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The fusion peptide EBO 16 (GAAIGLAWIPYFGPAA) comprises the fusion domain of an internal sequence located in the envelope fusion glycoprotein (GP2) of the Ebola virus. This region interacts with the cellular membrane of the host and leads to membrane fusion. To gain insight into the mechanism of the peptide-membrane interaction and fusion, insertion of the peptide was modeled by experiments in which the tryptophan fluorescence and 1 H NMR were monitored in the presence of sodium dodecyl sulfate micelles or in the presence of detergent-resistant membrane fractions. In the presence of SDS micelles, EBO 16 undergoes a random coil-helix transition, showing a tendency to self-associate. The three-dimensional structure displays a 3 10 -helix in the central part of molecule, similar to the fusion peptides of many known membrane fusion proteins. Our results also reveal that EBO 16 can interact with detergent-resistant membrane fractions and strongly suggest that Trp-8 and Phe-12 are important for structure maintenance within the membrane bilayer. Replacement of tryptophan 8 with alanine (W8A) resulted in dramatic loss of helical structure, proving the importance of the aromatic ring in stabilizing the helix. Molecular dynamics studies of the interaction between the peptide and the target membrane also corroborated the crucial participation of these aromatic residues. The aromatic-aromatic interaction may provide a mechanism for the free energy coupling between random coil-helical transition and membrane anchoring. Our data shed light on the structural "domains" of fusion peptides and provide a clue for the development of a drug that might block the early steps of viral infection.

Enveloped animal viruses must undergo membrane fusion to deliver their genome into the host cell. We demonstrate that high pressure inactivates two membrane-enveloped viruses, influenza and Sindbis, by trapping the particles in a fusion-intermediate state. The pressure-induced conformational changes in Sindbis and influenza viruses were followed using intrinsic and extrinsic fluorescence spectroscopy, circular dichroism, and fusion, plaque, and hemagglutination assays. Influenza virus subjected to pressure exposes hydrophobic domains as determined by tryptophan fluorescence and by the binding of bis-8-anilino-1-naphthalenesulfonate, a well established marker of the fusogenic state in influenza virus. Pressure also produced an increase in the fusion activity at neutral pH as monitored by fluorescence resonance energy transfer using lipid vesicles labeled with fluorescence probes. Sindbis virus also underwent conformational changes induced by pressure similar to those in influenza virus, and the increase in fusion activity was followed by pyrene excimer fluorescence of the metabolically labeled virus particles. Overall we show that pressure elicits subtle changes in the whole structure of the enveloped viruses triggering a conformational change that is similar to the change triggered by low pH. Our data strengthen the hypothesis that the native conformation of fusion proteins is metastable, and a cycle of pressure leads to a final state, the fusion-active state, of smaller volume.Enveloped viruses utilize regulated membrane fusion to introduce their genomes in the cytoplasm of the host cell. The fusion is mediated by surface envelope proteins of the virus in response to a trigger (1, 2). Once triggered, the fusion process leads to a conformational change that promotes the interaction of a specific sequence (fusion peptide) with the target membrane and initiates membrane fusion. Membrane fusion is crucial in other biological functions such as myotube formation, fertilization, and trafficking of endocytic and exocytic vesicles within eukaryotic cells (1, 3). Many enveloped animal viruses have been studied as models for understanding the mechanism of membrane fusion. While the fusion proteins of many viruses reveal significant similarity in their putative fusogenic conformation, such as those of influenza virus (hemagglutinin HA2), 1 human immunodeficiency virus (gp41 protein), Moloney murine leukemia virus (TM protein), and Ebola virus (GP2 protein) (4), the events of membrane fusion for other virus families (e.g. Flaviviridae and Togaviridae) are beginning to be understood. Alphavirus and the flavivirus fusion proteins appear to have evolved from a common ancestor (5) and possess a similar new class of membrane fusion proteins that do not form coiledcoils (6 -8).Sindbis and influenza are enveloped viruses that first enter a cell by endocytosis and then fuse with the cellular membrane in response to acidic conditions. Sindbis virus is the prototype of the Alphavirus genus, Togaviridae family. The Alphavirus spike is...

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Yellow fever is an acute infectious disease caused by prototype virus of the genus Flavivirus. It is endemic in Africa and South America where it represents a serious public health problem causing epidemics of hemorrhagic fever with mortality rates ranging from 20% to 50%. There is no available antiviral therapy and vaccination is the primary method of disease control. Although the attenuated vaccines for yellow fever show safety and efficacy it became necessary to develop a new yellow fever vaccine due to the occurrence of rare serious adverse events, which include visceral and neurotropic diseases. The new inactivated vaccine should be safer and effective as the existing attenuated one. In the present study, the immunogenicity of an inactivated 17DD vaccine in C57BL/6 mice was evaluated. The yellow fever virus was produced by cultivation of Vero cells in bioreactors, inactivated with β-propiolactone, and adsorbed to aluminum hydroxide (alum). Mice were inoculated with inactivated 17DD vaccine containing alum adjuvant and followed by intracerebral challenge with 17DD virus. The results showed that animals receiving 3 doses of the inactivated vaccine (2 μg/dose) with alum adjuvant had neutralizing antibody titers above the cut-off of PRNT50 (Plaque Reduction Neutralization Test). In addition, animals immunized with inactivated vaccine showed survival rate of 100% after the challenge as well as animals immunized with commercial attenuated 17DD vaccine.

During recent years, vaccination against hepatitis A has been implemented in several countries. It is expected that the increase in mass vaccination against hepatitis A will eventually result in a decreased prevalence of anti-HAV antibodies in the general population. For this reason, a suitable clinical sample for diagnosis of hepatitis A must be sufficiently sensitive to enable detection of lower antibodies titers. In this study, the feasibility of using dried blood spots (DBS) was assessed for the detection of anti-HAV antibodies after a natural infection and vaccination. Seventy-four DBS and paired plasma samples were obtained from a group of college students for a cross-sectional hepatitis A seroepidemiological study. Forty-six students seronegative for anti-HAV were selected randomly and immunized with an inactivated hepatitis A vaccine using an 0-6 month schedule. Seroconversion was monitored in paired plasma and DBS samples 6 months after the first dose followed by a period of 8 and 24 months after the second dose. A strong correlation between OD/CO rates of paired plasma and DBS samples for the detection of anti-HAV was observed. The sensitivity and specificity of the DBS compared with plasma for the detection of anti-HAV antibodies after natural infection was 100%. The sensitivity of DBS in samples collected 24 months after the second dose of hepatitis A vaccine was 95.4%. The results showed that DBS samples can be used for the detection of anti-HAV antibodies both after natural infection or vaccination.