The Egyptian Rousette Genome Reveals Unexpected Features of Bat Antiviral Immunity.

Bats harbor many viruses asymptomatically, including several notorious for causing extreme virulence in humans. To identify differences between antiviral mechanisms in humans and bats, we sequenced, assembled, and analyzed the genome of Rousettus aegyptiacus, a natural reservoir of Marburg virus and the only known reservoir for any filovirus. We found an expanded and diversified KLRC/KLRD family of natural killer cell receptors, MHC class I genes, and type I interferons, which dramatically differ from their functional counterparts in other mammals. Such concerted evolution of key components of bat immunity is strongly suggestive of novel modes of antiviral defense. An evaluation of the theoretical function of these genes suggests that an inhibitory immune state may exist in bats. Based on our findings, we hypothesize that tolerance of viral infection, rather than enhanced potency of antiviral defenses, may be a key mechanism by which bats asymptomatically host viruses that are pathogenic in humans.

Ebola Virus Glycoprotein with Increased Infectivity Dominated the 2013-2016 Epidemic.

The magnitude of the 2013-2016 Ebola virus disease (EVD) epidemic enabled an unprecedented number of viral mutations to occur over successive human-to-human transmission events, increasing the probability that adaptation to the human host occurred during the outbreak. We investigated one nonsynonymous mutation, Ebola virus (EBOV) glycoprotein (GP) mutant A82V, for its effect on viral infectivity. This mutation, located at the NPC1-binding site on EBOV GP, occurred early in the 2013-2016 outbreak and rose to high frequency. We found that GP-A82V had heightened ability to infect primate cells, including human dendritic cells. The increased infectivity was restricted to cells that have primate-specific NPC1 sequences at the EBOV interface, suggesting that this mutation was indeed an adaptation to the human host. GP-A82V was associated with increased mortality, consistent with the hypothesis that the heightened intrinsic infectivity of GP-A82V contributed to disease severity during the EVD epidemic.

Isolation, characterization, and circulation sphere of a filovirus in fruit bats.

Bats are associated with the circulation of most mammalian filoviruses (FiVs), with pathogenic ones frequently causing deadly hemorrhagic fevers in Africa. Divergent FiVs have been uncovered in Chinese bats, raising concerns about their threat to public health. Here, we describe a long-term surveillance to track bat FiVs at orchards, eventually resulting in the identification and isolation of a FiV, Dehong virus (DEHV), from Rousettus leschenaultii bats. DEHV has a typical filovirus-like morphology with a wide spectrum of cell tropism. Its entry into cells depends on the engagement of Niemann-Pick C1, and its replication is inhibited by remdesivir. DEHV has the largest genome size of filoviruses, with phylogenetic analysis placing it between the genera Dianlovirus and Orthomarburgvirus, suggesting its classification as the prototype of a new genus within the family Filoviridae. The continuous detection of viral RNA in the serological survey, together with the wide host distribution, has revealed that the region covering southern Yunnan, China, and bordering areas is a natural circulation sphere for bat FiVs. These emphasize the need for a better understanding of the pathogenicity and potential risk of FiVs in the region.

The Ebola Virus Nucleoprotein Recruits the Host PP2A-B56 Phosphatase to Activate Transcriptional Support Activity of VP30.

Transcription of the Ebola virus genome depends on the viral transcription factor VP30 in its unphosphorylated form, but the underlying molecular mechanism of VP30 dephosphorylation is unknown. Here we show that the Ebola virus nucleoprotein (NP) recruits the host PP2A-B56 protein phosphatase through a B56-binding LxxIxE motif and that this motif is essential for VP30 dephosphorylation and viral transcription. The LxxIxE motif and the binding site of VP30 in NP are in close proximity, and both binding sites are required for the dephosphorylation of VP30. We generate a specific inhibitor of PP2A-B56 and show that it suppresses Ebola virus transcription and infection. This work dissects the molecular mechanism of VP30 dephosphorylation by PP2A-B56, and it pinpoints this phosphatase as a potential target for therapeutic intervention.

Chaperone-assisted selective autophagy targets filovirus VP40 as a client and restricts egress of virus particles.

The filovirus VP40 protein directs virion egress, which is regulated either positively or negatively by select VP40-host interactions. We demonstrate that host BAG3 and HSP70 recognize VP40 as a client and inhibit the egress of VP40 virus-like particles (VLPs) by promoting degradation of VP40 via Chaperone-assisted selective autophagy (CASA). Pharmacological inhibition of either the early stage formation of the VP40/BAG3/HSP70 tripartite complex, or late stage formation of autolysosomes, rescued VP40 VLP egress back to WT levels. The mechanistic target of rapamycin complex 1 (mTORC1) is a master regulator of autophagy, and we found that surface expression of EBOV GP on either VLPs or an infectious VSV recombinant virus, activated mTORC1. Notably, pharmacological suppression of mTORC1 signaling by rapamycin activated CASA in a BAG3-dependent manner to restrict the egress of both VLPs and infectious EBOV in Huh7 cells. In sum, our findings highlight the involvement of the mTORC1/CASA axis in regulating filovirus egress.

Delayed treatment of cynomolgus macaques with a FVM04/CA45 monoclonal antibody cocktail provides complete protection against lethal Sudan virus infection.

Sudan ebolavirus (SUDV) is a member of the genus Ebolavirus (Family Filoviridae) and has caused sporadic outbreaks of Ebola disease (EBOD), or more specifically Sudan virus disease (SVD), with high mortality rates in Africa. Current vaccines and therapies that have been developed for filoviruses are almost all specific for Ebola virus (EBOV; of the species Zaire ebolavirus), and there is a current lack of therapeutics specific for SUDV. The recent SUDV outbreak in Uganda, which was distributed across multiple districts, including Kampala, a densely populated urban center, highlights the critical need for the development of novel SUDV-specific or pan-Ebola virus therapeutics. Previous work has characterized two monoclonal antibodies, FVM04 and CA45, which have neutralization capabilities against both EBOV and SUDV and have shown protective efficacy in animal challenge studies. Here, we expand upon this work, showing that treatment with a monoclonal antibody cocktail consisting of FVM04 and CA45 provides full protection against lethal SUDV infection in cynomolgus macaques. Studies that evaluate outcomes at late time points after infection, once clinical signs of illness are apparent, are vital for assessing the therapeutic efficacy of antibody therapeutics. We have shown that when treatment is initiated as late as 5 days after infection, with a second dose given on day 8, that treated groups showed few clinical signs or morbidity, with complete survival. This work provides further evidence that FVM04 and CA45 have strong therapeutic potential against SUDV and their development as a pan-Ebola virus therapeutic should be pursued. IMPORTANCE: There are currently no approved vaccines or therapeutics for Sudan virus, a filovirus which is highly related to Ebola virus and causes similar disease and outbreaks. In this study, a cocktail of two potent monoclonal antibodies that effectively neutralize Sudan virus was tested in a nonhuman primate model of Sudan virus disease. Treatment was highly effective, even when initiated as late as 5 days after infection, when clinical signs of infection were already evident. All treated animals showed complete recovery from infection, with little evidence of disease, while all animals that received a control treatment succumbed to infection within 8 days. The study further demonstrated the strong therapeutic potential of the antibody treatment and supported further development for use in Sudan virus outbreaks.

Macaque antibodies targeting Marburg virus glycoprotein induced by multivalent immunization.

Marburg virus infection in humans is associated with case fatality rates that can reach up to 90%, but to date, there are no approved vaccines or monoclonal antibody (mAb) countermeasures. Here, we immunized Rhesus macaques with multivalent combinations of filovirus glycoprotein (GP) antigens belonging to Marburg, Sudan, and Ebola viruses to generate monospecific and cross-reactive antibody responses against them. From the animal that developed the highest titers of Marburg virus GP-specific neutralizing antibodies, we sorted single memory B cells using a heterologous Ravn virus GP probe and cloned and characterized a panel of 34 mAbs belonging to 28 unique lineages. Antibody specificities were assessed by overlapping pepscan and binding competition analyses, revealing that roughly a third of the lineages mapped to the conserved receptor binding region, including potent neutralizing lineages that were confirmed by negative stain electron microscopy to target this region. Additional lineages targeted a protective region on GP2, while others were found to possess cross-filovirus reactivity. Our study advances the understanding of orthomarburgvirus glycoprotein antigenicity and furthers efforts to develop candidate antibody countermeasures against these lethal viruses. IMPORTANCE: Marburg viruses were the first filoviruses characterized to emerge in humans in 1967 and cause severe hemorrhagic fever with average case fatality rates of ~50%. Although mAb countermeasures have been approved for clinical use against the related Ebola viruses, there are currently no approved countermeasures against Marburg viruses. We successfully isolated a panel of orthomarburgvirus GP-specific mAbs from a macaque immunized with a multivalent combination of filovirus antigens. Our analyses revealed that roughly half of the antibodies in the panel mapped to regions on the glycoprotein shown to protect from infection, including the host cell receptor binding domain and a protective region on the membrane-anchoring subunit. Other antibodies in the panel exhibited broad filovirus GP recognition. Our study describes the discovery of a diverse panel of cross-reactive macaque antibodies targeting orthomarburgvirus and other filovirus GPs and provides candidate immunotherapeutics for further study and development.

A bivalent Adenovirus-Vectored Vaccine induces a robust humoral response, but does not protect cynomolgus macaques against a lethal challenge with Sudan virus.

The most recent Sudan virus (SUDV) outbreak in Uganda was first detected in September 2022 and resulted in 164 laboratory-confirmed cases and 77 deaths. There are no approved vaccines against SUDV. Here, we investigated the protective efficacy of ChAdOx1-biEBOV in cynomolgus macaques using a prime or a prime-boost regimen. ChAdOx1-biEBOV is a replication-deficient simian adenovirus vector encoding SUDV and Ebola virus (EBOV) glycoproteins (GPs). Intramuscular vaccination induced SUDV and EBOV GP-specific IgG responses and neutralizing antibodies. Upon challenge with SUDV, vaccinated animals showed signs of disease like those observed in control animals, and no difference in survival outcomes were measured among all three groups. Viral load in blood samples and in tissue samples obtained after necropsy were not significantly different between groups. Overall, this study highlights the importance of evaluating vaccines in multiple animal models and demonstrates the importance of understanding protective efficacy in both animal models and human hosts.

ICTV Virus Taxonomy Profile: Filoviridae 2024.

Filoviridae is a family of negative-sense RNA viruses with genomes of about 13.1-20.9 kb that infect fish, mammals and reptiles. The filovirid genome is a linear, non-segmented RNA with five canonical open reading frames (ORFs) that encode a nucleoprotein (NP), a polymerase cofactor (VP35), a glycoprotein (GP1,2), a transcriptional activator (VP30) and a large protein (L) containing an RNA-directed RNA polymerase (RdRP) domain. All filovirid genomes encode additional proteins that vary among genera. Several filovirids (e.g., Ebola virus, Marburg virus) are pathogenic for humans and highly virulent. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Filoviridae, which is available at www.ictv.global/report/filoviridae.

Characterization of Ebola Virus Mucosal Challenge Routes in Cynomolgus Macaques.

Zaïre ebolavirus (EBOV) causes Ebola virus disease (EVD), a devastating viral hemorrhagic fever in humans. Nonhuman primate (NHP) models of EVD traditionally use intramuscular infection with higher case fatality rates and reduced mean time-to-death compared to contact transmission typical of human cases of EVD. A cynomolgus macaque model of oral and conjunctival EBOV was used to further characterize the more clinically relevant contact transmission of EVD. NHPs challenged via the oral route had an overall 50% survival rate. NHPs challenged with a target dose of 1 × 102 PFU or 1 × 104 PFU of EBOV via the conjunctival route had 40% and 100% mortality, respectively. Classic signs of lethal EVD-like disease were observed in all NHPs that succumbed to EBOV infection including viremia, hematological abnormalities, clinical chemistries indicative of hepatic and renal disease, and histopathological findings. Evidence of EBOV viral persistence in the eye was observed in NHPs challenged via the conjunctival route. IMPORTANCE This study is the first to examine the Kikwit strain of EBOV, the most commonly used strain, in the gold-standard macaque model of infection. Additionally, this is the first description of the detection of virus in the vitreous fluid, an immune privileged site that has been proposed as a viral reservoir, following conjunctival challenge. The oral and conjunctival macaque challenge model of EVD described here more faithfully recapitulates the prodrome that has been reported for human EVD. This work paves the way for more advanced studies to model contact transmission of EVD, including early events in mucosal infection and immunity, as well as the establishment of persistent viral infection and the emergence from these reservoirs.

Sudan virus disease - A quick review.

The recent Sudan virus disease (SVD) outbreak in Uganda is a reminder of threat from Filovirus diseases. Unlike Ebola virus disease, no effective antiviral and vaccine is available for SVD. The outbreak was declared over after 115 days, with 142 confirmed cases and case fatality rate of 39%, before any dose of candidate vaccine could be used on contacts. We provide a quick review of up-to-date information on the Uganda outbreak, summary of previous outbreaks, and detail the existing SVD treatment and vaccine candidates. Evolution of disease attributes and the impact on public health were also discussed. For high consequence infectious disease like SVD, it takes international collaboration to be better prepared for the next outbreak.

A Single Case Observation: Is the Ebola Virus Soluble Glycoprotein an Indicator of Viral Recrudescence?

This case study investigated the long-term expression dynamics of Ebola virus (EBOV) soluble glycoprotein (sGP) in the serum of a patient who was infected with EBOV in West Africa and recovered from acute Ebola virus disease (EVD) at the National Institutes of Health Clinical Center in Bethesda, Maryland. Samples from this patient were collected during acute EVD and during convalescence up to day 361 following illness onset. Although blood samples were negative by reverse transcription-quantitative polymerase chain reaction after recovery from acute EVD, we detected small amounts of EBOV sGP in the serum of the patient long after recovery, potentially indicating viral recrudescence. As this was only observed in a single patient, additional longitudinal patient samples are needed to confirm our hypothesis that EBOV sGP may be an indicator of viral recrudescence long after recovery from acute EVD.

Atypical Ebola Virus Disease in a Rhesus Macaque.

Ebola virus (EBOV)-Makona infected more than 30 000 people from 2013 to 2016 in West Africa, among them many health care workers including foreign nationals. Most of the infected foreign nationals were evacuated and treated in their respective home countries, resulting in detailed reports of the acute disease following EBOV infection as well as descriptions of symptoms now known as post-Ebola syndrome, which occurred months after the infection. Symptoms associated with this syndrome include uveitis and neurological manifestations. In 1 of our EBOV-Makona nonhuman primate (NHP) studies, 1 NHP was euthanized on day 28 after infection having completely recovered from the acute disease. During convalescence, this NHP developed neurological signs and acute respiratory distress requiring euthanasia. The organ tropism had changed with high virus titers in lungs, brain, eye, and reproductive organs but no virus in the typical target organs for acute EBOV infection. This in part reflects sequelae described for EBOV survivors albeit developing quicker after recovery from acute disease.

Clinical and Immunologic Correlates of Vasodilatory Shock Among Ebola Virus-Infected Nonhuman Primates in a Critical Care Model.

BACKGROUND: Existing models of Ebola virus infection have not fully characterized the pathophysiology of shock in connection with daily virologic, clinical, and immunologic parameters. We implemented a nonhuman primate critical care model to investigate these associations. METHODS: Two rhesus macaques received a target dose of 1000 plaque-forming units of Ebola virus intramuscularly with supportive care initiated on day 3. High-dimensional spectral cytometry was used to phenotype neutrophils and peripheral blood mononuclear cells daily. RESULTS: We observed progressive vasodilatory shock with preserved cardiac function following viremia onset on day 5. Multiorgan dysfunction began on day 6 coincident with the nadir of circulating neutrophils. Consumptive coagulopathy and anemia occurred on days 7 to 8 along with irreversible shock, followed by death. The monocyte repertoire began shifting on day 4 with a decline in classical and expansion of double-negative monocytes. A selective loss of CXCR3-positive B and T cells, expansion of naive B cells, and activation of natural killer cells followed viremia onset. CONCLUSIONS: Our model allows for high-fidelity characterization of the pathophysiology of acute Ebola virus infection with host innate and adaptive immune responses, which may advance host-targeted therapy design and evaluation for use after the onset of multiorgan failure.

Single-Dose Treatment With Vesicular Stomatitis Virus-Based Ebola Virus Vaccine Expressing Ebola Virus-Specific Artificial Micro-RNA Does Not Protect Mice From Lethal Disease.

Although significant progress has been made in the development of therapeutics against Ebola virus (EBOV), we sought to expand upon existing strategies and combine an RNA interference-based intervention with the approved vesicular stomatitis virus-based Ebola virus (VSV-EBOV) vaccine to conjointly treat and vaccinate patients during an outbreak. We constructed VSV-EBOV vectors expressing artificial micro-RNAs (amiRNAs) targeting sequences of EBOV proteins. In vitro experiments demonstrated a robust decrease in EBOV replication using a minigenome system and infectious virus. For in vivo evaluation, mouse-adapted EBOV-infected CD-1 mice were treated 24 hours after infection with a single dose of the VSV-EBOV amiRNA constructs. We observed no difference in disease progression or survival compared to the control-treated mice. In summary, while amiRNAs decrease viral replication in vitro, the effect is not sufficient to protect mice from lethal disease, and this therapeutic approach requires further optimization.

Pathogenicity of Lloviu and Bombali Viruses in Type I Interferon Receptor Knockout Mice.

Type I interferon receptor knockout (IFNAR-/-) mice are not able to generate a complete innate immune response; therefore, these mice are often considered to assess the pathogenicity of emerging viruses. We infected IFNAR-/- mice with a low or high dose of Lloviu virus (LLOV) or Bombali virus (BOMV) by the intranasal (IN) or intraperitoneal (IP) route and compared virus loads at early and late time points after infection. No signs of disease and no viral RNA were detected after IN infection regardless of LLOV dose. In contrast, IP infections resulted in increased viral loads in the high-dose LLOV and BOMV groups at the early time point. The low-dose LLOV and BOMV groups achieved higher viral loads at the late time point. However, there was 100% survival in all groups and no signs of disease. In conclusion, our results indicate a limited value of the IFNAR-/- mouse model for investigation of the pathogenicity of LLOV and BOMV.

Establishing a Mouse Model for Sexual Transmission and Male Reproductive Tract Persistence of Ebola Virus.

Ebola virus disease (EVD) has resulted in the death of over 15 000 people since its discovery in 1976. At least 1 incident of re-emergence of EVD has been associated with persistent male reproductive tract infection in a patient surviving EVD greater than 500 days prior. To date, animal models of Ebola virus (EBOV) infection have failed to fully characterize the pathogenesis of reproductive tract infection. Furthermore, no animal model of sexual transmission of EBOV exists. In this study, we describe a roadmap to modeling sexual transmission of EBOV using a mouse-adapted EBOV isolate in immunocompetent male mice and female Ifnar-/- mice.

The Multiple Origins of Ebola Disease Outbreaks.

BACKGROUND: The origins of Ebola disease outbreaks remain enigmatic. Historically outbreaks have been attributed to spillover events from wildlife. However, recent data suggest that some outbreaks may originate from human-to-human transmission of prior outbreak strains instead of spillover. Clarifying the origins of Ebola disease outbreaks could improve detection and mitigation of future outbreaks. METHODS: We reviewed the origins of all Ebola disease outbreaks from 1976 to 2022 to analyze the earliest cases and characteristics of each outbreak. The epidemiology and phylogenetic relationships of outbreak strains were used to further identify the likely source of each outbreak. RESULTS: From 1976 to 2022 there were 35 Ebola disease outbreaks with 48 primary/index cases. While the majority of outbreaks were associated with wildlife spillover, resurgence of human-to-human transmission could account for roughly a quarter of outbreaks caused by Ebola virus. Larger outbreaks were more likely to lead to possible resurgence, and nosocomial transmission was associated with the majority of outbreaks. CONCLUSIONS: While spillover from wildlife has been a source for many Ebola disease outbreaks, multiple outbreaks may have originated from flare-ups of prior outbreak strains. Improving access to diagnostics as well as identifying groups at risk for resurgence of ebolaviruses will be crucial to preventing future outbreaks.

Identifying Paucisymptomatic or Asymptomatic and Unrecognized Ebola Virus Disease Among Close Contacts Based on Exposure Risk Assessments and Screening Algorithms.

BACKGROUND: There is limited evidence to evaluate screening algorithms with rapid antigen testing and exposure assessments as identification strategies for paucisymptomatic or asymptomatic Ebola virus (EBOV) infection and unrecognized EBOV disease (EVD). METHODS: We used serostatus and self-reported postexposure symptoms from a cohort study to classify contact-participants as having no infection, paucisymptomatic or asymptomatic infection, or unrecognized EVD. Exposure risk was categorized as low, intermediate, or high. We created hypothetical scenarios to evaluate the World Health Organization (WHO) case definition with or without rapid diagnostic testing (RDT) or exposure assessments. RESULTS: This analysis included 990 EVD survivors and 1909 contacts, of whom 115 (6%) had paucisymptomatic or asymptomatic EBOV infection, 107 (6%) had unrecognized EVD, and 1687 (88%) were uninfected. High-risk exposures were drivers of unrecognized EVD (adjusted odds ratio, 3.5 [95% confidence interval, 2.4-4.9]). To identify contacts with unrecognized EVD who test negative by the WHO case definition, the sensitivity was 96% with RDT (95% confidence interval, 91%-99%), 87% with high-risk exposure (82%-92%), and 97% with intermediate- to high-risk exposures (93%-99%). The proportion of false-positives was 2% with RDT and 53%-93% with intermediate- and/or high-risk exposures. CONCLUSION: We demonstrated the utility and trade-offs of sequential screening algorithms with RDT or exposure risk assessments as identification strategies for contacts with unrecognized EVD.

Fluorescent and Bioluminescent Reporter Mouse-Adapted Ebola Viruses Maintain Pathogenicity and Can Be Visualized in Vivo.

Ebola virus (EBOV) causes lethal disease in humans but not in mice. Here, we generated recombinant mouse-adapted (MA) EBOVs, including 1 based on the previously reported serially adapted strain (rMA-EBOV), along with single-reporter rMA-EBOVs expressing either fluorescent (ZsGreen1 [ZsG]) or bioluminescent (nano-luciferase [nLuc]) reporters, and dual-reporter rMA-EBOVs expressing both ZsG and nLuc. No detriment to viral growth in vitro was seen with inclusion of MA-associated mutations or reporter proteins. In CD-1 mice, infection with MA-EBOV, rMA-EBOV, and single-reporter rMA-EBOVs conferred 100% lethality; infection with dual-reporter rMA-EBOV resulted in 73% lethality. Bioluminescent signal from rMA-EBOV expressing nLuc was detected in vivo and ex vivo using the IVIS Spectrum CT. Fluorescent signal from rMA-EBOV expressing ZsG was detected in situ using handheld blue-light transillumination and ex vivo through epi-illumination with the IVIS Spectrum CT. These data support the use of reporter MA-EBOV for studies of Ebola virus in animal disease models.