Avian Leukosis Virus Subgroup J Infection Research Articles

Mutations in and Expression of the Tumor Suppressor Gene p53 in Egg-Type Chickens Infected With Subgroup J Avian Leukosis Virus.

To investigate the molecular mechanisms of the oncogenic effects of avian leukosis virus subgroup J (ALV-J), we examined mutations in and the expression of p53 in the myelocytomas distributed in the liver, spleen, trachea, and bone marrow, as well as in fibrosarcomas in the abdominal cavity and hemangiomas in skin from chickens that were naturally or experimentally infected with ALV-J. Two types of mutations in the p53 gene were detected in myelocytomas of both the experimentally infected and the naturally infected chickens and included point mutations and deletions. Two of the point mutations have not been reported previously. Partial complementary DNA clones with a 122-bp deletion in the p53 gene ORF and a 15-bp deletion in the C-terminus were identified in the myelocytomas. In addition, moderate expression of the mutant p53 protein was detected in the myelocytomas that were distributed in the liver, trachea, spleen, and bone marrow. Mutant p53 protein was not detected in the subcutaneous hemangiomas or in the abdominal fibrosarcomas associated with natural and experimental ALV-J infection, respectively. These results identify mutations associated with abnormal expression of p53 in ALV-J-associated myelocytomas, suggesting a role in tumorigenesis.

An avian leukosis virus subgroup J isolate with a Rous sarcoma virus-like 5'-LTR shows enhanced replication capability.

Avian leukosis virus subgroup J (ALV-J) was first isolated from meat-producing chickens that had developed myeloid leukosis. However, ALV-J infections associated with hemangiomas have occurred in egg-producing (layer) flocks in China. In this study, we identified an ALV-J layer isolate (HLJ13SH01) as a recombinant of ALV-J and a Rous sarcoma virus Schmidt-Ruppin B strain (RSV-SRB), which contained the RSV-SRB 5'-LTR and the other genes of ALV-J. Replication kinetic testing indicated that the HLJ13SH01 strain replicated faster than other ALV-J layer isolates in vitro. Sequence analysis indicated that the main difference between the two isolates was the 5'-LTR sequences, particularly the U3 sequences. A 19 nt insertion was uniquely found in the U3 region of the HLJ13SH01 strain. The results of a Dual-Glo luciferase assay revealed that the 19 nt insertion in the HLJ13SH01 strain increased the enhancer activity of the U3 region. Moreover, an additional CCAAT/enhancer element was found in the 19 nt insertion and the luciferase assay indicated that this element played a key role in increasing the enhancer activity of the 5'-U3 region. To confirm the potentiation effect of the 19 nt insertion and the CCAAT/enhancer element on virus replication, three infectious clones with 5'-U3 region variations were constructed and rescued. Replication kinetic testing of the rescued viruses demonstrated that the CCAAT/enhancer element in the 19 nt insertion enhanced the replication capacity of the ALV-J recombinant in vitro.

Genetic diversity and phylogenetic analysis of glycoprotein gp85 of avian leukosis virus subgroup J wild-bird isolates from Northeast China

Avian leukosis virus subgroup J (ALV-J), first isolated in 1989, preferentially infects meat-type birds. Chinese layer flocks have experienced outbreaks of this virus since 2008. To analyze the status of ALV-J infection in wild birds in China, 585 wild birds collected from three provinces of Northeast China from 2010 to 2012 were tested, and six ALV-J strains were isolated for the first time. Furthermore, the gp85 genes of the six strains were amplified, cloned, and sequenced. The results indicated that two different ALV-J strains coexisted in Chinese wild birds from 2010 to 2012. These results not only expand the epidemiological data available for ALV-J and provide necessary information for the further understanding of the evolution of ALV-J, but they also highlight the potential role of wild-bird migration in the spread of ALV-J.

The MYC , TERT , and ZIC1 Genes Are Common Targets of Viral Integration and Transcriptional Deregulation in Avian Leukosis Virus Subgroup J-Induced Myeloid Leukosis

The integration of retroviruses into the host genome following nonrandom genome-wide patterns may lead to the deregulation of gene expression and oncogene activation near the integration sites. Slow-transforming retroviruses have been widely used to perform genetic screens for the identification of genes involved in cancer. To investigate the involvement of avian leukosis virus subgroup J (ALV-J) integration in myeloid leukosis (ML) in chickens, we utilized an ALV-J insertional identification platform based on hybrid capture target enrichment and next-generation sequencing (NGS). Using high-definition mapping of the viral integration sites in the chicken genome, 241 unique insertion sites were obtained from six different ALV-J-induced ML samples. On the basis of previous statistical definitions, MYC, TERT, and ZIC1 genes were identified as common insertion sites (CIS) of provirus integration in tumor cells; these three genes have previously been shown to be involved in the malignant transformation of different human cell types. Compared to control samples, the expression levels of all three CIS genes were significantly upregulated in chicken ML samples. Furthermore, they were frequently, but not in all field ML cases, deregulated at the mRNA level as a result of ALV-J infection. Our findings contribute to the understanding of the relationship between multipathotypes associated with ALV-J infection and the molecular background of tumorigenesis. ALV-Js have been successfully eradicated from chicken breeding flocks in the poultry industries of developed countries, and the control and eradication of ALV-J in China are now progressing steadily. To further study the pathogenesis of ALV-J infections, it will be necessary to elucidate the in vivo viral integration and tumorigenesis mechanism. In this study, 241 unique insertion sites were obtained from six different ALV-J-induced ML samples. In addition, MYC, TERT, and ZIC1 genes were identified as the CIS of ALV-J in tumor cells, which might be a putative "driver" for the activation of the oncogene. In addition, the CIS genes showed deregulated expression compared to nontumor samples. These results have potentially important implications for the mechanism of viral carcinogenesis.

Selection of reference genes for quantitative real-time PCR analysis in chicken embryo fibroblasts infected with avian leukosis virus subgroup J

BackgroundThe selection of stably expressed reference genes is a prerequisite when evaluating gene expression, via real-time PCR, in cells in response to viral infections. The objective of our study was to identify suitable reference genes for mRNA expression analysis in chicken embryonic fibroblasts (CEF) after infection with avian leukosis virus subgroup J (ALV-J).FindingsThe expression levels of 11 potential reference genes in CEF infected with ALV-J were determined by real-time PCR. The expression stability of these genes were analyzed and ranked using the geNorm tool. Analysis indicated that the genes RPL30 (ribosomal protein L30) and SDHA (succinate dehydrogenase complex, subunit A) were the most stably expressed genes in the ALV-J infected CEF.ConclusionsThe RPL30 and SDHA were deemed suitable for use as reference genes for real-time PCR analysis of mRNA gene expression during ALV-J infection, whereas commonly used ACTB and GAPDH are unsuitable to be reference genes.

Multicentric Histiocytosis Related to Avian Leukosis Virus Subgroup J (ALV-J)-Infection in Meat-Type Local Chickens

ABSTRACTGross lesions characterized by swollen livers and spleens accompanied by diffuse white miliary spots, which resembled those of Marek’s disease, were detected in two flocks of local meat-type chickens at a Japanese poultry processing plant in June and August 2010. The microscopic examinations revealed proliferative foci consisting of spindle or polymorphic cells in the interstitium of livers, splenic follicles and the interstitium of kidneys. These cells were positive immunohistochemically with Iba1 antibody, indicating they were histiocytic cells. Some of them contained antigens of avian leukosis virus (ALV) by immunohistochemistry,and the env gene of ALV subgroup J was detected from the spleens by polymerase chain reaction (PCR). Phylogenetic analysis of the PCR product indicated that the env gene might be descended from the American ADOL-7501 strain of ALV-J. These results suggest that the swollen livers and spleens of the meat-type chickens may come from histiocytic proliferation caused by ALV-J infection.

Overexpression of microRNA gga-miR-1650 decreases the replication of avian leukosis virus subgroup J in infected cells

MicroRNAs (miRNAs) are a class of small regulatory non-coding RNAs that modulate gene expression at the post-transcriptional level, playing a crucial role in cell differentiation and development. Recently, some reports have demonstrated that a number of cellular miRNAs play a role during viral infection. In this study, a luciferase-reporter system carrying the 5' untranslated region (5' UTR) and 3' UTR of avian leukosis virus subgroup J (ALV-J) was used to determine whether cellular miRNAs are involved in ALV-J infection. The miRNA gga-miR-1650 was screened for its potential interaction with the 5' UTR of ALV-J and the ability to suppress luciferase-reporter activity. A mutational analysis of predicted gga-miR-1650-binding sites showed that the 5' and 3' ends of gga-miR-1650 contributed to the interaction between gga-miR-1650 and its target located at the 5' UTR. Overexpression of miRNA gga-miR-1650 was shown to downregulate the expression of the Gag protein and influence the replication of ALV-J through binding to the 5' UTR. Overall, this report provides the basis for the development of new strategies for anti-ALV-J intervention.

Detection of subgroup J avian leukosis virus gene by polymerase chain reaction from whole blood without DNA extraction

Avian leukosis virus subgroup J (ALV-J) is an exogenous avian leukosis virus that causes significant economic losses. In this study, a whole blood polymerase chain reaction (PCR) assay without DNA extraction was developed, optimized, and was compared with conventional methods for the detection of ALV-J infection. The evaluation assay indicated that the whole blood PCR had similar sensitivity and specificity when compared with virus isolation and conventional PCR. Taken together, the whole blood PCR method was very specific, easy to perform, rapid and of low cost, which will be useful for routine diagnosis of ALV-J, especially for large scale sampling. Key words: Av/ian leukosis virus subgroup J, polymerase chain reaction, whole blood.

Proteomics of DF-1 cells infected with avian leukosis virus subgroup J

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 years. The pathogenesis of virus infection and virus–host interactions are still not well elucidated. In this paper, we investigated the expression changes for cellular proteins in DF-1 cells infected with ALV-J. Comparative analyses revealed that the majority of the altered proteins in DF-1 cells appeared at 6–12h after ALV-J infection. Mass spectrometry identified 74 altered cellular proteins, including 30 up-regulated proteins and 44 down-regulated proteins. Some of these proteins are involved in cell cytoskeleton, metabolic processes, response to stimulus and immune responses. Other proteins, such as DJ-1, UCHL1, VDAC1 and HMGB1, have some relationship to apoptosis or oncogenesis. The changes in the transcriptional profile of DJ-1, UCHL1, VDAC1 and HMGB1 in infected as compared to uninfected DF-1 cells were confirmed by real-time RT-PCR. Our work gives some information about differential protein expression in cells infected with ALV-J, which will help us to understand viral pathogenicity.

Response of White Leghorn Chickens to Infection with Avian Leukosis Virus Subgroup J and Infectious Bursal Disease Virus

The effects of viral-induced immunosuppression on the infectious status (viremia and antibody) and shedding of avian leukosis virus (ALV) were studied. Experimental white leghorn chickens were inoculated with ALV subgroup J (ALV-J) and infectious bursal disease virus (IBDV) at day of hatch with the ALV-J ADOL prototype strain Hcl, the Lukert strain of IBDV, or both. Appropriate groups were exposed a second time with the Lukert strain at 2 wk of age. Serum samples were collected at 2 and 4 wk of age for IBDV antibody detection. Samples for ALV-J viremia, antibody detection, and cloacal shedding were collected at 4, 10, 18, and 30 wk of age. The experiment was terminated at 30 wk of age, and birds were necropsied and examined grossly for tumor development. Neoplasias detected included hemangiomas, bile duct carcinoma, and anaplastic sarcoma of the nerve. Control birds and IBDV-infected birds were negative for ALV-J-induced viremia, antibodies, and cloacal shedding throughout experiment. By 10 wk, ALV-J-infected groups began to develop antibodies to ALV-J. However, at 18 wk the incidence of virus isolation increased in both groups, with a simultaneous decrease in antibody levels. At 30 wk, 97% of birds in the ALV-J group were virus positive and 41% were antibody positive. In the ALV-J/IDBV group, 96% of the birds were virus positive at 30 wk, and 27% had antibodies to ALV-J. In this study, infection with a mild classic strain of IBDV did not influence ALV-J infection or antibody production.

Genetic diversity and phylogenetic analysis of glycoprotein GP85 of ALV-J isolates from Mainland China between 1999 and 2010: Coexistence of two extremely different subgroups in layers

Avian leukosis virus subgroup J (ALV-J), first isolated in 1989, preferentially infects meat-type birds. However, layer flocks in China have experienced outbreaks of this virus since 2008. To understand the genetic diversity of ALV-J in Chinese layers, we compared and analyzed the GP85 gene sequences of 106 ALV-J isolates that were isolated between 1999 and 2010 in Mainland China. The GP85 gene sequences of 41 layer isolates collected from 9 provinces of China between 2008 and 2010 belonged to two separate, highly diverse subgroups and were differentiated from meat-type chicken isolates. When compared to all meat-type isolates from China, Subgroup 1 exclusively contained current layer isolates and seemed to be dominant; all the isolates in this subgroup exhibited gene diversity, and many unique amino acid mutations were present. In contrast, the viruses in Subgroup 2 were perfectly conserved and shared high identity with the prototype meat-type chicken ALV-J strain HPRS-103. The two subgroups contained only two concurrent mutations at the same position. Moreover, most of the isolates in Subgroup 1 had two additional glycosylation sites (at positions 101 and 191) when compared with those in Subgroup 2. Our study provides evidence for the coexistence of two extremely different ALV-J subgroups in Chinese layers from 2008 to 2010, supporting the need for vaccine development and purification measures to prevent ALV-J infection in layers in China.

Avian Leukosis Virus Subgroup J in Layer Chickens, China

To the Editor: In recent years, cases of avian leukosis virus subgroup J (ALV-J) infection and tumors in commercial layer chickens and breeders of egg-type chickens have been emerging in the People’s Republic of China. ALV-J was first isolated from meat-type chickens with myeloid leukosis in 1988. Although egg-type chickens have been experimentally infected with ALV-J to induce tumors (1), field cases of ALV-J infection and tumors in commercial layer chickens were not found worldwide until 2004 (2). ALV-J has recently been found to have induced various tumors and caused production problems in commercial layer flocks and local chicken breeds in China (2,3). Many field cases of ALV-J infection and tumors have occurred in 15- to 29-week-old egg-type chickens in several provinces. Affected flocks had dramatically reduced egg production and hemorrhage in the skin surrounding the phalanges and feather follicles. Some birds had gray-white nodules in the liver, spleen, or kidneys, and liver and spleen were enlarged up to several times their normal size. Morbidity rates for some flocks reached 60%, and mortality rates for some flocks were >20%. Clinical samples from livers, spleens, whole blood, and tumors were collected from chickens in different provinces and sent for laboratory diagnosis. Results showed that the predominant virus in the samples was ALV-J. During 2007–2009, we conducted an epidemiologic investigation of ALV in layer flocks in China. All virus isolation was performed in DF-1 cells. Briefly, 233 clinical samples were collected from 44 layer flocks in different provinces and used to inoculate subconfluent cell cultures containing Dulbecco modified essential medium supplemented with 10% (vol/vol) fetal bovine serum and antimicrobial drugs. After a 7–9 day incubation period, the cells were frozen and thawed 3×. A group-specific antigen-capture ELISA was used to identify ALV. After proviral DNA was extracted directly from infected cell culture or tumors, PCR with strain-specific primers was used to detect ALV-A, ALV-B, or ALV-J (4). Of these samples, 150 (64.4%) were ALV-J positive, 28 (12.1%) were ALV-A positive, and 8 (3.4%) were ALV-B positive. Phylogenetic analysis showed an 87.3%–98.2% aa sequence identity of env genes in all ALV isolates compared with the HPRS-103 strain (5). All isolates had complete repeated transmembrane deletion and partial direct repeat–1 deletion but contained an intact E element. A mutation was found in the enhancer and promoter region of the U3 region in the 3′ long terminal repeat; this mutation is not found in ALV-J isolated from broiler chickens (6). The newly isolated ALV-J strain from layer chickens was used to examine the pathogenicity in 1-day-old White Leghorn specific pathogen–free chicks soon after hatching in separate incubators and rooms in the experimental animal house facilities at Harbin Veterinary Research Institute, Harbin, China. The chicks were inoculated intraabdominally with a 1,000-unit 50% tissue-culture infective dose of ALV-J propagated in the DF-1 cells. Blood samples were collected to check for viremia at 10 weeks of age. Experimental birds were reared until 27–30 weeks of age. Prolonged viremia developed in 15 (50%) of 30 chicks; hemangiomas developed in the skin surrounding phalanges and in the liver of 3 (10%); and myeloid leukosis, detected by gross or histologic examination, developed in 10 (30.3%). A previous study showed that meat-type birds infected with ALV-J retained a high level of viremia over their lifetime (7) but that layer chickens cleared the infection within a few weeks. Our study demonstrated that ALV-J infection can cause disease in layer chickens and can induce tumors and long-lasting viremia. For this reason, disease caused by ALV-J in layer chickens in China should be further investigated. Because ALV-J is vertically transmitted from dam to progeny by the embryo, it represents a potential threat for humans who receive vaccines that are produced in chicken embryonic fibroblasts or embryonated eggs (e.g., yellow fever vaccine and measles and mumps vaccine) (8). An effective vaccine against ALV is not available. Eradication of ALV-J has been difficult because of substantial genetic and antigenic variation among ALV-J isolates as well as high levels of vertical and horizontal transmission (9,10). Therefore, effective prevention and elimination measures should be developed as soon as possible.

Differentially expressed genes in a flock of Chinese local-breed chickens infected with a subgroup J avian leukosis virus using suppression subtractive hybridization.

Avian leukosis virus subgroup J (ALV-J) is a new type of virus that mainly induces myeloid leukosis (ML) in chickens. To further elucidate the pathogenesis of ALV-J infection and tumor development, expression profiles from the bone marrow tissue of 15 infected and 18 non-infected birds from a local-breed poultry-farm under naturally infected conditions, were analyzed by suppression-subtractive hybridization. The birds were diagnosed as ML+ (or ML-) by specific ALV-J detection methods, involving serological tests for antigens and antibodies, and RT-PCR to detect viral RNA. A total of 59 partial gene sequences were revealed by differential screening of 496 forward and 384 reverse subtracted cDNA clones. Of these, 22 identified genes, including 8 up-regulated and 14 down-regulated, were related to immune functions, these genes being, MHC B-G antigen, translationally-controlled tumor protein (TPT1/TPTC), transferrin and ferritin, hemoglobin and Carbonic anhydrase. Four of the down-regulated genes were selected for further analysis, in view of their predicted roles in infection and immunity by real-time qRT-PCR, using RNA collected from the same birds as those used for SSH. The four genes were expressed at significantly lower levels (p < 0.001) in ALV-J infected birds than in non-infected ones.

Detection of subgroup J avian leukosis virus infection in Australian meat‐type chickens

To determine the extent of avian leukosis virus subgroup J (ALV-J) infection in Australian broiler breeder flocks, using virus isolation and molecular biological detection. Any resultant ALV-J viral isolates to be characterised by neutralisation cross testing in order to determine antigenic relationships to overseas isolates of ALV-J. Samples of blood, feather pulp, albumen and tumours were obtained from broiler breeder flocks which represented four genetic strains of meat chickens being grown in Victoria, South Australia, NSW and Queensland. Dead and ailing birds were necropsied on farm and samples were collected for microscopic and virological examinations. Virus isolation was carried out in C/O and DF-1 CEF cultures and ALV group specific antigen was detected in culture lysates using AC-ELISA. Micro-neutralisation assay was used for antigenic characterisation of selected isolates. Genomic DNA was isolated from cultured cells, tumours and feather pulp. ALV-J envelope sequences were amplified by PCR using specific ALV-J primers while antibodies against ALV-J were detected by ELISA. A total of 62 ALV-J isolates were recovered and confirmed by PCR from 15 (31.3%) of 48 breeder flocks tested. Antibody to ALV-J was detected in 20 (47.6%) of the 42 flocks tested. Characteristic lesions of myeloid leukosis caused by ALV-J were found in affected flocks. The gross pathological lesions were characterised by skeletal myelocytomas located on the inner sternum and ribs, neoplastic enlargement of the liver, and in some cases gross tumour involvement of the spleen, kidney, trachea, skeletal muscles, bone marrow, skin and gonads. Microscopically, the tumours consisted of immature granulated myelocytes, and were present as focal or diffuse infiltrations in the affected organs. Virus micro-neutralisation assays demonstrated antigenic variation among Australian isolates and to overseas strains of ALV-J. ALV-J infection was prevalent in Australian broiler breeder flocks during 2001 to 2003. Australian isolates of ALV-J show a degree of antigenic variation when compared to overseas isolates.

Occurrence of avian leukosis virus subgroup J in commercial layer flocks in China

Mortality from myeloid leukosis was observed in commercial layers from 12 farms in northern China. Affected chickens were extremely thin and dehydrated, bleeding occurred in feather follicles and claws, combs were pale and anaemic, phalanges were swollen, and many yellowish–white tumours were seen on the visceral surface of the sternum. Focal tumour cells, with spherical eosinophilic granules in the cytoplasm, were found in the liver, spleen, kidney, ovary, oviduct, lung, bone marrow, proventriculus and gut by histopathological examination. Immunohistochemical studies with a monoclonal antibody to gp85 of avian leukosis virus subgroup J (ALV-J) revealed antigen in all organs examined. Polymerase chain reaction tests using a pair of ALV-J-specific primers H5/H7 (Smith et al., 1998) produced a 545 basepair fragment. The sequence of the Polymerase chain reaction product was compared with that of the ALV-J HPRS-103 prototype strain. The identity of nucleotides and predicted amino acids was 97.4% and 96.1%, respectively. On this basis the disease in the egg-type chickens was diagnosed as an ALV-J infection. This is the first report of field cases of myeloid leukosis caused by ALV-J in commercial egg-type chickens.

The effects of avian leukosis virus subgroup J on broiler chicken performance and response to vaccination.

The effects of avian leukosis virus subgroup J (ALV-J) infection on meat-type chickens reared in a simulated commercial setting were evaluated. Each of three ALV-J isolates was evaluated with both simulated horizontal transmission (SHT) and simulated vertical transmission (SVT). Mortality, morbidity, disease condemnations, and feed conversions were increased and body weights at processing were decreased in ALV-J infected birds as compared to sham inoculated hatch mates. The adverse effects of ALV-J infection were more severe in birds exposed by SVT than in birds exposed by SHT. At 8 weeks of age response to vaccination for infectious bronchitis virus and Newcastle disease virus or prior exposure to a pathogenic reovirus was assessed in the ALV-J and sham inoculated broiler chickens by challenge studies. Although not statistically significant, an overall trend of decreased protection to challenge after vaccination, or prior exposure, was observed in the ALV-J inoculates as compared to sham inoculated hatch mates. Differences in vaccine response were most evident in groups inoculated with ALV-J by the SVT route.

Polymerase chain reaction for detection of avian leukosis virus subgroup J in feather pulp.

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.

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.

Development and Characterization of Monoclonal Antibodies to Subgroup J Avian Leukosis Virus

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.

Immunohistochemical Localization of Avian Leukosis Virus Subgroup J in Tissues from Naturally Infected Chickens

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.