Simultaneous Cocirculation of 2 Genotypes of Dengue Virus Serotype 3 Causing a Large Outbreak in Sri Lanka in 2023

Abstract

Background: We observed a discrepancy between dengue NS1 antigen test and molecular diagnostics, with the emergence of dengue virus (DENV) serotype 3 in Sri Lanka, and sought to understand the cause for the rise in cases and high failure rates of molecular diagnostics.

Methods: Whole-genome sequencing was carried out in 22 DENV-3 samples. Phylogenetic and molecular clock analyses were done for genotype assignment and to understand the rate of evolution. Mutation analysis was done to understand the reasons for polymerase chain reaction (PCR) nondetection.

Results: We identified 2 DENV-3 genotypes (I and III) cocirculating. DENV-3 genotype III strains shared a common ancestor with a sequence from India collected in 2022, while DENV-3 genotype I, was found to share a common ancestor with DENV-3 sequences from China. DENV-3 genotype III was detected by the modified Centers for Disease Control and Prevention DENV-3 primers, whereas genotype I evaded detection due to key mutations at forward and reverse primer binding sites. We identified point mutations C744T and A756G in the forward primer binding sites and G795A in the reverse primer binding sites, which were not identified in DENV-3 genotype III. Furthermore, our Sri Lankan DENV-3 strains demonstrated a high root to tip ratio compared to the previous DENV-3 sequences, indicating a high mutation rate during the time of sampling (2017 to 2023).

Conclusions: The cocirculation of multiple genotypes associated with an increase in cases highlights the importance of continuous surveillance of DENVs to identify mutations resulting in nondetection by diagnostics and differences in virulence.

Keywords: dengue; genotypes; molecular diagnostics; mutations; sequencing; serotypes.

Figure 1.

Phylogenetic tree of the DENV-3 viruses sequenced from Sri Lanka in 2023 in comparison to global DENV-3 sequences. The Sri Lankan DENV-3 viruses sequenced were analyzed with 1316 sequences from 46 countries from 1956 to 2023. The Sri Lankan DENV-3 sequences that were not detected by the CDC DENV-3 primers were assigned to genotype I (highlighted in green) and the DENV-3 sequences that were detected by CDC primers assigned to genotype III (highlighted in pink). Abbreviations: CDC, Centers for Disease Control and Prevention; DENV, Dengue virus.

Figure 2.

The phylogenetic tree of genotype I DENV-3 sequences. The phylogenetic tree was generated with the Sri Lankan genotype I strains in comparison to the global genotype I strains. Three main clusters (clade I, clade II, clade III) were identified in the phylogenetic tree of DENV-3 genotype I. Three subclusters were identified within clade I, which includes sequences from China, Sri Lanka, Bangladesh, and Papua New Guinea. The Sri Lankan genotype I strain was assigned to the Southeast Asian subcluster of clade I, along with sequences from China (shaded in grey). Abbreviations: AICBU, Allergy Immunology and Cell Biology Unit, University of Sri Jayewardenepura; DENV, Dengue virus.

Figure 3.

The phylogenetic tree of genotype III DENV-3 sequences. The phylogenetic tree was generated with the Sri Lankan genotype III strains in comparison to the global genotype III strains. All the Sri Lankan sequences from 2017 onwards were assigned to clade I, which consists of both South Asian and Southeast Asian DENV-3 genomes. Three subclusters were identified within clade I, which included Indian sequences that were similar to the Sri Lankan sequences identified in 2023 (shaded in grey), a subcluster consisting of sequences from China and Singapore, and a subcluster with Indian sequences alone. Abbreviations: AICBU, Allergy Immunology and Cell Biology Unit, University of Sri Jayewardenepura; DENV, Dengue virus.

Figure 4.

Mutation within the primer and probe binding regions for the CDC DENV-3 primers in the DENV-3 genotype I and III strains detected in Sri Lanka in 2023. The mutations within the CDC DENV-3 primer and probe binding regions (nucleotides 740–813) of Sri Lankan DENV-3 genotype I and III were compared with previous Sri Lankan DENV-3 strains and global DENV-3 strains reported between 2013 and 2023. A, Point mutations were detected within the Sri Lankan genotype I (2023) strain at positions C744T and A756G of the forward primer binding sites (nucleotide region 741–760), while no mutations were detected in the Sri Lankan genotype I (2023) strain. B, Point mutations were also detected within the Sri Lankan genotype I (2023) strain at position G795A of the reverse primer binding sites (nucleotide region 789–814), while no mutations were detected in the Sri Lankan genotype I (2023) strain. C, No mutations were detected in either genotype I or III in the probe binding site (nucleotide region 762–787). Darker shades show higher number of reported strains with mutations in the primer and probe binding regions. Abbreviations: CDC, Centers for Disease Control and Prevention; Del, deletion; DENV, Dengue virus; G, genotype.