Bovine coronavirus (BCoV) is an important viral agent associated with neonatal calf diarrhea (NCD), winter dysentery, and bovine respiratory disease, contributing to economic losses in cattle production worldwide. To date, BCoV sequence data from Yanbian, Northeast China, are limited in public datasets. In this study, we report the first successful isolation of a BCoV strain (BCoV‐YBYJ) from a diarrheic calf in Yanbian using MDBK cells and characterize its replication in vitro and describe exploratory colon and lung findings following high‐dose oral exposure in a murine model. The isolate replicated efficiently in MDBK cells. In mice, viral RNA was detected in the colon and lung with mild‐to‐moderate histological changes under high‐dose exposure (nonnatural host). Phylogenetic analysis based on partial ORF1a, S, and M sequences showed that BCoV‐YBYJ clustered with contemporary Chinese isolates within the available reference set, supporting regional genetic relatedness based on partial loci. In parallel, three SYBR Green I‐based RT‐qPCR assays targeting BCoV, bovine rotavirus (BRV), and bovine parvovirus (BPV) were established and analytically validated. These assays demonstrated satisfactory analytical sensitivity, specificity, and reproducibility within the validated dynamic range, and BCoV RNA was detected in 70% (140/200) of targeted diarrheic calf specimens ( n = 200). This study provides a Yanbian BCoV isolate and analytically validated RT‐qPCR assays to support differential detection and surveillance of major viral agents associated with calf diarrhea. Because these were targeted submissions from diarrheic calves, this proportion is reported descriptively and should not be interpreted as population prevalence. The BRV and BPV assays underwent analytical validation only and require further clinical evaluation before routine diagnostic application.
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