2023–24 dengue outbreak in Valle del Cauca, Colombia caused by multiple virus serotypes and lineages

Global dengue cases rapidly rose to record levels in 2023–24. We investigated this trend in Valle del Cauca, Colombia to determine if specific dengue virus serotypes or lineages were responsible for the large outbreak. We detected all four serotypes and multiple lineages, suggesting that other factors, such as climatic conditions, are likely responsible.


Introduction
Reported cases caused by dengue virus (DENV: Flaviviridae; Orthoflavivirus), composed of four genetically distinct serotypes (DENV-1-4), are on the rise.In 2023, 4.6 million dengue cases were reported, a record at the time and a 64% increase over 2022 (1).Those numbers were quickly surpassed in 2024 with almost 10 million dengue cases through June.Of those, ~8.4 were reported from Brazil alone; however, large outbreaks have been reported by many countries, including Colombia.The Valle del Cauca State Health Department in Colombia reported ~56,000 dengue cases through May, 2024, compared to ~23,000 for all of 2023 and less than 5,000 in 2022 (Figure 1A).
The cause of the substantial increase in reported dengue cases is likely multifaceted.Warming temperatures caused by climate change are known to increase the transmission potential and expand the geographical range of the primary mosquito vector, Aedes aegypti (2).Moreover, Indian Ocean sea surface temperature anomalies, especially El Niño events, are associated with dengue epidemics (3).There was a powerful El Niño-Southern Oscillation event in 2023-24, the first since 2015-16.Moreover, new DENV introductions, perhaps due to the resumption of travel post the COVID-19 pandemic (4), could be reaching large susceptible populations.For example, DENV-3 was rarely detected in the Americas for the last 10 years before a recent introduction into the Caribbean from Asia (5,6).To investigate if there was a specific DENV serotype or lineage contributing to the recent surge in cases, we sequenced DENV infections diagnosed in Valle del Cauca, Colombia from April 2023 to May 2024.

Methods and Results
The We extracted RNA from 140 µL of serum per sample using the QIAamp Viral RNA Mini Kit.We initially determined the DENV serotype using a multiplexed RT-qPCR assay (7) before attempting pan-serotype wholegenome amplicon sequencing with DengueSeq (8).Bioinformatic analysis, including primer trimming and consensus sequence generation, was conducted using an iVar pipeline as described in Vogels et al. (8).We assigned DENV lineages to samples with at least 5% genome completeness using the new nomenclature system (9) implemented in Genome Detective (dengue-lineages.org).We assigned serotypes to 185 of the 266 samples (70%; Figure 1B) and assigned lineages to 171 samples (64%; Figure 1C; Appendix Table ).
Our DENV-1 phylogenetic analysis reveals the co-circulation of two distinct lineages, DENV-1V_D.1 and D.2 (Figure 2).Both have been previously detected in Colombia and elsewhere in South America (5,12), representing ongoing local and regional persistence and diversification of these lineages for the past ~15-20 years.Our phylogenetic analysis of DENV-2 presents a more complicated picture of three genetic clusters and three individual sequences dispersed among two defined lineages, DENV-2III_D.2and DENV-2II_F.1.2(Figure 3).Lineage 2III_D.2 is a descendent of the original DENV-2 genotype III ("Asian-American") introduced in the Americas during the late 1970s that subsequently became established throughout the region, including Colombia (13).DENV-2 genotype II ("Cosmopolitan") was recently introduced into the Americas from Asia, first detected during a dengue outbreak in Peru in 2019 (14).Detection of DENV-2II_F.1.2 in Valle del Cauca demonstrates that the emerging "Cosmopolitan" genotype can become established alongside the existing "Asian-American" genotype.One hypothesis for the sudden increase in dengue cases was the introduction and rapid spread of a new DENV-3 lineage from Asia (5).DENV-3 in the Americas can often go decade or more between detections (4,6,15).Therefore, detecting an emerging lineage (DENV-3III_B.3) in the Caribbean (5), Brazil (6), Nicaragua (4), and elsewhere in the Americas was of concern.While we detected one dengue case from Valle del Cauca in January 2024 with a likely 3III_B.3infection (18% genome coverage), 61 of 63 (97%) of DENV-3 infections were lineage 3III_C.1 (Figure 1C).DENV-3III_C was likely first introduced into the Americas in the early 1990s (13) and we show that it has persisted through long periods of low detection (Figure 4).This includes sporadic detections of 3III_C.1 in Colombia since the early 2000s.Therefore, our results suggest that populations in the Americas may be susceptible to DENV-3 in general and not just the emerging 3III_B.3lineage.

Conclusions
We investigated the DENV infections from Valle del Cauca, Colombia to determine if a specific virus serotype or lineage was in part driving the record number of dengue cases (1).We detected all four serotypes, with DENV-1, -2, and -3 sharing dominance, and at least eight separate defined lineages (9).These included multiple lineages of DENV-1 genotype V and DENV-2 genotype III ("Asian-America") which have circulated in the Americas for ~40 years (13), as well as the emerging DENV-2II_F.1.2("Cosmopolitan") lineage.Moreover, despite the rapid spread of a new DENV-3III_B.3lineage in the Americas (4-6), we found that the dominant DENV-3 lineage was 3III_C.1 that has been sporadically detected in Colombia for ~20 years.Our results show that the conditions for transmission in Valle del Cauca are highly suitable for a diversity of DENV serotypes and lineages, and thus that the specific viruses are not the primary driver of the large outbreak.

Figure 1 .
Figure 1.Dengue cases by virus serotype and lineage in Valle del Cauca, Colombia.(A) Monthly dengue cases reported by Valle del Cauca State Health Department in Colombia.Samples from confirmed dengue cases (n=266) sorted by DENV (A) serotype per month by RT-qPCR (unknown = below limits of PCR detection) and (B) lineage by ampliconbased sequencing (listed as serotype, genotype, and lineage).
The samples were shipped to Yale University in New Haven, Connecticut for molecular processing.Our study was approved by the Research Ethics Committee at HUV and Yale University Human Research Protection Program (Protocol ID: 2000033281) and all participants signed an informed consent.