Ebolavirus delta-peptide immunoadhesins inhibit marburgvirus and ebolavirus cell entry

With the exception of Reston and Lloviu viruses, filoviruses (marburgviruses, ebolaviruses, and “cuevaviruses”) cause severe viral hemorrhagic fevers in humans. Filoviruses use a class I fusion protein, GP_1,2, to bind to an unknown, but shared, cell surface receptor to initiate virus-cell fusion. In addition to GP_1,2, ebolaviruses and cuevaviruses, but not marburgviruses, express two secreted glycoproteins, soluble GP (sGP) and small soluble GP (ssGP). All three glycoproteins have identical N termini that include the receptor-binding region (RBR) but differ in their C termini. We evaluated the effect of the secreted ebolavirus glycoproteins on marburgvirus and ebolavirus cell entry, using Fc-tagged recombinant proteins. Neither sGP-Fc nor ssGP-Fc bound to filovirus-permissive cells or inhibited GP_1,2-mediated cell entry of pseudotyped retroviruses. Surprisingly, several Fc-tagged Δ-peptides, which are small C-terminal cleavage products of sGP secreted by ebolavirus-infected cells, inhibited entry of retroviruses pseudotyped with Marburg virus GP_1,2, as well as Marburg virus and Ebola virus infection in a dose-dependent manner and at low molarity despite absence of sequence similarity to filovirus RBRs. Fc-tagged Δ-peptides from three ebolaviruses (Ebola virus, Sudan virus, and Taï Forest virus) inhibited GP_1,2-mediated entry and infection of viruses comparably to or better than the Fc-tagged RBRs, whereas the Δ-peptide-Fc of an ebolavirus nonpathogenic for humans (Reston virus) and that of an ebolavirus with lower lethality for humans (Bundibugyo virus) had little effect. These data indicate that Δ-peptides are functional components of ebolavirus proteomes. They join cathepsins and integrins as novel modulators of filovirus cell entry, might play important roles in pathogenesis, and could be exploited for the synthesis of powerful new antivirals.     

Protective mAbs and Cross-Reactive mAbs Raised by Immunization with Engineered Marburg Virus GPs

The filoviruses, which include the marburg- and ebolaviruses, have caused multiple outbreaks among humans this decade. Antibodies against the filovirus surface glycoprotein (GP) have been shown to provide life-saving therapy in nonhuman primates, but such antibodies are generally virus-specific. Many monoclonal antibodies (mAbs) have been described against Ebola virus. In contrast, relatively few have been described against Marburg virus. Here we present ten mAbs elicited by immunization of mice using recombinant mucin-deleted GPs from different Marburg virus (MARV) strains. Surprisingly, two of the mAbs raised against MARV GP also cross-react with the mucin-deleted GP cores of all tested ebolaviruses (Ebola, Sudan, Bundibugyo, Reston), but these epitopes are masked differently by the mucin-like domains themselves. The most efficacious mAbs in this panel were found to recognize a novel “wing” feature on the GP2 subunit that is unique to Marburg and does not exist in Ebola. Two of these anti-wing antibodies confer 90 and 100% protection, respectively, one hour post-exposure in mice challenged with MARV.     

Development and Evaluation of a Panel of Filovirus Sequence Capture Probes for Pathogen Detection by Next-Generation Sequencing

A detailed understanding of the circulating pathogens in a particular geographic location aids in effectively utilizing targeted, rapid diagnostic assays, thus allowing for appropriate therapeutic and containment procedures. This is especially important in regions prevalent for highly pathogenic viruses co-circulating with other endemic pathogens such as the malaria parasite. The importance of biosurveillance is highlighted by the ongoing Ebola virus disease outbreak in West Africa. For example, a more comprehensive assessment of the regional pathogens could have identified the risk of a filovirus disease outbreak earlier and led to an improved diagnostic and response capacity in the region. In this context, being able to rapidly screen a single sample for multiple pathogens in a single tube reaction could improve both diagnostics as well as pathogen surveillance. Here, probes were designed to capture identifying filovirus sequence for the ebolaviruses Sudan, Ebola, Reston, Taï Forest, and Bundibugyo and the Marburg virus variants Musoke, Ci67, and Angola. These probes were combined into a single probe panel, and the captured filovirus sequence was successfully identified using the MiSeq next-generation sequencing platform. This panel was then used to identify the specific filovirus from nonhuman primates experimentally infected with Ebola virus as well as Bundibugyo virus in human sera samples from the Democratic Republic of the Congo, thus demonstrating the utility for pathogen detection using clinical samples. While not as sensitive and rapid as real-time PCR, this panel, along with incorporating additional sequence capture probe panels, could be used for broad pathogen screening and biosurveillance.     

Crystal Structure of Marburg Virus VP24

The VP24 protein plays an essential, albeit poorly understood role in the filovirus life cycle. VP24 is only 30% identical between Marburg virus and the ebolaviruses. Furthermore, VP24 from the ebolaviruses is immunosuppressive, while that of Marburg virus is not. The crystal structure of Marburg virus VP24, presented here, reveals that although the core is similar between the viral genera, Marburg VP24 is distinguished by a projecting β-shelf and an alternate conformation of the N-terminal polypeptide.     

Discovery of an ebolavirus-like filovirus in europe

Filoviruses, amongst the most lethal of primate pathogens, have only been reported as natural infections in sub-Saharan Africa and the Philippines. Infections of bats with the ebolaviruses and marburgviruses do not appear to be associated with disease. Here we report identification in dead insectivorous bats of a genetically distinct filovirus, provisionally named Lloviu virus, after the site of detection, Cueva del Lloviu, in Spain.     

Ad35 and Ad26 Vaccine Vectors Induce Potent and Cross-Reactive Antibody and T-Cell Responses to Multiple Filovirus Species

Filoviruses cause sporadic but highly lethal outbreaks of hemorrhagic fever in Africa in the human population. Currently, no drug or vaccine is available for treatment or prevention. A previous study with a vaccine candidate based on the low seroprevalent adenoviruses 26 and 35 (Ad26 and Ad35) was shown to provide protection against homologous Ebola Zaire challenge in non human primates (NHP) if applied in a prime-boost regimen. Here we have aimed to expand this principle to construct and evaluate Ad26 and Ad35 vectors for development of a vaccine to provide universal filovirus protection against all highly lethal strains that have caused major outbreaks in the past. We have therefore performed a phylogenetic analysis of filovirus glycoproteins to select the glycoproteins from two Ebola species (Ebola Zaire and Ebola Sudan/Gulu,), two Marburg strains (Marburg Angola and Marburg Ravn) and added the more distant non-lethal Ebola Ivory Coast species for broadest coverage. Ad26 and Ad35 vectors expressing these five filovirus glycoproteins were evaluated to induce a potent cellular and humoral immune response in mice. All adenoviral vectors induced a humoral immune response after single vaccination in a dose dependent manner that was cross-reactive within the Ebola and Marburg lineages. In addition, both strain-specific as well as cross-reactive T cell responses could be detected. A heterologous Ad26–Ad35 prime-boost regime enhanced mainly the humoral and to a lower extend the cellular immune response against the transgene. Combination of the five selected filovirus glycoproteins in one multivalent vaccine potentially elicits protective immunity in man against all major filovirus strains that have caused lethal outbreaks in the last 20 years.     

Ebola and Marburg virus antibody prevalence in selected populations of the Central African Republic

With the natural history of the filovirus family seemingly unknown, filovirus ecology in its natural environment remains a rudimentary field of research. In order to investigate the maintenance cycle of filovirus in Central Africa, a study was conducted within the rain forest of the Central African Republic. The epidemiological study determines the frequency and distribution of filovirus seroprevalence in a selected human population. Using an ELISA, serum samples from Pygmy and non-Pygmy populations were tested for Ebola-Zaire virus and Marburg (MBG) virus antibody. Filovirus antibody reacting sera were found in all zones investigated, and in all populations studied (Ebola virus IgG 5.3%; Marburg virus IgG 2.4%). Pygmies appeared to have a significantly higher seroprevalence (P < 0.03) against Ebola-Zaire virus (7.02%) than non-Pygmies (4.2%). MBG virus or related unknown filovirus strains also seem to be present in the western part of Central Africa. MBG virus antibodies were present in different Pygmy groups (ranging from 0.7 to 5.6%, mean 2.05%) and in several non-Pygmy populations (ranging from 0.0 to 3.9%, mean 3.4%) without an overall significant difference between the two groups (P = 0.14). The potentialities of nonpathogenic filovirus strains circulating in the Central African Republic are discussed.     

First laboratory confirmation and sequencing of Zaire ebolavirus in Uganda following two independent introductions of cases from the 10th Ebola Outbreak in the Democratic Republic of the Congo,June 2019

Uganda established a domestic Viral Hemorrhagic Fever (VHF) testing capacity in 2010 in response to the increasing occurrence of filovirus outbreaks. In July 2018, the neighboring Democratic Republic of Congo (DRC) experienced its 10^th Ebola Virus Disease (EVD) outbreak and for the duration of the outbreak, the Ugandan Ministry of Health (MOH) initiated a national EVD preparedness stance. Almost one year later, on 10^th June 2019, three family members who had contracted EVD in the DRC crossed into Uganda to seek medical treatment.Samples were collected from all the suspected cases using internationally established biosafety protocols and submitted for VHF diagnostic testing at Uganda Virus Research Institute. All samples were initially tested by RT-PCR for ebolaviruses, marburgviruses, Rift Valley fever (RVF) virus and Crimean-Congo hemorrhagic fever (CCHF) virus. Four people were identified as being positive for Zaire ebolavirus, marking the first report of Zaire ebolavirus in Uganda. In-country Next Generation Sequencing (NGS) and phylogenetic analysis was performed for the first time in Uganda, confirming the outbreak as imported from DRC at two different time point from different clades. This rapid response by the MoH, UVRI and partners led to the control of the outbreak and prevention of secondary virus transmission.     

Effectiveness of Personal Protective Equipment for Healthcare Workers Caring for Patients with Filovirus Disease: A Rapid Review

Background

A rapid review, guided by a protocol, was conducted to inform development of the World Health Organization’s guideline on personal protective equipment in the context of the ongoing (2013–present) Western African filovirus disease outbreak, with a focus on health care workers directly caring for patients with Ebola or Marburg virus diseases.

Methods

Electronic databases and grey literature sources were searched. Eligibility criteria initially included comparative studies on Ebola and Marburg virus diseases reported in English or French, but criteria were expanded to studies on other viral hemorrhagic fevers and non-comparative designs due to the paucity of studies. After title and abstract screening (two people to exclude), full-text reports of potentially relevant articles were assessed in duplicate. Fifty-seven percent of extraction information was verified. The Grading of Recommendations Assessment, Development and Evaluation framework was used to inform the quality of evidence assessments.

Results

Thirty non-comparative studies (8 related to Ebola virus disease) were located, and 27 provided data on viral transmission. Reporting of personal protective equipment components and infection prevention and control protocols was generally poor.

Conclusions

Insufficient evidence exists to draw conclusions regarding the comparative effectiveness of various types of personal protective equipment. Additional research is urgently needed to determine optimal PPE for health care workers caring for patients with filovirus.     

Durability of a Vesicular Stomatitis Virus-Based Marburg Virus Vaccine in Nonhuman Primates

The filoviruses, Marburg virus (MARV) and Ebola virus, causes severe hemorrhagic fever with high mortality in humans and nonhuman primates. A promising filovirus vaccine under development is based on a recombinant vesicular stomatitis virus (rVSV) that expresses individual filovirus glycoproteins (GPs) in place of the VSV glycoprotein (G). These vaccines have shown 100% efficacy against filovirus infection in nonhuman primates when challenge occurs 28–35 days after a single injection immunization. Here, we examined the ability of a rVSV MARV-GP vaccine to provide protection when challenge occurs more than a year after vaccination. Cynomolgus macaques were immunized with rVSV-MARV-GP and challenged with MARV approximately 14 months after vaccination. Immunization resulted in the vaccine cohort of six animals having anti-MARV GP IgG throughout the pre-challenge period. Following MARV challenge none of the vaccinated animals showed any signs of clinical disease or viremia and all were completely protected from MARV infection. Two unvaccinated control animals exhibited signs consistent with MARV infection and both succumbed. Importantly, these data are the first to show 100% protective efficacy against any high dose filovirus challenge beyond 8 weeks after final vaccination. These findings demonstrate the durability of VSV-based filovirus vaccines.     

Recombinant vesicular stomatitis virus vaccine vectors expressing filovirus glycoproteins lack neurovirulence in nonhuman primates

The filoviruses, Marburg virus and Ebola virus, cause severe hemorrhagic fever with high mortality in humans and nonhuman primates. Among the most promising filovirus vaccines under development is a system based on recombinant vesicular stomatitis virus (rVSV) that expresses an individual filovirus glycoprotein (GP) in place of the VSV glycoprotein (G). The main concern with all replication-competent vaccines, including the rVSV filovirus GP vectors, is their safety. To address this concern, we performed a neurovirulence study using 21 cynomolgus macaques where the vaccines were administered intrathalamically. Seven animals received a rVSV vector expressing the Zaire ebolavirus (ZEBOV) GP; seven animals received a rVSV vector expressing the Lake Victoria marburgvirus (MARV) GP; three animals received rVSV-wild type (wt) vector, and four animals received vehicle control. Two of three animals given rVSV-wt showed severe neurological symptoms whereas animals receiving vehicle control, rVSV-ZEBOV-GP, or rVSV-MARV-GP did not develop these symptoms. Histological analysis revealed major lesions in neural tissues of all three rVSV-wt animals; however, no significant lesions were observed in any animals from the filovirus vaccine or vehicle control groups. These data strongly suggest that rVSV filovirus GP vaccine vectors lack the neurovirulence properties associated with the rVSV-wt parent vector and support their further development as a vaccine platform for human use.     

Marburg and Ebola virus infections in laboratory non-human primates: a literature review

BACKGROUND AND PURPOSE: Several non-human primate species are used as laboratory animals for various types of studies. Although importation of monkeys may introduce different diseases, special attention has recently been drawn to Marburg and Ebola viruses. This review presented here discusses the potential risk of these viruses for persons working with non-human primates as laboratory animals by focusing on epidemiology, virology, symptoms, pathogenesis, natural reservoir, transmission, quarantine of non-human primates, therapy, and prevention. CONCLUSION: A total of 23 Marburg and Ebola virus outbreaks causing viral hemorrhagic fever has been reported among humans and monkeys since the first outbreak in Marburg, Germany in 1967. Most of the 1,100 human cases, with nearly 800 deaths, developed in Africa due mainly to direct and intimate contact with infected patients. Few human cases have developed after contact with non-human primates used for various scientific purposes. However, adequate quarantine should be applied to prevent human infections not only due to Marburg and Ebola viruses, but also to other infective agents. By following proper guidelines, the filovirus infection risk for people working with non-human primates during quarantine exists, but is minimal. There seems to be little risk for filovirus infections after an adequate quarantine period. Therefore, non-human primates can be used as laboratory animals, with little risk of filovirus infections, provided adequate precautions are taken.     

丝状病毒出血热的血清流行病学调查研究及现状

丝状病毒(包括马尔堡与埃波拉病毒)被发现已有40余年,对其自然流行病学尚不清楚。综述了该病血清流行病学调查结果,丝状病毒出现的频率,地理生态学分布,及病毒生物学特性,为该病流行因素提供了信息与框架。然而,对病毒宿主,传入人类的起源、地理分布、具体区域等流行生态学有待进一步研究。     

Association of the caveola vesicular system with cellular entry by filoviruses

The filoviruses Ebola Zaire virus and Marburg virus are believed to infect target cells through endocytic vesicles, but the details of this pathway are unknown. We used a pseudotyping strategy to investigate the cell biology of filovirus entry. We observed that specific inhibitors of the caveola system, including cholesterol-sequestering drugs and phorbol esters, inhibited the entry of filovirus pseudotypes into human cells. We also measured slower cell entry kinetics for both filovirus pseudotypes than for pseudotypes of vesicular stomatitis virus (VSV), which has been recognized to exploit the clathrin-mediated entry pathway. Finally, visualization by immunofluorescence and confocal microscopy revealed that the filovirus pseudotypes colocalized with the caveola protein marker caveolin-1 but that VSV pseudotypes did not. Collectively, these results provide evidence suggesting that filoviruses use caveolae to gain entry into cells.     

Human macrophage C-type lectin specific for galactose and N-acetylgalactosamine promotes filovirus entry

Filoviruses cause lethal hemorrhagic disease in humans and nonhuman primates. An initial target of filovirus infection is the mononuclear phagocytic cell. Calcium-dependent (C-type) lectins such as dendritic cell- or liver/lymph node-specific ICAM-3 grabbing nonintegrin (DC-SIGN or L-SIGN, respectively), as well as the hepatic asialoglycoprotein receptor, bind to Ebola or Marburg virus glycoprotein (GP) and enhance the infectivity of these viruses in vitro. Here, we demonstrate that a recently identified human macrophage galactose- and N-acetylgalactosamine-specific C-type lectin (hMGL), whose ligand specificity differs from DC-SIGN and L-SIGN, also enhances the infectivity of filoviruses. This enhancement was substantially weaker for the Reston and Marburg viruses than for the highly pathogenic Zaire virus. We also show that the heavily glycosylated, mucin-like domain on the filovirus GP is required for efficient interaction with this lectin. Furthermore, hMGL, like DC-SIGN and L-SIGN, is present on cells known to be major targets of filoviruses (i.e., macrophages and dendritic cells), suggesting a role for these C-type lectins in viral replication in vivo. We propose that filoviruses use different C-type lectins to gain cellular entry, depending on the cell type, and promote efficient viral replication.     

Ebola Virus Genome Plasticity as a Marker of Its Passaging History: A Comparison of In Vitro Passaging to Non-Human Primate Infection

To identify polymorphic sites that could be used as biomarkers of Ebola virus passage history, we repeatedly amplified Ebola virus (Kikwit variant) in vitro and in vivo and performed deep sequencing analysis of the complete genomes of the viral subpopulations. We then determined the sites undergoing selection during passage in Vero E6 cells. Four locations within the Ebola virus Kikwit genome were identified that together segregate cell culture-passaged virus and virus obtained from infected non-human primates. Three of the identified sites are located within the glycoprotein gene (GP) sequence: the poly-U (RNA editing) site at position 6925, as well as positions 6677, and 6179. One site was found in the VP24 gene at position 10833. In all cases, in vitro and in vivo, both populations (majority and minority variants) were maintained in the viral swarm, with rapid selections occurring after a few passages or infections. This analysis approach will be useful to differentiate whether filovirus stocks with unknown history have been passaged in cell culture and may support filovirus stock standardization for medical countermeasure development.     

DC-SIGN and DC-SIGNR interact with the glycoprotein of Marburg virus and the S protein of severe acute respiratory syndrome coronavirus

The lectins DC-SIGN and DC-SIGNR can augment viral infection; however, the range of pathogens interacting with these attachment factors is incompletely defined. Here we show that DC-SIGN and DC-SIGNR enhance infection mediated by the glycoprotein (GP) of Marburg virus (MARV) and the S protein of severe acute respiratory syndrome coronavirus and might promote viral dissemination. SIGNR1, a murine DC-SIGN homologue, also enhanced infection driven by MARV and Ebola virus GP and could be targeted to assess the role of attachment factors in filovirus infection in vivo.     

Laboratory detection and diagnosis of filoviruses

Ebola virus (EBOV) and Marburg virus (MARV), belonging to the Filoviridae family, emerged four decades ago and caused severe viral hemorrhagic fever in human and other primates. As high as 50–90% mortality, filoviruses can cause significant threats to public health. However, so far no specific and efficient vaccine has been available, nor have other treatment methods proved to be effective. It is of great importance to detect these pathogens specific, rapidly and sensitively in order to control future filovirus outbreaks. Here, recent progresses in the development of detection and diagnosis methods for EBOV and MARV are summarized.     

Lentivirus vectors pseudotyped with filoviral envelope glycoproteins transduce airway epithelia from the apical surface independently of folate receptor alpha

The practical application of gene therapy as a treatment for cystic fibrosis is limited by poor gene transfer efficiency with vectors applied to the apical surface of airway epithelia. Recently, folate receptor alpha (FR alpha), a glycosylphosphatidylinositol-linked surface protein, was reported to be a cellular receptor for the filoviruses. We found that polarized human airway epithelia expressed abundant FR alpha on their apical surface. In an attempt to target these apical receptors, we pseudotyped feline immunodeficiency virus (FIV)-based vectors by using envelope glycoproteins (GPs) from the filoviruses Marburg virus and Ebola virus. Importantly, primary cultures of well-differentiated human airway epithelia were transduced when filovirus GP-pseudotyped FIV was applied to the apical surface. Furthermore, by deleting a heavily O-glycosylated extracellular domain of the Ebola GP, we improved the titer of concentrated vector severalfold. To investigate the folate receptor dependence of gene transfer with the filovirus pseudotypes, we compared gene transfer efficiency in immortalized airway epithelium cell lines and primary cultures. By utilizing phosphatidylinositol-specific phospholipase C (PI-PLC) treatment and FR alpha-blocking antibodies, we demonstrated FR alpha-dependent and -independent entry by filovirus glycoprotein-pseudotyped FIV-based vectors in airway epithelia. Of particular interest, entry independent of FR alpha was observed in primary cultures of human airway epithelia. Understanding viral vector binding and entry pathways is fundamental for developing cystic fibrosis gene therapy applications.     

Phylogenetic assessment of filoviruses: how many lineages of Marburg virus?

Filoviruses have to date been considered as consisting of one diverse genus (Ebola viruses) and one undifferentiated genus (Marburg virus). We reconsider this idea by means of detailed phylogenetic analyses of sequence data available for the Filoviridae: using coalescent simulations, we ascertain that two Marburg isolates (termed the "RAVN" strain) represent a quite-distinct lineage that should be considered in studies of biogeography and host associations, and may merit recognition at the level of species. In contrast, filovirus isolates recently obtained from bat tissues are not distinct from previously known strains, and should be considered as drawn from the same population. Implications for understanding the transmission geography and host associations of these viruses are discussed.