Stewart H. Madin

Established kidney cell lines of normal adult bovine and ovine origin.

Summary Two newly established cell strains capable of continuous cultivation, one derived from adult bovine kidney (MDBK) and one from adult ovine kidney (MDOK), are discussed. It is suggested that the strains may be useful for those interested in veterinary virology in particular and virus-host relationships in general.

Cellular Resistance in Tissue Culture, Induced by Noncytopathogenic Strains, to a Cytopathogenic Strain of Virus Diarrhea Virus of Cattle:

Summary Three noncytopathogenic strains of virus diarrhea (VD) virus from cattle induced resistance to a plaque-forming strain of VD virus in agar overlay cell cultures of embryonic bovine kidney cells. Plaque inhibition made possible the measurement of virus concentration of noncytopathogenic VD virus strains. Cellular resistance was neutralized by VD virus antibodies.

Biological properties of human melanoma cells in culture

SummaryThree human melanoma cell lines derived from one primary and two metastatic tumors from three different patients were characterized for growth properties usually associated with malignant transformation; these include cell morphology, growth rate, saturation density, growth in semisolid media, colony-forming ability on contact-inhibited monolayers of normal fibroblasts and epithelial cells, and tumorigenicity in immunosuppressed mice. Variations in expression of aberrant properties were evident among the lines. One of the metastatic lines satisfied all the parameters of malignancy tested and the other showed a number of these properties, whereas the primary essentially fulfilled only one. These results suggest that cultured melanoma cells reflect the clinical variability often observed among melanoma patients and the metastatic melanoma seems to display a higher degree of malignant transformation than the primary.

The separation of infectious and autointerfering particles in vesicular stomatitis virus preparations

Abstract Vesicular stomatitis virus (VSV) infections produce two sizes of particles that differ not only in length, but also in infectivity and autointerference properties. The two forms have been separated by sucrose density gradient centrifugation after partial purification by differential centrifugation, enzyme, and fluorocarbon treatment. Peaks of radioactivity of 32 P-labeled preparations coincided with the two light-scattering bands. Infectivity was associated with the lower band which contained long (L) rods. Autointerference was associated with the upper band which contained short (S) rods.

Biochemical and biophysical properties of vesicular exanthema of swine virus

Abstract Vesicular exanthema of swine virus (VESV) was purified and some of its biochemical and biophysical properties measured. VESV grew in the presence of 2 μg/ml actinomycin D, was ether resistant, and was not stabilized by cations. Its sedimentation rate was approximately 207 S, and its composition was approximately 20% RNA and 80% protein. The RNA, which was not readily obtained in intact form, appeared to be single stranded with a molecular weight of about 2 × 10 6 . Heterogeneous distributions of VESV were observed upon centrifugation in CsCl density gradients. Crude virus, or virus concentrated by differential centrifugation or by ethanol precipitation, showed a major band at 1.36 g/ml, a secondary band at 1.38 g/ml, and some infectious virus of intermediate density. Sequential treatment with ethanol and chloroform produced a sharp peak at 1.38 g/ml. Chloroform treatment in conjunction with centrifugal concentration produced a sharp band of infectious virus at an intermediate density, 1.37 g/ml, and a band of low infectivity at 1.38 g/ml. The properties of VESV were different from those of the recognized groups of viruses, but most closely resembled properties of the picornaviruses, of which poliovirus and swine enteroviruses were employed for some direct comparisons in these studies.

Development of plaque assay for vesicular exanthema of swine virus (VESV) under methyl cellulose overlay

A plaque assay method, using 1.05% methyl cellulose overlay, was developed for vesicular exanthema of swine virus (VESV) types A48, H54, I55, and K54. The validity of this plaque method as an assay system was upheld by the well correlated dose response, and the specificity of plaque formation was demonstrated by the fact that a specific antiserum could reduce the plaque count by more than 98%. The concentration of methyl cellulose exerted an influence on the size of plaques formed by the virus, showing an inverse relationship between concentration of methyl cellulose in the overlay medium and plaque size. Adsorption of VESV to monolayers of pig kidney cells at 37° C was found to reach a maximum within 60 minutes.

Stability of Vesicular Stomatitis Virus at Varying Hydrogen Ion Concentrations

Discussion and Summary The present results are in conformity with the data of Galloway and Elford5 concerning the instability of vesicular stomatitis virus at pH values of 4.0 or less. The use of more sensitive experimental hosts and determination of 50% mortality endpoints, however, showed that in-activation of virus was achieved only at pH 2.0. Extension of studies beyond the pH value of 9.6 reported by Galloway and Elford (5) revealed the striking resistance of virus to inactivation by alkaline environments. It would appear that the wide range of stability of vesicular stomatitis virus may render it a likely prospect for purification by means of the ion exchange resins.

In vivo and in vitro studies of plaque type mutants of an RNA virus

Six plaque types all belonging to the same antigenic group (E54) of vesicular exanthema of swine virus (VESV) were studied for mutational changes in swine and in pig and canine cell cultures. The plaque types differed in virulence, the largest in size being the most virulent and the smallest the least virulent type. Neutralization tests revealed that only animals which showed frank disease developed specific viral antibodies. The inoculated type was generally mixed with other plaque types after recovery from the host. The contaminating new types were not the same for the different plaque type inoculations. Similar changes were observed in the two types of cell cultures. Since all inocula consisted of virus from single cell yields which were clonal in origin, it was concluded that mutational changes occurredin vivo andin vitro. Such mutations, together with the requisite selective forces, could lead to erroneous classification of a variant in regard to virulence.

In vitro Cultivation and Cytopathogenicity of Vesicular Exanthema Virus.

Discussion and Conclusions An agent capable of producing typical vesicular exanthema in swine has been propagated through 16 passages in a swine embryo tissue culture system. In the course of these passages the original material has been diluted to 10-19, so that it is highly unlikely that any infective material from the original inoculum was carried through all 16 passages. Multiplication of this agent in tissue culture was accompanied by extensive degeneration of epithelial cellular outgrowths which thus provided a simple means for in vitro assay of this virus. Considerable investigation is still required to determine the sensitivity and reliability of the system of tissue culture assay, whether in swine embryonic tissues or adult tissues such as kidney epithelium. Preliminary experiments have indicated that plaque formation on swine kidney epithelium using the method of Dulbecco7 is feasible. Evidence that the cytopathogenic agent was in fact the B type of the virus of vesicular exanthema was provided by the following observations: 1) The agent produced clinical vesicular esanthema in swine with wbiequent immunity to challenge with known 1I type virus, without loss of susceptibility to infection with the A type virus. 2) Specific complement fixation was obtained when tissue culture agent was mixed with serum from guinea pigs hyperimmunized with N type virus, but not when mixed with serum from guinea pigs hyperimmunized with the A type. 3) Neutralization of the cytopathogenic effect was observed in tissue culture when the agent was mixed with 13 type immune swine serum but not when mixed with A type antiserum.