Development Of Replicon-based Trans-packaging System Of Dengue Virus

OBJECTIVEWe developed the dengue virus replicon-based trans-packaging system by the use of reverse genetics operations. Then dengue virus single-round particles were produced in the system we have established.METHODS1. On the base of DV1 GZ-06 cDNA, dengue virus 5'terminal genome and GFP gene were fused together by overlap PCR technique first. Then the fusion gene was inserted into the corresponding region of dengue virus genome and replaces most of the structural gene to constructed dengue virus replicon with GFP gene reporter (DV1-EGFP-RP). In order to set up a matched control a lethal mutation replicon (DV1-EGFPâ–³GDD-RP) was constructed at the same time. The replicons were further detected and characterized by reporter gene system and RT-PCR, respectively.2. DV/JEV chimeric prME gene sequence was constructed by over-lap PCR technique. Then the chimeric gene was inserted into plasmid pcDNA3.1 to constructed eukaryotic expressed vector. The vectors were transfected into BHK-21 and detected and characterized by IFA and RT-PCR. The virus-like particles were purified by ultracentrifugation, and then identified by Western blot and electron microscope techniques.3. The DV1 reporting replicon DV1-EGFP-RP was transcription into infectious RNA and then electroporated into BHK-21 cells. After 48h, the BHK-21 cells were transfected with the helper packaging chimeric plasmid. Then dengue virus structural proteins and non-structural proteins were co-expressed in the host cells.48 hours later, the cell culture supernatant was collected. The dengue virus replicon-based virus-like particles in the supernatant were purified by ultracentrifugation, and then identified by RT-PCR, western blot and single-round infectious in cell cultures.RESULTS: 1. Constructed a dengue virus replicon vector included EGFP reporter gene which could highly expressed in host cells.2. Succussful construct pcDV/JEV prME which could secret high levels of virus-like partcles into the culture fluid of plasmid-transformed BHK-21 cells.3. Prepared single-round infectious particles of dengue virus.CONCLUSIONIn this research, we successfully established the DV1 replicon-based trans-packing system which can stably produce single-round infectious DV1 virus-like particles and lay a good foundation for the further research.