- Volume 33, Issue 3, 1976

Human lymphoid cells (NC-37) were infected with attenuated measles vaccine virus (Schwarz, AIK-C, and CAM-70 strains), subacute sclerosing panencephalitis virus (Mantooth and Halle strains), neurovirulent TYCSA strain, and wild type virus (Edmonston and Toyoshima strains) at an input multiplicity of 0.01. These strains were divided into two groups by their capacity to establish carrier states. CAM-70, Toyoshima, and Edmonston strains did not set up persistent infections in NC-37 cells, whereas AIK-C strain induced chronic cyclic infection and the Schwarz, TYCSA, Mantooth and Halle strains could set up persistent infections and furthermore two types of persistent infections were recognizable. Cells persistently infected with Schwarz strain contained nucleocapsid structures in both nucleus and cytoplasm, and produced infectious virus of 104 to 105 p.f.u./ml over 100 days after the inoculation of the virus but the cap-formation of measles antigens on the cell membrane was seldom observed. However, in cells persistently infected with TYCSA strain, nucleocapsid structures were rarely observed in the nucleus, but the cap-formation of measles antigens on the cell membrane was often observed. The titre of carried virus was always higher than the number of cells in the range of 106 to 107 p.f.u./ml. Mantooth strain was similar to Schwarz strain and Halle strain was similar to TYCSA strain in the properties of their carrier states. These carrier states were stable and the cells grew normally for over one year.

Significant protection to heterologous i.c. challenge with the flavovirus Langat occurred after a single i.c. injection of avirulent strains of the alpha viruses Semliki Forest or Sindbis given 1 day to 5 weeks before challenge. Some protection also occurred after an i.p. infection with these viruses. We consider that the protection afforded by the alpha viruses is due to interference with the multiplication of Langat virus and is related to the maximum level of brain infectivity reached in the alpha virus infection.

The glucosamine derivative, N-carbobenzoxy-d-glucosamine (NCBZG) inhibits the multiplication of Echovirus-12 and the synthesis of both virus RNA and protein at a stage in the virus growth cycle after attachment and penetration. However, the compound does not inhibit virus multiplication after the appearance of progeny virus nor after virus RNA has accumulated. Incorporation of radioactive glucosamine and choline into infected and uninfected cultures is inhibited by NCBZG as is the virus-induced increase in choline incorporation. The compound also prevents the appearance of radioactive choline in isolated membranous structures. The compound did not alter significantly the cellular RNA or protein synthesis, plating efficiency of the cells, their growth over a period of several days, nor the virus-directed inhibition of cellular RNA and protein. These findings suggest that the compound inhibits virus multiplication by its effect on the initiation of biosynthesis which appears to require membrane synthesis.

Three methods of pelleting, pelleting followed by Pronase treatment, polyethylene glycol (PEG)-Pronase, and diaflo ultrafiltration (diafiltration) were used to concentrate RSV (RAV-1) from tissue culture fluids. Sucrose-gradient fractions containing virus preparations which had been concentrated by diafiltration or pelleting were heavily contaminated with amorphous debris. This debris was not present in similar, gradient-purified preparations that had been concentrated by the PEG-Pronase or pellet-Pronase methods. Maximum recovery of radiolabelled virus particles and virion-associated RNA-dependent DNA polymerase activity was obtained in gradient fractions containing virus concentrates prepared by the pellet-Pronase and PEG-Pronase methods. Although there were slight differences in recovery by these two methods, the advantages of the PEG-Pronase method make it the preferred method, especially when large volumes of tissue culture fluids are used.

In the present study the extent of endogenous, type-C virus genome transcription in normal and regenerating mouse liver was analysed by using the technique of nucleic acid hybridization. The RNA preparations from regenerating liver tissues collected at various intervals following partial hepatectomy, and from normal liver samples of BALB/c mice, were hybridized to 3H-DNA complementary to 60 to 70S RNA of an endogenous, N-tropic virus, released spontaneously from BALB/c mouse cells in culture. Although partial transcription of the endogenous virus genome can be clearly detected in normal liver, a significant increase in the level of virus-specific RNA synthesis in the regenerating liver, in comparison to normal liver, is apparent, following partial hepatectomy. This increase in virus-specific RNA synthesis attains its highest level just before the level of DNA synthesis in the regenerating liver reaches its maximum. These observations may indicate a qualitative or quantitative change in the endogenous type-C virus genome transcription pattern in hepatocytes, in response to partial hepatectomy and suggest that this change in the transcription pattern and the initiation of cell proliferation, in regenerating livers, are probably sequential and related events.

The yields of Pichinde virus, a member of the arenavirus group, were markedly inhibited when infected BHK 21 cells were incubated in the presence of 0.4 to 4 µg/ml of actinomycin D. Maximal inhibition was observed when actinomycin D was added after the adsorption of virus to cultures; however, addition of drug as late as 12 h after infection reduced the 24 h yield by 50%. Virus antigen synthesis, as measured by complement fixation and immunodiffusion, was not dramatically reduced by actinomycin D. The expression of virus antigens on the surface of infected cells was greater on cells treated with actinomycin D than on untreated cells. Putative defective particles with a density of Pichinde virus were not detected in fluids of cultures incubated with actinomycin D and 3H-amino acids. Actinomycin D appears to inhibit Pichinde virus late in the replicative cycle. The observations raise the possibility that the drug inhibits the synthesis of proteins of the host cell membrane which are required for virus maturation.

When protoplasts are prepared from Chinese cabbage leaves 5 to 20 days after inoculation with turnip yellow mosaic virus (TYMV) a variable proportion of the cells possess the rounded and clumped chloroplasts typical of infection with this virus while others appear normal. On exposure to light normal chloroplasts remain unchanged while chloroplasts in diseased cells undergo a striking structural transformation. A large clear vesicle appears in the chloroplast, the chlorophyll-bearing structures being confined to a crescent-shaped fraction of the chloroplast volume. This ‘sickling’ effect occurs only in the light. Red and blue light are equally effective. There is a lag period of about 4 h in the light before protoplasts with sickled chloroplasts begin to appear. The proportion of protoplasts showing sickled chloroplasts after 24 h exposure increases with increasing light intensity up to about 2000 lux. The production of sickled cells is partially suppressed when glucose, fructose, sucrose, or certain other sugars are added to the incubation medium. The electron transport inhibitor DCMU completely suppresses the formation of sickled chloroplasts at a concentration of 10−6 m.

Cells infected with herpes simplex virus type 1 (HSV-1) DNA by the calcium phosphate precipitation technique produce virus which leads to the formation of plaques (Graham, Veldhuisen & Wilkie, 1973). In the study reported here we show that treatment of cell monolayers with dimethyl sulphoxide (DMSO) solutions after infection with DNA-calcium phosphate complexes leads to a considerable increase in the number of plaques obtained. The conditions for this enhancement of infectivity have been optimized for baby hamster kidney (BHK) cells, and increases in plaque numbers of over 100-fold have been obtained. The treatment appears to increase the proportion of cells which respond to DNA infection by initiating plaque formation, and results in a large increase in the measured specific infectivity of HSV-1 DNA. DMSO causes similar (but quantitatively different) responses in various other cell lines infected with HSV-1 DNA. BHK cells infected with either virus particles, or virus DNA by the DEAE-dextran technique (Laithier & Sheldrick, 1975), do not exhibit this massive enhancement following exposure to DMSO.

Pre-treatment of Newcastle disease virus (NDV) with fresh human plasma enhances its haemolytic (HL) capacity by several factors. The effect is due to complement activation by the heterophile anti-chick antibody present in human plasma. All the adult human plasmas tested were effective, also 91/100 human cord blood sera. The antibody was mainly of the IgM class. The enhanced HL was due to integration and transference of the complement ‘holed’ virus envelope membrane and subsequent leakage of haemoglobin. High concentration of activated complement destroys the integrity of the virus envelope. Treatment of chick erythrocytes and fibroblasts with human plasma also produced lysis of the cells.

An investigation of the activity of nuclear RNA polymerase following infection of LS cells with HSV-1 shows a decline in both major activities. This effect is not entirely due to inhibition of cellular protein synthesis, and the effect of α-amanitin-sensitive RNA polymerase is mediated by a protein(s) synthesized in the infected cell. Changes in the properties of this RNA polymerase activity include a reduction in the relative UTP/GTP incorporation ratio and an increased sensitivity to inhibition by actinomycin D, indicating that RNA polymerase II is involved in virus transcription.

RNA from a clone of polyoma virus-transformed hamster cells was fractionated by chromatography on oligo(dT)-cellulose. The proportion of virus-specific RNA in the polyadenylated and non-polyadenylated fractions was determined by hybridization of the labelled RNA with excess purified polyoma DNA, immobilized on filters. Seventy to 80% of the virus-specific RNA in both polysomal and total cell RNA was found in the polyadenylated fraction. Since it has been shown previously that more than 65% of the total virus-specific RNA is restricted to the nucleus in this cell line, these results indicate that a high proportion (at least 53%) of the nuclear virus-specific RNA is polyadenylated.

The sedimentation profile of total polyadenylated virus-specific RNA in dimethyl sulphoxide was comprised mainly of a broad band with a median sedimentation coefficient about 26S (relative to 28S rRNA). This profile was similar to that of total nuclear, and not cytoplasmic, virus-specific RNA.

To estimate the intramolecular proximity of virus-specific sequences to poly(A), total RNA was subjected to limited thermal scission to an average mol. wt. similar to that of mRNA. The RNA remaining attached to poly(A) was then isolated, using oligo(dT)-cellulose. It was found that 65% of the virus-specific RNA that was originally attached to poly(A) was released by the thermal scission. Most of the virus-specific sequence within polyadenylated RNA molecules therefore must have been located at some distance from the polyadenylated 3′-terminus. This observation, together with the results of sedimentation analysis, can most simply be explained by postulating the existence of ‘hybrid’ RNA molecules containing a host-specified sequence located between a virus-specific sequence and the 3′-terminal poly(A).

Fragments of foot-and-mouth disease virus RNA of decreasing size, containing the 3′ poly(A) sequence have been prepared by alkali treatment and sucrose gradient centrifugation followed by oligo(dT)-cellulose affinity chromatography. Polyacrylamide gel electrophoresis of the ribonuclease T1 resistant oligonucleotides from these polyadenylated fragments has enabled us to locate the position of some of the longer oligonucleotides on the RNA. In particular the poly(C) tract has been shown to be near the 5′ end of the RNA. The possible function of the poly(C) tract is discussed in the light of these findings.

The morphogenesis of a cytopathic bovine rotavirus isolate was examined in MDBK cells. Distension of cisternae of the rough endoplasmic reticulum was seen 14 h post-infection (p.i.), but few virus particles were present. Many virus particles were observed within distended cisternae of the endoplasmic reticulum 22 h p.i., and matrices of viroplasm were found close to developing virus particles. The virus particles measured 68 to 72 nm in diam. and consisted of an electron-dense nucleoid surrounded by a single less electron-dense shell. In contrast, many of the virus particles observed 54 h p.i. possessed an additional, outer, electron-dense shell. These double-shelled particles were 76 to 82 nm in diam. and acquired the outer shell by a budding process. Virus was released from the cell by discharge through breaks in the plasma membranes of damaged cells.

Uracil was incorporated into all three bacteriophage φ6 dsRNA segments throughout the infection cycle; the rates of incorporation into each of the three segments were approx. constant for the first 15 to 20 min and then increased rapidly until 50 min after infection. The medium and small dsRNA segments were produced in greater amounts than the large dsRNA segment at all times in the infection cycle. Inhibition of host RNA and protein synthesis with rifampin and chloramphenicol revealed that virus dsRNA synthesis immediately after infection was independent of either host function.

The Kuwait-5-67 strain of variola virus, although indistinguishable from variola virus in its other properties, behaved atypically in immuno-diffusion tests with antivaccinia serum. The precipitation line pattern produced was similar to that of cowpox virus but lacked one of the main precipitation bands given by all other strains of variola, alastrim and vaccinia viruses tested. Trypsin treatment of cowpox virus preparations revealed the missing precipitation line but did not do so with the Kuwait-5-67 strain. The antivaccinia serum absorbed by Kuwait-5-67 strain lost its ability to interact with this strain, but still gave precipitation lines with variola, vaccinia and cowpox viruses.

Human fibroblast and mouse L929 cell interferons can be purified by adsorption to and subsequent elution from Controlled Pore Glass. Purification of 40 to 90-fold to specific activities of 1 to 5 × 106 units/mg of protein can be achieved in a single step, with good recovery of activity. Human leukocyte interferon does not bind to the glass and cannot be purified in this way.

The 3 major lipoprotein fractions of human serum were investigated for their ability to inhibit the haemagglutination of 24 different togaviruses including rubella virus and arboviruses from 5 antigenic groups. All viruses were inhibited by the 3 fractions and this was most evident with low and very low density lipoproteins; inhibitory activity appeared to be associated with the lipid part of the lipoproteins. When artificial dispersions of the lipids cholesterol, cholesteryl ester and triglyceride were emulsified with egg lecithin three categories of inhibitory activity were observed which were independent of virus antigenic grouping — category 1, no activity; category 2, activity confined to cholesterol and category 3, activity for cholesterol and to a lesser extent the other lipids.

Poliovirus was treated with fluorescamine and disrupted with sodium dodecyl sulphate (SDS). The polypeptides were isolated by electrophoresis in a polyacrylamide gel (SDS-PAGE) and their amino acid compositions determined. The compositions of VP1, 2 and 3 were very similar but different from that of VP4.