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A novel single-stranded RNA (ssRNA) virus specifically infecting the bloom-forming diatom Rhizosolenia setigera (R. setigera RNA virus [RsRNAV]) was isolated from Ariake Sea, Japan. Viral replication occurred within the cytoplasm, and the virus particle was icosahedral, lacked a tail, and was 32 nm in diameter on average. The major nucleic acid extracted from the RsRNAV particles was an ssRNA molecule 11.2 kb in length, although smaller RNA molecules (0.6, 1.2, and 1.5 kb) were occasionally observed. The major structural proteins of RsRNAV were 41.5, 41.0, and 29.5 kDa. Inter-and intraspecies host specificity tests revealed that RsRNAV is not only species specific but also strain specific and that its intraspecies host specificity is diverse among virus clones. The latent period of RsRNAV was 2 days, and the burst sizes were 3,100 and 1,010 viruses per host cell when viruses were inoculated into the host culture at the exponential and stationary growth phases, respectively, at 15°C under a 12-h-12-h light-dark cycle of ca. 110 mol of photons m ؊2 s ؊1 with cool white fluorescent illumination. To our knowledge, this is the first report describing the biological properties of a virus infecting a diatom. Further studies on RsRNAV will be helpful in understanding the ecological relationship between diatoms and viruses in nature.

Cyanobacteria and their phages are significant microbial components of the freshwater and marine environments. We identified a lytic phage, Ma-LMM01, infecting Microcystis aeruginosa, a cyanobacterium that forms toxic blooms on the surfaces of freshwater lakes. Here, we describe the first sequenced freshwater cyanomyovirus genome of Ma-LMM01. The linear, circularly permuted, and terminally redundant genome has 162,109 bp and contains 184 predicted protein-coding genes and two tRNA genes. The genome exhibits no colinearity with previously sequenced genomes of cyanomyoviruses or other Myoviridae. The majority of the predicted genes have no detectable homologues in the databases. These findings indicate that Ma-LMM01 is a member of a new lineage of the Myoviridae family. The genome lacks homologues for the photosynthetic genes that are prevalent in marine cyanophages. However, it has a homologue of nblA, which is essential for the degradation of the major cyanobacteria light-harvesting complex, the phycobilisomes. The genome codes for a site-specific recombinase and two prophage antirepressors, suggesting that it has the capacity to integrate into the host genome. Ma-LMM01 possesses six genes, including three coding for transposases, that are highly similar to homologues found in cyanobacteria, suggesting that recent gene transfers have occurred between Ma-LMM01 and its host. We propose that the Ma-LMM01 NblA homologue possibly reduces the absorption of excess light energy and confers benefits to the phage living in surface waters. This phage genome study suggests that light is central in the phage-cyanobacterium relationships where the viruses use diverse genetic strategies to control their host's photosynthesis.The cyanobacterium Microcystis aeruginosa is a toxic, bloomforming bacteria found in eutrophic freshwaters throughout the world (12). The bacterium produces potent hepatotoxins, cyclic peptides called "microcystins," which inhibit eukaryotic protein phosphatase types 1 and 2A and can cause hepatocellular carcinoma (42,53,82). Blooms of M. aeruginosa can lead to the deaths of livestock and humans and pose serious problems for water management (12, 13).The mechanisms controlling bloom initiation and termination remain unclear; however, there have been many studies concerning the effects of environmental factors on M. aeruginosa growth (57). Recently, viral mortality of algae was recognized as one of the factors involved in the termination of algal blooms, including M. aeruginosa blooms (10,52,71,74,75). We previously reported culturing the lytic cyanophage Ma-LMM01 infecting the toxic M. aeruginosa strain NIES298 (81). Currently, M. aeruginosa NIES298 and Ma-LMM01 are the sole culture host/virus system to study interactions between a toxic cyanobacterium and its phage.Ma-LMM01 is a member of the Myoviridae family with a contractile tail (81), the distinctive morphological feature of this viral family. Myoviridae has at least six subgroups: T4-, P1-, P2-, Mu-, SPO1-, and H-like phages (23). These subgroups sh...

We isolated a cyanophage (Ma-LMM01) that specifically infects a toxic strain of the bloom-forming cyanobacterium Microcystis aeruginosa. Transmission electron microscopy showed that the virion is composed of an isometric head and a tail complex consisting of a central tube and a contractile sheath with helical symmetry. The morphological features and the host specificity suggest that Ma-LMM01 is a member of the cyanomyovirus group. Using semi-one-step growth experiments, the latent period and burst size were estimated to be 6 to 12 h and 50 to 120 infectious units per cell, respectively. The size of the phage genome was estimated to be ca. 160 kbp using pulse-field gel electrophoresis; the nucleic acid was sensitive to DNase I, Bal31, and all 14 restriction enzymes tested, suggesting that it is a linear double-stranded DNA having a low level of methylation. Phylogenetic analyses based on the deduced amino acid sequences of two open reading frames coding for ribonucleotide reductase alpha-and beta-subunits showed that Ma-LMM01 forms a sister group with marine and freshwater cyanobacteria and is apparently distinct from T4-like phages. Phylogenetic analysis of the deduced amino acid sequence of the putative sheath protein showed that Ma-LMM01 does not form a monophyletic group with either the T4-like phages or prophages, suggesting that Ma-LMM01 is distinct from other T4-like phages that have been described despite morphological similarity. The host-phage system which we studied is expected to contribute to our understanding of the ecology of Microcystis blooms and the genetics of cyanophages, and our results suggest the phages could be used to control toxic cyanobacterial blooms.Microcystis aeruginosa is one of the highly noxious cyanobacteria that frequently form dense blooms in eutrophic freshwaters throughout the world (9). M. aeruginosa produces potent hepatotoxins (microcystins) that specifically inhibit eukaryotic protein phosphatase types 1 and 2A and cause hepatocellular carcinoma (21,26,48). Hence, M. aeruginosa blooms are often responsible for the death of livestock and wildlife and cause serious problems in water management (9).Despite studies of the effects of various environmental factors on the growth of Microcystis species, the mechanisms that determine bloom dynamics and termination have not been studied sufficiently (27). Recent observations have shown that in addition to physical factors such as temperature and irradiation, chemical factors such as nutrients, and biological factors (predators), mortality induced by virus may be one of the important factors that control these algal blooms (8,23,44).There are a great number of viruses in natural waters, in both marine and freshwater environments (5), and it is suspected that a large proportion of these viruses are infectious for bacteria or cyanobacteria (30). The first isolation of freshwater cyanophages was reported about 40 years ago, and during the following two decades numerous cyanophage strains were isolated (2,3,14,(31)(32)(33)(34). In the...

Heterocapsa circularisquama RNA virus (HcRNAV) has at least two ecotypes (types UA and CY) that have intraspecies host specificities which are complementary to each other. We determined the complete genomic RNA sequence of two typical HcRNAV strains, HcRNAV34 and HcRNAV109, one of each ecotype. The nucleotide sequences of the viruses were 97.0% similar, and each had two open reading frames (ORFs), ORF-1 coding for a putative polyprotein having protease and RNA-dependent RNA polymerase (RdRp) domains and ORF-2 encoding a single major capsid protein. Phylogenetic analysis of the RdRp amino acid sequence suggested that HcRNAV belongs to a new previously unrecognized virus group. Four regions in ORF-2 had amino acid substitutions when HcRNAV34 was compared to HcRNAV109. We used a reverse transcriptionnested PCR system to amplify the corresponding regions and also examined RNAs purified from six other HcRNAV strains with known host ranges. We also looked at natural marine sediment samples. Phylogenetic dendrograms for the amplicons correlated with the intraspecies host specificities of the test virus strains. The cloned sequences found in sediment also exhibited considerable similarities to either the UA-type or CY-type sequence. The tertiary structure of the capsid proteins predicted using computer modeling indicated that many of the amino acid substitutions were located in regions on the outside of the viral capsid proteins. This strongly suggests that the intraspecies host specificity of HcRNAV is determined by nanostructures on the virus surface that may affect binding to suitable host cells. Our study shows that capsid alterations can change the phytoplankton-virus (host-parasite) interactions in marine systems.Only five RNA viruses are known to infect marine eukaryotic microorganisms. The following four viruses are singlestranded RNA (ssRNA) viruses: Heterosigma akashiwo RNA virus (HaRNAV) infects the noxious bloom-forming raphidophyte Heterosigma akashiwo (Raphidophyceae) (26); Rhizosolenia setigera RNA virus infects the bloom-forming diatom Rhizosolenia setigera (20); Heterocapsa circularisquama RNA virus (HcRNAV) infects the bivalve-killing bloom-forming dinoflagellate Heterocapsa circularisquama (30); and Schizochytrium sp. single-stranded RNA virus (SssRNAV) infects the marine fungoid protist Schizochytrium sp. (Labyrinthulae, Thraustochytriaceae) (27). One of the five viruses is a doublestranded RNA (dsRNA) virus (Micromonas pusilla RNA virus) that infects the cosmopolitan phytoplankton Micromonas pusilla (1). Detailed genomic analysis has been performed for two of these viruses, HaRNAV (18) and SssRNAV (Takao et al., unpublished data). Hence, genomic studies of the three other marine RNA viruses are required. In this paper we describe the genome of HcRNAV, which infects H. circularisquama.HcRNAV infection is strain specific rather than species specific because about 6,000 combinations of cross-infection tests between H. circularisquama strains and HcRNAV strains showed that HcRNAV strains are divided rou...