Optimization of parameters for expression and purification of G glycoprotein ectodomain of respiratory syncytial virus

Aim: G glycoprotein ectodomain (Ge) of BA genotype of group B respiratory syncytial virus was expressed and purified to achieve maximum yield of the protein. Materials & methods: We optimized different parameters like strains, temperature, inducer concentration and post induction time period for efficient protein expression in Escherichia coli. The protein was purified using affinity chromatography and confirmed by western blotting. Results: It was concluded that a 5-h induction with 0.75 mM isopropyl β-D-1-thiogalactopyranoside at 37°C in BL21(DE3) cells was the most favorable condition for maximal protein expression. The far-UV circular dichroism spectroscopy suggested that it is an α-helical protein. Conclusion: The purified Ge protein can be characterized by antigenic and biophysical methods in future studies, which will probably assist in vaccine development.

Ten Years of Global Evolution of the Human Respiratory Syncytial Virus BA Genotype with a 60-Nucleotide Duplication in the G Protein Gene

ABSTRACT The emergence of natural isolates of human respiratory syncytial virus group B (HRSV-B) with a 60-nucleotide (nt) duplication in the G protein gene in Buenos Aires, Argentina, in 1999 (A. Trento et al., J. Gen. Virol. 84:3115-3120, 2003) and their dissemination worldwide allowed us to use the duplicated segment as a natural tag to examine in detail the evolution of HRSV during propagation in its natural host. Viruses with the duplicated segment were all clustered in a new genotype, named BA (A. Trento et al., J. Virol. 80:975-984, 2006). To obtain information about the prevalence of these viruses in Spain, we tested for the presence of the duplicated segment in positive HRSV-B clinical samples collected at the Severo Ochoa Hospital (Madrid) during 12 consecutive epidemics (1996-1997 to 2007-2008). Viruses with the 60-nt duplication were found in 61 samples, with a high prevalence relative to the rest of B genotypes in the most recent seasons. Global phylogenetic and demographic analysis of all G sequences containing the duplication, collected across five continents up until April 2009, revealed that the prevalence of the BA genotype increased gradually until 2004-2005, despite its rapid dissemination worldwide. After that date and coinciding with a bottleneck effect on the population size, a relatively new BA lineage (BA-IV) replaced all other group B viruses, suggesting further adaptation of the BA genotype to its natural host.

Shoot Proliferation and Rooting in Vitro of Pulmonaria

Expanding shoot tips of Pulmonaria `Roy Davidson' and Pulmonaria saccharata `Margery Fish' were cultured in vitro on a modified Murashige and Skoog medium containing BA to establish proliferating cultures for use in comparing BA concentrations on shoot proliferation and rooting. The optimum level for shoot proliferation was 8.8 μm BA. Greatest rooting was on medium without BA. Genotype and time in culture influenced shoot and root counts. Chemical names used: N6-benzyladenine (BA)