## 3 Results  
Combinations of Ribavirin and Remdesivir or GS-441524 Result in Synergistic Antiviral Efficacy Against HPIV-3 Replication in LLC-MK2 Cells  
We first investigated the antiviral activity of single treatments of either ribavirin, remdesivir and GS-441524 against HPIV-3, using a reporter virus that expresses enhanced green fluorescent protein (GFP), referred to as HPIV-3 GFP [34]. Ribavirin, remdesivir and GS-441524 inhibit HPIV-3 replication in LLC-MK2 cells in a dose-dependent manner, with EC\({}_{50}\) of respectively 29 \(\upmu\)M, 0.21 \(\upmu\)M and 1.4 \(\upmu\)M (Figure S1A-C). Next, the antiviral activity and the toxicity effects of combinations of ribavirin with either remdesivir or GS-441524 were assessed in the same checkerboard format (Figure 1A,D). Both of the combinations presented additive effects between the two molecules (Figure S1D,E). Bliss scores reveal that both combinations resulted in significant synergistic antiviral efficacy against HPIV-3 across broad concentration ranges of either molecule (Figure 1B,E). For instance, the combination of 12.5 \(\upmu\)M ribavirin with 0.13 \(\upmu\)M remdesivir resulted in 100% inhibition of HPIV-3 replication, whereas the Bliss model predicts 47% inhibition based on respectively 19% and 34% inhibition of the single molecules (Figure 1A). Likewise, the combination of 5.0 \(\upmu\)M ribavirin with 0.37 \(\upmu\)M GS-441524 resulted in 93% inhibition of HPIV-3 replication, whereas the molecules alone resulted respectively in 16% and virtually no inhibition (Figure 1D). 3D maps of the combined efficacy of ribavirin with either remdesivir or GS-441524 visualize this synergism (red areas in Figure 1C,F). The Combined Treatment of Ribavirin and GS-441524 Potently Inhibits HPIV-3 Replication in Nasal Human Airway Epithelial Cell (HrnAEC) Cultures  
Next, the combined efficacy of ribavirin and GS-441524 against HPIV-3 in nasal human airway epithelial cell (HnAEC) cultures grown at the air-liquid interface was assessed. First, we determined which suboptimal antiviral concentrations of either molecule were to be used in the combination experiments; these were, respectively, 30 uM and 0.30 uM for ribavirin and GS-441524 (Figure 2A,B), which either alone or in combination did not result in toxic effects in the HnAEC cultures (as determined by quantifying the release of LDH per day and at the endpoint) (Figure 2C,D). Next, the combined antiviral efficacy was assessed over an extended period of 25 days whereby the molecules were added two hours before infection to the culture medium and wherein treatment was continued until day 18 p.i. ribavirin (100 uM) or GS-4415245 (0.3 uM) alone resulted in a slight antiviral effect during the first days of the experiment, but no inhibition of viral replication was observed between day 11 p.i. and 25 p.i., the end of the experiment, across two independent experiments. In contrast, the combined treatment of ribavirin and GS-441524 resulted in a potent reduction of HPIV-3 RNA levels (Figure 2E). In Experiment 1 (Figure 2E, left panel), HPIV-3 replication was suppressed to undetectable levels throughout the experiment in the presence of the combined treatment, resulting in a reduction of 3.5-4.5 log\({}_{10}\) compared to the control group. No viral rebound was observed even one week after cessation of treatment. In Experiment 2 (Figure 2E, right panel), HPIV-3 RNA levels remained suppressed below the lower limit of quantification until day 16 p.i., with a gradual rebound in 4 out of 6 cultures in the presence of combined treatment. However, RNA levels remained lower than those in the single treatment groups. Infectious virus titers were quantified as well and were found to  
Figure 1: The combined antiviral effect of ribavirin with either remdesivir or GS-441524 results in synergistic antiviral efficacy against HPIV-3 replication in LLC-MK2 cells. **(A,D)** The matrices of inhibition of HPIV-3 replication in the presence of combined treatments. Data are from five independent experiments.