Construction And Activity Detection Of Tick-borne Encephalitis Virus (TBEV) Report Virus Vector

Tick-borne encephalitis virus(TBEV)is an important tick-borne virus that belongs to the Flaviviridae genus Flaviviridae and is a positive-strand RNA virus.Members of this genus also include yellow fever virus(YFV),dengue virus(DENV),and Zika virus(ZIKV).Infection with TBEV may cause meningitis,encephalitis,and meningoencephalitis,which can severely cause life-long neurological complications.These symptoms are collectively referred to as tick-borne encephalitis(TBE).Since former Soviet scientists reported the disease in Soviet territories in 1937,the disease has been found in at least 34 countries including Europe,Siberia,Russia,northern China,and Japan.The incidence of TBE is increasing every year,and little is known about TBEV and other tick-borne viruses compared to other mosquito-borne flaviviruses.Therefore,it is still necessary to study the interaction between TBEV and host cells,and molecular biology about TBEV.Report virus is a powerful tool often used in monitoring viruses,studying their virulence,and screening for antiviral drugs.However,there have been no reports about TBEV report virus.In this paper,we have mainly carried out the following two aspects: TBEV Nluc reporter virus vector construction and activity detection;TBEV miRFP670 nano reporter virus vector construction and activity detection.?.Construction and activity detection of TBEV Nluc report virus vectorThe Nluc gene is a newly developed gene in the field of luciferase,which has the characteristics of small molecular weight and high sensitivity.The TBEV reporter virus containing the Nluc reporter gene was constructed,and the insertion site of the reporter gene was behind the 38 th amino acid of the TBEV capsid protein.Two cloning strategies are used.One is to connect a 2A sequence after the reporter gene to facilitate the cleavage of the reporter gene.Another strategy is to insert the 2A sequence before and after the reporter gene.After the construction of the Nluc reporter virus vector was completed,the luciferase activity and virus protein of the Nluc reporter virus were detected,and the virus growth curve was determined for all Nluc reporter viruses.The results showed that the TBEV Nluc reporter virus was successfully constructed,and the growth curve was consistent with the TBEV infectious clone.?.Construction and activity detection of TBEV miRFP670 nano reporter virus vectorAnother reporter gene used in the experiment is miRFP670 nano,a recently developed near-infrared protein that can combine with endogenous biliverdin in mammalian cells to produce fluorescence.When constructing the TBEV reporter virus vector containing miRFP670 nano,the 2A sequence was added before and after the reporter gene.After the construction of the miRFP670 nano reporter virus vector was completed,the expression of the reporter virus at the viral RNA and protein levels was detected,and the growth curve of the reporter virus was also detected.The results showed that the miRFP670 nano reporter virus of TBEV was successfully constructed.The growth curve and TBEV infection Sexual cloning is consistent.