in may 2005 , a severe outbreak of fatal systemic disease occurred in a pet shop in bari , italy . clinical symptoms were initially observed in 3 miniature pinschers ( 45 days of age ) and 1 cocker spaniel ( 53 days of age ) and consisted of fever ( 39.5c40c ) , lethargy , inappetence , vomiting , hemorrhagic diarrhea , and neurologic signs ( ataxia , seizures ) with death after 2 days . the same symptoms were observed 34 days later in 2 other miniature pinschers ( 45 days of age ) and 1 pekinese ( 56 days of age ) . necropsy of the dogs showed hemorrhagic enteritis , abundant serosanguineous fluid in the abdominal cavity , and severe lesions in the parenchymatous organs . livers were yellow - brown and congested , with hemorrhages on their surfaces , and spleens were enlarged with subcapsular hemorrhages . variable gross changes in other organs included multifocal hemorrhagic renal cortical infarcts and petechial hemorrhages on lymph node surfaces . virologic and bacteriologic investigations on the parenchymatous organs did not detect common canine pathogens , notably canine parvovirus type 2 , canine distemper virus , canine adenovirus type 1 and type 2 . ccov type i and type ii were identified in the intestinal contents of all puppies by genotype - specific real - time reverse transcription  polymerase chain reaction ( rt - pcr ) assays ( 9 ) . ccov type ii rna was also detected in lungs ( median 1.08  10 rna copies/l of template ) , spleen ( median 4.46  10 rna copies/l of template ) , liver ( median 9.02  10 rna copies/l of template ) , kidney ( median 7.54  10 rna copies/l of template ) , and brain ( median 5.23  10 rna copies/l of template ) . virus - induced cytopathic effect was observed in a-72 cells , and ccov type ii strain ( cb/05 ) was isolated from all tissues examined except brain tissue . immunohistochemical analysis with a ccov - specific monoclonal antibody detected ccov antigen in the organs with gross lesions that were examined ( lungs , kidneys , liver , spleen , gut , and lymph nodes ) ( figure 1 ) . immunohistochemical detection of canine coronavirus antigen ( arrows ) in canine lung tissue by a specific monoclonal antibody ( magnification 400 ) . the sequence of the 3  end of the genome ( 8.8 kb ) of the pantropic ccov strain was determined by rt - pcr amplification and sequencing of overlapping fragments . the s , envelope , and membrane proteins and nucleoprotein showed a high degree of amino acid identity with the cognate open reading frame ( orf ) of ccov type ii . the s protein of strain cb/05 had the highest identity to fcov type ii strain 79 - 1683 ( figure 2 ) . comparison with strain cb/05 was possible only with ccov type ii strains insavc-1 ( 10 ) and bgf ( 11 ) and ccov type i strains elmo/02 and 23/03 ( 3,12 ) because of a lack of data on the 3  end of the ccov genome in the genes encoding for nonstructural proteins ( nsps ) 3a , 3b , 3c , 7a , and 7b . nsp 3b ( 22 aa ) was 49 aa shorter than expected because of a 38-nucleotide deletion and a frame shift mutation in the downstream sequence that introduced an early stop codon . nsp 3c ( 244 aa ) was 6 aa shorter and 79 aa longer than the cognate proteins of the enteropathogen strain bgf and the attenuated strain insavc-1a , respectively . neighbor - joining tree of the spike protein of canine coronavirus ( ccov ) and feline coronavirus ( fcov ) . the following reference strains were used for phylogenetic analysis : ccov type i strains elmo/02 ( genbank accession no . ay307020 ) and 23/03 ( ay307021 ) ; ccov type ii strains insavc-1 ( d13096 ) and k378 ( x77047 ) ; fcov type i strains ku-2 ( d32044 ) , black ( ab088223 ) and ucd-1 ( ab088222 ) ; fcov type ii strains 79 - 1146 ( x06170 ) and 79 - 1683 ( x80799 ) ; and bovine coronavirus ( bcov ) strain ent ( nc_003045 ) . the following reference strains were used for phylogenetic analysis : ccov type i strains elmo/02 ( genbank accession no . type ii strains insavc-1 ( d13096 ) and k378 ( x77047 ) ; fcov type i strains ku-2 ( d32044 ) , black ( ab088223 ) and ucd-1 ( ab088222 ) ; fcov type ii strains 79 - 1146 ( x06170 ) and 79 - 1683 ( x80799 ) ; and bovine coronavirus ( bcov ) strain ent ( nc_003045 ) . the scale bar represents 20 substitutions per 100 sequence positions . to confirm the pathogenic potential of strain cb/05 , we experimentally infected two 6-month - old dogs ( authorization no . two milliliters of cryolysate of a lung - derived first - passage virus in a-72 cells were administered intranasally to the dogs . the cell cryolysate tested negative for other common canine pathogens and had a 50% tissue culture infectious dose of 10/50 l on a-72 cells and 1.18  10 rna copies/l of template by real - time rt - pcr . severe clinical symptoms characterized by pyrexia ( temperature 39.8c40.1c ) , anorexia , depression , vomiting , diarrhea , and leukopenia were observed that persisted 810 days . despite the severe symptoms , the dogs slowly recovered from their illness . point mutations or deletions in the s protein and nsps have been associated with changes in tropism and virulence of coronaviruses ( 47,13 ) . ccov strain cb/05 showed intact structural and nonstructural proteins , with an s protein closely related to that of other type ii ccovs . the only striking change was the truncated form of nsp 3b . whether the deletion in the orf of nsp 3b is involved in pathobiologic changes should be assessed with reverse genetic systems . the present study describes for the first time the occurrence of fatal infections in dogs by coronaviruses . experimental infection of dogs with the virus isolate resulted in a severe systemic disease that mimicked the clinical symptoms observed in the outbreak . however , the different ages at infection ( 6 months vs. < 2 months ) likely resulted in the disease being nonfatal . accordingly , the appearance of pathogenic ccov variants should always be regarded as a potential threat to domestic dogs and considered when unexplainable fatal disease outbreaks occur in puppies . epidemiologic studies are required to determine whether the pantropic ccov strain is a new coronavirus variant emerging in canine populations or a widespread infectious agent of dogs that usually goes undetected . vaccination trials could also help determine whether the ccov vaccines currently available are effective against the highly virulent ccov strain . the 20022003 sars epidemic has demonstrated that the study of animal coronaviruses is paramount to understanding the ecology and evolution of human coronaviruses . the coronaviruses of carnivores provide a paradigmatic model of how coronaviruses cross the species barriers , adapt to new host species , and change their pathogenicity .
canine coronavirus ( ccov ) is usually responsible for mild , self - limiting infections restricted to the enteric tract . 
 we report an outbreak of fatal disease in puppies caused by a pathogenic variant of ccov that was isolated from organs with severe lesions .