| Canine distemper(CD) is a highly contagious viral pathogen that caused by canine distemper virus(CDV), and characterized by fever, gastroenteritis, hyperkeratosis and neurological manifestations. CD outbreaks was observed in breeding fur-bearing farms in parts of Shandong province between August, 2013 and August, 2014. The present study was performed in the following aspects to reveal the prevalence and the genetic diversity of CDV and for providing theoretical foundation to prevent and control CD.1. The prevalence investigation were conducted in 25 large scale breeding fur-bearing farms in 10 regions, including Zhucheng, Qingdao, Wendeng, Rongcheng, Linyi, Linqu,Yantai, Liaocheng, Taian, Laiwu, in Shandong province from August, 2013 to August, 2014.665 clinical samples that submitted by private owners were analyzed for the difference of seasonal distribution and mixed infection, moreover, 100 slaughter mink serum samples that collected from Zhucheng city were used for the detection of CDV antibody(CDV-Ab) titers.Results showed that CDV positive rate is 22.6%(150/665), of which the positive rate of single infection of CDV is 46.7%(70/150), the CDV positive rate of multiple infection with bacterial disease is 53.5%(80/150), the rate of co-infection with Pseudomonas aeruginosa is as high as 31.3%(25/80). The average CDV-Ab positive rate is 35%, and a strong positive correction was found between the immunization times and CDV-Ab positive rates.2. The complete hemagglutinin(HA) gene were sequenced from 6 RNA-positive clinical specimens. Sequence analysis based on the HA gene demonstrated that the 6 new isolates were highly similar to each other(98.7%-99.5% nt and 97.9%-99.2% aa), while displayed lower identity(89.8%-90.6% nt and 88.2%-90.4% aa) to the vaccine strains.Phylogenetic analysis revealed that these CDV strains were characterized as Asia-1 genotype,clearly distinct from vaccine strains and other wild-type foreign CDV strains. Inspecting in the HA protein alignment, strain SD(13)9, which was detected in a fox, displayed 10 potential N-linked glycosylation sites(N-X-S/T), while the other 5 Shandong CDV strains exhibited 9,whereas vaccine strains have 4 or 7 potential sites. Moreover, a novel potential N-X-S/T at position 542-544 was observed in all CDV isolates. In the present study, all the 6 Shandong CDV strains were 530 G on HA protein, while possessed a tyrosine to histidine substitution at residue 549, regardless of host species.3. The full-length genome of strain CDV-ZC was amplified, sequenced and aligned withother CDV strains by RT-PCR. The complete genome nucleotide sequence of CDV-ZC(GenBank accession number, KJ994343) contains 15690 nt, exhibited highly homology to the CDV strain PS(97.8% at nt level), while identity to the vaccine strains Onderstepoort was the lowest(91.6% at nt level). The results of phylogenetic analysis based on the HA gene was consistent with that based on the complete genome. All CDV strains clustered into wild and vaccine strains. Phylogenetic analysis based on the whole genome demonstrated that CDV-ZC strain belonged to Asia-1 genotype, along with Chinese wild-type strain PS. CDV-ZC strain had the highest similarity with that of PS strain. Restriction Fragment Length Polymorphism(RFLP) analysis revealed that CDV-ZC was a wild strain. The results of viral neutralization assay(VNA) showed that there was antigetic differences among CDV-ZC strain and the vaccine strain. |
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