Study Design and Rationale
Coronaviruses have originated from many different animal species, including bats, cattle, camels, civets, and rodents . They are known to cause mild to serious respiratory, gastrointestinal, and neurological diseases. Efficient viral transmission appears to depend upon the viral proteins and unique cell surface receptor(s) on the invaded cell types. The present preclinical in vitro study was undertaken to better understand the broad-spectrum antiviral activity of PVP-I and to determine, if any, differential effectiveness for various PVP-I formulations against various coronavirus strains. In this study, the efficacy of different PVP-I formulations was compared with matching placebos against human and murine coronavirus surrogates for SARS-CoV-2.
Different test formulations, including active PVP-I (Betadine®, Avrio Health L.P., Stamford, USA) and placebo, were assessed: 0.5% (w/v) solution, placebo 0.5% (w/v) solution, 5.0% (w/v) solution, 7.5% (w/v) scrub, placebo 7.5% (w/v) scrub, and 10.0% (w/v) solution in study 1 and 0.5% (w/v) solution, placebo 0.5% (w/v) solution, 7.5% (w/v) scrub, placebo 7.5% (w/v) scrub, and 10.0% (w/v) solution in study 2.
Virus Strains and Host Cells
In study 1, murine coronavirus strain A59 (American Type Culture Collection [ATCC]® VR-764™), a betacoronavirus from the same genus that includes SARS-CoV-1, SARS-CoV-2, and MERS-CoV, was used as a surrogate for SARS-CoV-2; the strain was sourced from Dr. Nerea Irigoyen (Department of Pathology, University of Cambridge, UK). Mouse hepatitis virus A59 was used as a surrogate test virus for SARS-CoV-2 in study 1 since it is a representative member of the genus betacoronavirus within the subfamily Coronavirinae-2, of which SARS-CoV-2 is a member [26,27,28]. The National Collection of Type Cultures (NCTC) clone 1469 cells (ATCC® CCL-9.1™; newborn mouse liver cells) were used as host cells. In study 2, test formulations were challenged with three human viral strains that served as surrogates for SARS-CoV-2 : coronavirus strain OC43 (ZeptoMetrix Corp. #0810024CF), coronavirus strain NL63 (ZeptoMetrix Corp. #0810228CF), and coronavirus strain 229E (ATCC® VR-740™). MRC-5 cells (ATCC® CCL-171™; human lung fibroblast cells), Vero cells (ATCC® CCL-81™; green monkey epithelial kidney cells), and HCT-8 cells (ATCC® CCL-244™; human colon adenocarcinoma epithelial cells) were used as host cells.
The assays were performed at separate facilities: study 1 at BluTest Laboratories Ltd., Glasgow, UK, and study 2 at BioScience Laboratories, Inc. (BSLI), Bozeman, Montana, USA. Standard equipment and supplies were used; calibration was in accordance with the standard operating procedure (SOP) of each facility. This article is based on in vitro studies and did not include research on human participants or animals performed by any of the authors.
Preparation of Test Formulations, Virus Suspensions, and Host Cells
For study 1, higher-concentration PVP-I formulations (i.e., 5.0% (w/v) solution, 7.5% (w/v) scrub, and 10.0% (w/v) solution) were diluted 1:10 in sterile distilled water for neutralization validation and the in vitro time-kill assay; the remaining test formulations (i.e., 0.5% (w/v) solution, placebo 0.5% (w/v) solution, and placebo 7.5% (w/v) scrub) were used undiluted. For study 2, all test formulations were used as received from the study sponsor; final test formulations had a concentration of 90%.
Virus strains were propagated and stored per standard procedure for production of high-titer virus stock (BSLI SOP L-2102); aliquots of stock virus suspension stored at −70 °C were thawed before use. ATCC-obtained host cells were maintained as monolayers in disposable cell culture labware in accordance with standard procedure (BSLI SOP L-2084). Before testing, these cultures were seeded onto multiwell, cell culture-treated plates. Vero and HCT-8 cell monolayers were 80–90% confluent and less than 48 h old before virus inoculation; MRC-5 cells were 80% confluent and approximately 51 h old. The growth medium (i.e., RPMI-1640 or 1X Eagle’s Minimum Essential Medium with 10% fetal bovine serum and 1% antibiotic and l-glutamine) was replaced with the maintenance medium (i.e., RPMI-1640 or 1X Eagle’s Minimum Essential Medium with 2% fetal bovine serum and 1% antibiotic and l-glutamine) to support virus propagation.
Assessment of Cytopathic/Cytotoxic Effect
Cytopathic/cytotoxic effect in host cells was monitored to determine the lowest concentration of PVP-I that was noncytopathic and noncytotoxic.
Neutralization validation was performed to confirm the effectiveness of the procedure in neutralizing the active virucidal component with each formulation; this was performed for the median tissue culture infective dose (TCID50) input virus, positive control, negative control, and test formulations. Standard testing methods based on ASTM were employed. Neutralization was validated when virus recovery in the positive control matched that in the neutralized formulation.
Virucidal Suspension Test Using the Time-Kill Method
For study 1, triplicates were set up for each designated exposure time point. The first well contained 0.8 mL of the test formulation at a specific concentration and 0.1 mL of sterile water. The procedure was initiated when 0.1 mL of virus was added to this well and mixed. Samples were incubated for designated exposure times of 15 s, 30 s, 60 s, and 5 min. Upon the specified time indicated, a 0.1-mL volume from the well was sampled and applied gently to the surface of a prepared MicroSpin S-400 HR column and eluted per manufacturer’s instructions. The void volume eluent (0.1 mL) was applied to a well containing 0.9 mL of 5% bovine serum albumin in phosphate-buffered saline at 20 ± 1 °C and incubated for 5 min at 20 ± 1 °C. Following incubation, 0.1 mL of the mixture was diluted in 0.9 mL of cell culture media before being serially diluted up to a concentration of 10−6 and plated per the standard TCID50 reads/calculations; results were recorded. Subsequent analyses were performed using the well-established Spearman-Kärber method of quantification . Positive controls were set up alongside the test formulation being processed; the positive-control well contained 0.8 mL of cell culture media, 0.1 mL of sterile water, and 0.1 mL of the virus. Positive controls were assessed at 0 s and 5 min; negative controls were assessed at 15 s and 5 min. The negative control runs were identical to the test formulation runs, except water was substituted for the virus suspension.
For study 2, a virucidal suspension test based on ASTM E1052-11 (Standard Test Method to Assess the Activity of Microbicides against Viruses in Suspension) was also performed. A 0.5-mL aliquot of test virus was added to 4.5 mL of undiluted test formulation to obtain a 90% (w/v) concentration of the test formulation. The challenge virus suspension was exposed to the test formulation for the designated exposure times (i.e., 0 [< 15 s], 15 s, 30 s, 60 s, and 5 min). Immediately after exposure, the test virus suspension was neutralized in Dey–Engley neutralizing broth, mixed thoroughly, and serially diluted in the maintenance medium. Each dilution was plated in four replicates.
Viral titers in both studies were determined using the quantal test (Spearman-Kärber method)  and expressed as TCID50 using the following formula: log TCID50 = L − d(s − 0.5)/inoculum volume, where L is the negative log10 of the lowest dilution, d is the difference between the dilution steps, and s is the sum of proportions of the positive wells per test dilution. Reduction in test virus infectivity of each strain was calculated as the difference in log10 TCID50/mL between the virus control and the test virus; this was done at the designated exposure times (i.e., 15 s, 30 s, 60 s, and 5 min for study 1 and 0 [< 15 s], 15 s, 30 s, 60 s, and 5 min for study 2). The percentage reduction was also calculated and reported.