Construction of a Dengue NanoLuc Reporter Virus for In Vivo Live Imaging in Mice

Abstract

Since the first isolation in 1943, the dengue virus (DENV) has spread throughout the world, but effective antiviral drugs or vaccines are still not available. To provide a more stable reporter DENV for vaccine development and antiviral drug screening, we constructed a reporter DENV containing the NanoLuc reporter gene, which was inserted into the 5′ untranslated region and capsid junction region, enabling rapid virus rescue by in vitro ligation. In addition, we established a live imaging mouse model and found that the reporter virus maintained the neurovirulence of prototype DENV before engineering. DENV-4 exhibited dramatically increased neurovirulence following a glycosylation site-defective mutation in the envelope protein. Significant mice mortality with neurological onset symptoms was observed after intracranial infection of wild-type (WT) mice, thus providing a visualization tool for DENV virulence assessment. Using this model, DENV was detected in the intestinal tissues of WT mice after infection, suggesting that intestinal lymphoid tissues play an essential role in DENV pathogenesis.

NS1 Protein N-Linked Glycosylation Site Affects the Virulence and Pathogenesis of Dengue Virus

“…To construct infectious clones of the N-glycosylation site mutant strains of dengue virus NS1 protein, we selected fragments flanking the NS1 protein glycosylation sites with suitable restriction sites at the 5′ and 3′ ends, PCR-amplified them, and cloned them using In-Fusion cloning into a dengue virus infectious clone plasmid pSPTM-DENV (Ban18HK20) reported previously [ 29 , 30 ]. Table S1 shows the amplification primers and enzymatic sites for cloning.…”