Longitudinal sampling is required to maximize detection of intrahost A/H3N2 virus variants.

Seasonal human influenza viruses continually change antigenically to escape from neutralizing antibodies. It remains unclear how genetic variation in the intrahost virus population and selection at the level of individual hosts translates to the fast-paced evolution observed at the global level because emerging intrahost antigenic variants are rarely detected. We tracked intrahost variants in the hemagglutinin and neuraminidase surface proteins using longitudinally collected samples from 52 patients infected by A/H3N2 influenza virus, mostly young children, who received oseltamivir treatment. We identified emerging putative antigenic variants and oseltamivir-resistant variants, most of which remained detectable in samples collected at subsequent days, and identified variants that emerged intrahost immediately prior to increases in global rates. In contrast to most putative antigenic variants, oseltamivir-resistant variants rapidly increased to high frequencies in the virus population. Importantly, the majority of putative antigenic variants and oseltamivir-resistant variants were first detectable four or more days after onset of symptoms or start of treatment, respectively. Our observations demonstrate that de novo variants emerge, and may be positively selected, during the course of infection. Additionally, based on the 4-7 days post-treatment delay in emergence of oseltamivir-resistant variants in six out of the eight individuals with such variants, we find that limiting sample collection for routine surveillance and diagnostic testing to early timepoints after onset of symptoms can potentially preclude detection of emerging, positively selected variants.

The Molecular Basis for Antigenic Drift of Human A/H2N2 Influenza Viruses.

Influenza A/H2N2 viruses caused a pandemic in 1957 and continued to circulate in humans until 1968. The antigenic evolution of A/H2N2 viruses over time and the amino acid substitutions responsible for this antigenic evolution are not known. Here, the antigenic diversity of a representative set of human A/H2N2 viruses isolated between 1957 and 1968 was characterized. The antigenic change of influenza A/H2N2 viruses during the 12 years that this virus circulated was modest. Two amino acid substitutions, T128D and N139K, located in the head domain of the H2 hemagglutinin (HA) molecule, were identified as important determinants of antigenic change during A/H2N2 virus evolution. The rate of A/H2N2 virus antigenic evolution during the 12-year period after introduction in humans was half that of A/H3N2 viruses, despite similar rates of genetic change.IMPORTANCE While influenza A viruses of subtype H2N2 were at the origin of the Asian influenza pandemic, little is known about the antigenic changes that occurred during the twelve years of circulation in humans, the role of preexisting immunity, and the evolutionary rates of the virus. In this study, the antigenic map derived from hemagglutination inhibition (HI) titers of cell-cultured virus isolates and ferret postinfection sera displayed a directional evolution of viruses away from earlier isolates. Furthermore, individual mutations in close proximity to the receptor-binding site of the HA molecule determined the antigenic reactivity, confirming that individual amino acid substitutions in A/H2N2 viruses can confer major antigenic changes. This study adds to our understanding of virus evolution with respect to antigenic variability, rates of virus evolution, and potential escape mutants of A/H2N2.