Detection, Isolation And S And M Genes Anylasis Of Canine Coronavirus From Canidae Animals

Sixty-one fecal samples from healthy foxes and 24 from healthy raccoon dogs were examined by RT-nested PCR assays for the presence and genotypic identification of Canine coronaviruses (CCVs). A high incidence of CCV infection and a co-existence of two CCV genotypes (CCV type I and CCV type II.) were found. Sequence analysis showed that the M gene of CCV type I had a high similarity of 96.7%~98.1% between the fox- and raccoon dog strains and the reported Italian strain from diarrhea dogs .The two genotypes, with an identity of 88.3%~89.7%, formed two separate branches in phylogenetic tree. Interestingly, the sequence at several nucleic acid sites of CCV type II differed between foxes and raccoon dogs. The co-existence and popularity of the two CCV genotypes in healthy foxes and raccoon dogs were first confirmed in this article.Four strains of CCVs were isolated from feces of healthy dogs, foxes, raccoon dogs and the liver of an diarrhea fox which were all CCV positive. Genotypic identification confirmed that all the isolates were CCV type II, and no type I CCV was isolated. CPE of the four isolates were totally different from that of reference strain CCV 1-71.With three monoclonal antibodies and hyperimmune antisera to the reference strain, varying degrees of antigenic diversities were found among the strains by neutralization, indirect immunofluorescence assays, SDS-PAGE and Western-blot analysis.The complete membrane protein genes of the four CCV strains were cloned followed by sequence analysis, structure and antigenicity prediction. Results showed that (1) the M genes among the four isolates, the reference strain CCV 1-71 and the domestic strains CCV VI, V2 and DXMV shared a high similarity despite their original differences, indicating that domestic CCVs may have a common ancestor; (2)all the strains had a "CTTTAG" nucleotides on the M gene putative recombination region, meeting to "CTT (A/T) (A/T) G" that always appearing near to the recombination "hot spot" in infectious bronchitis virus RNA genome; And (3) all the CCV strains but CCV-BGF had a similar M protein structure: a secretory signal peptide cleavage, between the AA site 16, 17 or 17, 18; and 3 transmembrane regions.The 5' end of the S gene, a high variable region, of CCV isolates HF3 and DF werecloned and compared to the reference strain CCV-1-71. Sequence analysis showed that, in spite of their antigenicity differences, the three CCV strains shared a high homoiogy, and only a little few site mutations were found. The signal peptide cleavage site of HF3 is different to that of DF and 1-71, while DF isolate had one less N-linked glycosylation site than HF3 and 1-71. All these differences were caused by only one AA mutation. And it also can be assumed that the antigenicity differences in the S protein may due to mutations of some important nucleic acids.