Name: live imaging and quantification of viral infection in k18 hace2 transgenic mice using reporter-expressing recombinant sars-cov-2
Uploaded: 2021-11-05T13:00:00
Description: Watch this Scientific Journal Video about Live Imaging and Quantification of Viral Infection in K18 hACE2 Transgenic Mice Using Reporter-Expressing Recombinant SARS-CoV-2 at JoVE.com

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&#39;s laboratory is currently supported by the NIAID/NIH grants RO1AI161363-01, RO1AI161175-01A1, and R43AI165089-01; the Department of Defense (DoD) grants W81XWH2110095 and W81XWH2110103; the San Antonio Partnership for Precision Therapeutic; the Texas Biomedical Research Institute Forum; the University of Texas Health Science Center at San Antonio; the San Antonio Medical Foundation; and by the Center for Research on Influenza Pathogenesis and Transmission (CRIPT), a NIAID-funded Center of Excellence for Influenza Research and Response (CEIRR, contract # 75N93021C00014).

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...

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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...

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-CoV, Middle East respiratory syndrome coronavirus [MERS-CoV], and SARS-CoV-2)3,4,5,6. SARS-CoV, MERS-CoV, and SARS-CoV-2 are highly pathogenic, causing severe lower respiratory tract infection7. Prior to the emergence of SARS-CoV-2, there were two epidemic outbreaks caused by CoVs: SARS-CoV in Guangdong Providence, China, from 2002-2003, with a case fatality rate (CFR) of about 9.7%; and MERS-CoV in the Middle East from 2012 to present, with a CFR of about 34%7,8. SARS-CoV-2 has an overall CFR between 3.4%-49%, with underlying conditions contributing to a higher CFR8,9. Since its discovery in December 2019, in Wuhan, China, SARS-CoV-2 has been responsible for over 242 million human infections and more than 4.9 million human deaths worldwide7,10,11,12. Notably, since late 2020, new SARS-CoV-2 variants of concern (VoC) and variants of interest (VoI) have impacted virus characteristics, including transmission and antigenicity9,13, and the overall direction of the COVID-19 pandemic. For the treatment of SARS-CoV-2 infections, there is currently only one United States (U.S.) Food and Drug Administration (FDA) therapeutic antiviral (remdesivir) and one Emergency Use Authorization (EUA) drug (baricitinib, to be administered in combination with remdesivir)14. There are also 6 approved EUA monoclonal antibodies: REGEN-COV (casirivimab and imdevimab, administered together), sotrovimab, tocilizumab, and bamlanivimab and etesevimab administered together15,16,17,18,19. There is currently only one FDA-approved prophylactic vaccine, Pfizer-BioNTech, and two other prophylactic vaccines (Moderna and Janssen) have been EUA approved20,21,22,23,24. However, with the uncontrolled infection rate and the emergence of VoC and VoI, SARS-CoV-2 still poses a threat to human health. Therefore, new approaches are urgently needed to identify efficient prophylactics and therapeutics to control SARS-CoV-2 infection and the still ongoing COVID-19 pandemic.
Studying SARS-CoV-2 requires laborious techniques and secondary approaches to identify the presence of the virus in infected cells and/or validated animal models of infection. The use of reverse genetics has allowed for the generation of recombinant viruses to answer important questions in the biology of viral infections. For instance, reverse genetics techniques have provided means to uncover and understand the mechanisms of viral infection, pathogenesis, and disease. Likewise, reverse genetics approaches have paved the way to engineer recombinant viruses lacking viral proteins to understand their contribution in viral pathogenesis. In addition, reverse genetics techniques have been used to generate recombinant viruses expressing reporter genes for in vitro and in vivo applications, including identifying prophylactic and/or therapeutic approaches for the treatment of viral infections. Fluorescent and bioluminescent proteins are the most commonly used reporter genes due to their sensitivity, stability, and easy detection based on the improvement of new technologies25,26. In vitro, fluorescent proteins have been shown to serve as a better option for the localization of viruses in infected cells, while luciferases are more convenient for quantification studies27,28,29. In vivo, luciferases are preferred over fluorescent proteins for whole animal imaging, while fluorescent proteins are preferred for the identification of infected cells or ex vivo imaging30,31,32. The use of reporter-expressing recombinant viruses has served as a powerful tool for the study of viruses in many families, including, among others, flaviviruses, enteroviruses, alphaviruses, lentiviruses, arenaviruses, and influenza viruses28,33,34,35,36.
To overcome the need for secondary approaches to study SARS-CoV-2 and characterize real-time SARS-CoV-2 infection in vivo, we have generated replication-competent recombinant (r)SARS-CoV-2 that expresses bioluminescent (nanoluciferase, Nluc) or fluorescent (Venus) proteins using our previously described bacterial artificial chromosomes(BAC)-based reverse genetics, which are maintained as a single copy in E. coli in order to minimize toxicity of virus sequences during its propagation in bacteria37,38. Notably, rSARS-CoV-2/Nluc and rSARS-CoV-2/Venus showed rSARS-CoV-2/WT-like pathogenicity in vivo. The high level of Venus expression from rSARS-CoV-2/Venus allowed detecting viral infection in the lungs of infected K18 hACE2 transgenic mice using an in vivo imaging system (IVIS)39. The levels of Venus expression correlated well with viral titers detected in the lungs, demonstrating the feasibility of using Venus expression as a valid surrogate of SARS-CoV-2 infection. Using rSARS-CoV-2/Nluc, we were able to track the dynamics of viral infection in real-time and longitudinally assess SARS-CoV-2 infection in vivo using the same IVIS approach in K18 hACE2 transgenic mice.

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			<td>0.5% Triton X-100</td>
			<td>J.T.Baker</td>
			<td>X198-07</td>
			<td>Store at room temperature (RT)</td>
		</tr>
		<tr>
			<td>1% DEAE-Dextran</td>
			<td>MP Biomedicals</td>
			<td>195133</td>
			<td></td>
		</tr>
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			<td>10% Formalin solution, neutral buffered</td>
			<td>Sigma-Aldrich</td>
			<td>HT501128</td>
			<td></td>
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		<tr>
			<td>Agar</td>
			<td>Oxoid</td>
			<td>LP0028</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well Cell Culture Plate</td>
			<td>Greiner Bio-one</td>
			<td>662160</td>
			<td></td>
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			<td>5% Sodium bicarbonate</td>
			<td>Sigma Aldrich</td>
			<td>S-5761</td>
			<td></td>
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			<td>6-well Cell Culture Plate</td>
			<td>Greiner Bio-one</td>
			<td>657160</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well Cell Culture Plate</td>
			<td>Greiner Bio-one</td>
			<td>655-180</td>
			<td></td>
		</tr>
		<tr>
			<td>African green monkey kidney epithelial cells (Vero E6)</td>
			<td>ATCC</td>
			<td>CRL-1586</td>
			<td></td>
		</tr>
		<tr>
			<td>Ami HT</td>
			<td>Spectral Instruments Imaging</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Aura Imaging Software 3.2.0</td>
			<td>Spectral Instruments Imaging</td>
			<td></td>
			<td>Image analysis software</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA), 35%</td>
			<td>Sigma-Aldrich</td>
			<td>A9647</td>
			<td>Store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Cell culture grade water</td>
			<td>Corning</td>
			<td>25-055-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM)</td>
			<td>Corning Cellgro</td>
			<td>15-013-CV</td>
			<td>Store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Anesthesia gas machine</td>
			<td>Veterinary Anesthesia Systems, Inc.</td>
			<td>VAS 2001R</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Seradigm</td>
			<td>1500-050</td>
			<td>Store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Four- to six-week-old female K18-hACE2 transgenic mice</td>
			<td>The Jackson Laboratory</td>
			<td>34860</td>
			<td></td>
		</tr>
		<tr>
			<td>Graphpad Prism Version 9.1.0</td>
			<td>GraphPad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Baxter</td>
			<td>1001936040</td>
			<td>Store at RT</td>
		</tr>
		<tr>
			<td>MARS Data Analysis Software</td>
			<td>BMG LABTECH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MB10 tablets</td>
			<td>QUIP Laboratories</td>
			<td>MBTAB1.5</td>
			<td>Store at RT</td>
		</tr>
		<tr>
			<td>Nano-Glo Luciferase Assay Reagent</td>
			<td>Promega</td>
			<td>N1110</td>
			<td>This reagent is used to measure Nluc activity. Store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Nunc MicroWell 96-Well Microplates</td>
			<td>ThermoFisher Scientific</td>
			<td>269620</td>
			<td></td>
		</tr>
		<tr>
			<td>Nunc MicroWell 96-Well Microplates</td>
			<td>ThermoFisher Scientific</td>
			<td>269620</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin/L-Glutamine (PSG) 100x</td>
			<td>Corning</td>
			<td>30-009-CI</td>
			<td>Store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>PHERAstar FSX</td>
			<td>BMG LABTECH</td>
			<td>PHERAstar FSX</td>
			<td></td>
		</tr>
		<tr>
			<td>Precelleys Evolution homogenizer</td>
			<td>Bertin Instruments</td>
			<td>P000062-PEVO0-A</td>
			<td></td>
		</tr>
		<tr>
			<td>Soft tissue homogenizing CK14 &#8211; 7 mL</td>
			<td>Bertin Instruments</td>
			<td>P000940-LYSK0-A</td>
			<td></td>
		</tr>
		<tr>
			<td>T75 EasYFlask</td>
			<td>ThermoFisher Scientific</td>
			<td>156499</td>
			<td></td>
		</tr>
		<tr>
			<td>VECTASTAIN ABC-HRP Kit, Peroxidase</td>
			<td>Vector Laboratories</td>
			<td>PK-4002</td>
			<td>ABC kit and DAB Peroxidase Substrate kit</td>
		</tr>
	</tbody>
</table>

live imaging and quantification of viral infection in k18 hace2 transgenic mice using reporter-expressing recombinant sars-cov-2

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.

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...

Watch this Scientific Journal Video about Live Imaging and Quantification of Viral Infection in K18 hACE2 Transgenic Mice Using Reporter-Expressing Recombinant SARS-CoV-2 at JoVE.com

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

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 ...

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