Visualizing Trypsin-Activated Rotavirus Infection Using a Plaque Assay

Résumé

Source: Philip, A. A., et. al., Simplified Reverse Genetics Method to Recover Recombinant Rotaviruses Expressing Reporter Proteins. J. Vis. Exp. (2020)

This video demonstrates a plaque assay technique to monitor rotavirus infection in epithelial cells. Trypsin activation enhances viral entry, and staining highlights viable cells, enabling visualization of infection-induced plaques.

Protocole

1. Plaque isolation of recombinant viruses
   1. Activate viruses in 100 µL of clarified cell lysates by adding trypsin to a final concentration of 10 µg/mL and incubating at 37 °C for 1 h. Prepare a 10-fold serial dilution series ranging from 10^-1 to 10^-7 (1 mL each) in Dulbecco's Modified Eagle Medium (DMEM) incomplete medium.
   2. Rinse MA104 monolayers in 6-well plates 2x with 2 mL of phosphate-buffered saline (PBS) and once with DMEM incomplete medium. Add 400 mL of lysate dilutions in duplicate to the plates. Incubate the plates for 1 h in a 37 °C, 5% CO₂ incubator, rocking every 10−15 min to redistribute dilutions across the monolayer.
   3. Prepare an agarose-MEM overlay solution by combining equal volumes of 2x Eagle’s minimal essential medium (EMEM; prewarmed to 37 °C) with 1.5% agarose that has been melted in water using a microwave oven and pre-cooled to 45 °C. Maintain the overlay solution at 42 °C using a water bath and adjust to a final concentration of 0.5 µg/mL trypsin immediately before placing on cells.
   4. Draw off lysate dilutions from 6-well plates, then rinse cells once with 2 mL of incomplete DMEM. Gently overlay 3 mL of the agarose-MEM overlay solution onto the cell monolayer contained in each well. Allow the agarose overlay to harden at room temperature, then return the plates to the incubator.
   5. Three days later, prepare an agarose-MEM overlay solution and bring it to 42 °C. Immediately prior to use, adjust the overlay solution to a final concentration of 50 µg/mL neutral red.
   6. Add 2 mL of the overlay solution on top of the existing agarose layer in the 6-well plates. After allowing the new agarose layer to harden, return the plates to the incubator. Protect solutions and plates containing neutral red from exposure to light.
   7. Over the next 6 h, identify rotavirus plaques in the 6-well plates with the aid of a light box. Pick clearly defined plaques using disposable transfer pipets, recovering agarose plugs that extend fully to the cell layer.

Matériaux

Liste des matériaux utilisés dans cet article

Nom	Entreprise	Numéro de catalogue	Commentaires
Baby Hamster Kidney - T7 RdRP (BHK-T7) Cells			Contact: ubuchholz@niaid.nih.gov
Disposable Transfer Pipettes, Ultrafine Extended Tips	MTC Bio	P4113-11
Dulbecco's Modified Eagle Medium (DMEM)	Lonza	12-604F
Eagle's Minimal Essential Medium, 2x (2xEMEM)	Quality Biological	115-073-101
Ethanol, Absolute (200 proof)	Fisher Bioreagents	BP2818-500
Gibco FluroBrite DMEM	ThermoFisher	A1896701	DMEM with low background fluorescence
Medium 199 (M199) Culture Medium	Hyclone	Sh30253.01
Neutral Red Solution (0.33%)	Sigma-Aldrich	N2889-100ml
Non-Essential Amino Acid Solution (100x)	Gibco	11140-050
Phosphate Buffered Saline (PBS), 10x	Fisher Bioreagents	BP399-20
Porcine Trypsin, Type IX-S	Sigma-Aldrich	T0303
SeaKem LE Agarose	Lonza	50000	For gel electrophoresis
SeaPlaque agarose	Lonza	50100	For plaque assay
Zoe Fluorescent Live Cell Imager	Bio-Rad