Ebola Virus Makona Research Articles

Characterizing changes in transcriptome and kinome responses in testicular cells during infection by Ebola virus

Ebola virus (EBOV) is able to persist and actively replicate in the reproductive tract of male disease survivors months or years after recovery from Ebola virus disease (EVD)1. Persistent EBOV infections are usually asymptomatic and can be transmitted sexually, but the host and viral factors that mediate these infections have not been characterized2,3. We investigated the interaction between host and viral factors during EBOV infection of the blood testis barrier (BTB), with a focus on Sertoli cells as a potential reservoir for viral persistence. We assessed viral replication kinetics and host responses of mouse testicular Leydig cells and Sertoli cells infected with EBOV Makona (i.e. infectious EBOV) and collected samples up to 28 days post-infection. Viral replication was apparent in both cell lines, but intracellular early viral loads were much higher in Leydig cells compared to Sertoli cells. We used RNAseq analysis to characterize transcriptomic responses of Leydig cells and Sertoli cells to EBOV infection over time. Further investigation of early interactions between host cells and EBOV was performed using virus-like particles (EBOV trVLP) and assays of phosphorylation-based cell signaling. Our findings indicate that virus-treated Sertoli cells responded more rapidly and robustly than Leydig cells, and with a particular emphasis on detection of, and response to, external stimuli. We discuss how the roles played by Sertoli cells in immune privilege and spermatogenesis may affect their initial and continued response to EBOV infection in a manner that could facilitate asymptomatic persistence.

Sequence optimized diagnostic assay for Ebola virus detection

Rapid pathogen identification is a critical first step in patient isolation, treatment, and controlling an outbreak. Real-time PCR is a highly sensitive and specific approach commonly used for infectious disease diagnostics. However, mismatches in the primer or probe sequence and the target organism can cause decreased sensitivity, assay failure, and false negative results. Limited genomic sequences for rare pathogens such as Ebola virus (EBOV) can negatively impact assay performance due to undiscovered genetic diversity. We previously developed and validated several EBOV assays prior to the 2013–2016 EBOV outbreak in West Africa, and sequencing EBOV Makona identified sequence variants that could impact assay performance. Here, we assessed the impact sequence mismatches have on EBOV assay performance, finding one or two primer or probe mismatches resulted in a range of impact from minimal to almost two log sensitivity reduction. Redesigning this assay improved detection of all EBOV variants tested. Comparing the performance of the new assay with the previous assays across a panel of human EBOV samples confirmed increased assay sensitivity as reflected in decreased Cq values with detection of three positive that tested negative with the original assay.

Virucidal activity of three standard chemical disinfectants against Ebola virus suspended in tripartite soil and whole blood

Proper disinfection and inactivation of highly pathogenic viruses is an essential component of public health and prevention. Depending on environment, surfaces, and type of contaminant, various methods of disinfection must be both efficient and available. To test both established and novel chemical disinfectants against risk group 4 viruses in our maximum containment facility, we developed a standardized protocol and assessed the chemical inactivation of the two Ebola virus variants Mayinga and Makona suspended in two different biological soil loads. Standard chemical disinfectants ethanol and sodium hypochlorite completely inactivate both Ebola variants after 30 s in suspension at 70% and 0.5% v/v, respectively, concentrations recommended for disinfection by the World Health Organization. Additionally, peracetic acid is also inactivating at 0.2% v/v under the same conditions. Continued vigilance and optimization of current disinfection protocols is extremely important due to the continuous presence of Ebola virus on the African continent and increased zoonotic spillover of novel viral pathogens. Furthermore, to facilitate general pandemic preparedness, the establishment and sharing of standardized protocols is very important as it allows for rapid testing and evaluation of novel pathogens and chemical disinfectants.

Natural History of Nonhuman Primates After Oral Exposure to Ebola Virus Variant Makona.

The primary route of infection by Ebola virus (EBOV) is through contact of mucosal surfaces. Few studies have explored infection of nonhuman primates (NHPs) via the oral mucosa, which is a probable portal of natural infection in humans. To further characterize the pathogenesis of EBOV infection via the oral exposure route, we challenged cohorts of cynomolgus monkeys with low doses of EBOV variant Makona. Infection with 100 or 50 PFU of EBOV Makona via the oral route resulted in 50% and 83% lethality, respectively. Animals that progressed to fatal disease exhibited lymphopenia, marked coagulopathy, high viral loads, and increased levels of serum markers of inflammation and hepatic/renal injury. Survival in these cohorts was associated with milder fluctuations in leukocyte populations, lack of coagulopathy, and reduced or absent serum markers of inflammation and/or hepatic/renal function. Surprisingly, 2 surviving animals from the 100- and 50-PFU cohorts developed transient low-level viremia in the absence of other clinical signs of disease. Conversely, all animals in the 10 PFU cohort remained disease free and survived to the study end point. Our observations highlight the susceptibility of NHPs, and by extension, likely humans, to relatively low doses of EBOV via the oral route.

VSV-EBOV Induces Temporal and Dose-Dependent Transcriptional Responses in Non-human Primates

Zaire Ebola virus (EBOV), the causative agent of Ebola virus disease (EVD), is a member of the Filoviridae family. EVD is characterized by innate and adaptive immune dysregulation that leads to excessive inflammation, coagulopathy, lymphopenia, and multi-organ failure. Recurrent outbreaks of EBOV emphasize the critical need for effective and deployable anti-EBOV vaccines. The FDA-approved VSV-EBOV vaccine protects non-human primates (NHPs) and humans from EBOV when given at a 10–20 million PFU dose. We recently demonstrated that a dose as small as 10 PFU protected NHPs from lethal EBOV infection. Furthermore, 1 PFU of VSV-EBOV protected 75% of vaccinated NHPs. In this study, we performed a comparative transcriptional analysis of the whole blood transcriptome in NHPs vaccinated with doses of VSV-EBOV associated with complete protection (10M PFU), protection with mild EVD (10 PFU), and break-through protection (1 PFU) before and after challenge with a lethal dose of EBOV Makona. Transcriptional findings demonstrated that, regardless of dose, vaccination significantly attenuated the upregulation of genes associated with fatal EVD. Genes involved in T- and B-cell activation were more highly expressed in groups receiving 10 or 10M PFU than in 1 PFU–vaccinated animals. Furthermore, the singular vaccinated (1 PFU) non-survivor exhibited a transcriptional signature distinct from both surviving vaccinated animals and controls that received an irrelevant vaccine. These findings provide additional insight into mechanisms of vaccine-mediated protection and informing public policy on vaccine distribution during 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.

The SKI complex is a broad-spectrum antiviral drug target

Starting from a yeast suppressor screening platform, we have identified the SKI complex as a potential broad-spectrum antiviral target. We found that the NS1 protein of influenza A virus (IAV) and the ORF4a protein of Middle East respiratory syndrome coronavirus (MERS-CoV), which both function to bind double-strand RNA and inhibit cellular interferon responses, cause a slow growth phenotype when expressed in yeast. Knockout of the components of the yeast SKI complex caused a loss of this slow growth phenotype, suggesting a functional link between the viral proteins and the SKI complex. The SKI complex is a helicase that unwinds double-strand RNA and sends it to the RNA exosome for degradation. We next investigated whether the highly conserved human SKI complex was important for replication of IAV and MERS-CoV. RNAi based experiments showed that both viruses were inhibited when the SKI complex was removed, suggesting the complex has a proviral role in replication. Through in silico modelling using the published crystal structure of the SKI complex, we looked for potential binding pockets for chemical compounds. We screened a selection of these compounds for antiviral activity and have found four different chemicals capable of inhibiting IAV infection. Our most studied of these also inhibits not only MERS-CoV, but also Ebolavirus Makona. Our data suggests the SKI complex may be a target for broad-spectrum antiviral therapy and we have multiple chemical structures from which to work to develop therapeutic approaches.

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.

Early transmission and case fatality of Ebola virus at the index site of the 2013–16 west African Ebola outbreak: a cross-sectional seroprevalence survey

SummaryBackgroundTo date, epidemiological studies at the index site of the 2013–16 west African Ebola outbreak in Meliandou, Guinea, have been restricted in their scope. We aimed to determine the occurrence of previously undocumented Ebola virus disease (EVD) cases and infections, and to reconstruct transmission events.MethodsThis cross-sectional seroprevalence survey of the adult population of Meliandou used a highly specific oral fluid test and detailed interviews of all households in the village and key informants. Each household was interviewed, with all members prompted to describe the events of the outbreak, any illness within the household, and possible contact with suspected cases. Information for deceased individuals was provided by relatives living in the same household. Symptoms were based on Ebola virus Makona variant EVD case definitions (focusing on fever, vomiting, and diarrhoea). For antibody testing, we used an Ebola virus glycoprotein IgG capture enzyme immunoassay developed from a previously validated assay. A maximum exposure level was assigned to every participant using a predetermined scale. We used a generalised linear model (logit function) to estimate odds ratios for the association of sociodemographic variables and exposure level with Ebola virus infection. We adjusted estimates for age and maximum exposure, as appropriate.FindingsBetween June 22, and July 9, 2017, we enrolled 237 participants from 27 households in Meliandou. Two households refused to participate and one was absent. All adults in participating households who were present for the interview provided an oral fluid swab for testing, of which 224 were suitable for analysis. In addition to the 11 EVD deaths described previously, on the basis of clinical description and oral fluid testing, we found two probable EVD deaths and eight previously unrecognised anti-Ebola virus IgG-positive survivors, including one who had mild symptoms and one who was asymptomatic, resulting in a case fatality of 55·6% (95% CI 30·8–78·5) for adults. Health-care work (adjusted odds ratio 6·64, 1·54–28·56; p=0·001) and level of exposure (odds ratio adjusted for linear trend across five levels 2·79, 1·59–4·883; p<0·0001) were independent risk factors for infection.InterpretationEbola virus infection was more widespread in this spillover population than previously recognised (21 vs 11 cases). We show the first serological evidence of survivors in this population (eight anti-Ebola virus IgG seropositive) and report a case fatality lower than previously reported (55·6% vs 100% in adults). These data show the high community coverage achievable by using a non-invasive test and, by accurately documenting the beginnings of the west African Ebola virus outbreak, reveal important insight into transmission dynamics and risk factors that underpin Ebola virus spillover events.FundingUS Food and Drug Administration, Wellcome Trust, and German Research Council.

Growth-Adaptive Mutations in the Ebola Virus Makona Glycoprotein Alter Different Steps in the Virus Entry Pathway.

The Zaire ebolavirus (EBOV) glycoprotein (GP) is cleaved into two subunits (GP1 and GP2) that are both required for virus attachment and entry into cells. Sequence changes in the GP have been proposed to increase pathogenesis and to alter virus growth properties. Mutations in GP acquired during EBOV tissue culture passage have also been reported to change virus growth properties. Here, we report the isolation of six amino acid mutations in EBOV GP that spontaneously appeared during recovery and passage of an EBOV-Makona GP-pseudotyped vesicular stomatitis virus (VSV), two of which also occur during passage of EBOV clinical isolates in tissue culture. Each of the six mutations resulted in increased virus growth in monkey and human cell lines. All mutations are located in the GP2 fusion subunit and increase entry kinetics of EBOV virus-like particles (VLPs). The gain-of-entry function mapped to two mechanistic phenotypes. Mutations in heptad repeat 1 (HR1) decreased the requirement for cathepsin B activity for viral infection. Mutations directly within the fusion loop increased entry kinetics without altering the cathepsin B dependence. Several mutations in the fusion loop were substitutions of residues present in other ebolavirus glycoproteins, illustrating the evolutionary paths for maintaining an optimally functioning fusion loop under selection pressure.IMPORTANCEZaire ebolavirus (EBOV) is the causative agent of the highly lethal Ebola virus disease and poses a significant threat to the global health community. Approved antivirals against EBOV are lacking; however, promising therapies targeting the EBOV glycoprotein are being developed. Efficacy testing of these candidate therapeutics relies on EBOV laboratory stocks, which when grown in tissue culture may acquire mutations in the glycoprotein. These mutations can produce inaccurate results in therapeutic testing. Until recently, distinguishing between tissue culture mutations and naturally occurring polymorphisms in EBOV GP was difficult in the absence of consensus clinical GP sequences. Here, we utilize recombinant VSV (rVSV) pseudotyped with the consensus clinical EBOV Makona GP to identify several mutations that have emerged or have potential to emerge in EBOV GP during tissue culture passage. Identifying these mutations informs the EBOV research community as to which mutations may arise during preparation of laboratory virus stocks.

Structural Basis of Pan-Ebolavirus Neutralization by an Antibody Targeting the Glycoprotein Fusion Loop

Monoclonal antibodies (mAbs) with pan-ebolavirus cross-reactivity are highly desirable, but development of such mAbs is limited by a lack of a molecular understanding of cross-reactive epitopes. The antibody ADI-15878 was previously identified from a human survivor of Ebola virus Makona variant (EBOV/Mak) infection. This mAb demonstrated potent neutralizing activity against all known ebolaviruses and provided protection in rodent and ferret models against three ebolavirus species. Here, we describe the unliganded crystal structure of ADI-15878 aswell as the cryo-EM structures of ADI-15878 incomplex with the EBOV/Mak and Bundibugyo virus (BDBV) glycoproteins (GPs). ADI-15878 binds through an induced-fit mechanism by targeting highly conserved residues in the internal fusion loop (IFL), bridging across GP protomers via the heptad repeat 1 (HR1) region. Our structures provide a more complete description of the ebolavirus immunogenic landscape, as well as a molecular basis for how rare but potent antibodies target conserved filoviral fusion machinery.

Human Polyclonal Antibodies Produced by Transchromosomal Cattle Provide Partial Protection Against Lethal Zaire Ebolavirus Challenge in Rhesus Macaques.

Antibody therapy has been used to treat a variety of diseases and the success of ZMapp and other monoclonal antibody-based therapies during the 2014-2016 West African Ebola outbreak has shown this countermeasure can be a successful therapy for Ebola hemorrhagic fever. This study utilized transchromosomal bovines (TcB) vaccinated with a DNA plasmid encoding Ebola virus glycoprotein sequence to produce human polyclonal antibodies directed against Ebola virus glycoprotein. When administered 1 day postinfection, these TcB polyclonal antibodies provided partial protection and resulted in a 50% survival rate following a lethal challenge of Ebola virus Makona in rhesus macaques.

Fully Human Immunoglobulin G From Transchromosomic Bovines Treats Nonhuman Primates Infected With Ebola Virus Makona Isolate.

Transchromosomic bovines (Tc-bovines) adaptively produce fully human polyclonal immunoglobulin (Ig)G antibodies after exposure to immunogenic antigen(s). The National Interagency Confederation for Biological Research and collaborators rapidly produced and then evaluated anti-Ebola virus IgG immunoglobulins (collectively termed SAB-139) purified from Tc-bovine plasma after sequential hyperimmunization with an Ebola virus Makona isolate glycoprotein nanoparticle vaccine. SAB-139 was characterized by several in vitro production, research, and clinical level assays using wild-type Makona-C05 or recombinant virus/antigens from different Ebola virus variants. SAB-139 potently activates natural killer cells, monocytes, and peripheral blood mononuclear cells and has high-binding avidity demonstrated by surface plasmon resonance. SAB-139 has similar concentrations of galactose-α-1,3-galactose carbohydrates compared with human-derived intravenous Ig, and the IgG1 subclass antibody is predominant. All rhesus macaques infected with Ebola virus/H.sapiens-tc/GIN/2014/Makona-C05 and treated with sufficient SAB-139 at 1 day (n = 6) or 3 days (n = 6) postinfection survived versus 0% of controls. This study demonstrates that Tc-bovines can produce pathogen-specific human Ig to prevent and/or treat patients when an emerging infectious disease either threatens to or becomes an epidemic.

Recently Identified Mutations in the Ebola Virus-Makona Genome Do Not Alter Pathogenicity in Animal Models

Ebola virus (EBOV), isolate Makona, the causative agent of the West African EBOV epidemic, has been the subject of numerous investigations to determine the genetic diversity and its potential implication forvirus biology, pathogenicity, and transmissibility. Despite various mutations that have emerged over time through multiple human-to-human transmission chains, their biological relevance remains questionable. Recently, mutations in the glycoprotein GP and polymerase L, which emerged and stabilized early during the outbreak, have been associated with improved viral fitness in cell culture. Here, we infected mice and rhesus macaques with EBOV-Makonaisolates carrying or lacking those mutations. Surprisingly, all isolates behaved very similarly independent of the genotype, causing severe or lethal disease in mice and macaques, respectively. Likewise, we could not detect any evidence for differences invirus shedding. Thus, no specific biological phenotype could be associated with these EBOV-Makona mutations in two animal models.

Comparison of Transcriptomic Platforms for Analysis of Whole Blood from Ebola-Infected Cynomolgus Macaques

Ebola virus disease (EVD) is a serious illness with mortality rates of 20–90% in various outbreaks. EVD is characterized by robust virus replication and strong host inflammatory response. Analyzing host immune responses has increasingly involved multimodal approaches including transcriptomics to profile gene expression. We studied cynomolgus macaques exposed to Ebola virus Makona via different routes with the intent of comparing RNA-Seq to a NanoString nCounter codeset targeting 769 non-human primate (NHP) genes. RNA-Seq analysis of serial blood samples showed different routes led to the same overall transcriptional response seen in previously reported EBOV-exposed NHP studies. Both platforms displayed a strong correlation in gene expression patterns, including a strong induction of innate immune response genes at early times post-exposure, and neutrophil-associated genes at later time points. A 41-gene classifier was tested in both platforms for ability to cluster samples by infection status. Both NanoString and RNA-Seq could be used to predict relative abundances of circulating immune cell populations that matched traditional hematology. This demonstrates the complementarity of RNA-Seq and NanoString. Moreover, the development of an NHP-specific NanoString codeset should augment studies of filoviruses and other high containment infectious diseases without the infrastructure requirements of RNA-Seq technology.

High dose sertraline monotherapy fails to protect rhesus macaques from lethal challenge with Ebola virus Makona

The recent epidemic of Ebola virus disease in West Africa resulted in an unprecedented number of cases and deaths. Due to the scope of the outbreak combined with the lack of available approved treatment options, there was strong motivation to investigate any potential drug which had existing data reporting anti-Ebola activity. Drugs with demonstrated antiviral activity in the nonhuman primate models already approved for another indication or for which there was existing safety data were considered to be priorities for evaluation by the World Health Organization. Sertraline hydrochloride was reported to have anti-Ebola activity in vitro alone and in combination with other approved drugs. Although the efficacy was less than 100% in the murine model, the established safety profile of this product, the potential benefit alone and in combination, as well as the lack of other available options prioritized this compound for testing in the Ebola virus intramuscular rhesus macaque challenge model. Using a blinded dosing strategy, we demonstrated that high dose sertraline monotherapy provided no benefit for the prevention of Ebola virus disease in rhesus macaques with regards to reduction of viral load, morbidity, or survival highlighting the challenges of translating results between in vitro and in vivo models.

Spontaneous Mutation at Amino Acid 544 of the Ebola Virus Glycoprotein Potentiates Virus Entry and Selection in Tissue Culture.

Ebolaviruses have a surface glycoprotein (GP1,2) that is required for virus attachment and entry into cells. Mutations affecting GP1,2 functions can alter virus growth properties. We generated a recombinant vesicular stomatitis virus encoding Ebola virus Makona variant GP1,2 (rVSV-MAK-GP) and observed emergence of a T544I mutation in the Makona GP1,2 gene during tissue culture passage in certain cell lines. The T544I mutation emerged within two passages when VSV-MAK-GP was grown on Vero E6, Vero, and BS-C-1 cells but not when it was passaged on Huh7 and HepG2 cells. The mutation led to a marked increase in virus growth kinetics and conferred a robust growth advantage over wild-type rVSV-MAK-GP on Vero E6 cells. Analysis of complete viral genomes collected from patients in western Africa indicated that this mutation was not found in Ebola virus clinical samples. However, we observed the emergence of T544I during serial passage of various Ebola Makona isolates on Vero E6 cells. Three independent isolates showed emergence of T544I from undetectable levels in nonpassaged virus or virus passaged once to frequencies of greater than 60% within a single passage, consistent with it being a tissue culture adaptation. Intriguingly, T544I is not found in any Sudan, Bundibugyo, or Tai Forest ebolavirus sequences. Furthermore, T544I did not emerge when we serially passaged recombinant VSV encoding GP1,2 from these ebolaviruses. This report provides experimental evidence that the spontaneous mutation T544I is a tissue culture adaptation in certain cell lines and that it may be unique for the species Zaire ebolavirusIMPORTANCE The Ebola virus (Zaire) species is the most lethal species of all ebolaviruses in terms of mortality rate and number of deaths. Understanding how the Ebola virus surface glycoprotein functions to facilitate entry in cells is an area of intense research. Recently, three groups independently identified a polymorphism in the Ebola glycoprotein (I544) that enhanced virus entry, but they did not agree in their conclusions regarding its impact on pathogenesis. Our findings here address the origins of this polymorphism and provide experimental evidence showing that it is the result of a spontaneous mutation (T544I) specific to tissue culture conditions, suggesting that it has no role in pathogenesis. We further show that this mutation may be unique to the species Zaire ebolavirus, as it does not occur in Sudan, Bundibugyo, and Tai Forest ebolaviruses. Understanding the mechanism behind this mutation can provide insight into functional differences that exist in culture conditions and among ebolavirus glycoproteins.

Interferon-β and Interferon-γ Are Weak Inhibitors of Ebola Virus in Cell-Based Assays.

Previous studies have demonstrated little efficacy of interferons (IFNs) in animal models of Ebola virus disease. However, these studies were limited to a small number of type I IFNs and, during the most recent outbreak of Ebola virus, questions regarding the suitability of the animal models to evaluate IFNs were raised. To address the potential that anti-Ebola virus activity was overlooked, type I and type II IFNs (α-2a, α-2b, -β, -γ, and -universal) were tested in a variety of cell types (Vero E6, Huh 7 cells, and human macrophages). IFNs are weak inhibitors of Ebola virus Makona in these cell lines.

Functional mutations in spike glycoprotein of Zaire ebolavirus associated with an increase in infection efficiency.

Ebola virus (EBOV) is extremely virulent, and its glycoprotein is necessary for viral entry. EBOV may adapt to its new host humans during outbreaks by acquiring mutations especially in glycoprotein, which allows EBOV to spread more efficiently. To identify these evolutionary selected mutations and examine their effects on viral infectivity, we used experimental-phylogenetic-structural interdisciplinary approaches. In evolutionary analysis of all available Zaire ebolavirus glycoprotein sequences, we detected two codon sites under positive selection, which are located near/within the region critical for the host-viral membrane fusion, namely alanine-to-valine and threonine-to-isoleucine mutations at 82 (A82V) and 544 (T544I), respectively. The fine-scale transmission dynamics of EBOV Makona variants that caused the 2014-2015 outbreak showed that A82V mutant was fixed in the population, whereas T544I was not. Furthermore, pseudotype assays for the Makona glycoprotein showed that the A82V mutation caused a small increase in viral infectivity compared with the T544I mutation. These findings suggest that mutation fixation in EBOV glycoprotein may be associated with their increased infectivity levels; the mutant with a moderate increase in infectivity will fix. Our findings showed that a driving force for Ebola virus evolution via glycoprotein may be a balance between costs and benefits of its virulence.

Complete Genome Sequence of an Ebola Virus Isolate Imported from Sierra Leone to Germany Determined by Circle Sequencing.

We report here a complete genome sequence of Ebola virus Makona from a nonfatal patient sample that originated in Sierra Leone during the last Ebola virus outbreak in West Africa (species Zaire ebolavirus) using a highly accurate circle sequencing (Cir-seq) method.