RESUMEN
Early experiments have resulted in the establishment of an efficient methodology for the production of a yellow fever vaccine in chicken embryo fibroblasts (CEF) using the 17DD virus strain [Freire, M.S., Mann, G.F., Marchevsky, R.S., Yamamura, A.M., Almeida, L.F., Jabor, A.V., Malachias, J.M., Coutinho, E.S., Galler, R., 2005. Production of yellow fever 17DD vaccine virus in primary culture of chicken embryo fibroblasts: yields, thermo and genetic stability, attenuation and immunogenicity. Vaccine 23, 2501-2512]. To investigate the role of the interferon system in vaccine virus yields, CEF cultures seeded at high and low cell densities and infected with the yellow fever 17DD virus were used. The supernatants of these cultures were tested for the presence of interferon by an assay based on the reduction of cytopathic effect of a challenge virus (Sindbis), for the enzymatic activity of the interferon-induced 2',5'-oligoadenylate synthetase and for the expression of 2',5'-oligoadenylate synthetase mRNA. The presence of interferon and its influence in the replication of yellow fever 17DD virus in CEF cultures was clearly demonstrated.
Asunto(s)
Fibroblastos/virología , Interferones/biosíntesis , Vacuna contra la Fiebre Amarilla/biosíntesis , Virus de la Fiebre Amarilla/crecimiento & desarrollo , 2',5'-Oligoadenilato Sintetasa/genética , 2',5'-Oligoadenilato Sintetasa/metabolismo , Animales , Células Cultivadas , Embrión de Pollo , Pollos , Chlorocebus aethiops , Activación Enzimática , Fibroblastos/citología , Fibroblastos/enzimología , Fibroblastos/metabolismo , Regulación de la Expresión Génica , Interferones/genética , Interferones/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Virus Sindbis/metabolismo , Células Vero , Replicación Viral/fisiología , Vacuna contra la Fiebre Amarilla/inmunología , Virus de la Fiebre Amarilla/inmunologíaRESUMEN
Venezuelan equine encephalitis virus (VEEV) is an important, naturally emerging zoonotic pathogen. Recent outbreaks in Venezuela and Colombia in 1995, involving an estimated 100,000 human cases, indicate that VEEV still poses a serious public health threat. To develop a safe, efficient vaccine that protects against disease resulting from VEEV infection, we generated chimeric Sindbis (SIN) viruses expressing structural proteins of different strains of VEEV and analyzed their replication in vitro and in vivo, as well as the characteristics of the induced immune responses. None of the chimeric SIN/VEE viruses caused any detectable disease in adult mice after either intracerebral (i.c.) or subcutaneous (s.c.) inoculation, and all chimeras were more attenuated than the vaccine strain, VEEV TC83, in 6-day-old mice after i.c. infection. All vaccinated mice were protected against lethal encephalitis following i.c., s.c., or intranasal (i.n.) challenge with the virulent VEEV ZPC738 strain (ZPC738). In spite of the absence of clinical encephalitis in vaccinated mice challenged with ZPC738 via i.n. or i.c. route, we regularly detected high levels of infectious challenge virus in the central nervous system (CNS). However, infectious virus was undetectable in the brains of all immunized animals at 28 days after challenge. Hamsters vaccinated with chimeric SIN/VEE viruses were also protected against s.c. challenge with ZPC738. Taken together, our findings suggest that these chimeric SIN/VEE viruses are safe and efficacious in adult mice and hamsters and are potentially useful as VEEV vaccines. In addition, immunized animals provide a useful model for studying the mechanisms of the anti-VEEV neuroinflammatory response, leading to the reduction of viral titers in the CNS and survival of animals.
Asunto(s)
Encéfalo/virología , Virus de la Encefalitis Equina Venezolana/genética , Virus de la Encefalitis Equina Venezolana/inmunología , Encefalomielitis Equina Venezolana/prevención & control , Recombinación Genética , Virus Sindbis/genética , Vacunas Virales/administración & dosificación , Replicación Viral , Animales , Encéfalo/patología , Cricetinae , Replicación del ADN , Modelos Animales de Enfermedad , Virus de la Encefalitis Equina Venezolana/metabolismo , Encefalomielitis Equina Venezolana/inmunología , Encefalomielitis Equina Venezolana/patología , Encefalomielitis Equina Venezolana/virología , Femenino , Humanos , Masculino , Mesocricetus , Ratones , Virus Sindbis/inmunología , Virus Sindbis/metabolismo , Vacunación , Vacunas Atenuadas/administración & dosificación , Vacunas Atenuadas/genética , Proteínas Estructurales Virales/genética , Proteínas Estructurales Virales/inmunología , Proteínas Estructurales Virales/metabolismo , Vacunas Virales/genéticaRESUMEN
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.