Accumulation of mutations in nsp4, E, and the S2 subunit underlies mammalian cell tropism expansion and virulence attenuation of avian coronavirus.

Infectious bronchitis virus (IBV), a Gammacoronavirus with strict host tropism, infects chickens and avian-derived primary cells but not mammalian cells. The classical Beaudette strain, isolated in the 1930s, is a well-established IBV strain capable of replicating in Vero cells. Although sporadic reports have described similar adaptation in other strains, the molecular mechanisms underlying IBV cell tropism remain unclear. Here, a prevalent IBV strain was passaged in embryonated eggs and primary host cells to generate quasispecies diversity, followed by an alternating host-nonhost passage strategy that enabled efficient replication in BHK-21 cells within ten passages. Reverse genetics identified mutations in nsp4 (T53I), the E protein (E10G), and the S2 subunit as key contributors to mammalian cell tropism expansion. Notably, some of these mutations were detectable prior to BHK-21 passaging, suggesting that pre-existing variants laid the foundation for subsequent adaptation. Furthermore, introducing this minimal mutation set into the GI-1 H120 virus backbone similarly enabled efficient replication in BHK-21 cells, yielding titers of 105.4 TCID50/ml. In vivo, the nsp4-E-S2 mutant exhibited a 40% reduction in mortality compared with the wild type, and RNA sequencing revealed attenuated inflammatory responses. Collectively, our findings indicate that mammalian cell tropism expansion in IBV is associated with the stepwise selection of pre-existing variants, resulting in coordinated mutations in the S protein and other structural and non-structural proteins that reduced pathogenicity while maintaining protective efficacy, highlighting BHK-21 cells as a promising platform for next-generation vaccine development.