Research Article: Tracking dengue virus type 1 genetic diversity during lineage replacement in an hyperendemic area in Colombia

Date Published: March 7, 2019

Publisher: Public Library of Science

Author(s): Mauricio A. Salvo, Matthew T. Aliota, Louise H. Moncla, Ivan D. Velez, Andrea I. Trujillo, Thomas C. Friedrich, Jorge E. Osorio, George Dimopoulos.


Dengue virus (DENV) is a flavivirus responsible for the most common and burdensome arthropod-borne viral disease of humans[1]. DENV evolution has been extensively studied on broad geographic and time scales, using sequences from a single gene[2,3]. It is believed that DENV evolution in humans is dominated primarily by purifying selection due to the constraint of maintaining fitness in both humans and mosquitoes[4,5]. Few studies have explored DENV evolutionary dynamics using whole genome sequences, nor have they explored changes in viral diversity that occur during intra-epidemic periods. We used deep sequencing of the viral coding region to characterize DENV-1 evolution in a Colombian population sampled during two high-prevalence dengue seasons in which serotype dominance shifted. Our data demonstrate patterns of strain extinction and replacement within DENV-1 as its prevalence waned and DENV-3 became established. A comparison of whole-genome versus single-gene-based phylogenetic analyses highlights an important difference in evolutionary patterns. We report a trend of higher nonsynonymous to synonymous diversity ratios among non-structural (NS) genes, and statistically significantly higher values among these ratios in the NS1 gene after DENV-1 strain replacement. These results suggest that positive selection could be driving DENV evolution within individual communities. Signals of positive selection coming from distinct samples may be drowned out when combining multiple regions with differing patterns of endemic transmission as commonly done by large-scale geo-temporal assessments. Here, we frame our findings within a small, local transmission history which aids significance. Moreover, these data suggest that the NS1 gene, rather than the E gene, may be a target of positive selection, although not mutually exclusive, and potentially useful sentinel of adaptive changes at the population level.

Partial Text

Dengue virus (DENV; Flaviviridae; Flavivirus) is the cause of the most common and most important arthropod-borne viral disease of humans. The DENV complex is composed of four serotypes (DENV-1 to DENV-4) which cause the same clinical manifestations and show similar patterns of systemic dissemination, with tropism principally for monocytes, macrophages, and dendritic cells[1]. DENVs are predominantly transmitted by the mosquitoes Aedes aegypti and Aedes albopictus, facilitating heavy viral transmission in densely populated tropical and subtropical regions[6,7]. Furthermore, Ae. aegypti populations have reestablished themselves in the Americas since the cessation of eradication programs in the 1970s, and currently these mosquitoes can be found from the southern United States to north of the Southern Cone [1,8]. This expanding range increasingly potentiates DENV transmission in the United States. Although most DENV infections are asymptomatic, infection with any one of the four serotypes can result in a spectrum of clinical illness including a self-limited febrile illness termed dengue fever (DF). In a minority of cases it can manifest as a life-threatening vascular leakage syndrome, dengue hemorrhagic fever (DHF), and the often-fatal dengue shock syndrome (DSS). Severe disease is believed to be triggered by cross-reactive antibodies elicited by previous infection with a heterologous serotype[1,9]. For example, primary infection with any one of the four DENV serotypes provides a short initial period of cross-protection among all serotypes and later protective immunity against sequential reinfection with the same serotype [10]. In contrast, reinfection with a different DENV serotype can lead to unusually severe and potentially fatal disease[11,12]. This is particularly worrisome because co-circulation of multiple DENV serotypes has been increasingly reported within endemic areas[8], raising concern that more patients could develop severe disease.

To analyze DENV-1 between/within-host (inter/intra) viral diversity present in humans, we deep-sequenced 34 DENV-1 viruses from sera of infected patients from the Paris neighborhood of Bello, Colombia (Table 1). Our study period was characterized by an abundance of DENV-1 infections throughout 2014, an inter-epidemic period in 2015, and finally a decline in DENV-1 prevalence during a rise in DENV-3 prevalence throughout 2016 (Fig 1). We assembled E gene consensus sequences from each patient to determine the degree of genetic variability among viruses by Bayesian phylogenetic approaches. These sequences were aligned with 46 other DENV-1 E sequences available on NCBI GenBank representing all major DENV-1 global genotypes, frequently used for phylogenetic analyses. Our reconstructed phylogeny (Fig 2) clustered all 34 DENV strains within Genotype V (America/Africa), alongside other South American strains. Results from our analyses are in accordance with genotyping data reported from South America in previous years [21,26,34–36].

In general, DENV transmission cycles in South America are characterized by years of alternating dominance of a single serotype that fluctuate every 3–5 years [21,22]. The interplay between shifts in serotype dominance patterns and evolutionary drivers of serotype extinction as well as replacement of whole lineages within DENV serotypes has not been explored. Here, full genome, deep sequencing was used to characterize DENV-1 clade replacement coincident with a shift in serotype dominance from DENV-1 to DENV-3. Surprisingly, our analysis of within-host viral genetic diversity found evidence for diversification in the NS1 gene. In contrast to the hypothesis that the gene encoding the major glycoprotein would be the primary target of diversifying selection to avoid immune targeting, E genes in our dataset displayed levels of nonsynonymous to synonymous diversity indicative of purifying selection during seasons of both DENV-1 dominance and decline. Furthermore, the strain turnover observed in this study was associated with a significant increase in the ratio of nonsynonymous to synonymous variation within NS1. Interestingly, this increase in πN to πS ratio is largely due to a reduction in synonymous mutations in the latter clade. While one would expect diversifying selection to be associated with an increase in nonsynonymous mutations, this data opens questions regarding the attenuation of synonymous diversity, its possible mechanisms and interpretation.