Viruses in the Oceanic Basement

Abstract

Microbial life has been detected well into the igneous crust of the seafloor (i.e., the oceanic basement), but there have been no reports confirming the presence of viruses in this habitat. To detect and characterize an ocean basement virome, geothermally heated fluid samples (ca. 60 to 65°C) were collected from 117 to 292 m deep into the ocean basement using seafloor observatories installed in two boreholes (Integrated Ocean Drilling Program [IODP] U1362A and U1362B) drilled in the eastern sediment-covered flank of the Juan de Fuca Ridge. Concentrations of virus-like particles in the fluid samples were on the order of 0.2 × 10⁵ to 2 × 10⁵ ml^-1 (n = 8), higher than prokaryote-like cells in the same samples by a factor of 9 on average (range, 1.5 to 27). Electron microscopy revealed diverse viral morphotypes similar to those of viruses known to infect bacteria and thermophilic archaea. An analysis of virus-like sequences in basement microbial metagenomes suggests that those from archaeon-infecting viruses were the most common (63 to 80%). Complete genomes of a putative archaeon-infecting virus and a prophage within an archaeal scaffold were identified among the assembled sequences, and sequence analysis suggests that they represent lineages divergent from known thermophilic viruses. Of the clustered regularly interspaced short palindromic repeat (CRISPR)-containing scaffolds in the metagenomes for which a taxonomy could be inferred (163 out of 737), 51 to 55% appeared to be archaeal and 45 to 49% appeared to be bacterial. These results imply that the warmed, highly altered fluids in deeply buried ocean basement harbor a distinct assemblage of novel viruses, including many that infect archaea, and that these viruses are active participants in the ecology of the basement microbiome.IMPORTANCE The hydrothermally active ocean basement is voluminous and likely provided conditions critical to the origins of life, but the microbiology of this vast habitat is not well understood. Viruses in particular, although integral to the origins, evolution, and ecology of all life on earth, have never been documented in basement fluids. This report provides the first estimate of free virus particles (virions) within fluids circulating through the extrusive basalt of the seafloor and describes the morphological and genetic signatures of basement viruses. These data push the known geographical limits of the virosphere deep into the ocean basement and point to a wealth of novel viral diversity, exploration of which could shed light on the early evolution of viruses.

FIG 1

Location and schematic of the Juan de Fuca Ridge CORKs U1362A and U1362B. (A) Map illustrating the location (yellow dot) and relative orientation (expanded circle) of IODP boreholes U1362A and U1362B on the eastern flank of the Juan de Fuca Ridge. (B) Schematic of CORKs U1362A and U1362B illustrating the depths of the holes and the sampling intakes relative to the sediment and basement horizons. Titanium screen sampling inlet (green circle) is connected by PTFE line to a valved sampling port (green square) on the seafloor wellhead. Packers (black rectangles) just above each inlet isolate the lowest depth horizon in each hole. Depth (in meters below the seafloor [mbsf]) is shown on the y axis.

FIG 2

Concentrations of putative viruses (n = 8) and prokaryotic cells (n = 7) in basaltic basement fluids as determined by staining characteristics using epifluorescence microscopy. For each category, box-and-whisker plots are shown to the left and depict the mean (black square) and the 10th, 25th, 50th, 75th, and 90th percentiles of counts. The individual counts from 2013 (blue symbols) or 2014 (orange symbols) for CORK U1362A (squares) or U1362B (circles) are shown to the right of the box-and-whisker plots. In one instance (indicated by a small black arrow), only a filtered sample (<0.2 µm) was available, so only viruses were counted.

FIG 3

Morphologies of viruses observed in basalt-hosted crustal fluid. (A to H) Electron micrographs of particles harvested from borehole U1362A revealed tailed viruses similar to members of the order Caudovirales (A and B), untailed viruses (C), lemon-shaped viruses resembling members of the family Bicaudaviridae or Fuselloviridae (D), rod-shaped viruses resembling members of the family Rudiviridae (E), and other particles resembling filamentous (F) or spindle-shaped (G) viruses isolated from Archaea. Bilobate structures in the viral size range (H) were repeatedly observed, but they were unlike any classified viruses. These might represent a novel virus or may be membrane vesicles. Bars, 100 nm.

FIG 4

Taxonomic affiliations of the inferred hosts for the virus-like scaffolds identified by VirSorter (top) and of the CRISPR-containing scaffolds (bottom) in the assembled metagenomes (>0.2-µm fraction) prepared from basement fluid samples retrieved from U1362A or U1362B. MBG-E, marine benthic group E.

FIG 5

Consensus taxonomic affiliation of the best BLASTP hits for the genes on the virus-like scaffolds identified by VirSorter in metagenomes from U1362A or U1362B using only viral sequences from RefSeq as the target database and clustered by the most likely domain for the putative host of the virus. The data are presented as the fraction of scaffolds classified in each category after normalizing for read depth, but the absolute number of scaffolds (n) is also shown for each host category. “Unclassified (Unclass.)” indicates significant similarity (E value of 10⁻⁵) to specific viruses that are not yet classified in the NCBI taxonomy database (as of 2 September 2016). “Undetermined (Undeterm.)” indicates that there was no consensus in the taxonomy among the significant BLAST hits beyond the level of order. “Unknown” classifications indicate no significant hits to the viral RefSeq database. An exception to the NCBI-based taxonomy is the inclusion of Megavirales, which is currently a proposed, but not yet official, order (69).

FIG 6

Maximum-likelihood phylogeny of the DNA polymerase II gene (polB) from viral genome JdFR1000234 (in blue boldface font) and homologous viral and archaeal amino acid sequences. Values at the nodes indicate percent bootstrap support, and the bar indicates 0.05 substitutions per site. Bootstrap values at some internal nodes were omitted for clarity. Colored font indicates sequences that were obtained from the U1362A (red) or the U1362B (green) metagenomes. A cluster of the sequences derived from these metagenomes branch within archaeal polB genes and are likely derived from archaea. The DNA polymerase B gene from JdFR1000234 forms a monophyletic lineage with another sequence from borehole U1362A that is different from all other known polB sequences. Identification numbers for the sequences are listed in Table S3.