Dengue Virus Serotype 2 Intrahost Diversity in Patients with Different Clinical Outcomes

Abstract

Intrahost genetic diversity is thought to facilitate arbovirus adaptation to changing environments and hosts, and it might also be linked to viral pathogenesis. Dengue virus serotype 2 (DENV-2) has circulated in Brazil since 1990 and is associated with severe disease and explosive outbreaks. Intending to shed light on the viral determinants for severe dengue pathogenesis, we sought to analyze the DENV-2 intrahost genetic diversity in 68 patient cases clinically classified as dengue fever (n = 31), dengue with warning signs (n = 19), and severe dengue (n = 18). Unlike previous DENV intrahost diversity studies whose approaches employed PCR, here we performed viral whole-genome deep sequencing from clinical samples with an amplicon-free approach, representing the real intrahost diversity scenario. Striking differences were detected in the viral population structure between the three clinical categories, which appear to be driven mainly by different infection times and selection pressures, rather than being linked with the clinical outcome itself. Diversity in the NS2B gene, however, showed to be constrained, irrespective of clinical outcome and infection time. Finally, 385 non-synonymous intrahost single-nucleotide variants located along the viral polyprotein, plus variants located in the untranslated regions, were consistently identified among the samples. Of them, 124 were exclusively or highly detected among cases with warning signs and among severe cases. However, there was no variant that by itself appeared to characterize the cases of greater severity, either due to its low intrahost frequency or the conservative effect on amino acid substitution. Although further studies are necessary to determine their real effect on viral proteins, this heightens the possibility of epistatic interactions. The present analysis represents an initial effort to correlate DENV-2 genetic diversity to its pathogenic potential and thus contribute to understanding the virus’s dynamics within its human host.

In SilicoAnalysis of Dengue Virus Serotype 2 Mutations Detected at the Intrahost Level in Patients with Different Clinical Outcomes

“…It is worth mentioning that no mutation was selected as relevant for analysis in this gene. Indeed, NS2B was demonstrated in the preceding work to be the less variable gene irrespective of the clinical category, with just a few conservative amino acid substitutions or others at considerable low intrahost frequency ( 19 ). Hence, it could be considered a potential target for antivirals design.…”

“…Characteristics of each model can be found in Table S4. Finally, DENV-2 variants detected previously within the 68 patients with different clinical outcomes (Table S1) ( 19 ) were mapped on their respective models, and their effects were assessed by visual inspection in PyMol v9.25 ( https://pymol.org/2/ ) and molecular docking techniques. On the other hand, for viral proteins NS2A, NS2B, NS4A, and NS4B, a fold recognition and a de novo approach were employed instead, which will be addressed below.…”

“…Thirty variants detected previously within the UTRs ( 19 ) (Table S1) were checked for secondary structure alterations. Stem loops A and B (SLA and SLB) on the 5′UTR, plus SLI and SLII accompanied by the short hairpins, dumbbell structures (DB) 1 and 2, the short hairpin (sHP), and the 3′SL structures on the 3′UTR were determined for the baseline consensus sequences and plotted on Fig.…”

“…Considering all the above-mentioned details, and to better understand the association of viral features with severe dengue pathogenesis, we have previously explored DENV-2 intrahost genetic diversity in 68 Brazilian patients with different clinical outcomes with an amplicon-free deep-sequencing experimental approach. On these patients’ mutant swarms, we looked for any mutational pattern that could be correlated with the disease’s clinical outcome and detected a set of 141 intrahost single-nucleotide variants (NS-iSNV) and single-nucleotide polymorphisms (SNP; variants detected at consensus level, i.e., allele frequency higher than 50%) located along the viral genome that were identified consistently among the samples and were worthy of in-depth analysis ( 19 ). Therefore, to determine whether there is a potential structural or functional significance for these minor variants, we sought to assess their effect on viral proteins or RNA secondary structure employing molecular modeling techniques.…”

“…Therefore, to determine whether there is a potential structural or functional significance for these minor variants, we sought to assess their effect on viral proteins or RNA secondary structure employing molecular modeling techniques. In addition, under the assumption that recurrently mutated genomic regions, commonly known as hot spots, might be presumably functional and could help us understand evolutionary mechanisms that might affect virulence ( 20 ), we studied the presence of potential hot spots throughout the DENV-2 genome considering the entire mutational data set determined in our previous work, i.e., 10,180 insertions/deletions and synonymous and nonsynonymous substitutions ( 19 ).…”