HA222 polymorphism in Influenza A(H1N1) 2009 isolates from Intensive Care Units and ambulatory patients during three influenza seasons.

Amino acid substitutions which can affect the receptor binding specificity of the influenza virus, like the substitution of aspartic acid with glycine in position 222 of the haemagglutinin (HA) of influenza virus A(H1N1) 2009, have been associated with increased viral pathogenicity and increased tropism for the lower respiratory tract. In this paper, the polymorphic site 222 and the site 223 of the HA1 polypeptide of H1N1 2009 viruses were analyzed in order to better clarify the role of these substitutions in H1N1 2009 virus virulence. Viral strains included in this study were collected in Tuscany during 3 different influenza seasons from patients with severe as well as with mild forms of influenza caused by A(H1N1) 2009 virus. In addition, the oseltamivir resistance of the H1N1 2009 strains circulating during the same seasons was monitored with the aim to evaluate whether these changes in the HA and in neuraminidase (NA) tend to be linked and to influence each other. Altogether, the results indicate that in severe forms of influenza viral population is more variable than in mild influenza, as regards the site 222. The frequency of such substitutions varied among the three seasons, it was highest in the season 2010-2011 and very low in the season 2012-2013. However these differences were not significant.

A FRET based melting curve analysis to detect nucleotide variations in HA receptor-binding site of H5N1 virus.

Outbreaks of highly pathogenic H5N1 influenza A virus represent a major public health problem because of the possibility of direct transmission of these viruses from avian species to humans. For influenza H5N1 hemagglutinin, a switch from SA-a-2, 3-Gal to SA-a-2, 6-Gal receptor specificity is a critical step that could lead to inter-human transmission. The monitoring of the receptor-binding preference of H5N1 viruses represents an instrument to detect a potential pandemic virus. The aim of this study was to develop a method based on the fluorescence resonance energy transfer (FRET) technology and melting peaks analysis for rapid screening of pandemic H5N1 influenza A virus. Three selected probes corresponding to a 23bp nucleotide sequence of the avian receptor-binding site were used in a real-time RT-PCR to detect nucleotide variations. Five strains of avian influenza A viruses isolated from avian species and two synthesized HA gene were tested. The results showed that the melting peaks analysis is a reliable screening method for detecting the variability of the H5N1 receptor-binding site.