Researchers have previously generated recombinant influenza viruses that express either fluorescent or bioluminescent protein reporters. Part of our single expression of reporter genes in influenza virus severely limits its use in experiments. Our protocol describes the use of bireporter influenza virus that expresses both fluoresent and bioluminescent reporter genes, and therefore surmounts its limitation.
The main advantage of this technique is the use of a single recombinant influenza virus for in vitro and in vivo studies. When performing this technique, rapid injection of luciferase substrate and proper planning of mice handling is important, because the substrate is quickly metabolized. Also, shaving of mice is recommended to improve detection of bioluminescent signal.
To infect the mice, start by preparing the BIRFLU 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 intranasally with the prepared BIRFLU 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 BIRFLU, 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 one 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 four 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 80 degrees Celsius. To process the images, select the ROI tool, draw regions of interest around each of the individual lungs, and click measure. BIRFLU has been characterized in vitro by determining Venus, Nluc, and NP expression levels in infected cells.
Venus and Nluc expression are only detected in cells infected by BIRFLU, while NP is expressed in both wild-type PR8 and BIRFLU-infected cells. No expression is observed in mock-infected cells. Furthermore, Nluc activity has been measured in tissue culture supernatants at 24, 48, 72, and 96 hours post-infection.
It is detected as early as 24 hours post-infection, and the expression levels increase at 96 hours. It can also be demonstrated that replication kinetics of BIRFLU are comparable to the wild-type PR8 virus. BIRFLU has the ability to express both fluorescent and bioluminescent reporter genes, and the correlation between the two can be assessed using in vivo and ex vivo imaging of a mouse lung.
The lungs of mice infected with BIRFLU displayed high bioluminescence in vivo and fluorescence ex vivo. Viral titers and genetic stability of BIRFLU in vivo have also been determined. Plaque assays on the viruses from mouse lungs showed that the virus can form plaques and express both reporter genes.
BIRFLU could also be used to evaluate the effectiveness of antiviral and neutralizing antibodies against influenza. The output from these assays all were correlated with each other and are quick and easy to do.
