Susceptibility to an avian leukosis-sarcoma virus: close association with an erythrocyte isoantigen.

A dominant gene for susceptibility to early steps of cellular infection by subgroup B avian leukosis-sarcoma viruses is associated with the presence of an erythrocyte isoantigen. This gene may control both an isoantigen and a cell membrane receptor for an oncogenic virus.

Chicken genome mapping: a new era in avian genetics.

More than 460 loci representing either expressed or anonymous sequences have been mapped on to the first comprehensive molecular genetic linkage map of the chicken genome. Here, we review the current status of poultry genome mapping and discuss some of the new opportunities this provides.

Lymphoid neoplasms in chicken flocks free of infection with exogenous avian tumor viruses.

More than 4,500 breeding female chickens of nine inbred lines maintained under specific-pathogen-free conditions to approximately 500 days of age were studied. Routine monitoring and special assays indicated that they were free of infection by exogenous viruses of the leukosis-sarcoma and the reticuloendotheliosis groups. Some birds were maintained free of Marek's disease (MD) virus infection in plastic isolators, and others were maintained in conventional chicken houses and vaccinated with the herpesvirus of turkeys to prevent the lesions of MD. Ten birds bearing lymphoid tumors were observed in two sublines of one line of chickens known to produce embryos that spontaneously produce Rous-associated virus, type 0 (RAV-O), an endogenous virus of the chicken. Four tumors were found in chickens of one subline maintained free of MD virus infection in isolators. These tumors did not involve the bursa and had some histologic features different from those typical of lymphoid leukosis. Six tumors were found in chickens of the other subline that were vaccinated to prevent MD; these tumors involved the bursa and were typical of lymphoid leukosis but not MD. These results suggest that two types of tumors may have been observed. The fact that DNA extracted from both types of tumors did not contain exogenous lymphoid leukosis virus sequences confirms the virologic evidence that exogenous viruses were not involved. The fact that endogenous viral sequences were not increased in copy number suggests that the endogenous virus RAV-O did not directly induce the tumors. Two birds with tumors not involving the bursa were found alive, and transplantable lymphoid tumors were developed. These tumors were of T-cell origin rather than of bursa cell origin as would be expected of lymphoid leukosis. These are the first reported lymphoid tumors that have been observed in the absence of known exogenous tumor virus infection in chickens. Our evidence suggests that the endogenous virus RAV-O did not play a primary role in the induction of these tumors.

Low oncogenic potential of avian endogenous RNA tumor virus infection or expression.

Of chickens either spontaneously producing or exogenously infected in ovo with Rous-associated virus, type O (RAV-O), an endogenous virus of the chicken, only 1 died with lymphoid leukosis (LL), the most common neoplasm associated with the leukosis-sarcoma virus group. Because the chickens were not kept in strict isolation, it could not be assumed that the one LL was induced by RAV-O. In contrast, RAV-1-infected chickens from the same lines had a high incidence of LL and other neoplasms. Over 800 chickens of several inbred lines were maintained in plastic isolators free of exogenous avian leukosis-sarcoma virus infection for from 500 to nearly 1,000 days of age. No LL was observed, even though some lines are known to produce RAV-O spontaneously or to express inherited gs antigen. Three neoplasms of unknown etiology were observed, but none generally associated with leukosis virus infection. We concluded that avian endogenous virus expression had little, if any, oncogenic potential, and that exogenous avian leukosis viruses were responsible for most naturally occurring neoplasms.

The epidemiology of avian lymphoid leukosis.

Avian lymphoid leukosis can be induced by lymphoid leukosis viruses belonging to Subgroups A, B, C, and D. The endogenous virus of the chicken (Rous-associated virus type 0) belongs to Subgroup E and has little, if any, potential for inducing lymphoid leukosis. Nearly all chicken flocks are infected with Subgroup A lymphoid leukosis virus. This virus can be transmitted from dam to offspring or by contact with infected birds. Early infection, either by congential means, or soon after hatching, leads to the highest incidence of lymphoid leukosis. Maternal antibody or genetic resistance to infection delays or prevents infection, leading to a lower incidence of disease. In flocks segregating for genetic resistance to infection, continued infection is maintained through dynamic interactions between genetic resistance, acquired or maternal antibody ,and virus infection. Expression of endogenous viral information is controlled by dominant genes, but spontaneously produced Rous-associated virus type 0 can spread through a susceptible flock and be transmitted like an exogenous virus.

Gene insertion: current progress and long-term goals.

The methods for artificial gene insertion in the germline of the fly Drosophila and mice are now well established. In mice, cloned genes or retroviruses can be inserted by manipulation of newly fertilized ova, and intensive research is aimed at understanding the basis for regulation of gene expression using this technique. Manipulation of early embryos in the chicken is much more difficult. Therefore, we are concentrating on the use of avian retroviruses as vectors for gene insertion in this species. Some candidate genes are those controlling resistance to specific disease agents, those regulating humoral and cell-mediated immunity, and genes for immunogens that could be regulated to be expressed only after the development of immune competence, thus becoming an inherited vaccine. Basic research in these areas should lead to methods that will complement standard genetic selection for increased disease resistance in commercial chickens.

Studies of flocks with high mortality from lymphoid leukosis.

Two pairs of commercial white-egg parent flocks were selected for study, because one of each pair was observed to be dying with lymphoid leukosis at a high rate. The proportion of each flock producing eggs with lymphoid leukosis virus in the albumen was studied. In one pair the rate of birds shedding was no different in the high- and low-mortality flocks. The total rate of shedding was lower in the other pair, but the rate was higher in the high-mortality flock. These data indicate that the rate of virus infection is not always proportional to the development of lymphoid leukosis, and that other environmental factors may play a role. Direct complement-fixation tests on the albumens which were positive for lymphoid leukosis virus showed that group-specific antigen could be detected in 83%. Therefore, the direct complement-fixation test can be used on albumens as a rough estimate of shedding rate.

A comparison of test materials for differentiating avian leukosis virus group-specific antigens of exogenous and endogenous origin.

Three groups of pullets--those lacking endogenous viral (ev) genes, those carrying ev3, which codes for avian leukosis virus (ALV) group-specific (gs) antigen but not complete virus, and those carrying ev2, which codes for complete endogenous virus--were reared to maturity free of exogenous ALV infection or reared separately after inoculation at 1 day with ALV. The enzyme-linked immunosorbent assay (ELISA) was used to detect gs antigen in feather pulp, cloacal swabs, sera, white blood cells, and albumens from the pullets and in embryos, combs, and meconia from their progeny. These results were used to identify methods to distinguish between endogenous ALV expression and exogenous ALV infection. Although the frequency and levels of gs antigen detection were higher in most of the ALV-positive than in ev-positive ALV-negative materials, albumens and cloacal swabs had the lowest frequency of gs antigen detection in the ev-positive ALV-negative materials. These two materials had a further advantage in that detection of gs antigen in them has been shown to be highly correlated with congenital transmission. Further studies using ELISA absorbance values and titer to quantitate gs antigen showed that ev-positive ALV-negative albumens had much lower levels of gs antigen than ALV-positive albumens. The same criteria were not useful for distinguishing cloacal swabs of these two types. We conclude that in these lines, high levels of gs antigen in albumen is a sensitive and practical means of identifying dams congenitally transmitting ALV, because there is a very low frequency of "false positives" due to endogenous gs antigen in this material.

Pathogenicity of avian leukosis viruses.

Three methods were used in attempts to obtain non-oncogenic avian leukosis virus for possible use as an immunoprophylactic agent for the control of lymphoid leukosis in chickens. These were: 1) isolate a nononcogenic virus from commercial breeder flocks experiencing very little or no lymphoid leukosis; 2) obtain a non-oncogenic recombinant from mixed infection of a strain with low oncogenicity, Rous-associated virus-60 (RAV-60), with RAV-1 or RAV-2 in cell culture; and 3) attempt to attenuate subgroup A avian leukosis virus by serial passage in avian cell culture. Of 43 isolates obtained from field sources, all were pathogenic except one, and its pathogenicity was questionable because of the low amount of virus tested. All 42 clones from mixed infection of highly oncogenic and poorly oncogenic virus and all clones passaged serially in cell culture were oncogenic.

Avian leukosis virus infection and congenital transmission in lines of chickens resisting selection for reduced shedding.

Two lines (D and E) of three breeder lines of chickens that had resisted selection for reduced avian leukosis virus (ALV) congenital transmission on the breeder's premises did not resist the same selection procedures (tests for gs-antigen in albumen) under laboratory conditions. The incidence of ALV congenital transmission in the remaining third line (F) was spontaneously reduced from 13% to 0.9%. Environmental ALV exposure of uninfected chicks after hatching induced 7-10% of the progeny from lines E and F to become congenital transmitters but had negligible effects on line D. Neither errors in identifying dams nor horizontal transmission leading to congenital transmission were great enough to explain the lack of improvements in the three lines on the breeder's premises. Conditions of environmental exposure on the breeder's farm seem most likely to account for the resistance to reduced shedding. These findings suggest that the effectiveness of testing and selection procedures used to reduce ALV may be greatly influenced by the environment.

Low incidence of lymphoid tumors in chickens continuously producing endogenous virus.

A flock of 258 male and 243 female chickens of a cross of Regional Poultry Research Laboratory lines 15B and 7(2) were kept in a filtered-air positive-pressure house and observed for tumors from 100 to more than 729 days of age. These birds produced high titers of a subgroup E endogenous virus from the middle of the embryonic incubation period through the end of the experiment. No neoplasms were observed in the males. The females had two neoplasms indistinguishable from lymphoid leukosis and three other neoplasms not involving lymphoid cells. No evidence was found of infection with exogenous lymphoid leukosis viruses, Marek's disease virus, reticuloendotheliosis virus, or adenovirus (isolated on the isolation farm). Inoculation of another sample of this cross with a lymphoid leukosis virus of subgroup A resulted in 88% mortality with neoplasms (mostly lymphoid leukosis) by 167 days of age. The conclusion is that high levels of spontaneously produced endogenous virus do not induce high levels of neoplasms in chickens susceptible to lymphoid leukosis.

Tolerance, viral shedding, and neoplasia in chickens infected with non-defective reticuloendotheliosis viruses.

Chickens inoculated as embryos with non-defective reticuloendotheliosis viruses (ndREVs) generally developed a "tolerant" infection characterized by lack of immunofluorescent antibody and by a viremia that persisted through 93 weeks. Chickens inoculated at hatching generally developed a "non-tolerant" infection characterized by antibody development that gradually waned as the chickens aged and by a transient or intermittent viremia. Although chickens tolerantly infected with ndREV strain T were immunodepressed, tolerance to ndREVs did not depend on immunodepression, because 17-to-20-week-old chickens tolerantly infected with ndREV strain CS were normal in antibody response to sheep red blood cells and Brucella abortus and in mitogen-stimulated blastogenesis of blood lymphocytes. Tolerantly infected dams shed low levels of gs antigen and virus into eggs at high frequencies; however, in two trials, congenital transmission of virus by strain-CS-infected dams was documented in only 2 of 42 and 1 of 132 progeny chicks. Eggs and progeny chicks from non-tolerantly infected dams were always negative for virus and gs antigen. After long latent period (17 to 93 weeks), ndREV-infected chickens developed lymphomas involving the bursa of Fabricius and other visceral organs at high frequency and developed sarcomas, carcinomas, and inflammatory nerve lesions at a lower frequency. The ability of ndREVs to induce tolerant and non-tolerant infection, virus- and antigen-shedding into eggs, and chronic neoplastic disease resembled that of lymphoid leukosis virus, another common avian retrovirus. Certain differences in epidemiological properties of these 2 viruses are discussed.

Observations on an enzyme-linked immunosorbent assay for the detection of antibodies against avian leukosis-sarcoma viruses.

In the detection of antibodies against exogenous subgroup A avian leukosis viruses (ALVs) using a representative subgroup A virus, concordance between enzyme-linked immunosorbent assays (ELISAs) and serum neutralizations ranged from 83 to 95%. In ELISAs, subgroup A- and subgroup B-specific neutralizing antisera were equally reactive against ALVs of subgroups A, B, and E. Conversely, little cross-reactivity of high-titered subgroup E antisera was observed against subgroup A viruses. Significant cross-reactivities of spontaneously induced subgroup E-neutralizing antisera were observed when tested against a representative subgroup B ALV. Because some normal chickens spontaneously mount antibodies against infectious endogenous viruses, misleading results may be obtained if subgroup B or E ALVs are the source of target antigens in ELISAs.

Response of chickens carrying germline insert ALVA11 to challenge with a field strain of subgroup A avian leukosis virus.

The ALVA11 germline insert in chickens is a defective subgroup A avian leukosis virus (ALV) proviral insert that expresses a low-to-moderate level of subgroup A ALV envelope glycoprotein. Chicks carrying or lacking ALVA11 were evaluated for response to challenge by RPL-42, a pathogenic field strain of subgroup A ALV, by either exposure to chicks shedding RPL-42 or direct injection with various doses of RPL-42. Chicks carrying ALVA11 were significantly more resistant, as measured by infectious virus and viral antibody status, to horizontal infection and direct injection of RPL-42 than chicks lacking ALVA11.

Effect of endogenous leukosis virus genes on response to infection with avian leukosis and reticuloendotheliosis viruses.

We examined the effect of the presence or absence of endogenous viral gene (ev) 3, which controls expression of group-specific viral and envelope antigens (gs+chf+ phenotype), and ev2, which controls the production of a complete subgroup E virus (V-E+ phenotype), on the response of chickens to RAV-1, an exogenous avian leukosis virus (ALV) with an antigenic relationship to endogenous virus. After inoculation at one day of age, the chickens lacking either ev gene expression had a lower frequency of virus isolations and higher frequency and titer of neutralizing antibodies than those expressing ev genes. This relationship was not seen in groups inoculated with chick syncytial virus (CSV), a reticuloendotheliosis-associated virus with no relationship to endogenous virus, but the ev2+ birds tended to yield more CSV isolations than the ev2- birds. We suggest that chickens expressing ev genes may be immunologically tolerant to antigens common to exogenous and endogenous viruses. In addition, ev3- birds inoculated with RAV-1 at one day of age or as embryos died at a high rate between 6 and 12 weeks of age with a non-neoplastic syndrome characterized by severe atrophy of lymphoid organs, an inflammatory reaction in the liver, and a lower immune response to particulate antigens.

Absence of influence on immune competence by the sex-linked gene (K) determining slow feathering in white leghorn chickens.

Males of white leghorn strain crosses heterozygous (Kk) for the sex-linked feathering locus genes were mated to rapid-feathering (k-) females to produce rapid- and slow-feathering chicks of both sexes. K did not influence humoral-mediated immunity against challenge with sheep erythrocytes, killed Brucella abortus, or killed infectious bursal disease virus. Chicks challenged at 3 weeks of age had higher primary responses and higher titers of 2-mercaptoethanol-resistant antibody (IgG) than those challenged at 1 week of age. K had no influence on levels of cell-mediated immunity based on responses in in vitro phytohemagglutinin tests of donors 2 to 126 days of age, on responses in in vitro one-way mixed-lymphocyte cultures, and on rejection rates of skin grafts on young chicks. Feathering type did not influence viremia or antibody to avian leukosis virus; the level of lymphoid leukosis tumors was higher in rapid-feathering females than in slow-feathering females at 28 weeks of age (53% vs. 72%; P less than or equal to 0.10). We conclude that K does not influence general immune competence. The possibility that it may influence specific immunity to ALV under conditions not met in this study, because of an endogenous virus recently associated with the K locus, is discussed.

Influence of endogenous viral (ev) gene expression and strain of exogenous avian leukosis virus (ALV) on mortality and ALV infection and shedding in chickens.

Two experiments were conducted to determine the influence of endogenous viral (ev) gene expression on the response of chicken to day-old inoculation with two laboratory and two field strains of avian leukosis virus (ALV). The ev2- and ev3-negative semi-congenic chickens had much higher mortality from a unique non-neoplastic syndrome (NNS) than ev2- and ev3-positive semi-congenic chickens after inoculation with ALV strain RAV-1. The three other ALV strains induced little NNS. All four strains of ALV induced higher incidences of neutralizing antibody and lower incidences of viremia in ev2- and ev3-negative chickens than ev2- and ev3-positive chickens. The semi-congenic ev2- and ev3-negative chickens were only slightly less likely to shed ALV in the cloaca. Chickens of line 0, an unrelated line lacking ev genes, had a much higher rate of antibody production and lower rate of viremia and shedding than the semi-congenic chickens. Surprisingly, line 0 failed to get NNS after RAV-1 inoculation. There were major differences among strains of ALV in induction of antibody response, viremia, and shedding in the cloaca. The two field strains of ALV tended to be less immunogenic than the laboratory strains. We conclude that both the genetics of the host, including variation in ev gene expression, and the strain of ALV can influence the probability of ALV shedding and congenital transmission after horizontal infection.

Shedding of lymphoid leukosis virus in chickens following contact exposure and vaccination.

Chickens contact-exposed to lymphoid leukosis virus at various ages up to 32 weeks responded with relatively high rates of infection as determined by the presence of neutralizing antibody. Virus shedding as determined by cloacal swab and albumen testing occurred in 7 of 8 groups of such chickens, but the incidence was 10% or less and sporadic. Vaccination of chickens immediately before exposure with a low pathogenicity virus of subgroup A at 8 weeks of age did not eliminate subsequent shedding.

Characterization of monoclonal antibodies to avian leukosis viruses.

Hybridoma cell lines secreting monoclonal antibody (MCA) to avian leukosis virus (ALV) structural proteins p27 and p19 have been established. In an indirect enzyme-linked immunosorbent assay (ELISA), MCA 6AL20 (IgG1 isotype) reacted with RPL-40 (ALV subgroup A), avian myeloblastosis virus (AMV) (a mixture of subgroups A and B), Rous-associated virus (RAV)-2 (subgroup B), and Carr-Zilber strain of Rous sarcoma virus (CZ-RSV) (subgroup D) but not with Prague strain of RSV (PrC-RSV) (subgroup C) or the endogenous virus RAV-0 (subgroup E). MCA 6AL22 reacted as above and also reacted marginally with PrC-RSV. Both MCAs immunoprecipitated p19 from 35S-methionine-labeled chicken embryo fibroblasts (CEFs) infected with RPL-40 or RAV-1, but not from CEFs infected with RAV-0, thus identifying the viral structural protein p19 as a polypeptide with subgroup-specific epitopes. Both MCAs can be used to differentiate RPL-40 from RAV-0 infection either in an indirect antibody ELISA or by immunoprecipitation. A third MCA, 6AL42 (IgG2a isotype), reacted with the above viruses of subgroups A, B, C, and D at an antibody titer up to 1000-fold higher than with subgroup E RAV-0 virus in indirect ELISAs. MCA 6AL42 immunoprecipitated p27 from cells infected with RPL-40, RAV-1, or RAV-0. These MCAs are potentially useful in developing immunological tests for differentiation of ALV strains.

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.