- Volume 66, Issue 2, 1985

Thymidine kinase (TK) induced by varicella-zoster virus (VZV) was precipitated with ammonium sulphate and purified by Sephadex G-150, QAE-Sephadex and Blue Sepharose column chromatographies. The purified TK fraction also contained deoxycytidine kinase (dCK) activity and a 35 000 mol. wt. (35K) polypeptide as a major component. The TK and dCK activities were both neutralized by anti-VZV serum. Antiserum to an extract of cells infected with a bromodeoxyuridine (BUdR)-resistant mutant virus contained no antibody to the viral TK and dCK activities or to the 35K polypeptide. Antiserum to the purified viral TK fraction was prepared and absorbed with a lysate of BUdR-resistant mutant virus-infected cells. The resulting absorbed antiserum (anti-vTK serum) neutralized the viral activities of both TK and dCK, and specifically immunoprecipitated a 35K polypeptide from the lysate of parental virus-infected cells, but did not immunoprecipitate any detectable polypeptide from cells infected with BUdR-resistant mutant virus. Anti-vTK serum stained mainly the nuclei of cells infected with the parental virus strain, but did not stain those infected with BUdR-resistant mutant virus by an indirect fluorescent antibody test. These results suggest that the 35K polypeptide produced in VZV-infected cells is responsible for the viral TK and dCK activities, and that the TK and dCK are mainly localized in the nuclei of infected cells.

An antiserum was produced to the oligomeric form of glycoprotein B (gB) induced by herpes simplex virus type 1 (HSV-1) strain 17. This antiserum gave a single common precipitin line in agar gel immunodiffusion with HSV-1, HSV-2, bovine mammillitis virus (BMV) and equine herpesvirus type 1 (EHV-1). It also neutralized HSV-1, HSV-2 and BMV but not EHV-1. Absorption of the antiserum with excess HSV-2 or BMV antigen resulted in an HSV-1-specific neutralizing antiserum. In immunoprecipitation, two proteins, gB and pgB, were precipitated from HSV-1- and HSV-2-infected cells and at least three from BMV- and EHV-1-infected cells. Glycoprotein B and pgB of three HSV-1 and three HSV-2 strains and the corresponding antigenically related glycoproteins of BMV- and EHV-1-infected cells were labelled with 125I, digested with trypsin and the resulting peptides separated by two-dimensional thin-layer chromatography or high-pressure liquid chromatography. The resulting profiles were found to be almost identical, suggesting considerable structural conservation of the peptide backbone of the antigenically related glycoproteins of these four viruses.

Nineteen herpes simplex virus (HSV) antigen-specific human T lymphocyte clones were established from three volunteers with recent recurrent herpes labialis. All produced interferon gamma (IFN-γ) at titres of 200 to 700 units/ml when cultured in vitro with HSV antigen and irradiated peripheral blood mononuclear cells (PBMC) as filler cells. All 10 of those clones whose phenotype was determined were Leu 4+, Leu 2−, Leu 3+. Interleukin 2 alone failed to induce IFN-γ in titres greater than 10 units/ml from these clones cultured at 104/0.2 ml/well. However, the effect of different accessory or filler cells on IFN-γ production by clones was quite marked. For example, high titres were produced when irradiated PBMC or plastic-adherent cells (predominantly monocytes) were added and low titres when macrophages and irradiated Epstein-Barr virus-transformed B (EBV-B) cells were added. When tested for HSV antigen-stimulated IFN production alone, the irradiated PBMC and adherent cells produced low titres, but no detectable interferon was produced by the others. However, with higher concentrations of EBV-B cells, low concentrations of IFN-α were occasionally produced. Irradiation strikingly reduced IFN-α production by PBMC. The IFN-α and -γ produced by accessory cells may contribute to total IFN production by priming the production by cloned cells, and acting in synergy with IFN-γ produced by the cloned cells. Alternatively, the effect may be due to the presence of permissive concentrations of other lymphokines such as the interleukins. Interferon production by cloned T lymphocytes in the presence of non-producing macrophages was maximal within 24 h, much faster than with a similar polyclonal system, although attaining lower titres. EBV-B cells from only one of three patients supported antigen-specific lymphocyte activation. Almost all cells of the three cell lines expressed DR antigens, while DS/DC antigens were also expressed on nearly all cells of the antigen-presenting line and, at lower densities, on two-thirds of the cells of the other two lines.

The effect of herpes simplex virus type 2 (HSV-2) infection on the frequency of mutations at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus was studied in the non-permissive XC cell line. When the cells were infected with 20 to 800 p.f.u./cell, there was initially a lag in cell growth and cell death, but after 4 days there was no difference in growth rate between infected and control cultures. However, the mutation frequency, as determined by the number of 6-thioguanine-resistant colonies, was increased in infected cultures by factors ranging from 2.5 to 10.3. This effect was found to be dependent on the multiplicity of infection. The maximum effect was obtained between 20 and 100 p.f.u./cell while further increase in the amount of virus resulted in a drop in the yield of mutants. The optimum multiplicity of infection was a reproducible characteristic but was variable between viral stocks. When a number of mutant clones were examined they were found to have HGPRT activities ranging from undetectable to 6.9% of wild-type, indicating that the mutations were in the HGPRT gene. These results show that, in a non-productive infection, HSV-2 particles can increase the mutation frequency. The possible mechanisms by which this effect is brought about in the host genome are discussed.

In addition to its antiviral effect, interferon, at high concentrations, stimulates steroidogenesis and provokes cell rounding in Y-1 mouse adrenal tumour cells. This stimulation was inhibited by cytochalasin B and colchicine. In contrast, dibutyryl cAMP and cholera toxin, also able to induce steroid production and cell rounding, increased steroid production even in the presence of these cytoskeleton-disrupting agents. The initial trigger for interferon or cholera toxin thus probably involves a distinct receptor organization. However, since both inducers increased cAMP synthesis in this differentiated cell line, the further metabolic steps of ketosteroid production could be the same.

Intracytoplasmic type A particles known to be precursors to type B retroviruses in murine, hamster and marsupial cells are closely associated with microtubules and microtubule organizing centres. In this publication, the active participation of microtubules in the intracellular transport of the particles to the cell surface has been examined in NIH 3T3 cells infected with M432 virus using vincristine sulphate (VCR) as inhibitor of microtubule polymerization. The release of virus at different times after exposure to VCR was quantified by reverse transcriptase determinations of cell supernatants and by electron microscopic quantification of the number of virions at the cell surface using freeze-dried whole cell replicas. These studies indicate that VCR inhibits both microtubule polymerization and virus release, and thus suggest that intact cytoplasmic microtubules are necessary for intracellular transport and release of virus.

Ultrastructural studies of the uptake of enveloped and naked frog virus 3 (FV 3) particles by BHK-21 cells have shown that enveloped viruses are internalized by adsorptive endocytosis via coated pits. The enveloped particles then appear to move through endosomes and finally lysosomes. Naked viruses may also follow the same pathway but only rarely. Their more frequent mode of entry is by fusion between the virus shell and the cellular membranes, thus allowing the virus to shed its nucleoprotein content directly into the cytoplasm. This difference in the mechanism of penetration has been confirmed by the use of lysosomotropic agents: the inhibition of viral growth being far more drastic for enveloped FV 3 than for naked virus implies that a lysosomal step is required for the multiplication of enveloped viral particles.

The role of neuraminidase and the mechanism of low pH dependence in influenza virus-induced membrane fusion have been studied further using fowl plague virus (FPV, H7N1). Two specific anti-FPV neuraminidase antisera obtained from chickens immunized with recombinant virus strains inhibited viral neuraminidase activity without influencing its haemagglutinating activity. These sera totally inhibited the FPV-induced fusion of erythrocytes and partially reduced haemolysis. But both fusion and haemolysis activities could be restored by external addition of Vibrio cholerae neuraminidase, indicating participation of neuraminidase in FPV-induced membrane fusion. With regard to low pH-dependent fusion by influenza virus, it was found that erythrocytes of various species showed different pH optima for haemolysis by FPV and that erythrocytes could be sensitized for fusion and haemolysis by FPV at neutral pH if they had been pretreated with a low pH buffer. These results demonstrated that surface properties of erythrocytes rather than that of the virus are critical in the low pH-dependent fusion and haemolysis by influenza viruses.

When type 1 poliovirions were extracted from infected HeLa cells and heated at 56 °C in the presence of 1 mm-Mg2+, RNA-free, pluri-antigenic particles were formed. These particles possessed neutralization epitopes as well as epitopes associated with H antigen and with the isolated capsid protein VP3. The heating caused the formation of exclusively H antigenic particles when the Mg2+ concentration was 10−4 m or lower, or when the virus was further purified in order to remove remaining cellular components. The ability of purified virus to form pluri-antigenic particles upon heating could be restored by incubation at 37 °C with infected cell extract.

A tsRNA− intertypic recombinant, v3/al-25, which has the 5′ and 3′ halves of the genome derived from the neurovirulent type 3 poliovirus strain 452/62 3D and the attenuated type 1 poliovirus strain LSc-gr3, respectively, was previously shown to cause severe paralytic poliomyelitis after intracerebral inoculation of monkeys. To ascertain whether the illness was caused by the recombinant itself or by temperature-resistant trRNA+ mutants that might have arisen in the inoculated monkeys, five independent virus strains have been isolated from the spinal cord of the diseased animals. While two of these isolates exhibited RNA+ and RNA± phenotypes, respectively, the other three strains retained the parental RNA− character. Except for the RNA+ strain, the RNase T1 oligonucleotide maps of the genomes of all the isolates revealed only a minimal deviation from the parental pattern. These results were interpreted to mean that v3/a1-25 is intrinsically neurovirulent despite the presence of a tsRNA− mutation(s) in the 3′ half of its genome. Nevertheless, this mutation, or other peculiarities of the 3′ half of the recombinant genome, may somewhat alleviate the pathogenicity of the virus. This notion was inferred from the fact that, when used in a relatively small dose (about 103 p.f.u.), v3/a1-25 appeared to exhibit a lower level of neurovirulence compared to either the wild-type parent 452/62 3D, or a closely related intertypic recombinant having the genome 3′ half derived from a neurovirulent trRNA± type 1 poliovirus strain. The problem of genetic determination of poliovirus neurovirulence and attenuation is briefly discussed.

From a genomic DNA library of Sendai virus, we have identified and sequenced clones corresponding to the F glycoprotein gene. The limits of the F gene region were defined by mapping the 5′ and 3′ ends of the mRNA with S1 nuclease. The Sendai virus F gene is 1821 nucleotides long. The predicted primary translation product of the single long open reading frame would code for a protein of 565 amino acids, containing a putative signal peptide, three carbohydrate addition sites, a hydrophobic region corresponding to the known cleavage/activation site of F0, and a long, very hydrophobic region near the C-terminus which probably represents the transmembrane region of the protein. The signal peptide cleavage site of the mature protein was determined by mass spectrometry. Interestingly, the amino acid sequence surrounding the cleavage/activation site of the Sendai virus F protein shows significant homology to the same region of the influenza B and C virus HA proteins, suggesting that these genes may have evolved from a common ancestor. The ability of the Sendai virus F protein to fuse membranes relative to its primary structure is discussed.

Neutralizing monoclonal antibodies incorporated into plaque assay overlay medium were used to select antibody-resistant (AbR) mutants of both the Herts (using antifusion protein monoclonal 481) and Beaudette C (using anti-haemagglutinin-neuraminidase protein monoclonal 445) strains of Newcastle disease virus at the permissive temperature of 34 °C. Certain of the Herts, but none of the Beaudette C, AbR mutants were also temperature-sensitive (ts −) and failed to form plaques at the non-permissive temperature of 41.5 °C. [35S]Methionine-labelled proteins from chick embryo fibroblasts infected with wild-type, ts + AbR and ts − AbR virus when separated by two-dimensional polyacrylamide gel electrophoresis revealed a variety of changes in the isoelectric point of the fusion protein F (using monoclonal 481) and the haemagglutinin-neuraminidase protein HN (using monoclonal 445). The ts + ‘revertants’ of ts − AbR mutants remained AbR and also showed changed isoelectric points in the F protein.

The protease inhibitor leupeptin prevented multiplication of the human coronavirus strain 229E in cultures of MRC-C cells. The IC50 of leupeptin in plaque reduction tests was 0.4 µg/ml, whereas growth of host cells was unaffected by leupeptin at 50 µg/ml. Inhibition of plaque formation could be prevented by the addition of proteases to the overlay medium. In single-cycle growth experiments, leupeptin reduced virus yield only if added within 2 h of infection, indicating its action on an early stage of virus replication.

Several temperature-sensitive mutants of encephalomyocarditis virus were serially passed numerous times in HeLa, L929 and BHK-21 cells. The reversion of most of the mutants occurred most rapidly in BHK-21 cells and least rapidly in HeLa cells.

A defective subacute sclerosing panencephalitis (SSPE) virus which had been passaged in human embryonic lung cells was transferred to cultures of three neural cell types: neuroblastoma, oligodendroglioma and glioblastoma. The growth characteristics of the virus in these cells were essentially similar to those in non-neural cells. On the other hand, a marked difference in neurovirulence was noticed for the virus grown in neural cells when examined by intracerebral inoculation into mice. The virus passaged in neuroblastoma and oligodendroglioma cells showed high neurovirulence, inducing an acute encephalitis, whereas the virus passaged in human embryonic lung cells and that in glioblastoma cells did not show neurovirulence. These results suggest that the virus recovered its neurovirulence after passages in certain human neural cells.

In vitro cleavage of Gazdar murine sarcoma virus Pr65gag, which has all of the antigenic determinants of Moloney murine leukaemia virus Pr65gag, i.e. p15, p12, p30 and p10, by the Moloney murine leukaemia virus proteolytic activity yielded a p30 whose partial NH2-terminal sequence was identical to Moloney murine leukaemia virus. Both [3H]leucine-labelled and unlabelled Pr65gag were used to generate the cleaved p30.

The interaction of feline leukaemia viruses (FeLV) with erythrocytes was investigated. Haemadsorption (HAd) was observed on the surface of feline embryonic fibroblast cells infected with FeLV. HAd was detected in various degrees when cat, hamster or horse erythrocytes were incubated with cells infected with viruses of subgroup C (FeLV-C) and on cells infected with some FeLV subgroup A viruses (FeLV-A), but not on cells infected with FeLV subgroup B viruses (FeLV-B). HAd of sheep erythrocytes was detected on cells infected with some FeLV-C viruses. The HAd of hamster erythrocytes on cells infected with FeLV-C/Sarma virus was inhibited by antisera against gp70 or p15(E) but not by sera to the other FeLV structural polypeptides. HAd inhibition was also exhibited by cat sera which had FeLV-neutralizing activity but not by sera of specific pathogen-free cats. Haemagglutination by FeLV-C viruses was demonstrated after the virus was treated with neuraminidase and phospholipase C, or Tween-80 and ether. Contrary to expectations from the pattern observed by HAd, all FeLV-A viruses had similar haemagglutinin (HA) activity to FeLV-C viruses. FeLV-B viruses did not possess an HA.

Felid herpesvirus I (FHV I) is a respiratory pathogen of cats. Recovered animals remain latently infected but episodes of virus shedding may occur, particularly after stress. Previous attempts to identify the site(s) of latency of FHV I using co-culture, explant or tissue homogenization methods have been unsuccessful. Using a tissue fragment culture technique, however, FHV I was isolated from the trigeminal ganglia of three of 17 cats latently infected with field virus. No virus was found in four cats previously infected with a cold-adapted vaccine strain of FHV I, or in four cats given vaccine strain and field virus. The three trigeminal ganglion isolates had similar plaque size and morphology to the vaccine strain, the plaques being significantly smaller (P < 0.001) than those of the field viruses.