- Volume 41, Issue 3, 1978

The 20 to 25 nm particles of hepatitis B surface antigen were purified from the serum of a carrier chimpanzee. Five major polypeptide species were revealed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Treatment of the particles with neuraminidase (EC 3.2.1.18) and galactose oxidase (EC 1.1.3.9) followed by reduction with tritiated sodium borohydride labelled galactose residues in a single glycoprotein with an apparent mol. wt. of 28000. The glycoprotein was not labelled when neuraminidase treatment was omitted, indicating that the galactose residues are in subterminal positions in the oligosaccharide chains. There was no significant incorporation of radiolabel into lipid. The serological activity of the antigen, as measured by a competitive double-antibody radioimmunoprecipitation assay, was not altered by the labelling procedure nor by exposure to neuraminidase alone.

Virus replication, cytopathic effect and interferon production were measured in Namalva lymphoblastoid cells infected with measles viruses. Eight virus strains of different origin or passage history were compared. An inverse relationship seemed to exist between the abilities of strains to induce interferon and to replicate to high titres. Two representative strains were found to be highly sensitive to lymphoblastoid cell interferon, when tested in a line of monkey kidney cells (Vero). In contrast, Namalva cells were found to be highly insensitive to autologous interferon when challenged with measles or Semliki Forest virus (SFV).

Nuclei and chloroplasts isolated from cell-free extracts of Nicotiana glutinosa L. leaves by sedimentation at 1000 g contain DNA-directed RNA polymerase activity. Whereas only traces of RNA polymerase activity were detected in fractions prepared from the 1000 g supernatant of healthy leaf extracts, RNA-directed RNA polymerase was readily detected in similar extracts from lettuce necrotic yellows virus (LNYV)-infected plants. This virus-associated enzyme activity was strongly inhibited by fractions containing membrane material from ruptured leaf cells of both healthy and virus-infected plants. Fractions with LNYV-associated RNA polymerase activity were prepared after treating the 1000 g supernatant with the non-ionic detergent Nonidet P-40. RNA polymerase activities both of cell-free leaf extract fractions and of purified LNYV preparations were rapidly lost during storage; loss of activity was more rapid in the presence of the detergent.

Results presented in the paper are discussed in relation to the difficulties encountered in demonstrating RNA transcriptase activity associated with purified rhabdovirus preparations. We conclude that to detect such enzymes it is necessary to use rapid procedures to obtain virus preparations free of contaminating cellular membranes.

Thymidine kinase-deficient mouse cell cultures infected with herpes simplex virus type 1 exhibited a maximum of virus DNA synthesis around 8 h post-infection as determined by pulse labelling with 3H-thymidine. Cellular DNA synthesis was progressively inhibited, but still appreciable until 8 h post-infection and not completely abolished at any time during the infectious cycle. Phosphonoacetic acid was found to be a potent and selective inhibitor of virus DNA synthesis only when added to infected cultures before the onset of virus DNA synthesis. During the interval of increasing virus DNA synthesis the activity of cellular α polymerase decreased rapidly, whereas the β polymerase activity increased significantly; a slight increase was observed for the γ polymerase activity. When infected cells were kept in the presence of phosphonoacetic acid following virus adsorption the effect on cellular DNA polymerases was less pronounced.

The capacity of herpes simplex virus to kill cells and to form both infectious centres and progeny virus was studied in cell culture. Two virus stocks were compared at various m.o.i.: (i) a standard virus stock and (ii) a stock which contained interfering virus particles (I particles). The formation of infectious progeny virus as well as of infectious centres appeared to be reduced by the action of I particles. In the examined virus stock I particles exceeded infectious particles by at least a factor of 5 to 10. More than 50% of cells which could be demonstrated to be hit by I particles survived and formed colonies.

Exposure of the scrapie agent to u.v. light at various wavelengths has shown that light of 237 nm is 4 to 5 times as effective in inactivating it as ‘germicidal’ wavelengths (250 to 270 nm); whereas with systems that depend on RNA or DNA for function, inactivation is most effective by wavelengths in the germicidal range and there is a minimum of response in the wavelength region round 240 nm. The action spectrum for the scrapie agent is reminiscent of the absorption spectrum for purified bacterial endotoxin, identified as a lipopolysaccharide complex.

Dilute aqueous suspensions of scrapie agent were exposed to ionizing radiations in the presence or absence of oxygen. In dilute suspensions of test systems depending on the integrity of nucleic acid or protein, oxygen is almost invariably protective, but it was extremely sensitizing for inactivation of the scrapie agent, to an extent approached only in the case of membranous systems like lysosomes.

Results of these two methods argue against dependence of the scrapie agent on an intrinsic nucleic acid moiety for ability to replicate. They suggest that a lipid fraction is an important component and to that extent provide additional support for the ‘membrane hypothesis’.

Egg-grown mumps virus was used for purification of the glycoproteins of the virus. The two glycoproteins were separated by filtration on DEAE-Bio-Gel A columns and were used for preparation of rabbit hyperimmune sera. Antibodies directed against the large (mol. wt. 75 × 103) and small (mol. wt. 61 × 103) glycoprotein were immunologically distinct. Antiserum directed against the large glycoprotein inhibited haemagglutination, neuraminidase activity and virus infectivity, whereas antiserum against the small glycoprotein inhibited haemolysis, but not haemagglutination, neuraminidase activity or virus infectivity. It is concluded that mumps virus contains two glycoproteins on the envelope which are equivalent to the HN and F glycoproteins described for other paramyxoviruses.

Antibody activities of rabbit hyperimmune sera against untreated, Tween 80-ether and formalin treated purified virions were compared with those of sera against purified glycoproteins. In analogy with results obtained in studies on Sendai virus, these sera contained antibodies against both glycoprotein structures, but antibodies against the small glycoprotein blocking haemolysis could not be demonstrated.

The effect of formalin treatment on the two glycoprotein structures was studied in mixed haemadsorption experiments. Formalin treatment of mumps virus infected Vero cells resulted in destruction of the surface antigen of the small glycoprotein, whereas the surface antigen of the large glycoprotein was only moderately affected.

The structural polypeptides of egg grown mumps virus were analysed by SDS-polyacrylamide-slab-gel electrophoresis. Mumps virions contained eight major polypeptides with mol. wt. of 75, 73, 71, 61, 47, 44, 42 and 40 × 103. The 75 K and 61 K polypeptides were glycosylated. In virions treated with pronase and trypsin, the 75 K glycoprotein was removed more readily from the virus than the 61 K glycoprotein. The gradual removal of the 75 K glycoprotein was paralleled by a decrease of haemagglutinating activity. The large glycoprotein was cleaved into a 40 K glycoprotein by trypsin treatment. Pronase and trypsin treatment also removed the smallest 40 K non-glycosylated polypeptide. Thus this polypeptide appears to be located on the outside of the virion and probably represents a cleavage product of the large glycoprotein.

Treatment of virions with 2% Triton-X 100 under alkaline conditions in the absence or presence of 2 m-KCl solubilized the two glycoproteins and a fraction of the 71 and 44 K polypeptides, but not the 73 and 47 K polypeptides. The two smallest polypeptides were solubilized by treatment with 2% Triton X-100 in the presence of 2 m-KCl. Since the 40 K polypeptide was interpreted to represent a cleavage product of the large surface glycoprotein the 42 K polypeptide was proposed to represent the membrane protein of mumps virus. The 44 K polypeptide co-migrated with Vero cell actin. The nature of the 47 K polypeptide could not be determined, but it is probably located in the central part of the virus. The 73 K polypeptide and in some experiments also the 71 K polypeptide were found in purified nucleocapsid preparations.

It is concluded that mumps virus has a general polypeptide composition similar to other paramyxoviruses. However, the molecular weights of the different polypeptides of mumps virus differ markedly from the corresponding polypeptides in Newcastle disease virus and Sendai virus.

The addition of ammonium salts to cells treated with interferon prevents the development of the antiviral state and destroys it when already established. This treatment does not seem to act on the binding of interferon to the cells but blocks a further step of activation on the cell membrane. The anti-interferon effect of ammonium salts is reversible with a complete recovery of the antiviral state.

It is postulated that these salts may stabilize the interferon-receptor complex and thus prevent the changes in configuration necessary for the establishment and maintenance of its biological functions.

Large and small plaque variants of A12 foot-and-mouth disease virus were shown to have specific antigenic determinants. Large plaque virus antigenic specificity was destroyed by trypsin treatment, but the small plaque antigen was resistant despite cleavage of the trypsin-sensitive polypeptide. The cleavage of polypeptide VP3 by trypsin resulted in the formation of a new antigen not present on untreated virus. The effects of chymotrypsin and trypsin on the polypeptides of the plaque variants have been examined and related to changes in antigenicity, infectivity, and exposure of the polypeptides at the surface of the capsid. The results are discussed in relation to the orientation of the trypsin-sensitive polypeptide in the virus capsid.

The conditions for optimal transduction efficiency of the Bacillus subtilis phage SPP1 have been investigated. By irradiating transducing lysates with u.v. light we have been able to obtain a fivefold increase in the number of transductants and to reduce strongly the interference caused by infective particles. Any dependence of SPP1 transduction on PBSX induction has been ruled out by the use of xin mutants, which are unable to induce the defective phage. SPP1 mediated transduction is susceptible to the restriction and modification system of B. subtilis. The rec functions involved in the recombination of the SPP1 transduced DNA fragment are probably identical to those required in DNA transformation and heterologous PBS1 transduction.

Adenovirus type 5 DNA has low infectivity (Graham & van der Eb, 1973) which can be increased by various techniques, one of which is the dimethyl sulphoxide (DMSO) boost (Stow & Wilkie, 1976). In this report, it is shown that DMSO treatment of adenovirus 5 DNA-infected HeLa cells results in a 10-fold increase in plaque formation, and that this can be used to facilitate marker rescue experiments. Double DNA infections were performed by the calcium phosphate method, co-precipitating intact temperature-sensitive mutant DNA with purified wild-type DNA restriction endonuclease fragmens. Analysis of the plaquing ability of these mixtures and any progeny virus has resulted in the assignment of six temperature-sensitive mutations to discrete physical locations on the adenovirus type 5 genome. These locations are discussed with respect to the mutant phenotypes and the transcription-translation products of the appropriate regions.

Sera from 67 patients treated for renal diseases were assayed by as many as three different tests for activities against Mason–Pfizer virus (M–PV) antigens. Firstly, in patients studied before kidney transplantation, neutralizing activity against syncytium-forming units of M–PV was found in 50% of 24 cases of chronic glomerulonephritis but in only 10% of 20 cases with other diseases (P < 0.01). These proportions were higher after treatments accompanying transplantation since, of the 19 patients without antibodies before graft, 53% showed a seroconversion after this treatment. The incidence of M–PV antibodies did not correlate with the number of transfusions received by the patients. Neither did these antibodies correlate with the presence of antibodies to antigens associated with baboon endogenous virus or simian sarcoma virus; antibodies to the latter two viruses were found in 6 to 21% of the sera, with no specific distribution among the sera. Secondly, pseudotypes of vesicular stomatitis virus with M–PV antigens in their envelope were prepared; they were inactivated by 90% of the sera which neutralized M–PV syncytium forming units and by none of the negative sera. Thirdly, specific complement-dependent cytotoxic antibodies to HeLa cells producing M–PV were also found in 61% of the sera with neutralizing activity to M–PV and only in 12% of the sera which did not neutralize M–PV. Absorption experiments indicated that the serum activities against M–P V associated antigens were not due to anticellular antibodies directed against normal constituents of human cells. However, no evidence has been provided that the M–PV associated antigens reacting with the human sera were virus coded.

The course and outcome of intraperitoneally induced infections with the avirulent strain A7(74 ) of Semliki Forest virus have been studied in athymic ‘nude’ (nu/nu) mice, their heterozygous (nu/+) littermates and conventional Swiss A2G mice. The main distinguishing characteristics of the infection in the nu/nu mice were the persistence of virus in the brain after an initial phase of incomplete virus clearance and the apparent establishment of a secondary phase of virus replication in the brain which was associated with a falling neutralizing antibody response. This secondary phase of virus replication persisted until at least the 28th day after inoculation. In addition the typical histological lesions of encephalitis induced by this virus were rare and focal demyelination, which occurred at a light microscopy level in up to 26 % of nu/+ and Swiss A2G mice, was not observed. It is suggested that in immunocompetent mice the development of lesions including demyelination may be a result of an immunopathological response to virus infection which is related to the presence of thymus derived lymphocytes.

The structure of intracellular DNA extracted from phage T1 infected cells was analysed by sedimentation through sucrose gradients. DNA labelled with 3H-dThd during a short pulse given at any time during T1 DNA synthesis sedimented in neutral gradients as a broad heterogeneous band with a large fraction of the label sedimenting more rapidly than mature T1 DNA molecules. Rapidly-sedimenting label was also observed when pulse-labelled DNA was denatured and analysed on alkaline sucrose gradients. Electron microscopy of intracellular T1 DNA revealed linear molecules of variable length the longest of which were three to four times the mature genome length. The distribution of lengths derived from electron microscopy are consistent with the molecular length distributions calculated from the sedimentation coefficients. We conclude that the rapidly-sedimenting DNA is in the form of concatemers consisting of linear tandem repeats of the T1 genome. The concatemeric form of replicating T1 DNA is a precursor of progeny T1 genomes since in pulse-chase experiments it was converted efficiently into mature, infectious T1 phage particles. The identification of this concatemeric form of T1 DNA provides supporting evidence for the model proposed by Gill & MacHattie (1976) to account for the formation of the very limited number of cyclic permutations of gene sequence found for mature T1 DNA molecules.

Four isolates of infectious pancreatic necrosis virus showed ringed plaque morphology on chinook salmon embryo cells, while six other isolates showed clear plaque morphology. One isolate chosen from the ringed-plaque class yielded defective interfering (DI) particles after as few as two virus passages starting with plaque-purified material; another isolate from the clear-plaque class failed to yield detectable numbers of DI particles.

Analysis of the virus DNA isolated from arginine deprived cells revealed that the majority of DNA molecules are linear, with some molecules having one or more short branches. In addition, circular and circular-linear DNA molecules of genome size and smaller were observed.

Cells cultured from the breast muscles of 11 to 12-day-old chick embryos were infected in the undifferentiated mitotic myoblast stage or in the terminally differentiated non-mitotic myotube stage with one of two DNA viruses, vaccinia and herpes simplex virus type 1 (HSV-1). DNA synthesis was measured and production of virus-specific DNA detected in cells infected as myoblasts or myotubes by isotope labelling, autoradiographic and buoyant density centrifugation techniques. Furthermore, fully fused myotubes resemble myoblasts in their ability to support productive infection by these DNA viruses although DNA replication and nuclear division have ceased in myotubes and only minimum levels of host-cell DNA polymerase activity are present.

Indirect radioimmunoassays have been developed for the rapid detection of Sindbis virus. Dilutions of Sindbis virus from tissue culture fluids have been immobilized and allowed to react with rabbit anti-Sindbis virus antibodies. The bound antibodies were assayed either by 125I-labelled anti-rabbit IgG-antibodies or alternatively by addition of human complement and 125I-labelled anti-human C1q antibodies or 125I-labelled protein A.