- Volume 4, Issue 3, 1969

5-Aminoacridine in concentrations which had little or no effect on growth rate of the host organisms greatly reduced the yield of phage from bacteria infected with phage λ. The acridine reduced the number of bacteria releasing infective phage, and also the number of particles/bacterium. The inhibitory activity of the acridine became apparent at or just before the appearance of the first phage particles. The length of the eclipse period or latent period was not affected. Since 5-aminoacridine inhibited the synthesis of infective λ DNA, this could account for the decreased yield of complete infective phage particles.

Electron-microscopic evidence is presented for the infection of isolated tomato-fruit locule tissue protoplasts by tobacco mosaic virus. Uptake of tobacco mosaic virus by pinocytosis into vesicles in the cytoplasm was observed. Following the disappearance of virus from pinocytic vesicles and the regeneration of these isolated proplasts into cells the virus appeared in the cytoplasm in aggregates characteristic of tobacco mosaic virus infection in these cells. Adequate removal of ribonucleases and purification of the pectinase enzyme employed for the isolation of protoplasts was essential for the establishment of infection.

Polyacrylamide-gel electrophoresis of foot-and-mouth disease virus disrupted with 8m-urea revealed the presence of five major protein bands and one minor one. Evidence was obtained suggesting the likelihood of these bands being due to the presence of distinct polypeptides in the virus protein. Virus which had lost 3 to 4 log. of infectivity by treatment with trypsin also yielded six bands in polyacrylamide-gel electrophoresis. Five of these bands had the same mobility and intensity as those found in untreated virus but the slowest-moving band in the untreated virus had an enhanced mobility after enzyme treatment, suggesting that only one of the constituent polypeptides of the virus protein was affected by the enzyme. This polypeptide is necessary for the attachment of the virus to susceptible cells and for the immunizing activity of the inactivated virus particle.

The antigenic relationship between the viruses of varicella-zoster and herpes simplex was studied by complement-fixation, fluorescent antibody staining and neutralization tests. Twenty-three of 75 patients with herpes simplex infections showed significant heterologous increases in complement-fixing antibody titre to varicella-zoster virus. These heterologous increases occurred in patients with serological evidence of a prior infection with varicella-zoster virus, and the greatest proportion occurred in patients in the younger age groups who had probably experienced the most recent varicella-zoster virus infections. Five of 42 patients with varicella-zoster infections showed heterologous complement-fixing antibody responses to herpes simplex virus; all were patients with serological evidence of a prior herpes simplex virus infection. The patients with herpes simplex infection who showed heterologous complement-fixing antibody responses to varicella-zoster virus also showed marked increases in neutralizing antibody and antibody demonstrable by immunofluorescent staining. However, none of the patients with varicella-zoster infection who showed heterologous increases in complement-fixing antibody titre to herpes simplex virus had significant increases in neutralizing antibody.

Carrot mottle, a persistent aphid-borne virus transmissible by inoculation of sap, lost infectivity when Nicotiana clevelandii sap was diluted 10−3, heated for 10 min. at 70°, or stored at room temperature for 9 to 24 hr. Leaf extracts made using phenol contained infective RNA. Infectivity was abolished from leaf extracts by treatment with di-ethyl ether, chloroform or other organic solvents. Partially purified preparations, made by clarification with bentonite, followed by chromatography on calcium phosphate (brushite) columns and sucrose density gradient centrifugation, contained approximately spherical particles about 50 nm. in diameter. After equilibrium sedimentation in caesium chloride gradients, infectivity was found in fractions of density 1.14 to 1.17 g./ml., with a maximum at 1.154 g./ml. Ultrathin sections of infected N. clevelandii leaves revealed approximately spherical particles about 50 mµ in diameter in the cell vacuoles associated with the tonoplast. Some particles seemed in process of budding from the membrane. The particles seem unique among known plant viruses but resemble those of some viruses of vertebrates; they probably contain lipid. The cryptogram for carrot mottle is therefore R/*:*/*:S/(S):S/Ap.

Crystals of intracellular foot-and-mouth disease virus, type A1, were reacted with ferritin conjugated to an A1 antibody, an O2 antibody, and normal γ-globulin as well as unconjugated ferritin. Specific tagging was demonstrated with both the full and ‘empty’ capsid crystals with type A1 virus and anti-A1 globulin. Anti-O2 ferritin-conjugated globulin, normal ferritin-conjugated γ-globulin and unconjugated ferritin did not attach to the A1 virus and showed only minimal reaction with tissue fragments and debris. Immunodiffusion in agar gel was used to demonstrate the specificity of the reagents.

Residual infectivities of u.v.-irradiated preparations of potato virus X were assayed on three different kinds of plants. When the plants were kept in darkness to prevent photoreactivation of the inactivated virus, the residual infectivity appeared the same whether Chenopodium amaranticolor or either of two varieties of tobacco was used for assay. When the plants were exposed to daylight, photoreactivation occurred in all three kinds of plants, possibly to a somewhat greater extent in Chenopodium than in tobacco plants. Thus, in contrast to the results previously obtained with tobacco necrosis virus, no evidence was obtained for dark reactivation of u.v.-inactivated potato virus X in Chenopodium.

In the absence of photoreactivation, the quantum yields for inactivation were about 0.8 × 10−3 and 1.3 × 10−3 for the whole virus and for the RNA inside the virus, respectively.

Immunodiffusion and immunoelectrophoretic studies were made with rabbit antisera and purified influenza A viruses disrupted by sodium dodecyl sulphate. These techniques permitted the characterization of the type-specific ribonucleoprotein antigen and influenza A2 neuraminidase. A common precipitin line was obtained with different human and avian influenza A viruses when an antiserum for the ribonucleoprotein antigen of influenza A was used. A rabbit antiserum against purified influenza A2 neuraminidase gave identical precipitin lines with different influenza A2 viruses and with two recombinant strains known to contain influenza A2 neuraminidase, but not with influenza A0, A1 or fowl plague virus. No precipitin line corresponding to haemagglutinin could be detected with influenza A2 viruses treated with sodium dodecyl sulphate.

In a comparison of the effects of other virus disrupting agents on the detection of influenza virus antigens by immunodiffusion, only ‘Nonidet P40’ gave results as satisfactory as those obtained with sodium dodecyl sulphate in the characterization of the ribonucleoprotein and neuraminidase antigens.

The reticular structure previously described on the original taylor 1233 strain of influenza C was found on two further strains of influenza C.

In negatively stained preparations of influenza C three kinds of particles with different surface morphology have been found: particles with ‘spikes’ typical of myxovirus, particles with a hexagonal reticular surface and ‘naked’ particles with no reticular structure.

The fine structures of influenza viruses A and C as seen in thin sections were compared. In walls of influenza A the structures encountered from the surface inwards were: a layer of spikes hollow and open at both ends; an electron-transparent layer of uniform width; and a finely granulated layer. The electron-transparent layer of influenza C virus was much less prominent; the spikes were irregularly placed and often absent. On sectioned influenza C filaments a hexagonal pattern was seen. The internal component of influenza A virus measured about 6 nm. in diameter and sometimes appeared to be hollow. The internal component of influenza C measured about 9 nm. across and appeared to be helical.

On the basis of these results the taxonomic position of influenza C has been questioned.

A striking feature of the development of rubella virus in RK 13 cells was the presence of virus-like particles at the surface of the infected cells. These particles were not seen inside the cell cytoplasm or nucleus, nor were they identified in uninfected cultures. They were pleomorphic, with some evidence of substructure. Their appearance coincided with the development of new infective virus; they may, therefore, be mature virus particles.

In naturally occurring strains and laboratory selected variants of echovirus type 9, the association between virulence for infant mice (v) and the reproductive capacity at 40° (t) was of the v+t+ and v−t− types. By different selection methods the two other marker combinations, v+t− and v−t+, were obtained. The frequency of t+ in a t− echovirus type 9 population was 10−5. Of the four possible combinations between the v and c (cytopathic effect for human epithelial cell lines) markers, only three were found. The v+c+ covariation could not be obtained in spite of appropriate methods of selection.

The multiplication and inactivation of tomato bushy stunt virus (TBSV) and of bromegrass mosaic virus (BGMV), both of which have spherical particles, differed greatly at different temperatures. TBSV was inactivated in and disappeared from infected plants at 36°; BGMV maintained its content and virus produced at 36° was as infective as that produced at 20°. Infectivity of TBSV was lost in vivo or in vitro without any apparent change in the physical properties of the particles or of the nucleic acid.

BGMV was unaffected by heating in vitro for an hour at 45° at pH 5.8, but at pH 7.0 lost its infectivity within an hour at 36°. Even at 20° the particles were unstable and, in the presence of pancreatic ribonuclease, some but not all particles disintegrated to produce two new components. BGMV from plants infected for 1 week was, weight for weight, more infective than virus from plants infected for 3 weeks, and contained proportionally more 27 S nucleic acid than non-infective 22 S or 14S, RNAs. When virus was heated at 31° in buffer pH 6.8 the amounts of S27 and S22 nucleic acid diminished, but amounts of 14S nucleic acid diminished only at higher temperatures. Thus inactivation of BGMV, but not of TBSV, may reflect breaking of the nucleic acid.

Sequential cytological developments in primary dog kidney cells infected with a canine laryngotracheitis virus were studied by examining thin sections with the electron microscope. Early changes in the infected cells were enlargement of the nucleolus and the appearance of heterogeneous dark-staining granules in the nucleus. These granules developed into ‘initial bodies’ which were shown to contain DNA. The initial bodies increased in size to form ‘rings’ or more correctly spheres containing less densely staining central cores. The cores disappeared as the inclusions increased in size. Virus particles were occasionally seen at 14 hr and by 16 hr many of the cells contained virus. As the virus particles increased in number they usually aggregated and migrated towards the periphery of the nucleus. Release of the virus into the cytoplasm appeared to be by the formation of protrusions of the nucleus and the pinching off of membrane-bound virus aggregates.

Purified polyoma virus particles were degraded by exposure to carbonate + bicarbonate buffer, pH 10.5, at relatively low ionic strengths. The degradation was less when higher ionic strengths of the buffer were employed, i.e. 0.3 to 0.5m carbonate buffer. The degradation products were separated by caesium chloride density-gradient centrifugation and studied by infectivity, haemagglutination assays, and by electron microscopy. Electron microscopy demonstrated that treatment at pH 10.5 produced various stages of degradation, each stage dependent upon the ionic strength of the carbonate + bicarbonate solution. Polyoma particles were swollen when buffer of low ionic strength was used and, in addition, this swollen state allowed penetration of DNase. At slightly higher ionic strengths (0.1m-carbonate + bicarbonate buffer) the virus DNA was released from the polyoma virus coat and easily banded on caesium chloride gradients.

An early temperature-sensitive event which prevents the replication of variola-virus DNA in HeLa cells at 40° was studied in ‘temperature-shift’ experiments using 5-fluorodeoxyuridine or 5-bromodeoxyuridine. ‘Shift-up’ from 35° to 40° suppressed the replication of virus DNA, rapidly halting it even after it had begun at 35°. ‘Shift-down’ from 40° to 35° permitted the replication of virus DNA after a delay of not more than 2 hr. These results were confirmed in studies of the incorporation of [3H]thymidine into DNA in the cytoplasmic fraction. It was also shown that the incorporation which followed ‘shift-down’ from 40° could be inhibited by adding p-fluorophenylalanine at the time of shift. When cytoplasmic fractions from cells incubated at different temperatures were examined for enzyme activities, there was a marked increase in thymidine kinase activity in infected cells both at 35° and at 40°. DNA polymerase activity was increased five- to sixfold in infected cells at 35° but was not increased at 40° or in the presence of p-fluorophenylalanine. The increased polymerase activity in infected cells at 35° was unstable at 40° both in vivo and in vitro, in contrast to its greater stability at 35°. The behaviour of this enzyme was thought to explain the observed temperature-sensitivity of the replication of variola virus DNA.

Purified infectious preparations of cowpea mosaic virus consist of three centrifugal components with sedimentation coefficients of 58, 95 and 115S. These are referred to as top, middle and bottom component and contain 0, 23 and 34% RNA respectively (van Kammen, 1967). All three components are isometric particles with a diameter of 28 nm. and have serologically similar capsids. Recently van Kammen (1968) demonstrated that both RNA-containing components of the virus are necessary for infection. RNA prepared from purified middle component and bottom component is homogeneous in the analytical ultracentrifuge (u.v. optics) and has sedimentation coefficients of 26S and 34S respectively (L. J. L. D. van Griensven & A. van Kammen, unpublished). By applying Spirin’s (1963) relationship between the molecular weight of an RNA and its sedimentation coefficient (Mol. Wt = 1550 × S 2.1) the molecular weights were calculated as 1.4 × 106 and 2.5 × 106 respectively.

Treatment of monolayers of primary human amnion cells with homologous antiserum in sub-cytotoxic doses causes a reduction in the number of plaques formed in them by echoviruses and Coxsackievirus type A9 (Timbury, 1962). Polioviruses and Coxsackieviruses of group B are virtually insusceptible to the inhibitory effect of the antiserum which is due to a reaction of the antibody with the host cells and not to direct neutralization of the virus. This report describes further investigations, in HEp-2 cells, of the virus-inhibiting action of antibody to host cells.

Antiserum to HEp-2 cells was prepared in a rabbit as described for antiserum to amnion cells (Timbury, 1962). The antiserum had a titre of 1/1024 in complement-fixation tests with HEp-2 cells suspended in phosphate buffered saline at a concentration of 2.5 × 106 cells/ml. and a tire of 1/2048 in haemagglutination tests with human group O erythrocytes.