Differentiation of filoviruses by electron microscopy

Cultured monolayers of MA-104, Vero 76, SW-13, and DBS-FRhL-2 cells were infected with Marburg (MBG), Ebola-Sudan (EBO-S), Ebola-Zaire (EBO-Z), and Ebola-Reston (EBO-R) viruses (Filoviridae, Filovirus) and examined by electron microscopy to provide ultrastructural details of morphology and morphogenesis of these potential human pathogens. Replication of each filovirus was seen in all cell systems employed. Filoviral particles appeared to enter host cells by endocytosis. Filoviruses showed a similar progression of morphogenic events, from the appearance of nascent intracytoplasmic viral inclusions to formation of mature virions budded through plasma membranes, regardless of serotype or host cell. However, ultrastructural differences were demonstrated between MBG and other filoviruses. MBG virions recovered from culture fluids were uniformly shorter in mean unit length than EBO-S, EBO-Z, or EBO-R particles. Examination of filovirus-infected cells revealed that intermediate MBG inclusions were morphologically distinct from EBO-S, EBO-Z, and EBO-R inclusions. No structural difference of viral inclusion material was observed among EBO-S, EBO-Z, and EBO-R. Immunoelectron microscopy showed that the filoviral matrix protein (VP40) and nucleoprotein (NP) accumulated in EBO-Z inclusions, and were closely associated during viral morphogenesis. These details facilitate the efficient and definitive diagnosis of filoviral infections by electron microscopy.