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The major internal protein of the type C virus isolated from a Woolly monkey by Theilen and colleagues was purified by isoelectric focusing and used to raise specific antibody in guinea pigs. The protein has an isoelectric point of 6.5 to 6.6, and a molecular weight of 29,500 based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gel diffusion analyses showed a high degree of relationship to the type C virus isolated from a Gibbon ape by Kawakami and colleagues, while sera indicating this relationship were nonreactive with other mammalian type C viruses, except in highly sensitive radioimmunosassays. The “primate” viral proteins clearly contain determinants cross-reactive with the homologous protein in other mammalian type C viruses; however, heterogeneity of these cross-reactive determinants was indicated by differential reactivity of certain cross-reactive sera.

Summary Mouse cells made resistant to 6-azauridine were found to undergo syncytium formation upon infection with the Rauscher pseudotype of Moloney sarcoma virus (M-MSV(RLV)) and the Moloney strain of murine sarcoma virus (M-MSV(MLV)). The formation of syncytia was related to sarcoma virus replication since syncytia were never produced by leukaemia virus alone or by β-propiolactone-inactivated M-MSV(RLV).

BALB/3T3 cells transformed by the Kirsten sarcoma virus (nonvirus producer BALB/3T3 cells) and mutant cell lines derived therefrom by treatment with bromodeoxyuridine (BrdU) were analyzed for expression of virus-specific RNA using single-stranded DNA transcripts of Rauscher leukemia virus (RLV), a virus activated in one of the cell lines (58-2T), and Ki-SV-specific DNA transcript; the latter transcript after removal of all sequences cross-reactive with RLV RNA. The Rauscher virus DNA detected multiple copies of viral RNA in virus-producing cells (∼2.5 x 103/cell) whether infected with RLV or activated to produce virus with BrdU. Nonproducer (NP) cells and normal BALB cells showed small numbers of RNA genomes (70–250/cell) and only partial saturation of the transcript. The intracellular RNA sedimented at 35S (main peak) with a variable minor peak at 20S with the exception of one mutant cell, M-43-2 (main peak at 26–27S). The 58-2T transcript reacted preferentially in NP cells and their derivatives with biphasic kinetics suggesting the possibility of sequences specific for the original transforming virus. The size of Ki-SV specific sequences were 30S in mutant cells whether or not complete virus was being produced and independent of in vivo transplantability.

Human rhabdomyosarcoma cells (RD) productively infected with the transformation-defective feline virus, RD-114, contain viral-specific DNA integrated into the chromosomal DNA which specifies complete information for progency virus formation. This cellular DNA is infectious for permissive cells of human and canine origin, giving rise to progeny virus identical in assessed biochemical and biological properties to the parental virus. The host range for transfection with RD-114 DNA is similar to that of the intact virus. Although progeny virus could first be detected by reverse transcriptase assay at 2 to 4 weeks after transfection, virus is actually released at 3 days post-transfection, as shown by assay of culture media on sensitive indicator cells. The minimum dose of RD-114 DNA required for one infectious event in 2 × 10 6 RD cells is 0.10 to 0.20 μg. This gives a calculated efficiency of infection of 1 infectious unit per 5 × 10 4 to 5 × 10 5 viral gene copies, if there are 2 or 20 copies per infected cell, respectively. RD cells, productively infected with the transformation-defective mammalian viruses FeLV and GaLV, also contain viral-specific DNA, which gives rise to the respective virus of origin upon application to permissive RD cells.

Cultured human rhabdomyosarcoma (RD) cells were seeded on the chorioallantoic membrane of embryonated hens' eggs at the 10th to 12th day of incubation. When the membranes were harvested after 18 to 19 days' incubation, tumors had formed in eggs seeded with 5 × 105 to 10 × 106 RD cells. The microscopic appearance of the tumors was similar to that of the rhabdomyosarcoma of the patient from whom the cell line was derived. In contrast to cell lines derived from RD cell tumors which formed in a cat or an NIH-Swiss mouse and released the endogenous viruses of the cat and mouse, respectively, none of 36 cell lines derived from 13 tumors formed on the chorioallantoic membrane of hens' eggs released a type-C virus and none contained the avian virus group-specific antigen.

Infection of mouse embryo cells with two strains of murine sarcoma virus and one strain of murine leukemia virus was followed rapidly by synthesis of DNA in the cytoplasm. Persistently infected cells have not shown such synthesis, and ultraviolet-irradiated virus did not induce DNA synthesis. This new DNA presumably represents an intermediate in the virus replication cycle specified by the virion DNA polymerase(s). Failure to observe cytoplasmic DNA synthesis in persistently infected cells suggests, in keeping with the results of inhibitor experiments, that the new “viral” DNA becomes associated with cellular DNA in some form of stable interaction.

Recently we demonstrated the induction of a C-type RNA virus (XCIV) in tissue culture line XC derived from Wistar random-bred rats. In this article two other rat C-type virus isolates (NRKAV and RPLAV) are described. They were associated with, and isolated from, NRK and RPL lines originating in Osborne-Mendel and Lewis rat strains, respectively. The main biological characteristics shared by the three viruses were buoyant densities in sucrose gradients in the range of 1.14 to 1.15 g/ml, endogenous DNA polymerase activity, high molecular weight (60 to 70S) RNA, rat major speciesspecific (gs-1) antigen and gs-3 interspecies antigen. Two of the isolates (NRKAV and RPLAV) were able to rescue mouse sarcoma virus (MSV) genome from a rat MSV-transformed non-productive line. The NRK line was assayed for virus release at various in vitro passage levels. In lower passages (43–73) spontaneous virus release was absent or minimal but the virus could be induced by treatment of cells with 5-bromodeoxyuridine. At higher passage levels the cells produced a large amount of virus spontaneously, but in these treatment with 5-bromodeoxyuridine decreased virus production. The results of karyological analysis suggested that clonal selection was taking place in the NRK line during consecutive in vitro passages and simultaneously with the process of increasing spontaneous virus release.

Summary The group-specific antigen of two murine C-type RNA tumour virus types was purified to homogeneity by isoelectric-focusing of virus disrupted with tween-ether. The antigen has a molecular weight of 25,000 to 26,000 (estimated from sedimentation in sucrose relative to standards) and an isoelectric point of 6.7. The latter value was obtained from preparations of Friend, Moloney, Rauscher and Gross subgroups. The antigen was immunogenic in guinea-pigs inducing the synthesis of monospecific group-reactive antibodies. The antigen was relatively free of nucleic acid and appeared to be the only virus protein, based on labelling studies, with an isoelectric point near neutrality. Envelope antigen was localized at pH 4.5 by the electrofocusing technique.

A direct in vitro tissue culture method is described for the quantitation of bovine leukemia virus, utlizing a feline S+L-- cell line.

Gilden , R. V. (Wistar Institute, Philadelphia, Pa.), and R. I. Carp . Effects of cycloheximide and puromycin on synthesis of simian virus 40 T antigen in green monkey kidney cells. J. Bacteriol. 91: 1295–1297. 1966.—Synthesis of the simian virus 40 (SV40) T antigen in primary African green monkey kidney cells was abolished when cycloheximide was added up to 10 hr postinfection. In contrast, puromycin, another inhibitor of protein synthesis, did not suppress antigen production. The basis of this differential effect was the inability of puromycin to inhibit protein synthesis in the cells used. This was shown by the failure of the drug to depress the incorporation of labeled amino acid into protein and also failure to inhibit poliovirus synthesis. The puromycin preparation used was very effective in inhibiting poliovirus synthesis in HeLa cells. Thus, appearance of the SV40 T antigen is dependent on protein synthesis in infected cells.

Neutralization studies with human and simian cytomegaloviruses (CMV) are presently limited to sera obtained from naturally infected hosts or from monkeys immunized with specific CMV strains (1–3). The unavailability of reference antisera prepared in non-primate hosts raises the possibility that neutralization of human or simian CMV strains by human and monkey sera may result from a heterotypic response between the CMV strain under test and antibody directed against an inter- or intra-species cross-reacting virus. Cross-neutralization for example, has been reported between Herpesvirus hominis (herpes simplex) and Herpesvirus simiae (B-virus) (4, 5), and between Herpesvirus hominis and SA8 virus (6) isolated from vervet monkeys (7). The use of immune sera from non-primate hosts should minimize, although not completely exclude, the possibility of a heterotypic antibody response in serologic studies with human and simian CMV strains. This report concerns the preparation of antiserum in rabbits with neutralizing activity against a simian CMV (SA6) isolated from vervet monkeys (7).

A cell line (HT-1) derived from a hamster tumour induced by the Moloney sarcoma virus has been extensively used for preparation of murine pseudotype sarcoma viruses (Huebner et al. 1966; Hartley et al. 1969). Infectious virus could not be isolated from these cells nor was the group specific (gs) antigen of the murine C-type RNA viruses detectable in complement-fixation tests (Huebner et al. 1966). In further studies with this cell line, clonal sublines were derived and tested for infectious virus and virus particle production, gs antigen using both complement-fixation and precipitation-ingel assays, and the presence of virus genome by a highly reproducible rescue technique. HT-1 cells in the 80th in vitro passage were seeded into ten 60 × 15 mm. plastic Petri dishes (50 cells/dish). Each culture yielded 5 to 10 individual colonies, of which five were isolated by trypsinization within glass cloning rings. Each of the 50 clones was maintained separately in Eagle's basal medium with 10% foetal bovine serum.

Tumor virus RNAs from several mammalian and one reptilian species were purified; their 3′-terminal nucleoside was identified by separation of the trialcohols produced by periodate oxidation followed by reduction and tritiation with NaB 3 H 4 . Each virus contained uridine as the predominant terminal nucleoside. Molecular weight estimations based on the tritiation reactions were consistent with a structure consisting of four subunits.

A radioimmunoassay (RIA) for the major internal protein of the bovine leukemia virus (BLV p24) was established using anti-BLV p24 natural antibodies and purified 125I-labelled BLV p24. The final precipitation of the immune complexes was realized by a preparation of inactivated Staphylococcus aureau Cowan I. Sera from 363 cows belonging to (1) leukemic herds, (2) non-leukemic but BLV-exposed herds, and (3) apparently unexposed herds were studied comparatively in BLV p24 RIA, complement fixation and immunodiffusion. The BLV p24 RIA appeared much more senstive than the two other methods in the detection of positive sera. With this method 100% of the leukemic animals, excluding those with juvenile lymphosarcoma, presented very high antibody titers (greater than or equal to 10,000). Practically all cows with persistent lymphocytosis were also positive with slightly lower levels of antibodies, confirming the relationship between BLV infection and the persistent lymphycytosis. Moreover, about two-thirds of the hematologically suspect animals and one-third of the normal animals from BLV-exposed herds were found positive, whereas 100% of the sera from unexposed cows remained negative for anti-BLV p24 antibodies.

The NH2-terminal amino acid sequence of the major group-specific antigen, the major internal virion protein (p30; approximate molecular weight 30,000) of several mammalian type C RNA viruses was determined by the Edman degradation procedure using an automated protein sequenator. All of the proteins analyzed show a high degree of over-all sequence homology and also contain specific regions or single residues. All p30s begin with the sequence prolyl-leucylarginyl (Pro-Leu-Arg) and have an invariant, conserved region from residues 11 to 24. In this region only a single amino acid difference appears between the cat and mouse p30s. At position 17 alanine is found in the cat, and serine in all the mouse proteins. This homologous region starts at position 10 for RD-114 and baboon virus p30s, and at position 18 in the protein of the virus isolated from gibbon ape. The region extending from residue 4 to 10 shows considerable variability between p30s isolated from different mammalian species. Out of 24 residues compared, only a single amino acid difference was found between six different mouse p30s. At position 4, three have leucine, two have alanine, and one has serine. The comparative sequence data demonstrate that the viral p30s are products of related genes in the viruses from various mammalian species.

The present resolution (75-100 A) of the conventional scanning electron microscope (SEM) and its ability to image the surfaces of large numbers of whole cells in situ permits the approach of problems such as viral and cell surface antigen localization by immunological labeling with visual markers. Identification of virus and cell surface antigens in situ has been accomplished in indirect reactions by conjugated and unconjugated markers. Hemocyanin (Hcy) from whelk, Busycon canniculatum, has been developed as an immunospecific marker for virion and cell surface labeling in the electron microscope. Its size (approximately 30 x 50 nm) and distinct cylindrical shape permit easy visualization in the SEM and TEM. The Hcy method involves the preparation of antisera to Hcy in appropriate hosts for use in an unlabeled antibody macromolecular procedure based exclusively on antigen-antibody affinity to couple the macromolecule to the antigen site. Further correlative data from fluorescence microscopy can be obtained from similarly labeled samples by binding fluorescein to the bridging antibodies used in the Hcy technique. The usefulness of the Hcy marker system was demonstrated using antisera to the major envelope and cell surface glycoprotein (gp70) of Rauscher murine leukemia virus (R-MuLV), a type C retrovirus. The antiserum was shown to bind to the virion and cell surfaces of virus-infected cells in the homologous virus-infected cell system. It also demonstrated the expression of R-MuLV gp70-related antigens on a murine cell line Mm5mt/c1 which produces mouse mammary tumor virus (MuMTV), A type B retrovirus. Furthermore, when used in the Hcy marker system this antiserum was able to distinguish type B from type C budding virus on the same cell. Examples of other marker systems (ferritin, peroxidase, colloidal gold, and latex) used to show anti-gp70 serum reactivity will be presented to demonstrate their applicability to cell surface labeling studies. Methods for the preparation of immunoreagents and labeling of cells are discussed.

Using sensitive radiommunoprecipitation assays for highly purified type-C RNA tumor virus proteins, we found that 5 of 16 clinically normal gibbons (including 4 of 5 normal animals from a colony with 2 cases of lymphoma) and 4 of 4 experimentally inoculated gibbons formed antibodies to the major structural protein (p30) of gibbon ape leukemia virus (GaLV). An additional woolly monkey immunized with the closely related simian sarcoma virus also formed antibodies detectable with GaLV p30. Of 20 patients immunized with formalin-inactivated Rauscher murine leukemia virus (R-MuLV), 10 were previously reported to have antibodies to MuLV as determined by an internally labeled banded virus radioimmunoprecipitation assay. In comparison studies with purified R-MuLV proteins, 7 of 20 patients formed antibodies: 3/20 to R-MuLV p30 only, 1/20 to R-MuLV glycoprotein (gp) 70 only, and 3/20 to both p30 and gp70. Most responders were melanoma patients receiving immunotherapy with BCG. Additionally, rhesus monkeys produced antibodies to the endogenous cat virus RD114 and closely related endogenous baboon leukemia virus p30's. Thus these studies demonstrated the ability of primates (including humans) to form antibodies to well-characterized proteins from endogenous and exogenous type-C viruses and the potential utility of these assays for seroepidemiologic studies.

Genital warts (condylomata acuminata) are among the most frequent sexually transmitted infections. Human papillomavirus type 6 (HPV-6), which is etiologically related to a majority of these lesions, has not been propagated in tissue culture. We generated two forms of HPV-6 viral antigens: a chemically synthesized oligopeptide (referred to as the C-terminal synthetic peptide) corresponding to residues 482 to 495 of the 500-amino-acid-long L1 open reading frame (ORF), and a bacterially expressed 54-kilodalton (kDa) fusion protein containing the N-terminal 13 amino acids encoded by the lambda bacteriophage cII gene followed by one vector-insert junctional residue and 462 amino acids of the L1 ORF sequence (residues 39 to 500). The cII-L1 fusion protein was specifically recognized by an antipeptide serum directed against the N-terminal 13 amino acids derived from the cII gene, an antiserum raised against the C-terminal synthetic peptide, and a genus-specific serum prepared by immunization with disrupted viral capsids. The 54-kDa fusion protein was purified, and the sequence of its first 36 amino acids was determined and found to be as predicted by the DNA sequence. Both the genus-specific anticapsid serum and the antiserum raised against the fusion protein identified authentic L1 ORF proteins in HPV-1-induced (58 kDa) and HPV-6/11-induced (56 kDa) papillomas. The synthetic peptide antiserum recognized the 56- to 58-kDa protein in HPV-6-induced warts, but not in HPV-1- or HPV-11-infected specimens. Using the fusion protein as antigen in immunoassays, we were able to detect the corresponding antibodies in human sera.

A biophysical and immunological method for characterization of ribonucleic acid type C virus suspensions is described. The method provides a relationship to the total viral mass concentration of the particle titer, the gs antigen titer, and the ultraviolet absorbance (268 nm) of 2% sodium dodecyl sulfate digests. Data for murine, rat, feline, and hamster viruses are shown to be analogous within the test limitations. From these data, an assessment of the viral purity can be made, the structural integrity can be evaluated, an approximate molecular weight can be computed, and the mole ratio of gs antigen can be determined.

A low molecular weight, highly basic DNA-binding protein was purified from several oncornaviruses by the sequential procedures of gel filtration in guanidine-hydrochloride, DEAE-cellulose chromatography and affinity chromatography on single-stranded DNA sepharose. The binding protein from Rauscher and woolly monkey type-C viruses was the fastest migrating of the virion proteins in SDS-polyacrylamide gels and thus is designated p10 according to previous convention although our estimates of molecular weight were 8-9,000 daltons. The binding protein from these two viruses was resolved into two bands by acid-urea electrophoresis although only a single NH2 terminal amino acid was detected (S. Oroszlan, personal communication), thus indicating charge heterogeneity. Antibody to Rauscher virus p10 species-specific in gel diffusion and complement-fixation tests and did not exhibit cross-reactivity with other virion proteins. A DNA-binding protein was also detected in preparations of mouse mammary tumor virus. This purified protein had an apparent molecular weight of 12,500, was the second fastest migrating component in the virus preparations, and was antigenically unrelated to the mouse type-C virus p10.

Radioimmunoprecipitation was used to test cat sera for ability to bind to the purified major internal protein p30 of feline leukemia viurs (FeLV), to the endogenous cat virus (RD-114), and to murine leukemia virus (MuLV). The data were compared with results of tests for antibody to the feline oncornavirus-associated cell membrane antigen FOCMA and for the presence of viremia. In contrast to the general lack of free antibody to FeLV p30 in a random sample of healthy cats, high levels of antibody to FeLV p30 and FOCMA were found in normal animals from high-leukemia-cluster households. Titers of greater than or equal to 200 for p30 and greater than or equal to 32 for FOCMA were found in nonviremic animals; a percentage of animals with high FOCMA titers and lower or no p30 binding activity were viremic. Animals with neoplasms were low or negative for FOCMA antibody and did not have high titers of free p30 antibody. The p30 binding activity could be divided into three main categories: high binding with FeLV p30 and much lower activity with RD-114 and MuLV p30's, as seen with hyperimmune sera; high binding with FeLV and RD-114 p30's and low activity with MuLV p30, possibly indicative of specific antibody to both of the aforementioned proteins; and low level binding to all three p30's.

The human T-cell lymphotropic virus (HTLV) family includes members associated with T-cell cancers (HTLV-I and HTLV-II) as well as the etiological agent of the acquired immunodeficiency syndrome (HTLV-III). Molecular clones of these viruses were used in heteroduplex mapping experiments to study their structural and evolutionary relationships. The HTLV-I subgroup, despite some restriction enzyme site polymorphism, demonstrated a high degree of sequence conservation. Heteroduplexes of HTLV-I and HTLV-II demonstrated a significant amount of sequence homology, with the strongest region of conservation occurring in the 3'-most coding sequences, designated pX, and to a lesser, although substantial extent in the rest of the genome. Thus, the genomic organization of HTLV-II appears to be very similar to that of HTLV-I. All HTLV-III molecular clones appeared to be identical, with a single exception, which showed heterogeneity in the env gene region. In heteroduplexes between HTLV-I and HTLV-III, very little homology was observed, being confined to the gap/pol region. In contrast to the latter result, a striking amount of homology was detected between HTLV-III and a morphologically similar, pathogenic, nononcogenic lentivirus, visna virus. These data provide strong evidence for a close taxonomic and thus evolutionary relationship between HTLV-III and the lentivirus subfamily of retroviruses. A taxonomic tree, based on the genetic relatedness and biological properties of the HTLV family, is proposed.

DNA transcripts were prepared from three related viruses of hamster and analyzed by interviral hybridization and by reaction with cellular DNA. A virus (G-HaLV), isolated from a dimethylbenzanthraceneinduced tumor cell in Graffi hamsters, contained nucleic acid sequences highly specific for hamster cell DNA and did not react with mouse cell DNA nor did its transcript show homology (5%) with mouse or rat viral RNAs. The hamster-specific sarcoma virus, B-34, isolated by Bassin and coworkers from tumors induced by the Harvey strain of murine sarcoma virus, contained mouse-, hamster-, and possibly rat-specific sequences. B-34 transcripts were predominantly mouse-specific. GLOH(-), a lymphomagenic virus derived by dilution beyond the transforming endpoint of a hamster-specific sarcoma virus obtained from tumors induced by the Gross pseudotype of murine sarcoma virus, also contained hamster- and mouse-specific sequences. Only a portion of its hamster sequence (about 50%) was shared with B-34 and G-HaLV viruses. As expected, transcripts of GLOH(-) virus were reactive with mouse and hamster cellular DNA.

Natural and expreimental cat sera were tested in radioimmune precipitation assays vs purified p30s from FeLV, RD114 and MuLV. Antibodies with specificity for FeLV p30 comparable to hyperimmune sera from heterologous species but of low titer were found in a high percentage of normal cats from households with a high incidence of FeLV and neoplasia. Sera from cats with neoplasms were generally negative. Cats immunized with FeLV gave low-level immune response, also of the same general specificity as heterologous hyperimmune sera. Cat sera do not normally show antibody to RD114 p30 although two immunized weanling cats produced low titered but highly specific p30 antibody. Thus, for both classes of feline type-C virus p30s, there is an evident capability of the cat to mount an immune response to naturural or experimental exposure to the respective proteins. The magnitude of the response is between 100 and 1,000 fold below that seen in heterologous species. In contrast, cats immunized with MuLV p30 gave immune responses comparable to those seen in guineapigs, rabbits and goats. Several very old cats with carcinoma had antibody which preferentially precipitated MuLV p30. A competition assay using one such serum and labelled MuLV p30 was inhibited by FeLV, RD114, and MuLV p30s. This indicates that the assay is “interspecies” in nature. Among the possible explanations of this reaction category is that it represents antibody to the p30 of an as yet undefined class of feline type-C virus.

BALB/3T3 cells transformed by the Kirsten sarcoma virus (nonvirus producer BALB/3T3 cells) and mutant cell lines derived therefrom by treatment with bromodeoxyuridine (BrdU) were analyzed for expression of virus-specific RNA using single-stranded DNA transcripts of Rauscher leukemia virus (RLV), a virus activated in one of the cell lines (58-2T), and Ki-SV-specific DNA transcript; the latter transcript after removal of all sequences cross-reactive with RLV RNA. The Rauscher virus DNA detected multiple copies of viral RNA in virus-producing cells ( approximately 2.5 x 10(3)/cell) whether infected with RLV or activated to produce virus with BrdU. Nonproducer (NP) cells and normal BALB cells showed small numbers of RNA genomes (70-250/cell) and only partial saturation of the transcript. The intracellular RNA sedimented at 35S (main peak) with a variable minor peak at 20S with the exception of one mutant cell, M-43-2 (main peak at 26-27S). The 58-2T transcript reacted preferentially in NP cells and their derivatives with biphasic kinetics suggesting the possibility of sequences specific for the original transforming virus. The size of Ki-SV specific sequences were 30S in mutant cells whether or not complete virus was being produced and independent of in vivo transplantability.

Cell transformation and replication of the Rauscher pseudotype of Moloney murine sarcoma virus in mouse embryo fibroblasts were inhibited by hydroxyurea within a critical time period of 30 to 90 min postinfection. In cells infected by Rauscher leukemia virus, treatment with 1mM hydroxyurea during the critical time period resulted in the accumulation of minus-strand DNA (molecular weight, 3 x 10(6)) in association with the parental viral genoma RNA. This 5 to 6 x 10(6) dalton RNA:DNA hybrid was found in the cytoplasm. Positive-strand DNA of genomic or smaller size was not detected in the presence of hydroxyurea, but virus-specific DNA was found in the nucleus 30 min after removal of drug.

Four cell lines were derived from childhood malignancies: rhabdomyosarcoma, sarcoma, lymphosarcoma and an American Burkitt's lymphoma. Cells of the four lines formed tumors in immunosuppressed newborn hamsters. The tumors had a microscopic appearance like that of the tumors from which the cell lines were derived. No virus-like particles were detected by electron microscopy in the cell lines after treatment with bromodeoxyuridine; no hamster type-C virus expression was present in cell lines derived from the hamster tumors. Chromosome constitution and in vitro growth properties of the cell lines were studied as was the fibrinolytic function of the sarcoma lines.

DNA was extracted from two human sarcoma cell lines, TE-32 and TE-418, and the leukemic cells from five children with acute myelocytic leukemia, three children with acute lymphocytic leukemia and four adults with acute myelocytic leukemia. The DNAs, assayed for infectivity by transfection techniques, induced no measurable virus by methods which would detect known mammalian C-type antigens or RNA-directed DNA polymerase in TE-32, D-17 dog cells and other indicator cells, nor did they recombine with or rescue endogenous human or exogenous murine or baboon type-C virus. Model systems used as controls were human sarcoma cells, TE-32 and HT-1080, and human lymphoma cells, TE-543, experimentally infected with KiMuLV, GaLV or baboon type-C virus, all of which released infectious virus and whose DNAs were infectious for TE-32 and D-17 dog cells. Other model systems included. two baboon placentas and one embryonic cell strain spontaneously releasing infectious endogenous baboon virus and yielding DNAs infectious for D-17 dog cells but not for TE-32 cells. Four other baboon embryonic tissues and two embryonic cell strains, releasing either low levels of virus or no virus, did not yield infectious DNA.

Passive immunization with heterologous antivirus antiserum beginning at birth successfully suppressed infectious murine leukemia virus expression in Lake Casitas wild mice (Musmusculus) at 5-7 weeks of age.

The response of serologically mature fowls to antigens having varying degrees of relationship to a previously injected antigen was studied by quantitative precipitin methods and absorption analyses. After successive injections with related antigens three major antibody populations were detected: (1) antibodies reacting only with the second antigen, equivalent in amount to those obtained in a primary response to that antigen; (2) antibodies reacting only with the first antigen, lower in amount than those obtained during a primary response to that antigen; and (3) antibodies reacting with both antigens, in much greater amount than after successive injections with a single antigen.

ANTIBODY production after immunization with particulate antigens is known to persist over long periods of time. By contrast, immunization with soluble antigens in the absence of adjuvants is generally thought to give responses of relatively brief duration. Recently, it has been reported that antibody production continued for several hundred days in rabbits given one or several intravenous injections of a number of soluble antigens1. Antibody produced late in the course of the response gave negative ring tests, failed to sensitize the guinea pig intestine, and could be detected only by a passive haemagglutination technique. This communication is concerned with similar observations on the response of chickens to intravenous injections of bovine serum albumin (BSA). One major finding of this study is the persistence of precipitating antibody up to 313 days after injection.