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.

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.

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.

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.

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.

Challenge of Liquid Stressed Protective Materials and Environmental Persistence of Ebola Virus.

After the largest Ebola virus outbreak in history, experts have attempted to answer how the Zaire ebolavirus species emerged in West Africa and caused chains of human-to-human transmission. The widespread and untimely infection of Health Care Workers (HCW) in the affected countries accelerated spread of the virus within the community. Among the reasons attributed to this trend, it must be considered that HCW were exposed to the virus in their occupational environment. The contribution of environmental conditions to the spread of Ebola in West Africa was examined by investigating the effect of temperature/humidity on the virus's environmental persistence and by modeling if saturation (liquid stress) allows for penetration of Ebola virus through personal protective equipment (PPE). Ebola-Makona virus persisted on PPE and materials found in outbreak settings for less than 72 hours at 27 °C and 80% relative humidity (RH). A difference in virus penetration was observed between dry (5%, 1/21 tests) and saturated (33%, 7/21 tests) samples of PPE. Infectious virus particles penetrated through saturated coupons of Tyvek Micro Clean, Tychem QC, whole surgical masks and N95 respirators. These findings suggest inclusion of saturation or similar liquid stress simulation in protective equipment testing standards.

The Disinfection Characteristics of Ebola Virus Outbreak Variants.

The recent Ebola virus outbreak in West Africa has forced experts to re-evaluate their understanding of how to best disinfect areas contaminated with infectious bodily fluids. Recent research has found that Ebola virus remains viable in blood for 7-10 days making appropriate disinfection crucial to infection control. We sought to determine if the three most important outbreak variants of Zaire ebolavirus (Mayinga, Kikwit and Makona) exhibit separate phenotypes when challenged with a range of sodium hypochlorite (NaOCl) concentrations or 70% ethanol (EtOH) at average West African temperature. The time dependent killing of Ebola virus was evaluated by measuring infectious virus and viral RNA (vRNA), to determine if RNA detection is a viable method for decontamination measurement in areas without high containment laboratory access. Makona was less susceptible to weaker concentrations of NaOCl (0.05 and 0.1%) than Mayinga and Kikwit. At the recommended concentration of NaOCl (≥0.5%) all of the variants were inert after 5 minutes of contact time. Similarly, all variants were inactivated by 70% EtOH after 2.5 minutes, only Makona was detected at 1 minute. In multiple instances, high amounts of vRNA was detected in the absence of infectious virus, suggesting that it does not serve as an accurate measure of remaining infectivity after cleansing.

Development of a subgenomic clone system for Kyasanur Forest disease virus.

Emerging tropical viruses pose an increasing threat to public health because social, economic and environmental factors such as global trade and deforestation allow for their migration into previously unexposed populations and ecological niches. Among such viruses, Kyasanur Forest disease virus (KFDV) deserves particular recognition because it causes hemorrhagic fever. This work describes the completion of an antiviral testing platform (subgenomic system) for KFDV that could be used to quickly and safely screen compounds capable of inhibiting KFDV replication without the requirement for high containment, as the structural genes have been replaced with a luciferase reporter gene precluding the generation of infectious particles. The coordination of KFDV kinetics with the replication characteristics of the subgenomic system has provided additional insight into the timing of flavivirus replication events, as the genetically engineered KFDV genome began replication as early as 2h post cellular entry. Possession of such antiviral testing platforms by public health agencies should accelerate the testing of antiviral drugs against emerging or recently emerged viruses mitigating the effects of their disease and transmission.

Limited Effects of Type I Interferons on Kyasanur Forest Disease Virus in Cell Culture.

BACKGROUND: The tick-borne flavivirus, Kyasanur Forest disease virus (KFDV) causes seasonal infections and periodic outbreaks in south-west India. The current vaccine offers poor protection with reported issues of coverage and immunogenicity. Since there are no approved prophylactic therapeutics for KFDV, type I IFN-α/ß subtypes were assessed for antiviral potency against KFDV in cell culture. METHODOLOGY/PRINCIPAL FINDINGS: The continued passage of KFDV-infected cells with re-administered IFN-α2a treatment did not eliminate KFDV and had little effect on infectious particle production whereas the IFN-sensitive, green fluorescent protein-expressing vesicular stomatitis virus (VSV-GFP) infection was controlled. Further evaluation of the other IFN-α/ß subtypes versus KFDV infection indicated that single treatments of either IFN-αWA and IFN-αΚ appeared to be more effective than IFN-α2a at reducing KFDV titres. Concentration-dependent analysis of these IFN-α/ß subtypes revealed that regardless of subtype, low concentrations of IFN were able to limit cytopathic effects (CPE), while significantly higher concentrations were needed for inhibition of virion release. Furthermore, expression of the KFDV NS5 in cell culture before IFN addition enabled VSV-GFP to overcome the effects of IFN-α/ß signalling, producing a robust infection. CONCLUSIONS/SIGNIFICANCE: Treatment of cell culture with IFN does not appear to be suitable for KFDV eradication and the assay used for such studies should be carefully considered. Further, it appears that the NS5 protein is sufficient to permit KFDV to bypass the antiviral properties of IFN. We suggest that other prophylactic therapeutics should be evaluated in place of IFN for treatment of individuals with KFDV disease.

Influenza virus emitted by naturally-infected hosts in a healthcare setting.

BACKGROUND: The emergence of novel respiratory viruses such as avian influenza A(H7N9) virus and the Middle East Respiratory Syndrome Coronavirus (MERS-CoV) highlights the importance of understanding determinants of transmission to healthcare workers (HCWs) and the public. OBJECTIVES: We aim to determine the viral content of the air emitted by symptomatic inpatients or long-term care residents with laboratory-confirmed influenza virus infection (emitters), and in the breathing zones of healthcare workers who attend to them. DESIGN: A prospective pilot study of patients with laboratory-confirmed influenza virus infection was undertaken. Air within 1m of the patient was sampled using a high volume air sampler. In addition, a lower volume air sampler was placed <1 m from the patient, with another >1 m from the patient. Viral RNA was recovered from the samplers and submitted for quantitative real time PCR. In addition, personal button samplers were provided to HCWs. RESULTS: The air emitted by 15 participants with laboratory-confirmed influenza virus infection was sampled. Of the patients infected with influenza A, viral RNA was recovered from the air emitted by 9/12 patients using the low-volume sampler; no viral RNA was detected from air emitted by patients with influenza B (n=3). Influenza virus RNA was recovered from one HCW's sampler. CONCLUSIONS: Patients with respiratory virus infection emit virus into the air which disperses to >1 m and may reach the breathing zone of a HCW. This pilot study highlights the feasibility and importance of conducting a larger-scale study to identify determinants of exposure and transmission from patient to HCW.

Evaluating environmental persistence and disinfection of the Ebola virus Makona variant.

BACKGROUND: The current disease outbreak caused by the Ebola virus Makona variant (EBOV/Mak) has led to unprecedented morbidity and lethality given its geographic reach and sustained transmission. Sodium hypochlorite and ethanol are well-accepted decontamination agents, however little published evidence supports the selection of appropriate concentrations and contact times. The present study addresses the environmental robustness of EBOV/Mak and evaluates the effectiveness of sodium hypochlorite and ethanol as disinfectants. METHODS: EBOV/Mak was suspended in a simulated organic soil load and dried onto surfaces. Viability was measured at 1 hour, 24 hours, 72 hours, and 192 hours. For the evaluation of disinfectants, EBOV/Mak in a simulated organic soil was dried onto stainless steel carriers and disinfected with 0.01% (v/v), 0.1% (v/v), 0.5% (v/v) and 1% (v/v) sodium hypochlorite solutions or 67% (v/v) ethanol at contact times of 1, 5 or 10 minutes. RESULTS: EBOV/Mak persisted longer on steel and plastic surfaces (192 hours) than cotton (<24 hours). Dilute sodium hypochlorite (0.01% and 0.1%) showed little antiviral action, whereas 0.5% and 1% sodium hypochlorite solutions demonstrated recoverable virus at one minute but sterilized surfaces in five minutes. Disinfection with 67% ethanol did not fully clear infectious virions from 3/9 carriers at 1 minute but sterilized all carriers at 5 and 10 minutes. CONCLUSIONS: Sodium hypochlorite and ethanol effectively decontaminate EBOV/Mak suspended in a simulated organic load; however, selection of concentration and contact time proves critical.

The generation of a reverse genetics system for Kyasanur Forest Disease Virus and the ability to antagonize the induction of the antiviral state in vitro.

Kyasanur Forest Disease Virus (KFDV) is a tick-borne, hemorrhagic fever-causing member of the Flaviviridae virus family. With infections annually ranging from 50 to 1000 people in south-west India and the lack of effective treatments, a better understanding of this virus is needed. The development of a reverse genetics system (RGS) for KFDV would provide the opportunity to address these issues. The KFDV genome sequence was elucidated and the RGS was created. Utilizing this system, live infectious KFDV particles were produced from mammalian cell culture, thereby validating the success of the RGS. Flaviviruses have the ability to suppress the type 1 interferon response and indications are that the non structural (NS) proteins serve this role. Using luciferase bioassays, the NS5 protein of KFDV was determined to be the primary antagonist of the IFN response when compared to the other NS proteins, specifically NS4B and NS4B-2k. Moreover, our results indicate that this is attributed to a region, beginning before and including the RNA-dependent RNA polymerase (RdRp). With evasion of the interferon response by KFDV established, the further implementation of the reverse genetics system will enable investigation into pathogenesis and disease progression of KFDV with respect to the innate immune response, at the IFN and the NS5 protein levels.