DsRNA sequencing revealed a previously missed terminal sequence of a +ssRNA virus that infects dinoflagellate Heterocapsa circularisquama.

Heterocapsa circularisquama RNA virus (HcRNAV) is the only dinoflagellate-infecting RNA virus cultured. However, only two strains of HcRNAV have been registered with complete genome sequences (strains 34 and 109 for UA and CY types, respectively). To extend the genomic information of HcRNAV, we performed full-genome sequencing of an unsequenced strain of HcRNAV (strain A8) using the fragmented and primer-ligated double-stranded RNA (dsRNA) sequencing (FLDS) method. The complete genome of HcRNAV A8 with 4457 nucleotides (nt) was successfully determined, and sequence alignment of the major capsid protein gene suggested that A8 was a UA-type strain, consistent with its intraspecific host specificity. The complete sequence was found to be 80 nt longer at the 5' terminus than the registered sequences of HcRNAV strains (34 and 109), suggesting that FLDS is more reliable for determining the terminal sequence than conventional methods (5' Rapid Amplification of cDNA End). Our study contributes to a better understanding of dinoflagellate-infecting viruses with limited sequence data.

Isolation of viruses, including mollivirus, with the potential to infect Acanthamoeba from a Japanese warm temperate zone.

Acanthamoeba castellanii is infected with diverse nucleocytoplasmic large DNA viruses. Here, we report the co-isolation of 12 viral strains from marine sediments in Uranouchi Inlet, Kochi, Japan. Based on the morphological features revealed by electron microscopy, these isolates were classified into four viral groups including Megamimiviridae, Molliviridae, Pandoraviridae, and Pithoviridae. Genomic analyses indicated that these isolates showed high similarities to the known viral genomes with which they are taxonomically clustered, and their phylogenetic relationships were also supported by core gene similarities. It is noteworthy that Molliviridae was isolated from the marine sediments in the Japanese warm temperate zone because other strains have only been found in the subarctic region. Furthermore, this strain has 19 and 4 strain-specific genes found in Mollivirus sibericum and Mollivirus kamchatka, respectively. This study extends our knowledge about the habitat and genomic diversity of Molliviridae.

Dozens of toxin-related genes are expressed in a nontoxic strain of the dinoflagellate Heterocapsa circularisquama.

The dinoflagellate Heterocapsa circularisquama is lethal to a variety of marine organisms, in particular, commercially important farmed bivalves. Unlike most dinoflagellate toxins, which are polyketides, the only described toxin from H. circularisquama (H2-a) is a porphyrin derivative that functions in light. It is unknown whether H2-a is produced specifically for its lytic properties. We searched for toxin-related genes in the transcriptome of a nontoxic strain of H. circularisquama, and surprisingly found the richest set of toxin-related genes yet described in dinoflagellates. There are 87 distinct expressed sequence tag contigs that encode polyketide synthases and nonribosomal peptide synthases, as well as 8 contigs that are involved in porphyrin biosynthesis. Phylogenomic analysis shows that many toxin-related genes are widely distributed among dinoflagellates. Our data likely indicate a variety of unknown metabolic functions for the toxin-related genes in H. circularisquama because they were identified in a nontoxic strain raised in unialgal culture.

Diversity in Infection Specificity between the Bloom-forming Microalga Heterosigma akashiwo and Its dsDNA Virus, Heterosigma akashiwo Virus.

Heterosigma akashiwo virus (HaV) is a dsDNA virus that infects the bloom-forming raphidoflagellate Heterosigma akashiwo. Both the host and its virus are phenotypically diverse in terms of infection specificity. Their relationships have been examined based on the occurrence or absence of algal lysis following virus inoculation; however, variations in the strain-level host-virus relationship regarding infectivity and lysis rates remain unclear. Therefore, we performed a series of cross-infectivity tests using 60 H. akashiwo and 22 HaV strains isolated from the coastal waters of western Japan. The host strains were divided into 5 different groups and viruses into 4 groups. Using a representative strain from each group, algal lysis was observed in 14 of the (5×4=) 20 host-virus combinations; the concentration of infectious units in each HaV suspension was then assessed using the most probable number (MPN) assay on the five host strains. Virus titers ranged between 1.1×101 and 2.1×107 infectious units mL-1; the titer of each viral lysate was differently estimated using distinct H. akashiwo strains as hosts. These results suggest that (1) a clonal viral lysate comprises virions with different intraspecific infection specificities and/or (2) the efficiency and error rates of each intracellular replication process vary in each host-virus combination.

Visualization of RNA virus infection in a marine protist with a universal biomarker.

Half of the marine virosphere is hypothesized to be RNA viruses (kingdom Orthornavirae) that infect abundant micro-eukaryotic hosts (e.g. protists). To test this, quantitative approaches that broadly track infections in situ are needed. Here, we describe a technique-dsRNA-Immunofluorescence (dsRIF)-that uses a double-stranded RNA (dsRNA) targeting monoclonal antibody to assess host infection status based on the presence of dsRNA, a replicative intermediate of all Orthornavirae infections. We show that the dinoflagellate Heterocapsa circularisquama produces dsRIF signal ~ 1000 times above background autofluorescence when infected by the + ssRNA virus HcRNAV. dsRNA-positive virocells were detected across > 50% of the 48-h infection cycle and accumulated to represent at least 63% of the population. Photosynthetic and chromosomal integrity remained intact during peak replication, indicating HcRNAV infection does not interrupt these processes. This work validates the use of dsRIF on marine RNA viruses and their hosts, setting the stage for quantitative environmental applications that will accelerate understanding of virus-driven ecosystem impacts.

Degenerate PCR Targeting the Major Capsid Protein Gene of HcRNAV and Related Viruses.

Heterocapsa circularisquama RNA virus (HcRNAV) is the only dinoflagellate-infecting RNA virus that has been isolated to date. We herein investigated the diversity of the major capsid protein gene of HcRNAV and related viruses using degenerate PCR and in silico ana-lyses. Diverse sequences related to HcRNAV were successfully amplified from marine sediments. Amplicons contained conserved and variable regions; the latter were predicted to be located on the outer surface of the capsid. Our approach provides insights into the diversity of viruses that are difficult to isolate in the environment and will enhance rapidly growing metagenome sequence repositories.

RNA Virosphere in a Marine Zooplankton Community in the Subtropical Western North Pacific.

Zooplankton and viruses play a key role in marine ecosystems; however, their interactions have not been examined in detail. In the present study, the diversity of viruses associated with zooplankton collected using a plankton net (mesh size: 100| |µm) in the subtropical western North Pacific was investigated by fragmented and primer ligated dsRNA sequencing. We obtained 21 and 168 operational taxonomic units (OTUs) of ssRNA and dsRNA viruses, respectively, containing RNA-dependent RNA polymerase (RdRp). These OTUs presented average amino acid similarities of 43.5 and 44.0% to the RdRp genes of known viruses in ssRNA viruses and dsRNA viruses, respectively. Dominant OTUs mainly belonged to narna-like and picorna-like ssRNA viruses and chryso-like, partiti-like, picobirna-like, reo-like, and toti-like dsRNA viruses. Phylogenetic ana-lyses of the RdRp gene revealed that OTUs were phylogenetically diverse and clustered into distinct clades from known viral groups. The community structure of the same zooplankton sample was investigated using small subunit (SSU) rRNA sequences assembled from the metatranscriptome of single-stranded RNA. More than 90% of the sequence reads were derived from metazoan zooplankton; copepods comprised approximately 70% of the sequence reads. Although this ana-lysis provided no direct evidence of the host species of RNA viruses, these dominant zooplankton are expected to be associated with the RNA viruses detected in the present study. The present results indicate that zooplankton function as a reservoir of diverse RNA viruses and suggest that investigations of zooplankton viruses will provide a more detailed understanding of the role of viruses in marine ecosystems.

Horizontal and vertical distribution of Gambierdiscus spp. (Dinophyceae) including novel phylotypes in Japan identified by 18S rDNA metabarcoding.

The genus Gambierdiscus is a marine benthic/epiphytic dinoflagellate considered the causative agent of ciguatera poisoning (CP). Clarifying the geographical distribution of this genus to understand the potential risk of CP is important. Many studies have focused only on the species/phylotype composition of Gambierdiscus in shallow waters, but no study has investigated the species/phylotype composition of the genus in deep waters. In the present study, the distributions of Gambierdiscus species/phylotypes at two depths (2-8 and 30 m) and two sampling sites (temperate and subtropical) in Japan was investigated using high throughput sequencing (HTS) with a newly developed primer set that preferentially amplifies the 18S rDNA V8-V9 region of Alveolata. A phylogenetic analysis using 89 samples collected over three years revealed of ten Gambierdiscus species/phylotypes including not only two species that have not been reported in Japan (G. caribaeus and G. silvae) but also four novel phylotypes (Gambierdiscus spp. Clade II_1, Clade II_2, Clade II_3, and Clade VI_1). Uncorrected genetic distances also supported that these new phylotypes clearly diverged from other Gambierdiscus species. All four new phylotypes, G. caribaeus, and G. silvae were distributed in the subtropical region. Among them, Clade II_2, Clade VI_1, and G. silvae were also distributed in the temperate region. Four species/phylotypes previously reported from Japan showed a similar distribution as reported previously. Among the ten species/phylotypes, Gambierdiscus sp. type 3 and Clade VI_1 were found only in deep waters, whereas five species/phylotypes were observed only in shallow waters. The other three species/phylotypes were found in both deep and shallow waters. The results of the horizontal and vertical distribution suggest that the growth characteristics of each species/phylotypes found in Japan might adapt to the ambient environmental conditions. This study revealed an inclusive assemblage of Gambierdiscus species/phylotypes in Japan through metabarcoding using the Alveolata primer set. In the future, the abundance and toxicities/toxin productions of the newly reported species/phylotypes need to be clarified to understand the mechanism of CP outbreaks in Japan.

Year-round dynamics of amplicon sequence variant communities differ among eukaryotes, Imitervirales and prokaryotes in a coastal ecosystem.

Coastal microbial communities are affected by seasonal environmental change, biotic interactions and fluctuating nutrient availability. We investigated the seasonal dynamics of communities of eukaryotes, a major group of double-stranded DNA viruses that infect eukaryotes (order Imitervirales; phylum Nucleocytoviricota), and prokaryotes in the Uranouchi Inlet, Kochi, Japan. We performed metabarcoding using ribosomal RNA genes and viral polB genes as markers in 43 seawater samples collected over 20 months. Eukaryotes, prokaryotes and Imitervirales communities characterized by the compositions of amplicon sequence variants (ASVs) showed synchronic seasonal cycles. However, the community dynamics showed intriguing differences in several aspects, such as the recovery rate after a year. We also showed that the differences in community dynamics were at least partially explained by differences in recurrence/persistence levels of individual ASVs among eukaryotes, prokaryotes and Imitervirales. Prokaryotic ASVs were the most persistent, followed by eukaryotic ASVs and Imitervirales ASVs, which were the least persistent. We argue that the differences in the specificity of interactions (virus-eukaryote vs prokaryote-eukaryote) as well as the niche breadth of community members were at the origin of the distinct community dynamics among eukaryotes, their viruses and prokaryotes.

Three-dimensional reconstruction of Heterocapsa circularisquama RNA virus by electron cryo-microscopy.

Heterocapsa circularisquama RNA virus is a non-enveloped icosahedral ssRNA virus infectious to the harmful bloom-forming dinoflagellate, H. circularisquama, and which is assumed to be the major natural agent controlling the host population. The viral capsid is constructed from a single gene product. Electron cryo-microscopy revealed that the virus has a diameter of 34 nm and T = 3 symmetry. The 180 quasi-equivalent monomers have an unusual arrangement in that each monomer contributes to a 'bump' on the surface of the protein. Though the capsid protein probably has the classic 'jelly roll' ß-sandwich fold, this is a new packing arrangement and is distantly related to the other positive-sense ssRNA virus capsid proteins. The handedness of the structure has been determined by a novel method involving high resolution scanning electron microscopy of the negatively stained viruses and secondary electron detection.

Isolation and characterization of a single-stranded DNA virus infecting Chaetoceros lorenzianus Grunow.

Diatoms are one of the most significant primary producers in the ocean, and the importance of viruses as a potential source of mortality for diatoms has recently been recognized. Thus far, eight different diatom viruses infecting the genera Rhizosolenia and Chaetoceros have been isolated and characterized to different extents. We report the isolation of a novel diatom virus (ClorDNAV), which causes the lysis of the bloom-forming species Chaetoceros lorenzianus, and show its physiological, morphological, and genomic characteristics. The free virion was estimated to be ∼34 nm in diameter. The arrangement of virus particles appearing in cross-section was basically a random aggregation in the nucleus. Occasionally, distinctive formations such as a ring-like array composed of 9 or 10 spherical virions or a centipede-like array composed of rod-shaped particles were also observed. The latent period and the burst size were estimated to be <48 h and 2.2 × 10(4) infectious units per host cell, respectively. ClorDNAV harbors a covalently closed circular single-stranded DNA (ssDNA) genome (5,813 nucleotides [nt]) that includes a partially double-stranded DNA region (979 nt). At least three major open reading frames were identified; one showed a high similarity to putative replicase-related proteins of the other ssDNA diatom viruses, Chaetoceros salsugineum DNA virus (previously reported as CsNIV) and Chaetoceros tenuissimus DNA virus. ClorDNAV is the third member of the closed circular ssDNA diatom virus group, the genus Bacilladnavirus.

The genome of the diatom Chaetoceros tenuissimus carries an ancient integrated fragment of an extant virus.

Diatoms are one of the most prominent oceanic primary producers and are now recognized to be distributed throughout the world. They maintain their population despite predators, infections, and unfavourable environmental conditions. One of the smallest diatoms, Chaetoceros tenuissimus, can coexist with infectious viruses during blooms. To further understand this relationship, we sequenced the C. tenuissimus strain NIES-3715 genome. A gene fragment of a replication-associated gene from the infectious ssDNA virus (designated endogenous virus-like fragment, EVLF) was found to be integrated into each 41 Mb of haploid assembly. In addition, the EVLF was transcriptionally active and conserved in nine other C. tenuissimus strains from different geographical areas, although the primary structures of their proteins varied. The phylogenetic tree further suggested that the EVLF was acquired by the ancestor of C. tenuissimus. Additionally, retrotransposon genes possessing a reverse transcriptase function were more abundant in C. tenuissimus than in Thalassiosira pseudonana and Phaeodactylum tricornutum. Moreover, a target site duplication, a hallmark for long interspersed nuclear element retrotransposons, flanked the EVLF. Therefore, the EVLF was likely integrated by a retrotransposon during viral infection. The present study provides further insights into the diatom-virus evolutionary relationship.

Complete Genome Sequence of Helicobacter pylori Strain 3401, a Suitable Host for Bacteriophages KHP30 and KHP40.

Helicobacter pylori 3401, isolated from a patient with duodenal ulcers in Japan, is susceptible to the bacteriophages KHP30 and KHP40. In this study, we report the complete genome sequence of H. pylori 3401. This study may lead to the establishment of phage therapy against H. pylori infection.

Chronological distribution of dinoflagellate-infecting RNA virus in marine sediment core.

A bivalve-killing marine dinoflagellate, Heterocapsa circularisquama, is susceptible to the infectious single-stranded RNA virus, Heterocapsa circularisquama RNA virus (HcRNAV). The ecological relationship between H. circularisquama and HcRNAV was intensively studied from 2001 through 2005; however, only limited data are available for the ecological dynamics of HcRNAV before 2001. In this study, we applied radiometric dating and reverse transcription PCR (RT-PCR) to determine the chronological distribution of HcRNAV in a marine sediment core sampled from the Uranouchi Inlet, Kochi, Japan, where H. circularisquama was first discovered. Our results show that HcRNAV had existed in the inlet long before its first bloom in 1988. Furthermore, five HcRNAV variants, phylogenetically distinguishable based on the nucleotide sequence of the major capsid protein (MCP) gene, were identified. These variants were found to be distributed throughout the core over time, suggesting that the HcRNAV sequences registered in the NCBI database are only a portion of the variants that have emerged in the history of HcRNAV diversification. Herein, we have verified the applicability of the retrospective approach for speculating the distribution of algal RNA viruses over time in aquatic environments.

Crystallization and preliminary X-ray analysis of a marine diatom-infecting single-stranded RNA virus.

Crystals of a diatom-infecting virus (CtenRNAV) that diffracted to a resolution of 4.0 Šwere grown in a mixture of 2-methyl-2,4-pentanediol (MPD), calcium chloride and sodium acetate. It was possible to freeze the crystals directly at liquid-nitrogen temperature as the reservoir solution, which included about 30% MPD, acted as a cryoprotectant during X-ray diffraction data collection. A data set was collected from a single frozen crystal obtained using this method. The crystals belonged to space group P6(3)22, with unit-cell parameters a = b = 448.67, c = 309.76 Šand two virus particles in the unit cell. The virus-particle orientation was determined using a rotation function and the virus-particle centre was estimated on the basis of crystallographic considerations. The packing of CtenRNAV in the crystal lattice was revealed by this preliminary crystallographic study.

An Optimized Metabarcoding Method for Mimiviridae.

Mimiviridae is a group of viruses with large genomes and virions. Ecological relevance of Mimiviridae in marine environments has been increasingly recognized through the discoveries of novel isolates and metagenomic studies. To facilitate ecological profiling of Mimiviridae, we previously proposed a meta-barcoding approach based on 82 degenerate primer pairs (i.e., MEGAPRIMER) targeting the DNA polymerase gene of Mimiviridae. The method detected a larger number of operational taxonomic units (OTUs) in environmental samples than previous methods. However, it required large quantities of DNA and was laborious due to the use of individual primer pairs. Here, we examined coastal seawater samples using varying PCR conditions and purification protocols to streamline the MEGAPRIMER method. Mixing primer pairs in "cocktails" reduced the required amount of environmental DNA by 90%, while reproducing the results obtained by the original protocol. We compared the results obtained by the meta-barcoding approach with quantifications using qPCR for selected OTUs. This revealed possible amplification biases among different OTUs, but the frequency profiles for individual OTUs across multiple samples were similar to those obtained by qPCR. We anticipate that the newly developed MEGAPRIMER protocols will be useful for ecological investigation of Mimiviridae in a larger set of environmental samples.

Virus resistance in the toxic bloom-forming dinoflagellate Heterocapsa circularisquama to single-stranded RNA virus infection.

HcRNAV is the only known cultured dinoflagellate-infecting RNA virus. Lysis of its host dinoflagellate Heterocapsa circularisquama caused by HcRNAV is followed by apparent cell regrowth. Here we investigate the mechanism supporting the survival phenomenon. The proportion of normal cells with intact nucleus decreased to approximately 8% by 3 days post infection, and then, increased to > 90% at 15 days post infection. There were abnormal cells lacking an intact nucleus, and this was followed by propagation of virus-resistant survivor cells. The proportion of HcRNAV-resistant cells in three different subcultures and temporal fluctuations were compared: a clonal H. circularisquama culture without virus inoculation (virus-sensitive, VS), a surviving isolate from the HcRNAV-inoculated Culture-VS incubated in autoclaved medium (virus-resistant, VR) and a portion of Culture-VR incubated with HcRNAV (VR incubated with virus, VR + V). The proportion of HcRNAV-resistant cells in Culture-VS was 0% and in Culture-VR + V was > 94% during the experiment; and Culture-VR fluctuated from 4% to 71%. Hence, the virus resistance was assumed to be reversible. Using Northern hybridization, viral genome accumulation was not detected in Culture-VR + V cells either inoculated with HcRNAV or transfected with HcRNAV-genome; thus, intracellular viral RNA replication was assumed to be interrupted in the virus-resistant cells.

Characterization of the Chaetoceros salsugineum nuclear inclusion virus coat protein gene.

Analysis of the genome of Chaetoceros salsugineum nuclear inclusion virus (CsNIV) revealed the presence of six putative open reading frames (ORFs) in the genome. We further characterized ORF3, which encodes a putative coat protein. Polymerase chain reaction (PCR) using ORF3 gene-specific primers amplified a single DNA band nearly 1.2kb. This amplified product was gel-purified, cloned, sequenced, and expressed in Escherichia coli. Specific antiserum was raised against the recombinant protein and used for Western blotting to test whether the ORF3 protein is the CsNIV coat protein. One major CsNIV protein of approximately 46kDa reacted positively with the antiserum, suggesting that this antiserum is specific for the CsNIV coat protein. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) analysis of the 46kDa structural band revealed 14 peptide sequences that matched the ORF3 regions of CsNIV. The expression of ORF3 in host cells was examined by constructing a cDNA library of CsNIV-infected cells. Nucleotide sequences of the cDNA clones were complementary to various regions of both CsNIV ORF3 and ORF4; however, no clones containing only the ORF3 region were identified. Also, Northern blotting revealed a single 2.5-kb transcript, indicating that ORF3 could be transcribed together with ORF4.

Isolation and characterization of a single-stranded RNA virus infecting the bloom-forming diatom Chaetoceros socialis.

Diatoms are very significant primary producers in the world's oceans. Various environmental factors affect the depletion of diatom populations. The importance of viruses as a potential mortality source has recently been recognized. We isolated and characterized a new diatom virus (Chaetoceros socialis f. radians RNA virus [CsfrRNAV]) causing the lysis of the bloom-forming species Chaetoceros socialis Lauder f. radians (Schütt) Proschkina-Lavrenko. The virus infectious to C. socialis f. radians was isolated from water samples collected in Hiroshima Bay. Here we show the physiology, morphology, and genome characteristics of the virus clone. Virions were 22 nm in diameter and accumulated in the cytoplasm of the host cells. The latent period and the burst size were estimated to be <48 h and 66 infectious units per host cell, respectively. CsfrRNAV harbors a single-stranded RNA (ssRNA) genome and encodes at least three polypeptides of 32.0, 28.5, and 25.0 kDa. Sequencing analysis shows the length of the genome is 9,467 bases, excluding a poly(A) tail. The monophyly of CsfrRNAV and other diatom-infecting RNA viruses, Rhizosolenia setigera RNA virus and Chaetoceros tenuissimus RNA virus, was strongly supported by phylogenetic analysis based on the amino acid sequence of the RNA-dependent RNA polymerase domains. This suggested a new ssRNA virus family, Bacillariornaviridae. This discovery of CsfrRNAV may aid in further understanding the ecological dynamics of the C. socialis f. radians population in nature and the relationships between ssRNA diatom viruses and their hosts.

Remarkable sequence similarity between the dinoflagellate-infecting marine girus and the terrestrial pathogen African swine fever virus.

Heterocapsa circularisquama DNA virus (HcDNAV; previously designated as HcV) is a giant virus (girus) with a approximately 356-kbp double-stranded DNA (dsDNA) genome. HcDNAV lytically infects the bivalve-killing marine dinoflagellate H. circularisquama, and currently represents the sole DNA virus isolated from dinoflagellates, one of the most abundant protists in marine ecosystems. Its morphological features, genome type, and host range previously suggested that HcDNAV might be a member of the family Phycodnaviridae of Nucleo-Cytoplasmic Large DNA Viruses (NCLDVs), though no supporting sequence data was available. NCLDVs currently include two families found in aquatic environments (Phycodnaviridae, Mimiviridae), one mostly infecting terrestrial animals (Poxviridae), another isolated from fish, amphibians and insects (Iridoviridae), and the last one (Asfarviridae) exclusively represented by the animal pathogen African swine fever virus (ASFV), the agent of a fatal hemorrhagic disease in domestic swine. In this study, we determined the complete sequence of the type B DNA polymerase (PolB) gene of HcDNAV. The viral PolB was transcribed at least from 6 h post inoculation (hpi), suggesting its crucial function for viral replication. Most unexpectedly, the HcDNAV PolB sequence was found to be closely related to the PolB sequence of ASFV. In addition, the amino acid sequence of HcDNAV PolB showed a rare amino acid substitution within a motif containing highly conserved motif: YSDTDS was found in HcDNAV PolB instead of YGDTDS in most dsDNA viruses. Together with the previous observation of ASFV-like sequences in the Sorcerer II Global Ocean Sampling metagenomic datasets, our results further reinforce the ideas that the terrestrial ASFV has its evolutionary origin in marine environments.