| Canine kobuvirus(Canine kobuvirus,CaKoV)is a virus which associated with canine diarrhea.Since CaKoV first report in the United States in 2011,this virus has been reported in many countries in Europe and Asia,as well as in the southwest and northeast regions of China,posing a serious threat to the health of dogs.Up to now,there is no report of CaKoV infection in Anhui Province,and its genetic evolution background is still unclear,and further research is needed.In addition,rapid detection methods are of great significance for the early diagnosis of the disease.In this study,a total of 57 fecal samples were collected from diarrhea and healthy dogs which from different regions of Anhui province,and a total of 5 positive samples of CaKoV were detected by using specific primers.Subsequently,five VP1 gene sequences were successfully amplified,and the complete genome sequence of one of them was amplified.Using Mega X software,the phylogenetic analysis of 5 CaKoV positive samples VP1 genes were carried out.The analysis results based on the genetic evolution analysis of the VP1 gene,showed the homology of the VP1 gene of the 5CaKoV strains was between 94.0%-96.4%,among which the homology of AH-1 and AH-3 was the lowest 94%,and AH-1 The highest homology with AH-4 is 96.4%.The analysis of the phylogenetic tree shows that the global CaKoV can be divided into two large groups,among which the epidemic strains in Asia are a single group,and the epidemic strains in Europe and the Americas are the other group.The analysis results based on the complete genome sequence show that the Anhui strain of CaKoV has a close genetic relationship with the wild strains in Asia,which the homology with the wild strains in Asia can reach 93.3%-98.7%,the homology with the wild strains in Europe were 80.1%-85.5%,which indicated the genetic relationship with the strains in Europe and America is far,indicating that the prevalence of CaKoV may have the characteristics of local epidemics.Based on the results of the genetic evolution analysis of the multi-species Kobuvirus,it is shown that the genetic distance between CaKoV and the feline Kobuvirus is relatively close,and the genetic distance from the Kobuvirus of large animals such as cattle,sheep and pigs is far,indicating that the Kobuvirus of dogs and cats may exits the cases of cross-species transmission.Bioinformatics analysis of the VP1 protein of CaKoV revealed that the predicted dominant B cell epitopes of the VP1 protein are 60?67,112?117,and 240?247.The predicted dominant Th cell epitopes of the VP1 protein are LRISNPNGL at positions107-115,FYRLLPLGS at positions 49-57,FMAETPIP at positions 143-150 and VVKQGAHTS at positions 250-259.The predicted Th epitope of VP1 protein is LLIAYAPPG at positions 119-127.An isolated strain(AH01)of CaKoV was isolated by using MDCK and CRFK cells,which can provid biological material for subsequent researches on the molecular mechanism of pathogenic of CaKoV.In terms of detection method establishment,this study used Beacon Designer 7.0software to design a pair of specific primers for the CaKoV which target on conservative gene 3D,and established a real-time fluorescent quantitative RT-PCR detection method based on SYBR Green I.This method has good specificity and does not have non-specific amplification reactions with other common canine viruses;the minimum detection limit is 10 copies/?L,and its sensitivity is 100 times that of conventional PCR methods;the co-efficient of variation within and between groups both were less than 0.5%.The detection method has good specificity,high sensitivity and good repeat stability,and can provide effective technical support for the rapid diagnosis of CaKoV.The clinical sample test results show that the positive detection rate of real-time fluorescent quantitative PCR for CaKoV which established in this study is higher than the detection rate of ordinary PCR,indicating that real-time fluorescent quantitative PCR is more suitable for first-line clinical testing. |
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