Simultaneous co-circulation of two genotypes of dengue virus serotype 3 causing a large outbreak in Sri Lanka in year 2023

Abstract

As many other countries, Sri Lanka experienced a marked rise in the number of dengue cases in 2023, with an unusual pattern of disease epidemiology. This rise coincided with the emergence of dengue virus (DENV) serotype 3 in Sri Lanka as the predominant serotype after 2009. Interestingly, a discrepancy between NS1 rapid antigen test positivity and quantitative real time PCR positivity was observed, with 50% of NS1 positive samples being negative by molecular diagnostics. Following sequencing of the DENV-3 strains in 2023, we identified two DENV-3 genotypes (I and III) co-circulating. While DENV-3 genotype III was detected by the modified CDC DENV-3 primers, genotype I evaded detection due to key mutations at forward and reverse primer binding sites. The co-circulation of multiple genotypes associated with an increase in cases highlights the importance of continuous surveillance of DENVs to identify mutations resulting in non-detection by diagnostics and differences in virulence.

Keywords: CDC PCR primers; Dengue; genotype I; genotype III; molecular epidemiology; mutations; sequencing; serotype 3.

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 analysed with 1316 sequences from 46 countries from 1956 to 2023. The Sri Lankan DENV-3 sequences, which 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).

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 (Figure 2). Three sub clusters are 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 sub-cluster of Clade I, along with sequences from China (shaded in grey).

Figure 3:. The phylogenetic tree of genotype III DENV3DENV-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 sub-clusters 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.

Figure 4:. Evolution of the DENV-3 genotypes I and III.

A molecular clock analysis (Root-to-Tip analysis) was carried out to investigate the evolution of DENV-3 genotypes I and III in Sri Lanka in relation to the global DENV-3 strains, which shows that both genotypes present in Sri Lanka co-evolved at a similar rate to the global dengue strains rate.

Figure 5:. 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 2013–2023. Point mutations were detected within the Sri Lankan genotype I (2023) strain in 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 (A). Point mutations were also detected within the Sri Lankan genotype I (2023) strain in 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 (B), No mutations detected in either Genotype I or III in the probe binding site (nucleotide region 762–787).