Experimental Models for Investigating Influenza A Virus Evolution and Reassortment at the Human–Swine Interface

Abstract

Influenza A viruses (IAVs) pose a continual public health threat due to their segmented genome and capacity for rapid evolution through reassortment. The human–swine interface represents a critical environment for the generation of novel viral genotypes, yet the host cellular environments that influence reassortment dynamics and viral population evolution remain poorly defined. The research presented in this dissertation investigated the influence of human and swine airway epithelial cell cultures on influenza A virus replication and reassortment dynamics while developing biologically relevant in vitro models to study these processes. An immortalized swine respiratory cell line was first developed and characterized. The cell line retained key epithelial features, including physiologically relevant sialic acid receptor distribution, and supported replication of a diverse panel of human- and swine-origin IAVs without the need for exogenous protease. These findings establish a scalable platform for influenza A research in a swine-relevant context. Reassortment dynamics were then examined using fully differentiated human (NHBE) and swine (PBE) bronchial epithelial cell cultures. Co-infection with A/California/07/2009 (H1N1pdm09) and A/swine/Minnesota/2012 (H3N2) viruses generated high genotypic diversity at early passage, followed by a reduction in diversity during serial passaging. Host-specific differences were observed, with human epithelial cells selectively enriching reassortant genotypes containing the pandemic-derived matrix (M) gene within an H3N2 background, consistent with H3N2v-like viruses detected in humans. In contrast, swine epithelial cells maintained broader reassortant diversity. These results indicate that reassortment may be initially stochastic, subsequently becoming constrained by host-specific epithelial selection and segment compatibility. This work offers mechanistic elucidation of influenza evolution at the human–swine interface and thereby establishes an experimental framework for the assessment of zoonotic influenza virus emergence.