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SummaryThe antiviral activity induced in mice by MA-56 has been characterized as an interferon. Maximum serum concentration is attained within 16-18 hr after feeding a single oral dose ranging from 400 to 500 mg/kg body wt. MA-56 induces a hyporeactive period of at least 40 hr. Interferon-production kinetics and cycloheximide experiments indicate that MA-56, like tilorone, has an induction pattern that can be blocked by cycloheximide and resembles viral induction rather than that of synthetic polynucleotides and bacterial endotoxin. Although MA-56 apparently does not induce a very high concentration of titratable interferon when compared to other inducers, its ability to protect mice against the lethal challenge of several viruses has been demonstrated.The authors are indebted to Drs. M. Pollard and T. Merigan for their contributions in the total development of this study. We also thank our collaborators at Plum Island Laboratory for testing FMDV and Anna Huff of our laboratory for her excellent technical...

Pig kidney (1BRS-2) cells infected with vesicular exanthema virus (VEV), a calicivirus, did not contain any large precursor polypeptides similar to those found when they were infected with foot-and-mouth disease virus (FMDV). The largest induced protein found in the VEV-infected cells had a molecular weight identical with that of the virus structural polypeptide. This difference in strategy between VEV and FMDV, taken in conjunction with the morphological and structural differences described previously, provides strong evidence that the caliciviruses should not be included in the family Picornaviridae.

When suspended cultures of bovine omasum were cultured without agitation, the epithelium soon degenerated and foot-and-mouth disease virus multiplied mainly in the corium cells. Five days of preincubation were needed to reach a population of corium cells that could yield virus at a titer of 10 6.70 to 10 6.95 mean tissue culture infective doses per ml. The virus was freely released from the cells into the medium only when the degenerated epithelium was removed from the subepithelial tissue prior to virus inoculation. In agitated cultures, the viability of the epithelium was retained, the virus multiplied in all the layers of the epithelium and was freely released into the medium, and a virus titer of 10 6.95 mean tissue culture infective doses per ml was obtained without preincubation. The omasal laminae could be separated along the line of apposition of the two mucous membranes of the organ. The virus yield from these thin separated membranes was 0.5 to 1.0 log higher than that obtained from nonseparated laminae.

SUMMARYThe influence of the Open Air Factor (OAF) and daylight on the survival of foot-and-mouth disease (FMD) virus held as captured aerosols on spider micro-threads has been investigated. Virus inactivation due to OAF was slight. Similarly, the effect of daylight on the survival of virus was not marked. The results are discussed in relation to the airborne spread of FMD virus in nature.

Mice treated with divinyl ether-maleic anhydride (DVE/MA) 7 days before, 2 days before, or simultaneously with foot-and-mouth disease (FMD) viral antigens (aqueous or oil emulsified) were more resistant to virus infection and released antibody earlier than mice treated with antigen alone. Simultaneous treatment of swine with these antigens plus DVE/MA resulted in a slightly enhanced early antibody response, which, in a test with oil-emulsified antigen, was not associated with an early induced antiviral resistance to FMD. In general, the primary and secondary antibody responses to oil antigen plus DVE/MA were the same as in control swine receiving the antigen alone. However, swine sensitized with aqueous antigen plus DVE/MA responded faster and developed higher antibody titers after a secondary treatment with aqueous antigen than swine treated similarly but without DVE/MA.

A simple stainless-steel apparatus was designed to contain standard microscope slides on which were grown micro cell cultures in the form of 16 individual monolayers per slide. The application of this apparatus for the screening of serum samples by fluorescent antibody techniques is described.

Summary After purification of an 0 type of Foot-and-mouth disease virus by caesium chloride centrifugation we obtain purified fractions of viral structural proteins by chromatography on DEAE Sephadex A 25. We point out a partial loss of the polypeptide VP1 during viral purification and present an easy separation of this polypeptide using its ionisation state at pH 8.6. These observations should be correlated with the biological role of VP1 when giving rise to neutralizing antibodies against the total virus.

Summary Although the infection of BHK cells with foot-and-mouth disease virus did not cause a marked inhibition of cellular protein synthesis, the proteins synthesized gradually changed from host-specific to virus-specific. The synthesis of at least thirteen virus-induced proteins was demonstrated by polyacrylamide electrophoretic analysis of the infected cells. Only a small proportion of the virus-induced proteins was incorporated into the mature virus particles. The addition of amino acid analogues caused an accumulation of the larger mol. wt. proteins, suggesting that in infected cells some cleavages occurred, giving rise to the smaller mol. wt. proteins. The operation of the precursor-cleavage mechanism was more clearly shown in pulse-chase experiments, in which it was found that the proteins were degraded from larger to smaller mol. wt.

Sequences of amino acids at the N-termini of virus proteins VP1, VP2, and VP3 were determined for foot-and-mouth disease virus types A12 strain 119, O1Brugge and C3Resende. In the polyacrylamide gel electrophoresis system used to purify the proteins, VP3 migrated faster than VP1 or VP2; and in the virion, VP3 could be cleaved by trypsin into VP3a and VP3b. The N-terminal amino acids for each of the virus types were glycine in VP1, aspartic acid in VP2, and threonine in VP3. No divergences in sequence across the virus types were indicated until at least the fourth position in VP1, and the third in VP3. For virus types A12, O1 and C3, the sequences were, respectively: for VP1 (Gly-ile-phe,pro,val---), (Gly,ile,phe---) and Gly-ile-phe,ala---); for VP2 (Asp,X,met---), (Asp---) and Asp-leu---); and for VP3 (Thr-thr-ala-thr---), (Thr-thr-ser---) and (Thr-thr---). Unresolved mixtures of VP3a and VP3b, from either A12 or O1 viruses, appeared to have the N-terminal amino acids threonine, which is presumed to be the same threonine as in uncleaved VP3 and serine, which is generated by the tryptic cleavage.

SUMMARYAn examination was made of the relations between antigen, antibody and fixation of complement with foot-and-mouth disease virus (FMDV). It was found that complement fixation in this system follows the same principles as models developed in other antigen/antibody systems. The assumption that there is a relation of direct proportionality between the amount of complement fixed and the amount of antiserum reacting with constant antigen was found to be incorrect. An alternative method was proposed for the quantitative differentiation of FMDV strains by comparing the titres of an antiserum when reacting with optimum amounts of homologous or heterologous antigens.

Summary The chemical and serological properties of the full, naturally occurring empty and artificially produced empty particles of foot-and-mouth disease virus, serotype A (subtype 10, strain 61) have been studied. The full 146S particles comprised the virus RNA, three polypeptides (VP1 to VP3) mol. wt. about 30 × 103, one polypeptide (VP4) mol. wt. about 13.5 × 103, and a small amount of a polypeptide (VP0) mol. wt. about 43 × 103. The naturally occurring 75S empty particles contained no RNA and much less VP1 and VP4 than were found in the full particles. However they contained a much greater proportion of VPo than the full particles. Dialysis of purified full particles against tris-EDTA, pH 7.6, produced artificial 75S empty particles which contained only a small amount of RNA and no VP4; otherwise the polypeptide composition was similar to that of the full particles. Immunological and serological tests showed that the full particles were antigenically similar to the naturally occurring empty particles but distinct from the artificial empty particles. The latter particles, however, had serological properties similar to those of the 12S protein subunit of the virus. Both the full and naturally occurring empty particles attached efficiently to susceptible cells, whereas the artificial empty particles attached only to a limited extent. The results are related to the function of the individual polypeptides of the virus particle and compared with published work on other picornaviruses.

Summary A temperature-sensitive mutant of foot-and-mouth disease virus (FMDV) of the RNA- phenotype induces active RNA dependent RNA polymerase only at the permissive temperature. Once induced this enzyme is active when assayed under cell-free conditions, even at the non-permissive temperature. However, when the enzyme is induced at the permissive temperature and the cultures are shifted to the non-permissive temperature, a sharp decline in activity is observed. The RNA- character can be phenocopied by blocking protein synthesis. The evidence obtained suggests that virus RNA replication is dependent on continuous production of the enzyme.

After exposure for two hours to cattle with foot-and-mouth disease, each of the five species of deer found in the British countryside became infected. Clinical disease was typical and severe in the roe and muntjac deer, with some animals dying, less severe in the sika deer and usually subclinical in the fallow and red deer. Each species transmitted disease to its own species and to cattle and sheep. The amounts of virus present in the blood, and in oesophageal/pharyngeal samples and excreted as an aerosol during the course of the infection in the deer were similar to those recorded for the sheep and cattle in the same experiment. The fallow and sika deer commonly carried virus in the pharynx beyond 28 days after exposure; some red deer also became carriers. In epidemics of foot-and-mouth disease in the UK, it is likely that deer would have such intimate contact with farm animals as occurred in this study. The natural behavior of free-living deer in the UK suggests that, although the five species are susceptible to foot-and-mouth disease, they are unlikely to be an important factor in the maintenance and transmission of the virus during an epidemic of foot-and-mouth disease in domestic livestock.

Summary Three immuno-peroxidase techniques (direct, indirect and peroxidase-antiperoxidase) were compared for their potential in foot-and-mouth disease research. Each technique was shown to offer a simple and efficient means for the detection of the virus of foot-and-mouth disease and of virus-infection-associated (VIA) antigen in infected cells.

The physical and chemical properties of foot-and-mouth disease and swine vesicular disease viruses have been compared, and it is concluded that these viruses are structurally different. The purified S.V.D. and F.M.D. viruses were disrupted and analysed in polyacrylamide gels. Two different polypeptide chaines with molecular weight of approximately 38000 and 30000 daltons respectively were found in the virions as well as in the empty capsids of S.V.D. In contrast to FMDV, there was no observable cleavage proteins of the empty capsid yielling detectable shortened polypeptides, which may be associated with the viral RNA.

The association of the replicase of foot-and-mouth disease virus (FMDV) with cellular membranes was suggested following attempts to produce a soluble enzyme by the use of detergents (Arlinghaus & Polatnick, 1967, 1969; Delagneau, 1971), as described for the purification of poliovirus replicase (Ehrenfeld, Maizel & Summers, 1970). Subsequently, the association with cellular membranes of the replicase and the single-stranded and replicative forms of RNA was reported for poliovirus (Caliguiri & Tamm, 1969, 1970a, b), for Semliki Forest virus (Friedman et al. 1972) and for tobacco mosaic virus (Ralph, Bullivant & Wojcik, 1971). In each case, the replicase-membrane complex contained endogenous RNA template (Arlinghaus & Polatnick, 1969; Caliguiri & Tamm, 1969; Ehrenfeld et al. 1970; Friedman et al. 1972). We show in this communication that FMDV replicase is associated with ribosome-containing membranes and requires glycerol to stabilize its activity during sedimentation in the presence of detergents.

SUMMARYVariants of type A FMD virus from the Eastern Mediterranean region over the years 1964–72 have been shown to belong to a group distinct from the Western European strains as represented by A5Westerwald. This group appears to derive from the A22strain first recognized in 1964 and indicates the possibility of new strains supplanting old in the field.

Summary A cell-free system synthesizing FMDV RNA has been shown to be inhibited efficiently by purified FV3 particles. Neither vaccinia nor herpes virus particles produced this effect. The results obtained provide direct evidence that a structural component (or components) or FV3 is responsible for the inhibition.

Summary A strain of foot-and-mouth disease (FMD) virus (O1 BFS 1860) held in aerosols at high relative humidity (r.h.) was more unstable when suspended in bovine salivary fluid than when suspended in cell culture fluid. This instability was due to the suspending medium rather than to the passage history of the virus and was not related to the high pH of the salivary fluid or to surface inactivation. The inactivation at high r.h. was caused by an undefined organic molecule which was dialysable and sensitive to heating at 70 °C but not at 60 °C.

A study of protein and ribonucleic acid (RNA) synthesis in cells infected by foot-and-mouth disease virus has indicated possible mechanisms of viral control over host cell metabolism. Foot-and-mouth disease virus infection of baby hamster kidney cells resulted in 50% inhibition of host cell protein synthesis at 180 min postinfection. A viral-induced interference with host cell RNA methylation was observed to be more rapidly inhibited than protein synthesis. To determine the nature of methylation inhibition, the kinetics of several host cell methylated RNA species were examined subsequent to virus infection. Data from sucrose zonal centrifugation and methylated albumin kieselguhr chromatography showed that methylation of nuclear RNA was inhibited 50% at 60 min postinfection. Inhibition of nuclear ribosomal RNA precursors and formation of nascent ribosomes correlated with inhibition kinetics of nuclear RNA methylation. It is suggested that the viral interference with the host nuclear RNA methylation is directly responsible for the observed loss of nascent ribosome formation. Moreover, early in the infectious cycle, methylation inhibition of host cell RNA could, in part, account for the cessation of host protein synthesis.

Summary The polypeptides of several strains of the seven serotypes of foot-and-mouth disease virus have been examined by polyacrylamide gel electrophoresis. Most strains gave a distinctive pattern of separation in urea-polyacrylamide gels but all the viruses contained one polypeptide which migrated to the same position. The mol. wt. of this polypeptide (VP 4) was shown by co-electrophoresis in sodium dodecyl sulphate-polyacrylamide gels to be the same, 13.5 × 103, for all seven serotypes. Since VP 4 aggregates when it is dissociated from the virus, it can be separated readily from acid-disrupted virus particles by centrifuging. It has the properties of a group antigen since it reacts in complement fixation tests with both homotypic and heterotypic antisera. The reaction between VP 4 and heterotypic antisera has also been demonstrated by using [125I]-Fab fragments. The antigenic site of VP 4 is not located on the surface of virus particles since there is no reaction between intact particles and [125I]-labelled heterotypic IgG or its Fab fragments.

Foot-and-mouth disease virus (FMDV) was inactivated by ethylenimine (EI) at three concentrations and two temperatures. Comparison of inactivation kinetics and the antigenic and immunogenic potency of EI and N -acetylethylenimine (AEI)-inactivated FMDV indicates that EI has nearly optimal characteristics as an inactivant for FMDV vaccine preparation. Although AEI-inactivated FMDV has proved to be a potent specific immunogen, an equivalent percentage of EI inactivated FMDV at substantially faster rates and produced an equally potent immunogen. In addition, EI inactivated FMDV at rates that were essentially linear throughout the loss of nearly all measurable infectivity.

Because 7 S immunoglobulin (Ig) G antibodies of low type specificity were present in mixtures with highly specific 19 S IgM antibodies, many bovine antisera to foot-and-mouth disease virus (FMDV) type A 12 , strain 119 cross-reacted with type O of FMDV and to some degree with type C in the passive hemagglutination (HA) test. After 19 S IgM antibodies were separated by density gradient centrifugation or precipitated with 4% (w/v) polyethylene glycol, the antigen could be determined with “block” HA tests. Such tests used several antigen concentrations in the titration of each antiserum. Adding 4% (w/v) polyethylene glycol to the serum was especially convenient for rapid precipitation of 19 S IgM antibodies for the test. Similar results were obtained with bovine 19 S IgM antibodies to FMDV type O, subtype 1, strain Caseros and type C strain Rezende.

SummaryCell cultures of bovine embryonic lung were found to undergo morphological changes after inoculation with polyoma virus. The changes were characteristic for polyoma transformation with cells having stellate or triangular shape and lying at random, crisscrossing one another.Cell lines of rapidly-growing transformed cells were obtained. Attempts to isolate infectious virus from the lines were negative. The lines contained “tumor” antigen as demonstrated by complement fixation tests using serum from hamsters bearing polyoma-in-duced tumors.A comparison of the viral susceptibility between the polyoma-trans formed cells and SV40-transformed and normal cells derived from the same source revealed certain differences. Whereas the polyoma-transformed cells showed fairly high susceptibility to a wild strain of type C of foot-and-mouth disease virus (FMDV) the SV40-transformed and normal cells were less susceptible. The SV40-transformed cells showed a decreased susceptibility to bovine enterovirus (BEV) and p...

Simultaneous injection of divinyl ether-maleic amhydride (DVE/MA) or itaconic-acrylic acid and foot-and-mouth disease virus vaccine enhanced the survival of infant mice to subsequent injections of virus. This enhanced resistance was obtained even with doses of interferon inducers which, when administered alone, failed to protect the mice. There was some increase in serum-neutralizing antibody in mice 7 days after injection of DVE/MA and vaccine, as compared with mice given vaccine alone, but there was no clear connection between antibody level and amount of DVE/MA administered.

Summary Unfractionated harvests of foot-and-mouth disease virus grown in baby hamster kidney cells fixed complement with both heterotypic and homotypic antisera but the freshly prepared intact virus (25 nm. component) from these harvests fixed complement only with the homotypic antiserum. Storage at 4° or heating at 37° released an antigen from the 25 nm. component which fixed complement with heterotypic serum. This antigen could also be prepared by mixing the 25 nm. component with baby hamster kidney cells but it was obtained in greatest yield by disrupting with guanidine. It had a sedimentation coefficient of 14S in sucrose gradients. Serum from hyperimmunized infected guinea pigs which had been absorbed with excess homotypic 25 nm. component fixed complement with the disrupted virus but not with intact virus. The disrupted virus also reacted with heterotypic antiserum produced by inoculation of guinea pigs with inactivated 25 nm. component, providing further evidence that the antigen is a structural component of the virus particle.

SUMMARYCalf thyroid tissue cultures were found to be the most sensitive system in detecting virus collected in a large volume air sampler from boxes, where cattle, sheep and pigs infected with A and C strains of foot-and-mouth disease (FMD) virus were housed. It was confirmed with all strains of FMD virus tested that pigs excreted the most virus followed by cattle and sheep, but there was variation between strains, the highest virus recoveries being obtained from animals infected with O1and C Noville viruses. The results are discussed in relation to outbreaks of foot-and-mouth disease in Great Britain since 1954.

The use of ionizing radiation has given much information on the size and structure of virus particles (Ginoza, 1963; Contreras & Ohlbaum, 1968). The relation between virus particle size and sensitivity to inactivation by ionizing radiation was postulated by Lea (1940); later, the target theory was developed (Lea, 1946; Timofeeff-Ressovsky & Zimmer, 1947). The amount of genetic material in a virus can be determined by calculating the target molecular weight (Ginoza, 1967). Even though it is generally accepted that the RNA of picornaviruses has a molecular weight of about 2 × 106 (Allison & Burke, 1962), there is very little information about the molecular weight of the genome of many of them. The present study was undertaken to determine the size of the genome of foot-and-mouth disease virus by means of γ-irradiation. For the purpose of comparison, we irradiated simultaneously another enterovirus, poliovirus, under identical experimental conditions.

Foot-and-mouth disease virus (FMDV) isoelectric point (pI) values, determined by cellulose acetate electrophoresis, were linear with respect to the square root of the ionic strength. This effect for proteins is attributed to the preferential binding of anions. The Longsworth and Jacobsen equation, pI=pI0−(AΓ/2)/(1+BΓ/2), was evaluated for FMDV; Γ/2 is the ionic strength, pI0 is the isoionic point 7.6, and constants A and B are 21.5 and 4.2, respectively. With pI values of 3.25−7.5 estimated from this equation, it was found that FMDV was always inactivated at its pl, while at 2 pH units above its pI there was no loss of infectivity. Storing virus at conditions greater than 2 pH units above isoelectric points at alkaline pH also resulted in inactivation. Thus, virus inactivation or stability can be predetermined over a range of hydrogen ion concentrations and ionic strengths. Inactivation of FMDV below pH 7.0 in isotonic solution or degradation at pH 7.5 in hypotonic solution (Γ/2 0.002) which previously appeared to occur by two independent mechanisms are both the result of isoelectric inactivation.

Selected cationic and anionic surface-active compounds were tested to determine their virucidal effect on the foot-and-mouth disease virus, type O, strain M11, propagated in primary calf kidney cells. The chemical inactivation of the virus was tested with 0.5, 1.0, 2.0, and 5.0% concentrations of the selected compounds. Virus controls with p H adjusted to cover the expected range of the mixtures of the chemicals and virus were also tested. The absence of virus from the mixtures of chemical and virus after reaction at 28 C for 2 hr was assayed by inoculating suckling mice with the mixtures. One cationic compound, alkyl methyl isoquinilinium chloride, showed considerable antiviral activity due largely to p H effect. The use of the surface-active agents investigated in this study, in the presence of organic material, would not be recommended as virucides.

Several strains of foot-and-mouth disease virus yield vaccines of low potency when inactivated by the conventional methods with acetylethyleneimine or formaldehyde. Experiments with one of these strains (Kenya 227/66) have shown that a satisfactory vaccine can be produced by using 0.05% formaldehyde as inactivant. The increase in activity is probably due to stabilization of the 140 S virus particle with the formaldehyde.

SummaryProtein synthesis in FMDV-infected BHK cells was inhibited in cells incubated at 25 to 41°. The metabolism of infected cells was always highest at 33°. Infected cells incubated at 30 to 41° produced the same high levels of virus, but in times ranging from 12 to 4 hr, respectively. Virus-induced RNA polymerase activity was always highest at 37°, being about twice as great as that formed at any other temperature. The cell-free polymerase system was active from 25 to 50° and showed maximal activity after 60-min incubation at 37°. Sucrose gradient profiles of the products formed under all the conditions employed did not reveal any qualitative differences.

The effect of sodium and magnesium chloride in 1 and 2 m concentration at temperatures of 37 and 50 C on type C, strain 149, foot-and-mouth disease virus during storage for 6 days was studied. The exclusively passaged cattle strain and its tissue culture-adapted line were compared. Preparations of the various chemicals and their concentrations were made directly in suspensions of the virus, which, together with untreated control virus suspensions, were stored at indicated temperatures and tested daily for concentration of virus present. Both 1 and 2 m concentrations of Mg markedly slowed the degradation of the bovine-passaged virus, as compared with untreated virus stored at 37 or 50 C. Such was not the case with 1 and 2 m concentrations of Na at 37 and 50 C, in which instance the treated virus was degraded faster than the untreated controls at 37 C, and but slightly influenced at 50 C. The tissue culture-adapted virus at the 25th passage was not stabilized by any concentration of chemical additive either at 37 or 50 C, except for 1 and 2 m concentrations of Na at 37 C, which partially retarded degradation of the virus. After 91 passages of the virus in tissue culture, only a suggestion of the influence of 1 and 2 m concentrations of Na at 37 C remained to show a stabilizing effect. These responses tend to separate the bovine-passaged virus from the tissue culture-adapted virus under the conditions of this study.

Foot-and-mouth disease virus was passaged 20 times by alternately adsorbing the virus with homogenized calf kidney and propagating the adsorption-resistant fraction of virus in cell cultures. Passage of the virus was accompanied by a marked decrease in pathogenicity for 35-day-old mice and a reduction in its ability to adsorb to homogenized kidney from such mice. Adsorption by homogenized calf kidney and infant mouse kidney also decreased with passage, but pathogenicity for cattle and infant mice remained high. The passaged virus did not seem to change antigenically. When the 20th-passage virus was serially passaged 10 times in cell cultures, no reversion in pathogenicity for 35-day-old mice was observed.

The infectivity of suspensions of foot-and-mouth disease virus is greatly reduced by incubation with trypsin. Preliminary studies with unfractionated suspensions suggested that the viral RNA was not involved in the inactivation because the infectivity of the RNA extracted with phenol from the enzyme-treated suspensions was as high as that from the untreated virus (1). The reduction in infectivity was probably due to the failure of the virus to adsorb to the susceptible cells. This report describes a more detailed investigation of the mode of inactivation, using purified virus labelled with 32P- or 14C. Virus of type 0 was grown in BHK21 cells (2) using a phosphate-low medium containing 1 µg. actinomycin D/ml. and labelled with 14C, or Earle's saline containing protein hydrolysate phosphate labelled with 32P. The virus was purified by the method described by Brown & Cartwright (3). In a typical experiment with 32P-labelled virus, the infectivity was reduced from 6·3 × 108 plaque forming units (p.f.u.)/ml. to 1·8 × 105p.f.u./ml. by incubation with 1 mg. trypsin/ml. for 15 min. at 37°.

The effect of eleven derivatives of N-phenyl-N'-aryl- or alkylthiourea on the multiplication of Coxsackie B1, ECHO 19 and foot-and-mouth disease viruses in cell cultures has been tested. In one-step growth cycle experiments N-phenyl-N'-4-carboxy-5-hydroxyphenylthiourea, N-phenyl-N'-3-hydroxyphenylthiourea and γ-phenylthioureidobutyric acid inhibited 99.9% of the Coxsackie B1 and ECHO 19 virus yields. N-phenyl-N'-4-hydroxyphenylthiourea suppressed 99.65% of the foot-and-mouth disease virus yield.

SUMMARYIn animals exposed to foot-and-mouth disease virus by indirect contact, virus was recovered from the blood, milk, pharynx, vagina and rectum for variable periods of time before clinical disease was apparent. Virus instilled into the mammary gland multiplied rapidly and virus concentrations greater than 107 p.f.u./ml. were recorded within 8–32 hr., depending on the virus strain and dose inoculated. Virus multiplication was accompanied by clinical signs of mastitis but the classical signs of foot-and-mouth disease did not appear for 52–117 hr. Dissemination of virus from the mammary gland occurred within 4–24 hr. and in some animals samples taken from the pharynx, mouth, nose and vagina contained virus for periods up to 97 hr. before the appearance of vesicular lesions. Virus production in the udder declined with the appearance of virus neutralizing activity in the blood and the milk but persisted in some animals for periods of 3–7 weeks. The ability of foot-and-mouth disease virus to persist in mammary tissue was confirmed by the demonstration of virus multiplication in the udders of immune animals.

The present paper deals with the characterization of RNAs synthesized in vitro with the help of RNA polymerase (RNA nucleotidyl transferase EC 2.7.7.6) prepared from BHK 21 cells infected with foot-and-mouth disease virus (FMDV), strain a 2-spain. Polatnick & Arlinghaus (1967) reported an RNA-dependent RNA-polymerase in BHK cells infected with FMDV. The properties of this enzyme and the in vitro synthesized RNA were described by Arlinghaus & Polatnick (1969a, b), Polatnick & Arlinghaus (1967). The techniques published by Scholtissek (1969) were employed in our investigations. Using hybridization and nearest-neighbour analysis, Scholtissek (1969) was able to show for the fowl plague in vitro system, that the newly synthesized RNA consisted mainly of strands complementary to virus RNA. In contrast to these results the present work showed that the in vitro product synthesized with the help of a FMDV-induced polymerase consisted mainly of plus-stranded RNA. The FMDV polymerase was prepared as described by Polatnick & Arlinghaus (1967).

Foot-and-mouth disease virus and vesicular stomatitis virus both contain RNA and multiply in the cytoplasm (Mussgay, 1958; Mussgay & Weibel, 1963). It would be expected that their replication would not be affected by actinomycin D, which interferes with the template function of DNA (Reich et al. 1961a; Goldberg, Rabinowitz & Reich, 1962; Hurwitz et al. 1962; Kersten & Kersten, 1962), or by mitomycin C and u.v. light, which are known to destroy the cell genome (Lathja, 1960; Reich, Shatkin & Tatum, 1961b; Kersten et al. 1964). This communication presents evidence which shows that the replication of foot-and-mouth disease virus is inhibited by all three agents, whereas vesicular stomatitis virus replication is unaffected by mitomycin C or actonomycin D. Foot-and-mouth disease virus (type O) and vesicular stomatitis virus (type Indiana) were grown in monolayers of baby hamster kidney cells (BHK 21, clone 13; Macpherson & Stoker, 1962) and assayed by the plaque method (Mowat & Chapman, 1962).

Foot-and-mouth disease virus can be grown in baby hamster kidney cells with a chemically characterized medium containing only tris(hydroxymethyl)-amino-methane (Tris) buffer, glucose, glutamine, and salts. Virus infectivity was only 0.5 log unit less than in a complex cell growth medium containing serum, tryptose phosphate, and lactalbumin hydrolysate. At high multiplicity of infection, production was maximal in 5 hr, with the virus remaining largely intracellular. Glucose and glutamine appeared to act independently of each other although both were required at about the same time during the virus production cycle. Glutamine had the greater effect and could not be replaced by amino acids, purines, and pyrimidines. Glutamine also stimulated cellular oxygen uptake in both normal and infected cells. Serum and other organic components added singly to the defined medium did not increase the virus yield. Studies on uninfected cells over a 5-hr incubation period showed that the defined medium maintained protein and ribonucleic acid synthesis at rates similar to the complex cell growth medium. These rates were much lower in media containing only inorganic salts and Tris buffer. Glucose, however, was more important to uninfected cellular metabolism than was glutamine. Defined medium containing dialyzed calf serum produced the highest rate of protein synthesis.

SUMMARYSampling of human subjects, who had been in contact with animals infected with foot-and-mouth disease (FMD) virus, showed that virus could be recovered from the nose, throat, saliva and from air expelled during coughing, sneezing, talking and breathing. The amounts of virus recovered paralleled those collected with a large-volume sampler and multistage impinger and these findings confirmed that the highest recovery of airborne virus was from infected pigs followed by cattle and sheep. More virus was found in the noses of those examining infected animals than in those operating the samplers, but there was variation between the subjects. In the majority there was a 1·8 log fall in titre by 3·5 hr., but virus persisted in the nose of one subject for 28 hr. Nose blowing or washing the nostrils did not remove virus completely, nor were cloth or industrial masks completely effective in preventing inhalation of virus. It was possible to transmit virus from infected subjects to others on one occasion. No clinical cases of FMD in man resulted from exposure, nor was there any rise in antibody. Use was made of these findings in determining sites of aerosol excretion in animals, and the results are discussed in relation to FMD in man and to the spread of respiratory viruses by the airborne route.

1. The 37s RNA induced in baby-hamster kidney cells by infection with foot-and-mouth-disease virus was examined on sucrose gradients and by filtration through Sepharose 4B. 2. The RNA sedimented faster (37s) and as a broader band than the 35s RNA from purified virus. 3. Treatment with deoxyribonuclease, Pronase or amylase did not alter the sedimentation profile of the 37s RNA. 4. Treatment of individual fractions of the RNA with phenol, dimethyl sulphoxide or methylCellosolve did not decrease the sedimentation rate of the faster-sedimenting molecules. 5. Sedimentation in sucrose gradients of different ionic strengths or containing EDTA had no effect on the heterogeneous nature of the profile. 6. On filtration through Sepharose 4B columns, the 37s virus-induced RNA was eluted before viral RNA. 7. Only 20% of the rapidly sedimenting RNA was incorporated into complete virus particles.

Virus-like particles resembling those seen in baby hamster kidney (BHK-21) cell cultures and in mouse leukemia have been found in two stable pig kidney cell lines, PK-13 and IB-RS-2. The particle's average outer diameter is c. 90 mμ, while the core is c. 60 mμ.

Factors influencing interferon production by strains of foot-and-mouth disease virus in bovine tissue cultures were investigated. In calf thyroid cultures after low input of virus the amount of interferon produced by modified strains was regularly greater and the degree of cytopathogenic effect less than that produced by virulent strains suggesting the use of these properties as a marker for innocuity in the study of virus modification. In calf kidney cultures and in surviving cultures of bovine tongue epithelium interferon production by modified strains depended on virus multiplication. In all bovine tissue cultures increased interferon titres were noted with some modified strains after high input of virus, but with others interferon titre was low due to successful viral replication in the majority of cells. In contrast to primary thyroid cells little or no interferon was detected in passaged and cloned thyroid cells infected with virulent or modified virus. The ability of modified strains to produce higher titres of interferon than their virulent counterparts appeared to depend on qualitative differences between the strains rather than on quantitative differences in the amount of non-replicating or heat inactivated virus in the preparations.