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 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 ofZaire 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.

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.

Viral Infectious clone systems serve as robust platforms to study viral gene or replicative function by reverse genetics, formulate vaccines and adapt a wild type-virus to an animal host. Since the development of the first viral infectious clone system for the poliovirus, novel strategies of viral genome construction have allowed for the assembly of viral genomes across the identified viral families. However, the molecular profiles of some viruses make their genome more difficult to construct than others. Two factors that affect the difficulty of infectious clone construction are genome length and genome complexity. This work examines the available strategies for overcoming the obstacles of assembling the long and complex RNA genomes of coronaviruses and reports one-step construction of an infectious clone system for the Middle East Respiratory Syndrome coronavirus (MERS-CoV) by homologous recombination in S. cerevisiae. Future use of this methodology will shorten the time between emergence of a novel viral pathogen and construction of an infectious clone system. Completion of a viral infectious clone system facilitates further study of a virus's biology, improvement of diagnostic tests, vaccine production and the screening of antiviral compounds.

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/β subtypes were assessed for antiviral potency against KFDV in cell culture. 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/β 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/β 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/β signalling, producing a robust infection. 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. Since 1957 Kyasanur Forest disease virus (KFDV) has caused seasonal infections and periodic outbreaks in south-west India. It is estimated that nearly 500 people acquire KFDV annually and 3–5% of those infected succumb to the disease. The vaccine strategy is complicated by the lack of coverage, compliance and efficacy, highlighted by the fact that less than half of the target population received the recommended three dose-regimen. Besides the prevention of tick bites and vaccination, there are no approved antivirals for KFDV infection. Based on these observations, the commonly-used-IFN-α2a was assessed and was not capable of limiting KFDV virus titres. Further characterization of the other IFN-α/β subtypes used at different concentrations revealed that KFDV replication was insensitive to all subtypes, even though signs of cellular damage were reduced. Thus, infectious titre, rather than monolayer staining or cytopathic effect (CPE) monitoring, is more reliable for IFN analyses. The capability of KFDV to overcome the antiviral properties of IFN was attributed to the NS5 protein. Thus, other treatment options need to be evaluated for patients suffering with Kyasanur Forest disease.