Avian Tumor Research Articles

In vitro replication of infectious bursal disease virus in established lymphoid cell lines and chicken B lymphocytes

The in vitro susceptibility of chicken lymphocytes to a wild strains of infectious bursal disease virus was investigated by using immunofluorescence and virus assays as infection criteria. A variety of Marek's disease lymphoblastoid cell lines, all of thymus (T-cell) origin, were refractory to virus exposure. However, a bursa (B-cell)-derived lymphoblastoid cell line from an avian leukosis virus-induced tumor was highly susceptible. Viral antigen appeared in the cytoplasm of 20 to 30% of the cells, and large amounts of cell-free virus were released, with maximum yields occurring by 3 days postinfeciton. The virus also replicated in a small percentage of normal lymphocytes prepared from lymphoid tissues and peripheral blood of chickens. Pretreatment of the lymphocytes, with heat-inactivated anti-B-cell serum or with antiserum against fowl immunoglobulin M before inoculating them with the virus blocked the virus infection; no blocking occurred with anti-T-cell serum or with specific antiserum against fowl immunoglobulin G or immunoglobulin A. This suggests that surface immunoglobulin M-bearing B-lymphocytes were the target cells for infection.

Cellular and Humoral Immune Reactivity to Tumor-Associated Antigens in Chickens Infected with Rous Sarcoma Virus

Abstract With a leukocyte blastogenesis assay and 3 M KCl extracts of chicken cells infected or infected and transformed by various avian tumor viruses (ATV), the time course of development of cellular reactivity in RSV tumor-bearing chickens to antigens expressed on these cell types was examined. Leukocyte reactivity to mitogens in RSV-injected birds was also investigated. Similarly, humoral responses to ATV-antigens and virus-induced antigens were assessed over time, by measuring the development of antiviral neutralizing antibody, as well as antibody to cell surface antigens on various cell types, by means of an isotopic antiglobulin test (IAT). There were no differences in phytohemmaglutinin responsiveness between the leukocytes of RSV-injected birds and control birds, but responses to pokeweed mitogen were sometimes elevated in RSV-injected animals. Cellular immunity to 3 M KCl extracts, principally to those extracts containing structural components of the virus envelope, developed between 3 and 5 weeks post-injection, during tumor growth and regression, and then declined, although the immune response could be restimulated by virus rechallenge. For the most part, responses to nonvirion tumor-specific surface antigen (TSSA) was weak except after RSV rechallenge. Humoral reactivity as measured by IAT also seemed to correlate well with the presence of virus envelope antigens (VEA) on infected indicator cells and appeared before antiviral neutralizing antibody. The data indicated that, at least during the primary response, major immune reactivity in RSV-injected birds appeared to be directed against VEA and that TSSA appeared to be most immunogenic when VEA was also present.

Cells transformed by temperature-sensitive mutants of avian sarcoma virus cause tumors in vivo at permissive and nonpermissive temperatures

Chick embryo (CE) fibroblasts and normal rat kidney (NRK) cells transformed by temperature-sensitive ( ts) mutants of avian sarcoma virus (NY68, LA23, LA24, LA25, LA29, LA31, GI201, GI202, GI251, GI253) induce tumors on the chorioallantoic membrane (CAM) of chick eggs at temperatures that correspond to the permissive and nonpermissive temperatures used to induce conditional expression of the “transformed” phenotype in these cells when cultured in vitro. Chick embryo cells infected with transformation-defective mutants of ASV (td101, td108) or RAV-50 were nontumorigenic under the same conditions, as were nontransformed CE and NRK cells. This indicates that the CAM is not an unusually susceptible substrate for cell growth and that the ability of tsASV-transformed cells to form tumors at nonpermissive temperatures reflects their true tumorigenicity. In contrast, a ts mutant chemically transformed rat liver cell line, ts-223, only formed tumors on the CAM under permissive conditions. The wild-type parent cells (W-8) of this mutant produced tumors at both permissive and nonpermissive temperatures. Direct implantation of microprobe thermometers into tumors caused by tsASV-transformed cells at nonpermissive temperatures confirmed that tumor formation occurred in a stable temperature environment and was not due to temperature fluctuations which might have created semi-permissive or permissive conditions for tumor growth. Cells isolated from tumors formed at nonpermissive temperatures and recultured in vitro displayed temperature-dependent hexose transport and colony formation in agar similar to the original parent cell inoculum. Similarly, virus recovered from tumors at nonpermissive temperatures retained the ts mutation.

Avian acute leukemia viruses MC29 and MH2 share specific RNA sequences: Evidence for a second class of transforming genes

The genome of the defective avian tumor virus MH2 was identified as a RNA of 5.7 kilobases by its presence in different MH2-helper virus complexes and its absence from pure helper virus, by its unique fingerprint pattern of RNase T1-resistant (T1) oligonucleotides that differed from those of two helper virus RNAs, and by its structural analogy to the RNA of MC29, another avian acute leukemia virus. Two sets of sequences were distinguished in MH2 RNA: 66% hybridized with DNA complementary to helper-independent avian tumor viruses, termed group-specific, and 34% were specific. The percentage of specific sequences is considered a minimal estimate because the MH2 RNA used was about 30% contaminated by helper virus RNA. No sequences related to the transforming src gene of avian sarcoma viruses were found in MH2. MH2 shared three large T1 oligonucleotides with MC29, two of which could also be isolated from a RNase A- and T1-resistant hybrid formed between MH2 RNA and MC29 specific cDNA. These oligonucleotides belong to a group of six that define the specific segment of MC29 RNA described previously. The group-specific sequences of MH2 and MC29 RNA shared only the two smallest out of about 20 T1 oligonucleotides associated with MH2 RNA. It is concluded that the specific sequences of MH2 and MC29 are related, and it is proposed that they are necessary for, or identical with, the onc genes of these viruses. These sequences would define a related class of transforming genes in avian tumor viruses that differs from the src genes of avian sarcoma viruses.

Biochemical behavior of MC‐29 virus‐induced transplantable chicken hepatoma

Studies were performed to test the applicability of the molecular correlation concept developed in chemically induced transplantable rat hepatomas to virally induced transplantable hepatoma in chicken. In a comparison of the activities of carbohydrate, purine, and pyrimidine key enzymes in rat and chicken hepatomas, similar patterns were observed in rodent and avian tumors. These results show that the enzymatic imbalance elucidated in chemically induced rat hepatomas is applicable to the virus-induced hepatoma in chicken, independent of the nature of the carcinogenic agent and the species.

Avian Oncovirus Mill Hill No. 2: Pathogenicity in Chickens<xref ref-type="fn" rid="fn2">2</xref>

Mill Hill No. 2 (MH2), an avian tumor virus, was studied for its transforming and oncogenic effects. In tissue culture it induced transformation of chicken fibroblasts and yolk sac macrophages. When injected into the chicken, the main feature of this virus was its ability to cause liver and kidney carcinoma, in addition to sarcoma. MH2-associated viruses did not transform cell cultures but were able to cause only lymphoma in the birds. Light and electron microscopy were used in a detailed histologic study of the tumors induced by MH2 virus. An unclassified round cell sarcoma was produced in soft tissues at sites of injection; there was no evidence of origin from endothelium. In the kidney, carcinomas mixed with a malignant stroma were found. Hepatocarcinomas were the dominant tumors found in the liver. The lymphomas produced by the associated virus were poorly differentiated and highly malignant. The study illustrated the highly oncogenic potential of this virus and offered a model for the analysis of the carcinogenic events in a more specific way.

Antigenic determinants specific to the envelope glycoprotein of exogenous avian tumor viruses.

Antibody in the sera of 15B X 7(2) chickens was measured for reactivity to determinants of the envelope glycoprotein of endogenous and exogenous avian tumor viruses. The pattern of reactivity of animals infected with exogenous sarcoma virus served to operationally define a class of determinants present on the envelope glycoprotein of exogenous viruses and absent from that of the endogenous virus Rous-associated virus-0.

Analysis of integrated avian RNA tumor virus DNA in transformed chicken, duck and quail fibroblasts

The state of integration of avian sarcoma virus DNA in the genomes of transformed chicken, duck, and quail fibroblasts was deduced by means of restriction enzyme digestion of total cell DNA, gel electrophoresis, and subsequent analysis by the procedure of Southern. The cells used in these studies were either mass-infected cultures or clones of infected cells selected by their ability to form colonies in agar. For both mass-infected cultures and clones of cells of all three species, we found that integration occurred at a specific site on the viral genome but appeared to occur at many sites on the cell genome. At least some of the integrated viral DNA existed as intact nonpermuted species flanked by direct terminal repeats of at least 0.134 megadalton (217 base pairs). For each of 12 transformed quail clones studied, it was possible to detect, after digestion with Kpn I, unique junctions between viral and cellular DNA. That is, at our level of analysis, the integration site on the cell genome for each clone was different. However, within each of the 17 chicken and 9 duck clones of transformed cells, a heterogeneity presumably occurred during the outgrowth of the cell clone population, in that we could not readily detect identifiable cell-virus junction fragments.

Biosynthesis and maturation of cellular membrane glycoproteins.

The biosynthesis and the processing of asparagine-linked oligosaccharides of cellular membrane glycoproteins were examined in monolayer cultures of BHK21 cells and human diploid fibroblasts after pulse- and pulse-chase labeling with [2-3H]mannose. After pronase digestion, radiolabeled glycopeptides were characterized by high-resolution gel filtration, with or without additional digestion with various exoglycosidases and endoglycosidases. Pulse-labeled glycoproteins contained a relatively homogenous population of neutral oligosaccharides (major species: Man9GlcNAc2ASN). The vast majority of these asparagine-linked oligosaccharides was smaller than the major fraction of lipid-linked oligosaccharides from the cell and was apparently devoid of terminal glucose. After pulse-chase or long labeling periods, a significant fraction of the large oligomannosyl cores was processed by removal of mannose units and addition of branch sugars (NeuNAc-Gal-GlcNAc), resulting in complex acidic structures containing three and possibly five mannoses. In addition, some of the large oligomannosyl cores were processed by the removal of only several mannoses, resulting in a mixture of neutral structures with 5-9 mannoses. This oligomannosyl core heterogeneity in both neutral and acidic oligosaccharides linked to asparagine in cellular membrane glycoproteins was analogous to the heterogeneity reported for the oligosaccharides of avian RNA tumor virus glycoproteins (Hunt LA, Wright SE, Etchison JR, Summers DF: J Virol 29:336, 1979).

Avian RNA tumor virus DNA before and after integration into the host genome.

Avian RNA tumor virus DNA before and after integration into the host genome.

Inhibition by dihydrorifampicin of RNA polymerases I and II isolated from nuclei of Rous sarcoma cells and human tumor cells HEp-2

Dihydrorifampicin, a rifampicin derivative hydrogenated at the 18-19 carbon atoms of the aliphatic ansa chain of the rifampicin molecule, inhibits the enzymatic activity of RNA polymerases I and II, isolated from the nuclei of avian tumor cells (Rous sarcoma) and from the human tumor cell line HEp-2. The RNA polymerases from these tumors have been separated and partially purified by chromatography on DEAE Sephadex A-25 and characterized by the sensitivity to α-amanitin. The [3H]UMP-labeled ribonucleic acids synthesized in the isolated nuclei of Rous sarcoma cells in the presence and absence of DHR were analyzed by sedimentation analysis in sucrose density gradients. It was found that the synthesis of rRNAs and mRNAs is very significantly inhibited by dihydrorifampicin, whereas the synthesis of tRNAs is much less inhibited at the same DHR concentration (100μg/ml). The observed cytostatic effect of DHR on the growth of human tumor cells HEp-2 and embryonic cells in culture is apparently mediated by the selective inhibition of RNA polymerases I and II in human and avian cells. The relationship between the molecular structure of DHR and its affinity to RNA polymerases of eukaryotic cells is discussed.

The History of Avian Medicine in the United States V. Insights into Avian Tumor Virus Research

This historical review describes and discusses the discoveries that have been most important to the understanding of avian tumor viruses from the viewpoint of a biologist and a poultryman. It emphasizes the discoveries that were firsts in tumor virology, and, where appropriate, compares the state of the art in avian tumor virus research with that in tumor virus research of other animals. We will interject personal understandings, opinions, and philosophies, as well as a few experiences. This report is not a review of research on avian leukosis and related neoplasms. Such reviews are available elsewhere (9,16,22). Furthermore, to keep this article a reasonable length, we have omitted many reports and experiences.

Analysis of unintegrated avian RNA tumor virus double-stranded DNA intermediates

Previous studies by Guntaka et al. have shown that the unintegrated DNA intermediates of avian RNA tumor virus replication can be readily isolated from cultures of the quail tumor line QT-6 at 1 day after infection. The intermediates include double-stranded linear and covalently closed circular DNA species. Using the analysis procedure of Southern together with previously obtained information regarding the sites of action of certain restriction endonucleases on avian sarcoma virus DNA, we have further characterized the viral DNA intermediates. Evidence is presented that, relative to the RNA genome, most of the linear species possess a direct terminal sequence redundancy equivalent to 0.5 X 10(6) +/- 0.3 X 10(6) daltons of double-stranded DNA. Some of the circular forms also possess a sequence redundancy of 0.21 X 10(6) +/- 0.03 X 10(6) daltons.

Specific site of action for single-strand specific nuclease on the double-stranded circular DNA intermediates of an avian RNA tumor virus

Circular viral DNA intermediates obtained from the quail tumor line, QT6, at 1 day after infection, were opened at one specific location by the single-strand specific nuclease, S1, of Aspergillus oryzae. This site was no longer accessible to the S1 nuclease when circles were first opened at another location with a restriction endonuclease.

Specific RNA sequences and gene products of MC29 avian acute leukemia virus

The 28S RNA of the defective avian acute leukemia virus MC29 contains two sets of sequences: 60% are hybridized by DNA complementary to other avian tumor virus RNAs (group-specific cDNA) and 40% are hybridized only by MC29-specific cDNA. Specific and group-specific sequences of viral RNA, defined in terms of their large RNase T(1)-resistant oligonucleotides, were located on a map of all large T(1) oligonucleotides of viral RNA. Oligonucleotides representing MC29-specific sequences of viral RNA mapped between 0.4 and 0.7 unit from the 3'-poly(A) end. Oligonucleotides of group-specific sequences mapped between 0 and 0.4 and between 0.7 and 1 map unit. Cell-free translation of viral RNA yielded three proteins with approximate molecular weights of 120,000, 56,000, and 37,000, termed P120(mc), P56(mc), and P37(mc). P120(mc) contained both MC29-specific peptides and serological determinants and peptides of the conserved, internal group-specific antigens of avian tumor viruses. P120(mc) is translated only from full-length 28S RNA. Furthermore, MC29 RNA contains sequences related to the group-specific antigen gene (gag), near the 5' end, which are followed by MC29-specific sequences. We conclude that this protein is translated from the 5' 60% of the RNA, and that it includes a segment translated from the specific sequences. It is suggested that the transforming (onc) gene of MC29 may consists of the specific and some group-specific RNA sequences and that P120(mc), which is also found in transformed cells, may be the onc gene product.

Nucleotide sequence relationships between the genomes of an endogenous and an exogenous avian tumor virus

We have used mapping of large T1 oligonucleotides to examine the genome of Rous-associated virus-O (RAV-O), an endogenous virus of chickens, and to compare it with that of Prague strain Rous sarcoma virus, subgroup B, (Pr-RSV-B), an exogenous sarcoma virus. To extend the sensitivity of such comparisons, we have developed a system of nucleic acid hybridization and hybridization-competition combined with fingerprinting. This method allows us to estimate the relative degree of relatedness of various portions of the viral genomes. From the results of this study, we have concluded that the genomes of Pr-RSV-B and RAV-O are related in the following way. The 5'-terminal half of the genomes (corresponding to the gag and pol regions) is virtually identical, with only scattered single nucleotide differences. This region is followed by a region comprising 25 to 30% of the genome (the env region) which contains substantial nucleotide sequence differences, most or all of which are due to single base changes. The env-coding region can be further subdivided into three regions: a more variable region probably containing sequences coding for subgroup specificity, flanked by relatively common sequences on each side. To the 3' side of the env region, the RAV-O genome contains a very short sequence not found in Pr-RSV-B, whereas the Pr-RSV-B genome contains a much longer unrelated sequence. The central portion of this sequence comprises the src gene as defined by transformation-defective mutants. Particularly striking is the absence, in the RAV-O genome, of any nucleotide sequence related to the "c region" found very near the 3' end of all exogenous tumor viruses. Both the Pr-RSV-B and RAV-O genomes contain the identical terminally redundant sequence of 21 nucleotides near each end of the genome.

Differential mortality and lesion responses to reticuloendotheliosis virus infection in Marek's disease-resistant and susceptible chicken lines

The major histocompatibility complex of the chicken (Pazderka et al. 1975) resembles the mouse H-2 region in controlling many phenomena related to adaptive immune responsiveness. Immune response (Ir) loci within the B complex control cell-mediated and humoral immune responses to defined antigens (G/inther et al. 1974, Benedict et al. 1975, 1977). Lymphocyte response in the graft-versus-host and mixed lymphocyte reactions is also controlled by the B locus (Longenecker et al. 1972, Miggiano et al. 1974). Strain differences in host response to infection by Marek's disease virus (MDV) or avian leukosis virus (ALV) are well documented. Chickens heterozygous or homozygous for the B 21 allele show resistance to T-cell lymphomagenesis by MDV (Longenecker et al. 1976, Briles et al. 1977). Differences between chicken lines in regression of tumors induced by Rous sarcoma virus (Schierman et al. 1977, Collins et al. 1977), and susceptibility to induction of lymphoid leukosis (LL), a B-cell lymphoma, after ALV infection have also been described (Purchase and Gilmour, 1975, Crittenden et al. 1972). Reticuloendotheliosis virus (REV), the prototype of a newly described group of avian C-type retroviruses (Purchase et al. 1973), induces tumors composed of undefined hematopoietic cells in the spleen and liver in experimentally infected chickens (Purchase and Witter 1975). REV differs markedly from other avian tumor viruses in its virulence. The induction period may be as short as 5 days, and mortality may approach 100% (Purchase and Witter, 1975). REV induces the only 'short-incubation' lymphoreticular tumor caused by an avian C-type retrovirus, and therefore offers an attractive avian model system for studies of genetic control of tumorigenesis. The present report describes differences in mortality patterns and visceral lesion development in line N and line P chickens (B-locus genotype B21/B 21 and B19/B 19, respectively). These lines were developed by Cole (1972) on the basis of resistance and susceptibility to Marek's disease (MD). Differences between line N and P chickens in response to MDV infection may reflect genetically determined differences in the immune responses to virus infection and tumorigenesis (Briles et al. 1977). To investigate the possibility of genetic control of tumorigenesis in the REV system, we compared the mortality patterns and lesion responses of these two lines to REV infection. Chickens of line N (BZI/BEI) and line P (B19/B 19) were hatched on the same day and were inoculated intraperitoneally with REV (0.1 ml containing 5 • 103 plaque-

The Economics of Marek's Disease Control in the United States

SummaryA vaccine for the control of Marek's disease, a disease causing severe losses in poultry worldwide, was developed and made available for national use in the United States in 1971. This paper examines the benefits and costs of the control ol Marek's disease in the United States.The total public and private (including industry) expenditure on avian leukosis research for a 10-year period was estimated. Then the total benefits from the research up to 1974 were estimated from the reduced broiler and egg-type chicken losses and increased egg production and feed utilization. The total expenditure on research between 1965 and 1974 was about $32 million. Benefits from the control of the disease are estimated to total $168 million annually. If both costs and benefits are discounted at a reasonable rate of 7 per cent annually to the beginning of increased effort against Marek's disease in 1965, the ratio of benefits to costs is 22:1. Even if costs and benefits are discounted to the beginning of Federal work against avian leukosis in 1939 the ratio would be 4:1. In addition, there were many intangible benefits such as the more predictable growth of chickens and the impact of the research on science in other fields such as human cancer. By any measure, the investment in research on avian tumours has paid off handsomely. The greatest benefit was to the consumer in the form of a more uniform, less expensive product than would have been available had the technology not been developed.

Enhancement of avian sarcoma virus-induced tumor growth after pretreatment with BCG

Pretreatment of chickens with BCG 1 week before inoculation with avian sarcoma virus had a stimulatory effect on virus-induced tumor growth in 15 of 17 cases tested. This outcome was dependent upon the injection of both BCG and the oncogenic agent into the same wing web. Injection of BCG into the wing web contralateral to that used for virus inoculation or injection of a subcellular fraction of BCG into the same wing web did not affect tumor development. Administration of BCG also gave rise to heightened levels of tumor-associated, cell-mediated immunity, both on the part of chickens which received a subsequent injection of oncogenic virus as well as normal, BCG-inoculated controls.

Status of Endogenous Avian RNA Tumor Virus in Marek's Disease Lymphoblastoid Cell Lines and Susceptibility of Those Lines to Exogenous RNA Tumor Viruses

Three lymphoblastoid cell lines from Marek's disease (MD) tumors, two MD virus (MDV) producer lines (MSB-1 and HPRS-1), and a nonproducer line (RPL-1) were studied for the expression of avian leukosis sarcoma (ALS) viruses. The MSB-1 line was free of all known endogenous expressions, including replicating virus (RAV-O), group-specific (gs) antigens, and chick helper factor (chf). The RPL-1 and HPRS-1 were positive for gs antigens and chf. The RPL-1 and MSB-1 lines showed no evidence of an exogenous DNA provirus by nucleic acid hybridization (HPRS-1 line was not tested for that DNA). All three lymphoblastoid cell lines were susceptible to exogenous infection with both sarcoma and leukosis viruses of subgroup A but varied in susceptibility to subgroups B and C. All were resistant to subgroups D and E.