A phylogenetic study of dengue virus in urban Vietnam shows long-term persistence of endemic strains

Abstract

Dengue virus (DENV) causes repeated outbreaks of disease in endemic areas, with patterns of local transmission strongly influenced by seasonality, importation via human movement, immunity, and vector control efforts. An understanding of how each of these interacts to enable endemic transmission (continual circulation of local virus strains) is largely unknown. There are times of the year when no cases are reported, often for extended periods of time, perhaps wrongly implying the successful eradication of a local strain from that area. Individuals who presented at a clinic or hospital in four communes in Nha Trang, Vietnam, were initially tested for DENV antigen presence. Enrolled positive individuals then had their corresponding household members invited to participate, and those who enrolled were tested for DENV. The presence of viral nucleic acid in all samples was confirmed using quantitative polymerase chain reaction, and positive samples were then whole-genome sequenced using an amplicon and target enrichment library preparation techniques and Illumina MiSeq sequencing technology. Generated consensus genome sequences were then analysed using phylogenetic tree reconstruction to categorise sequences into clades with a common ancestor, enabling investigations of both viral clade persistence and introductions. Hypothetical introduction dates were additionally assessed using a molecular clock model that calculated the time to the most recent common ancestor (TMRCA). We obtained 511 DENV whole-genome sequences covering four serotypes and more than ten distinct viral clades. For five of these clades, we had sufficient data to show that the same viral lineage persisted for at least several months. We noted that some clades persisted longer than others during the sampling time, and by comparison with other published sequences from elsewhere in Vietnam and around the world, we saw that at least two different viral lineages were introduced into the population during the study period (April 2017-2019). Next, by inferring the TMRCA from the construction of molecular clock phylogenies, we predicted that two of the viral lineages had been present in the study population for over a decade. We observed five viral lineages co-circulating in Nha Trang from three DENV serotypes, with two likely to have remained as uninterrupted transmission chains for a decade. This suggests clade cryptic persistence in the area, even during periods of low reported incidence.

Keywords: dengue; persistence; phylogenetics; pransmission; sequencing; virus.

Figure 1.

A stacked bar chart of the number of full DENV genome sequences generated during the study and their breakdown by serotype and genotype.

Figure 2.

Midpoint-rooted maximum-likelihood trees of 2,446 publicly available full genome length DENV1 sequences (black terminal branches) with 193 DENV1 genomes generated in this study (red branches). The relevant region of the tree has been isolated as a separate tree. Branch supports, calculated as bootstrap scores from 1,000 replicates, are indicated on relevant branches. The sampling location of the publicly available sequences in a Nha Trang cluster is indicated by a coloured dot (see legend). The most likely country of origin of relevant internal nodes is indicated by a coloured node, with probability values given in brackets.

Figure 3.

Midpoint-rooted maximum-likelihood trees of 1,891 publicly available full genome length sequences with the 221 full-length D2 genomes generated in this study shown as red branches. The relevant regions of the tree have been isolated as separate trees, with the difference in genotypes of these sequences shown. The given country of isolation for the most genetically similar corresponding sequences is marked as a coloured dot at the end of the branch. Bootstrap support values over 70 and on relevant supporting branches have been shown except for genotype II, which is small enough to show all bootstrap supports.

Figure 4.

Midpoint-rooted maximum-likelihood trees of 407 publicly available full genome length sequences with the 96 D4 genomes generated in this study shown as red branches. The given country of isolation for the most genetically similar corresponding sequences is marked as a coloured dot at the end of the branch. Bootstrap support values over 70 and on relevant supporting branches have been shown.

Figure 5.

The number of sequences in each clade sampled each month from October 2016 to April 2019 with a total number of NS1-positive individuals recorded during the same time period.

Figure 6.

Dated phylogenies of five DENV clades sampled in Nha Trang between 2016 and 2019. Branch lengths correspond to time, with the year and month of the node shown in x-axis.

Figure 7.

The timescale of sampled and predicted historical samples based on linear regression analysis of phylogenetic diversity overlayed on historical and recently reported DENV cases in Nha Trang. Sequences have been divided into serotypes and then coloured by clade according to genetic similarity and putative node origin. Fixed lines denote that a sequence has been sampled from at least a 2-month time period, whilst dotted lines are the hypothesised TMRCA denoted as an X with 90 per cent confidence intervals denoted by square brackets.