Clinical, Virological, and Immunological Features in Cosmopolitan Genotype DENV-2-Infected Patients during a Large Dengue Outbreak in Sri Lanka in 2017

Abstract

In 2017, Sri Lanka experienced its largest dengue epidemic and reported severe and unusual presentations of dengue with high morbidity. This outbreak was associated with the reemergence of dengue virus-2 (DENV-2), with the responsible strain identified as a variant of the previously circulating DENV-2 cosmopolitan genotype. In this study, we characterized the DENV-2 cosmopolitan genotype from patients during this epidemic. Also, we identified host factors that contributed to the severity of dengue infection in patients infected with this particular virus. Ninety-one acute serum samples from patients at the National Hospital in Kandy were randomly selected. Of these, 40.2% and 48.9% were positive for dengue IgM and IgG, respectively. NS1 antigen levels were significantly higher in primary infections. The severe dengue (SD) and dengue with warning signs (DWWS) groups exhibited significantly higher viral genome and infectivity titers than the dengue without warning signs (DWoWS) group. The highest viremia level was observed in SD patients. As for host cytokine response, interferon α (IFN-α) levels were significantly higher in the DWoWS group than in the DWWS and SD groups, whereas interleukin (IL)-12p40 and tumor necrosis factor α (TNF-α) levels in SD patients were significantly higher than in the other two groups. The TNF-α, IL-4, and monocyte chemoattractant protein-1 concentrations were positively correlated with NS1 antigen levels. From whole-genome analysis, NS4 had the highest frequency of amino acid variants, followed by the E gene. Our study suggests that viremia levels and immune responses contributed to SD outcomes, and these findings may help in identifying an effective therapeutic strategy against SD infection.

Figure 1.

Map of Kandy, Sri Lanka, including National Hospital Kandy (red circle) where serum samples were collected from dengue-infected patients.

Figure 2.

Serological characteristics of patients according to types of infection and severity. (A) Mean NS1 Ag (P/N) ratios in different types of infection, (B) mean NS1 Ag (P/N) ratios in different severity groups, (C) mean IgM anti-dengue antibody (P/N) ratios in different severity groups, and (D) mean IgG anti-dengue antibody titer in different severity groups. Dotted line describes the IgG-positive value of 3,000 titers. Kruskal–Wallis one-way analysis of variance was performed among groups. Continuous variables were compared by using the Mann–Whitney U test between two groups (***P < 0.001). Error bars describe standard deviation of the mean. Ag = antigen; DWoWS = dengue without warning signs; DWWS = dengue with warning signs; P/N = positive/negative; SD = severe dengue.

Figure 3.

Viremia levels in different types of infection and in different severity groups. (A) Mean viral genome (copies/mL) levels in different types of infection. (B) Mean infectivity titer (FFU/mL) in different types of infection. (C) Mean viral genome (copies/mL) levels in different severity groups. (D) Mean infectivity titer (FFU/mL) in different severity groups. Kruskal–Wallis one-way analysis of variance was performed among groups. Continuous variables were compared using the Mann–Whitney U test between two groups (**P > 0.01; ***P < 0.001). Error bars describe standard deviation of the mean. DWoWS = dengue without warning signs; DWWS = dengue with warning signs; FFU = focus-forming units; SD = severe dengue.

Figure 4.

Nonsynonymous and synonymous variants in the sequenced regions. Reference strain (Gen Bank accession no: MT180479) was used. C = capsid; E = envelope; M = membrane.

Figure 5.

Cytokine expression patterns in healthy controls and different severity groups of dengue-infected patients. (A) IFN-α, (B) TNF-α, and (C) IL-12p40. Kruskal–Wallis one-way analysis of variance were performed among groups. Continuous variables were compared by using the Mann–Whitney U test between two groups (*P < 0.05, **P < 0.01). Error bars describe median with interquartile range. DWoWS = dengue without warning signs; DWWS = dengue with warning signs; IFN-α = interferon-α IL = interleukin; SD = severe dengue; TNF-α = tumor necrosis factor α.