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Sequential stages in the development and release of ECHO 9 virus have been illustrated and described. It is suggested that viral particles differentiate and become oriented in columns upon a fine filamentous lattice at cytoplasmic template sites which are distinct from the endoplasmic reticulum. Subsequently, virus is dispersed in the peripheral cytoplasm and gains egress from the cell through rents in the plasma membrane. Complete cellular disruption with viral release may supervene. The virus consists of a 13 to 15 mmicro dense core and a poorly defined outer membrane, 22 to 24 mmicro in diameter. Incomplete forms, lacking the core, are observed in the cytoplasm but have not been seen in the extracellular space.

The establishment of two strains of rhesus monkey kidney cells in continuous tissue culture, the development of a subline adapted to chemically defined medium, and the isolation of several clonal derivatives were described. Growth characteristics, chromosome numbers, malignant potentiality, and freeze storage data are presented. The cells were studied for their sensitivity to a large number of viruses and were extensively compared with primary cultures of monkey kidney cells for sensitivity to poliovirus. The cell strains were not sensitive to all the viruses which could be grown in primary cultures of the same tissue but were susceptible to most of them. In some instances an advantage to the use of the cell strain for certain viruses was noted.

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.

The virus of encephalomyocarditis (EMC), examined by the negative-contrast method, is indistinguishable from the serologically related Mengovirus. The particles are 270 to 280 A in diameter. The surface of EMC is composed of an undetermined number of subunits. Frequent sampling of infected cells was carried out throughout one-step cycles of viral multiplication to observe cytopathic changes occurring in L cells infected by these two related RNA viruses. EMC and Mengovirus, which multiply at equal rates, in most respects elicit similar alterations in cell fine structure. Rearrangement and changes in nuclear material accompanied by formation of small vesicles in the centrosphere region commence at 4 to 6 hours after infection. Thereafter a progressive degeneration of the nucleus and vesiculation of the cytoplasm are observed up to 18 to 20 hours. Increased numbers of small dense granules, indistinguishable from ribonucleoprotein particles, appear in the cytoplasm between 8 and 14 hours after infection. L cells infected with Mengovirus become permeable to Erythrocin more slowly than those infected with EMC. Only in the case of Mengovirus infection are large aggregates of dense material first observed in the cytoplasm at 8 hours, followed by the appearance of crystals probably composed of Mengovirus particles, at 12 hours. Differences in the rates of cell permeability after infection with EMC and Mengovirus are discussed in relation to formation of virus crystals and plaque-type mutants.

Hardy, Frank M. (U. S. Army Chemical Corps, Fort Detrick, Frederick, Maryland), and Arthur Brown. Growth of Venezuelan equine encephalomyelitis virus in L cells. II. Growth in submerged culture. J. Bacteriol. 82:449-457. 1961.-The detailed kinetics of Venezuelan equine encephalomyelitis virus multiplication and release and the progressive cell death due to the virus infection were studied in a uniformly susceptible L cell population. The adsorption phase was complete in approximately 15 min with multiplicities of 1, 10, and 100 MICLD(50). An eclipse phase showing the disappearance of some of the infectious virus activity lasted between 3 and 4 hr. An exponential increase of cell-associated and free virus then occurred until peak titer was reached (16 to 18 hr). Relatively constant titers of cell-associated or free virus were obtained from the time peak titer was attained until the 52nd hour of incubation. At that time the cell-associated titer declined more rapidly than the free virus titer. These constant titers, however, masked a continued production and release of new infectious virus. By viable and nonviable cell counts, it was determined that cell death occurred relatively late in the phase of constant growth and release.

A correlation of cytochemical changes with virus production has been studied in L cells infected with Mengovirus. After a latent period of about 2 hours, virus was produced rapidly, reaching maximum titers of up to 12,000 particles per cell in 6 to 8 hours. The earliest cytological change was in the nucleus and consisted of a slight condensation of chromatin. There is no evidence, however, for the multiplication of either the viral RNA or protein in the nucleus. RNA, of high molecular weight, accumulated in the perinuclear area of the cytoplasm and was later found in inclusions. The perinuclear RNA was digestible with RNase and may be located in or on ribosomes. The inclusion RNA was resistant to RNase but could be removed by pepsin or potassium permanganate; it is probably in completed virus particles. Viral antigen was first observed in a perinuclear location and later in the above-mentioned inclusions. Although the viral protein contains appreciable amounts of arginine and lysine, it is not a basic protein of the histone type. Phase-contrast microscopy of living cells clearly demonstrated the role of the inclusions in release of virus from infected cells. A comparison is made between these cytological changes in Mengo-infected cells and those which have been found by other workers in polio-infected cells. There are many very similar changes.