RESUMO
The entry of enveloped viruses into host cells is accomplished by fusion of the viral envelope with the target cell membrane. For the paramyxovirus parainfluenza virus type 5 (PIV-5), this fusion involves an attachment protein (HN) and a class I viral fusion protein (F). We investigated the effect of 20 different combinations of 12 amino-acid substitutions within functional domains of the PIV-5 F glycoprotein, by performing cell surface expression measurements, quantitative fusion and syncytia assays. We found that combinations of mutations conferring an autonomous phenotype with mutations leading to an increased fusion activity were compatible and generated functional PIV-5 F proteins. The addition of mutations in the heptad-repeat domains led to both autonomous and hyperfusogenic phenotypes, despite the low cell surface expression of the corresponding mutants. Such engineering approach may prove useful not only for deciphering the fundamental mechanism behind viral-mediated membrane fusion but also in the development of potential therapeutic applications.
Assuntos
Evolução Molecular Direcionada , Respirovirus/fisiologia , Proteínas Virais de Fusão/fisiologia , Internalização do Vírus , Substituição de Aminoácidos/genética , Animais , Linhagem Celular , Células Gigantes/virologia , Haplorrinos , Humanos , Mutagênese , Respirovirus/genética , Proteínas Virais de Fusão/genéticaRESUMO
BACKGROUND: Human parainfluenza viruses (hPIV) are respiratory pathogens responsible for upper and lower respiratory tract infections. In most labs, the clinical diagnosis of hPIV is routinely done using techniques based on the detection of viral antigens such as immunofluorescence assay or/and viral isolation. STUDY DESIGN: Five hPIV-2 isolated from respiratory samples exhibited unusual phenotypic and antigenic characteristics. These isolates showed important syncytial cytopathic effect and failed to react with one specific monoclonal antibody. These variant strains were subsequently compared with hPIV-2 prototype strain by cellular and molecular techniques. RESULTS: Both variant and prototype strains showed similar growth kinetics. Observation of plaque formation and syncytia assay indicated a more important fusogenic activity for the variant strains. Sequencing of fusion (F) and hemagglutinin-neuraminidase (HN) genes showed differences between the "atypical" hPIV-2 isolates and the Greer hPIV-2 prototype strain. These differences were analyzed with molecular modelling and structure prediction soft wares. A potential new glycosylation site in HN, in addition to minor changes observed in the predicted structure for the variant strains could explain their antigenic variation. Genetic changes in the fusion peptide and the cleavage site of F could also explain the difference observed in the fusion activity. CONCLUSIONS: Continuous global viral surveillance is essential to monitor antigenic changes that may occur in nature particularly with regards to the implementation of diagnostic assays. The differences observed in F and HN between the prototype strain and clinical hPIV-2 variants could also provide new data for the analysis of Paramyxovirus fusion mechanisms and their pathogenesis.
Assuntos
Proteína HN/genética , Vírus da Parainfluenza 2 Humana/fisiologia , RNA Viral , Infecções por Rubulavirus/virologia , Proteínas Virais de Fusão/genética , Adulto , Sequência de Aminoácidos , Animais , Variação Antigênica , Linhagem Celular , Criança , Glicosilação , Proteína HN/química , Proteína HN/imunologia , Haplorrinos , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Vírus da Parainfluenza 2 Humana/classificação , Vírus da Parainfluenza 2 Humana/genética , Vírus da Parainfluenza 2 Humana/isolamento & purificação , Filogenia , Conformação Proteica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/imunologia , Ensaio de Placa ViralRESUMO
Paramyxoviruses constitute a large family, including human animal pathogens with high prevalence and also major economic impact. During last fifteen years, development of reverse genetic systems, which allow generating viruses from cloned cDNA, revolutionized research on Paramyxoviruses. From the elaboration of minireplicons to the constitution of performing reverse genetic systems, this new strategy contributed to our understanding of viral cycle, pathogenicity or viral assembly, for example. This approach opened the way to the development of viral vectors and live-attenuated vaccines that could be useful tools in therapeutics and prophylaxis.
RESUMO
Reported here are two outbreaks of acute hemorrhagic conjunctivitis that occurred in the Democratic Republic of the Congo and in Morocco in the summers of 2003 and 2004, respectively, with a large impact on public health. Virus was isolated from the conjunctival swabs of 30 Congolese and 20 Moroccan patients. Enterovirus-specific cytopathic effect was observed in all samples. None of the strains could be typed using a conventional neutralization assay with the Melnick intersecting pools; however, by sequencing the VP1 region, the viruses could be identified as coxsackie A24 variants. Phylogenetic analysis of the 3C protease region revealed that these strains were closely related to each other as well as to genotype III isolates detected in Korea in 2002, thus proving their worldwide spread. This is the first report of an epidemic of acute hemorrhagic conjunctivitis due to a coxsackievirus A24 variant in Africa since 1987 and the first ever from Morocco.