02087nas a2200229   4500000000100000000000100001008004100002260001500043100001600058700001600074700002000090700001200110700002600122700002100148700002000169245009700189856005900286300001400345490000600359520147800365022001401843       2021                            d  c2021-10-311 aLonglong Si1 aHaiqing Bai1 aCrystal Yuri Oh1 aLei Jin1 aRachelle Prantil-Baun1 aDonald E. Ingber1 aHeba H. Mostafa00aClinically Relevant Influenza Virus Evolution Reconstituted in a Human Lung Airway-on-a-Chip  uhttps://journals.asm.org/doi/10.1128/Spectrum.00257-21  ae00257-210 v93 aHuman-to-human transmission of viruses, such as influenza viruses and coronaviruses, can promote virus evolution and the emergence of new strains with increased potential for creating pandemics. Clinical studies analyzing how a particular type of virus progressively evolves new traits, such as resistance to antiviral therapies, as a result of passing between different human hosts are difficult to carry out because of the complexity, scale, and cost of the challenge. Here, we demonstrate that spontaneous evolution of influenza A virus through both mutation and gene reassortment can be reconstituted in vitro by sequentially passaging infected mucus droplets between multiple human lung airway-on-a-chip microfluidic culture devices (airway chips). Modeling human-tohuman transmission of influenza virus infection on chips in the continued presence of the antiviral drugs amantadine or oseltamivir led to the spontaneous emergence of clinically prevalent resistance mutations, and strains that were resistant to both drugs were identified when they were administered in combination. In contrast, we found that nafamostat, an inhibitor targeting host serine proteases, did not induce viral resistance. This human preclinical model may be useful for studying viral evolution in vitro and identifying potential influenza virus variants before they appear in human populations, thereby enabling preemptive design of new and more effective vaccines and therapeutics.  a2165-0497