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The production of foot-and-mouth disease virus from BHK 21 C 13 cells grown on the surface of glass spheres.

In view of the advantages which are associated with the use of the BHK monolayer cell for the production of foot-and-mouth disease (FMD) virus, a unit system using glass spheres was developed to grow BHK monolayer cells and to test the susceptibility of such cells to FMD virus. The yield of cells and their susceptibility compares favorably with GREEN BHK monolayer cells which have been grown in Roux bottles.

Animals

The production of foot-and-mouth disease virus from BHK 21 C 13 cells grown on the surface of DEAE sephadex A50 beads.

Methods are described which make possible the production of foot-and-mouth disease (FMD) virus from BHK 21 C13 monolayer cells which have been grown on the surface of serum coated DEAE Sephadex A50 beads. The yield of cells and their susceptibility to infection by FMD virus are equivalent to conventional Roux monolayer systems. The potential for the commercial application of the DEAE Sephadex A50 system is discussed in relation to other unit process monolayer systems and in particular to the system in which cells are cultured in a deep bed of small glass spheres.

Animals

Enhancement by diethylamineothyl-dextran of the plaque-forming activity of foot-and-mouth disease virus-antibody complexes in pig kidney IB-RS-2 cells.

Mixtures of foot-and-mouth disease (FMD) virus and homologous antibody, assayed for surviving plaque-forming units (PFU) in pig kidney IB-RS-2 cells, demonstrated an average ten-fold greater recovery of infectivity when diethylaminoethyl-dextran (DEAE-dextran) was included in the overlay medium. This enhancement, which was not detected in baby hamster kidney BHK21 cells, was due to the ability of the polycation to potentiate attachment of virus-antibody complexes to the IB-RS-2 cells. In some instances the effect was so pronounced that the plaque-forming activity of virus in the presence of homologous antibody exceeded that of virus alone.

Animals

Susceptibility of various FMDV strains to urea.

Resistance to urea in vitro at 37 degrees C varied for each FMDV strain analysed. The urea marker did not correlate with other markers such as resistance to acid, resistance to acidity or size of plaques under agar on BHK21/13 cells. The resistance to urea of subtypes A24 Cruzeiro, O1 Caseros and C3 Resende varied in accordance with their antigenic potency when administered to swine as a trivalent water-in-oil emulsion type vaccine.

Antigens, Viral

Differentiation between specific and nonspecific reactions of bovine sera and foot and mouth disease virus (FMDV) in immunodiffusion tests.

The precipitating and neutralizing activities of normal bovine sera with FMDV were studied and compared. Twenty-two out of 79 normal bovine sera gave a positive reaction in micro neutralization tests with FMDV type O, while six did so with type A. In RID tests 32 sera were positive with type O and 28 with type A virus. Almost all of the 79 sera gave a positive reaction in the RID with trypsin treated virus of both types. After three to four fold concentration most sera also gave visible reactions in ID tests when tested against complete virus. When O virus was used the ID patterns produced by most normal sera clearly differed from those obtained with early and late convalescent sera from FMDV infected steers. When type A materials were employed this was also the case but to a lesser extent. The patterns obtained with concentrated normal sera showed, in general a strong line with trypsin treated virus and no line or a weaker one with complete virus. The substances in normal bovine sera precipitating trypsin treated O virus were different from those reacting with trypsinized virus of type A.

Animals

Binary ethylenimine as an inactivant for foot-and-mouth disease virus and its application for vaccine production.

Foot-and-mouth disease virus was inactivated with binary ethylenimine formed apart from or directly in the virus suspension by the cyclization of 2-bromoethylamine hydrobromide or 2-chloroethylamine hydrochloride under alkaline conditions. The inactivation rates with binary ethylenimine prepared apart from the virus suspension in dilute sodium hydroxide with either 2-bromoethylamine hydrobromide or 2-chlorethylamine hydrochloride were higher than with pure ethylenimine. When binary ethylenime was prepared directly in the virus suspension only 2-bromoethylamine hydrobromide gave acceptable inactivation rates. The reduced inactivation rates for binary ethylenimine directly prepared in the virus suspension are due to the different cyclization rates of 2-bromoethylamine hydrobromide and 2-chloroethylamine hydrochloride and to the interference of bicarbonate in the cyclization reaction. The complement fixing antigen of foot-and-mouth disease virus was not affected by binary ethylenimine inactivation. Vaccines prepared with foot-and-mouth disease virus inactivated by binary ethylenimine were comparable in their immunogenicity to vaccines prepared with ethylenimine or N-acetylethylenimine used as inactivants. Application of binary ethylenimine in the preparation of foot-and-mouth disease vaccines considerably reduces the potential danger associated with handling pure ethylenimine and other aziridines.

Animals

Ribonuclease activities associated with purified foot and mouth disease virus.

Ribonuclease activities internally and externally associated with purified foot-and mouth disease virus were detected. The outer activity was easily removed by cesium chloride or by detergent (Sarkosyl). The inner activity is not removable by any procedure used and could be the enzyme responsible for the heterogeneity normally observed in the extracted FMDV-RNA. It is not known at present if both activities are related to the same or to different enzymes.

Aphthovirus

Resolution of block neutralization test curves into components of the foot-and-mouth disease virus system.

Classic neutralization studies by Fazekas de St. Groth and Webster (8) on mixtures of influenza viruses and mixtures of rabbit antisera are reinterpreted in terms of a percentage contaminant in the stock used for the dilution series. A very small amount of a different virus changes the shape of quantal assay curves considerably, but even a large amount of a different antiserum has negligible effect on the shape and merely shifts the curve along the serum dilution axis. These conclusions are the reverse of the authors, who only considered an absolute amount of another component in all dilution tubes. An artificial mixture of 2.7 percent O8 in O1 foot-and-mouth disease virus strains was tested against anti-O1 serum assaying in suckling mice. The small amount of O8 virus greatly altered the shape of the neutralization curve in the direction expected from the reanalysis of the influenza literature data. Results from artificial mixtures are used to explain what were hitherto anomalously broad neutralization curves for some other foot-and-mouth disease strains given by Booth et al. (1) Many of the virus stocks studied can now be postulated as a natural mixture of related virus strains. In fact, the O1 and O8 stocks used might also be of themselves mixtures. These virus strains also exhibit still a further test complication in that the virus-antibody reaction appeared to shift away from complexes on dilution immediately prior to assay.

Antigen-Antibody Reactions

A new method for the isolation of undegraded FMDV-specific RNA from infected BHK cells.

Fractionation of Foot-and-Mouth disease virus infected cells by currently described procedures, leads to the appearance of variable amounts of heterogeneous single-stranded RNA fragments. A new method based upon the fractionation of cultured cells at extremely low temperatures has been developed to minimize the degradation of the viral RNAs by cellular nucleases. It was shown that the viral RNAs obtained by this procedure were almost non-degraded, and similar to those found in other picornavirus infected cells. More than 90 per cent of the polysomal RNAs were found as genome-size molecules, presumably being messenger RNA (mRNA). It was also found that the mRNA analyzed on sucrose gradients sedimented slightly ahead of the 35S genomic RNA. However, no differences were found when the analysis was performed on polyacrylamide gels.

Animals

Some investigations on the adjuvant mechanism of DEAE dextran.

In vitro it was shown that adsorption of inactivated FMDV onto DEAE-D kieselgur columns did not occur in the presence of 0.1--0.15M NaCl. These NaCl concentrations are present in DEAE-D/FMDV vaccines and in the tissues of animals. Therefore, adsorption of virus antigen does not appear to be responsible for the adjuvant effect of DEAE-D. In pigs it was demonstrated that DEAE-D exerts its optimal adjuvant effect, as measured by the formation of neutralizing antibodies and protection against challenge infection, when injected together with inactivated FMDV as vaccine. Apart from this, a good adjuvant effect (group immunity 75--100 per cent) was evoked in about one half and a moderate effect (group immunity 50--70 per cent) in about a quarter of the inoculated animals even if DEAE-D was separately injected locally and temporally from the inactivated virus. With regard to immunity it apparently does not matter whether DEAE-D or inactivated virus was given first, but an interval of 48 hours or 4 days between injection seemed to be more favourable than one of 24 hours. With regard to the formation of neutralizing antibodies the situation is comparable to that of immunity with the exception that a time interval of 24 hours between the application of DEAE-D and inactivated virus or vice versa was as good as that of 48 hours or 4 days. The results are discussed in regard to the possible mechanism of the adjuvant effect of DEAE-D on the cellular level.

Adjuvants, Immunologic

Inhibitors of foot-and-mouth disease virus. Temperature-dependence of the effect of guanidine on virus growth.

In suspended secondary calf kidney cells infected with foot-and-mouth disease virus (FMDV) the temperature range for optimal virus growth is shifted down by 3 to 5 degrees C in the presence of 1--2 mM guanidine. For some virus strains this shift is so effective that at infraoptimal temperatures virus yield in guanidine-treated cells exceeds that of the corresponding control by more than one log10. On the contrary, at supraoptimal temperatures inhibition of virus growth by the drug is strongly enhanced. At a concentration of 1 to 2 mM guanidine virus yield reduction or enhancement is based on a decrease in increase, respectively, of the number of virus producing cells (infective centers; I.C.), while virus yield per I.C. is less affected. Besides this "thermomimetic" effect virus production is inhibited by guanidine depending on the concentration of this substance. A mutant of FMDV strain O1L, resistant to 4.2 guanidine, did not differ from the original virus in its antigenic behaviour in the passive immunohemolysis test.

Aphthovirus

Stability and immunogenicity of empty particles of foot-and-mouth disease virus.

Three strains of foot-and-mouth disease virus were shown to contain significant amounts of naturally occurring 75S, empty particles as well as the infectious, 140S full particles. One of these strains--A Pando (1970)--was studied in detail. The empty particles from this virus strain were shown to have an observed sedimentation coefficient of 67S in 0.04 M phosphate buffer; they were labile in SDS, non-infectious and probably RNA-free and, on heating, they broke down to 12S subunits as did the 140S particles. The empty particles differed from the full particles in their polypeptide composition since they contained VP0, but there was no evidence for a diminished content of VP4. The 75S particles were shown to be present in significant amounts and to be stable to AEI inactivation. At 4 degrees C they were stable for at least two years. In guinea pigs they were as immunogenic as the 140S particles. The antisera raised against the 75S particles had the same serological specificity in neutralization tests as sera prepared against the 140S particle. It was concluded that the 75S particles from the A Pando (1970) strain of FMD virus may provide as important a contribution as 140S particles to the immunogenicity of inactivated vaccines prepared from this virus strain.

Antigens, Viral

Localisation on foot-and-mouth disease virus (FMDV) of an antigenic deficiency induced by passage in BHK cells.

Passage of FMDV in BHK suspended cells was confirmed to induce an antigenic deficiency on the virion. By immunodiffusion experiments with complete virus, with trypsin-treated virus and with 12S virus fraction it was shown that the induced antigenic deficiency is located on the trypsin-removable part of the virion. These results were confirmed by absorption experiments.

Animals

[Studies on the Relationship between immunity and the level of neutralizing antibodies in pigs vaccinated against foot-and-mouth disease (author's transl)].

The statistical relationship between the titers of neutralizing antibodies and the immunity of 706 pigs vaccinated against FMD was studied. This was done for each of four virus strains separately. Whereas no correlation between both test systems could be detected in case of the strains A5 Westerwald, C Detmold and O1Santander, a significant correlation was ascertained for the strain O1Kaufbeuren. Becuase of the different findings depending on the virus strain under study it was concluded that the antibody titer alone does not provide a useful measure for potency testing of FMD-vaccines for pigs.

Animals

The detection of antibody to virus-infection associated (VIA) antigen in various species of African wildlife following natural and experimental infection with foot and mouth disease virus.

The double immuno-diffusion (DID) test has been applied to detect antibody to VIA antigen in sera from various species of African wild ungulates. In conjunction with the serum neutralisation (SN) test it can be used to decide the degree of risk of movement of animals to other countries free from foot and mouth disease (FMD). The value of the test in assessing the history of infection is limited by its relatively low sensitivity and specificity in respect of virus type.

Africa