Bovine coronavirus enters PBIECs via membrane fusion and clathrin-, caveolin-mediated endocytosis, macropinocytosis.

Bovine coronavirus (BCoV) is an important pathogen that exhibits dual tropism for the respiratory and intestinal tracts, causing winter dysentery in adult cattle, diarrhea and respiratory infections in calves, thus imposing considerable economic losses on the global cattle industry. Our previous studies demonstrated that BCoV gains entry into susceptible HRT-18 cells through membrane fusion and clathrin-mediated endocytosis (CME). However, the precise mechanisms by which BCoV enters host cells remain incompletely elucidated, particularly in primary bovine intestinal epithelial cells (PBIECs). Importantly, as primary cells derived from the natural host, PBIECs more closely recapitulate the in vivo infection microenvironment than HRT-18 cell lines. In the present study, chemical inhibitors, RNA interference, and fluorescently labeled BCoV particles were used to define distinct entry pathways. Our data demonstrated that BCoV enters PBIECs via membrane fusion and three distinct endocytic pathways, including CME, caveolin-mediated endocytosis (CavME), and macropinocytosis. Dynamin, microtubules, cathepsins, and an acidic environment are essential for mediating endocytic entry, whereas cholesterol and TMPRSS2 are dispensable for this process. Furthermore, targeted interference with Rab5, Rab7, and Rab11 suppressed BCoV entry into PBIECs. Consistently, co-localization of fluorescently labeled BCoV with Rab5, Rab7, and Rab11 was observed by confocal microscopy, indicating that these Rab GTPases are involved in BCoV entry into PBIECs. These findings elucidate the entry mechanisms of BCoV and provide novel perspectives to enhance a more comprehensive understanding of the BCoV life cycle.