Analyses of selection pressure on the Hemagglutinin gene of influenza A/H3N2 Viruses circulating in Kenya 2007-2011

Background: The hemagglutinin (HA) gene of Influenza viruses, especially the HA1 portion, exhibits a rapid rate of change, largely in response to human immune surveillance in a partially immune human population. Mutations in influenza viral genes accumulate over time and are under selection pressure during epidemics or pandemics. Objective: To determine whether the Kenyan influenza A/H3N2 viruses are undergoing adaptive evolution to become epidemic threats. Methods: Nasopharyngeal samples from patients meeting the WHO ILI case definition were collected between 2007 and 2010 from across Kenya. The detection of H3N2 virus was carried out using real-time RT-PCR. Positive samples were then cultured in MDCK cells and confirmed using the HAI assay. 156 isolates from this period were selected for amplification of the HA1 portion of the HA gene and the resulting amplicons sequenced. Global estimates, ω, of dN and dS, averaged over the entire alignment, were compared to calculate the overall strength of selection using the HyPhy 2.0 software package implemented in datamonkey. Results: Analysis of neutrality using, Kumar’s method showed that ω varied from 0.50 in 2007, 0.36 in 2008, 0.32 in 2009, 0.61 in 2010 and 0.41 in 2011. Further site by site analysis identified amino acid positions 46, 158 and 173 to be under positive selection. Analysis of differential selection showed 7 sites that harbored two or more amino acid substitutions. Amino acid positions 158, 160 and 189 had the highest number of amino acid polymorphisms. Conclusions: Overall, this study shows that local Influenza A(H3N2) viruses have been evolving via a series of ‘adaptive bursts’ characterized by positive selection occurring largely in immunological epitopes B and D. In between these bursts there is little evidence for positive selection and newly-emergent strains slowly replace the existent strains. Based on this study alone, we propose that these bursts occur after every two years. Since in a single population, dN/dS <1does not follow monotonic function, it is difficult to infer selection pressure in these results. However, by focusing on the antigenic sites we were able to observe an evolutionary pattern in the Kenyan samples. Thus evolution of Kenyan Influenza A(H3N2) is characterized by non synonymous changes followed by a period of stasis that is then followed by another period of non synonymous changes which is followed by purification selection