George R. Dubes

Virulence of polioviruses in relation to variant characteristics distinguishable on cells in vitro.

Abstract Early terminal-dilution end point passages of the poliovirus strains Akron (antigenic type 1), Brooks (type 2), and Mabie (type 3) in monkey kidney tissue cultures resulted in the emergence of viruses of reduced virulence for monkeys. This reduction in virulence has not as yet been found to be associated with any change in the virus observable on cells in vitro . Through the use of specific selective environments, several variant viruses, which are characterizable on cells in vitro , were obtained from these viruses of reduced virulence. The distinguishing characteristics of these variant viruses are HeLa-adaptation, altered response to cystine, and cold-adaptation. Tests in monkeys showed that virulence is essentially independent of HeLa-adaptation and cystine-response. The cold-adapted viruses obtained by passage in monkey kidney tissue cultures at 30°, however, were found to be even less virulent than their only moderately virulent ancestors. Viruses further adapted to the cold were obtained by passage at 23° and were found not only to be more cold-adapted but also to be more avirulent than their progenitor 30°-adapted viruses. One of these 23°-adapted variants (Mabie) has shown no virulence for the monkey by the intraspinal route of inoculation. These cold adapted viruses are deadapted to monkey kidney tissue cultures at temperatures as high as 37° and the data indicate that the virulence of these viruses is negatively correlated with the degree of this deadaptation. The relative avirulence of a cold-adapted virus therefore appears to be due to its generally reduced capacity to propagate at 37° or above.

Facilitation of Infection of Monkey Cells with Poliovirus "Ribonucleic Acid"

The plaque titer of poliovirus ribonucleic acid on monkey kidney cells cultured in vitro is greatly increased by depleting these cells of calcium and treating the ribonucleic acid inoculum with a suspension of any one of several poorly water-soluble substances before inoculation. These undissolved substances apparently facilitate infection by serving as solid vehicles for the ribonucleic acid.

Poliovirus mutants with altered responses to cystine.

SUMMARY: Three strains of poliovirus (Akron, antigenic type 1; Brooks, type 2; Mabie, type 3), which require exogenously supplied cystine (or a cystine-substitute) for optimal cytopathogenic action on monkey kidney tissue cultures, were serially passaged ten times in cystine-deficient monkey kidney tissue cultures in tubes. From various passage levels in this system were isolated three mutants of Akron, four of Brooks and two of Mabie. These nine mutants are designated the cystine-response mutants and are distinguishable from their ancestral viruses used to initiate the passage series by being relatively cystine-non-requiring. Two criteria suffice to distinguish the mutants of any strain from each other: (1) the degree of loss of requirement for cystine, and (2) the degree of inhibition by relatively high concentrations of cystine. The compositions of the virus populations in most of the passage levels were determined, and from these determinations the patterns of emergence of the mutant viruses were delineated.

Differences among Strains of Poliomyelitis Viruses in Plaque Size on Monkey Kidney Cells.

Abstract On the basis of size of plaques developed on monkey kidney cell monolayers, six strains of type I poliomyelitis virus may be divided into three categories. The strain Slow Mahoney produces small plaques, Akron produces plaques of intermediate size, and Brunhilde, Fast Mahoney, and two independent reversions to Fast Mahoney from Slow Mahoney produce large plaques. Similarly, differences were found among five type II strains; Charbonneau and Theilers Lansing fall in the small category, Brooks in the intermediate, and Leyva and MEF1 in the large. No difference was found in plaque size between the two type III strains, Mabie and Saukett. The possibility of using some of these differences in plaque size as markers in mixed infection experiments has been discussed.

Cystine requirement for normal poliovirus action on monkey kidney tissue cultures.

Summary The cytopathogenic action of the poliovirus strains Akron. Brooks, and Mabie on either tube or Petri plate cultures of monkey kidney cells is considerably delayed when cystine is omitted from the maintenance medium. Omission of all the vitamins or of any one of the 12 other amino acids except glutamine normally present in the maintenance medium resulted in no delay in the appearance of the cytopathogenic effect of Akron PP1. Omission of glutamine resulted in a short delay or no delay. It has further been shown that the propagation of Akron is slower in the absence of cystine. The lowest concentration of L-cystine which will give optimal or near-optimal cytopaithogenic action by Akron is about 10-4 M. Glutathione and L-cysteine hydrochloride can replace cystine at about the same concentration; DL-homocysteine thiolactone hydrochloride and DL-cystathionine can replace also but only at higher molar concentrations. None of 13 other compounds was found to replace cystine. The adsorption of Akron onto monkey kidney cells is not affected by the absence of cystine.

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.

Chromatography of enteroviral ribonucleic acids on hydromagnesite

Abstract Preparations containing infective RNA, obtained by treatment of tissue culture-grown stocks of three types of enteroviruses with phenol, were chromatographed on columns of hydromagnesite using pH 8 phosphate buffer as eluent. The concentration of phosphate in the buffer was initially low and subsequently was gradually increased. Each infective RNA adsorbed to the column and then eluted when the concentration of phosphate reached about 0.11 M. As determined spectrophotometrically, most of the RNA eluted while the concentration of phosphate was still at its initial low value (0.04 M) and a small RNA peak was found at 0.07 M phosphate; but the infective RNA at 0.11 M phosphate was not detected spectrophotometrically. For each virus, infective RNA and intact virus were mixed together and chromatographed. The infective RNA again eluted as a peak at 0.11 M phosphate; most of the intact virus, however, eluted at 0.04 M phosphate.

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.

A comparative study of the prototypic Hill strain of ECHO virus, type 9, and several Coxsackie-like viruses related to it antigenically.

During the summer and the fall of 1955 there were, in different parts of I taly, but particularly in the region called Marche, several outbreaks of a disease characterized by purely meningeal symptoms in children, whereas signs of involvement of the central nervous system (CNS) like paresis, radicnloneuritis, and psychical disturbances could be found in a certain number of adults. From the eerebrospinal fluids, throat washings and stools of the patients a virus could be readily isolated either in monkey kidney (1, 2, 3, 4) or in human amnion (5) types of tissue culture. The association of the virus with the outbreak of disease was established by serological methods. After a single passage in tissue culture the virus was found to be paralytogenic for suckling mice; however, inoculation of suckling mice with samples obtained directly from human beings apparently did not have a pathological effect. Because of the effect in infant mice and because histological findings closely corresponded to the ones deseribcd by Dalldor] in mice infected with Coxsaekie group A viruses (6), the causative agent of the I tal ian epidemics was considered to be a Coxsackie virus, belonging to group A. Since the virus was not neutralized by any