Development of a quantitative real-time PCR for the detection of canine respiratory coronavirus

Canine respiratory coronavirus (CRCoV) has been detected recently in dogs with canine infectious respiratory disease and is involved in the clinical disease complex. CRCoV is a group 2 coronavirus most closely related to bovine coronavirus and human coronavirus OC43. A real-time PCR assay was developed for the detection of CRCoV. The assay was validated against cell culture grown virus and shown to have a high level of sensitivity. A range of tissue samples were collected from dogs at a re-homing centre with a history of endemic respiratory disease. The samples were tested using a conventional nested PCR assay and CRCoV was quantitated by real-time PCR. CRCoV was detected most frequently in the nasal mucosa, nasal tonsil and trachea. It was also detected in the lung, and bronchial lymph node. Of the enteric tissues, only one mesenteric lymph node sample was positive. In addition two colon samples were positive for CRCoV by nested PCR only. In conclusion, CRCoV appears to infect the upper respiratory tract preferentially. The CRCoV real-time PCR assay has proved to be a highly specific and sensitive assay that can be applied for diagnostic purposes as well as to investigate further the tissue tropism of CRCoV.