Naïve 4-week-old female BALB/c mice were purchased from the Jackson Laboratory (Bar Harbor, USA) and housed in the university biological resource laboratory for a period of 1 week for acclimatization before each one was subcutaneously injected with 2 mg of medroxyprogesterone (Depo-Provera). On day 5 after injection, mice were intravaginally infected with 1 × 106 PFU HSV-2 (333 strain). Similar to the ocular model of infection, DECON was applied topically (intravaginally) on alternate days, while ACV (50 mg/ml in PBS) was administered via an intraperitoneal injection. PBS was applied intravaginally as mock treatment for the control group. Vaginal swabs were collected using a Calgiswab (Calcium Alginate Mini-tip Urethro-Genital Swab, Puritan) dipped in Opti-MEM (Gibco, USA). Images of the anogenital region were taken on day 0 and day 7 after infection using a Carl Zeiss stereoscope at 7.5× magnification.

Experiments involving animals were performed under a UIC (University of Illinois at Chicago)–approved protocol ACC 14-091. The mice were monitored for weight loss, and disease scores were recorded in a blinded fashion for 14 days. Sick mice were euthanized according to the Institutional Animal Care and Use Committee protocol followed by the collection of ocular/vaginal tissue and lymph nodes. Ocular wash and vaginal swabs were used to assess viral titers using a plaque assay.

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