Genotype differentiation of highly pathogenic avian influenza viruses (HPAIV) of the goose/Guangdong lineage in Germany - Derivation and deployment of reference sequences

Genotype differentiation of highly pathogenic avian influenza viruses (HPAIV) of the goose/Guangdong lineage in Germany - Derivation and deployment of reference sequences. Summary Highly pathogenic avian influenza viruses (HPAIV) of the H5 goose/Guangdong (gs/GD) lineage have repeatedly emerged in Germany since 2006. Rooted in the respective gs/GD lineages, HPAI viruses in Germany have genetically diversified into a plethora of clades and subclades and evolved into an assortment of suband genotypes. This technical note summarizes the genotype differentiation procedure, lists the successfully assigned genotypes and supplies corresponding reference sequences. Methodology Genotype differentiation and derivation of reference sequence were established with a combined phylogenetic and similarity-based method. Full genome sequences from clinical samples and virus isolates were continuously screened for new genotypes and compared with the derivated reference sequences. For genotype assignment segment-specific multiple alignments were generated using MAFFT (Katoh & Standley, 2013). In addition, alignments of concatenated genomes were generated and included in the analysis. Subsequent maximum likelihood (ML) trees were calculated separately for all segments and for the concatenated genome alignment with RAxML (Stamatakis, 2014) utilizing model GTR GAMMA with rapid bootstrapping and search for the best scoring ML tree supported with 1000 bootstrap replicates. Genotypes were assigned, and new genotypes were differentiated if they clustered separately as a monophyletic group with robust bootstrapping values (>80) and if differences in normalized patristic distances were observed at segment level. The first complete genome sequence of a newly detected genotype is used as a reference sequence, and additional references for new genotypes were derived as needed. Genotype designation here is filed to include locality (ISO 3166-1 alpha-2), date of first discovery (year-month), and NA subtype. When multiple genotypes of the same subtype were assigned within the same locality and date, the names were numbered consecutively. Reference sequences were deployed along HA clades and HPAIV season and are available here. The nomenclature system currently in use here is different from that proposed by the European Reference Laboratory (EURL) for Avian Influenza, Padova, Italy; the corresponding EURL designation is shown in the last column of the table provided if applicable. 2 Overview of genotypes season year clade subtype genotype EURL US 2006 2.2.1 H5N1 DE-06-03-N1 2.2.2 H5N1 DE-06-02-N1 2007 2.2 H5N1 DE-07-06-N1 2008 2.2.1 H5N1 DE-08-10-N1 2014/2015 2014 2.3.4.4a H5N8 DE-14-11-N8 2016/2017 2016 2.3.4.4b H5N5 DE-16-12-N5.1 DE-16-12-N5.2 H5N8 DE-16-11-N8 DE-16-12-N8.1 DE-16-12-N8.2 2016/2017 2017 2.3.4.4b H5N6 DE-17-12-N6 2019/2020 2020 2.3.4.4b H5N8 DE-20-01-N8 Enzootic period 2.3.4.4b H5N8 DE-20-10-N8 A DE-20-10-N8.1 DE-20-10-N8.2 H5N5 DE-20-10-N5 2.3.4.4b H5N3 DE-20-12-N3 2021 2.3.4.4b H5N1 DE-21-02-N1 C DE-21-07-N1 C DE-21-10-N1.1 C A1A2 DE-21-10-N1.2 C A5 DE-21-10-N1.3 C DE-21-10-N1.4 DE-21-10-N1.5 AB DE-21-11-N1.1 DE-21-11-N1.2 DE-21-11-N1.3 DE-21-11-N1.4 A3 DE-21-11-N1.5 DE-21-11-N1.6 DE-21-12-N1.1 A4 DE-21-12-N1.2 DE-21-12-N1.3 DE-21-12-N1.4 DE-21-12-N1.5 DE-21-12-N1.6 H5N4 DE-21-02-N4 H5N8 DE-21-02-N8 DE-21-03-N8 DE-21-08-N8.1 DE-21-08-N8.2 2022 2.3.4.4b H5N1 DE-22-01-N1.1 3 DE-22-01-N1.2 DE-22-04-N1.1 CA DE-22-09-N1.1 CC DE-22-11-N1.1 CH DE-22-12-N1.1 CH H5N2 DE-22-01-N2.1 AU 2023 2.3.4.4b H5N1 DE-23-02-N1.1 BB H5N1 DE-23-11-N1.1 DB H5N1 DE-23-11-N1.2 AB H5N1 DE-23-11-N1.3 DG H5N1 DE-23-11-N1.4 DA 2024 2.3.4.4b H5N1 DE-24-01-N1.1 DJ H5N1 DE-24-01-N1.2 DO H5N1 DE-24-02-N1.1 DQ H5N1 DE-24-02-N1.2 DR H5N8 DE-24-03-N8.1 DS H5N1 DE-24-03-N1.1 DI.1 H5N1 DE-24-10-N1.1 DI.2 H5N5 DE-24-02-N5.1 I A6 H5N1 DE-24-11-N1.1 EF 2025 2.3.4.4b H5N1 DE-25-01-N1.1 EE H5N1 DE-25-02-N1.1 EJ H5N1 DE-25-04-N1.1 EK 2025 2.3.4.4.b H5N1 DE-25-10-N1.1 DI.2.1 References Katoh K & Standley DM (2013) MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol 30(4):772-780. Stamatakis A (2014) RAxML version 8: a tool for phylogenetic analysis and postanalysis of large phylogenies. Bioinformatics 30(9):1312-1313. Pohlmann, A. (2023) HPAIV Genotypes in Germany. Zenodo. doi.org/10.5281/zenodo.8135769