Cell Lines as Biological Models: Practical Steps for More Reliable Research.

Research in toxicology relies on in vitro models such as cell lines. These living models are prone to change and may be described in publications with insufficient information or quality control testing. This article sets out recommendations to improve the reliability of cell-based research.

Authentication of M14 melanoma cell line proves misidentification of MDA-MB-435 breast cancer cell line.

A variety of analytical approaches have indicated that melanoma cell line UCLA-SO-M14 (M14) and breast carcinoma cell line MDA-MB-435 originate from a common donor. This indicates that at some point in the past, one of these cell lines became misidentified, meaning that it ceased to correspond to the reported donor and instead became falsely identified (through cross-contamination or other means) as a cell line from a different donor. Initial studies concluded that MDA-MB-435 was the misidentified cell line and M14 was the authentic cell line, although contradictory evidence has been published, resulting in further confusion. To address this question, we obtained early samples of the melanoma cell line (M14), a lymphoblastoid cell line from the same donor (ML14), and donor serum preserved at the originator's institution. M14 samples were cryopreserved in December 1975, before MDA-MB-435 cells were established in culture. Through a series of molecular characterizations, including short tandem repeat (STR) profiling and cytogenetic analysis, we demonstrated that later samples of M14 and MDA-MB-435 correspond to samples of M14 frozen in 1975, to the lymphoblastoid cell line ML14, and to the melanoma donor's STR profile, sex and blood type. This work demonstrates conclusively that M14 is the authentic cell line and MDA-MB-435 is misidentified. With clear provenance information and authentication testing of early samples, it is possible to resolve debates regarding the origins of problematic cell lines that are widely used in cancer research.

Intra-family differences in efficacy of inactivation of small, non-enveloped viruses.

The use of specific model viruses for validating viral purification process steps and for assessing the efficacies of viral disinfectants is based, in part, on the assumption that viral susceptibilities to such treatments will be similar for different members, including different genera, within a given viral family. This assumption is useful in cases where cell-based infectivity assays or laboratory strains for the specific viruses of interest might not exist. There are some documented cases, however, where exceptions to this assumption exist. In this paper, we discuss some of the more striking cases of intra-family differences in susceptibilities to inactivation steps used for downstream viral purification steps in biologics manufacture (e.g. heat inactivation, low pH, and guanidinium hydrochloride inactivation) and to specific viral disinfectants (e.g. alcohols, hydrogen peroxide, and quaternary ammonium-containing disinfectants) that might be employed for facility/equipment disinfection. The results suggest that care should be taken when extrapolating viral inactivation susceptibilities from specific model viruses to different genera or even to different members of the same genus. This should be taken into consideration by regulatory agencies and biologics manufacturers designing viral clearance and facility disinfection validation studies, and developers and evaluators of viral disinfectants.

Differing Susceptibilities to Certain Microbicidal Chemistries among Three Representative Enveloped Viruses.

Three lipid-enveloped viruses (bovine viral diarrhea virus [BVDV], vaccinia virus, and severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2]) were evaluated in side-by-side liquid inactivation efficacy studies of low pH (3.0 to 3.1) treatment and of the non-formulated microbicidal actives sodium hypochlorite (100 ppm), ethanol (70%), quaternary ammonium compound BTC® 835 (100 ppm), and peracetic acid (100 ppm). Low pH was evaluated at 10 and 60 min contact times, and the microbicides were evaluated at 1 min contact time at room temperature per the ASTM E1052 standard. In each case, 5% animal serum was included in the viral inoculum as a challenge soil load. The three viruses displayed similar susceptibility to sodium hypochlorite and ethanol, with complete inactivation resulting. Significant differences in susceptibility to BTC® 835 and peracetic acid were identified, with the ordering of the three viruses for susceptibility to BTC® 835 being SARS-CoV-2 > vaccinia virus = BVDV, and the ordering for peracetic acid being vaccinia virus > SARS-CoV-2 > BVDV. The ordering for susceptibility to low pH treatment (60 min contact time) was vaccinia virus > SARS-CoV-2 > BVDV. Not all enveloped viruses display equivalent susceptibilities to inactivation approaches. For the chemistries evaluated here, BVDV appears to represent a worst-case enveloped virus.

Mechanisms of action of microbicides commonly used in infection prevention and control.

SUMMARYUnderstanding how commonly used chemical microbicides affect pathogenic microorganisms is important for formulation of microbicides. This review focuses on the mechanism(s) of action of chemical microbicides commonly used in infection prevention and control. Contrary to the typical site-specific mode of action of antibiotics, microbicides often act via multiple targets, causing rapid and irreversible damage to microbes. In the case of viruses, the envelope or protein capsid is usually the primary structural target, resulting in loss of envelope integrity or denaturation of proteins in the capsid, causing loss of the receptor-binding domain for host cell receptors, and/or breakdown of other viral proteins or nucleic acids. However, for certain virucidal microbicides, the nucleic acid may be a significant site of action. The region of primary damage to the protein or nucleic acid is site-specific and may vary with the virus type. Due to their greater complexity and metabolism, bacteria and fungi offer more targets. The rapid and irreversible damage to microbes may result from solubilization of lipid components and denaturation of enzymes involved in the transport of nutrients. Formulation of microbicidal actives that attack multiple sites on microbes, or control of the pH, addition of preservatives or potentiators, and so on, can increase the spectrum of action against pathogens and reduce both the concentrations and times needed to achieve microbicidal activity against the target pathogens.

Match criteria for human cell line authentication: where do we draw the line?

Continuous human cell lines have been used extensively as models for biomedical research. In working with these cell lines, researchers are often unaware of the risk of cross-contamination and other causes of misidentification. To reduce this risk, there is a pressing need to authenticate cell lines, comparing the sample handled in the laboratory to a previously tested sample. The American Type Culture Collection Standards Development Organization Workgroup ASN-0002 has developed a Standard for human cell line authentication, recommending short tandem repeat (STR) profiling for authentication of human cell lines. However, there are known limitations to the technique when applied to cultured samples, including possible genetic drift with passage. In our study, a dataset of 2,279 STR profiles from four cell banks was used to assess the effectiveness of the match criteria recommended within the Standard. Of these 2,279 STR profiles, 1,157 were grouped into sets of related cell lines-duplicate holdings, legitimately related samples or misidentified cell lines. Eight core STR loci plus amelogenin were used to unequivocally authenticate 98% of these related sets. Two simple match algorithms each clearly discriminated between related and unrelated samples, with separation between related samples at ≥80% match and unrelated samples at <50% match. A small degree of overlap was noted at 50-79% match, mostly from cell lines known to display variable STR profiles. These match criteria are recommended as a simple and effective way to interpret results from STR profiling of human cell lines.

Polyomavirus inactivation - a review.

Polyomavirus inactivation has been studied since the 1950s when it became apparent that certain polio vaccines were contaminated with SV40. Relatively high temperatures (≥70 °C) are required to effect thermal inactivation of the polyomaviruses. The chemical inactivants that are effective (ß-propiolactone, ethanol, sodium hydroxide, and formaldehyde) are those that have displayed efficacy for other small, non-enveloped viruses, such as the circoviruses. Low pH inactivation can be effective, especially at pH at or below 3 and at higher temperatures. Polyomaviruses are more resistant to UV-C irradiation than are other small non-enveloped viruses such as the parvoviruses and caliciviruses. The efficacy of photodynamic inactivation of polyomaviruses is very much dye-dependent, with toluidine blue, acridine orange, and methylene blue dyes being effective photosensitizers. Ionizing radiation can be effective, depending on the conditions employed and the inactivation matrix. Inactivation of the oncogenic properties of the polyomaviruses may require higher doses of inactivant than those required to inactivate infectivity. While the polyomaviruses are considered to be highly resistant to inactivation, the degree of resistance is dependent upon the specific approach under consideration. For certain approaches, such as UV-C and gamma-irradiation, the polyomaviruses appear to be more resistant than other small non-enveloped viruses.

Antiviral Natural Products, Their Mechanisms of Action and Potential Applications as Sanitizers and Disinfectants.

Plant extracts, natural products and plant oils contain natural virucidal actives that can be used to replace active ingredients in commercial sanitizers and disinfectants. This review focuses on the virucidal mechanisms of natural substances that may exhibit potential for indoor air and fomite disinfection. Review of scientific studies indicates: (1) most natural product studies use crude extracts and do not isolate or identify exact active antiviral substances; (2) many natural product studies contain unclear explanations of virucidal mechanisms of action; (3) natural product evaluations of virucidal activity should include methods that validate efficacy under standardized disinfectant testing procedures (e.g., carrier tests on applicable surfaces or activity against aerosolized viruses, etc.). The development of natural product disinfectants requires a better understanding of the mechanisms of action (MOA), chemical profiles, compound specificities, activity spectra, and the chemical formulations required for maximum activity. Combinations of natural antiviral substances and possibly the addition of synthetic compounds might be needed to increase inactivation of a broader spectrum of viruses, thereby providing the required efficacy for surface and air disinfection.

Efficacy of microbicidal actives and formulations for inactivation of Lassa virus in suspension.

The World Health Organization's R&D Blueprint list of priority diseases for 2022 includes Lassa fever, signifying the need for research and development in emergency contexts. This disease is caused by the arenavirus Lassa virus (LASV). Being an enveloped virus, LASV should be susceptible to a variety of microbicidal actives, although empirical data to support this expectation are needed. We evaluated the virucidal efficacy of sodium hypochlorite, ethanol, a formulated dual quaternary ammonium compound, an accelerated hydrogen peroxide formulation, and a p-chloro-m-xylenol formulation, per ASTM E1052-20, against LASV engineered to express green fluorescent protein (GFP). A 10-µL volume of virus in tripartite soil (bovine serum albumin, tryptone, and mucin) was combined with 50 µL of disinfectant in suspension for 0.5, 1, 5, or 10 min at 20-25 °C. Neutralized test mixtures were quantified by GFP expression to determine log10 reduction. Remaining material was passaged on Vero cells to confirm absence of residual infectious virus. Input virus titers of 6.6-8.0 log10 per assay were completely inactivated by each disinfectant within 1-5 min contact time. The rapid and substantial inactivation of LASV suggests the utility of these microbicides for mitigating spread of infectious virus during Lassa fever outbreaks.

Environmental dissemination of respiratory viruses: dynamic interdependencies of respiratory droplets, aerosols, aerial particulates, environmental surfaces, and contribution of viral re-aerosolization.

During the recent pandemic of COVID-19 (SARS-CoV-2), influential public health agencies such as the World Health Organization (WHO) and the U.S. Centers for Disease Control and Prevention (CDC) have favored the view that SARS CoV-2 spreads predominantly via droplets. Many experts in aerobiology have openly opposed that stance, forcing a vigorous debate on the topic. In this review, we discuss the various proposed modes of viral transmission, stressing the interdependencies between droplet, aerosol, and fomite spread. Relative humidity and temperature prevailing determine the rates at which respiratory aerosols and droplets emitted from an expiratory event (sneezing, coughing, etc.) evaporate to form smaller droplets or aerosols, or experience hygroscopic growth. Gravitational settling of droplets may result in contamination of environmental surfaces (fomites). Depending upon human, animal and mechanical activities in the occupied space indoors, viruses deposited on environmental surfaces may be re-aerosolized (re-suspended) to contribute to aerosols, and can be conveyed on aerial particulate matter such as dust and allergens. The transmission of respiratory viruses may then best be viewed as resulting from dynamic virus spread from infected individuals to susceptible individuals by various physical states of active respiratory emissions, instead of the current paradigm that emphasizes separate dissemination by respiratory droplets, aerosols or by contaminated fomites. To achieve the optimum outcome in terms of risk mitigation and infection prevention and control (IPAC) during seasonal infection peaks, outbreaks, and pandemics, this holistic view emphasizes the importance of dealing with all interdependent transmission modalities, rather than focusing on one modality.

Emerging SARS-CoV-2 Mutational Variants of Concern Should Not Vary in Susceptibility to Microbicidal Actives.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is evolving, with emergence of mutational variants due to the error-prone replication process of RNA viruses, in general. More recently, the Delta and Omicron variants (including sub-variants BA.1-5) predominate globally, and a Delta-Omicron recombinant termed Deltacron has emerged. The emergence of variants of concern (VOC) demonstrating immune evasion and potentially greater transmissibility and virulence naturally raises concern in both the infection control communities and the public at large, as to the continued suitability of interventions intended to mitigate the risk of viral dissemination and acquisition of the associated disease COVID-19. We evaluated the virucidal efficacy of targeted surface hygiene products (an ethanol/quaternary ammonium compound (QAC)-containing disinfectant spray, a QAC disinfectant wipe, a lactic acid disinfectant wipe, and a citric acid disinfectant wipe) through both theoretical arguments and empirical testing using international standard methodologies (ASTM E1053-20 hard surface test and EN14476:2013+A2:2019 suspension test) in the presence of soil loads simulating patients' bodily secretions/excretions containing shed virus. The results demonstrate, as expected, complete infectious viral inactivation (≥3.0 to ≥4.7 log10 reduction in infectious virus titer after as little as 15 s contact time at room temperature) by these surface hygiene agents of the original SARS-CoV-2 isolate and its Beta and Delta VOC. Through appropriate practices of targeted surface hygiene, it is expected that irrespective of the SARS-CoV-2 VOC encountered as the current pandemic unfolds (and, for that matter, any emerging and/or re-emerging enveloped virus), the chain of infection from virus-contaminated fomites to the hand and mucous membranes of a susceptible person may be disrupted.

Virucidal efficacy of laundry sanitizers against SARS-CoV-2 and other coronaviruses and influenza viruses.

The clothes laundering process affords numerous opportunities for dissemination of infectious virus from contaminated clothing to appliance surfaces and other household surfaces and eventually to launderer's hands. We have explored the efficacy of laundry sanitizers for inactivating coronaviruses and influenza viruses. Virucidal efficacy was tested using standardized suspension inactivation methods (EN 14476) or hard-surface inactivation methods (ASTM E1053-20) against SARS-CoV-2, human coronavirus 229E (HCoV 229E), influenza A virus (2009-H1N1 A/Mexico), or influenza B virus (B/Hong Kong). Efficacy was measured in terms of log10 reduction in infectious virus titer, after 15 min contact time (suspension studies) or 5 min contact time (hard surface studies) at 20 ± 1 °C. In liquid suspension studies, laundry sanitizers containing p-chloro-m-xylenol (PCMX) or quaternary ammonium compounds (QAC) caused complete inactivation (≥ 4 log10) of HCoV 229E and SARS-CoV-2 within 15 min contact time at 20 ± 1 °C. In hard surface studies, complete inactivation (≥ 4 log10) of each coronavirus or influenza virus, including SARS-CoV-2, was observed following a 5-min contact time at 20 ± 1 °C. Respiratory viruses may remain infectious on clothing/fabrics and environmental surfaces for hours to days. The use of a laundry sanitizer containing microbicidal actives may afford mitigation of the risk of contamination of surfaces during handling of the laundry and washing appliances (i.e., washer/dryer or basin), adjacent surfaces, the waste water stream, and the hands of individuals handling clothes contaminated with SARS-CoV-2, influenza viruses, or other emerging enveloped viruses.

Gamma irradiation of animal sera for inactivation of viruses and mollicutes--a review.

Animal-derived materials such as animal sera represent a low, but finite, risk for introduction of an adventitious agent (virus or mollicute) into a biological bulk harvest during upstream manufacturing processes involving mammalian cell substrates. Viral and mollicute (Mycoplasma sp. and Acholeplasma sp.) contamination events have been relatively rare, but many of those that have been reported have been attributed to use of infected animal sera in growth media during cell expansion. The risk of introduction of viruses and mollicutes may be mitigated by elimination of the use of animal sera and implementation instead of chemically defined or serum- and animal-derived material-free cell culture media. When use of animal sera is unavoidable, however, mitigation of the risk of introducing an adventitious contaminant may involve treatment of the sera to inactivate potential contaminants. Gamma irradiation is one of the most widely employed methods for viral and mollicute inactivation in animal sera. In this article, we review the inactivation results reported for viral and mollicute inactivation in frozen serum. Studies performed to assess the impact of gamma irradiation on serum quality and performance are also discussed. The available data indicate that inactivation of mollicutes in serum is essentially complete at the gamma radiation doses normally employed (25-40 kGy), while the efficacy and kinetics for viral inactivation in serum by gamma irradiation appear to be dependent in part upon the size of the target virus.

Soap, water, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2): an ancient handwashing strategy for preventing dissemination of a novel virus.

Public Health Agencies worldwide (World Health Organization, United States Centers for Disease Prevention & Control, Chinese Center for Disease Control and Prevention, European Centre for Disease Prevention and Control, etc.) are recommending hand washing with soap and water for preventing the dissemination of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections. In this review, we have discussed the mechanisms of decontamination by soap and water (involving both removal and inactivation), described the contribution of the various components of formulated soaps to performance as cleansers and to pathogen inactivation, explained why adherence to recommended contact times is critical, evaluated the possible contribution of water temperature to inactivation, discussed the advantages of antimicrobial soaps vs. basic soaps, discussed the differences between use of soap and water vs. alcohol-based hand sanitizers for hand decontamination, and evaluated the limitations and advantages of different methods of drying hands following washing. While the paper emphasizes data applicable to SARS-CoV-2, the topics discussed are germane to most emerging and re-emerging enveloped and non-enveloped viruses and many other pathogen types.

Comparison of the Efficacy of Disinfectant Pre-impregnated Wipes for Decontaminating Stainless Steel Carriers Experimentally Inoculated With Ebola Virus and Vesicular Stomatitis Virus.

The authors evaluated four disinfectant pre-impregnated wipes (DPW) for efficacy against Ebola virus Makona variant (EBOV) and vesicular stomatitis virus (VSV), Indiana serotype. Steel carriers were inoculated with the infectious virus and then were wiped with DPW in the Wiperator instrument per ASTM E2967-15. Following the use of J-Cloth impregnated with medium (negative control wipes) or the use of activated hydrogen peroxide (AHP)-, ethanol-, sodium hypochlorite (NaOCl)-, or single or dual quaternary ammonium compound (QAC)-based DPW, virus recovery from the carriers was assayed by titration assay and by two passages on Vero E6 cells in 6-well plates. The Wiperator also enabled the measurement of potential transfer of the virus from the inoculated carrier to a secondary carrier by the DPW or control wipes. The J-Cloth wipes wetted with medium alone (no microbicidal active) removed 1.9-3.5 log10 of virus from inoculated carriers but transferred ~4 log10 of the wiped virus to secondary carriers. DPW containing AHP, ethanol, NaOCl, or single or dual QAC as active microbicidal ingredients removed/inactivated ~6 log10 of the virus, with minimal EBOV or no VSV virus transfer to a secondary surface observed. In Ebola virus outbreaks, a DPW with demonstrated virucidal efficacy, used as directed, may help to mitigate the unintended spread of the infectious virus while performing surface cleaning.

Microbicidal actives with virucidal efficacy against SARS-CoV-2 and other beta- and alpha-coronaviruses and implications for future emerging coronaviruses and other enveloped viruses.

Mitigating the risk of acquiring coronaviruses including SARS-CoV-2 requires awareness of the survival of virus on high-touch environmental surfaces (HITES) and skin, and frequent use of targeted microbicides with demonstrated efficacy. The data on stability of infectious SARS-CoV-2 on surfaces and in suspension have been put into perspective, as these inform the need for hygiene. We evaluated the efficacies of formulated microbicidal actives against alpha- and beta-coronaviruses, including SARS-CoV-2. The coronaviruses SARS-CoV, SARS-CoV-2, human coronavirus 229E, murine hepatitis virus-1, or MERS-CoV were deposited on prototypic HITES or spiked into liquid matrices along with organic soil loads. Alcohol-, quaternary ammonium compound-, hydrochloric acid-, organic acid-, p-chloro-m-xylenol-, and sodium hypochlorite-based microbicidal formulations were evaluated per ASTM International and EN standard methodologies. All evaluated formulated microbicides inactivated SARS-CoV-2 and other coronaviruses in suspension or on prototypic HITES. Virucidal efficacies (≥ 3 to ≥ 6 log10 reduction) were displayed within 30 s to 5 min. The virucidal efficacy of a variety of commercially available formulated microbicides against SARS-CoV-2 and other coronaviruses was confirmed. These microbicides should be useful for targeted surface and hand hygiene and disinfection of liquids, as part of infection prevention and control for SARS-CoV-2 and emerging mutational variants, and other emerging enveloped viruses.

A 21st century view of infection control in everyday settings: Moving from the Germ Theory of Disease to the Microbial Theory of Health.

BACKGROUND: The growing understanding of the importance of a healthy microbiome is challenging traditional thinking that resulted in the general acceptance of the Germ Theory of Disease. We propose a more encompassing Microbial Theory of Health that will have implications for the way that we address our relationship with microbes, including hygiene policy and community-based infection control practices. METHODS: This paper considers theories over the last 30 years that have impacted hygiene policy and consumer practice, from the Germ Theory of Disease and the Hygiene Hypothesis, to the Microbial Theory of Health, including the concept of Bidirectional Hygiene. Here we present a high-level review of the literature on pathogen transmission and the cycle of infection in the home and everyday settings. RESULTS: Targeted hygiene is an evidence-based hygiene policy that is employed to prevent transmission of pathogens and the transmission of infectious diseases through targeting only sites, surfaces, and practices that are considered high risk for pathogen transmission. Targeted hygiene also discourages the indiscriminate use of broad-spectrum microbicides for lower-risk activities and surfaces. CONCLUSIONS: The Microbial Theory of Health, including age-appropriate and health-appropriate hygiene practices for home and everyday life, should usher in a new era in which pathogen reduction can be accomplished without indiscriminate elimination of potentially beneficial microbes from the human and environmental microbiomes.

Combating SARS-CoV-2: leveraging microbicidal experiences with other emerging/re-emerging viruses.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Wuhan City, China, late in December 2019 is an example of an emerging zoonotic virus that threatens public health and international travel and commerce. When such a virus emerges, there is often insufficient specific information available on mechanisms of virus dissemination from animal-to-human or from person-to-person, on the level or route of infection transmissibility or of viral release in body secretions/excretions, and on the survival of virus in aerosols or on surfaces. The effectiveness of available virucidal agents and hygiene practices as interventions for disrupting the spread of infection and the associated diseases may not be clear for the emerging virus. In the present review, we suggest that approaches for infection prevention and control (IPAC) for SARS-CoV-2 and future emerging/re-emerging viruses can be invoked based on pre-existing data on microbicidal and hygiene effectiveness for related and unrelated enveloped viruses.

Efficacy of microbicides for inactivation of Ebola-Makona virus on a non-porous surface: a targeted hygiene intervention for reducing virus spread.

Microbicides play critical roles in infection prevention and control of Ebola virus by decontaminating high-touch environmental surfaces (HITES), interrupting the virus-HITES-hands nexus. We evaluated the efficacy of formulations containing different microbicidal actives for inactivating Ebola virus-Makona strain (EBOV/Mak) on stainless-steel carriers per ASTM E2197-11. Formulations of sodium hypochlorite (NaOCl) (0.05-1%), ethanol (70%), chloroxylenol (PCMX) (0.12-0.48% by weight) in hard water, and a ready-to-use disinfectant spray with 58% ethanol (EDS), were tested at contact times of 0, or 0.5 to 10 min at ambient temperature. EBOV/Mak was inactivated (> 6 log10) by 70% ethanol after contact times ≥ 2.5 min, by 0.5% and 1% NaOCl or EDS (> 4 log10) at contact times ≥ 5 min, and by 0.12-0.48% PCMX (> 4.2 log10) at contact times ≥ 5 min. Residual infectious virus in neutralized samples was assessed by passage on cells and evaluation for viral cytopathic effect. No infectious virus was detected in cells inoculated with EBOV/Mak exposed to NaOCl (0.5% or 1%), PCMX (0.12% to 0.48%), or EDS for ≥ 5 min. These results demonstrate ≥ 6 log10 inactivation of EBOV/Mak dried on prototypic surfaces by EDS or formulations of NaOCl (≥ 0.5%), PCMX (≥ 0.12%), or 70% ethanol at contact times ≥ 5 min.

Assessing the Contributions of Inactivation, Removal, and Transfer of Ebola Virus and Vesicular Stomatitis Virus by Disinfectant Pre-soaked Wipes.

Disinfectant pre-soaked wipes (DPW) containing activated hydrogen peroxide (AHP) or quaternary ammonium compounds (QAC) were tested using ASTM E2967-15 to determine removal, transfer, and inactivation of Ebola virus Makona variant (EBOV/Mak) and vesicular stomatitis virus (VSV) from contaminated stainless steel prototypic environmental surfaces. The infectious virus-contaminated carriers were subjected to wiping in the Wiperator per the standard. Following the use of negative control (J-Cloth)-, AHP-, or QAC-based wipes, recovery of residual infectious virus was assayed. In the case of the J-Cloth wipes (negative control), although removal of virus from inoculated carriers was extensive i.e., ~99% (1.9-3.5 log10) transfer of virus by these wipes to a secondary surface amounted to ≤ 2% (~3.8 log10) of the initial virus load. In the case of each DPW, >6 log10 removal/inactivation of virus was observed, with limited (EBOV/Mak) or no (VSV) virus transfer observed. The efficacy of wipes for decontaminating high-touch environmental surfaces spiked with EBOV/Mak or VSV is discussed. In summary, removal of EBOV/Mak and VSV using wipes was extensive in this study. In the absence of a sufficient concentration and contact time of an appropriate microbicidal active in DPW (such as the AHP- and QAC-based DPW tested), transfer of a low, albeit significant (from an infectious unit/infectious dose perspective), quantity of infectious virus from the inoculated surface to a secondary surface was observed. In the case of Ebola virus, it is essential that a DPW with an appropriate microbicidal active, following the appropriate contact time, be used to prevent unintended transfer of infectious virus to a clean secondary surface (as observed in negative control /J-Cloth). Otherwise, there exists the possibility of dissemination of Ebola virus and the associated risk of transmission of Ebola virus disease.