Howard L. Bachrach

A plaque assay for foot-and-mouth disease virus and kinetics of virus reproduction.

A plaque assay for foot-and-mouth disease virus, type A, was developed in primary bovine kidney cultures, and the kinetic course of virus reproduction was established. The effect of environmental variables on plating efficiency was studied. Exposure of virus to cell layers for 90 minutes at 37° or 15 minutes at 43° prior to overlaying with agar and incubating for an additional 72 hours at 37° gave maximal plaque counts. Temperatures of 26° or 29° maintained during the adsorption period did not produce maximal numbers of plaques. Plating efficiency depended upon the volume of diluent added to virus samples before exposure to cell layers. For example, identical amounts of virus applied to cultures in volumes of 0.1, 0.4, and 6.4 ml produced plaques in the approximate relationship of 60:30:10. Virus diluent should contain serum. Without serum, calcium and magnesium ions were not fully effective. Washing cultures with phosphate-buffered saline before and after exposure to virus was not required, and it was shown that plaques developed only from infections initiated before agar was applied. Frequency distribution and dilution experiments showed that plaques were initiated by single virus particles. Where mean counts of replicate platings ranged from 47 to 66, twice the standard deviation of the means ranged from 16 to 31%. In virus reproduction experiments progeny appeared at 2.5 hours. At 11.5 hours there were 108.6 plaque-forming units (PFU) per millitier corresponding to a release from each cell of approximately 370 PFU.

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.

Early interactions of foot-and-mouth disease virus with cultured cells.

Abstract The adsorption of foot-and-mouth disease virus (FMDV) types A 12 119, O 1B , and C 3Res were studied in baby hamster kidney (BHK-21) cells by measuring the amount of radioactively labeled purified virus which remained attached to cells at various times after infection. At 4°, over half of the maximum adsorption of type A virus occurred within 15 min, and approximately 65% of the radioactivity bound by 45 min was removed by brief treatment with EDTA, indicating the assay measures primarily adsorption and not penetration. Upon shifting the temperature from 4 to 37°, about 60 to 70% of the bound virus eluted by 1 hr in an unmodified form. Only 140 S virus particles and 12 S subunits could be found associated with the infected cell 20 min after shifting the temperature from 4 to 37°. By 50 min, the number of 140 S particles decreased slightly. The number of viral receptors per BHK-21 cell was determined to be 1–2.5 × 10 4 for virus types A 12 and O 1B . Competition experiments between FMDV types A 12 , O 1B , and C 3Res indicated that they can utilize at least some common receptor sites on cells.

Foot-and-mouth disease virion RNA: Studies on the relation between the length of its 3′-poly(A) segment and infectivity

Abstract Intact 37 S RNA was extracted from foot-and-mouth disease virus (FMDV) (type A12 strain 119) and fractionated into oligo(dT)-cellulose unbound and bound fractions. The RNA in each fraction appeared to contain no hidden breaks as determined by sucrose gradient centrifugation under denaturing conditions. Combined pancreatic RNase A and T1 digestion of [3H]adenosine-labeled unbound and bound virion RNA yielded, in both cases, a poly(A) fragment that migrated considerably faster than 4 S RNA during gel electrophoresis. The length of the poly(A) fragment of unbound virion RNA is less than 10 residues of adenosine, whereas the length of the poly(A) fragment of bound virion RNA contains a segment of approximately 40 residues at its 3′ end. The specific infectivity of unbound virion RNA is essentially the same as bound virion RNA.

Foot-and-Mouth Disease Virus Immunogenic Capsid Protein VPT: N-Terminal Sequences and Immunogenic Peptides Obtained by CNBr and Tryptic Cleavages

The immunogenic capsid protein (VPT), circa 30 kiladaltons (kd), of foot-and-mouth disease virus was examined for (i) its ability to induce neutralizing antibody in guinea pigs after chemical modifications and CNBr or tryptic cleavages and (ii) N-terminal amino sequence homology across three virus types. The immunogenicity of VPT was inactivated by glutaraldehyde treatment, carboxymethylation and maleylation or citraconylation. However, de-citraconylation restored part of the lost activity. Cleavage of type A12 VPT with CNBr produced an immunogenic peptide of circa 13 kd. A slightly larger (ca. 16 kd) immunogenic doublet, VPTab, was obtained by tyrptic cleavage of VPT in the virion. Sequence homologies of circa 85% were found between the first 26 amino acids at the N-terminus of VP chains from virus types A12 strain 119 (A12), C3 Resende (C3R) and O1 Brugge (O1B).

Animal picornaviruses with a single major species of capsid protein.

Summary The caliciviruses—San Miguel sea lion virus (SMSV), vesicular exanthema of swine virus (VESV) and feline picornavirus (FPV)—were found by polyacrylamide gel electrophoresis to possess a single major species of capsid protein with a molecular weight of approximately 61,000 daltons. This differs from all other animal picornaviruses (cardio, entero and rhino), which have four types of proteins. The caliciviruses were also found to co-sediment as a single peak in sucrose-density gradients, and at a rate considerably faster than foot-and-mouth disease virus (FMDV), which is generally classified as a rhinovirus. Infectious ribonucleic acid could also be prepared from each of the caliciviruses, but in lower yield than from FMDV. These results lend support to the theory that VESV originated from SMSV.

FOOT-AND-MOUTH DISEASE VIRUS: STRUCTURE AND MECHANISM OF DEGRADATION AS DEDUCED FROM ABSORBANCE-TEMPERATURE RELATIONSHIPS.

The mechanism of degradation—denaturation and some structural details of FMDV† were revealed by absorbance—temperature relationships. Phenolderived RNA was also studied. Its denaturation at 0·08 μ Na+ , pH 7·5, was reversible from 8 to 96°C; the T m was 57°C and hyperchromieity 1·24. Native FMDV at 0·01 to 1·08 μ Na+ degraded to protein and RNA followed by denaturation of the RNA. Prior to degradation, virus strikingly resisted any change in u.v.-absorbance until the temperature reached 52 to 58°C. This reflected a strong stabilization of the RNA core by its protein coat. RNA released from the virus at 0·13 or lower μ Na+ denatured abruptly; at 0·20 or higher μ Na+ . it first renatured to a more structured form than in the virus core before denaturing as the temperature was raised further. This transient renaturation produced a hypochromic trough in the absorbance—temperature curve. Existence of this trough is in accord with the finding that the double-helical content of RNA in situ is only two-thirds of that attainable after its release. Redenaturations at 0·01 and 0·04 μ Na+ of once degraded-denatured virus produced reversible absorbance-temperature profiles identical to those of RNA. Their upper portions were nearly coincident with the hyperchromic portion of the initial profile. At these μ Na+ the protein was soluble. At higher μ Na+ , anomalously high absorbances due to protein aggregation displaced or completely masked the redenaturation curves. After clarification, however, reversibly-denaturable RNA was detected in the same quantity as at low μ Na+ .

Foot-and-mouth disease virus RNA. Presence of 3'-terminal polyriboadenylic acid and absence of amino acid binding ability.

Foot-and-mouth disease virus RNA contains a segment of polyriboadenylic acid (poly(A)) that is somewhat heterogenous in nature in polyacrylamide gels containing 8 M urea; however, a prominent peak of radioactive poly(A) migrates to the position of the 4 S marker RNA. The evidence derived from a number of experiments indicates that the poly(A) segment contains 60–80 nucleotides or less and is covalently attached to the 3′-terminus of the RNA. The RNA of the virus could not be aminoacylated with valine, histidine, tyrosine, or several other amino acids.

Ribonuclease contamination of crystalline deoxyribonuclease, trypsin, and of partially purified foot-and-mouth disease virus preparations

Abstract Using a microbiological assay for RNase based on the inactivation of viral RNA and capable of detecting RNase at a concentration as low as 0.1 mμg/ml, commercially available crystalline preparations of the pancreatic enzymes DNase and trypsin were found to be contaminated with 1 part in 100,000 and 1 part in 10,000, respectively, of RNase. A previous hypothesis that the susceptibility of infectious FMDV-RNA to crystalline DNase and to trypsin was due to the presence of an exceedingly small amount of RNase was also confirmed. The RNase content of partially purified preparations of FMDV was found to be about 0.013 μg/ml, attributable to the bovine serum and host cells used for viral propagation. This RNase contamination could be removed by chromatography on DEAE-cellulose, which allowed most of the enzyme to pass through unadsorbed. Similar chromatographic treatment of DNase decreased the amount of RNase present. Amberlite XE-64 treatment of trypsin was unsatisfactory for the separation of RNase activity.

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.