The RNA helicase DDX5 promotes viral infection via regulating N6-methyladenosine levels on the DHX58 and NFκB transcripts to dampen antiviral innate immunity.

Multi-functional DEAD-box helicase 5 (DDX5), which is important in transcriptional regulation, is hijacked by diverse viruses to facilitate viral replication. However, its regulatory effect in antiviral innate immunity remains unclear. We found that DDX5 interacts with the N6-methyladenosine (m6A) writer METTL3 to regulate methylation of mRNA through affecting the m6A writer METTL3-METTL14 heterodimer complex. Meanwhile, DDX5 promoted the m6A modification and nuclear export of transcripts DHX58, p65, and IKKγ by binding conserved UGCUGCAG element in innate response after viral infection. Stable IKKγ and p65 transcripts underwent YTHDF2-dependent mRNA decay, whereas DHX58 translation was promoted, resulting in inhibited antiviral innate response by DDX5 via blocking the p65 pathway and activating the DHX58-TBK1 pathway after infection with RNA virus. Furthermore, we found that DDX5 suppresses antiviral innate immunity in vivo. Our findings reveal that DDX5 serves as a negative regulator of innate immunity by promoting RNA methylation of antiviral transcripts and consequently facilitating viral propagation.

DEAD/DEAH-box helicase 5 is hijacked by an avian oncogenic herpesvirus to inhibit interferon beta production and promote viral replication.

DEAD-box helicase 5 (DDX5) plays a significant role in tumorigenesis and regulates viral replication of several viruses. An avian oncogenic herpesvirus, Marek's disease virus (MDV), is widely known to cause immunosuppression and lymphoma in chickens. However, the underlying mechanisms of how DDX5 plays a role in viral replication remain unclear. In this study, we show that MDV inhibits the production of interferon beta (IFN-ß) in chicken embryo fibroblasts (CEFs) by increasing the expression level and promoting the nuclear aggregation of DDX5. We further reveal how DDX5 down-regulates melanoma differentiation-associated gene 5/toll-like receptor 3 signaling through the fundamental transcription factor, interferon regulatory factor 1. MDV replication is suppressed, and the production of IFN-ß is promoted in the DDX5 absented CEFs. Taken together, our investigations demonstrate that MDV inhibits IFN-ß production by targeting DDX5-mediated signaling to facilitate viral replication, which offers a novel insight into the mechanism by which an avian oncogenic herpesvirus replicates in chicken cells.

Transcriptional Profiles Associated with Marek's Disease Virus in Bursa and Spleen Lymphocytes Reveal Contrasting Immune Responses during Early Cytolytic Infection.

Marek's disease virus (MDV), an alpha herpes virus, causes a lymphoproliferative state in chickens known as Marek's disease (MD), resulting in severe monetary losses to the poultry industry. Because lymphocytes of bursa of Fabricius and spleen are prime targets of MDV replication during the early cytolytic phase of infection, the immune response in bursa and spleen should be the foundation of late immunity induced by MDV. However, the mechanism of the MDV-mediated host immune response in lymphocytes in the early stage is poorly understood. The present study is primarily aimed at identifying the crucial genes and significant pathways involved in the immune response of chickens infected with MDV CVI988 and the very virulent RB1B (vvRB1B) strains. Using the RNA sequencing approach, we analyzed the generated transcriptomes from lymphocytes isolated from chicken bursa and spleen. Our findings validated the expression of previously characterized genes; however, they also revealed the expression of novel genes during the MDV-mediated immune response. The results showed that after challenge with CVI988 or vvRB1B strains, 634 and 313 differentially expressed genes (DEGs) were identified in splenic lymphocytes, respectively. However, 58 and 47 DEGs were observed in bursal lymphocytes infected with CVI988 and vvRB1B strains, respectively. Following MDV CVI988 or vvRB1B challenge, the bursal lymphocytes displayed changes in IL-6 and IL-4 gene expression. Surprisingly, splenic lymphocytes exhibited an overwhelming alteration in the expression of cytokines and cytokine receptors involved in immune response signaling. On the other hand, there was no distinct trend between infection with CVI988 and vvRB1B and the expression of cytokines and chemokines, such as IL-10, IFN-γ, STAT1, IRF1, CCL19, and CCL26. However, the expression profiles of IL-1ß, IL-6, IL8L1, CCL4 (GGCL1), and CCL5 were significantly upregulated in splenic lymphocytes from chickens infected with CVI988 compared with those of chickens infected with vvRB1B. Because these cytokines and chemokines are considered to be associated with B cell activation and antigenic signal transduction to T cells, they may indicate differences of immune responses initiated by vaccinal and virulent strains during the early phase of infection. Collectively, our study provides valuable data on the transcriptional landscape using high-throughput sequencing to understand the different mechanism between vaccine-mediated protection and pathogenesis of virulent MDV in vivo.

[Study on the influence of wearable lower limb exoskeleton on gait characteristics].

The purpose of this paper was to investigate the effects of wearable lower limb exoskeletons on the kinematics and kinetic parameters of the lower extremity joints and muscles during normal walking, aiming to provide scientific basis for optimizing its structural design and improving its system performance. We collected the walking data of subjects without lower limb exoskeleton and selected the joint angles in sagittal plane of human lower limbs as driving data for lower limb exoskeleton simulation analysis. Anybody (the human biomechanical analysis software) was used to establish the human body model (the human body model without lower limb exoskeleton) and the man-machine system model (the lower limb exoskeleton model). The kinematics parameters (joint force and joint moment) and muscle parameters (muscle strength, muscle activation, muscle contraction velocity and muscle length) under two situations were compared. The experimental result shows that walking gait after wearing the lower limb exoskeleton meets the normal gait, but there would be an occasional and sudden increase in muscle strength. The max activation level of main lower limb muscles were all not exceeding 1, in another word the muscles did not appear fatigue and injury. The highest increase activation level occurred in rectus femoris (0.456), and the lowest increase activation level occurred in semitendinosus (0.013), which means the lower limb exoskeletons could lead to the fatigue and injury of semitendinosus. The results of this study illustrate that to avoid the phenomenon of sudden increase of individual muscle force, the consistency between the length of body segment and the length of exoskeleton rod should be considered in the design of lower limb exoskeleton extremity.

An optimized aptamer-binding viral tegument protein VP8 inhibits the production of Bovine Herpesvirus-1 through blocking nucleocytoplasmic shuttling.

Bovine herpesvirus 1 (BoHV-1) is a major pathogen of infectious bovine rhinotracheitis in bovine. Previously, we generated the aptamer IBRV A4 using systemic evolution of ligands by exponential enrichment. This aptamer inhibited infectivity of BoHV-1 by blocking viral particle absorption onto cell membranes. In this study, we found that the major tegument protein VP8 of BoHV-1 was involved in inhibition of infectious virus production by IBRV A4. We improved the affinity of IBRV A4 for VP8 by optimizing aptamer's structure and repeat conformation. An optimized aptamer, IBRV A4.7, was constructed with quadruple binding sites and a new stem-loop structure, which had a stronger binding affinity for VP8 or BoHV-1 than raw aptamer IBRV A4. IBRV A4.7 bound to VP8 with a dissociation constant (Kd) value of 0.2054 ±â€¯0.03948 nM and bound to BoHV-1 with a Kd value of 0.3637 ±â€¯0.05452 nM. Crucially, IBRV A4.7 had improved antiviral activity compared to IBRV A4, with a half-maximal inhibitory concentration of 1.16 ±â€¯0.042 µM. Our results also revealed IBRV A4.7 inhibited BoHV-1 production in MDBK cells through blocking nucleocytoplasmic shuttling of viral VP8 in BoHV-1-infected MDBK cells. In conclusion, the aptamer IBRV A4.7 may have potency in preventing outbreaks in herds due to reactivation of latency.

Antiviral Immunotoxin Against Bovine herpesvirus-1: Targeted Inhibition of Viral Replication and Apoptosis of Infected Cell.

Bovine herpesvirus 1 (BoHV-1) is a highly contagious viral pathogen which causes infectious bovine rhinotracheitis in cattle worldwide. Currently, there is no antiviral prophylactic treatment available capable of mitigating the disease impact and facilitating recovery from latent infection. In this study, we have engineered a novel recombinant anti-BoHV-1 immunotoxin construct termed "BoScFv-PE38" that consists of a single-chain monoclonal antibody fragment (scFv) fused with an active domain of Pseudomonas exotoxin A as a toxic effector (PE38). The recombinant BoScFv-PE38 immunotoxin expressed in a prokaryotic expression system has specific binding affinity for BoHV-1 glycoprotein D (gD) with a dissociation constant (Kd) of 12.81 nM and for BoHV-1 virus particles with a Kd value of 97.63 nM. We demonstrate that the recombinant BoScFv-PE38 is internalized into MDBK cell compartments that inhibit BoHV-1 replication with a half-maximal inhibitory concentration (IC50) of 4.95 ± 0.33 nM and a selective index (SI) of 456 ± 31. Furthermore, the BoScFv-PE38 exerted a cytotoxic effect through the induction of ATP and ammonia, leading to apoptosis of BoHV-1-infected cells and the inhibition of BoHV-1 replication in MDBK cells. Collectively, we show that BoScFv-PE38 can potentially be employed as a therapeutic agent for the treatment of BoHV-1 infection.