Cloning And Expression Of H Gene And Molecular Epidemiology Of Canine Distemper Virus

Canine distemper (CD),caused by canine distemper virus (CDV), is an acute and highly contagious viral disease of dogs as well as some other animals. The virus genome consists of genes encoding N, P, M, L, F and H proteins. Many studies have shown that Hemagglutinin protein (H) of CDV is necessary during infection and a major target antigen for the host immunesystem. In addition, H protein is the most variable structural protein which can serve as the indicator in analysis of molecular evolution and epidemic study of CDV. The use of live attenuated vaccines has successfully prevented CDV infections in domestic dogs. However, in recent years, several episodes of CD in vaccinated animals have been reported throughout the world, which might be due to the frequent variation of epitopes especially in H protein and the failure of vaccination as well as the lower level of antibodies after uncorrected vaccination strategies. This study was aimed to find out the epidemic and evolutionary trend of CDV in Hangzhou, which was usefull for vaccine development. In addition, the H protein was expressed and analyzed. Thus, during January 2007 and April 2008, fourty-four blood samples or conjunctival swabs of diseased dogs suspected of canine distemper were collected. After detection for CDV, the H gene were cloned and sequenced for analysis of genetic characterization.1. The diagnosis of CD infection is usually based on clinical symptoms, which may result in misdiagnosis in cases. So a sensitive and specific method for detection of CDV infection is necessary. In this stuy, an one-step RT-PCR and a nested PCR are compared for their role in CDV detection. Both the methods reacted with the CDV vaccinal strain, but not with canine parvovirus. The expected fragment of the N gene was detected in 11.36% samples by one-step RT-PCR and 63.64% by nested PCR. So the nested PCR is more reliable than the one-step RT-PCR.2. To gain insight in epidemic and evolutionary trend of CDV in Hangzhou, phylogenetic analysis based on H gene was conducted. The results revealed that all the CDV isolates were segregated into seven groups which showed relationships with regard to geographic regions. Obviusly, most isolates of Asian country were in groupⅠand groupⅤ, but no Chinese isolates was in groupⅤ. Most isolates of our study (except one in group VII) were clustered in groupⅠt ogether with the most Chinese isolates. Prediction of potential N-glycosylation sites revealed that there were different sites in different genotypes. Compared to vaccine isolates of four to seven sites and other wildtype isolates of seven to nine sites, most of the isolates from Hangzhou contain nine potential N-glycosylation sites, except for HZ005 and HZ011 (eight and six respectively). Variability of the CDV H protein was analyzed by the differences between non-synonymous (dN) and synonymous (dS) rates and the entropy values. Majority of the positions within H were subject to negative selection. Our results suggest that recent isolates from Hangzhou are predominantly of groupⅠand they have shifted away from vaccine strains used in the area.3. To isolate CDV, canine epithelial kidney cells (MDCK) were inoculated with lung specimens from suspected dogs. Then one isolate of CDV, designated as HZ026, was found positive at three, six, nine, twelve passages by nested PCR. Then two fragments of H gene, containing 816bp and 459bp of its 3′end, were amplified and cloned into the prokaryotic expression vector pET30a for induction, which showed two bands of 36.4ku and 22.2ku respectively as analysed by SDS-PAGE. Moreover, both of the two fusion proteins could be recognized by CDV positive serum by Western blotting, indicating that the proteins had the antigenitic epitopes of H gene of CDV and could be used in future serological studies.