- Volume 53, Issue 1, 1981

Escherichia coli B exposed to high doses of bacteriophage T7 did not lyse. A similar effect was observed when the high dose was added within the first 7 min after primary infection. No viable phage was formed. DNA synthesis was inhibited rapidly and the nucleoid structure was absent. Protein synthesis was in general markedly reduced and so were the activities of the phage-specific enzymes endolysin and DNA polymerase. However, phage genes were transcribed both by the host RNA polymerase and by the phage-specific enzyme. We suggest that inhibition of phage development is due to structural alterations occurring in the cell wall/membrane such that replication is inhibited.

Phages for coagulase-negative staphylococci were adsorbed to heat-killed cells. The phages showed equal affinities for all the cells, which appeared to have an equal number of binding sites for all the phages tested. This number is estimated at 1.2 × 106 sites/cell. Competition for binding sites could be demonstrated between a pair of phages. It is concluded that coagulase-negative staphylococci have only a single series of binding sites for phage, probably the outer 20% or so of the wall teichoic acids. These organisms therefore bind all ‘coagulase-negative’ phages whether or not they are sensitive to them.

Partially purified fibroblast interferon and double-stranded RNA (dsRNA) of fungal origin were administered as single graded doses to A2G and Balb/c mice shortly before intraperitoneal infection by specified virulent or avirulent strains of representative togaviruses (Semliki Forest virus, Venezuelan equine encephalomyelitis virus and yellow fever virus). Changes of efficiency of infection arising from interference at the level of clearance or replication and changes of the expression of virulence or protective immunity, were compared for different initial doses of interferon or dsRNA in relation to different levels of infection by defined virus strains. Interferon and dsRNA, although acting through quantitatively different mechanisms, both reduced effective dose of virus and influenced only the extent of primary replication and host stimulation. Neither agent changed in any way the outcome of infection in terms of expression of virulence (regulatory immunity) or protective immunity. These results are discussed in terms of the control of virus infections at stages before or after immune stimulation can be effective.

Considerable differences exist in the lethality of herpes simplex virus type 1 (HSV-1) after intraperitoneal (i.p.) infection in different inbred strains of mice. In this study humoral and cellular immunity and interferon production were compared in resistant and susceptible mice. The serum IgG response, as determined by an enzyme-linked immunosorbent assay (ELISA), was the same in different strains of mice. There was also no difference in neutralizing antibodies between resistant C57BL/6 and susceptible DBA/2 mice. The production of macrophage migration inhibitory factor, obtained from spleen cells of mice 7 days after infection with different doses of HSV-1, was the same in resistant and susceptible mice. Serum interferon could be detected 8 h after i.p. injection of 107 p.f.u. of HSV-1, but not at lower virus doses. At 8 h, high interferon titres [>1000 reference research units (iu)/ml] were observed in the serum of C57BL/6 mice but low titres (about 100 iu/ml) were found in the serum of DBA/2 mice. At 24 h, the titres were low in both strains of mice. Interferon production was also measured in vitro in spleen cell cultures exposed to inactivated HSV-1. These studies also showed high interferon production in spleen cell cultures of resistant mice, whereas low titres were produced by spleen cells of susceptible mice. Thus, our study has failed to reveal any differences in humoral or cellular immunity in mice resistant or susceptible to HSV but a difference in HSV-induced interferon production.

Infection of cultures of three different normal rodent cell lines with murine sarcoma virus (MSV) resulted in very rapid loss of contact inhibition of growth and morphological transformation. In the case of two of these lines, anchorage dependence of growth was also rapidly lost but with the 3rd, C3H10T C1.8, an established line of mouse embryo fibroblasts, there was a delay of many cell generations before the cells became anchorage independent. This was despite 100% successful infection, as assessed by focus assays of the infected cells. The acquisition of anchorage independence was correlated with a substantial increase in tumorigenicity. A number of MSV-infected clones, isolated at random from C3H10T C1.8 cultures immediately after infection with MSV, also showed a progressive increase in anchorage independence and tumorigenicity, indicating that the progressive transformation of the uncloned cells could not be entirely due to selection of rare anchorage-independent, tumorigenic clones. It was concluded that neoplastic transformation of C3H10T C1.8 cells by MSV is a multi-step process.

Astrovirus, purified in aggregated form from epithelial cells of the small intestinal villi of infected gnotobiotic lambs, was shown to have a single-stranded RNA genome with an S value of 34 and poly(A) tract. Only two major capsid polypeptides were detected with similar mol. wt. of approx. 3.3 × 104.

The ribonucleoprotein (RNP) of avian infectious bronchitis virus (IBV) was examined by electron microscopy after shadowing with carbon/platinum. Linear RNP strands up to 6.7 µm in length, from three IBV strains, were sensitive to both pancreatic RNase and to proteases. These strands were obtained from spontaneously disrupted complete particles but not from disrupted incomplete particles that lacked RNP. They were also released from Nonidet P40-disrupted particles and could be isolated on sucrose density gradients at a density of 1.27 g/ml. In some cases, helical RNP complexes associated with virus particles were observed that were similar to RNPs of human coronavirus strain 229E and mouse hepatitis virus strain 3.

Prostaglandins of the A series (PGA) were shown to be potent inhibitors of Sendai virus replication in African green monkey kidney cells in culture. This antiviral activity was specific for PGA. The effective dose (4 µg/ml) was not toxic to the cells and did not alter either host cell metabolism or infectivity of the virus. PGA did not affect the first stages of virus replication and seemed to act by inhibiting virus maturation and/or budding from the cells. The antiviral action of PGA was pharmacological, was not mediated by cAMP and was completely reversible. Possible mechanisms of action include post-translational binding to virus proteins or interaction with interferon.

Subunit vaccines, containing the spike glycoproteins of Semliki Forest virus (SFV) in three different forms, have been prepared: detergent-solubilized monomers, detergent- and lipid-free octamers, and virosomes in which the spike proteins are reconstituted into phospholipid vesicles. Previous studies have shown that the octamers and the virosomes are very efficient in protecting mice against the encephalitis caused by virulent SFV (Morein et al., 1978). In this study we have characterized the specific antibody responses in mice vaccinated with the three SFV vaccines and correlated them with the protection against SFV encephalitis. The multimeric forms induced high humoral antibody titres; two doses of only 1 µg protein gave rise to specific antibody titres of 0.6 mg/ml. The monomeric form was much less immunogenic.

Avian infectious bronchitis virus (IBV) was grown and radiolabelled with 35S-methionine, 3H-leucine and 3H-glucosamine in de-embryonated chicken eggs. Approximately 12 different polypeptides were clearly detected by SDS-polyacrylamide gel electrophoresis of virus preparations. Growth of IBV in chorioallantoic membrane cells labelled with 35S-methionine indicated that most of these polypeptides, and additional ones, some of which were glycosylated, were host components. Five polypeptides appeared to be virus-coded, with apparent mol. wt. of 94 × 103, 84 × 103, 54 × 103, 30 × 103 and 28 × 103. Four of these, p94, p84, p30 and p28, were glycosylated. The virion spikes appeared to be composed of p94 and p84, while p30 and p28 were partially embedded in the virion membrane. By analogy with other reports, p54 is the nucleocapsid polypeptide.

Cultures of two human hepatoma cell lines were examined for expression of hepatitis B virus surface antigen (HBsAg). The PLC/PRF/5 cells secreted HBsAg continuously into the culture medium, whereas Mahlavu cells did not secrete the antigen. However, cytoplasmic antigen was detected in a low percentage (<5%) of the Mahlavu cells. The expression of HBsAg also was assayed in cultures treated with dexamethasone (DXM), 5-iodo-2′-deoxyuridine (IdUrd), or both. The results demonstrated that: (i) DXM stimulated secretion of HBsAg by PLC/PRF/5 cells but not by Mahlavu cells; (ii) the percentage of Mahlavu cells expressing cytoplasmic HBsAg was not increased in any cultures if the medium was replaced at 24 to 48 h intervals but was increased approx. fivefold within 4 days in cultures treated with DXM or IdUrd/DXM if the medium was not changed. However, no increase was noted in the intensity of the immunoperoxidase stain of PLC/PRF/5 cells that expressed cytoplasmic antigen in any DXM cultures; (iii) HBsAg expression was stimulated to a lesser extent in IdUrd/DXM cultures than in DXM cultures and was not enhanced in IdUrd cultures. Thus, DXM enhanced secretion of HBsAg by PLC/PRF/5 cells within 24 h and, after a delay, enhanced expression of cytoplasmic antigen by Mahlavu cells. However, antigen secretion by Mahlavu cells evidently was blocked.

A new procedure for the separation of influenza virus particle ribonucleoproteins (RNPs) by electrophoresis on a slab of polyacrylamide gel resolves five discrete bands. The largest is a triplet containing the three largest RNAs (1 to 3), the intermediate sized RNP is a doublet containing RNAs 5 and 6 and the others each contain a single RNA. In addition, each RNP is composed of NP protein and an amount of M which is independent of the size of the RNP. This suggests that some virus particle M is specifically associated with influenza ribonucleoprotein.

The properties of latent herpesvirus of turkey (HVT) and Marek’s disease virus (MDV) genomes have been studied in virus-non-producer MDCC-BO1(T) cells, a T-lymphoblastoid cell line derived from spleen cells of an HVT-vaccinated chicken. The numbers of the two virus genomes in BO1(T) cells remained stable at 1.6 to 1.8 HVT genome equivalents/cell and 3.4 to 3.8 MDV genome equivalents/cell throughout a number of passages and were not decreased by the presence of phosphonoacetic acid in the culture. When the culture temperature of the MDV-producer MDCC-MSB1 cell line was shifted from 41 to 37 °C, the cells cultured at 37 °C contained about five times as many virus genomes as those cultured at 41 °C. In contrast, the numbers of the two virus genomes in BO1(T) cells were not increased by culture at 37 °C. RNA extracted from BO1(T) whole cells and from the polyribosomal fraction hybridized to both MDV and HVT DNAs, indicating the expression of both latent virus genomes. Digestion of cell nuclei with micrococcal nuclease revealed that both latent HVT and MDV genomes possess a nucleosomal structure. Closed circular MDV DNA was demonstrated in BO1(T) by isopycnic centrifugation of DNA in ethidium bromide—CsCl gradients.

Coronavirus JHM contained six major proteins, four of which were glycosylated. The proteins were gp170, gp98, gp65, p60, gp25 and p23. Sac(-) cells infected with JHM shut off host cell protein synthesis, and the synthesis of three major (150K, 60K and 23K) and three minor (65K, 30K and 14K) polypeptides was detected by pulse-labelling with 35S-methionine. Antiserum directed against purified virus proteins specifically immunoprecipitated the three major intracellular species and also the 65K minor species. During a chase period, species 150K and 23K were processed and three new immunoprecipitable species, 170K, 98K and 25K appeared. The intracellular species 170K, 98K, 65K, 60K, 25K and 23K co-electrophoresed with virion proteins.

Two-dimensional gel electrophoresis of infected cell polypeptides showed that the 60K, 23K, 25K and 14K species were relatively basic polypeptides whilst the 98K and 170K were relatively acidic and heterogeneously charged polypeptides. Additionally, a charge-size modification of the 23K species during processing was detected, which was not apparent using one-dimensional gel analysis.

Human cytomegalovirus (HCMV) induces membrane antigens (MA) which are detectable by immunofluorescent techniques as early as 24 h after infection. These antigens appear on the surface of infected cells in non-permissive human epithelioid and animal cells in addition to permissive human cells. The synthesis of the MA occurs in the presence of a DNA synthesis inhibitor, whereas RNA and protein synthesis inhibitors prevent its formation completely. These results demonstrate that the MA is a newly synthesized protein coded by an early function of the viral genome.

The haemagglutinin chains HA1 and HA2 from the avian influenza virus A/duck/Ukraine/1/63 (Hav7, Neq2) have been subjected to amino acid analysis and N-terminal sequencing. Automated sequenator analysis of HA1 (40 cycles), after enzymic removal of the N-terminal pyroglutamic acid blocking group, and HA2 (43 cycles) showed that the Hav7 haemagglutinin closely resembled the human Hong Kong (H3) haemagglutinins including the presence of the characteristic extended 10 residue sequence at the N-terminus of HA1. These findings, together with the amino acid compositions for both chains, demonstrate that the Hav7 haemagglutinin is structurally similar to the Hong Kong (H3) haemagglutinins.

Four structural polypeptides of Hazara virus, an agent closely related to the Crimean—Congo haemorrhagic fever (C-CHF) viruses, were resolved by SDS—polyacrylamide gel electrophoresis. Three glycoproteins were identified (mol. wt. 84000, 45000 and 30000) and were found to be associated with the virion envelope. A fourth polypeptide (mol. wt. 52000) was non-glycosylated and associated with the nucleocapsid. The structural proteins of Hazara virus differ markedly from those reported for other bunyaviruses.

Clonally derived stocks of defective-interfering (DI) influenza virus prepared in chick embryo fibroblast (CEF) cells were all deficient in genomic RNAs 1, 2 and 3 but had different patterns of subgenomic RNAs. A single passage at high multiplicity in L cells altered the pattern of genomic RNAs independently of the subgenomic species, while in BHK cells the opposite situation prevailed. Therefore, there is no simple relationship in DI influenza virus between the loss of genomic RNA segments and the presence of subgenomic RNAs.