Isolation And Identification Of Canine Distemper Virus And The Pathogenicity Of Isolated Strain LNDL(17)M4

Background:Canine distemper?CD?is an acute and highly contagious infectious disease caused by canine distemper virus?CDV?.The spectrum of natural hosts for C DV includes several animals and CDV maintains a high incidence and high levels of lethality varied from 30%to 80%.Animals that most commonly suffer from this disea se are ferrets with 100%mortality.In recent years,due to environmental changes,hu man's intervention of wildlife animal's habitat,and the evolution of the virus,CDV has been shown to be a wider spectrum of natural hosts including families of the order c arnine?dog,fox,raccoon?,families of mustelidae?mink,sable,ferrets,badger,otter,a nd weasel?,families of raccoon?raccoons,panda?and most of the families members o f the cat?the lion,tiger,leopard,and cheetah?.Recently,the natural hosts for CDV h ave extended to 8 families such as carnivore,artiodactyla suidae animals,macaca prim ates and clasper suborder seals,and the numbers of the hosts have been discovered to have an expanding host-range.CD has caused serious damage to our country's kennel industry,fur animal breeding industry,and wild rare animals and resulted in huge ec onomic losses.Therefore,it has very great practical significance to investigate the curr ent CD epidemic strains and their pathogenicity.The results will have great significanc es for the effective prevention and control of CD.In this study,four CDV strains were successfully isolated from tissues of the minks and foxes suspected with CDV infection from the farms in Zhucheng,Shandong Province and Dalian,Liaoning Province from July to October,2017 using Vero-dogSLAM?VDS?cells.The isolated CDV strains were further confirmed and identified.In order to clarify the pathogenicity of the CDV,the isolated CDV strain LNDL?17?M4 was selected for the mink challenge experiment.Methods:?1?Virus isolation and titration:The tissue samples from the infected minks and foxes,homogenated with 2-fold of PBS medium with antibiotics,were sonicated and centrifuged.The supernatants were then sterilized by 0.22?m filter.A monolayer of Vero-dogSLAM?VDS?cells was infected with the homogenates for isolation.The CDV isolate strains were collected when 80%of CPE was occurred after infected 4 to 6 days and passaged 3 times in Vero-dogSLAM?VDS?cell.The isolated CDV isolates were titrated in a10-fold dilution using a 50%tissue culture infectious dose(TCID₅₀)assay and were detected by using RT-PCR.?2?RT-PCR identification:The RT-PCR was performed to clone the RBS sequence of CDV H gene using the virus cDNA as the templates,and the nucleotide sequence and homology analysis were also carried out based on the RBS sequences.?3?Indirect immunofluorescence assay?IFA?:CDV isolates at 3 passages were identified by IFA.The uninfected Vero-dogSLAM?VDS?cultures were taken as the negative controls.?4?Animal challenge experiment:CDV isolated strain LNDL?17?M4 was used for pathogenicity study by the animal challenge experiment.The clinical signs,weight and rectal temperatures of the minks were daily recorded.The tissue samples including lungs,spleen,and kidney were obtained from all dead mink after challenge.The viral load in each tissue and organ was detected by Real-time qPCR assay.Results:?1?Four CDV strains were isolated and the virus titers were determined.The LNDL?17?M4 isolated strain was shown to be the highest titer with 10^-5.23 TCID₅₀/mL.?2?The H RBS region sequences of CDV strains were cloned and sequenced.Analysis of H RBS region sequences of the isolated strains displayed more than 93%nucleotide sequence identity to other CDV strains.The sequences results confirmed the isolated strains are positive to CDV.?3?The four isolated CDV strains were confirmed to replicate in the cytoplasm by detecting the specific green fluorescence,but the normal Vero-dog SLAM?VDS?cells in the negative control group did not show specific green fluorescence.IFA res?Lts further confirmed that the four isolated strains are positive to CDV.?4?Minks infected with LNDL?17?M4 isolated strain showed obviously clinical signs of canine distemper including high body temperature,ocular discharge,nasal discharge,and conjunctivitis.The viruses were still detected in nasal and ocular discharge swabs,bloody diarrhea swabs after 7 and 12 days challenge.Conclusion:Four CDV strains were isolated and identified.LNDL?17?M4 isolated strain was identified to be a virulent strain.LNDL?17?M4 isolated strain was demonstrated to have the highest virus load in lymphatic tissue and display the strongest ability in infecting the tissue organs caused cause the death of mink with secondary infection.