- Volume 34, Issue 3, 1977

Some commonly used inducers of interferon such as viruses and double-stranded RNA are known to inhibit cellular protein synthesis. We have now shown that conventional inhibitors of macromolecular synthesis such as cycloheximide, 2-(4-methyl-2, 6-dinitroanilino)-N-methyl propionamide (MDMP) and 1-β-d-ribofuranosylbenzimidazole (DRB) can induce the production of significant amounts of interferon in human fibroblastoid cell lines. The interferon-inducing activity of these inhibitors depends on the concentration as well as the time of treatment with the inhibitors. These findings lead to the suggestion that the induction of human interferon may be mediated by a reduction in the critical concentration of a rapidly turning over repressor(s) which normally represses the interferon gene(s) in uninduced cells.

Marek’s disease virus (MDV) antigens, as detected by immunofluorescence, were induced in a lymphoblastoid cell line, MSB-1, in the presence of IdUrd. When treated with 20 µg/ml of IdUrd there was no increase in the number of cells producing virus particles. If IdUrd was removed, an increase in virus production followed. Activation of the MDV genome appeared to require incorporation of IdUrd into cellular DNA and occurred during the first 12 h of culture. Expression of the activated genome required de novo protein synthesis and occurred during the next 12 h. The MDV genome in high producer MSB-1 cells could be activated with low concentrations of IdUrd, whereas low producer MSB-1 cells could not be activated with IdUrd to any great extent.

The kinetics of equine herpesvirus type 3 (EHV-3) multiplication and of the synthesis of EHV-3 specific DNA and RNA were investigated. A one-step growth curve of EHV-3 in equine epithelial cells from a transitional cell carcinoma was characterized by: (1) a short eclipse period (4 h); (2) an exponential increase in infectious virus between 5 and 10 h post-inoculation; and (3) a slow, inefficient release of newly formed virus into the extracellular fluid. Two hours after infection of cells with EHV-3, the rates of incorporation of specific precursors into total cell RNA or DNA were reduced to 30% and 10%, respectively, of that seen in uninfected cells. With the aid of DNA-RNA hybridization and caesium chloride isopycnic centrifugation techniques, the rates of synthesis of EHV-3 specific nucleic acids at different stages of the virus replication cycle were determined. Virus RNA and DNA synthesis was detectable 2 h after infection and reached maximum levels at an interval (4 to 7 h post-inoculation) corresponding to that period of the virus replication cycle just preceding the time of maximal synthesis of infectious virus.

Three distinct particles were isolated from cell culture harvests of swine vesicular disease virus (SVDV) by sucrose and CsCl gradient centrifugation. Virions (148S), RNA-free empty capsids (81S), and a third particle (49S) also free of RNA showed immune reactivity with SVDV antiserum. The 81S and 49S particles had polypeptides typical of naturally occurring empty capsids. Injection of purified antigens into guinea pigs produced antisera which distinguished empty capsids from virions on immunodiffusion; the 49S antigen appeared similar to virions. Antisera produced to freshly prepared virus antigen grown in brains of baby mice distinguished SVDV from the serologically related Coxsackie B-5 virus but did not distinguish the individual S particle antigens. Partly purified virus preparations degraded to empty capsids when incubated in guinea pig serum. The possible origin of empty capsids and 49S particles and their relationship to antigenicity of virus preparations are discussed.

Analysis of purified human cytomegalovirus (CMV) by sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed 32 polypeptides with mol. wt. ranging from 13500 to 235000. Similar analysis of purified preparations of four strains of CMV showed a remarkable similarity in polypeptide composition. Results indicate that the four strains may be related.

The swine white blood cells sensitive to African swine fever (ASF) virus are monocytes differentiated in vitro to macrophages. These cells have been characterized by their morphology, phagocytic capacity and the presence of receptors for swine immunoglobulin G in their membranes.

ASF virus does not produce any detectable effect on macrophages from humans, rabbits, guinea pigs, hamsters or rats, whereas ASF virus-infected chicken macrophages show an enhancement of cellular DNA synthesis and an intense cytopathic effect.

ASF virus, adapted to grow in VERO cells, produces a strong cytopathic effect in human macrophages leading to cell destruction. This effect is not associated with the synthesis of infectious virus, cellular or virus DNA nor with the formation of detectable virus-related structures.

Dense poliovirus particles (DP) differ in buoyant density, sedimentation coefficient and lability from standard poliovirus particles. Dense particles band at a density of 1.44 g/ml in isopycnic CsCl gradients and sediment in sucrose gradients at 220S. However, when DP are centrifuged in sucrose gradients containing 1.5 m-KCl, NaCl or LiCl, two types of particles are observed, one sedimenting at 220S and the other at 160S. Particles sedimenting at 220S are converted into particles sedimenting at 160S by incubation at 37°C in 1.5 m-KCl.

The high buoyant density seems to be correlated with the high lability of DP. Dense particles are extremely labile in isotonic phosphate-buffered saline. Their degradation proceeds through an RNA-containing particle lacking polypeptide VP4, to RNA and empty capsids.

Some characteristics of a microplate method for the detection and assay of plant viruses using enzyme-labelled antibodies are described. The method enabled the highly sensitive detection of a number of morphologically different viruses in purified preparations and in unclarified extracts of herbaceous hosts and of infected crop plants. Virus concentrations were estimated by photometric measurement of the colour intensity of the hydrolysed substrate. The suitability of the technique for various field and research applications is considered.

Enzymic and biophysical studies with purified infantile gastroenteritis virus (IGV) nucleic acid indicated that the virion contained a double-stranded RNA genome of approx. 14 × 106 daltons which could be separated by gel electrophoresis into eight bands of RNA which were comprised of 15 RNA species. Two major virus proteins, VP2 (mol. wt. = 135000) and VP8 (mol. wt. = 40000), which composed about 85% of the total virion protein, were detected in IGV particles by polyacrylamide gel electrophoresis. Eight additional minor proteins were also resolved.

Phenotypic expression of different genes controlling resistance to tobacco mosaic virus (TMV) in tomato was analysed in the protoplast system using otherwise isogenic breeding lines. Genes Tm-2 and Tm-22 were not expressed and did not prevent TMV-L, a common tomato strain of TMV, infecting and multiplying. By contrast, homozygous gene Tm-1 was able to express its effect in protoplasts as well as in leaf discs; no virus progeny were detected by fluorescent antibody staining or by infectivity assay up to 3 days after inoculation with TMV-L. Protoplasts and leaf discs homozygous for Tm-1, however, became infected with TMV-CH2, a tomato strain able to overcome the effects of Tm-1 in intact plants.

Protoplasts from Lycopersicon peruvianum P. I. 128650, known to have a high level of resistance to TMV, were as readily infected with TMV-L, and synthesized progeny virus as rapidly as protoplasts from susceptible tomato. This genotype seems to have no resistance expressible in isolated protoplasts.

Herpes simplex virus (HSV) DNA molecules were isolated from infected BSC 1 cells and centrifuged in CsCl-ethidium bromide density gradients. Both newly labelled and mature virus DNA molecules were found to have a linear conformation. The morphology of virus DNA molecules at different stages of the virus growth cycle in BSC 1 cells, was studied by electron microscopy after separation of virus DNA from cellular DNA by centrifugation in CsCl gradients. In each sample, about 200 virus DNA molecules were photographed and the different morphological forms were studied. Four classes of virus DNA molecules were observed: (a) mature linear DNA molecules, 52.4 ± 3.3 µm in length, (b) DNA molecules that contain a replicative loop or are Y-shaped, resembling replicative intermediates, (c) virus DNA molecules having one or more single-stranded filaments attached to them and (d) molecules with collapsed regions or with branches. A few circular molecules as well as linear DNA molecules longer than unit length were also observed. The virus DNA molecules resembling replicative intermediates gradually increased in number and reached a maximal amount of about 5% of the virus DNA population at 12 h after infection. The other forms of virus DNA were found to persist after the number of replicating DNA molecules decreased.

Lectins of different specificities do not interfere with the maturation of myxoviruses; their inhibitory effect on virus replication is mainly due to prevention of the detachment of infectious virus particles from the host cell. In chick embryo fibroblasts infected with an influenza virus and treated with concanavalin A, budding occurs into intracytoplasmic vacuoles, but this phenomenon is not observed with a parainfluenza virus and with different cells.

The lipids of two cell types (primary pig kidney and secondary adult pig thyroid) and those of transmissible gastroenteritis virus (TGEV) grown in these cells were studied using 14C-palmitic acid. Differences were demonstrated between the incorporation of isotopically labelled lipid precursors in the two cell types and it was found that the phospholipid and glycolipid profiles of purified TGEV closely resembled those of the host cell in which it was grown.

When transformed human embryonic cells, RSa and RSb, were treated for 48 h with both 10−7 m-ouabain and 1000 units/ml of human leucocyte interferon, there was an additive suppression of cell growth. In contrast, the antiviral action of the human interferon was inhibited by this concentration of ouabain. Similar effects of combined treatment with interferon and ouabain were found with IFr cells (which are relatively resistant to the anticellular effect of interferon), and with mouse cells of the K3b line.

Treatment of adenovirus type 5-infected cells at late times after infection (48 to 72 h p.i.) by hypotonic buffer and subsequent manipulation of the crude extract has resulted in the crystallization and reaggregation of the adenovirus type 5 fibre and hexon components.

Treatment of purified preparations of tobacco rattle virus with 2 mm-EDTA decreased their infectivity, and that of RNA extracted from them, by about 90%. The effect of EDTA was apparently to remove divalent metal ions from the virus particles, making the RNA inside accessible to attack by a ribonuclease present in the preparations. Provided the enzyme activity was inhibited while the ions were absent, they could be put back with full restoration of full stability of infectivity.

Studies of the Uga 6/70 strain of foot-and-mouth disease (FMD) virus, type SAT 2, using the haemagglutination-inhibiting antibody consumption (HIC) test, have demonstrated the existence of trypsin-sensitive determinants on the intact virus particle and the 12S protein subunit.

Eosinophilic intranuclear filaments described previously in Rift Valley Fever infected cells, were shown to fluoresce specifically in an indirect technique with antiserum to the virus. Actinomycin D failed to suppress development of the filaments or replication of the virus.