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<strong>Abstract</strong><br />Ocean viruses are abundant, ubiquitous, and play important roles in global biogeochemical cycles through mortality, horizontal gene transfer and manipulation of host metabolism. However, the ability to link viruses to their hosts in a high-throughput manner bottlenecks our ability to understand virus-host interactions in complex communities. Here, we present viral tagging (VT), a method that combines mixtures of isotope labeled host cells and fluorescent viruses with flow cytometry. In a single experiment, we can screen 10<sup>7</sup> uncultivated ocean viruses with a single strain of Synechococcus. These viruses can then be sequenced to quantitatively link objectively<br />defined environmental viral populations, and their genomes, to their hosts.

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Li Deng<sup>1&dagger;</sup>, Bonnie T. Poulos<sup>1</sup>, Matthew B. Sullivan<sup>1,2,*</sup><br /><br />Departments of <sup>1</sup>Ecology and Evolutionary Biology, and <sup>2</sup>Molecular and Cellular<br />Biology, University of Arizona, Tucson, Arizona, 85719, USA<br /><br />* Corresponding Author, Email: mbsulli@email.arizona.edu<br /><sup>&dagger;</sup> Current address: Helmholtz Zentrum M&uuml;nchen-German Research Center for Environmental Health, Institute of Groundwater Ecology, Ingolstaedter Landstrasse 1, Neuherberg 85764, Germany.<br /><br />Protocol Exchange Laboratory Group: Sullivan Lab<br />Subject Terms: Microbiology<br />Keywords: cyanophage, uncultivated, flow cytometry, viral tagging<br /><br /><strong>Introduction</strong><br />&nbsp; &nbsp;As ocean ecology attempts to advance from observations to predictions, modeling becomes of fundamental importance. While two decades of viral ecology research has focused on community level understanding through measuring viral abundance, production, decay, and frequency of infected cells (<em>reviewed by Ref</em><sup>1</sup>), little is known&nbsp;about the fundamental question of &ldquo;who infects whom?&rdquo;.<br />&nbsp; &nbsp;Cultivation-based methods can directly link a virus strain to its host; however, it is&nbsp;impossible to characterize millions of viruses per milliliter of seawater and thousands to&nbsp;100s of thousands of &ldquo;viral types&rdquo; per sampling site<sup>2</sup> using traditional low-throughput cultivation methods. For example, Fluorescently Labeled Viruses (FLVs) have been used&nbsp;as probes to &ldquo;tag&rdquo; their host cells for examination under the microscope<sup>3-5</sup>, a method that&nbsp;is limited only by being low-throughput. Here, as illustrated in Fig 1, we expand upon the use of FLVs to tag host cells by (i) incorporating flow cytometry to enable high- throughput detection and sequencing of infected host cells, and (ii) isotope labeling host cells to minimize the bacterial DNA in the viral tagging metagenome.<a href="https://s3.amazonaws.com/pr-journal/src2xe.jpg" target="_blank"><br /></a><br /><a href="https://s3.amazonaws.com/pr-journal/stc2xe.jpg" target="_blank"><img src="https://s3.amazonaws.com/pr-journal/suc2xe.jpg" alt="" /></a><br /><strong>Figure 1. Overview for viral tagging (VT).</strong> <br />A. Environmental viruses are fluorescently-labeled green, then mixed with potential hostbacteria which are labeled with heavy isotope, but flow cytometrically green-negative. <br />B. Flow cytometry data triggered on forward scatter for the fluorescently labeled virus and host bacteria co-incubated at desire VBR. Tagged cells infecting by viruses with extra fluorescence can be sorted out from non-infected cells. <br />C. DNA is extracted from the sorted, viral-tagged population. Isotopically light viral DNA is then separated from heavy host DNA using a density gradient for the downstream amplification and sequencing&nbsp;<br /><br /><strong>Reagents</strong><br />1. <sup>15</sup>N ammonium chloride (Cambridge Isotope Laboratories, Inc. NLM-467-1).<br />2. Quant-iT Pico Green (Invitrogen P7589).<br />3. SYBR Gold (Invitrogen S11494)<br />4. TE buffer (10mM Tris, 1mM EDTA)<br />5. MTN buffer (0.6M NaCl, 0.1M Tris-Cl pH 7.5, 0.1M MgCl2)<br />6. Bovine serum albumin (BSA)<br />7. Phosphate buffered saline (PBS)<br />8. Fluorescent polystyrene FLOW CheckTM microspheres (1&micro;m yellow-green beads; Polysciences Inc., PA, cat# 23517-10)<br /><br /><strong>Equipment</strong><br />1. Ultracentrifuge, we used a Beckman L70 ultracentrifuge with Beckman VTi 65 vertical rotor and13x48 mm OptiSeal polyallomer tubes (4.9 ml capacity).<br />2. Appliskan plate reader (Thermo Electron, Vantaa, Finland).<br />3. Sonication bath, we used VWR Signature Ultrasonic cleaner B1500A-DTH.<br />4. Refrigerated centrifuge, we used Eppendorf 5417R.<br />5. Flow cytometer, we used iCyt Reflection flow cytometer (Sony Biotechnology) and a&nbsp;MoFlo&trade; XDP cytometer (Beckman Coulter).<br />6. Nanosep 10K centrifugal tubes (Pall OD010C33)&nbsp;<br /><br /><strong>Troubleshooting</strong><br />1. Increase in 520 nm fluorescence of the mixture of cells and the SYBR-Blank. The washing procedure for the viruses is probably not adequate.<br />2. No increase in 520 nm fluorescence of the mixture of cells and the strained and&nbsp;washed viruses. The washing procedure for the viruses might have failed and lost most of the viruses, count the number of viruses recovered from washing. Otherwise increase the VBR to add more viruses per cell.<br /><br /><strong>Time Taken</strong><br />The time required for isotope labeling of host cells is variable, for 3 inoculation of&nbsp;<em>Synechococcus</em> WH7803 are about 3-4 weeks. The viral wash steps usually take about&nbsp;3hs. The sorting takes about 1hour for E+08 cells, depending on the number collected and&nbsp;the experience of the investigator. Thus the whole procedure can be accomplished within&nbsp;4-5 weeks.<br /><br /><strong>Anticipated Results</strong><br />Following the viral tagging method and DNA isolation and extraction, resulting DNA can be sequenced and used for viral community analysis (see Figure 2).<br /><br /><a href="https://s3.amazonaws.com/pr-journal/sxc2xe.jpg" target="_blank"><img src="https://s3.amazonaws.com/pr-journal/syc2xe.jpg" alt="" /></a><br /><strong>Figure 2. Reference flow cytometry charts.</strong> <br />A-C. Reflection data of viral-tagged samples. <br />A. Bacteria alone: 15N-Syn WH7803 <br />B. No viruses staining control: 15N-Syn WH7803 + stained and washed buffer withoutadding viruses. A green negative and FSC negative noise population appeared in bottom left corner of plot, negative to bacterial primers. <br />C. Viral-tagging sample 1: 15N-Syn WH7803 + stained and washed viruses&nbsp;<br />D-F. MoFlo data of viral-tagged samples. <br />D. Bacteria alone: Syn WH7803 <br />E. No viruses staining control: Syn WH7803 + stained and washed buffer without adding viruses. <br />F. Viral-tagging sample 1: Syn WH7803 + stained and washed viruses <br /><br /><strong>References</strong><br />1. Breitbart, M. in <em>Annual Review of Marine Science, Vol</em> 4 Vol. 4 <em>Annual Review of&nbsp;Marine Science</em> (eds C. A. Carlson &amp; S. J. Giovannoni) 425-448 (2012).<br />2. Angly, F. et al. The marine viromes of four oceanic regions.<em> PLoS Biol</em> <strong>4</strong>, e368&nbsp;(2006).<br />3. Mosier-Boss, P. A. et al. Use of fluorescently labeled phage in the detection and&nbsp;identification of bacterial species. <em>Appl Spectrosc</em> <strong>57</strong>, 1138-1144 (2003).<br />4. Comeau, A. M. &amp; Noble, R. T. Preparation and application of fluorescently&nbsp;labeled virus particles. <em>S.W. Wilhelm, M.G. Weinbauer, and C.A. Suttle [eds.]. Manual of Aquatic Viral Ecology. Waco, TX:American Society of Limnology and Oceanography</em>. , 19-29 (2010).&nbsp;<br />5. Hennes, K. P., Suttle, S. A. &amp; Chan, A. M. Fluorescently labeled virus probes show that natural virus populations can control the structure of marine microbial communities. <em>Appl. Environ. Microbiol.</em> <strong>61</strong>, 3623-3627 (1995).<br />6. Waterbury, J. B., Watson, S. W., Valois, F. W. &amp; Franks, D. G. Biological and&nbsp;ecological characterization of the marine unicellular cyanobacterium&nbsp;<em>Synechococcus</em>. <em>Can. Bull. Fish. Aquat. Sci.</em> <strong>214</strong>, 71-120 (1986).<br />7. 'Cesium Chloride Virus Purification and Dialysis.' <em>Sullivan Lab Protocols</em>. http://www.eebweb.arizona.edu/faculty/mbsulli/protocols/<br />8. 'DNA Preparation and Linker Amplification.' <em>Sullivan Lab Protocols</em>. http://www.eebweb.arizona.edu/faculty/mbsulli/protocols/<br /><br /><strong>Acknowledgments</strong><br />Funding provided by DOE JGI Community Sequencing Program, Biosphere 2, BIO5,&nbsp;NSF OCE0940390, and Gordon and Betty Moore Foundation grants to MBS. We thank J.&nbsp;Fuhrman for suggesting stable-isotope-labeled host DNA; S.W. Chisholm for strains; Tucson Marine Phage Lab especially Christine Schirmer for technical/editorial support. We acknowledge iCyt and AZCC/ARL-Division of Biotechnology Cytometry Core Facility for cytometry support.<br /><br /><strong>Associated Publications</strong><br />Deng, L., Ann Gregory, Suzan Yilmaz, Bonnie T. Poulos, Philip Hugenholtz and Matthew B. Sullivan. Contrasting Life Strategies of Viruses that Infect Photo- and Heterotrophic Bacteria, as Revealed by Viral Tagging. <em>mBio</em> <strong>3</strong>, 6 e00373-12 (2012).<br />Deng, L., Ignacio-Espinoza, J.C., Gregory, A., Poulos, B.T., Weitz, J.S., Hugenholtz, P.,&nbsp;&amp; Sullivan, M.B. Viral tagging reveals discrete populations in&nbsp;<em>Synechococcus</em> viral&nbsp;genome sequence space. <em>in press</em>.<br /><br />&nbsp;<br /><br /><br /><br /><br />

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