Recombinant Lloviu virus as a tool to study viral replication and host responses.

Next generation sequencing has revealed the presence of numerous RNA viruses in animal reservoir hosts, including many closely related to known human pathogens. Despite their zoonotic potential, most of these viruses remain understudied due to not yet being cultured. While reverse genetic systems can facilitate virus rescue, this is often hindered by missing viral genome ends. A prime example is Lloviu virus (LLOV), an uncultured filovirus that is closely related to the highly pathogenic Ebola virus. Using minigenome systems, we complemented the missing LLOV genomic ends and identified cis-acting elements required for LLOV replication that were lacking in the published sequence. We leveraged these data to generate recombinant full-length LLOV clones and rescue infectious virus. Similar to other filoviruses, recombinant LLOV (rLLOV) forms filamentous virions and induces the formation of characteristic inclusions in the cytoplasm of the infected cells, as shown by electron microscopy. Known target cells of Ebola virus, including macrophages and hepatocytes, are permissive to rLLOV infection, suggesting that humans could be potential hosts. However, inflammatory responses in human macrophages, a hallmark of Ebola virus disease, are not induced by rLLOV. Additional tropism testing identified pneumocytes as capable of robust rLLOV and Ebola virus infection. We also used rLLOV to test antivirals targeting multiple facets of the replication cycle. Rescue of uncultured viruses of pathogenic concern represents a valuable tool in our arsenal for pandemic preparedness.

Identification of filovirus entry inhibitors targeting the endosomal receptor NPC1 binding site.

Filoviruses, mainly consisting of Ebola viruses (EBOV) and Marburg viruses (MARV), are enveloped negative-strand RNA viruses which can infect humans to cause severe hemorrhagic fevers and outbreaks with high mortality rates. The filovirus infection is mediated by the interaction of viral envelope glycoprotein (GP) and the human endosomal receptor Niemann-Pick C1 (NPC1). Blocking this interaction will prevent the infection. Therefore, we utilized an In silico screening approach to conduct virtual compound screening against the NPC1 receptor-binding site (RBS). Twenty-six top-hit compounds were purchased and evaluated by in vitro cell based inhibition assays against pseudotyped or replication-competent filoviruses. Two classes (A and U) of compounds were identified to have potent inhibitory activity against both Ebola and Marburg viruses. The IC50 values are in the lower level of micromolar concentrations. One compound (compd-A) was found to have a sub-micromolar IC50 value (0.86 µM) against pseudotyped Marburg virus. The cytotoxicity assay (MTT) indicates that compd-A has a moderate cytotoxicity level but the compd-U has much less toxicity and the CC50 value was about 100 µM. Structure-activity relationship (SAR) study has found some analogs of compd-A and -U have reduced the toxicity and enhanced the inhibitory activity. In conclusion, this work has identified several qualified lead-compounds for further drug development against filovirus infection.

A biaryl sulfonamide derivative as a novel inhibitor of filovirus infection.

Ebolaviruses and marburgviruses, members of the family Filoviridae, are known to cause fatal diseases often associated with hemorrhagic fever. Recent outbreaks of Ebola virus disease in West African countries and the Democratic Republic of the Congo have made clear the urgent need for the development of therapeutics and vaccines against filoviruses. Using replication-incompetent vesicular stomatitis virus (VSV) pseudotyped with the Ebola virus (EBOV) envelope glycoprotein (GP), we screened a chemical compound library to obtain new drug candidates that inhibit filoviral entry into target cells. We discovered a biaryl sulfonamide derivative that suppressed in vitro infection mediated by GPs derived from all known human-pathogenic filoviruses. To determine the inhibitory mechanism of the compound, we monitored each entry step (attachment, internalization, and membrane fusion) using lipophilic tracer-labeled ebolavirus-like particles and found that the compound efficiently blocked fusion between the viral envelope and the endosomal membrane during cellular entry. However, the compound did not block the interaction of GP with the Niemann-Pick C1 protein, which is believed to be the receptor of filoviruses. Using replication-competent VSVs pseudotyped with EBOV GP, we selected escape mutants and identified two EBOV GP amino acid residues (positions 47 and 66) important for the interaction with this compound. Interestingly, these amino acid residues were located at the base region of the GP trimer, suggesting that the compound might interfere with the GP conformational change required for membrane fusion. These results suggest that this biaryl sulfonamide derivative is a novel fusion inhibitor and a possible drug candidate for the development of a pan-filovirus therapeutic.

Filoviruses Infect Rhesus Macaque Synoviocytes in Vivo and Primary Human Synoviocytes in Vitro.

The most commonly reported symptom of post-Ebola virus disease syndrome in survivors is arthralgia, yet involvement of the joints in acute or convalescent Ebola virus infection is not well characterized in human patients or animal models. Through immunohistochemistry, we found that the lining synovial intima of the stifle (knee) is a target for acute infection by Ebola virus/Kikwit, Ebola virus/Makona-C05, and Marburg virus/Angola in the rhesus macaque model. Furthermore, histologic analysis, immunohistochemistry, RNAscope in situ hybridization, and transmission electron microscopy showed that synoviocytes of the stifle, shoulder, and hip are a target for mouse-adapted Ebola virus/Yambuku-Mayinga infection during acute disease in rhesus macaques. A time course of infection study with Ebola virus/Kikwit found that the large joint synovium became immunopositive beginning on postinfection day 6. In total, the synovium of 28 of 30 rhesus macaques with terminal filovirus disease had evidence of infection (64 of 96 joints examined). On the basis of immunofluorescence, infected cell types included CD68+ type A (macrophage-like) synoviocytes and CD44+ type B (fibroblast-like) synoviocytes. Cultured primary human fibroblast-like synoviocytes were permissive to infection with Ebola and Marburg viruses in vitro. Because synovial joints include immune privileged sites, these findings are significant for future investigations of filovirus pathogenesis and persistence as well as arthralgias in acute and convalescent filovirus disease.

Crystal structure of the Menglà virus VP30 C-terminal domain.

The family Filoviridae contains many important human viruses, including Marburg virus (MARV) and Ebola virus (EBOV). Menglà virus (MLAV), a newly discovered filovirus, is considered a potential human pathogen. The VP30 C-terminal domain (CTD) of these filoviruses plays an essential role in virion assembly. In common with other filoviruses, MLAV VP30 CTD mainly exists as a dimer in solution. In this work, we determined the crystal structure of recombinant MLAV VP30 CTD monomer, verifying that C-terminal helix-7 (H7) is critical for the dimerization process. This study provides a preliminary model for investigation of MLAV VP30 CTD as an anti-filovirus drug development target.

Serological Evidence for Henipa-like and Filo-like Viruses in Trinidad Bats.

Bat-borne zoonotic pathogens belonging to the family Paramxyoviridae, including Nipah and Hendra viruses, and the family Filoviridae, including Ebola and Marburg viruses, can cause severe disease and high mortality rates on spillover into human populations. Surveillance efforts for henipaviruses and filoviruses have been largely restricted to the Old World; however, recent studies suggest a potentially broader distribution for henipaviruses and filoviruses than previously recognized. In the current study, we screened for henipaviruses and filoviruses in New World bats collected across 4 locations in Trinidad near the coast of Venezuela. Bat tissue samples were screened using previously established reverse-transcription polymerase chain reaction assays. Serum were screened using a multiplex immunoassay to detect antibodies reactive with the envelope glycoprotein of viruses in the genus Henipavirus and the family Filoviridae. Serum samples were also screened by means of enzyme-linked immunosorbent assay for antibodies reactive with Nipah G and F glycoproteins. Of 84 serum samples, 28 were reactive with ≥1 henipavirus glycoprotein by ≥1 serological method, and 6 serum samples were reactive against ≥1 filovirus glycoproteins. These data provide evidence of potential circulation of viruses related to the henipaviruses and filoviruses in New World bats.

Seroreactivity against Marburg or related filoviruses in West and Central Africa.

A serological survey of 2,430 archived serum samples collected between 1997 and 2012 was conducted to retrospectively determine the prevalence of Marburg virus in five African countries. Serum samples were screened for neutralizing antibodies in a pseudotype micro-neutralization assay and confirmed by enzyme-linked immunosorbent assay (ELISA). Surprisingly, a seroprevalence for Marburg virus of 7.5 and 6.3% was found in Cameroon and Ghana, respectively, suggesting the circulation of filoviruses or related viruses outside of known endemic areas that remain undetected by current surveillance efforts. However, due to the lack of validated assays and appropriate positive controls, these results must be considered preliminary.

Insights into the Host Range, Genetic Diversity, and Geographical Distribution of Jingmenviruses.

Jingmenvirus is a recently identified group of segmented RNA viruses phylogenetically linked with unsegmented Flaviviridae viruses. Primarily identified in various tick genera originating in China, Jingmenvirus geographical distribution has rapidly expanded to cover Africa, South America, Caribbean, and Europe. The identification of Jingmen-related viruses in various mammals, including febrile humans, opens the possibility that Jingmenviruses may be novel tick-borne arboviruses. In this study, we aimed at increasing knowledge of the host range, genetic diversity, and geographical distribution of Jingmenviruses by reporting for the first time the identification of Jingmenviruses associated with Rhipicephalus microplus ticks originating in the French Antilles (Guadeloupe and Martinique islands), with Amblyomma testudinarium ticks in Lao PDR, and with Ixodes ricinus ticks in metropolitan France, and from urine of Pteropus lylei bats in Cambodia. Analyses of the relationships between the different Jingmenvirus genomes resulted in the identification of three main phylogenic subclades, each of them containing both tick-borne and mammal-borne strains, reinforcing the idea that Jingmenviruses may be considered as tick-borne arboviruses. Finally, we estimated the prevalence of Jingmenvirus-like infection using luciferase immunoprecipitation assay screening (LIPS) of asymptomatic humans and cattle highly exposed to tick bites. Among 70 French human, 153 Laotian human, and 200 Caribbean cattle sera tested, only one French human serum was found (slightly) positive, suggesting that the prevalence of Jingmenvirus human and cattle infections in these areas is probably low.IMPORTANCE Several arboviruses emerging as new pathogens for humans and domestic animals have recently raised public health concern and increased interest in the study of their host range and in detection of spillover events. Recently, a new group of segmented Flaviviridae-related viruses, the Jingmenviruses, has been identified worldwide in many invertebrate and vertebrate hosts, pointing out the issue of whether they belong to the arbovirus group. The study presented here combined whole-genome sequencing of three tick-borne Jingmenviruses and one bat-borne Jingmenvirus with comprehensive phylogenetic analyses and high-throughput serological screening of human and cattle populations exposed to these viruses to contribute to the knowledge of Jingmenvirus host range, geographical distribution, and mammalian exposure.

Filovirus-reactive antibodies in humans and bats in Northeast India imply zoonotic spillover.

Bats are reservoirs for several zoonotic pathogens, including filoviruses. Recent work highlights the diversity of bat borne filoviruses in Asia. High risk activities at the bat-human interface pose the threat of zoonotic virus transmission. We present evidence for prior exposure of bat harvesters and two resident fruit bat species to filovirus surface glycoproteins by screening sera in a multiplexed serological assay. Antibodies reactive to two antigenically distinct filoviruses were detected in human sera and to three individual filoviruses in bats in remote Northeast India. Sera obtained from Eonycteris spelaea bats showed similar patterns of cross-reactivity as human samples, suggesting them as the species responsible for the spillover. In contrast, sera from Rousettus leschenaultii bats reacted to two different virus glycoproteins. Our results indicate circulation of several filoviruses in bats and the possibility for filovirus transmission from bats to humans.

Cytokine Effects on the Entry of Filovirus Envelope Pseudotyped Virus-Like Particles into Primary Human Macrophages.

Macrophages are one of the first and also a major site of filovirus replication and, in addition, are a source of multiple cytokines, presumed to play a critical role in the pathogenesis of the viral infection. Some of these cytokines are known to induce macrophage phenotypic changes in vitro, but how macrophage polarization may affect the cell susceptibility to filovirus entry remains largely unstudied. We generated different macrophage subsets using cytokine pre-treatment and subsequently tested their ability to fuse with beta-lactamase containing virus-like particles (VLP), pseudotyped with the surface glycoprotein of Ebola virus (EBOV) or the glycoproteins of other clinically relevant filovirus species. We found that pre-incubation of primary human monocyte-derived macrophages (MDM) with interleukin-10 (IL-10) significantly enhanced filovirus entry into cells obtained from multiple healthy donors, and the IL-10 effect was preserved in the presence of pro-inflammatory cytokines found to be elevated during EBOV disease. In contrast, fusion of IL-10-treated macrophages with influenza hemagglutinin/neuraminidase pseudotyped VLPs was unchanged or slightly reduced. Importantly, our in vitro data showing enhanced virus entry are consistent with the correlation established between elevated serum IL-10 and increased mortality in filovirus infected patients and also reveal a novel mechanism that may account for the IL-10-mediated increase in filovirus pathogenicity.

Generation and Characterization of Anti-Filovirus Nucleoprotein Monoclonal Antibodies.

Filoviruses cause lethal hemorrhagic fever in humans. The filovirus nucleoprotein (NP) is expressed in high abundance in infected cells and is essential for virus replication. To generate anti-filovirus monoclonal antibodies (mAbs) against the NP, mice were immunized with peptides known as B-cell epitopes corresponding to different filovirus NPs, and hybridomas were screened using FLAG-tagged filovirus NP constructs. Numerous mAbs were identified, isotyped, and characterized. The anti-NP mAbs demonstrated different ranges of binding affinities to various filovirus NPs. Most of the clones specifically detected both recombinant and wild-type NPs from different filoviruses, including Ebola (EBOV), Sudan (SUDV), Bundibugyo (BDBV), Marburg (MARV), Tai Forest (TAFV), and Reston (RESTV) viruses in western blot analysis. The mAbs were also able to detect native NPs within the cytoplasm of infected cells by immunofluorescence confocal microscopy. Thus, this panel of mAbs represents an important set of tools that may be potentially useful for diagnosing filovirus infection, characterizing virus replication, and detecting NP⁻host protein interactions.

Characterization of Reston virus infection in ferrets.

Among the five currently recognized type viruses within the genus Ebolavirus, Reston virus (RESTV) is not known to cause disease in humans, although asymptomatic infections have been confirmed in the past. Intriguingly, despite the absence of pathogenicity in humans, RESTV is highly lethal to nonhuman primates and has been isolated from domestic pigs co-infected with other viruses in the Philippines and China. Whether infection in these animals can support the eventual emergence of a human-pathogenic RESTV remains unclear and requires further investigation. Unfortunately, there is currently no lethal small animal model available to investigate RESTV pathogenicity or pan-ebolavirus therapeutics. Here we show that wild type RESTV is uniformly lethal in ferrets. In this study, ferrets were challenged with 1260 TCID50 of wild type RESTV either intramuscularly or intranasally and monitored for clinical signs, survival, virus replication, alteration in serum biochemistry and blood cell counts. Irrespective of the route of challenge, viremia occurred in all ferrets on day 5 post-infection, and all animals succumbed to infection between days 9 and 11. Additionally, several similarities were observed between this model and the other ferret models of filovirus infection, including substantial decreases in lymphocyte and platelet counts and abnormalities in serum biochemistry indicating hepatic injury. The ferret model represents the first uniformly lethal model for RESTV infection, and it will undoubtedly prove useful for evaluating virus pathogenicity as well as pan-ebolavirus countermeasures.

Development, validation and clinical evaluation of a broad-range pan-filovirus RT-qPCR.

BACKGROUND: During the five decades since their discovery, filoviruses of four species have caused human hemorrhagic fever outbreaks: Marburg (MARV) marburgvirus, and Zaire (EBOV), Sudan (SUDV) and Bundybugyo (BDBV) ebolaviruses. The largest, devastating EBOV epidemic in West Africa in 2014-16, has been followed by outbreaks of MARV in Uganda, 2017, and EBOV in Democratic Republic of Congo, 2018, emphasizing the need to develop preparedness to diagnose all filoviruses. OBJECTIVES: The aim of this study was to optimize a new filovirus RT-qPCR to detect all filoviruses, define its limits of detection (LOD) and perform a field evaluation with outbreak samples. STUDY DESIGN: A pan-filovirus RT-qPCR targeting the L gene was developed and evaluated within the EbolaMoDRAD (Ebola virus: modern approaches for developing bedside rapid diagnostics) project. Specificity and sensitivity were determined and the effect of inactivation and PCR reagents (liquid and lyophilized format) were tested. RESULTS: The LODs for the lyophilized pan-filovirus L-RT-qPCR assay were 9.4 copies per PCR reaction for EBOV, 9.9 for MARV, 1151 for SUDV, 65 for BDBV and 289 for Taï Forest virus. The test was set at the Pasteur Institute, Dakar, Senegal, and 83 Ebola patient samples, with viral load ranging from 5 to 5 million copies of EBOV per reaction, were screened. The results for the patient samples were in 100% concordance with the reference EBOV-specific assay. DISCUSSION: Overall, the assay showed good sensitivity and specificity, covered all filoviruses known to be human pathogens, performed well both in lyophilized and liquid-phase formats and with EBOV outbreak clinical samples.

Post-exposure immunotherapy for two ebolaviruses and Marburg virus in nonhuman primates.

The 2013-2016 Ebola virus (EBOV) disease epidemic demonstrated the grave consequences of filovirus epidemics in the absence of effective therapeutics. Besides EBOV, two additional ebolaviruses, Sudan (SUDV) and Bundibugyo (BDBV) viruses, as well as multiple variants of Marburg virus (MARV), have also caused high fatality epidemics. Current experimental EBOV monoclonal antibodies (mAbs) are ineffective against SUDV, BDBV, or MARV. Here, we report that a cocktail of two broadly neutralizing ebolavirus mAbs, FVM04 and CA45, protects nonhuman primates (NHPs) against EBOV and SUDV infection when delivered four days post infection. This cocktail when supplemented by the anti-MARV mAb MR191 exhibited 100% efficacy in MARV-infected NHPs. These findings provide a solid foundation for clinical development of broadly protective immunotherapeutics for use in future filovirus epidemics.

A Two-Antibody Pan-Ebolavirus Cocktail Confers Broad Therapeutic Protection in Ferrets and Nonhuman Primates.

Recent and ongoing outbreaks of Ebola virus disease (EVD) underscore the unpredictable nature of ebolavirus reemergence and the urgent need for antiviral treatments. Unfortunately, available experimental vaccines and immunotherapeutics are specific for a single member of the Ebolavirus genus, Ebola virus (EBOV), and ineffective against other ebolaviruses associated with EVD, including Sudan virus (SUDV) and Bundibugyo virus (BDBV). Here we show that MBP134AF, a pan-ebolavirus therapeutic comprising two broadly neutralizing human antibodies (bNAbs), affords unprecedented effectiveness and potency as a therapeutic countermeasure to antigenically diverse ebolaviruses. MBP134AF could fully protect ferrets against lethal EBOV, SUDV, and BDBV infection, and a single 25-mg/kg dose was sufficient to protect NHPs against all three viruses. The development of MBP134AF provides a successful model for the rapid discovery and translational advancement of immunotherapeutics targeting emerging infectious diseases.

Small animal models of filovirus disease: recent advances and future directions.

INTRODUCTION: Small animal models have played a critical role in understanding the pathogenesis and transmission of disease caused by filoviruses. Notably, small animals have served to identify and validate many different approaches to countering infection with these highly pathogenic viruses. Nonetheless, predictive efficacy between each model does not appear to be equivalent as higher order animals seem to be more prognostic and therefore successful in the evaluation of medical countermeasures (MCM). Areas covered: This review comprehensively details the available small animal models of filovirus infection and discusses the benefits and shortcomings of each model with respect to the development of MCM. An up-to-date evaluation of mouse, hamster, guinea pig, and ferret models is provided. Expert opinion: The recent development of the domestic ferret model for ebolavirus offers a small animal model that faithfully reproduces most features of human disease without the need for viral adaptation or an immunocompromised host. That being said, choosing a small animal model to evaluate a particular MCM must consider potential confounders associated with each model. These confounding issues include incomplete host immune systems or mutations in the challenge virus that enables the disease.

[Innate immunity evasion mechanisms of filoviruses]. / Mécanismes d'échappement des filovirus à l'immunité innée.

Ebola virus is an important pathogen that emerged in Central Africa where it was responsible of numerous outbreaks of haemorrhagic fevers associated with a extremely high mortality rate (up to 90%). The filovirus pathogenicity is related to an inappropriate antiviral response. Indeed, this family of viruses has developed evasion strategies from early innate immunity mechanisms. As a result, a massive viral replication induces an unsuitable immune response causing an acute inflammatory reaction associated with the haemorrhagic syndrome. In this review, we describe the mechanisms adopted by filoviruses like Ebola virus to escape innate immunity response.

Postexposure Protective Efficacy of T-705 (Favipiravir) Against Sudan Virus Infection in Guinea Pigs.

Filoviruses such as Ebola virus (EBOV), Marburg virus (MARV), and Sudan virus (SUDV) cause deadly viral hemorrhagic fever in humans, with high case-fatality rates; however, no licensed therapeutic agent or vaccine has been clinically approved to treat or prevent infection. T-705 (favipiravir) is a novel antiviral drug that has been approved for the treatment of influenza in Japan. T-705 exhibits broad-spectrum antiviral activity against different viruses, including MARV and EBOV, and here, we are the first to report the in vitro and in vivo antiviral activity of T-705 against SUDV. T-705 treatment reduced SUDV replication in Vero E6 cells. Subcutaneous administration of T-705, beginning 1-4 days after infection and continuing for 7 days, significantly protected SUDV-infected guinea pigs, with a survival rate of 83%-100%. Viral RNA replication and infectious virus production were also significantly reduced in the blood, spleen, liver, lungs, and kidney. Moreover, early administration of low-dose T-705 and late administration (at 5 days after infection) of higher-dose T-705 also showed partial protection. Overall, our study is the first to demonstrate the antiviral activity of T-705 against SUDV, suggesting that T-705 may be a potential drug candidate for use during outbreaks.

Suspected Exposure to Filoviruses Among People Contacting Wildlife in Southwestern Uganda.

Background: Human and filovirus host interactions remain poorly understood in areas where Ebola hemorrhagic fever outbreaks are likely to occur. In the Bwindi region of Uganda, a hot spot of mammalian biodiversity in Africa, human livelihoods are intimately connected with wildlife, creating potential for exposure to filoviruses. Methods: We tested samples from 331 febrile patients presenting to healthcare facilities near Bwindi Impenetrable Forest, Uganda, by polymerase chain reaction (PCR) analysis and Western blot, using recombinant glycoprotein antigens for Ebola virus (EBOV), Sudan virus (SUDV), Bundibugyo virus (BDBV), and Marburg virus. Behavioral data on contact with wildlife were collected to examine risk factors for filovirus seropositivity. Results: All patients were negative for active filovirus infection, by PCR analysis. However, patients were seroreactive to SUDV (4.7%), EBOV (5.3%), and BDBV (8.9%), indicating previous exposure. Touching duikers was the most significant risk factor associated with EBOV seropositivity, while hunting primates and touching and/or eating cane rats were significant risk factors for SUDV seropositivity. Conclusions: People in southwestern Uganda have suspected previous exposure to filoviruses, particularly those with a history of wildlife contact. Circulation of filoviruses in wild animals and subsequent spillover into humans could be more common than previously reported.

Seroprevalence of Filovirus Infection of Rousettus aegyptiacus Bats in Zambia.

Bats are suspected to play important roles in the ecology of filoviruses, including ebolaviruses and marburgviruses. A cave-dwelling fruit bat, Rousettus aegyptiacus, has been shown to be a reservoir of marburgviruses. Using an enzyme-linked immunosorbent assay with the viral glycoprotein antigen, we detected immunoglobulin G antibodies specific to multiple filoviruses in 158 of 290 serum samples of R aegyptiacus bats captured in Zambia during the years 2014-2017. In particular, 43.8% of the bats were seropositive to marburgvirus, supporting the notion that this bat species continuously maintains marburgviruses as a reservoir. Of note, distinct peaks of seropositive rates were repeatedly observed at the beginning of rainy seasons, suggesting seasonality of the presence of newly infected individuals in this bat population. These data highlight the need for continued monitoring of filovirus infection in this bat species even in countries where filovirus diseases have not been reported.