Interaction between Avian Leukosis Virus Subgroup J Surface Protein and Doublecortin-Like Kinase 1 Accelerates Cell Proliferation and Epithelial-Mesenchymal Transition.

Avian leukosis virus subgroup J (ALV-J) induces myelocytomas, which can metastasize to multiple organs in diseased chickens. Although metastasis is the primary cause of death in such cases, the mechanism for it remains unclear. Here, we found that interaction between ALV-J surface protein (SU) and doublecortin-like kinase 1 (DCLK1) promotes epithelial-mesenchymal transition (EMT) and cell proliferation. We found that ALV-J can activate EMT in infected cells. Subsequently, proteomics analysis revealed that DCLK1, a well-established putative tumor stem cell marker, which is highly expressed in ALV-J-infected DF-1 cells and chickens, might be a potential factor mediating EMT. Furthermore, using immunofluorescence and immunoprecipitation, we verified that SU interacts with DCLK1. Functional studies suggested that overexpression of DCLK1 increased viral replication and promoted cell proliferation by accelerating the progression of cells from the G0/G1 phase to the S phase of cell cycle, whereas RNA interference of DCLK1 reduced viral replication and arrested cell proliferation by retarding cell cycle progression from the late G1 phase into the S phase in ALV-J-infected cells. Moreover, we demonstrate that the increased accumulation of DCLK1 promotes EMT by increasing the expression of N-cadherin, vimentin, MMP2, and transcription factor Snail1 and decreasing the expression of epithelial marker E-cadherin. These results suggest that ALV-J SU interacts with DCLK1, and accelerates cell proliferation, leading to increased viral replication and ultimately activating EMT, which paves the way for tumor metastasis. IMPORTANCE Tumor metastasis is a major challenge in cancer research, because of its systemic nature and the resistance of disseminated tumor cells to existing therapeutic agents. It is estimated that >90% of mortality from cancer is attributable to metastases. We found that ALV-J can activate EMT, which plays a critical role in cancer metastasis. Subsequently, we identified a tumor stem cell marker, DCLK1, in ALV-J infected cells, which interacts with surface protein (SU) of ALV-J to promote virus replication, activate EMT, and accelerate cell proliferation enabling ALV-J to obtain metastatic ability. Understanding the process of participation of ALV-J in EMT and the route of metastasis will help elucidate the mechanism of virus-induced tumor metastasis and help identify promising molecular targets and key obstacles for ALV-J control and clinical technology development.

Avian leukosis virus subgroup J and reticuloendotheliosis virus coinfection induced TRIM62 regulation of the actin cytoskeleton.

BACKGROUND: Coinfection with avian leukosis virus subgroup J (ALV-J) and reticuloendotheliosis virus (REV) is common in chickens, and the molecular mechanism of the synergistic pathogenic effects of the coinfection is not clear. Exosomes have been identified as new players in the pathogenesis of retroviruses. The different functions of exosomes depend on their cargo components. OBJECTIVES: The aim of this study was to investigate the function of co-regulation differentially expressed proteins in exosomes on coinfection of ALV-J and REV. METHODS: Here, viral replication in CEF cells infected with ALV-J, REV or both was detected by immunofluorescence microscopy. Then, we analyzed the exosomes isolated from supernatants of chicken embryo fibroblast (CEF) cells single infected and coinfected with ALV-J and REV by mass spectrometry. KEGG pathway enrichment analyzed the co-regulation differentially expressed proteins in exosomes. Next, we silenced and overexpressed tripartite motif containing 62 (TRIM62) to evaluate the effects of TRIM62 on viral replication and the expression levels of NCK-association proteins 1 (NCKAP1) and actin-related 2/3 complex subunit 5 (ARPC5) determined by quantitative reverse transcription polymerase chain reaction. RESULTS: The results showed that coinfection of ALV-J and REV promoted the replication of each other. Thirty proteins, including TRIM62, NCK-association proteins 1 (NCKAP1, also known as Nap125), and Arp2/3-5, ARPC5, were identified. NCKAP1 and ARPC5 were involved in the actin cytoskeleton pathway. TRIM62 negatively regulated viral replication and that the inhibition of REV was more significant than that on ALV-J in CEF cells coinfected with TRIM62. In addition, TRIM62 decreased the expression of NCKAP1 and increased the expression of ARPC5 in coinfected CEF cells. CONCLUSIONS: Collectively, our results indicated that coinfection with ALV-J and REV competitively promoted each other's replication, the actin cytoskeleton played an important role in the coinfection mechanism, and TRIM62 regulated the actin cytoskeleton.

Fertility and sperm quality of broiler breeder males infected with subgroup J avian leukosis virus.

In order to assess the effects of subgroup J avian leukosis virus (ALV-J) on semen quality, broiler breeder males were separated by ALV-J status (ALV-J positive = POS, ALV-J negative = NEG) at 44 wk of age. Of the 249 males originally placed at 1 day of age, 101 (40.6%) died by 43 wk of age. Observations of tumor expression and high mortality suggest that many of the males that died prior to 44 wk of age were infected with ALV-J. From 47 to 56 wk of age, hens were inseminated every third week with 7.5 x 10(7) sperm. Fertility and hatch data were collected by incubating eggs laid during the 2 wk postinsemination (WPI). The number of sperm that penetrated the perivitelline membrane of the ovum was determined from eggs laid on the eighth day postinsemination. Sperm mobility index (SMI) was determined at 58 and 60 wk of age from all males producing semen. Whereas SMI and sperm hole penetration measurements indicated that the sperm quality from treatments POS and NEG were similar, fertility was significantly greater in the POS treatment during the first (89.0% vs. 79.0%) and second WPI (59.3% vs. 45.0%). However, because of numerically higher hatch of fertile from the NEG group, the percentage of hatch of eggs set was similar between groups. These data suggest that ALV-J status of caged males has no influence on sperm quality or hatchability of eggs.

Body weight suppression in broilers naturally infected with avian leukosis virus subgroup J.

Broiler progeny were hatched from avian leukosis virus (ALV)-negative and ALV subgroup J (ALV-J)-positive breeders. ALV-J infection in progeny was identified by p27 antigen capture enzyme-linked immunosorbent assay and reverse transcriptase-polymerase chain reaction specific for ALV-J performed on serum or plasma samples obtained at hatch. Hatch weights did not differ between ALV-negative broilers and ALV-J-positive broilers. Body weights of ALV-J-positive broilers were 64.4% of those of ALV-negative broilers at 1 wk of age. At 8 wk of age, weights of broilers with congenital ALV-J infection were still only 63.8% of those of ALV-negative broilers. No other concurrent pathogen was detected in either group of broilers. These findings indicate that congenital ALV-J infection is associated with significant weight suppression in broilers in the absence of other pathogens.

Rapid induction of lymphoid leukosis and ascites by avian leukosis virus from a lymphoid leukosis cell line.

To examine whether a lymphoid leukosis (LL) cell line releases an LL-specific avian leukosis virus (ALV) or not, two viral materials, culture fluid and a concentrated viral material from an LL-cell line, were inoculated into a total of 74 day-old chicks of line 15I in 5 experiments. Spectrum of diseases induced, their incidence and incubation periods to onset were examined. Fifteen chicks were inoculated with the culture fluid and 9 (60%) developed ascites [59-119 days post inoculation (dpi); geometric mean (GM) of dpi, GM: 89.6)], but LL was not induced in any chicks inoculated. Fifty-nine chicks were inoculated with the concentrated viral material and LL was recognized in 13 (22.0%) (27-74 dpi; GM: 48.4), ascites with LL in 11 (18.6%) (34-75 dpi; GM: 41.3), ascites alone in 21 (35.6%) (32-83 dpi; GM: 48.2), erythroblastosis in 2 (3.4%) (70-102 dpi; GM: 84.5), and other diseases in 12 (20.3%) (43-102 dpi; GM: 61.8). LL lesions were frequently observed in the liver, spleen, kidneys, bursa of Fabricius (bursa), bone marrow and gonads. Mild lymphocytic foci in some visceral organs and perivascular cuffing in the central nervous system were observed mainly in several chicks diagnosed as having complication of ascites with LL or other diseases. In addition to these lesions, atrophy of bursa and thymuses was recognized in them. No antibodies against Marek's disease virus (MDV) and reticuloendotheliosis virus were detected in 36 sera taken from the chicks inoculated with the concentrated viral material. Serotype 2 MDV was isolated from the buffy coat of some inoculated chicks. These results suggest that the properties of ALV inoculated and immunosuppression caused by inoculation with high doses of ALV are involved in rapid induction of LL and expression of pathogenicity of serotype 2 MDV released from the LL cell line and included in the viral inoculum. This is the first report describing the rapid induction of LL and ascites in chicks.

Ducks: a new experimental host system for studying persistent infection with avian leukaemia retroviruses.

Long-term persistence of the avian leukosis virus (ALV), the transformation-defective mutant of Prague strain Rous sarcoma virus subgroup C (td PR-C) was established in heterologous duck hosts after infection in mid-embryogenesis. Transient viraemia was observed for about 4 weeks after hatching and was lost in most of the infected ducks by about 6 months. Loss of viraemia was accompanied by the increasing synthesis of virus-neutralizing antibodies. In spite of strong virus-neutralizing antibodies, virus was detected by the cocultivation assay in duck tissues throughout the observation period up to 5 years. In the viraemic phase of infection, we found integrated proviruses in various tissues, preferentially in stomach muscle tissue and in the thymus. The long-term persistence of virus was frequently accompanied by liver necrosis and neoplastic diseases. Injection of td PR-C virus into early embryos resulted in more pronounced infection accompanied by an increased copy number of viral DNA per cell, high mortality and remarkable atrophy of thymus tissue in infected ducklings.

Semen traits and fertility of White Leghorn males shown to be positive or negative for lymphoid leukosis virus in semen and feather pulp.

1. Males from strains selected for high egg production (and other economic traits) and from unselected control strains were used to determine the frequency of shedding of lymphoid leukosis virus (LLV) into semen. The effect of the male's LLV status on semen production, fertility and hatchability was also examined in males of the unselected control strains. 2. The frequency of detection of exogenous LLV in semen by the phenotypic mixing test, and high concentrations of the viral group specific antigen in feather pulp by the complement fixation test, were both higher in control strains than in strains selected for high egg production. 3. Semen production was not reduced in LLV-shedding males. 4. Significant associations of LLV shedding with higher incidence of abnormal spermatozoa and reduced fertility were found in some populations but not in others. No significant effect of LLV shedding on hatchability was detected. 5. Tests for group specific antigen in feather pulp proved useful in identifying males that shed LLV in semen.

Functional and structural alterations of the nervous system induced by avian retrovirus RAV-7.

Chickens infected as embryos with RAV-7 developed neurological signs including ataxia, lethargy, and imbalance. Evoked spinal cord potentials for RAV-7 infected SC chickens were considerably slower (64.8 m/s) than for uninfected SC (103.4 m/s), genetically hypothyroid (OS) (93.9 m/s) or special C (95.1 m/s) chickens. Conduction velocity measurements of sciatic nerves showed normal values for all the chickens examined in this study. Histopathological studies revealed non-suppurative meningoencephalomyelitis in RAV-7 infected SC chickens. The inflammatory infiltrate consisted of lymphocytes, macrophages, and occasional plasma cells. The cells in the infiltrate reacted with mouse monoclonal antibodies directed against la and T-cell antigens. Astrocytic hypertrophy and hyperplasia, demonstrated by the use of monoclonal antibody specific for glial fibrillary acidic protein (GFAP), was associated with the CNS lesions. The results of this investigation indicate that RAV-7 causes significant central nervous system lesions and functional impairment in the infected chicken. This system may serve as a useful model for studying retrovirus-induced neurological dysfunction.

The effects of geographical area, rearing method, caging density, and lymphoid leukosis infection on adult performance in egg stocks of chickens.

Birds were brooded reared, and laying house-tested at two widely separated sites: Animal Research Centre (ARC) and North Central Region Poultry Breeding Laboratory (NCRPBL). Two strain and a commercial stock were included only at NCRPBL. At ARC, all birds were brooded, reared, and laying house-tested in cages. At NCRPBL, half of the chicks were brooded and reared on floor and half in cages, and all hens were laying tested at one or two birds per cage. Most hens housed one per cage were tested for lymphoid leukosis virus (LLV) infection by the complement fixation test for group specific antigen in egg albumen. When all hens tested at NCRPBL were considered, the method of brooding and rearing affected only egg weight and body weight in favor of the hens reared in floor pens. The commercial stock and the strain crosses performed better than the control strains. Despite very similar genetic backgrounds, there were large differences between control strains 10 and NCR. Hens housed one per cage had lower mortality and higher egg production than those housed two per cage. Average performance of birds at ARC was somewhat better than that at NCRPBL, but there were genotype-site interactions for mortality, egg production, and sexual maturity. The frequency of shedders in the crosses (17.2%) was the same as that of the control strains (20.8%), but the frequency of shedders in the commercial stock (4.5%) was much lower. The method of brooding and rearing had no effect on the frequency of shedders. The LLV infection was associated with significant reductions in egg production and egg quality. For example, the difference between test-positive and test-negative hens was 25 eggs per hen housed and 16 eggs per surviving hen.

Association of lymphoid leukosis virus and performance in a randombred layer population.

The association of subclinical infections with lymphoid leukosis virus and performance was investigated in a randombred White Leghorn type population. The proportion of birds shedding group specific (gs) antigen into their egg albumen was high (23.8%). Birds with gs antigen in their albumen matured later, produced fewer and smaller eggs, and grew less rapidly tha their nonshedding counterparts.

Tumor latency in avian lymphoid leukosis.

Transfer of bursa cells from older chicken infected with lymphoid leukosis (LL) virus into young recipient chicks depleted of bursal lymphocytes by cyclophosphamide shortened the latency period for lymphoma development in recipient chickens by an amount equivalent to the age of the donor. Chickens were crosses between males of the Regional Poultry Research Laboratory, East Lansing, Michigan, inbred line 151, subline 5, and females of inbred line 7, subline 2. In a further experiment with intact infected chicks, artificial metastasis at various ages by surgical bursectomy and inoculation of bursa cells into the bloodstream of the host did not shorten the latency period. These results suggest that latency in avian LL is a property of target B-cells and is unrelated to maturational events of the host physiology.