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

Abstract

The four dengue virus (DENV) serotypes (DENV serotype 1 [DENV-1] to DENV-4) are transmitted by Aedes aegypti and A. albopictus mosquitoes, causing up to 390 million DENV infections worldwide each year. We previously reported a clade replacement of the DENV-2 Asian-American genotype NI-1 clade by the NI-2B clade in Managua, Nicaragua. Here, we describe our studies of the replicative ability of NI-1 and NI-2B viruses in an A. aegypti cell line (Aag2) and A. aegypti mosquitoes reared from eggs collected in Managua. In coinfection experiments, several different pairs of NI-1 and NI-2B clinical isolates were used to infect Aag2 cells or blood-fed A. aegypti mosquitoes. Results consistently showed a significant replicative advantage of NI-2B over NI-1 viruses early after infection in vitro, and in mosquitoes, NI-2B viruses attained a higher replicative index than NI-1 isolates 3 to 7 days postinfection (dpi). At 7 dpi, NI-2B viruses displayed a significantly higher replicative index in legs and salivary glands; however, this advantage was lost by 14 and 21 dpi. We also found that the percentage of mosquitoes in which NI-2B viruses were dominant was significantly higher than that in which NI-1 viruses were dominant on day 7 but not at later time points. Taken together, these data demonstrate that clade NI-2B holds a replicative advantage over clade NI-1 early in infection that wanes at later time points. This early fitness advantage of NI-2B viruses over NI-1 viruses in the native vector, A. aegypti, suggests a shorter extrinsic incubation period for NI-2B viruses, which could have contributed to the clade replacement event in Managua. IMPORTANCEDengue virus (DENV), one of the most medically important arthropod-borne viruses, is transmitted to humans by Aedes aegypti and A. albopictus mosquitoes in tropical and subtropical regions worldwide. Dengue epidemics continue to increase in frequency, geographic range, and severity and are a major public health concern. This is due to globalization, unplanned urbanization, and climate change, as well as host genetics and immune responses and viral genetic changes. DENV consists of four serotypes, in turn composed of genotypes and genetically distinct clades. What drives the frequent replacement of a previously circulating DENV clade by another is unclear. Here, we investigate the replicative fitness of two clades of DENV serotype 2 in Aedes aegypti cells and mosquitoes collected from the region where the viruses circulated and conclude that increased replicative fitness could have contributed to a DENV clade replacement event in Nicaragua. These findings provide insight into vectordriven evolution of DENV epidemics. Dengue virus (DENV) is an arbovirus of global importance comprised of four phylogenetically related serotypes, DENV serotype 1 (DENV-1) to DENV-4. DENV is transmitted by Aedes aegypti and A. albopictus mosquitoes, which are typically found in tropical and subtropical regions of the world. However, due to a complex combination of factors that include migratio...

Isolation and characterization of dengue virus serotype 2 from the large dengue outbreak in Guangdong, China in 2014

“…Amino acid substitutions at positions 71 and 390 in E protein were also identified; however, their biological effects remain unknown. A series of mutations increased DENV replication and viral fitness in mosquitoes have been well identified [20,24], while none of them was seen in GZ05/2014 and other Guangdong isolates (Table 2). Current sequence analysis failed to identify any amino acid mutations that would make an obvious connection between viral virulence and disease severity.…”

“…However, there were no reports about relationship between K126E substitution and dengue pathogenesis in humans. A few substitutions in NS1, NS4B, and NS5 protein were evidenced to increase viral replicative fitness in native mosquitoes, and contribute to DNEV genotype clade replacement [20,24]. None of these mutations existed in the new isolate GZ05/2014.…”

Dengue: knowledge gaps, unmet needs, and research priorities

“…52 The relative contribution of intrinsic fitness differences, 43,44,53 escape from neutralization, 54–56 cross-serotype enhancement, 50,57,58 or stochastic effects (e.g., climate, geography, genetic bottlenecks) 54,59 remain poorly understood. Estimating the contribution of these factors to DENV evolution could enable prediction of the strength and/or severity of epidemics.…”

Complexity of virus–vector interactions

“…For example, the ID50 for DENV-1 and DENV-2 were lower than for DENV-3 and DENV-4 in one study in Vietnam [22], and the American genotype of DENV-2 was more poorly transmitted by field populations of A. aegypti than the Asian genotype [23]. Even within the DENV-2 Asian-American genotype, one clade of virus had an early fitness advantage over another in A. aegypti , contributing to a clade replacement event [24] in Nicaragua.…”