## 4 Discussion
Depletion of GTP pools is a predominant mechanism of ribavirin's antiviral activity against many viruses, including paramysoviruses [11; 38; 39]. Depleted intracellular GTP pools may in turn potentially lead to lower ATP pools, since cells can consume ATP in an effort to maintain GPP pools [40; 41]. Furthermore, the 5'-triphosphate of ribavirin can result in mutation-induced viral error catastrophe and chain termination [42]. The simultaneous combined (i) inhibition of viral RNA replication by remdesivir (GS-441524), (ii) depletion of intracellular GTP pools by ribavirin, (iii) depletion of ATP pools and thus reduced competition at the level of the viral polymerase for the 5'-triphosphate of GS-441524, and (iv) error catastrophe induction by ribavirin may result in a more efficient (read synergistic) decline in viral replication than what may be expected from a solely additive antiviral effect of both molecules. In conclusion, we demonstrate that ribavirin combined with remdesivir or GS-441524 results in a markedly more pronounced antiviral effect than what would be expected from an additive antiviral effect. This finding provides preclinical evidence to explore in clinical studies the efficacy of co-administration of ribavirin with either remdesivir or obeldesivir (which can be seen as the oral form of remdesivir) to combat HPIV infections. Likewise, such combinations may also be explored against infections with other viruses that are also susceptible to these two drugs in vitro. **Supplementary Materials:** The following supporting information can be downloaded at: [[https://www.mdpi.com/article/10.3390/v17020172/s1](https://www.mdpi.com/article/10.3390/v17020172/s1)]([https://www.mdpi.com/article/10.3390/v17020172/s1](https://www.mdpi.com/article/10.3390/v17020172/s1)), Figure S1. (A-C) Percentage inhibition of HPIV-3 replication, measured as GFP-signal, normalized to the untreated-infected control. (D,E) The matrices of toxicity in the presence of combined treatments. Data are from three independent experiments. Figure S2. (A) Infectious virus titers in apical washes from Experiment 2 were determined by endpoint virus titration assay in the prophylactic setting. The lower limit of detection is \(-0.02\). (B,C) Neither ribavirin nor GS-441524 has obvious cytotoxicity in HnAEC cultures in both the prophylactic setting (B) and the therapeutic setting (C). Data presented for Experiment 1 and Experiment 2 are two independent experiments. The grey boxes indicate treatment windows; Figure S3. Effect of ribavirin and GS-441524 against HPIV-3 replication in AG129 mice. (A) AG129 mice were orally treated with ribavirin (either 25 mg/kg or 50 mg/kg) or the vehicle (i.e., the control group) twice daily (BID) for four consecutive days, starting one day before intranasal inoculation with HPIV-3. At day 3 p.i., mice were euthanized and lung samples were collected to quantify infectious virus titers. Data are from one experiment with 5 or 6 mice per group. (B) AG129 mice were orally treated with the vehicle and the combinations of ribavirin and various doses of GS-441524 (2.5 mg/kg, 10 mg/kg and 25 mg/kg) twice daily for four consecutive days, starting one day before intranasal inoculation. At day 3 p.i., lung samples were collected to quantify infectious virus titers. Data are from one experiment with 5 mice per group. (C) Weight change at day 6 p.i. is presented as a percentage of the body weight at day \(-1\) p.i., when treatment was started. Data are from two independent experiments with 9 mice in the combination-treated group and 11 mice in all other groups. LLOD presents the lower limit of detection. Individual data and median values (indicated by bars) are presented in all graphs. Data were analyzed with the Kruskal-Wallis test. ns, nonsignificant; \(p\) < 0.01, ***. J.N. acquired funding. [PERSON], [PERSON], and [PERSON] designed the study. [PERSON] and [PERSON] wrote the manuscript. [PERSON] performed experiments, analyzed data, and made graphs. [PERSON] assisted with animal experiments.