Plaque variant analysis of Entebbe bat virus reveals an envelope residue governing virus release from vertebrate cells

Entebbe bat virus (ENTV) is a bat-associated orthoflavivirus with no known arthropod vector. The ENTV isolate UGA125 produces plaques of distinct sizes, designated large (L) and small (S), suggesting underlying genetic heterogeneity. While such variation may influence viral phenotypes, biological differences beyond plaque morphology remain uncharacterized. In this study, we identified single-amino-acid substitutions in the envelope (E) responsible for each phenotype. Growth curve analysis using infectious-clone-derived viruses revealed that rL (H86N) had similar kinetics relative to rWT, whereas rS (D379N) was significantly attenuated in mammalian cells. Mechanistic investigation revealed impaired viral release in rS, a defect that can be rescued by neutralizing the pH of the secretory pathway. This finding suggests that D379N may elevate the pH threshold required for the E trimer to dimer conformational rearrangement, resulting in premature prM cleavage within early secretory compartments that are insufficiently acidic to stabilize pr-E association. Sequence analysis shows that the domain III FG loop, where D379N is located, is substantially longer in mosquito-borne flaviviruses and ENTV-clade no-known-vector flaviviruses than in tick-borne flaviviruses and other no-known-vector viruses. This structural difference may explain reported differences in pH-dependent conformational reversibility among immature particles across flavivirus groups. In experimental infections using the parental isolate, the D379N was lost in both mosquitoes (Aedes aegypti and Ae. albopictus) and Jamaican fruit bats (Artibeus jamaicensis). In contrast, the H86N was lost in mosquitoes but became dominant in a bat's brain following peripheral inoculation.