- Volume 2, Issue 1, 1968

Sap from plants infected with a strain (NDEMV) of dolichos enation mosaic virus (DEMV) contains many ringlike particles, but few particles of about 3000 Å, the normal length for viruses of the tobacco mosaic virus group. In purified preparations the mean length of normal particles of NDEMV was 3000 Å and that of DEMV, 2860 Å. Staining with uranyl formate clearly revealed the fine structure of these particles and in the ringlike particles it penetrated to the expected position of the ribonucleic acid.

The observed efficiency of plating (e.o.p.) is defined as the ratio of two plaque or colony counts and is in practice subject to substantial sampling fluctuations. The true e.o.p. is here defined as the ratio of the underlying mean values, of which the two counts are Poissonian estimates: thus, when the number of counts on each plate is assumed to increase indefinitely, the observed e.o.p. converges in probability to the true e.o.p. A contour map is provided, by means of which one may rapidly determine, for any given pair of counts in the range 50 to 1000, an interval which will in 95 % of all cases contain the true e.o.p.

The main effects of the proteolytic digestion of the Asian (A 2) strain of influenza virus were a loss of the characteristic surface morphology and a change in density. The untreated virus was found to have a density of 1·204 g./cm.3 on a sucrose gradient, but after protease action the density was 1·168 g./cm.3. The density change was correlated with a change in particle mass as indicated by a fall in sedimentation coefficient (S20 ) from the normal 788 Svedbergs to 562 Svedbergs.

From the data obtained with the protease-treated and control virus preparations it was calculated that the changes observed could be accounted for by a loss of 35 to 40 % of the particle mass. The action of the protease appeared to be confined to the surface of the virus. Concomitantly with the destruction of the haemagglutinating activity there was a release of neuraminidase activity, and on a density gradient this could be separated from the residual virus and was located at the top of the gradient tube. In addition to the changes described, there appeared to be a destruction of the virus V antigen.

One hundred and eighteen different strains of Proteus vulgaris were investigated for bacteriocinogeny. These and an additional 44 strains of P. vulgaris were used as indicators. Sixty-seven of the strains had a nontransmissible killing effect on one or more of the indicator organisms and 30 of these 67 bacteriocins with different spectra of activity were further investigated. Individual bacteriocins killed from 5 to 87 of the P. vulgaris indicators and a number of 44 different P. mirabilis strains but had no action on strains of other species of the family Enterobacteriaceae. Broth cultures of bacteriocinogenic strains are inducible by ultraviolet light and yield bacterio- cin titres of about 1/100. Activity is sedimentable by high-speed centrifugation. Electron microscopy of all 30 preparations revealed similar phage-tail-like structures with a contractile sheath round a hollow core. The structures consisted of protein and did not contain DNA. The particles resembled some pyocins and also the tail of a P. vulgaris transducing phage. In 2 preparations a few phage-like particles resembling other Proteus phages were also seen. Bacteriocin activity was always associated with uncontracted sheaths, and triggered tails did not adsorb to susceptible organisms. We conclude that the tail-like structures are the products of defective lysogeny. The high incidence of the latter state may be accounted for by the selection of genes favourable to the host which were originally acquired through transduction by lysogenization or lysogenic conversion.

Cultures of RK13 and BHK21 cells infected with rubella virus were examined by electron microscopy when the cultures showed maximal cyto-pathic effects. Infected RK13 cells contained crystalline inclusions (spacing 190 Å) as well as typical virus particles of total diameter 600 Å, with a dense 300 Å core. Identical particles also occurred in infected BHK21 cells, but in these no crystals were observed. Neither crystals nor particles were found in control cells. The particles did not resemble myxoviruses.

Preparations of nucleoproteins from stable variant infections of viruses of the tobacco rattle type were separated, by density gradient centrifugation, into ‘top’ and ‘bottom’ fractions containing predominantly ‘short’ or ‘long’ particles respectively. Comparisons of the products of infection when these fractions were used as inocula alone and mixed indicated that though lesion production and RNA replication were mediated by ‘long’ particles, the presence of ‘short’ particles was necessary for virus protein coat production. The coat-inducing function of ‘short’ particles could also be used to stabilize RNA from homologous or closely related unstable variant infections. Inoculating systemic hosts with ‘short’ particle preparations together with phenol extracts from the unstable variant infections, resulted in stable variants similar to those from which the unstable variants were initially derived.

In 15 to 60% sucrose density gradients the distribution of standard preparations of influenza viruses measured by haemagglutination or infectivity was always bimodal. Approximately 17% of virus particles sedimented in a diffuse leading peak (fraction 1), while the rest was confined to a second homogeneous peak (fraction 2). Virus particles comprising fraction 1 were less dense (1·250 g./cm.3) than those of fraction 2 (1·257 g./cm.3).

RNA labelled with 32P was extracted by phenol+sodium dodecyl sulphate from four strains of influenza A virus (pr8, bel, a2/singapre/1/57 and fowl plague) and from one strain of influenza B virus (lee). The sedimentation characteristics of each preparation of RNA on sucrose density gradients varied under different conditions of ionic strength. In the presence of 0·1 m-NaCl+0·001 m-EDTA, RNA from fraction 2 could be resolved into at least two species. The predominant species had a sedimentation coefficient of 18 to 21 S and (with the exception of the a2 RNA) about 10 to 15% of the total RNA sedimented as a diffuse band with a sedimentation coefficient of 32 to 42 S. The bel and lee strains contained a third species of RNA, with a sedimentation coefficient of 7 to 9 S. In 0·005 m-EDTA without NaCl the RNA from fraction 2 (pr8) sedimented principally as a 14 S component, and the 32 to 42 S component was not found. Fraction 1 virus particles also contained several RNA components when analysed under conditions of high ionic strength. A 7 to 9 S RNA species was found in amounts equal to, or greater than, the 18 to 21 S RNA component, together with a small amount of ‘38’ S RNA. In the presence of 0·005 m-EDTA the 38 S component was not found, but three components with sedimentation coefficients of 16 S, 9 S and 5 S were present in approximately equivalent amounts. From these findings it is concluded that the RNA of influenza viruses consists largely of 18 S molecules, under defined conditions of ionic strength, and that the 32 to 42 S component is an artifact caused by aggregation. It is further concluded that sucrose gradients containing 0·1 m-NaCl are unsatisfactory for studying the sedimentation characteristics of influenza RNA.

RNA was extracted from purified Sendai virus, labelled with 32P, and sedimented in sucrose density gradients. Under conditions of high ionic strength (0·1 m-NaCl) the sedimentation coefficient was 57 S. In the presence of 0·005 m-EDTA, the sedimentation coefficient was reduced to 40 S, indicating that the RNA of the virus exists as a single-stranded molecule.

The intracellular synthesis of virus-directed RNA during the latent period of infection of chick embryo cells by Sendai virus in the presence of actino- mycin was also studied. Cells were extracted by the phenol + sodium dodecyl sulphate method at 2,4, 6 and 7 hr after infection, having previously been exposed to [3H]uridine for at least 2 hr. It was found that the first newly formed RNA had a sedimentation coefficient of 18 S' and could be detected between 2 and 4 hr after infection. From 4 hr onwards a 57 S component was found in increasing amounts until 7 hr after infection. Substantially similar results were obtained when infected cells were treated with detergent alone, and the mixture immediately centrifuged on density gradients except that the principal RNA components now had sedimentation values of 57 S and 23 S.

Naples virus, a member of the Phlebotomus fever group of arboviruses, is able to multiply in cells derived from hamster embryos. It produces no cyto- pathic effect in these cells in a fluid medium and no plaques upon infection of confluent monolayers overlaid with a medium containing agar or the sodium salt of carboxymethylcellulose. Plaques are produced only when these cells are infected in suspension and plated to form monolayers under an overlay containing carboxymethylcellulose. A critical number of cells is required and the presence of carboxymethylcellulose is essential. The survival or death of a hamster embryo cell infected with this virus appears to depend upon the stage of growth of the cells at the time of infection and the environmental conditions.

A simple and rapid in vitro labelling technique is described for preparing tobacco mosaic virus labelled with 125I. The technique is versatile and has been used to obtain labelled virus of both high and low specific activity. High specific activity virus has been used in a pilot radioautographic experiment at the electron microscope level, and micrographs were obtained showing virus particles in close association with silver grains. At specific activities of 2 to 3 mc/mg. a rapid loss of infectivity was observed after labelling; this effect was not observed when the specific activity was 1 µc/mg. The sensitivity of detection for high specific activity preparations by scintillation counting and by electron microscope radioautography is discussed and the potentiality of 12SI-labelled virus of both high and low specific activity is indicated.

Four clones of the hamster cell line NIL-2 were transformed by adenovirus 12. The transformed cells formed foci of multilayered growth in monolayer cultures under agar medium and colonies when suspended in soft agar medium. A virus stock containing 5 × 109 particles and 1·1 × 109 p.f.u./ml. contained 2·8 × 102 focus-forming units (f.f.u.)/ml. in NIL-2 cells. The numbers of cell foci and of colonies induced by serial dilutions of virus were consistent with a linear dose response. Approximately 2 × 107 total virus particles or 4 × 106 infectious units were required to induce one focus of transformed cells. The highest transformation rate obtained was 0·002 % for cells exposed to about 80 p.f.u. of virus per cell. For comparison, primary rat embryo cells were transformed by adenovirus 12. The results obtained were approximately the same as those with NIL-2 cells except that the rat cells did not form colonies when infected and suspended immediately in agar. However, rat cells transformed in cultures under liquid medium formed colonies when suspended in agar medium.

Several plant viruses with filamentous particles ranging in modal lengths from 0·48 µ to 1·25 µ were negatively stained with uranyl formate, examined in the electron microscope, and the electron micrographs analysed in various ways. The particles of all the viruses were helically constructed with a basic pitch of 33 to 37 Å (mean 34 Å), but could be separated into groups by other features of their particles. Various measurements of the particles of five of the viruses suggest that there were 10 to 14 subunits in each turn of the basic helix of their particles.

All plant viruses with elongated particles seem to fall into one of two groups; those with modal lengths of 0·3 µ or less seem rigid and have a basic helix of pitch 23 to 25 Å, and those with longer particles are filamentous and have a basic helix of pitch 33 to 37 Å.

Baby hamster kidney cells were infected with herpes simplex virus under one-step growth conditions. Virus-specific antigens were traced by ‘direct’ and ‘indirect’ immunofluorescence and their times of appearance estimated by agar-gel diffusion using an antiserum made by immunization of rabbits with herpes-virus-infected rabbit cells. Twelve distinct precipitin lines were detected by agar-gel diffusion and a succession of fluorescent patterns was distinguished. Quantitative estimations indicated progressive accumulation of antigen in various patterns within the nucleus, in the cytoplasm and at the cell membrane. These patterns were correlated with the cellular distortions seen with a conventional cytological technique.

Five or possibly six different soluble adenovirus type 11 components have been identified. Zonal centrifugation separated, listed in order of decreasing sedimentation rates: (i) a complete haemagglutinin (HA), (2) an incomplete HA plus group-specific complement-fixation (CF) antigen, and (3) components absorbing haemagglutination inhibition (HI) and haemagglutination enhancement (HE) antibody. The complete HA carried toxin activity. The incomplete HA, which also exhibited toxin activity, could be separated from group-specific CF antigen by anion-exchange chromatography. The incomplete HA exhibited haemagglutination activity only in the presence of antibody which presumably interacts with vertex capsomere antigen. Antisera against members of all of Rosen’s subgroups contained HE antibody. The major portion of antigen capable of absorbing HE antibody was associated with incomplete HA, but a part of it appeared in a separate fraction. The incomplete HA also exhibited some capacity to absorb HI antibody.

Treatment with trypsin completely destroyed HE antibody-absorbing antigen and eliminated all toxin activity. Repeated erythrocyte absorptions removed all activities except group-specific CF antigen and some HE antibody-absorbing antigen.

By comparison with data obtained in parallel studies of adenovirus types 3, 4 and 5 it is suggested that the different type 11 components are of the following nature:

The incomplete HA might represent isolated penton components—vertex capsomeres plus projections—and the complete HA symmetrical aggregates of 12 such components. The group-specific CF antigen most likely is carried by non-vertex capsomeres (hexon components). HE antibody-absorbing structures not related to incomplete HA appeared heterogeneous and probably included both intact and fragments of free vertex capsomeres. Slowly sedimenting HI antibody-absorbing components presumably represent fibre components, i.e. isolated vertex projections.

The complete haemagglutinin (HA) of adenovirus type n sedimented more rapidly than the corresponding component of type 3. No significant difference was found between these two serotypes in the rates of sedimentation of their incomplete HAs, isolated haemagglutination-enhancement antibody absorbing components obtained by treatment with guanidine-HCl and slowly sedimenting haemagglutination-inhibition antibody absorbing components. These three products may be pentons (vertex capsomeres plus projections), isolated vertex capsomeres and projections (fibres), respectively.

Ultrastructural studies demonstrated that the architecture of complete HAs of adenovirus types 3 and 11 is principally the same, i.e. a symmetrical aggregate of 12 penton components located in the facets of a pentagonal dodecahedron. No difference was found in structural characteristics of either the projections or the capsomeres parts, in conformity with the results of the zonal centrifugation analyses. In spite of this the diameters of the two complete HAs, excluding their projections, were different: 225 to 275 Å for type 3 as compared to 280 to 330 Å for type 11. A variation in mass and structure of a postulated internal component may be of decisive importance for the difference in physical characteristics between the complete HAs of adenovirus types 3 and 11.

The incorporation of [14C]amino acids into the acid precipitable material of chick embryo fibroblasts was inhibited by both sparsomycin and gougero- tin with an efficiency which varied according to the amino acid and to the antibiotic. Of 16 amino acids tested, proline was the most susceptible to the action of sparsomycin and phenylalanine was the most resistant. With gougerotin, the order of decreasing susceptibilities of four amino acids were: phenylalanine, proline and glycine, lysine. Inhibition of virus growth by the two antibiotics was also specific. Synthesis of pseudorabies virus was very susceptible to inhibition by sparsomycin, while vaccinia virus synthesis was almost completely resistant. Newcastle disease virus, fowl plague and Western equine encephalomyelitis viruses showed intermediate susceptibilities to that drug.

In contrast, the synthesis of fowl plague virus was much more inhibited by gougerotin than that of Newcastle disease virus, and this antibiotic had an equal action on the growth of pseudorabies and vaccinia viruses. Most of the results may be explained by assuming that sparsomycin is an effective inhibitor of the activity of virus messenger RNAs which are rich in guanine and cytosine, while gougerotin is more effective on uracil-rich virus messenger RNAs.

The passive haemagglutination and bentonite flocculation tests were compared for detecting plant viruses, using antibody-sensitized tanned red cells or bentonite particles. Parsnip yellow fleck, raspberry ringspot, turnip yellow mosaic, narcissus mosaic, potato X and tobacco rattle viruses were detected by both tests in purified preparations and, where studied, in crude plant extracts. The highest antigen titres were obtained only when the red cells or bentonite particles were sensitized with optimal amounts of antibody; these had to be found experimentally because they differed between antisera and between the two tests. In comparative experiments the bentonite flocculation and passive haemagglutination tests were respectively about 2 to 5 and ioo to 125 times more sensitive than tube-precipitin tests for detecting the elongated viruses and about 10 to 20 and 500 to 600 times more sensitive for detecting the isometric viruses. The minimum concentrations of virus detected were 1·8 to 2·6 µg./ml. with the bentonite flocculation test and 0·04 to 0·24 µg./ml. with the passive haemagglutination test. Red cells treated with formalin before they were tanned and sensitized with antibody could be preserved at − 14° for up to 8 weeks without loss of activity.

In experiments with turnip yellow mosaic virus, extracts from healthy plants of eight species tested had little or no effect on either test but concentrated extracts from Chenopodium amaranticolor and Spinacia oleracea plants caused non-specific agglutination of unformalinized red cells, and dilute extracts from Petunia hybrida plants decreased eightfold the sensitivity of the passive haemagglutination test. These effects did not occur with for- malinized red cells.

Antibody-sensitized red cells were used in haemagglutination-inhibition tests to determine antiserum titres; antisera to turnip yellow mosaic and raspberry ringspot viruses gave titres 32 times higher than those obtained in tube- precipitin tests.

Molluscum contagiosum virus failed to inactivate encephalomyocarditis virus when incubated with it in the absence of cells. Adsorption and eclipse of encephalomyocarditis virus occurred normally in mouse embryo cell monolayers treated with molluscum virus but its growth was reduced according to the dose and duration of molluscum pretreatment. Encephalomyocarditis virus dose-response curves plotted by direct plaque count, by infective centre assay and by single cycle yields all showed a directly proportionate relationship, at low multiplicities of challenge, in both control and inhibited cultures. With high multiplicities of challenge, yield experiments suggested that interference might have been partially overcome at the cellular level but this was not confirmed in infective centre studies. Interference appeared to result in a uniform reduction of challenge virus growth in all the cells of a culture rather than in the complete protection of some of them. Certain features suggested the production and operation in inhibited cultures of a propagated interfering principle.

Guinea pig antisera were prepared against purified hexon, penton and fibre antigens of adenovirus type 5. Sera reacting specifically with particular antigens as judged by immunodiffusion, complement fixation and haemagglu- tination-inhibition tests were assayed for virus neutralizing capacity. The latter was exerted almost exclusively by anti-hexon sera. Type 5 anti-hexon but not anti-fibre sera neutralized type 1 virus provided the serum virus reaction took place at low pH values.

Since the hexon antigens contain the group-specific adenovirus antigen, demonstrable by complement fixation or immunodiffusion, the ability of heterotypic adenovirus rabbit antisera to combine with adenovirus type 5 was assayed. Evidence of such combinations was obtained by the demonstration of virus inactivation following addition of a goat anti-rabbit serum. It was shown that adenovirus type 1 and type 2 antisera combined with type 5 virus. The technique used did not reveal combinations between type 5 virus and antisera against type 3, type 12, type 21, nor against Cox- sackie B. The IgG fractions of rabbit antisera against type 1, type 2 and type 12 were shown to enhance inactivation of type 5 virus+antibody complexes. No enhancement was obtained with type 3 or Coxsackie B, IgG. The results suggest that although the hexon antigens contain common structures the configurational pattern differs among the different virus types.