- Volume 34, Issue 1, 1977

Cells infected with human adenovirus type 5 have been labelled with 32P-orthophosphate under various conditions and extracts examined, after denaturation in sodium dodecyl sulphate (SDS), by polyacrylamide gel electrophoresis (PAGE) followed by autoradiography. A number of polypeptides appear to be phosphorylated specifically as a result of infection. Early in infection, phosphorylation of a polypeptide of apparent mol. wt. 26 K associated with ribosomes can be detected. Two other phosphorylated polypeptides of apparent mol. wt. 72 K and 18 K can also be seen, the former being mainly confined to the nucleus and capable of being precipitated by the previously described virus-specific P antiserum. The 18 K phosphorylated polypeptide is found mainly in association with membrane fractions. Later in infection phosphorylated polypeptides of apparent mol. wt. 100 K and 39 K can be recognized, the former being associated with ribosomes but removed, however, with a high salt wash; the latter component is mainly detected in the nucleus. Analysis of the purified 32P-labelled virus by the SDS PAGE technique indicated that a structural polypeptide of apparent mol. wt. 66 K (IIIa) was also phosphorylated.

Two newly synthesized pyrimidine derivative were found to possess antiviral activity against Mengovirus in Fogh and Lund (FL) cells and in a cell-free system. The inhibitory effect on RNA-dependent RNA polymerase of Mengovirus-infected FL cells was assayed using 14C-UTP as precursor. Addition of 50 or 100 µM of the inhibitors in a cell-free system of crude enzyme and nucleoside triphosphate medium for 60 min incubation at 37 °C resulted in about 40 to 60% lower counting rates for drug-treated reaction mixtures. The analysis of the polymerase synthesis product (virus RNA extracted from the cell-free reaction mixture and deproteinized by the phenol-SDS method) was carried out by means of agarose-acrylamide gel electrophoresis. The main finding was a reduction of single-stranded Mengovirus RNA (RNase-sensitive and LiCl-precipitable). The rates of synthesis of the replicative intermediate (LiCl-precipitable) and the replicative form of RNA (LiCl-soluble) were not significantly influenced.

The most reliable method of distinguishing strains of low virulence amongst a collection of stock strains of Newcastle disease virus (NDV) was by their failure to produce a good c.p.e. in monolayers of baby hamster kidney cells and of chick embryo cells. This method was of no help in identifying avirulent mutants that emerged in the Essex 70 strain of NDV following ultraviolet light, nitrous acid, hydroxylamine or N-methyl-N′-nitro-N-nitrosoguanidine (NTG) treatment. A marked reduction in the ability to kill developing chick embryos at 41 °C was a much more reliable indicator. Several of these temperature-sensitive mutants, most of which had been isolated from NTG-treated virus, were non-lethal for young chicks but they did have a depressive effect on their growth rate. The immunity produced by three of these mutants in chicks free of NDV antibody, but not in chicks possessing appreciable amounts of antibody, was probably even better than that produced by Hitchner B1 strain. All three mutants reverted to virulence during passage in chicks, although in no case were the revertants as virulent as the original Essex 70 strain. The virulent revertants obtained from one of the mutants had lost their temperature-sensitivity and proliferated in large numbers in the tissues of infected chicks. Those obtained from the other two had either not lost, or only partly lost, their temperature-sensitivity; they were found only in low concentrations in the tissues of infected chicks, their concentrations being little different from that found in the tissues of chicks infected with the mutants from which they were derived.

Methods are described for the preparation and authentication of a highly specific antiserum against herpesvirus saimiri (HVS) capsid antigens. The antiserum was used in immunofluorescence tests to follow the development of capsid antigens in HVS-infected owl monkey kidney cells throughout the virus replication cycle in parallel with sequential titrations of virus infectivity in both cells and medium. Fluorescence was detected as a round or oval, bright green area of staining at the centre of the nucleus which was similar in outline to the Cowdry type A inclusion seen in HVS-infected cells stained by haematoxylin and eosin. The first detection of fluorescence towards the end of the eclipse phase of the virus growth cycle, and its abolition by the treatment of infected cultures with cytosine arabinoside confirmed the identity of HVS capsid antigens as late antigens. The failure to detect fluorescence in the cytoplasm of HVS-infected cells has brought to light a conflict between the site of accumulation of virus capsid antigens as determined by immunofluorescence and the finding, by electron microscopy, of cytoplasmic immature particles in intact cells during the early stages of the virus replication cycle. The significance of this discrepancy is discussed in relation to its possible existence for other members of the herpesvirus group.

Preparations of bacterial transfer RNA (tRNA), give dose-dependent protection of mice against encephalomyocarditis (EMC) virus infection at up to 1 mg tRNA per mouse with maximum response when the tRNA is administered around 6 h before infection. Protection occurs with intraperitoneally and intravenously administered tRNA against infections by both these routes. In some experiments significant protection occurs by single treatments of tRNA up to 24 h after infection with virus doses of 1 × LD100. Some tRNA preparations of eukaryotic origin do not give significant protection. Protection is not a feature of all species of bacterial tRNA; partially purified valine, tyrosine and phenylalanine tRNAs from Escherichia coli are not protective. tRNA treatment does not induce circulating interferon nor does it ‘hypo-reactivate’ the protective effect of poly (I). poly (C) treatment of mice. Humoral and cell mediated immune responses do not seem to be involved in tRNA mediated protection since first, cytosine arabinoside treatment does not affect protection by tRNA; second, serum from mice treated with tRNA and an EMC vaccine does not protect other mice against infection, and third, mice that survive normally lethal infections as a result of tRNA treatment are generally just as susceptible to re-infection as previously untreated, uninfected mice. Silica treatment abolishes protection of mice by tRNA implying that macrophages are necessary. However, tRNA does not seem to act by clearance of virus particles since vaccination of mice by inactivated EMC virus is not affected by tRNA treatment. These results are considered in relation to the presence of a tRNA-like structure in EMC virus RNA and protection of mice by other single stranded polynucleotides.

A comparison has been made of some of the serological and physicochemical properties of a virulent and an avirulent strain of foot-and-mouth disease virus, serotype SAT1. The avirulent strain (SAT1-82) was derived from the virulent strain (SAT1-7) by serial passage in BHK 21 cells. The viruses were indistinguishable in cross-neutralization tests. In immunodiffusion tests a clear spur line was obtained with the SAT1-82 antiserum but not with SAT1-7 antiserum. The major polypeptides of the two viruses were identical when examined by polyacrylamide gel electrophoresis.

Hybridization and thermal denaturation experiments failed to distinguish between the RNAs but two-dimensional electrophoresis of the oligonucleotides produced by ribonuclease T1 digestion revealed several differences. Possibly the most significant of these differences was the size of the polycytidylic acid [poly (C)] tract. There were about 170 nucleotides in the poly (C) tract of the SAT1-7 RNA compared with around 100 in the SAT1-82 RNA. Further evidence for this deletion was provided by the slightly different behaviour of the two RNAs when compared by sucrose gradient centrifugation and polyacrylamide gel electrophoresis.

The salivary glands of planthoppers, Laodelphax striatellus, transmitting barley yellow striate mosaic virus (BYSMV), cryptogram */*:*/*:U/U:S,I/Au, were sectioned and examined in the electron microscope. BYSMV was detected in the cytoplasm but not in the nuclei or other organelles of infected cells which did not show structural changes. The BYSMV virions, 300 to 320 nm long and about 40 nm wide, were frequently arranged in parallel aggregates bound by a membrane. Long flexuous tubules of variable length, 18 to 20 nm wide were found in close association with BYSMV. The tubules were typically found in bundles surrounded by a membrane which also contained virus particles in different stages of organization. The virion of BYSMV is believed to consist of two coaxial helices, the inner derived from the tubules and the outer being formed between the inner helix and the outer envelope of the virus. A hypothesis is advanced for the morphogenesis of BYSMV in insect tissue which differs from that occurring in plants.

Hydrangea ringspot virus has been purified to near homogeneity. Virus nucleic acid was identified as single-stranded RNA with an S value of 33.6 before and 21.1 after formaldehyde denaturation. Nucleic acid mol. wt. determinations made by linear log sucrose density gradient centrifugation gave values of 2.25 × 106 and 2.14 × 106, respectively, for native and formaldehyde-treated RNA. Electrophoresis on polyacrylamide gels before and after formaldehyde denaturation gave mol. wt. values of 2.64 × 106 and 2.03 × 106. The values from formaldehyde denatured RNAs are considered the most reliable. Thermal denaturation profiles showed a Tm of 55.2 °C in 0.1 m-sodium phosphate, pH 7.0, and a hyperchromicity of 22.4%.

A purification scheme is described for the M-protein of Sendai virus and an electron microscope study of the isolated protein is presented. The protein exists as subunits of 6 nm in diam., which possess a central hole; the subunits may be dimers of the polypeptide. They are able to form filamentous aggregates which wind around one another to form a helical structure. It is suggested that these filaments may be the form adopted by the protein in the virus, the filaments lying parallel to one another just beneath the virus membrane to form a shell, but that the helical form is likely to be a property only of the isolated protein.

The antineuraminidase activities of rabbit and human antisera were titrated by their ability to inhibit the elution of influenza virus from fowl red cells. The method was specific, sensitive and simple, and the results correlated well with those of conventional neuraminidase inhibition tests.

The biophysical and biochemical properties of Penicillium cyaneo-fulvum virus (Pc-fV) and Penicillium chrysogenum virus (PcV) have been compared. In sucrose density gradient sedimentation, purified virus preparations gave one major component, L, and three minor components E1, E2 and H with sedimentation coefficients of 145S, 80S, 102S and 172S respectively in each case. E1, E2 were shown to be empty particles. Pc-fV L particles contained only double-stranded RNA, which separated in polyacrylamide gel electrophoresis into four components with mol. wt. 2.21 × 106, 2.08 × 106, 1.98 × 106 and 1.93 × 106. PcV L particles gave three double-stranded RNA components, which were indistinguishable in polyacrylamide gel electrophoresis from the larger three RNA components of Pc-fV. In both viruses each RNA component was separately encapsidated. In both viruses H particles gave rise to the same double-stranded RNA components as their L particles, but contained, in addition, small amounts of single-stranded RNA.

Pc-fV and PcV have been shown to consist of isometric particles of the same size and to be serologically related, and the amino acid compositions of their capsid polypeptides were found to be very similar. The capsid polypeptides of the two viruses were examined by SDS-polyacrylamide gel electrophoresis. In each case E2, L and H particles gave one major polypeptide λ1, with mol. wt. 130000, whereas E1 particles contained one major polypeptide λ2 with mol. wt. 115000. The mol. wt. of L particles, determined from sedimentation and diffusion coefficients, was 9.8 × 106 for both Pc-fV and PcV. The capsid of the L particles of each virus was estimated to contain 60 molecules of polypeptide λ1.

Cells infected with pseudorabies virus excrete large amounts of a glycosylated sulphated protein, mol. wt. 89000, into the extracellular fluid. This paper reports the results of studies on the processing of this protein. Glycosylation occurs during, or very soon after, synthesis of the polypeptide chain. After a delay of several minutes the glycoprotein is sulphated; inhibition of glycosylation by high concentrations of glucosamine does not interfere with this process. The glycosylated sulphated polypeptide is then reduced in size from mol. wt. 99000 to 89000, possibly by proteolytic cleavage, and is excreted. Inhibition of glycosylation does not interfere with the excretion of this polypeptide, which is an energy-requiring process.

A solid-phase radioimmunoassay (RIA) was used to determine the presence of IgG and IgM antibodies to measles virus in human serum and cerebrospinal fluid (CSF). Purified measles virus was adsorbed on to polystyrene balls, which were then exposed to serial dilutions of test serum or CSF. The presence of antibody was measured by its capacity to bind 125I-labelled specific anti-human IgG or IgM.

Serum from a variety of patients as well as measles-immune clinically healthy persons were tested; binding ratios (using negative human serum controls) were usually between 10 and 30, but with subactue sclerosing panencephalitis (SSPE) ratios were as high as 50. Often CSF specimens tested, all but one, which was taken early in the convalescent phase of measles infection, had detectable IgG antibody. In six patients with acute measles, IgM antibodies were found in all serum specimens taken one or more days after the onset of rash. Maximal titres of 1:10000 to 1:40000 were found about 7 days later. Thereafter, IgM titres decreased rapidly but were still detectable at 40 days. A purified ribonucleoprotein of measles virus was also used successfully as an antigen in this RIA method.

RNA extracted from purified encephalomyocarditis (EMC) virus (EMC-RNA) can be aminoacylated with synthetase preparations from Escherichia coli, beef and rabbit liver. The extent of aminoacylation is between 0.024 and 0.080 moles per mole EMC-RNA and occurs only with serine. Neither removal of possible low mol. wt. contaminants with 3 m-sodium acetate nor periodate oxidation of the virus RNA affects its aminoacylation capacity.

The particles of bottom component of aphid-transmitted isolates of pea enation mosaic virus form multiple bands when electrophoresed in polyacrylamide gels; those of bottom component of non aphid-transmitted isolates form a single band. The particles in the multiple bands are separated on size, which is correlated with the presence of an extra protein. The structure of these particles is discussed as is their significance in relation to aphid transmissibility.

Bacteriophage PL-1 adsorbed specifically to fragments of the isolated cell walls of its host Lactobacillus casei ATCC 27092 and failed to adsorb to cell wall fragments of resistant strains. Soon after mixing, an equilibrium situation of phage adsorption was attained. The equilibrium position was dependent on the cell wall concentration, but was not affected by the incubation temperature. The adsorbed phages were not inactivated by the cell wall fragments, but formed phage-cell wall complexes maintaining original phage infectivity. The infectivity of phage-cell wall complexes was neutralized by antiphage sera in the same manner as free phages. When the phage-cell wall complexes were repeatedly washed by centrifuging and resuspending in a fresh medium, the adsorbed phages were eluted as infective virions, confirming that phage adsorption was reversible. When the reactants concerned were allowed to approach equilibrium from the opposite direction, the same equilibrium state was achieved. The value of the equilibrium constant (Keq) with respect to reversible adsorption was constant with various phage concentrations under the conditions used here. When a mixture of phages and cell walls at an equilibrium state was diluted, the unadsorbed phages increased in accordance with the decrease in the concentration of the reactants.

Preparations of highly concentrated simian foamy virus type 1 (SFV1) agglutinate guinea pig red blood cells. The agglutination occurs both at 22° and 37°C. It is inhibited by specific antisera against SFV1 but also by antisera prepared against several other types of simian foamy viruses.

Japanese encephalitis virus (JEV) infected chick embryo (CE) cells were treated with 4 µg actinomycin D/ml and 5 mm-d-glucosamine at 2 or 3 h before harvesting. Production of JEV was not affected by the short-time treatment of these drugs. The radioactivity in virus-specific RNA in the glucosamine-treated cells was apparently higher than in non-treated cells. Nuclear and cytoplasmic extracts were prepared from the JEV-infected cells pulse-labelled with 3H-uridine at 15 h after infection. Analysis of virus RNA in nuclear extracts on sucrose density gradients showed that most of the radioactivity was in 23S RNA, 26S RNA and 8 to 12S RNA. The radioactivity of virus RNA in cytoplasmic extracts was found in 42S RNA and RNA fragments sedimenting at less than 8S.