Ebola virus (EBOV) infection blocks cellular production of alpha/beta interferon (IFN-α/β) and the ability of cells to respond to IFN-α/β or IFN-γ. The EBOV VP35 protein has previously been identified as an EBOV-encoded inhibitor of IFN-α/β production. However, the mechanism by which EBOV infection inhibits responses to IFNs has not previously been defined. Here we demonstrate that the EBOV VP24 protein functions as an inhibitor of IFN-α/β and IFN-γ signaling. Expression of VP24 results in an inhibition of IFN-induced gene expression and an inability of IFNs to induce an antiviral state. The VP24-mediated inhibition of cellular responses to IFNs correlates with the impaired nuclear accumulation of tyrosine-phosphorylated STAT1 (PY-STAT1), a key step in both IFN-α/β and IFN-γ signaling. Consistent with this proposed function for VP24, infection of cells with EBOV also confers a block to the IFN-induced nuclear accumulation of PY-STAT1. Further, VP24 is found to specifically interact with karyopherin α1, the nuclear localization signal receptor for PY-STAT1, but not with karyopherin α2, α3, or α4. Overexpression of VP24 results in a loss of karyopherin α1-PY-STAT1 interaction, indicating that the VP24-karyopherin α1 interaction contributes to the block to IFN signaling. These data suggest that VP24 is likely to be an important virulence determinant that allows EBOV to evade the antiviral effects of IFNs.

The Ebola virus (EBOV) VP35 protein blocks the virus-induced phosphorylation and activation of interferon regulatory factor 3 (IRF-3), a transcription factor critical for the induction of alpha/beta interferon (IFN-α/β) expression. However, the mechanism(s) by which this blockage occurs remains incompletely defined. We now provide evidence that VP35 possesses double-stranded RNA (dsRNA)-binding activity. Specifically, VP35 bound to poly(rI) · poly(rC)-coated Sepharose beads but not control beads. In contrast, two VP35 point mutants, R312A and K309A, were found to be greatly impaired in their dsRNA-binding activity. Competition assays showed that VP35 interacted specifically with poly(rI) · poly(rC), poly(rA) · poly(rU), or in vitro-transcribed dsRNAs derived from EBOV sequences, and not with single-stranded RNAs (ssRNAs) or double-stranded DNA. We then screened wild-type and mutant VP35s for their ability to target different components of the signaling pathways that activate IRF-3. These experiments indicate that VP35 blocks activation of IRF-3 induced by overexpression of RIG-I, a cellular helicase recently implicated in the activation of IRF-3 by either virus or dsRNA. Interestingly, the VP35 mutants impaired for dsRNA binding have a decreased but measurable IFN antagonist activity in these assays. Additionally, wild-type and dsRNA-binding-mutant VP35s were found to have equivalent abilities to inhibit activation of the IFN-β promoter induced by overexpression of IPS-1, a recently identified signaling molecule downstream of RIG-I, or by overexpression of the IRF-3 kinases IKKε and TBK-1. These data support the hypothesis that dsRNA binding may contribute to VP35 IFN antagonist function. However, additional mechanisms of inhibition, at a point proximal to the IRF-3 kinases, most likely also exist.

In March 2005, the Centers for Disease Control and Prevention (CDC) investigated a large hemorrhagic fever (HF) outbreak in Uige Province in northern Angola, West Africa. In total, 15 initial specimens were sent to CDC, Atlanta, Ga., for testing for viruses associated with viral HFs known to be present in West Africa, including ebolavirus. Marburgvirus was also included despite the fact that the origins of all earlier outbreaks were linked directly to East Africa. Surprisingly, marburgvirus was confirmed (12 of 15 specimens) as the cause of the outbreak. The outbreak likely began in October 2004 and ended in July 2005, and it included 252 cases and 227 (90%) fatalities (report from the Ministry of Health, Republic of Angola, 2005), making it the largest Marburg HF outbreak on record. A real-time quantitative reverse transcription-PCR assay utilized and adapted during the outbreak proved to be highly sensitive and sufficiently robust for field use. Partial marburgvirus RNA sequence analysis revealed up to 21% nucleotide divergence among the previously characterized East African strains, with the most distinct being Ravn from Kenya (1987). The Angolan strain was less different (∼7%) from the main group of East African marburgviruses than one might expect given the large geographic separation. To more precisely analyze the virus genetic differences between outbreaks and among viruses within the Angola outbreak itself, a total of 16 complete virus genomes were determined, including those of the virus isolates Ravn (Kenya, 1987) and 05DRC, 07DRC, and 09DRC (Democratic Republic of Congo, 1998) and the reference Angolan virus isolate (Ang1379v). In addition, complete genome sequences were obtained from RNAs extracted from 10 clinical specimens reflecting various stages of the disease and locations within the Angolan outbreak. While the marburgviruses exhibit high overall genetic diversity (up to 22%), only 6.8% nucleotide difference was found between the West African Angolan viruses and the majority of East African viruses, suggesting that the virus reservoir species in these regions are not substantially distinct. Remarkably few nucleotide differences were found among the Angolan clinical specimens (0 to 0.07%), consistent with an outbreak scenario in which a single (or rare) introduction of virus from the reservoir species into the human population was followed by person-to-person transmission with little accumulation of mutations. This is in contrast to the 1998 to 2000 marburgvirus outbreak, where evidence of several virus genetic lineages (with up to 21% divergence) and multiple virus introductions into the human population was found.

Rift Valley fever (RVF) virus is a mosquito-borne human and veterinary pathogen associated with large outbreaks of severe disease throughout Africa and more recently the Arabian peninsula. Infection of livestock can result in sweeping “abortion storms” and high mortality among young animals. Human infection results in self-limiting febrile disease that in ∼1 to 2% of patients progresses to more serious complications including hepatitis, encephalitis, and retinitis or a hemorrhagic syndrome with high fatality. The virus S segment-encoded NSs and the M segment-encoded NSm proteins are important virulence factors. The development of safe, effective vaccines and tools to screen and evaluate antiviral compounds is critical for future control strategies. Here, we report the successful reverse genetics generation of multiple recombinant enhanced green fluorescent protein-tagged RVF viruses containing either the full-length, complete virus genome or precise deletions of the NSs gene alone or the NSs/NSm genes in combination, thus creating attenuating deletions on multiple virus genome segments. These viruses were highly attenuated, with no detectable viremia or clinical illness observed with high challenge dosages (1.0 × 10⁴PFU) in the rat lethal disease model. A single-dose immunization regimen induced robust anti-RVF virus immunoglobulin G antibodies (titer, ∼1:6,400) by day 26 postvaccination. All vaccinated animals that were subsequently challenged with a high dose of virulent RVF virus survived infection and could be serologically differentiated from naïve, experimentally infected animals by the lack of NSs antibodies. These rationally designed marker RVF vaccine viruses will be useful tools for in vitro screening of therapeutic compounds and will provide a basis for further development of RVF virus marker vaccines for use in endemic regions or following the natural or intentional introduction of the virus into previously unaffected areas.