2019-2020 H1N1 clade A5a.1 viruses have better in vitro fitness compared with the co-circulating A5a.2 clade.

Surveillance for emerging human influenza virus clades is important for identifying changes in viral fitness and assessing antigenic similarity to vaccine strains. While fitness and antigenic structure are both important aspects of virus success, they are distinct characteristics and do not always change in a complementary manner. The 2019-2020 Northern Hemisphere influenza season saw the emergence of two H1N1 clades: A5a.1 and A5a.2. While several studies indicated that A5a.2 showed similar or even increased antigenic drift compared with A5a.1, the A5a.1 clade was still the predominant circulating clade that season. Clinical isolates of representative viruses from these clades were collected in Baltimore, Maryland during the 2019-2020 season and multiple assays were performed to compare both antigenic drift and viral fitness between clades. Neutralization assays performed on serum from healthcare workers pre- and post-vaccination during the 2019-2020 season show a comparable drop in neutralizing titers against both A5a.1 and A5a.2 viruses compared with the vaccine strain, indicating that A5a.1 did not have antigenic advantages over A5a.2 that would explain its predominance in this population. Plaque assays were performed to investigate fitness differences, and the A5a.2 virus produced significantly smaller plaques compared with viruses from A5a.1 or the parental A5a clade. To assess viral replication, low MOI growth curves were performed on both MDCK-SIAT and primary differentiated human nasal epithelial cell cultures. In both cell cultures, A5a.2 yielded significantly reduced viral titers at multiple timepoints post-infection compared with A5a.1 or A5a. Receptor binding was then investigated through glycan array experiments which showed a reduction in receptor binding diversity for A5a.2, with fewer glycans bound and a higher percentage of total binding attributable to the top three highest bound glycans. Together these data indicate that the A5a.2 clade had a reduction in viral fitness, including reductions in receptor binding, that may have contributed to the limited prevalence observed after emergence.