Cultivation of human immunodeficiency virus from whole blood: effect of anticoagulant and inoculum size on virus growth.

Human immunodeficiency virus (HIV) was cultivated directly from whole blood treated with anticoagulant by cocultivation with phytohaemagglutinin-stimulated cord blood lymphocytes. When heparin was used as the anticoagulant, isolation rates were low (10% to 56%, depending on the patient group); but when EDTA was used, isolation rates were much higher (50% to 100%). Culture of whole blood gave results identical to those of culture of separated peripheral mononuclear cells, and in some cases virus could be isolated from as little as 10 microliters of unseparated EDTA anticoagulated blood.

The prevalence of serological markers for hepatitis B virus infection in Australian Naval personnel.

In order to determine the prevalence of hepatitis B virus infection in Royal Australian Navy personnel and to formulate policies for their education, screening and vaccination, a voluntary survey was undertaken. Seven hundred and forty-eight subjects completed a questionnaire that provided details of age, sex, rank, ethnic origin, service abroad, length of service, history of tattooing, liver disease and blood transfusions. Serum was assayed by radioimmunoassay for the presence of hepatitis B surface antigen (HBsAg), antibody to hepatitis B surface antigen (antiHBs) and antibody to hepatitis B core antigen (antiHBc). The presence of markers of hepatitis B virus infection was correlated with "risk factors" by means of the chi 2 test of independence. Fourteen (74%) of 19 personnel from the Pacific Islands, Asia and Africa who were excluded from analysis showed one-or-more such markers. The prevalence of any marker of hepatitis B virus infection was 4.4% (95% confidence interval [CI], 2.9%-5.9%). The individual markers HBsAg, antiHBs and antiHBc were found in 0.4% (95% CI, 0.1%-1.2%), 3.6% (95% CI, 2.2%-4.9%) and 3.9% (95% CI, 2.5%-5.3%) of subjects, respectively. The annual attack rate was estimated to be less than 1%. Factors which had a significant correlation with an increased prevalence of markers of hepatitis B virus infection included tattooing (especially if this had been performed in Asia), age, the duration of service and service abroad. Based on these data, and given the reduced cost of hepatitis B vaccines, vaccination now is considered worth while. A policy of vaccinating all personnel who may be required for service at sea, and others at risk, now is in effect. Educational programmes that are aimed at minimizing the risk of exposure to hepatitis B and other viral infections have been instituted.

The regulated expression of Epstein-Barr virus: evidence that the transition from primary to secondary protein synthesis in Raji cells superinfected with Epstein-Barr virus requires the synthesis of new RNA.

In the presence of canavanine and the absence of arginine, superinfected Raji cells synthesize a limited spectrum of proteins (primary proteins). After the removal of canavanine at 8 h post infection, the cells proceed after a lag phase of 2-3 h to synthesize a second group of proteins. The appearance of these proteins can be prevented by addition of actinomycin-D to the chase media, suggesting that active primary proteins are required for the synthesis of new mRNA coding for the secondary proteins.

Ultrastructural studies on cell fusion induced by Epstein-Barr virus or N-butyrate and 12-O-tetradecanoylphorbol-13-acetate. Brief report.

The morphological changes which occur in Raji cells during EBV induced fusion are described. Of particular interest is the formation of local contacts between cells, at these points the plasmalemmae of the two cells become disorganized and cytoplasmic bridges are formed.

Strategies for the economic preparation of Epstein-Barr virus proteins of diagnostic and protective value by genetic engineering: a new approach based on segments of virus-encoded gene products.

Immunoprecipitation of Epstein-Barr viral proteins with various sera from normal adults, patients with fresh infectious mononucleosis or nasopharyngeal carcinoma was used to identify antigens which are of importance in the determination of immune status and characteristic of a particular disease. Some genes coding for of these antigens have been localized on the Epstein-Barr virus (EBV) genome by hybrid-selected translation. With the use of sequence data, these genes could then be subcloned from EBV DNA and expressed in eukaryotic and prokaryotic cells. Data on the expression are presented and the application of the methods described for the production of diagnostic reagents and vaccines is discussed.

An immunoprecipitation blocking assay for the analysis of EBV induced antigens.

A technique for the analysis of EBV antigens in extracts of unlabelled EBV infected cells has been developed. Using this technique we have demonstrated that the EBV early antigen complex consists of several proteins and is not completely expressed in chemically induced Raji cells. Studies with a range of sera from patients with infectious mononucleosis and nasopharyngeal carcinoma have shown that despite similar titers by the indirect fluorescent antibody test different populations of proteins were precipitated by different sera.

Effect of the arginine analog canavanine on the synthesis of Epstein-Barr virus-induced proteins in superinfected Raji cells.

The addition of canavanine to cultures of superinfected Raji cells in the absence of arginine prevented the appearance of early antigens as defined by immunofluorescent staining. Addition of various amounts of arginine permitted the identification of at least three groups of proteins, each responding differently to the various concentrations of arginine-canavanine.

An Epstein--Barr virus early protein induces cell fusion.

Superinfection of Raji cells with Epstein--Barr virus (EBV) leads to syncytium formation. Studies using metabolic inhibitors and amino acid analogues suggest that the fusion-inducing factor belongs to the early group of virus-specified proteins. Induction of early EBV protein synthesis in Raji cells by using various chemicals also leads to syncytium formation, indicating that the fusion process is not caused by a virion membrane protein introduced into the cells upon infection. The relevance of these findings to the association of EBV with carcinoma of the nasopharynx is discussed.

The regulated expression of Epstein-Barr virus. III. Proteins specified by EBV during the lytic cycle.

The experiments show that 30 virus-induced or virus-specified proteins were synthesized in Raji cells after superinfection with Epstein-Barr virus (EBV) derived from P3HR1 cells. Using a combination of pulse labelling, application of cycloheximide blocks at different times post-infection, treatment with amino acid analogues and inhibition of DNA synthesis it was shown that three groups of proteins appear in Raji cells after superinfection; the synthesis of the proteins in any one group appears to be coordinately regulated. Amongst the six virus-induced proteins which were synthesized immediately after release from an early cycloheximide block one would expect to find those proteins essential for the transition from EBNA to EA synthesis. Using human sera with differing specificities for the various antigen groups 11 proteins were identified as being specifically precipitated by sera having high titres against the EBV-induced early antigen complex.

The spontaneous and induced synthesis of Epstein-Barr virus antigens in Raji cells immobilized on surface coated with anti-lymphocyte globulin.

Immobilization of Raji cells on surface coated with anti-lymphocyte globulin (ALG) at low cell densities lead to the synthesis of Epstein-Barr virus (EBV) early antigen (EA) in up to 5% of the cells. At higher cell densities the percentage of antigen-positive cells decreased and at confluency no antigen synthesis was observed. Addition of iododeoxyuridine (IdUrd) to low density cultures increased the expression of EA to 20%, whereas in confluent cultures the cells could not be induced to synthesize EA. Treatment of cells in suspension with ALG failed to induced EA synthesis and did not potentiate the effect of IdUrd. Immobilized Raji cells proved to be suitable targets for superinfection with EBV derived from P3HR1 cultures.

Epstein--Barr virus-induced cell fusion.

Serological and molecular biological studies have shown an association between Epstein--Barr virus (EBV) and nasopharyngeal carcinoma. Although it has been shown that the epithelioid tumour cells carry EBV genomes, they are apparently devoid of receptors for EBV (H.W., unpublished observations). Other have suggested that fusion of EBV carrying cells with epithelial cells may be the mode of entry of the virus into cells unable to absorb the virus and that this may be mediated by one of the known syncytium-forming viruses which inhabit the respiratory tract (for example, members of the paramyxovirus group). de Thé and colleagues suggested that intercellular bridges could be seen in NPC tumour material. We have developed a technique which permits the preparation of stable monolayers of viable human lymphoblastoid cell lines. Using this technique we have now demonstrated that EBV can induce fusion between EBV-superinfected lymphoblastoid cells and cells devoid of EBV receptors.

Lymphocyte activation by the tumor-promoting agent 12-O-tetradecanoylphorbol-13-acetate (TPA).

TPA, a highly active tumor-promoting agent, is an effective mitogen for primate peripheral blood lymphocytes. Optimal stimulation of human lymphocytes was obtained 4 days after the addition of TPA at a concentration of 7.5 ng/ml. Lymphocyte fractionation experiments demonstrated that both T and B cells incorporated 3H-thymidine significantly in response to TPA. Lymphocyte blastogenesis was not due to the reactivation of latent herpesviruses by the tumor promoter, since similar responses to TPA were obtained with virus-genome positive or negative cells. Increased levels of DNA synthesis were observed when TPA was added to marmoset, baboon, rhesus monkey, or chimpanzee peripheral blood lymphocytes. Canine peripheral blood lymphocytes and spleen cells from guinea pigs, rats, and mice were not stimulated by TPA. These observations suggest that TPA-induced lymphocyte blastogenesis may be useful for studies of lymphocyte activation and of the molecular mechanisms of action of tumor-promoting phorbol esters.