Molecular epidemiology of influenza A(H1N1)PDM09 hemagglutinin gene circulating in São Paulo State , Brazil: 2016 anticipated influenza season.

Compared to previous years, seasonal influenza activity commenced early in São Paulo State, Brazil, Southern hemisphere during the 2016 year. In order to investigate the genetic pattern of influenza A(H1N1)pdm09 in the State of Sao Paulo a total of 479 respiratory samples, collected in January by Sentinel Surveillance Units, were screened by real-time RT-PCR. A total of 6 Influenza viruses A(H1N1)pdm09 presenting ct values ≤ 30 were sequenced following phylogenetic analysis. The present study identified the circulation of the new 6B.1 subgroup (A/Sao Paulo/10-118/2016 and A/Sao Paulo/3032/2016). In addition, influenza A(H1N1)pdm09 group 6B has also been identified during January in the State of Sao Paulo. Despite amino acid changes and changes in potential glycosylation motifs, 6B.1 viruses were well inhibited by the reference ferret antiserum against A/California/07/2009 virus, the A(H1N1)pdm09 component of the vaccine for the 2016 influenza season.

Compared to previous years, seasonal influenza activity commenced early in São Paulo State, Brazil, Southern hemisphere. Influenza A(H1N1)pdm09 virus detection started in January 2016 during the summer season with hot temperatures, and it was the predominating strain in autumn , . In contrast, influenza seasonal activity commenced late in some countries in Western Europe, North America and Eastern Asia. Based on the WHO global influenza surveillance, in countries with influenza A(H1N1)pdm09 virus predominance, the hospitalization and intensive care unit (ICU) admission patterns seem to be similar to previous seasons when this virus predominated and young/middle-aged adults experienced severe disease . The aim of this study was to investigate the genetic pattern of influenza A(H1N1)pdm09 in the São Paulo State.

A total of 479 respiratory samples, collected in January by Sentinel Surveillance Units, were screened by real-time RT-PCR (qRT - PCR) . Among them, 30 Influenza virus A(H1N1)pdm09 presenting ct values ≤ 30 were identified. A total of 6 viruses were sequenced by using an Applied Biosystems BigDye(r) Terminator v3.1 Cycle Sequencing Kit with reaction products resolved on an Applied Biosystems Sequencer 3730 DNA Analyzer. Nucleotide sequences were aligned using MUSCLE . Sequences alignment results were further analyzed using the BioEdit program .

Our Institution is one of the National Influenza Centers accredited by the World Health Organization. The present follow up study of influenza surveillance has been approved by the Conselho Nacional de Ética em Pesquisa (CONEP) -135/2002.

Phylogenetic analyses

The TREESUB phylogenetic program (available from https://github.com/tamuri/treesub) was used to estimate the maximum likelihood phylogenetic trees using RAxML and PAML, followed by branch annotation of amino acid substitutions. The general time reversible+𝚪 (GTR+GAMMA) nucleotide substitution model was selected in RAxML v.7.3.0 for tree inference . Ancestral codon substitutions for each gene were estimated using baseml, as implemented in PAML using the ML trees inferred. Nonsynonymous substitutions were then transcribed onto the consensus gene phylogenies and visualized in Figure 1 Tree v1.4.2 (available from http://tree.bio.ed.ac.uk/software/figtree/).

Figure 1

Molecular epidemiology of influenza A(H1N1)pdm09 hemagglutinin gene circulating in São Paulo, state, Brazil: 2016 anticipated influenza season.

Influenza A(H1N1)pdm09 viruses identified in São Paulo State during November 2015 belong to the 6B genetic group, presenting AA changes in the HA at residue E179D and I183V in the HA2 region. Figure 1 shows two viruses from Brazil collected in January that belong to the new 6B.1 subgroup (A/Sao Paulo/10-118/2016 and A/Sao Paulo/3032/2016). The HA sequences from São Paulo State obtained in this study, which were also used in the phylogenetic analysis, were deposited in the EpiFlu database of the Global Initiative on sharing Avian Influenza Data (GISAID) under the following accession numbers: EPI725841, EPI725838.

Influenza A(H1N1)pdm09 group 6B has also been identified during January in São Paulo State as shown in Figure 1, which were also used in the phylogenetic analysis; A/Sao Paulo/3050/2016; A/Sao Paulo/1267/2016; A/Sao Paulo/815/2016; A/Sao Paulo/10-441/2016 deposited in the GISAID under the follow accession numbers EPI725839, EPI725844, EPI704027, EPI725842, respectively.

The phylogenetic analysis of the A(H1N1)pdm09 HA gene demonstrated that HA genes of the recent viruses diverged into genetic groups 6A, 6B or 6C, with all viruses collected since September 1st, 2015 belonging to the genetic subgroup 6B. Subgroups 6B and 6C share AA changes in the HA at residues K283E and E499K (mature A(H1N1)pdm09 numbering after the signal peptide). Subgroup 6B viruses also possess AA changes in the HA at residues K163Q and A256T. Within subgroup 6B, additional subgroups with shared amino acid changes have emerged. The majority of viruses share an AA change at residue S84N; among these more than half share two additional changes at residues S162N (adds a glycosylation motif) and I216T (new subgroup 6B.1). A smaller group of 6B viruses (new subgroup 6B.2) shares AA changes at residues V152T, V173I, E491G and D501E .

Despite amino acid changes and changes in glycosylation, 6B.1 viruses were well inhibited by the reference ferret antiserum against A/California/07/2009 virus the A(H1N1) pdm09 component of the vaccine for 2016 influenza season .

In addition, a pool of human post-vaccination sera collected from healthy adults in the United States of America who received influenza vaccine in the 2015-2016 seasons well inhibited all recent viruses tested in the WHO CC at the Centers for Disease Control and Prevention (CDC) in Atlanta .

According to the literature data, the United Kingdom (UK), as others countries in Europe, has experienced a season dominated by circulation of influenza A(H1N1)pdm09 with reports of increases in hospitalizations and ICU admissions mainly in younger adults. The epidemiological observations are consistent with earlier seasons in the UK dominated by circulation of A(H1N1)pdm09, in particular in 2010/11, the first post-pandemic season . In São Paulo State this influenza virus A(H1N1)pdm09 pattern of seasonality occurred in 2013 (personnel communication), and in the currently anticipated influenza virus season 2016, influenza A(H1N1)pdm09 predominated.

Influenza virological surveillance follow-up will provide the antigenic and phylogenetic patterns of influenza A(H1N1)pdm09 virus circulation during the coming winter and early spring period in São Paulo State. Taking into account that Brazilian vacation period corresponds to the North America and Western Europe influenza season (January/February), and also considering the late influenza virus seasonality in these regions, this pattern of seasonality may be considered to interpret the earlier influenza seasonality in São Paulo State in 2016.