Dynamic Variation and Reversion in the Signature Amino Acids of H7N9 Virus During Human Infection

Background: Signature amino acids of H7N9 influenza A virus play critical roles in human adaption and pathogenesis, but their dynamic variation is unknown during disease development. Methods: We sequentially collected respiratory samples from H7N9 patients at different timepoints and applied next-generation sequencing (NGS) to the whole genome of the H7N9 virus to investigate the variation at signature sites. Results: A total of 11 patients were involved, from whom 29 samples were successfully sequenced, including samples from multiple timepoints in 9 patients. Neuraminidase (NA) R292K, basic polymerase 2 (PB2) E627K, and D701N were the 3 most dynamic mutations. The oseltamivir resistance-related NA R292K mutation was present in 9 samples from 5 patients, including 1 sample obtained before antiviral therapy. In all patients with the NA 292K mutation, the oseltamivir-sensitive 292R genotype persisted and was not eliminated by antiviral treatment. The PB2 E627K substitution was present in 18 samples from 8 patients, among which 12 samples demonstrated a mixture of E/K and the 627K frequency exhibited dynamic variation. Dual D701N and E627K mutations emerged but failed to achieve predominance in any of the samples. Conclusions: Signature amino acids in PB2 and NA demonstrated high polymorphism and dynamic variation within individual patients during H7N9 virus infection.