- Volume 64, Issue 6, 1983

Liver sections from five patients with persistent hepatitis B virus (HBV) infection and active cirrhosis were shown to contain intracellular HBV DNA by in situ hybridization using cloned 3H-labelled HBV DNA probes. Two classes of infected cells, with different distributions throughout the liver, were distinguished: (i) cells containing a low copy number of double-stranded HBV nucleotide sequences, confined to the cell nucleus and thought to represent HBV DNA, and (ii) cells containing large amounts (estimated to be greater than 10 or 15 genome copies per cell) of HBV DNA, much of it in a single-stranded form and largely confined to the cell cytoplasm; these single-stranded regions represented widely separated regions of the HBV genome, in contrast to the structure of the DNA in mature virions. It is likely that these latter cells may be supporting viral DNA synthesis. Cells with large amounts of cytoplasmic HBV DNA invariably contained hepatitis B surface antigen (HBsAg) and in addition contained either no detectable hepatitis B core antigen (HBcAg), or cytoplasmic HBcAg or nuclear HBcAg in that order of frequency. Cytoplasmic HBcAg was highly predictive of the presence of large amounts of cytoplasmic HBV DNA in the same cell, while either nuclear HBcAg, or cytoplasmic HBsAg, were often seen both in cells with and without such levels of DNA. These patterns, relating HBV DNA and antigen content in naturally occurring asynchronous infection in a heterogeneous cell population, should provide a background to further studies of the virus replication cycle with a defined experimental system, when such a system becomes available.

Using the indirect immunoelectron microscopy technique, it was investigated whether during assembly of murine leukaemia virus (MuLV) and vesicular stomatitis virus (VSV), the glycoproteins (gp) Thy-1, H-2, Pgp-1 and T-200 present on the surface of BW5147 and BuEL4 leukaemia cell lines were incorporated into the virus envelopes. This work was done mainly with monoclonal antibodies against these gps to exclude the presence of antibodies against endogenous MuLV present in conventional mouse antisera. Thy-1 gps were incorporated into MuLV and VSV envelopes. In contrast, H-2, Pgp-1 and T-200 gps were excluded from the budding of both virus particles. To study whether the presence of Thy-1 gps on the viral envelopes is due to some lateral affinity of this molecule with viral gps, the physical association of Thy-1.1 antigens and MuLV antigens was studied with antibody-induced redistribution of both antigens on the BW5147 cell surface. Antibody-induced patching of the viral antigens did not result in co-patching of the Thy-1.1 antigens. In the reciprocal tests no co-redistribution of viral antigens with Thy-1.1 antigens was seen. These studies show that the presence of Thy-1.1 gp on the MuLV envelope cannot be due to a lateral affinity of this molecule with viral gps and that a selection of surface gps takes place during assembly of MuLV and VSV.

Thirteen IgG monoclonal antibodies to the envelope protein of 17D yellow fever virus (17D YF) were produced. All of the antibodies, whether type-specific to 17D YF or flavivirus cross-reactive, mediated antibody-dependent enhancement (ADE) of virus growth in P388D1 cells. There was no consistent relationship between ADE titres and the degree or pattern of neutralizing and/or haemagglutination inhibition activity. Monoclonal antibodies of different isotypes were used to investigate further the properties of P388D1 Fc receptors. The effects of trypsin treatment of P388D1 on ADE were similar to those previously described in experiments measuring direct binding of IgG proteins or rosetting of sheep red blood cells (SRBC) by macrophages, demonstrating sensitivity to digestion by trypsin of the Fc receptor for monomeric IgG2a but not for IgG2b. Aggregated myeloma proteins of IgG2a and IgG2b isotypes competed equally well with either IgG2a or IgG2b monoclonal antibodies to 17D YF in inhibition of ADE. However, selective inhibition by the homologous isotype was observed when rosetting by P388D1 of SRBC coated with IgG2a or IgG2b monoclonal antibodies was examined. These results may help to explain apparent discrepancies previously reported between experiments utilizing direct binding of IgG proteins and those using rosetting of antibody-coated SRBC to examine Fc receptor properties and indicate that immune complexes of virus and antibody resemble aggregated immunoglobulins with respect to macrophage Fc receptor function and differ from antibody-coated SRBCs.

Low doses of u.v. radiation rapidly inactivate poliovirus, and the virus is progressively converted into dense particles (DPs) of buoyant density 1.44 g/ml in CsCl. The DPs are structurally and antigenically related to standard virus (N-antigen), i.e. they are indistinguishable from virus in their RNA and protein content and in their sedimentation properties. Furthermore, there is no difference in reactivity of the structural proteins of virus and DPs with the monofunctional reagent [3H]N-succinimidyl propionate (3H-NSP). However, DPs differ from virus in that their capsids are permeable to several ions, and they can be degraded by RNase and protease. Increasing the radiation dose causes a successive transformation of DPs into 105S slow-sedimenting particles (SSPs). The SSPs are antigenically related to 76S artificial empty capsids (AECs) or H-antigen, but they differ physically and structurally from them. The SSPs have a higher S value than AECs and contain all the capsid proteins, including VP4, and the RNA, both of these macromolecules being absent from AECs. It is concluded, therefore, that transformation from N- to H-antigenicity by u.v. radiation does not require release of RNA and VP4. Conversion of virus particles to SSPs correlates with altered reactivity of VP2 and to a lesser extent VP1 and VP3, with the monofunctional reagent 3H-NSP.

RNA synthesis in Bunyamwera virus-infected cells was analysed either by sedimentation analysis in SDS-containing sucrose gradients or by hybridization procedures involving annealing with viral genome RNA (vRNA) followed by electrophoretic analysis. Using either procedure, none of the virus-specific RNAs from infected cells was found to be polyadenylated when analysed by oligo(dT)-cellulose chromatography. In addition, viral messenger RNA activity was found to be associated only with non-polyadenylated RNA species when assayed in an in vitro translation system. The infected cell RNAs could be partially resolved by sucrose gradient centrifugation, and virus-specific RNAs of each polarity were present in these preparations which indicated that the characteristic amplification of secondary transcription was occurring. In the presence of cycloheximide or puromycin, no detectable primary RNA transcription occurred. The same inhibitors, when used later in the infection cycle, caused a dramatic and almost complete inhibition of secondary RNA transcription. The inhibition of RNA synthesis caused by these drugs appeared to be fully reversible. Thus, these inhibitors of protein synthesis affect both primary and secondary RNA transcription by Bunyamwera virus indicating that this virus employs transcription mechanisms different from those known for other families of negative-stranded viruses. Hybridization of 32P-labelled vRNA from Bunyamwera virus with RNA extracted from virus-infected cells produced four duplex RNA molecules that were resolved by gel electrophoresis. Analysis by hybridization and oligonucleotide mapping showed that the two larger duplexes contained complementary (c)RNAs that were transcribed from the L and M segments of viral RNA while the cRNAs contained in the two smaller duplexes were both transcribed from the S RNA segment. Based on a comparison of their oligonucleotide fingerprints, the two latter cRNAs showed a considerable sequence overlap.

The presence of the gene Mx results in a greater interferon (IFN)-mediated antiviral resistance in cells challenged by orthomyxovirus. In the absence of IFN no difference in influenza virus yield was detected in Mx-bearing (Mx/Mx) or non-bearing (+/+) mouse embryo fibroblasts. Although the putative product of the gene Mx has been suggested to act in concert with IFN it is not clear whether Mx was itself induced by IFN. Binucleate heterokaryons were formed by the fusion of an Mx/Mx cell and an IFN-treated +/+ cell. Results show that no constitutive activity was present in the Mx cell that was not IFN-treated, which was capable of enhancing the marginal anti-influenza virus resistance characteristic of the IFN-treated +/+ cell. Heterokaryons between +/+ cells and IFN-treated Mx/Mx cells, however, showed resistance characteristic of the Mx-containing cell. Hybrids of fusions between IFN-pretreated +/+ cells and Mx/Mx cells that had been IFN-treated for 1 to 2 h revealed a graded response to influenza virus growth inhibition. Resistance in such hybrids increased commensurate with the development of the anti-influenza virus state in the Mx/Mx donor. No synergistic enhancement of influenza virus growth inhibition was observed. These data suggest that the product of the gene Mx is induced by IFN. Cybrids, constructed between nucleated +/+ or Mx/Mx cells and enucleated Mx/Mx cytoplasts, when subsequently treated with IFN and challenged with influenza virus, showed resistance characteristic only of the nucleated cell donor. Thus, no cytoplasmic component present in Mx-bearing cytoplasts is able to cooperate with the nuclear elements of a +/+ cell to induce a state of high influenza virus resistance. The simplest interpretation of these data is that specific resistance is not the result of modifications of virus growth or IFN activities but probably occurs by the IFN-mediated induction of the gene Mx, the product of which directly, or indirectly, interferes with orthomyxovirus replication.

A colorimetric assay for defective interfering (DI) particles of respiratory syncytial (RS) virus was developed. This quantitative biological assay is based on neutral red dye uptake by DI particle-protected cells that survive standard virus challenge. This assay was more sensitive than the reduction of infectious yield (RIY) assay and was capable of detecting 1 × 104 to 2 × 104 DI particles/ml. The coefficient of variation for parallel, simultaneous replicates (n = 10) was 23%. Cell-protecting activity in the colorimetric assay appeared simultaneously with activity in the RIY assay on undiluted passage of plaque-purified virus. Both activities were particulate, were inactivated by RS virus antiserum and exhibited similar ultraviolet-inactivation kinetics. The absolute values of the slopes of dilution curves for both assays were similar, and using regression analysis both assays enabled estimation of similar numbers of active particles. These results suggest that both activities are mediated by the same DI particle. The mechanism of cell protection does not appear to involve extracellular interferon because the inclusion of interferon antibody in the assay did not diminish DI particle cell protection. Finally, the colorimetric assay was used to reveal alternating cycles of infectious and DI virus production on serial undiluted passage.

We report the ultrastructural features associated with wild-type and temperature-sensitive (ts) mutant infection at non-permissive temperature for each of 18 herpes simplex virus type 1 (HSV-1) (strain 17) ts mutants. The mutants were classified by their ability to induce nucleocapsid-related structures:class I failed to induce any, class II induced empty and partially filled structures while class III induced all of the identified structures including those containing an electron-dense core. The time when expression of the ts lesion blocked virus replication was estimated for most mutants; this allows both mutant gene expression and the resulting ultrastructural features to be correlated with the sequence of virus replicative events. Three ts mutant-triggered features not previously described in HSV-1-infected cells were also recognized: a modification of rough endoplasmic reticulum, intranuclear accumulation of enveloped virus particles and cytoplasmic accumulation of novel doughnut-shaped particles having a concentric double ring appearance in thin section.

We have previously reported on the characterization of a novel ribonucleotide reductase induced in herpes simplex virus type 2 (HSV-2)-infected mammalian cells. The virus-induced enzyme was partially purified, free of the constitutive cell isozyme, by fractionation of infected cell extracts with ammonium sulphate. In this report we describe a further purification of the virus-induced enzyme and the development of a rabbit antiserum capable of specifically inhibiting its activity. Enzymically active salt fractions from infected cell extracts were sedimented through glycerol gradients; the virus-induced enzyme was found to sediment approximately 2.5 times faster than the constitutive, or control, enzyme and was separated from the majority of the protein in the sample. Immunization of rabbits with the partially purified enzyme preparation recovered from gradients resulted in the synthesis of antibodies which completely and specifically inhibited the virus-induced reductase, and precipitated one major polypeptide and a few minor species from both HSV-1- and HSV-2-infected cells. The antibodies, however, exhibited much greater affinity for the HSV-2 than the HSV-1 antigen. These results demonstrate that the virus-induced enzyme differs antigenically, as well as biochemically, from the constitutive cell isozyme and lend further support to the hypothesis that the enzyme is virus-coded.

Induction of Epstein–Barr virus antigens in Raji cells by 12-O-tetradecanoylphorbol 13-acetate (TPA), 5′-iodo-2′-deoxyuridine (IUdR), n-butyric acid (n-BA) antibody to IgM (anti-IgM) and a recently described serum factor was studied after single applications of individual inducers or of their combinations. Whereas individual inducers led to activation only in a limited number of cells, certain combinations of inducers greatly enlarged the reactive cell population. In synergistically acting combinations of drugs, the dose necessary for half-maximal induction of the first inducer was shifted to a much lower value by the second inducer. The serum factor cooperated with any of the four inducers TPA, IUdR, anti-IgM and n-butyric acid, whereas TPA only interacted with IUdR and n-butyric acid. The effects of the serum factor and of TPA showed some similarities but were not identical, whereas IUdR and n-BA showed identical effects.

The dye trypan blue inhibits infection of cells in tissue culture by measles and herpes simplex viruses at concentrations in excess of 0.01% (w/v) principally by direct inactivation of the virions. The inactivation is temperature-dependent (104 times greater at 37 °C than at 4 °C) and its mechanism appears to include inhibition of virus adsorption to cells.

The role of pseudorabies virus (PRV) thymidine kinase (TK) expression in the pathogenesis of PRV infection of mice was studied with TK-negative (TK−) mutants. Thymidine phosphorylation and arabinosylthymine inhibition of PRV replication and efficiency of plating were used to characterize TK+ and TK− PRV. In addition, a plaque autoradiography procedure was utilized to determine the TK phenotype of individual plaques. TK+ and TK− PRV replicated well in ocular tissues, while TK+ but not TK− did so in ganglion tissue. Mortality was absent after TK− PRV inoculation and widespread after inoculation of similar amounts of TK+ PRV. Latent infection in mice was not detected with either TK+ or TK− PRV. This study indicated the probable importance of PRV TK expression in acute trigeminal ganglion infection.

It was found that the virion terminal protein of chick embryo lethal orphan (CELO) virus had a molecular weight of 46000, and the hexon a molecular weight of 100000. 125I-labelled tryptic and chymotryptic peptide maps of the hexons and terminal proteins from CELO virus and human adenovirus type 5 (Ad5) differed. However, limited proteolysis of CELO virus and Ad5 terminal proteins by protease V8 showed similarities which were not detected in the case of the two hexons.

Different methods of molecular hybridization were used to study DNA sequences of the highly oncogenic simian adenovirus SA7 (C8) present in the genomes of two transformed rat cell lines and in cells from three hamster tumours induced by adenovirus SA7. The entire DNA or the left-hand terminal SalI C fragment (19.5% of the genome) were employed. All cell lines retained an intact left-hand region of the SA7 genome (0 to 12.4 map units). The blot hybridization technique failed to detect any site specificity of integration of SA7 DNA into the cell genome. In all cell lines the expression of the BglII D fragment (1.8 to 10 map units) of SA7 DNA was observed. As judged by the patterns of integration of virus sequences into the cell genome, the highly oncogenic simian adenovirus SA7 (C8) is similar to the non-oncogenic human adenoviruses of group C, and is different from the highly oncogenic human adenovirus type 12.

Administration of potent mouse interferon preparations or anti-mouse interferon globulin did not influence the evolution of scrapie in mice after intraperitoneal injection of the agent. We conclude that the interferon system is probably not involved in scrapie.

MG63 cells induced by Sendai virus produce human interferon (IFN)-β with a molecular weight of 22000. In the presence of tunicamycin, they produced non-glycosylated, biologically active, IFN-β with a molecular weight of 17000. It had slightly lower heat stability (at 56 °C) than normal glycosylated IFN, but the sensitivity to neutralization by antisera and the cross-species activities on murine, bovine and hamster cells were unaltered, indicating dispensability of the carbohydrate moiety for these parameters. However, the IFN yields were markedly reduced by the glycosylation inhibitor, suggesting an important role of glycosylation in production by cells of IFN-β.

Ibaraki virus core particles were purified from infected BHK-21 cells. The core particles displayed RNA transcriptase activity while the virion did not. The RNA transcriptase activity was stimulated about 15-fold by addition of S-adenosyl-l-methionine. The reaction product was single-stranded RNA which could be hybridized with heat-denatured Ibaraki virus RNA. The hybrids possessed the same electrophoretic mobility as Ibaraki virus double-stranded RNA.