Between- and within-host mutation of dengue virus

RNA viruses exhibit high mutation rates due to error-prone polymerases, leading to a diverse pool of viral haplotypes (also referred to as quasi-species) within infected hosts. While haplotypes have been well studied in chronic infections like HIV and HCV, diversity remains under-explored in acute infections like dengue (DENV), which are constrained by a short viremic phase. This study aimed to characterise the mutation hotspots in DENV genomes at both consensus and haplotype levels. Near full length DENV genomes were sequenced using Oxford Nanopore Technology (ONT) from the plasma of Sri Lankan patients with dengue fever recruited between 2017 -2020. Consensus sequences were mapped with Minimap-2, and haplotypes were reconstructed with Nano-Q, a tool designed for estimation of RNA virus haplotypes and their relative abundance. The genomic variability of DENV genomes was assessed by calculating Shannon Entropy (SE). Codons undergoing diversifying selection were identified with three phylogenetics-based algorithms (FEL, MEME, FUBAR) implemented within the Datamonkey suite. From 150 samples tested, both consensus and haplotype sequences were characterised in 90 samples (DENV1: 8, DENV2: 51, DENV3: 31). The genomic variability of consensus sequences measured by SE was higher in DENV2 compared to DENV3, and the reverse was true for haplotypes. At the consensus level, the NS2A gene had the greatest number of mutable sites when adjusted for gene length across all serotypes, while at the haplotype level the NS1 gene had the same. Overall, the haplotypes sequences revealed more sites with high mutability and codons under diversifying selection than those visible at consensus level. This provides proof-in-principle that in acute RNA viruses also have high mutability in haplotypes, which may be inapparent with a consensus-level analysis.