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 mice
<ol>
	<li>Purchase and maintain 4-6-week-old female B6.Cg-Tg(K18-ACE2)2Prlmn/J mice (K18 hACE2 transgenic mice) in a biosafety level (BSL)-2 animal care facility under specific pathogen-free conditions.
	<ol>
		<li>After arrival at the BSL2 facilities, allow the animals to acclimate for 7 days and transfer them to the BSL-3 animal facility for infections and other experimental procedures.</li>
		<li>Following IACUC protocols, place 4 mice per cage. To ensure that the animal is deceased, after mice infection with rSARS-CoV-2 and in vivo imaging, euthanize the animals with a lethal dose of Fatal-Plus (&#62;100 mg/kg).</li>
	</ol>
	</li>
</ol>
2. Biosafety
NOTE: In this manuscript, rSARS-CoV-2 is generated using the BAC-based reverse genetic systems for SARS-CoV-2 USA-WA1/2020 strain, as previously described37. All in vivo procedures involving rSARS-CoV-2/Nluc or rSARS-CoV-2/Venus infections must be performed in a biological safety cabinet under BSL-3 conditions.
<ol>
	<li>Clean the biosafety cabinet with 2% Wexicide and 70% Ethanol disinfectant sequentially before and after performing all the experimental procedures described in this article.</li>
	<li>Sterilize all dissection material (scissors, dissecting forceps, etc.) and the homogenizer before and after each use.</li>
	<li>Clean the isolation chamber and disinfect using MB10 tablets before and after each use according to the manufacturer&#39;s instruction.</li>
	<li>Discard all biological material produced during the procedures following IBC and IACUC guidelines.</li>
</ol>
3. In vivo characterization of rSARS-CoV-2
<ol>
	<li>Mouse infections
	<ol>
		<li>Place female 4-6-week-old K18 hACE2 transgenic mice in labeled cages, identify the mice in each cage using an ear punch code and place 4 mice per cage. Use a group of mice for body weight and survival and another group of animals for viral titration and imaging.</li>
		<li>Before infection, shave mice chest on the ventral side from the pectoral nipple to the inguinal nipple area to facilitate the bioluminescence signal.</li>
		<li>Prepare the rSARS-CoV-2 inoculum with 105 plaque-forming units (PFU)/mouse under sterile conditions in a total volume of 50 &#181;L using sterile 1x phosphate buffer saline (PBS). 
		NOTE: Calculate the amount of rSARS-CoV-2 to be used in the viral dilution with the formula: virus needed = (105 PFU per mouse / 50 &#181;L) x final volume) / stock viral titer.</li>
		<li>Keep the virus inoculum chilled on ice.</li>
		<li>Use the mock-infected (1x PBS) and rSARS-CoV-2/WT-infected mice as internal controls. Place the mock-infected mice in a separate cage than rSARS-CoV-2-infected mice.</li>
		<li>Anesthetize the mice using 5% isoflurane gaseous sedation in an anesthesia chamber and maintain with 3% isoflurane.</li>
		<li>Once postural reaction and righting reflex are confirmed, place the mouse in the dorsal recumbency position.</li>
		<li>Scruff the neck of the mouse between the index finger and thumb and hold the tail against the palm of the hand with the pinky finger to hold the animal in a dorsal position.</li>
		<li>Place the pipette tip containing 50 &#181;L of the virus inoculum in the nostril and slowly eject the solution. Ensure that the virus inoculum is inhaled and there is no reflux by observing the inoculum drop disappearing.</li>
	</ol>
	</li>
	<li>Bioluminescence monitoring K18 hACE2 transgenic mice infected with rSARS-CoV-2/Nluc (Figure 1 and Figure 2) 
	NOTE: This experiment follows the schematic representation and uses the viruses displayed in Figure 1. An isoflurane anesthesia attachment is required for the in vivo imaging system (IVIS). See Table of Materials for details instruments and systems required.
	<ol>
		<li>Initiate the imaging software and set up the parameters, click image mode to Bioluminescence, open filter, and set the exposure time to auto.</li>
		<li>Upon initializing the IVIS machine, place the mice in the isolation chamber while still inside the biosafety cabinet. Induce mice with isoflurane at a 5% concentration. Once loss of the postural reaction and righting reflex is confirmed, maintain with 3% isoflurane.</li>
		<li>Once mice are anesthetized, remove them from the isolation chamber and retro-orbitally administer the luciferase substrate diluted 1:10 in 1x PBS (final volume 100 &#181;L/mouse) with a 25 G needle.</li>
		<li>After luciferase substrate administration, place the mice back in the isolation chamber with their chests facing up and nasal cavity inside the manifold cone to keep the animal anesthetized during the imaging procedure.</li>
		<li>Transfer the isolation chamber to the IVIS machine, and instantaneously after closing the IVIS imager door, select Acquire in the software program to initialize (Figure 2A).</li>
		<li>To analyze bioluminescence images acquired, utilize the ROI (region of interest) tool in the software to designate the precise signal and measure the flux (Figure 2B).</li>
		<li>Click on Measure and allow the system to begin assessing the bioluminescence in photons to provide the absolute photon emission comparable to the output measurements provided by the various parameters.</li>
		<li>Image mice at 1-, 2-, 4- and 6-days post-infection.</li>
		<li>After&#160;imaging&#160;place mice back into cage.</li>
	</ol>
	</li>
	<li>Fluorescence analysis in K18 hACE2 transgenic mice infected with rSARS-CoV-2/Venus (Figure 3 and Figure 4) 
	NOTE: This experiment follows the schematic representation and rSARS-CoV-2 shown in Figure 3. See Table of Materials for details instruments and systems required.
	<ol>
		<li>Initiate the imaging software and set up the parameters, set excitation (500 nm) and emission filters (530 nm), click image mode to Fluorescence and set the exposure time to auto.</li>
		<li>Intraperitoneally euthanize the mice using a lethal dose of Fatal-Plus (&#62;100 mg/kg). Following euthanasia, disinfect the incision site with 70% ethanol.</li>
		<li>Using tweezers, pull the skin, make an incision from the sternum to the abdomen and cut the incision from the sides with scissors. Cut the hepatic vein to minimize the amount of blood in the lungs and avoid high background signals during imaging.</li>
		<li>Using scissors, cut the sternum and open the ribcage. Next, snip the end of the trachea with scissors and remove the lungs with tweezers.</li>
		<li>Place the excised lungs in a 6-well plate containing 2 mL of 1x PBS and rinse to remove excess blood. Minimize the possibility of contamination between samples by disinfecting and cleaning surgery tools between samples using 70% ethanol.</li>
		<li>After initializing the IVIS machine, place the lungs in a black tray and separate the tissues from each other.</li>
		<li>Place the tray inside the isolation chamber inside the biosafety cabinet, and then transfer the isolation chamber to the IVIS. Close the door and click on Acquire to initiate the imaging system (Figure 4A).</li>
		<li>To analyze fluorescence, utilize the ROI tool and draw ROIs around each of the individual lungs. Measure each ROI manually and then use the average radiant efficiency values given and subtract from those of the mock-infected mice (Figure 4B).</li>
		<li>Once imaging is complete, place the tissues on ice for same-day analysis or in a cryotube for dry ice freezing to store at -80 &#176;C for later processing.</li>
	</ol>
	</li>
	<li>Lung bright field imaging and pathology scoring of K18 hACE2 transgenic mice infected with rSARS-CoV-2/Nluc (Figure 2A-C) and rSARS-CoV-2/Venus (Figure 4A-C)
	<ol>
		<li>After imaging bioluminescence of mice infected with rSARS-CoV-2/Nluc, rSARS-CoV-2/WT, and mock-infected, return the mice to their cages. Proceed with euthanasia and the ex vivo bright field imaging of mice lungs (Figure 2A).</li>
		<li>After imaging ex vivo fluorescence of lungs of infected mice, take bright-field images of the lungs (Figure 4A).</li>
		<li>Analyze the gross lesions on the surface of the lung using ImageJ (Figures 2C and 4C).</li>
		<li>Open the lung image to be analyzed in ImageJ.</li>
		<li>Calculate the ratio of pixel to cm, use the Straight tool and measure the actual length of the image.</li>
		<li>After selecting, click on Analyze &#62; Measure to calculate the length of pixels for the length.</li>
		<li>Click on Analysis &#62; Set Scale and input the numbers calculated from the previous step.</li>
		<li>Use the Freehand Selections tool and select the entire lung surface. Click on Analyze &#62; Measure to measure total lung area.</li>
		<li>Click on Edit &#62; Selection &#62; Make Inverse to select the rest of the lung area, then press delete or backspace to remove the background.</li>
		<li>Remove the selected area, then click on Image &#62; Adjust &#62; Color Threshold.</li>
		<li>To select pathologic lesion area, adjust Brightness to a minimum (between 1 to 50) and a maximum (between 50 to 200), depending on the levels of congestion and hemorrhages present.</li>
		<li>Once pathologic lesions are selected, click on Select at the bottom of the threshold color panel, then click on Analyze &#62; Measure to measure pathologic areas.</li>
		<li>Calculate the percentage of gross lesions with the formula: % of pathologic area = (measurement of pathologic area/total lung surface) x 100.</li>
	</ol>
	</li>
	<li>Viral titrations (Figure 2D and Figure 4D)
	<ol>
		<li>Upon imaging, complete the mice euthanasia and collect lungs, brain, and nasal mucosa.</li>
		<li>Place the lungs, brain, and nasal mucosa into separate sterile tissue homogenizers and add 1 mL of cold 1x PBS.</li>
		<li>Homogenize the samples by centrifuging at 21,500 x g for 10 min to pellet cell debris. Collect and transfer the supernatants into a new sterile tube and discard the pellet.</li>
		<li>If viral titrations are performed the same day, store the supernatants at 4 &#176;C. Alternatively, freeze the supernatant of the homogenized samples at -80 &#176;C until being evaluated later.</li>
		<li>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 &#215; 106 cells/well, triplicates).</li>
		<li>Let the virus adsorb for 1 h at 37 &#176;C in a humidified 5% CO2 incubator.</li>
		<li>After viral adsorption, wash the cells with 1 mL of 1x PBS and incubate in 2 mL of post-infection media containing 1% Agar in the humidified 5% CO2 incubator at 37 &#176;C for 72 h.</li>
		<li>After the incubation, inactivate the plates in 10% neutral buffered formalin for 24 h at 4 &#176;C, ensure the entire plate is submerged.</li>
		<li>Take plates out of BSL3 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).</li>
		<li>Block the cells with 1 mL of 2.5% bovine serum albumin (BSA) in 1x PBS for 1 h at 37 &#176;C, followed by incubation in 1 mL of 1 &#181;g/mL of the SARS-CoV nucleocapsid (N) protein cross-reactive monoclonal antibody (1C7C7), diluted in 2.5% BSA for 1 h at 37 &#176;C.</li>
		<li>Wash the cells three times with 1 mL of 1x PBS and develop the plaques using the ABC kit and DAB Peroxidase Substrate kit according to the manufacturers&#39; instructions.</li>
		<li>Calculate the viral titers as PFU/mL. 
		&#8203;NOTE: Calculate with the formula PFU/mL = dilution factor x number of plaques x (1 mL/inoculum volume).</li>
	</ol>
	</li>
	<li>Nluc activity in tissues of K18 hACE2 transgenic mice infected with rSARS-CoV-2/Nluc (Figure 2E)
	<ol>
		<li>Quantify the presence of Nluc in the organ homogenates from mock, rSARS-CoV-2/WT, and rSARS-CoV-2/Nluc infected K18 hACE2 transgenic mice using a luciferase assay following the manufacturers&#39; instructions.</li>
	</ol>
	</li>
	<li>Evaluation of morbidity and mortality (Figure 2F, G and Figure 4E, F)
	<ol>
		<li>Intranasally infect 4-6-week-old female K18 hACE2 transgenic mice with 105 PFU of rSARS-CoV-2/WT, rSARS-CoV-2/Venus, rSARS-CoV-2/Nluc, or mock-infected as described in section 3.1.</li>
		<li>Monitor and weigh the mice over 12 days at the same time to minimize the weight variation due to food ingestion. Euthanize the mice that lose 25% of their initial body weight since they have reached a humane endpoint and note these mice as succumbing to viral infection.</li>
		<li>After 12 days, euthanize the mice that survive viral infection and calculate the % of body weight change and survival.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>V'Kovski, P., Kratzel, A., Steiner, S., Stalder, H., Thiel, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coronavirus+biology+and+replication:+implications+for+SARS-CoV-2.">Coronavirus biology and replication: implications for SARS-CoV-2.</a> Nature Reviews Microbiology. 19 (3), 155-170 (2021).</li><li>Pal, M., Berhanu, G., Desalegn, C., Kandi, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Severe+acute+respiratory+syndrome+coronavirus-2+(SARS-CoV-2):+An+update.">Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2): An update.</a> Cureus. 12 (3), 7423 (2020).</li><li>Su, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology,+genetic+recombination,+and+pathogenesis+of+coronaviruses.">Epidemiology, genetic recombination, and pathogenesis of coronaviruses.</a> Trends in Microbiology. 24 (6), 490-502 (2016).</li><li>Cui, J., Li, F., Shi, Z. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origin+and+evolution+of+pathogenic+coronaviruses.">Origin and evolution of pathogenic coronaviruses.</a> Nature Reviews Microbiology. 17 (3), 181-192 (2019).</li><li>Lu, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+characterisation+and+epidemiology+of+2019+novel+coronavirus:+implications+for+virus+origins+and+receptor+binding.">Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding.</a> Lancet. 395 (10224), 565-574 (2020).</li><li>Evans, J. P., Liu, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+host+factors+in+SARS-CoV-2+entry.">Role of host factors in SARS-CoV-2 entry.</a> Journal of Biological Chemistry. 297 (1), 100847 (2021).</li><li>Petersen, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparing+SARS-CoV-2+with+SARS-CoV+and+influenza+pandemics.">Comparing SARS-CoV-2 with SARS-CoV and influenza pandemics.</a> Lancet Infectious Diseases. 20 (9), 238-244 (2020).</li><li>Alfaraj, S. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+predictors+of+mortality+of+Middle+East+Respiratory+Syndrome+Coronavirus+(MERS-CoV)+infection:+A+cohort+study.">Clinical predictors of mortality of Middle East Respiratory Syndrome Coronavirus (MERS-CoV) infection: A cohort study.</a> Travel Medicine and Infectious Disease. 29, 48-50 (2019).</li><li>Harvey, W. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SARS-CoV-2+variants,+spike+mutations+and+immune+escape.">SARS-CoV-2 variants, spike mutations and immune escape.</a> Nature Reviews Microbiology. 19 (7), 409-424 (2021).</li><li>Dong, E., Du, H., Gardner, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+interactive+web-based+dashboard+to+track+COVID-19+in+real+time.">An interactive web-based dashboard to track COVID-19 in real time.</a> Lancet Infectious Diseases. 20 (5), 533-534 (2020).</li><li>Bar-On, Y. M., Flamholz, A., Phillips, R., Milo, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ARS-CoV-2+(COVID-19)+by+the+numbers.">ARS-CoV-2 (COVID-19) by the numbers.</a> Elife. 9, 57309 (2020).</li><li>Roussel, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SARS-CoV-2:+fear+versus+data.">SARS-CoV-2: fear versus data.</a> International Journal of Antimicrobial Agents. 55 (5), 105947 (2020).</li><li>Scialo, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SARS-CoV-2:+One+year+in+the+pandemic.+What+have+we+learned,+the+new+vaccine+era+and+the+threat+of+SARS-CoV-2+variants.">SARS-CoV-2: One year in the pandemic. What have we learned, the new vaccine era and the threat of SARS-CoV-2 variants.</a> Biomedicines. 9 (6), 611 (2021).</li><li>. Coronavirus (COVID-19) update: FDA authorizes additional monoclonal antibody for treatment of COVID-19 Available from: <a target="_blank" href="https://www.fda.gov/news-events/press-announcements/coronavirus-covid-19-update-fda-authorizes-additional-monoclonal-antibody-treatment-covid-19">https://www.fda.gov/news-events/press-announcements/coronavirus-covid-19-update-fda-authorizes-additional-monoclonal-antibody-treatment-covid-19</a> (2021)</li><li>Dougan, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bamlanivimab+plus+Etesevimab+in+Mild+or+Moderate+Covid-19.">Bamlanivimab plus Etesevimab in Mild or Moderate Covid-19.</a> New England Journal of Medicine. 385 (15), 1382-1392 (2021).</li><li>Ledford, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=COVID+antibody+treatments+show+promise+for+preventing+severe+disease.">COVID antibody treatments show promise for preventing severe disease.</a> Nature. 591 (7851), 513-514 (2021).</li><li>Tuccori, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+overview+of+the+preclinical+discovery+and+development+of+bamlanivimab+for+the+treatment+of+novel+coronavirus+infection+(COVID-19):+reasons+for+limited+clinical+use+and+lessons+for+the+future.">An overview of the preclinical discovery and development of bamlanivimab for the treatment of novel coronavirus infection (COVID-19): reasons for limited clinical use and lessons for the future.</a> Expert Opinion on Drug Discovery. , 1-12 (2021).</li><li>Phan, A. T., Gukasyan, J., Arabian, S., Wang, S., Neeki, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emergent+inpatient+administration+of+casirivimab+and+imdevimab+antibody+cocktail+for+the+treatment+of+COVID-19+pneumonia.">Emergent inpatient administration of casirivimab and imdevimab antibody cocktail for the treatment of COVID-19 pneumonia.</a> Cureus. 13 (5), 15280 (2021).</li><li>O'Brien, M. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcutaneous+REGEN-COV+antibody+combination+in+early+SARS-CoV-2+infection.">Subcutaneous REGEN-COV antibody combination in early SARS-CoV-2 infection.</a> medRxiv. , (2021).</li><li>Beigel, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remdesivir+for+the+treatment+of+Covid-19+-+Final+report.">Remdesivir for the treatment of Covid-19 - Final report.</a> New England Journal of Medicine. 383 (19), 1813-1826 (2020).</li><li>Li, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+convalescent+plasma+therapy+on+time+to+clinical+improvement+in+patients+with+severe+and+life-threatening+COVID-19:+A+randomized+clinical+trial.">Effect of convalescent plasma therapy on time to clinical improvement in patients with severe and life-threatening COVID-19: A randomized clinical trial.</a> Journal of the American Medical Association. 324 (5), 460-470 (2020).</li><li>Polack, F. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Safety+and+efficacy+of+the+BNT162b2+mRNA+Covid-19+vaccine.">Safety and efficacy of the BNT162b2 mRNA Covid-19 vaccine.</a> New England Journal of Medicine. 383 (27), 2603-2615 (2020).</li><li>Oliver, S. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+advisory+committee+on+immunization+practices'+interim+recommendation+for+use+of+Pfizer-BioNTech+COVID-19+vaccine+-+United+States,+December+2020.">The advisory committee on immunization practices' interim recommendation for use of Pfizer-BioNTech COVID-19 vaccine - United States, December 2020.</a> Morbidity and Mortality Weekly Report. 69 (50), 1922-1924 (2020).</li><li>Oliver, S. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+advisory+committee+on+immunization+practices'+interim+recommendation+for+use+of+Janssen+COVID-19+vaccine+-+United+States,+February+2021.">The advisory committee on immunization practices' interim recommendation for use of Janssen COVID-19 vaccine - United States, February 2021.</a> Morbidity and Mortality Weekly Report. 70 (9), 329-332 (2021).</li><li>Zhao, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emission+spectra+of+bioluminescent+reporters+and+interaction+with+mammalian+tissue+determine+the+sensitivity+of+detection+in+vivo.">Emission spectra of bioluminescent reporters and interaction with mammalian tissue determine the sensitivity of detection in vivo.</a> Journal of Biomedical Optics. 10 (4), 41210 (2005).</li><li>Shaner, N. C., Steinbach, P. A., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+guide+to+choosing+fluorescent+proteins.">A guide to choosing fluorescent proteins.</a> Nature Methods. 2 (12), 905-909 (2005).</li><li>Nogales, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+fluorescent+and+bioluminescent+bireporter+Influenza+A+Virus+to+evaluate+viral+infections.">A novel fluorescent and bioluminescent bireporter Influenza A Virus to evaluate viral infections.</a> Journal of Virology. 93 (10), 00032 (2019).</li><li>Nogales, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication-competent+fluorescent-expressing+influenza+B+virus.">Replication-competent fluorescent-expressing influenza B virus.</a> Virus Research. 213, 69-81 (2016).</li><li>Welsh, D. K., Noguchi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+bioluminescence+imaging.">Cellular bioluminescence imaging.</a> Cold Spring Harbor Protocols. 2012 (8), (2012).</li><li>Tran, V., Moser, L. A., Poole, D. S., Mehle, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+sensitive+real-time+in+vivo+imaging+of+an+influenza+reporter+virus+reveals+dynamics+of+replication+and+spread.">Highly sensitive real-time in vivo imaging of an influenza reporter virus reveals dynamics of replication and spread.</a> Journal of Virology. 87 (24), 13321-13329 (2013).</li><li>Schoggins, J. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dengue+reporter+viruses+reveal+viral+dynamics+in+interferon+receptor-deficient+mice+and+sensitivity+to+interferon+effectors+in+vitro.">Dengue reporter viruses reveal viral dynamics in interferon receptor-deficient mice and sensitivity to interferon effectors in vitro.</a> Proceedings of the National Academy of Sciences of the United States of America. 109 (36), 14610-14615 (2012).</li><li>Luker, G. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+bioluminescence+imaging+of+herpes+simplex+virus+type+1+infection+and+therapy+in+living+mice.">Noninvasive bioluminescence imaging of herpes simplex virus type 1 infection and therapy in living mice.</a> Journal of Virology. 76 (23), 12149-12161 (2002).</li><li>Li, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+rapid+antiviral+screening+assay+based+on+eGFP+reporter+virus+of+Mayaro+virus.">Development of a rapid antiviral screening assay based on eGFP reporter virus of Mayaro virus.</a> Antiviral Research. 168, 82-90 (2019).</li><li>Kirui, J., Freed, E. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+and+validation+of+a+highly+sensitive+bioluminescent+HIV-1+reporter+vector+that+simplifies+measurement+of+virus+release.">Generation and validation of a highly sensitive bioluminescent HIV-1 reporter vector that simplifies measurement of virus release.</a> Retrovirology. 17 (1), 12 (2020).</li><li>Shang, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+characterization+of+a+stable+eGFP+enterovirus+71+for+antiviral+screening.">Development and characterization of a stable eGFP enterovirus 71 for antiviral screening.</a> Antiviral Research. 97 (2), 198-205 (2013).</li><li>Zou, G., Xu, H. Y., Qing, M., Wang, Q. Y., Shi, P. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+characterization+of+a+stable+luciferase+dengue+virus+for+high-throughput+screening.">Development and characterization of a stable luciferase dengue virus for high-throughput screening.</a> Antiviral Research. 91 (1), 11-19 (2011).</li><li>Ye, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rescue+of+SARS-CoV-2+from+a+single+bacterial+artificial+chromosome.">Rescue of SARS-CoV-2 from a single bacterial artificial chromosome.</a> mBio. 11 (5), 02168 (2020).</li><li>Avila-Perez, G., Park, J. G., Nogales, A., Almazan, F., Martinez-Sobrido, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rescue+of+recombinant+Zika+virus+from+a+bacterial+artificial+chromosome+cDNA+clone.">Rescue of recombinant Zika virus from a bacterial artificial chromosome cDNA clone.</a> Journal of Visualized Experiments: JoVE. (148), e59537 (2019).</li><li>Chiem, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+bifluorescent-based+assay+for+the+identification+of+neutralizing+antibodies+against+SARS-CoV-2+variants+of+concern+in+vitro+and+in+vivo.">A bifluorescent-based assay for the identification of neutralizing antibodies against SARS-CoV-2 variants of concern in vitro and in vivo.</a> Journal of Virology. , (2021).</li><li>Committee for the update of the guide for the care and use of laboratory animals., Institute for laboratory animal research (U.S) &amp; National Academies Press (U.S.). <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guide+for+the+care+and+use+of+laboratory+animals.+8th+edn.">Guide for the care and use of laboratory animals. 8th edn.</a> National Research Council (US). , (2011).</li><li>Ye, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+SARS-CoV-2+infection+dynamic+in+vivo+using+reporter-expressing+viruses.">Analysis of SARS-CoV-2 infection dynamic in vivo using reporter-expressing viruses.</a> Proceedings of the National Academy of Sciences of the United States of America. 118 (41), (2021).</li></ol>

The authors declare that the research was conducted in the absence of any commercial, financial and non-financial, or personal conflict of interest in relation to the work described.

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<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>
		<tr>
			<td>10% Formalin solution, neutral buffered</td>
			<td>Sigma-Aldrich</td>
			<td>HT501128</td>
			<td></td>
		</tr>
		<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>
		</tr>
		<tr>
			<td>5% Sodium bicarbonate</td>
			<td>Sigma Aldrich</td>
			<td>S-5761</td>
			<td></td>
		</tr>
		<tr>
			<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 ...

live-imaging-quantification-viral-infection-k18-hace2-transgenic-mice

Research

JoVE Journal

Immunology and Infection

2.6K Views.  Texas Biomedical Research Institute. 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.

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

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most common forms of disease are visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). Mouse models of leishmaniasis are widely used, but quantification of parasite burdens during murine disease requires mice to be euthanized at various times after infection. Parasite loads are then measured either by microscopy, limiting dilution assay, or qPCR amplification of parasite DNA. The in vivo imaging system (IVIS) has an integrated software package that allows the detection of a bioluminescent signal associated with cells in living organisms. Both to minimize animal usage and to follow infection longitudinally in individuals, in vivo models for imaging Leishmania spp. causing VL or CL were established. Parasites were engineered to express luciferase, and these were introduced into mice either intradermally or intravenously. Quantitative measurements of the luciferase driving bioluminescence of the transgenic Leishmania parasites within the mouse were made using IVIS. Individual mice can be imaged multiple times during longitudinal studies, allowing us to assess the inter-animal variation in the initial experimental parasite inocula, and to assess the multiplication of parasites in mouse tissues. Parasites are detected with high sensitivity in cutaneous locations. Although it is very likely that the signal (photons/second/parasite) is lower in deeper visceral organs than the skin, but quantitative comparisons of signals in superficial versus deep sites have not been done. It is possible that parasite numbers between body sites cannot be directly compared, although parasite loads in the same tissues can be compared between mice. Examples of one visceralizing species (L. infantum chagasi) and one species causing cutaneous leishmaniasis (L. mexicana) are shown. The IVIS procedure can be used for monitoring and analyzing small animal models of a wide variety of Leishmania species causing the different forms of human leishmaniasis.

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

An in vivo imaging system is used to generate quantitative measurements of murine infection with the Trypanosomatid protozoan Leishmania. This is a non-invasive and non-lethal method for detecting parasites expressing luciferase within many tissues throughout the course of chronic Leishmania spp. infection.

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most ...

A Luciferase-fluorescent Reporter Influenza Virus for Live Imaging and Quantification of Viral Infection

Influenza A viruses (IAVs) cause human respiratory disease that is associated with significant health and economic consequences. As with other viruses, studying IAV requires the use of laborious secondary approaches to detect the presence of the virus in infected cells and/or in animal models of infection. This limitation has been recently circumvented with the generation of recombinant IAVs expressing easily traceable fluorescent or bioluminescent (luciferase) reporter proteins. However, researchers have been forced to select fluorescent or luciferase reporter genes due to the restricted capacity of the IAV genome for including foreign sequences. To overcome this limitation, we have generated a recombinant replication-competent bi-reporter IAV (BIRFLU) stably expressing both a fluorescent and a luciferase reporter gene to easily track IAV infections in vitro and in vivo. To this end, the viral non-structural (NS) and hemagglutinin (HA) viral segments of influenza A/Puerto Rico/8/34 H1N1 (PR8) were modified to encode the fluorescent Venus and the bioluminescent Nanoluc luciferase proteins, respectively. Here, we describe the use of BIRFLU in a mouse model of IAV infection and the detection of both reporter genes using an in vivo imaging system. Notably, we have observed a good correlation between the expressions of both reporters and viral replication. The combination of cutting-edge techniques in molecular biology, animal research and imaging technologies, provides researchers the unique opportunity to use this tool for influenza research, including the study of virus-host interactions and dynamics of viral infections. Importantly, the feasibility to genetically alter the viral genome to express two foreign genes from different viral segments opens up opportunities to use this approach for: (i) the development of novel IAV vaccines, (ii) the generation of recombinant IAVs that can be used as vaccine vectors for the treatment of other human pathogen infections.

Influenza A viruses (IAVs) are contagious respiratory pathogens that cause annual epidemics and occasional pandemics. Here, we describe a protocol to track viral infections in vivo using a novel recombinant luciferase and fluorescence-expressing bi-reporter IAV (BIRFLU). This approach provides researchers with an excellent tool to study IAV in vivo.

Influenza A viruses (IAVs) cause human respiratory disease that is associated with significant health and economic consequences. As with other viruses, ...

Visualization of SARS-CoV-2 using Immuno RNA-Fluorescence In Situ Hybridization

This manuscript provides a protocol for in situ hybridization chain reaction (HCR) coupled with immunofluorescence to visualize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in cell line&#160;and three-dimensional (3D) cultures of human airway epithelium. The method allows highly specific and sensitive visualization of viral RNA by relying on HCR initiated by probe localization. Split-initiator probes help amplify the signal by fluorescently labeled amplifiers, resulting in negligible background fluorescence in confocal microscopy. Labeling amplifiers with different fluorescent dyes facilitates the simultaneous recognition of various targets. This, in turn, allows the mapping of the infection in tissues to better understand viral pathogenesis and replication at the single-cell level. Coupling this method with immunofluorescence may facilitate better understanding of host-virus interactions, including alternation of the host epigenome and immune response pathways. Owing to sensitive and specific HCR technology, this protocol can also be used as a diagnostic tool. It is also important to remember that the technique may be modified easily to enable detection of any RNA, including non-coding RNAs and RNA viruses that may emerge in the future.

Here, we describe a simple method that combines RNA fluorescence in situ&#160;hybridization (RNA-FISH) with immunofluorescence to visualize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA. This protocol may increase understanding of the molecular characteristics of SARS-CoV-2 RNA-host interactions at a single-cell level.

This manuscript provides a protocol for in situ hybridization chain reaction (HCR) coupled with immunofluorescence to visualize severe acute respiratory ...

Detection of SARS-CoV-2 Neutralizing Antibodies using High-Throughput Fluorescent Imaging of Pseudovirus Infection

As the COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve, it has become evident that the presence of neutralizing antibodies against the virus may provide protection against future infection. Thus, as the creation and translation of effective COVID-19 vaccines continues at an unprecedented speed, the development of fast and effective methods to measure neutralizing antibodies against SARS-CoV-2 will become increasingly important to determine long-term protection against infection for both previously infected and immunized individuals. This paper describes a high-throughput protocol using vesicular stomatitis virus (VSV) pseudotyped with the SARS-CoV-2 spike protein to measure the presence of neutralizing antibodies in convalescent serum from patients who have recently recovered from COVID-19. The use of a replicating pseudotyped virus eliminates the necessity for a containment level 3 facility required for SARS-CoV-2 handling, making this protocol accessible to virtually any containment level 2 lab. The use of a 96-well format allows for many samples to be run at the same time with a short turnaround time of 24 h.

The protocol described here outlines a fast and effective method for measuring neutralizing antibodies against the SARS-CoV-2 spike protein by evaluating the ability of convalescent serum samples to inhibit infection by an enhanced green fluorescent protein-labeled vesicular stomatitis virus pseudotyped with spike glycoprotein.

As the COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve, it has become evident that the ...

Detection of SARS-CoV-2 Receptor-Binding Domain Antibody using a HiBiT-Based Bioreporter

The emergence of the COVID-19 pandemic has increased the need for better serological detection methods to determine the epidemiologic impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The increasing number of SARS-CoV-2 infections raises the need for better antibody detection assays. Current antibody detection methods compromise sensitivity for speed or are sensitive but time-consuming. A large proportion of SARS-CoV-2-neutralizing antibodies target the receptor-binding domain (RBD), one of the primary immunogenic compartments of SARS-CoV-2. We have recently designed and developed a highly sensitive, bioluminescent-tagged RBD (NanoLuc HiBiT-RBD) to detect SARS-CoV-2 antibodies. The following text describes the procedure to produce the HiBiT-RBD complex and a fast assay to evaluate the presence of RBD-targeting antibodies using this tool. Due to the durability of the HiBiT-RBD protein product over a wide range of temperatures and the shorter experimental procedure that can be completed within 1 h, the protocol can be considered as a more efficient alternative to detect SARS-CoV-2 antibodies in patient serum samples.

The outlined protocol describes the procedure for producing the HiBiT-receptor-binding domain protein complex and its application for fast and sensitive detection of SARS-CoV-2 antibodies.

The emergence of the COVID-19 pandemic has increased the need for better serological detection methods to determine the epidemiologic impact of severe ...

Quantifying Vaccine-Induced Neutralizing Antibodies in SARS-CoV-2 Variants

Application of Ha-CoV-2 Pseudovirus for Rapid Quantification of SARS-CoV-2 Variants and Neutralizing Antibodies

The coronavirus disease 2019 pandemic (COVID-19) has highlighted the need for rapid assays to accurately measure the infectivity of emerging SARS-CoV-2 variants and the effectiveness of vaccine-induced neutralizing antibodies against viral variants. These assays are essential for pandemic surveillance and validating vaccines and variant-specific boosters. This manuscript demonstrates the application of a novel hybrid alphavirus-SARS-CoV-2 pseudovirus (Ha-CoV-2) for quick quantification of SARS-CoV-2 variant infectivity and vaccine-induced neutralizing antibodies to viral variants. Ha-CoV-2 is a SARS-CoV-2 virus-like particle consisting of viral structural proteins (S, M, N, and E) and a fast-expressing RNA genome derived from an alphavirus, Semliki Forest Virus (SFV). Ha-CoV-2 also contains both green fluorescent protein (GFP) and luciferase reporter genes that allow for quick quantification of viral infectivity. As an example, the infectivity of the SARS-CoV-2 Delta (B.1.617.2) and the Omicron (B.1.1.529) variants are quantified, and their sensitivities to a neutralizing antibody (27VB) are also measured. These examples demonstrate the great potential of Ha-CoV-2 as a robust platform for rapid quantification of SARS-CoV-2 variants and their susceptibility to neutralizing antibodies.

Author Spotlight: A Pseudotype Virus System for Assessing Omicron Subvariants and Neutralizing Antibodies in SARS-CoV-2 Research

This protocol describes the application of a novel hybrid alphavirus-SARS-CoV-2 pseudovirus (Ha-CoV-2) as a platform for rapid quantification of infectivity of SARS-CoV-2 variants and their sensitivity to neutralizing antibodies.

The coronavirus disease 2019 pandemic (COVID-19) has highlighted the need for rapid assays to accurately measure the infectivity of emerging SARS-CoV-2 ...

Assessing Serum Neutralization Against SARS-CoV-2 with Pseudotyped Viruses

Pseudotyped Viruses As a Molecular Tool to Monitor Humoral Immune Responses Against SARS-CoV-2 Via Neutralization Assay

Pseudotyped viruses (PVs) are molecular tools that can be used to study host-virus interactions and to test the neutralizing ability of serum samples, in addition to their better-known use in gene therapy for the delivery of a gene of interest. PVs are replication defective because the viral genome is divided into different plasmids that are not incorporated into the PVs. This safe and versatile system allows the use of PVs in biosafety level 2 laboratories. Here, we present a general methodology to produce lentiviral PVs based on three plasmids as mentioned here: (1) the backbone plasmid carrying the reporter gene needed to monitor the infection; (2) the packaging plasmid carrying the genes for all the structural proteins needed to generate the PVs; (3) the envelope surface glycoprotein expression plasmid that determines virus tropism and mediates viral entry into the host cell. In this work, SARS-CoV-2 Spike is the envelope glycoprotein used for the production of non-replicative SARS-CoV-2 pseudotyped lentiviruses.
Briefly, packaging cells (HEK293T) were co-transfected with the three different plasmids using standard methods. After 48 h, the supernatant containing the PVs was harvested, filtered, and stored at -80 &#176;C. The infectivity of SARS-CoV-2 PVs was tested by studying the expression of the reporter gene (luciferase) in a target cell line 48 h after infection. The higher the value for relative luminescence units (RLUs), the higher the infection/transduction rate. Furthermore, the infectious PVs were added to the serially diluted serum samples to study the neutralization process of pseudoviruses' entry into target cells, measured as the reduction in RLU intensity: lower values corresponding to high neutralizing activity.

Author Spotlight: Studying Host-Virus Interactions with Pseudotyped Viruses 

Pseudotyped viruses (PVs) are replication-defective virions that are used to study host-virus interactions under safer conditions than handling authentic viruses. Presented here is a detailed protocol that shows how SARS-CoV-2 PVs can be used to test the neutralizing ability of patients' serum after COVID-19 vaccination.

Pseudotyped viruses (PVs) are molecular tools that can be used to study host-virus interactions and to test the neutralizing ability of serum samples, in ...

Analysis of SARS-CoV-2 Using Dual-Reporter and Multiplex Technology

Profiling of Surface Protein Epitopes on Viral Particles by Multiplex Dual-Reporter Strategy

Membrane proteins on enveloped viruses play an important role in many biological functions involving virus attachment to target cell receptors, fusion of viral particles to host cells, host-virus interactions, and disease pathogenesis. Furthermore, viral membrane proteins on virus particles and presented on host cell surfaces have proven to be excellent targets for antivirals and vaccines. Here, we describe a protocol to investigate surface proteins on intact severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) particles using the dual-reporter flow cytometric system. The assay exploits multiplex technology to obtain a triple detection of viral particles by three independent affinity reactions. Magnetic beads conjugated to recombinant human angiotensin-converting enzyme-2 (ACE2) were used to capture viral particles from the supernatant of cells infected with SARS-CoV-2. Then, two detection reagents labeled with R-phycoerythrin (PE) or Brilliant Violet 421 (BV421) were applied simultaneously. As a proof-of-concept, antibody fragments targeting different epitopes of the SARS-CoV-2 surface protein Spike (S1) were used. The detection of viral particles by three independent affinity reactions provides strong specificity and confirms the capture of intact virus particles. Dose-dependency curves of SARS-CoV-2 infected cell supernatant were generated with replicate coefficient variances (mean/SD) &#706;14%. Good assay performance in both channels confirmed that two virus surface target protein epitopes are detectable in parallel. The protocol described here could be applied for (i) high-multiplex, high-throughput profiling of surface proteins expressed on enveloped viruses; ii) detection of active intact viral particles; and (iii) assessment of specificity and affinity of antibodies and antiviral drugs for surface epitopes of viral antigens.The application can be potentially extended to any type of extracellular vesicles and bioparticles, exposing surface antigens in body fluids or other liquid matrices.

Author Spotlight: Advancing Antiviral Strategies Through Novel Immunocapture and Mass Spectrometry Techniques

Here, we describe a newly developed multiplex fluorescent immunoassay that uses a dual-reporter flow cytometric system to concurrently detect two unique spike protein epitopes on intact severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral particles that had been captured by angiotensin-converting enzyme-2-coupled magnetic microspheres.

Membrane proteins on enveloped viruses play an important role in many biological functions involving virus attachment to target cell receptors, fusion of ...