Intrahost Genetic Diversity of Dengue Virus in Human Hosts and Mosquito Vectors under Natural Conditions Which Impact Replicative Fitness In Vitro

Abstract

Dengue virus (DENV) is an arbovirus whose transmission cycle involves disparate hosts: humans and mosquitoes. The error-prone nature of viral RNA replication drives the high mutation rates, and the consequently high genetic diversity affects viral fitness over this transmission cycle. A few studies have been performed to investigate the intrahost genetic diversity between hosts, although their mosquito infections were performed artificially in the laboratory setting. Here, we performed whole-genome deep sequencing of DENV-1 (n = 11) and DENV-4 (n = 13) derived from clinical samples and field-caught mosquitoes from the houses of naturally infected patients, in order to analyze the intrahost genetic diversity of DENV between host types. Prominent differences in DENV intrahost diversity were observed in the viral population structure between DENV-1 and DENV-4, which appear to be associated with differing selection pressures. Interestingly, three single amino acid substitutions in the NS2A (K81R), NS3 (K107R), and NS5 (I563V) proteins in DENV-4 appear to be specifically acquired during infection in Ae. aegypti mosquitoes. Our in vitro study shows that the NS2A (K81R) mutant replicates similarly to the wild-type infectious clone-derived virus, while the NS3 (K107R), and NS5 (I563V) mutants have prolonged replication kinetics in the early phase in both Vero and C6/36 cells. These findings suggest that DENV is subjected to selection pressure in both mosquito and human hosts. The NS3 and NS5 genes may be specific targets of diversifying selection that play essential roles in early processing, RNA replication, and infectious particle production, and they are potentially adaptive at the population level during host switching.

Keywords: Aedes aegypti; dengue virus; host switching; intrahost genetic diversity; replication kinetics; selection pressure.

Figure 1

Intrahost genetic diversity of DENV. The variant frequency is plotted on the y-axis for DENV-1 (A) and DENV-4 (B), with genome position on the x-axis. This analysis was completed for each host type: humans (Hu) and mosquitoes (Ae. aegypti Mo). C: capsid, prM: precursor membrane, E: envelope, NS: non-structural protein, SNV: single-nucleotide variant.

Figure 2

The crystal structure of DENV NS2A, NS3, and NS5 proteins. (A) Ribbon representation of the protein structure of NS2A (first transmembrane segment, PDB 2M0S). (B) Ribbon representation of complete NS3 (PDB 2VBC) protein structure. A small yellow beta sheet represents the NS2B cofactor, purple represents the NS3 protease domain, and a large blue molecule represents the helicase domain of the NS3 protein. (C) Ribbon representation of the complete NS5 (PDB 4V0Q) protein structure: pink represents the MTase domain and green represents the RdRp domain. The locations of substitutions and residues on each protein are denoted in red. Structure figures were prepared using PyMOL.

Figure 3

Infectivity of infectious clones containing genome length RNA of wild type (WT), NS2A (K81R), NS3 (K107R), and NS5 (I563V) mutants in C6/36 cells and Vero cells. The viral copy numbers were measured from culture supernatants by RT-qPCR at 1 to 10 d.p.t. Results are expressed as the mean and SD of triplicate experiments. Statistically significant differences between each group are indicated by * p < 0.05 (one-way ANOVA with multiple comparisons, Tukey’s post-test).

Figure 4

Replication kinetics of DENV-4 wild type (WT), NS2A (K81R), NS3 (K107R), and NS5 (I563V) viruses in C6/36 cells and Vero cells. In vitro experiments were conducted at an MOI of 0.01. The viral titer of the cell culture supernatant was determined from 1 to 10 d.p.i. by immunofluorescence focus assays (IFA). Results are expressed as the mean and SD of triplicate experiments. Statistically significant differences (one-way ANOVA test) between each group are indicated by * p < 0.05 (one-way ANOVA with multiple comparisons, Tukey’s post-test).