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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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.......

Representative Results

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.......

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.......

Acknowledgements

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 ....

Materials

NameCompanyCatalog NumberComments
0.5% Triton X-100J.T.BakerX198-07Store at room temperature (RT)
1% DEAE-DextranMP Biomedicals195133
10% Formalin solution, neutral bufferedSigma-AldrichHT501128
AgarOxoidLP0028
24-well Cell Culture PlateGreiner Bio-one662160
5% Sodium bicarbonateSigma AldrichS-5761
6-well Cell Culture PlateGreiner Bio-one657160
96-well Cell Culture PlateGreiner Bio-one655-180
African green monkey kidney epithelial cells (Vero E6)ATCCCRL-1586
Ami HTSpectral Instruments Imaging
Aura Imaging Software 3.2.0Spectral Instruments ImagingImage analysis software
Bovine Serum Albumin (BSA), 35%Sigma-AldrichA9647Store at 4 °C
Cell culture grade waterCorning25-055-CV
Dulbecco’s modified Eagle’s medium (DMEM)Corning Cellgro15-013-CVStore at 4 °C
Anesthesia gas machineVeterinary Anesthesia Systems, Inc.VAS 2001R
Fetal Bovine Serum (FBS)Seradigm1500-050Store at -20 °C
Four- to six-week-old female K18-hACE2 transgenic miceThe Jackson Laboratory34860
Graphpad Prism Version 9.1.0GraphPad
IsofluraneBaxter1001936040Store at RT
MARS Data Analysis SoftwareBMG LABTECH
MB10 tabletsQUIP LaboratoriesMBTAB1.5Store at RT
Nano-Glo Luciferase Assay ReagentPromegaN1110This reagent is used to measure Nluc activity. Store at -20 °C
Nunc MicroWell 96-Well MicroplatesThermoFisher Scientific269620
Nunc MicroWell 96-Well MicroplatesThermoFisher Scientific269620
Penicillin/Streptomycin/L-Glutamine (PSG) 100xCorning30-009-CIStore at -20 °C
PHERAstar FSXBMG LABTECHPHERAstar FSX
Precelleys Evolution homogenizerBertin InstrumentsP000062-PEVO0-A
Soft tissue homogenizing CK14 – 7 mLBertin InstrumentsP000940-LYSK0-A
T75 EasYFlaskThermoFisher Scientific156499
VECTASTAIN ABC-HRP Kit, PeroxidaseVector LaboratoriesPK-4002ABC kit and DAB Peroxidase Substrate kit

References

  1. V'Kovski, P., Kratzel, A., Steiner, S., Stalder, H., Thiel, V. Coronavirus biology and replication: implications for SARS-CoV-2. Nature Reviews Microbiology. 19 (3), 155-170 (2021).
  2. Pal, M., Berhanu, G., Desalegn, C., Kandi, V.

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