Recombinant Rabies Virus With a Multiple Cloning Site: A Platform Capable of Immunological Alterations

Abstract

Background: Rabies is fatal encephalitis, i.e., preventable by appropriate vaccination. Reverse genetics has proved promising for manipulating the rabies virus immunological characteristics. The insertion or deletion of a gene from the rabies genome could render specific functions to the rabies virus. Materials and Methods: A multiple cloning site including 111 nucleotides long harboring 10 single-cut restriction sites have been designed. The designed fragment was cloned between the G and L genes of the rabies virus genome. The recombinant rabies virus was rescued, and its infectivity was confirmed in the BHK-21 cell line. The recombinant virus propagation was compared with the initial rabies virus strain. Statistical analysis was performed using GraphPad Prism. Results: The cloning and localization of the multiple cloning site were verified by nucleotide sequencing. The recombinant virus properly propagated and rescued in the BHK-21 cell line. Comparing the recombinant virus with the initial rabies virus has shown that both viruses had similar functionality and propagation rate. Conclusion: The recombinant virus obtained in the present study could facilitate further cloning experiments. Examples include constructing a marker virus, carrying green fluorescent protein to be used either in rabies immunity assays or tracking the virus infection in relevant tissues.