2.2. Guinea Pig Inoculation, Clinical Assessment and Tissue Collection

3–4-week-old female Hartley guinea pigs (Charles River Laboratories, Wilmington MA) were inoculated under anesthesia (ketamine/xylazine) vaginally (VAG) by pipette or intradermally (ID) by tuberculin syringe (n = 16 each group) with RVx201 (2 × 10⁷ PFU/mL, n = 16 ID, n = 3 VAG)), RVx202 (2 × 10⁷ PFU/mL, n = 16 ID, n = 3 VAG)), WT HSV-2 MS (2 × 10⁵ PFU/mL, n = 16 ID, n = 5 VAG)); or were inoculated ID with an equivalent volume of Vero cell lysate (n = 3), or were mock infected with PBS (n = 3). Guinea pigs were assessed daily for weight gain/loss and signs of distress and/or disease for 28 days post inoculation (dpi). Genital disease was assessed on a well-established 4-point severity scale, with 0 = no disease, 1 = redness and/or swelling, 2 = 1–2 lesions, 3 = 3–5 lesions, 4 = 6 or more lesions or coalescence of lesions, and recurrences were defined as presence of vesicular lesions on the first day of appearance. Clinical disease following intradermal inoculation was assessed on a similar 4-point severity scale, assessing for presence of clinical symptoms on the inner thigh at or near the site of inoculation. Neurological signs were assessed as presence of urinary retention and hindlimb weakness or paralysis each day. Guinea pigs were vaginally swabbed daily for quantification of viral shedding in vaginal secretions using FLOQSwabs (Copan Diagnostics, Murrieta, CA, USA) moistened with Dulbecco’s Modified Eagle Medium (DMEM, Thermo Fisher, Waltham MA, USA). Swabs in DMEM were stored at −80 °C for assessment via plaque assay. At study end, following euthanasia, sensory and autonomic ganglia including lumbosacral dorsal root ganglia (LS-DRG) sacral sympathetic ganglia (SSG), trigeminal ganglia (TG), superior cervical ganglia (SCG), brain and injection site skin were collected to assess for the presence of HSV-2 by qPCR and immunofluorescence (IF), expression of LAT by qRT-PCR and fluorescent in situ hybridization (FISH), and the ability to reactivate by explant reactivation. Blood was collected by cardiac puncture into serum separator tubes and centrifuged for 10 min at 3000 rpm. Sera and tissues, including brain, liver, kidney, and injection site skin, were collected in 4% PFA and subjected to immunological and histological analyses.