Reduction of Neuraminidase Activity Exacerbates Disease in 2009 Pandemic Influenza Virus-Infected Mice.

During the first wave of the 2009 pandemic, caused by a H1N1 influenza virus (pH1N1) of swine origin, antivirals were the only form of therapeutic available to control the proliferation of disease until the conventional strain-matched vaccine was produced. Oseltamivir is an antiviral that inhibits the sialidase activity of the viral neuraminidase (NA) protein and was shown to be effective against pH1N1 viruses in ferrets. Furthermore, it was used in humans to treat infections during the pandemic and is still used for current infections without reported complication or exacerbation of illness. However, in an evaluation of the effectiveness of oseltamivir against pH1N1 infection, we unexpectedly observed an exacerbation of disease in virus-infected mice treated with oseltamivir, transforming an otherwise mild illness into one with high morbidity and mortality. In contrast, an identical treatment regime alleviated all signs of illness in mice infected with the pathogenic mouse-adapted virus A/WSN/33 (H1N1). The worsened clinical outcome with pH1N1 viruses occurred over a range of oseltamivir doses and treatment schedules and was directly linked to a reduction in NA enzymatic activity. Our results suggest that the suppression of NA activity with antivirals may exacerbate disease in a host-dependent manner by increasing replicative fitness in viruses that are not optimally adapted for replication in that host. IMPORTANCE: Here, we report that treatment of pH1N1-infected mice with oseltamivir enhanced disease progression, transforming a mild illness into a lethal infection. This raises a potential pitfall of using the mouse model for evaluation of the therapeutic efficacy of neuraminidase inhibitors. We show that antiviral efficacy determined in a single animal species may not represent treatment in humans and that caution should be used when interpreting the outcome. Furthermore, increased virulence due to oseltamivir treatment was the effect of a shift in the hemagglutinin (HA) and neuraminidase (NA) activity balance. This is the first study that has demonstrated that altering the HA/NA activity balance by reduction in NA activity can result in an increase in virulence in any animal model from nonpathogenic to lethal and the first to demonstrate a situation in which treatment with a NA activity inhibitor has an effect opposite to the intended therapeutic effect of ameliorating the infection.

Development and Characterization of a Guinea Pig-Adapted Sudan Virus.

UNLABELLED: Infections with Sudan virus (SUDV), a member of the genus Ebolavirus, result in a severe hemorrhagic fever with a fatal outcome in over 50% of human cases. The paucity of prophylactics and therapeutics against SUDV is attributed to the lack of a small-animal model to screen promising compounds. By repeatedly passaging SUDV within the livers and spleens of guinea pigs in vivo, a guinea pig-adapted SUDV variant (SUDV-GA) uniformly lethal to these animals, with a 50% lethal dose (LD50) of 5.3 × 10(-2) 50% tissue culture infective doses (TCID50), was developed. Animals infected with SUDV-GA developed high viremia and died between 9 and 14 days postinfection. Several hallmarks of SUDV infection, including lymphadenopathy, increased liver enzyme activities, and coagulation abnormalities, were observed. Virological analyses and gross pathology, histopathology, and immunohistochemistry findings indicate that SUDV-GA replicates in the livers and spleens of infected animals similarly to SUDV infections in nonhuman primates. These developments will accelerate the development of specific medical countermeasures in preparation for a future disease outbreak due to SUDV. IMPORTANCE: A disease outbreak due to Ebola virus (EBOV), suspected to have emerged during December 2013 in Guinea, with over 11,000 dead and 28,000 infected, is finally winding down. Experimental EBOV vaccines and treatments were administered to patients under compassionate circumstances with promising results, and availability of an approved countermeasure appears to be close. However, the same range of experimental candidates against a potential disease outbreak caused by other members of the genus Ebolavirus, such as Sudan virus (SUDV), is not readily available. One bottleneck contributing to this situation is the lack of a small-animal model to screen promising drugs in an efficient and economical manner. To address this, we have generated a SUDV variant (SUDV-GA) that is uniformly lethal to guinea pigs. Animals infected with SUDV-GA develop disease similar to that of SUDV-infected humans and monkeys. We believe that this model will significantly accelerate the development of life-saving measures against SUDV infections.

Inactivation of Zaire ebolavirus Variant Makona in Human Serum Samples Analyzed by Enzyme-Linked Immunosorbent Assay.

Personnel deployed to remote areas during infectious disease outbreaks have limited access to mechanical and chemical inactivation resources. The inactivation of infectious agents present in diagnostic samples is critical to ensure the safety of personnel and the containment of the disease. We evaluated the efficacy of thermal inactivation (exposure to 56°C for 1 hour) and chemical inactivation with 0.5% Tween-20 against a high titer of Ebola virus (species Zaire ebolavirus) variant Makona in spiked human serum samples. No surviving virus was revealed by a 50% tissue culture infective dose assay after the combined treatment under laboratory conditions. In-field use of this inactivation protocol during the 2013-2016 West Africa Ebola outbreaks demonstrated readily detectable levels of immunoglobulin G and/or immunoglobulin M in human plasma samples after treatment.

Inactivating Zaire Ebolavirus in Whole-Blood Thin Smears Used for Malaria Diagnosis.

Malaria is an important mimic or coinfection in potential Ebolavirus disease patients. Here, we evaluated the efficacy of the 100% methanol-inactivating Zaire Ebolavirus Makona variant for malaria thin-smear preparation. We determined that 100% methanol completely inactivated the virus after 15 min.

Ebola virus transmission in guinea pigs.

UNLABELLED: Ebola virus (EBOV) transmission is currently poorly characterized and is thought to occur primarily by direct contact with infectious material; however transmission from swine to nonhuman primates via the respiratory tract has been documented. To establish an EBOV transmission model for performing studies with statistical significance, groups of six guinea pigs (gps) were challenged intranasally (i.n.) or intraperitoneally (i.p.) with 10,000 times the 50% lethal dose (LD50) of gp-adapted EBOV, and naive gps were then introduced as cage mates for contact exposure at 1 day postinfection (p.i.). The animals were monitored for survival and clinical signs of disease and quantitated for virus shedding postexposure. Changes in the duration of contact of naive gps with infected animals were evaluated for their impact on transmission efficiency. Transmission was more efficient from i.n.- than from i.p.-challenged gps, with 17% versus 83% of naive gps surviving exposure, respectively. Virus shedding was detected beginning at 3 days p.i. from both i.n.- and i.p.-challenged animals. Contact duration positively correlated with transmission efficiency, and the abrogation of direct contact between infected and naive animals through the erection of a steel mesh was effective at stopping virus spread, provided that infectious animal bedding was absent from the cages. Histopathological and immunohistochemical findings show that i.n.-infected gps display enhanced lung pathology and EBOV antigen in the trachea, which supports increased virus transmission from these animals. The results suggest that i.n.-challenged gps are more infectious to naive animals than their systemically infected counterparts and that transmission occurs through direct contact with infectious materials, including those transported through air movement over short distances. IMPORTANCE: Ebola is generally thought to be spread between humans though infectious bodily fluids. However, a study has shown that Ebola can be spread from pigs to monkeys without direct contact. Further studies have been hampered, because an economical animal model for Ebola transmission is not available. To address this, we established a transmission model in guinea pigs and determined the mechanisms behind virus spread. The survival data, in addition to microscopic examination of lung and trachea sections, show that mucosal infection of guinea pigs is an efficient model for Ebola transmission. Virus spread is increased with longer contact times with an infected animal and is possible without direct contact between an infected and a naive host but can be stopped if infectious materials are absent. These results warrant consideration for the development of future strategies against Ebola transmission and for a better understanding of the parameters involved in virus spread.

Establishment and characterization of a lethal mouse model for the Angola strain of Marburg virus.

UNLABELLED: Infections with Marburg virus (MARV) and Ebola virus (EBOV) cause severe hemorrhagic fever in humans and nonhuman primates (NHPs) with fatality rates up to 90%. A number of experimental vaccine and treatment platforms have previously been shown to be protective against EBOV infection. However, the rate of development for prophylactics and therapeutics against MARV has been lower in comparison, possibly because a small-animal model is not widely available. Here we report the development of a mouse model for studying the pathogenesis of MARV Angola (MARV/Ang), the most virulent strain of MARV. Infection with the wild-type virus does not cause disease in mice, but the adapted virus (MARV/Ang-MA) recovered from liver homogenates after 24 serial passages in severe combined immunodeficient (SCID) mice caused severe disease when administered intranasally (i.n.) or intraperitoneally (i.p.). The median lethal dose (LD50) was determined to be 0.015 50% TCID50 (tissue culture infective dose) of MARV/Ang-MA in SCID mice, and i.p. infection at a dose of 1,000× LD50 resulted in death between 6 and 8 days postinfection in SCID mice. Similar results were obtained with immunocompetent BALB/c and C57BL/6 mice challenged i.p. with 2,000× LD50 of MARV/Ang-MA. Virological and pathological analyses of MARV/Ang-MA-infected BALB/c mice revealed that the associated pathology was reminiscent of observations made in NHPs with MARV/Ang. MARV/Ang-MA-infected mice showed most of the clinical hallmarks observed with Marburg hemorrhagic fever, including lymphopenia, thrombocytopenia, marked liver damage, and uncontrolled viremia. Virus titers reached 10(8) TCID50/ml in the blood and between 10(6) and 10(10) TCID50/g tissue in the intestines, kidney, lungs, brain, spleen, and liver. This model provides an important tool to screen candidate vaccines and therapeutics against MARV infections. IMPORTANCE: The Angola strain of Marburg virus (MARV/Ang) was responsible for the largest outbreak ever documented for Marburg viruses. With a 90% fatality rate, it is similar to Ebola virus, which makes it one of the most lethal viruses known to humans. There are currently no approved interventions for Marburg virus, in part because a small-animal model that is vulnerable to MARV/Ang infection is not available to screen and test potential vaccines and therapeutics in a quick and economical manner. To address this need, we have adapted MARV/Ang so that it causes illness in mice resulting in death. The signs of disease in these mice are reminiscent of wild-type MARV/Ang infections in humans and nonhuman primates. We believe that this will be of help in accelerating the development of life-saving measures against Marburg virus infections.

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.

Mechanical Wiping Increases the Efficacy of Liquid Disinfectants on SARS-CoV-2.

High-touch environmental surfaces are acknowledged as potential sources of pathogen transmission, particularly in health care settings where infectious agents may be readily abundant. Methods of disinfecting these surfaces often include direct application of a chemical disinfectant or simply wiping the surface with a disinfectant pre-soaked wipe (DPW). In this study, we examine the ability of four disinfectants, ethanol (EtOH), sodium hypochlorite (NaOCl), chlorine dioxide (ClO2), and potassium monopersulfate (KMPS), to inactivate SARS-CoV-2 on a hard, non-porous surface, assessing the effects of concentration and contact time. The efficacy of DPWs to decontaminate carriers spiked with SARS-CoV-2, as well as the transferability of the virus from used DPWs to clean surfaces, is also assessed. Stainless steel carriers inoculated with approximately 6 logs of SARS-CoV-2 prepared in a soil load were disinfected within 5 min through exposure to 66.5% EtOH, 0.5% NaOCl, and 1% KMPS. The addition of mechanical wiping using DPWs impregnated with these biocides rendered the virus inactive almost immediately, with no viral transfer from the used DPW to adjacent surfaces. Carriers treated with 100 ppm of ClO2 showed a significant amount of viable virus remaining after 10 min of biocide exposure, while the virus was only completely inactivated after 10 min of treatment with 500 ppm of ClO2. Wiping SARS-CoV-2-spiked carriers with DPWs containing either concentration of ClO2 for 5 s left significant amounts of viable virus on the carriers. Furthermore, higher titers of infectious virus retained on the ClO2-infused DPWs were transferred to uninoculated carriers immediately after wiping. Overall, 66.5% EtOH, 0.5% NaOCl, and 1% KMPS appear to be highly effective biocidal agents against SARS-CoV-2, while ClO2 formulations are much less efficacious.

Efficacy Testing of Personal Protective Filters on Biosafety Level 4 Positive Pressure Suits.

Introduction: Positive pressure breathing air-fed protective suits from three vendors are commonly used in biosafety level 4 (BSL-4) laboratories: they are Dover Chemturion suits (ILC Dover, DE), Delta suits (Honeywell Safety, NC), and HVO suits (HVO-ISSI-Deutschland GmbH, Germany). To address the potential risk of infectious agents being introduced through the supplied breathing air stream, some suit manufacturers incorporate protective filters on the suits themselves. However, these integrated filters are not amenable to in situ testing for efficacy verification. We have been using external filters from Matheson USA on the positive pressure suits since our BSL-4 laboratories were commissioned two decades ago. As part of our BSL-4 protective suit management program, we test these filters before them being put into use, and annually thereafter. In the past few years, we have observed these filters failing at a higher rate, as high as two out of three of the new filters tested at one point. Objective: The purpose of this study was to procure personal protective filters from other sources and validate their efficacy long-term. Methods: Filters from Respirex, HVO, and Honeywell were validated for filter integrity and filter loading. Results: Respirex filters performed well during the initial testing and periodic testing thereafter. Regular testing of the Respirex filters has now been ongoing for 30 months with continued successful performance. Conclusion: Filters from Respirex are a suitable option to protect personnel wearing positive pressure suits in BSL-4 laboratories.

Stability of SARS-CoV-2 on critical personal protective equipment.

The spread of COVID-19 in healthcare settings is concerning, with healthcare workers representing a disproportionately high percentage of confirmed cases. Although SARS-CoV-2 virus has been found to persist on surfaces for a number of days, the extent and duration of fomites as a mode of transmission, particularly in healthcare settings, has not been fully characterized. To shed light on this critical matter, the present study provides the first comprehensive assessment of SARS-CoV-2 stability on experimentally contaminated personal protective equipment (PPE) widely used by healthcare workers and the general public. Persistence of viable virus was monitored over 21 days on eight different materials, including nitrile medical examination gloves, reinforced chemical resistant gloves, N-95 and N-100 particulate respirator masks, Tyvek, plastic, cotton, and stainless steel. Unlike previous reports, viable SARS-CoV-2 in the presence of a soil load persisted for up to 21 days on experimentally inoculated PPE, including materials from filtering facepiece respirators (N-95 and N-100 masks) and a plastic visor. Conversely, when applied to 100% cotton fabric, the virus underwent rapid degradation and became undetectable by TCID50 assay within 24 h. These findings underline the importance of appropriate handling of contaminated PPE during and following use in high-risk settings and provide interesting insight into the potential utility of cotton in limiting COVID-19 transmission.

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.

Simulated sunlight decreases the viability of SARS-CoV-2 in mucus.

The novel coronavirus, SARS-CoV-2, has spread into a pandemic since its emergence in Wuhan, China in December of 2019. This has been facilitated by its high transmissibility within the human population and its ability to remain viable on inanimate surfaces for an extended period. To address the latter, we examined the effect of simulated sunlight on the viability of SARS-CoV-2 spiked into tissue culture medium or mucus. The study revealed that inactivation took 37 minutes in medium and 107 minutes in mucus. These times-to-inactivation were unexpected since they are longer than have been observed in other studies. From this work, we demonstrate that sunlight represents an effective decontamination method but the speed of decontamination is variable based on the underlying matrix. This information has an important impact on the development of infection prevention and control protocols to reduce the spread of this deadly pathogen.

Aerosol SARS-CoV-2 in hospitals and long-term care homes during the COVID-19 pandemic.

BACKGROUND: Few studies have quantified aerosol concentrations of SARS-CoV-2 in hospitals and long-term care homes, and fewer still have examined samples for viability. This information is needed to clarify transmission risks beyond close contact. METHODS: We deployed particulate air samplers in rooms with COVID-19 positive patients in hospital ward and ICU rooms, rooms in long-term care homes experiencing outbreaks, and a correctional facility experiencing an outbreak. Samplers were placed between 2 and 3 meters from the patient. Aerosol (small liquid particles suspended in air) samples were collected onto gelatin filters by Ultrasonic Personal Air Samplers (UPAS) fitted with <2.5µm (micrometer) and <10 µm size-selective inlets operated for 16 hours (total 1.92m3), and with a Coriolis Biosampler over 10 minutes (total 1.5m3). Samples were assayed for viable SARS-CoV-2 virus and for the viral genome by multiplex PCR using the E and N protein target sequences. We validated the sampling methods by inoculating gelatin filters with viable vesicular stomatitis virus (VSV), and with three concentrations of viable SARS-CoV-2, operating personal samplers for 16hrs, and quantifying viable virus recovery by TCID50 assay. RESULTS: In total, 138 samples were collected from 99 rooms. RNA samples were positive in 9.1% (6/66) of samples obtained with the UPAS 2.5µm samplers, 13.5% (7/52) with the UPAS 10µm samplers, and 10.0% (2/20) samples obtained with the Coriolis samplers. Culturable virus was not recovered in any samples. Viral RNA was detected in 15.1% of the rooms sampled. There was no significant difference in viral RNA recovery between the different room locations or samplers. Method development experiments indicated minimal loss of SARS-CoV-2 viability via the personal air sampler operation.

Standard hospital blanket warming cabinets can be utilized for complete moist heat SARS-CoV2 inactivation of contaminated N95 masks for re-use.

Shortages of personal protective equipment for use during the SARS-CoV-2 pandemic continue to be an issue among health-care workers globally. Extended and repeated use of N95 filtering facepiece respirators without adequate decontamination is of particular concern. Although several methods to decontaminate and re-use these masks have been proposed, logistic or practical issues limit adoption of these techniques. In this study, we propose and validate the use of the application of moist heat (70 °C with humidity augmented by an open pan of water) applied by commonly available hospital (blanket) warming cabinets to decontaminate N95 masks. This report shows that a variety of N95 masks can be repeatedly decontaminated of SARS-CoV-2 over 6 h moist heat exposure without compromise of their filtering function as assessed by standard fit and sodium chloride aerosol filtration efficiency testing. This approached can easily adapted to provide point-of-care N95 mask decontamination allowing for increased practical utility of mask recycling in the health care setting.

The interaction between thymine DNA glycosylase and nuclear receptor coactivator 3 is required for the transcriptional activation of nuclear hormone receptors.

The T:G mismatch specific DNA glycosylase (TDG) is known as an important enzyme in repairing damaged DNA. Recent studies also showed that TDG interacts with a p160 protein, steroid receptor coactivator 1 or nuclear receptor coactivator 1 (SRC1), and is involved in transcriptional activation of the estrogen receptor. However, whether other members of the p160 family are also involved in TDG-interaction and signal transduction regulation remains to be seen. In this study, we employed the mammalian two-hybrid system to investigate the interaction between TDG and another member of the p160 family, nuclear receptor coactivator 3 (NCoA-3). We found that a DXXD motif from aa 294-297 within TDG was responsible for the TDG-NCoA-3 interaction, we also found that a LLXXXL motif (X means any amino acid) from aa 1029-1037 (LLRNSL) and a merged LLXXL motif (LLDQLHTLL) from aa 1053-1061 in NCoA-3 were important for the TDG-NCoA-3 interactions. Mutation of the two aspartic acids (aa 294 and 297) into two alanines in TDG significantly affected the interaction and subsequent transcriptional activation of several steroid hormone receptors including, estrogen-, androgen- and progesterone- receptors in Huh7 cells. We also identified that mutations of NCoA-3 at either leucines 1029-1030 or 1053-1054 (replaced by alanines) also reduced the interaction activity between TDG and NCoA1. These data indicated that the TDG-NCoA-3 interaction is important for broad range activation of steroid hormone nuclear receptors, and may also contribute significantly to further understanding of TDG-related nuclear receptor regulation.

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.

Decontamination of N95 masks for re-use employing 7 widely available sterilization methods.

The response to the COVID-19 epidemic is generating severe shortages of personal protective equipment around the world. In particular, the supply of N95 respirator masks has become severely depleted, with supplies having to be rationed and health care workers having to use masks for prolonged periods in many countries. We sought to test the ability of 7 different decontamination methods: autoclave treatment, ethylene oxide gassing (ETO), low temperature hydrogen peroxide gas plasma (LT-HPGP) treatment, vaporous hydrogen peroxide (VHP) exposure, peracetic acid dry fogging (PAF), ultraviolet C irradiation (UVCI) and moist heat (MH) treatment to decontaminate a variety of different N95 masks following experimental contamination with SARS-CoV-2 or vesicular stomatitis virus as a surrogate. In addition, we sought to determine whether masks would tolerate repeated cycles of decontamination while maintaining structural and functional integrity. All methods except for UVCI were effective in total elimination of viable virus from treated masks. We found that all respirator masks tolerated at least one cycle of all treatment modalities without structural or functional deterioration as assessed by fit testing; filtration efficiency testing results were mostly similar except that a single cycle of LT-HPGP was associated with failures in 3 of 6 masks assessed. VHP, PAF, UVCI, and MH were associated with preserved mask integrity to a minimum of 10 cycles by both fit and filtration testing. A similar result was shown with ethylene oxide gassing to the maximum 3 cycles tested. Pleated, layered non-woven fabric N95 masks retained integrity in fit testing for at least 10 cycles of autoclaving but the molded N95 masks failed after 1 cycle; filtration testing however was intact to 5 cycles for all masks. The successful application of autoclaving for layered, pleated masks may be of particular use to institutions globally due to the virtually universal accessibility of autoclaves in health care settings. Given the ability to modify widely available heating cabinets on hospital wards in well-resourced settings, the application of moist heat may allow local processing of N95 masks.

Effectiveness of Dettol Antiseptic Liquid for Inactivation of Ebola Virus in Suspension.

The efficacy of Dettol Antiseptic Liquid (DAL) for inactivating Ebola virus (Makona C07 variant) (EBOV/Mak) within an organic load in suspension was evaluated per ASTM E1052-11. Three DAL lots were evaluated at dilutions of 1:10, 1:20, and 1:40, with contact times of 0.5, 1, 5, and 10 min. Approximately 7 log10 tissue culture infectious dose50 (TCID50) of EBOV/Mak was exposed to DAL at ambient temperature. Each dilution tested reduced the infectious EBOV/Mak titer by ~5 log10 within one min. Detectable virus was still present after an 0.5-min exposure, but each DAL dilution caused >4 log10 reduction within this time. Detection of virus below the limit of detection of the TCID50 assay was assessed by inoculating flasks of Vero E6 cells with undiluted neutralized sample and evaluating the cultures for cytopathic effect after 14 days incubation. No infectious virus was detected with this non-quantitative method in samples subjected to DAL for 5 or 10 min, regardless of the dilution evaluated. The rapid and substantial inactivation of EBOV/Mak by DAL suggests that use of this hygiene product could help prevent the spread of Ebola virus disease during outbreaks.