The anti-influenza virus drug, arbidol is an efficient inhibitor of SARS-CoV-2 in vitro

Dear editor, Since December 2019, a novel disease COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) rapidly spread to over 200 countries and infected over 1.50 million people including 92,798 deaths (data as of April 10, 2020). On March 11, the World Health Organization (WHO) characterized COVID-19 as a pandemic, and called for accelerating diagnostics, vaccines, and drugs developments to combat this novel disease. Apart of the new coronavirus, influenza virus infections have been a consistent threat to the global public health over the years. In the United States alone, the Centers for Disease Control and Prevention (CDC) estimates that, so far during the 2019–2020 winter season, there have been at least 39 million illnesses, 400,000 hospitalizations and 24,000 deaths from influenza (https://www.cdc.gov/flu/weekly/index.htm). Considering the current concomitant circulation of SARS-CoV-2 and influenza virus infections, the exploration of available and viable anti-influenza drugs to treat both diseases is of great interest. Actually, in the early stages of the outbreak of COVID19, some anti-flu drugs (for example, oseltamivir) have been applied for the treatment of COVID-19 patients. Previously, we reported that favipiravir (T705), an antiinfluenza drug approved in Japan and China, showed a certain efficacy against SARS-CoV-2 in vitro. In addition, arbidol, an anti-influenza drug targeting the viral hemagglutinin (HA) is being used in a clinical trial against COVID-19 (ChiCTR2000029573) and has been recently added to the Guidelines for the Diagnosis and Treatment of COVID-19 (sixth and seventh editions) in China. A recent retrospective study suggested that arbidol treatment showed tendency to improve the discharging rate and decrease the mortality rate of COVID-19 patients. However, to our knowledge, there has been no systematical analysis about the efficacy of anti-influenza drugs against SARS-CoV-2. In this study, we evaluated six currently available and licensed anti-influenza drugs against SARS-CoV-2. The drugs include arbidol, baloxavir, laninamivir, oseltamivir, peramivir, and zanamivir. The M2 inhibitors (amantadine and rimantadine) were not considered in this study since they were not recommended for treating influenza by WHO due to drug resistance. First, the cytotoxicity of the compounds in African green monkey kidney cells, Vero E6 (ATCC-1586) was measured by a standard cell counting kit-8 (CCK8) assay. Then, the cells were infected with SARS-CoV-2 at a multiplicity of infection (MOI) of 0.05 in the presence of either compound or dimethyl sulfoxide (DMSO) control. The dose–response curves were determined by quantification of viral RNA copy numbers in the supernatant of infected cell at 48 h post infection (p.i.). As demonstrated in Fig. 1a, arbidol efficiently inhibited virus infection in vitro. The 50% maximal effective concentration (EC50) and the 50% cytotoxic concentration (CC50) of arbidol was 4.11 (3.55–4.73) and 31.79 (29.89–33.81) μM, respectively, and the selectivity index (SI=CC50/EC50) was 7.73. Baloxavir partially inhibited SARS-CoV-2 infection (~29%) at a high concentration of 50 μM (Fig. 1a). In contrast, laninamivir, oseltamivir, peramivir, and zanamivir did not exhibit antiSARS-CoV-2 activity even at the highest drug concentrations (Fig. 1a). The antiviral effect of the compounds was also evaluated by observing cytopathic effects

The corresponding concentration (0.25%) of DMSO diluted with cell culture medium was used as control treatments.

CCK-8 assay
The cytotoxicity of the anti-influenza virus drugs on Vero E6 Cells were determined by a cell counting kit-8 (CCK8) (Beyotime, China) according to the manufacturer's instructions. Briefly, 1 × 10 4 cells were pre-seeded in 96-well plate and a series of concentrations (two fold or three fold diluted) of the compounds were added. DMSO was used as the negative control. Culture medium was set as the blank control. After drug incubation for 24 h, 20 μL CCK-8 reagent (Beyotime, China) was added and mixed thoroughly by gentle shaking, and followed by two hours incubation at 37 o C.
Subsequently, OD450 was measured in a microplate reader and the drug cytotoxicity was calculated. Cytotoxicity (%) = 1 -(A(drug)-A(blank))/ (A(negative)-A(blank)) × 100%. The concentration of each drug which showed ≤ 10% cytotoxicity was chosen the starting point for evaluation of anti-SARS-CoV-2 activities. The experiments were performed in triplicate with two independent repeats and the data represent the mean ± standard deviation (SD) from two independent repeats.

Evaluation of antiviral activities
To evaluate the antiviral potential of the drugs, Vero E6 cells (1 × 10 5 cells/well) were in triplicate with two independent repeats and the data represent the mean ± SD from two independent repeats.

Time-of-addition experiment
To determine the replication stages of SARS-CoV-2 targeted by arbidol, time-of-addition experiment was performed as previously described 2 . In all the experimental groups, Vero E6 cells (1 × 10 5 cells/well) were infected with SARS-CoV-2 at an MOI of 0.05 for 2 h. For "Full-time" group, cells were pretreated with Arbidol (10 μM) for 1 h prior to viral attachment. Then, the supernatant was replaced with the drug-containing medium until the end of the experiment. For "Entry" group, a procedure similar to that of the "Full-time" group was exploited, except for the cells were maintained in drug-free medium after removal of the virus inoculum.
For "Post-entry" treatment, the drug was added to the cells after viral attachment.
At 16 h p.i., virus production in the culture supernatants was quantified by qRT-PCR.
The expression of viral NP in the infected cells was explored by immunofluorescence analysis (IFA) and Western blotting.

Immunofluorescence analysis
To detect the expression viral NP protein, infected Vero E6 cells were fixed with 4%

The mechanism of arbidol in inhibition of virus entry
To investigate the impact of arbidol on virus binding, Vero E6 cells (2 × 10 5 cells/well) cultured in 24-well cell-culture plates were pre-treated with arbidol (10 μM) or DMSO (0.5% v/v) for 1 h before virus attachment, and then incubated with SARS-CoV-2 (MOI = 0.05) at 4 °C to allow virus attachment for 1 h. After that, 100 μL of cell supernatant (unbound virions) was collected to determining viral RNA copy numbers by qRT-PCR as described above. The cells (bound virions) were collected after being washed three times with pre-chilled PBS, and the total cellular RNA was extracted with TRIzol Reagent (Invitrogen). One μg RNA was used for cDNA synthesis and a relative quantification was performed with viral spike gene as the target and the cellular glyceraldehyde-phosphate dehydrogenase (GAPDH) gene as an internal control, respectively. The fold change of RNA quantity in each group was calculated by the 2 -ΔΔCt method 3 . The data was normalized to the DMSO group, and P values were calculated by unpaired two-tailed t test.
The impact of arbidol on viral intracellular trafficking was investigated by co-localization analysis of virions with endosomes as previously described 4 . Briefly, Vero E6 cells (

Fig. S3. Effect of arbidol on SARS-CoV-2 co-localization with early endosomes
(EEs). Vero E6 cells were pre-treated with arbidol (10 μM) or DMSO for 1 h, followed by virus binding (MOI = 5) at 4°C for 1 h. After removal of virus-drug mixture, virus infection was allowed to proceed in the presence of arbidol or DMSO at 37°C for 30, 60, and 90 min before being collected for immunofluorescence assay using anti-NP polyclonal antibody for virions (red), and antibodies against EEA1 for EEs (green). The nuclei (blue) were stained with Hoechst 33258 dye. Virions co-localized with EEs in the representative confocal microscopic images of virions (red) and EEA1+ EEs (green) in each group were indicated by white arrows. Bars are