- Volume 52, Issue 1, 1981

Three aspects of the adsorption of the defective phage PBS Z1 to Bacillus subtilis 168 Wt have been investigated. These are the kinetics, the number of receptors on the cell wall and the character of these receptors. The reaction constants for the binding of phages onto receptors, for the dissociation of the phage-receptor complex and for the transition from reversible to irreversible binding of the phages were calculated from adsorption curves obtained by an enzyme-linked immunosorbent assay (ELISA). They were 1.8 × 10−13, 6.7 × 10−2 and 9.0 × 10−3 respectively. The maximum number of phages adsorbed per cell was 2700, a number limited by the surface area of the cells. Apart from the receptors on the cell wall, receptors on the cell membrane were found. This was concluded from additional adsorption experiments with stable L-forms and contracted phages. Based on these results, together with data from the literature on bacteriocins, phage ghosts and yeast killer factors, a hypothesis concerning the first stage of killing by defective phages has been formulated.

A virulent bacteriophage was isolated from lysates of Bacillus stearothermophilus strain NU-10 induced either by treatment with mitomycin C or by shifts in temperature. Optimum conditions for induction, morphology and other properties of the virus are described. Chloroform treatment and shifts in temperature of producer cells released approximately similar amounts of phage as did mitomycin induction, suggesting an effect on release rather than on synthesis of the virus.

The effect of mitomycin C was tested on Epstein-Barr virus (EBV) antigen and virus DNA synthesis in producer and non-producer cell lines. We found that producer, but not non-producer cell lines, are inducible for early antigen and virus capsid antigen synthesis. Although host DNA synthesis is effectively inhibited by over 99% in the presence of mitomycin C, virus DNA synthesis in the producer lines is unaffected and induced in parallel with the virus antigens.

Vaccinia virus mRNAs carry the cap structure m7G5′pppAm- or m7G5′pppGm- at the 5′-terminus, which is synthesized by a series of RNA polymerase and capping enzymes contained in the virus particle. The process of the cap formation at the 5′-terminus of mRNA was studied using an in vitro system under similar conditions to those of vaccinia virus multiplication in its host cell. After adding a methyl-group donor, methyl-3H-S-adenosylmethionine, the oligonucleotides, which were the de novo synthesized 5′-terminal part of mRNA, were isolated from the RNA-synthesizing virion at appropriate time intervals, and were analysed. The 5′-5′ confronting nucleotides with 2′-O-methylation, G5′pppAm and G5′pppGm, were found with the completed cap structure, m7G5′pppAm and m7G5′pppGm. The confronting nucleotides with only 7-methyl guanine as a methylated component, m7G5′pppA and m7G5′pppG, were not detected at any incubation time, and it was concluded that methylation at the 2′-position of the 5′-terminal purine nucleoside of mRNA precedes methylation of the 7-position of the blocking guanosine. This result is different from that obtained using the enzymes isolated from vaccinia virus (Moss et al., 1976) and also from the results obtained using other kinds of virus particles, which carry RNA polymerase and capping enzymes. These differences may be due to the specific organization of a series of capping enzymes and RNA polymerase in each virus particle.

Analysis of poliovirus-infected cells by isoelectric focusing in urea resolves at least 25 bands. Combination of isoelectric focusing and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) in a two-dimensional analysis (2D-analysis) revealed a much better resolution than each method separately. By 2D-analysis most of the bands could be correlated with the known poliovirus-specific polypeptides generated in the infected cell. With this technique it was possible to determine the apparent isoelectric point (pI) of the identified poliovirus polypeptides. They are in the range from pH 5.6 for polypeptide 7d to pH 8.8 for polypeptide X, which is the most basic found so far.

When athymic nude (nu/nu) and heterozygous littermate (nu/+) mice were injected intraperitoneally (i.p.) with a mouse-adapted strain of dengue virus (DV), the following differences were noted in the course of infection. (i) The average survival time of nu/nu mice was longer than that of nu/+ mice, although the mortality ratios were not significantly different. (ii) DV persisted in some of the nu/nu mice for long periods of time without exhibiting any symptoms but they died after prolonged incubation periods. These aspects were not observed in the nu/+ mice. (iii) Infected nu/nu mice produced IgM antibody only transiently in the early stage of infection but they did not subsequently show regular IgG antibody production which normally occurred in nu/+ mice. (iv) Piamatral and perivascular mononuclear cell infiltration in the infected brain was more intense in nu/+ than in nu/nu mice. It is suggested from these data that the course of DV infection in mice is affected by the availability of thymus-derived lymphocytes (T-cells).

Infectious virus was detected in various organs and tissues of infected mice. The hearts of nu/nu mice tended to show higher virus titres than those of nu/+ mice, whereas the virus concentrations in the brain, skeletal muscle and lymph node were the same in both groups of mice. Specific DV antigen was revealed by the fluorescent antibody (FA) technique in cells located in the infected tissues.

Two hundred and thirty virus-induced polypeptides have been detected in BHK cells infected with herpes simplex virus type 1 (HSV-1; strain 17) by means of two-dimensional gel electrophoresis. The polypeptides have been characterized by both relative mobility following isoelectric focusing and apparent mol. wt. in SDS-polyacrylamide gels. Some polypeptides, visualized as a single band on a one-dimensional SDS-polyacrylamide gel, were resolved into several spots. Three were identified in V mw43, the band thought to contain thymidine kinase activity.

Not all the observed polypeptides are unique species: some appear to be related and have altered mobilities as a consequence of post-translational modification events. Pulse-chase experiments and treatment of infected cells with neuraminidase suggested that glycoproteins gB, gC and gD contain sialic acid and that synthesis of gB and gD occurs by at least 15 and 10 discrete steps respectively.

Seventeen avian paramyxoviruses consisting of ten reference strains and seven recent isolates were classified by immunodiffusion into six species on the basis of the antigenic specificity of their M protein (MP) antigens. The species were represented by the following viruses. (1) Newcastle disease virus, (2) Chicken/California/Yucaipa/56, (3) Turkey/Wisconsin/68, (4) Duck/Mississippi/75, (5) Pigeon/Otaru/76 and (6) Budgerigar/Kunitachi/1/74. The differences in the HN antigens between viruses which were classified into the same species on the basis of the antigenic specificity of their MP antigens, may provide a basis for classifying them into types.

Human embryonic fibroblasts infected with u.v.-irradiated herpes simplex virus type 2 (HSV-2, strain 186) and maintained at 40.5 °C did not yield detectable virus. Virus synthesis was induced by temperature shift-down to 36.5 °C. The induced virus grew very poorly and was inactivated very rapidly at 40.5 °C. Non-irradiated virus failed to establish latency at 40.5 °C in infected cells. Enhanced reactivation of HSV-2 was observed when latently infected cultures were superinfected with human cytomegalovirus (HCMV) or irradiated with a small dose of u.v. light at the time of temperature shift-down. HCMV did not enhance synthesis of HSV-2 during a normal growth cycle but did enhance synthesis of u.v.-irradiated HSV-2. These observations suggest that in this in vitro latency system, some HSV genomes damaged by u.v. irradiation were maintained in a non-replicating state without being destroyed or significantly repaired.

The physical mapping of six ts mutations of herpes simplex virus type 2 (HSV-2) is presented. The results were obtained from 14 separate intratypic marker rescue experiments and the analysis of 20 HSV-1/HSV-2 intertypic recombinants. The order of these mutations on the physical map of HSV-2 is unambiguous and correlates almost exactly with the previously published genetic map of Timbury & Calder (1976). One of the mutants studied (HSV-2 ts 12) has apparently two distinct conditionally lethal ts mutations, one in the long and the other in the short region of the HSV genome.

Treatment of mouse embryo (ME) cells with 5-iodo-2′-deoxyuridine (IdUrd) before infection with SV40 virus, enhances T-antigen (T-Ag) production as detected by immunofluorescence and complement fixation. Cellular DNA and RNA synthesis are inhibited in both SV40 and mock-infected cells after IdUrd treatment. The analogue pretreatment significantly increases the amount of radiolabelled nuclear and cytoplasmic SV40-specific RNA and the RNA polymerase activity of the viral transcriptional complexes of the Sarkosyl supernatants, suggesting that the enhancement of SV40 T-Ag production in infected pretreated ME cells results from an increased synthesis of early virus RNA.

The expression of Forssman-type heterophile antigen on Marek’s disease (MD) virus (MDV)-transformed cell lines, MDCC-MSB1, -HP1, -RP1 and -BP1, and avian leukosis virus (ALV)-transformed cell lines, LSCC-1104B1 and -1104X5 was investigated by membrane immunofluorescence and complement-dependent antibody cytotoxicity tests. Forssman antigen was detected on a high percentage of the cells in two ALV-transformed cell lines and on a smaller percentage of splenic lymphocytes from normal chicken. Of the MDV-transformed cell lines tested only the RP1 and BP1 cell lines, derived from transplantable MD tumours, expressed Forssman antigen, while the MSB1 and HP1 cell lines, derived from MD lymphomas, did not. Forssman antigen appears to be unrelated to MD tumour-associated surface antigen (MATSA).

Canine coronavirus (CCV) isolate 1–71 was grown in secondary dog kidney cells and purified by rate zonal centrifugation. Polyacrylamide gel electrophoresis revealed four major structural polypeptides with apparent mol. wt. of 203800 (gp204), 49800 (p50), 31800 (gp32) and 21600 (gp22). Incorporation of 3H-glucosamine into gp204, gp32 and gp22 indicated that these were glycopolypeptides. Comparison of the structural polypeptides of CCV and porcine transmissible gastroenteritis virus (TGEV) by co-electrophoresis demonstrated that TGEV polypeptides corresponded closely, but not identically, with gp204, p50 and gp32 of CCV and confirmed that gp22 was a major structural component only in the canine virus. The close similarities in structure of the two coronaviruses augments the relationship established by serology.

DBA/2 and C57B1/6 mice were infected intraperitoneally with two different strains of herpes simplex virus type 1, HSV-1 ANG and HSV-1 WAL. Unlike HSV-1 WAL, HSV-1 ANG was apathogenic by this mode of infection. Furthermore, infection with HSV-1 ANG protected mice of both inbred strains against infections with lethal doses of pathogenic HSV-1 WAL. This protection was observed when the apathogenic virus was given with the pathogenic virus or 4 to 24 h before it.

The oligosaccharide sidechains attached to the major polypeptide, HA1 of the haemagglutinin of influenza virus were examined for antigenic activity using a solid-phase radioimmunoassay. Cross-reactivity between the HA1 of different human subtypes was clearly demonstrable with IgG raised against purified virus but was abrogated if anti-carbohydrate antibodies were first removed by passage of the IgG through an immunoadsorbent column containing haemagglutinin (HA) from an unrelated avian influenza strain. Antibodies eluted from the column were found to cross-react with the HA1 of all subtypes tested. ‘Host antigen’ extracted from chick chorioallantoic membrane and coupled to Sepharose was also able to remove cross-reactive antibodies from antiviral sera, while antibodies raised against host antigen bound to the HA1 isolated from each subtype tested. It is concluded that, although there are qualitative and quantitative differences between the oligosaccharide sidechains of influenza haemagglutinins, the antigenically active side-chains are cross-reactive.

150 lymphoblastoid cell lines derived from normal individuals or from subjects with various clinical conditions were induced to form interferon by treatment with Sendai virus. Irrespective of the status of the donor, most of the lines produced some interferon and 22 produced considerable amounts (more than 3000 international units/ml). Lines derived from infectious mononucleosis patients were good interferon producers while those from leukaemic donors were poor producers. The data suggest that the clinical conditions of the donor and the source of transforming virus may influence the quantity of interferon produced by a given cell line.

The virus isolated from the black beetle, Heteronychus arator, is an icosahedral ribovirus with a diam. of 30 nm and contains one major structural polypeptide of mol. wt. 40000. The virus can be grown in Drosophila melanogaster cells and also titred in this line using a plaque assay method. Infected Drosophila cells pulsed with 35S-methionine in the presence of actinomycin D induced three virus polypeptides of mol. wt. 110000, 40000 and 8000. Short pulses, chases with excess non-radioactive methionine and pre-pulse treatments with protease inhibitors failed to demonstrate processing of virus-induced polypeptides.