To study the correlation between 50% tissue-culture infective dose (TCID50) value and p27 antigen S/P value of Avian leukosis virus subgroup J and discuss their significance, chicken embryo fibroblast (CEF) cells were inoculated with Avian leukosis virus subgroup J strain NX0101 and samples were tested continuously for ten days after changing maintenance media. The correlation between TCID50 and p27 antigen S/P value of ten days were then analysized. Simultaneously, DF-1 cells were inoculated with NX0101 and passaged to 20 generations. Samples taken from 1st generation, 5th generation, 10th generation, 15th generation and 20th generation were tested for the TCID50 titer and the p27 antigen S/P value separately. A significant Pearson correlation was found between them in CEF cells (r = 0.85277; P < 0.0001) and in DF-1 cells (r = 0.93000; P = 0.0220). This study provided an important parameter for predicting TCID50 by detecting the p27 antigen S/P value.
Animals ; Avian Leukosis ; virology ; Avian Leukosis Virus ; immunology ; pathogenicity ; Chick Embryo ; Fibroblasts ; virology ; Proliferating Cell Nuclear Antigen ; analysis ; immunology ; Viral Load ; immunology

Two subgroup J avian leukosis viurses (ALVs) were isolated from broiler breeder flocks, in which myeloid leukosis had occurred. The isolates could be classified as subgroup J ALV. by the positive reaction in polymerase chain reaction (PCR) with primers specific for subgroup J ALV. Two isolates replicated in chicken embryo fibroblast (CEF) cells from the alv6 chicken line in which cells are resistant to subgroup A and E ALVs. In in vitro serum neutralization tests with other subgroup ALVs including ADOL-Hc1, the prototype of subgroup J ALVs isolated in the United States of America, two isolates were partially neutralized by antibody to ADOL-Hc1, indicating that Korean isolates and ADOL-Hc1 may be antigenically related, but not identical. When the PCR was done with a primer pair designed to amplify genes of E element and long terminal repeat of proviral DNA, the PCR product size of one isolate (KOAL-PET) was smaller than that of ADOL-Hc1, suggesting that some sequences in these regions are deleted.
Animals ; Antibodies, Viral/immunology ; Antigens, Viral/immunology ; Avian Leukosis/virology ; Avian leukosis virus/*classification/genetics/immunology/*isolation & purification ; Cell Line ; Chick Embryo ; Chickens/*virology ; Korea ; Neutralization Tests ; Polymerase Chain Reaction ; Poultry Diseases/virology

To study the correlation between ELISA and IFA tests in detection of ALV-A/B antibody in chicken sera, ELSA S/P values and IFA titers for different serum samples were measured and statistically analyzed. The results indicated that there was a strong positive correlation between ELISA S/P values and IFA titers (r = 0.97435, P < 0.001). Because the positive correlation between ELISA and IFA was so strong and antibody positive rates were identical in two tests, it suggested that IFA could be used as a alternative method to replace ELISA kit when only limited numbers of samples to be tested to reduce the cost and increase the sensitivity.
Animals ; Antibodies, Viral ; blood ; immunology ; Avian Leukosis ; diagnosis ; immunology ; virology ; Avian Leukosis Virus ; classification ; immunology ; isolation & purification ; Cell Line ; Chickens ; Enzyme-Linked Immunosorbent Assay ; methods ; Fluorescent Antibody Technique, Indirect ; methods ; Poultry Diseases ; diagnosis ; immunology ; virology ; Species Specificity

In this study, we investigated the effects of T-cell suppression on the pathogenesis of subgroup J avian leukosis virus (ALV-J). Chickens were treated with cyclosporin A (CSP) 50 mg/Kg body weight or a corresponding volume of olive oil per every three days after hatching until the end of experiment. Some of the chickens from each treatment group were infected with an isolate of ALV-J, ADOL-7501, at 2 weeks of age. The effects of viral infection were compared to uninfected birds in same treatment group. Intramuscular injection of CSP induced significant T-cell specific immunosuppression determined by decreased cutaneous basophilic hypersensitivity response and decreased lymphocyte mitogenic activity using concanavalin A. Most of the chickens examined had Marek's disease virus infection prior to 3 weeks of age. The percentage of antibody-positive birds and antibody titers were similar in infected chickens between both treatment groups. The ratio of viremic chickens was significantly higher in CSP treated group than that of the Oil treated group. Microscopically, one CSP treated chicken had a nephroblastoma at 10 weeks post infection. At 7 and 10 weeks post-infection, more chickens had myeloid cell infiltrations in multiple organs including heart, liver and occasionally lung. Expression of ALV-J viral antigen determined by immunohistochemical staining was significantly higher in CSP treated chickens than Oil treated chickens at 10 weeks post-infection. This study indicated that chemically-induced T-cell suppression may enhance pathogenicity of the AVL-J virus in broilers.
Animals ; Antibodies, Viral/blood ; Avian Leukosis/*immunology/virology ; Avian leukosis virus/genetics/*immunology ; Body Weight ; *Chickens ; Cyclosporine/*pharmacology ; Dermatitis, Contact/immunology/virology ; Flow Cytometry ; Immunocompromised Host ; Immunohistochemistry/veterinary ; Immunophenotyping ; Immunosuppressive Agents/*pharmacology ; Lymphocyte Activation/immunology ; Marek Disease/*immunology/virology ; RNA, Viral/chemistry/genetics ; Reverse Transcriptase Polymerase Chain Reaction/veterinary ; T-Lymphocytes/*immunology/virology ; Viremia/veterinary

Studies were performed to determine the effects of Bcell suppression on the pathogenesis of Subgroup J avian leukosis virus (ALV-J) in broiler chickens. Neonatal chickens were treated with cyclophosphamide (CY) or PBS, and then infected with ALV-J (ADOL-7501) at 2 weeks of age. CY treatment induced B cell specific immunosuppression throughout the experiment confirmed by decreased bursal weight, intact lymphocyte mitogenetic activity stimulated by Con A and increased relative subpopulation of CD3-positive cells as measured by flow cytometry. Chickens in this experiment had Mareks disease virus exposure prior to three weeks of age as determined by the presence of lymphocytic infiltration and antibody. Virus neutralizing antibody against ALV-J was first observed at 6 weeks post-infection in some of the infected chickens in the PBS group. As expected, none of the chickens from the CY group and uninfected chickens developed virus-neutralizing antibody. The viremic status was measured by real time RT-PCR using SYBR green I dye. The percentage of viremic chickens was significantly higher, and more chickens had high titered viremia, in the CY treated group. No neoplastic foci consistent with ALVJ infection were observed in any of the experimental chickens. The frequency and intensity of viral antigen expression determined by immunohistochemistry was significantly higher in tissues from CY treated birds than those of PBS treated chickens at 3 weeks post-infection. This study showed that B cell specific immunosuppression with CY treatment in chickens resulted in increase in viremia and viral antigen load in tissues.
Animals ; Avian Leukosis/*immunology/virology ; Avian leukosis virus/genetics/*immunology ; Body Weight/physiology ; Bursa of Fabricius/immunology ; *Chickens ; Concanavalin A/immunology ; Cyclophosphamide/*pharmacology ; Flow Cytometry/veterinary ; Immunocompromised Host ; Immunohistochemistry/veterinary ; Immunophenotyping/veterinary ; Immunosuppressive Agents/*pharmacology ; Lymphocyte Activation/drug effects/immunology ; Organic Chemicals/chemistry ; Poultry Diseases/immunology/*virology ; RNA, Viral/chemistry/genetics ; Random Allocation ; Reverse Transcriptase Polymerase Chain Reaction/veterinary ; Spleen/immunology/virology ; Statistics, Nonparametric ; Viremia/veterinary

The critical time of avian leukosis virus subgroup J (ALV-J)-mediated immunosuppression was determined by body weight, relative immune organ weight, histopathology, and presence of group specific antigen and antibodies in specific pathogen-free (SPF) chickens. CD4+ and CD8+ cell activity in the spleen, total and differential leukocyte counts in blood, and viral RNA levels in spleen were measured. Significant growth suppression was observed in the two ALV-J-infected groups. A strong immune response by infected groups was present in spleen at 2-weeks-of-age, but after 4-weeks-of-age, the response decreased quickly. The thymus and bursa showed persistent immunosuppression until 4-weeks-of-age. Proliferation of fibroblasts and dendritic cells were observed in immune organs at 4- and 5-weeks-of-age. However, the granulocyte cell number was markedly lower in the infected groups than in the control group. In group 1 (day 1 infection) CD4+ cells increased during the second week but significantly decreased during the fourth week, while group 2 (day 7 infection) showed the opposite effect. Viral RNA increased significantly by the fourth week. These data identify 3~4 weeks post-infection as the key time at which the ALV-J virus exerts its immunosuppressive effects on the host.
Animals ; Antibodies, Viral/blood ; Antigens, CD4/blood ; Antigens, CD8/blood ; Avian Leukosis/*immunology/transmission/virology ; Avian leukosis virus/classification/*immunology ; Body Weight ; *Chickens ; China ; Enzyme-Linked Immunosorbent Assay/veterinary ; Immune Tolerance ; Leukocyte Count/veterinary ; Poultry Diseases/*immunology/transmission/virology ; RNA, Viral/genetics ; Real-Time Polymerase Chain Reaction/veterinary ; Reverse Transcriptase Polymerase Chain Reaction/veterinary ; Specific Pathogen-Free Organisms ; Spleen/immunology

The purpose of this study was to compare the whole genome sequences and replication dynamics in cell cultures of two Avian leukosis viruses of subgroup B (ALV) isolates, SDAU09E3 and SDAU09C2. Comparison of the amino acid sequences indicated that the gp85 identity of these two subgroup B isolates was 95.4%, the identity with other three ALV-B reference strains was 91.0%-94.9%, and less than 87.9% with ALV subgroup A, C, D, E and J. Comparison of the nucleotide sequence of gag and pol genes indicated that homologies of gag gene and pol gene of these two ALV-B isolates with all compared reference strains of different subgroups were above 93%. Homologies of LTR sequence of these two ALV-B isolates with other exogenous ALVs subgroups A, B, C, D and J were 72.6%-88.3%, but only 51.5% when compared with endogenous ALV subgroup E. The identity of LTR between these two ALV-B strains was only 74.8%, which was far lower than the identity of other genes. The identity of U3 region of LTR between these two ALV-B isolates was only 68.8% and there were obvious differences in the number CAAT Boxes. Replication dynamics in DF-1 cell indicated that the value of TCID50 was similar between 2 isolates but the concentration of nucleocapsid protein p27 antigen of SDAU09E3 was significantly higher than SDAU09C2 in cell culture supernatant, which indicated there was no parallel relationship between p27 antigen concentration and infectious virus particles. Whether such difference was resulted from the diversity of U3 region of LTR, further studies with their recombinant infectious clones is necessary.
Animals ; Antibodies, Viral ; immunology ; Avian Leukosis Virus ; classification ; genetics ; physiology ; Base Sequence ; Cell Line ; Cells, Cultured ; Chick Embryo ; Chickens ; Genome, Viral ; genetics ; Molecular Sequence Data ; Phylogeny ; Poultry Diseases ; virology ; Sequence Alignment ; Sequence Homology, Nucleic Acid ; Viral Matrix Proteins ; genetics ; Virus Replication ; physiology