key: cord-0823840-cihkbzby authors: Pagani, Isabel; Ghezzi, Silvia; Clementi, Massimo; Poli, Guido; Bussi, Mario; Pianta, Luca; Trimarchi, Matteo; Vicenzi, Elisa title: Vinegar and Its Active Component Acetic Acid Inhibit SARS-CoV-2 Infection In Vitro and Ex Vivo date: 2020-07-20 journal: bioRxiv DOI: 10.1101/2020.07.08.193193 sha: 22cd8340faa42ed2693dc247a2fd98cab6c0efbe doc_id: 823840 cord_uid: cihkbzby Effective and economical measures are needed to either prevent or inhibit the replication of SARS-CoV-2, the causative agent of COVID-19, in the upper respiratory tract. As fumigation of vinegar at low concentration (0.34%) ameliorated the symptoms of mild SARS-CoV-2 infection, we tested in vitro the potential antiviral activity of vinegar and of its active component, acetic acid. We here demonstrate that both vinegar and acetic acid indeed strongly inactivate SARS-CoV-2 infectivity in Vero cells. Furthermore, vinegar treatment caused a 90% inhibition of the infectious titer when directly applied to a nasopharyngeal swab transfer medium of a COVID-19 patient. These effects were potentiated if conduced at a temperature of 45 °C vs. 37 °C, a condition that is transiently generated in the upper respiratory tract during fumigation. Our findings are consistent and extend the results of studies performed in the early and mid-20th century on the disinfectant capacity of organic acids and can provide an affordable home-made aid to prevent or contain SARS-CoV-2 infection of the upper respiratory tract. Based on ancient medical tradition 6 , in order to mitigate virus replication and its consequences in the upper respiratory tract mucosa, including acute anosmia (a frequent early symptom of SARS-CoV-2 infection 7 ), fumigation of vinegar diluted in boiling water at a concentration of 0.34% for 15 min has been adopted as an empirical practice in COVID-19 patients. Indeed, improvement of symptoms such as coughing and fever was observed in >90% of individuals 8 . In this regard, vinegar (derived from the biochemical processing of acetobacter species converting the ethanol of wine into acetic acid 9 ) has been used as a disinfectant for thousands of years and is commonly used to eliminate bacteria from fresh products 10 or as topical treatment for otitis externa 11 . Acetic acid, the active component of vinegar, is also an effective disinfectant against mycobacterial infection 12 . Herein, we tested the potential antiviral activity of commercial vinegar and of its active component acetic acid against SARS-CoV-2 infection and replication in vitro and ex vivo. Vero cells were seeded at 2.5x10 5 cell/well in 24-well plates in EMEM supplemented with 10% fetal calf serum (complete medium). Twenty-four h later, 50 plaque forming units (PFU) of a previously titrated SARS-CoV-2 isolate (GISAID accession ID: EPI_ISL_413489) were added to vinegar (from 0.28% to 0.008%) or acetic acid (from 0.1 to 0.5%) serially diluted (1:2) in PBS and incubated for 15 min at either 37 °C or 45 °C before addition to confluent Vero cells. Cell supernatants were discarded after 60 min and 1% methylcellulose (500 µl/well) dissolved in complete medium was added to each well. After 3 days, cells were fixed with formaldehyde/PBS solution (6%) and stained with crystal violet (1%; Sigma Chemical Corp.) in 70% methanol. Viral plaques were counted under a stereoscopic microscope (SMZ-1500, Nikon), as published 13 . A nasopharyngeal flocked swab (UTM® viral transport, COPAN Diagnostics Inc.) was obtained from a COVID-19 patient with severe symptoms. Fifty µl of the COPAN transport medium were diluted 1:2 with a vinegar solution at 0.28% either at 37°C or 45 °C for 15 min. The vinegar solution was then added to a monolayer of Vero cells that were seeded 24 h earlier at 2.5x10 5 cell/well in 24-well plates in complete medium. As for in vitro infection, the supernatants were discarded after 60 min and 1% methylcellulose (500 µl/well) dissolved in complete medium was added to each well. After three days, plaques were stained and counted as described above. All infection experiments were performed in a biosafety level-3 (BLS-3) . Ten μl samples of culture supernatant were transferred on a half black 96 well plate (Costar). To each well, 50 μl of the adenylate kinase detection reagent (ToxiLight ® BioAssay, Lonza) was added and the plate was incubated for 10 min at room temperature. Luminescence was measured in a Mithras LB940 Microplate Reader (Berthold Technologies). The results were expressed as Relative Light Unit (RLU). Prism GraphPad software v. 8.0 (www.graphpad.com) was used for all statistical analyses. Comparison among groups were performed using the one-way analysis of variance with the Bonferroni's multiple comparison test. According to the concentration used for fumigation in SARS-CoV-2 infected patients 8 , a concentration-response curve of viral inhibition by vinegar (diluted from 0.28% to 0.008% in PBS) was generated by a plaque assay previously optimized for Zika virus infection 13 . Diluted vinegar effectively inhibited SARS-CoV-2 plaque formation by 80% at the top concentration of 0.28% with an inhibitory effective concentration 50 (IC50) of 0.08% ( Figure 1A ); of note is the fact that this vinegar dilution was not toxic to cells (Figure 1B) as revealed by the levels of adenylate kinase activity released in culture supernatants as an indicator of plasma membrane damage and cell necrosis 13 . As the active component of vinegar is acetic acid, we next evaluated its anti-SARS-CoV-2 activity within the concentration range of vinegar. Indeed, incubation of SARS-CoV-2 with 0.5% acetic acid in PBS for 15 min at 37 °C reduced SARS-CoV-2 plaque formation by 89% with an IC50 of 0.14% ( Figure 1C ) without evidence of cytotoxicity ( Figure 1D ). As vinegar has been topically administered by fumigation in boiling water 8 , an empirical determination of the t° was performed with a thermometer placed in the nostrils for 15 min during fumigation. The starting nose temperature was 54 °C and quickly decreased to 42 °C. Based on these empirical data, we tested the antiviral effect of vinegar diluted at 0.28% in PBS at 45 °C for 15 min. A significant decrease of plaque formation was observed by infecting Vero cells with virus treated at 45 °C as compared to untreated control at 37 °C ( Figure 2 ). In addition, when the vinegar solution was heated at 45°C for 15 min, a significant additive reduction to 90% as compared to the virus inoculum treated at 37 °C was observed. We next evaluated whether vinegar could inactivate the infectivity of SARS-CoV-2 present in a nasopharyngeal swab transport medium. To this purpose, the flocked swab transport medium was directly tested in a plaque assay either in the presence of a vinegar solution at the concentration of 0.14%. When either 50 µl or 10 µl of transport medium were added to Vero cells, plaque formation proportional to the volume of transport medium was observed and vinegar significantly reduced the number of plaques ( Figure 3A) . When tested at 45 °C, viral plaques generated by 10 µl of transport medium were reduced to almost zero ( Figure 3B) . As the plaque number were 5-fold less with 10 µl of transport medium than with 50 µl, the results were pooled as shown in Figure 4 . The mean number of plaques in the control sample was 248±18 at 37 °C whereas at 45°C a significantly decreased (ca. 50%) number of plaques was observed. Vinegar reduced the plaque yield to ca. 50% at 37 °C and up to 90% at 45 °C. In the present study we demonstrate that both commercial vinegar and its active The concentration of acetic acid present in commercial vinegar varies from brand-tobrand and country to country. In Italy, it is typically found at 6-7% in vinegar derived from white wine, as used in this study, whereas in the United States it is usually 5%. Despite these differences, the IC50 as calculated from a non-linear fitting is relatively low (0.08%) and obtainable with vinegar sold at almost any percentage. As fumigation of vinegar has been used in mild COVID-19 patients in hot, boiling water, we here show that a temperature of 45 °C had an additive effect in terms of inactivation of virus infectivity. Importantly, we here demonstrate that vinegar is effective in inhibiting the viral infectivity by lowering the viral titer present in a nasopharyngeal swab. **** Clinical Characteristics of Covid-19 in China. Reply Virological assessment of hospitalized patients with COVID-2019 SARS-CoV-2 Viral Load in Upper Respiratory Specimens of Infected Patients Viral dynamics in mild and severe cases of COVID-19 Suppression of a SARS-CoV-2 outbreak in the Italian municipality of Vo' The history of vinegar and of its acetification systems Sudden and Complete Olfactory Loss Function as a Possible Symptom of COVID-19 Acid Acetic disinfection as a potential adjunctive therapy for non-severe COVID-19 Biotechnological applications of acetic acid bacteria Antibacterial action of vinegar against food-borne pathogenic bacteria including Escherichia coli O157:H7 Interventions for acute otitis externa Acetic Acid, the active component of vinegar, is an effective tuberculocidal disinfectant Human Endometrial Stromal Cells Are Highly Permissive To Productive Infection by Zika Virus Defectiveness of interferon production and of rubella virus interference in a line of African green monkey kidney cells (Vero) Transcriptional and posttranscriptional regulation of exogenous human beta interferon gene in simian cells defective in interferon synthesis Mutations Conferring Increased Sensitivity to Tripartite Motif 22 Restriction Accumulated Progressively in the Nucleoprotein of Seasonal Influenza A (H1N1) Viruses between Coronaviridae and SARS-associated coronavirus strain HSR1 Heparin inhibits cellular invasion by SARS-CoV-2: structural dependence of the interaction of the surface protein (spike) S1 receptor binding domain with heparin