Evolutionary pathways to antiviral resistance for influenza virus - influenza virus presents a recurring threat

The National Institute of Allergy and Infectious Diseases awarded the University of Wisconsin - Madison $414,861 under Project Grant R21AI195857 on September 1, 2026, through the Allergy and Infectious Diseases Research program (CFDA 93.855).

The award funds research into the evolutionary pathways by which influenza virus variants acquire antiviral resistance. The investigator will use engineered viral libraries carrying random nucleotide barcodes on the PA segment of the influenza genome to create populations with over 100,000 uniquely identifiable viral members, enabling fine-grained analysis of how adaptive variants arise and become selected within individual hosts. In Aim 1, the research will identify and quantify variant lineages that emerge when influenza replicates in the presence of an experimental small-molecule inhibitor, using evolutionary analysis to reveal adaptive mechanisms. The work addresses a critical knowledge gap: naturally occurring viral diversity within individual hosts is too low to allow detailed study of how fitter variants arise and reach sufficient frequency to enable transmission, selection, and potential fixation at the population level. Understanding this process is necessary for predicting antigenic variation, drug resistance emergence, seasonal epidemics, and pandemic risk.

Performance will occur at Madison, Wisconsin. The period of performance runs from September 1, 2026, through August 31, 2028.

Evolution at the global scale results in genetic drift and seasonal epidemics, necessitating annual updates to the influenza vaccine. New, evolutionarily advanced variants that acquire beneficial phenotypes like drug resistance or immune escape must arise and be transmitted from individual infected hosts, and yet, such variants detected on the global scale are rarely seen in acute infections. Instead, diversity within in an individual is low and variants with a potential fitness advantage rarely rise to high frequency. This leave a major knowledge GAP - how do fitter variants arise within an individual to allow their ultimate transmission, selection and potential fixation at the population level? Answering this question is a prerequisite for accurately predicting antigenic variation, the emergence of drug resistance, seasonal epidemics, and occasional global pandemics. To begin to address this, we seek to obtain a clear understanding of the very first step in the process when variants arise and are then selected. These types of questions have been out of reach, as the relatively low levels of naturally occurring variation within viral populations do not provide sufficient information for fine-grained analysis of population dynamics. We overcame this limitation by engineering viral libraries encoding random nucleotide barcodes on the PA segment of the influenza virus genome, creating rich viral populations with over 105 uniquely quantifiable members. This technical innovation has US poised to understand how adaptive variants arise, potentially get selected, and alter phenotypes in viral populations. In aim 1 we will define the processes that allow influenza virus to replicate in the presence of an experimental small-molecule inhibitor. We will use our barcoded viruses to identify and quantify variant lineages. Evolutionary analysis will also reveal how these variants emerge at the population level, and possible tradeoffs the viruses faces. Having established these "ground rules" in an idealized setting, aim 2 will then investigate how selection drives the emergence of viral variants in vivo. These results will inform how variants arise in an individual establishing the potential to become the dominant lineage in the global population. Together, this work will elucidate fundamental properties of evolutionary selection during influenza virus infection, and how these rules are reshaped during replication in vivo.