Genome-Wide Patterns of Intrahuman Dengue Virus Diversity Reveal Associations with Viral Phylogenetic Clade and Interhost Diversity

Abstract

Analogous to observations in RNA viruses such as human immunodeficiency virus, genetic variation associated with intrahost dengue virus (DENV) populations has been postulated to influence viral fitness and disease pathogenesis. Previous attempts to investigate intrahost genetic variation in DENV characterized only a few viral genes or a limited number of full-length genomes. We developed a whole-genome amplification approach coupled with deep sequencing to capture intrahost diversity across the entire coding region of DENV-2. Using this approach, we sequenced DENV-2 genomes from the serum of 22 Nicaraguan individuals with secondary DENV infection and captured ϳ75% of the DENV genome in each sample (range, 40 to 98%). We identified and quantified variants using a highly sensitive and specific method and determined that the extent of diversity was considerably lower than previous estimates. Significant differences in intrahost diversity were detected between genes and also between antigenically distinct domains of the Envelope gene. Interestingly, a strong association was discerned between the extent of intrahost diversity in a few genes and viral clade identity. Additionally, the abundance of viral variants within a host, as well as the impact of viral mutations on amino acid encoding and predicted protein function, determined whether intrahost variants were observed at the interhost level in circulating Nicaraguan DENV-2 populations, strongly suggestive of purifying selection across transmission events. Our data illustrate the value of high-coverage genome-wide analysis of intrahost diversity for high-resolution mapping of the relationship between intrahost diversity and clinical, epidemiological, and virological parameters of viral infection. N early three billion people worldwide are at risk for infection with dengue virus (DENV), a Flavivirus transmitted by the mosquitoes Aedes aegypti and A. albopictus (for reviews, see references 24 and 25). DENV is an RNA virus with a single-stranded, nonsegmented RNA genome ϳ10.7 kb in length, which encodes three structural proteins (capsid [C], premembrane/membrane [prM/M], and envelope ]E]) and seven nonstructural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). There are four closely related serotypes of DENV (DENV-1 to DENV-4) that can cause asymptomatic infections or clinical illnesses ranging from the self-limiting but debilitating dengue fever to the lifethreatening dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS), which are characterized by vascular leakage (76). Even though several factors, such as viral genetics, preexisting heterotypic immunity (i.e., immunity to a different serotype due to a prior infection), the sequence of infection with distinct serotypes, and host genetics appear to influence disease severity (23,35,53,54,58,61,69), the precise causes for progression to severe disease remain unknown.The four DENV serotypes share limited identity, with 25 to 40% variability observed between serotypes at the amino acid level (30, 68)....

Mosquito Saliva Serine Protease Enhances Dissemination of Dengue Virus into the Mammalian Host

“…An alignment with tissue culture-adapted DENV2 NGC, DENV2 PLO46, and DENV2 E124/E128 envelope protein with 21 envelope proteins sequenced directly from the serum of patients showed that residues proposed to interact with heparan sulfate were conserved (22,24,25). This analysis suggested that low-and high-affinity heparan sulfate binding viruses exist in nature.…”

“…Because low affinity to heparan sulfate increases in vivo pathogenesis of dengue and a number of other viruses (22), and because high-affinity interactions with heparan sulfate can evolve during tissue culture passaging, we aligned tissue culture-adapted DENV2 NGC, DENV2 PLO46, and DENV2 E124/E128 envelope protein with 21 envelope proteins previously sequenced directly from the serum of patients with secondary dengue fever, dengue hemorrhagic fever, and dengue septic shock 1 to 5 days postinfection (dpi) and analyzed residues previously proposed to interact with heparan sulfate (22,24,25). The resolving of the crystal structure of the dengue virus envelope, in addition to mutational studies, has identified multiple basic residues that may interact with heparan sulfate (22,25).…”

Genotype-Specific Evolution of Hepatitis E Virus

“…ORF1, in contrast, contains housekeeping genes, which are less likely to be host specific. Cyclical host jumps seen in arboviruses, e.g., West Nile virus, are associated with purifying selection (33,34). The concept behind this is that only substitutions conferring a selection advantage in both hosts are preserved.…”

Increased Replicative Fitness of a Dengue Virus 2 Clade in Native Mosquitoes: Potential Contribution to a Clade Replacement Event in Nicaragua

“…These replacements may begin with a cloud of closely related virus strains (i.e., intrahost viral diversity), generated in part by the error-prone RNA-dependent RNA polymerase of DENV (15,16). The intrahost diversity of viruses is typically described in chronic infections, such as those caused by HIV and hepatitis C virus (17)(18)(19), but has also been found in acute infections with DENV and other RNA viruses (20)(21)(22)(23). During arbovirus infections, such populations are subject to natural selection in both the mosquito and the human host.…”