Genomic sequencing and neutralizing serological profiles during acute dengue infection: A 2017 cohort study in Nepal

Abstract

Dengue virus (DENV) is a mosquito-borne flavivirus that poses a threat to nearly 50% of the global population. DENV has been endemic in Nepal since 2006; however, little is known about how DENV is evolving or the prevalence of anti-DENV immunity within the Nepalese population. To begin to address these gaps, we performed a serologic and genetic study of 49 patients from across Nepal who presented at central hospitals during the 2017 dengue season with suspected DENV infection. Of the 49 subjects assessed, 21 (43%) were positive for DENV NS1 antigen; of these; 5 were also anti-DENV IgM+ IgG+; 7 were DENV IgM+ IgG-, 2 were IgM- IgG+, and 7 were IgM-IgG- by specific ELISAs. Seven of the 21 NS1 positive sera were RNA positive by RT-PCR (six DENV2, one DENV3), suggesting that DENV2 was the dominant serotype in our cohort. Whole-genome sequencing of two DENV2 isolates showed similarity with strains circulating in Singapore in 2016, and the envelope genes were also similar to strains circulating in India in 2017. DENV-neutralizing antibodies (nAbs) were present in 31 of 47 sera tested (66%); among these, 20, 24, 26, and 12 sera contained nAbs against DENV1, 2, 3, and 4 serotypes, respectively. Additionally, 27 (58%) samples had nAbs against multiple serotypes (2 or more). Serology analysis suggested that 12 (26%) and 19 (40%) of the 47 subjects were experiencing primary and secondary DENV infections, respectively. Collectively, our results provide evidence for current and/or past exposure to multiple DENV serotypes in our cohort. These data suggest that expanded local surveillance of circulating DENV genotypes and population immunity will be important to effectively manage and mitigate future dengue outbreaks in Nepal.

Fig 1. Geographic distribution of the home residences of the study subjects.

Patients originated from across Nepal and were seen at hospitals in Kathmandu (green dot) and Chitwan (yellow dot). White borders demarcate the seven provinces in Nepal. Abbreviation: No, number. Image modified from Wikipedia (https://en.m.wikipedia.org/wiki/File:Provinces_of_Nepal.png).

Fig 2. Distribution of nAb titers in samples collected during the 2017 outbreak.

(A and B) NT₉₀ titers were determined according to DENV1–4 reference serotypes (A) and sample collection site (B). Mean values for n = 47 (n = 21 Kathmandu, n = 26 Chitwan) with circles representing individual samples. Dashed lines represent the cutoff value for nAb positivity (NT₉₀ = 40). Mean values were compared using the non-parametric Kruskal–Wallis test.

Fig 3. Phylogenetic analysis of full-genome sequences of the Nepal 2017 DENV2 isolates generated here and other DENV2 isolates.

Phylogenetic tree comparing the DENV2 isolates from this study (blue, PP152366|2017|NP and PP152367|2017|NP), with other DENV2 full-genome sequences present in NCBI. Strains are labeled by GenBank ID, as well as year and country of isolation (CN, China; SG, Singapore; NP, Nepal).