Reston Virus Research Articles

Reemergence of Reston ebolavirus in Cynomolgus Monkeys, the Philippines, 2015.

In August 2015, a nonhuman primate facility south of Manila, the Philippines, noted unusual deaths of 6 cynomolgus monkeys (Macaca fascicularis), characterized by generalized rashes, inappetence, or sudden death. We identified Reston ebolavirus (RESTV) infection in monkeys by using serologic and molecular assays. We isolated viruses in tissues from infected monkeys and determined viral genome sequences. RESTV found in the 2015 outbreak is genetically closer to 1 of the 4 RESTVs that caused the 2008 outbreak among swine. Eight macaques, including 2 also infected with RESTV, tested positive for measles. Concurrently, the measles virus was circulating throughout the Philippines, indicating that the infection of the macaques may be a reverse zoonosis. Improved biosecurity measures will minimize the public health risk, as well as limit the introduction of disease and vectors.

Broad Neutralizing Activity Against Ebolaviruses Lacking the Mucin-Like Domain in Convalescent Plasma Specimens From Patients With Ebola Virus Disease.

BackgroundIn Ebola virus (EBOV) infection, the specific neutralizing activity of convalescent plasma against other members of the Ebolavirus genus has not been extensively analyzed.MethodsWe measured the neutralizing activity in plasma from 3 survivors of the recent outbreak due to the Makona variant of EBOV and tested its neutralizing potency against other variants of EBOV (ie, Mayinga and Kikwit) and against Sudan virus (SUDV), Bundibugyo virus (BDBV), and Reston virus (RESTV), using a glycoprotein (GP)–pseudotyped lentiviral system both with full-length GP and in vitro–cleaved GP (GPCL).ResultsConvalescent plasma specimens from survivors of EBOV infection showed low neutralizing activity against full-length GPs of SUDV, BDBV, RESTV, and EBOV variants Mayinga and Kikwit. However, broad and potent neutralizing activity was observed against the GPCL forms of SUDV, BDBV, and RESTV.DiscussionRemoval of the mucin-like domain and glycan cap from the GP of members of the Ebolavirus genus presumably exposes conserved epitopes in or in the vicinity of the receptor binding site and internal fusion loop that are readily amenable to neutralization. These types of broad neutralizing antibodies could be induced by using immunogens mimicking GPCL.

Broadly neutralizing antibodies from human survivors target a conserved site in the Ebola virus glycoprotein HR2-MPER region.

Summary paragraphEbola virus (EBOV) in humans causes a severe illness with high mortality rates. Several strategies have been developed in the past to treat EBOV infection, including the antibody cocktail ZMappTM that has been shown to be effective in nonhuman primate models of infection1 and has been used under compassionate-treatment protocols in humans2. ZMappTM is a mixture of three chimerized murine monoclonal antibodies (mAbs)3–6 that target EBOV-specific epitopes on the surface glycoprotein (GP)7,8. However, ZMappTM mAbs do not neutralize other species from the Ebolavirus genus, such as Bundibugyo virus (BDBV), Reston virus (RESTV) or Sudan virus (SUDV). Here we describe three naturally-occurring human cross-neutralizing mAbs, from BDBV survivors, that target an antigenic site in the canonical heptad repeat 2 (HR2) region near the membrane proximal external region (MPER) of GP. The identification of a conserved neutralizing antigenic site in the GP suggests that these mAbs could be used to design universal antibody therapeutics against diverse ebolavirus species. Furthermore, we found that immunization with a peptide comprising the HR2/MPER antigenic site elicits neutralizing antibodies in rabbits. Structural features determined by conserved residues in the antigenic site described here could inform an epitope-based vaccine design against infection caused by diverse ebolavirus species.

Ebolavirus diagnosis made simple, comparable and faster than molecular detection methods: preparing for the future

BackgroundThe 2014/2015 Ebolavirus outbreak resulted in more than 28,000 cases and 11,323 reported deaths, as of March 2016. Domestic transmission of the Guinea strain associated with the outbreak occurred mainly in six African countries, and international transmission was reported in four countries. Outbreak management was limited by the inability to rapidly diagnose infected cases. A further fifteen countries in Africa are predicted to be at risk of Ebolavirus outbreaks in the future as a consequence of climate change and urbanization. Early detection of cases and reduction of transmission rates is critical to prevent and manage future severe outbreaks. We designed a rapid assay for detection of Ebolavirus using recombinase polymerase amplification, a rapid isothermal amplification technology that can be combined with portable lateral flow detection technology. The developed rapid assay operates in 30 min and was comparable with real-time TaqMan™ PCR.MethodsDesigned, screened, selected and optimized oligonucleotides using the NP coding region from Ebola Zaire virus (Guinea strain). We determined the analytical sensitivity of our Ebola rapid molecular test by testing selected primers and probe with tenfold serial dilutions (1.34 × 1010− 1.34 × 101 copies/μL) of cloned NP gene from Mayinga strain of Zaire ebolavirus in pCAGGS vector, and serially diluted cultured Ebolavirus as established by real-time TaqMan™ PCR that was performed using ABI7500 in Fast Mode. We tested extracted and reverse transcribed RNA from cultured Zaire ebolavirus strains – Mayinga, Gueckedou C05, Gueckedou C07, Makona, Kissidougou and Kiwit. We determined the analytical specificity of our assay with related viruses: Marburg, Ebola Reston and Ebola Sudan. We further tested for Dengue virus 1–4, Plasmodium falciparum and West Nile Virus (Kunjin strain).ResultsThe assay had a detection limit of 134 copies per μL of plasmid containing the NP gene of Ebolavirus Mayinga, and cultured Ebolavirus and was highly specific for the Zaire ebolavirus species, including the Guinea strain responsible for the 2014/2015 outbreak. The assay did not detect related viruses like Marburg, Reston, or Sudan viruses, and other pathogens likely to be isolated from clinical samples.ConclusionsOur assay could be suitable for implementation in district and primary health laboratories, as only a heating block and centrifuge is required for operation. The technique could provide a pathway for rapid screening of patients and animals for improved management of outbreaks.

A Critical Domain of Ebolavirus Envelope Glycoprotein Determines Glycoform and Infectivity

Ebolaviruses comprises 5 species that exert varying degrees of mortality/infectivity in humans with Reston ebolaviruses (REBOV) showing the lowest and Zaire ebolaviruses (ZEBOV) showing the highest. However, the molecular basis of this differential mortality/infectivity remains unclear. Here, we report that the structural features of ebolavirus envelope glycoproteins (GPs) and one of their counter receptors, macrophage galactose-type calcium-type lectin (MGL/CD301), play crucial roles in determining viral infectivity. The low infectivity of REBOV mediated by the interaction between GPs and MGL/CD301 dramatically increased when the N-terminal 18 amino acids (33rd through 50th) of GPs were replaced with that of ZEBOV. Furthermore, structural analysis of glycans of GPs revealed that N-glycans were more extended in REBOV than in ZEBOV. N-glycan extension was reversed by the replacement of aforementioned N-terminal 18 amino acid residues. Therefore, these data strongly suggest that extended N-glycans on GPs reduce MGL/CD301-mediated viral infectivity by hindering the interaction between GPs and MGL/CD301 preferentially binds O-glycans.

Different effects of two mutations on the infectivity of Ebola virus glycoprotein in nine mammalian species.

Ebola virus (EBOV), which belongs to the genus Ebolavirus, causes a severe and often fatal infection in primates, including humans, whereas Reston virus (RESTV) only causes lethal disease in non-human primates. Two amino acids (aa) at positions 82 and 544 of the EBOV glycoprotein (GP) are involved in determining viral infectivity. However, it remains unclear how these two aa residues affect the infectivity of Ebolavirus species in various hosts. Here we performed viral pseudotyping experiments with EBOV and RESTV GP derivatives in 10 cell lines from 9 mammalian species. We demonstrated that isoleucine at position 544/545 increases viral infectivity in all host species, whereas valine at position 82/83 modulates viral infectivity, depending on the viral and host species. Structural modelling suggested that the former residue affects viral fusion, whereas the latter residue influences the interaction with the viral entry receptor, Niemann–Pick C1.

A comparative computational genomics of Ebola Virus Disease strains: In-silico Insight for Ebola control

Abstract Ebola Virus Disease (EVD), is a national epidemic in Countries affected. It is also a potential global public health pandemic. The menace of the disease outbreak among West and Central African nations, in recent years, has resulted in the death of many unsuspecting victims. The present study was conducted to present a systematic review of the literature, focusing on the control of Ebola Virus Disease (EVD), among human subjects. It also centered on a bioinformatics analysis of five different strains of Ebola virus. Research articles published between 2008 and 2018, on EVD control studies, were systematically reviewed. Four online databases were searched for the purpose of this review. These include: Science Direct, Google Scholar, SpringerLink and PubMed. Study outcomes were extracted. The outcomes were summarized and categorized. Five different strains of Ebola virus were obtained from the NCBI database, specifically the Entrez Genome database. Bioinformatics analysis was performed using Muscle software, RawXL, Treview, iTOL and Clustal X. Bioinformatics analysis was performed on five selected strains of Ebola virus (Reston, Bundingbugyo, Zaire, Sudan and Tai forest). Evaluation of the phylogenetic tree was performed by using MEGA X and PHYLIP software. 237,498 publications were identified, out of which 104 research articles, from different regions of the world, fulfilled our inclusion criteria. Insight was gained for the control of EVD from these studies. Of the studies reviewed, 23 articles focused on vaccines/vaccination-related Ebola control research, 12 studies on modeling and simulation-related Ebola control research, 41 on drugs and therapeutics-related Ebola control research, and 28 focused on other experimental studies (such as biological experiments, bioinformatics experiments, travel border control measures, educational campaign measures, hand and environmental sanitization, amongst others). Very few modeling and simulation studies have been conducted on the control of EVD in the last 10 years. Thus, there is the need for more modeling and simulation-related ebola control research. Comparative computational genomics of the five Ebola virus strains produced phylogenetic trees in different shapes. An evaluation of the phylogenetic tree was performed. Results showed that Taiforest Ebola virus and Bundibugyo Ebola virus are closely related. The results also revealed that Sudan and Reston Ebola virus are closely related. Zaire Ebola virus stood out from all the others. It may be possible to adopt similar Ebola control measures against Ebola virus strains that are closely related. Insight from these results, can facilitate the development and production of multi-protective, multi-treatment drugs, multi-protective vaccines and antivirals, against these ebola virus disease strains. The results of the evaluations of the phylogenetic tree can be assistive in providing insight into the origin, evolution, and possible structural and genetic mutations of the Ebola virus. It can also provide insight for inferring the structural and functional properties of each Ebola virus. The knowledge of such inference can be useful for EVD control. This can bring about a radical transformation in control efforts for disease.

Cooperation of the Ebola Virus Proteins VP40 and GP1,2 with BST2 To Activate NF-κB Independently of Virus-Like Particle Trapping.

BST2 is a host protein with dual functions in response to viral infections: it traps newly assembled enveloped virions at the plasma membrane in infected cells, and it induces NF-κB activity, especially in the context of retroviral assembly. In this study, we examined whether Ebola virus proteins affect BST2-mediated induction of NF-κB. We found that the Ebola virus matrix protein, VP40, and envelope glycoprotein, GP, each cooperate with BST2 to induce NF-κB activity, with maximal activity when all three proteins are expressed. Unlike human immunodeficiency virus type 1 Vpu protein, which antagonizes both virion entrapment and the activation of NF-κB by BST2, Ebola virus GP does not inhibit NF-κB signaling even while it antagonizes the entrapment of virus-like particles. GP from Reston ebolavirus, a nonpathogenic species in humans, showed a phenotype similar to that of GP from Zaire ebolavirus, a highly pathogenic species, in terms of both the activation of NF-κB and the antagonism of virion entrapment. Although Ebola virus VP40 and GP both activate NF-κB independently of BST2, VP40 is the more potent activator. Activation of NF-κB by the Ebola virus proteins either alone or together with BST2 requires the canonical NF-κB signaling pathway. Mechanistically, the maximal NF-κB activation by GP, VP40, and BST2 together requires the ectodomain cysteines needed for BST2 dimerization, the putative BST2 tetramerization residue L70, and Y6 of a potential hemi-ITAM motif in BST2's cytoplasmic domain. BST2 with a glycosylphosphatidylinositol (GPI) anchor signal deletion, which is not expressed at the plasma membrane and is unable to entrap virions, activated NF-κB in concert with the Ebola virus proteins at least as effectively as wild-type BST2. Signaling by the GPI anchor mutant also depended on Y6 of BST2. Overall, our data show that activation of NF-κB by BST2 is independent of virion entrapment in the case of Ebola virus. Nonetheless, BST2 may induce or amplify proinflammatory signaling during Ebola virus infection, potentially contributing to the dysregulated cytokine response that is a hallmark of Ebola virus disease.IMPORTANCE Understanding how the host responds to viral infections informs the development of therapeutics and vaccines. We asked how proinflammatory signaling by the host protein BST2/tetherin, which is mediated by the transcription factor NF-κB, responds to Ebola virus proteins. Although the Ebola virus envelope glycoprotein (GP1,2) antagonizes the trapping of newly formed virions at the plasma membrane by BST2, we found that it does not inhibit BST2's ability to induce NF-κB activity. This distinguishes GP1,2 from the HIV-1 protein Vpu, the prototype BST2 antagonist, which inhibits both virion entrapment and the induction of NF-κB activity. Ebola virus GP1,2, the Ebola virus matrix protein VP40, and BST2 are at least additive with respect to the induction of NF-κB activity. The effects of these proteins converge on an intracellular signaling pathway that depends on a protein modification termed neddylation. Better mechanistic understanding of these phenomena could provide targets for therapies that modulate the inflammatory response during Ebola virus disease.

Severity of Disease in Humanized Mice Infected With Ebola Virus or Reston Virus Is Associated With Magnitude of Early Viral Replication in Liver.

Both Ebola virus (EBOV) and Reston virus (RESTV) cause disease in nonhuman primates, yet only EBOV causes disease in humans. To investigate differences in viral pathogenicity, humanized mice (hu-NSG-SGM3) were inoculated with EBOV or RESTV. Consistent with differences in disease in human infection, pronounced weight loss and markers of hepatic damage and disease were observed exclusively in EBOV-infected mice. These abnormalities were associated with significantly higher EBOV replication in the liver but not in the spleen, suggesting that in this model, efficiency of viral replication in select tissues early in infection may contribute to differences in viral pathogenicity.

Investigating Ebola virus pathogenicity using molecular dynamics

BackgroundEbolaviruses have been known to cause deadly disease in humans for 40 years and have recently been demonstrated in West Africa to be able to cause large outbreaks. Four Ebolavirus species cause severe disease associated with high mortality in humans. Reston viruses are the only Ebolaviruses that do not cause disease in humans. Conserved amino acid changes in the Reston virus protein VP24 compared to VP24 of other Ebolaviruses have been suggested to alter VP24 binding to host cell karyopherins resulting in impaired inhibition of interferon signalling, which may explain the difference in human pathogenicity. Here we used protein structural analysis and molecular dynamics to further elucidate the interaction between VP24 and KPNA5.ResultsAs a control experiment, we compared the interaction of wild-type and R137A-mutant (known to affect KPNA5 binding) Ebola virus VP24 with KPNA5. Results confirmed that the R137A mutation weakens direct VP24-KPNA5 binding and enables water molecules to penetrate at the interface. Similarly, Reston virus VP24 displayed a weaker interaction with KPNA5 than Ebola virus VP24, which is likely to reduce the ability of Reston virus VP24 to prevent host cell interferon signalling.ConclusionOur results provide novel molecular detail on the interaction of Reston virus VP24 and Ebola virus VP24 with human KPNA5. The results indicate a weaker interaction of Reston virus VP24 with KPNA5 than Ebola virus VP24, which is probably associated with a decreased ability to interfere with the host cell interferon response. Hence, our study provides further evidence that VP24 is a key player in determining Ebolavirus pathogenicity.

Novel Cross-Reactive Monoclonal Antibodies against Ebolavirus Glycoproteins Show Protection in a Murine Challenge Model.

Out of an estimated 31,100 cases since their discovery in 1976, ebolaviruses have caused approximately 13,000 deaths. The vast majority (∼11,000) of these occurred during the 2013-2016 West African epidemic. Three out of five species in the genus are known to cause Ebola Virus Disease in humans. Several monoclonal antibodies against the ebolavirus glycoprotein are currently in development as therapeutics. However, there is still a paucity of monoclonal antibodies that can cross-react between the glycoproteins of different ebolavirus species, and the mechanism of these monoclonal antibody therapeutics is still not understood in detail. Here, we generated a panel of eight murine monoclonal antibodies (MAbs) utilizing a prime-boost vaccination regimen with a Zaire ebolavirus glycoprotein expression plasmid followed by infection with a vesicular stomatitis virus expressing the Zaire ebolavirus glycoprotein. We tested the binding breadth of the resulting monoclonal antibodies using a set of recombinant surface glycoproteins from Reston, Taï Forest, Bundibugyo, Zaire, Sudan, and Marburg viruses and found two antibodies that showed pan-ebolavirus binding. An in vivo Stat2-/- mouse model was utilized to test the ability of these MAbs to protect from infection with a vesicular stomatitis virus expressing the Zaire ebolavirus glycoprotein. Several of our antibodies, including the broadly binding ones, protected mice from mortality despite lacking neutralization capability in vitro, suggesting their protection may be mediated by Fc-FcR interactions. Indeed, three antibodies displayed cellular phagocytosis and/or antibody-dependent cell-mediated cytotoxicity in vitro Our antibodies, specifically the two identified cross-reactive monoclonal antibodies (KL-2E5 and KL-2H7), might add to the understanding of anti-ebolavirus humoral immunity.IMPORTANCE This study describes the generation of a panel of novel anti-ebolavirus glycoprotein monoclonal antibodies, including two antibodies with broad cross-reactivity to all known ebolavirus species. The antibodies were raised using a heterologous DNA-viral vector prime-boost regimen, resulting in a high proportion of cross-reactive antibodies (25%). Similar vaccination regimens have been used successfully to induce broad protection against influenza viruses in humans, and our limited data indicate that this might be a useful strategy for filovirus vaccines as well. Several of our antibodies showed protective efficacy when tested in a novel murine challenge model and may be developed into future therapeutics.

Insights into Reston virus spillovers and adaption from virus whole genome sequences.

Reston virus (family Filoviridae) is unique among the viruses of the Ebolavirus genus in that it is considered non-pathogenic in humans, in contrast to the other members which are highly virulent. The virus has however, been associated with several outbreaks of highly lethal hemorrhagic fever in non-human primates (NHPs), specifically cynomolgus monkeys (Macaca fascicularis) originating in the Philippines. In addition, Reston virus has been isolated from domestic pigs in the Philippines. To better understand virus spillover events and potential adaption to new hosts, the whole genome sequences of representative Reston virus isolates were obtained using a next generation sequencing (NGS) approach and comparative genomic analysis and virus fitness analyses were performed. Nine virus genome sequences were completed for novel and previously described isolates obtained from a variety of hosts including a human case, non-human primates and pigs. Results of phylogenetic analysis of the sequence differences are consistent with multiple independent introductions of RESTV from a still unknown natural reservoir into non-human primates and swine farming operations. No consistent virus genetic markers were found specific for viruses associated with primate or pig infections, but similar to what had been seen with some Ebola viruses detected in the large Western Africa outbreak in 2014–2016, a truncated version of VP30 was identified in a subgroup of Reston viruses obtained from an outbreak in pigs 2008–2009. Finally, the genetic comparison of two closely related viruses, one isolated from a human case and one from an NHP, showed amino acid differences in the viral polymerase and detectable differences were found in competitive growth assays on human and NHP cell lines.

Ebolaviruses Associated with Differential Pathogenicity Induce Distinct Host Responses in Human Macrophages.

Ebola virus (EBOV) and Reston virus (RESTV) are members of the Ebolavirus genus which greatly differ in their pathogenicity. While EBOV causes a severe disease in humans characterized by a dysregulated inflammatory response and elevated cytokine and chemokine production, there are no reported disease-associated human cases of RESTV infection, suggesting that RESTV is nonpathogenic for humans. The underlying mechanisms determining the pathogenicity of different ebolavirus species are not yet known. In this study, we dissected the host response to EBOV and RESTV infection in primary human monocyte-derived macrophages (MDMs). As expected, EBOV infection led to a profound proinflammatory response, including strong induction of type I and type III interferons (IFNs). In contrast, RESTV-infected macrophages remained surprisingly silent. Early activation of IFN regulatory factor 3 (IRF3) and NF-κB was observed in EBOV-infected, but not in RESTV-infected, MDMs. In concordance with previous results, MDMs treated with inactivated EBOV and Ebola virus-like particles (VLPs) induced NF-κB activation mediated by Toll-like receptor 4 (TLR4) in a glycoprotein (GP)-dependent manner. This was not the case in cells exposed to live RESTV, inactivated RESTV, or VLPs containing RESTV GP, indicating that RESTV GP does not trigger TLR4 signaling. Our results suggest that the lack of immune activation in RESTV-infected MDMs contributes to lower pathogenicity by preventing the cytokine storm observed in EBOV infection. We further demonstrate that inhibition of TLR4 signaling abolishes EBOV GP-mediated NF-κB activation. This finding indicates that limiting the excessive TLR4-mediated proinflammatory response in EBOV infection should be considered as a potential supportive treatment option for EBOV disease.IMPORTANCE Emerging infectious diseases are a major public health concern, as exemplified by the recent devastating Ebola virus (EBOV) outbreak. Different ebolavirus species are associated with widely varying pathogenicity in humans, ranging from asymptomatic infections for Reston virus (RESTV) to severe disease with fatal outcomes for EBOV. In this comparative study of EBOV- and RESTV-infected human macrophages, we identified key differences in host cell responses. Consistent with previous data, EBOV infection is associated with a proinflammatory signature triggered by the surface glycoprotein (GP), which can be inhibited by blocking TLR4 signaling. In contrast, infection with RESTV failed to stimulate a strong host response in infected macrophages due to the inability of RESTV GP to stimulate TLR4. We propose that disparate proinflammatory host signatures contribute to the differences in pathogenicity reported for ebolavirus species and suggest that proinflammatory pathways represent an intriguing target for the development of novel therapeutics.

Immunization-Elicited Broadly Protective Antibody Reveals Ebolavirus Fusion Loop as a Site of Vulnerability

While neutralizing antibodies are highly effective against ebolavirus infections, current experimental ebolavirus vaccines primarily elicit species-specific antibody responses. Here, we describe an immunization-elicited macaque antibody (CA45) that clamps the internal fusion loop with the N terminus of the ebolavirus glycoproteins (GPs) and potently neutralizes Ebola, Sudan, Bundibugyo, and Reston viruses. CA45, alone or in combination with an antibody that blocks receptor binding, provided full protection against all pathogenic ebolavirusesinmice, guinea pigs, and ferrets. Analysis of memory B cells from the immunized macaque suggests thatelicitation of broadly neutralizing antibodies (bNAbs) for ebolaviruses is possible but difficult, potentially due to the rarity of bNAb clones and their precursors. Unexpectedly, germline-reverted CA45, while exhibiting negligible binding to full-length GP, bound a proteolytically remodeled GP with picomolar affinity, suggestingthat engineered ebolavirus vaccines could trigger rare bNAb precursors more robustly. These findings have important implications for developing pan-ebolavirus vaccine and immunotherapeutic cocktails.

Detection of Zaire ebolavirus in swine: Assay development and optimization.

Ebolaviruses (family Filoviridae, order Mononegavirales) cause often fatal, haemorrhagic fever in primates including humans. Pigs have been identified as a species susceptible to Reston ebolavirus (RESTV) infection, with indicated transmission to humans in the Philippines; however, their role during Ebola outbreaks in Africa needs to be clarified. To perform surveillance studies, detection of ebolavirus requires a prerequisite validation of viral RNA and antibody detection methods in swine samples. These diagnostic tests also need to be suitable for deployment to low-level containment laboratories. In this study, we developed a set of tests for detection of antibodies against Zaire ebolavirus (EBOV) in swine. Recombinant EBOV nucleoprotein was produced using a baculovirus expression system for indirect ELISA development. Evaluation of this assay was performed using laboratory and field samples, achieving a diagnostic specificity of 99%. Importantly, the indirect ELISA was able to detect antibodies to EBOV at 7dpi, 3days earlier than virus neutralization tests (VNT). The format of the VNT in this work was modified to a microtitre plaque reduction neutralization assay (miPRNT) complemented with immunostaining to provide a more rapid and highly specific assay. Finally, a confirmatory immunoblot assay was generated to supplement the indirect ELISA results.

Rapid detection of all known ebolavirus species by reverse transcription-loop-mediated isothermal amplification (RT-LAMP)

Ebola virus disease (EVD), a highly virulent infectious disease caused by ebolaviruses, has a fatality rate of 25–90%. Without a licensed chemotherapeutic agent or vaccine for the treatment and prevention of EVD, control of outbreaks requires accurate and rapid diagnosis of cases. In this study, five sets of six oligonucleotide primers targeting the nucleoprotein gene were designed for specific identification of each of the five ebolavirus species using reverse transcription-loop mediated isothermal amplification (RT-LAMP) assay. The detection limits of the ebolavirus species-specific primer sets were evaluated using in vitro transcribed RNAs. The detection limit of species-specific RT-LAMP assays for Zaire ebolavirus, Sudan ebolavirus, Taï Forest ebolavirus, and Bundibugyo ebolavirus was 256 copies/reaction, while the detection limit for Reston ebolavirus was 64 copies/reaction, and the detection time for each of the RT-LAMP assays was 13.3±3.0, 19.8±4.6, 14.3±0.6, 16.1±4.7, and 19.8±2.4min (mean±SD), respectively. The sensitivity of the species-specific RT-LAMP assays were similar to that of the established RT-PCR and quantitative RT-PCR assays for diagnosis of EVD and are suitable for field or point-of-care diagnosis. The RT-LAMP assays were specific for the detection of the respective species of ebolavirus with no cross reaction with other species of ebolavirus and other viral hemorrhagic fever viruses such as Marburg virus, Lassa fever virus, and Dengue virus.The species-specific RT-LAMP assays developed in this study are rapid, sensitive, and specific and could be useful in case of an EVD outbreak.

Changes associated with Ebola virus adaptation to novel species.

Ebola viruses are not pathogenic but can be adapted to replicate and cause disease in rodents. Here, we used a structural bioinformatics approach to analyze the mutations associated with Ebola virus adaptation to rodents to elucidate the determinants of host-specific Ebola virus pathogenicity. We identified 33 different mutations associated with Ebola virus adaptation to rodents in the proteins GP, NP, L, VP24 and VP35. Only VP24, GP and NP were consistently found mutated in rodent-adapted Ebola virus strains. Fewer than five mutations in these genes seem to be required for the adaptation of Ebola viruses to a new species. The role of mutations in GP and NP is not clear. However, three VP24 mutations located in the protein interface with karyopherin α5 may enable VP24 to inhibit karyopherins and subsequently the host interferon response. Three further VP24 mutations change hydrogen bonding or cause conformational changes. Hence, there is evidence that few mutations including crucial mutations in VP24 enable Ebola virus adaptation to new hosts. Since Reston virus, the only non-human pathogenic Ebolavirus species circulates in pigs in Asia, this raises concerns that few mutations may result in novel human pathogenic Ebolaviruses. m.n.wass@kent.ac.uk , m.michaelis@kent.ac.uk or j.s.rossman@kent.ac.uk. Supplementary data are available at Bioinformatics online.

VP24-Karyopherin Alpha Binding Affinities Differ between Ebolavirus Species, Influencing Interferon Inhibition and VP24 Stability.

The interaction of Ebola virus (EBOV) VP24 protein with host karyopherin alpha (KPNA) proteins blocks type I interferon (IFN) signaling, which is a central component of the host innate immune response to viral infection. Here, we quantitatively compared the interactions of VP24 proteins from EBOV and two members of the Ebolavirus genus, Bundibugyo virus (BDBV) and Reston virus (RESTV). The data reveal lower binding affinity of the BDBV VP24 (bVP24) for KPNAs and demonstrate that the interaction with KPNA modulates inhibition of IFN signaling and VP24 stability. The effect of KPNA interaction on VP24 stability is a novel functional consequence of this virus-host interaction, and the differences identified between viral species may contribute to differences in pathogenesis.

Sequence of Reston Virus Isolate AZ-1435, an Ebolavirus Isolate Obtained during the 1989-1990 Reston Virus Epizootic in the United States.

ABSTRACTReston virus (RESTV) was discovered in 1989–1990 during three connected epizootics of highly lethal viral hemorrhagic fever among captive macaques in primate housing facilities in the United States and Philippines. Currently, only one RESTV isolate from that outbreak (named Pennsylvania) has been sequenced. Here, we report the sequence of a second isolate, Reston virus/M.fascicularis-tc/USA/1990/Philippines89-AZ1435.

Guide to the Correct Use of Filoviral Nomenclature.

The International Committee on Taxonomy of Viruses (ICTV) currently recognizes three genera and seven species as part of the mononegaviral family Filoviridae. Eight distinct filoviruses (Bundibugyo virus, Ebola virus, Lloviu virus, Marburg virus, Ravn virus, Reston virus, Sudan virus, and Taï Forest virus) have been assigned to these seven species. This chapter briefly summarizes the status quo of filovirus classification and focuses on the importance of differentiating between filoviral species and filoviruses and the correct use of taxonomic and vernacular filovirus names and abbreviations in written and oral discourse.