2.3. Construction of Chimeric Virus

The JEV GI/GIII intertypic virus was constructed with the K05GS envelope protein in the SA14-14-2 backbone. The prM/E proteins of the K05GS strain were codon-optimized and cloned into the pBHA vector with Bioneer to effectively express the target genes in humans. The cDNA of JEV SA14-14-2 was copied into the pACYC184 vector, and the E region of the infectious cDNA clone SA14-14-2 was changed with the in vitro-synthesized E region of Syn_GI. The plasmid that the prM/E region of the K05GS strain codon-optimized was Syn_GI, and it was subcloned into the pACYC184 vector by Bioneer. The E regions of K05GS and SA14-14-2 backbones were amplified with specific primers (Table S1), and each DNA fragment was cloned into the corresponding region with the In-Fusion Cloning System (Takara Bio, Seoul, Korea). After the cloning, plasmids were amplified from Escherichia coli HST08 cells.

To create the virus, we conducted transfection in BHK-T7 cells with Lipofectamine^® 2000 Reagent (Invitrogen™, Carlsbad, CA, USA). Cells were maintained in DMEM with 10% heat-inactivated FBS before transfection. After three days, the transfected cells were thawed thrice to obtain the viral supernatants. The complete recombinant infectious clone was termed SA14-GI env.