- Volume 51, Issue 2, 1980

An Epstein-Barr virus (EBV)-like herpesvirus has been isolated from a baboon cell line (594S/F9) by induction with the tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). The herpesvirus papio (HVP) DNA was mixed with B95 EBV DNA and was characterized by sedimentation in neutral glycerol gradient as 55S DNA, with a buoyant density of approx. 1.718 g/ml after equilibrium centrifugation in caesium chloride. DNA–DNA reassociation kinetics between B95 EBV and HVP DNA showed that HVP DNA shares about 40% homology with B95 EBV DNA. Blot hybridization of EcoRI fragments of HVP DNA with a 32P-B95 EBV DNA probe showed that most of the EcoRI fragments of HVP DNA were hybridized to B95 EBV DNA, suggesting that the homologous sequences were dispersed throughout the virus DNA.

The effect of inhibitors of RNA (actinomycin D, Act. D) and protein synthesis (cycloheximide, CH) on induction of Epstein-Barr virus (EBV) antigens by the tumour promoter TPA and by EBV superinfection has been analysed. The data indicate that (i) concomitant treatment of cells with Act. D and TPA leads to complete suppression of virus antigen induction. Subsequent treatment of the cells with Act. D after prior exposure to TPA results in some virus antigen induction, the amount depending on the time of TPA treatment. (ii) Simultaneous treatment of the cells with TPA and CH blocks antigen expression completely. Removal of the inhibitor results in antigen expression at a comparable rate to that of CH-untreated cells. (iii) If CH treatment is followed by addition of Act. D, virus antigen induction by TPA is completely inhibited. In contrast, superinfection of the cells with P3HR-1 EBV in the presence of CH for the same period followed by removal of the inhibitor and addition of Act. D leads to virus antigen expression by 3 h after Act. D addition. (iv) Concomitant treatment with CH and TPA followed by addition of either iododeoxyuridine or n-butyric acid results in ‘superinduction’. Virtually all cells exhibit EBV-specified antigens. This implies that induction of virus antigens by tumour promoters requires the synthesis of a specific RNA, that this RNA increases in concentration during the induction period and that the same RNA is not required for EBV transcription after exogenous infection.

Bovine rotavirus infectivity for continuous green monkey kidney (BSC-1) cells was enhanced in hypertonic medium and following incorporation of cortisol, retinoic acid and vitamin B12 into the cell culture maintenance medium. The virus yields produced under these conditions were similar whether obtained in the presence or absence of trypsin. Infectivity titres were increased following the incorporation of trypsin in the maintenance medium throughout the infection cycle but remained unchanged after trypsin treatment of infected cell extracts. Bovine rotavirus infectivity was not affected by incorporation of phytohaemagglutinin, thyroid gland extracts or foetal calf serum in similar experiments. Unexpectedly, serum promoted virus growth when used with cells treated with actinomycin D. Marked differences in the stability of the newly produced infectious rotavirus were observed, suggesting that cell permissiveness to rotavirus infection may vary following the physiological state of the host cell.

The virus proteins, reverse transcriptase (RT) and p30, were found to increase with time in the subcellular fractions of lymphocytic tissue from either the thymus or spleen of AKR mice with spontaneous lymphocytic leukaemia. Significant levels of RT activity were first detected in the microsomal fractions of the two tissues at 15 and 20 weeks old, respectively. Although low amounts of p30 could be found in both tissues within the first week of life, the overall increase in the amount of p30 within each tissue followed much the same course as that shown by RT. In addition, a protein complex consisting of p30 and RT was first found in thymus and spleen lymphocytes of 15 and 20 week-old animals, respectively. The complex increased in amount in both organs as the animals aged, reaching a maximum level in 30 week-old mice. Repeated attempts to detect other virus proteins such as gp70 in association with the complex by immunological means were unsuccessful. The complex could not be found in lymphocytic tissue taken from younger animals or in ‘non-target’ organs, such as liver or kidney, of animals with leukaemia. In animals treated with antiviral IgG, which delayed the development of spontaneous leukaemia, the complex did not appear until much later in life (45 weeks) and then in considerably smaller amounts.

RNA from the Snyder–Theilen feline sarcoma–leukaemia virus complex (ST-FeSV–FeLV) sedimented in a double-peaked band between 50 and 70S, but Gardner-Arnstein (GA) FeSV–FeLV RNA sedimented in a single 70S peak. FeLV isolated from the ST virus mixture contained RNA which sedimented in a 70S band like GA-FeSV–FeLV RNA, but F422 FeLV RNA sedimented more slowly, at 50 to 60S. After thermal denaturation, resedimentation revealed three classes of RNA subunits in ST-FeSV–FeLV RNA: the first class, 35 to 37S, was also found in ST-FeLV and other FeLVs (except F422 FeLV), in the endogenous feline virus, RD114 and in GA-FeSV–FeLV; the second class, 32 to 34S, was similar to subunits in F422 FeLV and minor components of GA-FeSV–FeLV and ST-FeLV; the third class, 25S, was detected only in ST-FeSV–FeLV RNA. Electrophoresis of RNA species in buffered formamide provided evidence that the three classes of RNA subunits distinguishable on the basis of sedimentation rates actually represent three size classes of subunits. The ST virus mixture was shown to contain about equal titres of infectious FeLV and transforming FeSV whereas GA-FeSV–FeLV had at least a 10-fold excess of FeLV over FeSV. These observations are discussed in terms of possible origins of the three sizes of FeSV–FeLV RNA subunits and their relationships to three species of FeSV–FeLV proviral DNA described recently (Sherr et al., 1979).

Measles virus nucleoprotein (NP) and matrix (M) components were purified by two different procedures. Antigens were prepared by sedimenting material from 1% Cutscum extracts of infected cells into the interphase between 65 and 40% sucrose and further fractionation of the interphase material in a linear CsCl gradient, density range 1.20 to 1.33 g/ml. NP components contaminated with some M material and cellular actin banded at 1.30 to 1.32 g/ml, but at the low density range of 1.20 to 1.22 g/ml pure M component was demonstrable. Partially denatured antigens were obtained by elution of the 60K NP and 36K M polypeptides after SDS-polyacrylamide slab gel electrophoresis. Rabbit hyperimmune sera were prepared against both purified antigens and isolated polypeptides. All sera reacted only with homologous antigen except the antiserum against NP components isolated from CsCl gradients, which also contained antibodies to the M component. Antibodies against NP antigen stained both intranuclear inclusions and cytoplasmic material in immune fluorescence tests. In contrast, antisera against M antigen only stained the cytoplasm. Since intranuclear nucleocapsids are smooth, whereas intracytoplasmic nucleocapsids are ‘fuzzy’, this may infer that the fuzziness, at least in part, is caused by M antigen adhering to nucleocapsid components.

Organs of RIII mice at various physiological stages were tested for mouse mammary tumour virus (MMTV) antigen expression. Indirect immunofluorescence was used with three monospecific antisera to localize one envelope glycoprotein, gp47, and two core proteins, p28 and p8. These virus-specific antigens gave characteristic fluorescent patterns in the mammary tissues and were also detected in thymus and salivary gland sections of some mice. The amounts of antigens gp47 and p28 were measured by immunoassay in sera and organ extracts of corresponding samples of mice. Sera of mice of both sexes contained virus antigens from the suckling age onwards. Although ingested virus could be traced in suckling mice, weanlings were characterized by the absence of virus expression except in lymph nodes. This location points to the possible role of lymphoid tissue in producing the antigens of the blood and in disseminating the infection. In adult animals, virus antigens were present in salivary glands, digestive tract, lymph nodes, male genital organs and female mammary glands. Antigen expression, even found in the mammary glands of virgin mice, was strikingly increased by pregnancy, lactation and (or) ageing, the highest values being found in mammary tumours. The results for milk-borne MMTV infection in RIII mice are compared with those obtained previously in Swiss albino mice.

Two kinds of hepatitis B virus-specific particles are present in cytoplasmic extracts of hepatoma cells synthesizing hepatitis B virus (HBV) surface antigen. One class of particle contains the surface antigen of the virus, is 20S in size and has a buoyant density (in CsCl) of 1.2 g/ml. The second class of particle is a deoxyribonucleoprotein (DNP) with 1.3 g/ml buoyant density (in CsCl) and is 30S in size, the DNA of which contains HBV sequences thus proving virus specificity.

Mice immunized with Sindbis virus, strains HR (heat-resistant) or AR339, by a dual injection given intracerebrally (i.c.) and intraperitoneally (i.p.) at day zero, were cross-protected from challenge at day 10 with Semliki Forest virus (SFV). Neutralizing and haemagglutination-inhibition antibodies to Sindbis are detected in high titre by day 6 after immunization but no cross-reacting antibodies to SFV were found up to day 42. Immunized mice that were challenged with SFV showed an 8 to 10 day delay in the appearance of antibody to SFV compared to mice that were sham-inoculated. Thus, cross-protection in Sindbis-immunized animals was correlated with a temporary suppression of antibody formation to the challenge virus (SFV), while the appearance of high titre antibody to SFV in sham-immunized mice after challenge with SFV did not protect.

To facilitate an in vitro study of bluegill lymphocystis virus, a new cell line was established from the fins and caudal tissue of bluegill sunfish and designated BF-W. Bluegill lymphocystis virus was accurately and reproducibly assayed by an enlarged-cell enumeration assay and was propagated in BF-W cell cultures to give maximum titres of about 106 cell-enlarging units/ml in 21 days at 25 °C. The infectivity titre of preparations of bluegill lymphocystis virus was not affected by ultrasonic treatment, freezing and thawing, or long-term storage in maintenance medium at -70 or +4 °C, but decreased during storage at -20 °C in 50% glycerol. The infectivity of virus preparations was reduced by treatment with diethyl ether. Virus cultured in BF-W cells was entirely cell-associated and the growth of virus was completely inhibited by 1 mm-5-BrdUrd.

Murine cytomegalovirus (MCMV) may be rapidly and reproducibly attenuated by passage in tissue culture. A high proportion of CDI mice neonatally infected with the virulent strain of CMV can be shown to have replicating virus in the kidneys for up to 90 weeks p.i. and this high rate of isolation is not altered in pregnancy. A much lower rate of isolation from the kidneys is seen in CDI mice neonatally infected with the attenuated strain. However, this isolation rate was significantly increased during pregnancy and higher titres of virus were recovered.

An established model for latency by mouse cytomegalovirus (MCMV) was used to assess the effects of leukaemia on reactivation of MCMV. After inoculation of 400 spleen focus-forming units of Friend leukaemia virus (FLV), a significant number of mice yielded MCMV in salivary glands and kidneys 4 to 5 weeks later. Administration of cyclophosphamide during FLV infection increased the percentage of MCMV-positive animals. These results suggest that cytomegalovirus infections in leukaemia patients may reactivate as a result of the leukaemia itself and may be exacerbated by chemotherapy.

Bee virus Y (BVY), isolated from dead adult honey bees (Apis mellifera) collected in the field, is a commonly occurring serotype of bee virus X (BVX) in Great Britain. The viruses are very similar physically, although BVY aggregates in low salt concentrations and its single protein has a slightly lower mol. wt. than that of BVX. The viruses form separate bands when centrifuged to equilibrium in CsCl.

Preparations of the 30 × 103 mol. wt. protein (p30) of Rauscher murine leukaemia virus (R-MuLV) which had been purified to homogeneity as judged by gel electrophoresis in the presence of SDS and by amino-terminal amino acid analysis, showed considerable isoelectric heterogeneity. It was found that R-MuLV p30 polypeptide chains are easily converted in vitro into chains with more acidic isoelectric points. R-MuLV p30 polypeptides with different isoelectric points displayed the same set of 125I-labelled tryptic peptides. It is concluded that the charge heterogeneity of R-MuLV p30, as revealed in isoelectric focusing experiments, is not caused by genetic heterogeneity of the virus genome but by post-translational modification.

The double-stranded DNA of an adenovirus of tupaia (TAV) which has a mol. wt. of 21.5 × 106 was analysed as follows: the cleavage sites of restriction endonucleases BamHI, EcoRI, KpnI, SmaI, BstEII, ClaI and HpaI were determined by complete, partial and double digestion followed by gel electrophoretic separation of the resulting fragments. Terminal HpaI fragments were determined by hydrolysing the intact DNA with exonuclease III before restriction enzyme cleavage. Partial denaturation mapping of uncleaved DNA and EcoRI fragments revealed the cleavage sites of EcoRI as well as A+T-rich regions at both termini of the genome.

Adsorption of murine leukaemia virus (MLV) to NIH/3T3 cells, as determined by analysing its reverse transcriptase activity in the cell membrane, was found to be unaffected by interferon (IFN). Virus penetration and uncoating were followed by quantifying intracellular virions in terms of sedimentable reverse transcriptase activity in the cytoplasmic fraction. The penetrating virions were found to accumulate to a higher level in IFN-treated cells than in untreated controls. Intracellular virions were uncoated in untreated cells shortly after their penetration, whereas their uncoating was delayed in the IFN-treated cells for 2 to 3 h. Neither virus uncoating nor the effect of IFN on this process appeared to require new protein synthesis, since both were unaffected by cycloheximide (CH).

The effect of human fibroblast interferon (IFN), a heterologous IFN, on the uncoating of murine leukaemia virus (MLV) was investigated in exogenously infected NIH/3T3 mouse cells. Virus uncoating was determined by following the disappearance of the penetrating virus particles from the cytoplasm of the infected cells. Intracellular virus particles were estimated by sedimenting them at high speed from the cytoplasmic fraction of the infected cells and assaying their reverse transcriptase activity. In untreated control cells, uncoating started immediately after penetration, but in cells treated with human IFN, uncoating was delayed for 2 to 3 h. This delay led to prolongation of the infectious cycle, with delayed release of progeny virus. The delay in release did not result from inhibition by IFN of the process of release, since in NIH/3T3 cells chronically infected with MLV, treatment with IFN had no effect on virus release.