TITLE: Holobiont Virus Extraction Protocol (Veglia et al.)
AUTHORS: Alex Veglia

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>The decribed holobiont virus extraction protocol contains five steps: i) Rinse and rest, ii) Tissue separation, iii) Physical cell disruption and homogenization, iv) Homogenate cleaning, and v) Virus particle concentration and extraction validation. These protocol stages were informed by previous virology studies applying common techniques for virus particle and nucleic acid extraction from biotic and abiotic materials (Williamson et al. 2003; Davy et al. 2006; Thurber 2009; Weynberg et al. 2014).The use of polyethylene glycol removed the need for ultracentrifugation making the protocol more practical for trips to the field and is designed to remove some level of impurities (BioVision Research Products).Overall, it is expected that most RNA viruses and double and single-stranded DNA viruses including cyanophages will be retained in the pellet. The speed of the final centrifugation step (Stage 4, Fig. 2) can be altered to target larger virus size classes (e.g. the proposed order </span><span style = "font-style:italic;">Megavirales</span><span>). </span></div></div>

[STEPS]
?. [Rinse and rest]
First, invertebrate samples taken at reef or from aquarium must be rinsed with sterile seawater to mitigate potential contamination from ambient seawater.
?. [Rinse and rest]
Before beginning the protocol, all bench areas should be sterilized using either 70% ethanol and/or lysol (or both)1) Remove reef sourced sea water from sampling container     **wipe down outside of containers and area around drain/waste storage container (if on benchtop)                                                                                              ↓2) Refill the sampling container with sterile seawater (0.2 micron (cellulose nitrate membrane) filter and autoclaved)    **wipe down outside of containers and area around drain/waste storage container (if on benchtop)3) Now, vigorously shake and/or swirl the sampling container, to remove/loosen any particulates or external mucus sheets from the invertabrate sample, for ~4) After resting for around  , swirl  sample once more prior to decanting particulate- or mucus-containing seawater into a waste beaker5) Repeat Steps 2-4 two more times 6) After final round of rinsing, carefully move the invertebrate sample to a sterile glass beaker containing sterile seawater OPTIONAL STEP -> Post-rinse, allow invertebrate to sit in sterile beaker with sterile seawater for 5 to 10 minutes to allow fitlering organisms like sponges or tunicates to "flush out" particles sourced from the reef prior to tissue processing
?. [Tissue Separation ]
Depending on the organism, tissue can be seperated/processed in several ways. Generally, with fleshy organisms with or without a skeleton, a sterile razor blade can be used. A scalpel is sufficient as well, especially for softer organisms like sponges or seaslugs. **This step is usually done in triplicate, for example, three tissue aliquots of 0.2-0.3 mL processes per sampled invertebrate to allow for ample volume of virus particles for downstream manipulation (i.e. isolation)
?. [Tissue Separation ]
7) ~0.2-0.3 mL of tissue was removed from organism using a sterile scalpel or razorblade and placed in a sterile 2 mL centrifuge tube       ** for organisms with very thin tissue layers over a calcium carbonate skeleton, whole samples can be processed using a mortar and pestle 8) On top of the tissue, 0.5 mL of a sterilized glass bead mixture (e.g. 0.1 g of 0.5 mm beads, 0.15 g of 0.1 mm beads, 0.4 g of 1.0 mm beads and three 1.3 mm beads) is added to the 2 mL centrifuge tube      ** the glass bead mixture should contain three-four different sizes to target a majority of cellular organisms within invertebrate tissue 9) Fill 2 mL centrifuge tube containing glass beads and invertebrate tissue with sterile seawater to the 2 mL mark10) Seal the tops of 2 mL centrifuge tubes with parafilm prior to homogenization step to mitigate the chance of them opening and spilling
?. [Physical cell disruption and homogenization]
For the homogenization step a vortex or a FastPrep machine can be used. Between cycles of disruption, be sure to put samples on ice.
?. [Physical cell disruption and homogenization]
11) Secure the 2 mL centrifuge tubes containing, glass beads + tissue + sterile seawater, to a vortex machine or a FastPrep™ instrument (Thermo Electron Corporation; FP120) 12) Vortex -> On highest setting (10), run vortex for        OR         FastPrep  -> Run for 13) Let samples sit on ice for 14) Repeat Steps 12-13 three times (example result is pictured above)
?. [Homogenate cleaning]
Instead of physical filters being used, cellular debris or any remaining intact cells were then removed from the supergntant using sequential centrifugation steps. Any bench top centrifuge capable of reaching speeds of 3,200 xg and 16,000 x g is ok to use for this step. Ideally, there would be temperature control, however, extractions have been successful without. 15) Centrifuge samples for  at 16) Transfer  as much supernatant as possilbe to a new, sterile 1.5 mL centrifuge tube and if low volume, dilute to ~1.2 mL mark with sterile seawater17) Centrifuge sample again for  at 18) Transfer supernatant to new, sterile 1.5 mL centrifuge tube and dilute to 1.0 mL mark, with sterile seawater, before moving to the next step
Centrifuge: 3.200 34, 4 10
Centrifuge: 16.000 34, 4 10
?. [Virus particle concentration and extraction validation]
To seperate virus particles from the resultant supernatant, polyethylene glycol (5X; BioVision Research Products, CA) is added to solution.19) Add 250-300 ul of PEG (polyethylene glycol 5x; BioVision - Catalog #K904) to the newly tranferred supernatant in the sterile 1.5 mL centrifuge tube.20) Let the 1.5 mL centrifuge tube with the supernatant + PEG sit overnight (no less than 12 hours) at 4 degrees C21) After the overnight incubation, centrifuge the sample for  at 22) Very carefully, remove the supergnatant without disturbing the pellet using a pipette, or by carefully decanting to a waste container. 23) Re-suspend pellet in atleast ~1 mL of sterile seawater or to keep in high concetration, in ~200 ul of virus resuspension liquid (BioVision - Catalog #K904). At this point, depending on size of pellet, this solution can be diluted further to increase the volume usable for isolation efforts.
Centrifuge: 3.200 34, 4 10