| Canine distemper (CD) is an acute, febrile, contagious disease, which is caused by the canine distemper viruses (CDV), threaten to dogs, mink, fox and raccoon dog, etc. Along with canine distemper continuing to spread among different hosts, the host range has expanded to the entire carnivore from Canidae, Mustelidae. In recently, the fatal infection cases with CDV among nonhuman primate rhesus monkey (Macaca mulatta) and crab-Eating monkey (Macaca fascicularis) were reported. In order to understand canine distemper virus genetic evolution characteristics and pathogenic properties, we perform serial researches included virus isolation, sequence analysis, animal infected with CDV isolates, serum treatment, and the evaluation of interferon against CDV wild type virus.Five CDV were isolated, two canine distemper virus strains from canine lung and spleen (CDV-TM-CC, the CDV-Dog-SCh), two strains from fox lung and spleen (CDV-Fox-WF, the CDV-Fox-SY) and one strains from monkey lung (CDV-Monkey-BJ). All isolates were identified by electron microscopy, indirect immunofluorescence and PCR methods. The results showed that Vero-DST cells are effective for CDV isolated from the dog, fox and monkey samples. No cytopathic effect were oberserved on the Vero-DST and vero cells infected with1-10passages CDV-TM-CC. The titers of virus isolated from monkey replication in Vero-DST cell lines the expression of canine SLAM receptor was markedly lower than the virus isolated from dog did, the CDV-Monkey-BJ102.5TCID50/ml while the CDV-TM-CC103.5TCID50/ml. The results indicated that the CDV virus isolated from monkey is not susceptible to the canine SLAM receptor.According to the standard strain sequence AF164967(A75/17), the whole genome sequenced primers were designed, the whole genome of the five viruses were sequenced. The evolution analysises of The HA of the5isolated viruses and GenBank sequences showed that all isolates blong to Asia Ⅰ virulent strain, and the N-terminal potential glycosylation sites, cysteine signal and the location of the F gene and H gene was the same as the virulent strain. The F1fragment of the canine distemper virus from the monkey had seven unique amino acids sites:317R,3471,465I,466T,471P,472W,586N. The canine distemper virus (CDV-TM-CC) were passaged for25generations in Vero cells, the full genome sequence of the fifth, tenth, twentyith generations were respectively sequenced, and twenty-five site differences were found, three sites which was related to the F protein changed:Y99S, H115Q, S116Y, and they were the same in tenth, twentyth generation viruses, so these three sites may be associated with the formation of the cell lesions. Ⅴ gene analysis also showed that they had a close phylogenetic relationship with the virulent strain, and reported that Ⅴ protein amino acid110Y play an important role in the anti-interferon pathway. The single-amino acid mutant Ⅴ protein Y110D retained slight STAT2 binding but almost completely lost STAT1interaction. There was diversity at272amino acid residue between the canine distemper virus and measles virus Though the configuration analysis, C272R mutation can make Zn2+binding capacity decrease, and the zinc finger structure of the spatial configuration is influenced. Throuth the research of measles virus, C272R V protein rendered the V protein IFN-insuppressible and the R272C conversion conferred an IFN-suppressive function on the V protein. Only V proteins possessing the272C residue accelerate nuclear translocation of IRF-3. V protein does not affect TICAM-1or RIG-I-induced IFN-β promoter activation.Hence, the272C residue is crucial for the V protein to block MDA5function and MDA5is the molecule which V protein targets for inhibition of the initial induction of IFN-β.The results of pathogenicity studies different generations of CDV in the Vero, the results indicate that Vero cells have a strong attention effect to CDV,20passages can not lead to dog’s morbidity. Gene sequencing of canine distemper virus in Vero passaged25generations, A total of25sites differences were found in5th passage,10th passage and20th passage between which related to integration of the F protein changes in the three loci:Y99S, H115Q, S116Y. and10generations,20on behalf of sites in the same, so these three animo acid loci may be associated with the formation of cytopathic effect.Using dogs as experimental animals, though the screen of the infection routes and optimization of the infection dose, the canine distemper virus Vero isolated from the Tibetan mastiff strain is determined as a standard strain, the dogs of three to four months were infected by104TCID50though intranasal and eye infection routes, we established a stable model of the infection of dogs. Infected animals shows typical CDV clinical symptoms, the detoxification after three to five days, high fever, diarrhea, the purulent secretions in the eyes and noses, but rarely neurological symptoms. Based on this model, we determined the distribution of the virus in the body and detoxification states. The data show that the nasal turbinates, the submandibular lymph nodes, lung, thymus, spleen, mesenteric lymph nodes, intestines were strongly positive. Nearby tissues of the cerebellum, hypothalamus and herpes were weakly positive. Brain, pancrea, liver, bladder were negative. Canine distemper virus was detected from the decrees and nasal after three days of the infection, and the presence of the eyes and pharynx secretions need about seven days. The lung, intestine, and spleen were strongly positive though the pathological observation.In the different proposals of treatment trials using the model of the dog infection, the survival rate of the high-dose treatment group was33.3%, the disease course extended one to two days more than the control group; and low-dose group, one days; in the treatment group in serum supplemented with poly and Vit E, the survival rate was25%, duration of one to four days longer than the control group; the survival rate of the vaccine treatment group was20%, and canine distemper virus, which was detected was negative. After Checkouting. dead animals above, we find that all lesions are slighter than the control group. In summary, the single-factor treatment does not completely cure the infected dogs; it can only prolong the disease course, needing reasonable antibiotics, improved immunity and nutrition drugs added.In the ferrets and monkeys trials based on the dog animal model, the canine distemper virus from the ferrets and the monkeys make the ferrets develop disease and produce significant clinical symptoms. The infected tests of the canine distemper virus in cells from macaques show no animal infection and the presence of anti bodies in the blood; the lung and spleen tissues milled liquor from dogs and monkeys infected assay, finding that virus detection were positive in all experimental animal blood after eight days of the infection. The serum antibody of Tissue liquor from monkeys is1:256after twenty-two days, but the dog tissues were negative. And two groups were no obvious clinical symptoms, only the clinical fever phenomenon. Demonstrating that the monkeys only were infected canine distemper virus, but did not cause disease, The results show that the canine distemper virus can infect non-human primates monkeys infected with canine distemper virus, and thus the cross-species spread of the phenomenon should be of concern. Therefore, the pathogenicity of canine distemper virus for monkeys and the dead reasons has yet been further research.It reported that canine distemper virus has a strong anti-interferon capacity. We examined the inhibitory effect of interferon virulent strain of canine distemper, domesticated strains of virulent in Vero cells and vaccine strain. The experiment used250IU,1IU, OIU the interferon sense of experiment3groups in Vero cells. The supernatant was removed after24hours in each group, respectively add1000TCID50of the CDV-TM-CC5passages, the20th passages in Vero cells and the vaccine strain (CDV-HL). The results show that a significant inhibitory effect of interferon on the vaccine strains and domesticated strains, but significantly attenuated the inhibition of the virulent strain, and sequence analysis revealed that V gene of CDV-TM5passages and20passages associated with the interferon signaling pathway exist4Amino acid differences, these sites have not been reported.In conclusion, we have isolated five wildtype CDV strains and explored the genitic information and pathogenicity of wildtype CDV. We also have established the CDV infection model and confirmed the virus cross-species infections from canide to non-human primate. Our results provied the theoretical and technological basis for CDV prevention and control. |
PDF Full Text Request