N-linked glycosylation of the hemagglutinin protein influences virulence and antigenicity of the 1918 pandemic and seasonal H1N1 influenza A viruses.

The hemagglutinin (HA) protein is a major virulence determinant for the 1918 pandemic influenza virus; however, it encodes no known virulence-associated determinants. In comparison to seasonal influenza viruses of lesser virulence, the 1918 H1N1 virus has fewer glycosylation sequons on the HA globular head region. Using site-directed mutagenesis, we found that a 1918 HA recombinant virus, of high virulence, could be significantly attenuated in mice by adding two additional glycosylation sites (asparagine [Asn] 71 and Asn 286) on the side of the HA head. The 1918 HA recombinant virus was further attenuated by introducing two additional glycosylation sites on the top of the HA head at Asn 142 and Asn 172. In a reciprocal experimental approach, deletion of HA glycosylation sites (Asn 142 and Asn 177, but not Asn 71 and Asn 104) from a seasonal influenza H1N1 virus, A/Solomon Islands/2006 (SI/06), led to increased virulence in mice. The addition of glycosylation sites to 1918 HA and removal of glycosylation sites from SI/06 HA imposed constraints on the theoretical structure surrounding the glycan receptor binding sites, which in turn led to distinct glycan receptor binding properties. The modification of glycosylation sites for the 1918 and SI/06 viruses also caused changes in viral antigenicity based on cross-reactive hemagglutinin inhibition antibody titers with antisera from mice infected with wild-type or glycan mutant viruses. These results demonstrate that glycosylation patterns of the 1918 and seasonal H1N1 viruses directly contribute to differences in virulence and are partially responsible for their distinct antigenicity.

Novel copper (Cu) loaded core-shell silica nanoparticles with improved Cu bioavailability: synthesis, characterization and study of antibacterial properties.

We report synthesis of a novel core-shell silica based antimicrobial nanoparticles where the silica shell has been engineered to accommodate copper (Cu). Synthesis of the core-shell Cu-silica nanoparticle (C-S CuSiO2NP) involves preparation of base-hydrolyzed Stöber silica "seed" particles first, followed by the acid-catalyzed seeded growth of the Cu-silica shell layer around the core. The Scanning Electron Microscopy (SEM) and the Transmission Electron Microscopy (TEM) measured the seed particle size to be -380 nm and the shell thickness to be -35 nm. The SEM particle characterization confirms formation of highly monodispersed particles with smooth surface morphology. Characterization of particle size distribution in solution by Dynamic Light Scattering (DLS) technique was fairly consistent with the electron microscopy results. Loading of Cu to nanoparticles was confirmed by the SEM-Energy Dispersive X-Ray Spectroscopy (EDS) and Atomic Absorption Spectroscopy (AAS). The Cu loading was estimated to be 0.098 microg of metallic copper per mg of C-S CuSiO2NP material by the AAS technique. Antibacterial efficacy of C-S CuSiO2NP was evaluated against E. coli and B. subtilis using Cu hydroxide ("Insoluble" Cu compound, sub-micron size particles) as positive control and silica "seed" particles (without Cu loading) as negative control. Bacterial growth in solution was measured against different concentrations of C-S CuSiO2NP to determine the Minimum Inhibitory Concentration (MIC) value. The estimated MIC values were 2.4 microg metallic Cu/mL for both E. coli and B. subtilis. Bac-light fluorescence microscopy based assay was used to count relative population of the live and dead bacteria cells. Antibacterial study clearly shows that C-S CuSiO2NP is more effective than insoluble Cu hydroxide particles at equivalent metallic Cu concentration, suggesting improvement of Cu bioavailability (i.e., more soluble Cu) in C-SCuSiO2NP material due to its core-shell design.