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.
SummaryEarly work on marine algal viruses focused exclusively on those having DNA genomes, but recent studies suggest that RNA viruses, especially those with positive-sense, single-stranded RNA (1ssRNA) genomes, are abundant in tropical and temperate coastal seawater. To test whether this was also true of polar waters, we estimated the relative abundances of RNA and DNA viruses using a mass ratio approach and conducted shotgun metagenomics on purified viral samples collected from a coastal site near Palmer Station, Antarctica on six occasions throughout a summer phytoplankton bloom (NovemberMarch). Our data suggest that RNA viruses contributed up to 65% of the total virioplankton (8-65%), and that, as observed previously in warmer waters, the majority of RNA viruses in these Antarctic RNA virus metagenomes had 1ssRNA genomes most closely related to viruses in the order Picornavirales. Assembly of the metagenomic reads resulted in five novel, nearly complete genomes, three of which had features similar to diatom-infecting viruses. Our data are consistent with the hypothesis that RNA viruses influence diatom bloom dynamics in Antarctic waters.
Reverse transcription, quantitative PCR (RT-qPCR) is a sensitive method for quantification of specific RNA targets, but the first step of the assay, reverse transcription, is notoriously variable and sensitive to reaction conditions. In this study, we used purified Bacteriophage MS2 genomic RNA as a model virus target to test two different RT enzymes (SuperScript II and SuperScript III), two RT-priming strategies (gene-specific primers and random hexamers), and varying background RNA concentrations (0-50ngμl) to determine how these variables influence the efficiency of reverse transcription over a range of target concentrations (10-10 copies μl). The efficiency of the RT reaction was greatly improved by increasing both background RNA and primer concentrations, but the benefit provided by background RNA was source dependent. At a given target concentration, similar RT efficiencies were achieved with gene-specific primers and random hexamers, but the latter required much higher concentrations. With random hexamers, we observed a systematic variation in RT reaction efficiency as a function of target concentration. Using an RNA standard curve that was also subject to RT effectively normalized for this systematic variability, but the assay accuracy depended critically on the length of the standard RNA extending to the 3' end of the qPCR target site. Our results shed some light on previous contradictory conclusions in the literature, and provide insights that may aid in the design of RT-qPCR assays and the design of synthetic RNA standards when full-length material is not available.
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