Multiple Group-specific Antigen Components of Avian Tumor Viruses Detected with Chicken and Hamster Sera

Multiple group-specific (gs) components of the avian leukosis-sarcoma viruses were detected by immunodiffusion (Ouchterlony) tests with sera from hamsters bearing tumors induced by sarcoma viruses and with sera from adult chickens immunized with avian sarcoma or leukosis viruses. Immune hamster sera detected up to four components, whereas chicken sera detected at least one. The hamster and chicken sera identified a similar antigen, as indicated by reactions of identity. Relatively few chicken sera containing neutralizing antibody to avian sarcoma or leukosis viruses reacted in immunodiffusion with the gs antigen. The gs components were released from the virion by various means of disruption, including freezing and thawing. Tests with tissues from normal chickens and from chickens with Marek's disease failed to demonstrate any reactions with hamster or chicken gs antiserum.     

Performance of 3 successive generations of specified-pathogenfree chickens maintained as a closed flock

No antibodies against Salmonella pullorum, Mycoplasma gallisepticum, Mycoplasma synoviae, Haemophilus gallinarum, fowl pox virus, Marek's disease virus, herpes virus of turkey, infectious laryngotracheitis virus, avian adenovirus, avian reovirus, infectious bursal disease virus, reticuloendotheliosis virus, avian leukosis virus, avian encephalomyelitis virus and Newcastle disease virus were detectable in the sera obtained from these chickens in 3 generations at various ages. Antibodies against infectious bronchitis virus were detected in the sera of the 3rd generations at 66, 74 and 108 weeks of age. The performances of these chickens was nearly the same as that of conventional healthy chickens in the poultry industry, with no tendency to decline.     

Tumor growth and antibodies after RSV-challenge in normal chickens and in chickens congenitally infected with avian leukosis virus.

Normal chickens and chickens congenitally infected with an avian leukosis virus (ALV) of antigenic subgroup A were challenged with strains of Rous sarcoma virus (RSV) of two different antigenic subgroups (B and C) and tumor induction and growth as well as humoral antibody to viral envelope antigen (VEA) and tumor-specific surface antigen (TSSA) were measured. There was no effect of congenital ALV infection on RSV tumor incidence or latent period but the growth rate and size of the tumors were much higher in congenitally infected birds as compared to controls. Whereas most tumors in the RSV-challenged normal birds regressed, tumors in ALV-infected birds grew progressively. There were no striking differences in the number of birds in either group in the incidence of anti-TSSA or anti-VEA antibodies nor did the presence of either type of antibody reflect the tumor status of the host.     

Induction of Group-specific Interspecies Antibody in a Cat by Immunization with Disrupted Feline Leukaemia Virus

“ALL mice, cats and virtually all chickens seem to be completely refractory to developing antibody to the group-specific, gs, antigens characteristic of the RNA tumour viruses of their own species.”¹ This is explained on the basis of an immune tolerance induced in early embryonic life by the expression of these antigens before the development of immune competence. Avian group-specific (gs) antibody has been demonstrated in the sera of immunized chickens by the immunodiffusion (Ouchterlony)² and complement fixation inhibition³ tests. This report is to record the production of gs antibody in a cat which had been immunized with gs antigen from disrupted feline leukaemia virus (FeLV).     

Reaction of Chick Embryo Cells Infected with Leukosis Strain MC29 Virus with Fluorescein-Labeled Antibody

Immune serum was prepared in the rabbit with BAI strain A leukosis virus isolated by centrifugal fractionation from the plasma of chickens with myeloblastic leukemia and further purified on a potassium tartrate gradient. Antibody to group-specific antigen was demonstrated in the serum by immunoelectrophoresis and immunodiffusion. Fluorescein-conjugated serum was used unabsorbed and absorbed with chick cells for study of acetone-fixed chick embryo cells uninfected or infected with strain MC29 avian leukosis virus. With unabsorbed serum, large numbers of cytoplasmic particles stained in a few cells within 2 hr after exposure to virus, and the cell number increased greatly in 24 hr. Absorption of the serum abolished the early reaction. Staining with absorbed serum was delayed until about 14 hr after culture exposure to virus, but essentially all cells were stained within 72 hr at the time when all cells were morphologically altered. Differences between the responses to unabsorbed and absorbed serum suggested cytoplasmic formation or concentration of chick tissue antigen similar to that incorporated in leukosis virus particles. The characteristics of staining with absorbed serum were similar to those observed by others in analogous studies with avian tumor viruses.     

Avian leukosis virus infection: analysis of viremia and DNA integration in susceptible and resistant chicken lines

Avian leukosis viruses induce lymphoid leukosis, a lymphoma which develops within the bursa of Fabricius several months after virus infection. Chickens from the Hyline SC and FP lines are, respectively, susceptible and resistant to avian leukosis virus-induced lymphoid leukosis. We examined plasma and cellular DNA obtained from avian leukosis virus-infected chickens for the presence of viremia and integrated viral sequences to determine whether the extent of virus infection is comparable in individuals of both lines. A less than twofold difference in the frequency of viremia was detected between chickens of the two different lines. Although the analysis of plasma samples, which were obtained at different times postinfection, demonstrated that the duration of viremia was comparable in both susceptible and resistant chickens, the onset of the viremia observed in susceptible chickens generally preceded by 1 week that observed in resistant chickens. Moreover, integrated viral sequences were detected in approximately 90% of the SC and 40% of the FP chickens. The appearance of infectious virus in the plasma was, in general, associated with the presence of integrated viral sequences in both the bursal cells and the erythrocytes obtained from the same chicken. The presence of both the viremia and the integrated viral DNA sequences was transient, suggesting a mechanism for the elimination of virus-infected cells in both susceptible and resistant chickens. Furthermore, at 5 weeks postinfection no integrated exogenous viral sequences were detected in splenic lymphocytes obtained from either chicken line, regardless of whether these chickens were viremic or had integrated viral sequences detectable in other tissues. Our results indicate that extensive avian leukosis virus replication occurs in approximately 50% of the FP and 100% of the SC chickens. Although it appears that the viral infection spreads more quickly in the SC chickens, our results afford no obvious explanation of the resistance to the development of lymphoma exhibited by FP chickens.     

Persistence of Marek''s disease virus in a subpopulation of B cells that is transformed by avian leukosis virus, but not in normal bursal B cells.

Previous studies have described an augmentation of avian leukosis virus (ALV)-induced lymphoid leukosis in chickens that were coinfected with a serotype 2 Marek's disease virus (MDV) strain, SB-1. As a first step toward understanding the mechanism of this augmentation, we have analyzed the tropism of the MDV for the ALV-transformed B cell. After hatching, chickens were coinfected with ALV and a nonpathogenic strain of MDV, SB-1. Seventy primary and metastatic ALV-induced lymphomas that developed in chickens between 14 and 20 weeks of age were found, with only one exception, to carry SB-1 DNA. The MDV genome was maintained in cell lines derived from the tumors. However, MDV DNA could not be detected in nontransformed bursal B cells from chickens carrying ALV lymphomas. Moreover, during and after the lytic phase of MDV infection, SB-1 DNA was near or below the level of detection in bursal cells, suggesting that MDV most likely infects only a small subpopulation of bursal cells. By contrast, ALV-transformed B cells from MDV-free chickens could be persistently infected with MDV in vitro. These findings indicate that ALV lymphoma cells, unlike nontransformed bursal B cells, are susceptible to persistent MDV infection and can serve as a reservoir of MDV that can potentially influence the physiology of the transformed cell.     

Detection of the Viral Sarcoma Gene Product in Cells Infected with Various Strains of Avian Sarcoma Virus and of a Related Protein in Uninfected Chicken Cells

Genetic analyses have defined a single gene (src) as that portion of the avian sarcoma virus (ASV) genome which encodes the protein directly responsible for ASV-induced neoplastic transformation. We have recently identified the polypeptide product of the src gene of the Schmidt-Ruppin (SR) strain of ASV, a 60,000-dalton phosphoprotein designated pp60(src), and have further determined that pp60(src) acts as a protein kinase. Essential to the identification and characterization of the pp60(src) protein of SR-ASV was the use of serum (TBR serum) from rabbits bearing SR-ASV-induced tumors. TBR serum was, however, strain specific, recognizing pp60(src) from SR-ASV-transformed cells only. We report here that sera from marmosets bearing tumors induced by the Bryan or SR strains of ASV (TBM sera) contain antibody which precipitates the transforming gene product from cells transformed by the SR, Bryan, Prague, or Bratislava strains of ASV. In contrast, rabbits bearing tumors induced by either the Bratislava or Bryan strains of ASV, or hamsters with SR-ASV-induced tumors did not produce antibody to pp60(src) from any strain of ASV. The 60,000-dalton polypeptides immunoprecipitated with TBM serum from cells transformed by each of the above virus strains are phosphoproteins. One-dimensional peptide mapping by limited proteolysis revealed that the pp60(src) proteins are structurally very similar, but not identical. Furthermore, all of the viral pp60(src) proteins have an associated phosphotransferase activity. In addition to detecting the viral src proteins, TBM serum was able to immunoprecipitate an antigenically related protein from normal uninfected avian cells.     

Lack of Serological Relationship Among DNA Polymerases of Avian Leukosis-Sarcoma Viruses, Reticuloendotheliosis Viruses, and Chicken Cells

Antibodies against a large and a small DNA polymerase isolated from chicken embryos and against avian myeloblastosis virus DNA polymerase were used to study the serological relationships of the DNA polymerase activities of three avian systems with RNA and a DNA polymerase-avian leukosis-sarcoma viruses, reticuloendotheliosis viruses, and a fraction from uninfected chicken cells. The DNA polymerase activity of disrupted virions of all avian leukosis-sarcoma viruses tested was neutralized to the same extent by antibody against avian myeloblastosis virus DNA polymerase and was not neutralized by the antibodies against chicken cellular DNA polymerases. The viruses tested included induced leukosis viruses and Rous-associated virus-O. The DNA polymerase activity of disrupted virions of all of the reticuloendotheliosis viruses was not neutralized by any of the antibodies. The chicken endogenous RNA-directed DNA polymerase activity was neutralized partially or completely, in different experiments, by antibody against the small DNA polymerase isolated from chicken embryos, but was not neutralized by the other two antibodies.     

Radioimmunoassay for Avian C-Type Virus Group-Specific Antigen: Detection in Normal and Virus-Transformed Cells

A radioimmunoassay has been developed for detection of avian C-type virus (30,000 mol wt) group-specific (gs) antigen. The method is 10- to 1,000-fold more sensitive than immunological methods previously available. By the radioimmunoassay technique, normal chicken embryo cells, which have previously been classified as gs negative or weakly gs positive, contain clearly detectable amounts of gs antigen. The assay has been used to study the effect of chemical induction and superinfection by mammalian C-type viruses on the expression of avian gs antigen in mammalian cells nonproductively transformed by avian sarcoma viruses.     

应用跨膜区置换的血凝素蛋白建立H7N9禽流感病毒抗体间接ELISA检测方法

【目的】由于H7N9禽流感病毒能够感染鸡,并且已经变异成了高致病性毒株,因此,鸡群中H7N9禽流感疫苗的免疫是一个趋势,而鸡群免疫后抗体检测方法的建立也十分必要。本研究旨在建立一种灵敏、高效、高通量的鸡群H7N9亚型禽流感病毒抗体间接酶联免疫吸附试验(ELISA)检测方法。【方法】通过昆虫杆状病毒表达系统分别表达属于W1、W2-A和W2-B分支H7N9流感病毒的3种野生型血凝素(HA)蛋白,以及跨膜区(TM)置换为H3 HA TM的W2-B分支HA蛋白(H7-53TM)。4种HA蛋白经过离子交换层析纯化后作为抗原,通过ELISA检测H7N9禽流感病毒抗体。【结果】ELISA特异性、敏感性和重复性试验结果显示,跨膜区置换主要影响HA蛋白ELISA检测的重复性,以H7-53TM为抗原的ELISA方法具有较好的重复性,其批内和批间变异系数小于10%,然而3种野生型HA蛋白与部分血清反应批内和批间变异系数大于10%,重复性较差,因此选择H7-53TM蛋白作为ELISA包被抗原。通过受试者工作特征曲线(ROC曲线)分析,以H7-53TM为抗原的ELISA能够精准地区分H7N9亚型流感病毒抗体阳性和阴性血清。通过相关性分析,该ELISA方法与134份鸡血清HI试验结果具有显著强相关性(r=0.854 6,P0.000 1),并且与3个分支疫苗株免疫血清的HI试验结果也具有显著相关性(r0.5,P0.05)。【结论】跨膜区置换能够提高HA蛋白抗原检测H7N9禽流感病毒抗体的重复性,并应用跨膜区置换的HA蛋白建立了一种能够检测不同分支疫苗株免疫的H7N9亚型禽流感病毒抗体间接ELISA检测方法。     

Intracellular restriction on the growth of induced subgroup E avian type C viruses in chicken cells.

Subgroup E avian type C viruses produced by bromodeoxyuridine-treated 100 X 7, line 7, or line C chicken cells were restricted in their intracellular growth on K28 chicken cells but not on line 15 chicken cells. Cells from embryos of line 15 chickens bred with K28 chickens did not restrict the growth of the subgroup E induced leukosis viruses (ILVs). This result indicates that the phenotype for the intracellular restriction of the growth of subgroup E ILVs found in K28 cells is recessive. Long-term growth of the subgroup E ILVs in K28 cells resulted in the appearance of subgroup E virus that grew well on K28 cells. No change in growth characteristics was observed for subgroup E ILVs grown in line 15 cells indicating that appearance of nonrestricted virus occurred only during growth of the subgrouo E ILVs on a restrictive host. RAV-0, a subgroup E virus closely related to the ilvs, had the same growth characteristics as the subgroup E ILVs. RAV-60, a subgroup E virus formed by recombination of exogenous avian leukosis virus with endogenous subgroup E virus coat information, grew well on both line 15 and K28 cells.     

Infrequent involvement of c-fos in avian leukosis virus-induced nephroblastoma.

To determine whether c-fos is involved in avian leukosis virus-induced nephroblastoma, 28 tumors from chickens were analyzed for novel fos fragments. DNA from 1 of 16 clonal outgrowths (in chicken 6561) contained novel fos-related EcoRI and KpnI fragments which hybridized to both v-fos and viral probes. Oncogenicity tests using filtered 6561 tumor cell homogenates did not reveal a tumor-inducing transduction of c-fos. We conclude that c-fos is only an occasional target for proviral insertions or new transductions in avian leukosis virus-induced nephroblastoma. The results also identify a polymorphism in c-fos in K28 chickens and demonstrate that unintegrated viral DNA is not a general characteristic of avian leukosis virus-induced nephroblastoma.     

Augmentation of retrovirus-induced lymphoid leukosis by Marek''s disease herpesviruses in White Leghorn chickens.

Our objective was to determine whether the cell-associated herpesvirus vaccines used in chickens to control Marek's disease tumors can augment development of lymphoid leukosis (LL) induced by exogenous avian leukosis virus (ALV). Various single or mixed Marek's disease vaccines were inoculated at day 1, and ALV was injected at 1 to 10 days, with chickens of several experimental or commercial strains. Development of LL was monitored at 16 to 48 weeks in various experiments. In several strains of chickens we repeatedly found that the widely used serotype 3 turkey herpesvirus vaccine did not augment LL in comparison with unvaccinated controls. However, LL development and incidence were prominently augmented in several chicken strains vaccinated with serotype 2 vaccines, used alone or as mixtures with other serotypes. In one chicken strain, augmentation was demonstrated after natural exposure to ALV or serotype 2 Marek's disease virus viremic shedder chickens. Augmentation of LL by virulent or attenuated Marek's disease viruses of serotype 1 was intermediate in effect. Serotype 2 Marek's disease virus augmentation of LL was prominent in three laboratory lines and one commercial strain of White Leghorns, but it was not observed in an LL-resistant laboratory line or four commercial strains susceptible to ALV infection. Chickens developed similar levels of viremia and neutralizing antibodies to ALV regardless of the presence of augmentation of LL, suggesting that the mechanism of enhanced LL did not result from differences in susceptibility or immune response to ALV. We postulate that the serotype 2 herpesviruses may augment LL through one of several possible influences on bursal cells that are subsequently transformed by exogenous ALV.     

Genetic determinants of neoplastic diseases induced by a subgroup F avian leukosis virus.

Two subgroup F avian leukosis viruses, ring-necked pheasant virus (RPV) and RAV-61, were previously shown to induce a high incidence of a fatal proliferative disorder in the lungs of infected chickens. These lung lesions, termed angiosarcomas, appear rapidly (4 to 5 weeks after infection), show no evidence of proto-oncogene activation by proviral integration, and are not induced by avian leukosis viruses belonging to other subgroups. To identify the viral sequences responsible for induction of these tumors, we constructed recombinant viruses by exchanging genomic segments of molecularly cloned RPV with those of a subgroup A leukosis virus, UR2AV. The ability to induce rapid lung tumors segregated only with the env sequences of RPV; the long terminal repeat of RPV was not required. However, recombinants carrying both env and long terminal repeat sequences of RPV induced lung tumors with a shorter latency. In several cases, recombinant viruses exhibited pathogenic properties differing from those of either parental virus. Recombinants carrying the gag-pol region of RPV and the env gene of UR2AV induced a high incidence of a muscle lesion termed infiltrative intramuscular fibromatosis. One recombinant, EU-8, which carries the gag-pol and LTR sequences of RPV, and the env gene of UR2AV, induced lymphoid leukosis after an unusually short latent period. The median time of death from lymphoid leukosis was 6 to 7 weeks after infection with EU-8 compared with approximately 5 months for UR2AV.     

Cross-reactive cellular and humoral immunity to carcinogen-induced chicken fibrosarcomas. II. Effect of prior immunization with transplantable tumors on primary tumor incidence

Primary carcinogen-induced (7,12-dimethyl-benz[a]anthracene; DMBA) tumor-bearing SC chickens (B2/B2) frequently showed antibodies in their sera which reacted with cells from their autochthonous tumors, chicken embryo fibroblasts (CEF), tumor cells from some transplantable tumor lines, and from approximately 10% of other primary tumors. Similar results were obtained by ELISA on glutaraldehyde-fixed cells and by immunofluorescence on viable cells. The serum antibody reactivity could be removed by absorption with CEF but not with non-cross-reacting primary tumor cells or a variety of normal tissues. Although sera from normal chickens never showed significant reactivity, a high percentage of sera from chickens that had been injected with DMBA but failed to develop detectable tumors showed antibody activity to a transplantable DMBA-induced tumor and to CEF. On the basis of previously established cross-reactivity patterns in protective immunity to transplantable carcinogen-induced fibrosarcomas, attempts were made to protect against chemical carcinogenesis by prior immunization with selected DMBA-induced transplantable tumors. Tumor-immune chickens showed a significant decrease in the development of tumors during the first 3 mo after injection of DMBA (p = 0.001) or methylcholanthrene (p = 0.033) when compared to controls. This resistance to tumor induction in immune chickens was correlated to the degree of tumor immunity to the immunizing tumor present 1 mo after carcinogen injection (p = 0.046). There was, however, no detectable difference in the incidence of tumors arising later than 3 mo after carcinogen injection. The reduction in tumor incidence in immune as compared to control chickens at 5 mo was therefore less striking than the reduction seen at 3 mo. Immunization with CEF and adjuvants or with adjuvants alone afforded no protection to tumor induction.     

Isolation and Identification of a Subgroup A Avian Leukosis Virus from Imported Meat-type Grand-parent Chickens

An exogenous avian leukosis virus (ALV) strain SDAU09C1 was isolated in DF-1 cells from one of 240 imported 1-day-old white meat-type grand parent breeder chicks. Inoculation of SDAU09C1 in ALV-free chickens induced antibody reactions specific to subgroup A or B. But gp85 amino acid sequence comparisons indicated that SDAU09C1 fell into subgroup A; it had homology of 88.8%-90.3% to 6 reference strains of subgroup A, much higher compared to other subgroups including subgroup B. This is the first report for A...     

Pathogenesis of virus-induced osteopetrosis in the chicken

Infection of chicken embryos with a nondefective avian leukosis virus MAV-2(0) induced bone hyperplasia and profound suppression of bursal and thymic lymphoid cell development within 2- to 3 weeks after hatching. A time-course study of infectivity during embryonal life showed a gradual loss of susceptibility between the 10th day of incubation and hatching. This finding suggests that an embryonal stem cell either before or early after seeding into the bone and primary lymphoid organs is a virus-susceptible target cell. Histologic observations of the bursa indicated only mild damage during the 1st week and severely impaired proliferation of follicular lymphoid cells during subsequent weeks of development. Thus, viral infection conferred a cytostatic rather than a cytolytic effect upon the lymphoid target cell. Infection of stem cells, which are progenitors of lymphoid cells as well as of bone osteoclasts, is compatible with the manifestations of the disease. A model of cellular disorders of the bone and lymphoid tissues is proposed. Adoptive transfer of lymphoid cells from adult MAV-2(0)-immunized chickens into histocompatible virus-infected juvenile recipients abrogated the manifestation of osteopetrosis. Passive injection of antiserum containing virus-neutralizing antibodies was also effective. Anti-gp85 viral surface glycoprotein-binding antibodies in sera from adoptively protected chickens receiving immune cells were exclusively of cell donor IgG allotype.     

一株具有急性致瘤特性的马立克氏病病毒的分离与鉴定

应用鸭胚成纤维细胞(DEF)从免疫过CVI988/Rispens疫苗株患马立克氏病(MD)的三黄鸡中分离到一株马立克氏病病毒(MDV)(命名为YL040920株)。从该分离株蚀斑克隆获得的9个克隆在蚀斑形成时间及其形态大小上均无明显差别,表明它较为单一;应用聚合酶链式反应(PCR)技术扩增并测定了毒株的致瘤相关基因meq的核苷酸序列,并与其他MDV-1参考毒株的序列进行比较分析,发现其序列具有MDV-1强毒株的特征;用基于抗MDV-1 MEQ蛋白的单克隆抗体3G12E6的免疫荧光试验(FA)对毒株的DEF培养物进行检测,发现有特异性的荧光定位于细胞核内;应用毒株感染霞烟鸡,最早在接种后(PI)21d即可诱发明显的内脏器官,在各器官肿瘤中以心脏、肝脏和皮肤的肿瘤发生率最高;用禽肿瘤病三重PCR鉴别诊断技术对毒株的DEF培养物以及感染鸡的内脏器官组织进行检测,均能扩增到MDV-1强毒株的特异性带,而网状内皮增生症病毒(REV)和禽白血病病毒(ALV)的检测则均为阴性。研究的结果表明,分离株YL040920为单一的MDV-1强毒株,无REV、ALV以及疫苗株CVI988/Rispens的混杂,并具有以心脏、肝脏和皮肤肿瘤为主的急性致瘤特性。