Isolation, identification, and whole genome sequencing analysis of influenza A virus clinical isolates from Shanghai, China, during 2022–2023

Influenza A virus remains a persistent threat, continuously evolving and contributing to seasonal epidemics and potential pandemics. In this study, we analyzed clinical samples collected from pediatric patients in Shanghai between 2022 and 2023, following the SARS-CoV-2 pandemic, to assess the genetic diversity of circulating influenza A virus strains. Out of 88 clinical samples that were positive for influenza A virus, seven isolates were successfully cultured and subjected to whole-genome sequencing. Whole-genome sequencing was performed using the Illumina high-throughput sequencing platform, while missing genomic segments, which were identified in the whole-genome sequence of isolates, were later amplified via standard PCR and Sanger sequencing. Phylogenetic analysis revealed a significant divergence between H3N2 strains from 2023 to those from 2022, suggesting a potential decline in vaccine efficacy over successive seasons. A H1N1 strain isolated from 2022 exhibited close genetic similarity to the vaccine strain. Mutational analysis identified key substitutions in the hemagglutinin and neuraminidase regions, including the I222V mutation in the neuraminidase segment of sample B623080303, which is associated with strong resistance to oseltamivir and peramivir. Another highly drug-resistant N206D mutation in the hemagglutinin region, detected in four clinical isolates, was found to alter host specificity by reducing binding affinity to human-like α2,6-linked receptors while increasing affinity for avian-like α2,3-linked receptors, thereby influencing host adaptation dynamics. The findings provide critical insights into the evolutionary trends of influenza A virus strains post-COVID-19 pandemic and their potential implications for vaccine effectiveness and antiviral resistance.