Assessing Rotavirus Growth Curve by Plaque Assay

Résumé

Source: Kadoya, S., Sano, D. Assays for the Specific Growth Rate and Cell-binding Ability of Rotavirus. J. Vis. Exp. (2019).

This video demonstrates the use of a plaque assay to assess the rotavirus growth curve. The method involves infecting host cells with diluted virus, overlaying with agarose to restrict spread, and staining to visualize plaques formed by viral replication. By counting plaque-forming units at defined time points, the resulting growth curve reveals the lag, exponential, and stationary phases of viral replication.

Protocole

1. Specific Growth Rate of Rotavirus

NOTE: Rhesus rotavirus (RRV, genotype: G3P) is utilized in this protocol because RRV can rapidly and easily form plaques with MA104 cells. 

1. Place a tube containing 1 mL of the virus suspension (10⁷ pfu/mL) in a serum-free medium stored at -80 °C in a water bath at 37 °C to thaw. Add 1 µg/µL trypsin from porcine pancreas to 1 mL of the virus suspension (final trypsin concentration is 4 µg/mL) and then vortex. Incubate the virus suspension at 37 °C and 5% CO₂ under the saturated vapor for 30 min.
   NOTE: Trypsin from the other sources can be used, but the effect on rotavirus infectivity needs to be tested in advance.
2. Dilute the activated virus suspension with a serum-free medium to adjust the multiplicity of infection (MOI) to 0.1 pfu/cell.
3. Add 1 mL of diluted virus suspension to MA104 cell lines (80% confluent) in a T75 flask 3 days after the cell plating, incubate at 37 °C for 1 h, and gently shake the flask every 15 min.
4. Then, add 30 mL of a serum-free medium containing 0.13 µg/mL of trypsin from a porcine pancreas to the flask. Incubate the flask at 37 °C and 5% CO₂ under the saturated vapor.
5. Collect 1 mL of the supernatant in the flask at 0, 6, 12, 18, 24, and 36 (and/or 48) h post-infection (hpi) and replace the supernatant in the 1.5 mL tubes using a pipette.
6. Conduct the freeze (-80 °C) and melt in a water bath at 37 °C cycle three times. Then centrifuge the tubes at 12,600 x g for 10 min at 4 °C. Collect the supernatant.
7. Filter the supernatant with a distilled 0.2 µm filter to remove the cell fraction. Store the supernatant -80 °C in the refrigerator until applying it to the plaque assay for measuring the virus titer.
8. Place the tubes containing the collected supernatant (step 1.5) in a water bath at 37 °C. Add 4 µg/mL trypsin to a 1 mL of 10-fold diluted sample and incubate at 37 °C for 30 min.
9. During the 30 min incubation in 3.8, to begin the plaque assay for measuring the virus titer obtained from time course samples (step 1.5), wash the MA104 cells twice in a 6-well plate with 2 mL of 1x PBS after removing the serum-containing medium.
10. Serially dilute the incubated samples with a serum-free medium and inoculate 1 mL of the diluted sample into each well. Incubate the plate for 90 min at 37 °C and 5% CO₂ under the saturated vapor, and gently shake the plate every 15 min.
11. After incubation, remove the inoculum from the 6-well plate. Add 4 µg/mL trypsin to the medium prepared in (step 1.3). Gently but immediately add 3 mL of the medium mixed with agarose gel (the ratio is 1:1) to each well.
12. Keep the plate at room temperature for more than 10 min (until the agarose gel becomes solid) and incubate for 2 days at 37 °C and 5% CO₂ under the saturated vapor.
    NOTE: Pour the medium mixed with agar from the edge of the well.
13. Add 1 mL of the 0.015% neutral red solution diluted with 1x PBS to each well and incubate at 37 °C and 5% CO₂ under the saturated vapor. Remove the dye after 3 h and incubate for 1 day at 37 °C and 5% CO₂ under the saturated vapor.
14. The next day, count the number of plaques in each well and calculate the pfu/mL. Carefully check the cell confluence before the plaque assay to assure the plaque numbers.

Matériaux

Liste des matériaux utilisés dans cet article

Nom	Entreprise	Numéro de catalogue	Commentaires
Agar-EPI	Nakalai Tesque, Inc	01101-34	Plaque assay
Disodium Hydrogenphosphate	Wako Pure Chemical Corporation	194-02875	Cell binding assay
Eagle's MEM "Nissui"	Nissui Pharmaceutical Co., Ltd	5900	Cell culture
Eagle's MEM "Nissui"	Nissui Pharmaceutical Co., Ltd	5901	Plaque assay
EasYFlask 75 cm2	Thermo Scientific	156499	Cell culture
Fetal bovine Serim, qualified, USDA-approved regions	Gibco	10437028	Cell culture and Plaque assay
Forward / Reverse primers	Eurofins Genomics		qPCR
L-Glutamine, 200 mM Solution	Gibco	2530081	Cell culture and Plaque assay
Neutral Red	Wako Pure Chemical Corporation	140-00932	Plaque assay
PBS (-) "Nissui"	Nissui Pharmaceutical Co., Ltd	5913	Cell culture and Plaque assay
Penicillin-Streptomycin, Liguid	Gibco	15140122	Cell culture and Plaque assay
Potassium Chloride	Wako Pure Chemical Corporation	163-03545	Cell binding assay
Sodium Bicarbonate	Wako Pure Chemical Corporation	199-05985	Cell culture and Plaque assay
Sodium Chloride	Wako Pure Chemical Corporation	198-01675	Cell binding assay
Tissue culture plates 24-well plate	TPP	92024	Cell binding assay
Tissue culture plates 6-well plate	TPP	92006	Plaque assay
Trizma base	SIGMA-ALDRICH	T1503	Cell binding assay
Trypsin from porcine pancrease	SIGMA-ALDRICH	T0303-1G	Activate for rotavirus
Trypsin-EDTA (0.05 %), phenol red	Gibco	25300054	Cell culture