Chick Embryo Fibroblast Monolayers Research Articles

Plaque assay of Toxoplasma on monolayers of chick embryo fibroblasts.

SummaryThe RH strain of Toxoplasma gondii induces formation of necrotic plaques on monolayers of chick embryo fibroblasts overlaid with nutrient agar. Correlation between number of organisms plated and number of plaques produced indicates that each plaque is induced by a single toxoplasma. Survival of toxoplasmata in various media has been tested, and it has been found that Hanks BSS containing chick embryo extract in concentrations of 5 to 50% preserves 80-100% of plaque forming units (pfu) for 3 to 4 hours, 50-60% for 6 hours. Adsorption of pfu on monolayers is maximal after 70 to 90 minutes (Kc = 0.024), can be retarded by increased viscosity of the medium, speeded by centrifugation. Artificial lysis of chick embryo fibroblasts infected at low multiplicity yields populations with a predictable efficiency of plating of about 50% of the counted organisms.

Studies on persistent infections of tissue cultures. I. General aspects of the system.

Inoculation of the MCN and Lung-To lines of human cells in continuous culture with Newcastle disease (NDV), mumps, or 6-6 viruses led to slight cytopathic effects (CPE) if the multiplicity of infection exceeded one. On second passage or with smaller initial inocula no CPE became apparent. The viruses multiplied, however, as determined by titrations in HeLa cultures or chick embryos. Indeed, persistently infected sublines of MCN and Lung-To were readily established without resort to special manipulations and some of these have been carried now for over 18 months on the same media and schedules as the uninfected parent strains. The viruses were found to be associated mainly with the cells and only 1, or at most 10 per cent of it was detectable in the media. The titers obtained were always low in relation to the available cell population. Reduction or even omission of the horse serum component in the media or ultraviolet irradiation of the cultures did not increase the yield of virus, and CPE became apparent only when similarly treated, uninfected cultures were, likewise, affected by the manipulations. The persistently infected cultures differed from their uninfected counterparts in that they exhibited (a) decreased cellular growth rates and ultimate yields; (b) increased aerobic glycolysis; and (c) a high degree of resistance to cytopathogenic viruses, influenza A (PR8), herpes simplex and, especially vesicular stomatitis (VSV) viruses. Prolonged treatment of persistently infected cultures by addition of specific antiviral immune sera to the media reduced significantly the amount of virus present and the degree of resistance to VSV. However, upon removal of the sera after as many as 187 days of treatment the viruses reappeared in all but one instance. The cured culture, on reinfection, became again persistently infected. No evidence was obtained to indicate genetic inhomogeneity of the cell populations. Of 50 cloned MCN lines none was destroyed by NDV and all became persistently infected. None were initially resistant to VSV but all after establishment of persistent NBV infection. All 39 cloned lines derived from MCN(NDV) cultures in the presence of anti-NDV serum, were free of virus and susceptible to VSV, and all acquired persistent infections and with it resistance to VSV following inoculation of NDV. NDV maintained in MCN cultures differed from the parent, chick embryo-adapted strain with respect to its plaque morphology. Whereas the former yielded only plaques on monolayers of chick embryo fibroblasts which were of pin-point size and hazy, those obtained with the latter were rarely of this type and mostly large and clear. This apparent selection of virus particles did not alter significantly their behavior with respect to cytopathogenicity for uninfected MCN cultures.

Dynamics of heterologous interference between viable viruses in chick embryo fibroblast monolayers

A study of heterologous interference in chick embryo cells infected with two viruses has shown: Western equine encephalomyelitis virus (WEE) can suppress the production of Newcastle disease virus (NDV) in chick embryo fibroblast monolayers if added as late as 5 to 6 hours after the cells have been initially infected with NDV. Single-cell experiments to determine the maximum latent period for NDV indicate that by 5 to 6 hours after infection, the majority of cells have passed through the latent period for NDV production. The ability of NDV-infected cells to produce WEE decreases exponentially with time after NDV infection. Evidence is presented that would suggest that the reduced yield of WEE in NDV-infected cells results from a reduction in the amount produced by each cell. This information is discussed in terms of its relationship to the site of interference. In contrast to NDV-infected cells, cells infected with the PR8 strain of influenza virus do not lose their ability to produce WEE for 4 to 5 hours. This period of normal WEE yield is followed by one of continuous decline in WEE production.