Whole Genome Sequencing and Phylogenetic Analysis of Dengue Virus in Central Nepal from 2022 and 2023

Background: In Nepal, dengue is an emerging disease of growing concern as outbreaks are continuously increasing in size and geographic reach, and beginning to affect areas previously thought dengue-free. Dengue genomic surveillance has previously been limited within Nepal, however, with the increase in accessibility to sequencing technologies since the COVID-19 pandemic, it has recently become more feasible. Methods: This hospital-based retrospective study utilized banked samples from the 2022 and 2023 dengue seasons from Dhulikhel Hospital/Kathmandu University Hospital in central Nepal. Next generation sequencing was performed to obtain whole genome sequences of dengue virus which were analyzed phylogenetically using a maximum likelihood GTR+G model. Mutations were evaluated across viral particle regions using the GISAID DengueServer. Results : We obtained 41 full-length sequences of DENV from 80 PCR+ samples–24 from 2022 (58.5%) and 17 from 2023 (41.5%). We identified a shift in the majority serotype of our samples from DENV-1 in 2022 to DENV-3 in 2023, though 3 out of the 4 serotypes were identified in both years. Phylogenetic analysis revealed clusters within genotype III of DENV-1 and genotype III of DENV-3 closely related to strains from an outbreak of DENV in northern India in 2018-19. DENV-2 sequences fell into the Cosmopolitan genotype IV-A1 and IV-B2 clades and were related to sequences from South and Southeast Asia and the US, pointing to the global nature of dengue transmission. The NS3 showed the highest frequency of mutation whereas NS2B, NS4, NS5 and E were the most conserved. The most common mutations found were substitutions L17M and T20I in the 2K peptide. A high number of mutations were observed in DENV3, followed by DENV2 and DENV1, with some mutations being unique to specific serotypes and others matching previously reported strains. Conclusions : We identified possible clade shifts in the DENV-1 and -2 populations and a rising prevalence of DENV-3. Our study showed a high level of serotype diversity of DENV circulating in central Nepal. Furthermore, our results indicate that DENV populations in Nepal are related to a geographically diverse set of sequences but are most strongly influenced by Indian strains of DENV.