2019-20 H1N1 clade A5a.1 viruses have betterin vitroreplication compared with the co-circulating A5a.2 clade

Abstract

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 2019-20 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 2019-20 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 2019-20 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.

Evolutionary analysis of seasonal influenza A viruses in Pakistan 2020–2023

“…Mutations in the H1N1pdm09 viruses resulted in changes in the HA1 polypeptide, specifically K54Q, A186T, Q189E, E224A, R259K, and K308R. Notably, several of these substitutions occupy both antigenic and receptor binding sites, suggesting that they may have served some compensatory function that allowed the virus to improve its fitness and/or antigenic structure 18,26,27 …”

“…Notably, several of these substitutions occupy both antigenic and receptor binding sites, suggesting that they may have served some compensatory function that allowed the virus to improve its fitness and/or antigenic structure. 18,26,27 F I G U R E 1 Number of influenza cases in Pakistan by month and year (January 2020-January 2023).…”

Clade-defining mutations in human H1N1 hemagglutinin protein from 2021-2023 have opposing effects on in vitro fitness and antigenic drift

“…The emergence of the 5a.2a clade of H1N1, and the speed with which it predominated, was surprising considering the failure of its parental clade, 5a.2 (Figure 1A). The 5a.2 clade had significantly reduced in vitro fitness compared with the cocirculating 5a.1 clade and their parental 5a clade, including reduced receptor binding diversity, replication, and plaque formation (8). The contrasting success of its progeny 5a.2a clade suggested that there were phenotypic advantages conferred by the 5a.2a clade-defining mutations.…”

“…Mutations can alter numerous aspects of viral phenotype, including changes to both fitness and antigenic profile. Changes to fitness can include differences in receptor binding (5)(6)(7)(8), virus replication (8)(9)(10), interactions with innate immunity (11), transmissibility (12)(13)(14), and other characteristics (15). Antigenic profile refers to the regions of a virus which are recognized by host antibodies, and changes to this profile through antigenic drift can alter how the virus appears to the immune system and result in a loss of antibody binding.…”

“…In the 2019-20 Northern Hemisphere (NH) influenza season the 5a.1 and 5a.2 clades both emerged from the 5a parental clade (18). The 5a.1 clade quickly predominated over 5a.2, and the 5a.2 clade was shown to have impaired in vitro fitness compared with 5a.1 which may have contributed to its limited spread (8).The 5a.2 clade rapidly acquired a series of HA mutations at the start of 2021 and branched into a novel subclade, 5a.2a, which quickly became the predominant H1N1 clade. 5a.2a is defined by mutations in the receptor binding site and antigenic site mutations A186T, Q189E, and E224A (H1 numbering).…”