Molecular Characterization of the Predominant Influenza A(H1N1)pdm09 Virus in Mexico, December 2011–February 2012

<div><p>When the A(H1N1)pdm09 pandemic influenza virus moved into the post-pandemic period, there was a worldwide predominance of the seasonal influenza A(H3N2) and B viruses. However, A(H1N1)pdm09 became the prevailing subtype in the 2011–2012 influenza season in Mexico and most of Central America. During this season, we collected nasopharyngeal swabs of individuals presenting with influenza-like illness at our institution in Mexico City. Samples were tested for seasonal A(H3N2) and B influenza viruses, as well as A(H1N1)pdm09 by real-time reverse transcription–polymerase chain reaction. Of 205 samples tested, 46% were positive to influenza, all of them A(H1N1)pdm09. The clinical characteristics of patients showed a similar pattern to the 2009 pandemic cases. Using next generation sequencing, we obtained whole genome sequences of viruses from 4 different patients, and in 8 additional viruses we performed partial Sanger sequencing of the HA segment. Non-synonymous changes found in the Mexican isolates with respect to the prototype isolate H1N1 (A/California/04/2009) included HA S69T, K163R and N260D unique to 2012 Mexican and North American isolates and located within or adjacent to HA antigenic sites; HA S143G, S185T, A197T and S203T previously reported in viruses from the 2010–2011 season, located within or adjacent to HA antigenic sites; and HA E374K located in a relevant site for membrane fusion. All Mexican isolates had an oseltamivir-sensitive genotype. Phylogenetic analysis with all 8 influenza gene segments showed that 2012 Mexican sequences formed a robust, distinct cluster. In all cases, 2012 Mexican sequences tended to group with 2010–2011 Asian and European sequences, but not with 2009 Mexican sequences, suggesting a possible recent common ancestor between these latter regions and the 2012 Mexican viruses. It remains to be defined if these viral changes represent an important antigenic drift that would enable viral immune evasion and/or affect influenza vaccine effectiveness.</p> </div

Maximum likelihood (ML) phylogenetic trees for the 8 influenza genetic segments.

<p>ML trees from 136 to 162 A(H1N1)pdm09 viruses registered in GenBank were produced with 1,000 bootstrap replicates, for the indicated genetic segments as explained in the Methods section. The four 2012 Mexican fully sequenced by next generation sequencing are included. Red dots at nodes show branches with >50% bootstrap support leading to the 2012 sequences described in this work. Branches are colored according to WHO influenza transmission zones <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050116#pone.0050116-WHO3" target="_blank">[13]</a>.</p

Genetic distances between Mexican 2012 isolates and viruses from all over the world.

<p>On the right side of the plot the average distance between the Mexican 2012 sequences and the sequences included for each geographical transmission zone <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050116#pone.0050116-WHO3" target="_blank">[13]</a> in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050116#pone-0050116-g003" target="_blank">Figure 3</a> is shown, for each influenza gene segment. On the left a histogram is shown with the distribution of the 20 closest sequences to the Mexican 2012 cluster for each viral segment. The four 2012 Mexican viruses sequenced by NGS are considered. A single virus from Central America and Caribbean and one from Central Asia were omitted from the distance graphs. SEA, South Eastern Asia; SWE, South Eastern Europe; EE, Eastern Europe; CA, Central Asia; OCP, Oceania, Micronesia and Polynesia; NA, North America; SA, Tempered South America; MX09, Mexican sequences from 2009 to 2011.</p

Baseline clinical data of the study population.

a<p>Patients presenting with influenza-like illness (ILI), A(H1N1)pdm2009 influenza virus not detected.</p>b<p>Patients presenting with ILI, A(H1N1)pdm2009 influenza virus detected.</p>c<p>Chi-square test.</p>d<p>9 patients, 4 confirmed and 5 suspect cases ignored vaccination status. Vaccinated cases confirmed by medical records.</p

Amino acid substitutions in the HA antigenic sites of the 2012 influenza A H1N1 Mexican isolates.

<p>Amino acid sequences are shown for the HA antigenic region of the four fully sequenced 2012 Mexican isolates (MX2012a-d, GenBank accession numbers JQ714072–JQ714075 respectively), the reference California 2009 isolate (CA2009, accession number CY054707) and a reference 2009 Mexican isolate (MX2009, accession number GQ402189). Amino acid positions are numbered without considering the HA signal peptide. Amino acid substitutions in the 2012 Mexican isolates are marked in red. Antigenic sites are shaded: Sa – purple, Sb – yellow, Ca – green, Cb – blue. Substitutions S69T, S143G, K163R, S185T, S203T and A197T are shown to be within or adjacent to antigenic sites.</p

Number of influenza like illness (ILI) cases (middle track) and proportion positive for the A(H1N1)pdm09 virus (upper track, 3 week moving average) during epidemic weeks 48–2011 to 07–2012 at the National Institute of Respiratory Diseases (INER) in Mexico City.

<p>Dates are for onset of symptoms. The lower track shows the number of influenza A cases reported in Mexico according to the WHO (<a href="http://apps.who.int/globalatlas/dataQuery/default.asp" target="_blank">http://apps.who.int/globalatlas/dataQuery/default.asp</a>, last accessed on February 27, 2012). Week 7 was omitted due to a lag in reporting to this database.</p