Avian influenza viruses of the H5N1 subtype pose a serious global health threat due to the high mortality (>60%) associated with the disease caused by these viruses and the lack of protective antibodies to these viruses in the general population. The factors that enable avian H5N1 influenza viruses to replicate in humans are not completely understood. Here we use a high-throughput screening approach to identify novel mutations in the polymerase genes of an avian H5N1 virus that confer efficient polymerase activity in mammalian cells. Several of the identified mutations (which have previously been found in natural isolates) increase viral replication in mammalian cells and virulence in infected mice compared with the wild-type virus. The identification of amino-acid mutations in avian H5N1 influenza virus polymerase complexes that confer increased replication and virulence in mammals is important for the identification of circulating H5N1 viruses with an increased potential to infect humans.

Ebola virus (EBOV) is the causative agent of a severe hemorrhagic fever in humans with reported case fatality rates as high as 90%. There are currently no licensed vaccines or antiviral therapeutics to combat EBOV infections. Heme oxygenase-1 (HO-1), an enzyme that catalyzes the rate-limiting step in heme degradation, has antioxidative properties and protects cells from various stresses. Activated HO-1 was recently shown to have antiviral activity, potently inhibiting the replication of viruses such as hepatitis C virus and human immunodeficiency virus. However, the effect of HO-1 activation on EBOV replication remains unknown. To determine whether the upregulation of HO-1 attenuates EBOV replication, we treated cells with cobalt protoporphyrin (CoPP), a selective HO-1 inducer, and assessed its effects on EBOV replication. We found that CoPP treatment, pre-and postinfection, significantly suppressed EBOV replication in a manner dependent upon HO-1 upregulation and activity. In addition, stable overexpression of HO-1 significantly attenuated EBOV growth. Although the exact mechanism behind the antiviral properties of HO-1 remains to be elucidated, our data show that HO-1 upregulation does not attenuate EBOV entry or budding but specifically targets EBOV transcription/replication. Therefore, modulation of the cellular enzyme HO-1 may represent a novel therapeutic strategy against EBOV infection. E bola virus (EBOV) is an enveloped, nonsegmented, negativestrand RNA virus that, together with Marburg virus (MARV), makes up the family Filoviridae (filovirus) (1). There are five antigenically distinct species of EBOV: Zaire ebolavirus, Sudan ebolavirus, Taï Forest ebolavirus (previously Côte d'Ivoire ebolavirus),Reston ebolavirus, and Bundibugyo ebolavirus (2). Sudan ebolavirus and Zaire ebolavirus are associated with severe outbreaks of hemorrhagic fever in humans, with case fatality rates ranging from 55% to 90% (3). Budibugyo ebolavirus, a recently identified species associated with a 2007 outbreak, produced a case fatality rate of about 25% (4).The EBOV genome includes seven structural genes. The single surface glycoprotein (GP) mediates virus entry into a variety of different cell types (5-7). Four structural proteins, nucleoprotein (NP), RNA-dependent RNA polymerase (L), VP30, and VP35, are essential for amplification of the viral genome (8). The primary membraneassociated viral protein, VP40, is critical for viral budding (9). The secondary membrane-associated protein, VP24, and VP35 effectively antagonize interferon (IFN) pathways by inhibiting the Janus kinasesignal transducer and activator of transcription (JAK-STAT) signaling cascade and by suppressing IFN regulatory factor 3 (IRF3) phosphorylation, respectively (10-13). This efficient suppression of IFN pathways may contribute to EBOV pathogenesis and renders IFN therapeutic treatments ineffective (14, 15).Currently, there are no approved vaccines or antiviral therapeutics to combat EBOV infection. Given the limited number of biosafety level 4 (BSL4) contai...

Zaire ebolavirus is the causative agent of the current outbreak of hemorrhagic fever disease in West Africa. Previously, we showed that a whole Ebola virus (EBOV) vaccine based on a replication-defective EBOV (EBOVΔVP30) protects immunized mice and guinea pigs against lethal challenge with rodent-adapted EBOV. Here, we demonstrate that EBOVΔVP30 protects nonhuman primates against lethal infection with EBOV. Although EBOVΔVP30 is replication-incompetent, we additionally inactivated the vaccine with hydrogen peroxide; the chemically inactivated vaccine remained antigenic and protective in nonhuman primates. EBOVΔVP30 thus represents a safe, efficacious, whole-EBOV vaccine candidate that differs from other EBOV vaccine platforms in that it presents all viral proteins and the viral RNA to the host immune system, which might contribute to protective immune responses.

The West Africa Ebola outbreak was the largest outbreak ever recorded, with over 28,000 reported infections; this devastating epidemic emphasized the need to understand the mechanisms to counteract virus infection. Here, we screen a library of nearly 400 interferonstimulated genes (ISGs) against a biologically contained Ebola virus and identify several ISGs not previously known to affect Ebola virus infection. Overexpression of the top ten ISGs attenuates virus titers by up to 1000-fold. Mechanistic studies demonstrate that three ISGs interfere with virus entry, six affect viral transcription/replication, and two inhibit virion formation and budding. A comprehensive study of one ISG (CCDC92) that shows anti-Ebola activity in our screen reveals that CCDC92 can inhibit viral transcription and the formation of complete virions via an interaction with the viral protein NP. Our findings provide insights into Ebola virus infection that could be exploited for the development of therapeutics against this virus.

The inflammasome is part of the innate immune system that regulates the secretion of proinflammatory cytokines such as interleukin-1β (IL-1β). Ebola virus (EBOV) infection of monocytes and macrophages (primary target cells early during infection) leads to the production of proinflammatory cytokines; however, the mechanism behind the activation and release of these cytokines is not fully understood. Here, we demonstrate that EBOV infection leads to the activation of the NLRP3 inflammasome and the subsequent secretion of IL-1β and IL-18. This process is dependent on protease caspase-1, a component of the NLRP3 inflammasome complex, but is independent of virus replication. These findings may lead to the development of novel drugs that impede the pathogenesis of EBOV infection.