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

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.

Dengue virus in humans and mosquitoes and their molecular characteristics in northeastern Thailand 2016-2018.

Dengue is hyperendemic in most Southeast Asian countries including Thailand, where all four dengue virus serotypes (DENV-1 to -4) have circulated over different periods and regions. Despite dengue cases being annually reported in all regions of Thailand, there is limited data on the relationship of epidemic DENV infection between humans and mosquitoes, and about the dynamics of DENV during outbreaks in the northeastern region. The present study was conducted in this region to investigate the molecular epidemiology of DENV and explore the relationships of DENV infection in humans and in mosquitoes during 2016-2018. A total of 292 dengue suspected patients from 11 hospitals and 902 individual mosquitoes (at patient's houses and neighboring houses) were recruited and investigated for DENV serotypes infection using PCR. A total of 103 patients and 149 individual mosquitoes were DENV -positive. Among patients, the predominant DENV serotypes in 2016 and 2018 were DENV-4 (74%) and DENV-3 (53%) respectively, whereas in 2017, DENV-1, -3 and -4 had similar prevalence (38%). Additionally, only 19% of DENV infections in humans and mosquitoes at surrounding houses were serotypically matched, while 81% of infections were serotypically mismatched, suggesting that mosquitoes outside the residence may be an important factor of endemic dengue transmission. Phylogenetic analyses based on envelope gene sequences showed the genotype I of both DENV-1 and DENV-4, and co-circulation of the Cosmopolitan and Asian I genotypes of DENV-2. These strains were closely related to concurrent strains in other parts of Thailand and also similar to strains in previous epidemiological profiles in Thailand and elsewhere in Southeast Asia. These findings highlight genomic data of DENV in this region and suggest that people's movement in urban environments may result in mosquitoes far away from the residential area being key determinants of DENV epidemic dynamics.