Marguerite Vogt

A Study of the Basic Aspects of Neutralization of Two Animal Viruses, Western Equine Encephalitis Virus and Poliomyelitis Virus.

Abstract A study of the process of neutralization of two animal viruses, Western equine encephalomyelitis (WEE), and poliomyelitis type 1 (P1), was carried out by using the plaque technique as assay method. In all experiments the antibody acted on the virus in a test tube and the proportion of the virus escaping neutralization was determined after having removed the remaining free antibody either by dilution or by washing the assay plates. The neutralization was found to proceed with time as a first order reaction; its rate was proportional to the concentration of the antibody and was affected by temperature as a process whose activation energy is of the order of 6000 calories. In numerous tests, the virus-antibody complexes were found to be stable at 37°, pH 7.5 and physiological ionic strength. No reaction (P1 virus) or only a slight reactivation (WEE virus) occurred upon dilution of an incubated virus-antiserum mixture. Reactivation occurred, on the contrary, if high titer virus, either active or inactivated by ultraviolet light, was added to an incubated WEE virus-antiserum mixture. This type of reactivation is attributed to the collision of the virus-antibody complexes with free virus particles. When mixtures of virus and antibody were incubated for a long time, a fraction of the virus remained active, even at highest concentrations of antibody. This persistent fraction could not be attributed to virus particles reactivated by dissociation of the antibody, to absence of cofactors or to presence of inhibitors of virus action, and was found to be nonhereditary. At low antibody concentrations, the final virus survival depended on the antibody: virus ratio. This dependence suggested the introduction of an antibody equivalent of the virus as a measure of the antibody titer. A theory of neutralization was developed and on its basis some parameters of neutralization were introduced and determined in actual cases. A hypothesis was proposed to explain certain features of experiments of neutralization carried out by the end-point technique, in which the antibody is present in the cultures where the surviving virus is titrated.

The nucleic acid of polyoma virus

The nucleic acid of polyoma (PY) virus was identified as DNA by the isolation of P32-labeled deoxyribonucleotides from purified P32-labeled PY virus. The inhibition of the multiplication of PY virus by aminopterin and bromodeoxyuridine (5-BDU) is described. Since both inhibitors are known to affect the synthesis of DNA, the results with both metabolic inhibitors support the conclusion that PY virus contains DNA as the essential nucleic acid.

Kinetics of the Release of Poliomyelitis Virus from Single Cells.

Abstract A technique is described which allows the handling and observation of single animal cells in microdrops. The kinetics of the release of poliomyelitis virus type 1 (Brunhilde strain) by individual monkey kidney cells was studied. After a latent period of a few hours, the bulk of the virus was released in less than 1 hour. Characteristic morphological changes appear to be correlated with the release of the virus.

Mutants of poliomyelitis viruses with reduced efficiency of plating in acid medium and reduced neuropathogenicity.

Abstract A class of mutants of poliomyelitis virus is described for which the efficiency of plating is markedly reduced under an acid agar-overlay. These mutants are designated as d (delayed) mutants. The d character is caused by a gradual decrease—under acid agar—of the susceptibility of the host cells to d virus. The optimal growth conditions for d virus are thus restricted to a narrow pH range. This physiological restriction of d virus may be the basis for its reduced neuropathogenicity.

Studies on cells rendered neoplastic by polyoma virus: the problem of the presence of virus-related materials

Abstract The continued virus release in mouse or hamster embryo cultures, rendered in vitro neoplastic by polyoma (Py) virus, is due to a “virus-carrier” state. All its characteristics can be reproduced by infecting nonvirus-releasing clonal sublines with Py virus. Since Py-transformed cells do not require the presence of a multiplying, plaque-forming virus to maintain their neoplastic character, the presence of the virus in a deficient state in the cells was looked for. The possible states tested were: the presence of a transforming, but noncytocidal virus; the presence of an incomplete virus detectable by fluorescent antibody or by its serum-blocking power; the presence of an infectious viral nucleic acid; and the presence of a provirus, susceptible to the inducing conditions effective in lysogenic bacteria. All attempts to demonstrate the virus in any of the states mentioned, failed. It is therefore concluded that either the virus is present in the neoplastic cells in a highly integrated state or, the cells do not contain the viral genome.

BIOLOGICAL PROPERTIES OF POLIOMYELITIS VIRUSES AS STUDIED BY THE PLAQUE TECHNIQUE

The plaque technique for animal viruses was developed for the purpose of providing a method suitable for a quantitative analysis of the interrelations between animal viruses and their host cells. So far, this method has been applied to a number of problems in the field of poliomyelitis viruses, such as the intracellular growth of poliomyelitis virus, its release from infected cells, its genetic properties, and its inactivation by antibody. In the present communication, we shall discuss some results obtained in the study of the growth and genetic properties of poliomyelitis virus. Before describing the experimental results, let us discuss a point of theoretical importance, namely the characterization of the virus particle. We detect a virus particle by its effect on a monocell layer, namely that of giving rise to a localized necrosis of the cell layer, a so-called plaque. Each plaque contains a viral population stemming from a single virus particle. This is proved by the following two findings. According to the first finding, the number of plaques produced by a virus sample is proportional to the amount of the sample deposited on the cell layer in dmilution experiments (R. Dulbecco and M. Vogt, 1 9 5 4 ~ ) . This result proves that either a single virus particle or a clump of particles, indivisible upon dilution, is sufficient to produce a plaque. The second finding is the linearity of the virus inactivation curves obtained by ultraviolet light (FIGURE 1). In these curves, the survival of the plaque-forming ability of the virus is measured as a function of the dose of ultraviolet light to which the virus was exposed. If the virus particles were aggregated in clumps, the plaque-forming ability of the clump would be destroyed only after destroying the plaque-forming ability of all the individual particles present in the clump. It can be demonstrated, and is intuitively obvious that, under these circumstances, the resulting inactivation curves would have a lag at low doses. Such a lag is not found experimentally. The results just mentioned clearly define the concept of the plaque-forming particle as a physical and biological unit. Since the plaque-forming titer of a virus preparation is of the same order of magnitude as the infectious titer determined by other methods, the plaque-forming particle can be identified with the infectious particle. Furthermore, since the operational definition of a virus particle rests on its infectivity, this infectious particle is the virus particle. I t has now to be discussed whether the counting of plaques gives us an accurate estimate of all the virus particles present in a given preparation. This discussion is the more required since purified preparations of poliomyelitis virus have been shown by electron microscopy (C. E. Schwerdt et al., 1955) to contain 100 to 1000 times more particles than the corresponding estimate from plaque counts. We shall discuss the problem for a system constituted by * This work was aided by a grant from the National Foundation for Infantile Paralysis, Inc., New York, N . Y.

Properties of a HeLa cell culture with increased resistance to poliomyelitis virus

Abstract A culture of increased resistance to poliovirus was obtained from the cloned S3 cell line of strain HeLa after repeated exposure to type 3 poliovirus. The increased resistance of the culture could be attributed to a decreased probability for a given virus particle to initiate an infection after adsorption to a “resistant” cell. A subculturing of the “resistant culture” in the presence of antiserum cured the culture from an apparent temporary virogenicity. A study of the fate of individual cells in drops under paraffin oil failed to provide evidence that the culture was or could become permanently virogenic. It is suggested that the “resistant culture” was obtained by a selection by the virus of pre-existing “resistant” variants in the S3 population.

Study of the mutability of d lines of polioviruses

Abstract Various d lines of poliovirus give rise to mutants with an efficiency of plating under low bicarbonate agar (eop-ac) more similar to that of wild type virus. These “reverse” mutants are produced during the growth of d virus under conditions in which they are not selected for as shown by fluctuation tests carried out according to Luria and Delbruck. The mutation frequency at which these mutants are produced is of the order of 2–3 × 10 −5 per particle per duplication. “High efficiency” d lines with an eop-ac > 10 −5 revert, probably in a single step, to virus having the same eop-ac as wild type ( d + ) virus. “Low efficiency” d lines with an eop-ac −5 give rise to mutants with an eop-ac smaller than that of the corresponding wild type virus. In this case, mutants with an eop-ac nearer but not equal to that of wild type virus can be obtained by successive reversion steps.

Studies on the induction of mutations in poliovirus by proflavin

Abstract The effect of proflavin on the mutability of d virus to d + virus was studied. No mutagenic effect of proflavin was observed if the host cells were exposed to proflavin prior to the infection with d virus. In contrast, the presence of proflavin during virus growth increased the proportion of d + mutants in the virus yield 15- to 25-fold over that in control d virus grown in the absence of proflavin.