- Volume 17, Issue 2, 1972

Chicory yellow mottle virus (CYMV), an isometric plant virus containing RNA, is degraded when infected tissues or purified virus suspensions are frozen and thawed. Virus preparations obtained from frozen tissues contain substantial proportions of empty capsids (top component) which increase in relation to the length of time for which plant material is kept frozen before processing. Virus suspensions frozen in vitro after purification behave similarly in that, after exposure to −25 °C, they are almost totally dissociated into RNA and protein shells. Most of the dissociation takes place within the first 10 min of freezing at −25 °C. Lower temperatures (−78 °C and −196 °C) are less effective in inducing virus dissociation.

The extent of dissociation varies with the chemical composition of the suspending medium. Na-acetate and NaCl confer complete protection from freezing injury, whereas no protection is given by water and (K-Na)phosphate buffer plus NaCl. (K-Na)phosphate alone, (Na-Na)phosphate and KCl afford only partial protection.

It is possible that several factors, i.e. increased molarity and lowering of pH of solutions at the eutectic point and removal of water associated with protein macromolecules have a bearing in explaining the influence of the various chemicals.

We have investigated the capacity of the avian adenovirus CELO to induce interferon in primary chicken embryo fibroblasts. The multiplicity of infection appeared to be an important factor in the induction of interferon.

U.v. irradiation of the virus at 1 or 2 × 104 ergs/mm2 completely inhibited virus replication, but had no effect on interferon synthesis. Interferon production was decreased by 50% when CELO virus was irradiated with a dose of 5 × 104 ergs/mm2.

Treatment of the infected cells with cytosine arabinoside inhibited new DNA synthesis and virus replication, but had little effect on interferon production. Cytosine arabinoside, also, did not inhibit interferon production in chicken cells infected with human adenovirus type 7. It can be concluded that at appropriate multiplicities of infection the parental CELO virus was responsible for interferon induction.

Although infective Newcastle disease virus (NDV) did not produce interferon in chick cells, brief heat treatment converted it to an inducer. Experiments in which mixtures of heated and unheated virus were used to induce interferon showed that a substance was produced in infected cells that inhibited interferon formation. Heat treatment of NDV caused the same rate of loss of infectivity and of virus particle RNA polymerase activity, and it was concluded that polymerase activity was essential for virus infectivity. It was also shown that some polymerase activity still existed in heat-inactivated virus able to induce interferon. Appropriate inactivation of the virus by u.v. irradiation or by treatment with β-propiolactone or at pH 2.5, also made the virus into an inducer of interferon, and in each case some virus polymerase activity was present in the inactivated particles. Both u.v. irradiation and β-propiolactone inactivated infectivity more rapidly than polymerase activity, while pH 2.5 treatment had the reverse effect.

The tobacco necrosis satellite viruses SV1 and SV2 interfere with one another’s replication, and the larger the dose of the interfering satellite the greater the degree of interference produced. The amount of interference also depends on the strain of tobacco necrosis virus (TNV) used as helper. Suppression of SV2 by SV1 is greater than that of SV1 by SV2, although SV2 is the more infective. SVc differs serologically from SV1 and SV2 no more than these two differ from each other, but it needs a different strain of TNV for replication. Nevertheless, there is no interference between SVc and either SV1 or SV2. The interference between SV1 and SV2 takes place in the first 2 h after inoculation. Satellite viruses inoculated 3 days after TNV do not interfere with one another provided the TNV strain is one that aids their multiplication. The results suggest that SV1 and SV2 compete for an early metabolite.

Although previous studies have shown that Colorado tick fever virus occurs only transiently in the serum, but persists for prolonged periods in the blood cell fraction of man and experimental animals, the exact nature of this cell-associated viraemia was unknown. Using virus isolation, fluorescent antibody staining, histological and electron-microscopic techniques, we have shown that persistent viraemia is predominantly due to virus contained within erythrocytes. Erythrocyte precursor cells are presumably infected in the bone marrow and are subsequently released into the blood stream, persisting for prolonged periods despite the presence of serum antibody. Virus either continues to replicate slowly or is stabilized and remains viable within the erythrocyte. Colorado tick fever virus may be a useful model virus for further study of the mechanism of blood cell infection, with implications for other arboviruses or viruses believed to be involved in leukaemia or other haematological disorders.

A study was made of the virus-specific proteins present in Krebs 2 ascites cells infected with encephalomyocarditis (EMC) virus. About 15 virus-specific polypeptides, mol. wt. varying from 13000 to 147000, were detected by acrylamide gel analysis of extracts from infected cells briefly pulsed with [14C]-amino acids. Three of the components were probably capsid proteins, while the fourth capsid protein (7300 mol. wt.) was not detected in the infected cell and possibly arose after maturation by cleavage of the largest capsid protein. Pulse-chase experiments showed that only two medium-sized (mol. wt. 36000 and 54000) and some smaller (mol. wt. 13500 to 17000) non-capsid proteins were stable in vivo; the remaining larger non-capsid proteins lost radioactivity during the chase with cold amino acids, and were probably intermediates in the formation of smaller polypeptides by some post-translational cleavage process. Various methods were employed to block this cleavage process, utilizing elevated temperatures, protease inhibitors and amino acid analogues.

Pulse-chase experiments with [3H]-uridine were performed with glucosamine-treated chick cells infected by RNA viruses. It was established that in cells infected by fowl plague virus both virus RNA and the RNA with the complementary base sequence were metabolically stable. Newcastle disease virus-specific RNA was also metabolically stable in chick fibroblasts. In cells infected by Semliki Forest virus, radioactivity was not lost from the virus-induced 26 S RNA, while the activity of the partially RNase-resistant 20 S RNA could be chased to RNase-sensitive RNA of high mol. wt.

Replication of RNA tumour viruses appears to involve a DNA intermediate (provirus), as was postulated by Temin (1964) and is suggested notably by the discovery of virus-associated RNA-dependent DNA polymerase able to transcribe virus RNA into DNA (Baltimore, 1970; Duesberg & Canaani, 1970; Green et al. 1970; Mizutani & Temin, 1970; Spiegelman et al. 1970) and the recent finding, in RSV-transformed cells, of infectious DNA able to prime virus production in permissive cells (Hill & Hillova, 1971, 1972; Montagnier & Vigier, 1972).

New rifamycin derivatives have now been synthesized which are powerful inhibitors of the RNA-dependent DNA polymerase of murine sarcoma virus (MSV) (Gurgo, Ray & Green, 1972). Especially active are the 3-oxime derivatives such as AF/05 and AF/013 which also have been shown to inhibit DNA-dependent RNA polymerase by preventing initiation of the RNA chains (Butterworth, Cox & Chesterton, 1971; Meilhac, Tysper & Chambon, 1972).

Mammalian cells are able to repair damage to their DNA by a non-semiconservative mode of replication (repair replication, Rasmussen & Painter, 1964, 1966). Mengovirus and Newcastle disease virus are two cytocidal RNA viruses that inhibit semiconservative DNA replication soon after infection (Ensminger & Tamm, 1970a). Neither inhibits non-S phase or unscheduled DNA synthesis (Hand & Tamm, 1971), which probably represents a repair process (Painter & Cleaver, 1969). We have now examined repair replication in cells infected with mengovirus or Newcastle disease virus using more direct methods of measurement. If damaged DNA is labelled during repair with [3H]-bromodeoxyuridine (BrdUrd), the repaired regions are too short to produce a shift in density of the DNA and thus sediment as DNA of normal density when analysed by isopycnic sedimentation in caesium chloride (Pettijohn & Hanawalt, 1964; Cleaver, 1969b).

One of the important biological activities of EB virus (Epstein, Achong & Barr, 1964) is the transformation of human leucocytes in vitro (Henle et al. 1967; Pope, Horne & Scott, 1968). Transformation in this context is defined as morphological change in a proportion of the cells, associated with continued cell proliferation leading to establishment of a lymphoid cell line. The transformation factor was identified as EB virus by showing that it had some of the general properties of herpes viruses and was neutralized specifically by human sera containing antibody to EB virus (Pope, Horne & Scott, 1969).

The all-or-none nature of the transformation response to EB virus makes it a suitable basis for assay of virus infectivity. Some experiments have been reported of titration of EB virus by transformation assay in 1 ml leucocyte cultures (Pope et al. 1969, 1971) and of the distribution of infectivity in a sucrose density gradient (Walters & Pope, 1971).

One of the first successful demonstrations of the presence of rhinoviruses in vitro was by the interference technique (Hitchcock & Tyrrell, 1960). The use of this technique became unnecessary following the subsequent finding that rhinoviruses readily produced c.p.e. in cell cultures if maintenance medium contained a low bicarbonate concentration and cultures were rolled at 33 °C (Tyrrell & Parsons, 1960). The development and use of human diploid cell strains (Hayflick & Moorehead, 1961) provided even more sensitive systems for the recovery of viruses of this group (Hamparian, Kettler & Hilleman, 1961).

Recent evidence, however, suggested that not all rhinoviruses are cytopathogenic even when cultivated under these ‘common cold’ conditions. Hoorn & Tyrrell (1966) described a strain of rhinovirus isolated in organ cultures of human embryo trachea which could not be adapted to grow in cultures of human diploid cells.

During studies of respiratory illnesses in military recruits, three virus strains were isolated in human embryonic kidney (HEK) cells by use of the interference technique.

Determinations of the efficiency of the process of mechanical inoculation of tobacco mosaic virus (TMV) have usually been made with local lesion hosts. To our knowledge, the greatest efficiencies were reported by Steere (1955), who found that one lesion was produced for every 50000 characteristic particles in the inoculum, and by Schramm & Engler (1958), who reported that the minimal TMV concentration detectable on the local lesion test plant Nicotiana glutinosa was 10-12 to 10-13 g/ml (about 1500 to 15000 particles/ml). In the latter paper, Schramm & Engler (1958) also reported that 50% infection of N. tabacum cv. Samsun, a plant which reacts with systemic symptoms, could be obtained by rubbing leaves with 5 ml of an inoculum which contained 10-16 g/ml of TMV (about 7.5 particles/plant). This indicated that the minimal infective dose for systemically infectible hosts might be less than that for local lesion hosts.