Sydney S. Breese

Electron microscope observations of African swine fever virus in tissue culture cells.

African swine fever virus, Hinde isolate, grown in a stable pig kidney cell line and primary pig kidney cells, was examined for successive stages in the development of the virus particle. The mature particle has a hexagonal outer membrane structure (diameter 175-215 micron) surrounding an electron lucent region and a dense nucleoid (diameter 72-89 micron). The increase in particle production in the cell cytoplasm is consistent with both the rise in infectivity as measured in pig kidney cell cultures and the rise of hemadsorption titer as measured in leukocyte cultures.

Inactivation of Foot-and-Mouth Disease Virus by pH and Temperature Changes and by Formaldehyde

Summary 1) Rates of inactivation of tissue-culture-derived, foot-and-mouth disease virus, type A, strain 119 (FMDV–A119), at various pH levels and temperatures and by formaldehyde were determined. Ranges of pH and temperature investigated were 2.0 through 10.0 and 4°C through 61°C. respectively: formaldehyde was employed at 0.009%. The results are interpretable by first-order kinetics. However, at pH 5 and 6 and also at 55° and 61°C small fractions of the virus population had much lower first-order inactivation rates than the bulk of the virus. Possibilities concerning the nature of the fractions with higher resistance are discussed. 2) Rates of inactivation at various pH levels were determined at 4°C. Below pH 4 the virus was totally destroyed within a few seconds. At pH 5 and 6 infectivity was lost at a rate of about 90% per second and minute, respectively, until only one-millionth of the virus remained. This residual virus was very stable to further inactivation. At pH 6.5 and 10, 90% of the virus was inactivated every 14 hours. The virus showed marked stability only at pH 7 and 7.5. losing little infectivity during a 5-week period. At pH 8 and 9, a 90% reduction of infectivity occurred within a 3- and a 1-week period, respectively. 3) Rates of thermal inactivation were determined at pH 7.5. The time intervals required for the inactivation of 90% of the virus existing at any time were as follows: 18 weeks at 4°; 11 days at 20°; 21 hours at 37°; 7 hours at 43°; 1 hour at 49°; 20 seconds at 55° to a survival of 0.001, 7 minutes thereafter; and 3 seconds at 61°C to a survival of 0.00001, 11 minutes thereafter. Activation energies calculated for loss of infectivity below and above 43°C were 27.200 and 120,600 calories per mole of FMDV. respectively. 4) Virus treated with formaldehyde at a concentration of 0.009% was inactivated at a rate of 90% per day of storage at 4°C.

Electron microscopic characterization of duck plague virus

A virus [duck plague Long Island virulent virus (DPLIVV)] was isolated from ducks in a recent disease outbreak on farms on Long Island, New York. This disease exhibited characteristics similar to duck plague, a disease in ducks endemic to the Netherlands and India and not previously reported in the United States. An attenuated strain of the duck plague virus from the Netherlands was studied morphologically by electron microscopy. Its infectivity for duck embryo cell cultures was also studied. The DPLIVV and the Holland strain were morphologically identical. Furthermore, enzymatic digestion of methacrylate-embedded thin sections of mature virus particles showed that both Holland duck plague-attenuated virus and DPLIVV contained deoxyribonucleic acid. The dimensions of the mature cytoplasmic particle; nucleocapsid diameter ca. 75 micron, envelope diameter ca. 181 micron, and other characteristics would presumably place duck plague virus in the herpesvirus group.

Purification and electron microscopy of foot-and-mouth disease virus.

Summary Foot-and-mouth disease virus (FMDV), type A, from bovine-kidney tissue cultures has been purified successively by methanol precipitation, extraction with organic solvents, and differential centrifugation. The FMDV particle in the resulting purified virus concentrates has been identified by analytical electron microscopy in air-dried specimens as a uniform-sized 22-mμ particle. This identification was made through correlative experiments relating particle counts to infectivity and to aggregation of the particles with antiviral bovine serum. While an average of 690 virus particles was present in one plaque-forming unit for bovine-kidney monolayer cultures, a value as low as 33 was obtained.

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.

Electron microscopy of the interaction of african swine fever virus with ferritin-conjugated antibody

Abstract Two isolates of African swine fever virus were studied by electron microscopy of interactions with ferritin-conjugated antibody, complement fixation, and agar gel diffusion precipitin tests. The viruses, Lisbon-57 and Tengani, were tested with anti-body to the Lisbon-57, Tengani, Salamanca, and Hinde isolates. Correlation of the data from these three independent methods indicates that the similarities between isolates are greater than the differences and that, perhaps, no differences exist.

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.

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.

Electron microscopy of rinderpest virus in bovine kidney tissue culture cells

Abstract The early development of rinderpest virus (Pendik strain) in bovine kidney tissue culture monolayers was examined by thin sectioning and electron microscopy. The virus-cell culture system which was used, permitted visualization of viral structures over a period of 8 days. Studies were made of morphological changes taking place at 3 hours, 1, 3, 4, 7, and 8 days after the cultures were made. The cells were grown in infective tissue culture fluids at a ratio of 250 ml of fluid (average titer 10 6.5 TC ID 50 /ml) to 1 ml of packed trypsinized bovine kidney epithelial cells. The effects of infection were visible at 3 hours, but development of virus bodies and possible precursor structures took longer with various stages of development visible at all times. After 8 days, the cells no longer adhered to the glass. Rinderpest virus developed within or contiguous to mitochondria and had a small-particle structure of 40–60 mμ which preceded the complete virus of diameter 150–300 mμ. The cytoplasmic sacs or inclusions which contained both small and large particles were 300–700 mμ in diameter.

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.