- Volume 25, Issue 1, 1974

Highly purified plasma membranes were isolated from chicken embryo fibroblasts infected with Marek’s disease virus (MDV) or turkey herpes virus (HVT). The purification was monitored by the incorporation of fucose and marker enzymes specific for plasma membranes. Polyacrylamide gel electrophoresis showed that the membrane preparations contained two new virus-induced proteins. When reacted in the double immunodiffusion test solubilized plasma membranes from MDV-infected cells formed two specific precipitation bands with Marek’s disease immunoglobulins.

Antisera prepared against plasma membranes from MDV- or HVT-infected cells neutralized extracellular infectious HVT. After incubation of plasma membranes from MDV-infected cells with Marek’s disease antibodies the buoyant density of the membranes increased from 1.05 g/ml to 1.08 g/ml due to the binding of immunoglobulins to the virus-induced membrane proteins. The Marek’s disease mortality of chickens twice vaccinated with a plasma membrane preparation from HVT-infected cells was reduced by 94%.

From the results it was concluded that the plasma membranes acquire new immunologic specificities after infection with MDV or HVT.

The ability of wheat germ cell-free extracts to translate several virus RNAs has been surveyed, using RNA from viruses with different host ranges, and which do not normally infect wheat. Synthesis of specific coat protein-like products was achieved with RNAs from cowpea chlorotic mottle virus, broad bean mottle virus, cucumber mosaic virus, and alfalfa mosaic virus. It appears to be mainly the small RNAs (which contain the coat protein cistron) that are translated with most fidelity. The larger RNA components of these multicomponent viruses direct amino acid incorporation quite efficiently, but the products are heterogenous. Other larger RNAs, such as tobacco mosaic virus RNA, also produce a heterogenous (polydisperse) mixture of polypeptides, rather than virus products. The RNAs of Maus–Elberfeld virus and avian myoblastosis virus stimulate a little amino acid incorporation without directing the synthesis of any recognizable virus protein-sized products. The relationship of RNA size with translatability is discussed.

Newly formed gs-1 antigen of RD-114 virus was detected by the indirect fluorescent antibody (IFA) procedure in the cytoplasm of the D-17 dog cell line at 24 h post-infection in approx. 30% of the cells. Virus-induced membrane antigen was present in approx. 50% of the cells by 48 h, at which time new infectious virus was being produced. Polykaryons were observed in infected cells. Their formation paralleled production of virus, as determined by the IFA membrane staining technique.

Neither tobacco mesophyll protoplasts nor tobacco leaves could be infected with wild type brome mosaic virus (BMV wt) or its RNA, but both were infected when inoculated with 50 µg/ml or more of a variant of BMV (BMV V5). This is about 100 × the amount required for efficient infection by cowpea chlorotic mottle virus (CCMV). Poly-l-ornithine stimulated the infection of protoplasts with BMV V5, but was not essential. BMV V5. RNA inoculum caused infection of protoplasts under conditions similar to those needed for their infection with CCMV-RNA.

Incubation in medium lacking arginine of monkey kidney cells infected with simian virus 40 resulted in a marked inhibition of production of both infectious virus and empty capsids. Virus DNA synthesis and formation of a virus DNA-protein complex were unaffected. All the virus structural polypeptides were made, although in reduced amounts (42%) when compared with infected cells incubated in medium containing arginine. Protein synthesis was inhibited in uninfected cells deprived of arginine. All the newly synthesized virus proteins were found in the nuclei of arginine-deprived cells. Addition of arginine to arginine-deprived cells resulted in the assembly of pre-synthesized virus components into infectious virus and empty capsids, as well as the production of virus from virus proteins and DNA synthesized after the addition of arginine. Studies with inhibitors of protein and DNA synthesis suggested that two late virus functions were affected in the absence of arginine: the assembly of capsids and the encapsidation of DNA by capsids to form infectious virus.

Mengovirus-induced, double-stranded RNA (RF) is infective, but its infectivity, unlike that of mengovirus, is strictly dependent upon host cell macromolecular synthesis. The treatment of cells with actinomycin D, α-amanitin or cordycepin 1 h before infection with mengovirus RF results in a drastic reduction of virus yield, whereas the same treatment has no effect on mengovirus infectivity. The kinetics of sensitivity to inhibitors suggest that the cellular macromolecule necessary for RF to initiate its infective cycle is involved only during the very early steps of replication, and probably has a very rapid turn-over. The cellular uptake of the infecting molecule seems not to be alterated by actinomycin treatment. Analysis of the intracellular distribution of [3H]- or [32P]-labelled mengovirus RF indicates that up to 40% of incoming molecules accumulate within the nuclear fraction (4 to 5% in nucleoli). Sedimentation velocity analyses of labelled RF recovered from each subcellular compartment show that the input molecule becomes heavier and polydisperse in gradients as the cycle of infection proceeds. A replication mechanism is proposed in which infective RF is transformed into replicative intermediate (RI), by a cellular RNA polymerase transcribing the first virus messenger RNAs with RF as abnormal template.

The DNA molecules synthesized in herpes simplex virus (HSV) infected cells were analysed by electrophoresis in polyacrylamide gels. It was found that part of the virus DNA, isolated from infected nuclei, migrated into the gels in a manner similar to the virus DNA molecules isolated from purified herpes simplex virus particles. Most of the virus DNA molecules, labelled in vivo for 60 min with [3H]-thymidine as well as labelled cellular DNA molecules, were retained at the top of the gel during electrophoresis. The amount of HSV-DNA which migrated into the gel gradually increased when the labelled cells were reincubated after removal of the isotope (chase). Most of the DNA molecules, which were retained at the top of the gel, had the density of virus DNA but differed in their sedimentation behaviour in sucrose gradients from the intact DNA molecules isolated from purified virus particles. The presence of replicating as well as mature virus DNA molecules in infected nuclei is discussed.

The structure of bacteriophage T1 was examined after inactivation by rotating the virus suspension in a rotating bulb or by bubbling gas through the suspension. After inactivation the [3H]-label of the nucleic acid was located in CsCl and sucrose gradients at the site of free DNA. The small fraction of phage which is resistant to surface inactivation had the same sedimentation coefficient and temperature sensitivity as the control phage, indicating that resistance is not due to agglomerate formation. No morphological differences between phage particles before and after inactivation could be demonstrated by electron microscopy.

When human rhinovirus type 2 attaches to HeLa cells, it first forms a reversible complex which can be dissociated by addition of EDTA to the medium. After prolonged incubation at 25 °C, most of the cell-associated virus progresses to an irreversible complex from which it cannot be eluted by EDTA, although some noninfectious material elutes spontaneously. The addition of a sufficient concentration of SDS to block cell-mediated eclipse of the virus does not block the formation of the irreversible complex at 25 °C, while virus adsorbed and incubated at 0 °C remains reversibly bound. The number of receptor sites per cell is not influenced by SDS, but at 0 °C cells appear to have fewer receptor sites than at physiological temperature.

Visna virus particles disrupted by exposure to 0.05 or 0.1% SDS release an internal nucleic acid component in the form of rings or short curvilinear rods. The ring structures have a mean circumference of 3.0 µm and are three times wider than single-stranded RNA. Incubation of detergent-disrupted virus particles with dimethylsulphoxide for 5 min causes uncoiling of rings and produces a heterogeneous population of single, unbranched filaments up to 9.3 µm long, similar in size to strands observed in 60 to 70S visna virus RNA recovered from glycerol velocity gradients. Treatment with dimethylsulphoxide for 30 min results in complete denaturing of the virus RNA into short fragments that average 3.2 µm in length. The visna virus genome consists of a molecule 9.3 µm long, apparently composed of subunits, which assumes a coiled configuration within the virus particle.

A virus was isolated from larvae of the rhinoceros beetle (Oryctes rhinoceros), which resembled particles observed in thin sections of the midgut epithelium of diseased insects. The virus was rod-shaped, enveloped, and measured approx. 220 × 120 nm. Purified particles had a density in sucrose of 1.18 g/ml and contained eleven protein components as determined by electrophoresis in 10% polyacrylamide gels. The two major proteins were of low mol. wt. (9.7 and 12.5 × 103). The nucleic acid had a density in caesium chloride characteristic of double-stranded DNA, with an estimated guanosine:cytosine content of 43%. A small proportion (9%) of the DNA was present as covalently-closed molecules. The virus DNA contained molecules with different sedimentation velocities, the major component having a sedimentation coefficient of 57.2S and an estimated mol. wt. of 87 × 106. It is proposed that this virus be included in the Baculovirus group.

When a monolayer culture of HeLa cells persistently infected with haemadsorption type 2 virus (HeLa/HA2) was dispersed by trypsin and plated on a monolayer culture of uninfected HeLa cells, syncytia developed. For induction of the syncytia, it was specifically pre-requisite to treat the carrier cells with trypsin. The formation of syncytia started at 1 h and was completed by 4 h, followed by the synthesis of virus antigens in the syncytia. The presence of cycloheximide at a concentration of 50 µg/ml did not inhibit the syncytium formation but the presence of antiviral serum completely suppressed it. The results indicated that the principle responsible for the cell fusion is not only of virus nature but also localized in a masked form on the surface of the carrier cells and that the trypsin treatment efficiently activates it. The mechanism of cell fusion in this system and the applicability of the above procedure to the isolation of causative agents from the lesions with which paramyxovirus-like structures are latently associated are discussed.

Properly purified preparations of poliovirus particles are practically free of d-galactose and N-acetyl-d-glucosamine. These sugars, however, are found in crude virus preparations, obtained by high and low speed sedimentation and by CsCl-gradient fractionation from infected HeLa cells. They are separated from the virus particle by extraction with chloroform and subsequent isopycnic sedimentation of the virus preparation. Since d-galactose and N-acetyl-d-glucosamine are regular constituents of glycoproteins, the absence of significant amounts of these sugars demonstrates the lack of glycoproteins in poliovirus particles.

Radioactive monosaccharides are recommended for the detection of cellular impurities in carbohydrate-free viruses.

A simple and rapid method for the purification of large quantities of poliovirus by ‘precipitation’ with polyethylene glycol, resuspension in CsCl solutions, extraction with chloroform and isopycnic sedimentation is described.

The kinetic data obtained from examination of restriction enzyme fragments give a direct determination of the position of the origin of replication in polyoma virus DNA. The results obtained are consistent with the previous estimates based on electron microscopic measurement of replicating molecules cleaved by endonuclease EcoR1 (29 ± 2% from the EcoR1 site). The position of the terminus is diametrically opposite the origin, at approx. 79% clockwise from the EcoR1 site.

Mouse L cells can be primed for enhanced interferon production by interferon covalently attached to beads of Sepharose. The induction of interferon synthesis by MM virus in primed cells can occur after removal of the interferon suggesting that interferon does not have to be in contact with the cell at the time of virus attachment. It is concluded that, like induction of the antiviral state, L cells can be primed at the cell surface for induction of interferon.

U.v. irradiation of human diploid cells enhanced interferon production after induction with poly I.C. Moreover, interferon production was even further increased by DEAE-dextran in cultures already enhanced by u.v.

Twenty-four strains of Herpesvirus hominis (HVH) were tested for their ability to produce large focal necrotic lesions in the liver of mice inoculated intraperitoneally with herpes virus. A close relationship was found between this property and infection with the type 2 strains and it is hoped that this observation may be used as a simple and reliable biological marker for the differentiation of HVH type 1 and type 2. Besides, it may draw the attention to a specific pathogenic distinction between these two types.

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.

Infection of chicken embryonic heart, lung and liver cells with parainfluenza 1 and 3 viruses revealed that the most susceptible (S) cell is the heart cell and the least susceptible (LS) cell is the liver cell. The results of these experiments are similar to observations where the S and LS cell for another paramyxovirus, mumps virus, were also cultivated from the chicken embryonic heart and liver, respectively.