Detection of co-circulating HA-222 variants of influenza A(H1N1)pdm09 viruses identified during 2009/2010 and 2010/2011 by PSQ analysis.

NA: not applicable.

P: p-value of difference of fatal/fatal and severe versus mild cases with HA-222 mutation, calculated by Fisher's exact test, two-sided.

For identification of HA-222 variants, RNA was extracted from patient specimens. Mild cases collected in 2009/2010 constituted an exception, because only 10% were represented by patient specimens and 90% by cell culture isolates, respectively.

PM: polymorphism. In 2009/2010, HA-222 polymorphisms were detected using the PSQ primer SF662 and the cyclic entry (ACGT)₁₀. All HA-222 variants in mixtures could not be clearly identified by the applied cyclic entry. Two quasispecies (222D/G, 222D/G/N/V/Y) could be confirmed by TA cloning and subsequent pyrosequencing. In 2010/2011, direct pyrosequencing of all circulating 222 variants and quasispecies could be realized by the application of the PSQ primer SF663 and the customized entry ATATGTAT(ACGT)₅.