RNA Viruses in Aquatic Unicellular Eukaryotes

Increasing sequence information indicates that RNA viruses constitute a major fraction of marine virus assemblages. However, only 12 RNA virus species have been described, infecting known host species of marine single-celled eukaryotes. Eight of these use diatoms as hosts, while four are resident in dinoflagellate, raphidophyte, thraustochytrid, or prasinophyte species. Most of these belong to the order Picornavirales, while two are divergent and fall into the families Alvernaviridae and Reoviridae. However, a very recent study has suggested that there is extraordinary diversity in aquatic RNA viromes, describing thousands of viruses, many of which likely use protist hosts. Thus, RNA viruses are expected to play a major ecological role for marine unicellular eukaryotic hosts. In this review, we describe in detail what has to date been discovered concerning viruses with RNA genomes that infect aquatic unicellular eukaryotes

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 ~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

Evidence in the Japan Sea of microdolomite mineralization within gas hydrate microbiomes

This study was conducted under the commission of AIST (National Institute of Advanced Industrial Science and Technology, Japan) from 2013–2015 as part of the methane hydrate research project funded by METI (the Ministry of Economy, Trade and Industry, Japan). Ongoing work is currently being carried out thanks to a Grant-in-aid provided by the JSPS and MEXT (Kaken Project # 17K05712). The authors also would like to acknowledge laboratory assistance provided by A. Hiruta, T. Oi, N. Ishida, and R. Warabi (GHRL, Meiji University), Y. Kusaba (AORI, University of Tokyo), S. Motai (Kochi Inst. Core Sample Research, JAMSTEC), and Y. Nakajima (Joetsu Environmental Science Centre).Peer reviewedPublisher PD

Novel Protocol for Estimating Viruses Specifically Infecting the Marine Planktonic Diatoms

Since their discovery, at least 15 diatom viruses have been isolated and characterised using a culture method with two cycles of extinction dilution. However, the method is time consuming and laborious, and it isolates only the most dominant virus in a water sample. Recent studies have suggested inter-species host specificity of diatom viruses. Here, we describe a new protocol to estimate previously unrecognised host-virus relationships. Host cell cultures after inoculation of natural sediment pore water samples were obtained before complete lysis. The proliferated viral genomes in the host cells were amplified using degenerate primer pairs targeting protein replication regions of single-stranded RNA (ssRNA) and single-stranded DNA (ssDNA) viruses, and then sequenced. Diverse ssRNA virus types within known diatom virus group were detected from inoculated Chaetoceros tenuissimus and C. setoensis cells. A previously unknown ssDNA virus type was detected in inoculated C. tenuissimus cells, but not in C. setoensis cells. Despite the possible protocol biases, for example non-specific adsorptions of virions onto the host cells, the present method helps to estimate the viruses infectious to a single host species. Further improvements to this protocol targeting the proliferated viral genomes might reveal unexpected diatom–virus ecological relationships

Isolation and characterization of a single-stranded DNA virus infecting the marine diatom Chaetoceros sp. strain SS628-11 isolated from western Japan.

Diatoms are significant organisms for primary production in the earth's aquatic environment. Hence, their dynamics are an important focus area in current studies. Viruses are a great concern as potential factors of diatom mortality, along with other physical, chemical, and biological factors. We isolated and characterized a new diatom virus (Csp07DNAV) that lyses the marine planktonic diatom Chaetoceros sp. strain SS628-11. This paper examines the physiological, morphological, and genomic characteristics of Csp07DNAV. The virus was isolated from a surface water sample that was collected at Hiroshima Bay, Japan. It was icosahedral, had a diameter of 34 nm, and accumulated in the nuclei of host cells. Rod-shaped virus particles also coexisted in the host nuclei. The latent period and burst size were estimated to be <12 h and 29 infectious units per host cell, respectively. Csp07DNAV had a closed circular single-stranded DNA genome (5,552 nucleotides), which included a double-stranded region and 3 open reading frames. The monophyly of Csp07DNAV and other Bacilladnavirus group single-stranded DNA viruses was supported by phylogenetic analysis that was based on the amino acid sequence of each virus protein. On the basis of these results, we considered Csp07DNAV to be a new member of the genus Bacilladnavirus

Visualization of a Dinoflagellate-Infecting Virus HcDNAV and Its Infection Process

HcDNAV (a type species of Genus Dinodnavirus) is a large double-stranded DNA virus, which lytically infects the bloom-forming marine microalga Heterocapsa circularisquama Horiguchi (Dinophyceae). In the present study, detailed observation of the HcDNAV particle and its infection process was conducted via field emission scanning electron microscopy (FE-SEM) and epifluorescence microscopy (EFM). Each five-fold vertex of the icosahedral virion was decorated with a protrusion, which may be related to the entry process of HcDNAV into the host. The transverse groove of host cells is proposed to be the main virus entry site. A visible DAPI-stained region, which is considered to be the viroplasm (virus factory), appeared in close proximity to the host nucleus at 11 h post infection (hpi); the putative viral DAPI signal was remarkably enlarged at 11&ndash;30 hpi. It was kidney-shaped at 13&ndash;15 hpi, horseshoe-shaped at 20 hpi, doughnut-shaped at 30 hpi, and changed into a three-dimensionally complicated shape at 51&ndash;53 hpi, by which time most parts of the host cell were occupied by the putative viral DAPI signal. While the virions were within the viroplasm, they were easily distinguishable by their vertex protrusions by FE-SEM

Nucleic acids analysis of Csp07DNAVgenome.

<p>(A) Csp07DNAV genome. Extracts of DNA (lane 1) and RNA (lane 2). (B) Nucleic acids of Csp07DNAV without treatment (lane 1), 100°C for 5 min (lane 2), treated with DNase I (lane 3), RNase A (lane 4), and S1 nuclease (lane 5). The samples were electrophoresed on a formaldehyde-agarose gel.</p

Transmission electron micrographs of ultrathin sections of <i>Chaetoceros</i> sp. strain SS628-11 and negatively stained Csp07DNAV particles.

<p>(A, B) A healthy cell. (A) A whole cell. (B) Higher magnification of a nucleus. (C–F) Cells that were infected with Csp07DNAV at 2 dpi. (C) A whole cell. (D) Higher magnification of a nucleus with virus-like particles (VLPs). The arrow denotes the accumulation of VLPs. (E) Crystalline array aggregation of VLPs in an infected host nucleus. (F) Random aggregation of VLPs with rod-shaped particles in an infected host nucleus. Arrows: rod-shaped particles. (G) Negatively stained Csp07DNAV particles in culture lysate. Arrow heads: virus particles. (H) Negatively stained rod-shaped particles in culture lysate. N, nucleus; C, chloroplast; M, mitochondrion; and VP, virus particle.</p