Live attenuated vaccine is one of the most effective vaccines against flavivirus. Recently, site-directed mutation of the flavivirus genome using reverse genetics techniques has been used for the rapid development of attenuated vaccines. However, this technique relies on basic research of critical virulence loci of the virus. To screen the attenuated sites in dengue virus, a total of eleven dengue virus type four mutant strains with deletion of N-glycosylation sites in the NS1 protein were designed and constructed. Ten of them (except for the N207-del mutant strain) were successfully rescued. Out of the ten strains, one mutant strain (N130del+207-209QQA) was found to have significantly reduced virulence through neurovirulence assay in suckling mice, but was genetically unstable. Further purification using the plaque purification assay yielded a genetically stable attenuated strain #11-puri9 with mutations of K129T, N130K, N207Q, and T209A in the NS1 protein and E99D in the NS2A protein. Identifying the virulence loci by constructing revertant mutant and chimeric viruses revealed that five amino acid adaptive mutations in the dengue virus type four non-structural proteins NS1 and NS2A dramatically affected its neurovirulence and could be used in constructing attenuated dengue chimeric viruses. Our study is the first to obtain an attenuated dengue virus strain through the deletion of amino acid residues at the N-glycosylation site, providing a theoretical basis for understanding the pathogenesis of the dengue virus and developing its live attenuated vaccines.

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.