RESUMEN
We previously described the use of an established reverse genetics system for the generation of recombinant human influenza A viruses from cloned cDNAs. Here, we have assembled a set of plasmids to allow recovery of the avian H5N1 influenza virus A/Turkey/England/50-92/91 entirely from cDNA. This system enables us to introduce mutations or truncations into the cDNAs to create mutant viruses altered specifically in a chosen gene. These mutant viruses can then be used in future pathogenesis studies in chickens and in studies to understand the host range restrictions of avian influenza viruses in humans.
Asunto(s)
Técnicas Genéticas , Subtipo H5N1 del Virus de la Influenza A/genética , Animales , Línea Celular , Cricetinae , ADN Complementario , ADN Viral , ARN Polimerasas Dirigidas por ADN/metabolismo , Perros , HumanosRESUMEN
BACKGROUND: Although H5N1 avian influenza viruses pose the most obvious imminent pandemic threat, there have been several recent zoonotic incidents involving transmission of H7 viruses to humans. Vaccines are the primary public health defense against pandemics, but reliance on embryonated chickens eggs to propagate vaccine and logistic problems posed by the use of new technology may slow our ability to respond rapidly in a pandemic situation. OBJECTIVES: We sought to generate an H7 candidate vaccine virus suitable for administration to humans whose generation and amplification avoided the use of eggs. METHODS: We generated a suitable H7 vaccine virus by reverse genetics. This virus, known as RD3, comprises the internal genes of A/Puerto Rico/8/34 with surface antigens of the highly pathogenic avian strain A/Chicken/Italy/13474/99 (H7N1). The multi-basic amino acid site in the HA gene, associated with high pathogenicity in chickens, was removed. RESULTS: The HA modification did not alter the antigenicity of the virus and the resultant single basic motif was stably retained following several passages in Vero and PER.C6 cells. RD3 was attenuated for growth in embryonated eggs, chickens, and ferrets. RD3 induced an antibody response in infected animals reactive against both the homologous virus and other H7 influenza viruses associated with recent infection by H7 viruses in humans. CONCLUSIONS: This is the first report of a candidate H7 vaccine virus for use in humans generated by reverse genetics and propagated entirely in mammalian tissue culture. The vaccine has potential use against a wide range of H7 strains.
Asunto(s)
Virus de la Influenza A/genética , Virus de la Influenza A/inmunología , Vacunas contra la Influenza/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Antivirales/sangre , Secuencia de Bases , Línea Celular , Embrión de Pollo , ADN Viral/genética , Brotes de Enfermedades/prevención & control , Europa (Continente)/epidemiología , Femenino , Hurones , Humanos , Gripe Humana/epidemiología , Gripe Humana/prevención & control , Masculino , Ratones , Ratones Endogámicos BALB C , Conejos , Virus Reordenados/inmunología , Proteínas Virales/química , Proteínas Virales/genéticaRESUMEN
The role of the herpes simplex virus tegument protein VP22 is not yet known. Here we describe the characterization of a virus in which the entire VP22 open reading frame has been deleted. We show that VP22 is not essential for virus growth but that virus lacking VP22 (Delta22) displays a cell-specific replication defect in epithelial MDBK cells. Virus particles assembled in the absence of VP22 show few obvious differences to wild-type (WT) particles, except for a moderate reduction in glycoproteins gD and gB. In addition, the Delta22 virus exhibits a general delay in the initiation of virus protein synthesis, but this is not due to a glycoprotein-related defect in virus entry. Intriguingly, however, the absence of VP22 has an obvious effect on the intracellular level of the immediate-early (IE) protein ICP0. Moreover, following translocation from the nucleus to the cytoplasm, ICP0 is unable to localize to the characteristic cytoplasmic sites observed in a WT infection. We demonstrate that, in WT-infected cells, VP22 and ICP0 are concentrated in the same cytoplasmic sites. Furthermore, we show that, while ICP0 and ICP4 are components of WT extracellular virions, the altered localization of ICP0 in the cytoplasm of Delta22-infected cells correlates with an absence of both ICP0 and ICP4 from Delta22 virions. Hence, while a role has not yet been defined for virion IE proteins in virus infection, our results suggest that their incorporation is a specific event requiring the tegument protein VP22. This report provides the first direct evidence that VP22 influences virus assembly.
Asunto(s)
Herpesvirus Humano 1/fisiología , Proteínas Inmediatas-Precoces/metabolismo , Proteínas Estructurales Virales/fisiología , Virión/metabolismo , Animales , Bovinos , Línea Celular , Cricetinae , Citoplasma/metabolismo , Eliminación de Gen , Glicoproteínas , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/metabolismo , Humanos , Proteínas Estructurales Virales/genética , Ensamble de VirusRESUMEN
Glycoprotein D (gD) of herpes simplex virus type 1 is a type 1 membrane protein in the virus envelope that binds to receptor molecules on the cell surface and which induces cell-cell fusion when co-expressed with gB, gH and gL. A chimeric gD molecule in which the membrane anchor and cytoplasmic tail domains were replaced with analogous regions from the human CD8 molecule was as competent as wild-type gD at mediating membrane fusion and virus entry. However, when gD was tethered to the membrane by means of a glycosylphosphatidylinositol (gpi)-anchor sequence, which binds only to the outer leaflet of the lipid bilayer, it was unable to function in cell-cell fusion assays. This chimera was incorporated into virions as efficiently as wild-type gD and yet virus particles containing gpi-linked gD entered cells more slowly than virions containing wild-type gD in their envelopes, suggesting that gD must be anchored in both leaflets of a lipid bilayer for it to function in both cell fusion and virus entry.
Asunto(s)
Herpesvirus Humano 1/patogenicidad , Fusión de Membrana , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/metabolismo , Fusión Celular , Línea Celular , Membrana Celular/metabolismo , Citoplasma/metabolismo , Glicosilfosfatidilinositoles/metabolismo , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/metabolismo , Humanos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas del Envoltorio Viral/genéticaRESUMEN
The subcellular localization of herpes simplex virus tegument proteins during infection is varied and complex. By using viruses expressing tegument proteins tagged with fluorescent proteins, we previously demonstrated that the major tegument protein VP22 exhibits a cytoplasmic localization, whereas the major tegument protein VP13/14 localizes to nuclear replication compartments and punctate domains. Here, we demonstrate the presence of a second minor population of VP22 in nuclear dots similar in appearance to those formed by VP13/14. We have constructed the first-described doubly fluorescence-tagged virus expressing VP22 and VP13/14 as fusion proteins with cyan fluorescent protein and yellow fluorescent protein, respectively. Visualization of both proteins within the same live infected cells has indicated that these two tegument proteins localize to the same nuclear dots but that VP22 appears there earlier than VP13/14. Further studies have shown that these tegument-specific dots are detectable as phase-dense bodies as early as 2 h after infection and that they are different from the previously described nuclear domains that contain capsid proteins. They are also different from the ICP0 domains formed at cellular nuclear domain 10 sites early in infection but, in almost all cases, are located in juxtaposition to these ICP0 domains. Hence, these tegument proteins join a growing number of proteins that are targeted to discrete nuclear domains in the herpesvirus-infected cell nucleus.