) and the burst size (that is the number of progeny viruses released per infected host cell). These viral parameters vary depending on the virus isolate and the host they infect. Collectively these variables constitute important components to model virus–host interactions in the ocean. For example, the infection strategy of a given virus largely determines the ecological impact of virus interactions. Gaining fundamental knowledge on the infection strategy of relevant virus-host model systems, the conditions that induce the transition between these different modes of infection, and the potential influence environmental factors have on the intensity and the kinetics of infection are thus a prerequisite for understanding the impact of viral infection on microbial assemblages.

The infection range (the range of action of a phage measured in terms of the varieties of bacteria in which it can grow) has been a major conundrum of virus studies. The host specificity of bacteriophages (i.e., viruses that infect bacteria; there are also referred to as phage or bacteriovirus) was recognized very soon after their discovery in the early twentieth century (Abedon 2000). The idea of phage therapy was based on this early discovery and was the main driver of the original interest on bacteriophages, later displaced by the enormous role played by phages in the development of genetics and molecular biology. The success or failure of adsorption and subsequent lysis of the host typically, but not always, determines the host infection range. There is a wide diversity of mechanisms within the cell to prevent phage replication, including receptor variation, restriction–modification, abortive infection, lysogenic immunity, or innate immunity conferred by CRISPRs (see for example Stern and Sorek 2011). In the laboratory, host specificities are determined by pairwise infection, in which a clonal virus lineage is added to a collection of host cultures, infection is scored positive if lysis occurs. These infection assays suggest that marine viruses exist along a continuum of specialists to generalists. The specialist viruses can only infect a restricted range of strains, sometimes even only the one used for their initial isolation. The generalist viruses can infect many different strains and occasionally can infect also isolates from different species and even different genera (e.g., Baudoux et al. 2012; Johannessen et al. 2015; Matsuzaki et al. 1992; Sullivan et al. 2003; Suttle et al. 1995). However, in general the genus is the taxonomic unit that encompasses the most sensitive strains for the vast majority of phages. It is sensible to say that the boundary of natural host range of most phages is the genus.

Cross-infections between virus and hosts are challenging to interpret. In spite of 25 years of investigation, there is as yet no consensus on either the global pattern of viral infection or the environmental drivers that shape these patterns of interactions. For example, to what extent does virus–host infection vary in space and/or time, are there factors that favor generalists upon specialists, and are virus–host interactions structured at all (Avrani et al. 2012)? It is presently inconceivable to bring into culture and test the specificity pattern of all individual virus-host model systems dispersed in marine systems. As an alternative, theoretical approaches that rely on empirical data have been proposed to study of virus–host interactions as networks (Flores et al. 2011, 2013; Weitz et al. 2013). Network-based analysis tests whether virus–host interactions are structured (i.e., they possess a structured pattern) or random. The most common patterns in ecological networks are being nested or modular. Nested virus–host interaction networks are characterized by a hierarchy of resistance among hosts and infection ability among viruses (Flores et al. 2011; Weitz et al. 2013). This pattern is thought to result from coevolutionary arms race (Red Queen hypothesis) where viruses evolve to broaden host ranges and hosts evolve to increase the number of viruses to which they are resistant (Flores et al. 2011). In contrast, modularity is characterized by groups of viruses and hosts (referred to as modules) that preferentially interact with another (Flores et al. 2011; Weitz et al. 2013). This ecological pattern should emerge if virus–host interactions result from evolutionary processes that lead to specialization.

A network-based analysis of cross-infections of 215 phages against 286 bacteria collected across the Atlantic Ocean (Moebus and Nattkemper 1981) revealed that infection network possessed a multiscale structure (Flores et al. 2013). At a global network scale, virus–host interactions network displayed a modular structure. This modularity was explained, at least in part, by geography. In other words, viruses isolated from a given sampling site were more likely to infect cooccurring hosts, supporting the need to account for biogeography in the analysis of viral diversity. However, individual modules were either further modular or nested, which suggests that different coevolutionary processes drive virus–host interaction at different scale (Flores et al. 2013). Network-based analyses of virus–host interactions have emerged quite recently. Yet, these analyses represent a promising tool to quantify the functional complexity of viral infections in natural systems and to identify the drivers of microbial species interactions.

4 Virus and Host Diversity

The role of viruses in shaping the evolution of their hosts is acquiring new dimensions both as selective pressure that keeps populations diverse (Rodriguez-Valera et al. 2009) and as sources of genetic innovation (Suttle 2007). Viruses may carry metabolic genes of the host to enhance infection performance, but can also introduce genetic diversity to the cognate pathways. Microbiology has failed to provide a sensible representation of the diversity of microbes using only the pure culture approach. Although great advances are being made, until recently the vast majority of microbes in nature has been inaccessible to microbiologists. If this is the situation of cellular microbes, the status of the viruses preying on them is even direr. Most of what we know about viruses derives from the studies of human, cattle and plant crop viral pathogens. Lately large efforts to sequence more virus genomes of different hosts have been made (Pope et al. 2015), and the approaches based on high-throughput sequencing metagenomics and single cell genomics are also applied to marine viruses (Angly et al. 2006; Rodriguez-Brito et al. 2010).

The advent of high-throughput sequencing technologies permitted to determine the complete genome sequence of more than one hundred cultured marine viruses over the past 15 years and many more are currently being analyzed. Virus genomics provides invaluable insights into the understanding of how viral infection and virus evolutionary relationships function. One notable feature is that marine viruses encode remarkable panoplies of biological functionalities. They display core genes, found in most viruses, involved into the replication of viral DNA and the structure and the assembly of virions. Genome analyses also indicate that viruses encode and transfer a variety of auxiliary metabolic genes (AMG) derived from their hosts (Rohwer et al. 2000; Sullivan et al. 2005; Wilson et al. 2005). Although the ability to shuffle genetic information through HGT is commonly reported in viruses, the repertoire of host-derived genes encode by marine viruses differ from those of non-marine origins. The most emblematic AMG is probably the psbA photosynthesis gene reported in cyanophage genomes (Mann et al. 2003; Sullivan et al. 2005). This gene encodes the protein D1, which forms part of the photosystem II reaction center in the host cell. During the course of viral infection, the D1 proteins derived from the host decline dramatically while those encoded by the cyanophages increase (Clockie et al. 2006). This led to the hypothesis that the expression of virus-derived psbA helps maintaining photosynthesis throughout the lytic cycle, which, in turn, ‘boosts’ the host metabolism to support virus replication. The analysis of AMG in virus genomes suggests that marine viruses have evolved a wide range of strategies to hijack host metabolism towards their own replication (Hurwitz et al. 2013). AMG are widespread and they appear to be involved into phosphate scavenging, carbon, or nitrogen metabolism (Brum and Sullivan 2015; Hurwitz et al. 2013; Rohwer et al. 2000; Sullivan et al. 2005). The genome analysis of viruses that infect the bloom-forming alga Emiliania huxleyi (EhV) also revealed a unique cluster of sphingolipid biosynthetic genes (Wilson et al. 2005) that facilitate virus replication and assembly (Rosenwasser et al. 2014). Genomics of marine virus isolates provides unprecedented knowledge about complex metabolic pathways that play a pivotal in role in host–virus interactions.

In parallel to genome analysis of cultured isolates, the development of culture-independent tools was a breakthrough in virus ecology in the ocean. Metagenomics largely contributed to unveil the extent of viral diversity and the dynamics of viral communities (Angly et al. 2006; Breitbart et al. 2004; Brum et al. 2015). The genome sequence of relevant virus models is thus essential to build a reference database in order to identify the members of the viral communities and, importantly, whom they infect (Kang et al. 2013; Zhao et al. 2013). Despite its success, a general problem in the application of metagenomics techniques is that although the number of viral particles is high, the amount of viral DNA in natural environments is much smaller than cellular DNA. Therefore, since the beginning, DNA amplification approaches were applied to retrieve the required amount for Next Generation Sequencing (NGS) (Angly et al. 2006). In addition, the short NGS reads complicate enormously the already shaky grounds of viral genome annotation. Finally, one classical major limitation of viral metagenomics or metaviromics was assembly. By assembly of high coverage metagenomes large genome contigs can be reconstructed that are much more reliably annotated and classified. However, for unknown reasons, assembly works much more poorly with phage genomes in metaviromes. This problem can be partially by-passed by using cellular metagenomes. Cellular DNA is also rich in viral DNA derived from cells that are undergoing the lytic cycle. In metabolically active communities such as the deep chlorophyll maximum (DCM) 10–15 % of the bacterial and archaeal DNA derives from such phages (Ghai et al. 2010). For example, by using advanced binning approaches, a bacteroidetes phage that seems to be extremely widespread and conserved in the human microbiome could be assembled (Dutilh et al. 2014). By using metagenomic fosmid libraries Mizuno et al. (2013) described large genomic fragments from more than a 1000 viral genomes obtained from a single sample from the Mediterranean DCM. Fosmids have similar size to typical Caudovirales genomes and many of these could be retrieved. They could be gathered by genome comparison into 21 major sequence groups and a vast genomic diversity was present within each group. Host could be assigned to some of the 10 totally novel groups by comparison to putative host genomes what allowed assigning the first phages detected to marine Verrucomicrobia and the recently described marine actinobacteria Actinomarinales (Ghai et al. 2013). This work also revealed high microdiversity among the highly related sequence groups (Rodriguez-Valera et al. 2014), particularly in the host recognition modules. Specific single stranded (ss) DNA amplification has allowed the reconstruction of ssDNA viruses that are prevalent in marine habitats and have small genomes that simplify their reconstruction from metaviromes (Labonté and Suttle 2013a, b). The newly developed technology of single cell genomics is also providing data about viruses taking advantage of the same phenomenon, i.e., sorted cells are often undergoing lysis and eventually the active virus genome is abundantly represented in the resulted amplified genome (Labonté et al. 2015). This technique allowed the description of the first genome of a virus preying on the ubiquitous group I marine archaea or Thaumarchaeota.

5 Ecological Importance

Under natural conditions the richness of microbial species is high, while laboratory research indicates that competition for the same resources ultimately will lead to the dominance of the best competitor and the extinction of the other competitor(s). The most renowned example is the Plankton Paradox (Hutchinson 1961), whereby according to the competitive exclusion principle only a small number of plankton species should be able to coexist on the growth-limiting resources while in reality large numbers of plankton species are found to coexist within small regions of open sea. Because ecological factors stay hardly ever constant, spatio-temporal heterogeneity, but also selective grazing, symbiosis and commensalism have been suggested as factors responsible for resolving the Plankton Paradox. Another top-down controlling factor more recently acknowledged is viral infection. Marine viruses have typically narrow host ranges, which makes them highly relevant as underlying cause of coexistence of microbial species (Suttle 2007; Thingstad 2000). As viral infection is dependent on contact with the host, higher abundances of susceptible host species are expected to favor successful infection. This Killing-the-Winner (KtW) model describes how the abundance of the competition strategist is top-down controlled by viruses, thereby ensuring coexistence of competing species since the growth-limiting resources can be used by others (Thingstad 2000). Then again, a high abundance of host is not necessarily the result of rapid growth as host species might be resistant to top-down control (by zooplankton predators and/or viral lysis), or there might be substantial loss of viral infectivity or virus particles that prevent further infection of the developing host population. The traditional rank-abundance curves (illustrating the relative abundance of species) have to be read differently then, i.e., the least abundant taxa are not rare (or dormant) but are instead actively growing but at the same time also predated and/or lost by viral lysis (Suttle 2007). They illustrate r-selection, with high reproductive output, while the few most abundant taxa are K-selected with low maximum growth rates, better competitors for resources and less sensitive to cell loss (Suttle 2007; Våge et al. 2013). At the same time, the marine viruses rank-abundance curve shows a contrasting pattern with most viruses r-selected, being virulent with small genome sizes and high decay rates. The low abundance virus taxa are K-selected with larger genomes, longer-lived, and potentially forming stable associations with their host.

Blooms of phytoplankton exemplify situations where there is an unbalance between growth and loss processes, either because the algal species is able to outgrow infecting (at least temporarily) or because it is (largely) resistant to infection. Blooms of E. huxleyi illustrate the concept of KtW well, because during the development of the bloom the percentage of infected cells is increasing to a point that causes the collapse of the bloom (Bratbak et al. 1993; Brussaard et al. 1996a; Martínez-Martínez et al. 2007). Interestingly, the cell morphotype of E. huxleyi seems to affect the chance of infection. A laboratory study showed that the diploid calcifying coccolith-bearing cells were virally infected but the haploid cells were not (Frada et al. 2008). This finding led to the proposition of the Cheshire Cat escape strategy, where viral infection indirectly promotes a resistant haploid phenotype and thus sexual cycling (Frada et al. 2008). However, the genetically distinct communities of the E. huxleyi host and the co-occurring viruses under natural conditions were stable over several years, which seems to be inconsistent with the Cheshire Cat hypothesis (sexual reproduction would actually reshuffle the genes in E. huxleyi; Morin 2008). Moreover, a study of a natural bloom of E. huxleyi showed that the haploid organic scale-bearing cells (haploid by nature, Laguna et al. 2001) were also visibly infected by viruses (using transmission electron microscopy; Brussaard et al. 1996a). It needs to be seen whether these differences between the studies (1) indicate that sex does not act as an antiviral strategy (Morin 2008), (2) are due to growth-controlling factors (i.e., the natural bloom was nitrogen depleted while the laboratory study used nutrient replete cultures), (3) different virus types (the natural study showed co-infection of two different viruses), (4) genetically distinct algal host strains (and thus potentially different sensitivity to infection), or (5) the haploid E. huxleyi cells in the laboratory study being different than the natural scale-bearing cells.

Another example of how blooms are formed by (largely) viral infection-resistant cell morphotypes is the algal blooms of the phytoplankton genus Phaeocystis (Brussaard et al. 2005). Phaeocystis can make colonies provided that there is enough light available to allow the excess organic carbon to be excreted in the form of carbohydrates forming a colonial matrix (Schoemann et al. 2005). The colonial cell morphotype has a much lower chance of becoming infected (or grazed) than a single cell (Jacobsen et al. 2007; Ruardij et al. 2005) and therefore a considerable increase in biomass is possible. At the moment, the cells become shed from the colonial matrix (due to low light or nutrient depletion; Brussaard et al. 2005) the liberated single cells are readily infected and eaten and the bloom crashes (Baudoux et al. 2006).

Viruses can be so specific that they only infect certain strains. This provides the potential for intraspecies succession of host and virus within a phytoplankton bloom (Baudoux et al. 2006; Tarutani et al. 2000). For example, the different sensitivities of Heterosigma akashiwo clones to viral infection resulted not only in changes in the total abundance of the virus infecting H. akashiwo but also the clonal diversity of the algal host. Another example of high differential sensitivity to viral infection within a species is the blooms of Synechococcus spp. that were composed of many (genetically) different populations of host and cyanophage (Mühling et al. 2005; Suttle and Chan 1994; Waterbury and Valois 1993). Differences in infectivity properties of the host strains may be directly due to changes in the composition of the attachment sites on the host’s cell surface (Marston et al. 2012), or may be the indirect result of altered virus proliferation as a consequence of variation in host cell metabolic traits between the host strains (Brussaard, unpublished data). Resistance is thought to come with a cost, i.e., a resource competitive disadvantage compared to viral infection susceptible strains (Lenski 1988; Thingstad et al. 2015). Furthermore, host growth-controlling factors have been shown to affect virus latent period, viral burst size, the level of infectivity of the viral offspring, and even viral life strategy (Baudoux and Brussaard 2008; Bratbak et al. 1998; Maat et al. 2014; Wilson et al. 1996). Altogether, variations in host susceptibility to viral infection and viral production rate and yield add not only to biodiversity but also to spatial and temporal variability of microbial hosts and viruses.

Although many studies have indicated that viral infection is a significant loss factor, accurate measurements of viral lysis rates of microorganisms in the field are still few (Suttle 2005). Furthermore, rates of virally mediated mortality are assessed using different approaches (results are therefore not necessarily comparable), and most methods rely on different assumptions and conversion factors. The first approach, using visibly infected cells obtained from transmission electron microscopy analysis, is dependent on good estimation of the proportion of the lytic cycle during which virus particles are visible in the infected host cells (Brussaard et al. 1996a; Proctor and Fuhrman 1990). This factor is difficult to assess as the lytic virus growth cycle is highly variable between viruses and it can also differ within a virus depending on host metabolism. Estimating viral lysis rates using (fluorescent) virus tracers requires estimates of burst sizes (Heldal and Bratbak 1991; Noble and Fuhrman 2000; Suttle and Chen 1992). Using viral decay rates to estimate production rates also requires the assumption that there is a steady-state situation. The use of synthesis rates of viral DNA requires many conversion factors (Steward et al. 1992). Measuring viral production rates is most widely used but does need an estimate of burst size and comes with substantial sample handling (Weinbauer et al. 2010). Many of these methods, except the visibly infected host cell assay, measure viral lysis of the total community, because the different virus (and often also host) populations cannot be discriminated. Because algal viruses typically represent a few percent of the total virus abundance, the same methods are not well suited for phytoplankton viral lysis rate estimates unless the algal virus can be separately counted. For phytoplankton, viral lysis rate can be assessed using a modified dilution assay (Baudoux et al. 2006; Evans et al. 2003; Kimmance and Brussaard 2010), but here the restriction is that the algal host cells are newly infected and lysed within the time of incubation (typically 24 h because of synchronized cell division of many phytoplankton species).

The available viral lysis rate measurements in the field demonstrate that viral lysis is similar to predation rates (Baudoux et al. 2006, 2007; Mojica et al. 2015). The pathways of cellular organic matter and energy through the food web will, therefore, follow different ways (Suttle 2005). Upon predation organic carbon and nutrients are transferred to higher trophic levels, while upon viral lysis the host’s cellular content is released into the surrounding waters as dissolved and detrital particulate organic matter. Viruses are as mortality agents causing accelerated transformation of organic matter from the particulate (lysed host cells) state to the dissolved phase, upon which heterotrophic microbial communities mineralize the largely (semi-) labile organic matter directly (Lønborg et al. 2013). The diversion of organic matter towards the microbial loop (microbial recycling) results in enhanced respiration (Suttle 2005). Cell lysis of microorganisms has been reported to sustain the heterotrophic bacterial carbon demand (Brussaard et al. 1995, 1996b, 2005). Globally, viral lysis seems to negatively affect the efficiency of the biological pump (i.e., the transfer of photosynthetically fixed carbon to the deep ocean as dead organisms or feces). The rapid release and remineralization of nutrients in the photic zone may increase the ratio of carbon relative to nutrients exported to the deep. Conversely, viral lysis may potentially short-circuit the biological pump by stimulating the formation of transparent exopolymer particles (TEP) and aggregates (Brussaard et al. 2008; Mari et al. 2005), as well as release elements that could as such stimulate primary production (e.g., ligand-bound iron and organic phosphorus; Gobler et al. 1997; Løvdal et al. 2008; Poorvin et al. 2004). With respect to the global anthropogenic climate change (greenhouse effect but also alterations in the physicochemical characteristics of the water masses, e.g., temperature, salinity, light, nutrients), it is urgent to improve our understanding of how viral activity affects the processes involved in the natural sequestration of carbon dioxide. A study in the northeast Atlantic Ocean showed for instance that the share of viral lysis (as compared to grazing) of phytoplankton increased under conditions of temperature-induced vertical stratification (Mojica et al. 2015).

6 Isolation, Culture, and Characterization of Marine Viruses

Almost from their first discovery in 1917, cultured virus–host systems (VHS) were viewed as relevant models for microbiologists and geneticists who investigated the nature of genes and heredity. This early era of virus research constituted the foundations of molecular biology. It was not until the mid-1950s that marine viruses were discovered with the isolation and the description of a phage lytic to the fish gut associated bacterium, Photobacterium phosphoreum (Spencer 1955). Although the potential of bacteriophages to control host populations emerged quite rapidly, virus isolation from oceanic settings was only reported sporadically and most studies focused on the biology rather than the ecological implications of these model systems (Hidaka and Fujimura 1971; Hidaka 1977; Spencer 1960). The breakthrough discovery that viruses are the most abundant biological entities in the ocean marked a turning point in the field of virus ecology and literally renewed interest in isolating, culturing, and characterizing marine viruses.

Several protocols describing procedures for the isolation of lytic viruses have been reported from various laboratories. These protocols include three main steps: (1) A natural sample is added to a selection of prospective host cultures, (2) the lysis of this host is visualized and, (3) in case of positive lysis, the lytic agent is cloned and stored appropriately until characterization. In the following section, we provide a thorough description of these steps and recommendations to optimize successful virus isolation. Although these features are applicable to most viruses, viral strains may differ in their development, and therefore isolation protocols may have to be adapted accordingly. In theory, at least one, and usually multiple, viruses, can infect any given organism. However, empirical data have shown that some strains are more permissive to viral infection than others. It is, therefore, preferable to use several clonal host lineages in order to increase the chance of successful isolation. When possible, one should isolate the host strains just prior to virus isolation from the same location in order to increase success rate in case the virus has a narrow host range. Furthermore, owing to their parasitic nature, viruses largely rely on the metabolism of their hosts for production. In most cases, well-growing hosts tend to be more susceptible to viral infection.

Prior to the isolation, it is recommended to remove particles larger than viruses by low-speed centrifugation and/or pre-filtration through low protein binding membrane filters (e.g., polycarbonate, polyethylsulfone, polyvinylidene fluoride) with a preferable pore size of 0.45–0.8 µm (filtration through 0.2 µm could exclude larger virus particles). However, this may result in loss of viruses and therefore it is recommended to also use a non-treated sample. For sediment or any other type of porous samples, viral particles need to be transferred into a buffer solution prior to centrifugation and/or pre-filtration (Danovaro and Middelboe 2010). If the targeted virus population is expected to be low in abundance, tangential flow filtration/ultrafiltration or ultra-centrifugal units with a retention cutoff of at least 100 kDa can concentrate viral particles. As an alternative to these techniques that can become time consuming and costly, the host-enrichment technique also increases the probability of successful isolation considerably. This enrichment step consists of amending the viral community to be screened with 1–5 prospective host strains and host growth medium. Upon incubation this mixture, viruses lytic to these hosts should propagate and hence this procedure facilitates isolation. After incubation, the suspension can be clarified by centrifugation or pre-filtration and, if necessary, further concentrated by ultrafiltration as described above. Once prepared, the extracted viral community can be stored at 4 °C until the isolation procedure.

6.1 Detection of Lytic Viral Lysis and Virus Purification

There are several approaches to visualize cell lysis of the targeted host due to lytic viral infection. One common technique is the plaque assay, which has been successfully applied to the isolation of viruses of bacteria and microalgae (Fig. 5.2). This technique relies on combining an inoculum of the viral sample and the targeted host culture in soft agar overlay on agar plates. On incubation, the host develops a homogenous lawn, except where virus lysis occurs, resulting in localized translucent plaques, referred to as a plaque-forming unit (PFU, Fig. 5.2). PFU typically originates from one infectious virus and displays a well-defined morphology. Different viruses infecting the same host can coexist in the same sample. It is therefore recommended to pick as many individual plaques as possible and to repeat the plaque assay for another two times to produce as many clonal lineages of these isolates. Alternatively, the lysis of infected host can be detected in liquid medium. In this case, the propagation of viruses is not limited by the diffusion in semi-solid agar media. Upon attachment on a susceptible host, the viral progeny is released in liquid medium and the newly produced virions can initiate another lysis cycle and propagate until complete lysis of the cultured host. Lysis is detected by complete clearing of the host suspension as shown in Fig. 5.3. The resulting lysate can comprise a mixture of viruses that need to be separated as soon as possible to obtain clonal viral lineages. This can be achieved by extinction dilution methods in multiwell-plates (Fig. 5.3). The principle of this procedure is to serially dilute (usually using tenfold dilution increment) the lytic agents in vigorously growing host culture until complete extinction (i.e., no lysis detected anymore). The lysed culture from the most diluted dilution is selected and the complete procedure is repeated twice to ensure a clonal virus. While the plaque assay can lead to the isolation of several viral strains infecting the same host, isolation in liquid medium generally selects for the most abundant viral strain lytic for the targeted hosts. However, this latter procedure requires only non-specialized equipment, consumables and minimal sample handling.

Fig. 5.2
figure 2

Petri dish showing a plaque assay for viruses infecting the picoeukaryotic phytoplankton Micromonas sp. (photo courtesy Nigel Grimsley)

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Fig. 5.3
figure 3

Aquatic serial dilution set-up using microwell plates whereby a suspension of Micromonas virus RCC4265 was diluted from ×101 down to ×10−14 in Micromonas host culture RCC829. Lysis is scored positive until dilution ×108. Non-infected controls (first column) were taken along for reference

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6.2 Isolation of Temperate Viruses

The most common procedure to induce lysogens and isolate temperate viruses consists of inducing lysogens with the antibiotic mitomycin-C (Paul and Weinbauer 2010). The recommended mitomycin-C concentration varies depending on the lysogen strains but usually ranges between 0.1 and 2 µg mL−1. This treatment usually induces the collapse of lysogenized culture and the release of temperate virus within 12 h. Cellular debris and larger particles can be clarified from the suspension by low-speed centrifugation and/or filtration through 0.45 µm low protein binding membrane filters. The filtrate containing temperate viral isolates can then be stored at 4 °C in the dark until further characterization. Alternatively, lysogens can also be induced upon short (30 s) exposure to UV-C radiation (Jiang and Paul 1998; Lohr et al. 2007; Weinbauer and Suttle 1996). These procedures demonstrated that lysogeny was frequent among cultured marine bacteria (Jiang and Paul 1998; Lossouarn et al. 2015; Stopar et al. 2004) yet it has been seldom reported in microalgae (Lohr et al. 2007) and archaea. However, one should bear in mind that lysogeny represents a complex and stable relationship between the virus genome and that of its hosts. The induction of lysogens upon chemical or physical induction does not take into account the biological principle of this symbiosis (Paul 2008). It is thus likely that some lysogens are not induced following these treatments.

6.3 Isolation of Chronic Viruses

Chronic viruses that are released by the host cell by continuous or intermittent budding or extrusion are less investigated than their lytic and temperate counterparts, yet they have been reported in marine eukaryotic photoautotrophs Emiliania huxelyi (Mackinder et al. 2009) and Ostreococcus sp. (Thomas et al. 2011), but also for viruses of the hyperthermophilic euryarchaeote Pyrococcus abyssi (Geslin et al. 2003) and Thermococcus prieurii (Gorlas et al. 2012). Chronic viruses are typically detected by the examination of host culture by transmission electron microscopy. The isolation of these viruses mostly relies on extracting the virus particles from the host culture. This can be done either by low-speed centrifugation and/or filtration through low protein binding membrane filters with pore size ranging from 0.2 to 0.45 µm.

6.4 Maintenance and Storage

The most common procedure to store virus stocks is to keep them refrigerated (4 °C) in the dark. However, viral strains differ considerably with respect to their stability in the cold. While some isolates can be kept for years, others decay when stored refrigerated. Therefore, appropriate storage conditions should be designed for each individual type of virus. For the most challenging isolates, repeated transfers into fresh host culture can be considered for maintaining viruses. Finally, cryopreservation can be an alternative for, at least, some isolates. Bacteriophages affiliated to the order Caudovirales are often stored at −80 °C in 10–20 % sucrose, 10-20 % dimethyl sulfoxide (DMSO). These concentrations of cryoprotectant were also appropriate to store viruses of phytoplankton; however, storage temperature has to be modified according to the isolate stability (Nagasaki and Bratbak 2010). Conversely, to viruses pathogenic to human, animals or plants, marine virus isolates are rarely deposited in culture collections. Except for the Roscoff Culture Collection (RCC), which curates and supplies viruses of marine microbes, most VHS are maintained in personal culture collections of individual laboratories.

7 Marine Virus-Host Model Systems in Culture

During the past three decades few hundreds of viruses have been isolated from marine systems and brought into culture. Cultured viral isolates have been reported for many species emblematic of heterotrophic and phototrophic marine bacteria such as Proteobacteria, Bacteroidetes, Cyanobacteria (e.g., Holmfeldt et al. 2013, 2014; Moebus and Nattkemper 1983; Sullivan et al. 2003, 2005, 2009) and algae (Brussaard and Martínez-Martínez 2008; Short 2012; Tomaru et al. 2015 and references therein), whereas thus far only few viruses infecting archaea (Geslin et al. 2003; Gorlas et al. 2012), or heterotrophic protozoa (Arslan et al. 2011; Fischer et al. 2010; Garza and Suttle 1995) were brought into culture. Among the latter, the virus of the protozoa Cafeteria roenbergensis (CroV) was even parasitized by a small virophage (Mavirus) during CroV host infection (Fischer and Suttle 2011).

The isolation and characterization of these VHS unveiled an extraordinarily high level of morphological, taxonomical, and functional novelty. The characterization of 31 cellulophages demonstrated considerable divergence with known counterparts (Holmfeldt et al. 2013). The morphological and genomic analysis of these isolates showed that they represent 12 novel genera globally distributed in the ocean and together they comprise the largest diversity of phages associated to a single marine host (Holmfeldt et al. 2013). Likewise, marine protists host a wide diversity of viral pathogens. The majority of these viruses is complex, possesses a large dsDNA genome, and belongs to the Nucleo-Cytoplasmic Large DNA Viruses (NCLDV), for which the order Megavirales has been proposed (Colson et al. 2013). NCLDV from marine environments belong to 2 main families. The Phycodnaviridae family includes the majority of viruses that infects photosynthetic protists among which the haptophytes E. huxleyi (Schroeder et al. 2002; Wilson et al. 2005), P. globosa (Baudoux and Brussaard 2005; Brussaard et al. 2004b), the prasinophytes Micromonas, Bathycoccus, and Ostreococcus (Baudoux et al. 2015; Derelle et al. 2008, 2015; Martínez-Martínez et al. 2015; Zingone et al. 2006), or the raphidophyte H. akashiwo (Nagasaki et al. 1999).

Marine NCLDV also classify within the Mimiviridae family. This clade, named after the discovery of Mimivirus (Raoult et al. 2004), originally included viruses with exceptionally large dsDNA genomes. With a sophisticated shell 0.7 µm and a huge genome of 1.2 Mb, Megavirus chilensis, isolated from coastal waters of Chile, represents the largest Mimiviridae and it surprisingly propagates on a freshwater acanthamoeba (Arslan et al. 2011). Viruses with smaller genome sizes but sharing several genetic features with these giant viruses were added to this virus family; Mimiviridae includes now the virus CroV that infects the marine protozoa Cafeteria roenbergensis (750 kb, Fischer et al. 2010), but also viruses that infect photosynthetic protists such as the haptophytes Phaeocystis pouchetii (485 kb, Jacobsen et al. 1996), Phaeocystis globosa (460 kb PgV-16T, Santini et al. 2013), Haptolina ericina (formely named Chrysochromulina ericina, 530 kb, Sandaa et al. 2001; Johannessen et al. 2015), Prymnesium kappa (507 kb, Johannessen et al. 2015), the prasinophyte Pyramimonas orientalis (560 kb, Sandaa et al. 2001) and the pelagophyte Aureococcus anophagefferens (371 kb, Moniruzzaman et al. 2014) are awaiting assignment to this virus family. The discovery of Mimiviridae initiated intensive debates on the concept virus. Their huge and astonishingly complex genome was found to encode trademark cellular functions, which literally blurs the boundaries between viruses and cellular organisms. The upper limits of the viral world both in terms of particle size and genome complexity have been pushed out with the isolation of Pandoraviruses from coastal marine waters and a freshwater pond (Philippe et al. 2013). These amoeba viruses exhibit dsDNA genome of 2.5 Mb lacking similarities with known NCLDV.

Not all protist viruses are of the dsDNA type. The prasinophyte Micromonas, which hosts many Phycodnaviridae, can also be infected by dsRNA virus (Brussaard et al. 2004b). This reovirus displays unusual morphology and genetic features compared to known Reoviridae, which led to the proposition of a new genus, Mimoreovirus, within the family Reoviridae (Attoui et al. 2006). Moreover, diatom viruses exhibit tiny particle diameter (< 40 nm) compared to Phycodnaviridae and belong to two main groups: the Bacilladnavirus (ssDNA viruses, Kimura and Tomaru 2013, 2015; Nagasaki et al. 2005; Tomaru et al. 2008, 2011a, b, 2013b) and the Bacillarnavirus (ssRNA, Kimura and Tomaru 2015; Nagasaki et al. 2004; Shirai et al. 2008; Tomaru et al. 2009, 2012, 2013a).

Together, these novel VHS demonstrate that we have barely scratched the surface of viral diversity. Characterizing these model systems is essential to gaining knowledge about the functional role of this tremendous taxonomic diversity. Importantly, marine environments have been inadequately sampled. Most VHS cultured thus far originate from temperate coastal marine environments, while open-ocean, tropical and extreme marine systems have been largely under-explored. Likewise, VHS mostly include dsDNA virus that infect emblematic marine bacteria or protists. By comparison archaeal viruses, RNA viruses, or ssDNA are under-represented while some of these groups seem to comprise an important fraction of the viral community (Culley et al. 2006; Labonté and Suttle 2013a, b; Labonté et al. 2015). Hence, there is no question that more efforts for isolating novel viruses need to be pursued.

8 Marine Viruses and Biotechnological Applications

Metagenomic studies of marine viruses reveal the presence of many new genes and novel proteins and as such the vast amount of genetically diverse marine viruses is a largely untapped genetic resource for biotechnological applications. Interesting findings from studying marine viruses thus far involve the algal virus EhV infecting E. huxleyi, which has a unique cluster of seven genes that are involved in the sphingolipid biosynthesis, leading to ceramide formation (Wilson et al. 2005). Ceramides are the key structural lipids of skin, nails and hair as an effective epidermal barrier against water evaporation and entry of microorganisms. Use of ceramides by the cosmetic industry depends still largely on biomolecules obtained from natural sources, and thus the expression and production of viral ceramides may be a promising alternative source of natural ceramides (Wilson et al. 2005).

Other exciting recent discoveries are the viruses infecting viruses which showed that the infection with such virophage (‘eaters of viruses’) not only helps the survival of the cellular organism by decreasing the yield of the larger virus, but it also illustrated the potential of the virophages as source of biotechnologically interesting features (Fischer and Suttle 2011; La Scola et al. 2008). For example, the name of the virophage Mavirus is derived from Maverick virus as its genome encoding 20 predicting coding sequences of which seven have homology to the Maverick/Polinto family of DNA transposons. Furthermore, the DNA genome of the virophage Sputnik contains genes related to those in viruses infecting the three domains of life, and may as such serve the transfer of genetic material among viruses.

In the marine aquaculture industry, viruses are typically directly linked to (lethal) diseases and therefore are considered a major economic burden (Suttle 2007). However, the beneficial use of lytic viruses as mortality agents and source of lytic enzymes has been explored for multiple purposes. In aquaculture, Chlorella viruses were exploited to improve the extraction efficiency of algal lipids for biodiesel production (Sanmukh et al. 2014). The stringent host specificity of virus is another feature that has been exploited in aquaculture. Cultured fish, shellfish, or crustaceans, like any animal, are the target of bacterial infections that can have a dramatic economic impact. The use of chemotherapy is a rapid and efficient way to limit these infections. Yet these treatments are also lethal for beneficial bacteria and, importantly, they have led to the evolvement of multidrug-resistant bacterial strains that are a serious threat for farming industries. By comparison, lytic viruses are efficient antimicrobial agents that specifically infect and propagate on a limited number of host strains, leaving the co-occurring microbial community untouched. They have thus emerged as potential therapeutic agents to prevent and treat bacterial infections. Phage therapy has already been implemented to treat several fish pathogens and its effectiveness is currently tested for the treatment of other cultured animals in experimental conditions (Karunasagar et al. 2007; Martínez-Díaz and Hipólito-Morales 2013; Nakai and Park 2002; Oliveira et al. 2012 and references therein).

The application of viruses as therapeutic agents goes beyond aquaculture. In natural ecosystems, the combined effect of anthropogenic pressure and global change severely threatens the health of coral reefs. The increased surface temperature and pollution has been associated with the development of infectious diseases that lead to the demise of infected reefs. Phage therapy reached a new milestone with promising tests accomplished on the scleractinian corals Favia favus infected by Thalassomonas loyana, the causative agent of the white plague disease (Atad et al. 2012). Inoculation of the marine phage BA3 (Efrony et al. 2007, 2009) with diseased corals prevented the progression of the white plague disease and its transmission to healthy corals in the Gulf of Aqba, Red Sea. This promising study constitutes the first application of phage therapy to cure diseased corals in situ.

Another emerging field of interest is the detection and identification of viruses in ballast water. Ballast water discharges from ships are responsible for introducing invasive species and their viruses to other marine regions. The knowledge of marine microorganisms spread by ballast water discharge is still largely understudied but has potential of spreading disease agents. There are a few examples (already) of specific viruses that were introduced via ships’ ballast water, i.e., the Infectious Salmon Anemia Virus (ISAV) in salmon farms of Scotland were linked to vessel visits, and marine cyanophages were introduced to the Great Lakes, USA (Murray et al. 2002; Wilhelm et al. 2006). Given the increasing volume of shipping and rapidly growing aquaculture industry, introduction of pathogenic viruses to near-coast ecosystems and aquaculture farms is a realistic threat. At the same time, increasingly strict regulations on ballast water discharge force the shipping industry to make use of on-board ballast water treatment systems in order to decrease the abundance of organisms and viruses. Effective methods of virus concentration and virus detection are needed to comply with the new quality standards.

The high yield genes found as AMG can be used by synthetic biology to create more efficient artificial cells in the future. It is clear that the vast genetic diversity contained in the viral biosphere compartment will eventually be used for biotech applications or nanotechnology (Fischlechner and Donath 2007).

9 Future Perspectives

Viruses play a major role in the natural history and population dynamics of most living organisms and this is even truer for Bacteria, Archaea, and protists. Marine viruses might contribute decisively to the marine ecosystem functioning and to the stability and performance of the microbial communities. Therefore, it is important to strive to get a meaningful representation of all the groups involved in the community behavior and that includes viruses. If we are to understand the biology of these microbes we have to advance in the knowledge of their viruses. Culture-independent approaches are allowing fast advances, but do not exclude the need for in situ viral lysis rate measurements, as well as experimental studies of viral–host interactions.

Viruses are probably the largest reservoir of novel genes in the biosphere and this novelty is important for their host biology. The role that viruses play in the evolution of microbes is considered to be fundamental. Cells can outsource their most innovative evolutionary experiments to their viruses that are not under the pressure of optimizing growth parameters to compete. The number of genes shared by (microbial) hosts and their viruses has been continuously increasing. As more viral genomes become available, the list of AMG genes increases and is now including multiples aspects of the host physiology (Breitbart 2012).

It has been proposed that viruses are a part of the bacterial and archaeal pan-genomes (Rodriguez-Valera and Ussery 2012) and that as such are impossible to extricate from the cellular lineages from an evolutionary perspective, i.e., to form a single selection unit. In the formidable task that microbiologists face when struggling to describe microbial diversity and its ecological function, viral diversity and activity can be considered the last but important frontiers that need to be overcome.