CONTEXTO INTERNACIONAL

Viruses are now widely accepted as the most abundant biological entities in natural waters and are an integral component of the marine ecosystem. The first evidence of infective algal viruses in seawater was reported in 1979, when a virus infecting the marine eukaryotic algal species Micromonas pusilla was successfully isolated (Mayer and Taylor, 1979). These researchers observed that M. pusilla cells, isolated during a study of nanoplankton in British Colombia coastal waters, declined rapidly in culture. Addition of filtrates generated from the medium of these cultures produced the lysis of healthy M. pusilla cells. Transmission electron microscopy (TEM) analysis of the lysate revealed polyhedral virus-like particles (VLPs) of 130 - 135 nm in diameter. This initial report on M. pusilla viruses was followed by the isolation and characterisation of a number of other marine eukaryotic algal viruses. Viruses infecting M. pusilla have been isolated and described since this initial report (Cottrell and Suttle, 1995; Sahlsten, 1998; Sahlsten and Karlson, 1998; Brussaard et al., 2004; Zingone et al., 2006).

Since the 1970s, the significance of viruses as agents of mortality for phytoplankton in aquatic systems has been highlighted. Viruses have been found in algal cells from all major taxonomic classes comprising some key species, such as Synechococcus sp. (Suttle and Chan, 1993; Wilson et al., 1993), Emiliania huxleyi (Bratbak et al., 1993), Phaeocystis pouchetti (Jacobsen et al., 1996) and harmful species such as Aureococcus anophagefferens (Milligan and Cosper, 1994; Gastrich et al., 1998) and

Heterosigma akashiwo (Nagasaki and Yamaguchi, 1997). At present, a number of algal viruses have been isolated and characterised and are now widely accepted as

being diverse and dynamic within the microbial community (Cottrell and Suttle, 1991; Short and Suttle, 2002; Schroeder et al., 2003; Brussaard, 2004b).

The isolation and characterisation of a virus that infects a Chlorella-like species led to the formation of the Phycodnaviridae family (Van Etten et al., 1983b; Van Etten and Meints, 1999). Paramecium bursaria Chlorella virus (PBCV-1) is the prototype of this family of large viruses, which infect both freshwater and marine eukaryotic algae. Members and prospective members of the Phycodnaviridae are continually being discovered. They all have icosahedral morphology, an internal lipid membrane, and large dsDNA genomes of 157 to 560 kb (Van Etten et al., 2002; Wilson et al., 2005b; Schroeder et al., 2009).

Availability of experimental host-virus systems is the key to understanding more about the features and ecological significance of algal viruses and to develop molecular methods that may be applied in field studies. Therefore, it has become necessary to bring more host-virus systems into the laboratory, where they can be characterised and described. Virus-like particles have now been observed in over 44 taxa of marine algae (reviewed by Van Etten et al., 1991). However, only a small number have been isolated and even fewer brought into culture and characterised in detail. Those that have been adequately described have been putatively assigned to the family Phycodnaviridae. The taxonomic divisions within this family are supported by sequence analysis of DNA polymerase genes (Chen and Suttle 1995a; b; 1996).

Generally, the isolation of lytic algal viruses follows a relatively simple protocol. A prefiltered natural seawater sample is added directly or, following concentration, to one, or various strains, of the algal species of interest. A loss in chlorophyll autofluorescence is then monitored. Natural samples can be concentrated by ultrafiltration (Suttle et al., 1991) and/or prefiltered, or centrifuged at low speed, to remove cellular material. A loss of autofluorescence indicates a loss of cells in comparison to a noninfected control culture (Suttle et al., 1990). An overview of the various steps that can be employed in algal virus isolation, and were indeed used in this present study, is presented in Figure 3.1.

Screening with whole-water samples ensures no viruses are lost, as may occur during filtration or concentration. The use of whole-water samples has led to the isolation of a range of algal viruses (Nagasaki and Yamaguchi, 1997; Tarutani et al., 2001; Castberg et al., 2002). This method is the simplest and quickest approach to virus isolation. Large volumes of water are required, however, as no concentration steps are included.

Figure 3.1. Flow diagram of procedure for detection and isolation of viruses infecting phytoplankton.

Although a number of viruses may be removed by prefiltration and concentration steps, the use of concentrated water samples greatly increases the detection limits of screening. Viruses have been concentrated using techniques such as tangential flow filtration, vortex flow filtration and ultracentrifugation (Paul et al., 1991; Suttle et al., 1991; Wommack et al., 1995). Although large viruses are selectively removed using these techniques, the possibility of detecting less common viruses is enhanced. Serial dilutions of lysate are screened for the propagation of viruses. In theory, only one virus is required to cause the eventual lysis of an entire population, so the highest dilution that results in lysis will contain the one virus and this will give a clonal isolate. Clonal isolation is important as this ensures a single virus type is present. However, endpoint dilutions are not 100% accurate and the process must be repeated a minimum of three consecutive times to ensure the clonal status of the isolate. To obtain purified clonal cultures of infectious viruses, typically several cycles of a liquid serial dilution procedure are used. This process can be time-consuming and labour-intensive, although is helped if cultures can be grown in microtitre plates.

The most efficient method for obtaining virus clones is the use of plaque purification assays, since individual plaques represent the propagation of a single virus. Plaque assays were originally developed to count and measure infectivity of bacteriophages (Safferman and Morris, 1963). The plaque assay technique has since been adapted for the isolation of viruses infecting Chlorella species (Van Etten et al., 1983b), cyanobacteria (Suttle and Chan, 1993; Wilson et al., 1993) and Emiliania huxleyi (Wilson et al., 2002b). The use of plaque formation in the isolation of viruses has advantages. If a single plaque is isolated it can be treated as having originated from a single virus particle. Therefore, the number of infectious particles can be calculated. The plaque assay method enables a discrete form of virus growth, which can be

distinguished from a background of uninfected cells. The host cells therefore support growth of the virus and provide a contrasting background against which virus growth can be recognised. The basis of the plaque assay technique used in the isolation and purification of viruses and to determine viral titres, is to measure the ability of a single infectious virus to form a plaque on a confluent monolayer of host cells. A plaque is formed as a result of infection of one cell by a single virus particle followed by the replication of that virus and eventually the death of the cell. The newly replicated cell then proceeds to infect and kill the surrounding cells forming a clear plaque on the lawn of host cells. The plaque can be visualised by the naked eye or light microscopy. Each plaque represents a single virus. Therefore, clonal virus populations can be purified by isolating individual plaques. Individual plaques obtained from varying dilutions of a viral stock can be counted to determine the viral titre.

Most algal viruses isolated to date infect hosts that generally do not grow, or grow poorly, on agar plates. Plaque assay purification methods, analogous to those used in the purification of bacteriophages, are therefore inapplicable in the isolation of viruses infecting such hosts. Instead viruses are isolated and purified in liquid cultures and many viruses, including those that infect dinoflagellates (Tarutani et al., 2001), prymnesiophytes (Jacobsen et al., 1996; Sandaa et al., 2001) and raphidophytes (Nagasaki and Yamaguchi, 1997; Lawrence et al., 2001), have been isolated using this technique. This end-point method gives an all-or-none response and the virus is diluted until only a few inoculants contain a single infective particle. To generate a pure viral stock by end-point dilution, the aim is to dilute the virus, such that, if multiple cultures are exposed to the diluted inoculants, any cultures that

virus should be diluted to the point where only 10% of the cultures become infected. The enumeration of infectious units may be achieved by adding serial dilutions of the inoculated culture to replicate cultures and monitoring for lysis at each dilution level. In this assay it must be assumed the lysed cultures contain at least one infectious virus particle. The number of viruses present in the dilution series is then estimated using statistical analysis.

Algal viruses are detected by techniques routinely used in virology, such as TEM, which can also provide information about morphology of the virus particles. Certain algal viruses can also be discriminated using analytical flow cytometry (AFC) (Brussaard et al., 2000; 2004a). AFC is a high throughput method and can enable many of the fluorescent stained viruses to be distinguished from bacteriophages. The use of nucleic acid-specific stains can allow viruses to be enumerated by AFC (Brussaard, 2004a) and also epifluorescence microscopy (Hennes and Suttle, 1995; Noble and Fuhrman, 1998). AFC allows the discrimination of subpopulations, based on their fluorescence and scatter characteristics.

In this chapter, the screening of seawater from natural samples is described in order to isolate novel viruses infecting phytoplankton and to characterise them further. The host phytoplankton strains used to test for susceptibility to viruses were representative of the nano- and picophytoplankton assemblages. Picoeukaryotes (cells smaller than 3 µm in diameter) and nanophytoplankton (cells 3 µm to 20 µm in diameter) are widely distributed in aquatic environments. Their significant biomass and high productivity suggest that they play a major role in oceanic, coastal, and freshwater systems (Diez et al., 2001; Massana et al., 2002). A total of 12 viruses

were isolated in this study that infect the picoeukaryote species Ostreococcus tauri and Micromonas pusilla.