Molecular Epidemiology Investigation And Preliminary Isolation Of Canine Coronavirus In Three Cities Of Heilongjiang Province From 2014 To 2015

Canine coronavirus disease is a highly contagious,enteric disease of canine caused by canine coronavirus(Canine coronavirus,CCoV).In 1971,CCoV was first recognized as an intestinal pathogen of dogs in Germany.CCoV present global trends in Asia,Europe,South America,North America and other regions.The prevalence and genetic diversity of CCoV was very little understanding in the last five years.Therefore,to trace the evolution of canine coronavirus,genetic evolution analysis,the preliminary isolation,test collecting feces of dogs with diarrhea symptoms in three cities of Heilongjiang Province from May 2014 to April 2015.This study clarifies the prevalence of the three cities in Heilongjiang Province,and provide theoretical basis for prevention and control of CCoV and the subsequent related research.To trace the evolution of canine coronavirus(CCoV),201 stool samples from diarrheic dogs in the three cities Heilongjiang province of northeast China were subjected to reverse transcription-polymerase chain reactions(RT-PCRs)targeting the partial M and S genes of CCoV,followed by an epidemiological analysis.M gene RT-PCRs showed that(28.36%,57/201)of the samples were positive for CCoV.In the 57 positive samples,CCoV-? and CCoV-? accounted for 15.79%(9/57)and 84.21%(48/57),respectively.A sequence comparison of the partial M gene revealed nucleotide homologies of 88.4%~100% among the 57 CCoV strains,and88.7%~96.2% identity between the 57 CCoV strains and the Chinese reference strain HF3.The CCoV-? and CCoV-? strains exhibited genetic diversity when compared with reference strains from China and other countries.The 57 CCoV strains exhibited high co-infection rates with canine kobuvirus(Ca KV)(33.33%)and canine parvovirus-2(CPV-2)(31.58%).The CCoV prevalence in diarrheic dogs differed significantly with immunization status,regions,seasons,and ages.Moreover,28 S genes were amplified from the 57 CCoV-positive samples,including26 CCoV-?a strains,one CCoV-?b strain,and one CCoV-? strain.A sequence comparison of the partial S gene revealed 86.3%~100% nucleotide identity among the 26 CCoV-?a strains,and89.6%~92.2% identity between the 26 CCoV-?a strains and the Chinese reference strain V1.The26 CCoV-?a strains showed genetic diversity when compared with reference strains from China and other countries.In this study,23 strains of CCoV simple infection positive samples were chosen for virusisolation through MDCK cells.When blind to the 4th generation,9 samples appeared obvious CPE and continued into the 7th generation.At last CPE gradually weakened until it disappeared.9 samples were isolated in MDCK cells again,there were 4 strains appeared CPE at the 5th generation which were identified by nested RT-PCR,4 strains were amplified target band.I solation was been continued and CPE disappeared at the 6th generation.The data indicated that CCoV-?,CCoV-?a,and CCoV-?b strains has high co-infection rates with Ca KV and CPV-2 in the diarrheic dogs in northeast China,and the CCoV-? strains exhibited high prevalence and genetic diversity.Even though CPE could occured in MDCK cells,but the virus was extremely unstable.