Margaret Chapin

Characteristics of Five Rhesus Monkey Kidney Cell Lines

Summary Five lines of rhesus monkey kidney cells were established. Four of the 5 became more resistant than primary rhesus kidney cultures to wild-type polioviruses; the sensitivity of the remaining line (κ) to wild-type polioviruses was roughly equal to that of primary cultures. All 5 lines were susceptible to poliovirus RNA, though some were more susceptible than others. Passages of polioviruses on the lines selected 3 minute-plaque and 5 rapid poliovirus mutants. The authors thank Drs. Herbert A. Wenner, Hassan Rouhandeh, and Tom D. Y. Chin for critical reading of the manuscript.

The genetic relationship between thermostability of poliovirus and its in vivo response to cystine

The relationships among three genetic properties (inhibition by cystine, thermostability, and cystine-requirement) of antigenic type 1 poliovirus were investigated. Partial or complete loss through mutation of the property of being inhibited by cystine was accompanied by changes in the other two properties, indicating that the genetic element(s) which determines inhibition by cystine also determines, at least partially, thermostability and cystine-requirement. Inhibition by cystine and thermostability were found to be closely and positively correlated, suggesting that they are simply different manifestations of a single more basic virus property.

Relationship Between Inactivation of Poliovirus by Phenol and Appearance of Ribonuclease-Labile Infectivity.∗

Summary The influence of phenol concentration, time, and pH on phenolic inactivation of poliovirus at 0° was studied. A minimal phenol concentration of about 0.5 M was required for removal of all the original ribonuclease-stable poliovirus infectivity. This minimal requirement was influenced slightly by pH; it was lower at pH 4.0 than at pH 6.2 and lower at pH 6.2 than pH 7.4. Amount of inactivation was independent of time of exposure to phenol over range of 0.5 to 240 minutes. Poliovirus populations studied were heterogeneous with respect to phenol-stability, some particles requiring higher phenol concentrations than others for inactivation. Under conditions giving phenolic inactivation of all ribonuclease-stable infectivity, the ribonuclease-labile infectivity was present 0.5 minute after start of reaction and its titer remained essentially constant for at least 4 hours.

Effect of L-cystine and sulfated polysaccharides on replication of echovirus type 32 in monkey kidney cells.

Summary In order to form visible plaques on PRMK cells echovirus type 32 requires(l) the incorporation of DEAE-dextran into the agar overlay to counteract the inhibitory effect of sulfated polysaccharide occurring in agar and (2) the presence of L-cystine in the maintenance medium. A method employing a combination of these two compounds as described affords efficient plaque assay in PRMK cells.

EFFECT OF ETHER AND CA++ ON INFECTIVITY OF POLIOVIRUS RNA.

Using conventional procedures of extraction with diethyl ether to remove phenol, the infective poliovirus RNA titer was found to be 5 to 100 times greater when the virus stock was diluted about one-hundredfold into certain diluents prior to treatment with phenol (560 mM) than when undiluted stock was treated. Diluents giving this result were calcium-free, magnesium-free, mildly alkaline (pH 7–11) solutions, notably, e. g., 10 mM NaHCO3. The milieu afforded by such diluents was not necessary for the treatment with phenol but was necessary primarily to prevent “inactivation” of the poliovirus RNA by the ether; however, even without exposure to ether and with no extractions to remove phenol, mildly acid conditions or a moderate concentration of Ca++ resulted in some loss of infective RNA titer. Assay of ethereal phase detected no infectivity. Even with added base and chelating agent, extractions of phenol-treated essentially undiluted virus stocks with ether resulted in large loss of infective RNA titer. Extractions with benzene in place of ether had little or no effect on RNA titer using diluted systems; but with undiluted systems, a large titer loss was found. Treatment with ether without extractions with ether also inactivated RNA. Using heat shock (70° C, 20 seconds) in place of phenol to obtain infective RNA, the nature of the diluent required to give high infective RNA titer was very different from that required for the prephenol dilution when the conventional extractions with ether were employed.

The quenching of reovirus plaques by serum

Demonstration of reovirus antibodies by hemagglutination inhibition (HI) alone is not always satisfactory since many sera contain nonspecifie inhibitors (1, 2). Neutralization of virus infectivity on the other hand by plaque reduction is more sensitive and specific. The inhibition of plaque formation may be shown basically by two methods: a) treating the virus with serial serum dilutions and determining the portion of non-neutralized virus, or b) incorporating the serum at various concentrations in the agar overlay to prevent the spread of infection and hence the formation of plaques. In the following we report the effect of immune and nonimmune sera incorporated in the agar overlay on the development of reovirus plaques. The three reovirus prototypes were grown in monkey kidney tissue cultures (MKTC) grown in roller tubes or Roux bottles. Plaque assays were carried out on secondary MKTC propagated in 50 mm Petri dishes. The agar overlay contained high cystine altered Eagles medium (3) and 0.6% ionagar No. 2 (4). Pancreatin at various concentrations was presen~ in the agar overlay (5). The stock solution of pancreatin was prepared as suggested by the manufacturer (4) and appropriate dilutions were made therefrom. The adsorption period of the virus was 1 hour at 37~ and the plaque development period was 6 days in a continuous

Some effects of enzymatic digestions of fixed monkey kidney tissue cultures on their staining patterns.

Summary A large decrease in cytoplasmic pyroninophilia and thioninophilia resulted from digestion of formalin-fixed monkey kidney tissue culture with either chymotrypsin or ribonuclease. This pair of enzymes decreased cytoplasmic pyroninophilia and thioninophilia more than either enzyme alone. Digestion of similar cultures with desoxyribonuclease gave a large decrease in nuclear pyroninophilia and thioninophilia. The effects of these 3 enzymes on pyroninophilia of these cells were more clearly shown when a purified pyronin was used.