*2.2. Virus*

African lineage ZIKV MR766 (GenBank accession number: AY632553), recovered from a sentinel rhesus macaque in Uganda in 1947, was used for inoculations (BEI Resources, Manassas, VA, USA). Viral stocks were produced by passage once in Vero E6 cells (CRL-1586, ATCC, Manassas, VA, USA) [60,61]. Viral titer was determined by standard plaque assay on Vero E6 cells [60,61].

#### *2.3. Guinea Pig Subcutaneous and Vaginal Infection*

After a three day acclimation period, three-week-old female Hartley guinea pigs (Charles River Laboratories, Wilmington, MA, USA) were inoculated under anesthesia with 1 × 10<sup>6</sup> plaque forming units (PFU) ZIKV MR766 diluted to a total volume of 50 uL in PBS, subcutaneously (SQ, *n* = 6) by injection in the nape of the neck using a tuberculin syringe, or intravaginally (VAG, *n* = 6) by pipette.

Guinea pigs were monitored daily for 24 days for temperature (tympanic), weight, and development of clinical signs of ZIKV infection (fever, lethargy, hunching, ru ffled fur, and decreased mobility), using a numerical value (0–5) based on severity of clinical signs, with 0 representing normal parameters and 5 representing severe clinical signs. Vaginal, salivary, and ocular samples were collected daily for 21 days using pre-moistened FLOQSwabs (Copan Diagnostics, Murrieta, CA, USA). Swabs were maintained on ice for the duration of swabbing all animals each day, and then stored at −80 ◦C until analysis by plaque assay.

Seven days post infection (dpi), two guinea pigs from each group were euthanized to assess acute infection characteristics. Blood samples were collected and centrifuged to separate serum, which was stored at −80 ◦C until analysis via RT-qPCR. Necropsy was performed immediately following euthanasia and tissues were collected, including spleen, lymph nodes, genitourinary tract (GUT), ovaries, lumbosacral DRG (LS-DRG, innervates the GUT), cervical DRG (C-DRG, innervates the neck), trigeminal ganglia (TG, innervates the face), superior cervical ganglion (SCG, innervates head and neck), ciliary ganglion (CG, motor and sensory innervation to the eye), brain, and eyes. Half of the tissues were fixed in 4% paraformaldehyde (PFA), embedded in optimum cutting temperature (OCT) media, and sectioned for immunofluorescence. The other half of the tissues were frozen for RNA isolation for RT-qPCR. At 37 dpi, allowing five weeks for viral clearance to ensure we had reached convalescence, the remaining guinea pigs were euthanized, necropsied, and samples were collected as described above to assess convalescent characteristics and viral persistence.
