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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Mouse foot pad abrasion
- Anesthetize a C57BL/6 mouse (5–7 weeks old) with isoflurane gas anesthetic, delivered at a dosage of 3% via a small anesthesia system (chamber).
- Place the mouse in the surgical plane of anesthesia on its back, prior to inoculation, in the hind footpad. Attach a nose cone with a diaphragm (a slit sufficiently sized to fit the muzzle of the animal) to the mouse and deliver isoflurane gas anesthetic at a constant dosage of 1.5%–2.0%.
- Closely monitor the mouse for a response to painful stimulus created by forceful pinching of the toe with forceps.
- Lightly grasp one hind footpad with flat forceps.
- Gently abrade the glabrous skin of the hind footpad approximately 20x, between the heel and walking pads, with an emery board (100–180 grit). Do not induce bleeding by abrading too frequently or applying too much pressure.
- Using fine forceps, slowly peel off the stratum corneum detached by abrasion to expose the stratum basale.
2. PRV footpad inoculation
- Prepare the virus inoculum diluted to the desired titer in DMEM media containing 2% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Table of Materials).
- Quantitate the number of plaque-forming units (PFU) in the virus stock on PK15 cells to calculate and standardize the viral dose and dilute the viral inoculum accordingly.
NOTE: In this study, virulent strain of PRV (PRVBecker) was used at a dose of 8 x 106 PFU. This dose was optimized in a previous preliminary experiment to ensure that all inoculated animals showed clinical symptoms at 82 h post-inoculation (hpi).
- Keep the virus inoculum on ice and gently mix before use.
- Add a 20 μL droplet of virus inoculum (8 x 106 PFU) onto the abraded footpad (topical administration). Carry out mock inoculations (medium only) in parallel.
- Gently rub 10x with the shaft of a needle to facilitate adsorption of the virus. Avoid scratching with the needle point. Repeat this step every 10 min.
- Keep the mouse under anesthesia for 30 min until the abraded footpad is dry.
- After stopping anesthesia, monitor the mouse until it can maintain sternal recumbency and place it in an individual cage for clinical follow-up and sampling.