Propagation of marine eukaryotic viruses (Prasinoviruses)
Cleaning and Concentration of Lysate.
Once the primary infection culture has cleared, filter entire volume of lysate through a 0.45-µm-pore-size PES membrane sterile disposable filter unit to remove large cell debris.
Add 20 mL filtered lysate to the upper reservoir of a 100,000 Dalton MWCO PES membrane Vivaspin20 ultrafiltration unit.
NOTE: Appropriate MWCO will depend on virus capsid size.
For maximum recovery select a MWCO at least 50% smaller than the molecular size of the particle of interest.
200 kDa is the equivalent of ~10 nm.
Place unit with counter-balance in swinging bucket rotor in pre-cooled (4°C) benchtop centrifuge so that the printed side faces upwards/outwards.
Centrifuge at 1,000 x g (do not exceed!) for 5-10 minutes.
Centrifuge time will depend on the amount of material in the sample.
Do not let the filter go dry.
Record the volume retained in the upper reservoir.
Discard the filtrate by separating the upper reservoir from the bottom of the Vivaspin20 unit.
Repeat 2-6 (adding filtered lysate to the retentate in the upper reservoir up to 20 mL before re-centrifuging) until the entire volume of filtered lysate has been processed.
NOTE: Several Vivaspin20 units can be used in parallel for large lysate volumes, and pooled after concentration.
Transfer the concentrated virus sample (retentate) from the upper reservoir to a sterile 15 or 50 mL conical tube.
Remove the upper reservoir from the Vivaspin20 unit and cover the bottom with a double-layer of Parafilm.
Add 2 mL of 0.02-µm-filtered 1X TE buffer (pH 8.0) to the upper reservoir.
Vortex the upper reservoir (Parafilm side down) for ~20 sec on 60% power to wash the Vivaspin20 filter.
Add the washed sample to the recovered viral concentrate.
Repeat 10-12 twice more for a total of 3 washes with TE buffer.
Store concentrated virus sample at 4°C protected from light.
Primary Infection.
Ethanol-clean culture hood as usual, then UV pipettes, tips, and tube rack for 10 min.
Work with one virus at a time, transferring the master stock from 4°C to the culture hood.
Initiate the primary infection by adding the master virus stock at 1% of the total culture volume (e.g., 5 mL virus to 500 mL exponentially-growing host culture).
Mix the infected flask and incubate at standard growth conditions until culture is mostly lysed (e.g., ~5 days for O. lucimarinus viruses).
Thoroughly clean pipette and gloves with ethanol before repeating for additional viruses.
Ethanol-clean hood and UV pipettes, tips, and tube rack after use.
