- Volume 35, Issue 2, 1977

The expression of virus, thymus and tumour-specific antigens was studied in two Marek’s disease lymphoblastoid cell lines (MSB-1 and HPRS-line 2).

The proportions of cells which spontaneously expressed virus antigens or which formed infective centres in vitro were considerably higher in MSB-1 than in HPRS-line 2, but did not exceed 10%. In contrast to virus antigens, thymus-specific and tumour-specific antigens were expressed on the majority of the cells. Treatment with IdUrd increased the proportion of cells forming infective centres in both cell lines and the proportion of cells expressing virus antigens in MSB-1. A decline in the proportion of cells forming infective centres in IdUrd-treated and in untreated cultures was noted during continuous subculture of both cell lines.

Direct evidence for the presence of virus-specific antigens in MSB-1 cells was obtained by immunodiffusion. The results suggested further that lymphoblastoid cells are unable to synthesize a major precipitating glycoprotein antigen (A antigen) normally associated with infection of permissive cells with MDV.

Analysis of surface proteins of normal thymus cells labelled by lactoperoxidase-catalysed iodination showed that thymus-specific determinants are associated with iodinated polypeptides in the mol. wt. range 45000 to 47000, 50000 to 58000 and 90000 to 150000. The major thymus-specific polypeptide exposed at the surface of normal thymus cells was in the mol. wt. range of 50000 to 58000. Surface proteins of lymphoblastoid cells were not accessible to iodination.

Inoculation of wild-type (wt) VSV intracerebrally (i.c.) in Swiss weanling mice results in a rapidly fatal illness with death in two to three days. In contrast, i.c. inoculation of temperature-sensitive (ts) VSV mutants G31 and G22, but not ts G11 or ts G41, results in a more slowly progressive central nervous system (CNS) disease with distinct neurological signs. Studies undertaken to evaluate the neurovirulence of ts VSV mutants indicated that the ability of ts mutants to produce pathological changes in the CNS of mice appeared related to their ability to replicate to high titre in brain and spinal cord. However, replication of ts VSV mutants in brain alone was not sufficient to produce clinical illness. More importantly, the ability of ts VSV mutants to replicate at non-permissive temperatures in vitro did not appear to correlate with neurovirulence. VSV harvests from brains and spinal cords of mice infected with each of the ts mutants were temperature-insensitive. In spite of their temperature-insensitivity, the biological behaviour of viruses recovered from CNS tissues was, surprisingly, not that which was characteristic of revertant clones. Virus isolates recovered from infected CNS tissues, despite their temperature-insensitivity, behaved biologically like the original stocks of ts mutant virus. These data suggest that temperature-sensitivity is not directly correlated with the unique pathogenesis elicited by infection with ts VSV mutants.

The effects of various reagents on the disassembly of potato virus Y are described and discussed. The virus can be disassembled in acetic acid, guanidine, LiCl, NaSCN and in a variety of other salts but is stable in NaCl, CsCl and NaF. Polymerization of coat protein from pH 3 to 11 in 0.1 to 0.5 m-NaCl was followed by analytical centrifugation. Extensive polymerization (with major proportions being 100 to 200S) was only found between pH 6 and pH 9 in 0.1 m-NaCl. A nucleoprotein with structural, density and stability properties similar to those of the virus, but less than one third as long, was obtained by the addition of RNA to polymerized protein at 20 °C at pH 7 to 8 at very low ionic strength. Possible modes of assembly are presented.

Infection of interferon-treated L cells with VSV led frequently to the establishment of L cells persistently infected with VSV (Lvsv cells). These cells were characterized by the following properties; (1) no supplement of antiviral factors such as anti-VSV antiserum, interferon, was required for their maintenance; (2) virus antigens were detected in about 5 to 30% of the cells by immunofluorescence staining; (3) the cells were not only resistant to superinfection by homologous virus, but also resistant to challenge by heterologous viruses such as Mengo virus; (4) the cells were destroyed by co-cultivation with heterologous cells susceptible to VSV infection; (5) the cells could be cured by serial cultivation in medium containing antiviral antibody, and the cured cells were as susceptible to VSV as normal L cells.

It was shown that at least three factors (interferon, defective interfering [DI] particles and a selection of small-plaque temperature-sensitive [ts] mutants) took part in the maintenance of Lvsv cells although it was difficult to evaluate exactly the relative importance of these factors. The effect of antiviral antibody, interferon and incubation temperature upon the maintenance of Lvsv cells are discussed further.

Four primary cleavage products, mol. wt. 103 × 100, 88, 56 and 52 (P100, P88, P56 and P52 respectively) are present in BHK 21 cells infected with foot-and-mouth disease virus (FMDV). However, no precursor polyprotein equal to the sum of their mol. wt. was detected, even when amino acid analogues and proteolytic enzyme inhibitors were used. Three of the primary products were shown to cleave to smaller polypeptides, including the capsid polypeptides of the virus. Polypeptide P88, which was shown to be the precursor of the capsid polypeptides, is translated from the gene located at the 5′-end of the genome. The order of the structural polypeptides, determined by the use of emetine, is VP4, VP2, VP3, VP1.

The order of the remaining primary cleavage products is P52, P56 and P100. P56 is a stable product, identical with the virus infection associated (VIA) antigen found in virus harvests. The function of the other two products P52 and P100 is not known.

FMDV thus differs from other picornaviruses in that there is an extra primary cleavage product, appearently resulting from translation of more of the virus genome.

The antigenic variation of swine vesicular disease virus (SVDV) and Coxsackie B5 virus (CB5) has been examined at the molecular level by analysing the protein and nucleic acid of the virus particles. The tryptic peptides of carboxymethylated 35S-methionine labelled virus particles were very similar, although some minor differences were apparent. Competition hybridization experiments confirmed that there is variation in the RNA sequence of antigenically distinct SVD viruses and some limited homology of these RNAs to the CB5 virus RNAs. Competition hybridization using mixtures of two RNAs as competitors showed that the sequences shared by the CB5 virus RNAs were largely the same as those shared by the CB5 virus RNAs and the SVDV RNAs. Similar experiments with the SVDV RNAs established that the homologous regions in these RNAs were also shared. Thermal denaturation curves of SVDV RNA-RNA hybrids generally supported the competition hybridization results but the hybrids between SVDV RNA and CB5 virus RNA were shown to be mismatched. Ribonuclease T1 oligonucleotide maps of the virus RNAs were also compared to obtain another measure of relatedness. A number of long oligonucleotides were shared by the SVDV RNAs but few of these were found in either of the CB5 RNAs. Further ways of investigating the antigenic variation of SVDV at the molecular level are discussed.

The indirect ferritin-labelled antibody technique was used to determine the reactivity of an antiserum prepared against the NZB xenotropic virus with three murine xenotropic viruses, a feline xenotropic virus and a murine ecotropic virus. The envelope antigens of the xenotropic type C viruses isolated from the NZB, NIH Swiss and C57L mice were tagged with ferritin. The feline RD114 virus was not. Gross murine leukaemia virus was tagged, but only at high serum concentrations. The cross-reactivity titre of Gross virus to anti-NZB serum was removed by a serum dilution which was still reactive to xenotropic viruses. This difference in reactivity titres between a xenotropic and an ecotropic virus was sufficient to distinguish one from the other in doubly infected cultures. Specific tagging of membranes of cells infected by xenotropic virus was also observed.

The effect of interferon on the synthesis of the RNA species and proteins of vesicular stomatitis virus has been studied in two cell types. Virus protein synthesis is inhibited by interferon despite the apparent presence of near normal amounts of virus RNA with sedimentation values characteristic of virus messenger RNA. The synthesis of those virus RNA species which are completely dependent on virus protein synthesis is preferentially inhibited in interferon-treated cells. These results are most consistent with a model of interferon action postulating a primary effect on translation of virus messenger RNA.

Forty-six arboviruses were tested for c.p.e. and/or plaque formation in an amphibian cell line. C.p.e. was observed with a high proportion of the viruses tested. Comparative plaque assay, in the XTC-2 cells at 28 °C and Vero cells at 37 °C, suggests that these systems are comparable in sensitivity and susceptibility to infection. Practical uses of this cell line are discussed.

Human leukocyte interferon, purified approximately 1000-fold by affinity chromatography on immobilized anti-interferon globulins and SDS-Sephadex filtration, was resolved into one major and one minor component by adsorption chromatography on hydroxylapatite and electrophoresis in polyacrylamide gels. These components were indistinguishable in their capacity to protect bovine, porcine and murine cells, and the antiviral activities of both were equally susceptible to reduction by β-mercaptoethanol. They were neutralized to the same degree by rabbit anti-leukocyte interferon but were not neutralized by rabbit antifibroblast interferon serum. Mice immunized with either component developed antibodies to both but failed to form antibodies against human fibroblast interferon. Our present evidence indicates that the two components possess at most only minor structural and antigenic dissimilarities.

Pseudomonas phaseolicola cells synchronously infected with φ6 were fixed and embedded by several procedures. The diameter of φ6 measured 75 nm and its nucleocapsid 60 nm. Virus nucleocapsids were icosahedral and surrounded by a double membrane. In planar sections the nucleic acid appeared as a hexagonal ring and in cross sections as two angular structures.

The inhibitory effects of interferon (IF) on the replication of varicella-zoster (VZ) virus in human foreskin fibroblast (HFF) cultures inoculated with infected cells or with cell-free virus were assayed by measuring (1) yields of infected cells, (2) plaques, (3) microfoci and (4) cytopathic effects. More If was needed to reduce yields of infected cells at high input multiplicities of challenge than at low input multiplicities, and still more IF was needed to prevent cytopathology due to VZ virus. A cell-free virus inoculum was more sensitive to the inhibitory effects of IF than an inoculum of infected cells. With the latter, but not with cell-free virus, the continuous presence of IF in the medium was necessary for it to express its maximum antiviral activity. To explain these results, it is suggested that some herpesviruses may establish ‘reservoirs’ of infectivity and thus provide a prolonged challenge to IF-treated cells which are not uniformly resistant to infection.

Bacteriophages were isolated from twelve lysogenic strains of Streptococcus equi. Based on sensitivity data and antiserum neutralization tests, the phage isolates were divided into two distinct but related groups. All twelve phage changed the colonial morphology of S. equi from mucoid to matt. Possible phage-mediated effects on S. equi virulence are discussed.

Protoplasts were isolated from leaves of three tomato cultivars and inoculated with citrus exocortis viroid (CEV), potato spindle tuber viroid (PSTV) and cucumber pale fruit viroid (CPFV), respectively, using glycine-KOH-buffered mannitol at pH 9. CPFV multiplied detectably in protoplasts from the tomato cultivar Hilda 72 but replication of the other two viroids was rarely detectable. The minimum viroid concentration for infection was 10 µg RNA/ml using 106 protoplasts/ml. Newly synthesized viroid RNA was detected 36 h after inoculation by bioassay, and 48 h after inoculation by 3H-uridine incorporation into the viroid RNA band obtained by polyacrylamide gel electrophoresis. Incorporation of radioactive uridine into CPFV in inoculated protoplasts reached about 0.6% of that into tRNA during 72 h after inoculation. In protoplasts isolated from systemically infected leaves, however, incorporation of 3H-uridine into CPFV was only 0.1% of that into tRNA. In inoculated protoplasts, incorporation of 3H-uridine into CPFV was much more rapid at 35 °C than at 25 °C but incorporation into tRNA was similar at the two temperatures.

Particles similar in appearance to the virions of the invertebrate baculoviruses have been found in large numbers in hyphae of the insect-parasitic fungus, Strongwellsea magna, growing in the fly, Fannia canicularis. The particles (100 × 390 nm) consisted of a densely staining core (50 × 340 nm) within a poorly staining envelope. The envelope of some of the particles was contiguous with vesicles derived from unit membranes originating at the hyphal plasma membrane. The structure and aspects of the apparent morphogenesis of these particles suggest they may belong to a virus of the genus Baculovirus.

Cricket paralysis virus, an insect picorna-like virus, caused a distinct c.p.e. in cultured Drosophila melanogaster cells, allowing the development of titration methods based on end-point dilution or plaque assay methods. The end-point dilution (TCD50) method is more sensitive and economical than plaque assays and is easily scored. The data indicate a minimum infectivity/particle ratio of about 1/2000.

A French isolate of beet necrotic yellow vein virus has rod-shaped particles of four predominant lengths (85, 100, 265 and 390 nm), and a width of about 20 nm. Virus preparations yielded four nucleic acid components of apparent mol. wt. 2.3 × 106, 1.8 × 106, 0.7 × 106 and 0.6 × 106 respectively, and a single protein of mol. wt. 21000.