Dengue virus surveillance in Nepal yields the first on-site whole genome sequences of isolates from the 2022 outbreak

Abstract

Background: The 4 serotypes of dengue virus (DENV1-4) can each cause potentially deadly dengue disease, and are spreading globally from tropical and subtropical areas to more temperate ones. Nepal provides a microcosm of this global phenomenon, having met each of these grim benchmarks. To better understand DENV transmission dynamics and spread into new areas, we chose to study dengue in Nepal and, in so doing, to build the onsite infrastructure needed to manage future, larger studies.

Methods and results: During the 2022 dengue season, we enrolled 384 patients presenting at a hospital in Kathmandu with dengue-like symptoms; 79% of the study participants had active or recent DENV infection (NS1 antigen and IgM). To identify circulating serotypes, we screened serum from 50 of the NS1⁺ participants by RT-PCR and identified DENV1, 2, and 3 - with DENV1 and 3 codominant. We also performed whole-genome sequencing of DENV, for the first time in Nepal, using our new on-site capacity. Sequencing analysis demonstrated the DENV1 and 3 genomes clustered with sequences reported from India in 2019, and the DENV2 genome clustered with a sequence reported from China in 2018.

Conclusion: These findings highlight DENV's geographic expansion from neighboring countries, identify China and India as the likely origin of the 2022 DENV cases in Nepal, and demonstrate the feasibility of building onsite capacity for more rapid genomic surveillance of circulating DENV. These ongoing efforts promise to protect populations in Nepal and beyond by informing the development and deployment of DENV drugs and vaccines in real time.

Keywords: Capacity building; Dengue outbreak; Dengue virus; Genomic surveillance; Whole genome sequencing.

Fig. 1

(a) Project overview (n values in parentheses). At Sukraraj Tropical and Infectious Disease Hospital in Nepal, 384 dengue-suspected patients were enrolled, blood samples collected, and NS1⁺ (dengue-confirmed) samples isolated (light blue boxes). At Tribhuvan University, also in Nepal, 50 samples were serotyped and whole-genome sequences obtained for 23 of them by NGS (yellow boxes). Finally, bioinformatic analyses were performed at Tribhuvan University in Nepal and Washington University/St Louis in the USA (green boxes). (b) The vast majority of study participants were from temperate districts.Demographic distribution of 296 of the 384 dengue-suspected cases. Enlargement, part of Bagmati province showing the highest density and number of dengue-suspected cases in Kathmandu and Lalitpur districts

Fig. 2

Phylogenetic tree of DENV1 full-length genomes. A maximum likelihood tree of DENV1 created using IQ-tree2, and dated to infer ancestral sequences with TreeTime. Sequences were named by accession number, country of origin, and year, and nodes labelled with the predicted year of the common ancestor; unrelated clades were collapsed. Red, 3 DENV1 genomes from this study (Table S1). Blue, 3 recently reported DENV1 genomes

Fig. 3

Phylogenetic tree of DENV2 full-length genomes. A maximum likelihood tree of DENV2 created using IQ-tree2, and dated to infer ancestral sequences with TreeTime. Sequences were named by accession number, country of origin, and date, and nodes labelled with the predicted date of the common ancestor; unrelated clades were collapsed. Red, 2 DENV2 genomes from this study (Table S1)

Fig. 4

Phylogenetic tree of DENV3 full-length genomes. A maximum likelihood tree of DENV3 created using IQ-tree2 and dated to infer ancestral sequences with TreeTime. Sequences were named by accession number, country of origin, and year, and nodes labelled with the predicted year of the common ancestor; unrelated clades were collapsed. Red, 1 DENV3 genome from this study (Table S1). Blue, 3 recently reported DENV3 genomes