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The temporal relationships of the production of infectious dengue-2 virus and its antigens were investigated in intracerebrally infected suckling mice. Infectious virus, a slowly sedimenting noninfectious hemagglutinin (SHA), and a noninfectious soluble complement-fixing antigen (SCF) were found in the brain. Serum contained high concentrations of SCF antigen relative to infectivity when compared to SCF to infectivity ratios in the brain. Degradation of virions by Tween-80 and ether produced two antigens with sedimentation characteristics similar to the noninfectious antigens occurring naturally in infected tissues. However, the virionderived SHA differed from native SHA when examined by electron microscopy and by equilibrium centrifugation in cesium chloride. Virion-derived SCF (as well as the virions and both SHA antigens) was denatured by sodium lauryl sulfate (SLS) and 2-mercaptoethanol (2-ME), whereas native SCF retained its complement-fixing activity. SLS and 2-ME treatment of dengue-1 and dengue-2 sucrose-acetone antigens increased their serotypic specificity. The hemagglutinin present in sucrose-acetone antigens was predominantly native SHA.

Immunofluorescence and electron microscopy were used to study the development and cytological distribution of dengue-2 virion and nonvirion antigens in monkey kidney cells. The type and combination of fixatives were found to affect the intensity of fluorescence. Paraformaldehyde fixation alone resulted in a low level of fluorescence, but an additional fixation step with ethanol or acetone resulted in maximum staining. No fluorescence was obtained after fixation with glutaraldehyde alone or in combination with other solvents. Methanol caused selective ablation of fluorescence by antibody to purified virions. Dengue-specific fluorescence that was most intense in the perinuclear area radiated in a granular pattern of decreasing intensity into the cytoplasm. The perinuclear fluorescence was associated with nonvirion antigens and the cytoplasmic fluorescence was associated with virion antigens. Electron micrographs of infected cells revealed vesicular bodies with reticular electron-dense centers, clusters of structurally complete virions deep within the endoplasmic reticulum, and single virus particles in vacuoles near the periphery of the cell.