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. 2023 Sep 19;11(5):e0025523.
doi: 10.1128/spectrum.00255-23. Online ahead of print.

Highly divergent CRESS DNA and picorna-like viruses associated with bleached thalli of the green seaweed Ulva

Affiliations

Highly divergent CRESS DNA and picorna-like viruses associated with bleached thalli of the green seaweed Ulva

Luna M van der Loos et al. Microbiol Spectr. .

Abstract

Marine macroalgae (seaweeds) are important primary producers and foundation species in coastal ecosystems around the world. Seaweeds currently contribute to an estimated 51% of the global mariculture production, with a long-term growth rate of 6% per year, and an estimated market value of more than US$11.3 billion. Viral infections could have a substantial impact on the ecology and aquaculture of seaweeds, but surprisingly little is known about virus diversity in macroalgal hosts. Using metagenomic sequencing, we characterized viral communities associated with healthy and bleached specimens of the commercially important green seaweed Ulva. We identified 20 putative new and divergent viruses, of which the majority belonged to the Circular Rep-Encoding Single-Stranded (CRESS) DNA viruses [single-stranded (ss)DNA genomes], Durnavirales [double-stranded (ds)RNA], and Picornavirales (ssRNA). Other newly identified RNA viruses were related to the Ghabrivirales, the Mitoviridae, and the Tombusviridae. Bleached Ulva samples contained particularly high viral read numbers. While reads matching assembled CRESS DNA viruses and picorna-like viruses were nearly absent from the healthy Ulva samples (confirmed by qPCR), they were very abundant in the bleached specimens. Therefore, bleaching in Ulva could be caused by one or a combination of the identified viruses but may also be the result of another causative agent or abiotic stress, with the viruses simply proliferating in already unhealthy seaweed tissue. This study highlights how little we know about the diversity and ecology of seaweed viruses, especially in relation to the health and diseases of the algal host, and emphasizes the need to better characterize the algal virosphere. IMPORTANCE Green seaweeds of the genus Ulva are considered a model system to study microbial interactions with the algal host. Remarkably little is known, however, about viral communities associated with green seaweeds, especially in relation to the health of the host. In this study, we characterized the viral communities associated with healthy and bleached Ulva. Our findings revealed the presence of 20 putative novel viruses associated with Ulva, encompassing both DNA and RNA viruses. The majority of these viruses were found to be especially abundant in bleached Ulva specimens. This is the first step toward understanding the role of viruses in the ecology and aquaculture of this green seaweed.

Keywords: DNA viruses; RNA viruses; Ulva; chlorophyta; seaweed.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig 1
Fig 1
Possible roles and relationships between the algal host, viruses, and the other components of the microbiome. Image concept based on Peixoto et al. (14).
Fig 2
Fig 2
Heatmap showing mean Ct (cycle threshold) qPCR values for the 20 putative new viruses associated with the green seaweed Ulva. Low Ct values correspond to high viral load. From each culture or site, 1–2 cm2 tissue from two different individuals was sampled.
Fig 3
Fig 3
Eukaryotic viral read numbers. (A) Total eukaryotic viral reads per sample (n = 8; two replicates per sample type). (B) Total eukaryotic viral reads from bleached Ulva australis viromes that were assigned to the phyla Pisuviricota, Cressdnaviricota, Kitrinoviricota, Lenarviricota, Nucleocytoviricota, and unclassified viruses (sum of two replicates, total eukaryotic viral reads = 474,578). (C) Total eukaryotic viral reads from healthy Ulva australis samples from natural populations (sum of two replicates, total eukaryotic viral reads = 110,460). Identifications are based on BLAST with the NCBI database. Colors represent different orders.
Fig 4
Fig 4
The genome organization of the five Ulva CRESS viruses, including replicase and capsid protein genes. Open reading frames (ORFs) were predicted using ORFfinder (https://www.ncbi.nlm.nih.gov/orffinder/).
Fig 5
Fig 5
Unrooted maximum likelihood phylogenetic tree of Rep proteins from CRESS DNA viruses estimated using IQ-Tree with ultrafast bootstrap replicates (values below 70 are not displayed). Closely related sequence groups are collapsed into triangles, whose side lengths are proportional to the distances between the closest and farthest leaf nodes. The locations of the Ulva-associated CRESS DNA viruses are marked with numbered arrows. The numbers correspond to Ulva CRESS DNA virus 1–5. Branch lengths are scaled according to the number of amino acid substitutions per site.
Fig 6
Fig 6
Details of the phylogeny of CRESS DNA viruses based on the Rep protein. (A) The Smacoviridae and unclassified clade, (B) CRESS5 group, (C) Circoviridae, and (D) Repensiviricetes (Genomoviridae and Geminiviridae). Newly discovered viruses from Ulva spp. are highlighted in bold. Each viral sequence’s putative eukaryotic host (or sample habitat) is displayed in brackets. Maximum likelihood tree estimated using IQ-Tree with ultrafast bootstrap replicates (values below 70 are not displayed). The tree is mid-point rooted. Branch lengths are scaled according to the number of amino acid substitutions per site.
Fig 7
Fig 7
Phylogeny and genome organization of the Ulva durnaviruses. (A) Phylogentic tree of the order Durnavirales based on the RdRp protein. Newly discovered viruses from Ulva spp. are highlighted in bold and indicated with a triangle. Each viral sequence’s putative eukaryotic host (or sample habitat) is displayed in brackets. Maximum likelihood tree estimated using IQ-Tree with ultrafast bootstrap replicates (values below 70 are not displayed). The tree is mid-point rooted. Branch lengths are scaled according to the number of amino acid substitutions per site. (B) Genome organization, including RNA-dependent RNA polymerase (RdRp) genes. Open Reading Frames (ORFs) were predicted using ORFfinder (https://www.ncbi.nlm.nih.gov/orffinder/).
Fig 8
Fig 8
Phylogeny and genome organization of Ulva ghabrivirus 1. (A) Phylogenetic tree of the Ghabrivirales based on the RdRp protein. The newly discovered Ulva ghabrivirus is highlighted in bold and indicated with a triangle. Each viral sequence’s putative eukaryotic host (or sample habitat) is displayed in brackets. Maximum likelihood tree estimated using IQ-Tree with ultrafast bootstrap replicates (values below 70 are not displayed). The tree is mid-point rooted. Branch lengths are scaled according to the number of amino acid substitutions per site. (B) Genome organization, including the RNA-dependent RNA polymerase (RdRp) gene. The genomes of the Ghabrivirales are segmented, and it is therefore likely that the contig of Ulva ghabrivirus 1 only represents a partial genome. Open Reading Frames (ORFs) were predicted using ORFfinder (https://www.ncbi.nlm.nih.gov/orffinder/).
Fig 9
Fig 9
Phylogeny and genome organization of Ulva mitoviruses and mito-like viruses. (A) Phylogenetic tree of the Narnaviridae, Botourmiaviridae, and Mitoviridae based on the RdRp protein. Newly discovered viruses from Ulva spp. are highlighted in bold and indicated with a triangle. Each viral sequence’s putative eukaryotic host (or sample habitat) is displayed in brackets. Maximum likelihood tree estimated using IQ-Tree with ultrafast bootstrap replicates (values <70 are not displayed). The tree is mid-point rooted. Branch lengths are scaled according to the number of amino acid substitutions per site. (B) Genome organization, including RNA-dependent RNA polymerase (RdRp) genes. Open Reading Frames (ORFs) were predicted using ORFfinder (https://www.ncbi.nlm.nih.gov/orffinder/).
Fig 10
Fig 10
Phylogeny and genome organization of Ulva tombus-like virus 1. (A) Phylogenetic tree of the Tolivirales (Tombusviridae and Carmotetraviridae) and the Nodamuvirales based on the RdRp protein. The newly discovered Ulva tombus-like virus is highlighted in bold and indicated with a triangle. Each viral sequence’s putative eukaryotic host (or sample habitat) is displayed in brackets. Maximum likelihood tree estimated using IQ-Tree with ultrafast bootstrap replicates (values <70 are not displayed). The tree is mid-point rooted. Branch lengths are scaled according to the number of amino acid substitutions per site. (B) Genome organization, including RNA-dependent RNA polymerase (RdRp) genes. Open Reading Frames (ORFs) were predicted using ORFfinder (https://www.ncbi.nlm.nih.gov/orffinder/).
Fig 11
Fig 11
Phylogeny of the Picornavirales based on the proteinase/polymerase gene region. Newly discovered viruses from Ulva spp. are highlighted in bold. Each viral sequence’s putative eukaryotic host (or sample habitat) is displayed in brackets. Maximum likelihood tree estimated using IQ-Tree with ultrafast bootstrap replicates (values <70 are not displayed). The tree is mid-point rooted. Branch lengths are scaled according to the number of amino acid substitutions per site.
Fig 12
Fig 12
The genome organization and conserved picornaviral motifs of the six Ulva picorna-like viruses. Arrows indicate conserved motifs (Walker A and B motifs, proteinase active site motif, polymerase motifs, and drug-binding pockets of capsid proteins). Rhinovirus-like (rhv-like) domains, including conserved amino acids of the drug-binding pocket, were also identified. Conserved motifs were identified with Pfam and Phyre2.

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