Immunofluorescence Focus Assay for Ebola Virus

The recently discovered (1976) Ebola virus is responsible for one type of human hemorrhagic fever on the African continent (10). The virus is highly pathogenic, and no vaccine is available; therefore, it is classified as a class 4 viral agent (2). We initiated a program to investigate the ecology, biology, immunology, and chemistry of this virus and its relatives, and have recently reported on the RNA and protein composition of purified Ebola virions (9, 14). These studies and others have indicated that this virus possesses unique properties which may qualify it for consideration as a member of a new family of animal viruses. To facilitate further studies, we developed a rapid, quantitative test to measure viral infectivity and antibodies. Several assays have been described for negative-strand viruses (1, 3, 4, 6-8, 12, 16) and were considered for the needs described above. These include hemagglutination, plaque formation, noncytopathic hemadsorption plaques, and immunoperoxidase staining, among others. Although the plaque assay recently reported for Ebola virus (11) is quantitative, we were unable to obtain consistent results. In addition, it requires at least 1 week to obtain results with this assay. Immunofluorescence assay techniques for viruses have been widely used. An immunofluorescence focus assay for adenoviruses 1, 2, and 12, adeno-associated virus type 1, and reovirus type 1 used human amnion cells (17). Fluorescent cell counting has been used as an assay for respiratory syncytial virus (15). In addition, extensive studies using immunofluorescence focus assay techniques have been carried out with persistent mumps virus infections and other agents (18, 19). We describe here a rapid, quantitative test, the immunofluorescence focus unit (IFU) assay, which we adapted for use with Ebola virus. We also demonstrate that the test can be used for the quantitation of Lassa virus, a class 4 arenavirus.