Isolation And Identification Of Canine Coronavirus And Establishment Of Two Detection Methods

Canine coronavirus(CCo V)is usually considered as a self-limiting intestine pathogen,which generally causes mild diarrhea in young dogs with a high morbidity and low mortality.When co-infection with other intestinal pathogens,it can lead to a high mortality.In 1971,CCoV was first reported in Germany,and it is a worldwide epidemic now.In recent years,with the reports of many cases of fatal CCoV highly pathogenic variant strains in European,widespread concern is caused in the global research scholars.The test was designed to detect the dog feces samples collected from Beijing,and Viral isolation was performed on positive samples.After being identified as CCoVs,the SYBR Green?real-time RT-PCR assay was established to detect CCoV and the double nanometer RT-PCR method for identification of CCo V-?and CCoV-?was established by using the isolated virus.This study can provide an in-depth understanding of the prevalence of CCoV in Beijing and lay a foundation for the diagnosis,prevention and treatment of canine coronavirus disease and follow-up research.Canine coronavirus was detected and genotyped by optimized RT-PCR method.Three samples with high concentration of virus particles and no other canine common virus infection were screened out from the positive samples.The CPE of the 3 isolates were obvious before the 4th generation,and they were positive by RT-PCR.The results of RT-PCR showed that CCoV could be found in MDCK cells,but the proliferation of CCoV was unstable.The indirect immunofluorescence test was performed on the isolated virus using the polyclonal antibody of the reference strain.Combined with the results of morphological examination,the virus was identified as canine coronavirus.The genotype of isolate was type II by RT-PCR.The gene fragment of C-terminal 9kb of CCoV isolate was cloned and sequenced.By comparing with other CCoV genomes provided by GenBank,the results showed that there was the highest homology with CCoVfc1 strain and high homology with domestic isolates.It indicates that there is a common source of evolution in China,CCoV isolate is the lowest with CCoV 23/03 strain.By using BLAST comparison,the homology was the highest with CCoV strain 430/07 strain in Italy,and the homology was 96%.A real-time fluorescence quantitative RT-PCR method for rapid detection of CCo V was established by using the isolated virus.The method was used to detect a total of 76 fecal samples from a dog farm and animal hospital in Beijing.The results showed that intensive farming could easily lead to the spread of CCoV.At the same time,a double nanometer RT-PCR method for the identification of CCoV-? and CCoV-? was established.The method had good specificity and sensitivity,and it was simple and fast to operate.The sensitivity test showed that the lowest nucleic acid detection method was 100 times higher than that of the ordinary RT-PCR method.The results of 60 fecal samples collected in Beijing showed that the infection rate of CCoV-? in canine population was higher than that of CCoV-?,and the results showed that there were two genotypes of co-infection in dogs.