TITLE: Viral Tagging and Grow: a scalable approach to capture and characterize infectious virus-host pairs
AUTHORS: Ho Bin Jang, Lauren Chittick, Fen Li ., Courtney M Sanderson

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Viral tagging (‘VT’), as shown in the </span><span style = "font-weight:bold;">Fig. 1A and 1B</span><span>, entails fluorescently labeling the DNA of wild virus particles (upper, </span><span style = "font-weight:bold;">A</span><span>), mixing these with a target, cultivable host bacterium (lower, </span><span style = "font-weight:bold;">A</span><span>), and assessing the population of cells via flow cytometry for a fluorescent shift attributed to ‘viral tagging’ (VT) (</span><span style = "font-weight:bold;">B</span><span>). VT was developed with culturable marine isolates (1, 2) and more recently applied to deduce virus-host pairing with unknown hosts in the human gut (3). However, all virus-host pairs detected (via phage adsorption) do not necessarily represent a productive lytic infection due to reversible and/or non-specific binding of lysogeny (phage genome replicates with the host genome). Additionally, post-adsorption cellular defenses could all prevent successful infection. To capture and characterize infectious, individual virus-host pairs, we developed ‘Viral Tag and Grow (VT+Grow)’ by incorporating a plate-based ‘Grow’ step (</span><span style = "font-weight:bold;">C</span><span>) to VT (</span><span style = "font-weight:bold;">A and B</span><span>).</span></div><br/></div>

[STEPS]
?. [Amicon tube pre-treatment:]
Prepare 0.02µm-filtered 1% bovine serum albumin. * The BSA should be freshly prepared (1-2 days, stored at 4°C) before viral washing. *The purpose of this is to better cushion the viruses during the subsequent wash steps and to aid in recovery
?. [Amicon tube pre-treatment:]
Wet Amicon tubes by adding 500μl MSM buffer; let stand for 10 min, then spin until almost dry (5,000 x g, 10 min) and discard the flow-through.
?. [Amicon tube pre-treatment:]
Add 500μl of 0.02μm filtered 1% BSA, and let stand for 1 hr at room temperature. Proceed to viral staining during this period
?. [Viral staining and washing]
Thaw prepared 10,000X SYBR Gold in the dark.*We recommend making 1uL aliquots of stock SYBR Gold to avoid freeze-thawing of the reagent.
?. [Viral staining and washing]
In a clean 1.5mL tube, stain the viruses by adding SYBR Gold to a final concentration of 10X. Perform in low light as SYBR Gold will degrade upon exposure to light. * Add 1μL stock SYBR Gold per 1mL of phage; if using a more concentrated phage stock, use 1uL of 1:10 diluted SYBR Gold to 100uL of concentrated phage * We do not recommend adding more than 1010 viruses to the Amicon unit as this will greatly decrease the efficiency of excess SYBR Gold removal, which will result in more false-positive shifts during cell sorting * If using a low titer of viruses, we recommend concentrating them to 108-109 prior to staining to reduce the amount of SYBR Gold required. However, concentrating can be done concurrently with the initial wash step by repeating Step 11 until the entire volume of stained phages has flowed through the membrane.
?. [Viral staining and washing]
Prepare a blank without virus to evaluate the efficiency of excessive SYBR Gold removal in later steps. This will be used as a negative control (SYBR-Blank, SYBR-Gold without viruses) for flow cytometric detection of bacterial cells. See step #6 in the Viral tagging and grow part below
?. [Viral staining and washing]
Vortex the sample briefly to mix the dye and viruses.
?. [Viral staining and washing]
Incubate for 20 min in the dark at room temperature. * Practically speaking, viruses can be stained anywhere between 20-60 minutes; 20 minutes is a minimum
?. [Viral staining and washing]
Spin down the BSA-treated Amicon tube (5,000 x g, 10-20 minutes) until all BSA has flowed through; discard the flow-through.
?. [Viral staining and washing]
Rinse excess BSA from the Amicon filter by centrifuging 500uL filtered MSM buffer through the membrane (5,000 x g, 10 minutes)
?. [Viral staining and washing]
Add the stained virus preparations and the SYBR-Blank in the pre-treated Amicon devices and centrifuge 3,000 x g for 30 min, 18℃. * Following each centrifugation step, the volume remaining in the Amicon device should be reduced to 50-100uL for adequate washing. Depending on the phage concentration, you may need to adjust the length of centrifugation (may range between 20-60 minutes)
?. [Viral staining and washing]
Wash the reduced volume of stained virus or SYBR-Blank by adding 1mL MSM buffer to the Amicon filter and gently pipetting up and down against the membrane. Add an additional 1mL of MSM and centrifuge as in step #11. * If using a 0.5mL capacity Amicon filter, add 500μL MSM and wash the membrane by very gently pipetting up and down; proceed to centrifugation step
?. [Viral staining and washing]
Repeat #9 3 times so that a total of 8mL of MSM has flowed through the membrane (be sure volume falls
?. [Viral staining and washing]
Following the final wash step, remove the Amicon filter from the collection tube, and vortex each side of the membrane for 5 seconds. Collect all liquid from the bottom of the membrane into a clean tube.
?. [Viral staining and washing]
Add 100μL MSM to the filter and gently pipet up and down against the membrane to dislodge additional viruses. Without the collection tube, vortex the membrane as in Step 11, and collect all liquid. * Depending on the starting concentration of viruses, the resuspension volume can be adjusted to maximize recovery. We found that recovering ~109 stained viruses with three final recovery washes of 300μL, for an estimated final volume of 1mL averaged the best recovery (70-80%) and simplified subsequent VBR calculations
?. [Viral staining and washing]
Repeat Step 12 twice.
?. [Viral tagging and grow]
Quantify your recovered SYBR-stained viruses to determine the amount to add. The multiplicity of infection (MOI, see below) will depend on the virus-host systems used (e.g., MOIs of 0.5 to 10). Cells are most susceptible during mid-log phase, so timing of your culture is crucial. Transfer 200uL of your overnight culture in 19.8mL of media prior to FCM setup with enough time to allow your culture to reach mid-log phase. We recommend completing an OD-CFU correlation of your target bacterium prior to this experiment.* For environmental virus system where the phage’s infection efficiency is unknown, measure the virus-to-bacterium ratio (VBR, see the reference #2)
?. [Viral tagging and grow]
Before viral tagging, flow cytometric (FCM) conditions and parameters should be adequately optimized as below. For more details, see the sections below: ‘BD influx Cell sorter Start Up and Shut Down for Viral Tagging and Grow’ and ‘Flow cytometer optimization for cell detection and sorting’.* Stream/laser alignment, drop-charge delay, and sorting calibration are optimized using Ultra Rainbow particles, Accudrop Beads, and 8-Peak Beads, respectively (see the reagents). * Finely align the 96-well plate tray for single-cell sorting. * Set up the flow cytometric gating and conditions (e.g., forward and side scatters and SYBR-Gold, respectively) to avoid the overlap of the cell signals with background noises. * Format your 96-well plate to allow the appropriate number of wells for VT and NT populations, as well as sufficient control wells (media blank, and non-infection (-) and positive-infection (+) controls) (Fig. 2)
?. [Viral tagging and grow]
Infect ~105-106 cells with stained viruses depending on your desired MOI and the estimated titer from Step 1 of this section *The appropriate incubation time will vary between systems, and should be determined by latent periods of known infectious phages * Ideally, the infection volume should be kept * Particularly for single-cell assay for burst size (4) using VT+G, the one-step growth curve for target bacteria using specific phages helps to decide the proper time for incubation, sorting, and plating (see Kirzner et al., (4)), respectively.
?. [Viral tagging and grow]
Immediately after incubation of viruses with hosts, add MSM buffer to bring the infection volume up to 1 ml. To remove free viruses, centrifuge the infection (12,000 ×g for 1 min at room temperature) and resuspend the pellet in 1mL MSM buffer.
?. [Viral tagging and grow]
Repeat Step 20 twice for a total of three centrifugations
?. [Viral tagging and grow]
Resuspend the infected cells to final concentrations ranging from ~5 x 105 ml-1 to 106 ml-1. This process will decrease the coincident sorting of multiple cells per droplet and achieve optimal spacing between sorted cells.
?. [Viral tagging and grow]
Before sorting, add 100 µl of Zobell media to each well of a 96-well plate to prevent desiccation of sorted droplets during processing. *We recommend sorting NT populations in parallel with VT populations to assess the false-positive cell lysis of VT-containing wells by the free virus(es).
?. [Viral tagging and grow]
Following FCM sorting of cells into the 96-well plate, monitor bacterial growth by measuring OD600 nm at 15-min intervals for 24 h. Remove any contaminated blanks, and determine the lysis curves of your wells by plotting OD against time. *Details about FCM gating strategy and single-cell sorting are described in ‘Flow cytometer optimization for cell detection and sorting’ below.
?. [Viral tagging and grow]
Compare the lysis patterns of the wells containing sorted VT and NT cells to those of positive and negative controls (Fig. 3).
?. [Flow cytometer optimization for cell detection and sorting]
The instrument we used is a BD influx cell sorter (Becton Dickson) equipped with two high-power lasers at 488 nm (blue) and 642 nm (red) and nine optical detectors, respectively. Each detector uses a high-performance photomultiplier (PMT) to amplify the low-intensity signals of the nano-sized particles (e.g., viruses, (5)). This influx sorter has the capability of 2- to 6-way population sorting into tubes and single-cell sorting directly into plates or slides. The fluidic system was only run using sterile solutions as a sheath (e.g., 0.02-µm-filtered and autoclaved MSM, see Reagents) and is always cleaned and air dried before shutdown. Additional details are described in the ‘BD influx Cell sorter Start Up and Shut Down for Viral Tagging and Grow’
?. [Flow cytometer optimization for cell detection and sorting]
Details about system startup, alignment, optimization, shutdown, and maintenance are described in the ‘BD influx Cell sorter Start Up and Shut Down for Viral Tagging and Grow’
?. [Flow cytometer optimization for cell detection and sorting]
Detect the cells using hierarchical gating. For example, as shown in Fig. 4A, a parent gate is drawn on Pseudoalteromonas sp. H71 cells in the forward (FSC) vs. side scatter (SSC), from which viral tagged (VTs, green positive) and nontagged (NTs, green-negative) cells are further gated as a sub-fraction in the green fluorescence (SYBR-Gold) vs. SSC. Events were detected using a forward scatter trigger, and data were obtained in logarithmic mode then analyzed with BD FACS software version 1.2.0.142 (Becton Dickinson, San Jose, CA).
?. [Flow cytometer optimization for cell detection and sorting]
Adjust trigger and the PMT voltages for relevant parameters (FSC and SSC) to avoid the overlap of the cell signals with background noises from the instrument (electronic noise), micro-particles in the buffer, and/or cellular debris (5) (Fig. 4B and 4C).
?. [Flow cytometer optimization for cell detection and sorting]
Sort single VTs or NTs into each well of the 96-well plate using the single sort mode of 1.0 drop purity. * To prevent the coincidence of free viruses sorted with VT or NT cells, which would be too close to be separated, adjust the event rate for sorted particles from the VT and NT cell populations to ~40 events per second. The estimated ratio of sorted particles to generated drops (piezoelectric at 43.2 kHz, above) of ~1/1,180, ensuing a separation enough between sorted particles (6).  * To maximize the sorting purity of the false-positive population, we use the biased gating strategy (7).
?. [Flow cytometer optimization for cell detection and sorting]
After sorting, monitor bacterial growth as previously described (see #8 and Fig. 3 in ‘Viral tag and grow’).
?. [Flow cytometer optimization for cell detection and sorting]
4. Before sorting, finely align the 96-well plate by visually inspecting the deposition of droplets into the center of each well.