Biophysical Measurement of Molecular Weight of Foot-and-Mouth Disease RNA Fragments

Author(s):  
Sydney S. Breese ◽  
Howard L. Bachrach

Continuing studies on the physical and chemical properties of foot-and-mouth disease virus (FMDV) have included electron microscopy of RNA strands released when highly purified virus (1) was dialyzed against demlneralized distilled water. The RNA strands were dried on formvar-carbon coated electron microscope screens pretreated with 0.1% bovine plasma albumin in distilled water. At this low salt concentration the RNA strands were extended and were stained with 1% phosphotungstic acid. Random dispersions of strands were recorded on electron micrographs, enlarged to 30,000 or 40,000 X and the lengths measured with a map-measuring wheel. Figure 1 is a typical micrograph and Fig. 2 shows the distributions of strand lengths for the three major types of FMDV (A119 of 6/9/72; C3-Rezende of 1/5/73; and O1-Brugge of 8/24/73.

Author(s):  
Sydney S. Breese ◽  
Howard L. Bachrach

In the continuing study of the physical and chemical properties of purified foot-and-mouth disease virus (FMDV), particles were treated to release RNA strands by dialysis against demineralized distilled water. The RNA strands were mounted by loop drop on formvar carbon coated specimen grids pretreated with 0.1% bovine plasma albumin to give a protein spreading surface. Dilutions were made in demineralized distilled water to achieve the best concentration for subsequent measurements. Specimen grids were rotary shadowed at low angle with uranium. 2.5 to 3.0 X prints of electron micrographs taken of random fields at 8,000 to 11,000 X were used for measurements with a map-measuring device.Samples were selected from supplies in long term storage which had been made according to methods outlined previously.


1927 ◽  
Vol 45 (4) ◽  
pp. 685-699 ◽  
Author(s):  
Peter K. Olitsky ◽  
Louis Boëz

Cataphoresis experiments show that the virus of foot-and-mouth disease carries, under ordinary conditions, an electropositive charge. Its isoelectric range is at the high point of pH = about 8. Although cultivable bacteria are, as a rule, electronegative, yet certain protozoa, such as trypanosomes and spirochetes are also electropositive. In respect to charge, then, the virus is different from ordinary bacteria, but there is nothing in this finding to indicate an inanimate character of the incitant. A knowledge of the charge, however, aids in the interpretation of certain filtration phenomena, and indirectly in delimiting the size of the virus. In addition, it serves to explain its remarkable resistance to certain chemicals—a subject to be dealt with in the next paper of this series. Finally, cataphoresis indicates the possible separation of the virus from protein particles. Filtration experiments were made with different types of filters: Seitz, Berkefeld V and N, and Chamberland, of practically all sizes, collodion membranes, and Bechhold's ultrafilter. The results confirm the electropositive charge of the virus, as well as the minuteness of its size. Filtration was effected through the Seitz, Berkefeld, and Chamberland filters. In regard to the latter, the active agent passed through the L 11 only when its charge was shifted to negative: under ordinary conditions, carrying an electropositive charge, it failed to traverse this more dense wall, and was completely adsorbed in the oppositely charged barrier. Filtrations through electronegative collodion membranes, prepared in different ways and of varying thicknesses, resulted, as a rule, in failure, unless the thinnest and most permeable membranes were employed. But in these, the complication of microscopic holes was to be considered. Hence this method was regarded as impracticable. Success, however, was obtained with Bechhold's ultrafilter membranes of the most permeable type, and with these it was possible to measure relatively, by a system of "molecular" filtrations, the size of the incitant. This was found to be, in relation to other particles of like charge, between 20 and 100 mµ in diameter. The filtration phenomena of the foot-and-mouth disease virus can be accounted for on the basis of the minute size of the particles of the incitant carrying an electropositive charge, and no evidence can be deduced therefrom that the virus is of a fluid character. For the relative size of the particles is constant and the invariability of the limits of measurement contradicts the notion that the incitant may be a "solute" varying in dimensions in different "solvents."


1927 ◽  
Vol 45 (5) ◽  
pp. 815-831 ◽  
Author(s):  
Peter K. Olitsky ◽  
Louis Boëz

The virus of foot-and-mouth disease exhibits a remarkable resistance to such bactericidal agents as the narcotic solvents (alcohol, ether, chloroform), or such antiseptics as phenol, bichloride of mercury, or cresol, as shown by tests made by others and ourselves. We have shown, however, that the resistance of the incitant to these chemicals is really masked. It is due to the fact that the reagents coagulate the proteins of the medium in which the virus is, as a rule, suspended. As a result the active agent is protected by the coagula which prevents direct contact with the chemicals. On the other hand, if advantage is taken of the periodic phenomenon attending such processes, and coagulation is prevented, the virus can then be brought under direct action of the antiseptics. Under these conditions, the incitant is more sensitive to destruction by the chemicals than is the living staphylococcus. As a corollary, the virus is destroyed as rapidly, or even more so, than are staphylococci by substances such as sodium hydrate (1 to 2 per cent solutions), or antiformin (1 per cent solution) which do not form coagula. We are therefore compelled to contradict the opinion that the extraordinary resistance to certain chemicals of the virus of foot-and-mouth disease, as it ordinarily occurs admixed with proteins, is an indication of its inanimate character. The results of a large series of experiments lead to the conclusion that of a number of antiseptics employed the sodium hydrate in 1 to 2 per cent solutions is an effective virucide. It is capable of killing the virus within 1 minute as shown by tests on cattle and guinea pigs. Furthermore, its effectiveness is not diminished even when the virulent material is admixed with cattle's urine, with manure, or with garden soil. The experimental evidence and the cheapness suggest its use in field practice as a disinfectant.


Author(s):  
S. S. Breese ◽  
H. L. Bachrach

Models for the structure of foot-and-mouth disease virus (FMDV) have been proposed from chemical and physical measurements (Brown, et al., 1970; Talbot and Brown, 1972; Strohmaier and Adam, 1976) and from rotational image-enhancement electron microscopy (Breese, et al., 1965). In this report we examine the surface structure of FMDV particles by high resolution electron microscopy and compare it with that of particles in which the outermost capsid protein VP3 (ca. 30, 000 daltons) has been split into smaller segments, two of which VP3a and VP3b have molecular weights of about 15, 000 daltons (Bachrach, et al., 1975).Highly purified and concentrated type A12, strain 119 FMDV (5 mg/ml) was prepared as previously described (Bachrach, et al., 1964) and stored at 4°C in 0. 2 M KC1-0. 5 M potassium phosphate buffer at pH 7. 5. For electron microscopy, 1. 0 ml samples of purified virus and trypsin-treated virus were dialyzed at 4°C against 0. 2 M NH4OAC at pH 7. 3, deposited onto carbonized formvar-coated copper screens and stained with phosphotungstic acid, pH 7. 3.


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