Within-Host Influenza Viral Diversity in the Pediatric Population as a Function of Age, Vaccine and Health Status

Seasonal influenza virus predominantly evolves through antigenic drift, marked by the slow accumulation of mutations at antigenic sites. Because of antigenic drift, influenza vaccines are frequently updated, though their efficacy may still be limited due to strain mismatches. Despite the high levels of viral diversity observed across populations, most human studies reveal limited intrahost diversity, leaving the origin of the population-level viral diversity unclear. Previous studies show host characteristics, such as immunity, might affect within-host viral evolution. Here we investigate influenza A viral diversity in children aged between 6 months and 18 years. Influenza virus evolution in children is less well characterized than in adults, yet may be associated with higher levels of viral diversity given the lower level of pre-existing immunity and longer durations of infection in children. We obtained influenza isolates from banked influenza A-positive nasopharyngeal swabs collected at the Children's Hospital of Philadelphia during the 2017-2018 iinfluenza season. Using next-generation sequencing, we evaluated the population of influenza viruses present in each sample. We characterized within-host viral diversity using the number and frequency of intrahost single nucleotide variants detected in each sample. We related viral diversity to clinical metadata, including subjects' age, vaccination status, and comorbid conditions, as well as sample metadata such as virus strain and cycle threshold. Consistent with previous studies, most samples contained low levels of diversity. While we observed no association between diversity and subjects' age or vaccination status, there was a significant association between diversity and subjects' health status, as categorized by the Pediatric Medical Complexity Algorithm. Additionally, for the H3N2 viral isolates, we observed clustering of iSNVs on the HA gene segment, with further enrichment of non-synonymous iSNVs at known antigenic sites. These latter observations were primarily driven by the two subjects with higher levels of diversity. These observations suggest the presence of antibody-mediated, positive selection operating in a small subset of our study population. However, further studies involving serial viral isolates and the collection of paired serum samples will be necessary to better characterize the complex interplay between humoral immunity and within-host influenza virus evolution.