SARS-CoV-2 infection enhancement by amphotericin B: implications for disease management

Abstract

Severe coronavirus disease 2019 (COVID-19) patients who require hospitalization are at high risk of invasive pulmonary mucormycosis. Amphotericin B (AmB), which is the first-line therapy for invasive pulmonary mucormycosis, has been shown to promote or inhibit replication of a spectrum of viruses. In this study, we first predicted that AmB and nystatin had strong interactions with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteins using in silico screening, indicative of drugs with potential therapeutic activity against this virus. Subsequently, we investigated the impact of AmB, nystatin, natamycin, fluconazole, and caspofungin on SARS-CoV-2 infection and replication in vitro. Results showed that AmB and nystatin actually increased SARS-CoV-2 replication in Vero E6, Calu-3, and Huh7 cells. At optimal concentrations, AmB and nystatin increase SARS-CoV-2 replication by up to 100- and 10-fold in Vero E6 and Calu-3 cells, respectively. The other antifungals tested had no impact on SARS-CoV-2 infection in vitro. Drug kinetic studies indicate that AmB enhances SARS-CoV-2 infection by promoting viral entry into cells. Additionally, knockdown of genes encoding for interferon-induced transmembrane (IFITM) proteins 1, 2, and 3 suggests AmB enhances SARS-CoV-2 cell entry by overcoming the antiviral effect of the IFITM3 protein. This study further elucidates the role of IFITM3 in viral entry and highlights the potential dangers of treating COVID-19 patients, with invasive pulmonary mucormycosis, using AmB.IMPORTANCEAmB and nystatin are common treatments for fungal infections but were predicted to strongly interact with SARS-CoV-2 proteins, indicating their potential modulation or inhibition against the virus. However, our tests revealed that these antifungals, in fact, enhance SARS-CoV-2 infection by facilitating viral entry into cells. The magnitude of enhancement could be up to 10- or 100-fold, depending on cell lines used. These findings indicate that AmB and nystatin have the potential to enhance disease when given to patients infected with SARS-CoV-2 and therefore should not be used for treatment of fungal infections in active COVID-19 cases.

Keywords: SARS-CoV-2; amphotericin B; disease management; infection enhancement.

Fig 1

SARS-CoV-2 infection enhancement by AmB and nystatin. (A through C) Effects of antifungals on SARS-CoV-2 infection in Vero E6, Calu-3, and Huh7 cells. (D and E) Time course of SARS-CoV-2 infection at optimal concentrations of AmB and nystatin in Vero E6 and Calu-3 cells. (F) MTT assay testing the cytotoxicity of AmB and nystatin in Vero E6 and Calu-3 cells with the dotted line indicating 100% cell viability. (G) Cell staining with crystal violet shows different levels of cell death (detachment) at 24, 48, and 72 hpi. (H) Titration of supernatant from wells infected with the virus in panel G.

Fig 2

AmB promotes SARS-CoV-2 cell entry without affecting cellular gene expression. (A and B) Vero E6 or Calu-3 cells were treated with AmB at 3.12 and 0.78 µM, respectively, at different time points before (pre-treated) or after infection. AmB was removed 1 h after addition and replaced with medium without AmB. At 24 hpi, the supernatant was harvested, and SARS-CoV-2 was titrated. (C) Gene expression changes in Calu-3 cells treated with AmB at 0.78 µM compared to the cell control. The horizontal dashed line represents a q value of 0.05. The vertical dashed line represents a fold change of 2. (D) Infection of Vero E6 and Calu-3 cells with SARS-CoV-2 pseudovirus in the presence of AmB at 3.2 and 0.78 µM, respectively. (E) Vero E6 cells were transfected with pCSFLW plasmid carrying the reporter gene and incubated with or without AmB 3.2 µM. Luciferase assay was performed at 48 h post transfection. **P < 0.01, ***P < 0.001, ****P < 0.0001. ns, non-significant.

Fig 3

Effects of IFITM siRNAs on SARS-CoV-2 infection in different cell lines. (A) Quantification of IFITM gene expression in Calu-3 and Huh7 cells transfected with siRNA control or respective IFITM siRNAs. Values were normalized to GAPDH and calculated relative to the control (set to 100%). (B) Extracellular virus titration from Calu-3 and Huh7 cells transfected with siRNA control or IFITM siRNAs before infection with SARS-CoV-2. Values were calculated relative to the control (set to 100%). (C) Extracellular virus titration from Huh7 cells transfected with siRNA control or IFITM3 siRNA before infection with SARS-CoV-2, in the presence or absence of AmB. Values were calculated relative to the control (set to 100%). ***P < 0.001, ****P < 0.0001.