Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replicates throughout human airways. The polarized human airway epithelium (HAE) cultured at an airway-liquid interface (HAE-ALI) is an in vitro model mimicking the in vivo human mucociliary airway epithelium and supports the replication of SARS-CoV-2. However, previous studies only characterized short-period SARS-CoV-2 infection in HAE. In this study, continuously monitoring the SARS-CoV-2 infection in HAE-ALI cultures for a long period of up to 51 days revealed that SARS-CoV-2 infection was long lasting with recurrent replication peaks appearing between an interval of approximately 7-10 days, which was consistent in all the tested HAE-ALI cultures derived from 4 lung bronchi of independent donors. We also identified that SARS-CoV-2 does not infect HAE from the basolateral side, and the dominant SARS-CoV-2 permissive epithelial cells are ciliated cells and goblet cells, whereas virus replication in basal cells and club cells was not detectable. Notably, virus infection immediately damaged the HAE, which is demonstrated by dispersed Zonula occludens-1 (ZO-1) expression without clear tight junctions and partial loss of cilia. Importantly, we identified that SARS-CoV-2 productive infection of HAE requires a high viral load of 2.5 × 10 ⁵ virions per cm ² of epithelium. Thus, our studies highlight the importance of a high viral load and that epithelial renewal initiates and maintains a recurrent infection of HAE with SARS-CoV-2.

Bourbon virus (BRBV) was first isolated from a patient hospitalized at the University of Kansas Hospital in 2014. Since then, several deaths have been reported to be caused by BRBV infection in the Midwest and Southern United States. BRBV is a tick-borne virus that is widely carried by lone star ticks. It belongs to genus Thogotovirus of the Orthomyxoviridae family. Currently, there are no treatments or vaccines available for BRBV or thogotovirus infection caused diseases. In this study, we reconstituted a replicon reporter system, composed of plasmids expressing the RNA-dependent RNA polymerase (RdRP) complex (PA, PB1 and PB2), nucleocapsid (NP) protein, and a reporter gene flanked by the 3’ and 5’ UTR of the envelope glycoprotein (GP) genome segment. By using the luciferase reporter, we screened a few small molecule compounds of anti-endonuclease that inhibited the nicking activity by parvovirus B19 (B19V) NS1, as well as FDA-approved drugs targeting the RdRP of influenza virus. Our results demonstrated that myricetin, and an anti-B19V NS1 nicking inhibitor, efficiently inhibited the RdRP activity of BRBV and virus replication. The IC ₅₀ and EC ₅₀ of myricetin are 2.22 μM and 4.6 μM, respectively, in cells. Myricetin had minimal cytotoxicity in cells, and therefore the therapeutic index of the compound is high. In conclusion, the BRBV replicon system is a useful tool to study viral RNA replication and to develop antivirals, and myricetin may hold promise in treatment of BRBV infected patients.