Studies on the Measles Virus in Tissue Culture

II. Cytochemical and Fluorescent Antibody Studies on the Growth of Measles Virus in FL cells

Abstract

Experiments on the growth of measles virus have been hitherto carried out by tube titration method. In the first report it was made clear that the plaque assay of measles virus was easily to perform with stable cell lines (FL, HeLa, HEp-2 or KB cells). Thus, reliable growth curves of measles virus were obtained by our plaque assay method and at the same time morphological observations were also made by hema-toxylin-eosin or acridinorange staining. The following results were obtained:
1) The cell-associated virus appeared 10-12 hours after inoculation, reached the peak titer within about 30 hours and then declined to low levels. The supernatant virus appeared a little later, compared with the former; it appeared between the 14th and the 18th hour after inoculation and then increased logarithmically until 72 hours.
2) The growth experiments of measles virus were made in FL cell culture with YLE medium. It can be found no difference of growth rate between calf serum-free media and 3 or 10% serum media. Phase-contrast observations showed that cytopathogenic effects appeared rapidly when a large amount of serum was used.
3) Though the curve of CF antigen titer paralleled that of infective virus, the amouts of extracellular CF antigen were less than those of intracellular antigen. But in the condition of complete celldrop the obtained results were totally converse.
4) The cytopathogenic effect of the infected cells consisted mainly of multinuclear giant cells and cytoplasmic inclusion bodies, and rarely of intranuclear inclusion bodies, though the latter appeared at the late stage of infection.
5) As for the localization of virus antigen, it was shown that distinct fluorescent foci were observed. around the nucleus starting at about 8 hours after inoculation (first early satge). 12-20 hours after inoculation (second stage) these foci gradually increased in size and brightness, sometimes forming a typical & ldquo;corona & rdquo; around the nucleus. 30 hours after inoculation and thereafter (the late satge of the infectious cycle) fluorescent foci could be observed spreading through the whole region of the cytoplasm. In preparations treated in combination with immune fluorescent statining and hematoxylin-eosin staining, specific fluorescence was observed at the very place of cytoplasmic includion bodies.
6) In preparations stained with acridinorange a considerable increase of RNA amount in the cytoplasm was not found. In cytoplasmic inclusion bodies the existence of green or dark regions was observed.