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

Figures

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  Fig 1. PEDV was identified from the uterine organs of sows infected with PEDV. (A) Bayesian phylogenetic tree of the full-length genome of PEDV and related coronaviruses, the blue square represents the PEDV strain, while the red circle represents human coronavirus strains. (B) PEDV-N mRNA copies were detected in the uterine tissue of sow A, sow B, and sow C infected with PEDV. (C) PEDV nucleocapsid protein was detected by western blot in uterine tissue of sows infected with PEDV. The Sow-A, Sow-B, and Sow-C indicate uterine tissues of sow A, sow B, and sow C infected with PEDV, the Mock indicates negative sows not infected with PEDV, and P represents a positive control. (D) PEDV infects uterine tissue of sows with CD10 as a marker. CD10 (green), PEDV-N (red), DAPI (blue).

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  Fig 2. PEDV efficiently replicates in primary porcine endometrial cells (PPEC). (A) The flow chart of isolation and culture of primary porcine endometrial cells (PPEC). (B) Identification and analysis of the PPEC by IFA. Images of PPEC are representative of cells marked with porcine endometrial cells, Pans-KRT (red). (C) The epithelial α-smooth muscle actin rabbit monoclonal antibody (ACTA2) (green) positive phenotype is evident in PPEC. (D) PPEC infected with PEDV CV777 and PEDV GH at MOI = 1 at 48 h post infection. The red arrow represents the dead cells. (E) IFA indicates PEDV-N protein positive signals in PPEC infected with either CV777 strain and GH strain at MOI = 1 without trypsin solution, respectively. (F) Western blot indicates that the PEDV nucleocapsid protein in PPEC infected with PEDV. (G) PEDV-N mRNA copies of PPEC infected with PEDV were analyzed by RT-PCR. (H) The TCID50 of PEDV CV777 strain in Vero, IPEC-J2, and PPEC.

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  Fig 3. Single-cell transcriptomic analysis reveals uterine stromal cells as the primary target of PEDV infection. (A) Uniform manifold approximation and projection (UMAP) visualization of single cells isolated from PPEC. (B) Uniform manifold approximation and projection (UMAP) visualization of single cells isolated from PPEC, colored according to annotated cell types, including stromal cells, epithelial cells, endothelial cells, vascular endothelial cells, and stem cells. (C) Dot plot showing the mean expression of marker genes used to classify cell clusters. The color intensity indicates gene expression levels, whereas dot size represents the fraction of cells expressing the gene within each cell type. (D) Feature plot showing the distribution of PEDV-derived viral transcripts across uterine cell populations. PEDV RNA signals are predominantly localized to stromal cells. (E) Proportion of PEDV-infected cells within each uterine cell type, indicating selective enrichment of viral infection in stromal cells. Proportion of PEDV-positive cells across different cell types. (F) The bubble size indicates the absolute gene counts of all stromal cells enriched in a GO term, and the color bar…

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  Fig 4. Cell–cell communication network analysis reveals uterine stromal cells as a central signaling hub following PEDV infection. (A) Global overview of intercellular communication networks among major uterine cell populations inferred from ligand-receptor pair analysis. Line thickness represents interaction strength, and arrow direction indicates signal flow from sender to receiver cells. (B) Quantification of outgoing and incoming signaling strength for each uterine cell type, highlighting enhanced communication activity of stromal cells. (C) Heatmap (or dot plot) showing the relative strength of interactions between stromal cells and other uterine cell populations, including endothelial cells, and smooth muscle cells. (D) Inferred LAMININ signaling intercellular communication network between mock group and PEDV-infected group shown in a circular plot (left). Thicker lines show a higher number of interactions and greater interaction strength between cell types. Expression levels of LAMININ pathway genes in each cell type are compared between control (orange) groups and PEDV-infected (cyan). Normalized expression levels are determined in a violin plot (right). (E) Inferred FN1…

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  Fig 5. PEDV infection significantly alters multiple cells signaling in PPEC. The bubble size indicates the absolute gene counts enriched in a GO term, and the color bar indicates the minus logarithm of P values (A and B). C–J Heatmaps indicate the relative expression level for sets of cell adhesion (C), cell death (D), cytokines (E), ISGs (F), blood vessel development (G), extracellular matrix (H), connective tissue development (I), and embryonic morphogenesis (J).

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  Fig 6. PEDV infection induces immune activation and cellular homeostasis disruption in PPEC. Chemokine expression in PPEC infected with PEDV: PEDV infection enhanced the mRNA expression of IFIT2 (A), TNF-α (B), IL-1α (C), IL8 (D), IL-1β (E), and IL-6 (F) in PPEC at 12, 24, 48, and 72 hpi analyzed by RT-PCR. Effects of PEDV infection on cell cycle regulation of PPEC: the design of the flow cytometry experiment involved three samples per group at 24, 48, and 72 hpi (A). The number of PPEC in the G0/G1 phase was significantly less than that of PPEC infected with PEDV CV777 or PEDV GH at 24, 48, and 72 hpi (G). The number of PPEC infected with PEDV in the S phase at 24, 48, and 72 hpi (H). The number of PPEC infected with PEDV in the G2 phase at 24, 48, and 72 hpi (I). PEDV infection disrupts the epithelial tight junctions of PPEC: The levels of occludin in PPEC infected with PEDV were assessed by IFA (J). The average fluorescence intensity of occludin in PPEC infected with PEDV was analyzed (K). The levels of ZO-1 in PPEC infected with PEDV were assessed by IFA (L). The average fluorescence intensity of ZO-1 in PPEC infected with PEDV was analyzed (M).

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  Fig 7. Correlation analysis between PEDV gene expression and known coronavirus receptor expression across uterine cell populations. Single-cell RNA sequencing data from PEDV-infected porcine uteri were used to assess the relationship between viral gene expression and host receptor genes associated with coronavirus entry. The UMAP visualization showing the expression levels of ANPEP (A), CTSB (B), FURIN (C), and NANS (D) in control and PEDV-infected groups. Each dot represents an individual cell, colored by cell type. Correlation coefficients were calculated across all cells to evaluate the association between PEDV expression levels and receptor gene expression.

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  Fig 8. Overview of pregnant sow infected with porcine epidemic diarrhea virus. The placental barrier of sows in the late gestation period is mainly composed of these six types of tightly linked cells (maternal vascular endothelial cell, stromal cell, uterine epithelium, trophoblast, fetal placental mesenchyme, and fetal vascular endothelial cell). PEDV can successfully infect stromal cells and replicate efficiently, which may contribute to reproductive disorders through induction of inflammatory responses, extracellular matrix disruption, and altered intercellular communication.