Transport and budding at two distinct sites of visible nucleocapsids of West Nile (Sarafend) virus

It has been difficult to detect and visualize the physical nucleocapsid particles during the replication process of the flaviviruses. The use of cryo-immunoelectron microscopy has clearly revealed the capsid proteins and nucleocapsid particles of West Nile (Sarafend) virus (a flavivirus) for the first time. Physical nucleocapsid particles accumulated in large numbers from 8 hr postinfection. Double immunolabeling of the envelope and capsid proteins showed a close association of these structural proteins for most of the replication cycle. By 10 hr postinfection, budding of nucelocapsids from the plasma membrane was very obvious. Although maturation at the plasma membrane was the dominant mode, during late infection, intracellular maturation into large vacuoles was also observed.