Measles virus genetic evolution throughout an imported epidemic outbreak in a highly vaccinated population.

Measles virus circulates endemically in African and Asian large urban populations, causing outbreaks worldwide in populations with up-to-95% immune protection. We studied the natural genetic variability of genotype B3.1 in a population with 95% vaccine coverage throughout an imported six month measles outbreak. From first pass viral isolates of 47 patients we performed direct sequencing of genomic cDNA. Whilst no variation from index case sequence occurred in the Nucleocapsid gene hyper-variable carboxy end, in the Hemagglutinin gene, main target for neutralizing antibodies, we observed gradual nucleotide divergence from index case along the outbreak (0% to 0.380%, average 0.138%) with the emergence of transient and persistent non-synonymous and synonymous mutations. Little or no variation was observed between the index and last outbreak cases in Phosphoprotein, Nucleocapsid, Matrix and Fusion genes. Most of the H non-synonymous mutations were mapped on the protein surface near antigenic and receptors binding sites. We estimated a MV-Hemagglutinin nucleotide substitution rate of 7.28 × 10-6 substitutions/site/day by a Bayesian phylogenetic analysis. The dN/dS analysis did not suggest significant immune or other selective pressures on the H gene during the outbreak. These results emphasize the usefulness of MV-H sequence analysis in measles epidemiological surveillance and elimination programs, and in detection of potentially emergence of measles virus neutralization-resistant mutants.

Membrane cell fusion activity of the vaccinia virus A17-A27 protein complex.

Vaccinia virus enters cells by endocytosis and via a membrane fusion mechanism mediated by viral envelope protein complexes. While several proteins have been implicated in the entry/fusion event, there is no direct proof for fusogenic activity of any viral protein in heterologous systems. Transient coexpression of A17 and A27 in mammalian cells led to syncytia formation in a pH-dependent manner, as ascertained by confocal fluorescent immunomicroscopy. The pH-dependent fusion activity was identified to reside in A17 amino-terminal ectodomain after overexpression in insect cells using recombinant baculoviruses. Through the use of A17 ectodomain deletion mutants, it was found that the domain important for fusion spanned between residues 18 and 34. To further characterize A17-A27 fusion activity in mammalian cells, 293T cell lines stably expressing A17, A27 or coexpressing both proteins were generated using lentivectors. A27 was exposed on the cell surface only when A17 was coexpressed. In addition, pH-dependent fusion activity was functionally demonstrated in mammalian cells by cytoplasmic transfer of fluorescent proteins, only when A17 and A27 were coexpressed. Bioinformatic tools were used to compare the putative A17-A27 protein complex with well-characterized fusion proteins. Finally, all experimental evidence was integrated into a working model for A17-A27-induced pH-dependent cell-to-cell fusion.