Lujo virus (LUJV), a new member of the family Arenaviridae and the first hemorrhagic fever-associated arenavirus from the Old World discovered in three decades, was isolated in South Africa during an ...outbreak of human disease characterized by nosocomial transmission and an unprecedented high case fatality rate of 80% (4/5 cases). Unbiased pyrosequencing of RNA extracts from serum and tissues of outbreak victims enabled identification and detailed phylogenetic characterization within 72 hours of sample receipt. Full genome analyses of LUJV showed it to be unique and branching off the ancestral node of the Old World arenaviruses. The virus G1 glycoprotein sequence was highly diverse and almost equidistant from that of other Old World and New World arenaviruses, consistent with a potential distinctive receptor tropism. LUJV is a novel, genetically distinct, highly pathogenic arenavirus.
GS-5734 is a monophosphate prodrug of an adenosine nucleoside analog that showed therapeutic efficacy in a non-human primate model of Ebola virus infection. It has been administered under ...compassionate use to two Ebola patients, both of whom survived, and is currently in Phase 2 clinical development for treatment of Ebola virus disease. Here we report the antiviral activities of GS-5734 and the parent nucleoside analog across multiple virus families, providing evidence to support new indications for this compound against human viruses of significant public health concern.
The most recent Ebola virus outbreak in West Africa, which was unprecedented in the number of cases and fatalities, geographic distribution, and number of nations affected, highlights the need for ...safe, effective, and readily available antiviral agents for treatment and prevention of acute Ebola virus (EBOV) disease (EVD) or sequelae. No antiviral therapeutics have yet received regulatory approval or demonstrated clinical efficacy. Here we report the discovery of a novel small molecule GS-5734, a monophosphoramidate prodrug of an adenosine analogue, with antiviral activity against EBOV. GS-5734 exhibits antiviral activity against multiple variants of EBOV and other filoviruses in cell-based assays. The pharmacologically active nucleoside triphosphate (NTP) is efficiently formed in multiple human cell types incubated with GS-5734 in vitro, and the NTP acts as an alternative substrate and RNA-chain terminator in primer-extension assays using a surrogate respiratory syncytial virus RNA polymerase. Intravenous administration of GS-5734 to nonhuman primates resulted in persistent NTP levels in peripheral blood mononuclear cells (half-life, 14 h) and distribution to sanctuary sites for viral replication including testes, eyes, and brain. In a rhesus monkey model of EVD, once-daily intravenous administration of 10 mg kg(-1) GS-5734 for 12 days resulted in profound suppression of EBOV replication and protected 100% of EBOV-infected animals against lethal disease, ameliorating clinical disease signs and pathophysiological markers, even when treatments were initiated three days after virus exposure when systemic viral RNA was detected in two out of six treated animals. These results show the first substantive post-exposure protection by a small-molecule antiviral compound against EBOV in nonhuman primates. The broad-spectrum antiviral activity of GS-5734 in vitro against other pathogenic RNA viruses, including filoviruses, arenaviruses, and coronaviruses, suggests the potential for wider medical use. GS-5734 is amenable to large-scale manufacturing, and clinical studies investigating the drug safety and pharmacokinetics are ongoing.
There are no approved therapies for Ebola virus infection. Here, to find potential therapeutic targets, we perform a screen for genes essential for Ebola virus (EBOV) infection. We identify GNPTAB, ...which encodes the α and β subunits of N-acetylglucosamine-1-phosphate transferase. We show that EBOV infection of a GNPTAB knockout cell line is impaired, and that this is reversed by reconstituting GNPTAB expression. Fibroblasts from patients with mucolipidosis II, a disorder associated with mutations in GNPTAB, are refractory to EBOV, whereas cells from their healthy parents support infection. Impaired infection correlates with loss of the expression of cathepsin B, known to be essential for EBOV entry. GNPTAB activity is dependent upon proteolytic cleavage by the SKI-1/S1P protease. Inhibiting this protease with the small-molecule PF-429242 blocks EBOV entry and infection. Disruption of GNPTAB function may represent a strategy for a host-targeted therapy for EBOV.
TMEM41B Is a Pan-flavivirus Host Factor Hoffmann, H.-Heinrich; Schneider, William M.; Rozen-Gagnon, Kathryn ...
Cell,
01/2021, Volume:
184, Issue:
1
Journal Article
Peer reviewed
Open access
Flaviviruses pose a constant threat to human health. These RNA viruses are transmitted by the bite of infected mosquitoes and ticks and regularly cause outbreaks. To identify host factors required ...for flavivirus infection, we performed full-genome loss of function CRISPR-Cas9 screens. Based on these results, we focused our efforts on characterizing the roles that TMEM41B and VMP1 play in the virus replication cycle. Our mechanistic studies on TMEM41B revealed that all members of the Flaviviridae family that we tested require TMEM41B. We tested 12 additional virus families and found that SARS-CoV-2 of the Coronaviridae also required TMEM41B for infection. Remarkably, single nucleotide polymorphisms present at nearly 20% in East Asian populations reduce flavivirus infection. Based on our mechanistic studies, we propose that TMEM41B is recruited to flavivirus RNA replication complexes to facilitate membrane curvature, which creates a protected environment for viral genome replication.
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•TMEM41B is required for flavivirus infection, but autophagy is not required•TMEM41B associates with flavivirus proteins and may facilitate cell membrane remodeling•TMEM41B single nucleotide polymorphisms reduce flavivirus infection•TMEM41B-deficiency confers a heightened innate immune response to flavivirus infection
Hoffmann et al. determine that the transmembrane protein, TMEM41B, is required for infection by members of the Flaviviridae family of viruses. Loss of TMEM41B reduces viral RNA replication and increases innate immune activation in response to flavivirus infection. Thus, TMEM41B is a potential host target for antiviral therapeutics.
Two men from northwestern Missouri independently presented to a medical facility with fever, fatigue, diarrhea, thrombocytopenia, and leukopenia, and both had been bitten by ticks 5 to 7 days before ...the onset of illness. Ehrlichia chaffeensis was suspected as the causal agent but was not found on serologic analysis, polymerase-chain-reaction (PCR) assay, or cell culture. Electron microscopy revealed viruses consistent with members of the Bunyaviridae family. Next-generation sequencing and phylogenetic analysis identified the viruses as novel members of the phlebovirus genus. Although Koch's postulates have not been completely fulfilled, we believe that this phlebovirus, which is novel in the Americas, is the cause of this clinical syndrome.
The ongoing Ebola virus outbreak in the Ituri and North Kivu Provinces of the Democratic Republic of the Congo, which began in July, 2018, is the second largest ever recorded. Despite civil unrest, ...outbreak control measures and the administration of experimental therapies and a vaccine have been initiated. The aim of this study was to test the efficacy of candidate therapies and diagnostic tests with the outbreak strain Ituri Ebola virus. Lacking a virus isolate from this outbreak, a recombinant Ituri Ebola virus was compared with a similarly engineered Makona virus from the 2013–16 outbreak.
Using Ebola virus sequences provided by organisations in DR Congo and a reverse genetics system, we generated an authentic Ebola virus from the ongoing outbreak in Ituri and North Kivu provinces. To relate this virus to other Ebola viruses in DR Congo, we did a phylogenetic analysis of representative complete Ebola virus genome sequences from previous outbreaks. We evaluated experimental therapies being tested in clinical trials in DR Congo, including remdesivir and ZMapp monoclonal antibodies, for their ability to inhibit the growth of infectious Ituri Ebola virus in cell culture. We also tested diagnostic assays for detection of the Ituri Ebola virus sequence.
The phylogenetic analysis of whole-genome sequences from each Ebola virus outbreak suggests there are at least two Ebola virus strains in DR Congo, which have independently crossed into the human population. The Ituri Ebola strain initially grew slower than the Makona strain, yet reached similar mean yields of 3 × 107 50% tissue culture infectious dose by 72 h infection in Huh-7 cells. Ituri Ebola virus was similar to Makona in its susceptibility to inhibition by remdesivir and to neutralisation by monoclonal antibodies from ZMapp and other monoclonal antibodies. Remdesivir inhibited Ituri Ebola virus at a 50% effective concentration (EC50) of 12nM (with a selectivity index of 303) and Makona Ebola virus at 13nM (with a selectivity index of 279). The Zmapp monoclonal antibodies 2G4 and 4G7 neutralised Ituri Ebola virus with a mean EC50 of 0·24 μg/mL and 0·48 μg/mL, and Makona Ebola virus with a mean EC50 of 0·45 μg/mL and 0·2 μg/mL. The Xpert Ebola and US Centers for Disease Control and Prevention real-time RT-qPCR diagnostic assays detected Ituri and Makona Ebola virus sequences with similar sensitivities and efficiencies, despite primer site binding mismatches in the Ituri Ebola virus.
Our findings provide a rationale for the continued testing of investigational therapies, confirm the effectiveness of the diagnostic assays used in the region, and establish a paradigm for the use of reverse genetics to inform response activities in an outbreak.
US Centers for Disease Control and Prevention.
Remdesivir is a broad-spectrum antiviral nucleotide prodrug that has been clinically evaluated in Ebola virus patients and recently received emergency use authorization (EUA) for treatment of ...COVID-19. With approvals from the Federal Select Agent Program and the Centers for Disease Control and Prevention’s Institutional Biosecurity Board, we characterized the resistance profile of remdesivir by serially passaging Ebola virus under remdesivir selection; we generated lineages with low-level reduced susceptibility to remdesivir after 35 passages. We found that a single amino acid substitution, F548S, in the Ebola virus polymerase conferred low-level reduced susceptibility to remdesivir. The F548 residue is highly conserved in filoviruses but should be subject to specific surveillance among novel filoviruses, in newly emerging variants in ongoing outbreaks, and also in Ebola virus patients undergoing remdesivir therapy. Homology modeling suggests that the Ebola virus polymerase F548 residue lies in the F-motif of the polymerase active site, a region that was previously identified as susceptible to resistance mutations in coronaviruses. Our data suggest that molecular surveillance of this region of the polymerase in remdesivir-treated COVID-19 patients is also warranted.
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