Development Of Viral Vectors Based On Attenuated Rabies Virus CTN-181Strain

The highly attenuated rabies virus CTN-181strain has been derived from the parental CTN-1strain, which was approved to be used for production of human rabies vaccines in China. It will be promising to be used as rabies vaccine for animals and as viral vectors carring foreign genes. Therefore, in this study, we aims to construct viral vectors based on CTN-181strain and recombinant vector vaccines against HCVand RV.Section#1-Development of Viral Vectors Based on Attenuated Rabies Virus CTN-181StrainCompared with its parental strain, twenty-eight nucleotides and13amino acids substitutions located in whole genome of CTN-181strain, especially the amino acid substitution from pathogenic Arginine to nonpathogenic Glutamine in position333of glycoprotein. So CTN-181strain has been selected as virus seed for vector construction and vectors encoding the secreted gene marker Gaussia luciferase (Gluc) were generated based on it. Vectors included replication competent CTN-Gluc and CTN/GQ333r-Gluc in which the amino acid in position333of glycoprotein was mutated from Glutamine (Q.) to Arginine (R), and replication constrained CTNAG-Gluc in which the glycoprotein encoding gene (G) was deleted. The growth of recombinant RVs in transfected cells were confirmed through biochemical assays of Gluc activities. Gluc expression in recombinant CTNAG-Gluc virus was highest while that in CTN/GQ333R-Gluc virus was lowest. The optimal time to harvest recombinant RVs was determined that72hrs post-transfection is optimal to harvest CTN-Gluc and CTNAG-Gluc, while96hrs is optimal to harvest CTN/GQ333R-Gluc. The function of pathogenic and nonpathogenic rabies glycoprotein in virus recovery was examined that the titer of rescued virus was lowered even when the amino acid in G333position of glycoprotein was mutated from nonpathogenic Gln to pathogenic Arg. Finally, the addition of glycoprotein in the process of transfection was slightly beneficial for virus recovery. In short, viral vectors based on a human rabies vaccine strain CTN-181were successful. Glue was useful as an in vitro gene marker for monitoring the growth of recombinant RVs iteratively in cell culture.Section#2-Construction of chimeric rabies viruses carring envelop proteins E1E2of Hepatitis C VirusOn the basis of the established vector systems, the E1E2genes of HCV HeBei strain were cloned and the chimeric viruses CTN-E1E2, CTNAG-E1E2and CTN/GQ333r-E1E2were recovered. The results of DFA and RT-PCR demonstrated that the chimeric viruses were rescued successfully. All the chemiric viruses have the ability to re-infect normal sensitive cell lines and express HCV E1E2genes detected in the level of mRNA. The establishment of chimeric RVs expressing HCV E1E2genes provides the evidence that it is feasible to develop novel HCV vaccines based on viral vectors in theory and in practice.Section#3-Construction of recombinant rabies viruses carring two copies of glycoprotein encoding geneRecombinant RVs including CTN, CTN/GQ333R, CTN/2G and CTN/2GQ333R were also rescued the same way as above. Compared with parental CTN-181, the recombinant CTN is defective in pseudogene, CTN/GQ333R is derived from CTN with GQ333R mutation, CTN/2G and CTN/2GQ333R encodes two copies of G or GQ333R gene in genome. The results of DFA, RT-PCR and virus titration demonstrated that the recombinant RVs were recoveried successfully. All the recombinant RVs were able to infect normal cells and the titer of these RVs kept slowly increasing generation by generation in infected cultured cells. The findings make a foundation for developing genetic engineering live rabies vaccines.ConclusionsThe replication competent and replication constrained viral vectors based on CTN-181strain were successfully established through two subclones. Both of them have the ability to express foreigh genes and the replication competent virus can propagate in normal BHK-21cells. Followed by, the chemiric RV/HCV viruses expressing envelop proteins E1E2of HCV and recombinant RVs expressing two copies of RV GP were rescued. Although the further studies on evaluation of immunization effect was hampered owing to the low titer of rescued viruses, this study opens a way to discover vector vaccines against HCV and recombinant RV vaccines, and provides a molecule model to study the mechanism of virus pathogenicity.