Kinetic and Ultrastructural Study of Rubella Virus Replication

An Application of Enzyme Labeled Antibody Techniq u e

Abstract

It is generally accepted that Rubella virus (RV) causes weak cytopathogenecity. Cytopathic effect (CPE) by RV has been rather irregular and difficult to recognize in tissue culture cells. Therefore, investigation of RV replication must be taken much effort.
In the present study, the author applied Enzyme focus forming a ssay (EFFA) which was developed for the titration of such weak cytopathogenic viruses and studied RV replication morphologically.
Serially diluted RV was inoculated on Vero cells which were cultured in multiwell plates. Cells were added mainteance medium containing 1.5% methylcellulose. After 7-10 days incubation at 37°C, the infectious foci were stained with either hoseradish peroxidase conjugated anti RV IgG, Fab' fraction (HRP-Fab'), or Giemsa's solution. Though the infectious foci were not shown by Giemsa staining, immunoperoxidase positive foci were stained clearly and it was easy to count the RV titer as EFFU. The immunoperoxidase positive foci were embeded as immunoelectron microscopic specimen. Following results were obtained in the present study.
1. RV, M-33 strain, did not cause clear CPE on Vero cells.
2. RV titer, measured by 50% tissue culture interference dose (TCInD₅₀), increased logarithmically from 2 to 8 days after infection. The TCInD50 of 8 and 10 days after in f e ction were 10^6-3 and 10^6-0 respectively and kept the same titer after 10 days. Instead o f these viral titers, clear CPE was not found. RV infeced Vero cells were resemble to the cel l s in which the other virus infected persistently.
3. RV specific fluorescence was observed around the nucleus of Vero cells 3 days after infection. The specific fluorescence spread from perinuclear area to cytoplasm by lineal y or spotted pattern according to the infections time has passed.
4. Similar findings were obtained when the cells were stained with HRP-Fab'.
5. Replicated RV formed infectious foci when assayed by EFFA. These foci were easily observed and caliculated macroscopically. Uniform foci,0.8 mm and 1.0 ^-1.2 mm in diameter, were formed on the plates of 7 days and 10 days after the inoculation respectivey. The titers of specimen 4 and 10 days after the infection were well correspond e d to the titers of T CInD₅₀. In addition, the titer of EFFA was obtained earlier than the tite r of TCInD₅₀.
6. Aggregates of electron dense particles,20-30 nm in diameter, were observed beneath the Vero cell membrane and cytoplasmic vacuoles in the specimen of 3 days after infection b y electron microscope. Mature RV particles were observed 4 days after infection. Further more, RV, budding from the cell membrane, were observed.
7. In the specimen of immunoenzyme electron micrscope (IEM), which was processed from the foci of EFFA, heavyly stained particles of 20-30 nm in diameter were observed at the peripheral area of the Vero cells. These particles corresponded to the particles found o n the conventioanl electron microscocopic study. Therefore, the electrondense particles foun d in the cells 3 days after RV infection were identified as the immature form of RV i n the cytoplasm.
8. In the IEM study, microvilli of RV infected Vero cells were stained heavyly. Furthermore, particles of 60-100 nm in diameter, outside of the microvilli were stained. T h ese particles were thought to be mature RV which budded out from the microvilli.
9. From these findings, the author conclude that the proteins which concerned RV were formed perinucleally in the Vero cells. RV precursor particles were most recongnized j ust underneath the cell membrane or vacuole membrane and they bud through these membra n e and form mature RV particles.