The FDA-approved Drug Ivermectin inhibits the replication of SARS-CoV-2 in vitro Highlights • Ivermectin is an inhibitor of the COVID-19 causative virus (SARS-CoV-2) in vitro. • A single treatment able to effect ∼5000-fold reduction in virus at 48h in cell culture. • Ivermectin is FDA-approved for parasitic infections, and therefore has a potential for repurposing. • Ivermectin is widely available, due to its inclusion on the WHO model list of essential medicines. Abstract Although several clinical trials are now underway to test possible therapies, the worldwide response to the COVID-19 outbreak has been largely limited to monitoring/containment. We report here that Ivermectin, an FDA-approved anti-parasitic previously shown to have broad-spectrum anti-viral activity in vitro, is an inhibitor of the causative virus (SARS-CoV-2), with a single addition to Vero-hSLAM cells 2 hours post infection with SARS-CoV-2 able to effect ∼5000-fold reduction in viral RNA at 48 h. Ivermectin therefore warrants further investigation for possible benefits in humans. Ivermectin is an FDA-approved broad spectrum anti-parasitic agent that in recent years we, along with other groups, have shown to have anti-viral activity against a broad range of viruses in vitro. Originally identified as an inhibitor of interaction between the human immunodeficiency virus-1 (HIV-1) integrase protein (IN) and the importin (IMP) α/β1 heterodimer responsible for IN nuclear import6, Ivermectin has since been confirmed to inhibit IN nuclear import and HIV-1 replication. Other actions of ivermectin have been reported, but ivermectin has been shown to inhibit nuclear import of host and viral proteins, including simian virus SV40 large tumour antigen (T-ag) and dengue virus (DENV) non-structural protein. Importantly, it has been demonstrated to limit infection by RNA viruses such as DENV 1-44, West Nile Virus, Venezuelan equine encephalitis virus (VEEV) and influenza, with this broad spectrum activity believed to be due to the reliance by many different RNA viruses on IMPα/β1 during infection. Ivermectin has similarly been shown to be effective against the DNA virus pseudorabies virus (PRV) both in vitro and in vivo, with ivermectin treatment shown to increase survival in PRV-infected mice. Efficacy was not observed for ivermectin against Zika virus (ZIKV) in mice, but the authors acknowledged that study limitations justified re-evaluation of ivermectin’s anti-ZIKV activity. Finally, ivermectin was the focus of a phase III clinical trial in Thailand in 2014-2017, against DENV infection, in which a single daily oral dose was observed to be safe and resulted in a significant reduction in serum levels of viral NS1 protein, but no change in viremia or clinical benefit was observed (see below). The causative agent of the current COVID-19 pandemic, SARS-CoV-2, is a single stranded positive sense RNA virus that is closely related to severe acute respiratory syndrome coronavirus (SARS-CoV). Studies on SARS-CoV proteins have revealed a potential role for IMPα/β1 during infection in signal-dependent nucleocytoplasmic shutting of the SARS-CoV Nucleocapsid protein, that may impact host cell division19,20. In addition, the SARS-CoV accessory protein ORF6 has been shown to antagonize the antiviral activity of the STAT1 transcription factor by sequestering IMPα/β1 on the rough ER/Golgi membrane21. Taken together, these reports suggested that ivermectin’s nuclear transport inhibitory activity may be effective against SARS-CoV-2. To test the antiviral activity of ivermectin towards SARS-CoV-2, we infected Vero/hSLAM cells with SARS-CoV-2 isolate Australia/VIC01/2020 at an MOI of 0.1 for 2 h, followed by the addition of 5 μM ivermectin. Supernatant and cell pellets were harvested at days 0-3 and analysed by RT-PCR for the replication of SARS-CoV-2 RNA (Fig. 1A/B). At 24 h, there was a 93% reduction in viral RNA present in the supernatant (indicative of released virions) of samples treated with ivermectin compared to the vehicle DMSO. Similarly a 99.8% reduction in cell-associated viral RNA (indicative of unreleased and unpackaged virions) was observed with ivermectin treatment. By 48h this effect increased to an ∼5000-fold reduction of viral RNA in ivermectin-treated compared to control samples, indicating that ivermectin treatment resulted in the effective loss of essentially all viral material by 48 h. Consistent with this idea, no further reduction in viral RNA was observed at 72 h. As we have observed previously, no toxicity of ivermectin was observed at any of the timepoints tested, in either the sample wells or in parallel tested drug alone samples. https://www.sciencedirect.com/science/article/pii/S0166354220302011