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.

Investigation into scalable and efficient enterotoxigenic Escherichia coli bacteriophage production.

As the demand for bacteriophage (phage) therapy increases due to antibiotic resistance in microbial pathogens, strategies and methods for increased efficiency, large-scale phage production need to be determined. To date, very little has been published on how to establish scalable production for phages, while achieving and maintaining a high titer in an economical manner. The present work outlines a phage production strategy using an enterotoxigenic Escherichia coli-targeting phage, 'Phage75', and accounts for the following variables: infection load, multiplicity of infection, temperature, media composition, harvest time, and host bacteria. To streamline this process, variables impacting phage propagation were screened through a high-throughput assay monitoring optical density at 600 nm (OD600) to indirectly infer phage production from host cell lysis. Following screening, propagation conditions were translated in a scalable fashion in shake flasks at 0.01 L, 0.1 L, and 1 L. A final, proof-of-concept production was then carried out in a CellMaker bioreactor to represent practical application at an industrial level. Phage titers were obtained in the range of 9.5-10.1 log10 PFU/mL with no significant difference between yields from shake flasks and CellMaker. Overall, this suggests that the methodology for scalable processing is reliable for translating into large-scale phage production.

Combining bacteriophage and vancomycin is efficacious against MRSA biofilm-like aggregates formed in synovial fluid.

Background: Biofilm formation is a major clinical challenge contributing to treatment failure of periprosthetic joint infection (PJI). Lytic bacteriophages (phages) can target biofilm associated bacteria at localized sites of infection. The aim of this study is to investigate whether combination therapy of phage and vancomycin is capable of clearing Staphylococcus aureus biofilm-like aggregates formed in human synovial fluid. Methods: In this study, S. aureus BP043, a PJI clinical isolate was utilized. This strain is a methicillin-resistant S. aureus (MRSA) biofilm-former. Phage Remus, known to infect S. aureus, was selected for the treatment protocol. BP043 was grown as aggregates in human synovial fluid. The characterization of S. aureus aggregates was assessed for structure and size using scanning electron microscopy (SEM) and flow cytometry, respectively. Moreover, the formed aggregates were subsequently treated in vitro with: (a) phage Remus [∼108 plaque-forming units (PFU)/ml], (b) vancomycin (500 µg/ml), or (c) phage Remus (∼108 PFU/ml) followed by vancomycin (500 µg/ml), for 48 h. Bacterial survival was quantified by enumeration [colony-forming units (CFU)/ml]. The efficacy of phage and vancomycin against BP043 aggregates was assessed in vivo as individual treatments and in combination. The in vivo model utilized Galleria mellonella larvae which were infected with BP043 aggregates pre-formed in synovial fluid. Results: Scanning electron microscopy (SEM) images and flow cytometry data demonstrated the ability of human synovial fluid to promote formation of S. aureus aggregates. Treatment with Remus resulted in significant reduction in viable S. aureus residing within the synovial fluid aggregates compared to the aggregates that did not receive Remus (p < 0.0001). Remus was more efficient in eliminating viable bacteria within the aggregates compared to vancomycin (p < 0.0001). Combination treatment of Remus followed by vancomycin was more efficacious in reducing bacterial load compared to using either Remus or vancomycin alone (p = 0.0023, p < 0.0001, respectively). When tested in vivo, this combination treatment also resulted in the highest survival rate (37%) 96 h post-treatment, compared to untreated larvae (3%; p < 0.0001). Conclusion: We demonstrate that combining phage Remus and vancomycin led to synergistic interaction against MRSA biofilm-like aggregates in vitro and in vivo.

Implementation of a Rapid RT-LAMP Saliva-Based SARS-CoV-2 Testing Program in the Workplace.

Rising SARS-CoV-2 cases, testing delays, and the risk of pre-symptomatic and asymptomatic transmission provided the impetus for an in-house rapid testing program. Employees and their household contacts were encouraged to self-collect saliva samples that were pooled for routine testing using an established colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay. In brief, individual or a maximum of four saliva samples were pooled and heat-inactivated to render microorganisms, especially SARS-CoV-2, non-infectious prior to being added to RT-LAMP assay tubes containing either the human sample control gene, RNase P, or a region of the SARS-CoV-2 gene, ORF1ab. During the second wave of SARS-CoV-2 infections in November 2020, two samples from an employee and a member of their household tested positive via RT-LAMP within two days of each other. A delayed clinical qRT-PCR test confirmation of both individuals 5 days later underscored the power of routine rapid testing with within-the-hour turnaround times. Workplace rapid testing programs using RT-LAMP are flexible in their design, have a reduced cost compared to qRT-PCR, may involve non-invasive self-saliva collection for increased safety for the testing personnel, and can be performed with minimal training.

Characterization of Ebola Virus Risk to Bedside Providers in an Intensive Care Environment.

BACKGROUND: The 2014-2016 Ebola outbreak in West Africa recapitulated that nosocomial spread of Ebola virus could occur and that health care workers were at particular risk including notable cases in Europe and North America. These instances highlighted the need for centers to better prepare for potential Ebola virus cases; including understanding how the virus spreads and which interventions pose the greatest risk. METHODS: We created a fully equipped intensive care unit (ICU), within a Biosafety Level 4 (BSL4) laboratory, and infected multiple sedated non-human primates (NHPs) with Ebola virus. While providing bedside care, we sampled blood, urine, and gastric residuals; as well as buccal, ocular, nasal, rectal, and skin swabs, to assess the risks associated with routine care. We also assessed the physical environment at end-point. RESULTS: Although viral RNA was detectable in blood as early as three days post-infection, it was not detectable in the urine, gastric fluid, or swabs until late-stage disease. While droplet spread and fomite contamination were present on a few of the surfaces that were routinely touched while providing care in the ICU for the infected animal, these may have been abrogated through good routine hygiene practices. CONCLUSIONS: Overall this study has helped further our understanding of which procedures may pose the highest risk to healthcare providers and provides temporal evidence of this over the clinical course of disease.

Characterization of the Enterobacter Phage vB_EclM_CIP9.

Enterobacter cloacae is an opportunistic pathogen that causes hospital-acquired infections in immunocompromised patients. Here, we describe vB_EclM_CIP9, a novel Enterobacter phage that infects a multidrug-resistant isolate of E. cloacae Phage vB_EclM_CIP9 is a myovirus that has a 174,924-bp genome, with 296 predicted open reading frames.

Impact of intensive care unit supportive care on the physiology of Ebola virus disease in a universally lethal non-human primate model.

BACKGROUND: There are currently limited data for the use of specific antiviral therapies for the treatment of Ebola virus disease (EVD). While there is anecdotal evidence that supportive care may be effective, there is a paucity of direct experimental data to demonstrate a role for supportive care in EVD. We studied the impact of ICU-level supportive care interventions including fluid resuscitation, vasoactive medications, blood transfusion, hydrocortisone, and ventilator support on the pathophysiology of EVD in rhesus macaques infected with a universally lethal dose of Ebola virus strain Makona C07. METHODS: Four NHPs were infected with a universally lethal dose Ebola virus strain Makona, in accordance with the gold standard lethal Ebola NHP challenge model. Following infection, the following therapeutic interventions were employed: continuous bedside supportive care, ventilator support, judicious fluid resuscitation, vasoactive medications, blood transfusion, and hydrocortisone as needed to treat cardiovascular compromise. A range of physiological parameters were continuously monitored to gage any response to the interventions. RESULTS: All four NHPs developed EVD and demonstrated a similar clinical course. All animals reached a terminal endpoint, which occurred at an average time of 166.5 ± 14.8 h post-infection. Fluid administration may have temporarily blunted a rise in lactate, but the effect was short lived. Vasoactive medications resulted in short-lived improvements in mean arterial pressure. Blood transfusion and hydrocortisone did not appear to have a significant positive impact on the course of the disease. CONCLUSIONS: The model employed for this study is reflective of an intramuscular infection in humans (e.g., needle stick) and is highly lethal to NHPs. Using this model, we found that the animals developed progressive severe organ dysfunction and profound shock preceding death. While the overall impact of supportive care on the observed pathophysiology was limited, we did observe some time-dependent positive responses. Since this model is highly lethal, it does not reflect the full spectrum of human EVD. Our findings support the need for continued development of animal models that replicate the spectrum of human disease as well as ongoing development of anti-Ebola therapies to complement supportive care.

Correction to: Impact of intensive care unit supportive care on the physiology of Ebola virus disease in a universally lethal non-human primate model.

Please note that four authors (Logan Banadyga, Alixandra Albietz, Brad Pickering, and Gary Wong) have been erroneously omitted from the author list in the published original article [1].

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.

Rapid one-step construction of a Middle East Respiratory Syndrome (MERS-CoV) infectious clone system by homologous recombination.

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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 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.

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.

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.

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.