- Volume 34, Issue 2, 1977

The synthesis of herpes simplex virus DNA in isolated nuclei under in vitro conditions was found to be dependent on the addition of ATP and an ATP generating system to the reaction mixture. In vitro DNA synthesis was stimulated and prolonged when p-hydroxymercuribenzoate was added to the isolated nuclei. Under these improved conditions virus DNA molecules which were initiated in vivo were completed in vitro, but most of the DNA molecules synthesized in vitro sedimented in sucrose gradients more slowly than herpes virion DNA. Denaturation of the in vitro labelled DNA molecules produced short single-stranded labelled DNA chains. Thus, under our improved in vitro conditions there was prolonged synthesis of DNA at a high rate, with the formation of both complete and incomplete virus DNA molecules.

The RNA from infectious pancreatic necrosis virus has been purified and had a sedimentation velocity of 14S on sucrose gradients, a buoyant density of 1.60 g/ml in Cs2SO4 and pyrimidine to purine ratios near unity. The RNA had the appearance of a linear double stranded molecule with an average length of 0.92 µm and a standard deviation of 0.07 µm when observed under the electron microscope using the Kleinschmidt protein film technique. This would correspond to a mol. wt. of 2.4±0.2 × 106. The RNase A resistance of IPN virus RNA exhibited a marked salt dependence; it was 92% resistant in 0.1 m-NaCl, but only 9% resistant, or less, in 0.01 m-NaCl. The RNA was resistant to denaturation by boiling at NaCl concentrations of 0.04 m or higher, but did denature at lower concentrations. Polyacrylamide gel electrophoresis of the RNA indicated that two RNA species were present and the standard deviation of lengths in the electron microscope indicated that they could not differ by more than 4 × 105 in mol. wt.

Addition of concanavalin A to BHK cell monolayers infected with vesicular stomatitis virus prevented the formation of mature virus particles. In these cells the virus glycoprotein (G) was inserted into the plasma membrane and the protein that is in close association with the ribonucleic acid, protein N, was found in the cytoplasm. At times when cells infected in the absence of the lectin were liberating virus into the supernatant medium, the M or matrix protein was found in association with the plasma membrane of the lectin-treated cells.

The removal of the lectin from the cells with α-methyl-d-glucoside 3 h after infection was followed by the immediate release of mature virus particles. The rate of virus release from these cells was the same as that from cells infected in the absence of the lectin. Addition of cycloheximide, an inhibitor of protein synthesis, immediately after α-methyl-d-glucoside treatment of the cells did not alter the rate of virus production, suggesting that the proteins required for virus synthesis were available in the lectin-treated cells and that virus assembly took place without further protein synthesis on removal of the lectin.

The structural polypeptides of five bunyaviruses, snowshoe hare, Lumbo and La Crosse viruses (members of the California encephalitis subgroup of bunyaviruses), Bunyamwera and Main Drain viruses (members of the Bunyamwera subgroup of bunyaviruses), have been compared by polyacrylamide-SDS gel electrophoresis. Each virus was found to possess three major structural polypeptides, two glycoproteins (G1 and G2), and one nucleocapsid protein (N). Although the sizes of the G1 polypeptides (mol. wt. approx. 115 × 103) and G2 polypeptides (mol. wt. approx. 38 × 103) of the five viruses were found to be essentially similar, the sizes of the N polypeptides of the various viruses differed (mol. wt. range 19 to 24 × 103). The RNA genomes of four bunyaviruses (snowshoe hare, La Crosse, Bunyamwera and Main Drain) have also been compared. Each virus has three RNA species of mol. wt. approx. 3 × 106, 1.9 × 106 and 0.4 × 106. Minor size differences were observed for the smallest RNA species of the four viruses (mol. wt. range 0.34 to 0.50 × 106). For snowshoe hare virus the RNA segments have a 5′ sequence of pppAp … which suggests that the RNA is linear and not circular.

Some properties of Drosophila C virus (DCV), a non-occluded isometric virus, have been studied. The virus particles were 30 nm in diam., their sedimentation coefficient was 153S and their buoyant density was 1.34 g/ml in caesium chloride in the pH range 7 to 9. These particles contained about 31% ribonucleic acid (RNA) and 69% protein. The reaction of formaldehyde with DCV particles suggested that the RNA in situ is single-stranded. The infectivity of DCV was stable at pH 3. The virus capsid contained three major polypeptides with mol. wt. of 31000, 30000 and 28000, and two minor components of mol. wt. 37000 and 8500.

Virus preparations also contained a small number of infective particles 24 nm in diam. banding at a density of 1.44 g/ml. Preliminary results indicated that these heavy particles probably correspond to the dense particles recently reported in several vertebrate picornaviruses.

RNA extracted from DCV was single-stranded and infectious. Its mol. wt. was calculated to be approx. 3 × 106. It is proposed that this virus be included in the enterovirus group. The cryptogram of the virus is R/1:3.0/31:S/S:I/O.

The synthesis of proteins has been studied in tobacco protoplasts infected with cowpea chlorotic mottle virus (CCMV). Three new proteins were found with mol. wt. 19000 (P1), 34000 to 36000 (P2) and 100000 (P3). Protein P2 reached a maximum steady level about 16 to 20 h after infection; it was not a precursor of P1, which is virus coat protein. P3 appeared later than the other two proteins, reached its maximum about 44 h after infection and then declined. P2 and P3 were localized in the 1000 g and 20000 g pellets of a cell homogenate, in contrast to P1 (coat) which was largely found in the supernatant fraction after centrifuging at 20000 g for 30 min. The three proteins account for about 60% of the tripartite genome of CCMV.

Repeated passage of the ‘Chandler’ strain of scrapie in female golden hamsters using the intracerebral route of inoculation reduces the minimum incubation period to 60 days, about half of the minimum incubation period so far found in any of the mouse models of scrapie. The infectivity titres in brain in the clinical stage of the disease are considerably higher (> 8.0 -log10 LD50 i.c. units/0.05 g) than those found in mouse scrapie. The biological characteristics of this model of hamster scrapie are reported, including the effects on incubation period of route of inoculation, dose of agent, sex of hamster, ambient temperature (hibernation) and splenectomy. Some general and specific applications of this experimental model of scrapie are discussed.

Vesicular stomatitis virus (VSV) induces polykaryocytes in rat embryonic fibroblasts transformed by the Prague strain of Rous sarcoma virus (XC cells). The cell fusion requires the uncoating of the virus in the cell, the synthesis of normally structured G and M proteins and their incorporation into the cell membrane. The synthesis of fully infectious virus is unnecessary. In addition to these antigens, a special yet undefined constitution of the host membrane is also important. With thermosensitive mutants non-defective for G and M antigens, cell fusion is much more extensive at the non-permissive temperature (39.6°C) than at the permissive one (31°C). The importance of the two antigens is also shown using rifampicin-sensitive mutants. We postulate that these two antigens induce in the cell membrane an imbalance in the distribution of phospholipids which then diffuse through membrane junctions to surrounding cells, provoking thereafter the cell fusion.

Palisade parenchyma protoplasts isolated from the tobacco varieties Samsun and Samsun NN were inoculated in vitro with cowpea mosaic virus (CPMV). By fluorescent antibody staining it was demonstrated that about 75% of the protoplasts became infected. Poly-l-ornithine was required for infection. The cytopathic structures shown previously to be characteristic for CPMV-infected cowpea cells were also produced in CPMV-infected tobacco protoplasts. The optimum inoculum concentration for CPMV was 4 to 20 µg/ml. Compared with CPMV multiplication in cowpea protoplasts and tobacco mosaic virus multiplication in tobacco protoplasts, the growth of CPMV in tobacco protoplasts had a longer lag period but the virus concentrations in all three kinds of infection finally reached similar values. The difficulty with which tobacco leaves are infected with CPMV contrasts with the ease of infection of tobacco mesophyll protoplasts.

It has been found by immune electron microscopy that rotavirus-infected faeces, calf or human, contain an antigenic subunit associated with the inner of the two virus capsids. This internal component represents the group specific antigen for the rotavirus group and the subunit reacts with both homologous and heterologous antiserum. It can therefore be used in diagnostic tests and in this paper its use as a reagent for immunodiffusion is described.

Encephalomyocarditis (EMC) virus RNA, selected by its affinity for oligo(dT)-cellulose, contains poly(A) of size: (i) about 14 nucleotide residues long, based on the percentage of radioactivity in the RNA resistant to digestion by a mixture of pancreatic and T1 RNases; (ii) about 15 residues long, as measured by the ratio of the amount of terminal adenosine to internal adenylic acid in isolated poly(A); and (iii) in the range 12 to 45 residues, the majority of tracts being about 16 to 18 residues long, based upon electrophoretic mobility on polyacrylamide gels using poly(A) molecules of known size as mol. wt. markers.

The poly(A) appears to be located at the 3′-terminus of the virus genome since the tract, liberated by digestion with a mixture of pancreatic and T1 RNases, was shown by compositional analysis to contain a non-phosphorylated 3′-terminus and only adenine residues. The size heterogeneity in the poly(A) tracts revealed by gel electrophoresis is also consistent with a terminal location.

Comparison of our data for EMC virus with published data for other picornaviruses suggests that the sizes of poly(A) tracts in polio- and Mengovirus RNA have been overestimated; poly(A) tracts in cardioviruses appear to be smaller than those in poliovirus; the minimum size of poly(A) required for full infectivity of picornavirus RNA has also been overestimated; a tract of at least 13 adenine residues long is required for full infectivity of EMC virus RNA.

The ‘b’ protein components specific to virus infected tobacco leaves (Gianinazzi, Martin & Vallée, 1970) can be partially purified by preferential extraction at pH 2.8. Evidence is presented that they are rich in aromatic amino acids. Results of treatment of the proteins with SDS and subsequent separation by gel electrophoresis in the presence of SDS suggest that b1, b2 and b3 are composed of the same monomer of mol. wt. about 16000 whilst b4 is composed of a monomer of mol. wt. about 29000. By purifying and concentrating the soluble protein extracts of water inoculated leaves, further evidence is provided that the ‘b’ proteins are not normal constituents of healthy tobacco leaves.

In vitro cultured mouse peritoneal macrophages are inefficient host cells for both alpha and flaviruses tested. Production of infectious virus ceased 6 to 24 h after infection, except for virulent and avirulent SF strains. This limited growth is unrelated to interferon production. No correlation was found between the LD50 of different virus strains for mice and the virus yields of in vitro infected macrophages therefrom. When macrophages from SF immunized mice were inoculated in vitro with SF or EEE, a cytopathic effect occurred, while the multiplication of SF, but not of EEE, was decreased. Virus multiplication in proteose peptone induced macrophages was enhanced for SF strains only. These results are discussed in relation to the virulence of several arboviruses for mice.

Scanning electron microscopy of AKR cells chronically infected with AKR mouse leukaemia virus revealed that the number of budding virions was greatly increased in interferon-treated cells. These results, together with previous biochemical findings, suggest that in this system, interferon inhibits a late stage of virus assembly or release.

Multinucleated giant cell formation in a clone of BHK-21 cells, BHK-21-528, was tested with certain arboviruses. Eleven out of 19 viruses tested, Chikungunya, Getah, Sagiyama, Sindbis, Western equine encephalitis, St. Louis encephalitis, Japanese encephalitis, West Nile, Bunyamwera, Germiston and California encephalitis virus induced cell fusion after infection. All cases where giant cells were observed during the experiments, involved the B type cell fusion (fusion from within).

A method was established to obtain a high yield of Epstein-Barr virus (EBV) DNA for nucleic acid hybridization studies on latent virus DNA in transformed cells. Superinfection of Raji cells with EBV concentrated from HRI cell cultures produced a 600-fold higher yield of EBV DNA than direct isolation of EBV from HRI cell cultures. The virus DNA thus prepared from Raji cells superinfected with EBV was radioisotopically and spectrophotometrically pure and served as a satisfactory template for the preparation of cRNA specific to EBV DNA.

Using pairs of smooth and rough forms of Enterobacteriaceae, six smooth-specific bacteriophages were isolated from sewage and another was obtained from Dr Hedda Milch, Budapest. Upon incubation of the individual extracted (smooth) host cell wall lipopolysaccharides with the homologous purified viruses, liberation of reducing groups (i.e. of about di- to nonasaccharides) was observed in four cases, indicating the action of glycanases — but no liberation of acetic acid, indicating the absence of esterase activity. Under the electron microscope, all phages were seen to exhibit Bradley group B or C morphology and to carry tail spikes.

Iodination of the surface of poliovirus and its artificial empty capsid demonstrated predominant labelling of polypeptide VP 1 on the intact particle and an increased labelling of polypeptide VP 2 on the artificial empty capsid.

The effects of actinomycin D on the production of genomic RNA, messenger RNA and protein in measles virus-infected cells were investigated. It was found that whereas high concentrations of the drug inhibited genome synthesis, the production of messenger RNA was unaffected and in some cases enhanced. These effects were shown, at least in part, to be due to a secondary effect of the drug which caused an inhibition of virus-specific protein synthesis.