Enteroviral Ribonucleic Acid

Abstract

Recognition of the infectivity of ribonucleic acid (RNA) of tobacco mosaic (1, 2) and of animal virus (3) initiated investigation of the molecular aspects of viral reproduction. Study of replication of infectious RNA depends upon employment of efficient and reproducible biological assay procedures. Reception of viral genetic material normally depends upon specific interaction of cellular and viral receptors (4--6) while phenol extracted virus has lost this specificity (7). Therefore, assay of infectious RNA may require extracellular environments different from those suitable for assay of whole virus.Plaque formation of infectious RNA was greatly improved by use of 1 sodium chloride solution as described by Alexander et al. (8), but considerable variation among enteroviral RNA titers was noted when different RNA preparations were tested in a number of established human cell strains. The general procedure of Gierer and Schramm (1) consistently yielded infectious poliovirus RNA and minor alterations in the extraction procedure were ineffective. Much of the inefficiency of viral RNA action apparently results from failure to introduce RNA into replicating sites of cells. This paper reports factors affecting the biological activity of enteroviral RNA, and a plaque assay system that has proved sensitive and reproducible. Production of infectious RNA from highly purified poliovirus is reported, in confirmation of the observations of others (9, 10), and evidence is presented that only a small fraction of total extracted virus RNA is adsorbed to cells, and is responsible for infectivity. Material and MethodsCell Cultures and Methods.--Methods and media used for routine cultivation of HeLa cells and monkey kidney, human amnion, and rabbit skin fibroblast cells in primary culture have

The Assay, Extraction and Storage of Infective Ribonucleic Acid from Foot and Mouth Disease Virus

“…Outside a 16-fold dilution range and plaque counts 160-10, plaque formation was no longer a linear function of the RNA dilution and at very low dilutions inhibition may occur. This may be due to the aggregation of the RNA in the presence of ZM-M~SO,, as suggested by Holland et al (1960), or to inhibition by cellular DNA and RNA present in the extract (Franklin, Wecker & Henry, 1959;Holland et al 1960). The first possibility is contra-indicated in our system by the fact that RNA diluted in ZM-M~SO, could be held for up to ' 1 hr a t 0' without apparent loss in titre.…”

“…As with pig kidney cells, very few plaques were obtained when the RNA was added in isotonic solution. When the cell sheets were washed with hypertonic solutions before the addition of the RNA (Koch, Koenig & Alexander, 1960) or the RNA was diluted in hypertonic solution (Alexander, Koch, Holland, Hoyer, McLaren & Syverton, 1960), more plaques were formed. These observations led us to make a more detailed study of the optimal conditions for plaque formation.…”

The Role of Interferon in Persistent Infection with Foot-and-Mouth Disease Virus

“…a t 18" in different buffers and tested for infectivity by the plaque method. Dilution and adsorption in ~M -M~S O , according to Holland, Hoyer, McLaren & Syverton (1960) gave 20-fold higher titres than other methods tested ( Table 1). The nucleic acid infectivity was completely inactivated by treatment with ribonuclease which did not affect the titre of intact virus.…”

Strontium phosphate transfection of human cells in primary culture: stable expression of the simian virus 40 large-T-antigen gene in primary human bronchial epithelial cells.

“…The efficacy of transfection could be predicted from the quality of the precipitate. After 90 min (NHBE), 2 h (CV-1), or 3 h (NIH 3T3), the cells were rinsed twice with serum-free medium at 37°C, incubated with 1.5 ml of 15% glycerol for 30 s at room temperature (NHBE), 3 min at 37°C (CV-1), or 1.5 min at room temperature (NIH 3T3), rinsed three times with serum-free medium, and refed (11,29).…”