Abstract
In March/April 2009, a new pandemic influenza A virus (A(H1N1)pdm09) emerged and spread rapidly via human-to-human transmission, giving rise to the first pandemic of the 21th century. Influenza virus may be present in the infected host as a mixture of variants, referred to as quasi-species, on which natural and immune-driven selection operates. Since hemagglutinin (HA) and non-structural 1 (NS1) proteins are relevant in respect of adaptive and innate immune responses, the present study was aimed at establishing the intra-host genetic heterogeneity of HA and NS1 genes, applying ultra-deep pyrosequencing (UDPS) to nasopharyngeal swabs (NPS) from patients with confirmed influenza A(H1N1)pdm09 infection. The intra-patient nucleotide diversity of HA was significantly higher than that of NS1 (median (IQR): 37.9 (32.8–42.3) X 10−4 vs 30.6 (27.4–33.6) X 10−4 substitutions/site, p = 0.024); no significant correlation for nucleotide diversity of NS1 and HA was observed (r = 0.319, p = 0.29). Furthermore, a strong inverse correlation between nucleotide diversity of NS1 and viral load was observed (r = - 0.74, p = 0.004). For both HA and NS1, the variants appeared scattered along the genes, thus indicating no privileged mutation site. Known polymorphisms, S203T (HA) and I123V (NS1), were observed as dominant variants (>98%) in almost all patients; three HA and two NS1 further variants were observed at frequency >40%; a number of additional variants were detected at frequency <6% (minority variants), of which three HA and four NS1 variants were novel. In few patients multiple variants were observed at HA residues 203 and 222. According to the FLUSURVER tool, some of these variants may affect immune recognition and host range; however, these inferences are based on H5N1, and their extension to A(H1N1)pdm09 requires caution. More studies are necessary to address the significance of the composite nature of influenza virus quasi-species within infected patients.
Highlights
In early 2009, influenza A virus (H1N1)pdm09 emerged in Mexico and spread rapidly via human-to-human transmission, giving rise to the first pandemic of the 21th century [1]
No significant correlation (r = 0.319, p = 0.29) for nucleotide diversity of non-structural 1 (NS1) and HA genes was observed; no correlation between nucleotide diversity and age was found both for HA (r = -0.46, p = 0.13) and NS1 (r = 0.041, p = 0.89); a strong inverse correlation between nucleotide diversity of NS1 and viral load (r = -0.74, p = 0.004) was observed (Fig 1), but not between nucleotide diversity of HA and viral load (r = 0.017, p = 0.96)
Concerning selective pressure, the results indicated just the codon 103 of NS1 to be under negative selective pressure (p = 0.0045), while no selective pressure seems to act on HA gene, at least within the analyzed regions
Summary
In early 2009, influenza A virus (H1N1)pdm emerged in Mexico and spread rapidly via human-to-human transmission, giving rise to the first pandemic of the 21th century [1]. This virus was a unique combination of influenza virus genes never previously identified in either animals or people [2,3]. HA and NS1 Variability in A(H1N1)pdm Infected Patients particular that of RNA viruses, is characterized by high turnover, high mutational rates and large population sizes These conditions lead to a constant production of a large number of viral variants, creating high genetic diversity on which natural and immune-driven selection operates. Recent studies, based on generation sequencing (NGS), revealed for influenza virus patterns of intra-host viral variability in a variety of in vitro and ex vivo systems, such as swine respiratory cells [8], infected poultry [9] and human patients [10,11]
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