TITLE: Influenza virus plaque assay
AUTHORS: Steven F Baker

[DESCRIPTION]
Determine virus concentration from cell culture, lung homogenates, and more. This protocol measures virus in plaque forming units per ml solution. 

This protocol differs from classic flu plaque assays by using a semi-solid overlay (Avicel) as opposed to agarose. This is beneficial in avoiding reproducibility issues that can arise from burning monolayers with hot overlay, and is faster to make and aspirate. The negative is that you cannot plaque purify viral isolates as you can with agarose. 

Theoretically one infectious virion can spawn one plaque by initiating in a cell and infecting neighboring cells. This is the gold standard for virology, and for influenza, the plaque:particle ration is ~ 1:100. Thus there are lots of non/semi-infectious or defective interfering particles that are produced that are not on their own infectious.

[BEFORE_START]
Bring DMEM, VGM, trypsin, PBS, 2X DMEM to 37 C in water bath

[GUIDELINES]
This protocol uses 12-well plates. Best practice is to use technical triplicates (or duplicates at the minimum). Serial 10-fold dilutions across the plate commonly account for 4-logs (horizontal) or 3-log (vertical) resolution.

[STEPS]
SECTION: Seed cells (day 1)
1. Collect and count MDCK cells

For the following steps, MDCK cells are maintained in 10 cm dishes in 10 ml volume of D10
SECTION: Seed cells (day 1)
1.1. Wash & collect cells 

- Aspirate D10, add 4 ml sterile DPBS, aspirate
- Trypsinize cells with 1 ml 0.25% trypsin for 5-15 min at 37 C, tap gently to lift off
- Resuspend in 10 ml D10
- Count cells by adding 10 ul to counting slide and measure on TC20 or hemacytometer
SECTION: Seed cells (day 1)
1.2. Prepare enough cells to seed 12 well plates with 1 ml per well

- Resuspend cells to 3E5 cells/ml
- Add 1 ml per well in a 12 well plate
    Always fill all wells in a plate with liquid, even if all wells are not needed. This prevents evaporation effects
- Incubate cells at 37C, 5% CO2 overnight
SECTION: Prepare virus dilutions (day 2)
2. Prepare virus dilutions

Enveloped viruses are sticky and can adhere to plastic pipet tips. It's crucial to change tips between each dilution step, and pipet up & down a consistent number of times. For example, pipet up/down once, move liquid to next dilution, pipet up/down 8 times; discard tips, repeat.
SECTION: Infect cells (day 2)
3. Infect cells 60 min 

SECTION: Infect cells (day 2)
3.1. Inoculate

- Wash MDCK cells by aspirating, adding ~0.5 ml VGM per well
- Aspirate, add 0.1 ml virus inoculum
- Incubate at 37 C
    Critical to label plate with accurate description of dilution series/virus used
SECTION: Infect cells (day 2)
3.2. Infect for 1 h at 37C

- Rock plate every ~15 min by drastically tipping forward-back and side-side. Don't allow inoculum only to sit on the well edges or monolayer to dry out
SECTION: Infect cells (day 2)
3.4. Stop infection by adding overlay

- Add 1 ml overlay per well (no need to aspirate virus inoculum!)
    Critical to vigorously swirl plate after inoculum is added. The goal is to get an even mixture of inoculum/overlay. If you don't do this and maintain a liquid interface at the cell surface, plaques will be amorphous and hard to differentiate.
SECTION: Infect cells (day 2)
3.3. Prepare overlay

Each well will receive 1 ml overlay, determine final volume needed
- Add 2X DMEM into 50 ml falcon tubes, add TPCK-trypsin to ~1 ug/ml (volume calculated after 2X diluted with Avicel). However, you need to first mix TPCK-trypsin with 2X DMEM before adding avicel, or the mixture won't be uniform concentration
- Dilute 2X DMEM 1:1 with Avicel
- Store these 50 ml tubes in 37 C water bath until ready to stop infection

SECTION: Infect cells (day 2)
3.5. Incubate at 37 C for 3 days

4320 min 

Some fast growing viruses like PR8 or WSN can be developed after 2 days, but 3 days is best to be safe.
SECTION: Develop (day 5)
4. Stop infection and visualize plaques with Crystal Violet
SECTION: Develop (day 5)
4.1. Aspirate wells

- Avicel-containing overlay can be aspirated into the trap (hooray!). In BSC, tip plate towards you and aspirate overlay, trying to collect it all.
SECTION: Develop (day 5)
4.2. Wash

- Add ~0.5 ml PBS (does not have to be sterile) to each well. I just lightly dispense across the plate without measuring
- Swirl the liquid in the wells to dislodge remaining overlay
- Aspirate
SECTION: Develop (day 5)
4.3. Fix

- Add ~0.5 ml 70% ethanol to each well
- Incubate for 10 minutes at room temperature
- Aspirate

10 min 

SECTION: Develop (day 5)
4.4. Stain

- Add ~0.5 ml Crystal Violet to each well, making sure that liquid completely coats the surface
- Incubate for 10 minutes at room temperature
- Aspirate into Crystal Violet trap in the Kajon lab fume hood

10 min 

SECTION: Develop (day 5)
4.5. Wash

- Add tap water to fill the wells of the plate and dump down the sink
- Repeat for a total of 4 washes
SECTION: Develop (day 5)
4.6. Dry

- Dry plates by leaving upside down at an angle to encourage air flow
- On top of bench pad in the fume hood
SECTION: Count and titer
5. Titers are represented in plaque forming units (pfu) per ml stock
From animal infections, often represented as per g tissue. Be sure to weigh tissue so that you can back calculate later
SECTION: Count and titer
5.1. Aim to count 10-30 plaques per well
SECTION: Count and titer
5.2. Calculate titer by multiplying average number of plaques by reciprocal dilution factor and by inoculum volume

For example, and average of 3 plaques are observed in the 10-5 dilution well
3 X 105 [10-5 dilution] X 10 [0.1 ml inoculum]

Save yourself some time by developing a spreadsheet template like this one
SECTION: Prepare virus dilutions (day 2)
2.2. From tissue culture-grown virus stock

Infection inoculums for 12-well plates use 0.1 ml virus, when preparing dilutions, be sure to have adequate volume. Prepare 10-fold serial dilution series either in 96-well V-bottom plates or eppendorf tubes.

It may feel like less work to dilute in eppendorf tubes (less dilutions!) but by using a plate, you can use a multichannel pipette to simultaneously dilute samples. Plus the "technical replicate" portion is more faithful. E.g. if I'm just quick tittering 1-2 viruses I'll opt for eppendorf tubes. Most other situations I use 96-well plates.

    Prepare in 96-well V-bottom plates (use each column as a technical singlet, ie, use 3 columns for triplicate):
        Add 135 ul VGM to all wells
        Add 15 ul stock solution to row A
        Dilute stock solution serially, transferring 15 ul stepwise
    Prepare in eppendorf tubes (pull from eppendorf tubes to inoculate technical replicates):
        Add 360 ul VGM to eppendorf tubes
        Serially dilute 40 ul
SECTION: Prepare virus dilutions (day 2)
2.1. From mouse lungs

Lungs should be collected in individual Eppendorf tubes and flash frozen. To flash freeze, submerge closed tube in liquid nitrogen. Frozen samples are stored at -80 C.

On day of infection, prepare lung homogenates as follows:
- Thaw lungs on ice for >1 h
- Weigh lungs, move to bead buster 2 ml eppie tubes that each contain 1 magnetic ball bearing
- Add 0.5 ml VGM to rinse original lung tube and add to 2 ml tube. Up to 1 ml liquid volume can be used
- Lyse lungs using Qiagen Tissue Lyser II
- Program 4: 3 cycles, 30 Hz, 30 s. In between cycles, move tubes to ice for ~1 minute
- Spin homogenates 10,000 x g at 4 C for 10 min, transfer supernatant (containing virus) to new eppie tube, continue to dilutions in step 2.2 below
- Transfer beads to 70% ethanol to sterilize, rinse thoroughly with DI H2O, and store for later autoclaving