- Volume 15, Issue 2, 1972

Light and electron microscopy were used to compare two flexuous-rod viruses: cowpea virus (CV) and bean yellow mosaic virus (BYMV). Inclusions induced by CV were consistently different from those induced by BYMV. In the light microscope, BYMV-induced cylindrical inclusions appeared as a group of plates, whereas CV-induced cylindrical inclusions appeared as a group of tubes. Electron microscopy showed that CV induced fibrous nuclear inclusions, while BYMV induced non-fibrous nucleolar inclusions in Crotalaria spectabilis. Thin section studies showed that CV-induced cylindrical inclusions contained circles and tubes, but no laminated aggregates, while BYMV-induced cylindrical inclusions contained laminated aggregates, but no circles or tubes. The shapes of extracted inclusions induced by CV were consistently different from those induced by BYMV. These viruses are cytologically different and are believed to be distinct.

The pre-treatment of mouse L cells with interferon or polyinosinic-polycytidylic acid potentiated the interferon response of these cells to subsequent stimulation with suboptimal doses of polyinosinic-polycytidylic acid. This potentiation was manifested as an earlier production of interferon, a faster attainment of maximum interferon production, and a greater yield of interferon. Pre-treatment of cells with either low (5 units/ml.) or high (1000 units/ml.) doses of interferon never caused a reduced interferon response to stimulation by polyinosinic-polycytidylic acid. Primary rabbit kidney cells which were pre-treated with interferon (500 units/ml.) responded to stimulation with polyinosinic-polycytidylic acid in the same manner as did mouse L cells. In contrast, under certain conditions mouse L cells pre-treated with interferon became resistant to stimulation with Newcastle disease virus. The extent of the reduced response depended on the pre-treatment dose of interferon and the stimulating dose of Newcastle disease virus. It was concluded that the presence of interferon and its virus resistant state were not directly correlated with hyporesponsiveness to repeated induction of interferon, at least by rI.rC.

L cells in which interferon had been induced by u.v. irradiated Newcastle disease virus were exposed for varying periods of time to [3H]-l-methionine, [3H]- or [14C]-d-glucosamine, [3H]-protein hydrolysate or [14C]-l-fucose. Interferon was subsequently purified by SE-Sephadex chromatography and polyacrylamide gel electrophoresis to contain in excess of 1 × 106 units/mg. of protein. Electropherograms disclosed that methionine, amino acid mixture and, to a lesser degree, glucosamine, were incorporated into proteins associated with the dominant interferon fraction. No incorporation of fucose was noted. Methionine and glucosamine labels were absent from the major interferon band in control preparations from non-induced cells, co-electrophoresed in acrylamide gel with non-labelled chromatographed marker interferon.

The induction of interferon in chick embryo cells by human adenovirus types 1, 5 and 12 has been examined. Rifampicin, at a concentration of 100 µg./ml., added either immediately or within 24 hr after infection, completely inhibited interferon induction by all three adenoviruses. Up to 24 hr after infection, the inhibitory effect was reversible, and when the drug was removed, interferon production resumed. Rifampicin at 100 µg./ml. also inhibited interferon induction by Semliki Forest virus in chick cells at 37° and 42°, although it had no effect on virus growth at 37°. We conclude that the antibiotic inhibits either a non-essential virus function involved in induction, or alternatively a cellular function. The antiviral action of chick cell interferon against Semliki Forest virus or vaccinia virus was not affected by rifampicin. The drug inhibited chick cell growth, but did not appear to injure cell viability over 48 to 72 hr periods. Rifampicin significantly depressed the incorporation of [3H]-thymidine into chick cell DNA, but had a much lesser effect on incorporation of [3H]-uridine and [3H]-leucine into RNA and protein respectively.

A modification of the peroxidase-labelled antibody technique was applied to study the distribution of virus antigens in chicken cells infected with two serotypes (A and B) of avian leukosis virus (ALV). Type-specific chicken antisera reacted only with cells infected with virus of the homologous envelope serotype. When unfixed cells were exposed to type-specific antivirus serum, only antigens located at the cell surface were stained, while cells exposed to type-specific antibodies after fixation revealed both surface and intracytoplasmic virus antigen. Cytoplasmic antigen was usually concentrated in discrete granules which often had a vesicular structure.

Hamster antibodies against ALV group-specific (gs) antigen reacted with fixed infected cells regardless of envelope serotype, and the distribution of antigen was as shown with chicken type-specific antibodies. Intranuclear gs antigen may have been present in a few cells. Unfixed infected cells did not react with hamster gs antibody and confirmed the location of ALV gs antigen within the virus particle.

Some chicken antisera with neutralizing antibodies against a single envelope serotype of ALV contained both type-specific and gs antibodies. These antisera reacted with fixed cells infected with virus of either the homologous or heterologous serotype and stained both surface and cytoplasmic antigens. With unfixed infected cells, these antisera combined only with the surface of cells infected with virus of the same serotype as the neutralizing antibodies in the serum. Thus, the reaction in heterologously infected fixed cells was with internal gs antigen. This confirms independently that chickens are not naturally tolerant to their homologous ‘C type’ gs antigens.

The protein composition of several members of the animal picornavirus group is remarkably similar. Thus, the enteroviruses polio type 1, Echo 12 and bovine entero VG-5-27 (Maizel & Summers, 1968; Korant, Lonberg-Holm & Halperen, 1970; Johnston & Martin, 1971), the cardioviruses Maus-Elberfeld and encephalomyocarditis (Rueckert, Dunker & Stoltzfus, 1969; A. T. H. Burness, personal communication), the rhinoviruses 1A and HGP (Medappa, McLean & Rueckert, 1971; E. J. Stott & R. Killington, personal communication) and several of the seven immunological types of foot-and-mouth disease virus (Burroughs et al. 1971; P. Talbot, unpublished observations) contain three polypeptides with molecular weights ranging from 37 to 24 × 103 (VP1, VP2, VP3) and a smaller polypeptide with a molecular weight in the range of 7 to 13.5 × 103 (VP4). In several instances the molar ratio of the four polypeptides has been determined and, with the exception of rhinovirus 1A (Medappa et al. 1971), the three larger polypeptides are present in equimolar amounts and the smaller polypeptide is present in a half molar amount relative to the others.

Seven serologically and immunologically distinct types of foot-and-mouth disease virus (FMDV) are known; these types can be subdivided into numerous subtypes. Recently, we reported on polynucleotide sequence homologies among the ribonucleic acids (RNA’s) of FMDV types A, O and C (Dietzschold et al. 1971). Making use of saturation and competition experiments various degrees (44 to 65%) of base sequence homologies between different types were obtained. This report is concerned with similar studies but using subtypes and mutants of FMDV, type O. We felt that there is a need especially for quantitative data on genetic relationships between different subtypes, because the serological classification is not always congruent with a classification based on immunological properties (Wittmann, 1964). Furthermore, it appeared interesting to compare in cross-annealing experiments wild-type virus with mutants reacting identically in serological tests.

The experiments were performed with radioactive labelled FMDV RNA of five O-subtypes and five mutants of subtype O1 which were hybridized with denatured unlabelled FMDV-specific double-stranded RNA of the respective virus-strains.