Single-cell RNA sequencing identifies uterine stromal cells as a previously unrecognized target of an alphacoronavirus underlying reproductive disorders

ABSTRACT Porcine epidemic diarrhea virus (PEDV), an alphacoronavirus, is primarily recognized as an enteric pathogen causing severe gastrointestinal disease in newborn piglets. PEDV has been endemic in most parts of the world, leading to high mortality in neonatal piglets and causing massive economic losses to the pig industry. However, reproductive disorders have been repeatedly observed in sows following PEDV outbreaks, and the underlying mechanisms remain poorly understood. Here, we provide evidence that PEDV can infect the porcine uterus in vivo and exhibits a pronounced cellular tropism toward uterine stromal cells. Using single-cell RNA sequencing, immunofluorescence analyses, and primary cell infection models, we show that PEDV-derived viral transcripts and proteins are preferentially detected in stromal cells, whereas epithelial and other uterine cell populations display minimal viral signals. PEDV efficiently replicates in primary porcine uterine endometrial cells in a trypsin-independent manner, indicating tissue-specific differences in viral entry requirements. Transcriptomic profiling reveals that PEDV infection is associated with extensive inflammatory activation, disruption of extracellular matrix organization, and altered intercellular communication. Notably, stromal cells emerge as a central signaling hub within the infected primary uterine endometrial cell culture system and likely within the uterine microenvironment as well. Correlation analyses further suggest that PEDV infection is linked to stromal cell-restricted expression patterns of candidate host factors rather than canonical enteric coronavirus receptors. Together, these findings redefine the cellular landscape of PEDV infection beyond the intestine and provide a mechanistic framework linking PEDV infection of uterine stromal cells to PEDV-associated reproductive disorders. IMPORTANCE This study identifies uterine stromal cells as a novel target of porcine epidemic diarrhea virus (PEDV), breaking the long-held notion that PEDV is strictly enteric and expanding its known tissue tropism beyond the intestine. Leveraging single-cell RNA sequencing and multi-layered functional validation, it uncovers that PEDV replicates efficiently in uterine stromal cells via a trypsin-independent mechanism—distinct from its intestinal infection mode. This infection triggers inflammatory activation, extracellular matrix disruption, and altered intercellular communication, which are potential key contributors of PEDV-associated reproductive disorders plaguing the pig industry. These findings redefine alphacoronavirus pathogenesis and provide a critical mechanistic framework for developing targeted interventions. Additionally, this work highlights the value of single-cell approaches in unraveling extraintestinal viral infection, advancing both coronavirus biology research and veterinary disease control.