A Rapid Method for the Localization of Lassa Virus in Fixed Tissue Using Labeled Riboprobes Visualized by Enzyme-Linked Immunoassay
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Date
1994-12
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Hood College Biology
Program
Hood College Biomedical Science
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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.