Abstract

Human rhinoviruses (HRVs) are major etiological agents of upper respiratory tract infections and are strongly associated with asthma exacerbations and chronic obstructive pulmonary disease, yet no approved antiviral therapies are currently available. Reverse genetics systems, particularly reporter viruses, are critical for facilitating antiviral discovery; however, robust and broadly applicable HRV infectious clones remain limited. Here, we recovered a viral genome from a clinical sample and generated infectious cDNA clones of an HRV-A1 strain, designated HRV-fd. Both the wild-type virus and a Nanoluc (Nluc) reporter virus exhibited efficient replication in HeLa cells without inducing pronounced cytopathic effects and were capable of infecting HeLa-ICAM1 cells at extremely low inocula. Leveraging the reporter virus, we conducted a drug-repurposing screen and identified pyrvinium pamoate as an antiviral compound that targets multiple steps of the HRV infection cycle, exhibiting an IC 50 of 58.6 nM, an IC 90 of 193.1 nM, and a CC 50 of 3.4 μM. Notably, pyrvinium pamoate conferred sustained antiviral activity following transient exposure and subsequent withdrawal, suggesting the induction of host cellular alterations that restrict viral amplification. Collectively, these findings establish a robust HRV-A1 reverse genetics and reporter platform for virological studies and antiviral screening, and identify pyvinium pamoate as a promising lead compound for the development of effective anti-HRV therapeutics.