Detection of antibodies against hepatitis B core antigen using the avidin-biotin system.

An enzyme avidin-biotin assay for the detection of anti-HBcAg antibody in human sera was developed. The assay uses genetically engineered HBcAg. HBcAg is immobilized on the surface of the wells of microtitre plates and the test serum sample, biotin-labelled HBcAg and streptavidin-labelled horseradish peroxidase are added. The assay was found to be specific and was compared with a commercial radioimmunoassay kit for sensitivity by testing 96 human clinical sera for anti-HBcAg antibody. Both assays gave identical results.

Monoclonal antibodies against hepatitis B e antigen: production, characterization, and use for diagnosis.

Five different hybridoma clones secreting anti-HBeAg antibody were constructed by fusing cells of mouse myeloma line SP2/0 with splenocytes from BALB/c mice immunized with recombinant HBeAg. The monoclonal antibodies obtained were characterized immunologically and one was used to develop ELISA for detection of HBeAg and anti-HBeAg antibody. These monoclonal assays enabled the detection of 3 U HBeAg/ml and 1 U anti-HBeAg/ml with reference to standards of the Paul Ehrlich Institute, Frankfurt, F.R.G. Both assays compared well with a commercially available kit (Abbott Laboratory) and were used for detection of HBeAg and anti-HBeAg antibody in clinical serum samples.

Characterization of tumors induced in adult Macaca mulata monkeys with Carr-Zilber strain of Rous sarcoma virus.

The Carr-Zilber strain of Rous sarcoma virus is highly oncogenic for adult monkeys of the species Macaca mulata. In the present study, we examined some characteristics of tumor growth when the tumor either regressed or progressed until it killed its host. We followed the kinetics of antibody formation to antigens coded by CZ-RSV in animals with a different course of tumor disease. The sera of animals tested contained virus-neutralizing antibodies, in addition to gs antibodies. The results are discussed with regard to the possible virus-productive type of interaction between RSV and the monkey cell.

Transformation of Chinese hamster embryo cells with an avian sarcoma virus ts mutant.

Chinese hamster embryo cells were transformed with temperature-sensitive mutant of the Schmidt-Ruppin strain of Rous sarcoma virus-subgroup A (ts NY68S Ra-A). The developed cell line elicited the morphological transformation at a permissive temperature of 35 degrees C, whereas after a shift to a non-permissive temperature of 40 degrees C the cells reverted to a normal phenotype. Tests for the presence of avian sarcoma group-specific antigen showed that the antigen was not expressed both at permissive and non-permissive temperature. However, the transient expression of gs antigen could be achieved by treatment with 5-IUdR only at the permissive temperature. There was a difference in the rate of 14C-2-deoxy-D-glucose uptake between cells cultured at the permissive and non-permissive temperature.

Effect of large doses of turkey herpes virus on antibody response in chickens.

During vaccination of chickens, repeated administration of large doses of a cell-associated turkey herpes virus compared to a single large dose did not affect markedly the virus-neutralizing antibody titre but accelerated the formation of agaroprecipitating antibodies. The formation of antibody types proceeded independently of each other. Health condition of chickens was not influenced.

Large-scale production, concentration and purification of Rous Sarcoma Virus in tissue culture.

A method is described for long-term standard production of purified virus from cultures of PR-RSV-C-transformed chicken cells. The mean yields were 4.91 mg of purified virus per liter of culture medium. Cells were grown in long-term cultures for 26 weeks. A combination of polyethylene glycol 6 000 precipitation and zonal centrifugation has been found satisfactory for virus concentration and purification.

Investigation of group-specific antigen during the culture of MC-29 hepatoma.

The short-time culture established from MC-29 chicken hepatoma was used for the analysis of cell morphology and expression of avian gs antigen. The content of gs antigen was not changed during the period of cultivation. Two morphologically different types of cells were present in tested tumour cells. Fluorescent antibody test showed the presence of gs antigen in both types of cells.

A rapid detection of avian oncovirus group-specific antigens in feather pulp by the enzyme-linked immunosorbent assay.

An ELISA procedure for the detection of avian sarcoma-leukosis gs antigen in feather pulp of adult birds is described. The test can be used for identifying gs+ and gs- chickens in leukosis-free populations. The titres of exogenous and endogenous virus-coded gs antigens overlap and the ELISA can be used only for a preliminary screening of unknown chicken populations.

A suppressive mechanism counteracts the production of anti-idiotype antibody.

BALB/c mice were repeatedly, at two-week intervals, immunized with monoclonal antibody against hepatitis B surface antigen and their sera were titrated for anti-idiotype antibody. The highest titres appeared after 1-4 immunizations. Further immunizations led to fast disappearance of the anti-idiotype antibody. These data suggest that anti-idiotype antibody production is a rather complicated process in which some suppressive mechanism is involved.

Role of replaced v-src and env genes in the duck-adapted variant of Rous sarcoma virus.

The env and v-src genes of a duck-adapted variant of Rous sarcoma virus were replaced for corresponding genes from parental chick-derived virus. The generation of viral constructs with replaced genes is described. DNAs of viruses with replaced genes were assayed on chick and duck embryo fibroblasts by transfection assays. Transformation efficiency was measured by the focus assay and multiplication of virus by the reverse transcriptase assay. Duck-adapted virus with replaced env gene lost the higher transformation efficiency for duck cells, whereas replacement of the v-src gene had no effect on its host-specific transformation activity.

Expression of major histocompatibility complex antigens (H-B) on haemopoietic cells, lymphoid cells, and chick embryo fibroblasts from congenic lines of chickens.

The expression and distribution of chicken major histocompatibility antigens (H-B) was examined by electron microscopy. Using an indirect method of ferritin and peroxidase labelling with monospecific heteroantisera and with monoclonal antibodies, the H-B alloantigens could be localized at the plasma membrane of various cell types. The antigenic binding sites were quantified on mature erythrocytes, erythropoietic cells, heterophils, lymphoid cells of both thymus and bursa origin, and on cultured normal and Rous sarcoma virus-transformed chick embryo fibroblasts. The degree of labelling was higher in mature normoblastic erythrocytes and lymphoid cells than in other haemopoietic cells and fibroblasts when the heterospecific antisera were used. A larger number of antigenic binding sites was labelled by the monomeric monoclonal antibodies than by the polyclonal antisera. The distribution and aggregation of the specific label on the plasma membrane of cells other than erythrocytes, however, differed in the fixed and unfixed cells.

Anti-idiotype antibody as a prospective vaccine against hepatitis B.

Rabbit and mouse sera containing anti-idiotype antibodies against a monoclonal and against some polyclonal anti-HBsAg antibodies were prepared. The immunoglobulin fractions of these sera induced the formation of anti-HBsAg antibodies when injected into BALB/c mice. The sera of humans repeatedly immunized with human anti-HBsAg antibodies were shown to contain anti-idiotype antibodies capable of eliciting an anti-HBsAg response in BALB/c mice and Syrian hamsters. Such sera may be a potential source of human anti-idiotype vaccine.

Inhibition of hepatitis B virus surface gene expression by antisense oligodeoxynucleotides in a human hepatoma cell line.

We have studied the inhibitory effect of antisense oligodeoxynucleotides on the expression of hepatitis B virus surface antigens. Human hepatoma cell line PLC/PRF/5 harbors several integrated copies of the HBV genome and produces and secretes hepatitis B virus surface antigen (HBsAg) to the medium. Synthetic antisense oligodeoxynucleotides complementary to various regions of the surface antigen gene were synthesized and their ability to block its expression was tested. Oligodeoxynucleotides (17- and 21-mers) complementary to regions covering ATG codons of both preS2 and S genes significantly inhibited preS2 and S protein production. Less efficient inhibition was achieved when the oligonucleotide complementary to the inside S gene region was assayed.

Replication of transformation-defective mutants of the Prague strain of Rous sarcoma virus and isolation of a td mutant from duck-adapted PR-RSV-C.

The replication of transformation-defective mutants of the Prague strain of Rous sarcoma virus subgroup C was studied using roller cultures. Under such conditions, 10(5)--10(6) infectous units of virus per 0.2 ml were produced, as revealed in both the reverse transcriptase and 16Q complementation tests. A new td daPR-RSV-C mutant was isolated from duck-adapted PR-RSV-C. This mutant replicated in roller cultures with equal efficiency as the original td PR-RSV-C. It was verified that td daPR-RSV-C does not transform chicken fibroblasts, is not oncogenic for 3-week-old chickens and has subgroup C host-range specificity. Both td mutants replicate in duck cells and reach the same titres.

Use of the hybridoma technique for antibody production to group-specific antigens of avian oncornaviruses.

Two clones of cell hybrids producing antibodies against the gag gene product of RSV were obtained using the hybridoma technique. Antibody production was tested by RIP test with subsequent SDS-PAGE analysis.

The expression of avian gs antigen in mammalian Rous sarcoma virus-transformed cells after treatment with 5-iododeoxyuridine and dexamethasone.

The content of avian gs antigen in RSV-transformed rat cells LW13-RsK1 and LW13-RsK4 showed a transient increase after treatment with 5-iododeoxyuridine. Higher levels of gs antigen were found when treated cells were cultivated in the presence of dexamethasone. In no case was the production of RSV found in treated RSV-transformed rat cell lines when, in addition, XC cells were tested.

In vitro cleavage of gag-myc fused protein P110 of a defective leukaemia virus MC29 by retroviral protease p15.

Gag-related proteins were precipitated from 35S-methionine-labelled cells of the PR-2 line derived from a hepatoma induced by virus MC29. Immunoprecipitates were incubated with viral protease p15 and the cleavage products of P110 were analysed in SDS-PAGE. The major cleavage fragment of P110 is a protein with mol. wt. of 56 000. Preparative isolation of the non-gag fragments from protein P110 by means of a gag immunosorbent showed that the 56K fragment did not contain any gag specific antigenic determinants. This finding was also confirmed by performing immunization with this fragment. Cleavage of protein P110 and the properties of cleavage products are discussed.

Genetic linkage of endogenous viral loci with the B (MHC) and C histocompatibility loci in chickens.

Genetic linkage of endogenous viral loci and histocompatibility loci B (MHC) and C has been demonstrated serologically in backcross progeny of inbred lines of chickens. The endogenous viral locus linked to the B complex is the first case of the localization of an endogenous viral gene to the microchromosomes in chickens.

Amino acid sequence homology between protein products of oncogenes and hormones (v-myc--gastrin and oxytocin; v-sis--secretin).

Computer comparisons of amino acid sequences from 8 retroviral oncogenes and 26 protein hormones were done with respect to structure similarity and so-called uninterrupted structure similarity. Sequence homology was found between v-myc, the transforming protein of MC29 virus, and human gastrin and oxytocin on the one hand, and between v-sis, the transforming protein of simian sarcoma virus, and secretin on the other.

Effect of the expression of an endogenous viral gene on the growth of tumours induced by Rous sarcoma virus in chickens.

The endogenous group-specific antigen of avian retroviruses has been detected in feather pulp of chickens of the inbred CB line (WL breed). Expression of the gs antigen has been demonstrated by ELISA, whereas the classical methods have classified the CB line as gs-. The number of gs+ chickens decreased with increasing age after hatching, and all chickens were gs- already at 20 weeks of age. The longer duration of gs antigen expression in line CB chickens had an adverse effect on their ability to regress Rous sarcomas. Chickens from a closed flock of the BL breed that segregated to gs+ and gs- also significantly differed in the ability to regress Rous sarcomas.