## 3 Results
Data from 3–4 inserts per group are presented as mean \(\pm\) SD in Experiment 1 and 2. (**C**) HPIV-3 RNA levels and infectious virus titers at 14 days post-infection (14 d.p.i.). (**D**) Infectious virus titers of area under the curve over the treatment period in Experiment 1 and 2. Data from seven or eight inserts per group across two independent experiments are presented as mean \(\pm\) SD. Data were analyzed using one-way ANOVA with [PERSON]’s multiple comparisons test. ns, nonsignificant; \(p<0.05\), *; \(p<0.01\), **; \(p<0.001\), ***; \(p<0.0001\), ***. The grey boxes indicate treatment windows. The Combined Treatment of Ribavirin and GS-441524 Potentially Reduces HPIV-3 Replication and Virus-Induced Lung Pathology in AG129 Mice  
We next determined the antiviral effect of either ribavirin or GS-441524 (both dosed orally) alone or in combination in a mouse HPIV-3 infection model that we recently developed and validated for antiviral studies, wherein the mice are intranasally inoculated with a clinical isolate of the virus [27]. Ribavirin, at doses of either 25 mg/kg or 50 mg/kg twice daily BID, could not significantly reduce infectious viral titers in the lungs of infected AG129 mice at day 3 p.i. (a time point when peak viral titers were noted in vehicle-treated mice) (Figure S3A). Because of a large variation in infectious viral lung titers observed in the 50 mg/kg, but not in the 25 mg/kg dose group, the latter dose was selected. We previously demonstrated that GS-441524 at a dose of 50 mg/kg reduces infectious HPIV3 titers to undetectable levels in the lungs of all infected mice [27]. Therefore, lower doses [2.5 mg/kg, 10 mg/kg or 25 mg/kg (BID)] of GS-441524 were selected for testing in combination with 25 mg/kg of ribavirin. The combination of 25 mg/kg ribavirin with 25 mg/kg GS-441524 was found to result in the most potent antiviral activity at day 3 p.i. (Figure S3B). We next directly compared the antiviral efficacy of either ribavirin (25 mg/kg, per dose) or GS-441524 (25 mg/kg, per dose) alone with their combination. Treatment was started one day before intranasal inoculation with HPIV-3 and was continued for either 4 or 7 consecutive days. At day 3 p.i. or day 6 p.i., mice were euthanized and lung samples were collected for quantification of viral loads and assessment of lung histopathology, respectively (Figure 4A). These timepoints were chosen since infectious viral lung titers peak at day 3 p.i., whereas lung pathology develops prominently from day 5 p.i. [27]. Ribavirin monotherapy did not result in an antiviral effect; GS-441524 monotherapy reduced infectious viral lung titers by 1.2 log\({}_{10}\) TCID\({}_{50}\)/mg lung sample (\(p\) = 0.02) as compared to the vehicle-treated group. The combined treatment resulted in a reduction of 1.9 log\({}_{10}\) TCID\({}_{50}\)/mg lung sample (\(p\) < 0.001) in infectious virus titers, with no detectable infectious virus in 4 out of 11 mice (Figure 4B). No significant weight loss or signs of adverse effects were observed in any group at day 6 p.i. (Figure S3C). The lungs of uninfected mice were histologically normal, whereas the vehicle-treated mice presented with intra-alveolar hemorrhage, significant peri-vascular and peri-bronchial inflammation and bronchopneumonia foci. Most of the mice from both the ribavirin and GS-441524 monotherapy groups presented with limited but significant peri-vascular and peri-bronchial inflammation and intra-alveolar hemorrhage. In contrast, only mild peri-vascular inflammation in 6 out of 9 mice and mild intra-alveolar hemorrhage in 2 out of 9 mice were observed in the combined treatment group (Figure 4C). The median lung histopathology score was 5.5 in the vehicle-treated group versus 1.5 in the combined treatment group (Figure 4D); the median lung score in the uninfected untreated mice was 1.0.