TITLE: Modified persistence (mP) assay
AUTHORS: Eva JP Lievens

[DESCRIPTION]
This protocol describes a modified persistence assay, as presented in the manuscript "Life history diversity and signals of trade-offs in a large group of chloroviruses" (Lievens et al., bioRxiv). It is designed to quantify the specific infectivity and mortality rate of chloroviruses (large, lytic dsDNA viruses). The protocol should also be applicable to other lytic viruses that are large enough to be counted by flow cytometry. For viruses that are not large enough to be counted by flow cytometry, this protocol could be adjusted to start with different MOIs. It would then measure the mortality rate only.

Compared to a classic persistence assay (or survival assay, inactivation assay, ...), this protocol uses predetermined virion concentrations and non-linear statistical modeling to increase efficiency. Briefly, solutions containing 50000, 5000, 500, or 50 virions*/ml are prepared for each virus, these solutions are split into aliquots, and independent aliquots are used to do an MPN-like assay after 0, 7, 14, ... days. The MPN-like assay involves distributing the aliquots across 16 replicate liquid cultures of host cells (algae) and observing the number of cultures that are lysed. Statistical models are then used to estimate the initial concentration and decline of infectious virions. See the associated manuscript for a full description and schematic figure, as well as the statistical model.

[BEFORE_START]
This version is for two virus plates, which corresponds to max. 47 different viruses, and 5 time points (including time point 0). It is assumed that the virion concentration of the virus samples is known (see dx.doi.org/10.17504/protocols.io.6qpvr6q93vmk/v1).

Use sterile techniques throughout this protocol. Using materials that don't bind viruses is important; see Materials for more information.

Definitions:
BBM = modified Bold's Basal Medium, see the associated publication
persistence conditions = the conditions under which persistence is being tested (e.g. a specific temperature)
growth conditions = the conditions that allow algae to grow (e.g. 20°C, constant light, on an orbital shaker)
culture plates = 96-well flat-bottomed tissue culture plates (see Materials)
deep well plates = 96-well 2.2ml PP deep well plates (see Materials)
transparent seal = transparent PCR plate seal (see Materials)

[STEPS]
SECTION: time point 0 - mP setup
1. Mix virus solutions at 50000, 5000, 500, or 50 virions/ml.
SECTION: time point 0 - mP setup
1.1. In two deep well plates ("1" and "2"), mix viral solutions so they have a volume of 1.5ml and 50000, 5000, 500, or 50 virions/ml. Avoid reusing tips for virus pipetting; reusing tips leads to lower accuracy (possibly because viruses bind to the plastic) (EJPL, unpublished data).
Notes: If the virus samples are highly concentrated, it may be necessary to make preparatory dilutions of the virus samples.
 
                plate  column  row  strain  MOP  virus to add (ul)  BBM to add (ul)    1  1  A  NE-JV4  0.5  1.03  49.0    1  1  B  NE-JV4  1  2.07  47.9    1  1  C  NE-JV4  2  4.14  45.9    1  1  D  NE-JV4  5  10.3  39.7    1  1  E  NE-JV4  10  20.7  29.3    1  1  F  MA-1E  0.5  1.04  49.0    1  1  G  MA-1E  1  2.07  47.9    1  1  H  MA-1E  2  4.15  45.9    1  2  A  MA-1E  5  10.4  39.6    1  2  B  MA-1E  10  20.7  29.3    1  2  C  PBCV-1  0.5  0.72  49.3    1  2  D  PBCV-1  1  1.44  48.6    1  2  E  PBCV-1  2  2.89  47.1    1  2  F  PBCV-1  5  7.21  42.8    1  2  G  PBCV-1  10  14.4  35.6    1  2  H  ctrl        50  

SECTION: time point 0 - mP setup
1.2. Mix the deep well plates by using a 1000μl multichannel pipette to pipette each column up and down 3x.
SECTION: time point 0 - mP setup
2. Prepare the virus plates.
SECTION: time point 0 - mP setup
2.1. Using a multichannel pipette, aliquot 200μl from deep well plate "1" into five culture plates. These are the virus plates "1". Do the same for deep well plate "2" and the virus plates "2".
SECTION: time point 0 - mP setup
2.2. Seal all but one of the virus plates "1" and all but one of the virus plates "2" with a transparent seal, and store under the persistence conditions.
SECTION: time point 0 - MPN-like assay
3. Prepare an intermediate algal solution.
SECTION: time point 0 - MPN-like assay
3.1. Count the concentration of algal cells in the algal culture using a haemocytometer.
Notes: If necessary, centrifuge the algae for 5min at 4500rpm, gently pour off the supernatant, resuspend the pellet, and count the concentration of algal cells in the pellet.
SECTION: time point 0 - MPN-like assay
3.2. Using the algal culture and BBM, prepare 590ml of an intermediate algal solution at 1.05*106 cells/ml.
Background info: This concentration is late exponential phase for our algae; the idea is that the algae are concentrated enough for the viruses to lyse quickly, but aren't yet in stationary phase.
SECTION: time point 0 - MPN-like assay
4. Prepare the MPN plates.
SECTION: time point 0 - MPN-like assay
4.1. Aliquot 190μl algae into each well of 32 culture plates. These are the MPN plates.
SECTION: time point 0 - MPN-like assay
4.2. Using a multichannel pipette (ideally a 12-channel multichannel), distribute 10μl from the each row of the remaining virus plate "1" into two MPN plates as shown. Do the same for the remaining virus plate "2". It is possible to use the same tips for all the aliquots of a given row. Label each MPN plate with the virus plate number ("pl1" or "pl2"), virus plate row, and suffix 1 or 2.

  
Background info: This step makes it clear why the initial concentrations maximize the power of the MPN-like assay:
50000 virions/ml concentration = 500 virions in 10μl -> if the initial proportion of infectious virions (the specific infectivity) is 0.001, half of the 16 replicate wells will be virus-positive at time point 0
5000 virions/ml concentration = 50 virions in 10μl -> if the initial proportion of infectious virions (the specific infectivity) is 0.01, half of the 16 replicate wells will be virus-positive at time point 0
500 virions/ml concentration = 5 virions in 10μl -> if the initial proportion of infectious virions (the specific infectivity) is 0.1, half of the 16 replicate wells will be virus-positive at time point 0
50 virions/ml concentration = 0.5 virions in 10μl -> if the initial proportion of infectious virions (the specific infectivity) is 1, half of the 16 replicate wells will be virus-positive at time point 0
SECTION: time point 0 - MPN-like assay
4.3. Close the MPN plates with their lids, wrap the edges in Parafilm to reduce evaporation, and store them under the growth conditions.
SECTION: time point 0 - MPN-like assay
5. After 4 days, check the algal growth in the MPN plates. This can be done by eye, by measuring optical density at 680nm, etc.
SECTION: time point 7/14/21/28 - MPN-like assay
6. Collect one virus plate "1" and one virus plate "2" from the persistence conditions. Vortex the plates and spin down.
SECTION: time point 7/14/21/28 - MPN-like assay
7. Use these virus plates to repeat steps 3-5.