Abstract: This study aims to establish the culture method of human primary airway epithelial cell (hAEC) and to investigate the anti-respiratory syncytial virus (RSV) activity and mechanism of 3-thioindole compound RSVA-4 and immunosuppressive metabolite 6-MMPR using hAEC system, which intends to construct a cell model for RSV drug screening and efficacy evaluation. The respiratory tract epithelial cells from volunteers were collected and cultured, then the morphology, activity and purity were identified. The anti-RSV activity and cytotoxicity of RSVA-4 and 6-MMPR were further verified in hAEC system. The mechanism of RSVA-4 and 6-MMPR accounting for the suppression of RSV replication on hAEC was explored by using fluorescence real-time quantitative PCR (RT-qPCR) and time-of-addition assay. The survival rate of cultured hAEC was 93.51% as determined by trypan blue staining. The half maximal inhibitory concentrations (IC50) of RSV-A-4 and 6-MMPR were （207.30±4.77） μmol/L and （3191.00±6.11） μmol/L, respectively. The half maximal cytotoxic concentration (CC50) of 6-MMPR was （95526.00±10.97） μmol/L, while no toxicity of RSVA-4 was observed on hAEC. Mechanistically, RSV-A-4 and 6-MMPr inhibited RSV replication in the genome replication/transcription phase. The hAEC culture method was successfully established, which could be used to screen and evaluate the anti-RSV drugs in vitro. RSVA-4 and 6-MMPR could effectively inhibit RSV replication at the cellular level. Altogether, the result could provide an experimental basis for the research and development of RSV drug and pathogenesis.

Key words: human airway epithelial cell, human respiratory syncytial virus, antiviral compound, antiviral activity, antiviral mechanism