Specific and Sensitive Visual Proviral DNA Detection of Major Pathogenic Avian Leukosis Virus Subgroups Using CRISPR-Associated Nuclease Cas13a.

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.

BiTiS3 bio-transducer with explosive on-demand generation of NO gas for synergetic cancer therapy.

Combined photothermal therapy and nitric oxide (NO)-mediated gas therapy has shown great potential as a cancer treatment. However, the on-demand release of NO at a high concentration presents a challenge owing to the lack of an ideal bio-transducer with a high loading capacity of NO donors and sufficient energy to induce NO release. Here, we present a new 2D BiTiS3 nanosheet that is synthesized, loaded with the NO donor (BNN6), and conjugated with PEG-iRGD to produce a multifunctional bio-transducer (BNN6-BiTiS3-iRGD) for the on-demand production of NO. The BiTiS3 nanosheets not only have a high loading capacity of NO donors (750%), but also exhibit a high photothermal conversion efficiency (59.5%) after irradiation by a 1064-nm laser at 0.5 W/cm2. As a result of the above advantages, the temporal-controllable generation of NO within a large dynamic range (from 0 to 344 µM) is achieved by adjusting power densities, which is among the highest efficiency values reported for NO generators so far. Moreover, the targeted accumulation of BNN6-BiTiS3-iRGD at tumor sites leads to spatial-controllable NO release. In vitro and in vivo assessments demonstrate synergistic NO gas therapy with mild photothermal therapy based on BNN6-BiTiS3-iRGD. Our work provides insights into the design and application of other 2D nanomaterial-based therapeutic platforms.

miR-155 facilitates the synergistic replication between avian leukosis virus subgroup J and reticuloendotheliosis virus by targeting a dual pathway.

IMPORTANCE: The synergy of two oncogenic retroviruses is an essential phenomenon in nature. The synergistic replication of ALV-J and REV in poultry flocks increases immunosuppression and pathogenicity, extends the tumor spectrum, and accelerates viral evolution, causing substantial economic losses to the poultry industry. However, the mechanism of synergistic replication between ALV-J and REV is still incompletely elusive. We observed that microRNA-155 targets a dual pathway, PRKCI-MAPK8 and TIMP3-MMP2, interacting with the U3 region of ALV-J and REV, enabling synergistic replication. This work gives us new targets to modulate ALV-J and REV's synergistic replication, guiding future research on the mechanism.

Biodegradable FePS3 nanoplatform for efficient treatment of osteosarcoma by combination of gene and NIR-II photothermal therapy.

As a common tumor with high incidence, osteosarcoma possesses extremely poor prognosis and high mortality. Improving the survival of osteosarcoma patients is still a great challenge due to the precipice of advancement in treatment. In this study, a combination strategy of gene therapy and photothermal therapy (PTT) is developed for efficient treatment of osteosarcoma. Two-dimensional (2D) FePS3 nanosheets are synthesized and functionalized by poly-L-lysine-PEG-folic acid (PPF) to fabricate a multifunctional nanoplatform (FePS@PPF) for further loading microRNAs inhibitor, miR-19a inhibitor (anti-miR-19a). The photothermal conversion efficiency of FePS@PPF is up to 47.1% under irradiation by 1064 nm laser. In vitro study shows that anti-miR-19a can be efficiently internalized into osteosarcoma cells through the protection and delivery of FePS@PPF nanaocarrier, which induces up-regulation of PTEN protein and down-regulation p-AKT protein. After intravenous injection, the FePS@PPF nanoplatform specifically accumulates to tumor site of osteosarcoma-bearing mice. The in vitro and in vivo investigations reveal that the combined PTT-gene therapy displays most significant tumor ablation compared with monotherapy. More importantly, the good biodegradability promotes FePS@PPF to be cleared from body avoiding potential toxicity of long-term retention. Our work not only develops a combined strategy of NIR-II PTT and gene therapy mediated by anti-miR-19a/FePS@PPF but also provides insights into the design and applications of other nanotherapeutic platforms.

Molecular characteristics and pathogenicity of a Tibet-origin mutant avian leukosis virus subgroup J isolated from Tibetan chickens in China.

Tibetan chicken is found in China Tibet (average altitude; ˃4500 m). However, little is known about avian leukosis virus subgroup J (ALV-J) found in Tibetan chickens. ALV-J is a typical alpharetrovirus that causes immunosuppression and myelocytomatosis and thus seriously affects the development of the poultry industry. In this study, Tibet-origin mutant ALV-J was isolated from Tibetan chickens and named RKZ-1-RKZ-5. A Myelocytomatosis outbreak occurred in a commercial Tibetan chicken farm in Shigatse of Rikaze, Tibet, China, in March 2022. About 20% of Tibetan chickens in the farm showed severe immunosuppression, and mortality increased to 5.6%. Histopathological examination showed typical myelocytomas in various tissues. Virus isolation and phylogenetic analysis demonstrated that ALV-J caused the disease. Gene-wide phylogenetic analysis showed the RKZ isolates were the original strains of the previously reported Tibetan isolates (TBC-J4 and TBC-J6) (identity; 94.5% to 94.9%). Furthermore, significant nucleotide mutations and deletions occurred in the hr1 and hr2 hypervariable regions of gp85 gene, 3'UTR, Y Box, and TATA Box of 3'LTR. Pathogenicity experiments demonstrated that the viral load, viremia, and viral shedding level were significantly higher in RKZ-1-infected chickens than in NX0101-infected chickens. Notably, RKZ-1 caused more severe cardiopulmonary damage in SPF chickens. These findings prove the origin of Tibet ALV-J and provide insights into the molecular characteristics and pathogenic ability of ALV-J in the plateau area. Therefore, this study may provide a basis for ALV-J prevention and eradication in Tibet.

TRIM25 participates in the fibrous tissue hyperplasia induced by ALV-J infection in chickens by targeting 14-3-3σ protein.

ALV-J-SD1005 strain was subcutaneously inoculated into the necks of 1-day-old HY-Line Brown chickens and caused severe growth retardation, viremia and subcutaneous fibrosarcomas in the necks of all infected chickens from 14 days post inoculation (DPI) to 21 DPI, and also significantly increased the expressions of TRIM25, P53, etc., but significantly decreased the expressions of 14-3-3σ, etc. Overexpression of chicken TRIM25 (chTRIM25) significantly promoted cell proliferation and improved the expressions of P53, CDC2, and CDK2 tumor factors; and significantly inhibited the expression of 14-3-3σ in ALV-J-SD1005-infected DF1 cells; but knockdown of chTRIM25 caused the opposite effects. The results of co-immunoprecipitation (Co-IP) and confocal microscopy confirmed that chTRIM25 can recognize and bind 14-3-3σ protein in ALV-J-SD1005-infected cells, and they were co-located in the cytoplasm. It can be concluded that chTRIM25 participates in the fibrous tissue hyperplasia induced by ALV-J-SD1005 infections in chickens by binding 14-3-3σ protein and regulating the expressions of 14-3-3σ, P53, CDC2, and CDK2.

Serum amyloid A regulates TLR2/4-mediated IFN-ß signaling pathway against Marek's disease virus.

Serum amyloid A (SAA), an acute response phase protein (APP), is crucial for the innate immune response during pathogenic microorganisms' invasion. Marek's disease virus (MDV) is a highly oncogenic alphaherpesvirus that activates multiple innate immune molecules, including SAA, in the host during infection. However, the pathway through which SAA participates in MDV-induced host innate immunity remains unknown. The present study aimed to elucidate the pathway through which SAA exerts its anti-MDV function. We observed that MDV infection in vivo and in vitro significantly elevated SAA expression. Furthermore, through SAA overexpression and knockdown experiments, we demonstrated that SAA could inhibit MDV replication. Subsequently, we found that SAA activated Toll-Like Receptor 2/4 (TLR2/4) -mediated Interferon Beta (IFN-ß) promoter activity and IFN regulatory factor 7 (IRF7) promoter activity. During MDV infection, SAA enhanced TLR2/4-mediated IFN-ß signal transduction and messenger RNAs (mRNAs) expression of type I IFN (IFN-I) and interferon-stimulated genes (ISGs). Finally, TLR2/4 inhibitor OxPAPC inhibits the anti-MDV activity of SAA. These results demonstrated that SAA inhibits MDV replication and enhancing TLR2/4-mediated IFN-ß signal transduction to promote IFNs and ISGs expression. This finding is the first to demonstrate the signaling pathway by which SAA exerts its anti-MDV function. It also provides new insights into the control of oncogenic herpesviruses from the perspective of acute response phase proteins.

Musashi-1 and miR-147 Precursor Interaction Mediates Synergistic Oncogenicity Induced by Co-Infection of Two Avian Retroviruses.

Synergism between avian leukosis virus subgroup J (ALV-J) and reticuloendotheliosis virus (REV) has been reported frequently in co-infected chicken flocks. Although significant progress has been made in understanding the tumorigenesis mechanisms of ALV and REV, how these two simple oncogenic retroviruses induce synergistic oncogenicity remains unclear. In this study, we found that ALV-J and REV synergistically promoted mutual replication, suppressed cellular senescence, and activated epithelial-mesenchymal transition (EMT) in vitro. Mechanistically, structural proteins from ALV-J and REV synergistically activated the expression of Musashi-1(MSI1), which directly targeted pri-miR-147 through its RNA binding site. This inhibited the maturation of miR-147, which relieved the inhibition of NF-κB/KIAA1199/EGFR signaling, thereby suppressing cellular senescence and activating EMT. We revealed a synergistic oncogenicity mechanism induced by ALV-J and REV in vitro. The elucidation of the synergistic oncogenicity of these two simple retroviruses could help in understanding the mechanism of tumorigenesis in ALV-J and REV co-infection and help identify promising molecular targets and key obstacles for the joint control of ALV-J and REV and the development of clinical technologies.

Fabricating Black-Phosphorus/Iron-Tetraphosphide Heterostructure via a Solid-Phase Solution-Precipitation Method for High-Performance Nitrogen Reduction.

Although constructing heterostructures is considered as one of the most successful strategies to improve the activity of a catalyst, the heterostructures usually suffer from the cumbersome preparation treatments and low-yield. Inspired by a solid-phase solution-precipitation (SPSP) process, an approach for interface intensive heterostructures with high yield is developed. Herein, a black-phosphorus/iron-tetraphosphide (BP/FeP4 ) heterostructure is prepared mechanochemically with high transient pressure by the solid-phase ball milling approach. The BP/FeP4 heterostructure delivers excellent catalytic performance in the nitrogen reduction reaction (NRR) as exemplified by an NH3 yield of 77.6 µg h-1 mg cat . - 1 \[{\rm{mg}}_{{\rm{cat}}{\rm{.}}}^{{\bm{ - }}1}\] and Faradic efficiency of 62.9% (-0.2 V), which are superior to that of most NRR catalysts recently reported. Experimental investigation and density-functional theory calculation indicate the importance of excess phosphorus in the heterostructures on the NRR activity, which assists the Fe atom to activate N2 via adsorbing the H atom. The results demonstrate the great potential of this new type of heterostructures prepared by the SPSP approach. Benefiting from the simple preparation process and low cost, the heterostructures offer a new insight into the development of highly efficient catalysts.

Effect of Platycodon grandiflorus Polysaccharide on M1 Polarization Induced by Autophagy Degradation of SOCS1/2 Proteins in 3D4/21 Cells.

M1-polarized macrophages can improve the body's immune function. This study aimed to explore the mechanism of Platycodon grandiflorus polysaccharide (PGPSt) degrading SOCS1/2 protein through autophagy and promoting M1 polarization in 3D4/21 cells. Immunoprecipitation, confocal laser scanning microscopy, flow cytometry, and intracellular co-localization were used to detect the expression of related phenotypic proteins and cytokines in M1-polarized cells. The results showed that PGPSt significantly promoted the mRNA expression of IL-6, IL-12, and TNF-α and enhanced the protein expression of IL-6, IL-12, TNF-α, IL-1ß, iNOS, CD80, and CD86, indicating that PGPSt promoted M1 polarization in 3D4/21 cells. Next, the effect of the PGPSt autophagy degradation of SOCS1/2 on the M1 polarization of 3D4/21 cells was detected. The results showed that PGPSt significantly downregulated the expression level of SOCS1/2 protein, but had no obvious effect on the mRNA expression level of SOCS1/2, indicating that PGPSt degraded SOCS1/2 protein by activating the lysosome system. Further research found that under the action of 3-MA and BafA1, PGPSt upregulated LC3B II and downregulated SOCS1/2 protein expression, which increased the possibility of LC3B, the key component of autophagy, bridging this connection and degrading SOCS1/2. The interaction between SOCS1/2 and LC3 was identified by indirect immunofluorescence and Co-IP. The results showed that the co-localization percentage of the two proteins increased significantly after PGPSt treatment, and LC3 interacted with SOCS1 and SOCS2. This provides a theoretical basis for the application of PGPSt in the treatment or improvement of diseases related to macrophage polarization by regulating the autophagy level.

RIOK3-Mediated Akt phosphorylation facilitates synergistic replication of Marek's disease and reticuloendotheliosis viruses.

Co-infection of Marek's disease virus (MDV) and reticuloendotheliosis virus (REV) synergistically drives disease progression, yet little is known about the mechanism of the synergism. Here, we found that co-infection of REV and MDV increased their replication via the RIOK3-Akt pathway. Initially, we noticed that the viral titres of MDV and REV significantly increased in REV and MDV co-infected cells compared with single-infected cells. Furthermore, tandem mass tag peptide labelling coupled with LC/MS analysis showed that Akt was upregulated in REV and MDV co-infected cells. Overexpression of Akt promoted synergistic replication of MDV and REV. Conversely, inhibition of Akt suppressed synergistic replication of MDV and REV. However, PI3K inhibition did not affect synergistic replication of MDV and REV, suggesting that the PI3K/Akt pathway is not involved in the synergism of MDV and REV. In addition, we revealed that RIOK3 was recruited to regulate Akt in REV and MDV co-infected cells. Moreover, wild-type RIOK3, but not kinase-dead RIOK3, mediated Akt phosphorylation and promoted synergistic replication of MDV and REV. Our results illustrate that MDV and REV activated a novel RIOK3-Akt signalling pathway to facilitate their synergistic replication.

TMT-based proteomic analysis reveals integrins involved in the synergistic infection of reticuloendotheliosis virus and avian leukosis virus subgroup J.

BACKGROUND: Co-infection with the avian leukosis virus subgroup J (ALV-J) and the reticuloendotheliosis virus (REV) increases mutual viral replication, causing a more serious pathogenic effect by accelerating the progression of neoplasia and extending the tumor spectrum. However, the molecular mechanism underlying the synergistic replication of ALV-J and REV remains unclear. RESULTS: Here, we performed this study to compare the differentially expressed proteins among CEF cells infected with ALV-J, REV or both at the optimal synergistic infection time using TMT-based quantitative proteomics. We identified a total of 719 (292 upregulated and 427 downregulated) and 64 (35 upregulated and 29 downregulated) proteins by comparing co-infecting both viruses with monoinfecting ALV-J and REV, respectively. GO annotation and KEGG pathway analysis showed the differentially expressed proteins participated in virus-vector interaction, biological adhesion and immune response pathways in the synergistic actions of ALV-J and REV at the protein levels. Among the differentially expressed proteins, a large number of integrins were inhibited or increased in the co-infection group. Further, eight integrins, including ITGα1, ITGα3, ITGα5, ITGα6, ITGα8, ITGα9, ITGα11 and ITGß3, were validated in CEF cells by qRT-PCR or western blot. CONCLUSIONS: These findings proved that integrins may be key regulators in the mechanism of synergistic infection of REV and ALV-J, which will provide more insight into the pathogenesis of synergism of REV and ALV-J at protein level.

Interaction of p10/p27 with macrophage migration inhibitory factor promotes avian leukosis virus subgroup J infection.

Avian leukosis virus subgroup J (ALV-J), an oncogenic retrovirus, induces myelocytoma and various other tumors in broilers and layers. Many recent studies have shown that ALV-J can hijack host molecules to facilitate infection. However, the molecular mechanisms of this process are not clear. Here, we aimed to elucidate the molecular mechanisms contributing to ALV-J infection. ALV-J hijacked MIF via p10 and p27 to facilitate ALV-J infection. ALV-J persistently activated MIF expression in DF-1 cells, and MIF significantly facilitated ALV-J internalization and replication, which demonstrated by MIF overexpression and knockdown experiments and treatment with the MIF antagonist ISO-1. Furthermore, we found that the two subunit proteins of Gag, p10 and p27, interacted with MIF in the cytoplasm, respectively. These results suggested that the p10 and p27 subunit in Gag protein recruited MIF to promote ALV-J infection, providing insights into the roles of the p10/p27 and the host factor MIF in ALV-J infection. The finding may facilitate the development of new strategies for controlling ALV-J or retrovirus infections.

Marek's disease virus serine/threonine kinase Us3 facilitates viral replication by targeting IRF7 to block IFN-ß production.

Marek's disease virus (MDV) is a highly oncogenic alphaherpesvirus that induces malignant T-cell lymphomas in chickens, leading to great economic loss in poultry industry. The unique-short kinase 3 (Us3), a serine/threonine kinase encoded by three MDV types (MDV-1, MDV-2 and HVT), is important for MDV replication. However, the mechanism of Us3 facilitating MDV replication has not been completely elucidated. In the present study, we report that Us3 significantly facilities MDV replication via inhibition of ß interferon (IFN-ß) production at the late phase of MDV infection. Overexpression or interference of Us3 significantly promoted or inhibited the replication of MDV, and accordingly inhibited or promoted the expression of IFN-ß. Further, Us3 was shown to suppresses interferon stimulatory DNA (ISD)-triggered IFN-ß production by targeting IFN regulatory factor 7 (IRF7) rather than NF-κB signaling. Moreover, Us3 but not kinase-dead (KD) Us3 mutant K220A blocked the nuclear translocation of IRF7 by inhibiting dimerization. Importantly, Us3 phosphorylated and interacted with IRF7. Furthermore, Us3-deficient MDV weakened viral replication and increased IFN-ß production in infected cells or chickens. These results indicated that Us3 interrupts the cytosolic DNA sensing pathway, thereby leading to inhibition of IFN-ß production by targeting IRF7, promoting MDV replication. Our finding expands the knowledge about the role of Us3 in MDV replication.

Molybdenum Diphosphide Nanorods with Laser-Potentiated Peroxidase Catalytic/Mild-Photothermal Therapy of Oral Cancer.

Chemodynamic therapy (CDT) is an emerging treatment that usually employs chemical agents to decompose hydrogen peroxide (H2O2) into hydroxyl radical (•OH) via Fenton or Fenton-like reactions, inducing cell apoptosis or necrosis by damaging biomacromolecules such as, lipids, proteins, and DNA. Generally, CDT shows high tumor-specificity and minimal-invasiveness in patients, thus it has attracted extensive research interests. However, the catalytic reaction efficiency of CDT is largely limited by the relatively high pH at the tumor sites. Herein, a 808 nm laser-potentiated peroxidase catalytic/mild-photothermal therapy of molybdenum diphosphide nanorods (MoP2 NRs) is developed to improve CDT performance, and simultaneously achieve effective tumor eradication and anti-infection. In this system, MoP2 NRs exhibit a favorable cytocompatibility due to their inherent excellent elemental biocompatibility. Upon irradiation with an 808 nm laser, MoP2 NRs act as photosensitizers to efficiently capture the photo-excited band electrons and valance band holes, exhibiting enhanced peroxidase-like catalytic activity to sustainedly decompose tumor endogenous H2O2 to •OH, which subsequently destroy the cellular biomacromolecules both in tumor cells and bacteria. As demonstrated both in vitro and in vivo, this system exhibits a superior therapeutic efficiency with inappreciable toxicity. Hence, the work may provide a promising therapeutic technique for further clinical applications.

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.

The CCCH-Type Zinc Finger Antiviral Protein Relieves Immunosuppression of T Cells Induced by Avian Leukosis Virus Subgroup J via the NLP-PKC-δ-NFAT Pathway.

The CCCH-type zinc finger antiviral protein (ZAP) can recognize and induce the degradation of mRNAs and proteins of certain viruses, as well as exerting its antiviral activity by activating T cells. However, the mechanism of ZAP that mediates T cell activation during virus infection remains unclear. Here, we found a potential function of ZAP that relieves immunosuppression of T cell induced by avian leukosis virus subgroup J (ALV-J) via a novel signaling pathway that involves norbin-like protein (NLP), protein kinase C delta (PKC-δ), and nuclear factor of activated T cell (NFAT). Specifically, ZAP expression activated T cells by promoting the dephosphorylation and nuclear translocation of NFAT. Furthermore, knockdown of ZAP weakened the reactivity and antiviral response of T cells. Mechanistically, ZAP reduced PKC-δ activity by upregulating and reactivating NLP by competitively binding with viral protein. Knockdown of NLP decreased the dephosphorylation of PKC-δ by ZAP expression. Moreover, we show that knockdown of PKC-δ reduced the phosphorylation levels of NFAT and enhanced its nuclear translocation. Taken together, these data revealed that ZAP relieves immunosuppression caused by ALV-J and mediates T cell activation through the NLP-PKC-δ-NFAT pathway. IMPORTANCE The evolution of the host defense system is driven synchronously in the process of resisting virus invasion. Accordingly, host innate defense factors effectively work to suppress virus replication. However, it remains unclear whether the host innate defense factors are involved in antiviral immune responses against the invasion of immunosuppressive viruses. Here, we found that CCCH-type zinc finger antiviral protein (ZAP) effectively worked in resistance to immunosuppression caused by avian leukosis virus subgroup J (ALV-J), a classic immunosuppressive virus. Evidence showed that ZAP released the phosphatase activity of NLP inhibited by ALV-J and further activated NFAT by inactivating PKC-δ. This novel molecular mechanism, i.e., ZAP regulation of the antiviral immune response by mediating the NLP-PKC-δ-NFAT pathway, has greatly enriched the understanding of the functions of host innate defense factors and provided important scientific ideas and a theoretical basis for research on immunosuppressive viruses and antiviral immunity.

Comparative analysis of the exosomal contents of DF-1 cells infected by ALV-J.

Exploration of the abnormal expression of exosomal molecules during the infection of avian leukosis virus subgroup J (ALV-J) is essential to provide a deeper understanding of the exosome's role in the viral pathogenesis involved. The study aimed to investigate the differentially expressed proteins and miRNAs of the exosomes derived from DF-1 cells infected by ALV-J, their gene function and involved signal pathways. We isolated exosomes from DF-1 cells infected by ALV-J. The differentially expressed proteins and miRNAs of the exosomes were determined by proteomics and transcription detection technology. A Gene Ontology (GO) analysis and a Kyoto Encyclopedia of Genes and Genomes (KEGG) signal pathway analysis identified the miRNAs target genes and the signal pathways regulated by the different proteins or/and miRNAs. A total of 116 proteins (58 upregulated and 58 downregulated) and 3 miRNAs (all upregulated) were determined. These proteins were involved in 155 signal pathways, in which the highest number of proteins involved in the cancer pathway was (up to) seven. The target genes of the miRNAs were involved in 3 signal pathways. Both the proteins and target genes of the miRNAs were involved in the Ribosome pathway and ECM-receptor interaction pathway. The results suggested that the ALV-J infection changed the proteins and miRNAs of the exosomes significantly.

High-frequency and activation of CD4+CD25+ T cells maintain persistent immunotolerance induced by congenital ALV-J infection.

Congenital avian leukosis virus subgroup J (ALV-J) infection can induce persistent immunotolerance in chicken, however, the underlying mechanism remains unclear. Here, we demonstrate that congenital ALV-J infection induces the production of high-frequency and activated CD4+CD25+ Tregs that maintain persistent immunotolerance. A model of congenital infection by ALV-J was established in fertilized eggs, and hatched chicks showed persistent immunotolerance characterized by persistent viremia, immune organ dysplasia, severe imbalance of the ratio of CD4+/CD8+ T cells in blood and immune organs, and significant decrease in CD3+ T cells and Bu-1+ B cells in the spleen. Concurrently, the mRNA levels of IL-2, IL-10, and IFN-γ showed significant fluctuations in immune organs. Moreover, the frequency of CD4+CD25+ Tregs in blood and immune organs significantly increased, and the frequency of CD4+CD25+ Tregs was positively correlated with changes in ALV-J load in immune organs. Interestingly, CD4+CD25+ Tregs increased in the marginal zone of splenic nodules in ALV-J-infected chickens and dispersed to the germinal center. In addition, the proliferation and activation of B cells in splenic nodules was inhibited, and the number of IgM+ and IgG+ cells in the marginal zone significantly decreased. We further found that the mRNA levels of TGF- ß and CTLA-4 in CD4+CD25+ Tregs of ALV-J-infected chickens significantly increased. Together, high-frequency and activated CD4+CD25+ Tregs inhibited B cells functions by expressing the inhibitory cytokine TGF-ß and inhibitory surface receptor CTLA-4, thereby maintaining persistent immunotolerance in congenital ALV-J-infected chickens.

Kinetic analysis of pathogenicity and tissue tropism of gyrovirus 3 in experimentally infected chickens.

Gyrovirus 3 (GyV3), the third novel emerging species of the genus Gyrovirus of the Anelloviridae family, has been described in multiple hosts. Epidemiologically, there are suggestions that GyV3 is associated with diarrhea/proventriculitis, however, no direct causal evidence exists between GyV3 infection and specific clinical diseases. Herein, we infected special pathogen-free (SPF) chickens with GyV3, and then assessed the pathogenicity and tissue tropism. The results revealed that GyV3 induced persistent infection characterized by diarrhea, aplastic anemia, immunosuppression, and persistent systemic lymphocytic inflammation. Clinically, the infected chickens presented ruffled feathers, diarrhea, anemia, and weight loss. Aplastic anemia was characterized by progressive depletion of hematopoietic cells in the bone marrow, immunosuppression was associated with atrophy of the thymus, spleen, and bursa of Fabricious, progressive lymphocytic inflammations were characterized by proventriculitis, adrenalitis, pancreatitis, hepatitis, nephritis, and bronchitis. Viral loads of GyV3 in tissues exhibited "M", "N", "W" or "V" type dynamic changes. The highest level of viral loads was reported in bone marrow at 7dpi, followed by the adrenal gland at 2 dpi, the sciatic nerve at 7 dpi, and bile at 35 dpi. The bone marrow and kidney demonstrate the strongest immunostaining of GyV3-VP1 antigen and were suggested as the target tissues of GyV3. Collectively, GyV3 is an immunosuppressive pathogenic virus that targets the bone marrow and kidney in chickens. Exploring the pathogenicity and tissue tropism of GyV3 will guide the basic understanding of the biology of GyV3 and its pathogenesis in chickens.