Expression of endogenous ALV antigens and susceptibility to subgroup E ALV in three strains of chickens (endogenous avian C-type virus).

Cells from three strains of Kimber Farms chickens, K16, K18, and K28, have been characterized for the expression of endogenous avian C-type virus (ALV) antigens and for susceptibility to subgroup E ALV. In K16 the coordinate dominant expression of chick helper factor (chf), the type specific antigen of endogenous subgroup ALV, and the group specific (gs) antigen of ALV was observed. The expression of chf and gs antigen in K16 chf⁺gs⁺ cells was similar to that observed in SPAFAS and H &; N gs⁺ cells. In K18 chf but not gs antigen was expressed. The expression of chf in K18 chf⁺gs⁺ cells was distinct from that observed for the chf⁺gs⁺ pedigrees but similar to that found in SPAFAS chf⁺gs^? helper extremely high (h_E) cells. Cells from K16 and K18 chickens were uniformly resistant to subgroup E ALV. In cells from K28 chickens, susceptibility to subgroup B virus correlated with susceptibility to subgroup E virus. The efficiency of plating of subgroup E virus on susceptible K28 cells was 10³–10⁴-fold lower on chf⁺ cells than on chf^? cells.     

Geese not susceptible to virulent subgroup J avian leukosis virus isolated from chickens

ABSTRACT

To determine whether geese are susceptible to infection by avian leukosis virus (ALV), 702 serum samples from domestic and foreign goose breeds were screened for p27 antigen as well as being inoculated into DF-1 cell cultures to isolate ALV. Although 5.7% of samples were positive for p27 antigen, reactivity appeared to be non-specific because no ALV was detected in the corresponding DF-1 cultures. To further determine whether geese are susceptible to ALV-J isolated from chickens, ALV-J strain JS09GY7 was artificially inoculated into 10-day-old goose embryos, with one-day-old hatched goslings then screened for p27 antigen and the presence of ALV. In all cases, the results of both tests were negative. Liver tissues from the 1-day-old goslings were screened using a polymerase chain reaction-based assay, which failed to amplify ALV-J gene fragments from any of the samples. Further, no histopathological damage was observed in the liver tissues. ALV-J was further inoculated intraperitoneally into one-day-old goslings, with cloacal swabs samples and plasma samples then collected every 5 days for 30 days. All samples were again negative for the presence of p27 antigen and ALV, and liver tissues from the challenged geese showed no histopathological damage and were negative for the presence of ALV-J gene fragments. Furthermore, p27 antigen detection, PCR-based screening, and indirect immunofluorescence assays were all negative following the infection of goose embryo fibroblasts with ALV-J. Together, these results confirm that virulent chicken-derived ALV-J strains cannot infect geese, and that p27 antigen detection in goose serum is susceptible to non-specific interference.     

Methods for evaluating and developing commercial chicken strains free of endogenous subgroup E avian leukosis virus.

The genome of nearly all chickens contains various DNA proviral insertions of retroviruses of subgroup E avian leukosis virus (ALVE). However, the elimination or control of ALVE gene expression is desirable to improve productivity, to improve resistance to avian leukosis virus (ALV)-induced tumours, and to develop safer live virus vaccines in chick embryos and cultured chick cells. Restriction fragment length polymorphism and polymerase chain reaction methods are used to define the presence of ALVE genes; and the expression of ALVE in chicken plasma or on cells, and the susceptibility of cells to ALVE is determined by flow cytometry using a specific (R2) antibody. ADOL line 0 chickens have been selected to be free of ALVE genes, while being resistant (i.e. lack receptors to ALVE), but susceptible to exogenous ALV (i.e. ALVA, ALVB, ALVC and ALVJ). To develop improved line 0-type chickens, ADOL line 0 was outcrossed to a commercial line that had one ALVE gene and evidence for ALVE resistance. Rous sarcoma virus (RSV) challenge was used to confirm resistance of F1 chickens to ALVE, and susceptibility of F2 breeders to ALVA and ALVB using test chicks produced by matings to line 7(2). Selected F2 breeders were resistant to ALVE, but susceptible to exogenous ALVA, ALVB, ALVC and ALVJ, based on challenge tests of progeny chick cells using an enzyme-linked immunosorbent assay. The new line, 0(1), has evidence for improved egg size, productivity, fertility and hatchability. Similar procedures may be used for development of productive ALVE free chicken lines with preferred ALV susceptibility traits.     

Detection of endogenous avian leukosis virus envelope in chicken plasma using R2 antiserum

Immunologic tolerance to oncogenic avian leukosis virus (ALV) is mediated, in part, by the interaction of endogenous ALV (EV) envelope and immune competent cells. A flow cytometry method is described for detecting the EV envelope in chicken plasma or serum. The method employs two types of target red blood cells (RBC) obtained from chickens lacking EV; RBC susceptible to EV infection (containing EV receptors), and those resistant to EV infection (lacking EV receptors). RBC from susceptible chickens will bind EV envelope glycoprotein (gp85) when present in plasma. The gp85-bound RBC are subsequently incubated with a highly specific chicken alloantibody, termed R2. Using flow cytometry, gp85 is detected indirectly with a fluoresceine-tagged antibody to chicken immunoglobulin; plasmas lacking gp85 are nonreactive and fluorescence remains at a background level. Because RBC from resistant chickens are nonreactive regardless of the presence or absence of EV gp85, a specific binding index was calculated to compare relative binding of EV gp85 on susceptible and resistant RBC, and thus identify chickens that express EV gp85. The specificity of the assay was demonstrated using plasma from chickens of 14 standard laboratory lines previously defined for EV envelope expression including two sets of highly congenic lines that differ in EV expression. This assay detects differences attributable to EV gp85 in chickens of commercial breeding lines of White Leghorns and broilers. Moreover, if chickens lack EV, the R2 plasma assay can differentiate between EV-susceptible and EVresistant siblings.     

Synergistic pathogenic effects of co-infection of subgroup J avian leukosis virus and reticuloendotheliosis virus in broiler chickens

To study interactions between avian leukosis virus subgroup J (ALV-J) and reticuloendotheliosis virus (REV) and the effects of co-infection on pathogenicity of these viruses, 1-day-old broiler chicks were infected with ALV-J, REV or both ALV-J and REV. The results indicated that co-infection of ALV-J and REV induced more growth retardation and higher mortality rate than ALV-J or REV single infection (P < 0.05). Chickens co-infected with ALV-J and REV also showed more severe immunosuppression than those with a single infection. This was manifested by significantly lower bursa of Fabricius and thymus to body weight ratios and lower antibody responses to Newcastle disease virus and H9-avian influenza virus (P < 0.05). Perihepatitis and pericarditis related to severe infection with Escherichia coli were found in many of the dead birds. E. coli was isolated from each case of perihepatitis and pericarditis. The mortality associated with E. coli infection in the co-infection groups was significantly higher than in the other groups (P < 0.05). Among 516 tested E. coli isolates from 58 dead birds, 12 serotypes of the O-antigen were identified in two experiments. Different serotypes of E. coli strains were even isolated from the same organ of the same bird. Diversification of O-serotypes suggested that perihepatitis and pericarditis associated with E. coli infection was the most frequent secondary infection following the immunosuppression induced by ALV-J and REV co-infection. These results suggested that the co-infection of ALV-J and REV caused more serious synergistic pathogenic effects, growth retardation, immunosuppression, and secondary E. coli infection in broiler chickens.     

一株A和B亚群禽白血病病毒特异性单克隆抗体的制备及其特性

目的:制备A和B亚群禽白血病病毒(Avian leukosis virus,ALV)特异性单克隆抗体。方法:用ALV-A-SDAU09C1株的env-gp85基因的PCR产物构建重组表达性质粒pET32a-SDAU09C1-gp85,经IPTG诱导后,表达分子量为53 kD的ALV-A囊膜gp85蛋白与GST的融合蛋白,将表达产物免疫BALB/c小鼠,取其脾脏细胞与骨髓瘤细胞SP2/0进行融合,筛选杂交瘤细胞株。结果:获得了1株(A6D1株)能与A和B亚群ALV发生反应但不与J亚群ALV反应的杂交瘤细胞株。Western blot试验结果表明,单克隆抗体识别的A和B亚群ALV囊膜糖蛋白的分子量为53 kD。在IFA中,这株单克隆抗体可以与所试验的3株ALV-A和1株ALV-B毒株反应,而与4株ALV-J亚群的毒株不反应。结论:A6D1株单克隆抗体可以用于A和B亚群ALV感染的诊断和流行病学调查,弥补了目前只有J亚群ALV特异性单克隆抗体可用的不足。     

Occurrence of subgroup J avian leukosis virus in Taiwan

There are three grandparent farms for three different chicken breeds in Taiwan. One of these farms, populated by breast meat yield chickens (yield type), suffered from a severe subgroup J avian leukosis virus (ALV-J) infection in mid-1997. The affected flocks at that farm had a weekly mortality of more than 1% and a 15% drop in egg production. The broilers from that breed had a 10% condemnation rate during the first week of age, and 5% of the remaining broilers were stunted afterwards. Some chickens had myeloid leukosis lesions at 6 weeks old. Their survivability was about 85%. However, chickens at the other two grandparent farms, populated with non-yield chickens (regular type), were also infected by ALV-J and showed myeloid leukosis. However, chickens at these farms produced progeny whose survivability reached more than 95%. ALV-J caused greater economic loss in yield type chickens than in regular type chickens in Taiwan.     

Comparative response of turkeys and chickens to avian lymphoid leukosis virus

Response of turkeys and 151(5)x7(1) chickens to prenatal or neonatal inoculation with the avian leukosis virus RAV-1 was compared. Virus-inoculated turkeys and chickens developed viraemia and antibody to. RAV-1. Many of the chickens remained persistently viraemic through the duration of the experiment, whereas in turkeys viraemia was transient. Circulating antibodies were detected earlier in turkeys than in chickens. Inoculation of turkeys with RAV-1 resulted in a high incidence of inflammatory and lymphoproliferative, but non-neoplastic, lesions in various visceral organs, including spleen, pancreas, heart, bursa and thymus, 3 to 5 weeks after virus inoculation. The lesions in chickens were those typical after RAV-1 infection, i.e. neoplastic, and appeared after a latent period of 9 weeks.     

Genetic control of susceptibility to an a subgroup sarcoma virus in commercial chickens

Sykes Line A White Leghorn and Line B Rhode Island Red embryos had differences in the average pock counts on the chorio-allantoic membrane (CAM) after infection with the Bryan strain of Rous sarcoma Virus (BS-RSV). By crossing the two lines 2610 embryos comprising 38 sire families were obtained. After challenge of their CAMs with BS-RSV analysis of the number of resulting pocks was made to determine whether or not the CAM response, a numerical trait, was controlled by simple Mendelian inheritance in commercial fowl. It was shown by appropriate statistical models that the trait was mainly controlled by a single pair of genes. However evidence in favour of other genes having a minor influence was also present in these lines of chicken.     

Development of loop-mediated isothermal amplification for rapid detection of avian leukosis virus subgroup A

This study aimed to establish a loop-mediated isothermal amplification (LAMP) method for distinguishing avian leukosis virus (ALV) subgroup A from other subgroups of the virus. On the basis of the results of sequence comparison and the sequence characteristics of ALV subgroups, a LAMP method was designed to target the gp85 segment for detection of ALV-A. Under optimal reaction conditions, ALV-A LAMP produced neither cross-reactions with other major subgroups (including subgroups J, B, C, and E) nor nonspecific reactions with other common avian infectious diseases. A sensitivity test showed that this method can detect 20 copies of proviral nucleic acid sequence within 45 min, which is 100 times more sensitive than the conventional polymerase chain reaction (PCR). This method can detect subgroup A virus rapidly and the results can be assessed based on color changes. The whole reaction process can be performed without opening the lid of the reaction tube, which reduces the possibility of contamination greatly and simplifies the detection process, indicating the considerable potential of this method for in situ application in the future.     

Endogenous leukosis viral antigen in eggs from meat-type chickens on an avian leukosis virus eradication programme

In an effort to eliminate exogenous avian leukosis virus (ALV) from a susceptible population, albumen of one egg per hen from each of four generations was tested by ELISA for group-specific antigen (gsa) of leukosis/sarcoma viruses. From 1510 to 2099 hens were tested in each generation. Hens were not used as breeders if optical density readings for gsa were 0.4 or greater. Despite this procedure, there was no appeciable change in the occurrence of gsa in eggs from one generation to the next and in the sire population the percentage of hens with levels of 0.4 or greater was 7.1 and 8.7 in the first and fourth generations tested, respectively. There were no consistent differences in antigen levels in any of the eight strains that comprised the two populations. For the most part, antigen detected in eggs resulted from expression of endogenous viral genes since there was a low incidence of exogenous infection. Six of 234 hens of the second generation tested from the sire population were positive for ALV in serum and three of 161 were positive for antibody to subgroup A virus; no antibody to subgroup B was detected. Five of the six hens that tested positive for ALV were from the same strain and had been reared together. With one exception these hens would have been eliminated as breeders based on tests for antigen in eggs. In the fourth generation tested, no virus or subgroup A antibody was found in serum from approximately 250 hens from the two populations.     

Full-length genome sequence analysis of four subgroup J avian leukosis virus strains isolated from chickens with clinical hemangioma

Since 2014, cases of hemangioma associated with avian leukosis virus subgroup J (ALV-J) have been emerging in commercial chickens in Guangxi. In this study, four strains of the subgroup J avian leukosis virus (ALV-J), named GX14HG01, GX14HG04, GX14LT07, and GX14ZS14, were isolated from chickens with clinical hemangioma in 2014 by DF-1 cell culture and then identified with ELISA detection of ALV group specific antigen p27, the detection of subtype specific PCR and indirect immunofluorescence assay (IFA) with ALV-J specific monoclonal antibody. The complete genomes of the isolates were sequenced and it was found that the gag and pol were relatively conservative, while env was variable especially the gp85 gene. Homology analysis of the env gene sequences showed that the env gene of all the four isolates had higher similarities with the hemangioma (HE)-type reference strains than that of the myeloid leukosis (ML)-type strains, and moreover, the HE-type strains’ specific deletion of 205-bp sequence covering the rTM and DR1 in 3′UTR fragment was also found in the four isolates. Further analysis on the sequences of subunits of env gene revealed an interesting finding: the gp85 of isolates GX14ZS14 and GX14HG04 had a higher similarity with HPRS-103 and much lower similarity with the HE-type reference strains resulting in GX14ZS14, GX14HG04, and HPRS-103 being clustered in the same branch, while gp37 had higher similarities with the HE-type reference strains when compared to that of HPRS-103, resulted in GX14ZS14, GX14HG04, and HE-type reference strains being clustered in the same branch. The results suggested that isolates GX14ZS14 and GX14HG04 may be the recombinant strains of the foreign strain HPRS-103 with the local epidemic HE-type strains of ALV-J.     

Response of meat-type chickens to infection with RAV-1 avian leukosis virus

Meat-type and White Leghorn chickens were inoculated with the RAV-1 strain of avian leukosis virus at 1 day of age and the severity of infection was assessed by clinical illness, haematology and post-mortem findings. The following were examined from selected birds: histological section for chronic mononuclear myocarditis, immunohistochemically-stained sections of myocardium, spleen, bursa of Fabricius and kidney for group-specific viral antigen, and ultrathin sections of these tissues for virus particles by electron microscopy. The experiment was terminated at 115-122 days. Approximately one-third of the 52 inoculated White Leghorns appeared anaemic at 3-4 weeks of age whereas there was no evidence of anaemia in 177 inoculated meat-type birds or in uninoculated birds of either type. Haemograms confirmed these observations. The first tumour found was a myelocytoma and it was in a meat-type bird at 65 days. Of the 151 meat-type birds of the inoculated group alive at 65 days, 8.5% developed nephroblastomas, but there were no cases of lymphoid leukosis. Myocarditis and virus replication in myocardium were usually more extensive in chickens that developed nephroblastomas than in those without such lesions. In 17 uninoculated control chickens examined between 26 and 122 days of age there were no virus particles or lesions in myocardium.     

Role of cell-mediated immunity (CMI) in chickens inoculated with avian leukosis/sarcoma virus

Immunosuppression of chickens infected in ovo with avian leukosis virus (RAV-1) by the injection of antilymphocyte serum (ALS) did not significantly (P > 0.05) alter the incidence or distribution of lesions in chickens between 3 and 6 months of age as compared to control groups. Antilymphocyte serum treatment of Rous sarcoma virus [RSV (RAV-2)]-infected chickens significantly (P < 0.05) inhibited tumour regression and enhanced tumour metastasis. It was concluded that cell-mediated immunity was not a significant factor in effecting the survival of viraemic chickens Viraemic leukosis virus infected-chickens responded as well as normal chickens to sensitization to Mycobacterium tuberculosis H37Ra. The results were based on in vivo wattle tests and in vitro cell-mediated cytotoxicity assays. It was concluded that subclinical avian leukosis virus infection had no effect on the thymus-dependent lymphocyte (T-cell) population associated with cutaneous delayed hypersensitivity and cell-mediated cytotoxicity.     

Karyotype analysis of the acute fibrosarcoma from chickens infected with subgroup J avian leukosis virus associated with v-src oncogene

To understand the cytogenetic characteristics of acute fibrosarcoma in chickens infected with the subgroup J avian leukosis virus associated with the v-src oncogene, we performed a karyotype analysis of fibrosarcoma cell cultures. Twenty-nine of 50 qualified cell culture spreads demonstrated polyploidy of some macrochromosomes, 21 of which were trisomic for chromosome 7, and others were trisomic for chromosomes 3, 4, 5 (sex chromosome w), and 10. In addition, one of them was trisomic for both chromosome 7 and the sex chromosome 5 (w). In contrast, no aneuploidy was found for 10 macrochromosomes of 12 spreads of normal chicken embryo fibroblast cells, although aneuploidy for some microchromosomes was demonstrated in five of the 12 spreads. The cytogenetic mosaicism or polymorphism of the aneuploidy in the acute fibrosarcoma described in this study suggests that the analysed cells are polyclonal.     

Epidemiological and pathological studies of subgroup J avian leukosis virus infections in Chinese local "yellow" chickens.

Epidemiological, pathological and molecular studies indicate that subgroup J avian leukosis virus (ALV-J) infections are widely spread in "yellow chickens" of local breeds in China. ALV-J induced tumour mortality and the serological conversion rates to ALV-J were very high in some breeder flocks. Typical myelocytomatosis was demonstrated not only in livers, spleens, kidneys, and sternums, as in white meat-type chickens, but also in thymuses and the bursa of Fabricius. Especially, severe myeloid cell infiltration was found throughout the whole enlarged thymuses of some birds. ALV-J was isolated at high positive rates from both liver tumour samples and embryos collected from breeder flocks with tumours. At the same time, reticuloendotheliosis virus was also co-isolated with ALV-J in some tumour samples and embryos. Sequence analysis of env genes demonstrated that the gp85 and gp37 among six ALV-J isolates from "yellow chickens" of Chinese local breeds varied as highly as among ALV-J strains isolated from white meat-type chickens worldwide. But strain GD0512 isolated in 2005 from a "yellow chicken" farm in southern China had high identity of 95.1% for gp85 or 99.5% for gp37 to strain HN0001 isolated in 2000 from a white meat-type breeder farm in northern China, a much higher identity than to other yellow chicken and white chicken strains. This is the first report of the isolation and identification of ALV-J from yellow chickens of Chinese local breeds and also the first report of vertical co-infection of ALV-J and reticuloendotheliosis virus. The significance of co-infection of ALV-J and reticuloendotheliosis virus in pathogenesis is discussed.     

ev22, a new endogenous avian leukosis virus locus found in chickens with spontaneous autoimmune thyroiditis

Summary Chickens of the Obese strain (OS) develop a hereditary spontaneous autoimmune thyroiditis (SAT) which closely resembles human Hashimoto's disease. Analysis of the endogenous viruses harboured by these animals revealed a new endogenous virus (ev22) detected as a 5.5kb Sac I fragment.Crossbreeding experiments showed thatev22 is vertically transmitted as an autosomal trait not associated with major histocompatibility (MHC) alleles. Preliminary experiments indicate thatev22 does not necessarily cause SAT, however, it may have a modulatory role in the development of the disease.     

Cellular uptake of avian leukosis virus subgroup B is mediated by clathrin

Avian leukosis virus (ALV) requires endocytosis and a low pH step for successful viral entry. Here we report that transient treatment with lysosomotropic agents was not sufficient to block ALV subgroup B (ALV-B) entry, while it completely inhibited uptake of the pH-dependent Semliki Forest virus. Extended incubations with lysosomotropic agents were required to block ALV-B entry, suggesting that ALV particles are stable in endosomal compartments. We analyzed endocytic pathways involved in the uptake of ALV-B into target cells. The ALV-B receptor TVB(S3) was not associated with detergent-resistant membranes (DRMs) in the presence or absence of ALV-B particles. This result suggested that DRM-associated endocytic pathways were not required for ALV-B entry. Using several approaches, we found that clathrin mediates endocytosis of ALV-B particles into target cells. By means of confocal microscopy, we established that the ALV-B receptor TVB(S3) colocalized with clathrin in TVB(S3)-expressing quail QT-6 cells. In addition, chlorpromazine, an inhibitor of clathrin-mediated endocytosis, blocked uptake of soluble ALV-B Env into chicken embryo fibroblasts. To examine ALV-B uptake into clathrin-negative cells, we used a chicken DT40 B cell line containing a tetracycline-regulatable clathrin gene. Clathrin depletion significantly reduced ALV-B entry into the chicken DT40 cell line. Taken together, our results suggest that clathrin is involved in uptake of ALV-B particles into target cells.     

Reduction of horizontal transmission of avian leukosis virus subgroup J in broiler breeder chickens hatched and reared in small groups

Transmission of avian leukosis virus, subgroup J (ALV-J), from donor chickens inoculated as embryos to simulate congenital infection to uninfected hatchmates was studied in two strains of commercial broiler breeder chickens. Chicks of two commercial lines free of ALV-J became infected when hatched (1/2 lots positive) or reared (8/8 lots positive) in direct physical contact with ALV-J-infected donors. Infection also occurred when chicks were exposed in the hatchery to ALV-J-infected donors by cloacal swab transfer (2/2 lots positive), needle transfer during subcutaneous inoculation (2/2 lots positive), or ingestion of infected meconium (2/2 lots positive). However, transmission was delayed or prevented by wire partitions in the hatcher and rearing of small groups in cubicles, and rarely (1/10 lots positive) resulted from short-term direct or indirect contact. In a simulated field test, a flock of 503 broiler breeder chickens with an initial embryo infection rate of 4.6% was hatched and reared as 48 small groups to 4 weeks of age. Groups were tested at hatch and at 3 weeks, and 14 infected groups were eliminated. This flock tested negative for ALV-J infection from 4 to 32 weeks and did not transmit infection to progeny or develop tumours. A control group of 377 chickens with a similar initial infection rate was hatched and reared as a single group. This control flock transmitted virus to 5.7% of its progeny and about 5% of the hens developed tumours. The small-group hatching and rearing practices employed in these studies allowed for the accurate identification and removal of groups containing chickens infected prior to hatching and prevented horizontal transmission of ALV-J between uninfected and infected groups for at least 4 weeks. More importantly, application of these procedures successfully eradicated ALV-J in a single generation under laboratory conditions. This suggests that similar procedures could be a valuable adjunct to virus eradication programmes in the field.