## 3 Results
**(B,E)** The interaction between two molecules was determined by using the Bliss independence model. Data are presented as mean values of BLISS scores. Data are analyzed using the one-sample Student’s _t_-test. \(p\) < 0.05, *; \(p\) < 0.01, **; \(p\) < 0.001, ***._ **(C,F)** 3D synergy maps. be markedly decreased in the combined treatment group compared to those in the control and single treatment groups during the treatment period (Figure S2A). No cytotoxicity was observed in any of the groups, as determined by measuring the levels of released LDH (Figure S2B). We next studied the antiviral effect of the single and combined treatments when the molecules were first added to the cultures at 4 days p.i. and after which treatment was continued for 18 consecutive days. A reduction in HPIV-3 RNA levels was observed in the combination group, but it did not reach undetectable levels (Figure 3A, Experiment  
Figure 2: The combination of ribavirin and GS-441524 potentially inhibits HPIV-3 replication in nasal human airway epithelial (HnAEC) cultures in a prophylactic setting. (**A**,**B**) The antiviral effect of ribavirin and GS-441524 at various concentrations against HPIV-3 replication. Molecules were added to the basal medium of HnAEC cultures 2 h prior to infection, with treatments continuing for 16 consecutive days. Viral RNA levels in the apical washes were quantified by RT-qPCR. Individual data from two inserts of uninfected DMSO control and three inserts of each of the other groups are presented. (**C**,**D**) The toxicity of the molecules was determined by LDH assay per day (**C**) or at the endpoint (**D**). The maximum LDH activity was determined by lysing two fresh HnAE culture inserts using Triton X-100 for 24 h. Data from four inserts are presented as mean \(\pm\) SD and analyzed using one-way ANOVA with [PERSON]’s multiple comparisons test. ns, nonsignificant. (**E**) Kinetics of HPIV-3 replication in the presence of single treatments with each of GS-441524, ribavirin or their combination; two independent experiments (Experiment 1 and Experiment 2). Molecules were added for 18 consecutive days, followed by further culturing with basolateral medium that did not contain inhibitors. Levels of viral RNA in apical washes were quantified by RT-qPCR. Data from three inserts of uninfected DMSO control and four inserts from each of the other groups, and data from six inserts of the combination group and four inserts from each of the other groups are presented as mean \(\pm\) SD in Experiment 1 and Experiment 2, respectively. The grey boxes indicate the treatment window. LLOQ presents the lower limit of quantification. “eq” stands for equivalent, as vRNA was measured but TCID\({}_{50}\)-equivalent was reported. 1 and Experiment 2). No cytotoxicity was observed in any of the groups, as determined by measuring the levels of released LDH (Figure S2C). Next, infectious virus titers were quantified and shown to decline in the combined therapy group in both experiments 1 and 2, reaching undetectable levels at certain time points, unlike the limited reduction observed in the single-treatment groups (Figure 3B). Notably, both HPIV-3 RNA levels and infectious virus titers in the combined therapy group were significantly lower than those in the single-treatment groups at day 14 p.i. and when analyzing the area under the curve over the treatment period (Figure 3C,D). Figure 3: The combination of ribavirin and GS-441524 potently inhibits HPIV-3 replication in HnAEC cultures in a therapeutic setting. At day 4 p.i., the HnAEC cultures were treated with GS-441524, ribavirin or their combination. Treatment continued until day 22 p.i., after which the basal medium without molecules was refreshed. (**A**) HPIV-3 RNA levels in apical washes were quantified by RT-qPCR. LLOQ presents the lower limit of quantification. (**B**) Infectious virus titers in apical washes were determined by endpoint virus titration assay. The lower limit of detection is \(-0.02\). Experiment 1 and 2 are two independent experiments.