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8?��?103?RNA copies/mL) (p 0.01) (Fig.?1a). In two of these patients (pt#5 and #6), the HA sequence from the nasal swab could not be obtained because of low virus load ([https://en.wikipedia.org/wiki/E-64 E-64] out of four (75.0%) patients with 222G/N variants and available HA sequence in both nasal swab and BAL, the mutant viruses were detected only in the lower respiratory tract, whereas in a single patient (pt#2) a 222N variant was detected both in nasal swab and BAL samples (Fig.?1b). In these four patients (pt#2, #3, #8 and #9), the relative proportion of wild-type and mutant viruses in the upper vs lower respiratory tract was investigated by clonal analysis of HA sequences. For comparison, ten HA plasmid clones were also obtained from the nasal swab and [http://www.selleckchem.com/products/r428.html R428 ic50] BAL samples of pt#4, who did not show the presence of 222G/N variants in either specimen (Fig.?1b). The 222G/N variants were detected at clonal level in all patients. However, a significantly (p?[http://www.selleckchem.com/products/jq1.html JQ1 cost] who showed by direct sequencing 222G/N variants in both nasal swab and BAL, a comparable number of mutant variant specimens was observed by clonal analysis in both specimens (six of ten clones, 60% and five of ten clones, 50%, respectively). Although direct sequencing of HA amplicons from nasal swabs of pt#3, #8 and #9 did not detect mutant viruses, a minor proportion of 222G/N variants was shown in pt#3 and #9 by clonal analysis (one of ten clones, 10%). Similarly, a minor proportion of 222G/N variants not detected by direct sequencing was shown in nasal swab (one of ten, 10%) and BAL (two of ten, 20%) clones of pt#4. Using a stringent clinical definition of the severity of ILI in a multicentre prospective study, the association between the emergence of influenza A/H1N1/09v 222G/N virus variants and increased virulence was documented. Frequency of 222G/N variants in patients with severe ILI was strikingly higher than that in patients with moderate or mild ILI. Similarly, patients with moderate ILI more frequently harboured mutant viruses. In addition, it was shown that mutant virus variants segregated preferentially in the lower respiratory tract, where increased virus replication and higher virus load were also shown.