Live Imaging and Quantification of Viral Infection in K18 hACE2 Transgenic Mice Using Reporter-Expressing Recombinant SARS-CoV-2

Summary

This protocol describes the dynamics of viral infections using luciferase- and fluorescence-expressing recombinant (r)SARS-CoV-2 and an in vivo imaging systems (IVIS) in K18 hACE2 transgenic mice to overcome the need of secondary approaches required to study SARS-CoV-2 infections in vivo.

Abstract

The coronavirus disease 2019 (COVID-19) pandemic has been caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To date, SARS-CoV-2 has been responsible for over 242 million infections and more than 4.9 million deaths worldwide. Similar to other viruses, studying SARS-CoV-2 requires the use of experimental methods to detect the presence of virus in infected cells and/or in animal models. To overcome this limitation, we generated replication-competent recombinant (r)SARS-CoV-2 that expresses bioluminescent (nanoluciferase, Nluc) or fluorescent (Venus) proteins. These reporter-expressing rSARS-CoV-2 allow tracking viral infections in vitro and in vivo based on the expression of Nluc and Venus reporter genes. Here the study describes the use of rSARS-CoV-2/Nluc and rSARS-CoV-2/Venus to detect and track SARS-CoV-2 infection in the previously described K18 human angiotensin-converting enzyme 2 (hACE2) transgenic mouse model of infection using in vivo imaging systems (IVIS). This rSARS-CoV-2/Nluc and rSARS-CoV-2/Venus show rSARS-CoV-2/WT-like pathogenicity and viral replication in vivo. Importantly, Nluc and Venus expression allow us to directly track viral infections in vivo and ex vivo, in infected mice. These rSARS-CoV-2/Nluc and rSARS-CoV-2/Venus represent an excellent option to study the biology of SARS-CoV-2 in vivo, to understand viral infection and associated COVID-19 disease, and to identify effective prophylactic and/or therapeutic treatments to combat SARS-CoV-2 infection.

Introduction

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an enveloped, positive-sense, single-stranded RNA virus that belongs to the Betacoronavirus lineage in the Coronaviridae family¹. This viral family is divided into Alpha-, Beta-, Gamma-, and Delta-coronavirus¹. Alpha- and Betacoronaviruses mainly infect mammals, whereas Gamma- and Deltacoronavirus infect almost exclusively birds². To date, seven coronaviruses (CoV) have crossed species barriers and emerged as human coronaviruses (HCoV): two alpha-CoVs (HCoV-229E and HCoV-NL63) and five beta-CoVs (HCoV-OC43, HCoV-HKU1, SARS-Co....

Protocol

Protocols involving K18 hACE2 transgenic mice were approved by the Texas Biomedical Research Institute (TBRI) Institutional Biosafety Committee (IBC) and the Institutional Animal Care and Use Committee (IACUC). All experiments follow the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Research Council⁴⁰. The appropriate Personal Protection Equipment (PPE) is required when working with mice.

1. Use of K18 hACE2 transgenic mic.......

Discussion

This protocol demonstrates the feasibility of using these rSARS-CoV-2 expressing reporter genes to monitor viral infections in vivo. Both reporter-expressing recombinant viruses provide an excellent tool for studying SARS-CoV-2 infections in vivo. The described ex vivo (rSARS-CoV-2/Venus) and in vivo (rSARS-CoV-2/Nluc) imaging systems represent an excellent option to understand the dynamics of SARS-CoV-2 infection, viral pathogenesis and to identify infected cells/organs at different t.......

Materials

Name	Company	Catalog Number	Comments
0.5% Triton X-100	J.T.Baker	X198-07	Store at room temperature (RT)
1% DEAE-Dextran	MP Biomedicals	195133
10% Formalin solution, neutral buffered	Sigma-Aldrich	HT501128
Agar	Oxoid	LP0028
24-well Cell Culture Plate	Greiner Bio-one	662160
5% Sodium bicarbonate	Sigma Aldrich	S-5761
6-well Cell Culture Plate	Greiner Bio-one	657160
96-well Cell Culture Plate	Greiner Bio-one	655-180
African green monkey kidney epithelial cells (Vero E6)	ATCC	CRL-1586
Ami HT	Spectral Instruments Imaging
Aura Imaging Software 3.2.0	Spectral Instruments Imaging		Image analysis software
Bovine Serum Albumin (BSA), 35%	Sigma-Aldrich	A9647	Store at 4 °C
Cell culture grade water	Corning	25-055-CV
Dulbecco’s modified Eagle’s medium (DMEM)	Corning Cellgro	15-013-CV	Store at 4 °C
Anesthesia gas machine	Veterinary Anesthesia Systems, Inc.	VAS 2001R
Fetal Bovine Serum (FBS)	Seradigm	1500-050	Store at -20 °C
Four- to six-week-old female K18-hACE2 transgenic mice	The Jackson Laboratory	34860
Graphpad Prism Version 9.1.0	GraphPad
Isoflurane	Baxter	1001936040	Store at RT
MARS Data Analysis Software	BMG LABTECH
MB10 tablets	QUIP Laboratories	MBTAB1.5	Store at RT
Nano-Glo Luciferase Assay Reagent	Promega	N1110	This reagent is used to measure Nluc activity. Store at -20 °C
Nunc MicroWell 96-Well Microplates	ThermoFisher Scientific	269620
Nunc MicroWell 96-Well Microplates	ThermoFisher Scientific	269620
Penicillin/Streptomycin/L-Glutamine (PSG) 100x	Corning	30-009-CI	Store at -20 °C
PHERAstar FSX	BMG LABTECH	PHERAstar FSX
Precelleys Evolution homogenizer	Bertin Instruments	P000062-PEVO0-A
Soft tissue homogenizing CK14 – 7 mL	Bertin Instruments	P000940-LYSK0-A
T75 EasYFlask	ThermoFisher Scientific	156499
VECTASTAIN ABC-HRP Kit, Peroxidase	Vector Laboratories	PK-4002	ABC kit and DAB Peroxidase Substrate kit