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

All protocols involving mice have been approved by the Institutional Animal Care and Use Committee (IACUC) and the Institutional Biosafety Committee (IBC) at the University of Rochester, School of Medicine and Dentistry. All the experiments performed in animals follow the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Research Council⁵⁸. The Vivarium and Division of Laboratory Animal Medicine facilities at the School of Medicine and Dentistry at University of Rochester is accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care (AALAC) International and conform to federal and state laws and National Institutes of Health (NIH) policy. Proper Personal Protection Equipment (PPE) is required when working with mice. Similar policies and requirements should be implemented when performing experiments outlined within this manuscript at each institution.

1. Use of Small Vertebrate Animals

1. Purchase five to seven-week-old female BALB/c mice and maintain them in the animal care facility under specific pathogen-free conditions. Upon mice arrival to the determined facilities, allow period of rest for 4–5 days to allow animals acclimate to their new environment.
2. Following IACUC protocols, place a maximum of 5 mice per cage.
   NOTE: At experiment conclusion, in order to ensure that the animal is dead, mice were euthanized using two approved procedures, taking into consideration that the second must be a physical method. In this study, after mice infection with BIRFLU and in vivo imaging, animals are euthanized with a lethal dose of 2,2,2-tribromoethanol (TBE), and by cutting the hepatic vein as the physical secondary method as we have previously shown²³.

2. Biosafety

NOTE: In this manuscript, BIRFLU was generated in the backbone of influenza A/Puerto Rico/08/34 H1N1 (PR8), which is a common mouse-adapted laboratory IAV strain^23,32,33,56. The virus was generated using previously described plasmid-based reverse genetics approaches and a complete description of the generation, and in vitro and in vivo characterization of BIRFLU can be found in our recent publication⁵⁵. All procedures that involve IAV infections (in vitro or in vivo) were performed in a biological safety cabinet under biosafety level (BSL)-2 conditions.

CAUTION: An appropriate biosafety level should be determined in accordance with a biosafety risk assessment. Additional information on performing biosafety risk assessments and establishing effective biosafety containment should be consulted with the institution where the experiments will be performed.

1. Clean the biosafety cabinets with 70% ethanol or chlorine dioxide disinfectant before and after performing all the experimental procedures. For mice work, sterilize all dissection material (scissors, dissecting forceps, etc.) and the Dounce homogenizer before and after their use.
2. Discard all biological material produced during the procedures under proper IBC and IACUC guidelines.

3. In Vitro Characterization of BIRFLU (Figure 2)

NOTE: Refer to Table 1 for all buffer and media compositions.

1. Analyze protein expression by fluorescence (Figure 2A) and indirect immunofluorescence (Figure 2B)
   1. One day before infection, seed 24-well plates with Madin-Darby Canine Kidney (MDCK) epithelial cells (1 x 10⁵ cells/well, triplicates) in tissue culture media and maintain the plates in a 37 °C incubator with 5% CO₂. Prepare enough wells to evaluate all the chosen antibodies in both mock-infected and BIRFLU-infected cells.
      NOTE: We recommend visualizing the cells under a light microscope to confirm a monolayer before starting the viral infection. A 90% confluency monolayer of MDCK cells by the time of infection is recommended.
   2. Prepare dilutions of WT or BIRFLU IAVs in infection media and infect the seeded MDCK cells with a multiplicity of infection (MOI) of 0.1 plaque-forming units (PFU) per cell in a final volume of 0.25 mL/well of infection media.
   3. Remove the tissue culture medium from step 3.1.1 and wash the MDCK cells twice with 1x phosphate-buffered saline (PBS). Add the virus dilutions from 3.1.2 to the MDCK cells and place the plates on a rocking platform for 1 h at room temperature to allow viral adsorption.
      NOTE: Mock-infected cells are incubated only with infection media in the absence of virus.
   4. After viral adsorption (step 3.1.3), remove the viral inoculum by aspiration and add 1 mL of post-infection media containing 1 μg/mL of TPCK-treated trypsin to each well. Incubate cells for 18 h in a 5% CO₂ humidified incubator at 33 °C.
   5. After 18 h of infection, remove the tissue culture supernatant from the 24-well plates (3.1.4). Fix and permeabilize the cells with 0.25 mL/well of fixation/permeabilization solution for 20 min at room temperature.
      NOTE: Prepare the fixation/permeabilization solution in a fume hood to prevent formaldehyde exposure.
   6. Remove the fixation/permeabilization solution from step 3.1.5, wash the cells twice with 1x PBS, and incubate the cells with 0.25 mL/well of blocking solution for 1 h at room temperature.
      NOTE: Proceed to the next step or store cells in blocking solution at 4 °C overnight.
   7. Remove the blocking solution (step 3.1.6) and add 0.25 mL/well (1 µg/mL) of a mouse monoclonal antibody (MAb) against the IAV nucleoprotein, NP (HB-65) or a 1:1,000 dilution of a rabbit polyclonal antibody (pAb) against NLuc (see the Table of Materials), both diluted in antibody dilution solution (1x PBS, 2.5% BSA), and incubate the cells for 1 h at 37 °C.
   8. Remove the primary antibody from step 3.1.7, wash the cells three times with 1x PBS and incubate them with 0.25 mL/well of a Texas Red-conjugated anti-mouse or anti-rabbit secondary antibodies (see the Table of Materials) diluted 1:200 in antibody dilution solution.
      NOTE: Cell nuclei can be stained at the same time by adding 0.5 µg/mL of 4’,6’-diamidino-2-phenylindole (DAPI) to the same secondary antibody solution. Incubate the cells for 1 h at 37 °C in the dark. Other secondary antibodies conjugated with other fluorophores can be used.
   9. Remove the secondary antibody and DAPI from step 3.1.8, wash the cells three times with 1x PBS. After washing, leave the cells in 0.25 mL/well of 1x PBS.
   10. Place the plate on the stage of a fluorescence microscope to detect Venus and Nluc reporter expression, and NP from infected cells using the proper fluorescent filters. Capture images using a fluorescence microscope (20x magnification) and merge them using an image editing software (Figure 2A,B).
2. Analyze the Nluc activity (Figure 2C) and viral replication (Figure 2D).
   1. One day before infection, seed 12-well plates with MDCK cells (2 x 10⁵cells/well, triplicates) using tissue culture media in a 37 °C incubator with 5% CO₂ to reach approximately 90% confluency by the time of infection.
      NOTE: Before infection, check the cells under a microscope to verify a monolayer of MDCK cells.
   2. The day of infection prepare dilutions of the WT and BIRFLU viruses in infection media to infect the MDCK cells (step 3.2.1.) in triplicate with a MOI of 0.001 PFU in 0.5 mL/well. Remove the tissue culture medium and wash the MDCK cells twice with 1x PBS.
   3. Add the virus dilutions to the MDCK monolayers and allow viral adsorption at room temperature for 1 h on a rocking platform. After viral adsorption, remove the virus inoculum and add 1.5 mL of post-infection media containing 1 μg/mL of TPCK-treated trypsin, to each well. Incubate infected cells in a 5% CO₂ humidified incubator at 33 °C and collect supernatants at the indicated time points indicated in step 3.2.4.
   4. At 24, 48, 72, and 96 h post infection (p.i.) collect 150 µL of tissue culture supernatant from each well and store the samples in a microcentrifuge tube at -80 °C to perform the Nluc assays and viral titrations.
   5. To perform the Nluc activity assay, follow the manufacturer’s indications. First, thaw the tissue culture supernatant stored at -80 °C (step 3.2.4) on ice.
      NOTE: Refer to the recommendations from the manufacturer and optimize the assay if required.
   6. Prepare the luciferase assay solution (see the Table of Materials) by diluting the Nluc substrate 1:50 with the dilution buffer. In a white flat-bottom 96-well microplate for luciferase assays, mix 25 µL of luciferase assay solution with 10 to 25 µL of the collected tissue culture supernatant samples. Incubate the mix for 2–3 min before measuring the luminescence using a luminometer (Figure 2C).
      NOTE: Presence of infectious viral particles in the tissue culture supernatants is determined by fluorescence immune-focus assay (Venus) or indirect immunofluorescence (viral antigen staining) as previously described^23,32,33,56.
   7. A day before viral titrations, seed MDCK cells in a 96-well plate (2 x 10⁴ cells/well, triplicates) with tissue culture media and allow cells to reach 90% confluence by the time of infection in an incubator set at 37 °C with 5% CO₂.
   8. Use the tissue culture supernatant samples that were thawed on ice (step 3.2.4.). Add 90 μL of infection media to each of the wells in a new 96-well plate, then add 10 μL of the thawed tissue culture supernatant (step 3.2.4) to the first well (row A). Use a multichannel pipet to mix and transfer 10 μL from row A to row B. Change the tips between dilutions and mix well.
      NOTE: This procedure should be repeated until the last row (H). We recommend performing viral titrations in triplicates for accurate measure of viral titers.
   9. Remove the tissue culture medium from the MDCK cells in the 96-well plates (step 3.2.7) and wash twice with 1x PBS. Add 50 µL of the supernatant dilutions of the replica 96-well plate containing the virus dilutions (step 3.2.8) to each well in the 96-well plate containing MDCK cells.
   10. Incubate the 96-well plate on a rocking platform for 1 h at room temperature to allow viral adsorption. After viral adsorption, remove the inoculum and add 100 μL/well of post-infection media containing 1 μg/mL of TPCK-treated trypsin. Incubate infected cells for 12 h in a 33 °C incubator with 5% CO₂.
   11. Since BIRFLU expresses Venus, the number of infected cells can be directly counted using a fluorescence microscope. Alternatively, viral titers can be determined by indirect immunofluorescence as previously described using an antibody against a viral protein^23,56,57,59. For the latter, proceed to fix/permeabilize the cells and stain using the anti-NP mAb HB-65 as described in section 4.4.
   12. Determine the viral titers by counting the number of fluorescent-forming units (FFU)/mL using the formula: ((number of FFU) x 20 x 1/dilution (Figure 2D).

4. In Vivo Characterization of BIRFLU (Figure 3 and Figure 4)

1. Mouse infection
   NOTE: Intranasal infection of mice was done as previously described²³. For a more detailed protocol of IAV infection in vivo using the mouse model of infection, we recommend viewing the video associated with the previous publication²³. This section only summarizes the steps required for mouse infection with BIRFLU.
   1. Inspect the mice to evaluate their health and overall physical appearance. Prepare the dilution of BIRFLU in 1x PBS to inoculate mice with 1 x 10⁶ PFU of BIRFLU in a total volume of 30 μL/mouse. Maintain the virus on ice until mouse inoculation.
      NOTE: Mock-infected (1x PBS) mice will be needed as internal control for imaging. Place mock-infected mice in a different cage than BIRFLU-infected animals.
   2. Anesthetize five-to-seven-week-old female BALB/c mice intraperitoneally with 240–250 mg/kg of tribromoethanol (TBE) by inserting the needle into the caudal 2/3 of the right side of the abdomen. Then, return the mouse to the cage and wait for around 5 min. Check that the mouse is anesthetized before virus inoculation by the absence of the toe-pinch reflex.
      NOTE: Injectable sedatives are recommended over inhaled sedatives for influenza infections in vivo, as the latter can modify the behavior and uptake of the virus by the airway epithelium. Moreover, they can also affect local immune responses of the lung and interfere with the in vivo imaging of infected mice. Finally, inhaled sedatives have reduced duration of effect, which would be a problem for in vivo imaging of the infected animals. 
   3. Inoculate the mice intranasally with the 30 μL of the prepared BIRFLU dilution. Check that the mice are breathing properly before returning them to the cage.
2. Bioluminescence monitoring of mice infected with BIRFLU (Figure 4A)
   NOTE: In this manuscript reporter expression of Nluc or Venus and viral replication in the lungs of mice infected with BIRFLU are determined at 3 days post-infection. However, the nature of bioluminescence imaging allows repeated monitoring of Nluc expression in individual BIRFLU-infected animals without the need to euthanize them for each experimental time-point. An in vivo imaging system with an isoflurane anesthesia manifold is required to perform the described experimental procedures. See the Table of Material for details regarding the imaging instrument and image software used for the image acquisition and data analysis.
   1. Shave the mice chest to improve the bioluminescence signal. Open the imaging software and press Initialize. Next, set the parameters that will be used, including setting the imaging mode to bioluminescence, auto saving, exposure time to auto, open filter, etc.
   2. Once the machine is fully initialized, turn on the isoflurane anesthesia system. Place animals in the anesthesia chamber. Mice are simultaneously and lightly anesthetized with a mixture of oxygen gas and vaporized 1–2% isoflurane.
   3. Once mice are anesthetized, administer the Nluc substrate (see the Table of Materials) diluted 1:10 in 1x PBS (final volume 100 µL/mouse) via retro-orbital route using a syringe with a 22 G needle.
   4. Immediately after Nluc reagent administration, place the animals in the imaging instrument with their chests facing up and snout inside the manifold cone to keep animal anesthetized during imaging. Immediately after closing the imager door, click Acquire in the software program (Figure 4A, top).
   5. After imaging, return the mice to their cages monitoring them until they have fully recovered and turn off the isoflurane vaporizer. Then, proceed with the ex vivo imaging of mice lungs to evaluate Venus reporter gene expression (section 4.3).
   6. Use the imaging software tools to analyze the acquired bioluminescence data. Utilize the tool ROI (region of interest) to designate the specific signal and perform flux measurements in the region of interest (usually around the chest) (Figure 4A, bottom). Although ROI shape is irrelevant, larger ROIs are generally preferred to capture the entire signal diffusion area.
   7. Click Measure. Assess the bioluminescence in photons since it provides an absolute photon emission measurement that is comparable to output measurements provided by different parameters or imaging instruments.
3. Fluorescence analysis in mice infected with BIRFLU (Figure 3 and Figure 4B)
   1. Collect the mouse lungs after in vivo imaging, as previously described²³.
      1. Briefly, euthanize mice with a lethal dose of TBE (500 mg/kg). Disinfect the incision site with 70% ethanol. With a scalpel, make an incision from the sternum to the base of the abdomen and then cut from the bottom of the incision to the sides with scissors. Next, cut the hepatic vein (second physical euthanasia method) to bleed the animal.
         NOTE: To avoid high background signal during imaging, it is important to minimize the amount of blood in the lungs (see below).
   2. Place the mouse in dorsal recumbency, and use the scissors to cut the pleura and open the rib cage. Subsequently, remove the lungs by snipping the end of the trachea with scissors while gently holding the lungs with forceps.
      1. Place the lungs in a six-well plate with 2 mL of 1x PBS and wash the lungs three times with 1x PBS.
         NOTE: To avoid contamination between samples, clean and disinfect the dissecting tools between each animal.
   3. Start the image acquisition software by click initialize and set the parameters for imaging, including setting imaging mode to fluorescence, auto saving, exposure time to auto, excitation (500 nm) and emission (540 nm) filters.
   4. When the machine is initialized, place the lungs in a black background tray, ensuring that the tissues are separated from one another, introduce the tray into the imager, and click Acquire on the imaging system program after closing the imager door (Figure 4B, top).
   5. After imaging, remove the tissues immediately and store them on ice (4 °C) if the samples are processed the same day; or on a tube and dry ice to freeze them quickly, before storing them at -80 °C, if the samples will be processed later on a different day.
   6. For image processing, select the ROI tool and draw ROIs around each of the individual lungs. Click Measure. Then, using the resulting average radiant efficiency measurements, subtract the values from the mock-infected mice (Figure 4B, bottom).
      NOTE: With BIRFLU, there is a good correlation of the levels of Nluc and Venus expression, and signal distribution. Therefore, it is important to analyze Nluc (whole mouse) and Venus (excised lungs) expression from the same animal, and maintain the same orientation.
4. Evaluation of BIRFLU replication in mice lungs (Figure 3 and Figure 4C)
   1. Homogenize mice lungs for viral titration by plaque assay as previously described²³.
5. Place the lungs into a Dounce homogenizer with 1 mL of chilled infection media. Homogenize the lungs with the pestle for 1 min at room temperature until it has fully disintegrated and store the samples in a sterile tube at 4 °C.
   1. Centrifuge the samples at 300 x g for 10 min and collect the supernatant in a sterile tube. Store the samples on ice if they will be used on the same day or freeze them at -80 °C to evaluate viral titers at a later time.
      NOTE: A new Dounce homogenizer should be used for each lung sample.
   2. To perform the plaque assay, the day prior to performing viral titration, seed six-well plates with MDCK cells (5 x 10⁵ cells/well) in tissue culture media. Incubate the cells overnight at 37 °C with 5% CO₂ to reach 90% confluence next day. Before infection, confirm that the cells form a monolayer under a light microscope.
   3. Prepare 1:10 serial dilutions of the supernatant from the homogenized samples (step 4.4.1) in infection media. Prepare microcentrifuge tubes with 540 µL of infection media, add 60 µL from the homogenized lungs sample to the first tube, mix by pipetting up and down, and then using a new tip, transfer 60 µL to the next tube. Repeat this serial dilution process until the last dilution.
      NOTE: In our experience 7 dilutions are sufficient to determine viral titers by plaque assay from the lungs of infected mice.
   4. Wash the MDCK cells (step 4.4.3) twice with 1x PBS and transfer 500 µL of the serially diluted samples to each well in the six-well plate. Place the plates on a rocking platform and allow viral absorption to occur for 1 h at room temperature.
   5. After 1 h of viral absorption, remove the viral inoculum, add 2 mL/well of overlay medium containing 1 μg/mL of TPCK-treated trypsin, and then incubate the cells at 33 °C under 5% CO₂ for 3 days.
   6. Fix infected cells (step 4.4.6) with 1 mL/well of 4% formaldehyde diluted in 1x PBS at room temperature for 2 h, then carefully remove the overlay medium and add 1 mL of 1x PBS to each well. For visualization of Venus, image the six-well plates with an imaging system for the detection of fluorescence.
      NOTE: Viral plaques can be colored using an image editing software (see the Table of Materials).
   7. To evaluate Nluc expression, visualize viral plaques by immunostaining using an anti-Nluc pAb. For this, remove the 1x PBS, permeabilize cells using 0.5 mL/well of permeabilization solution for 15 min at room temperature.
   8. Remove the permeabilization solution, wash the cells three times with 1x PBS and block with 0.5 mL/well of blocking solution for 1 h at room temperature.
   9. Remove the blocking solution from step 4.4.9 and incubate the cells with 0.5 mL/well of the pAb anti-Nluc diluted 1:1,000 in dilution solution for 1 h at 37 °C.
   10. Use ABC Alkaline Phosphatase kit and DAB Peroxidase Substrate Kit following manufacturer’s recommendation for visualization of Nluc-expressing plaques.
       1. Briefly, wash cells from 4.4.10 three times with 1x PBS and incubate them with 0.5 mL/well of biotinylated anti-rabbit secondary antibody for 1 h at 37 °C.
       2. Remove the secondary antibody, wash the cells three times with 1x PBS, and incubate with the ABC solution for 1 h at 37 °C. Wash the cells three times with 1x PBS and visualize the viral plaques with the DAB HRP substrate Kit. Scan the immunostained plaques using a conventional scanner.
          NOTE: Other similar kits for immunostaining could be used.
   11. Stain the viral plaques with crystal violet solution for 1 h at room temperature. Discard the crystal violet, wash the plates three times with water, allow plates to dry, and scan the plates again.
   12. To determine viral titers, count the plaques revealed after crystal violet staining. Scan the plaques using a conventional scanner. Calculate virus titer as plaque forming units (PFU) per mL (PFU/mL).
   13. To assess BIRFLU stability in vivo, calculate the percentage of reporter-expressing viruses by counting the number of crystal violet-stained plaques (number of infectious viruses, step 4.4.12) and compare with the number of Venus- and Nluc-expressing plaques (step 4.4.7 and step 4.4.11, respectively).