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Avian Leukosis Virus (ALV) is a retrovirus that induces immunosuppression and tumor formation in poultry, posing a significant threat to the poultry industry. Currently, there are no effective vaccines or treatments for ALV. Therefore, the early diagnosis of infected flocks and farm sanitation are crucial for controlling outbreaks of this disease. To address the limitations of traditional diagnostic methods, which require sophisticated equipment and skilled personnel, a dual-tube detection method for ALV-J based on reverse transcription isothermal amplification (RAA) and the CRISPR-Cas13a system has been developed. This method offers the advantages of high sensitivity, specificity, and rapidity; it is capable of detecting virus concentrations as low as 5.4 × 10 0 copies/μL without cross-reactivity with other avian viruses, with a total testing time not exceeding 85 min. The system was applied to 429 clinical samples, resulting in a positivity rate of 15.2% for CRISPR-Cas13a, which was higher than the 14.7% detected by PCR and 14.2% by ELISA, indicating superior detection capability and consistency. Furthermore, the dual-tube RAA-CRISPR detection system provides visually interpretable results, making it suitable for on-site diagnosis in remote farms lacking laboratory facilities. In conclusion, the proposed ALV-J detection method, characterized by its high sensitivity, specificity, and convenience, is expected to be a vital technology for purification efforts against ALV-J., Competing Interests: The authors declare no conflict of interest.

Avian leukosis viruses (ALVs) include a group of avian retroviruses primarily associated with neoplastic diseases in poultry, commonly referred to as avian leukosis. Belonging to different subgroups based on their envelope properties, ALV subgroups A, B, and J (ALV-A, ALV-B, and ALV-J) are the most widespread in poultry populations. Early identification and removal of virus-shedding birds from infected flocks are essential for the ALVs' eradication. Therefore, the development of rapid, accurate, simple-to-use, and cost effective on-site diagnostic methods for the detection of ALV subgroups is very important. Cas13a, an RNA-guided RNA endonuclease that cleaves target single-stranded RNA, also exhibits non-specific endonuclease activity on any bystander RNA in close proximity. The distinct trans-cleavage activity of Cas13 has been exploited in the molecular diagnosis of multiple pathogens including several viruses. Here, we describe the development and application of a highly sensitive Cas13a-based molecular test for the specific detection of proviral DNA of ALV-A, B, and J subgroups. Prokaryotically expressed LwaCas13a, purified through ion exchange and size-exclusion chromatography, was combined with recombinase polymerase amplification (RPA) and T7 transcription to establish the SHERLOCK (specific high-sensitivity enzymatic reporter unlocking) molecular detection system for the detection of proviral DNA of ALV-A/B/J subgroups. This novel method that needs less sample input with a short turnaround time is based on isothermal detection at 37 °C with a color-based lateral flow readout. The detection limit of the assay for ALV-A/B/J subgroups was 50 copies with no cross reactivity with ALV-C/D/E subgroups and other avian oncogenic viruses such as reticuloendotheliosis virus (REV) and Marek's disease virus (MDV). The development and evaluation of a highly sensitive and specific visual method of detection of ALV-A/B/J nucleic acids using CRISPR-Cas13a described here will help in ALV detection in eradication programs.

Nanoparticle-assisted polymerase chain reaction (nanoPCR) is a novel method for the rapid detection of pathogens. A sensitive and specific multiple nanoPCR assay was developed for simultaneous detection of avian leucosis virus (ALV) subgroups A, B and J. In this study, three pairs of primers were designed, based on the conserved region of the gp85 gene. An exploration of the optimal primer concentration and annealing temperature were carried out, for better performance of the nanoPCR assay. According to the results, the multiple nanoPCR assay amplified 336 pb, 625 bp and 167 bp fragments of ALV-A, -B and -J, respectively, and showed no cross-reactivity with irrelevant pathogens, suggesting the excellent specificity of the assay. The constructed standard DNA templates were used to estimate the limit of detection. As shown by the results, the detection limit of the nanoPCR assay was nearly 10 copies/μL. To further evaluate the detection ability of the assay, 186 clinical samples were detected using the nanoPCR assay, among which, 14 samples were confirmed as ALV positive; the results were further confirmed by sequencing. In conclusion, a highly specific and sensitive nanoPCR assay was successfully developed, which could be a useful tool for clinical diagnosis as well as for the discrimination of ALV-A, -B and -J.

Avian leukosis (AL), caused by avian leukosis virus (ALV), is a contagious tumor disease that results in significant economic losses for the poultry industry. Currently, ALV-A, B, J, and K subgroups are the most common in commercial poultry and cause possible coinfections. Therefore, close monitoring is necessary to avoid greater economic losses. In this study, a novel multiplex quantitative polymerase chain reaction (qPCR) assay was developed to detect ALV-A, ALV-B, ALV-J, and ALV-K with limits of detection of 40, 11, 13.7, and 96 copies/µL, respectively, and no cross-reactivity with other ALV subtypes and avian pathogens. We detected 852 cell cultures inoculated with clinical samples using this method, showing good consistency with conventional PCR and ELISA. The most prevalent ALV strain in Hubei Province, China, was still ALV-J (11.74%). Although single infections with ALV-A, ALV-B, and ALV-K were not found, coinfections with different subgroup strains were identified: 0.7% for ALV-A/J, 0.35% for ALV-B/J, 0.25% for ALV-J/K, and 0.12% for ALV-A/B/K and ALV-A/B/J. Therefore, our novel multiplex qPCR may be a useful tool for molecular epidemiology, clinical detection of ALV, and ALV eradication programs.

Subgroup J Avian leukosis virus (ALV-J) is an important pathogen of poultry tumor diseases. Since its discovery, it has caused significant economic losses to the poultry industry. Thus, the rapid detection of molecular level with strong specificity is particularly important whether poultry are infected with ALV-J. In this study, we designed primers and probe for real-time fluorescent reverse-transcription recombinase-aided amplification assay (RT-RAA) based on the ALV-J gp85 sequence. We had established a real-time fluorescent RT-RAA method and confirmed this system by verifying the specificity and sensitivity of the primers and probe. In addition, repeatability tests and clinical sample regression tests were used for preliminary evaluation of this detection method. The sensitivity of established method was about 10 1 copies/μL, and the repeatability of the CV of the C T value is 4%, indicating repeatability is good. Moreover, there was no cross-reactivity with NDV, IBV, IBDV, H9N2, MDV, and REV, and other avian leukosis virus subgroups, such as subgroups A, B, C, D, K and E. Importantly, the real-time fluorescent RT-RAA completed the test within 30 min at a constant temperature of 41°C. Forty-two clinical samples with known background were tested, and the test results were coincided with 100%. Overall, these results suggested that the real-time fluorescent RT-RAA developed in this study had strong specificity, high sensitivity, and good feasibility. The method is simple, easy, and portable, that is suitable for clinical and laboratory diagnosis, and provides technical support for the prevention and control of ALV-J., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Avian erythroblastosis (AE; erythroid leukosis) was detected in a 78-day-old Japanese native chicken. At necropsy, the liver was enlarged and diffusely dark red in color. Moderate splenomegaly was observed. Histologically, round to polygonal tumor cells were observed only in the blood vessels of the liver and other organs. Immunohistochemistry (IHC) was performed on formalin-fixed, paraffin-embedded (FFPE) sections to characterize tumor cells. Tumor cells, as well as normal erythrocytes as positive controls, were consistently positive for IHC by using the commercially available anti-human hemoglobin antibody. Exogenous avian leukosis virus (ALV) subgroup B and endogenous ALV-E genes were detected by PCR, although ultrastructural observation revealed no viral particles associated with tumor cells. Results suggest that the commercial anti-human hemoglobin antibody used in the study cross-react to chicken erythrocytes on FFPE sections by IHC and can also be used for definitive diagnosis of the spontaneous case of AE in chickens.

During the past two decades, avian leukosis virus (ALV) caused tremendous economic losses to poultry industry in China. ALV-K as a newly found subgroup in recent years, which made the control and eradication of ALV more difficult as they were originated from the recombination of different subgroups. To date, specific rapid detection methods refer to ALV-K are still missing. Gp85 is the main structural protein of the virus, which mediates the invasion of host cells by the virus and determinates the classification of subgroups. In this study, we prepared a monoclonal antibody (Mab) named Km3 against Gp85 of ALV-K. Immunofluorescence assay showed that Km3 specifically recognized the strains of ALV-K rather than the strains of ALV-A or ALV-J. To explain the subgroups specificity of Km3, the epitope cognized by the Mab was identified by Western blotting using 15 overlapping fragments spanning the Gp85. Finally, the peptide 129 AFGPRSIDTLSDWSRPQ 145 was identified as the minimal linear epitope recognized by Km3. Alignment of Gp85 from different subgroups showed that the epitope was highly conserved among ALV-K strains, which was quite different from that of the strains from ALV -A, -B and -J. In conclusion, the Mab Km3 may serve as a useful reagent for ALV-K detection and diagnosis in the future., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Avian leucosis (AL) is a disease characterized by tumors and is caused by the avian leukosis virus (ALV). Because of the high variability of viruses and complex pathogenic mechanisms, screening and breeding J subgroup of ALV (ALV-J) resistant avian breeds is one of the strategies for prevention and treatment of AL, thus screening of significant immune markers is needed to promote the development of disease-resistant breeds. In this study, data-independent acquisition (DIA) technology was used to detect the DEPs of three breeds of chicken according to different comparison to investigate the potential markers. Results showed special DEPs for spleen development of each breed were detected, such as PCNT, DDB2, and ZNF62. These DEPs were involved in intestinal immune network used in production of IgA signaling pathways and related to immune response which can be used as potential markers for spleen development in different breeds. The DEPs such as RAB44 and TPN involved in viral myocarditis, transcriptional misregulation in cancer, and tuberculosis can be used as potential markers of spleen immune response after ALV-J infection in chickens. Pair-wise analysis was performed for the three breeds after the infection of ALV-J. The proteins such as RFX1, TAF10, and VH1 were differently expressed between three breeds. These DEPs involved in antigen processing and expression, acute myelogenous leukemia, and viral carcinogenesis can be used as potential immune markers after ALV-J infection of different genetic backgrounds. The screening of potential markers at protein level provides a strong theoretical research basis for disease resistance breeding in poultry.

We developed a rapid fluorescent microsphere immunochromatographic test strip (FM-ICTS) assay for the quantitative detection of avian leukosis virus (ALV). A monoclonal antibody specific for the ALV major capsid protein encoded by the gag gene was coupled to label fluorescent microspheres. ALV antibodies were coated on a nitrocellulose membrane to prepare a test line for sample detection. The fluorescence signals of the test and control lines can be read either visually by exposure to UV light or using a fluorescence analyzer. ALV could be detected quantitatively using the ratio of fluorescence signals of the test and control lines (T/C). The assay threshold was determined as a T/C value of 0.0606. The fitting curve equation was established between 1 and 2,048 ng/mL P27 protein with an r2 value of 0.9998. The assay showed no cross reactivity with Newcastle disease virus, infectious laryngotracheitis virus, infectious bronchitis virus, Marek's disease virus, infectious bursal disease, Reoviridae virus, or avian influenza virus. The repeatability was satisfactory with an overall average CV of 8.65%. The Kappa coefficient between a commercial ELISA kit was 0.7031 using clinical chicken meconium samples. Thus, a simple, rapid, sensitive, and specific fluorescent microsphere immunochromatographic test strip was developed based on specific anti-capsid protein p27 monoclonal antibodies., (© 2019 Poultry Science Association Inc.)

A sensitive and specific photoelectrochemical (PEC) immunosensor was fabricated for subgroup J avian leukosis viruses (ALV-J) analysis based on a dual signal-on strategy. Gold nanoparticles (AuNPs) decorated graphitic carbon nitride (AuNPs/g-C 3 N 4 ) as photoelectrochemical species and primary antibody (Ab 1 ) against ALV-J were immobilized onto ITO electrode in turn. An ALP-CdTe-Ab 2 bio-conjugant was fabricated by assembling second antibody (Ab 2 ) and alkaline phosphatase (ALP) to CdTe quantum dots (QDs) surface. The PEC immunosensor was fabricated by successively anchoring the target ALV-J and ALP-CdTe-Ab 2 bio-conjugants onto electrode surface via the immune recognition. By virtue of the matched energy levels between CdTe QDs and AuNPs/g-C 3 N 4 , ALP-CdTe-Ab 2 bio-conjugants could serve as the PEC active probes for photocurrent enhancement. Moreover, the photocurrent response could be further enhanced attributed to the ALP catalytic chemistry to in situ produce ascorbic acid for electron donating, achieving an effective dual signal-on mode for PEC assay. On the basis of the ALV-J titers-dependent photocurrent increment, the fabricated PEC immunosensor showed high sensitivity, specificity and stability for ALV-J assay in a wide linear range with a low detection limit of 85 TCID 50 /mL. This PEC immunosensor with the dual signal-on strategy may open up a promising platform for more target analytes in novel immune analysis and clinical diagnostics., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Avian leukosis virus (ALV) is an avian oncogenic retrovirus that induces leukemia-like proliferative diseases in chickens. ALV infection can result in the development of immunological tolerance and persistent viremia. Since effective vaccines against ALV are not yet available, its current prevention primarily depends on detection and eradication to establish exogenous ALV-free poultry flocks. In this study, a rapid and simple colloidal gold test strip method, specific for the group-specific antigen, p27 protein, was developed and systematically evaluated for the detection of ALV from different samples. The detection limit of this assay was as low as 6.25 ng/ml for p27 protein and 80 TCID 50 /ml for different subgroups of ALV. Besides, the test strip showed high specificity in the detection of different subgroups of ALV, including ALV-A, ALV-B, ALV-J, and ALV-K, with no cross-reaction with other avian pathogens. Furthermore, we artificially infected specific pathogen-free (SPF) chickens with ALV-J, collected cloacal swabs, and examined viral shedding using both test strips and ELISA. Results from the test strip were highly consistent with that from ELISA. In addition, 1104 virus isolates from anti-coagulant blood samples, 645 albumen samples, and 4312 meconium samples were tested, and the test strip results agreed with those of ELISA kit up to 97.1%. All the results indicated that the colloidal gold test strip could serve as a simple, rapid, sensitive, and specific diagnostic method for eradication of ALV in poultry farms.

A novel pH-responsive sandwich-type electrochemiluminescence immunosensor was successfully developed for the detection of avian leukosis virus subgroup J (ALV-J). The immunosensor consisted of graphene oxide functionalized with tannic acid (rGO-TA) and primary antibodies (Ab 1 ) as the main ALV-J sensing platform. For signal amplification, pH-responsive hollow MnO 2 (hMnO 2 ) nanospheres encapsulating Ru(bpy) 3 Cl 2 and carrying secondary antibodies (Ab 2 ) were employed. Under optimal conditions, the immunosensor exhibited a wide ALV-J detection range of 10 1.80 to 10 4.30 TCID 50 /mL (TCID 50 : 50% tissue culture infective dose) and a low detection limit of 10 1.71 TCID 50 /mL (S/N = 3). Importantly, the immunosensor showed high sensitivity, good reproducibility and excellent operational stability. Finally, the immunosensor was evaluated using a real avian serum sample containing ALV-J, with excellent results being achieved., (Copyright © 2018. Published by Elsevier B.V.)

In this study, we identified a chicken liver cell line (LMH) which could strongly support the replication of ALV-J (Subgroup J of avian leukosis virus) with high viral titer. Notably, ALV-J was efficiently detected by ELISA in LMH cells 1 day before DF1 cells. In comparison with DF1 cells, LMH cells not only expressed higher levels of ALV-J receptor chNHE-1, but also possessed a more efficient protein expression system for foreign genes. Thus, LMH cells could be a novel tool to shorten the ALV-J eradication approach and accelerate studies on the pathogenesis and oncogenesis of ALV-J.

A sensitive sandwich-type electrochemical immunosensor was developed for the detection of avian leukosis virus subgroup J (ALV-J), which benefitted from multiple signal amplification involving graphene-perylene-3,4,9,10-tetracarboxylic acid nanocomposites (GR-PTCA), nanocellulose-Au NP composites (NC-Au) and the alkaline phosphatase (ALP) catalytic reaction. GR-PTCA nanocomposites on glassy carbon electrodes served as the immunosensor platform. Due to their excellent electrical conductivity and abundant polycarboxylic sites, the GR-PTCA nanocomposites allowed fast electron transfer and good immobilization of primary antibodies, thereby affording a strong immunosensor signal in the presence of ALV-J. The detected signal could be further amplified by the introduction of NC-Au composites as a carrier of secondary antibodies (Ab 2 ) and by harnessing the catalytic properties of Au and ALP. Under optimized testing conditions, the electrochemical immunosensor displayed excellent analytical performance for the detection of ALV-J, showing a linear current response from 10 2.08 to 10 4.0 TCID 50 /mL (TCID 50 : 50% tissue culture infective dose) with a low detection limit of 10 1.98 TCID 50 /mL (S/N = 3). In addition to high sensitivity, the immunosensor showed very good selectivity, reproducibility and operational stability, demonstrating potential application for the quantitative detection of ALV-J in clinical diagnosis., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Avian leukosis virus subgroup J (ALV-J) is an avian oncogenic retrovirus that has led to severe economic losses in the poultry industry in China in recent decades. Here, using high throughput transcriptome sequencing of HD11 and CEF cells infected with ALV-J, a set of 4804 novel long non-coding transcripts and numerous differentially expressed long non-coding RNAs (lncRNAs) were identified. We also found that they share relatively shorter transcripts and fewer exon numbers compared to mRNA. Correlation analysis suggested that many lncRNAs may activate gene expression in an enhancer-like manner other than through transcriptional regulation. Expression level analyses in vivo showed that three lncRNAs (NONGGAT001975.2, NONGGAT005832.2 and NONGGAT009792.2) may be associated with immune response regulation and could function as novel biomarkers for ALV-J infection. Our findings provides new insight into the pathological process of ALV-J infection and should serve as a high-quality resource for further research on epigenetic influences on disease-resistance breeding as well as immune system and genomic studies., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Avian leukosis virus (ALV) is an important pathogen for various neoplasms, including lymphoid, myeloid, and erythroid neoplasms, and it causes significant economic loss in the poultry industry. Several efficient methods for the detection of ALV have been reported. However, these previously developed approaches are based on either PCR or immunoassays. Here, we used a proximity ligation technique and combined PCR with the immunoassay to develop a novel immuno-PCR (Im-PCR) approach for the detection of ALV. Our data showed that the Im-PCR had high specificity and sensitivity to ALV. The Im-PCR method selectively reacted to ALV but not to the other avian viruses tested. The limit of detection of Im-PCR could reach 0.5 TCID50. Moreover, the results of Im-PCR were in agreement with results from commercial ELISA when the clinical cloaca samples were used for ALV detection. The present results demonstrate that the novel Im-PCR method can be efficiently applied to detect ALV in a clinical setting. Our data also highlight that Im-PCR may have promising applications in the diagnosis of pathogens., (Copyright © 2016 Elsevier B.V. All rights reserved.)

To elucidate the molecular basis for the rapid oncogenicity of an acutely transforming avian leukosis virus (ALV), isolated from fibrosarcomas in Hy-Line Brown commercial layer chickens infected with ALV subgroup J (ALV-J), the complete genomic structure of the provirus was determined. In addition to ALV-J replication-complete virus SDAU1102, five proviral DNA genomes, named SJ-1, SJ-2, SJ-3, SJ-4 and SJ-5, carrying different lengths of the v-src oncogene were amplified from original tumours and chicken embryo fibroblasts (CEFs) infected with viral stocks. The genomic sequences of the SJ-1-SJ-5 provirus were closely related to that of SDAU1102 but were defective. The results of Western blot analysis and immunohistochemical staining also showed overexpression of the p60v-src protein in infected CEFs and tumour tissue. To the best of our knowledge, this is the first report of the isolation and identification of acutely transforming viruses carrying the v-src oncogene with ALV-J as the helper virus. It also offers insight into the generation of acutely transforming ALVs carrying the v-src oncogene.

The subgroup J avian leukosis virus favors the myelocytic series cells and causes myeloid leukosis (myeloblastosis and myelocytomatosis). Natural cases of myeloblastosis (myeloblastic myeloid leukosis) are uncommon and usually occur in adult chickens. This paper describes clinical signs and gross and histopathologic features of myeloblastosis in an adult female budgerigar (Melopsittacus undulatus) that was infected naturally. At necropsy, the spleen was greatly enlarged (enlarged seven or eight times normal) while the other visceral organs were normal. Histologic examination of the spleen indicated a massive intravascular and extravascular accumulation of myeloblasts with a variable proportion of promyelocytes and myelocytes in the red pulp of the spleen.

Background: Subgroups A, B, E and J are the major subgroups of avian leukosis virus (ALV) infecting chickens. ALV infection has become endemic in China and has a significant negative effect on the poultry industry. Consequently, there is an urgent need for a specific, sensitive and rapid method for diagnosis and eradication of ALV. Therefore, we developed a simple and rapid real-time loop-mediated isothermal amplification (LAMP) reaction for the timely detection of the common ALV subgroups, whereby the amplification can be obtained in 35 min under isothermal conditions at 63 °C, ability to specific, sensitive and rapid detect all the common ALV subgroups., Methods: A set of four specific primers was designed to target the sequences of the pol gene of ALV, and the loop-mediated isothermal amplification (LAMP) assay were developed and compared with PCR and virus isolation methods., Results: The results from specificity of the LAMP assay showed that only target ALVs DNA was amplified. The LAMP assay demonstrated a sensitivity of 20 copies/reaction of ALV DNA, which was 10 times higher than the conventional PCR measurement. To further evaluate the reliability of the method, the assay was evaluated with ALV DNA from a panel of 81 clinical samples suspected of ALV infection. The results verify that the LAMP method was more sensitive than the conventional PCR and virus isolation method., Conclusion: In conclusion, the developed LAMP assay was a simple, inexpensive, sensitive method for the rapid detection of the most common subgroups of ALV, and it provided a useful and practical tool in the eradication program for ALV in the poultry industry.

A rapid immunochromatographic strip for detecting capsid protein antigen p27 of avian leukosis virus was successfully developed based on two high-affinity monoclonal antibodies. The test strip could detect not only 600pg purified recombinant p27 protein but also quantified avian leukosis virus as low as 70 TCID50, which has comparative sensitivity to the commercial enzyme-linked immunosorbent assay (ELISA) kit. For the evaluation of this test strip, 1100 samples consisting of cloacal swabs, meconium collected from the earliest stool of one day old chicken and virus isolates were assessed both by the strip and by the commercial ELISA kit. The agreement between these two tests was 93.91%, 93.42% and 100%, respectively. The sensitivity and specificity of the strip were also calculated by using the ELISA kit as the standard. This immunochromatographic strip provides advantages of rapid and simple detection of capsid protein antigen p27 of avian leukosis virus, which could be applied as an on-site testing assay and used for control and eradication programs of avian leukosis disease., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Background: Subgroup A, B, and J ALVs are the most prevalent avian leukosis virus (ALV). Our study attempted to develop two SYBR Green I-based real-time PCR (RT-PCR) assays for specific detection of ALV subgroup J (ALV-J) and multiplex detection of ALV subgroups A and B (ALV-A/B), respectively., Results: The two assays showed high specificity for ALV-J and ALV-A/B and the sensitivity of the two assays was at least 100 times higher than that of the routine PCR assay. The minimum virus detection limit of virus culture, routine PCR and real-time PCR for detection of ALV-A strain was 10(3) TCID50 units, 10(2) TCID50 units and fewer than 10 TCID50 units, respectively. In addition, the coefficients of variation for intra- and inter-assay were both less than 5%. Forty clinical plasma samples were evaluated by real-time PCR, routine PCR, and virus culture with positive rates of 80% (32/40), 72.5% (29/40) and 62.5% (25/40), respectively. When the assay for detection of ALV-J was used to quantify the viral load of various organ tissues in chicken inoculated by ALV-J strains CHN06 and NX0101, the results exhibited that ALV-J genes could be detected in all organ tissues examined and the highest copies of ALV-J were mainly in heart and kidney samples at 30 weeks post-infection. Except in lung, the virus copies of CHN06 group were higher than that of NX0101 group in various organ tissues., Conclusions: The SYBR Green I-based real-time RT-PCR assay provides a powerful tool for the detection of ALV and study of virus replication and infection.

The diagnosis of avian leukosis virus subgroup J (ALV-J) infection in Chinese Partridge Shank chickens was confirmed by necropsy, histopathological examinations, antibody tests, viral isolation, immunofluorescence assays, and sequence analysis. Myelocytoma, myeloma, and fibrosarcoma were simultaneously found in Partridge Shank flock with ALV-J infection. Sequence analysis of the env genes of ALV-J demonstrated that both gp85 and gp37 were highly homologous among the three strains from local chickens of those among ALV-J strains isolated from white meat-type chickens. The phylogenetic trees indicated that the three strains isolated in this study were closely related to reference strains isolated in so-called Chinese yellow chickens and some strains isolated from white meat-type chickens, both from the USA and China. The observed ALV-J infection was the first report on Partridge Shank chickens, and myelocytoma, myeloma, and fibrosarcoma were found at the same time in this batch of local chickens., (© 2015 Poultry Science Association Inc.)

Avian oncogenic viruses include the avian leukosis virus (ALV), reticuloendotheliosis virus (REV) and Marek's disease virus (MDV). Multiple oncogenic viral infections are frequently seen, with even Marek's disease vaccines reported to be contaminated with ALV and REV. The gross lesions caused by avian oncogenic viruses often overlap, making differentiation diagnosis based on histopathology difficult. The objective of this study is to develop a rapid approach to simultaneously differentiate, subgroup and pathotype the avian oncogenic viruses. The oligonucleotide microarray was employed in this study. Particular DNA sequences were recognized using specific hybridization between the DNA target and probe on the microarray, followed with colorimetric development through enzyme reaction. With 10 designed probes, ALV-A, ALV-E, ALV-J, REV, MDV pathogenic and vaccine strains were clearly discriminated on the microarray with the naked eyes. The detection limit was 27 copy numbers, which was 10-100 times lower than multiplex PCR. Of 102 field samples screened using the oligonucleotide microarray, 32 samples were positive for ALV-E, 17 samples were positive for ALV-J, 6 samples were positive for REV, 4 samples were positive for MDV, 7 samples were positive for both ALV-A and ALV-E, 5 samples were positive for ALV-A, ALV-E and ALV-J, one sample was positive for both ALV-J and MDV, and 3 samples were positive for both REV and MDV. The oligonucleotide microarray, an easy-to-use, high-specificity, high-sensitivity and extendable assay, presents a potent technique for rapid differential diagnosis of avian oncogenic viruses and the detection of multiple avian oncogenic viral infections under field conditions., (Copyright © 2014 Elsevier B.V. All rights reserved.)

A novel pH-controlled immunosensor using hollow mesoporous silica and apoferritin combined system has been reported for the first time. The goal of this study was to introduce more electroactive probes into the electrochemical immunosensor. Under such background, we focused our attention on hollow mesoporous silica and apoferritin, both of which have admirable accommodating performances and can be applied for encapsulating electroactive probes. Based on the pH-controlled disassociation-reconstitution process of apoferritin and the mesoporous channels of silica, after the appropriate chemical modification, apoferritin could be fabricated with silica. The results showed that the hollow mesoporous silica and apoferritin combined system was successfully assembled. The excellent performance of the combined system can be applied for ultrasensitive detection of a target virus., (Crown Copyright © 2013 Published by Elsevier B.V. All rights reserved.)

A universal and effective photoelectrochemical (PEC) immunosensing device was fabricated on an indium tin oxide (ITO) electrode for sensitive and specific detection of subgroup J of avian leukosis virus (ALVs-J) based on a signal-on strategy. Bismuth sulfide (Bi2S3) nanorods, with good morphology, high crystallinity and differentiated PEC properties, were selected as the photoelectrochemical species and synthesized by a facile hydrothermal method. On the basis of alkaline phosphatase catalytic chemistry to in situ produce ascorbic acid for electron donating, an enhanced photocurrent was obtained. Due to the dependence of the photocurrent signal on the concentration of generated electron donor, an exquisite immunosandwich protocol was successfully constructed for PEC detection of ALVs-J with a linear range from 10(2.14) to 10(3.65) TCID50/mL. The detection limit was 10(2.08) TCID50/mL (S/N=3), and high stability and specificity were obtained. The strategy provides a fast and sensitive method for ALVs-J analysis and opens a general format for future development of PEC immunoanalysis., (© 2013 Published by Elsevier B.V.)

Avian leukosis virus subgroup A (ALV-A) is a retrovirus which infects egg-type chickens and is the main pathogen of lymphoid leukosis (LL) and myeloid leukosis (ML). In order to greatly enhance the diagnosis and treatment of clinical avian leukemia, two monoclonal antibodies (MAbs) to ALV-A were developed by fusion between SP2/0 and spleen cells from mice immunized with expressed ALV-A env-gp85 protein. Using immunofluorescence assay (IFA), two MAbs reacted with ALV-A, but not with subgroups B and J of ALV. Western blot tests showed that molecular weight of ALV-A envelope glycoprotein recognized by MAbs was about 53 kD. Isotyping test revealed that two MAbs (A5C1 and A4C8) were IgG1 isotypes. These MAbs can be used for diagnosis and epidemiology of ALV-A., (© 2013 John Wiley & Sons Ltd.)

Avian leukosis virus (ALV) subgroups A, B, and J are very common in poultry flocks and have caused serious economic losses in recent years. A multiplex PCR (mPCR) method for the detection of these three subgroups was developed and optimized in this study. We first designed a common forward primer, PF, and three downstream primers, AR, BR, and JR, which can amplify 715 bp for subgroup A, 515 bp for subgroup B, and 422 bp for subgroup J simultaneously in one reaction. The mPCR method produced neither cross-reactions with other subgroups of ALVs nor nonspecific reactions with other common avian viruses. The detection limit of the mPCR was as low as 1 × 10(3) viral DNA copies of each of the three subgroups. In animal experiments, the mPCR detected ALVs 2 to 4 days earlier than did virus isolation from whole-blood samples and cloaca swabs. Furthermore, a total of 346 clinical samples (including 127 tissue samples, 86 cloaca swabs, 59 albumen samples, and 74 whole-blood samples) from poultry flocks with suspected ALV infection were examined by mPCR, routine PCR, and virus isolation. The positive sample/total sample ratios for ALV-A, ALV-B, and ALV-J were 48% (166/346) as detected by mPCR and 48% (166/346) as detected by routine PCR. However, the positive sample/total sample ratio detected by virus isolation was 40% (138/346). The results of the mPCR and routine PCR were confirmed by sequencing the specific fragments. These results indicate that the mPCR method is rapid, specific, sensitive, and convenient for use in epidemiological studies of ALV, clinical detection of ALV, and ALV eradication programs.

The ALV-J gp37 pocket region located in transmembrane (TM) protein is an ideal viral target because it is extracellular, highly conserved, and essential for viral entry. Although it has been a target of drug and vaccine design, there are not any polyclonal antibody tools to specifically probe the antigenicity and immunogenicity of the pocket region. Our goal was to elicit a neutralizing polyclonal antibody that targets this pocket region. A conserved peptide designated R73 that is derived from the pocket region of TM of ALV-J was synthesized. New Zealand rabbits were immunized with R73 conjugated with keyhole limpet hemocyanin (KLH), and the antiserum was gained 10 days after the third immunity boost. The antibody titers were tested by endpoint ELISA, and the specificity and affinity were tested by Western blot analysis. The results showed that R73 has good antigenicity (antibody titers≈1:64,000∼1:128,000), immunogenicity, and affinity. The results of the indirect fluorescent assay (IFA) and real-time RT-PCR test showed that R73 polyclonal antibody can recognize ALV-J antigen and completely inhibit viral replication in DF-1 cells. The results of the neutralization assay showed that neutralization titer of the antiserum was 1:16. Therefore, a polyclonal antibody against the neutralizing epitope on an existing pocket region may be a useful tool in ALV-J diagnosis or for preventing infection.

To analyze the status of wild birds infected with avian leukosis virus (ALV) in China, we collected 300 wild birds from various areas. Virus isolation and PCR showed that wild birds were infected by ALV-A and ALV-B. Two ALV-A and 4 ALV-B env sequences were obtained by PCR using primers designed to detect ALV-A and -B respectively. Our results showed that the gp85 genes of the 2 ALV-A strains have the highest homology with RAV-1, 99.8%, and more than 92% homology with other American strains. However, the gp85 genes of the two ALV-A strains showed slightly lower homology with Chinese strains (87.2-92.6%). Additionally, the 4 ALV-B strains have high homology with the prototype strain (RAV-2), from 99.1 to 99.4%, but they have slightly lower identity with Schmidt-Ruppin B and Prague subgroup B, from 93.3 to 98.4%. The 4 ALV-B strains showed the lowest identity with SDAU09C2 and SDAU09E3 (90%). In total, these results suggested that avian leukosis virus has infected wild birds in China., (Copyright © 2013 Elsevier B.V. All rights reserved.)

An antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) employing monoclonal and polyclonal antibodies against p27 was developed for the detection of the avian leukosis virus (ALV). The specificity of the optimized AC-ELISA was evaluated using avian leukosis virus subgroup J (ALV-J), avian leukosis virus subgroup A (ALV-A), avian leukosis virus subgroup B (ALV-B), avian infectious bronchitis virus (IBV), Marek's disease virus (MDV), avian infectious laryngotracheitis virus (ILTV), Fowlpox virus (FPV), infectious bursal disease virus (IBDV), Newcastle disease virus (NDV), avian reovirus (ARV), reticuloendotheliosis virus (REV), avian influenza virus (AIV) and Escherichia coli. The only specimens that yielded a strong signal were ALV-J, ALV-A and ALV-B, indicating that this assay is suitable for the detection of ALV. The limit of detection of this assay was 1.25 ng/ml of rp27 protein and 10(1.79)TCID(50) units of HLJ09MDJ-1 (ALV-J). Moreover, this AC-ELISA can detect ALV in cloacal swabs of chickens experimentally infected as early as 12 days post-infection. The AC-ELISA detected the virus in the albumin and cloacal swabs of naturally infected chickens, and the results were confirmed by PCR, indicating that the AC-ELISA was a suitable method for the detection of ALV. This test is rapid and sensitive and could be convenient for epidemiological studies and eradication programs., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Subgroup J avian leukosis virus (ALV-J) is an avian retrovirus that causes severe economic losses in the poultry industry. The early identification and removal of virus-shedding birds are important to reduce the spread of congenital and contact infections. In this study, a TaqMan-based real-time PCR method for the rapid detection and quantification of ALV-J with proviral DNA was developed. This method exhibited a high specificity for ALV-J. Moreover, the detection limit was as low as 10 viral DNA copies. The coefficients of variation (CVs) of both interassay and intra-assay reproducibility were less than 1%. The growth curves of ALV-J in DF-1 cells were measured by real-time PCR, yielding a trend line similar to those determined by 50% tissue culture infective dose (TCID(50)) and p27 antigen detection. Tissue samples suspected of ALV infection were evaluated using real-time PCR, virus isolation, and routine PCR, and the positivity rates were 60.1%, 41.6% and 44.5%, respectively. Our data indicated that the real-time PCR method provides a sensitive, specific, and reproducible diagnostic tool for the identification and quantification of ALV-J for clinical diagnosis and in laboratory research.

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.

A novel electrochemical immunosensor for ultrasensitive detection of subgroup J of avian leukosis virus (ALVs-J) was designed by using graphene sheets (GS)-layered double hydroxides (LDHs) composites modified electrode with multifunctional Fe(3)O(4) core/Ni-Al LDHs shell (LDHs@Fe(3)O(4)) nanospheres as labels. At first, the GS-LDHs were used for the immunosensor platform for improving the electronic transmission rate as well as increasing the surface area to capture a large amount of primary antibodies (Ab(1)). After that, ferrocene (Fc), secondary antibodies (Ab(2)) and horseradish peroxidase (HRP) multifunctional LDHs@Fe(3)O(4) nanospheres were used as labels with high load amount and good biological activity. Subsequently, in presence of H(2)O(2), amplified signals were obtained by an electrochemical sandwich immunoassay protocol. To embody the signal amplification property of the protocol, the analytical properties of various immunosensor platform and labels were compared in detail. Under optimal conditions, the reduction peak currents of the electrochemical immunosensor were proportional to the ALVs-J concentration over the range from 10(2.32) to 10(5.50) TCID(50)/mL with a low detection limit (180 TCID(50)/mL, S/N=3). The resulting immunosensor also displayed a good selectivity, reproducibility and stability., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Avian leukosis virus subgroup J poses a great threat to the poultry industry in China. To reduce the economic losses, a quick method for detection of ALV-J antigen is required for diagnosis and identification of the congenitally transmitting hens. In this study, we report the production and evaluation of one monoclonal antibody (MAb) suitable for achieving these goals. The gp85 gene of avian leukosis virus subgroup J CAUHM01 China isolates was subcloned into the expression vectors pGEX-6p-1 and pET28a and successfully expressed in E. coli. After immunizing BALB/c mice with recombinant His-Jgp85 protein, splenic cells from immunized mice were fused with SP2/0 myeloma cells to produce hybridomas. We isolated and characterized one ALV-J gp85-specific MAb by determining its titer, affinity and IgG subclass. In addition, we performed epitope mapping and determined the epitope for the MAb 1E3 to be 81-92 aa of ALV-J gp85 protein (LPWDPQELDILG). Bioinformatics analysis and IFA studies revealed that this epitope is conserved among all ALV-J isolates and that this antibody could serve as a useful reagent for ALV-J detection and diagnosis.

Avian leukosis virus (ALV) is associated with tumor development and growth retardation in poultry. Eradication of virus infection at the primary breeder level is the principal method for controlling ALV infection in chickens. An indirect enzyme-linked immunosorbent assay (iELISA) method that utilized the prokaryotically expressed and affinity-purified viral capsid protein antigen p27 was developed for the detection of ALV-specific antibodies in chicken sera. The protocol of iELISA was validated and resulted in a higher agreement value than fluorescent antibody test (FAT) and was shown to be more sensitive and specific compared to the commercial ALV antibody test kit when FAT was used as a reference test. The main advantage of this method is the use of a single immunogenic protein to detect antibodies against all ALV exogenous subgroups. The results show that the developed iELISA is an inexpensive alternative and can potentially be used as a confirmatory test for the presence of anti-ALV antibodies on a large scale.

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., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Objective: In order to get a rapid specific diagnostic reagent for subgroup A Avian Leukosis Virus detection., Methods: The Avian Leukosis Virus Subgroup A (ALV-A) SDAU09E1 strain was inoculated into DF1 cells, an ALV-A- gp85 DNA fragment of 1023 bp was amplified from infected cells and inserted into PET-32a(+) plasmid at the location between restriction endonucleases BamH I and Not I sites. The recombinant plasmid PET-SDAU09E1 -gp85 was transformed into E coli. BL21 (Rosetta) for gp85 gene expression. Then we used the purified recombinant fusion protein to immunize 6 weeks old Kunming white mice, and the antiserum were prepared., Results: The recombinant ALV-A gp85 fusion protein with a molecular weight of 52.8 kDa demonstrated a good antigenecity. Mon-specific serum produced by vaccinated mice came out reactive with subgroups A and B ALV (ALV-A and ALV-B but not subgroup J ALV) by the indirect immunofluorescence (IFA) method., Conclusion: This was the first time to demonstrate a mono-specific antiserum specific to ALV-A and ALV-B, it could be used for differential diagnosis of exogenous ALV infections in CEF cultures when in complement with ALV-J specific monoclonal antibodies. Chickens in our country are now distressed by both classic ALV-A/B and emerging ALV-J, making differential diagnosis necessary, so studying this reagent has high practical value.

Avian leukosis viruses (ALVs) belong to Alpharetrovirus genus of the family Retroviridae that are widespread in nature. Different subgroups of ALV commonly infect egg-laying hens. They are responsible for economic losses due to both mortality and depressed performance in chickens. To investigate the presence of these viruses in chickens in Iran, 560 egg albumens were selected from different farms of Fars province, Iran. These eggs were obtained from flocks of two research centers of native fowl production (60 eggs), a broiler grandparent farm (100 eggs), three broiler breeder farms (300 eggs), and a commercial layer flock (100 eggs). Firstly, for primary screening a degenerative primer set (PU1 and PU2) were used in reverse transcriptase-polymerase chain reaction (RT-PCR). Positive cases were detected in 47 of 300 (15.7%) samples from three broiler breeders, 40 of 100 (40%) samples from commercial layer, 53 of 60 (88.3%) samples from flocks of two research centers of native fowl production, and none from the samples of broiler grandparent. Then RT-PCR was undertaken with primers PA1 and PA2 on the positive samples. RT-PCR analysis detected ALVs in two of 47 (4.3%) samples from three broiler breeders, 13 of 40 (32.5%) samples from commercial layer, and 19 of 53 (35.8%) samples from flocks of two research centers of native fowl production. The sequencing results showed that subgroup E of ALV was the most detected virus among chicken eggs and subgroup B was more prevalent in the eggs of native fowls. This is the first report of the ALV subgroup B and E in egg albumen in Iran.

Infection of breeder flocks in China with subgroup J avian leukosis virus (ALV-J) has increased recently. In this study, we have developed a loop-mediated isothermal amplification (LAMP) assay for rapid detection of ALV-J from culture isolates and clinical samples. The ALV-J-specific LAMP assay efficiently amplified the target gene within 45 min at 63 degrees C using only a simple laboratory water bath. To determine the specificity of the LAMP assay, various subgroup ALVs and other related viruses were detected. A ladder pattern on gel electrophoresis was observed for ALV-J isolates but not for other viruses. To evaluate the sensitivities of the LAMP assay and conventional PCR, the NX0101 isolate plasmid DNA was amplified by them. The detection limit of the LAMP assay was 5 target gene copies/reaction, which was up to 20 times higher than that of conventional PCR. To evaluate the application of the LAMP assay for detection of ALV-J in clinical samples, 49 samples suspected of ALV infection from breeder flocks were tested by the LAMP assay and PCR. Moreover, virus isolation from these samples was also performed using cell culture. The positive-sample ratios were 21/49 (43%) by conventional PCR, 26/49 (53%) by the LAMP assay, and 19/46 (41%) by virus isolation. Additionally, a positive LAMP reaction can be visually ascertained by the observation of turbidity or a color change after addition of SYBR green I dye. Consequently, the LAMP assay is a simple, rapid, and sensitive diagnostic method and can potentially be developed for rapid detection of ALV-J infection in the field.

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 ALV of subgroup A isolated from imported breeders.

A polymerase chain reaction (PCR) assay that utilizes nested primers to amplify a fragment of the long terminal repeat of exogenous avian leukosis virus (ALV) was developed and evaluated for detection of ALV subgroup J directly from clinical samples. Compilation of sequence data from different endogenous and exogenous ALVs allowed the selection of a conserved set of nested primers specific for the amplification of exogenous ALV subgroups A, B, C, D, and J and excluded amplification of endogenous viruses or endogenous viral sequences within the chicken genome. The nested primers were successfully used in both PCR and reverse transcriptase (RT)-PCR assays to detect genetically diverse ALV-J field isolates. Detection limits of ALV-J isolate ADOL-Hc1 DNA by nested PCR and RNA by RT-nested PCR were superior to detection of group-specific antigen by enzyme-linked immunosorbent assay (ELISA) in cell culture. Detection of ALV-J in cloacal swabs by RT-nested PCR was compared with direct detection by antigen-capture (ac)-ELISA; RT-nested PCR detected fewer positive samples than ac-ELISA, suggesting that RT-nested PCR excluded detection of endogenous virus in clinical samples. Detection of ALV-J in plasma samples by RT-nested PCR was compared with virus isolation in C/E chicken embryo fibroblasts; the level of agreement between both assays as applied to plasma samples ranged from low to moderate. The main disagreement between both assays was observed for a group of plasma samples found positive by RT-nested PCR and negative by virus isolation, suggesting that RT-nested PCR detected ALV-J genome in plasma samples of transiently or intermittently infected birds. ALV-J transient and intermittent infection profiles are characterized by inconsistent virus isolation responses throughout the life of a naturally infected flock.

Feather pulp from experimentally infected chickens was used as a source of DNA for polymerase chain reaction (PCR) amplification of avian leukosis virus subgroup J (ALV-J) proviral DNA. A primer set that produces a large amplicon (approximately 2,125) was used to detect ALV-J proviral DNA. This primer set was used in lieu of previously published primers because it allows for sequencing of the entire envelope gene and because it was able to detect diagnostically a number of North American ALV-J isolates that could not be detected with previously published primers and PCR conditions. ALV-J proviral DNA was detected in feather pulp at 7 days of age in more than 90% of birds infected as embryos and 7 days postinoculation in over 50% of chickens infected at 3 days of age. The results obtained with PCR on feather pulp were compared with those of virus isolation. In the embryo-inoculated birds, the percentages of agreement between PCR and virus isolation were 92.5% at 7 days of age and 100% at 28, 42, 49, and 56 days of age. However, the overall sensitivity of virus isolation in embryo-infected birds was higher, particularly at 7 and 56 days of age. In chickens inoculated at 3 days of age, the percentages of agreement of detection between PCR and virus isolation ranged from 75% at 10 days of age to 100% at 42 days of age. Agreement of negative results of ALV-J detection by PCR and virus isolation in chickens infected posthatch ranged between 66.6% and 100% between the ages of 10 and 42 days. Virus isolation requires chicken embryo fibroblasts of specific genetic lines, and the process takes onaverage 7-9 days. Aseptic collection of blood and tissues for virus isolation and molecular detection of ALV-J requires sterile necropsy instruments as well as syringes and needles for each individual chicken, whereas sterile microcentrifuge tubes and gloves are the only equipment necessary for aseptic feather pulp collection for ALV-J detection by PCR. PCR-based detection of ALV-J in feather pulp is especially suitable when ALV-J infection must be diagnosed rapidly and unequivocally without killing the chicken(s) and in situations where crucial reagents or suitable virus propagation substrates are not readily available for isolation and propagation of ALV-J in cell culture.

Subgroup J avian leukosis virus (ALV-J) causes serious economic losses in the commercial poultry industry. Measuring group-specific antigen (GSA) by enzyme-linked immunosorbent assay (ELISA) has been used to identify chickens infected with this virus. However, the inability of ELISA to discriminate the GSA from endogenous ALV (subgroup E ALV [ALV-E]) or ALV-J infection has limited its usage. The purpose of the present study was to develop a method to discriminate between uninfected flocks having ALV-E and ALV-J-infected flocks by ELISA. The GSA and anti-ALV-J antibody in the plasma samples from chickens at different ages in three grandparent farms were measured by ELISA. Infected flocks were confirmed by reverse transcription-polymerase chain reaction with different subgroup-specific primers and sequence analysis. The results indicated that the GSA of ALV-J-infected flocks increased, but that of the uninfected flocks decreased during young ages. The anti-ALV-J antibody of infected flocks was higher and increased earlier than that of uninfected flocks. Thus, measuring GSA in blood at the ages of 1 and 6 wk by ELISA is suitable to discriminate between ALV-J-infected flocks and uninfected flocks having ALV-E.

Commercial white leghorn egg layer flocks being used to produce fertile eggs for human vaccine production exhibited dramatically low peaks in egg production, two to four times higher than normal weekly mortality, and high numbers of cull, nonlaying birds after the onset of sexual maturity. These lower production characteristics could not be associated with management-related problems. Gross lesions of cull and fresh dead birds necropsied showed approximately 60% lacked ovarian activity and had lesions of a bacterial bursitis or synovitis, whereas the other 40% had tumors of the viscera but not of the bursa of Fabricius. Histologic examination of tumor-containing tissues showed lesions typical of myelocytomatosis. The diagnosis of myeloid leukosis was confirmed by the isolation of a recombinant avian leukosis virus (ALV) containing the LTR of subgroup J and the envelope of subgroup B ALV. A positive polymerase chain reaction with primers specific for the 3' untranslated region LTR confirmed the presence of LTR of ALV-J. The source of infection with this recombinant ALV was not determined; however, it is likely that commingling of the day-old egg-type chicks with ALV-J-infected meat-type chicks in a common hatchery had contributed to this outbreak.

Marek's disease virus (MDV), a herpesvirus, and avian leukosis virus, subgroup J (ALV-J), a retrovirus, are oncogenic viruses of poultry. The present report describes a case-report study aimed at examining the efficacy of amplifying MDV and/or ALV-J from feather-tip DNA as compared with DNA purified from liver and spleen. We show that the polymerase chain reaction for MDV and ALV-J env using DNA from feather tips was more effective for diagnosis of naturally infected commercial chickens than using the liver and spleen.

Subgroup J avian leukosis viruses (ALVs), which are a recombinant virus between exogenous and endogenous ALVs, can spread by either vertical or horizontal transmission. Exogenous and endogenous ALVs can be detected in feather pulp. In this study, virus titers in feather pulp of chickens infected with subgroup J ALV were compared with those of plasma and cloacal swab. All of the broiler chickens inoculated with subgroup J ALV at 1 day old were positive for virus from feather pulp during the experimental period of between 2 wk and 8 wk of age. Virus titers in feather pulp of some broiler chickens infected with subgroup J ALV were very high, ranging from 10(7) to 10(8) infective units per 0.2 ml. Virus titers in feather pulp were usually the highest among the samples of plasma, cloacal swab, and feather pulp tested. In another experiment in which layer chickens were inoculated with subgroup J ALV at 1 day old, virus was detected in feather pulp from 2 wk until 18 wk of age, and virus persisted longer in feather pulp than in plasma. Almost all of the layer chickens tested were positive for virus by polymerase chain reaction (PCR) with DNA extracted from feather pulp samples at 2, 4, and 10 wk of age, and the PCR from feather pulp was more sensitive than virus isolation from plasma, cloacal swab, and feather pulp. All above results indicate that samples of feather pulp can be useful for virus isolation and PCR to confirm subgroup J ALV infection.

The tissue tropism of avian leukosis virus (ALV) subgroup J (ALV-J) was investigated in congenitally infected broiler chickens by an immunohistochemistry technique detecting gp85 viral glycoprotein. All organs examined contained detectable antigen. The most intense staining was in the adrenal gland, heart, kidney, and proventriculus. Intense staining for viral antigen in the heart may explain the ability of ALVs to cause cardiomyopathy. Although recent investigations failed to demonstrate specific viral staining in bone marrow from infected chickens, we were able to show moderate staining in myelocytic precursor cells in bone marrow. This finding agrees with previous work showing cell cultures of bone marrow are susceptible to ALV-J infection and the tendency of subgroup J to predominantly induce myeloid rather than lymphoid neoplasms.

In an attempt to develop a specific diagnostic test for avian leukosis virus (ALV) subgroup J (ALV-J) strain Hc1, four monoclonal antibodies (MAbs), JE9, G2, 145, and J47, were generated that are specific for ALV-J envelope glycoprotein, gp85. Polymerase chain reaction (PCR) was used to amplify genomic pro-viral DNA of Avian Disease and Oncology Laboratory (ADOL)-Hc1 and ADOL-4817 envelope genes. Both open reading frames encoding glycoproteins gp85 and gp37 were cloned into baculoviruses. Abundant expression of gp85 and gp37 was detected in the recombinant viruses with specific antibody to Hc1 strain of the ALV-J. The expressed proteins were used for immunization of mice to produce hybridoma cell lines secreting MAbs specific to ALV-J envelope protein. A panel of MAbs was generated by fusing NS1 myeloma cells and spleen cells from mice immunized with the recombinant baculoviruses. With the use of an immunofluorescence assay, three MAbs (JE9, G2, 145) reacted with ALV-J but not with subgroups A, B, C, D, or E of ALV. MAb J47 reacted with all exogenous subgroups of ALV including A, B, C, D, and J but not with endogenous subgroup E viruses. Western blot analysis was performed with all four MAbs against recombinant baculovirus and Hc1-infected chicken embryo fibroblast (CEF) lysates. A major band with a molecular weight about 90 kD corresponding to the size of ALV-J envelope was consistently obtained. With these MAbs, we detected the Hc1 antigen in CEFs infected with several ALV-J viruses isolated in the United States and also in tissue sections from chickens infected with Hc1 strain of ALV-J. These MAbs will be useful reagents for the diagnosis of ALV-J infection because they recognize a common antigenic epitope in six isolates tested thus far.