| Rabies, which is caused by rabies virus(RABV), is a serious worldwide anthropozoonosis.Since 2007, epidemic situation of human rabies has gradually become better in China, but approximate 1000 people every year still suffer from the torture of rabies, and its prevalence area is unceasingly expanding, which is quite severe for developing countries like China with large areas unimmunized.Humans possess the last position during the spread of rabies, and the human rabies is largely attributed to animals, so the root control of its source is the first priority, especially stray dogs and wild animals. To vaccinate these animals, oral immunization, would be the only feasible method.There are two kinds of oral vaccines, attenuated and recombinant live vaccine, the former is mainly used in Europe while is reletively danger with certain residual virulence and is not appropriate for stray dogs and wild animals. But the latter would be ideal, and America, Canada and Europe have licenced and used largely the recombinant RABV vaccines respectively based on vaccina virus and human adenovirus 5(V-RG and AdRG1.3-ONRAB?) in various wild animals to date, and these vaccine are proved to be effective. However, such vaccine is not developed yet in our country. To eliminate rabies, the research and application about oral vaccine in stray dogs and wild carnivores would be the only resort.Parainfluenza virus 5(PIV5), formerly known as simian virus(SV5) because of its discovery in primary monkery kidney cells in 1956, is a monopartite negative-sense RNA virus and belongs to the Paramyxovirus family, Rubulavirus genus. PIV5 can not only cause clinically “Kennel Cough” in cannine but also cause infectious respiratory symptoms in weaning calves(isolated strain: PV5-BC14) which is found by our lab in in Baicheng City, Jilin Province. With the development of reverse genetics of negative RNA virus, PIV5 have many obvious advantages such as direct infection, broad spectrum of host animals, stable genome and easy accesibility of high titer virus, and so on, so the superiority of PIV5 as a ideal recombinant vaccine vector that shows a great practical and financial potential value has made it used for preventing and controlling many important pathogens such as influenza virus and RABV.This study tries to establish the reverse genetics of PIV5 CC-14 vaccine strain, and express the glycoprotein(G) of RABV BD06 strain whose immunogenicity is much better in order to develope a superior recombinant rabies oral vaccine. In addition, this research that the highest nucleotide identity was observed between PV5-BC14 strain and PIV5 CC-14 strain based on the CDS fragment of NP gene. And we have confirmed that sporadic cattle rabies is usually ascribed to the bite of foxes and stray dogs in Xinjiang, Inner Mongolia and Heilongjiang, and so on.Accordingly, the research on PIV5 CC-14 strain-vectored rabies vaccine has practical significance for the prevention of canine and cattle rabies.Protocols and results:1. The complete genome sequencing and analysis of PIV5 CC-14 strain: Primers were designed based on the complete genome of SV5 which was derived from Gene Bank. The target genes were amplified by RT-PCR and ligated into pMD18-T vector, then sequenced, assembled and analyzed. The results indicated that the length of PIV5 CC-14 strain genome is 15246bp(GenBank accession NO. KP893891). The difference between CC-14 strain and other most PIV5 strain is the lack of SH gene. Based upon available GenBank sequences, the CDS fragment of NP gene was used for homologous alignment and phylogenetic analysis. The former reveals that NP gene possesses 1530 bp in length with 97.25%~99.80% homology with other strains, and98.62%~99.80% homology in amino acid, and the later reveals that this strain was closely related to PV5-BC14 strain of bovine from China.2. Construction of infectious clone of PIV5 CC-14 strain: According to the restriction enzyme analysis of the full genome of PIV5 CC-14 strain, seven primers were designed and plasmids were constructed using standard molecular biology techniques, then the full genome cDNA clone vector named pPIV5 preinstalled a foreign gene insertion site(NotⅠ) was obtained.To verify the completeness of the reverse genetics system, we inserted the EGFP gene which contained the transcription and replication components of PIV5 into NotⅠ site, then pPIV5-EGFP was constructed. And we inserted the RABV BD06 strain glycoprotein gene(G) with better immunogenicity into the same site, then pPIV5-BD06-G was obtained. To increase the rescue efficiency, the full genome cDNA clone vector were flanked at its left end by a T7 bacteriophage RNA polymerase promoter and at the right end by a hepatitis delta virus ribozyme(HdvRz). In addition, three helper plasmids named respectively pc DNA3.1-NP、pcDNA3.1-P and pcDNA3.1-L were constructed as methods mentioned above. All of the plasmids were sequenced, assembled and analyzed, the results show that these plasmids were correct.3. Rescue of recombinant PIV5 CC-14 strain: The plasmids of pPIV5-EGFP, pc DNA3.1-NP、pc DNA3.1-P and pc DNA3.1-L were co-transfected into BHK-T7 cells. At 48 h posttransfection,passage the cell into cell culture 25cm2 flask for 3-4 days, then observe whether there is fluorescence or not, and this work is still under way.4. Construction of helper cell line using for rescue of recombinant virus: We tried to establish a MDCK cell line stably expressing T7 RNA polymerase(named MDCK-T7) used to package the virus particle for PIV5 CC-14 strain can grow well in this cell. First, the G418 optimal concentration was confirmed to 1300μg/ml by G418 selection. Second, tranfect the pcDNA3.1-T7 plasmid into MDCK cell, and exert 1300μg/ml G418 next day, then change the G418 culture every two days. Third, after two week, the monoclonal cell were selected from the new-grown cells by limiting dilution method. The last, verify the monoclonal cell by tranfecting pUC57-IRES-T7-EGFP and so on. This work is still under way.5. Establishment of dectection method of PIV5 CC-14 strain: For lack of CPE, four methods including RT-PCR, indirect immunofluorescence(IFA), hemabsorption and hemagglutination test were performed to detect this virus. The results indicated the convenience of hemabsorption.Conclusions:1. This study completed the full genome sequencing and analysis of PIV5 CC-14 strain( GenBank NO. KP893891).2. Three helper plasmids, including pc DNA3.1-N, P, L and three full length cDNA clone plasmids consist of pPIV5, pPIV5-EGFP, pPIV5-BD06-G were constructed.3. We established a convenient and effective hemabsorption method to detect PIV5 CC-14 strain.4. The construction of MDCK-T7 cell line and the rescue of recombinant virus are still in progress.Innovations:1. It is the first time to date that PIV5-based vector in China is used for developing a effective rabies recombinant vaccine in order to explore a way of rabies oral immunization. This research has practical implications in preventing rabies in cannine and cattle.2. RABV BD06 strain which was isolated by our lab and is characterized by a classical epidemic rabies virus strain in China possesses glycoprotein(G) with better immunogenicity, so we chose the BD06-G as foreign gene and inserted it into pPIV5 in order to obtain a novel rabies oral vaccine with better and stronger immunity. |
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