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.

Our protocol described the use of fluorescent or luminescent-expressing recombinant SARS-CoV-2 for in vivo and ex vivo live imaging viral detection and viral tracking in the K18 human ACE2 transgenic mouse model of SARS-CoV-2 infection. The main advantage of this technique is the application and feasibility of viral detection and tracking for in vivo and ex vivo studies of SARS-CoV-2 in the K18 human ACE2 transgenic mouse model. The use of the in vivo imaging of SARS-CoV-2 in the K18 human ACE2 transgenic mouse model represent an excellent option to evaluate therapeutics for the treatment of SARS-CoV-2 infection in vivo.

Implementation of reporters SARS-CoV-2 represent an excellent option to assess pathogenicity, viral replication, and trophism in vivo. For in vivo studies with luciferase expressing SARS-CoV-2, shaving the mice and prompt imaging after substrate injection is recommended to improve of bioluminescence signal. Begin bioluminescence monitoring by initiating the in vivo imaging system, or IVIS, and setting up the parameters, click the image mode to Bioluminescence then open the filter and set the exposure time to Auto.

Next, remove an already shaved and anesthetized mouse from the isolation chamber and use a 25 gauge needle to retro-orbitally administer 100 microliters of the luciferase substrate diluted 1 to 10 in PBS to the mouse. Then place the mouse back into the isolation chamber, transfer the isolation chamber to the IVIS machine, which is equipped with a heating component to prevent hypothermia, and close the IVIS imager door to initialize imaging by selecting Acquire in the software program. To analyze the acquired bioluminescence images, utilize the ROI or region of interest tool in the software to designate the precise signal and measure the flux.

Initiate the imaging software and set up the parameters. Click the image mode to Fluorescence, set excitation at 500 nanometers and emission filters at 530 nanometers, and set the exposure time to Auto. Conduct necroscopy and excise lungs from mice.

Place excised lungs in a 6-well plate containing two milliliters of 1X PBS and rinse to remove excess blood. Disinfect and clean surgery tools between the samples using 70%ethanol. After initializing the IVIS machine, place the lungs on a black sheet and separate the tissues from each other.

Then place the tray inside the isolation chamber inside the biosafety cabinet and then transfer the isolation chamber to the IVIS. Close the IVIS imager door and click on Acquire to initiate the imaging system. 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 the mock-infected mice. Once imaging is complete, place the tissues in a cryotube for dry ice freezing to store at minus 80 degrees Celsius for later processing. Utilizing the supernate obtained from the tissue homogenates, perform tenfold serial dilution and infect confluent monolayers of Vero E6 cells with one milliliter of each supernatant dilution in triplicates.

Let the virus adsorb for one hour at 37 degrees Celsius in a humidified 5%carbon dioxide incubator. Once done, wash the cells with one milliliter of 1X PBS and then incubate the cells in two milliliters of post-infection media containing 1%agar in the humidified 5%carbon dioxide incubator at 37 degrees Celsius for 72 hours. After the incubation, inactivate the plates in 10%neutral buffered formalin for 24 hours at four degrees Celsius, ensuring the entire plate is submerged.

Take plates out of biosafety level three and wash the cells three times with one milliliter of 0.5%Triton X-100 for 10 minutes at room temperature. Next, block the permeabilized cells with one milliliter of 2.5 bovine serum albumin or BSA and PBS for one hour at 37 degrees Celsius followed by incubation in one milliliter of one microgram per milliliter of the SARS-CoV nucleocapsid protein cross-reactive monoclonal antibody 1C7C7 diluted in 2.5 BSA for one hour at 37 degrees Celsius. Wash the cells three times with one milliliter of 1X PBS and develop the plaques using the ABC and diaminobenzene, or DAB, peroxidase substrate kits according to the manufacturer's instructions, then calculate the viral titers as plaque forming units per milliliter.

In the study, it was observed that the bioluminescent nanoluciferase, or Nluc, was easily detected and mice infected with the rSARS-CoV-2 Nluc, but not those infected with rSARS-CoV-2 wild-type or mock infected. The quantitative analyses showed Nluc intensity at different days post-infection. Gross lesions on the lung surface of mice infected with rSARS-CoV-2 Nluc were comparable to those in the rSARS-CoV-2 wild-type infected group.

Additionally, viral titers detected and the rSARS-CoV-2 Nluc infected mice were comparable to those infected with rSARS-CoV-2 wild-type in all organs at different days post-infection. At the same time, Nluc activity was only detected in the organs from rSARS-CoV-2 Nluc infected mice. A separate group of mock infected and virus infected mice was monitored for 12 days for changes in body weight and survival.

Mice infected with rSARS-CoV-2 Nluc and rSARS-CoV-2 wild-type lost up to 25%of their body weight and all succumb to viral infection between seven to eight days post-infection. After rSARS-CoV-2 Venus infection, Venus expression was readily detected in all lungs from mice infected with rSARS-CoV-2 Venus but not those infected with rSARS-CoV-2 wild-type or mock infected. The quantitative analyses showed that Venus intensity peaks at two days post-infection and decreases over the course of infection and the lungs of infected mice.

Images of the lung surface revealed gross lesions of mice infected with rSARS-CoV-2 Venus was comparable to that of rSARS-CoV-2 wild-type infected mice. Also, infection with rSARS-CoV-2 Venus resulted in comparable viral titers to those observed in mice infected with rSARS-CoV-2 wild-type in all organs. The mice infected with rSARS-CoV-2 Venus and rSARS-CoV-2 wild-type lost up to 25%of their body weight and succumb to viral infection by day nine post-infection with no survival.

The results from reporter expressing SARS-CoV-2 have shown to be valid surrogates of viral infection. Thus, this could lead to rapid identification and evaluation of countermeasures for the treatment of SARS-CoV-2 infection.