Plaque Assay–Based Quantification of Viral Load in an Infected Mouse Tissue Sample

Abstract

Source: Morales Vasquez, D., et al. Live Imaging and Quantification of Viral Infection in K18 hACE2 Transgenic Mice Using Reporter-Expressing Recombinant SARS-CoV-2. J. Vis. Exp. (2021)

This video demonstrates the protocol for carrying out plaque assay–based viral titration for quantifying infectious virus levels in tissue sample collected from a virus–infected mouse model.

Protocol

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.

1. Viral titrations
   1. Utilizing the supernatants obtained from the tissue homogenates, make 10-fold serial dilutions and infect confluent monolayers of Vero E6 cells with 1 mL of each dilution of the supernatant (6-well plate format, 1.2 × 10⁶ cells/well, triplicates).
   2. Let the virus adsorb for 1 h at 37 °C in a humidified 5% CO₂ incubator.
   3. After viral adsorption, wash the cells with 1 mL of 1x phosphate-buffered saline (PBS) and incubate in 2 mL of post-infection media containing 1% Agar in the humidified 5% CO₂ incubator at 37 °C for 72 h.
   4. After the incubation, inactivate the plates in 10% neutral buffered formalin for 24 h at 4 °C, ensure the entire plate is submerged.
   5. Take plates out of biosafety level 3 (BSL3) cabinet and wash the cells three times with 1 mL of 1x PBS and permeabilize with 1 mL of 0.5% Triton X-100 for 10 min at room temperature (RT).
   6. Block the cells with 1 mL of 2.5% bovine serum albumin (BSA) in 1x PBS for 1 h at 37 °C, followed by incubation in 1 mL of 1 µg/mL of the SARS-CoV nucleocapsid (N) protein cross-reactive monoclonal antibody (1C7C7), diluted in 2.5% BSA for 1 h at 37 °C.
   7. Wash the cells three times with 1 mL of 1x PBS and develop the plaques using the Avidin-biotin complex (ABC) kit and 3,3′-Diaminobenzidine (DAB) Peroxidase Substrate kit according to the manufacturers' instructions.
   8. Calculate the viral titers as plaque-forming units (PFU)/mL.
      NOTE: Calculate with the formula PFU/mL = dilution factor x number of plaques x (1 mL/inoculum volume).

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.5% Triton X-100	J.T.Baker	X198-07	Store at room temperature (RT)
10% Formalin solution, neutral buffered	Sigma-Aldrich	HT501128
Agar	Oxoid	LP0028
6-well Cell Culture Plate	Greiner Bio-one	657160
African green monkey kidney epithelial cells (Vero E6)	ATCC	CRL-1586
Bovine Serum Albumin (BSA), 35%	Sigma-Aldrich	A9647	Store at 4 °C
Dulbecco’s modified Eagle’s medium (DMEM)	Corning Cellgro	15-013-CV	Store at 4 °C
VECTASTAIN ABC-HRP Kit, Peroxidase	Vector Laboratories	PK-4002	ABC kit and DAB Peroxidase Substrate kit