Isolation and Characterization of a Novel Single-Stranded RNA Virus Infectious to a Marine Fungoid Protist, Schizochytrium sp. (Thraustochytriaceae, Labyrinthulea)

Microorganism cultures.Strains of thraustochytrids and other microorganisms used in this study are listed in Table 1. All of these organisms are clonal, as established by the micropipetting method or an extinction dilution method. Thraustochytrids were grown at 20°C in 10× medium H (medium H is 0.2% glucose, 0.02% yeast extract, and 0.05% monosodium glutamate in seawater) (20). Other organisms were grown at 20°C in IMK medium (Wako Co., Ltd.) or f/2 medium (19). For cultivation of phytoplankton, the light conditions were 12 h of light (55 μmol photons m⁻² s⁻¹; cool white fluorescent illumination) and 12 h of darkness.

Isolation of lytic viruses.Surface water was collected in Kobe Harbor, Hyogo Prefecture, Japan, on 26 July 2000. It was filtered through a 0.2-μm-pore-size filter (Nuclepore) to remove eukaryotic microorganisms and most bacteria. Aliquots (100 μl) of the filtrate were inoculated into exponentially growing cultures (150 μl) of the three thraustochytrid strains shown in Table 1 and incubated at 20°C. Cultures inoculated with filtrates treated at 121°C for 15 min served as controls. Test cultures were checked by optical microscopy for 14 days to examine whether cell lysis occurred. In the Schizochytrium sp. strain NIBH N1-27 culture inoculated with the filtrate, apparent growth inhibition was detected, although no lysis was observed in cultures of the other two strains and control cultures. Then a clonal lytic agent was obtained through two cycles of the extinction dilution procedure (43, 60). The lysate in the most diluted well of the second assay was inoculated into a 50-ml fresh culture of Schizochytrium sp. strain NIBH N1-27, and the resultant lysate was filtered through a 0.2-μm-pore-size filter (Nuclepore); then 0.3 ml of the filtrate was mixed with 1 ml of 10% glycerol in 10× medium H and cryopreserved at −80°C as the original pathogen suspension. Serial transfers of a lysed culture to an exponentially growing culture of Schizochytrium sp. strain NIBH N1-27 were performed more than twice to verify the transferability. The concentration of the pathogenic agent was estimated by the extinction dilution method (43, 60) using the computer program described by Nishihara et al. (45).

Host range test.The host range of the pathogenic agent was examined by adding 100-μl portions of the original pathogen suspensions to 1-ml cultures of the exponentially growing microorganisms listed in Table 1. The cultures were observed by optical microscopy. Cultures that were not lysed after 10 days were considered to be unsuitable hosts for the pathogen.

Growth experiment.In the one-step growth experiments, an exponentially growing culture of Schizochytrium sp. strain NIBH N1-27 was inoculated with the pathogen suspension at multiplicities of infection of 20.5, 21.2, and 30.1. Control cultures, to which an autoclaved (121°C, 15 min) filtrate was added, were also used for comparison. Aliquots of the cell suspension were removed every 8 h; then the cell density was estimated by optical microscopy, and the pathogen density was measured by the extinction dilution method (43, 60). Each experiment was performed in triplicate.

TEM.For transmission electron microscopy (TEM) observations of the pathogen, exponentially growing cultures of Schizochytrium sp. strain NIBH N1-27 were inoculated with the pathogen, and samples (2 ml) were removed at 0, 8, 16, and 24 h postinoculation. Each cell suspension was mixed with an equal volume of a fixing cocktail (5% glutaraldehyde, 0.2 M sucrose, 0.2 M cacodylate buffer) and kept on ice for 2 h. Cells were harvested by centrifugation at 640 × g for 2 min; then the pellet was rinsed three times with 0.2 M cacodylate buffer and postfixed with 1% buffered OsO₄ for 1.5 h on ice. Following three rinses with 0.2 M cacodylate buffer, the pellet was dehydrated in a graded ethanol series (30 to 100%) and embedded in Spurr's resin (NISSHIN EM Co., Ltd). Thin sections were stained with 1% uranyl acetate and 3% lead citrate and observed at 80 kV using a JEOL JEM-1010 transmission electron microscope. Negatively stained pathogens were also observed by TEM. Briefly, a pathogen suspension was mounted on a grid (no. 780111630; JEOL DATUM Ltd.) for 30 s, and excess water was removed with filter paper (no. 1; TOYO Co., Ltd.). Then 4% uranyl acetate was added to the grid for 10 s, and the excess dye was removed with filter paper. After the grid was dried in a desiccator for 10 min, negatively stained pathogens were observed by TEM at an acceleration voltage of 80 kV. Particle diameters were estimated based on the negatively stained images.

Analysis of nucleic acids and proteins.A Schizochytrium sp. strain NIBH N1-27 culture (1.5 liters) was inoculated with 5 ml of a fresh pathogen suspension and lysed. Then the lysates were centrifuged at 14,000 × g for 15 min to remove the cellular debris. The supernatant was added to polyethylene glycol 6000 (Wako Co., Ltd.) at a final concentration of 10% (wt/vol) and stored at 4°C overnight. After centrifugation at 3,600 × g for 1.5 h, the pellet was suspended in 10 mM phosphate buffer (10 mM Na₂HPO₄ and 10 mM KH₂PO₄ in distilled water) and centrifuged at 100,000 × g for 2 h. This purification process was repeated twice. The pellet was resuspended in 750 μl of distilled water; then the pathogen suspension was treated with proteinase K (final concentration, 1 mg ml⁻¹; Nippon Gene) at 55°C for 1.5 h. Nucleic acids were extracted from the pellet by using TRIzol LS (Invitrogen), precipitated with ethanol, and then suspended in 50 μl of distilled water. Aliquots (2 μl) of the suspension were digested at 37°C for 1 h with RNase A (final concentration, 0.1 μg μl⁻¹; Nippon Gene) in 0.01× SSC or 2× SSC (1× SSC is 0.15 M NaCl plus 0.015 M trisodium citrate, pH 7.0) or with DNase I (final concentration, 10 U μl⁻¹; TAKARA Bio Inc.). Extract kept on ice with no enzymatic treatment served as a control. A formaldehyde-agarose gel (1%; 15 by 20 cm; Seakem Gold Agarose; BMA Inc.) was loaded with 20 μl of nucleic acid per lane and electrophoresed at 50 V for 14.5 h. Nucleic acids were visualized by SYBR Green II staining (Molecular Probes, Inc.).

In addition, the pathogen suspension was mixed with one-third volume of 4× sample buffer (250 mM Tris-HCl, pH 6.8, 8% 2-mercaptoethanol, 8% sodium dodecyl sulfate [SDS], 40% glycerol, 0.04% bromophenol blue) and boiled for 5 min; then the proteins were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) using 15% polyacrylamide gels at 150 V for 20 min. Proteins were visualized by Coomassie brilliant blue staining. SDS-PAGE standards (Bio-Rad Laboratories, Inc.) with molecular masses ranging from 14.4 to 97.4 kDa were used for size calibration.

Virus isolation and host range.A pathogenic agent causing lysis of Schizochytrium sp. strain NIBH N1-27 was isolated from the surface water collected in Kobe Harbor. This pathogen was serially transferable to fresh host cultures, in which more than 95% of the host cells were lysed within 36 h after virus inoculation (Fig. 1). In the exponential growth stage, Schizochytrium sp. strain NIBH N1-27 exhibits two distinct forms: vegetative growth and formation of zoospores. Based on the present observations, young zoospores appeared to be highly sensitive to viral infection because settlement to the bottom of vessels was immediately followed by cell lysis (data not shown). Further investigation is required to elucidate the relationship between the host's life cycle and virus sensitivity.

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FIG. 1.

Optical microphotographs of Schizochytrium sp. strain NIBH N1-27. (A) Intact cells; (B) lysed cells 48 h after inoculation of SssRNAV.

Based on the TEM observations, small VLPs were observed in the lysate of a Schizochytrium sp. strain NIBH N1-27 culture inoculated with the pathogen (Fig. 2A). Although healthy cells of Schizochytrium sp. strain NIBH N1-27 in the control cultures had cytoplasmic structures diagnostic of thraustochytrids and showed no symptoms of viral infection (Fig. 2B), VLPs whose sizes were similar were also observed in the cytoplasm of Schizochytrium sp. strain NIBH N1-27 cells inoculated with the pathogen (Fig. 2C, D, and E). Based on these results, it was demonstrated that (i) the pathogen was transferable to a fresh culture and caused cell lysis, (ii) the VLPs specifically appeared in lysed cultures, and (iii) the VLPs were not detected in healthy cultures, thus fulfilling Koch's postulates. Therefore, we concluded that the VLP was a lytic virus infecting Schizochytrium sp. strain NIBH N1-27. We designated the virus SssRNAV (Schizochytrium single-stranded RNA virus).

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FIG. 2.

Transmission electron microphotographs of Schizochytrium sp. strain NIBH N1-27 infected by SssRNAV. (A) Negatively stained SssRNAV particles in the culture lysate; (B) thin section of a healthy cell of Schizochytrium sp. strain NIBH N1-27; (C) thin section of an SssRNAV-infected cell at 8 h postinoculation (the arrowheads indicate fibrils within vacuoles); (D) crystalline arrays of SssRNAV; (E) unordered aggregation of SssRNAV; (F) virus particles concentrated along the intracellular membranes. N, nucleus; G, Golgi body; Mt, mitochondrion.

SssRNAV caused lysis of four thraustochytrid strains tested in the present experiments (NIBH N1-27, SEK 0209, MBIC 11066, and MBIC 11072) but had no effect on the other thraustochytrid strains and some unialgal strains (Table 1). As thraustochytrid strains from different localities showed sensitivity to SssRNAV, we predicted that this host-virus system is common at least along the central to western coast of Japan. Because the SssRNAV-sensitive Schizochytrium sp. strain SEK 0209 was isolated from the same sampling site as SssRNAV, it is likely that SssRNAV has some impact on the dynamics of thraustochytrids in natural environments.

Because the classifications of thraustochytrids based on morphology and molecular phylogeny do not necessarily agree with each other (21), it is difficult to discuss whether SssRNAV is species specific or strain specific based only on the present results. However, it is notable that the four SssRNAV-sensitive strains (NIBH N1-27, SEK 0209, MBIC 11066, and MBIC 11072) belong to a particular group (Table 1) based on molecular phylogeny (22; R. Yokoyama, personal. communication).

Morphology of SssRNAV.The particles of SssRNAV were 25 ± 2 nm in diameter (average ± standard deviation) and angular and lacked a tail and an external membrane (Fig. 2A). SssRNAV often formed crystalline arrays (Fig. 2C and D) or random aggregations (Fig. 2E) in the host cell's cytoplasmic area. Both crystalline array formation and unordered aggregation within the cytoplasm are common features of several ssRNA viruses infectious to plants (36, 56), picornaviruses infectious to animals (11, 24), and marine algal ssRNA viruses (HaRNAV and HcRNAV) (61, 63). It was also notable that the virus particles were concentrated along the intracellular membrane structures (Fig. 2F). In SssRNAV-infected cells, vacuolation of the cytoplasm and the appearance of fibrils within small vacuoles were apparent (Fig. 2C). Vacuolation of the cytoplasm and the appearance of fibrils within vacuoles were also observed with HaRNAV (61).

Genome of SssRNAV.Denaturing gel electrophoresis revealed that SssRNAV has a single molecule of nucleic acid that is approximately 10.2 kb long and is sensitive to RNase A treatment under both low- and high-salt conditions but is resistant to DNase I (Fig. 3). These data indicate that the SssRNAV genome is ssRNA. Based on these observations, SssRNAV is thought to be related to the picornavirus-like superfamily, the vertebrate virus families Picornaviridae (genomic RNA size, 7 to 8 kb) and Caliciviridae (7 to 8 kb), the plant virus family Sequiviridae (9 to 12 kb), and the invertebrate virus family Dicistroviridae (9 to 12 kb), all of which have a poly(A) tail. Partial sequencing of the SssRNAV genome is now under way (data not shown), and the data reveal some similarity (E values, 1e-25 to 3e-22) with Triatoma virus, Drosophila C virus (DCV), Acute bee paralysis virus, and Taura syndrome virus belonging to the family Dicistroviridae. Further characterization of the viral genome, however, is necessary to determine the taxonomic position of SssRNAV. Considering that the hosts of the family Dicistroviridae are mainly crustaceans (insects and shrimps), the process of host range expansion and evolution of SssRNAV and related viruses is of great interest.

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FIG. 3.

Formaldehyde-agarose gel electrophoresis of viral nucleic acids. SssRNAV nucleic acids were not treated (lane 2), were treated with RNase A at 37°C under low-salt conditions (lane 3) or high-salt conditions (lane 4), or were treated with DNase I at 37°C (lane 5). Lane 1 contained an RNA molecular marker.

Proteins of SssRNAV.The protein analysis showed that SssRNAV has three major proteins (37, 34, and 32 kDa) and two minor proteins (80 and 18 kDa) (Fig. 4). The strength of the 16-kDa band was variable in the experiments (more than five experiments); thus, we could not verify if it originated from the host cells or SssRNAV particles.

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FIG. 4.

SDS-PAGE of SssRNAV structural proteins. The gel was stained with Coomassie brilliant blue.

The band pattern of SssRNAV resembles that of DCV belonging to the family Dicistroviridae, which has three major capsid proteins (31, 30, and 28 kDa) and one minor capsid protein (8.5 kDa) (25, 27). In addition, considering that capsid proteins of DCV are processed out of a precursor protein (100 kDa) (31, 38), the functions of the 80-kDa protein of SssRNAV are also of interest. Precise interpretation of the present results awaits future studies.

Replication of SssRNAV.In the triplicate one-step growth experiments, a rapid decrease in host cell abundance within 8 h after virus inoculation was accompanied by a rapid increase in the viral titer (Fig. 5); thus, the latent period of SssRNAV was estimated to be <8 h. The burst sizes estimated from the experiments ranged from 5.8 × 10³ to 6.4 × 10⁴ infectious units cell⁻¹. When SssRNAV was compared with the other ssRNA viruses infecting microalgae, the latent period of SssRNAV was found to be much shorter than those of HaRNAV (<12 days) (61) and HcRNAV (1 to 2 days) (63), and the burst size was found to be similar to that of HcRNAV (3.4 × 10³ to 16 × 10³ infectious units cell⁻¹) (63). TEM observations revealed that >3.4 × 10³ SssRNAV particles were scattered in a thin section of an infected cell 8 h after virus inoculation (Fig. 2C), which suggests that >6.0 × 10⁵ virus particles can be present in a whole cell based on geometric analysis. Thus, the possibility of underestimation of the burst sizes should be noted. Although the reasons for the underestimation have not been elucidated, the most likely explanations are that (i) a cluster of crystallized virus particles was counted as one infectious unit by the extinction dilution method and (ii) incomplete virus particles (lacking infectivity) accounted for a considerable proportion. In addition, it is also possible that PUFA excreted from burst cells interfered with the adsorption of viruses to host cells, because an increase in the viral titer of host lysates was often observed when the PUFA fraction was excluded by filtration through 0.2-μm-pore-size filters (data not shown).

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FIG. 5.

Changes in abundance of Schizochytrium sp. strain N1-27 cells with (▪) or without (□) viral inoculation and the viral titer (○). SssRNAV inoculation was performed in the exponential growth phase of host cultures (arrow). Results for only one of the triplicate experiments are shown. The error bars indicate the 95% confidence limits for the viral titer.

Ecological implications.In the present study, we examined the characteristics of a novel ssRNA virus, SssRNAV, which infects the marine fungoid protist Schizochytrium sp. The characteristics of SssRNAV are quite different from those of the herpes-type VLPs found in Thraustochytrium sp. reported by Kazama and Schornstein (28, 29). As far as we know, this is the first report of an ssRNA virus infecting marine unicellular protists.

Because of the recent progress in studies of marine viruses, the importance of viral impact on marine phytoplankton and bacteria has been highlighted. The present results emphasize the possibility that fungoid protists are also exposed to viral attack in marine systems. From the viewpoint of marine microbial ecology, thraustochytrids are considered to have a role as decomposers; i.e., they presumably feed on dying and dead cells of microorganisms in marine environments. Actually, it has been reported that Schizochytrium sp. cells prey on a bloom-forming diatom (Thalassiosira sp.) (14), suggesting that this organism may have a role as a decomposer in the terminal stage of algal blooms. These considerations invite further empirical investigation of the dynamics of decomposers and their viruses, in addition to blooming algae and their viruses. Comparisons of the dynamics of these microalgal components in bloom events are of great interest.