Selection and Preparation of Drugs

To rapidly identify a pharmacologic agent that could be used to treat SARS, a collection of antiviral drugs was tested against SARS-CoV, the etiologic agent of the atypical pneumonia. To investigate a wide spectrum of potential molecular targets, we decided to cover the entire pharmacologic range of commercially available antiviral agents, including agents not expected to be active against coronaviruses. Information on antiviral drugs provided here was obtained from prescribing information sheets or from communications with the manufacturer.

Nucleoside analogues are a diverse class of compounds; in general, they inhibit viral RNA or DNA polymerases or other enzymes, interfering with nucleic acid synthesis. In this study, the selected compounds that target DNA viruses such as herpes simplex virus (HSV) and varicella-zoster viruses (VZV) were acyclovir, ganciclovir, and foscarnet. Ribavirin has activity against a range of DNA and RNA viruses; in different cell lines, ED₅₀ ranges from 1 to100 μg/mL. Antiretroviral (HIV) drugs include reverse transcriptase (RT) inhibitors and protease inhibitors. Selected HIV nucleoside RT inhibitors studied were zidovudine and lamivudine, while HIV protease inhibitors studied were indinavir, nelfinavir, and saquinavir. The third group of antivirals studied were the neuraminidase inhibitors, both commercially available preparations, zanamivir and oseltamivir were used in this study. Interferons were the next major class of antivirals studied. Various subtypes of interferon α (2a, 2b, n1, and n3, human leukocyte) and β (1a and 1b) were used. Amantadine, an old antiviral compound, was also studied. Different terms have been used to express antiviral activity, namely, EC₅₀, 95% effective concentration (EC₉₅), and 50% inhibitory concentration (IC₅₀); Table 1 illustrates the range of activity against selected viruses.

Tenfold dilutions of the drug were tested to cover a broad range of concentrations above and below inhibitory dosages as reported by the manufacturer for other viral-host combinations. Compounds already present in aqueous injections were made up to volume by using Hank’s buffered saline solution. For tablet and capsule formulations with soluble active ingredients, the outer coat was removed wherever applicable, and the preparation was ground in a mortar and pestle. The contents were dissolved in water, vortexed, and centrifuged thereafter at 3,000 g. The required volume was pipetted from the supernatant and diluted accordingly. When the active ingredients were insoluble in water (nelfinavir and saquinavir), the contents were dissolved in dimethylsulphoxide (DMSO); care was taken to ensure that the final concentration of DMSO in the dilutions would not exceed 1%. For plaque assays, fivefold drug dilutions were prepared by using growth media as specified below.

SARS-CoV Production and Infection

Vero E6 cells (American Type Culture Collection, Manassas, VA) were propagated in 75 cm² cell culture flasks in growth medium consisting of medium 199 (Sigma, St Louis, MO) supplemented with 10% fetal calf serum (FCS; Biological Industries, Kibbutz Beit Haemek, Israel). SARS-CoV 2003VA2774 (an isolate from a SARS patient in Singapore), which has been previously sequenced (14), was propagated in Vero E6 cells. Briefly, 2 mL of stock virus was added to a confluent monolayer of Vero E6 cells and incubated at 37°C in 5% CO₂ for 1 h; 13 mL of medium 199 supplemented with 5% FCS was then added. The cultures were incubated at 37^oC in 5% CO₂, and the supernatant was harvested after 48 h; in >75% of cultures, inhibition of CPE (3+) in each well was observed with an inverted microscope. The supernatant was clarified at 2,500 rpm and then divided into aliquots, placed in cryovials, and stored at –80°C until use.

Virus Handling and Titration

All virus culture and assays were carried out in the biosafety level-3 laboratory at the Environmental Health Institute, according to the conditions set out in Biosafety in Microbiological and Biomedical Laboratories (15). Virus titer in the frozen culture supernatant was determined by using a plaque assay. Briefly, 100 μL of virus in 10-fold serial dilution was added, in duplicates, to a monolayer of Vero E6 cells in a 24-well plate. After 1 h of incubation at 37°C in 5% CO₂, the viral inoculum was aspirated, and 1 mL of carboxymethylcellulose overlay with medium 199, supplemented with 5% FCS, was added to each well. After 4 days of incubation, the cells were fixed with 10% formalin and stained with 2% crystal violet. The plaques were counted visually, and the virus titer in plaque-forming units per mL (PFU/mL) was calculated.

Cytopathic Endpoint Assay

The protocol used was adapted from Al-Jabri et al. (16), and all drugs were tested in quadruplicate. Briefly, 100 μL of serial 10-fold dilutions of the drugs were incubated with 100 μL of Vero E6 cells, giving a final cell count of 20,000 cells per well in a 96-well plate. The incubation period was 1 h at 37°C in 5% CO₂, except for the interferons, which were incubated overnight with the cells. Ten μL of virus at a concentration of 10,000 PFU/well was then added to each of the test wells. The plates were incubated at 37°C in 5%CO₂ for 3 days and observed daily for CPE. The end point was the drug dilution that inhibited 100% of the CPE (CIA₁₀₀) in quadruplicate wells. To determine cytotoxicity, 100 μL of serial 10-fold dilutions of the drugs was incubated with 100 μL of Vero E6 cells, giving a final cell count of 20,000 cells per well in a 96-well plate, without viral challenge. The plates were then incubated at 37°C in 5% CO₂ for 3 days and examined for toxicity effects by using an inverted microscope.

Plaque Reduction Assay

Trypsinized Vero E6 cells were resuspended in growth medium and preincubated with interferons (serial fivefold dilution) in quadruplicate wells in 24-well plates. The next day, the medium was aspirated, and 100 μL of virus was added to each well at a titer of 100 PFU/well. After incubation for 1 h, the virus inoculum was aspirated, and a carboxymethylcellulose overlay containing maintenance medium and the appropriate interferon concentration was added. After 4 days’ incubation, the plates were fixed and stained as described previously. The number of plaques was then counted visually, and the concentration of drug that inhibits 50% of plaques in each well (IC₅₀) was determined. Results were plotted in Microsoft Excel, and a polynomial of order three was used to approximate the data and extrapolate IC₅₀ and IC₉₅ values.

Cell-based Assay of SARS-CoV Infection

Figure 1

Figure 1. Microscopic appearance of control (a) and infected (b) Vero E6 cells demonstrating cytopathic effects.

High titers of infectious SARS-CoV, originally derived from a respiratory sample of a SARS patient, were propagated on Vero E6 cells. The CPE of SARS-CoV on Vero E6 was evident within 24 hours after infection (Figure 1). SARS-CoV–infected cells display a CPE characterized by the appearance of rounded cells and the destruction of the monolayer.

Antiviral Drug Activity

A collection of 19 antiviral drugs was tested in the SARS-CoV CPE inhibition assay (Table 2). The set of drugs tested included seven interferons, five nucleoside analogs, three protease inhibitors, two RT inhibitors, and two neuraminidase inhibitors. Complete inhibition of the CPE was observed for four of the seven interferons in the initial screen when very high viral challenge of 10⁴ PFU/well and a high multiplicity of infection (MOI = 0.5) rate were used. Complete inhibition, expressed as CIA₁₀₀, was observed for interferon β-1b (Betaferon) at 5,000 IU/mL, interferon α-n3 (Alferon) at 5,000 IU/mL, interferon α-n1 (Wellferon) at 250,000 IU/mL, and human leukocyte interferon α (Multiferon) at 500,000 IU/mL. Ribavirin also completely inhibited the CPE at 5,000 μg/mL (Table 3). None of the other drugs showed complete inhibition of CPE, even at the highest concentration of drug tested (Table 2).

Rebif (IFN-β-1a) showed slight inhibition of CPE at 250,000 IU/mL, but the inhibition was not complete at the screening virus load of 10,000 PFU/well. Likewise, Roferon (IFN-α-2a) showed slight, incomplete inhibition at 50,000 IU/mL. Because the criteria for ascertaining anti-SARS-CoV activity in this screen were set at 100% inhibition of CPE, and as high doses of interferons may result in severe clinical side effects, we chose to conduct further evaluations only in the interferons that showed complete inhibition from initial screen, namely, Wellferon, Multiferon, Betaferon, and Alferon.

Based upon results of the primary screen, the four active interferons and ribavirin were retested at two lower viral challenges, 10³ and 10² PFU/ well. All four drugs again showed inhibitory effect, although the CIA₁₀₀ were dependent on viral loads (Table 3). At the lowest viral load the CIA₁₀₀ were 5 IU/mL for both interferon β-1b (Betaferon) and human leukocyte interferon α (Multiferon); and 50 and 250 IU/mL for interferon α-n3 (Alferon) and interferon α-n1 (Wellferon), respectively. No cytotoxicity of the interferons was observed at or near inhibitory concentrations. Ribavirin showed inhibitory activity at all three viral loads, but only at high concentrations of the drug, 0.5–5 mg/mL. At high concentrations of ribavirin (0.2–1 mg/mL) cytotoxic effects were observed on VeroE6 cells, as has been reported for other cell types (17,18). As such, we consider ribavirin to be inactive against SARS-CoV.

Figure 2

Figure 2. Dose-response curves for Alferon (a), Betaferon (b), and Multiferon (c) as determined by plaque reduction assays. IC₅₀ (50% inhibitory concentration) and IC₉₅ (95% inhibitory concentration) values were calculated by using the...

A plaque reduction assay format with 100 PFU of SARS-CoV (MOI = 0.0005) was conducted to determine the IC₅₀ for Betaferon, Alferon, and Multiferon, the three compounds that showed greatest potency for inhibition of CPE. Additional supply was not available for testing interferon α-n1 (Wellferon), as production of this drug has been discontinued. Cells were preincubated for 15 h with fivefold dilutions of drug. Viral-induced plaques, which developed in 3 days, were counted to determine the inhibitory effect of the drugs at various concentrations. All three interferon preparations displayed a dose-dependent inhibition of SARS-CoV plaque formation in this assay (Figure 2). The IC₅₀ and IC₉₅ were determined to be 0.2 and 8 IU/mL for Betaferon, 0.8 and 200 IU/mL for Alferon, and 2 and 44 IU/mL for Multiferon.