Multiple mosquito species serve as competent vectors to carry and transmit numerous flaviviruses1,2. Several long-standing scientific questions remain to be answered, including identification of the fundamental factors that facilitate flavivirus infectivity in mosquitoes and the genetic basis that contributes to the naturally occurring interspecies specificity of mosquitoes to flaviviruses3–8, such as Aedes aegypti mosquitoes to dengue virus (DENV). Here we report that circulating mature virions are inactivated by the acidity of mosquito haemolymph; thus, extracellular vesicles carrying replication-competent viral nucleocapsids serve as the predominant means of intercellular viral dissemination. Mechanistically, mosquito valosin-containing protein (VCP) binds to the viral capsid, thereby allowing the incorporation of nucleocapsids into extracellular vesicles. The capsid of a flavivirus (such as DENV) selectively binds to the VCP of its natural vector (Ae. aegypti), but not to that of an incompetent vector (for example, Culex quinquefasciatus). Replacing the DENV capsid with that of Japanese encephalitis virus (JEV) renders DENV infectious in the haemolymph of the natural JEV vector, Cx. quinquefasciatus. Furthermore, two amino residues in Aedes (D723/N728) and Culex (E723/E728) VCP determine its binding specificity for viral capsid, thus contributing to interspecies specificity of mosquitoes to flaviviruses. In vivo ectopic expression of the Cx. quinquefasciatus VCP mutant E723D/E728N renders Cx. quinquefasciatus susceptible to DENV2 via intrathoracic microinjection. Our study provides a major molecular mechanism contributing to the selectivity and compatibility between mosquito vectors and flavivirus species, enabling systemic virus dissemination after the virus reaches the haemocoel. Upstream mechanisms that determine specificity at the midgut level remain to be determined.
