Rodes Trautman

Determination of density gradients in isodensity equilibrium ultracentrifugation.

Abstract Formulas are developed to convert conventional activity coefficients of a binary solution to a density scale of concentration. These enable precise calculation of the effect of activity coefficient variation with concentration on the density gradient at equilibrium in an ultracentrifuge. Results are given for CsCl at 25 °C. One value is verified from the displacement of a density marker in simultaneous experiments at slightly different initial density.

Detection and properties of a genomic masked viral particle consisting of foot-and-mouth disease virus nucleic acid in bovine enterovirus protein capsid

Abstract Foot-and-mouth disease virus (FMDV) and bovine enterovirus (BEV) were used for simultaneous dual infection of calf kidney tissue cultures. In the harvest fluid a viral agent was found that had the FMDV genome, but (1) was neutralized by BEV-antiserum and not by FMDV-antiserum; (2) had the acid-stability of BEV and not FMDV; (3) had the buoyant density of BEV (1.35 class) and not FMDV (1.45 class); and (4) had the s-rate class (140–150 S) of both parental viruses. Such a particle meets criteria for simple genomic masking. Its ultracentrifugal properties show that the protein coat influenced the buoyant density in CsCl more than nucleic acid, presumably because of permeability to salt.

Absorption optics data processing with standard errors for sedimentation and diffusion coefficients from moving boundary ultracentrifugation

Abstract A data processing protocol is presented for moving boundary ultracentrifugation in which the boundary position and width are determined from absorption optics scanner traces. The best fitting Gaussian (normal probability) function is determined for each trace by weighted probit analysis. The boundary positions are used to determine the s-rate. The boundary spreading is used to determine the diffusion coefficient after correction for s-rate concentration dependence obtained elsewhere. Both linear and quadratic least-squares curve fittings are used. If the quadratic term is significant, it yields an estimate of concentration dependence from the boundary movement analysis and an estimate of the s-rate heterogeneity from the boundary spreading analysis. The carrying along of the internal standard errors at each step is emphasized. Suggestions for data processing and complete least-squares formulas are given.

Rotational Symmetry in Foot-and-Mouth Disease Virus and Models

With small, animal viruses for which electron-microscope images show little penetration by negative stain, the rotation technique for structure determination is useful. Foot-and-mouth disease virus resembles a 32-unit model when rotational images of virus and models simulating virus in negative stain are compared.

Immunochemical studies of foot-and-mouth disease: II. Characterization of RNA-free viruslike particles

Abstract A new antigen of foot-and-mouth disease virus (FMDV) is described. It has a sedimentation rate of 75 S, and a density of 1.31 g/ml. It is noninfectious and appears to be devoid of RNA. It has an estimated diameter of 21 mμ. It fixes complement when used as an antigen in the complement-fixation test and forms a precipitin line with homotypic serum in agar plates. It differs from the 140 S FMDV antigen in that: (a) it cannot be disrupted to the 12 S subunit at pH 6.0; and (b) it appears to react with specific antibody to the 12 S subunit as well as with specific antibody to the 140 S intact virus. It occurs as approximately 85% of the total complement-fixing activity of one of the four foot-and-mouth disease viruses studied.

Examination of Spray-Droplet Particle Counting as a Measure of Virus Concentration.

Abstract Model systems using known mixtures of polystyrene latex solutions have been used to study the spray-droplet method for determining particle concentration. Neither the binomial nor the Poisson distributions are applicable to the counts of particles in individual droplet residues. Both distributions are useful, however, for mathematical deductions about counting errors in the totality of data. The chi-square test for the homogeneity of a series of droplets is verified. The relationship of the critical region to the value of the ratio of the numbers of particles present is demonstrated, and the reasons for selecting the 5 % level are given. A chart is presented from which, in the case of determining the concentration of an unknown particle such as a virus, the level of error to be expected may be determined. This level is in terms of the total number of particles counted and the ratio of the numbers of known to unknown particles.

Carrier-free zone electrophoresis of infectious ribonucleic acid derived by phenol and sodium dodecylsulfate methods from purified foot-and-mouth disease virus

Abstract Infectious ribonucleic acid (RNA) from foot-and-mouth disease virus collected during electrophoresis had negative mobilities at ionic strength 0.1, pH 8.6, of 11.65 and 10.94 × 10 −5 cm2/V. second when prepared by phenol or acidic-sodium dodecylsulfate (SDS) methods, respectively. At pH 5.0, these mobilities wore increased to 12.46 and 11.79. Phenol treatment of SDS-prepared RNA increased the mobility of the latter to that of RNA prepared by phenol extraction only. But SDS extraction following phenol extraction did not lower the mobility. By schlieren optical measurements, more than 90% of the viral RNA in all preparations was degraded and of higher mobility than infectious RNA. Portions of this breakdown occurred during both extraction of RNA from the virus and equilibration of the RNA against electrophoretic buffer solutions.

Analysis by electron microscopy and infectivity of foot-and-mouth disease virus in moving-boundary and zone ultracentrifugation☆

Abstract Several techniques were used in the study of ultracentrifugal separation of foot-and-mouth disease virus, type A, from partially purified suspensions. A moving-boundary preparative ultracentrifuge method was used to confirm the sedimentation rate of 140 S. Zone centrifugation of the virus was done in a D2O and ammonium acetate medium. Direct analysis by infectivity and virus particle counts of the various fractions was effective in determining the optimum region for virus recovery. The number of droplets required to determine reliable virus concentration was reduced by the chi-square statistical analysis of particle counts.

Free diffusion measured by biological assay in multilayered cells: II. Diffusion coefficient of foot-and-mouth disease virus determined by infectivity☆

Abstract A multilayered glass diffusion cell was used to measure the diffusion coefficient of foot-and-mouth disease virus (FMDV) type A, strain 119, propagated in bovine kidney tissue culture. Diffusion was followed by infectivity changes rather than by optical methods. The cell was arranged either for free diffusion in an essentially double-infinite system or for restricted diffusion in a closed system separable into five layers. Infectivity of each fraction was measured by plaque assay to determine the concentration of virus in each layer after diffusion. The influence of the assay error was reduced by using fractions at a distance from the initial boundary where the concentration was several orders of magnitude less than the starting material. All data were weighted to adjust for errors of assay and the location of the layer in the cell. The weighted mean diffusion coefficient D , expressed in 10−7 sq cm/sec at 4°C, was determined for the various replicate experiments. In nine replicate runs in the double-infinite cell, with a diffusion time of 94 hr and an assay error as high as ±90%, D was 1.02. In a similar series of 10 runs in the five-layer cell, with a diffusion time of 66 hr and an assay error of about ±100%, D was 1.01. The weighted mean value of D for this total of 19 runs, representing 76 individual values, was 1.020 ± .067. This corresponds to a particle diameter for FMDV of 22.7 ± 1.5 mμ which compares favorably with the electron microscope value of 23 ± 2 mμ.

Free diffusion measured by biological assay in multilayered cells: I. Tables of the average concentration in successive layers and the effect of assay errors☆

Abstract The expressions for free diffusion from an initial infinitely sharp boundary were considered in terms of the type of concentration measurement made and its error. The percentage error in the computed diffusion coefficient D will be less than the percentage concentration error if c c o h / (2 Dt ) > 0.8 . Here co is the initial concentration below the boundary, t the time and h the height of the measurement plane above the boundary. If the concentration can be measured where c c o ≈ 10 −5 , then a 100% error in c c o causes only a 10% error in D. A table is given of the integral of the error function complement (ierfc x) to seven figures for 0.05 increments in the argument to 2.55 and six figures to 5.40. The average relative concentration c c o of successive, finite volume fractions was computed from ierfc x for a multilayered, double-infinite cell intended for nonoptical diffusion measurements. Using this table and the method of reflection, an appropriate table can be constructed for any finite cell. A special case of such restricted diffusion is presented for a five-layer cell in which the initial solution occupies the lowest layer. For both the double-infinite and the five-layer cells the weighting factor for each entry in the table of c c o is given for use in obtaining a weighted mean diffusion coefficient from all the data from several fractions in one experiment or replicate experiments.