Construction And Application Of Tick-borne Encephalitis Virus Reverse Genetic System

Tick-borne encephalitis(TBE)is a central nervous system disease caused by tickborne encephalitis virus(TBEV)infection,which can lead to death or residual neurological sequelae in severe cases.There are TBE natural epidemic areas in our country.Due to low vaccine coverage of risk groups and social activities,new cases are continued recorded which pose a threat to public health.In comparison to the widely studied mosquito-borne flavivirus,as a tick-borne flavivirus,TBEV still has many research gaps,and there is currently no specific antiviral drug available in clinical practice.Therefore,in-depth study on TBEV is required.The RNA virus reverse genetics system refers to the technology of obtaining the complementary DNA of the full-length viral genome through molecular biology methods,and thus rescuing infectious virus particles from the cells.The reverse genetic system allows us to modify the virus genome at the DNA level and further characterize the viral pathogenesis.According to the "List of Pathogenic Microorganisms Infected Among Humans",TBEV belongs to the first category of pathogenic microorganisms and needs to be operated in biosafety level 3(BSL-3)laboratory,which has stringent requirements for experimental facilities and operators,limits the TBEV-related research.In order to facilitate study on the molecular mechanisms,drug screening,and vaccine development of TBEV,this project aim to establish TBEV research platform based on RNA virus reverse genetic system which is not relying on BSL-3 facilities.The platform consists of four systems: The replicon was generated by deleting majority of the viral structural genes which can reflect the process of viral replication and translation without producing infectious virus particles.The replicon system that relies on transient transfection can be flexibly used to explore the molecular mechanism of TBEV replication,while the stable transfection cell line that can stably carry the TBEV replicon is suitable for high-throughput drug screening targeting the replication process of TBEV.The chimeric YF17D/TBEV virus using the yellow fever virus attenuated vaccine strain YF17 D as the backbone expressing TBEV membrane protein and envelope protein,which can be operated in BSL-2 facilities.Since it’s containing the TBEV-specific envelope,it is suitable for serum antibody neutralization detection and drug screening against TBEV viral envelope.The TBEV single-round infection particle(SRIP)system established through trans-complementation can simulate the entire life cycle of TBEV and can be used for both structural and non-structural function analysis while it can only initiate single round of infection in cells.Due to its single round infection,we can operate the SRIP in BSL-2 facilities.The systems mentioned above can work coordinately with each other in the application and do not rely on BSL-3 level facilities.We can use the platform to explore TBEV pathogenesis,anti-TBEV compound screening,and neutralizing antibody detection;at the same time,the insertion of reporter genes in the viral genome can achieve real-time tracking of virus activity in the cells,which represents time-saving,labor-saving and the detection approach can be automated,establishing a foundation for high-throughput screening.Using established single-round infection particle reporter system,the molecular mechanism of the TBEV membrane protein affecting virus infection was explored.The results showed mutation of a highly conserved amino acid position on the TBEV membrane protein would lead to significant decrease in virus titer.The inhibitory effect could be due to decreased cleavage efficiency of the TBEV precursor membrane protein.Next,a small molecule compound library was used to screen anti-TBEV compound by TBEV single-round infection particles,and 8 compounds with antiviral activity and low cell cytotoxicity were obtained,further enriching the candidate drug library for TBEV treatment.In summary,this project has established the platform that can conduct research on TBEV without relying on BSL-3 laboratories.The platform includes four TBEV reporter systems that can be applied to different fields and suitable to various experimental demands,provides convenience for the subsequent deepening of TBEV research.