- Volume 1, Issue 4, 1967

Hybridization reactions between the DNAs of the six members of the ‘weakly’ oncogenic adenovirus group (types 3, 7, 11, 14, 16 and 21) show that they are closely related to each other, sharing 70 to 100 % of their nucleotide sequences. The ‘weakly’ oncogenic adenoviruses are but distantly related to ‘strongly’ oncogenic types 12 and 18, showing only 11 to 22% homology. Thus, two groups of oncogenic adenoviruses exist, differing remarkably in nucleotide sequence as well as in base composition and degree of oncogenicity.

An electron microscopic study was made of the plasma lactic dehydrogenase elevating virus of Riley et al. (1960) in cultures of mouse peritoneal macrophages. Four morphologically different forms of particle were seen: (1) large round particles, 510 Å in diameter, consisting of a nucleoid en-closed by dense double membranes (310 Å) and a less dense halo, which was occasionally seen to be bounded by a thin external membrane; (2) large elongated particles of similar morphology to type 1; (3) small round particles, 330 Å in diameter, very similar in appearance to the nucleoid of the large particle; (4) small rod-shaped particles, also surrounded by double membranes and varying in length between 640 and 1240 Å and in width between 200 and 380 Å. Similar particles were observed in pellets prepared by ultracentrifugation from viraemic mouse plasma and the medium of infected cultures.

Dolichos enation mosaic virus is a serotype of tobacco mosaic virus infecting leguminous plants; it produces a large proportion of particles shorter than 3000 Å, the accepted length of infective tobacco mosaic virus.

A new strain of dolichos enation mosaic virus isolated from a single necrotic lesion on French bean caused local and systemic necroses in French bean plants. It produced many more defective particles than the parent strain when infected plants were kept at 20° but not at temperatures above 32°. Both strains multiplied faster at 32° than at 20°, but the effect of increasing temperature was greater with the new strain. The effect on the new strain appeared greater when virus content was assayed hy infectivity tests than when it was assayed serologically, suggesting that infectivity per unit weight of virus was also greater at 32° than at 20°. No evidence was found that leaves infected with the new strain contained free infective RNA.

The ultraviolet absorption spectra of purified preparations of the new strain produced at 20° had a greater optical density at 280 mαt than at 260 mα, whereas the parent strain had greater density at 260 mα, showing that some of the particles of the new strain were without RNA. The type of particles produced by the two strains differed when purified preparations in 0·06 m- phosphate buffer at pH 8 were subjected to analytical ultracentrifugation in sucrose gradient columns or fractionation through agar columns. When produced in plants at 20°, the new strain consisted mainly of ring-like particles, virus protein, some free RNA and very few infective particles. By contrast, the parent strain consisted mainly of infective virus and broken particles of various lengths, of which a particle 400 Å long was plentiful enough to give a peak in the analytical centrifuge and a zone in sucrose gradient columns. The new strain produced in plants at 32 to 36° did not differ in appearance or infectivity from the parent strain. The distribution of particle lengths in sprayed droplets, measured in the electron microscope, confirmed the difference found by other means between the type of particle in the two strains.

Changing the pH value of a purified preparation of the new strain (produced at 20°) from 5·2 to 8 released some RNA in amounts suggesting that about a third of the particles in the preparation released their RNA.

Loss of infectivity by tobacco mosaic virus exposed to ultraviolet radiation appeared to result entirely from changes in the RNA of the virus. Any changes that may have occurred in the virus protein did not seem to contribute to loss of infectivity of the virus, but there was an interaction between the protein and the RNA because free RNA was equally sensitive to inactivation by absorbed radiation energy of any wavelength (i.e. the extent of inactivation depended entirely on the number of absorbed quanta irrespective of the wavelength), while inside the virus the RNA was about 24 times more sensitive to inactivation at 230 mµ than at 280 mµ. Inside the virus the RNA seemed to be largely protected by the protein from damage by radiation of 280 mµ and 254 mµ, but not of 230 mµ to which it was about as sensitive as when free.

Ultraviolet irradiation at any wavelength caused at least two kinds of damage to free RNA, one of which was photoreversible, but the photoreversible damage did not occur in the RNA irradiated when inside the virus.

Mice in our scrapie-infected colony were found to have high levels of plasma lactic dehydrogenase activity. The cause of this appears to be contamination of mouse-passaged scrapie material with a plasma lactic dehydrogenase-elevating virus, similar to that normally found as a contaminant of transplantable mouse neoplasia. Mice inoculated with the Compton strain of scrapie virus or with sheep scrapie virus had normal plasma lactic dehydrogenase activities.

Two kinds of assay, particle counts by electron microscopy and infectivity for leaves of Phaseolus vulgaris were used to estimate the amounts of virus in extracts made from tobacco leaves kept at 14 to 34° for 1 to 6 days after inoculation, and in extracts from uninoculated tip leaves sampled 14 days after the plants were inoculated. One, 2 and 6 days after inoculation, virus isolate prn was obtained in largest amount from leaves at 22°, 18° and 14° respectively. Isolate cam showed the same trend, but equivalent temperatures were 4 to 6° higher. At 14° infectivity and particle numbers continued to increase between 1 and 6 days after inoculation but at 30° infectivity decreased after 1 day. At some temperatures the number of particles first increased and then decreased. For instance, at 26° the number of particles of isolate cam tripled between 1 and 2 days, and then decreased at least 100-fold between 2 and 6 days after inoculation. Temperature had only small effects on the ratio of long (c. 1900 Å) to short (mainly 450 to 900 Å) virus particles. Specific infectivity increased slightly when particle number was increasing and decreased greatly when particle number was decreasing. Specific infectivity sometimes decreased slightly before particle number. As with some small isometric plant viruses, the amount of tobacco rattle virus extracted from leaves apparently represents the resultant between synthesis and degradation, and the two processes are differently affected by temperature. At 14 to 22° but not at 26°, isolate prn produced necrotic lesions in inoculated leaves, whereas isolate cam did not cause visible lesions. This difference seemed to have little effect on the changes in virus titre but although isolate prn multiplied optimally at a lower temperature than cam, it was more stable in vivo at 26°. Isolate prn invaded uninoculated leaves only sporadically and accumulated in only small amounts. Isolate cam readily invaded uninoculated leaves, in which it reached its greatest concentration at 22°.

Partially purified preparations of the kpeve strain of cocoa mottle leaf virus were inactivated after 10 min. at 6o°, but not after 10 min. at 55°, All undiluted preparations were infective, most were still infective when diluted 1/10 and some at 1/100; they were still infective after storing for 96 hr at 0 to 4°, or 25°,. The virus retained infectivity after freezing in vitro but lost some infectivity when frozen in leaves. The virus was precipitated from clarified extracts by adding 150 to 200 g./l. ammonium sulphate. Infective preparations contained straight rod−shaped particles with rounded ends having a mean length of 1430 Å and apparent width 256 to 260 Å.

Arginine is essential for the replication of herpes simplex virus. Omission of arginine from the culture medium did not affect the synthesis of viral DNA but prevented the formation of virions. Addition of arginine immediately stimulated protein synthesis which was followed by virus formation.

The RNA-containing components of vesicular stomatitis virus were labelled with 32P by growing the virus in a phosphate-deficient medium containing 32P04 and Actinomycin-D. Centrifugation of the virus suspensions in a sucrose gradient gave four radioactive fractions. The most rapidly sedimenting fraction contained the infective component of the virus, whereas most of the interfering activity was in the second fraction, which contained ‘caps’, filaments and rosette-like structures. The interfering activity was associated with the ‘cap’, which could be separated from the filaments and rosettes by centrifuging in a potassium tartrate gradient.

The viral RNA sedimented predominantly in the 36 to 40 S position in sucrose gradients prepared in o-iM-acetate buffer but frequently exhibited a more heterogeneous profile. The percentage distribution of 32P in the four nucleotides was A = 29·3, C = 21·1, G = 20·9 and U = 28·7. Ribonucleic acid from the interfering component invariably gave a sharp profile with a sedimentation coefficient of 18 to 20 S and the percentage distribution of 32P was A = 27·1, C = 21·9, G = 20·3 and U = 30·7. Both nucleic acids were hydrolysed to low molecular weight materials by ribonuclease 0·01 µg./ml.

The interaction of vaccinia virus with embryonic chick skin was studied in vitro using an organ culture technique. Skin explants were taken from chick embryos between 5½ to 6 days’ and 17 to 18 days’ incubation. Susceptibility of the epidermis of both the skin and the feather germ to vaccinia virus at these different stages remained unchanged; a generalized pattern of infection was observed with epidermal hyperplasia followed by necrosis. An exception to this behaviour pattern was seen in cultures from the 8½- to 9-day embryo where a ‘honeycomb’ type of infection occurred; the epidermis in the apical zone of the feather germ presented a characteristic appearance with neither hyperplastic reaction nor normal thickening. The dermis showed pronounced susceptibility to vaccinial infection in explants from 5½- to 6-day embryos, but became progressively and rapidly resistant at embryo ages between 7⅛ and 10 days. Dermal sensitivity was again marked in explants from 11- to 18-day embryos. The mesenchymal cells forming the mesodermal core of the feather germs were resistant to the pathogenic effect of the vaccinia virus in 6½- to 11-day explants; feather germ development was nevertheless modified. The dermal papilla, however, was grossly sensitive to viral infection from the time of its initial differentiation at 12 to 13 days’ incubation. The susceptibility of the cellular components of the skin to the viral infection appeared to be influenced by the inductive processes responsible for the differentiation of the normal skin. The pathogenic effect of vaccinia virus varied according to the stage of development of the embryonic skin.

Antisera were prepared which reacted specifically with different antigens in the capsid of adenovirus type 5, viz. hexons, pentons, fibres and penton bases. A rabbit antiserum was obtained which reacted with another antigen (designated P) formed during the early stages of the infectious process. This antigen is similar to the T antigen associated with infection by the oncogenic adenoviruses. Using the specific antisera and the P antiserum a study was made of the properties and sequence of development of the antigens in single-step infection. The time of appearance of the antigens was a function of the added multiplicity of infection. The P antigen was detected early in infection and was followed 4 hr later by the fibres and hexons and a further 2 hr later by the penton bases. The appearance of the penton bases was accompanied by an increase in infectivity. Haemagglutination and the ability of the antigens to inhibit cell spreading were measured and it is concluded that relatively late in infection the capsid antigens are no longer incorporated into virus particles and can be detected as free antigens.

When purified preparations of adenovirus type 5 were heated the icosa- hedral capsid of the virus was ruptured at the twelve vertices, allowing the viral DNA to become accessible to the action of DNAse. The viral DNA could then be substantially separated from protein by digestion with trypsin or pronase. An analysis of the distribution of viral DNA by radioactivity and the various capsid complement-fixing antigens after density gradient centrifugation of heated virus suggested the presence of some other hitherto undescribed protein within the capsid.

SUMMARY

Complement-fixation and radioactive-agglutination tests were performed on purified preparations of adenovirus type 5 using specific antisera against capsid antigens and an antiserum which can detect an antigen (the P antigen) in infected cells which has many of the properties of the T antigen associated with infection by the oncogenic adenoviruses. A serological reaction was detected between preparations containing intact virions and antihexon serum but not with antisera against fibres, penton bases and the P antigen. However, virus preparations which had been ‘aged’ at 4°, and in which electron microscopic examination revealed the presence of many broken capsids, did react in complement fixation and radioactive agglutination with all the antisera. It was concluded that within the adenovirus capsid there is a hitherto undescribed protein related to the P antigen.

Sixteen bacteriophages for several strains of Neisseria perflava were isolated from throat washings and lysogenic organisms. The phages are placed into five groups based on serological and host-range characteristics. Group I, host strain c r, has phages n1, n2, mutant n2c, n5, n6, and n13. n1 is virulent, all others temperate for the host. Plaque size is about 1·5 mm., the largest for Neisseria phages. Group II, host strains s, t and u, has phage n4 and mutant n4c. The neutralizable antigens of these phages are modified when the host is changed. The group III phages, n3, n7, n8, n9 and n11, are temperate for host strain t. Group III A has a single member, n10, which is temperate for its host, strain u. The group IV phages, n12, n155, n159 and n208, were all isolated from strains of N.flavescens on host strain s. Plaque size is about o-i mm., the smallest for the phages. All phages within a group are apparently serologically identical. Groups II, III and III A are serologically related, the others are distinct. The most remarkable feature is the extremely limited host range of each phage. Since the original chance isolations of the hosts given above, no additional phage-sensitive Neisseria have been found in over 300 strains tested.

The Pasteur strain of fixed rabies virus was inactivated by most common organic solvents, but survived a single treatment with trifluorotricholoro- ethane. The virus was stable at pH values between 5·0 and 10·0. Inactivation by heat was similar to that of other viruses. The virus was destroyed by trypsin and damaged by phospholipase C but not by ribonuclease; enzyme inhibitors did not preserve the infectivity of mouse-brain suspensions. Viral infectivity was diminished only after repeated freezing and thawing or long exposure to ultrasonic vibration. Equilibrium centrifugation in sucrose solutions gave two peaks of infectivity corresponding to densities of 1·13 g./ cm.3 and 1·19g./cm.3. Chromatographic purification on ECTEOLA-cellulose was unsuccessful. Partially purified virus was readily inactivated by the photodynamic effect of methylene blue. Potent suspensions of virus did not agglutinate red cells of several mammalian and avian species under different conditions of pH and temperature.

Adenovirus types 13, 15, and 19 and their soluble antigens were studied by electron microscopy, applying the negative staining technique to purified material. Virus particles showed projections of 160 to 190Å length with a final knob arising from the 12 vertices of the icosahedron. Incomplete particles were also seen. The group-specific antigen consisted of free (‘ hexon ’) capsomeres. The type-specific antigen, which showed the phenomenon of haemagglutination in the presence of a heterologous immune serum, had the appearance of fibres with final knobs (‘fibre antigen’). The soluble haemag- glutinin appeared as a star with a diameter of 450 to 600 A. It was composed of presumably 12 dumb-bell-shaped subunits, arranged in a regular fashion. The virus types studied were morphologically more closely related to type 3 (group I) than to type 5 (group III).

Bacteriophages which transduce genetic markers in Proteus mirabilis (1, 2) P. morganii (3) and Providence (4) have been reported.

In the course of an investigation of lysogeny in local strains of Proteus vulgaris, strain 107 was found lysogenic for P. vulgaris strain 69. The phage (named 107/69) produces turbid plaques on strain 69 and its ability to transduce markers into the strain was investigated using methods previously described (1, 4). A chloroform- sterilized lysate of strain 69 str-r can transduce the marker to wild strain 69 at a rate of 3 × 10−6 plaque forming units adsorbed, and similar lysates of wild strain 69 convert auxotrophs to prototrophy at the same rates showing that the phage is capable of generalized transduction. As in transduction with other proteus phages (4) abortive transductants were not detected.