Figure 3: Phylogenetic analysis of HA (A) and NA (B) genes of A(H1N1)pdm09 viruses circulating in 2019-2020 (cw 42/2019 – 05/2020).
Virus genomes were analyzed by NGS and were phylogenetically evaluated with Mega7 (Neighbor-Joining method, bootstrap test with1000 replicates, Kimura 2-parameter method, partial deletion/site coverage cutoff 5%). 164 influenza viruses were characterized, of which 59 (HA gene) and 58 (NA gene) viruses are shown in the phylogenetic analyzes. Almost all identical sequences are removed. Numbering of nucleotide positions (nt) starting from the first nucleotide of the gene (HA nt33 – nt1730, NA nt21 – nt1430). Substitutions in the deduced HA and NA amino acid sequences were identified by FluSurver database. The identified HA substitutions are displayed according to H1-numbering. H1-numbering starts after the signal peptide (-17). Mutations in the signal peptide are marked as sig pep. Polymorphisms are indicated as e.g. F242F/L. Virus specific substitutions are indicated for HA and NA sequences from Germany and additionally for the vaccine strain (2019-2020) A/Brisbane/02/2018. Furthermore, the identified substitutions are color-coded and assigned to the protein domains according to Igarashi (2010, 5) and Yang (2010, 12): HA1 (black), HA2 (blue), antigenic site (red), receptor binding site (brown), both: antigenic site/receptor binding site (green). According to FluSurver the substitutions are highlighted that are significant for/associated with creation or removal of potential N-glycosylation site (pNLG) or antiviral drug resistance/reduced sensitivity.
The HA and NA sequences are marked as follows: viruses from 2019-2020-sentinel (black), 2019-2020-non-sentinel (additionally NRZ), 2019-2020-N156K-variant (red), viruses from outbreaks in hospitals and old people’s homes (outbreak), from vaccination breakthroughs after administration of trivalent (TIV) or quadrivalent (QIV) vaccine (2019-2020), viruses with reduced or no vaccine strain reactivity (HI40-640, noHI), viruses from patients with (respiratory) chronic pre-existing conditions (resp) chronicD), pneumonia, antiviral therapy, double infections with influenza and other viruses. The WHO reference sequences are marked in black / italics and the vaccine strains that were included in the 2019-2020 vaccine are red / italics. Intra-clade reassortants are framed.
The following abbreviations are used for the names of the virus isolates: BWB: Baden-Württemberg, BAY: Bavaria, BLN: Berlin, BBG: Brandenburg, BRE: Bremen, HAM: Hamburg, HES: Hessen, MVP: Mecklenburg-Vorpommern, NSA: Niedersachsen, NRW: North Rhine-Westphalia, RPF: Rhineland-Palatinate, SAS: Saxony, SAT: Saxony-Anhalt, SAL: Saarland, SHO: Schleswig-Holstein, THR: Thuringia.
Figure 4: Structural models of HA and NA proteins of A(H1N1)pdm09 viruses detected in upper respiratory specimens. HA (A-C), NA (D-F). Viruses with HA-N156K substitution are analyzed by FluSurver database: A/MVP/2/2020/cluster 1 (A/C), A/MVP/12/2019/cluster 3 (B/E), A/NRW/142/2019/cluster 4 (C/F). Substitutions are relative to the closest reference A/Brisbane/02/2018 and the position of substitutions (H1-numbering) are highlighted if mutations are close to common drug, host receptor or antibody binding sites or if a glycosylation motif is lost or created through a mutation (black=no known effect/interestlevel 0, green=subtype marker/interestlevel 0, blue=site of interaction/ interestlevel 1, orange=drug binding/host-cell specificity/antigenic shifts/mild drug resistance/ interestlevel 2, magenta= create/remove potential glycosylation site/ interestlevel 2, red=alter virulence/strong drug resistance/ interestlevel 3).