A Rapid Method for the Localization of Lassa Virus in Fixed Tissue Using Labeled Riboprobes Visualized by Enzyme-Linked Immunoassay

Abstract

Lassa fever is a hemorrhagic disease of West Africa affecting as many as 300,000 people per year (Fisher-Hoch, S.P., et al., 1989). Lassa fever, is difficult to diagnose because it has a wide range of clinical manifestations and variable severity (McCormic, J.B., et al., 1987a). Lassa virus, the etiologic agent of Lassa fever, is an Arenavirus p the Family Arenaviridae. An assay for the detection of Lassa virus in guinea pig tissue would provide a means for testing the efficacy of potential vaccines as well as contribute to the overall understanding of the pathogenicity of Lassa fever. Conventional immunoassay detection of Lassa virus antigen in formalin fixed, paraffin embedded guinea pig tissue has not been successful in our laboratory. Thus, the aim of this study was to develop and standardize a nonisotopic in situ hybridization assay to detect Lassa virus mRNA in the guinea pig tissue. Digoxigenin labeled riboprobes were transcribed from T7/SP6 multicloning vectors, bearing Lassa virus cDNA inserts. Riboprobes were hybridized to Lassa virus mRNA and tagged with alkaline phosphatase conjugated anti-digoxigenin polyclonal sheep antibody Fab-fragments. Nitroblue tetrazolium and 5-bromo-4-chloro-3-indoyl phosphate generated a dark blue precipitate in tissues from infected animals but not in uninfected controls. The assay thus provided detection of Lassa virus mRNA with minimal background and clear delineation of cellular detail.