A Luciferase-Fluorescent Reporter Influenza Virus to Track Viral Infections

Start with a pathogenic recombinant influenza virus with a genome modified to incorporate compact reporter genes.

These reporter genes encode a small luciferase bioluminescent enzyme and a monomeric fluorescent protein.

Inoculate the virus intranasally into an anesthetized mouse, appropriately shaved for subsequent imaging.

The virus enters the host cell and releases its genetic contents.

Inside the nucleus, the viral genome transcribes into mRNA molecules and subsequently translates to produce luciferase and fluorescent protein in the host cell.

Inject a luciferin substrate retro-orbitally into the mouse.

The luciferase enzyme oxidizes luciferin to oxyluciferin, which on relaxation, emits a bioluminescent signal.

Measure the signal to estimate luciferase levels, enabling viral infection monitoring in a live animal.

For ex vivo analysis, excise the lungs and capture the fluorescence signal through a suitable imaging technique.

Unlike regular variants, monomeric fluorescent proteins do not form aggregates, yielding a clear image to track viral infection accurately.

To infect the mice, start by preparing the bird flu in 1X PBS. Maintain the virus on ice until inoculation. Check for the absence of the toe pinch reflex to make sure that the mouse is anesthetized, and inoculate it intranasal with the prepared bird flu dilution. Ensure all mice are breathing properly before returning them to their cages.

Open the imaging software and press Initialize, then, set the parameters that will be used for the imaging. To monitor the mice infected with bird flu, shave their chest to improve detection of the bioluminescent signal.

Once the mice are anesthetized, use a 22-gauge needle to retro-orbitally administer 100 microliters of NLuc luciferase substrate diluted 1 to 10 in PBS. Immediately after injection, place the animals in the isolation chamber with their chest facing up, then, place the isolation chamber in the imaging instrument, close the door, and acquire images.

After imaging, return the mice to their cages, and monitor them until they have fully recovered. Use the imaging software ROI tool to analyze the acquired bioluminescence data by designating a region of interest and clicking Measure.

To perform ex vivo imaging of mouse lungs, collect the lungs according to the manuscript directions, start the image acquisition software, and set the parameters for imaging. Once the machine is initialized, place the lungs in a black background tray and ensure that the tissues are separated from one another. Introduce the tray into the imager, close the door, and acquire images.

After imaging, remove the tissues immediately and store them on ice at 4 degrees Celsius if the samples will be processed on the same day. If they will be processed later, freeze them quickly in a tube on dry ice, and store them at minus 80 degrees Celsius. To process the images, select the ROI tool, draw regions of interest around each of the individual lungs, and click Measure.