Virucidal Activity of World Health Organization–Recommended Formulations Against Enveloped Viruses, Including Zika, Ebola, and Emerging Coronaviruses

Siddharta, Anindya; Pfaender, Stephanie; Vielle, Nathalie Jane; Dijkman, Ronald; Friesland, Martina; Becker, Britta; Yang, Jaewon; Engelmann, Michael; Todt, Daniel; Windisch, Marc P.; Brill, Florian H.; Steinmann, Joerg; Steinmann, Jochen; Becker, Stephan; Alves, Marco P.; Pietschmann, Thomas; Eickmann, Markus; Thiel, Volker; Steinmann, Eike

Based on the obtained virucidal activities of the WHO formulations against the different enveloped viruses, we next analyzed the inactivation profiles in a comparative analysis ( Figure 2C and 2D) . The most susceptible viruses to the WHO formulation I were the bovine and emerging CoVs (BCoV, SARS-CoV, MERS-CoV) and ZIKV ( Figure 2C ). With a shift to increasing WHO forumation I concentration, the more stable viruses included the full infectious EBOV (trVLPs excluded in this analysis) and HCV ( Figure 2C ). The highest alcohol-based concentrations of WHO formulation I (>40%) were required for H1N1 and MVA, which displayed nearly identical inactivation response curves ( Figure 2C ). The results for the isopropanol-based WHO formulation II are depicted in Figure 2D ; it demonstrated a similar pattern of susceptibility for the different enveloped viruses with an obvious shift toward lower concentrations ( Figure 2D ). The CoVs and ZIKV showed the highest susceptibility to WHO formulation II, whereas HCV, EBOV, H1N1 and MVA demonstrated a more resistant inactivation profile ( Figure 2D ). To also directly compare the performance of the 2 WHO formulations, we determined the concentrations at which the products reached the EC 50 (Supplementary Figure 2) . World Health Organization formulation II showed a significantly higher virucidal activity against the different viruses compared with WHO formulation I (P = .008). In summary, CoVs and ZIKV showed the highest susceptibility to WHO formulations. Ebola virus and HCV were observed to be less susceptible than the CoVs, whereas H1N1 and MVA were the most stable viruses. In addition, WHO formulation II demonstrated a higher virucidal effect compared with WHO formulation I. The WHO has recommended 2 formulations in Guidelines on Hand Hygiene in Health Care, a document proposing the use of cheap alcohol-based hand rubs to reduce the transmission of pathogens [1] . We aimed in this study to analyze the virucidal efficacies of these products, particularly against emerging or re-emerging viruses that caused severe epidemics in the recent past [2] . Importantly, both WHO formulations inactivated all tested viruses, including ZIKV, EBOV, and emerging CoVs, in a suspension test with 30-second exposure time, implicating the formulations I and II were tested for their efficacy in inactivating EBOV transcription-and replication-competent virus-like particles (trVLPs) (A) and EBOV (B). The biocide concentrations ranged from 0% to 80% with an exposure time of 30 seconds. For this inactivation assay, 1 part virus and 1 part organic load were mixed with 8 parts of biocide. For determination of the EBOV trVLP infectivity, luciferase activity was measured 72 hours later. For EBOV, residual infectivity was determined by a limiting dilution assay. Viral titers are displayed as 50% tissue culture infectious dose (TCID 50 ) values. The cytotoxicity was calculated in analogy to the determination of virus titer (TCID 50 /mL) and is depicted as a dashed line. The means of 2 independent experiments with standard deviations are shown. Normalized values of percentage inactivation of viral infectivity (y-axis) were plotted against WHO formulations I (C) or II (D) in dose-response curves (x-axis, log representation). Viruses are listed in each panel and are ranked from the most to the least stable. Normalization and nonlinear regression calculation of all data were performed using GraphPad Prism version 6.07 for Windows. Abbr