ArticleViruses2025
Influenza PA Substitutions and Genetic Diversity of A(H1N1)pdm09, A(H3N2), and B/Victoria Viruses in Japan During the 2023-2024 Season.
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Abstract
We characterized influenza A(H1N1)pdm09, A(H3N2), and B/Victoria viruses circulating in Japan during 2023-2024, focusing on lineage placement relative to WHO-recommended vaccine strains and on baloxavir resistance (PA/I38T substitutions). We enrolled 210 outpatients with influenza-like illness across eight clinics in six prefectures (October 2023-September 2024). Of these, 209 had an analyzable pre-treatment respiratory specimen for RT-PCR; hemagglutinin (HA) and neuraminidase (NA) genes were sequenced by next-generation sequencing (NGS). PA/I38T substitutions that confer baloxavir resistance were assessed by cycling-probe RT-PCR, Sanger sequencing, and NGS. HA phylogenies were constructed with global datasets and WHO vaccine reference strains. Of 209 pre-treatment specimens, 181 were influenza-positive (A(H1N1)pdm09 44.2%, A(H3N2) 37.6%, B/Victoria 18.2%); 51 follow-up specimens were collected ≈4-5 days after baloxavir or neuraminidase inhibitor therapy. HA phylogeny placed A(H1N1)pdm09 in clades 5a.2a/5a.2a.1 with predominance of subclade D.2. A(H3N2) clustered exclusively in clade 2a.3a.1 (J lineage, mostly J.1), indicating a mismatch with the season's A/Darwin/9/2021 vaccine component and supporting the subsequent J-lineage update. All B/Victoria genomes fell within V1A.3a.2 on a C.5 backbone (C.5.1 and C.5.7). No PA/I38T variant was detected in any pre-treatment specimen. Post-baloxavir, PA/I38T emerged in one A(H3N2) case (confirmed by all three methods) and in one B/Victoria case detected by NGS only (minority variant in a low-load sample). NA genes showed no substitutions associated with reduced susceptibility to laninamivir (e.g., E119A, G147E). During 2023-2024, A(H1N1)pdm09 and B/Victoria remained genetically aligned with their vaccine components, whereas A(H3N2) shifted to the J lineage, consistent with the 2024-2025 vaccine update. Although pre-treatment PA/I38T was absent, low-frequency on-therapy selection was observed, including a rare PA/I38T in influenza B/Victoria detected by NGS, suggesting the value of deep sequencing when viral loads are low. These integrated genomic-clinical data support vaccine strain realignment for H3N2 and continued monitoring of baloxavir resistance in outpatient care.
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