- Volume 3, Issue 2, 1968

Freeze-dried preparations of the scrapie agent and of 5 strains of virus were used to determine the extent of protection afforded against ionizing radiation by anoxia. The five viruses tested were bacteriophages T1 and T2 (DNA) and µ2 (RNA); and the animal viruses herpes simplex (DNA) and yellow fever (RNA). Oxygen enhancement ratios (measured in terms of doses required to give the same effect in the absence and presence of oxygen) varied from about 1.4 for the scrapie agent to 2.5 for yellow fever virus. With the viruses there was no obvious correlation of oxygen enhancement ratio with type or size of nucleic acid core.

‘Target sizes’ of the viruses were calculated from the doses required to give an average of one lethal event per particle. These agreed well with independent estimates of size of nucleic acid core; except that for bacteriophages T1 and T3 the calculated molecular weight was 0.3 of that determined by Bresler et al. (1967). Results with the scrapie agent confirmed the previous estimate (Alper, Haig & Clarke, 1966) of a molecular weight of about 1.5 × 105 daltons.

A non-occluded virus of Galleria mellonella was purified using a chloroform+butanol treatment followed by sucrose gradient centrifugation. Purified virus and top component were produced in proportions of 1:3; their diameters were approximately 200 Å, and their sedimentation coefficients 119S and 58S respectively. The virus had an approximate DNA:protein ratio of 37:63.

Virus and top component were serologically identical in gel diffusion tests and both were different antigenically from insect proteins with sedimentation coefficients of 2 to 3S, 17S, 30S and 70 to 75S, present in both healthy and infected larvae. The amino acid composition of the protein from the virus and top component was very similar, and differed from that of the 17S component. No soluble antigens were detected in the extracts of infected larvae.

When chick embryo cells were infected with Semliki Forest virus, six new proteins were formed. All six were first detectable 3 hr after infection and were present in the same proportions thereafter. Their distribution between different subcellular fractions was correlated with the presence of viral components in these fractions. Two of the proteins were identified as those of the complete virus particle; one was associated with the lipoprotein envelope and the other with the ribonucleoprotein core.

Biological and serological properties of a simian agent (strain mk 5) were investigated and it was shown to be a type 1 foamy virus. Grids prepared from cells lysed with sodium phosphotungstate were satisfactory for demonstrating the virus and, using this technique, the standard virus particle was found to be 1100 Å in diameter. It was composed of an envelope with 125 Å projections with an internal component 725 Å in diameter. Variations in the morphology of the virus are described. The structure of foamy agents types 1 to 3 was indistinguishable from that of mk 5.

The production of interferon by chick embryo cells infected with Semliki Forest virus and incubated at 42° is dependent on a period of incubation at 36°. During incubation at 36° virus-directed RNA synthesis occurs and it is suggested that the temperature-sensitive event in interferon production is an early stage in virus multiplication rather than a process controlled by the cell genome.

Serological cross-reactions were detected between four avian influenza viruses and a human A 2/57 virus and between an avian (TURKEY/CANADA/63) and an equine (EQUI 2/MIAMI/63) strain. The reactions were detected by strain-specific complement fixation tests and neuraminidase inhibition but not by haemagglutination-inhibition. Specific antiserum to the neuraminidase of an A 2/57 virus also reacted with the four avian viruses showing that it was the neuraminidase antigen on the avian and human A 2 virus that were antigenically closely related. The possibility that these viruses may have arisen by recombination between human and avian influenza viruses in nature is discussed.

A direct experimental comparison of the Tipula iridescent virus and the Sericesthis iridescent virus has shown them to be very similar but not identical. After extensive purification the viruses were differentiated by electrophoretic mobility, tryptic peptide patterns, serological cross-reactivity, and pathogenicity in a group of host insects. The viruses were identical in gross chemical composition and in a number of physical properties. A particle weight of 1.2 to 1.3 × 109 daltons was calculated by three methods. A slower sedimenting top component consisting of empty protein shells was found during the purification of each virus; similar empty shells could be produced by succinylation or formic acid-diphenylamine treatment of whole virus.

Larvae of the small tortoiseshell butterfly, Aglais urticae (Lepidoptera: Nymphalidae), were infected orally with a nuclear polyhedrosis virus. Sections of larvae which had been killed and fixed at intervals after infection were examined by light and electron microscopy. Typical virus development was observed in the fat body cells. However, in the nuclei of infected columnar cells of the mid-gut no incorporation of the virus particles into the polyhedral inclusion body was observed although both virus particles and crystalline polyhedron protein were present. Subsequently virus particles were found in the cytoplasm of these cells between the nucleus and the basal membrane. It is proposed that this unusual virus development is significant in the pathway of infection of a nuclear polyhedrosis virus.

None of four isolates of Olpidium brassicae (Wor.) Dang. (Olpidia 1, 2, 3 and 6) transmitted the original strain of satellite virus (sv 1). Two strains of satellite virus recently isolated (sv 2 and sv 3) were both transmitted, but sv 2 only by Olpidium 6 and sv 3 only by Olpidium 3. Hence different isolates of Olpidium seem to be specific vectors for different strains of satellite virus.

Heating high-titre preparations of reovirus particles for approximately 3 hr at temperatures of 52° to 53° converted the entire population to inner capsid components as monitored by electron microscopy. There was a tenfold loss in infectivity after this conversion. The residual infectivity could be accounted for only by the presence of the inner capsids, indicating that the outer layer of capsomeres is not mandatory for reovirus infectivity, but may be responsible for the stability of the complete virion.

At 25° all components of fowl plague virus were synthesized in chick embryo fibroblasts. The rate of formation was about a quarter of that at 37°, but the final yield was the same. Infectious virus, however, was not formed at 25°. If infected cells were preincubated at 25° for 20 hr and thereafter kept at 37°, a normal yield of infectious progeny was found. With longer preincubation at 25° a decreasing yield of infectious virus was found. This effect may have been due to the synthesis at 25° of a viral component responsible for the cytopathic effect which acts only at the higher temperature. Preincubation of infected cells at 37° for different times and further incubation at 25° revealed a virus-specific precursor synthesized early in the infectious cycle which was rate limiting at 25°.

The structure and infective process of a contractile Pseudomonas aeruginosa bacteriophage (PB-1) was studied in the electron microscope using negative staining and thin sections. The octahedral form of the phage head was demonstrated by comparing electron micrographs with models. Sections of infected cells showed condensation of the nucleoplasm up to 30 min. after infection followed by its gradual dispersion. The first intracellular phage appeared 20 min. after infection; the individual particles were surrounded by clear regions. Lysis began 30 min. after infection. First, bulges appeared in the cell wall which then peeled off. The plasma membrane disintegrated, causing the cell to split open, releasing virus particles. The length of the phage nucleic acid was found to be about 24µ by electron microscopy. It contained double-stranded DNA as indicated by acridine orange staining.

The multiplication of variola virus in HeLa cells was examined under one-step growth conditions at 35° and at 40°. At 35°, after an eclipse of 10 hr, exponential growth proceeded for 10 to 12 hr with final yields of 200 to 500 pk.f.u. per cell; at 40° there was no growth of infectious virus. In contrast, vaccinia virus grew equally well at either temperature. The temperature-sensitive phase of the growth cycle was delineated by experiments involving temperature-shift. The first sensitive event was at 4 hr after infection, the time at which virus DNA synthesis normally begins at 35°, and the last was within 1 hr of the onset of virus maturation. Other features of the growth cycle at 40° were the absence of virus DNA production, the delayed appearance of the LS-antigen complex and the haemagglutinin, and the absence of any evidence of particle formation. Analysis of the temperature-shift experiments suggested that production of the LS-antigen complex and particle development were both directly involved in the late event.

The taxonomic position of the virus of transmissible gastroenteritis of swine is not yet clear. On the basis of ether sensitivity, size determination and electron microscopy, it has been suggested that it may be related either to the myxoviruses or to the herpesvirus group (Cartwright et al. 1962; Ristic, Sibinovic & Alberts, 1965; Sheffy, 1965; Okaniwa et al. 1966). Nikolsky et al. (1967) stated that particles of the virus isolated in the Ukraine, from pigs suffering from infectious gastroenteritis, are icosahedral with 5:3:2 symmetry and have 252 capsomeres, characteristics of the adenovirus group. However, the relationship between the viruses that have been isolated is not clear and it is possible that more than one agent is being described under the designation of transmissible gastroenteritis. Sheffy (1965) examined the effect of the metabolic inhibitors 5-fluorodeoxyuridine and 5-iododeoxyuridine on the multiplication of the Japanese sh strain of the virus and concluded that it contains RNA and belongs to the myxovirus group.

We present evidence that 1% (v/v) acetone is capable of inhibiting multiplication of vaccinia and rabbitpox viruses in chick embryo fibroblast cell cultures; and when infections with these viruses were treated with both acetone and N-methyl-isatin-β-thiosemicarbazone the extent of inhibition of virus multiplication was much greater than in experiments using each preparation separately.

Addition to agar overlays of acetone in concentration of 1% reduced by 1.3 to 1.5 log. the capacity of vaccinia and rabbitpox viruses to produce plaques in chick embryo fibroblast cultures. ‘Aceton zur Analyse’, DDR, VEB Laborchemie Apolda was used. The virus was adsorbed at 36° for 60 min., then agar overlay containing 1% acetone was added and the vials were immediately stoppered tightly with rubber stoppers. About the same degree of plaque formation by these viruses was observed upon addition to the agar of 1 µg./ml. N-methyl-isatin-β-thiosemicarbazone, which has a known inhibiting effect on the variola group of viruses (Bauer & Sadler, 1961; Appleyard & Way, 1966).

Human cells of different origins have been morphologically transformed by SV40. Among these are cells from newborn kidney (Shein & Enders, 1962), skin and buccal mucosa (Koprowski et al. 1962), embryonic lung (Ashkenazi & Melnick, 1963; Jensen, Koprowski & Pontén, 1963; Moyer, Wallace & Cox, 1964) and adult thyroid tissue (Rabson et al. 1962). This report describes transformation of human embryonic liver cells by SV40.

Cells were prepared from the liver of a human embryo of an estimated three months gestation. After one washing in phosphate buffered saline the liver was minced, trypsinized and seeded in 16 × 150 mm. tubes. The tubes were incubated at 37° in a stationary position. Monolayer cultures were inoculated with 0.1 ml. of undiluted SV40 virus. Cultures were initiated in Eagle’s basal medium with Hanks’s balanced salt solution (bicarbonate 0.35 g./l.), 10% calf serum, 100 u./ml. penicillin and 100 µg./ml. streptomycin.

Electron-microscopic studies have revealed intracytoplasmic virus particles and unusual intracytoplasmic and intranuclear fibrillar structures in BHK 21 and KB cells infected with Colorado tick fever (CTF) virus. This communication briefly describes these findings.

BHK 21 cells were grown in Eagle’s basal medium (BME) in Hanks’s balanced salt solution (BSS) + 10% foetal bovine serum. Fortified BME in Earle’s BSS containing 10% foetal bovine serum was used as the outgrowth medium for KB cells. The monolayers were grown in 8 oz prescription bottles and were infected with 0.5 ml. of the florio strain of CTF virus (106.5 TCD 50/ml.) in 10 ml. of maintenance medium. The maintenance medium for BHK 21 cells consisted of BME in Earle’s BSS and 3% foetal bovine serum. Leibovitz 15 (L-15) medium (Leibovitz, 1963) containing 2% inactivated foetal bovine serum was used as the maintenance medium for KB cells.

The transmissible agent of scrapie has not yet been characterized but it is commonly referred to as a virus (Andrewes & Pereira, 1967). However, on the basis of its resistance to acetylethyleneimine, Stamp et al. (1959) suggested that the agent might not contain nucleoprotein. Pattison (1965), reporting on the effect of formalin, expressed the opinion that the agent might not be a virus and now believes it to be, or to be associated with, a small basic protein (Pattison & Jones, 1967). In a study of the effect of ionizing and ultraviolet irradiation Alper, Haig & Clarke (1966) and Alper et al. (1967) suggested that the agent may lack nucleic acid. On the basis of these results and the inactivation of the scrapie agent with urea and phenol, Gibbons & Hunter (1967) have proposed a membrane hypothesis for its nature. Other workers (e.g. Adams & Caspary, 1967) still believe that the agent is essentially viral in character.

Several plaque tests for influenza viruses have been described in the past (Granoff, 1955; Ledinko, 1955; Gotlieb & Hirst, 1956; Henry & Youngner, 1957; Wright & Sagik, 1958; Zimmermann & Schäfer, 1959; Choppin, 1962; Lehmann-Grube, 1963; Suguira & Kilbourne, 1965). For various reasons none of these tests has found general acceptance as a routine procedure. Using chicken kidney cells, a plaque test was developed for virus n, an influenza A virus isolated from chicken (Babiker & Rott, 1968). Subsequently we used this system for other influenza viruses.

The viruses tested were type A, strain pr8, type A1, strain fm1, type A2, strain singapore/1957 and type B, strain lee. These strains were routinely propagated in chick embryos. The preparation of monolayers of kidney cells from 5- to 6-day-old chickens and the composition of the growth medium were described by Babiker & Rott (1968).