Genetic Evolution Analysis Of Canine Distemper Virus In Guangzhou And Its Regulation On IFN-β

Canine distemper（CD）is an acute,highly contagious disease caused by canine distemper virus（CDV）,which mainly infects carnivores.CDV has a strong ability to cross the species barrier and has been found in at least 20 families within seven orders,including non-human primates.At present,vaccination is an effective way to prevent and control CDV from infecting canines,but immune failures were reported frequently,and the specific mechanism of CDV infection causing immunosuppression is still unclear.Therefore,the genetic evolution tree analysis of CDV H gene was carried out through the etiology investigation of CDV in Guangzhou,combined with the sequence information of public databases,and 5 CDV field strains have been isolated.Then,in order to understand the interaction between CDV and innate immunity,this study preliminarily explored the regulation effect of CDV on IFN-βresponse.Three aspects of work were mainly carried out in this research.First,13 field CDV strains were detected from clinical samples,their H gene was amplified,and their genetic characteristics were analyzed.H gene sequence analysis indicated that the similarity of H gene nucleotides and amino acid sequences of 13 CDV strains was 98.4%～100.0%and99.1%～100.0%respectively;Compared with Onderstepoort,Lederle,Convac and other vaccine strains,the sequence similarity was 90.1%～91.5%and 89.3%～90.8%respectively;Evolutionary tree analysis indicates that the 13 CDV field strains belong to Asia typeⅠand are far away from the vaccine strain branches;Studies have shown that there are 4～7potential N-glycosylation sites in the H protein of vaccine strains,while the field strains identified in this study evolved into 9 potential N-glycosylation sites.Therefore,Asia typeⅠCDV is still an epidemic genotype in Guangzhou,which has certain evolutionary distance and appears significant difference compared with the vaccine strains.Second,five CDV field strains were isolated from positive samples by Vero-SLAM cell lines,two vaccine strains were isolated from vaccines,and the isolated strains were identified by virology.After inoculation on Vero-SLAM cells,typical syncytial lesions can be observed,and the results of RT-PCR and IFA were also positive.The titers of five field strains including GD188,GD189,GD1810,GD1811,GD1812 and canine vaccine strains CDV-11 and mink vaccine strains CDV3 were 10^3.67/m L、10^2.75/m L、10^3.20/m L、10^3.13/m L、10^3.45/m L、10^6.00/m L、10^4.53/m L respectively.Primers were designed according to the N gene of CDV,SYBR GreenⅠfluorescence quantitative RT-PCR detection method of CDV was established,and then the viral loads were measured and the growth curves were plotted in terms of the data.According to the one-step growth curve,the field strains and CDV-11 showed the same trend,gradually increasing from a slow growth rate to an exponential growth rate,and then reaching a peak at 48h and starting to decline.The early trend of CDV3 was similar to that of the other two strains,but firstly reached the peak and entered the plateau stage at36h.Third,the SYBR GreenⅠfluorescence quantitative RT-PCR detection method of IFN-βm RNA and the dual-luciferase reporting system of canine IFN-βpromoter were established.CDV field strain GD1812,vaccine strains and sendai virus（Se V）（MOI=0.1）were inoculated on MDCK-SLAM cells,and the expression levels of IFN-βm RNA in the cells were detected 24h later.It was found that field strains includng GD1812,CDV-11 and CDV3 had no significant activation effect on IFN-βm RNA expression in MDCK-SLAM cell line.The dual-luciferase reporting system plasmid of IFN-βpromoter was transfected into MDCK-SLAM and Vero-SLAM cells,which after 24h were inoculated with CDV field strain GD1812,vaccine strains and sendai virus（MOI=0.1）.The activation of canine IFN-βpromoter by several strains was detected 24h after infection.Corresponding to the expression of IFN-βm RNA,CDV field strain,canine vaccine strain CDV-11 and mink vaccine strain CDV3 also had no significant activation on IFN-βpromoter in MDCK-SLAM and Vero-SLAM cells.The result is consistent with the fact that no expression of IFN-βhas been detected in the clinical analysis of blood samples from CDV infected dogs,and similar to the regulatory effect of measles virus（Me V）in the same genus on expression of IFN-β.Now the mechanism of CDV acting on the expression of IFN-βis still not clear and this study adds reference data for the mechanism of CDV escaping the host’s natural immune response.