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Poultry Science Apr 2022Subgroup 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...
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 copies/μL, and the repeatability of the CV of the C 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.
Topics: Animals; Avian Leukosis; Avian Leukosis Virus; Chickens; DNA Primers; Influenza A Virus, H9N2 Subtype; Poultry Diseases; Recombinases; Sensitivity and Specificity
PubMed: 35240352
DOI: 10.1016/j.psj.2022.101743 -
Viruses Feb 2022A modified SELEX (Systematic Evolution of Ligands by Exponential Enrichment) pr,otocol (referred to as PT SELEX) was used to select primer-template (P/T) sequences that...
A modified SELEX (Systematic Evolution of Ligands by Exponential Enrichment) pr,otocol (referred to as PT SELEX) was used to select primer-template (P/T) sequences that bound to the vaccinia virus polymerase catalytic subunit (E9) with enhanced affinity. A single selected P/T sequence (referred to as E9-R5-12) bound in physiological salt conditions with an apparent equilibrium dissociation constant (K) of 93 ± 7 nM. The dissociation rate constant () and binding half-life (t) for E9-R5-12 were 0.083 ± 0.019 min and 8.6 ± 2.0 min, respectively. The values indicated a several-fold greater binding ability compared to controls, which bound too weakly to be accurately measured under the conditions employed. Loop-back DNA constructs with 3'-recessed termini derived from E9-R5-12 also showed enhanced binding when the hybrid region was 21 nucleotides or more. Although the sequence of E9-R5-12 matched perfectly over a 12-base-pair segment in the coding region of the virus B20 protein, there was no clear indication that this sequence plays any role in vaccinia virus biology, or a clear reason why it promotes stronger binding to E9. In addition to E9, five other polymerases (HIV-1, Moloney murine leukemia virus, and avian myeloblastosis virus reverse transcriptases (RTs), and and Klenow DNA polymerases) have demonstrated strong sequence binding preferences for P/Ts and, in those cases, there was biological or potential evolutionary relevance. For the HIV-1 RT, sequence preferences were used to aid crystallization and study viral inhibitors. The results suggest that several other DNA polymerases may have P/T sequence preferences that could potentially be exploited in various protocols.
Topics: Avian Myeloblastosis Virus; Base Sequence; DNA, Viral; DNA-Directed DNA Polymerase; HIV Reverse Transcriptase; Moloney murine leukemia virus; Protein Binding; SELEX Aptamer Technique; Vaccinia virus; Viral Proteins; Virus Replication
PubMed: 35215961
DOI: 10.3390/v14020369 -
Molecular Therapy. Nucleic Acids Mar 2022DNA-modifying technologies, such as the CRISPR-Cas9 system, are promising tools in the field of gene and cell therapies. However, high and prolonged expression of...
DNA-modifying technologies, such as the CRISPR-Cas9 system, are promising tools in the field of gene and cell therapies. However, high and prolonged expression of DNA-modifying enzymes may cause cytotoxic and genotoxic side effects and is therefore unwanted in therapeutic approaches. Consequently, development of new and potent short-term delivery methods is of utmost importance. Recently, we developed non-integrating gammaretrovirus- and MS2 bacteriophage-based Gag.MS2 (g.Gag.MS2) particles for transient transfer of non-retroviral CRISPR-Cas9 RNA into target cells. In the present study, we further improved the technique by transferring the system to the alpharetroviral vector platform (a.Gag.MS2), which significantly increased CRISPR-Cas9 delivery into target cells and allowed efficient targeted knockout of endogenous genes in primary murine fibroblasts as well as primary human fibroblasts, hepatocytes, and cord-blood-derived CD34 stem and progenitor cells. Strikingly, co-packaging of mRNA and multiple single guide RNAs (sgRNAs) into a.Gag.MS2 chimera displayed efficient targeted knockout of up to three genes. Co-transfection of single-stranded DNA donor oligonucleotides during CRISPR-Cas9 particle production generated all-in-one particles, which mediated up to 12.5% of homology-directed repair in primary cell cultures. In summary, optimized a.Gag.MS2 particles represent a versatile tool for short-term delivery of DNA-modifying enzymes into a variety of target cells, including primary murine and human cells.
PubMed: 35141043
DOI: 10.1016/j.omtn.2021.12.033 -
Journal of Virology Mar 2022Avian leukosis virus subgroup J (ALV-J) induces myelocytomas, which can metastasize to multiple organs in diseased chickens. Although metastasis is the primary cause of...
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 G/G 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 G 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. 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.
Topics: Animals; Avian Leukosis; Avian Leukosis Virus; Cell Proliferation; Chickens; Doublecortin-Like Kinases; Epithelial-Mesenchymal Transition; Membrane Proteins
PubMed: 35080427
DOI: 10.1128/JVI.01657-21 -
MBio Feb 2022Accumulating evidence suggests that p53 is involved in viral infection. However, it remains elusive whether avian p53 orchestrates avian leukosis virus (ALV)...
Accumulating evidence suggests that p53 is involved in viral infection. However, it remains elusive whether avian p53 orchestrates avian leukosis virus (ALV) replication. We showed that p53 recruits the histone deacetylase 1 and 2 (HDAC1/2) complex to the ALV promoter to shut off ALV's promoter activity and viral replication. HDAC1/2 binding to the ALV promoter was abolished in the absence of p53. Moreover, we collected samples in ALV-infected chickens and found that the acetylation status of ALV-bound H3 and H4 histones correlated with ALV viremia. HDAC inhibitors (HDACi) potently increase ALV replication, but HDACi-promoted viral replication is dramatically reduced in cells with p53 depletion. These data demonstrate that p53 is critical for inhibition ALV replication and suggest that future studies of ALV replication need to account for the potential effects of p53 activity. Rous sarcoma virus (RSV)/ALV was the first retrovirus to be discovered, which was really the first hint that cancer, or a tumor, could be transmitted by a virus. The specific mechanisms that regulate ALV replication during infection remain poorly understood. Here, we show that avian p53 and HDAC complex inhibit ALV promoter activity and replication, and p53 inhibits ALV replication through binding to the ALV promoter. We demonstrated that the acetylation status of ALV-bound H3 and H4 histones correlates with ALV viremia level using clinical samples collected from commercial poultry. These findings identify both p53-mediated inhibition on ALV replication and a potential role for virus-induced tumorigenesis.
Topics: Animals; Chickens; Avian Leukosis Virus; Tumor Suppressor Protein p53; Antiviral Agents; Viremia; Histones; Neoplasms; Carcinogenesis; Poultry Diseases
PubMed: 35038897
DOI: 10.1128/mbio.03287-21 -
BMC Veterinary Research Jan 2022Subgroup J avian leukosis virus (ALV-J) is an oncovirus which can induce multiple types of tumors in chicken. In this report, we found novel ALV-J infection is closely...
BACKGROUND
Subgroup J avian leukosis virus (ALV-J) is an oncovirus which can induce multiple types of tumors in chicken. In this report, we found novel ALV-J infection is closely associated with serious hepatomegaly and splenomegaly in chicken.
CASE PRESENTATION
The layer chickens from six flocks in Jiangsu province, China, showed serious hemoperitoneum, hepatomegaly and splenomegaly. Histopathological results indicated focal lymphocytic infiltration, cell edema and congestion in the liver, atrophy and depletion of lymphocyte in the spleen. Tumor cells were not detected in all the organs. avian hepatitis E virus (aHEV), which is thought to be the cause of a very similar disease, big liver and spleen disease (BLS), was not detected. Other viruses causing tumors or liver damage including Marek's disease virus (MDV), reticuloendotheliosis virus (REV), fowl adenovirus (FAdV) and chicken infectious anemia virus (CIAV) were also proved negative by either PCR or RT-PCR. However, we did detect ALV-J in those chickens using PCR. Only novel ALV-J strains were efficiently isolated from these chicken livers.
CONCLUSIONS
This is the first report that chicken hepatomegaly and splenomegaly disease was closely associated with novel ALV-J, highlighting the importance of ALV-J eradication program in China.
Topics: Animals; Avian Leukosis; Avian Leukosis Virus; Chickens; China; Hepatomegaly; Neoplasms; Poultry Diseases; Splenomegaly
PubMed: 35027055
DOI: 10.1186/s12917-022-03139-1 -
Veterinary Research Jan 2022To understand the differences in immune responses between early feathering (EF) and late feathering (LF) chickens after infection with avian leukosis virus, subgroup J...
To understand the differences in immune responses between early feathering (EF) and late feathering (LF) chickens after infection with avian leukosis virus, subgroup J (ALV-J), we monitored the levels of prolactin, growth hormone and the immunoglobulins IgG and IgM in the serum of LF and EF chickens for 8 weeks. Moreover, we analysed the expression of immune-related genes in the spleen and the expression of PRLR, SPEF2 and dPRLR in the immune organs and DF-1 cells by qRT-PCR. The results showed that ALV-J infection affected the expression of prolactin, growth hormone, IgG and IgM in the serum. Regardless of whether LF and EF chickens were infected with ALV-J, the serum levels of the two hormones and two immunoglobulins in EF chickens were higher than those in LF chickens (P < 0.05). However, the expression of immune-related genes in the spleen of positive LF chickens was higher than that in the spleen of positive EF chickens. In the four immune organs, PRLR and SPEF2 expression was also higher in LF chickens than in EF chickens. Furthermore, the dPRLR expression of positive LF chickens was higher than that of negative LF chickens. After infection with ALV-J, the expression of PRLR in DF-1 cells significantly increased. In addition, overexpression of PRLR or dPRLR in DF-1 cells promoted replication of ALV-J. These results suggested that the susceptibility of LF chickens to ALV-J might be induced by dPRLR.
Topics: Animals; Avian Leukosis; Avian Leukosis Virus; Chickens; Growth Hormone; Immunity; Immunoglobulin G; Immunoglobulin M; Poultry Diseases; Prolactin; Receptors, Prolactin
PubMed: 34998433
DOI: 10.1186/s13567-021-01016-7 -
Viruses Dec 2021The chicken Tva cell surface protein, a member of the low-density lipoprotein receptor family, has been identified as an entry receptor for avian leukosis virus of...
Knock-Out of Retrovirus Receptor Gene in the Chicken Confers Resistance to Avian Leukosis Virus Subgroups A and K and Affects Cobalamin (Vitamin B)-Dependent Level of Methylmalonic Acid.
The chicken Tva cell surface protein, a member of the low-density lipoprotein receptor family, has been identified as an entry receptor for avian leukosis virus of classic subgroup A and newly emerging subgroup K. Because both viruses represent an important concern for the poultry industry, we introduced a frame-shifting deletion into the chicken locus with the aim of knocking-out Tva expression and creating a virus-resistant chicken line. The knock-out was prepared by CRISPR/Cas9 gene editing in chicken primordial germ cells and orthotopic transplantation of edited cells into the testes of sterilized recipient roosters. The resulting -/- chickens tested fully resistant to avian leukosis virus subgroups A and K, both in in vitro and in vivo assays, in contrast to their susceptible +/+ and +/- siblings. We also found a specific disorder of the cobalamin/vitamin B metabolism in the tva knock-out chickens, which is in accordance with the recently recognized physiological function of Tva as a receptor for cobalamin in complex with transcobalamin transporter. Last but not least, we bring a new example of the de novo resistance created by CRISPR/Cas9 editing of pathogen dependence genes in farm animals and, furthermore, a new example of gene editing in chicken.
Topics: Animals; Avian Leukosis Virus; Avian Proteins; Chick Embryo; Chickens; Female; Frameshift Mutation; Gene Editing; Gene Knockout Techniques; Male; Methylmalonic Acid; Receptors, Virus; Vitamin B 12
PubMed: 34960774
DOI: 10.3390/v13122504 -
Journal of Virology Feb 2022Glycans on envelope glycoprotein (Env) of the subgroup J avian leukosis virus (ALV-J) play an essential role in the virion integrity and infection process. In this...
Glycans on envelope glycoprotein (Env) of the subgroup J avian leukosis virus (ALV-J) play an essential role in the virion integrity and infection process. In this study, we found that, among the 13 predicted N-linked glycosylation sites (NGSs) in of Tibetan chicken strain TBC-J6, N17, and N193/N191 are pivotal for virus replication. Further research illustrated that a mutation at N193 weakened Env-receptor binding in a blocking assay of the viral entrance, coimmunoprecipitation, and ELISA. Our studies also showed that N17 was involved in Env protein processing and later virion incorporation based on the detection of p27 and Env protein in the supernatant and in the cell culture. This report is systematic research on clarifying the biological function of NGSs on ALV-J , which would provide valuable insight into the role of in the ALV life cycle and anti-ALV-J strategies. ALV-J is a retrovirus that can cause multiple types of tumors in chickens. Among all the viral proteins, the heavily glycosylated envelope protein is especially crucial. Glycosylation plays a major role in Env protein function, including protein processing, receptor attachment, and immune evasion. Notably, viruses isolated recently seem to lose their 6 and 11 NGS, which proved to be important in receptor binding. In our study, the 1 (N17) and 8 (N193) NGS of of the strain TBC-J6 can largely influence the titer of this virus. Deglycosylation at N193 weakened Env-receptor binding while mutation at N17 influenced Env protein processing. This study systemically analyzed the function of NGSs in ALV-J in different aspects, which may help us to understand the life cycle of ALV-J and provide antiviral targets for the control of ALV-J.
Topics: Animals; Avian Leukosis Virus; Cell Line; Chickens; Glycosylation; Mutation; Protein Binding; Protein Processing, Post-Translational; Receptors, Virus; Viral Envelope Proteins; Viral Load; Virion
PubMed: 34878920
DOI: 10.1128/JVI.01549-21 -
Frontiers in Cellular and Infection... 2021It is well known that chicken CD8 T cell response is vital to clearing viral infections. However, the differences between T cell subsets expressing CD8 receptors in...
It is well known that chicken CD8 T cell response is vital to clearing viral infections. However, the differences between T cell subsets expressing CD8 receptors in chicken peripheral blood mononuclear cells (PBMCs) have not been compared. Herein, we used Smart-Seq2 scRNA-seq technology to characterize the difference of chicken CD8, CD8 αα, CD8 αβ, CD8, and CD4CD8 T cell subsets from PBMCs of avian leukosis virus subgroup J (ALV-J)-infected chickens. Weighted gene co-expression network analysis (WGCNA) and Trend analysis revealed that genes enriched in the "Cytokine-cytokine receptor interaction" pathway were most highly expressed in the CD8 αα T cell population, especially T cell activation or response-related genes including CD40LG, IL2RA, IL2RB, IL17A, IL1R1, TNFRSF25, and TNFRSF11, suggesting that CD8 αα T cells rather than other CD8 subpopulations were more responsive to ALV-J infections. On the other hand, genes involved in the "FoxO signaling pathway" and "TGF-beta signaling pathway" were most highly expressed in the CD4CD8 (CD8) T cell population and the function of CD4CD8 T cells may play roles in negatively regulating the functions of T cells based on the high expression of CCND1, ROCK1, FOXO1, FOXO3, TNFRSF18, and TNFRSF21. The selected gene expressions in CD8 T cells and CD4CD8 double-positive T cells confirmed by qRT-PCR matched the Smart-Seq2 data, indicating the reliability of the smart-seq results. The high expressions of Granzyme K, Granzyme A, and CCL5 indicated the positive response of CD8 T cells. Conversely, CD4CD8 T cells may have the suppressor activity based on the low expression of activation molecules but high expression of T cell activity suppressor genes. These findings verified the heterogeneity and transcriptional differences of T cells expressing CD8 receptors in chicken PBMCs.
Topics: Animals; Avian Leukosis; Avian Leukosis Virus; CD8-Positive T-Lymphocytes; Chickens; Leukocytes, Mononuclear; Poultry Diseases; Reproducibility of Results
PubMed: 34858872
DOI: 10.3389/fcimb.2021.747094