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

<p>NA: not applicable.</p><p>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.</p>A<p>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.</p>B<p>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.</p

Prevalence of co-circulating HA-222 variants and HA-222 quasispecies of influenza A(H1N1)pdm09 viruses in the upper and lower respiratory tract identified in 2009/2010 and 2010/2011.

<p>Data represented mainly single upper or lower airway specimens of one patient.</p

Tissue distribution of the HA-222D/G quasispecies associated with a fatal clinical outcome.

<p>A nasal swab, lung and heart tissue samples were collected (specimens 2158–2160/11). The pyrograms demonstrate a 222D/G (GAT/GGT) polymorphism in the nasal swab (A), an increase of 222G in the lung (B) and in the heart tissue (C) relative to the wild type 222D.</p

PSQ analysis of a HA-222D/G/N/V/Y polymorphism identified in a fatal case.

<p>The pyrogram represents a HA-222 polymorphism (specimen 3070/10) (A). After amplification and TA cloning of the HA-222 fragment, 23 clones were analyzed using the cyclic entry (CTGA)₁₀. Three of them show the wild-type 222D with codon G<u>GAT</u> (B), seven 222G (G<u>GGT)</u> (C), four 222N (G<u>AAT)</u> (D), one 222V (G<u>GTT)</u> (E) and eight clones 222Y (G<u>TAT)</u> (F).</p

Clinical characteristics of patients with influenza A(H1N1)pdm09 virus infection associated with fatal and severe outcomes.

<p>The characteristics were indicated as provided by the attending physicians.</p>*<p>In 2009/2010 and 2010/2011 the A(H1N1)pdm09 PCR-positive samples associated with fatal and severe outcomes which were provided by hospitals were analyzed in the current study. Additionally, in 2010/2011 all sentinel samples sent by the medical practices were screened and the A(H1N1)pdm09 PCR-positive specimens that were associated with pneumonia were included in the group of severe outcomes.</p

PSQ analysis of a HA-222D/G polymorphism detected in a severe case.

<p>The pyrogram shows a HA-222 polymorphism (specimen 3004/10) (A). After amplification and TA cloning of the HA-222 fragment, 30 clons were picked and subsequently pyrosequenced using the cyclic entry (CTGA)₁₀. Fifteen clones are identified as wild-type 222D with codon G<u>GAT</u> (B) and 15 clones as variant 222G with codon G<u>GGT</u> (C).</p

Detection of pure and heterogeneous HA-222 variants using the cyclic dispensation order (ACGT)₁₀ and the customized dispensation order ATATGTAT(ACGT)₅.

<p>The pyrograms represent the wild-type HA-222D (G<u>GAT</u>) using the PSQ primer SF662 and (ACGT)₁₀ (A), HA-222D/G (G<u>GAT</u>/G<u>GGT</u>) polymorphism of a fatal case (448/11) utilizing the PSQ primer SF662A and (ACGT)₁₀ (B) and HA-222D/G/N polymorphism of the same specimen (448/11) as in (B) applying the PSQ primer SF663 and ATATGTAT(ACGT)₅ (C).</p

Primer and probe sequences.

<p>All oligonucleotide sequences are listed in 5′–3′ orientation. M = Matrix, HA = Hemagglutinin, NA = Neuraminidase, n.a. = not applicable.</p

Emergence of the HA-222D/G quasispecies during a severe course of disease.

<p>The BAL samples were collected 22, 29 and 43 days after the onset of ILI symptoms (specimens 3340/11, 3341/11, 3343/11). The pyrograms show the wild-type 222D (GAT) (A), the emergence of the 222G variant (B) and the increase of the 222G variant (C) relative to the wild-type 222D.</p

PCR assay validation results.

a<p>validation data was obtained on a Stratagene Mx3000 instrument for the assays indicated with an asterisk. All other assays were validated on a Stratagene Mx3005 instrument.</p>b<p>slope, efficiency (E) and correlation (R²) of standard curve; PCR efficiency was calculated as E = 10^(−1/slope)−1.</p>c<p>Limit of detection (LOD) was calculated as 95% detection probability by probit analyses applying the SPSS 17.0 Statistics software.</p>d<p>reproducibility was calculated by examination of indicated plasmid copy numbers; intraassay: sixfold examination in a single run, interassay: twofold examination of double reactions plus inclusion of intraassay data; standard deviation of obtained Ct values are listed.</p