Abstract

Viruses are major pathogens in all biological systems. Virus propagation and downstream analysis remains a challenge, particularly in the ocean where the majority of their microbial hosts remain recalcitrant to current culturing techniques. We used a cultivation-independent approach to isolate and sequence individual viruses. The protocol uses high-speed fluorescence-activated virus sorting flow cytometry, multiple displacement amplification (MDA), and downstream genomic sequencing. We focused on ‘giant viruses’ that are readily distinguishable by flow cytometry. From a single-milliliter sample of seawater collected from off the dock at Boothbay Harbor, ME, USA, we sorted almost 700 single virus particles, and subsequently focused on a detailed genome analysis of 12. A wide diversity of viruses was identified that included Iridoviridae, extended Mimiviridae and even a taxonomically novel (unresolved) giant virus. We discovered a viral metacaspase homolog in one of our sorted virus particles and discussed its implications in rewiring host metabolism to enhance infection. In addition, we demonstrated that viral metacaspases are widespread in the ocean. We also discovered a virus that contains both a reverse transcriptase and a transposase; although highly speculative, we suggest such a genetic complement would potentially allow this virus to exploit a latency propagation mechanism. Application of single virus genomics provides a powerful opportunity to circumvent cultivation of viruses, moving directly to genomic investigation of naturally occurring viruses, with the assurance that the sequence data is virus-specific, non-chimeric and contains no cellular contamination.

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Acknowledgements

We acknowledge the support from NSF grants EF-0949162 (to WHW, RS, and SWW) and OCE-0821374 (to RS and WHW). Support for this project was also received from the Kenneth & Blaire Mossman endowment to the University of Tennessee. SK was supported by the Intramural Research Program of the National Human Genome Institute, National Institutes of Health. JMM was supported by the Gordon and Betty Moore Foundation through Grant GBMF5334.

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Affiliations

  1. Bigelow Laboratory for Ocean Sciences, Boothbay, ME, USA

    • William H Wilson
    • , Ilana C Gilg
    • , Erin K Field
    • , Joaquín Martínez Martínez
    • , Nicole J Poulton
    • , Brandon K Swan
    •  & Ramunas Stepanauskas
  2. School of Marine Science and Engineering, Plymouth University, Plymouth, UK

    • William H Wilson
  3. Department of Microbiology, The University of Tennessee, Knoxville, TN, USA

    • Mohammad Moniruzzaman
    • , Gary R LeCleir
    •  & Steven W Wilhelm
  4. Department of Biology, Howell Science Complex, East Carolina University, Greenville, NC, USA

    • Erin K Field
  5. Genome Informatics Section, Computational and Statistical Genomics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, USA

    • Sergey Koren
  6. National Biodefense Analysis and Countermeasures Center, Frederick, MD, USA

    • Brandon K Swan

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Correspondence to William H Wilson.

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https://doi.org/10.1038/ismej.2017.61

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