*3.1. Clinical Observations*

The guinea pigs were observed for 24 days post infection (dpi) for clinical signs of ZIKV infection. Although none of the guinea pigs' temperatures exceeded the normal range (37.2–39.5 ◦C), vaginally (VAG)-infected guinea pigs had higher temperatures than subcutaneously (SQ)-infected guinea pigs, which was statistically significant at 4 dpi (Figure 1A, *p* < 0.01). All guinea pigs gained weight at similar rates following infection (Figure 1B).

**Figure 1.** Clinical signs of infection following subcutaneous (SQ) and intravaginal (VAG) inoculation with 1 × 10<sup>6</sup> PFU ZIKV MR766. (**A**) Temperature (average/group, \* *p* < 0.01); (**B**) Weight (average/group); (**C**) Percentage of guinea pigs exhibiting clinical signs, including conjunctivitis, ear sensitivity, vocalization, hyperactivity, vaginal discharge, or eye sensitivity; cyclical peaks began 8 dpi and repeated every 5 days; (**D**) 291 total clinical observations were recorded over 24 days. The number of times each clinical sign was observed was recorded and is shown as percentage of guinea pigs in each group exhibiting specific clinical signs (\* *p* < 0.05, \*\* *p* < 0.01); (**E**) Total clinical signs (excluding "normal" observations) recorded for each guinea pig.

Based on clinical scoring, using a 5-point severity scale, ZIKV inoculation resulted in mild infections in both groups, although wide variability occurred among individual guinea pigs. Although mild, clinical signs were most apparent from 3–8 dpi and cyclical peaks of clinical signs occurred every five days thereafter (e.g., 8, 13, 18, 23 dpi, Figure 1C). While mild clinical signs of infection have been reported in previous guinea pig models, ours is the first to demonstrate a cyclical pattern to their nature. Over the 24-day observation period, 291 clinical observations were recorded; 39% of the 158 observations from the SQ group and 38% of the 133 observations from the VAG group were categorized as "no clinical signs" or "normal" (Figure 1D). Although we did not observe many classical overt signs of severe infection (e.g., ruffled fur, immobility, weight loss), some discrete signs of distress were observed in multiple animals. Conjunctivitis was observed in both SQ (12%) and VAG (19%) infected animals. However, guinea pigs infected SQ had a more frequent occurrence of ear sensitivity when tympanic temperatures were taken (*p* = *0.014*), vocalization during handling (*p* = *0.008*), and hyperactivity (*p* = *0.002*) compared to the VAG infected group. In contrast, VAG infected guinea pigs had a significantly greater occurrence of vaginal discharge compared to SQ infected animals (*p* < 0.001 Figure 1D). Substantial variability was observed among all guinea pigs, regardless of inoculation route, with some animals showing minimal signs (Figure 1E). One guinea pig, GP#3, displayed the greatest number of observed signs of infection in the SQ group (GP#1-6), while GP#1 and GP#2 showed minimal signs of infection, which is consistent with ~20% of humans developing clinical disease from ZIKV infection. In the VAG group (GP#7-12), all but one guinea pig (GP#7) displayed signs of infection (Figure 1E). Determining the specific route of infection of humans in epidemic and endemic areas is rarely possible, thus the percentage of humans developing clinical symptoms following vaginal infection is not known. Therefore, the significance of these differences in relation to humans is challenging to interpret.
