Selected PB2 amino acid positions showing cosubstitution with K627E.

<p>Selected PB2 amino acid positions showing cosubstitution with K627E.</p

Stereography of influenza A H7N9 PB2 residue-varying positions listed in Table 3.

<p>(A and B) Simulated H7N9 PB2 stereography for A/Taiwan/4-CGMH2/2014(H7N9) viral PB2 is shown in the ribbon mode. (C and D) The distribution of electrostatic potential on the protein surface (blue = relative positive charge; red = relative negative charge). Protein modeling was performed using SWISS-MODEL by applying Coulomb’s law in Chimera according to the full-length PB2 of A/little yellow-shouldered bat/Guatemala/060/2010(H17N10) (PDB ID 4WSB).</p

Statistics for the RNP activity assay of the influenza A H7N9 PB2.

<p>(A) RNP activity for the H7N9 virus in 293T cells. The RNP complexes exhibiting combinations of the avian signature PB2 627E and its covarying residues (positions labeled with asterisks in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148432#pone.0148432.t003" target="_blank">Table 3</a>) from the A/Anhui/1/2013(H7N9) virus were cotransfected into the 293T cells. CAT ELISA was performed 48 h after transfection to detect protein expression levels of the virus-like CAT reporter, thereby signaling the viral RNP activity. The error bars show the SEM from 3 independent experiments. Two-way ANOVA and the GraphPad Prism Software were used to statistically support the observed disparity. (B) Each component of the viral RNP complex and NP was detected using Western blotting.</p

Web logos for H7N9 genome displaying at least 7 substitutions.

<p>Positions accompanied by red dots are among the 47 species-associated signature positions listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148432#pone.0148432.t002" target="_blank">Table 2</a>. The leading three logos for PA-X and PA are identical, as the two proteins share the 191 amino acids at the N terminal. Sequence counts used in each of these logos are included in the parentheses as PB2 (147), PB1 (137), PB1-F2 (134), PA (139), PA-X (132), HA (142), NP (140), NA (141), M1 (143), M2 (142), NS1 (141), NS2 (142). The Y-axis shows the residue frequency for each residue, which adds up to 1.0.</p

Amino acid compositions at 47 species-associated signature positions for H7N9 viruses.

<p>Amino acid compositions at 47 species-associated signature positions for H7N9 viruses.</p

Amino acid variations of the four Taiwanese H7N9 genomes since 2013.

<p>Amino acid variations of the four Taiwanese H7N9 genomes since 2013.</p

Amino Acids Transitioning of 2009 H1N1pdm in Taiwan from 2009 to 2011

<div><p>A swine-origin influenza A was detected in April 2009 and soon became the 2009 H1N1 pandemic strain (H1N1pdm). The current study revealed the genetic diversity of H1N1pdm, based on 77 and 70 isolates which we collected, respectively, during the 2009/2010 and 2010/2011 influenza seasons in Taiwan. We focused on tracking the amino acid transitioning of hemagglutinin (HA) and neuraminidase (NA) genes in the early diversification of the virus and compared them with H1N1pdm strains reported worldwide. We identified newly emerged mutation markers based on A/California/04/2009, described how these markers shifted from the first H1N1pdm season to the one that immediately followed, and discussed how these observations may relate to antigenicity, receptor-binding, and drug susceptibility. It was found that the amino acid mutation rates of H1N1pdm were elevated, from 9.29×10⁻³ substitutions per site in the first season to 1.46×10⁻² in the second season in HA, and from 5.23×10⁻³ to 1.10×10⁻² in NA. Many mutation markers were newly detected in the second season, including 11 in HA and 8 in NA, and some were found having statistical correlation to disease severity. There were five noticeable HA mutations made to antigenic sites. No significant titer changes, however, were detected based on hemagglutination inhibition tests. Only one isolate with H275Y mutation known to reduce susceptibility to NA inhibitors was detected. As limited Taiwanese H1N1pdm viruses were isolated after our sampling period, we gathered 8,876 HA and 6,017 NA H1N1pdm sequences up to April 2012 from NCBI to follow up the dynamics of mentioned HA mutations. While some mutations described in this study seemed to either settle in or die out in the 2011–2012 season, a number of them still showed signs of transitioning, prompting the importance of continuous monitoring of this virus for more seasons to come.</p> </div

NA mutation dynamics of Taiwanese H1N1pdm viruses versus publicly available H1N1pdm viruses.

<p>Horizontal axis represents the year/month the sampling took place, and the vertical axis represents the frequency (in percentage) that one particular mutation occurred in the month. Taiwanese data range from June 2009 to February 2011, and are graphed by various markers (circles, squares, triangles, and diamonds). A total of 6,017 H1N1pdm NA sequences are collected from NCBI which cover a 3-year span from April 2009 to April 2012, and are graphed in thick lines. Mutations are grouped according to the transitioning types described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045946#pone-0045946-t002" target="_blank">Table 2</a>, and are displayed from top to bottom as type I, II.a, II.b and II.c.</p

Correlation of significant mutation sites on HA and NA genes between severe and non-severe cases of H1N1pdm infection.

a<p>Upper respiratory tract infection.</p>b<p>Pneumonia, Acute respiratory distress syndrome, Expired.</p>*<p>Fisher’s exact test.</p

Chronological surveillance of influenza viruses in Chang Gung Memorial Hospital from 2007 to 2012.

<p>Chronological surveillance of influenza viruses in Chang Gung Memorial Hospital from 2007 to 2012.</p