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Applied and Environmental Microbiology, July 2005, p. 3528-3535, Vol. 71, No. 7
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.7.3528-3535.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Previously Unknown Virus Infects Marine Diatom

Keizo Nagasaki,1* Yuji Tomaru,1 Yoshitake Takao,2 Kensho Nishida,1,{dagger} Yoko Shirai,1 Hidekazu Suzuki,3 and Tamotsu Nagumo4

Harmful Algal Bloom Division, National Research Institute of Fisheries and Environment of Inland Sea, Fisheries Research Agency, Hiroshima, Japan,1 Department of Biology, Faculty of Science and Engineering, Konan University, Kobe, Japan,2 Aoyama Gakuin Senior High School, Tokyo, Japan,3 Department of Biology, Nippon Dental University, Tokyo, Japan4

Received 16 November 2004/ Accepted 4 January 2005


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
Diatoms are a major phytoplankton group that play important roles in maintaining oxygen levels in the atmosphere and sustaining the primary nutritional production of the aquatic environment. Among diatoms, the genus Chaetoceros is one of the most abundant and widespread. Temperature, climate, salinity, nutrients, and predators were regarded as important factors controlling the abundance and population dynamics of diatoms. Here we show that a viral infection can occur in the genus Chaetoceros and should therefore be considered as a potential mortality source. Chaetoceros salsugineum nuclear inclusion virus (CsNIV) is a 38-nm icosahedral virus that replicates within the nucleus of C. salsugineum. The latent period was estimated to be between 12 and 24 h, with a burst size of 325 infectious units per host cell. CsNIV has a genome structure unlike that of other viruses that have been described. It consists of a single molecule of covalently closed circular single-stranded DNA (ssDNA; 6,005 nucleotides), as well as a segment of linear ssDNA (997 nucleotides). The linear segment is complementary to a portion of the closed circle creating a partially double-stranded genome. Sequence analysis reveals a low but significant similarity to the replicase of circoviruses that have a covalently closed circular ssDNA genome. This new host-virus system will be useful for investigating the ecological relationships between bloom-forming diatoms and other viruses in the marine system. Our study supports the view that, given the diversity and abundance of plankton, the ocean is a treasury of undiscovered viruses.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
Diatoms are the major phytoplankton group that plays important roles in maintaining the oxygen levels in the atmosphere and the carbon cycle that sustains the primary nutritional production in the aquatic environments (6, 30). The genus Chaetoceros is one of the most frequently found diatoms in both fresh and marine aquatic environments. The genus Chaetoceros includes more than 400 species; most of them are harmless and are considered to be the key primary photosynthetic producers that sustain higher forms of life (19). There are also a small number of species that have negative impacts on fisheries. For example, some species of the genus Chaetoceros (e.g., C. convolutus and C. concavicornis) are most clearly demonstrated to cause finfish and crab mortality, and their damage is considered caused by mechanical irritation of gill tissue (1, 2, 8); blooms of C. pseudocurvisetum and C. sociale often cause short-term depletion of nutrients that damage seaweed laver (Porphyra yezoensis) cultures due to discoloration of the thalli, especially in the Ariake Sea of Japan (S. Oda, unpublished data). Hence, the genus Chaetoceros plays significant roles from both the viewpoint of the marine ecosystem and the diatom's effects on fisheries.

C. salsugineum Takano is a bloom-forming diatom that occurs in brackish lakes and estuarine waters. It is small (2.0 to 9.5 µm wide) and forms short or long straight chains (22). The occurrence of C. salsugineum has been reported in Tachibana-ura (Tokushima Prefecture), Osaka Bay (Osaka Prefecture), Atsumi Bay (Aichi Prefecture), Lake Hamana (Shizuoka Prefecture), and Ariake Sea (Fukuoka Prefecture) in Japan (22; the present study), Vostok Bay in Russia (16), and Urdaibai estuary in Spain (27). Since there have been no reports of its negative impacts on fisheries, the main ecological implication of this species is as primary photosynthetic producers in brackish lakes and estuarine waters.

Since the late 1970s, the isolation of more than 13 viruses infectious to marine eukaryotic microalgae has been reported (4, 28). As their characteristics have been observed from the viewpoint of physiology, ecology, and molecular biology, so has their significance as mortality agents for phytoplankton in marine systems (4). Although the relationship between viruses and diatoms was not known for a long time, the first virus infecting diatoms, Rhizosolenia setigera RNA virus (RsRNAV), was recently isolated. RsRNAV is a single-stranded RNA virus specifically infectious to the bloom-forming diatom R. setigera Brightwell (12). We describe here the isolation and characterization of a second diatom-infecting virus, C. salsugineum nuclear inclusion virus (CsNIV).


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Algal cultures and growth conditions.
The algal strains used in the present study are shown in Table 1 and were maintained at the National Research Institute of Fisheries and Environment of the Inland Sea (Japan). Algal cultures were grown in modified SWM3 medium enriched with 2 nM Na2SeO3 (5, 9, 10) under a 12/12-h light-dark cycle of ca. 110 to 150 µmol of photons m–2 s–1 with cool white fluorescent illumination at the temperatures given in Table 1.


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  		TABLE 1. Infection specificities of CsNIV against 58 strains of marine phytoplankton

 
Isolation of pathogen.
Sediment sample (0- to 3-cm depth) was collected by using an Ekman Berge sampler (15 by 15 cm) at the mouth of the Shiotsuka River in the Ariake Sea, Japan, on 18 April 2003. Collected sediment samples were sent to the laboratory without fixation within 24 h of sampling and stored at 4°C. Twelve grams of the sediment sample was shaken with 12 ml of Na2SeO3-enriched SWM3 (400 rpm, 23°C, for 30 min) and centrifuged (860 x g, 4°C, for 10 min). The supernatants were sequentially passed through GF/F filters (Whatman) and 0.2-µm-pore-size Dismic-25cs filters (Advantec) to remove eucaryotic microorganisms and most bacteria. Aliquots (0.2 ml) of the filtrate were then inoculated into exponentially growing cultures (0.8 ml) of the 38 diatom strains shown in Table 1, followed by incubation at 15°C under the light conditions stated above. Algal cultures inoculated with SWM3 served as controls. The C. salsugineum Ch42 (Fig. 1A and B) axenic culture inoculated with the filtrate showed apparent inhibition of algal growth. In the present experiments, the pathogen lytic to C. salsugineum Ch42 was examined and characterized.



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  		FIG. 1. C. salsugineum. (A) Optical micrograph of intact cells; (B) scanning electron micrograph of intact cells; (C) C. salsugineum cultures with or without inoculation of CsNIV; (D) optical micrograph of CsNIV-infected cells of C. salsugineum; (E) scanning electron micrograph of the setae having ellipse pores (white arrowheads) of an uninfected C. salsugineum cell.

 
From the lysed culture of C. salsugineum Ch42, we cloned the pathogen through two cycles by using the extinction dilution procedure (14, 21). The lysate in the most diluted well of the second assay was sterilized by filtration through a 0.1-µm-pore-size polycarbonate membrane filter (Nuclepore) and transferred to an exponentially growing culture of C. salsugineum Ch42. The resultant lysate was regarded as the clonal pathogen suspension. For the pathogen, serial transfers of a lysed culture in an exponentially growing culture of C. salsugineum Ch42 were performed more than twice to verify transferability.

Storage.
An exponentially growing culture of C. salsugineum Ch42 was inoculated with the virus and incubated for 3 days under the conditions given above. The resulting lysates were sequentially passed through 8.0, 0.8, and 0.2-µm-pore-size filters (Nuclepore) to remove cellular debris. The titer of the resultant fresh lysate was then estimated by means of the extinction dilution method, and an aliquot of the lysate was kept at 20, 10, 4, –20, or –196°C (in liquid nitrogen) in the dark without the addition of cryoprotectants. A viability titration was conducted after 28 days of storage to verify the stability of the pathogen at each temperature.

Analysis of CsNIV nucleic acids.
A total of 450 ml of exponentially growing C. salsugineum Ch42 cultures (2.48 x 107 ml–1) was inoculated with 22.5 ml of the virus (9.82 x 107 infectious units ml–1) and lysed. The resultant lysates were centrifuged at 4,500 x g, 4°C for 10 min; the supernatants were then sequentially passed through 8.0-, 0.8-, and 0.2-µm pore-sized polycarbonate membrane filters (Nuclepore) to remove cellular debris. Polyethylene glycol 6000 (Wako Pure Chemical Industries, Ltd.) was added to the filtrates to obtain a final concentration of 10% (wt/vol), and the resultant suspension was stored at 4°C in the dark overnight. After centrifugation at 57,000 x g and 4°C for 1.5 h, the viral pellet was washed with 10 mM phosphate buffer (pH 7.2) and centrifuged again at 217,000 x g and 4°C for 4 h to collect virus particles. They were then resuspended in 500 µl of ultrapure water. The viral suspension was treated with proteinase K (1 mg ml–1; Wako Pure Chemical Industries, Ltd.) and sarcosyl (1%; International Technologies, Inc.) at 55°C for 1.5 h. Nucleic acids were extracted from the pellet by using the phenol-chloroform extraction method (31). With or without treatment at 100°C for 10 min, followed by cooling on ice, nucleic acids were electrophoresed in a formaldehyde-agarose gel (1%, 15 by 20 cm; SeaKem Gold Agarose; BMA, Inc.) at 50 V for 14.5 h. Nucleic acids were visualized by SYBR-Green II staining (Molecular Probes, Inc.). The nucleic acid samples were then digested with RNase A (Nippon Gene Co., Ltd.) at 0.05 µg µl–1 or DNase I (Promega Co., Ltd.) at 0.5 U µl–1 at 37°C for 1 h or with S1 nuclease (Takara Bio, Inc.) at 0.7 U µl–1 at 23°C for 15 min. The samples were then electrophoresed in 2% (wt/vol) Agarose S gels (Nippon Gene Co., Ltd.). Nucleic acid extractions held on ice without enzymatic treatment served as controls. Nucleic acids were visualized by using ethidium bromide staining.

The S1 nuclease-resistant fragment (~1 kbp) was excised from the gel by using Quantum PrepTM Freeze 'N Squeeze DNA gel extraction spin columns (Bio-Rad Laboratories, Inc.), extracted by using phenol-chloroform extraction, and dissolved in ultrapure water. It was then blunt ended, phosphorylated using a TaKaRa BKL kit (Takara Bio, Inc.), and ligated into the bacterial alkaline phosphatase-treated pBluescript SK(+) vector digested with EcoRV by using a Ligation High Kit (Toyobo Co., Ltd.) according to the manufacturer's recommendations. After the resultant plasmids were transformed into Escherichia coli DH5{alpha}-competent cells (Toyobo Co., Ltd.), sequencing was conducted by using the dideoxy method with an ABI Prism 3100 Genetic Analyzer (Applied Biosystems). In order to confirm that the S1 nuclease-resistant fragment originated from circular DNA, two primer sets were designed based on its nucleotide sequence. L53 (5'-TTA AGT CCT AAG TAT TGT TAT TGC-3') and R35 (5'-CCG TTA GCA CGT GCT TC-3') were designed for amplifying a part of the S1 nuclease-resistant fragment; R53 (5'-TAA CCC GAA GCA CGT GCT AAC-3') and L35 (5'-ATA GCA ATA ACA ATA CTT AGG AC-3') were designed for amplifying the remaining (S1 nuclease-sensitive) region of the circular DNA. For the former primer set, PCR amplification was performed with 20-µl mixtures containing ~130 ng of template viral DNA, 1x ExTaq buffer (Takara Bio, Inc.), each deoxynucleoside triphosphate at a concentration of 200 µM, 20 pmol of each primer, 1 U of ExTaq DNA polymerase with a GeneAmp PCR System 9700 (Applied Biosystems) according to the following cycle parameters: denaturation at 98°C (40 s), annealing at 45°C (30 s), and extension at 72°C (90 s). For the latter primer set, PCR amplification was performed with 20-µl mixtures using 1x Z-Taq (Takara Bio, Inc.) buffer containing 3 mM Mg2+ and a 200 µM concentration of each deoxynucleoside triphosphate with Z-Taq using a GeneAmp PCR system 9700 (Applied Biosystems) according to the cycle parameters given above. In both cases, after 30 rounds of amplification the PCR products were electrophoresed in 1% (wt/vol) Agarose S gels in which the nucleic acids were visualized by ethidium bromide staining. The amplicons were ligated into the appropriate vectors (pCR-XL-TOPO vector or the TOPO TA cloning vector [Invitrogen]) according to the manufacturer's recommendations.

Analysis of CsNIV proteins.
The virus suspension was mixed with a fourfold volume of the sample buffer (62.5 mM Tris-HCl, 5% 2-mercaptoethanol, 2% sodium dodecyl sulfate [SDS], 20% glycerol, 0.005% bromophenol blue) and boiled for 5 min. The proteins were then separated by using SDS-polyacrylamide gel electrophoresis (80 by 40 by 1.0 mm, 12.5% polyacrylamide gel, 150 V) using the XV Pantera System (DRC Co., Ltd.). Proteins were visualized by Coomassie brilliant blue staining. Protein molecular mass standards (DRC Co., Ltd.) ranging from 6.5 to 200 kDa were used for size calibration.

Host range analysis.
The interspecies host specificity of the pathogen was tested by adding 5% (vol/vol) aliquots of fresh virus suspension to duplicate cultures of exponentially growing clonal algal strains that belong to the families of Bacillariophyceae, Chlorophyceae, Dinophyceae, Eustigmatophyceae, and Raphidophyceae (Table 1). They were cultured under the conditions given above at the temperatures shown in Table 1. The growth and evidence of lysis in each algal culture were monitored by optical microscopy and compared to control cultures inoculated with SWM3. Cultures not lysed after 14 days were considered to be unsuitable hosts for the viral pathogen.

One-step growth experiment.
A one-step growth experiment was designed according to the method of Sandaa et al. (20). An exponentially growing culture of C. salsugineum Ch42 (530 ml) was inoculated with the pathogen at a multiplicity of infection of 10.8. As a control, a C. salsugineum Ch42 culture was inoculated with an autoclaved viral suspension. An aliquot of cell suspension was sampled from each culture at 0, 12, 24, 30, 36, 48, 54, and 76 h postinoculation and used to determine the number of host cells and lytic agents by transmission electron microscopy (TEM) observations. The number of lytic agents was determined by the extinction dilution method (14, 21). Incubation conditions were as described above.

TEM.
For TEM observations, C. salsugineum Ch42 cells were harvested by centrifugation at 860 x g at 4°C for 10 min and fixed with 1% glutaraldehyde in SWM3 for >4 h at 4°C. The cell pellets were postfixed for 3 h in 2% osmic acid in 0.1 M phosphate buffer (pH 7.2 to 7.4), dehydrated in a graded ethanol series (50 to 100%), and embedded in Quetol 653 resin (Nisshin EM Co., Ltd.). Thin sections were stained with 4% uranyl acetate and 3% lead citrate and observed at 80 kV by using a JEOL JEM-1010 transmission electron microscope.

The algicidal pathogens negatively stained with uranyl acetate were also observed by using TEM. Briefly, the algicidal pathogen suspension was mounted on a grid (no. 780111630; JEOL Datum, Ltd.) for 30 s, and excess water was removed by using filter paper (no. 1; TOYO Co., Ltd.). Then, 4% uranyl acetate was put on the grid for 10 s and the excess dye was removed by using 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 from the negatively stained images.


   RESULTS AND DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
Isolation of the viral pathogen.
A cloned pathogen causing lysis of C. salsugineum Ch42 was successfully isolated. Because the viral pathogen retained algicidal activity after filtration through a 0.1-µm-pore-size filter, it was easily made free of bacterial contamination. The algicidal activity was lost by treatment at 121°C for 15 min (data not shown). These data demonstrate that the algicidal effect is heat labile and caused by a substance or particle smaller than 0.1 µm in diameter. The algicidal activity was serially transferable to exponentially growing C. salsugineum Ch42 cultures that consistently resulted in lysis (data not shown). Cultures and cells of C. salsugineum Ch42 lysed by the pathogen became pale in color, presumably due to the loss or degradation of photosynthetic pigments (Fig. 1C and D). Although viral attack of many microalgal cultures allows some host cells to survive and thus regrowth of the culture to occur within a few days (15, 23, 26, 29), it was distinctive that this pathogen caused almost complete lysis of the host cultures and not detectable regrowth after 12 weeks.

Morphological features.
Thin sections of healthy C. salsugineum Ch42 cells indicated that the cytoplasmic organization and frustules were diagnostic of diatoms (Fig. 2A). In contrast, electron micrographs of thin-sectioned C. salsugineum Ch42 cells inoculated with the viral pathogen revealed the presence of small virus-like particles (VLPs) randomly assembled in the nucleus (Fig. 2B and C). In cells in which the nuclear envelope was partly ruptured, VLPs were also observed in the cytoplasm (Fig. 2D). Formation of crystalline arrays of particles was not observed in these experiments. No particles were evident within healthy cells of the control cultures (Fig. 2A). Moreover, VLPs were observed in culture lysates by means of the negative staining method. They were icosahedral in shape, 38 ± 3 nm (n = 25) in diameter, and lacked a tail or an outer membrane (Fig. 2E).



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  		FIG. 2. Transmission electron micrographs of C. salsugineum. (A) Thin section of a healthy cell; (B) thin section of a cell 24 h after inoculation with CsNIV; (C) close-up view of intranuclear virus particles in B; (D) thin section of a CsNIV-infected cell in which the nuclear envelope was partially ruptured (arrowheads); (E) negatively stained virus particles in the culture lysate. CH, a chloroplast; NU, a nucleus; NS, a nucleolus.

 
Since (i) the algicidal pathogen was transferable to a fresh algal culture, (ii) VLPs were observed in the lysed culture, and (iii) the VLPs were not found in healthy cultures, Koch's postulates are fulfilled. We conclude that the VLPs observed within the infected cells and in the algal lysates were a pathogenic virus for C. salsugineum. This new virus was termed C. salsugineum nuclear inclusion virus (i.e., CsNIV) after its host species and site of propagation.

In the case of RsRNAV, the frustule pores of its host R. setigera are larger than RsRNAV, so they are considered a possible route of viral infection (12), whereas the frustule pores of C. salsugineum were too small for CsNIV to penetrate (ca. 6 nm in diameter). A small number of larger ellipse pores (ca. 85 by 47 nm) were sporadically observed on the setae (Fig. 1E) that may be one of the possible routes of infection for CsNIV. However, no direct evidence for this speculation has been obtained at present.

Stability.
A CsNIV suspension containing 3.5 x 108 (95% confidence intervals of 1.4 x 108 to 8.3 x 108) infectious units ml–1 was subjected to a storage test. The infectious titers of virus suspension after 28 days of storage at 20, 10, 4, –20, and –196°C in the dark were 5.1 x 108 (2.0 x 108 to 1.2 x 109), 3.9 x 108 (1.6 x 108 to 9.6 x 108), 1.9 x 108 (8.7 x 107 to 4.2 x 108), 3.9 x 108 (1.6 x 108 to 9.6 x 108), and 2.1 x 108 (8.6 x 107 to 5.0 x 108) infectious units ml–1, respectively (with 95% confidence intervals in parentheses). Thus, interestingly, there was no significant loss of infectivity at any of the conditions tested, indicating that the virus is very stable in natural environment.

After the isolation of CsNIV from the sediment sample, 10 other virus clones infecting C. salsugineum were isolated from both sediments and seawaters (data not shown). Based on these data, sediments might work as an important environmental reservoir, where CsNIV might be long-lived due to its high thermal stability. These results support the hypothesis that sediments are a reservoir for viruses infecting microalgae (11, 13, 26).

Genome.
The intact CsNIV genome exhibited three major bands of nucleic acids using denaturing gel electrophoresis, where the band showing the lowest mobility was much stronger than the second band (Fig. 3A, lane 1). Heat treatment at 100°C for 10 min reduced the first band and strengthened the second (Fig. 3A, lane 2). These results suggest that part of the first band is sensitive to heat treatment and was changing into the second. On the assumption that the first and second bands are "covalently closed circular form having lower mobility" and "linear form of the same molecule having higher mobility," respectively, and were ~6 kb in length (Fig. 3A), the following experiments were designed to confirm the genome structure as given in Materials and Methods. Because all bands were sensitive to DNase I but not to RNase A (Fig. 3, lanes 3, 4, and 6), the viral genome is considered DNA. In addition, the genome was digested with S1 nuclease; however, a double-stranded DNA (dsDNA) of ~1.0 kbp remained undigested; a faint second band of ~800 bp, presumably overdigested by S1 nuclease, was also detected (Fig. 3B, lane 5). The undigested dsDNA fragment was sequenced. PCR experiments with the two primer sets designed to confirm the structure of CsNIV-DNA (see Materials and Methods) resulted in amplification of the expected product sizes: 890 bp (Fig. 3C, lane 8) and ~5 kbp (Fig. 3C, lane 9, and Fig. 4). Based on these data, we concluded that the viral genome consists of a single strand of circular DNA (~6 kb) that is partly double-stranded (~1 kb) and covalently closed (Fig. 4). As far as we know, this genome structure is unlike that of any other viruses that have been described to date.



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  		FIG. 3. (A) Nucleic acids of CsNIV with (lane 1) or without (lane 2) heat treatment at 100°C for 10 min electrophoresed in formaldehyde agarose gel; (B) nucleic acids of CsNIV either untreated (lane 3) or treated with DNase I (lane 4), S1 nuclease (lane 5), or RNase A (lane 6) electrophoresed in TAE agarose gel; (C) nucleic acids extracted from CsNIV treated with S1 nuclease (lane 7) or PCR products amplified with L53 and R35 (lane 8) and L35 and R53 (lane 9). Band I in lane 9 is the targeted amplicon, and bands II and III are probably amplified due to overlaps in the nucleotide sequence.

 


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  		FIG.4. Scheme of the CsNIV genome structure. Primers used in the experiments are shown as arrowheads.

 
Full sequencing of CsNIV genome revealed that the circular DNA and the dsDNA regions are 6,005 and 997 nucleotides in length, respectively (DDBJ accession number AB193315). If we assume that CUG or ACG are its initiation codon in addition to the universal initiation codon AUG (3, 7, 17, 18), one of the open reading frames showed slight similarity with the putative replicase-associated protein of beak and feather disease virus (E-value = 2.5E–2) and the replication protein V1 of goose circovirus (E-value = 2.2E–1), both belonging to the family Circoviridae (24, 25). Despite the low E-value, a similarity to replication-associated proteins of circoviruses, which harbor single-stranded circular genomic DNA, was noticeable. However, according to the universal codon usage, the corresponding sequence is located outside of the open reading frames. The codon usage system of CsNIV awaits future study. Preliminary analysis of secondary structure of CsNIV genome revealed that the dsDNA region (997 nucleotides) is located between significant loop structures (data not shown), which may be related to its specific structure. Further characterization of the CsNIV genome and its replication system will be explained elsewhere.

Proteins.
The sizes and numbers of structural proteins making the virus particles were determined by SDS-polyacrylamide gel electrophoresis. CsNIV contains two major polypeptides of 46.0 and 43.5 kDa and two minor polypeptides of 42.0 and 36.0 kDa (Fig. 5). The number of major proteins of CsNIV was much smaller than those of other DNA viruses infecting algae (see, for example, reference 28). The results may reflect differences in genome size since CsNIV is the smallest among the DNA viruses infecting algae.



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  		FIG. 5. Major structural proteins of CsNIV.

 
Host range.
The host range of CsNIV was tested on 58 phytoplankton strains, including 38 strains of diatoms isolated from the coastal waters of western Japan. CsNIV was found only to cause lysis of C. salsugineum Ch42 but not of any of the other microalgal species tested (Table 1), showing that its infection specificity was high, a common viral property. In the present study we could not determine whether the virus also was strain specific due to the lack of multiple strains of C. salsugineum.

Replication.
In the one-step growth experiment, an increase in virus number was noticeable 12 to 24 h after inoculation (Fig. 6). Thus, the latent period of CsNIV was estimated to be <24 h, whereas the decrease in host cell number was obvious 36 to 76 h postinoculation (Fig. 6). Considering that the multiplicity of infection (10.8) was high enough to make all of the sensitive cells infected, it is presumable that viral infection does not necessarily interrupt algal binary fissions all at once, as was observed in the case of RsRNAV (12), because we observed a slight increase in host cell numbers even after virus inoculation (Fig. 6). Hence, a precise estimation of the burst size was considered impossible. The hosts/virus ratio at 12 to 48 h postinoculation was used to calculate the burst size and was estimated to be 325 infectious units cell–1. This was considered an underestimate compared to the particle numbers found in thin section views of infected cells. Possible explanations for the small burst size are crystallization of virus particles causing an underestimation of the most probable number, difficulties in distinguishing dead cell and living cell by optical microscopy, or a dominance of defective particles lacking infectivity.



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  		FIG. 6. Changes in abundance of C. salsugineum Ch42 used for the one-step growth experiments with (•) or without virus inoculation ({circ}), and the virus titer ({blacksquare}). Virus inoculation was performed in the exponentially growing phase (arrow) at multiplicities of infection of 10.8. The error bars indicate the standard deviations for host abundance and the 95% confidence limits for virus titer.

 
Implications.
CsNIV is the first DNA virus shown to infect diatoms. Our investigation emphasizes that viruses which infect and cause lysis of diatoms are also a component of the natural marine viral community and affect the hosts' dynamics.

There are a lot of virus groups containing DNA genome whose virion assembly occurs in the nucleus of the hosts, e.g., Adenoviridae, Baculoviridae, Geminiviridae, Herpesviridae, Papillomaviridae, Polyomaviridae, Parvoviridae, etc. Also, in the case of the family Circoviridae, viral particles have been detected within the nucleus of the feather epithelium of birds (24, 25). Among the viruses infecting eukaryotic algae, Heterosigma akashiwo nuclear inclusion virus is the only known virus that propagates in its host's nucleus. In H. akashiwo nuclear inclusion virus-infected H. akashiwo cells, margination of heterochromatin within the nucleoplasm was observed (11). Also, in the present experiments, C. salsugineum cells infected by CsNIV revealed degradation of the nucleolus (Fig. 2A and B), but we do not understand the reason for this.

CsNIV occurred naturally at <98.2 infectious units ml–1 in the Ariake Sea, Japan (Y. Tomaru, unpublished data). Studies in the natural environment on interaction between the virus and its host are still under way. The reason for this interest is (i) CsNIV is infectious to a species of the most abundant diatom genus, Chaetoceros: (ii) the virus's genome structure is distinctive, i.e., consisting of a single molecular of circular ssDNA that is partly double stranded and covalently closed; and (iii) this virus is the first DNA virus shown to propagate within the nucleus of diatom cells. The host-virus system described here will be useful in investigating the relationships between the earth's breadbasket, diatoms, and their viral diseases, as well as the unique aspects of viral replication biology.


   ACKNOWLEDGMENTS
 
This study was supported by Grants-in-Aid for Scientific Research (A)(2)(16208019) from the Ministry of Education, Science, and Culture in Japan, by the Industrial Technology Research Grant Program in 2004 from the New Energy and Industrial Technology Development Organization of Japan (NEDO), by the Society for Techno-Innovation of Agriculture, and by the Ministry of Agriculture, Forestry, and Fisheries of Japan.

We thank T. Uchida (Hokkaido National Fisheries Research Institute); I. Imai (Kyoto University); M. Yamaguchi, S. Itakura, S. Nagai, and Y. Matsuyama (National Research Institute of Fisheries and Environment of Inland Sea); T. Nishikawa (Fisheries Technology Institute, Hyogo Prefectural Technology Center for Agriculture, Forestry, and Fisheries); and R. A. Lewin (Scripps Institute of Oceanography) for providing some of the algal cultures used in this study. We also thank S. Oda (Fukuoka Fisheries and Marine Technology Research Center) for providing water and sediment samples, C. A. Suttle (University of British Columbia) for critical reading of the manuscript, and N. Katanozaka (Hitec Co., Ltd.) for technical assistance.


   FOOTNOTES
 
* Corresponding author. Mailing address: National Research Institute of Fisheries and Environment of Inland Sea, Fisheries Research Agency, 2-17-5 Maruishi, Ohno, Saeki, Hiroshima 739-0452, Japan. Phone: 81-829-55-0666. Fax: 81-829-54-1216. E-mail: nagasaki{at}affrc.go.jp. Back

{dagger} Present address: Hiroshima Prefectural Institute of Industrial Science and Technology, 3-10-32 Kagamiyama, Higashi-Hiroshima 739-0046, Japan. Back


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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Applied and Environmental Microbiology, July 2005, p. 3528-3535, Vol. 71, No. 7
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.7.3528-3535.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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