Evolutionarily distinct dengue virus type 2 with a unique type-specific neutralization determinant

Dengue viruses 1-4 (DENV1-4) mostly circulate in endemic cycles involving humans and Aedes mosquitoes. However, rare dengue outbreaks originate from non-human primates. These spillovers are caused by sylvatic virus genotypes that are closely related to endemic strains of the same serotype, but some are caused by strains that fall between the four canonical serotypes on phylogenetic trees. It is not clear whether these genotypic differences are reflected in corresponding antigenic differences. Here, we characterized the antigenic features of QML-22, a divergent strain most closely related to DENV2. On an antigenic map, QML-22 clustered with other globally representative DENV2 strains. QML-22 was not as well neutralized by antisera raised against endemic DENV2 and was better neutralized by antisera from DENV1, 3, and 4 infections than other strains of DENV2. QML-22 had a cluster of distinct amino acid residues in domain I of the envelope (E) protein, with only scattered point mutations elsewhere in the envelope. A potently neutralizing anti-DENV2 antibody that targets this site in domain I of the E protein, 3F9, had >1000-fold lower neutralization of QML-22 than other endemic DENV2 strains, whereas other cross-reactive and DENV2-specific antibodies neutralized QML-22 and endemic strains to similar titers. When antisera from non-human primates infected with QML-22 or DENV2 reference strain S16803 were depleted with the heterologous virions, homologous neutralization was unaffected but heterologous neutralization was reduced, suggesting homologous neutralization was due to distinct epitopes. Evolutionary analyses showed the 3F9 epitope was under relaxed selection pressure compared to other neutralizing epitopes and that QML-22 had unique amino acids in this epitope relative to the likely common ancestor of all DENV2. These findings provide insight into the antigenic diversification of DENV and the possible evolution of new serotypes.