- Volume 48, Issue 2, 1980

Four major polypeptides with mol. wt. of 22000, 25000, 52000 and 68000 were isolated from solubilized preparations of hepatitis type B surface antigen (HBsAg). These four populations, referred to as P22, P25, P52 and P68, respectively, were used to immunize guinea-pigs. Guinea-pigs were also inoculated with HBsAg and with purified human serum albumin (HuSA). These antisera were utilized to establish that intact HBsAg particles are associated with HuSA antigenic reactivity. HuSA antigenic determinants were associated with purified preparations of P68. HuSA antigenic activity was not detected with purified preparations of P22, P25 and P52 or with the respective specific antisera to each of the above. However, purified P68 contained the antigenic determinants of both host protein and hepatitis B virus-specified protein origin.

In a search for additional antigens associated with virus-induced human liver disease a radioimmunoassay (RIA) was developed using IgG from sera of a multiply transfused person. Polystyrene beads coated with IgG F(ab)′2 fragments, dinitrophenylated F(ab)′2 fragments and 125I-labelled anti-2,4-dinitrophenyl antibodies (Neurath & Strick, 1979) were used in the RIA. An apparently new antigen or the corresponding antibodies were detected in 155 serum specimens from 35/37 (94%) individuals who developed non-A, non-B hepatitis. The antigen was also present in hepatitis B surface antigen-negative sera of blood donors with normal (13.2%) and elevated levels of alanine aminotransferase (34%). The antigen has an approximate mol. wt. of 45000, a buoyant density of 1.23 g/ml and an isoelectric point of 7.

Agar immunodiffusion tests demonstrated that BHK 21 cells infected with either HSV 1 or HSV 2 release only a few HSV-specified antigens into the extra-cellular fluid (infected cell released polypeptides-ICRP). Neutralization blocking experiments showed that the majority of antigens/(including the Band II common antigen) involved as target sites in antibody-mediated virus neutralization are present in the ICRP of both HSV 1 and HSV 2. SDS-PAGE identified six regions of virus-specified proteins in the ICRP from both HSV 1- and HSV 2-infected BHK cells. All these specifically released proteins are glycosylated, although to varying degrees. The SDS-PAGE profiles of HSV 1 and HSV 2 ICRP are different but do show some similarities, the most notable being a highly glycosylated protein with an estimated mol. wt. of 50000 to 54000 in HSV 1 ICRP and 52000 to 56000 in HSV 2 ICRP. Immune precipitation demonstrated that these two proteins contain the Band II antigenic site. Similar studies showed that the major type 1 specific antigenic site, which is involved as a target site in the neutralization of virus infectivity, is located in the highest mol. wt. glycoprotein region of HSV 1 ICRP and has a similar mobility to the VP7/8 region of purified enveloped virus.

The nature of the refractoriness of C6 glioblastoma cells to herpes simplex virus type 1 (HSV-1) infection has been studied. The cells were restricted in susceptibility to HSV-1 since only a small proportion of the cells could be infected by HSV-1 and the virus yield per cell was low. The susceptibility to infection was increased by treating the cells with trypsin-EDTA prior to infection. The cells so treated recovered resistance to the virus when incubated at 37 °C, their resistance being restored to the initial level in 2 days. This restoration was inhibited by addition of cycloheximide or puromycin. Trypsin-EDTA treatment of C6 cells increased the efficiency of adsorption of HSV-1 and the formation of stable cell-virus complexes from which the virus could not be dissociated by heparin.

The Pr65gag proteolytic factor obtained from Moloney (MoLV) or Rauscher (RLV) leukaemia virus has been characterized. We found that it was present in small amounts in virions and was extremely unstable. Although it eluted at the trailing edge of p12 on Sephadex G-75 columns, it could clearly be separated from p12 on DEAE-Sephadex A-50M columns, making it unlikely that the factor is p12 or any other major murine leukaemia virus (MuLV) protein. This fact also distinguishes the murine factor from the avian p15-associated protease which is present in large amounts in avian tumour viruses and is stable to column purification methods (von der Helm, 1977; Dittmar & Moelling, 1978). We further observed that: (i) the murine proteolytic factor had an estimated mol. wt. of 20000 to 22000, relative to MuLV p12, which eluted as a dimer on Sephadex G-75 columns in the presence of 0.1% NP-40; and (ii) in vitro cleavage of an iodinated Pr65gag-rich, p30-deficient substrate yielded a clear increase in both p30 and p12, which suggests that the in vitro cleavage of Pr65gag is similar to its processing in vivo.

A technique for the isolation and characterization of newly transcribed murine leukaemia virus RNA in chronically infected cells has been developed. Cellular RNA was pulse labelled with 3H-uridine and virus-specific sequences were annealed with an excess of mercurated complementary DNA. Based on the affinity between mercurated cDNA and sulphydryl-Sepharose, the hybrid was specifically selected by affinity column chromatography. The specificity of this method was dependent on the purity of the cDNA and it was necessary to remove non-viral sequences from the cDNA in order to isolate virus-specific RNA. Between 0.5 and 0.8% of the labelled RNA in Moloney MuLV-infected rat cells and 1.5% of the labelled RNA in Moloney MuLV-infected NIH Swiss mouse cells were virus-specific. Using this methodology, the effect of the cell cycle on the transcriptional activity of proviral genes was investigated. Cultures of Moloney MuLV-infected rat cells arrested in G0 phase of the cell cycle released reduced quantities of virus, but continued to synthesize virus RNA. The pools of virus RNA and p30 antigen in the G0-arrested cells equalled the pools in actively dividing cells. These results suggested that post-transcriptional events controlled virus production in the G0-arrested cells.

Delayed type hypersensitivity (DTH) was induced in mice sensitized with an intradermal inoculation of herpes simplex virus type 1 (HSV-1). The reaction was observed 4 to 5 days p.i. and could still be induced up to 18 months later. In contrast, the adoptive transfer of DTH using draining lymph node cells was only possible during the period 6 to 10 days p.i. The cells taken at these times also contained mediators of antiviral immunity, as determined by a marked reduction of virus titres in the ears of infected animals 1 to 3 days after transfer. Draining lymph node cells taken at later times contained mediators of virus immunity, but titres were not reduced until day 5 after the transfer. The cell type involved in both the DTH and antiviral activity was a T lymphocyte, although the particular T cell subsets involved have yet to be determined.

Lymph node cells, regional to the site of infection with herpes simplex virus type 1 (HSV-1), when taken 1 to 9 months p.i. and transferred to HSV-1 immune recipients, suppressed the delayed type hypersensitivity (DTH) reaction to the virus. The suppressive activity was specific for HSV-1 and was transferred by a thy-1.2-negative, Ig-positive, lymphocyte population. Anti-HSV-1 serum did not suppress the HSV-1-induced DTH response. Contralateral lymph nodes contained little or no suppressor cells activity but in infected, adult thymectomized mice, these lymph node cells were as effective as the draining node in transferring suppression. The significance of these observations for the pathogenesis of herpes infections is discussed.

Three mouse cell lines (L, 3T3, and SV 3T3) were studied with respect to the elevation of cellular cAMP levels following interferon treatment, and the effect of stimulators of cAMP levels on the antiviral activity of interferon. Interferon treatment resulted in increased cAMP levels in L and 3T3 cells but not in SV 3T3. The antiviral activity of interferon in cells treated with epinephrine and 1-methyl-3-isobutyl xanthine (stimulators of cAMP levels) was potentiated in L cells, but not in 3T3 cells and was lost in SV 3T3 cells.

Seventeen of twenty-six influenza A virus isolates of the H1N1 antigenic subtype and two of eleven H3N2 virus isolates from the 1977–78 season exhibited a ts phenotype, were restricted in plaquing in MDCK cells at 38.5 °C compared to 34 °C and appeared to be naturally occurring ts mutants. The cut-off temperature for two such ts H1N1 virus isolates was established as 38 °C. The ts viruses were as thermostable as non-ts isolates and no complementation was detected between the twelve ts viruses tested. Cloning studies with an H1N1 virus isolate with minimal ts properties indicated the presence of a mixed population of ts + and ts particles. Analysis of seven recombinants of A/HK/117/77 (N1N1) virus indicated that the ts lesion(s) was not located in the NA or HA proteins.

A defective-interfering (DI) particle of Sindbis virus was generated from a ts mutant of RNA− complementation group A by serially undiluted passages at 30 °C. This mutant induced interferon at a permissive temperature (30 °C), but not at a non-permissive temperature (40.5 °C); it also expressed homotypic interference throughout the range 30 to 40.5 °C. This demonstrates for the first time in a DI particle a ts function, namely, the ability to induce interferon. In addition, our data provide further evidence that the RNA genome of a Sindbis DI particle can be translated within the cell. We postulate that the products of translation function to produce the putative inducer of interferon, namely a molecule of dsRNA.

The morphological heterogeneity of the chick embryo-adapted Enders strain of mumps virus was examined in relation to biological and biochemical properties of the virus. The range of virion sizes increased as multiplicity of infection (m.o.i.) increased, with virions of 800 to 1000 nm in diam. occurring in preparations grown at the higher multiplicities. There was no evidence for a distinct class of non-infectious haemagglutinating particles. Dense fractions from isopycnic gradients of virus were enriched in larger virions, but virus RNA was predominantly a single 50S species. Despite evidence that many virions were multiploid, u.v. inactivation kinetics were single-hit, suggesting only a single biologically active genome per virion.

The antigenicity of the avian sarcoma virus (ASV)-coded src-gene product pp60src, which is responsible for fibroblast transformation after ASV infection, has been investigated in STU mouse fibrosarcoma cell lines and the corresponding immune response in syngeneic mice has been determined. The development of effective anti-pp60src antibody titres depends on the mode and site of injection of tumour cells and parallels tumour growth. It was found that mouse immunoglobulin heavy chains are unable to serve as substrate for the protein kinase activity of pp60src. Therefore, an indirect protein kinase absorption (PKA) test was initiated to demonstrate recognition of the protein kinase activity associated with the src-gene product. The availability of syngeneic mice and the corresponding ASV-transformed tumour cells should facilitate studies designed to elucidate the possible relationship between the cytoplasmic pp60src and ASV-induced tumour-specific surface antigens (TSSA), for example, by allowing the production of stable mouse hybridomas synthesizing antibodies specific for pp60src and TSSA.

A comparative study on the infectivity and biological activities of parainfluenza virus type 2 revealed that one strain (Toshiba) showed c.p.e. with cell fusior and produced plaques in Vero cells, an established line of African green monkey kidney cells. Another strain (62-M786) of this virus, however, showed minimum c.p.e. and produced almost no plaques in Vero cells, although c.p.e. appeared and plaques were observed following addition of trypsin. The infectivity titre of the latter strain, estimated by 50% tissue culture infectious doses (TCID50), was increased 10 times and plaque counts more than 6400 times by addition of an adequate concentration of trypsin; however, trypsin did not affect the infectivity of the former strain. Trypsin also increased the haemolytic activity of strain 62-M786 but not that of the Toshiba strain. These results show that the two isolates of parainfluenza virus type 2 were affected very differently by trypsin as regards infectivity, cell fusion and haemolytic activity.

The influence of relative humidity (r.h.) on the survival of vesicular exanthema virus (VEV) in aerosols at 1 s and during the next 5 min when generated from phosphate buffer solution containing polyhydroxy compounds, dimethyl sulphoxide, salt or protein has been examined. VEV was sensitive to r.h. in the range of 40 to 60% in the presence of bovine serum albumin, glucose, inositol or phosphate buffer. Addition of sodium chloride stabilized the virus in aerosols at mid-range r.h. both immediately after generation and after a period of storage for 5 min. In the presence of dimethyl sulphoxide or glycerol, virus survival was reduced at 20% r.h. at 1 s and at all r.h. during the first 5 min. Pre-humidification did not produce any significant difference in virus recovery.

Acyclovir (ACV) was effective in preventing recurrence of herpes simplex in mice whose skin was stripped with cellophane tape. Treatment with ACV did not eliminate latent herpes simplex virus from the cervical ganglia.

Bovine leukaemia virus (BLV) induces two types of cell fusion. Cells inoculated at high multiplicity show cell fusion within 2 h. This type of fusion can be induced by non-infectious virus and does not require protein synthesis for induction. Low m.o.i. also induces cell fusion but this depends on production of infectious virus.

A hybrid between a murine myeloma cell line and spleen cells from a mouse immunized with measles has been produced. Two stable clones produce antibody with identical immunochemical and biological properties. This antibody reacts with the 76000 mol. wt. protein present in the lysates and on the surface of cells persistently infected with measles. It exhibits HAI and neutralizing activity.