ABSTRACT. Ten strains, eight field and two reference laboratory strains, of canine coronavirus (CCV) were comparatively examined with respect to antigenic relationships and pathogenic potential in dogs. With monoclonal antibodies and hyperimmune antisera to feline coronavirus and CCV, respectively, varying degrees of antigenic diversities were found among the strains by neutralization and immunofluorescence assays, but it was felt that they belong to one serotype. Specific-pathogen-free puppies experimentally inoculated with some CCV strains manifested clinical symptoms, but there was a difference in their virulence. In order to elucidate the prevalence of CCV infections in dogs in Japan, we tested for neutralizing antibodies to CCV in 467 field dogs, and found a prevalence of 44.1%. Moreover, by using nested reverse transcriptase-polymerase chain reaction on rectal swabs of 100 diarrheic dogs recently presented in veterinary clinics, evidence of CCV in 16% of these specimens was found. The results suggested that CCV infection is more widespread than expected in dogs, and that CCV is a significant etiologic factor in canine diarrhea also in Japan.-KEY WORDS: canine, canine coronavirus, diarrhea, enteritis.J. Vet. Med. Sci. 61 (7): [731][732][733][734][735][736] 1999 we examined eight strains newly isolated from diarrheic dogs, and two American strains as reference with respect to their antigenic and pathogenic characteristics. For serological epidemiology of CCV infection, neutralizing antibodies to CCV in dog serum samples recently collected were determined. In addition, we applied nested reverse transcriptase-polymerase chain reaction (RT-nPCR) for detection of CCV in the recent field diarrheic dog feces. MATERIALS AND METHODS Virus isolation and reference CCVs:Isolation of CCV was tried for feces of diarrheic dogs collected from various parts of Japan during the years 1990 to 1997. A rectal swab was placed in 1 ml of Eagle's minimal essential medium (Eagle's MEM), the extract was clarified by centrifugation at 8,500 g for 10 min, and supernatant was inoculated onto the felis cutus whole fetus-4 (fcwf-4) cell monolayer in a culture dish. After 1hr of adsorption, the inoculum was removed and the conditioned Eagle's MEM containing 5% fetal calf serum, 10% tryptose phosphate broth and antibiotics (penicillin 200 U, streptomycin 200 µg and kanamycin 20 µg/ml) was added. Then they were incubated at 37°C in a humidified chamber containing 5% CO2. When a cytopathic effect (CPE) was observed, an isolate was identified by an indirect immunofluorescence assay (IFA) with specific monoclonal antibodies (MAb) to FCoV, as described previously [13].Reference CCV strains 1-71 and TN449 were obtained from Tokyo University, Tokyo and Fort Dodge Laboratories, Iowa, respectively.MAbs and hyperimmune antisera: A MAb reagent, which is a mixture of MAbs against each nucleocapsid, integral membrane and spike protein of FCoV, was used for