## 4 Discussion  
HPIV-3 is an important causative agent of lower respiratory tract illnesses in vulnerable populations, particularly children under 5 years, the elderly and immunocompromised individuals. There are no vaccines nor antiviral drugs available for preventing and treating HPIV-3 or other HPIVs infections. We here demonstrate that ribavirin combined with remdesivir or its parent nucleoside, GS-441524, results in potent antiviral activity against HPIV-3 infection both in vitro and in vivo. In LLC-MK2 cells, the combined treatment of ribavirin with either remdesivir or GS-441524 resulted in a marked antiviral synergism against HPIV-3 over a relatively broad  
Figure 4: The combination of ribavirin and GS-441524 potentially reduces viral loads and lung pathology upon intranasal inoculation of HPIV-3 in AG129 mice (**A**) Setup of the study. AG129 mice were orally dosed with either vehicle, ribavirin (25 mg/kg, per dose), GS-441524 (25 mg/kg, per dose) or their combination twice daily from day 1 p.i. to day 2 p.i. or day 5 p.i. At day 0, the mice were intranasally inoculated with 1.5 \(\times\) 10\({}^{6}\) TCID\({}_{50}\) of HPIV-3. Lung samples were collected at day 3 p.i. to measure viral loads and at day 6 p.i. to assess lung histopathology. (**B**) Infectious virus titers are expressed as \(\log_{10}\) TCID\({}_{50}\) per milligram of lung sample. LLOD presents the lower limit of detection. Data are from two independent experiments with 10 mice in the vehicle-treated group and 11 mice in drug-treated groups. (**C**) Representative H&E-stained images revealed normal lung parenchyma in uninfected mice, intra-alveolar hemorrhage (left upper corner), significant peri-vascular (red arrows) and peri-bronchial (blue arrows) inflammation, and focus of bronchopneumonia (green arrows) in the vehicle group, focal but significant peri-vascular (red arrows) and peri-bronchial (blue arrows) inflammation in the ribavirin-treated group, very limited peri-vascular (red arrow) and peri-bronchial (blue arrows) inflammation in the GS-441524 group, and very limited and slight peri-vascular inflammation in the combination group. The samples of uninfected mice and infected mice were from the same experiment, but the staining procedure was performed at different moments. The scale bar is 100 \(\upmu\)m. (**D**) Cumulative severity scores of lungs of all infected mice. Data are from two independent experiments with 9 mice in the combination-treated group and 11 mice in all other groups. Individual data and median values (indicated by bars) are presented in all graphs. Data were analyzed with the Kruskal-Wallis test. ns, nonsignificant; \(p<0.05\), \({}^{\star}\); \(p<0.01\), \({}^{\star\star}\); \(p<0.001\), \({}^{\star\star\star}\). (**A**) was designed with BioRender. concentration range. In human nasal airway epithelial cell cultures grown at the air-liquid interface, the combined treatment of ribavirin and GS-441524, each at suboptimal or even inactive concentrations, resulted in pronounced inhibition of HPIV-3 replication. In AG129 mice, the combined treatment of ribavirin and GS-441524 resulted in a potent antiviral effect against HPIV-3, with lower or undetectable levels of infectious viruses in the lungs of more animals as compared with the single treatment groups. All of the mice in the combination group had virtually normal lung histology. Ribavirin and remdesivir inhibit viral RNA synthesis through different mechanisms. GS-441524, the parent nucleoside of remdesivir, is intracellularly converted to its 5'-triphosphate metabolite (which can be considered a mimic of the natural substrate ATP) and inhibits viral replication by incorporation into the viral RNA by the RNA-dependent RNA polymerase followed by chain termination [35; 36]. Ribavirin, once converted to its 5'-monophosphate, acts as a potent competitive inhibitor of inosine monophosphate dehydrogenase resulting in depletion of intracellular GTP pools [37].