Assessing Rotavirus Growth Curve by Plaque Assay

Abstract

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.

Protocollo

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 s....

Materiali

Elenco dei materiali utilizzati in questo articolo

Nome	Azienda	Numero di catalogo	Commenti
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