APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 2005, p.

45164522
0099-2240/05/$08.000 doi:10.1128/AEM.71.8.45164522.2005
Copyright 2005, American Society for Microbiology. All Rights Reserved.

Vol. 71, No. 8

Isolation and Characterization of a Novel Single-Stranded RNA Virus

Infectious to a Marine Fungoid Protist, Schizochytrium sp.
(Thraustochytriaceae, Labyrinthulea)
Yoshitake Takao,1 Keizo Nagasaki,2 Kazuyuki Mise,3 Tetsuro Okuno,3 and Daiske Honda1*
Department of Biology, Faculty of Science and Engineering, Konan University, 8-9-1 Okamoto, Higashinada, Kobe 658-8501,
Japan1; National Research Institute of Fisheries and Environment of Inland Sea, Fisheries Research Agency, 2-17-5
Maruishi, Ohno, Saeki, Hiroshima 739-0452, Japan2; and Laboratory of Plant
Pathology, Graduate School of Agriculture, Kyoto University,
Sakyo-ku, Kyoto 606-8502, Japan3
Received 5 September 2004/Accepted 3 March 2005

and demonstrated that the biovolume of thraustochytrids in

coastal waters could reach 43% of the bacterial biovolume.
The wide distribution and high abundance of these organisms
indicate their ecological importance as decomposers (39, 52).
In addition, thraustochytrids are known to produce large
amounts of polyunsaturated fatty acids (PUFA), such as docosahexaenoic acid and docosapentaenoic acid (44), which are
considered important food resources for higher organisms in
marine systems (30, 34, 50). Furthermore, some species of
thraustochytrids are known to be pathogens of mollusks, such
as octopuses and bivalves (1, 46, 49). Because of these distinctive features, the ecological significance of thraustochytrids in
the coastal ecosystems has been highlighted (50, 51).
On the other hand, viruses are very abundant in marine
environments (3, 48). Viruses and virus-like particles (VLPs)
have been discovered in a variety of phytoplankton and bacteria (48, 54, 64) and have been recognized as important agents
in controlling bacterial and algal biomass (4, 41, 48) and nutrient cycling (17, 66) and in maintaining the biodiversity of
bacteria and microalgae (5, 10, 12). So far, more than 13
viruses infecting marine microalgae have been isolated and
characterized (5). The majority of these viruses are large (100to 200-nm) double-stranded DNA viruses, and they are either
included in the family Phycodnaviridae (5, 58, 65) or considered
most likely to belong to this family based on some characteristics (7, 16, 23, 43, 57, 62).
In contrast, there have been only a small number of reports
describing RNA viruses infecting marine eukaryotic microor-

Thraustochytrids are marine fungoid protists classified in the

class Labyrinthulea in the kingdom Chromista (8, 9). They are
comprised of six genera (33, 47), Althornia (26), Aplanochytrium (2), Japonochytrium (32), Schizochytrium (18), Thraustochytrium (59), and Ulkenia (13). However, it has been shown
that the current classification of these genera based on morphology does not agree with the molecular phylogenetic relationships based on the 18S rRNA gene sequences (21). Currently, in order to resolve the confusion regarding the
classification and nomenclature of the thraustochytrids, further
comparative studies based on morphology, molecular phylogeny, and chemotaxonomy are under way (R. Yokoyama, personal communication). Hence, some of the thraustochytrid
strains tested in the present study have not been fully identified
yet (Table 1).
Thraustochytrids are distributed in saline lakes and in marine, estuarine, and deep-sea waters throughout the world (35,
47, 52), and they have been isolated from algal and plant
material, as well as from sediments and water (14, 37, 55, 59).
Recently, a rapid direct detection technique for thraustochytrids using the fluorogenic acriflavine dye was developed
(53). Using this method, Naganuma et al. (39) estimated the
abundance of thraustochytrids in the Seto Inland Sea of Japan
* Corresponding author. Mailing address: Department of Biology,
Faculty of Science and Engineering, Konan University, 8-9-1 Okamoto, Higashinada, Kobe 658-8501, Japan. Phone: 81 78-435-2515.
Fax: 81 78-435-2539. E-mail: dhonda@konan-u.ac.jp.
4516

Downloaded from http://aem.asm.org/ on April 9, 2016 by guest

Thraustochytrids are cosmopolitan osmoheterotrophic microorganisms that play important roles as decomposers, producers of polyunsaturated fatty acids, and pathogens of mollusks, especially in coastal ecosystems.
SssRNAV, a novel single-stranded RNA (ssRNA) virus infecting the marine fungoid protist Schizochytrium sp.
(Labyrinthulea, Thraustochytriaceae) was isolated from the coastal water of Kobe Harbor, Japan, in July 2000,
and its basic characteristics were examined. The virus particle is icosahedral, lacks a tail, and is ca. 25 nm in
diameter. SssRNAV formed crystalline arrays and random assemblies within the cytoplasm of host cells, and
it was also concentrated along the intracellular membrane structures. By means of one-step growth experiments, the lytic cycle and the burst size were estimated to be <8 h and 5.8 103 to 6.4 104 infectious units
per host cell, respectively. SssRNAV had a single molecule of ssRNA that was approximately 10.2 kb long, three
major proteins (37, 34, and 32 kDa), and two minor proteins (80 and 18 kDa). Although SssRNAV was
considered to have some similarities with invertebrate viruses belonging to the family Dicistroviridae based on
its partial nucleotide sequence, further genomic analysis is required to determine the detailed classification
and nomenclature of SssRNAV. Our results indicate that viral infection is one of the significant factors
controlling the dynamics of thraustochytrids and provide new insights into understanding the ecology of these
organisms.

ssRNA VIRUS INFECTING SCHIZOCHYTRIUM SP.

VOL. 71, 2005

4517

TABLE 1. Infection specificities of SssRNAV with 19 strains of marine microorganisms

Taxon

Kingdom Chromista
Phylum Sagenista
Class Labyrinthulea

Phylum Ochrophyta
Class Bacillariophyceae
Class Raphidophyceae
Class Chrysophyceae
Kingdom Protozoa
Phylum Euglenozoa
Phylum Dinozoa

Groupb

Schizochytrium sp. strain NIBH N1-27d

Schizochytrium sp. strain SEK 0209
Schizochytrium limacinum IFO 32693d
Thraustochytrium aureum ATCC 34304d
Thraustochytriaceae sp. strain MBIC 11066
Thraustochytriaceae sp. strain MBIC 11071
Thraustochytriaceae sp. strain MBIC 11072
Thraustochytriaceae sp. strain SEK 0210
Thraustochytriaceae sp. strain SEK 0211
Thraustochytriaceae sp. strain SEK 0212
Thraustochytriaceae sp. strain SEK 0213
Thraustochytriaceae sp. strain SEK 0214
Cafeteria sp. strain SEK 0124

1
1
1
2
1
1
1
2
2
2
2
2

Isolation locality

Sensitivity
to
SssRNAVc

Nakaminato Harbor, Ibaragi, Japan

Kobe Harbor, Hyogo, Japan
Colonia, Yap Island
Woods Hole, Mass.
Iriomote Island, Okinawa, Japan
Iriomote Island, Okinawa, Japan
Iriomote Island, Okinawa, Japan
Okinawa Island, Okinawa, Japan
Ishigaki Island, Okinawa, Japan
Ishigaki Island, Okinawa, Japan
Iriomote Island, Okinawa, Japan
Hiroshima Bay, Hiroshima, Japan
Awaji Island, Hyogo, Japan

Nitzschia sp. strain SEK 0215

Skeletonema sp. strain SEK 0135
Heterosigma akashiwo SEK 0023
Sulcochrysis biplastida MBIC 10502

Kobe Harbor, Hyogo, Japan

Kobe Harbor, Hyogo, Japan
Kure Bay, Hiroshima, Japan
Yokohama Harbor, Kanagawa, Japan

Bodo sp. strain SEK 0126

Prorocentrum sp. strain SEK 0112

Awaji Island, Hyogo, Japan

Takamatsu Harbor, Kagawa, Japan

a
ATCC, American Type Culture Collection, (United States); MBIC, Marine Biotechnology Institute Culture Collection (Japan); IFO, Institute for Fermentation,
Osaka (Japan); NIBH, National Institute of Bioscience and Human Technology (Japan); SEK, Laboratory of Systematics and Evolution, Konan University (Japan).
b
Results of phylogenetic grouping for the strains tested (R. Yokoyama, personal communication).
c
, lysed; , not lysed.
d
Strain used for virus isolation.

ganisms. So far, three RNA viruses infecting marine eukaryotic

microalgae have been isolated; two of them are single-stranded
RNA (ssRNA) viruses (HaRNAV and HcRNAV), and one is
a double-stranded RNA (dsRNA) virus (MpRNAV).
HaRNAV is infectious to one of the most noxious bloomforming phytoflagellates, Heterosigma akashiwo (Raphidophyceae), and has an ssRNA genome that is 9.1 kb long (61).
HcRNAV is infectious to the bivalve-killing dinoflagellate Heterocapsa circularisquama and has an approximately 4.4-kb ssRNA genome (63). MpRNAV is infectious to the cosmopolitan phytoplankter Micromonas pusilla and harbors 11 segments
of dsRNA as the viral genome, the total length of which is 25.5
kbp (6).
In addition, the relationship between protists and their viruses is poorly understood. Nagasaki et al. (40, 42) observed
VLPs in marine apochlorotic flagellates and suggested that
viral infection might be one of the factors affecting their dynamics. Garza and Suttle (15) isolated and characterized a
large dsDNA virus infecting a marine heterotrophic
nanoflagellate, Bodo sp., which also shared some characteristics with viruses belonging to the family Phycodnaviridae. For
thraustochytrids, Kazama and Schornstein (28, 29) found herpes-type VLPs in Thraustochytrium sp. (Thraustochytriaceae,
Labyrinthulea) which were roundish, enveloped, 110 nm in
diameter, and predicted to have a DNA genome. However,
because the VLPs were not successfully brought into culture,
further study could not be completed.
In the present report, we describe the isolation and characterization of a novel ssRNA virus infecting Schizochytrium sp.

(Thraustochytriaceae, Labyrinthulea). To our knowledge, this

is the first report describing the biological properties of an
RNA virus infecting marine fungoid protists.

MATERIALS AND METHODS

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 20C 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 20C in IMK medium (Wako Co., Ltd.) or
f/2 medium (19). For cultivation of phytoplankton, the light conditions were 12 h
of light (55 mol photons m2 s1; 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-m-pore-size
filter (Nuclepore) to remove eukaryotic microorganisms and most bacteria. Aliquots (100 l) of the filtrate were inoculated into exponentially growing cultures
(150 l) of the three thraustochytrid strains shown in Table 1 and incubated at
20C. Cultures inoculated with filtrates treated at 121C 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-m-poresize filter (Nuclepore); then 0.3 ml of the filtrate was mixed with 1 ml of 10%
glycerol in 10 medium H and cryopreserved at 80C 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 esti-

Downloaded from http://aem.asm.org/ on April 9, 2016 by guest

Class Bicoecea

Straina

4518

TAKAO ET AL.

RESULTS AND DISCUSSION

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

FIG. 1. Optical microphotographs of Schizochytrium sp. strain

NIBH N1-27. (A) Intact cells; (B) lysed cells 48 h after inoculation of
SssRNAV.

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 hosts life cycle and virus sensitivity.
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 Kochs 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).
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).

Downloaded from http://aem.asm.org/ on April 9, 2016 by guest

mated 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-l 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 (121C, 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 Spurrs 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 4C
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 l of distilled water; then the
pathogen suspension was treated with proteinase K (final concentration, 1 mg
ml1; Nippon Gene) at 55C for 1.5 h. Nucleic acids were extracted from the
pellet by using TRIzol LS (Invitrogen), precipitated with ethanol, and then
suspended in 50 l of distilled water. Aliquots (2 l) of the suspension were
digested at 37C for 1 h with RNase A (final concentration, 0.1 g l1; 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 l1; 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 l 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 (BioRad Laboratories, Inc.) with molecular masses ranging from 14.4 to 97.4 kDa
were used for size calibration.

APPL. ENVIRON. MICROBIOL.

VOL. 71, 2005

ssRNA VIRUS INFECTING SCHIZOCHYTRIUM SP.

4519

Downloaded from http://aem.asm.org/ on April 9, 2016 by guest

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.

4520

TAKAO ET AL.

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 cells 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 expan-

FIG. 4. SDS-PAGE of SssRNAV structural proteins. The gel was

stained with Coomassie brilliant blue.

sion and evolution of SssRNAV and related viruses is of great

interest.
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.
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.

FIG. 5. Changes in abundance of Schizochytrium sp. strain N1-27

cells with () or without () viral inoculation and the viral titer (E).
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.

Downloaded from http://aem.asm.org/ on April 9, 2016 by guest

FIG. 3. Formaldehyde-agarose gel electrophoresis of viral nucleic

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

APPL. ENVIRON. MICROBIOL.

ssRNA VIRUS INFECTING SCHIZOCHYTRIUM SP.

VOL. 71, 2005

ACKNOWLEDGMENTS
This work was supported by the Kato Memorial Bioscience Foundation and the Asahi Glass Foundation.
We are grateful to Toro Nakahara, Toshihiro Yokochi (National
Institute of Advanced Industrial Science and Technology, Japan), and
Rinka Yokoyama (Konan University, Japan), who kindly provided the
thraustochytrid strains. We thank Hiroshi Kawai, Akio Murakami,
Mitsunobu Kamiya (Kobe University, Japan), and Yuji Tomaru (Na-

tional Research Institute of Fisheries and Environment of Inland Sea,

Japan) for their technical advice concerning transmission electron microscopy. We also thank Tokushiro Takaso (University of the Ryukyus,
Japan), who kindly provided seawater samples.
REFERENCES
1. Azevedo, C., and L. Corral. 1997. Some ultrastructural observations of a
thraustochytrid (Protoctista, Labyrinthulomycota) from the clam Ruditapes
descussatus (Mollusca, Bivalvia). Dis. Aquat. Org. 31:7378.
2. Bahnweg, G., and F. K. Sparrow. 1972. Aplanochytrium kerguelensis gen. nov.
spec. nov., a new phycomycete from subantarctic marine waters. Arch. Microbiol. 81:4549.
3. Bergh, ., K. Y. Brsheim, G. Bratbak, and M. Heldal. 1989. High abundance of viruses found in aquatic environments. Nature 340:467468.
4. Bratbak, G., J. K. Egge, and M. Heldal. 1993. Viral mortality of the marine
alga Emiliania huxleyi (Haptophyceae) and termination of algal blooms.
Mar. Ecol. Prog. Ser. 93:3948.
5. Brussaard, C. P. D. 2004. Viral control of phytoplankton populationsa
review. J. Eukaryot. Microbiol. 51:125138.
6. Brussaard, C. P. D., A. A. M. Noordeloos, R.-A. Sandaa, M. Heldal, and G.
Bratbak. 2004. Discovery of a dsRNA virus infecting the marine photosynthetic protist Micromonas pusilla. Virology 319:280291.
7. Brussaard, C. P. D., S. M. Short, C. M. Frederickson, and C. A. Suttle. 2004.
Isolation and phylogenetic analysis of novel virus infecting the phytoplankton Phaeocystis globosa (Prymnesiophyceae). Appl. Environ. Microbiol. 70:
37003705.
8. Cavalier-Smith, T. 1997. Sagenista and Bigyra, two phyla of heterotrophic
heterokont chromists. Arch. Protistenkd. 148:253267.
9. Cavalier-Smith, T., M. T. E. P. Allsopp, and E. E. Chao. 1994. Thraustochytrids are chromists, not fungi: 18S r RNA signature of Heterokonta.
Phil. Trans. R. Soc. Lond. B Biol. Sci. 346:387397.
10. Cottrell, M. T., and C. A. Suttle. 1991. Wide-spread occurrence and clonal
variation in viruses which cause lysis of a cosmopolitan, eukaryotic marine
phytoplankter, Micromonas pusilla. Mar. Ecol. Prog. Ser. 78:19.
11. Dales, S., H. J. Eggers, I. Tamm, and G. E. Palade. 1965. Electron microscopic study of the formation of poliovirus. Virology 26:379389.
12. Fuhrman, J. A.. 1999. Marine viruses and their biogeochemical and ecological effects. Nature 399:541548.
13. Gaertner, A. 1977. Revision of the Thraustochytriaceae (lower marine
fungi). I. Ulkenia nov. gen., with description of three new species. Vero
ff.
Inst. Meeresforsch. Bremerh. 16:139157.
14. Gaertner, A.. 1979. Some fungal parasites found in the diatom populations of
the Rusfjord area (South Norway) during March 1979. Veroeff. Inst. Meeresforsch. Bremerhav. 18:2933.
15. Garza, D. R., and C. A. Suttle. 1995. Large double-stranded DNA viruses
which cause the lysis of a marine heterotrophic nanoflagellate (Bodo sp.)
occur in natural marine viral communities. Aquat. Microb. Ecol. 9:203210.
16. Gastrich, M. D., O. R. Anderson, S. S. Benmayor, and E. M. Cosper. 1998.
Ultrastructural analysis of viral infection in the brown-tide alga, Aureococcus
anophagefferens (Pelagophyceae). Phycologia 37:300306.
17. Gobler, C. J., D. A. Hutchins, N. S. Fisher, E. M. Cosper, and S. A. SanudoWilhelmy. 1997. N release and bioavailability of C, P, Se, and Fe following
viral lysis of marine chrysophyte. Limnol. Oceanogr. 42:14921504.
18. Goldstein, S., and M. Belsky. 1964. Axenic culture studies of a new marine
phycomycete possessing an usual type of asexual reproduction. Am. J. Bot.
51:7278.
19. Guillard, R. R. L., and J. H. Ryther. 1962. Studies of marine planktonic
diatoms. I. Cyclotella nana Hustedt, and Detonula confervacea (Cleve) Gran.
Can. J. Microbiol. 8:229239.
20. Honda, D., T. Yokochi, T. Nakahara, M. Erata, and T. Higashihara. 1998.
Schizochytrium limasinum sp. nov., a new thraustochytrid from a mangrove
area in the west Pacific Ocean. Mycol. Res. 102:439448.
21. Honda, D., T. Yokochi, T. Nakahara, S. Raghukumar, A. Nakagiri, K.
Schaumann, and T. Higashihara. 1999. Molecular phylogeny of labyrinthulids and thraustochytrids based on the sequence of 18S ribosomal RNA gene.
J. Eukaryot. Microbiol. 46:637647.
22. Huang, J., T. Aki, T. Yokochi, T. Nakahara, D. Honda, S. Kawamoto, S.
Shigeta, K. Ono, and O. Suzuki. 2003. Grouping newly isolated docosahexaenoic acid-producing thraustochytrids based on their polyunsaturated
fatty acid profiles and comparative analysis of 18S rRNA genes. Mar. Biotechnol. 5:450457.
23. Jacobsen, A., G. Bratbak, and M. Heldal. 1996. Isolation and characterization of a virus infecting Phaeocystis pouchetii (Prymnesiophyceae). J. Phycol.
32:923927.
24. Jesequel, A.-M., and J. M. Steiner. 1966. Some ultrastructural and histochemical aspects of coxackie virus-cell interactions. Lab. Investig. 15:1055
1083.
25. Johnson, K. N., and P. D. Christian. 1998. The novel genome organization
of the insect picorna-like virus Drosophila C virus suggests this virus belongs
to a previously undescribed virus family. J. Gen. Virol. 79:191203.

Downloaded from http://aem.asm.org/ on April 9, 2016 by guest

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 cell1. 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 cell1) (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-mpore-size filters (data not shown).
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.

4521

4522

TAKAO ET AL.

48. Proctor, L. M., and J. A. Fuhrman. 1990. Viral mortality of marine bacteria
and cyanobacteria. Nature 343:6062.
49. Ragan, M. A., G. S. MacCallum, C. A. Murphy, J. J. Cannone, R. R. Gutell,
and S. E. McGladdery. 2000. Protistan parasite QPX of hard-shell clam
Mercenaria mercenaria is a member of Labyrinthulomycota. Dis. Aquat. Org.
42:185190.
50. Raghukumar, S. 1996. Morphology, taxonomy, and ecology of thraustochytrids and labyrinthulids, the marine counterparts of zoosporic fungi, p.
3560. In R. Dayal (ed.), Advances in zoosporic fungi. Publications Pvt. Ltd.,
New Delhi, India.
51. Raghukumar, S. 2002. Ecology of the marine protists, the Labyrinthulomycetes (thraustochytrids and labyrinthulids). Eur. J. Protistol. 38:127145.
52. Raghukumar, S., N. Ramaiah, and C. Raghukumar. 2001. Dynamics of
thraustochytrid protists in the water column of the Arabian Sea. Aquat.
Microb. Ecol. 24:175186.
53. Raghukumar, S., and K. Schaumann. 1993. An epifluorescence microscopy
method for direct detection and enumeration of the fungi-like marine protists, the thraustochytrids. Limnol. Oceanogr. 38:182187.
54. Reisser, W. 1993. Viruses and virus-like particles of freshwater and marine
eukaryotic algaea review. Arch. Protistenkd. 143:257265.
55. Riemann, F., and M. Schrage. 1983. On the mass occurrence of a thraustochytroid protist (fungi or rhizopodan protozoa) in an Antarctic anaerobic
sediment. Veroeff. Inst. Meeresforsch. Bremerhav. 19:191202.
56. Roberts, D. A., R. G. Christie, and M. C. Archer, Jr. 1970. Infection of apical
initials in tobacco shoot meristems by tobacco ringspot virus. Virology 42:
217220.
57. Sanddaa, R. A., M. Heldal, T. Castberg, R. Thyrhaug, and G. Bratbak. 2001.
Isolation and characterization of two viruses with large genome size infecting
Chrysochromulina ericina (Prymnesiophyceae) and Pyramimonas orientalis
(Prasinophyceae). Virology 290:272280.
58. Schroeder, D. C., J. Oke, G. Malin, and W. H. Wilson. 2002. Coccolithovirus
(Phycodnaviridae): characterization of a new large dsDNA algal virus that
infects Emiliania huxleyi. Arch. Virol. 147:16851698.
59. Sparrow, F. K., Jr. 1936. Biological observations on the marine fungi of
Woods Hole waters. Biol. Bull. 70:236263.
60. Suttle, C. A. 1993. Enumeration and isolation of viruses, p. 121137. In P. F.
Kemp, B. Sherr, E. Sherr, and J. J. Cole (ed.), Handbook of methods in
aquatic microbial ecology. Lewis Publishers, Boca Raton, Fla.
61. Tai, V., J. E. Lawrence, A. S. Lang, A. M. Chan, A. I. Culley, and C. A. Suttle.
2003. Characterization of HaRNAV, a single-stranded RNA virus causing
lysis of Heterosigma akashiwo (Raphidophyceae). J. Phycol. 39:343352.
62. Tarutani, K., K. Nagasaki, S. Itakura, and M. Yamaguchi. 2001. Isolation of
a virus infecting the novel shellfish-killing dinoflagellate Heterocapsa circularisquama. Aquat. Microb. Ecol. 23:103111.
63. Tomaru, Y., N. Katanozaka, K. Nishida, Y. Shirai, K. Tarutani, M. Yamaguchi, and K. Nagasaki. 2004. Isolation and characterization of two distinct
types of HcRNAV, a single-stranded RNA virus infecting the bivalve-killing
microalga Heterocapsa circularisquama. Aquat. Microb. Ecol. 34:207218.
64. Van Etten, J. L., L. C. Lane, and R. H. Meints. 1991. Viruses and virus-like
particles of eukaryotic algae. Microbiol. Rev. 55:584620.
65. Van Etten, J. L. 2000. Phycodnaviridae, p. 183193. In M. H. V. Van Regenmortel, C. M. Fauquet, D. H. L. Bishop, et al. (ed.), Virus taxonomy,
classification and nomenclature of viruses, 7th report. Academic Press, San
Diego, CA.
66. Wilhelm, S. W., and C. A. Suttle. 1999. Viruses and nutrient cycles in the sea.
Virus play critical roles in the structure and function of aquatic food webs.
BioScience 49:781788.

Downloaded from http://aem.asm.org/ on April 9, 2016 by guest

26. Jones, E. B. G., and D. J. Alderman. 1971. Althornia crouchii gen. et sp. nov.,
a marine biflagellate fungus. Nova Hedwigia 21:381400.
27. Jousset, F., M. Bergoin, and B. Revet. 1977. Characterization of the Drosophila C virus. J. Gen. Virol. 34:269285.
28. Kazama, F. Y., and K. L. Schornstein. 1972. Herpes-type virus particles
associated with a fungus. Science 177:696697.
29. Kazama, F. Y., and K. L. Schornstein. 1973. Ultra-structure of a fungus
herpes-type virus. Virology 52:478487.
30. Kimura, H., T. Fukuba, and T. Naganuma.. 1999. Biomass of thraustochytrid
protoctists in coastal water. Mar. Ecol. Prog. Ser. 189:2733.
31. King, L. A., J. S. K. Pullin, and N. F. Moore. 1984. Characterization of a
picornavirus isolated from a tumorous blood cell line of Drosophila melanogaster. Microbios Lett. 26:121127.
32. Kobayashi, Y., and M. Ookubo. 1953. Studies on marine Phycomycetes [1].
Bull. Nat. Sci. Mus. (Tokyo) 33:5365.
33. Leander, C. A., and D. Porter. 2000. Redefining the genus Aplanochytrium
(phylum Labyrinthulomycota). Mycotaxon 76:439444.
34. Lewis, T. E., P. D. Nichols, and T. A. McMeekin. 1999. The biotechnological
potential of thraustochytrids. Mar. Biotechnol. 1:580587.
35. Lopez-Garcia, P., F. Rodriguez-Valera, C. Pedros-Alio, and D. Moreira.
2001. Unexpected diversity of small eukaryotes in deep-sea Antarctic plankton. Nature 409:603607.
36. Martelli, G. P., and M. Russo. 1972. Pelargonium leaf curl virus in host leaf
tissues. J. Gen. Virol. 15:193203.
37. Miller, J. D., and E. B. G. Jones. 1983. Observation on the association of
thraustochytrid marine fungi with decaying seaweed. Bot. Mar. 26:345351.
38. Moore, N. F., and J. S. K. Pullin. 1983. Heat shock used in combination with
amino acid analogues and protease inhibitors to demonstrate the processing
of proteins of an insect picorna virus (Drosophila C virus) in Drosophila
melanogaster cells. Ann. Virol. 134:285292.
39. Naganuma, T., H. Takasugi, and H. Kimura. 1998. Abundance of thraustochytrids in coastal plankton. Mar. Ecol. Prog. Ser. 162:105110.
40. Nagasaki, K., M. Ando, I. Imai, S. Itakura, and Y. Ishida. 1993. Virus-like
particles in an apochlorotic flagellate in Hiroshima Bay, Japan. Mar. Ecol.
Prog. Ser. 96:307310.
41. Nagasaki, K., M. Ando, S. Itakura, I. Imai, and Y. Ishida. 1994. Viral
mortality in the final stage of Heterosigma akashiwo (Raphidophyceae) red
tide. J. Plankton Res. 16:15951599.
42. Nagasaki, K., M. Ando, I. Imai, S. Itakura, and Y. Ishida. 1995. Virus-like
particles in unicellular apochlorotic microorganisms in the coastal water of
Japan. Fish. Sci. (Tokyo) 61:235239.
43. Nagasaki, K., and M. Yamaguchi. 1997. Isolation of a virus infectious to the
harmful bloom causing microalga Heterosigma akashiwo (Raphidophyceae).
Aquat. Microb. Ecol. 13:135140.
44. Nakahara, T., T. Yokochi, T. Higashihara, S. Tanaka, T. Yaguchi, and D.
Honda. 1996. Production of docosahexaenoic and docosapentaenoic acid by
Schizochytrium sp. isolated from Yap Islands. JAOCS (J. Assoc. Oil Chem.
Soc.) 73:14211426.
45. Nishihara, T., N. Kurano, and S. Shinoda. 1986. Calculation of most probable number for enumeration of bacteria on a microcomputer. Eisei Kagaku
32:226228. (In Japanese with English abstract.)
46. Polglase, J. L. 1980. A preliminary report on the thraustochytrid(s) and
labyrinthurid(s) associated with a pathological condition in lesser octopus
Eledone cirrhosa. Bot. Mar. 23:699706.
47. Porter, D. 1989. Phylum Labyrinthulomycota, p. 388398. In L. Margulis,
J. O. Corliss, M. Melkonian, and D. Chapman (ed.), Handbook of protoctista. Jones and Bartlett Publishers, Boston, Mass.

APPL. ENVIRON. MICROBIOL.