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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.
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.
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....
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.......
rSARS-CoV-2/Nluc infection in K18 hACE2 transgenic mice (Figures 1 and 2)
Figure 1A shows a schematic representation of the rSARS-CoV-2/WT (top) and rSARS-CoV-2/Nluc (bottom) used to assess infections in vivo. Figure 1B shows the schematic flow chart applied to assess rSARS-CoV-2/Nluc infection dynamics in K18 hACE2 transgenic mice. Four-to-six-week-old female K18 hACE2 transgenic mic.......
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.......
We would like to thank members at our institute (Texas Biomedical Research Institute) for their efforts in keeping our facilities fully operational and safe during the COVID-19 pandemic. We would also like to thank our Institutional Biosafety Committee (IBC) and spell (IACUC) for reviewing our protocols in a time-efficient manner.We thank Dr. Thomas Moran at the Icahn School of Medicine at Mount Sinai for providing the SARS-CoV cross-reactive 1C7C7 nucleocapsid (N) protein monoclonal antibody. SARS-CoV-2 research in the Martinez-Sobrido's laboratory is currently supported by the NIAID/NIH grants RO1AI161363-01, RO1AI161175-01A1, and R43AI165089-01; the Department ....
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 |
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