Identification of canine coronavirus strains from feces by S gene nested PCR and molecular characterization of a new Australian isolate

A nested PCR (nPCR) assay for the detection of canine coronavirus (CCV) in fecal samples is described. The target sequence for the assay was a 514-bp fragment within the spike (S) glycoprotein gene. The sensitivity of the ass ay is extremely high, detecting as little as 25 50% tissue culture infectiv e doses per g of unprocessed feces. A clinical trial using dogs challenged orally with CCV SA4 and CCV NVSL was used to compare viral isolation and th e nPCR assay as detection techniques over a 2-week period of infection. Vir us isolation defected CCV shedding from day 4 to 9 postchallenge, while the nPCR assay detected CCV shedding from day 4 to 13 postchallenge. Cloning a nd sequencing of the nPCR assay product enabled investigation of the evolut ionary relationships between strains within the S gene. The simple and rapi d procedure described here makes this assay an ideal alternative technique to electron microscopy and viral isolation in cell culture for detection of CCV shedding in feces. The described assay also provides a method of ident ifying new strains of CCV without the complicated and time-consuming practi ce of raising antibodies to individual strains. This is illustrated by the identification, for the first time, of an Australian isolate of CCV (UWSMN- 1).