A Limited Structural Modification Results in a Significantly More Efficacious Diazachrysene-Based Filovirus Inhibitor

Ebola (EBOV) and Marburg (MARV) filoviruses are highly infectious pathogens causing deadly hemorrhagic fever in humans and non-human primates. Promising vaccine candidates providing immunity against filoviruses have been reported. However, the sporadic nature and swift progression of filovirus disease underlines the need for the development of small molecule therapeutics providing immediate antiviral effects. Herein we describe a brief structural exploration of two previously reported diazachrysene (DAAC)-based EBOV inhibitors. Specifically, three analogs were prepared to examine how slight substituent modifications would affect inhibitory efficacy and inhibitor-mediated toxicity during not only EBOV, but also MARV cellular infection. Of the three analogs, one was highly efficacious, providing IC₅₀ values of 0.696 µM ± 0.13 µM and 2.76 µM ± 0.21 µM against EBOV and MARV infection, respectively, with little or no associated cellular toxicity. Overall, the structure-activity and structure-toxicity results from this study provide a framework for the future development of DAAC-based filovirus inhibitors that will be both active and non-toxic in vivo.     

Niemann-Pick C1 (NPC1)/NPC1-like1 Chimeras Define Sequences Critical for NPC1’s Function as a Filovirus Entry Receptor

We recently demonstrated that Niemann-Pick C1 (NPC1), a ubiquitous 13-pass cellular membrane protein involved in lysosomal cholesterol transport, is a critical entry receptor for filoviruses. Here we show that Niemann-Pick C1-like1 (NPC1L1), an NPC1 paralog and hepatitis C virus entry factor, lacks filovirus receptor activity. We exploited the structural similarity between NPC1 and NPC1L1 to construct and analyze a panel of chimeras in which NPC1L1 sequences were replaced with cognate sequences from NPC1. Only one chimera, NPC1L1 containing the second luminal domain (C) of NPC1 in place of its own, bound to the viral glycoprotein, GP. This engineered protein mediated authentic filovirus infection nearly as well as wild-type NPC1, and more efficiently than did a minimal NPC1 domain C-based receptor recently described by us. A reciprocal chimera, NPC1 containing NPC1L1’s domain C, was completely inactive. Remarkably, an intra-domain NPC1L1-NPC1 chimera bearing only a ~130-amino acid N–terminal region of NPC1 domain C could confer substantial viral receptor activity on NPC1L1. Taken together, these findings account for the failure of NPC1L1 to serve as a filovirus receptor, highlight the central role of the luminal domain C of NPC1 in filovirus entry, and reveal the direct involvement of N–terminal domain C sequences in NPC1’s function as a filovirus receptor.     

Host Cell Factors in Filovirus Entry: Novel Players,New Insights

Filoviruses cause severe hemorrhagic fever in humans with high case-fatality rates. The cellular factors exploited by filoviruses for their spread constitute potential targets for intervention, but are incompletely defined. The viral glycoprotein (GP) mediates filovirus entry into host cells. Recent studies revealed important insights into the host cell molecules engaged by GP for cellular entry. The binding of GP to cellular lectins was found to concentrate virions onto susceptible cells and might contribute to the early and sustained infection of macrophages and dendritic cells, important viral targets. Tyrosine kinase receptors were shown to promote macropinocytic uptake of filoviruses into a subset of susceptible cells without binding to GP, while interactions between GP and human T cell Ig mucin 1 (TIM-1) might contribute to filovirus infection of mucosal epithelial cells. Moreover, GP engagement of the cholesterol transporter Niemann-Pick C1 was demonstrated to be essential for GP-mediated fusion of the viral envelope with a host cell membrane. Finally, mutagenic and structural analyses defined GP domains which interact with these host cell factors. Here, we will review the recent progress in elucidating the molecular interactions underlying filovirus entry and discuss their implications for our understanding of the viral cell tropism.     

Nomenclature- and Database-Compatible Names for the Two Ebola Virus Variants that Emerged in Guinea and the Democratic Republic of the Congo in 2014

In 2014, Ebola virus (EBOV) was identified as the etiological agent of a large and still expanding outbreak of Ebola virus disease (EVD) in West Africa and a much more confined EVD outbreak in Middle Africa. Epidemiological and evolutionary analyses confirmed that all cases of both outbreaks are connected to a single introduction each of EBOV into human populations and that both outbreaks are not directly connected. Coding-complete genomic sequence analyses of isolates revealed that the two outbreaks were caused by two novel EBOV variants, and initial clinical observations suggest that neither of them should be considered strains. Here we present consensus decisions on naming for both variants (West Africa: “Makona”, Middle Africa: “Lomela”) and provide database-compatible full, shortened, and abbreviated names that are in line with recently established filovirus sub-species nomenclatures.     

Response to Imported Case of Marburg Hemorrhagic Fever, the Netherlands

On July 10, 2008, Marburg hemorrhagic fever was confirmed in a Dutch patient who had vacationed recently in Uganda. Exposure most likely occurred in the Python Cave (Maramagambo Forest), which harbors bat species that elsewhere in Africa have been found positive for Marburg virus. A multidisciplinary response team was convened to perform a structured risk assessment, perform risk classification of contacts, issue guidelines for follow-up, provide information, and monitor the crisis response. In total, 130 contacts were identified (66 classified as high risk and 64 as low risk) and monitored for 21 days after their last possible exposure. The case raised questions specific to international travel, postexposure prophylaxis for Marburg virus, and laboratory testing of contacts with fever. We present lessons learned and results of the follow-up serosurvey of contacts and focus on factors that prevented overreaction during an event with a high public health impact.     

Viewpoint: Filovirus haemorrhagic fever outbreaks:much ado about nothing?

The recent outbreak of Marburg haemorrhagic fever in the Democratic Republic of Congo has put the filovirus threat back on the international health agenda. This paper gives an overview of Marburg and Ebola outbreaks so far observed and puts them in a public health perspective. Damage on the local level has been devastating at times, but was marginal on the international level despite the considerable media attention these outbreaks received. The potential hazard of outbreaks, however, after export of filovirus from its natural environment into metropolitan areas, is argued to be considerable. Some avenues for future research and intervention are explored. Beyond the obvious need to find the reservoir and study the natural history, public health strategies for a more timely and efficient response are urgently needed.     

Serological Evidence of Filovirus Infection in Nonhuman Primates in Zambia

Ebolaviruses and marburgviruses are filoviruses that are known to cause severe hemorrhagic fever in humans and nonhuman primates (NHPs). While some bat species are suspected to be natural reservoirs of these filoviruses, wild NHPs often act as intermediate hosts for viral transmission to humans. Using an enzyme-linked immunosorbent assay, we screened two NHP species, wild baboons and vervet monkeys captured in Zambia, for their serum IgG antibodies specific to the envelope glycoproteins of filoviruses. From 243 samples tested, 39 NHPs (16%) were found to be seropositive either for ebolaviruses or marburgviruses with endpoint antibody titers ranging from 100 to 25,600. Interestingly, antibodies reactive to Reston virus, which is found only in Asia, were detected in both NHP species. There was a significant difference in the seropositivity for the marburgvirus antigen between the two NHP species, with baboons having a higher positive rate. These results suggest that wild NHPs in Zambia might be nonlethally exposed to these filoviruses, and this emphasizes the need for continuous monitoring of filovirus infection in wild animals to better understand the ecology of filoviruses and to assess potential risks of outbreaks in humans in previously nonendemic countries.     

Cellular and humoral immunity to Ebola Zaire glycoprotein and viral vector proteins following immunization with recombinant vesicular stomatitis virus-based Ebola vaccine (rVSVΔG-ZEBOV-GP)

While effective at preventing Zaire ebolavirus (ZEBOV) disease, cellular immunity to ZEBOV and vector-directed immunity elicited by the recombinant vesicular stomatitis virus expressing ZEBOV glycoprotein (rVSVΔG-ZEBOV-GP) vaccine remain poorly understood. Sera and peripheral blood mononuclear cells were collected from 32 participants enrolled in a prospective multicenter study [ClinicalTrials.gov NCT02788227] before vaccination and up to six months post-vaccination. IgM and IgG antibodies, IgG-producing memory B cells (MBCs), and T cell reactivity to ZEBOV glycoprotein (ZEBOV-GP), vesicular stomatitis virus-Indiana strain (VSV-I) matrix (M) protein, and VSV-I nucleoprotein (NP) were measured using ELISA, ELISpot, and flow cytometry, respectively. 11/32 (34.4%) participants previously received a different investigational ZEBOV vaccine prior to enrollment and 21/32 (65.6%) participants were ZEBOV vaccine naïve. Both ZEBOV vaccine naïve and experienced participants had increased ZEBOV-GP IgG optical densities (ODs) post-rVSVΔG-ZEBOV-GP vaccination while only ZEBOV vaccine naïve participants had increased ZEBOV-GP IgM ODs. Transient IgM and IgG antibody responses to VSV-I M protein and NP were observed in a minority of participants. All participants had detectable ZEBOV-GP specific IgG-producing MBCs by 6 months post-vaccination while no changes were observed in the median IgG-producing MBCs to VSV-I proteins. T cell responses to ZEBOV-GP differed between ZEBOV vaccine experienced and ZEBOV vaccine naïve participants. T cell responses to both VSV-I M protein and VSV-I NP were observed, but were of a low magnitude. The rVSVΔG-ZEBOV-GP vaccine elicits robust humoral and memory B cell responses to ZEBOV glycoprotein in both ZEBOV vaccine naïve and experienced individuals and can generate vector-directed T cell immunity. Further research is needed to understand the significance of pre-existing vector and target antigen immunity on responses to booster doses of rVSVΔG-ZEBOV-GP and other rVSV-vectored vaccines.