An upstream open reading frame modulates ebola virus polymerase translation and virus replication

Ebolaviruses, highly lethal zoonotic pathogens, possess longer genomes than most other non-segmented negative-strand RNA viruses due in part to long 5' and 3' untranslated regions (UTRs) present in the seven viral transcriptional units. To date, specific functions have not been assigned to these UTRs. With reporter assays, we demonstrated that the Zaire ebolavirus (EBOV) 5'-UTRs lack internal ribosomal entry site function. However, the 5'-UTRs do differentially regulate cap-dependent translation when placed upstream of a GFP reporter gene. Most dramatically, the 5'-UTR derived from the viral polymerase (L) mRNA strongly suppressed translation of GFP compared to a β-actin 5'-UTR. The L 5'-UTR is one of four viral genes to possess upstream AUGs (uAUGs), and ablation of each uAUG enhanced translation of the primary ORF (pORF), most dramatically in the case of the L 5'-UTR. The L uAUG was sufficient to initiate translation, is surrounded by a "weak" Kozak sequence and suppressed pORF translation in a position-dependent manner. Under conditions where eIF2α was phosphorylated, the presence of the uORF maintained translation of the L pORF, indicating that the uORF modulates L translation in response to cellular stress. To directly address the role of the L uAUG in virus replication, a recombinant EBOV was generated in which the L uAUG was mutated to UCG. Strikingly, mutating two nucleotides outside of previously-defined protein coding and cis-acting regulatory sequences attenuated virus growth to titers 10-100-fold lower than a wild-type virus in Vero and A549 cells. The mutant virus also exhibited decreased viral RNA synthesis as early as 6 hours post-infection and enhanced sensitivity to the stress inducer thapsigargin. Cumulatively, these data identify novel mechanisms by which EBOV regulates its polymerase expression, demonstrate their relevance to virus replication and identify a potential therapeutic target

Immunization with GP1 but Not Core-like Particles Displaying Isolated Receptor-Binding Epitopes Elicits Virus-Neutralizing Antibodies against Junín Virus

New World arenaviruses are rodent-transmitted viruses and include a number of pathogens that are responsible for causing severe human disease. This includes Junín virus (JUNV), which is the causative agent of Argentine hemorrhagic fever. The wild nature and mobility of the rodent reservoir host makes it difficult to control the disease, and currently passive immunization with high-titer neutralizing antibody-containing plasma from convalescent patients is the only specific therapy. However, dwindling supplies of naturally available convalescent plasma, and challenges in developing similar resources for other closely related viruses, have made the development of alternative antibody-based therapeutic approaches of critical importance. In this study, we sought to induce a neutralizing antibody response in rabbits against the receptor-binding subunit of the viral glycoprotein, GP1, and the specific peptide sequences in GP1 involved in cellular receptor contacts. While these specific receptor-interacting peptides did not efficiently induce the production of neutralizing antibodies when delivered as a particulate antigen (as part of hepatitis B virus core-like particles), we showed that recombinant JUNV GP1 purified from transfected mammalian cells induced virus-neutralizing antibodies at high titers in rabbits. Further, neutralization was observed across a range of unrelated JUNV strains, a feature that is critical for effectiveness in the field. These results underscore the potential of GP1 alone to induce a potent neutralizing antibody response and highlight the importance of epitope presentation. In addition, effective virus neutralization by rabbit antibodies supports the potential applicability of this species for the future development of immunotherapeutics (e.g., based on humanized monoclonal antibodies). Such information can be applied in the design of vaccines and immunogens for both prevention and specific therapies against this and likely also other closely related pathogenic New World arenaviruses.Fil: Roman Sosa, Gleyder. Ulm University Hospital; AlemaniaFil: Leske, Anne. Friedrich-Loeffler-Institut; AlemaniaFil: Ficht, Xenia. Ulm University Hospital; AlemaniaFil: Dau, Tung Huy. Friedrich-Loeffler-Institut; AlemaniaFil: Holzerland, Julia. Friedrich-Loeffler-Institut; AlemaniaFil: Hoenen, Thomas. Friedrich-Loeffler-Institut; AlemaniaFil: Beer, Martin. Friedrich-Loeffler-Institut; AlemaniaFil: Kammerer, Robert. Friedrich-Loeffler-Institut; AlemaniaFil: Schirmbeck, Reinhold. Friedrich-Loeffler-Institut; AlemaniaFil: Rey, Felix A.. Friedrich-Loeffler-Institut; AlemaniaFil: Cordo, Sandra Myriam. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Groseth, Allison. Friedrich-Loeffler-Institut; Alemani

SHARC II: a Caltech Submillimeter Observatory facility camera with 384 pixels

SHARC II is a background-limited 350 μm and 450 μm facility camera for the Caltech Submillimeter Observatory undergoing commissioning in 2002. The key component of SHARC II is a 12×32 array of doped silicon 'pop-up' bolometers developed at NASA/Goddard. Each 1 mm × 1 mm pixel is coated with a 400 Ω/square bismuth film and located λ/4 above a reflective backshort to achieve >75% absorption efficiency. The pixels cover the focal plane with >90% filling factor. At 350 μm, the SHARC II pixels are separated by 0.65 λ/D. In contrast to the silicon bolometers in the predecessor of SHARC II, each doped thermistor occupies nearly the full area of the pixel, which lowers the 1/f knee of the detector noise to <0.03 Hz, under load, at the bath temperature of 0.36 K. The bolometers are AC-biased and read in 'total power' mode to take advantage of the improved stability. Each bolometer is biased through a custom ~130 MΩ CrSi load resistor at 7 K and read with a commercial JFET at 120 K. The JFETs and load resistors are integrated with the detectors into a single assembly to minimize microphonic noise. Electrical connection across the 0.36 K to 4 K and 4 K to 120 K temperature interfaces is accomplished with lithographed metal wires on dielectric substrates. In the best 25% of winter nights on Mauna Kea, SHARC II is expected to have an NEFD at 350 μm of 1 Jy Hz-1/2 or better. The new camera should be at least 4 times faster at detecting known point sources and 30 times faster at mapping large areas compared to the prior instrument

Tacaribe Virus but Not Junin Virus Infection Induces Cytokine Release from Primary Human Monocytes and Macrophages

The mechanisms underlying the development of disease during arenavirus infection are poorly understood. However, common to all hemorrhagic fever diseases is the involvement of macrophages as primary target cells, suggesting that the immune response in these cells may be of paramount importance during infection. Thus, in order to identify features of the immune response that contribute to arenavirus pathogenesis, we have examined the growth kinetics and cytokine profiles of two closely related New World arenaviruses, the apathogenic Tacaribe virus (TCRV) and the hemorrhagic fever-causing Junin virus (JUNV), in primary human monocytes and macrophages. Both viruses grew robustly in VeroE6 cells; however, TCRV titres were decreased by approximately 10 fold compared to JUNV in both monocytes and macrophages. Infection of both monocytes and macrophages with TCRV also resulted in the release of high levels of IL-6, IL-10 and TNF-α, while levels of IFN-α, IFN-β and IL-12 were not affected. However, we could show that the presence of these cytokines had no direct effect on growth of either TCRV of JUNV in macrophages. Further analysis also showed that while the production of IL-6 and IL-10 are dependent on viral replication, production of TNF-α also occurs after exposure to UV-inactivated TCRV particles and is thus independent of productive virus infection. Surprisingly, JUNV infection did not have an effect on any of the cytokines examined indicating that, in contrast to other viral hemorrhagic fever viruses, macrophage-derived cytokine production is unlikely to play an active role in contributing to the cytokine dysregulation observed in JUNV infected patients. Rather, these results suggest that an early, controlled immune response by infected macrophages may be critical for the successful control of infection of apathogenic viruses and prevention of subsequent disease, including systemic cytokine dysregulation

Serologic Cross-Reactivity of Human IgM and IgG Antibodies to Five Species of Ebola Virus

Five species of Ebola virus (EBOV) have been identified, with nucleotide differences of 30–45% between species. Four of these species have been shown to cause Ebola hemorrhagic fever (EHF) in humans and a fifth species (Reston ebolavirus) is capable of causing a similar disease in non-human primates. While examining potential serologic cross-reactivity between EBOV species is important for diagnostic assays as well as putative vaccines, the nature of cross-reactive antibodies following EBOV infection has not been thoroughly characterized. In order to examine cross-reactivity of human serologic responses to EBOV, we developed antigen preparations for all five EBOV species, and compared serologic responses by IgM capture and IgG enzyme-linked immunosorbent assay (ELISA) in groups of convalescent diagnostic sera from outbreaks in Kikwit, Democratic Republic of Congo (n = 24), Gulu, Uganda (n = 20), Bundibugyo, Uganda (n = 33), and the Philippines (n = 18), which represent outbreaks due to four different EBOV species. For groups of samples from Kikwit, Gulu, and Bundibugyo, some limited IgM cross-reactivity was noted between heterologous sera-antigen pairs, however, IgM responses were largely stronger against autologous antigen. In some instances IgG responses were higher to autologous antigen than heterologous antigen, however, in contrast to IgM responses, we observed strong cross-reactive IgG antibody responses to heterologous antigens among all sets of samples. Finally, we examined autologous IgM and IgG antibody levels, relative to time following EHF onset, and observed early peaking and declining IgM antibody levels (by 80 days) and early development and persistence of IgG antibodies among all samples, implying a consistent pattern of antibody kinetics, regardless of EBOV species. Our findings demonstrate limited cross-reactivity of IgM antibodies to EBOV, however, the stronger tendency for cross-reactive IgG antibody responses can largely circumvent limitations in the utility of heterologous antigen for diagnostic assays and may assist in the development of antibody-mediated vaccines to EBOV

Cytomegalovirus-based vaccine expressing Ebola virus glycoprotein protects nonhuman primates from Ebola virus infection.

Ebolaviruses pose significant public health problems due to their high lethality, unpredictable emergence, and localization to the poorest areas of the world. In addition to implementation of standard public health control procedures, a number of experimental human vaccines are being explored as a further means for outbreak control. Recombinant cytomegalovirus (CMV)-based vectors are a novel vaccine platform that have been shown to induce substantial levels of durable, but primarily T-cell-biased responses against the encoded heterologous target antigen. Herein, we demonstrate the ability of rhesus CMV (RhCMV) expressing Ebola virus (EBOV) glycoprotein (GP) to provide protective immunity to rhesus macaques against lethal EBOV challenge. Surprisingly, vaccination was associated with high levels of GP-specific antibodies, but with no detectable GP-directed cellular immunity

Demonstration of Cross-Protective Vaccine Immunity against an Emerging Pathogenic Ebolavirus Species

A major challenge in developing vaccines for emerging pathogens is their continued evolution and ability to escape human immunity. Therefore, an important goal of vaccine research is to advance vaccine candidates with sufficient breadth to respond to new outbreaks of previously undetected viruses. Ebolavirus (EBOV) vaccines have demonstrated protection against EBOV infection in nonhuman primates (NHP) and show promise in human clinical trials but immune protection occurs only with vaccines whose antigens are matched to the infectious challenge species. A 2007 hemorrhagic fever outbreak in Uganda demonstrated the existence of a new EBOV species, Bundibugyo (BEBOV), that differed from viruses covered by current vaccine candidates by up to 43% in genome sequence. To address the question of whether cross-protective immunity can be generated against this novel species, cynomolgus macaques were immunized with DNA/rAd5 vaccines expressing ZEBOV and SEBOV glycoprotein (GP) prior to lethal challenge with BEBOV. Vaccinated subjects developed robust, antigen-specific humoral and cellular immune responses against the GP from ZEBOV as well as cellular immunity against BEBOV GP, and immunized macaques were uniformly protected against lethal challenge with BEBOV. This report provides the first demonstration of vaccine-induced protective immunity against challenge with a heterologous EBOV species, and shows that Ebola vaccines capable of eliciting potent cellular immunity may provide the best strategy for eliciting cross-protection against newly emerging heterologous EBOV species

Establishment of Fruit Bat Cells (Rousettus aegyptiacus) as a Model System for the Investigation of Filoviral Infection

Marburg virus and several species of Ebola virus are endemic in central Africa and cause sporadic outbreaks in this region with mortality rates of up to 90%. So far, there is no vaccination or therapy available to protect people at risk in these regions. Recently, different fruit bats have been identified as potential reservoirs. One of them is Rousettus aegyptiacus. It seems that within huge bat populations only relatively small numbers are positive for filovirus-specific antibodies or filoviral RNA, a phenomenon that is currently not understood. As a first step towards understanding the biology of filoviruses in bats, we sought to establish a model system to investigate filovirus replication in cells derived from their natural reservoir. Here, we provide the first insights into this topic by monitoring filovirus infection of a Rousettus aegyptiacus derived cell line, R06E. We were able to show that filoviruses propagate well in R06E cells, which can, therefore, be used to investigate replication and transcription of filovirus RNA and to very efficiently perform rescue of recombinant Marburg virus using reverse genetics. These results emphasize the suitability of the newly established bat cell line for filovirus research

Mucosal Immunization of Cynomolgus Macaques with the VSVΔG/ZEBOVGP Vaccine Stimulates Strong Ebola GP-Specific Immune Responses

(ZEBOV) produces a lethal viral hemorrhagic fever in humans and non-human primates.We demonstrate that the VSVΔG/ZEBOVGP vaccine given 28 days pre-challenge either intranasally (IN), orally (OR), or intramuscularly (IM) protects non-human primates against a lethal systemic challenge of ZEBOV, and induces cellular and humoral immune responses. We demonstrated that ZEBOVGP-specific T-cell and humoral responses induced in the IN and OR groups, following an immunization and challenge, produced the most IFN-γ and IL-2 secreting cells, and long term memory responses.We have shown conclusively that mucosal immunization can protect from systemic ZEBOV challenge and that mucosal delivery, particularly IN immunization, seems to be more potent than IM injection in the immune parameters we have tested. Mucosal immunization would be a huge benefit in any emergency mass vaccination campaign during a natural outbreak, or following intentional release, or for mucosal immunization of great apes in the wild

Elucidating variations in the nucleotide sequence of Ebola virus associated with increasing pathogenicity

Background Ebolaviruses cause a severe and often fatal haemorrhagic fever in humans, with some species such as Ebola virus having case fatality rates approaching 90%. Currently, the worst Ebola virus outbreak since the disease was discovered is occurring in West Africa. Although thought to be a zoonotic infection, a concern is that with increasing numbers of humans being infected, Ebola virus variants could be selected which are better adapted for human-to-human transmission. Results To investigate whether genetic changes in Ebola virus become established in response to adaptation in a different host, a guinea pig model of infection was used. In this experimental system, guinea pigs were infected with Ebola virus (EBOV), which initially did not cause disease. To simulate transmission to uninfected individuals, the virus was serially passaged five times in naïve animals. As the virus was passaged, virulence increased and clinical effects were observed in the guinea pig. An RNAseq and consensus mapping approach was then used to evaluate potential nucleotide changes in the Ebola virus genome at each passage. Conclusions Upon passage in the guinea pig model, EBOV become more virulent, RNA editing and also coding changes in key proteins become established. The data suggest that the initial evolutionary trajectory of EBOV in a new host can lead to a gain in virulence. Given the circumstances of the sustained transmission of EBOV in the current outbreak in West Africa, increases in virulence may be associated with prolonged and uncontrolled epidemics of EBOV