- Volume 6, Issue 1, 1970

A system is described in which rhinovirus 2 may be grown in suspension cultures of L 132 cells. The virus adsorbed to the cells more efficiently at 33° than at 4°, probably by means of specific trypsin-sensitive lipoprotein receptors on the cells. Virus penetration demonstrated using a potent virus antiserum was temperature-sensitive and rapid at 33°. During single-cycle virus growth at 33° the latent period was about 4 hr and maximum virus yields were obtained about 10 hr after infection. Addition of 30 mm-MgCl2 in the medium enhanced virus release from the cells but did not affect the total virus yield. Highest virus yields were obtained at pH 7·7 and a temperature of 33°. Lower temperatures increased the latent period. At higher temperatures up to 37° the latent period was shorter and the virus titre ceased to increase earlier. At 39°the latent period was longer than at 33° and there was a severe inhibition of virus production. Shift-up and shift-down experiments indicated that the temperature-sensitive event or events at 39° occurred late in the growth cycle.

A quantitative analysis was made of the inactivation of MS 2 bacteriophage. Data were obtained relating the fraction of MS 2 surviving to the number of rabbit antibody molecules bound, from the minimum of one antibody molecule adsorbed per virus particle to a maximum of 80 to 85 per virus particle. Between 20 and 40 binding sites on the phage particle were critical, the covering of any one of which resulted in inactivation. In extreme antibody excess, approximately 10−4 of the phage population survived. In this antibody range, a maximum of 80 to 85 antibody molecules were bound to a phage particle leaving 95 to 100 antigenic sites free, explaining the small but constant probability of survival in extreme antibody excess (the persistent fraction).

The nearest neighbour frequency pattern analyses of the DNAs of a minute virus of mice, Kilham rat virus and H-1 virus were very similar to each other. They were also similar to the pattern of the host cell DNA, apart from differences related to structure, the virus DNAs being single-stranded and the host double-stranded. All three DNAs exhibited the low frequency of the dinucleotide sequence CpG previously found in mammalian DNAs and in the nucleic acids of small mammalian viruses. The sizes and densities of the virus particles were measured, and the molecular weight of H-1 DNA was determined.

Retinal cultures from newborn rabbits were infected with herpes simplex virus and neuro- and non-neuroadapted strains of vaccinia virus. Herpes simplex virus gave rise to characteristic nuclear changes of mesenchymal cells, and rod cells and a rapid destruction of neurons. The vaccinia virus strains all caused development of cytoplasmic inclusion bodies in the mesenchymal cells, which then degenerated. Changes in neuroectodermal cells were observed relatively late and might be secondary to the deterioration of mesenchymal cells. The implication of these findings for the study of the problem of selective cellular susceptility of the central nervous system to viruses is discussed.

Ten out of 29 temperature-sensitive (ts) mutants of poliovirus lost infecti-vity in vitro at 45° more rapidly than did ts +, and therefore had structural protein defects. This division into structural protein and non-structural protein defectives accorded very closely with their position in the genetic map and with previous tests for maturation defects, enabling the region of the genetic map specifying virus structural protein to be defined. Not all mutants mapping in this region were thermolabile, however. Three out of five ts + revertants from the thermolabile mutants were thermostable and so their thermolability and ts characters almost certainly resulted from mutations in the same gene, i.e. the ts defects of these thermolabile mutants were confirmed as residing in structural protein. Four of the thermolabile mutants were sufficiently unstable at the restrictive temperature to explain their ts character. Three mutants mapping in the structural protein region produced only small amounts of RNA-containing particles and so their defect may act before maturation. Of five structural protein mutants examined, none showed any antigenic difference from ts +. Marked covariation was found between cystine requirement for growth and structural protein defects suggesting that cystine requirement is a property of the structural protein of the virus.

Sixteen ts mutants were isolated from influenza virus type A0, strain wsn, after treatment with either fluorouracil or hydroxylamine. Cells doubly infected with several pairs of ts mutants yielded a high proportion of wild-type virus, probably because of genetic recombination. Recombination frequencies with particular pairs of mutants were very variable, even in replicate tubes of the same experiment, until infected cells were treated with receptor-destroying enzyme (rde) after adsorption of virus. Day-to-day variation between experiments could not be eliminated, but was allowed for by including a standard cross in all experiments, and relating recombination frequencies to the values obtained in this cross. Standardized recombination frequencies were then reproducible and characteristic of each pair of mutants.

The recombination frequencies thus obtained allowed certain of the mutants to be placed in a sequence with additivity between the intervals, suggesting that influenza virus may have a linear genetic map.

The nucleic acid of rubella virus was shown to be RNA by the incorporation of [3H]-uridine into virus particles, and by the presence of ribonuclease-sensitive material, sedimenting mainly at 38 to 40 s, in sodium dodecyl sulphate extracts of purified virus. There were at least two virus-specific RNAs in cytoplasmic extracts of BHK21 cells infected with rubella virus. One, possibly the structural RNA of the virus, was sensitive to ribonuclease and sedimented at 38 to 40 s like the RNA extracted from virus particles. The other had a heterogeneous sedimentation pattern with main peak at 20 s. It was partially resistant to ribonuclease and might be the replicative intermediate form of the RNA of rubella virus.

The lytic bacteriophage ϕW-14 was isolated from sewage using Pseudomonas acidovorans no. 14 as host. The phage had an icosahedral head some 85 nm. in diameter and a contractile tail some 140 nm. long. ϕW-14 formed plaques on only a few strains of P. acidovorans. The phage gave biphasic absorption kinetics, with an adsorption constant of 1·9 × 10−9 ml./min. The latent period was 60 min. and the burst size was about 300. The burst size was dependent upon culture age. The k u.v.. for inactivation of ϕW-14 was 4·35 min.−1. P. acidovorans was shown to possess a potent photoreactivating system. The heat of inactivation of ϕW-14 was calculated to be 75,700 cal./mole. The phage gave biphasic thermal inactivation kinetics at 55° and 60° but not at 65°. The phage mutated spontaneously to a different plaque type. This mutation affected the adsorptive properties, the thermal sensitivity and the burst size of the phage.

Both fowl plague and Newcastle disease viruses failed to produce interferon in chick-embryo cells because of the inhibitory effect of infection on host-cell protein synthesis. When this inhibitory effect was destroyed by ultraviolet irradiation or by hydroxylamine inactivation, interferon was formed. No virus RNA synthesis could be detected in the interferon-producing cells and it was concluded that the virus single-stranded RN A was the stimulus for interferon formation.

The sedimentation properties of adenovirus 12 induced antigens were studied by rate-zonal centrifugation in linear sucrose gradients. Adenovirus 12 hexon and fibre antigens were estimated to have molecular weights of 22–25 × 104 and 6–7 × 104 daltons, respectively. In addition, two species of T antigen were separated by rate-zonal centrifugation. The two T antigen species had molecular weights of 8–9 × 104 and 4–5 × 104 daltons and were distinct from adenovirus 12 hexon and fibre antigens. Results were identical whether the adenovirus 12 antigens were extracted from infected H.Ep-2 or HEK cells. Similar studies of tumour antigen from transplanted adenovirus 12 induced tumours in hamsters and CBA mice, and two hamster cell lines grown in vitro, revealed the presence of two species of tumour antigen. Molecular weight estimates for the two species of tumour antigen indicated that these antigens were similar in molecular weight to the T antigens. Differences in the relative proportions of the two species of antigen in tumour extracts and in T antigen extracts were found. Treatment of tumour extracts with sodium deoxycholate or ribonuclease did not alter the sedimentation properties of the tumour antigens.

Two components, interferon and residual particles of input virus, contributed to the interfering activity recoverable from mouse embryo cultures treated with molluscum contagiosum virus. Physical and biological techniques were used to distinguish and allow the independent biological assay of each of these components. Cultural conditions greatly affected the induction of interference by molluscum virus. Under optimal conditions, interferon was first detected in treated cultures 6 hr after virus adsorption, reached a maximal value at about 18 hr and then declined rapidly. Virus itself, distinguished from interferon by its acid lability, sedimentability and failure to induce interference in L cells, declined rapidly in activity, during the early hours after adsorption to mouse embryo cells, to about 25% of that calculated to have been adsorbed. Although a direct correlation was not established between the amount of interferon produced and the decline in recoverable virus, a substantial part of this decline was interpreted as a significant biological event akin to ‘eclipse’ of infective virus in a permissive host.

Non-crystalline inclusions, referred to as X-bodies, developed in leaf-hair cells of Nicotiana clevelandii infected with a RNA-producing defective isolate (cam/df) of tobacco rattle virus but not in those infected with the nucleo- protein particle-producing strain (cam) from which cam/df was derived. The X-bodies appeared 3 days after inoculation; some persisted for at least 10 weeks. They were digested by pronase and were rich in RNA, which was protected by proteinaceous material from digestion by pancreatic ribo- nuclease; they did not contain virus coat-protein detectable by fluorescent antibody tests. Electron microscopy of thin sections showed that mitochondria in infected cells developed two abnormalities—peripheral membranous sacs and membrane bounded vacuoles—and then aggregated, together with ribosomes and material containing small darkly staining granules, to form small X- bodies. These fused to form larger X-bodies, in which the mitochondria were further modified and eventually became barely recognizable. Finally, the contents of the X-bodies became increasingly amorphous, and the bodies disintegrated.

X-bodies also developed in leaf-hair cells infected with the virus particle- producing strain prn, but were less common and mostly persisted for only a few days. These X-bodies were formed in the same way as those produced by cam/df, but contained in addition small aggregates of virus particles resembling the aggregates found in the cytoplasm in other parts of the same cells. The frequency of formation, and persistence, of X-bodies induced by these tobacco rattle viruses paralleled the severity and persistence of their macroscopic effects on N. clevelandii. The possibility that mitochondria are sites of synthesis of tobacco rattle virus RNA is discussed.

The intact particles and nucleocapsids of mumps, Sendai and measles viruses are of closely similar appearance, size and structure. The intact particles are about 150 nm. in diameter. The filamentous nucleocapsids have a modal length of about 1·1 µm., and are constructed of subunits arranged as a single start helix of pitch 5·0 µm. for Sendai virus, and about 6·0 µm. for mumps and measles viruses. A (helical) projection of the structure of the Sendai nucleocapsid calculated from an electron micrograph showed that the structural subunits are hour-glass-shaped and are arranged in the helix with their long axes inclined at an angle of about 60° to the long axis of the particle. There are probably 11 or 13 subunits in each turn of the basic helix. Optical diffraction patterns of electron micrographs of mumps and measles nucleocapsids show that they have closely similar structures.

The viral inhibitors guanidine hydrochloride and 2-(α-hydroxybenzyl)- benzimidazole (HBB) were used to study the relationship between lysosomal enzyme release and the development of poliovirus-induced cytopathic effect (CPE) in HEp-2 cells. Lysosomal enzyme release, the development of CPE and virus replication were inhibited or delayed if these antiviral agents were added up to 2 to 3 hr after infection. When added later the agents were no longer effective. It is suggested that a virus-induced protein produced during 3 hr after infection is responsible for lysosomal enzyme release.

SUMMARY

The effect of different doses of viral RNA extracted from avian myeloblastosis virus was tested on RIF-free chick embryo cells. The results showed that the number of cells with positive gs-antigen was probably related to the dose of RNA. The specificity of viral RNA action was shown by a fluorescent antibody method and by the use of ribonuclease.