Innate antiviral immunity is the first line of host defense against invading viral pathogens. Immunity activation primarily relies on the recognition of pathogen‐associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs). Viral proteins or nucleic acids mainly engage three classes of PRRs: Toll‐like receptors (TLRs), retinoic acid‐inducible gene I (RIG‐I)‐like receptors (RLRs), and DNA sensor cyclic GMP‐AMP (cGAMP) synthase (cGAS). These receptors initiate a series of signaling cascades that lead to the production of proinflammatory cytokines and type I interferon (IFN‐I) in response to viral infection. This system requires precise regulation to avoid aberrant activation. Emerging evidence has unveiled the crucial roles that the ubiquitin system, especially deubiquitinating enzymes (DUBs), play in controlling immune responses. In this review, an overview of the most current findings on the function of DUBs in the innate antiviral immune pathways is provided. Insights into the role of viral DUBs in counteracting host immune responses are also provided. Furthermore, the prospects and challenges of utilizing DUBs as therapeutic targets for infectious diseases are discussed. This review systematically summarizes the role and mechanisms of deubiquitinating enzymes (DUBs) in regulating innate antiviral signaling via the Toll‐like receptors (TLRs), RIG‐I like receptors (RLRs), and cyclic GMP‐AMP synthase (cGAS). The role of viral DUBs counteracting host immune responses is also concluded. Moreover, the prospects and challenges of utilizing DUBs as therapeutic targets for treatment of infectious diseases like COVID‐19 are also discussed. Upon recognition, PRR initiates a series of signaling cascades that ultimately lead to the synthesis and secretion of type I interferons (IFN‐I) and proinflammatory cytokines, thereby exhibiting antiviral immune responses.Our living environment is surrounded by various infectious viruses. To counteract these pathogens, hosts have evolved diverse innate immune systems. This is achieved by pattern recognition receptors (PRRs) expressed in cells that recognize molecules specific to the pathogens, namely pathogen‐associated molecular patterns (PAMPs).1, 2 This raises the question of how host cells limit innate immune signals to extremely low levels and meanwhile ensure its timely activation to deal with the challenge of pathogens. The rapid switch on of innate antiviral signaling requires certain posttranslational modifications (PTMs) of crucial sensors for viral RNA, such as ubiquitination.As insufficient interferon production results in chronic infection whereas excessive interferon leads to autoimmune and/or inflammatory disease, innate immunity must be tightly regulated to efficiently respond to invading pathogen and meanwhile avoid excessive harmful immune pathology.5 This To date, three classes of E3s have been identified: really interesting new gene (RING), homologous to E6AP C‐terminus (HECT), and RING‐between‐RING (RBR). RING E3s directly transfer the ubiquitin from E2s to the substrate, whereas HECT and RBR E3s first receive ubiquitin from E2s through a catalytic cysteine, after which the ubiquitin is transferred to the substrate. An isopeptide bond is then formed between the carboxyl group of the ubiquitin's glycine and the epsilon‐amino group of the substrate's lysine  and an N‐terminus methionine (M1) serving as points of ubiquitination, thereby resulting in complex linkage types. A substrate protein can be attached with a mono‐ or poly‐ubiquitin molecule. The resulting ubiquitinated proteins vary based on the linkage type of the ubiquitin chains. The most common linkage types are K48‐ and K63‐linked polyubiquitin chains, with the former recognized by the proteasome, leading to the degradation of the substrate, whereas the latter is mainly involved in nondegradative roles, including cellular signaling, intracellular trafficking, and DNA damage response. Those not linked via canonical K48 linkages or K63 linkages are known as atypical ubiquitin chains, whose physiological roles have only started to accumulate. For example, linear ubiquitin chains are linked through M1 and have been established to play crucial roles in inflammatory signaling and apoptotic cell death. Accumulating evidence has uncovered the role of both typical and atypical ubiquitin chains in the initiation, maintenance, and termination of antiviral immune responses.Ubiquitination is an enzymatic posttranslational modification in which a ubiquitin protein with 76 amino acids is conjugated to a target molecule. Ubiquitin is so named because it occurs ubiquitously in cellular processes. Ubiquitination requires a cascade consisting of three enzymes: ubiquitin‐activating enzymes (E1), ubiquitin‐conjugating enzymes (E2), and ubiquitin ligases (E3). In this cascade, E1 can bind with many E2s, which further bind with hundreds of E3s in a hierarchical way.7 To d DUBs cleave ubiquitin molecules from substrates to generate a free ubiquitin pool, thereby ensuring the ubiquitin cycle .18 DUB22.1 To date, DUBs have been divided into seven families, based on their structural features: six families of cysteine proteases, namely ubiquitin‐specific proteases (USPs), ovarian tumor proteases (OTUs), ubiquitin C‐terminal hydrolases (UCHs), the Josephin family, the motif interacting with ubiquitin (MIU)‐containing novel DUB family (MINDYs), zinc finger with UFM1‐specific peptidase domain protein/C6orf113/ZUP1 (ZUFSP), and the JAB1/MPN/MOV34 metalloenzyme family , which belongs to the zinc‐metalloprotease group. DUBs generally contain a catalytic domain surrounded by one or more accessory domains, some of which contribute to target recognition. These additional domains include zinc finger (ZnF) domain, ubiquitin‐like domain (UBL), coiled‐coil (CC) domain, and motif interacting with ubiquitin (MIU) domain  was the first identified DUB to exhibit linkage specificity, and many JAMMs are reported to be K63 specific. MINDY DUBs are K48 specific. Most OTU family DUBs can be linkage specific, whereas most USPs show little or no linkage preference. The underlying mechanisms of linkage specificity have been reviewed elsewhere and will therefore not be covered herein.DUBs cleave ubiquitin from ubiquitinated substrates in different ways, either by binding to a protein substrate that they deubiquitinate (target‐specific) or by directly binding to the ubiquitin chain that they cleave (linkage‐specific).25 In Ubiquitin chains are cleaved by DUBs either through endo‐ or exocleavage activity. While endo‐cleavage activity efficiently removes ubiquitin chains from a substrate, the released chain undergoing further processing to regenerate monoubiquitin, exo‐cleavage directly generates monoubiquitin, but requires multiple steps for the DUB to remove the entire polyubiquitin chain. Although it seems complicated to determine whether a DUB cleaves with endo‐ or exocleavage activity, several studies have elucidated that the activity depends on both the DUB structure and the ubiquitin linkage type.252.3 inflammatory diseases, and neurological diseases. DUBs can be regulated by a multifaceted approach, including the control of their abundance, catalytic activity, as well as their localization.Dysregulation of DUBs may lead to aberrant functions that can cause a variety of diseases, such as cancer,32 infκB (NF‐κB) activation. Once expressed, DUBs can also be negatively regulated, as exemplified by the stimulation of the expression of OTUD‐6B in B lymphocytes by IL‐3, IL‐4, IL‐13, or granulocyte‐macrophage colony‐stimulating factor (GM‐CSF); however, an opposing effect occurs with sustained induction, leading to a decrease in OTUD‐6B expression.The abundance of DUBs is modulated at the transcriptional level and the expression level of DUBs can be induced in a stimulation‐dependent manner. For example, the abundance of A20 is low in unstimulated cells, yet is upregulated in response to TLR4‐mediated nuclear factor tivation.38 OncκB essential modulator (NEMO). Second, DUB activity is commonly adjusted in an allosteric fashion. This occurs especially when DUBs function in complexes with other molecules. For instance, USP1 by itself is an ineffective enzyme, but its activity is boosted when bound to USP1‐associated factor 1 (UAF‐1) and it undergoes conformational changes. Additionally, substrate binding may promote DUB activation, thereby enabling fine‐tuning of linkage specificity. For example, USP7 alters its inactive configuration toward an active one upon ubiquitin binding, which enables full activation of the USP7 catalytic domain. Third, DUB catalytic activity can be modulated by posttranslational modifications, such as phosphorylation, ubiquitination, and SUMOylation. For instance, our previous studies demonstrated that poly‐SUMOylation is required for OTUB2 to interact with and deubiquitinate Yes‐associated protein (YAP) and transcriptional coactivator with PDZ‐binding motif (TAZ). Intriguingly, these modifications can play dual roles in DUB activity, either inhibitory or activating, thereby revealing the fascinating intricacy of posttranslational regulation. Moreover, the cellular microenvironment can also influence DUB activity. DUBs that are cysteine proteases harbor a reactive cysteine residue that can be oxidized by reactive oxygen species (ROS), thus attenuating DUB activity.DUB catalytic activity can be regulated in a number of ways: binding to regulatory proteins, allosteric regulation, and posttranslational modification. First, DUBs can target a specific substrate by recruiting other factors, as demonstrated by USP10 recruiting and interacting with the monocyte chemotactic protein induced protein 1 (MCPIP1) to deubiquitinate its substrate, nuclear factor r (NEMO).40 Sec DUB localization can be regulated by various approaches. Posttranslational modifications, apart from affecting DUB catalytic activity, have also been reported to affect the nuclear localization of DUBs. To illustrate, phosphorylation of USP4 by protein kinase B  results in its redistribution from the nucleus to the cytoplasm, which enables USP4 to reach the cell membrane and deubiquitinate the transforming growth factor‐β (TGF‐β) receptor I (TβR‐I). Furthermore, localization can also be indirectly modulated by changing the protein interactions that a DUB engages in ref. . For example, hydroxylation of OTUB1 by factor inhibiting HIF (FIH) leads to a profound change in the interaction of OTUB1 with proteins important in cellular metabolism. This alteration of OTUB1 interactome affects OTUB1 localization and substrate accessibility.DUB regulation is also exerted through subcellular localization, which is necessary for substrate accessibility. DUBs are often found to form complexes with other proteins, including adaptors and scaffolds. Interactions with these proteins can assure proper localization of the DUB so that the specific substrate is delivered to the DUB in the right place for its catalytic action.48 DUBThe diversity of the mechanisms for DUB regulation enables their function to be finely tuned, which ensures appropriate responses in different contexts, including innate immune response against viruses.33.1 TLRs have evolved to recognize a variety of PAMPs. To date, at least ten human TLRs have been identified, with TLRs 1, 2, 4, 5, 6, and 10 localizing at the cell surface, whereas TLRs 3, 7, 8, and 9 are anchored to endolysosomes. Of the identified TLRs, several have been linked to innate antiviral immunity. Among them, TLR2 and TLR4 sense both structural and nonstructural viral proteins, whereas TLR3, TLR7/8, and TLR9 detect virus‐derived dsRNA, ssRNA, and unmethylated CpG DNA, respectively, which together is responsible for the effectiveness of the antiviral immune surveillance. TLRs share similar domain structures, including an ectodomain comprising leucine‐rich repeats that recognize PAMPs, a transmembrane domain, and a C‐terminal cytoplasmic domain, known as Toll‐interleukin‐1 receptor (IL‐1R) homology (TIR) domain. The TIR domain in the cytoplasm is crucial for signal transduction.As the most widely studied PRRs, TLRs are transmembrane glycoproteins that are mainly expressed in immune cells. TLRs were first acknowledged to be critical for defending fungal infections.55 TLRFigure β (TRIF), all TLRs involved in antiviral immunity recruit myeloid differentiation primary response 88 (MyD88).TLRs initiate downstream signaling cascades by recruiting their respective adaptor proteins upon ligand binding Figure 3. Apart TRAF6, cIAP1, and cIAP2 can also be recruited to Myddosome, functioning as ubiquitin ligases that mediate K48‐linked polyubiquitination of the tumor necrosis factor receptor‐associated factor 3 (TRAF3) and consequent degradation by the proteasome. In response to stimuli, Myddosome triggers IRAK4 autophosphorylation and activation, followed by IRAK4‐mediated phosphorylation and activation of IRAK1/2. IRAKs are then dissociated from MyD88, resulting in the activation of TRAF6. TRAF6 forms a complex with two E2 enzymes, Ubc13 and Uev1A, which promotes the synthesis of K63‐linked polyubiquitin chains, thereby acting as a scaffold to recruit the downstream signaling molecules, TGF‐β‐activated kinase 1 (TAK1) and κB kinase (IKK) complexes, through their ubiquitin‐binding subunits, TGF‐β‐activated kinase 1 and MAP3K7‐binding protein 2/3 (TAB2/3) and NEMO, respectively. As a homologous binding partner of both IKKα/IKKβ complex and TANK‐binding kinase 1 (TBK1)/IKKε complex, NEMO can be catalyzed with K27‐, K29‐, and M1‐linked ubiquitin chains, thereby regulating the phosphorylation of NF‐κB and interferon regulatory factor 3 (IRF3) and subsequent translocation into the nucleus. Notably, linear ubiquitin chain assembly complex (LUBAC), which consists of heme‐oxidized IRP2 ubiquitin ligase 1L (HOIL‐1L), HOIL‐1‐interacting protein (HOIP), and Sharpin, catalyzes the M1‐linked ubiquitination  of NEMO, which is a prerequisite for recruiting the IKK complex (IKKα and IKKβ). TAK1 not only induces the phosphorylation of IKKβ, leading to the activation of IKK, but also activates the mitogen‐activated protein kinase (MAPK) kinase family. These kinases lead to the activation of transcription factors NF‐κB and activator protein 1 (AP1) and induction of proinflammatory cytokines. Of note, in the TLR7/8/9‐mediated signaling pathway, IRF7 can be directly recruited by both MyD88 and IRAK1, followed by IRAK1‐mediated phosphorylation and activation. TRAF6, TRAF3, and IKKα are proved to be necessary for this activation. Besides, IRF5 downstream of MyD88 and TRAF6 is indispensable for TLR7/8/9‐mediated production of proinflammatory cytokines.TLR2/4, as well as TLR7/8/9 activation, induces the recruitment of MyD88 and the assembly of the MyD88 signaling complex, termed Myddosome, which consists of MyD88 itself and the IL‐1 receptor‐associated kinase (IRAK) family kinases IRAK4 and IRAK1/2.64 TRA Activated TRIF binds TRAF3, a ubiquitin ligase crucial for the recruitment of TBK1 and the inhibitor of the IKKε complex, following modification of TRAF3 with K63‐linked polyubiquitin. The TBK1/IKKε complex recruits and phosphorylates IRF3. The activated IRF3 dimerizes and enters the nucleus and induces type I IFN production. Additionally, TRIF also activates the NF‐κB pathway. TRIF recruits and binds to receptor‐interacting serine/threonine‐protein kinase 1 , which activates TAK1. The activation of NF‐κB is then mediated by a similar mechanism downstream of TRAF6. Therefore, TAK1 results in a variety of signaling cascades and ultimately initiate the transcription of proinflammatory cytokines.In contrast, TLR3 activation leads to the recruitment and subsequent activation of TRIF through TIR–TIR interaction.78 Act The E3 ubiquitin ligase Parkin negatively regulates the antiviral signaling pathway by targeting TRAF3 for degradation; however, the process is inhibited by the mitochondrial protein PTEN‐induced kinase 1 (PINK1), which associates with TRAF3 via the kinase domain. Smurf1/2 interacts with MyD88, leading to its K48‐linked ubiquitination and degradation, thus limiting the inflammatory response. Neuregulin receptor degradation protein 1 (Nrdp1) is another E3 ubiquitin ligase that leads to MyD88 degradation through K48‐linked ubiquitination. Intriguingly, Nrdp1 can also conjugate K63‐linked polyubiquitin on TBK1, thereby causing effects that are opposite. Similarly, TRIF can be targeted for K48‐linked polyubiquitination and subsequent degradation by the E3 ligase WW domain‐containing protein 2 (WWP2) and tripartite motif‐containing 38 (TRIM38). BICP0, a viral E3 ubiquitin ligase encoded by bovine herpes virus‐1, is another negative regulator that promotes the K48‐linked ubiquitination and degradation of TRAF6. TRAF6 itself is the E3 ligase that can target IRF7 for K63‐linked polyubiquitination. This process is negatively regulated by TARBP2 by its participation in the interaction between IRF7 and TRAF6.Ubiquitination plays a critical role in TLR‐triggered MyD88 and TRIF signaling. Multiple E3 ligases have been identified to negatively regulate TLR signaling through K48‐linked polyubiquitination. Notably, Triad3A has been reported to target not only certain TLRs, including TLR3, 4, 5, and TLR9 but also RIP1 and TRAF3, ultimately leading to their degradation.81 The Nevertheless, the role of A20 is not that simple due to its structural features, including the presence of an OTU domain as well as seven zinc‐finger (ZnF) motifs, which confer both DUB activity and E3 ubiquitin ligase activity on A20, respectively. Whereas A20 removes K63‐linked polyubiquitin chains from its substrates, such as RIPK1, A20 also introduces K48‐linked polyubiquitin chains in the same substrate, thereby tagging it for proteasomal degradation. Additionally, A20 can bind polyubiquitin chains through its zinc‐finger motif, which leads to its specific binding to ubiquitinated NEMO. This allows phosphorylation of IKK to be sufficiently blocked by its upstream kinase TAK1, which inhibits NF‐κB activation. Another study has shown that A20, with the help of the regulatory molecule Tax1 Binding Protein 1 (TAX1BP1), interacts with E2 enzymes Ubc13 and UbcH5c and triggers their ubiquitination and subsequent degradation, which antagonizes their interactions with the E3 ligases TRAF6, TRAF2, and cIAP1. The activities of these E3 ligases are consequently dampened. These findings together reveal the diverse and intricate roles of A20 in the inhibition of TLR signaling pathways. CYLD is another well‐studied DUB. In TLR‐mediated signaling, CYLD negatively regulates NF‐κB signaling by cleaving K63‐linked polyubiquitin chains and M1‐linked polyubiquitin chains from RIPK1, TRAF2, and NEMO.Deubiquitination precisely controls the abundance and activity of signaling molecules in TLR signaling, thereby avoiding dysregulation Figure . Over th91 Nev OTUD4 was previously shown as a K48‐specific DUB that is important for maintaining the stability of alkylation repair enzymes. In another study, OTUD4 has been reported to harbor K63‐specific DUB activity following phosphorylation at Serine202 and Serine204. Furthermore, this activity requires an adjacent ubiquitin‐interacting motif that increases the affinity of OTUD4 for K63‐linked chains. OTUD4 can target MyD88, thereby negatively regulating the TLR‐mediated NF‐κB pathway. Indeed, OTUD4‐deficient mice have been shown to exhibit increased inflammatory signaling in response to TLR stimulation. TRAF6 and TRAF3 are essential for mediating downstream signaling, and their activities are tightly regulated by several DUBs. Myb‐like, SWIRM, and MPN domains 1 (MYSM1) targets both TRAF3 and TRAF6 for K63‐linked deubiquitination and subsequent inactivation, subsequently terminating TLR‐mediated pathways for antiviral responses, which is evidenced genetically. Specifically, the SWIRM and metalloproteinase domains have been identified as responsible for the interaction with both TRAF3 and TRAF6, and the removal of K63‐linked polyubiquitin chains, respectively. USP4 has been identified as a potent negative regulator of TLR/IL‐1R signaling. USP4 interacts with and deubiquitinates TRAF6, thereby preventing the activation of NF‐κB and AP‐1 transcription factors and subsequent immune responses. Moreover, usp4‐depleted zebrafish (Danio rerio) larvae have been shown to produce more proinflammatory cytokines and were more susceptible to endotoxic challenge following LPS treatment. Ubiquitin carboxyl‐terminal hydrolase L1 (UCHL1) cleaves K63‐linked polyubiquitin chains on TRAF3, which ultimately leads to reduced production of type I IFN, proinflammatory cytokines, and chemokines in response to high‐risk human papillomavirus (hrHPV) infection. TRAF family member‐associated NF‐κB activator (TANK) interacts with both monocyte chemotactic protein‐1‐induced protein‐1 (MCPIP1) and USP10, leading to the cleavage of ubiquitin chains on TRAF6 and termination of NF‐κB activation in response to TLR activation. Of note, USP10 also interacts with NEMO via MCPIP1 and leads to the removal of NEMO‐attached M1‐linked polyubiquitin chains, thus inhibiting the genotoxic NF‐κB signaling cascade. In addition to interacting with USP10, MCPIP1 itself regulates cellular inflammation pathways with distinct mechanisms. A study showed that MCPIP1 negatively regulates c‐Jun N‐terminal kinase (JNK) and NF‐κB activities, induced by LPS, IL‐1β, and tumor necrosis factor‐α (TNF‐α), by removing ubiquitin moieties from TRAF2, TRAF3, and TRAF6. Consistently, the fatal inflammatory syndrome was observed in MCPIP1‐deficient mice. In addition to its DUB activity, MCPIP1 restricts cellular inflammation via its RNase activity. Interestingly, several studies have shown that MCPIP1 RNase activity renders specific mRNA degradation of IL‐6, IL‐1β, and IL‐2. Conversely, MCPIP1 can also be a positive regulator of antiviral immunity. MCPIP1 ribonuclease exhibits broad‐spectrum antiviral effects via the binding and degradation of viral RNA. Studies have found that MCPIP1 not only inhibits RNA viruses, such as Japanese encephalitis (JEV), dengue virus (DEN), sindbis virus, encephalomyocarditis, influenza A, hepatitis C virus (HCV), and human immunodeficiency virus (HIV), but also DNA viruses, such as the hepatitis B virus (HBV). Moreover, while MCPIP1 can also promote IFN‐I‐mediated antiviral efficacy in a manner independent of its RNase and DUB activity, the underlying mechanism is not clear. Similarly, USP18 acts as a negative regulator of IFN responses by counteracting ISG15 conjugation upon viral infection. Additionally, it has been reported that USP18 also regulates TLR‐induced NF‐κB activation by removing K63‐linked polyubiquitin chains of TAK1 and NEMO.Several DUBs in TLR signaling perform nondegradative roles. For example, USP19 was identified to play a role in downregulating TLR3/4‐mediated signaling. USP19 interacts with TRIF and removes the K27‐linked polyubiquitin moieties of TRIF, which inhibits the recruitment of TRIF to TLR3/4. In vitro and in vivo assays further demonstrated that USP19 negatively regulates TLR3/4‐mediated innate immune and inflammatory responses.98 OTU In response to noncanonical NF‐κB stimuli, OTUD7B displays K48‐specific DUB function, which inhibits TRAF3 proteolysis and prevents aberrant non‐canonical NF‐κB activation through binding and deubiquitination of TRAF3.It has been shown that some DUBs are related to the stability of signaling molecules in the TLR pathway. In response to LPS, USP25 prevents the K48‐linked ubiquitination of TRAF3 that is mediated by cIAP2. Deficiency in USP25 accelerates its degradation following TLR4 activation. Indeed, USP25 knockout mice exhibited increased susceptibility to LPS‐induced septic shock.117 InAlthough a large number of host DUBs that control TLR signaling have been characterized over the years, much of the data attempting to elucidate whether DUBs function in the context of defending viral infections or other pathogen infections are inconclusive. Hence, in this Review, we present a summary of the host DUBs that target key molecules in the TLR signaling pathway. It should be noted that the specific function of the host DUBs differs, depending on the PAMPs, cell type, and infection model examined. Further studies are needed to confirm the antiviral function of DUBs in TLR signaling.3.2 melanoma differentiation‐associated protein 5 (MDA5), and laboratory of genetics and physiology 2 (LGP2), all of which are distributed in the cytoplasm. All RLRs contain a C‐terminal domain (CTD) and an intermediate RNA helicase domain. At the N‐termini, all RLRs, except for LGP2, possess tandem caspase recruitment domains (CARDs), which are necessary for downstream signal transduction. Although sharing similar domain architectures, they each have different ligand preferences: RIG‐I recognizes ssRNAs bearing a 5′‐triphosphate moiety, as well as short dsRNA (∼1kb) bearing 5′‐triphosphate or with a lower affinity 5′‐diphosphate, whereas MDA5 prefers to sense longer dsRNA. LGP2 has been shown to engage in the recognition of and interaction between RNA and RIG‐I/MDA5, thus exerting a regulatory role in downstream signaling.While TLR‐mediated signaling exerts extensive antiviral immune responses, this system has its limitations due to a lack of TLR expression in most nonimmune cells. Therefore, alternative PRRs, such as RLRs, are needed. RLRs are cytosolic RNA sensors that consist of three members: RIG‐I,119 me MAVS further activates TBK1 and IKK by recruiting TRAF proteins. TBK1 and IKK complexes activate and phosphorylate IRF3 and NF‐κB, respectively, leading to the induction of interferon and proinflammatory cytokines  through CARD–CARD interaction.133 MA Moreover, the E3 ligases TRIM25 and Riplet/RING finger protein 135 (RNF135) have been reported to conjugate K63‐linked polyubiquitin chains to RIG‐I CARDs, thus stabilizing this complex and boosting its activation. RIG‐I can also be conjugated with K63‐linked ubiquitin chains by E3 ligase TRIM4 and Mex‐3 RNA binding family member C (MEX3C), thus enhancing virus‐mediated type I IFN production. Similarly, unanchored K63 polyubiquitin chains are indispensable in the oligomerization of MDA5, the resulting oligomer complex being stabilized by the covalent binding of polyubiquitin chains. The E3 ligase TRIM31 interacts and catalyzes the K63‐linked polyubiquitination on MAVS, which promotes the formation of prion‐like aggregates of MAVS after viral infection. Furthermore, the E3 ligase TRIM21 positively regulates innate antiviral immunity through K27‐linked polyubiquitination of MAVS. LUBAC negatively regulates RIG‐I and TRIM25‐mediated type I IFN induction by catalyzing M1‐linked ubiquitination and subsequent degradation of RIG‐I, as well as competing with TRIM25 for its association with RIG‐I, thus inhibiting RLR‐mediated signaling.Ubiquitination is also crucially involved in regulating RLR signaling. Structural remodeling of RIG‐I into active conformations occurs upon RNA binding, which allows RLRs undergoing oligomerization to be active. RIG‐I forms tetramers with the help of E3 ligase TRIM25, which can catalyze and form free K63‐linked polyubiquitin chains that bind to its CARDs.136 Mo RNF122, carboxyl terminus of Hsc70‐interacting protein (CHIP), and STIP1 homology and U‐box containing protein 1 (STUB1) have been reported to attach K48‐linked ubiquitin chains on RIG‐I, and TRIM13 works on MDA5. TRIM40 has been reported to interact with both RIG‐I and MDA5, catalyzing K27‐ and K48‐linked polyubiquitination, respectively, thereby promoting their degradation. A few E3 ligases, such as RNF5, SMAD specific E3 ubiquitin protein ligase 1(Smurf1), Smurf2, TAX1BP1, membrane‐associated RING finger (C3HC4) 5 (MARCH5), poly(rC)‐binding protein 1 (PCBP1), PCBP2, TRIM25, and receptor for activated C kinase 1 (RACK1), are responsible for the K48‐linked ubiquitination on MAVS.K48‐linked polyubiquitination is needed for proteasomal degradation, thus negatively regulating the MAVS signaling cascade. The E3 ligases RNF125,147 RN Upon viral infection, or in the presence of a tumor necrosis factor, the CYLD expression level is downregulated to enhance IFN production. Notably, it has been shown that CYLD targets not only RIG‐I but also downstream TBK1, the kinase that phosphorylates and activates IRF3. In this case, Optineurin (Optn) was shown to dampen the response via targeting CYLD to TBK1 to impair its enzymatic activity. Similarly, USP3 also acts as a negative regulator of RIG‐I‐mediated signaling by removing K63‐linked ubiquitination on RIG‐I. Upon viral infection or ligand stimulation, USP3 binds to the CARD domain of RIG‐I and then removes its polyubiquitin chains through both the zinc‐finger Ub‐binding domain and USP catalytic domains. However, whether USP3 regulates RIG‐I in vivo remains unknown. Moreover, USP21 has been reported to remove K63‐linked ubiquitination of RIG‐I, resulting in impaired antiviral responses. Interestingly, USP21 presumably removes polyubiquitin chains from more than one lysine sites in either CARD or CTD domain, since USP21 can reverse the polyubiquitination mediated by both RNF135 and TRIM25. USP21‐deficient mice are more resistant to vesicular stomatitis virus (VSV) infection, concomitated with elevated IFN production. USP14, in another study, has also been shown to remove K63‐linked ubiquitin chains on RIG‐I. USP14‐specific inhibitor, IU1, was demonstrated to increase RIG‐I‐mediated type I IFN production and antiviral responses in vitro and in vivo. Recently, siRNA library screening identified USP27X as a negative regulator of type I IFN signaling by cleaving K63‐linked polyubiquitin chains from RIG‐I. While USP15 also plays a negative role in RIG‐I signaling, its activities are more difficult to decipher as it exhibits an opposite function in other contexts (see below).Since K63‐linked ubiquitination of RIG‐I is crucial for its activation and signaling, it is not surprising that several DUBs are involved in downregulating RLR signaling to avoid overactivation Figure . As the 162 Upβ production. It is worth mentioning that both YOD1 and TRIM31 interact with the proline‐rich domain of MAVS, indicating competition between these two enzymes with opposing roles. Recently, our previous research identified OTUD3 as an acetylation‐dependent DUB that directly deconjugates the K63‐linked polyubiquitin chains of MAVS and terminates downstream antiviral response. Intriguingly, the catalytic activity of OTUD3 is dependent on the acetylation of Lysine129. To confirm this, acetyl‐lysine is incorporated into a recombinant OTUD3 to generate a fully Lys129‐acetylated OTUD3 (OTUD3Lys129‐Ac) with chemical biology approaches. In vitro Ubiquitin‐AMC assay and deubiquitination assay demonstrated that OTUD3Lys129‐Ac exhibits robust deubiquitinase activity toward K63‐linked ubiquitin chains and is more effective in targeting MAVS. Besides, this acetylated residue can be removed by sirtuin 1 (SIRT1) upon virus infection, which promptly inactivates OTUD3 and triggers the innate antiviral immune response promptly. Consistently, intensive antiviral immune response, diminished viral load, and morbidity were observed in OTUD3 knockout mice, and acetyl‐OTUD3 levels are negatively correlated with IFN‐β expression in influenza patients. These findings collectively establish OTUD3 as a new repressor of MAVS and unveil a novel regulatory mechanism through which the catalytic activity of OTUD3 is tightly tuned in the control of the timely antiviral defense.YOD1 , a DUB of the OTU family, was the first identified DUB responsible for downregulating the K63‐linked polyubiquitination of MAVS. Following a Sendai virus infection, YOD1 was recruited to mitochondria and was shown to interact with MAVS. Subsequently, YOD1 removes the K63‐linked ubiquitin chain and abrogates the aggregation of MAVS, thus inhibiting not only IRF3 and p65 activation, but also IFN‐oduction.170 It Because various components are involved in virus‐induced signaling, biochemical assays have demonstrated that OTUB1 and OTUB2 inhibit RIG‐I‐, MDA5‐, and MAVS‐mediated but not TBK1‐ and IRF3‐mediated signaling. Notably, the negative regulation of OTUB1 in TRAF3‐elicited antiviral innate immunity is enhanced by HSCARG , which fosters the recruitment of OTUB1 to TRAF3 and abolishes its ubiquitination.Negative regulation of RLR signaling is also accomplished by targeting downstream molecules of MAVS. Coimmunoprecipitation analyses indicate that OTUB1 and OTUB2 associate with TRAF3 and TRAF6, negatively regulating antiviral responses by deubiquitinating their respective K63‐linked polyubiquitin chains.172 Be OTUD1 was also reported to inhibit IFN production during RNA virus infection. Mechanistic studies demonstrated that OTUD1 stabilizes Smurf1 by removing its ubiquitin chains, which allows the binding of Smurf1 to MAVS, TRAF3, and TRAF6 with subsequent degradation of each component of the MAVS/TRAF3/TRAF6 signalosome. Notably, OTUD1‐deficient mice were consistently shown to be more resistant to RNA virus infections when more antiviral cytokines were produced.Modulating the stability of key signaling molecules is another approach to inhibit RLR‐mediated signal transduction. A systematic functional screening revealed that USP5 can interact with and recruit STUB1 to mediate the degradation of RIG‐I, thus enhancing VSV replication.174 OT USP4 interacts with and cleaves K48‐linked polyubiquitin chains from RIG‐I, thereby promoting RIG‐I‐mediated IFN‐β signaling and inhibiting VSV replication. Following virus‐induced RIG‐I activation, USP4 expression was shown to attenuate. Additionally, overexpression of USP4 dramatically enhanced the RIG‐I protein level, while knockdown of USP4 had an opposite effect. Although multiple E3 ligases responsible for the K48‐linked ubiquitination of MAVS have been identified over the past years, the corresponding DUBs remained elusive until the identification of OTUD4. It has been shown by DUB screening and biochemical analyses that OTUD4 removes K48‐linked polyubiquitin chains on MAVS in response to viral infection, thereby inhibiting the proteasomal degradation of MAVS and triggering downstream signaling. This is supported by the observation that knockout or knockdown of OTUD4 impairs the RNA virus‐triggered expression of type I interferon and enhances VSV replication, both in vitro and in vivo. Interestingly, as mentioned before, OTUD4 cleaves K63‐linked polyubiquitin chains after it is phosphorylated. Therefore, it might be the in vivo protein modification that strongly altered the chain specificity of OTUD4. TRAF6 and TRAF3 play crucial roles in the RLR‐mediated antiviral signaling pathway by recruiting other related proteins to form a complex that facilitate NF‐κB signaling. A study has demonstrated that USP4 is capable of targeting TRAF6 for K48‐linked deubiquitination and inhibiting enterovirus 71 (EV71) replication. USP25 interacts with TRAF3 and TRAF6 following RNA virus or DNA virus infection and prevents TRAF3 and TRAF6 from proteasomal degradation via deubiquitinating K48‐linked polyubiquitin chains. Furthermore, USP25 deficiency results in enhanced ubiquitination and turnover of TRAF6 and TRAF3. Consistently, USP25 deficient mice were shown to be more susceptible to H5N1 or HSV‐1 infection compared to their wild‐type counterparts.Over the years, a growing number of DUBs have been identified to upregulate the RLR‐mediated signaling pathway. Since K48‐linked polyubiquitination may bring signaling molecules to proteasomal degradation, several DUBs were identified to reverse the process. Among them, USP17 and USP4 are the only two identified DUBs that are responsible for upregulating the stability of RIG‐I. USP17 facilitates RIG‐I‐ and MDA5‐mediated induction of type I IFN via the deubiquitination of RIG‐I, as well as MDA5. Furthermore, USP17‐deficiency inhibits the RNA‐virus‐induced expression of type I IFN and antiviral responses.176 US Mechanistically, USP15 interacts with TRIM25 during the late stage of a viral infection, removes the LUBAC‐induced K48‐linked polyubiquitination of TRIM25 at its SPRY domain, and ultimately leads to sustained type I IFN expression. Combined with the negative role of USP15 mentioned above, the opposing roles suggest that USP15 exerts disparate effects depending on substrate recognition or infection severity, thereby underscoring the tight regulation of cellular immune responses.In addition to preventing signaling proteins from degradation by removing K48‐linked polyubiquitin chains, USP15 was shown to upregulate RLR signal transduction by indirectly attaching K63‐linked polyubiquitin chains on RIG‐I. As mentioned previously, the E3 ligase TRIM25 is responsible for K63‐linked polyubiquitination of RIG‐I and it can be degraded by LUBAC‐mediated polyubiquitination. A study has demonstrated that USP15 deubiquitinates TRIM25, thus counteracting the LUBAC‐dependent degradation of TRIM25.181 Me3.3 The main pathway that mediates the immune response to DNA is governed by cGAS. cGAS recognizes DNA and is activated through direct binding, which catalyzes the formation of cGAMP. cGAMP then binds to and activates the stimulator of interferon genes , an endoplasmic reticulum (ER)‐localized adaptor. Upon activation, STING translocates to the Golgi and interacts with TBK1 and IKK, which phosphorylates IRF3 and NF‐κB inhibitor IκBα, respectively. The TBK1/IKKε and IKK pathways are subsequently activated, thus leading to the induction of type I interferons and proinflammatory cytokines  catalyzes K27‐linked polyubiquitination of STING, which serves as an anchoring platform for recruiting TBK1 and promotes its translocation to the perinuclear microsomes. In addition to STING, cGAS can also be ubiquitinated to facilitate signaling. For example, the E3 ligase RNF185 attaches K27‐linked polyubiquitination to cGAS, thereby promoting its enzymatic activity. TRIM56 targets cGAS through monoubiquitination, which results in a remarkable increase in its dimerization, DNA‐binding activity, and cGAMP production. Another E3 ubiquitin ligase that mediates the monoubiquitination of cGAS is RING finger‐interacting protein with C kinase , which is critical for cGAS activation.Ubiquitination is equally important in STING signaling. TRIM56, an interferon‐induced E3 ubiquitin ligase, interacts with STING and targets it for K63‐linked ubiquitination, which induces STING dimerization and recruitment of TBK1.197 Li TRIM29, and TRIM30, all of which target STING for K48‐linked ubiquitination and subsequent proteasomal degradation. Interestingly, the E3 ligase RNF26 competitively promoted K11‐linked polyubiquitination of STING at the same residue targeted by RNF5, thus protecting it from RNF5‐mediated degradation. Besides, K48‐linked ubiquitination of cGAS has been reported as a recognition signal for p62‐dependent selective autophagic degradation, although the E3 ubiquitin ligase(s) involved has not been determined yet.The negative role of ubiquitination in the cGAS‐STING pathway is executed by the E3 ligases RNF5,203 TR Apart from targeting RIG‐I, USP27x was previously identified as another DUB that regulates the stability of cGAS. Unlike USP14, USP27x directly interacts with cGAS and releases the K48‐linked polyubiquitin chains during viral infection. Recently, a study reported that USP29 interacts with cGAS, hydrolyzes K48‐linked polyubiquitin chains on cGAS, and stabilizes cGAS in uninfected cells, or after HSV‐1 stimulation. Following HSV‐1 infection, USP29 knockout mice were shown to be hypersensitive to HSV‐1 and produced less type I IFNs and proinflammatory cytokines than the wildtype control. Moreover, Trex1 deficiency resulted in the hyperproduction of type I IFNs and proinflammatory cytokines and have been associated with autoimmune disorders, such as Aicardi‐Goutieres syndrome (AGS) and systemic lupus erythematosus (SLE). Additionally, Trex1 knockout mice have been previously characterized to exhibit severe systemic inflammation and even lethal autoimmune responses. In the study, genetic ablation of USP29 in Trex1 knockout mice attenuated the related pathological symptoms, highlighting the crucial role of USP29 in the regulation of cGAS. Similar to cGAS, extensive studies have been performed on the discovery of DUBs involved in the cleavage of the K48‐linked ubiquitin chain on STING. For instance, USP20 was shown to interact with and remove K48‐linked ubiquitin chains from STING after HSV‐1 infection, thereby stabilizing STING and promoting cellular antiviral responses. Although deubiquitinating STING alone, USP20 can be recruited by USP18, thus causing USP20 to remove K48‐linked polyubiquitin chains more efficiently, even at a low dose. Furthermore, a deficiency of either USP20 or USP18 has been reported to affect the stability of STING, and subsequent production of type I IFN, upon viral infection. Notably, this process is independent of the enzymatic activity of USP18. CYLD is another DUB that removes the K48‐linked polyubiquitin chains from STING at K150. CYLD knockout mice were shown to be more susceptible to HSV‐1 infection than their wild‐type littermates. More recently, a study identified USP44 as a novel positive regulator of STING. Upon DNA virus infection, USP44 was recruited to the membrane‐localized STING and selectively removed its K48‐linked polyubiquitin moieties at K236. Additionally, USP44‐deficient mice were more susceptible following HSV‐1 infection. All three DUBs seem to prevent STING from proteasome‐dependent degradation through uncoupling K48‐linked ubiquitination after DNA virus infection. However, compared to USP20 and USP44, CYLD‐mediated deubiquitination lacks target specificity in the control of innate immune signaling, as it also targets TRAF2 and RIG‐I. USP20 seems more complicated, as it has also been shown to deubiquitinate and stabilize Unc‐51 like autophagy activating kinase 1 (ULK1), which is a negative regulator of STING. Besides, reconstitution experiments demonstrated that USP44‐mediated regulation of STING signaling is independent of USP20 and CYLD. In light of these, it is likely that USP20, CYLD, and USP44 are not functionally redundant. They may target STING spatially and temporally to fine‐tune the onset and termination of innate immune responses against DNA viruses.The K48 ubiquitin linkage type of cGAS is further supported by USP14, which can be recruited by the E3 ligase TRIM14 to cleave the K48‐linked ubiquitin chain of cGAS, thus promoting its stability Figure . TRIM14 207 Ap DUBs can also cleave atypical polyubiquitin chains on STING, as demonstrated by USP13 deconjugating the K27/33‐linked polyubiquitin chains from STING, which impairs the recruitment of TBK1 to STING. In line with this, USP13‐deficient mice restricted HSV‐1 replication in vivo and were more resistant to HSV‐1 infection. USP21 is another DUB that hydrolyzed the K27/K63‐linked polyubiquitin chain on STING following p38‐mediated phosphorylation, thus negatively regulating the production of type I interferons induced by the DNA virus. STING signaling can also be negatively regulated by USP22, which interacts with USP13 and specifically cleaves K27‐linked ubiquitin chains on STING, while the catalytic activity of USP22 is dispensable for this effect.DUBs also play a role in downregulating the STING pathway Figure . K63‐lin217 DUIn light of these studies, it is conceivable that STING is regulated by numerous DUBs in a spatial and temporal manner so as to initiate and terminate innate antiviral responses more accurately against non‐self. However, it remains to be investigated how these ubiquitination and deubiquitination processes are spatially and temporally coordinated.4κB and IRF3. IKK phosphorylates IκB, the inhibitory subunit of NF‐κB that sequesters NF‐κB in the cytoplasm, and the phosphorylated IκB leads to its own ubiquitination and subsequent proteasomal degradation, thus enabling the release of NF‐κB to the nucleus and the transcription of proinflammatory cytokines. Conversely, TBK1, along with its homolog IKKε, mediates phosphorylation and activation of IRF3. Upon activation, IRF3 dimerizes and translocates to the nucleus, thus leading to the production of type I IFN , and TRAF‐interacting protein (TRIP) conjugate K48‐linked polyubiquitin chains on TRAF3 and TBK1, resulting in their proteasomal degradation and inhibition of type I IFN production. DUBs targeting TBK1 cannot be ignored as several DUBs have been shown to be involved in the regulation of TBK1 activity , a protein of the Importin α family that is involved in the import of proteins into the cell nucleus, thus efficiently boosting nuclear translocation of IRF3 and subsequent antiviral responses.As for the downstream molecule of TBK1, increasing evidence has shown the regulation of IRF3 activity via the ubiquitin system. Multiple E3 ubiquitin ligases, such as TRIM26, Pin1, RBCC protein interacting with PKC 1 (RBCK2), Ro52, and RTA‐associated ubiquitin ligase  target IRF3 for K48‐linked polyubiquitination, thus negatively regulating the antiviral innate immune response.243 To5Table Table Our discussion in the preceding sections of this review focused on how the host performs antiviral immune responses with DUBs. A list of host DUBs targeting key molecules in three major innate immune pathways is summarized in USP). Specifically, UL36USP has been reported to act as a viral protein‐mediated immune antagonist by deubiquitinating TRAF3 and dampening IFN‐β signaling. The UL36USP knockout virus leads to increased production of IFN‐β compared to wild‐type virus. UL36USP was also shown to restrict NF‐κB activation in the DNA sensing signaling pathway to evade host antiviral immunity. Mechanistically, UL36USP cleaves polyubiquitin chains from IκBα and abrogates proteasomal degradation of IκBα, thus inhibiting NF‐κB activation. In addition, UL36USP dampens the IFN‐β‐induced activation of interferon‐sensitive response element (ISRE) promoter and the transcription of ISGs, by specifically binding to the interferon α and β receptor subunit 2 (IFNAR2) subunit, and blocks the association between Janus kinase 1 (JAK1) and IFNAR2. This study underscores the UL36USP‐IFNAR2 interaction as a novel evasion strategy exploited by HSV‐1. Homologs of UL36USP have also been identified in other viruses in the Herpesviridae family, such as human cytomegalovirus (HCMV), murine γherpes virus 68 (MHV‐68), Marek's disease virus (MDV), and Kaposi's sarcoma virus (KSHV). Although the DUB activity was identified, the role of these viral DUBs in hijacking host immune systems remains unknown. ORF64USP is the tegument protein of Kaposi's sarcoma‐associated herpesvirus (KSHV). ORF64USP was found to process K48‐ and K63‐linked polyubiquitin chains in vitro, and to target and deubiquitinate RIG‐I, thus leading to an inhibition of IFN‐I signaling. HBx, encoded by hepatitis B virus, another DNA virus, deubiquitinates both RIG‐I and TRAF3 for K63‐linked ubiquitin chains and inhibits the production of type I interferon. Epstein‐Barr virus (EBV) is a human oncogenic herpesvirus that presents a prolonged latent infection in the host. It has been found that EBV‐encoded BPLF1 interacts with and deubiquitinates TRAF6, inhibiting NF‐κB signaling, and leads to viral lytic DNA replication upon lytic infection. BPLF1 was also shown to inhibit TLR signaling via the deubiquitination of downstream signaling components, such as NEMO and IκBα.Virus‐encoded DUBs have been identified in DNA viruses. An example of a DNA virus‐encoded DUB that interferes with the host antiviral immune responses is the herpes simplex virus 1 (HSV‐1) ubiquitin‐specific protease  encoded PLP2 was reported to strongly inhibit cellular type I interferon expression. Further investigation revealed that MHV PLP2 achieves this by targeting both IRF3 and TBK1, and affects both activities. Additional CoV PLPs include Porcine epidemic diarrhea virus (PEDV) replicase encoded PLP2, which was identified as an antagonist to both RIG‐I‐ and STING‐mediated IFN signaling by the reduction of RIG‐I and STING ubiquitination via its deubiquitinating activity that cleaves both K48‐ and K63‐linked polyubiquitin chains.In addition to DNA viruses, RNA viruses have also evolved DUBs that involve in virus‐host interplay. Coronaviruses (CoVs) are a large family of RNA viruses that cause respiratory tract infections, ranging from the mild common cold to serious diseases such as severe acute respiratory syndrome (SARS), middle east respiratory syndrome (MERS), and the recent outbreak of the novel coronavirus, coronavirus disease 2019 (COVID‐19). These three diseases result from SARS‐CoV, MERS‐CoV, and SARS‐CoV‐2, respectively. Both SARS‐CoV and MERS‐CoV encode a papain‐like protease (PLP), featuring both deubiquitinating and deISGylating activities, which subsequently blocks the innate immune signaling of the proinflammatory cytokines.260 Se Previous studies have reported that the arterivirus‐ and nairovirus‐encoded DUBs resemble the OTU family of DUBs, and in the case of arteriviruses, the domain was characterized as PLP2. Notably, PRRSV‐encoded PLP2 also targets IκBα. The foot‐and‐mouth disease virus (FMDV) is among the picornavirus family. Its product, Lpro, targets not only RIG‐I, but also TBK1, TRAF3, and TRAF6, thereby collectively suppressing the host innate immune signaling pathways. For the Seneca Valley virus (SVV), another picornavirus, a study has shown that SVV 3C protease (3Cpro) harbors deubiquitinating activity and acts on both K48‐ and K63‐linked polyubiquitin chains. The 3Cpro deubiquitinates RIG‐I, TBK1, and TRAF3, and consequently abolishes IFN‐β induction and IFN stimulated gene 54 (ISG54) mRNA expression. We have listed these viral DUBs in Table In addition to coronaviruses, several other RNA virus‐encoded DUBs identify RIG‐I as solid targets. These DUBs are encoded by the Equine arteritis virus (EAV), porcine reproductive and respiratory syndrome virus (PRRSV), simian hemorrhagic fever virus (SHFV), and lactate dehydrogenase‐elevating virus (LDV), all belonging to the arterivirus family, as well as the Crimean‐Congo hemorrhagic fever virus (CCHFV), belonging to the nairovirus family.273 Pr M1‐linked ubiquitination is indispensable in antiviral responses elicited by hosts. Recently, a unique M1‐linked ubiquitin chain‐specific effector DUB was identified in pathogenic bacteria; however, whether viral pathogens have evolved DUBs to cleave M1‐linked ubiquitin chains remains a question to be answered. The interactions between hosts and invading viruses are an emerging research field that provides deeper insights into viral pathogenic mechanisms. Thus, further research to elucidate the viral manipulation of DUBs is of particular importance.Although it has been shown that various viral DUBs regulate the same target protein or pathway as their host cell counterpart, these virus‐encoded DUBs share no sequence or structural homology, which reflects the diversity and complexity of host‐virus interplay.279 M16Given the important roles of DUBs in innate antiviral immune response, and since DUBs harbor a catalytic domain or active center functioning as the target of inhibitors, it is not surprising that DUBs will be considered as potential therapeutic targets for a wide range of diagnoses and treatments. The compound IU1, a reversible small‐molecule inhibitor of USP14, was shown to target the USP14 catalytic site. Functional assays revealed that IU1 inhibits replication of several flaviviruses, especially the Dengue virus. Similarly, WP1130, directly inhibiting the DUB activity of USP5, UCH‐L1, USP9x, USP14, and UCH37, shows broad‐spectrum antiviral activity for several RNA viruses. Evidence showed that WP1130 inhibits USP9x activity through covalent modification of critical cysteine residues important for USP9x catalytic activity. PR‐619 is a general DUB inhibitor that nonetheless does not target other cysteine proteases like cathepsin B or calpain 1. In addition, P22077 was shown to covalently modify the catalytic cysteine of USP7 and induce a conformational switch in the enzyme associated with active site rearrangement, thus impairing HIV Gag processing and reducing the infectivity of released virions.In emerging basic studies, DUBs have shown promising prospects as drug targets for infectious diseases.33 The To illustrate, GRL‐0617 was shown to be a potent, selective, and competitive noncovalent inhibitor of SARS‐CoV PLP. Several compounds, both clinical and preclinical, targeting SARS‐CoV PLP have been previously reviewed. The worldwide outbreak of SARS‐CoV‐2 compelled researchers to struggle with countermeasures to fight infection and prevent the spread of the virus. Since PLP inhibitors may be effective in reducing coronavirus infections, we propose that PLP inhibitors should be considered as a latent therapeutic strategy for COVID‐19. Recently, sequence analysis showed that the PLP present in SARS‐CoV‐2 has a high degree of similarity to that in the extensively studied SARS‐CoV. Given that many active compounds against SARS‐CoV PLP have been clearly identified, evaluation of these inhibitors in targeting SARS‐CoV‐2 PLP should be put on the agenda. Intriguingly, several recent studies demonstrated that GRL‐0617 not only inhibits SARS‐CoV PLP but also inhibits the activity of SARS‐CoV‐2 PLP, thus impairing the virus‐induced cytopathogenic effect, facilitating the antiviral signaling, and reducing viral replication in infected cells.As mentioned above, viral DUBs play pivotal roles in the regulation of host cellular processes, which provides us with novel insights into the possibility and validity of developing inhibitors that target the viral DUBs rather than the host DUBs. Indeed, several studies support the idea that PLP inhibitors may be effective in reducing coronaviruses infection.294 To Third, previous inhibitors against viruses were designed to inhibit viral replication and inherent survival. The targets of these inhibitors are essential proteins for virus replication, assembly, and survival, which are effective in killing viruses in a short time. However, due to strong selection pressure, the virus mutates or evolves to obtain long‐term resistance. Therefore, how to improve antiviral drugs to counter drug resistance is a key point.Despite the promising insights on targeting both host and viral DUBs as an antiviral strategy, challenges exist in the following aspects. First, as previously mentioned in this review, DUBs function in delicate mechanisms with complexity. For instance, different DUBs may target the same substrate, while some may target more than one substrate. DUBs may perform opposing roles depending on the context. As an example, USP7 would be a plausible target in the oncology context, but inhibiting USP7 may have a potential impact on inflammatory responses. Hence, given the complexity and pleiotropy of DUBs, comprehensive approaches and diverse strategies are needed to develop these inhibitors. Second, there are still many unknowns about how DUBs work and are modulated. To date, many studies merely link DUBs to their functions in vitro, which is unconvincing, as the bona fide DUB activity in vivo may not be reflected.300 ThNevertheless, based on the pivotal role of DUBs in pathogenesis, the druggable feature, and the existing positive evidence against viral infection, developing inhibitors targeting DUBs, especially viral DUBs, should not be underestimated and might deserve at least a further evaluation.7Figure As a key regulator of the ubiquitin system, DUBs play a pivotal role in antiviral innate immunity at different layers. Generally, DUBs act alone or in a complex to strengthen their catalytic activities. Specifically, the mechanisms underlying the function of DUBs in the antiviral signaling pathway can be divided into six working models, according to the pattern of altering the ubiquitination status Figure 7: i) moThe effects of DUBs are intricate. While some DUBs positively regulate antiviral pathways, others may function as negative regulators. Still, some DUBs play dual roles (either inhibitory or activating) depending on specific contexts, such as subcellular localization or the type of substrate proteins. Some even act on identical substrates with opposing outcomes (such as USP4 and USP15).Significant efforts have been dedicated to unveiling the virus‐encoded DUBs that are responsible for hijacking the cellular ubiquitin system to counteract host defense and evade host surveillance. While every effort was made to include in this Review all DUBs that have been identified, to date, in innate antiviral immunity, many more undoubtedly remain to be characterized. Many studies included in this Review relied on molecular and cellular approaches, which fail to accurately reflect the authentic circumstances that occur in vivo. Hence, further research is needed to ascertain the cellular targets and function of DUBs with in vivo animal experiments.Given the growing evidence showing the importance of DUB activity in innate antiviral immunity, it is reasonable to consider DUBs as promising drug targets for infectious disease treatment. Such strategies should focus on tightly regulating the antiviral immune responses and enhancing infected cell death by modulating the DUBs involved. Additionally, virus‐encoded DUBs may provide attractive therapeutic targets for antiviral therapy. Given the importance and urgency of finding treatments for COVID‐19, the recommendation to evaluate potential drugs targeting SARS‐CoV‐2 PLP should not be underestimated. Overall, given the success of DUB inhibitors for cancer therapies, it is promising that the development of small‐molecule inhibitors or agonists targeting DUBs is on the right path.There are still many gaps in our current knowledge and understanding of DUB mechanisms, which hinders progress in the therapeutic application of research in this area. First, unlike the designated roles of the canonical K63‐ and K48‐linked polyubiquitin chains, some atypical linkage types of polyubiquitin chains may exhibit quite distinctive roles. Yet, these atypical polyubiquitin chains are rarely reported in innate antiviral immune signaling. Thus, further research endeavors to elucidate the atypical polyubiquitin chains recognized by DUBs are required. Second, the activation and inhibition of key signaling molecules are determined by both E3 ligases and DUBs. How these E3 ligases and DUBs cooperate in a dynamic, temporal, and spatial manner to strictly regulate the immune response needs to be clarified further and deeper. Moreover, compared with the vast number of human DUBs, the number of virus‐encoded DUBs in control of host immunity appears limited. Therefore, identifying more viral DUBs that are responsible for modulating host immune system warrants increasing efforts to propel this field forward.The authors declare no conflict of interest.Z.Z., Z.K.Z., and L.W. contributed equally to this work. Z.Z. and Z.K.Z. conceived and drafted the manuscript. Z.Z. drew the figures. L.W. discussed the concepts of the manuscript. F.Z. and L.Z. provided valuable discussion and revised the manuscript. The authors apologize to those researchers whose related work were not able to cite in this review.


The aim of the present study was to identify factors associated with h delayed initiation of post-exposure prophylaxis (PEP) among animal bite victims.This cross-sectional study assessed biting patterns among 3032 cases that were referred to Tabriz Rabies Center. The delay was described as the initiation of PEP more than 48 hours (h) after possible exposure to the rabies virus. Determinants of delay in initiating PEP were recognized by a decision tree model.Totally, 8.5% of the victims who were bitten by an animal had a delay of more than 48 h in the initiation of PEP. The relative frequency of a delay more than 48 h in females was higher than in males (12.9% compared to 8.5%) . Relative frequency of a delay of more of 48 h from carnivorous  was significantly less than others . Of the decision tree, the overall classification accuracy was 89.5%, with 44.1% sensitivity and 92.3% specificity. The identified variables included gender, biting place , and type of animal.according to the results of the present study, among the various variables that affect the delayed initiation of PEP, rural residents and being female, in particular, were the major factors associated with a delay in the initiation of PEP for rabies prevention. We found relatively low rates of vaccine completion. Our findings indicat that providing training and patient education are required to ensure the completion of appropriate treatment. Animals attack is still a major health and social issue worldwide. An animal bite is the main source of transmission of rabies to humans, which has not yet been controlled in most parts of the world . ApproxiAfter the onset of clinical symptoms, there is no effective treatment for rabies. Therefore, the currently recommended intervention strategy is to remove and neutralize the infectious virus before it enters the nervous system . InapproAnnually, 180000 animal bite cases are reported in Iran, all of whom receive PEP because all biting animals such as cats, dogs, and wolf are considered to be reservoirs of rabies .In this regard, the community needs to be adequately educated to ensure them to take care of themselves in preventing rabies and the consequences caused by animal bites . BesidesTreating a rabies exposure, where the average cost of rabies PEP is US$ 40 in Africa, and US$ 49 in Asia, can be a catastrophic financial burden on affected families whose average daily income is around US$ 1–2 per person . PEP is Study designThis cross-sectional study investigated the patterns of animal bites among the cases referred to the rabies center of Tabriz between March 1 2013 and February 29 2019. A sequential sampling of 3032 patients with animal bite history visiting the rabies center was performed. The data were taken from rabies surveillance forms used in the district. These forms are used to identify and follow-up the suspected bites. When a bite case is admitted to a health center, a health worker for communicable disease control performs an examination, decides whether prophylaxis is necessary, fills out the form and follows-up the case until the end of PEP. Data mining was used via the decision tree model to identify factors affecting delay in vaccination 48 h subsequent to bites to prevent rabies. Study Site Post-prophylactic rabies centers have been established in provinces and even in small towns in Iran under the supervision of the Ministry of Health and Medical Education. The study was conducted at the rabies center of Tabriz which is affiliated to Tabriz University of Medical Sciences.Study populationAll cases of animal bite in all age and gender groups which had been referred to the Rabies Center of Tabriz were investigated.An animal bite was defined as any animal bites caused by mammals. Data were extracted for the patients who were registered at the health center and referred to the health center of Tabriz city to receive a rabies vaccination. Ethics approval and consent to participateThe institutional ethical review board reviewed and approved the study protocol in Tabriz, Iran (IR.TBZMED.REC.1397.1096). All data have been anonymized and treated confidentially.Data collectionWe collected data per span (year) on the number of animal bite injuries. To collect the data, we used the following a structured data abstraction tool to extract the required data form recoded forms: age ; sex ; occupation ; biting place ; being stray (yes/ no); place of injury in human body ; puncture wounds ; animal status ; time of event . The dependent variable was: PEP initiation delay which was defined as the initiation of PEP more than 48 h after an animal bite. We looked into the time of injury and time to visit with the respondents. In the present study, PEP delay was defined as the initiation of PEP more than 48 h after a possible exposure to the rabies virus. The variable was coded according to: 0= “prompt PEP”, 1= “delay of initiation of PEP”.Input variablesAfter cleaning and preparation of the data, the final dataset consisted of 3032 records. The outcome of interest was the delay more than 48 h in initiation of PEP which was assessed by the model based on several input variables presented in Construction of decision treeAs the number of instances in this study was enough, the decision tree was built with the holdout method in which data are randomly partitioned into two independent sets, a training set and a test set.We divided the original dataset into two parts using stratified random sampling based on the target variable, with the training dataset containing about 70%, and 30% of the participants as the testing dataset. The Gini impurity index was chosen as the attribute selection measure. Where pi is the probability that an example in D belongs to class Ci, and is estimated by [Ci, D|/|D|. The sum is computed over m classes. The Gini index considers a binary split for each variable. In the decision tree, the first variable (root) is the most important factor and variables far away from the root are the next important factors in classifying the data. For easy understanding, the decision tree can be converted to a set of If-Then rules by tracing the path from the root node to each terminal (leaf) node. All the variables in one path are considered as predictors (If part) and the class label of the leaf node is expected outcome (Then part). These rules are extracted just by top down tracing of the path and there is no rank or weight for the rules. For classifying a new person, we should start with the root node of the decision tree and moving along the path that the person belong it until the leaf node is reached. The decision about the person is determined based on value of leaf node which is usually positive or negative with a certain probability .In addition, missing values for numerical features were handled by setting their values to average value, and replacing the most frequent value for nominal features.Classification is the process of finding a model (or function) describing and distinguishing data classes on concepts using the model to predict object classes . ClassifWhere J is the number of classes or the target variables, π(j) is the prior probability for class j, p (j|m) is the probability that node m includes observations of class j, and Gini (m) is the Gini index, which indicates impurity in node m. The Gini index is 0, if all the observations in a single node belong to a unique class that displays the least impurity, and is equal to 1-1/i, if results in different classes in one node are of the same proportion. In this situation, the maximum tree that overfits the training data has been created. Reducing the complexity of the end tree and generating simpler trees, based on a cost-complexity algorithm, the tree will be “pruned”. In the CART method, the decision tree is getting bigger and more so until each terminal node has the same observations. Gini index the degree or probability of a particular variable being wrongly classified when it is randomly chosen.Statistical analysisMean ± standard deviation (SD) for continuous variables and frequencies (%) for categorical variables were used to demonstrate baseline characteristics of the participants. Factors associated with PEP treatment with dichotomized and categorical variables were tested using chi-square test as a univariate study.In total, 3032 animal bites were recorded through 2013-2019; with no human rabies cases. The mean age of subjects was 33.71± 18.50 years. Cases ranged in age from 1 to 91 years, and 2438 (80.4%) were males. Demographic and injury Characteristics of the study population, and the number of victims bitten by the animals are shown in According to the results of the chi-square test, the relative frequency of a delay of more than 48 h in females was higher than the males (12.9% vs. 8.5%) . Relative frequency of a delay of more of 48 h from carnivorous  was significantly less than others . There was a significant difference between the animal status and delay in initiation of PEP according to the chi-square test  to be more specific the relative frequency of delay was higher in cases that the biter animal is dead after bite., animal status and biting place, respectively [The Gini index, as an impurity function of the CART algorithm, showed that the most important variables for predicting the delay of PEP include: type of animal, genderectively .We developed a decision tree with instruction set (3032 records). The criteria for building the tree included minimum record number per node, the pruning process and attribute selection measures; minimum number represents a stopping condition for further data partitioning at decision nodes. Briefly, various decision trees with different ‘minimum records’ were built and the value of 50 was chosen which resulted in best performance. The Gini index was selected as measure of attribute selection, and the tree were kept unpruned. The overall classification accuracy was 89.5%, with 44.1% sensitivity and 92.3% specificity.According to If the biting animal is other animals, 20.5% will have a delay of more than 48 h.If the biter animal is a dog or cat and gender of the victim is female, 10.9% had a delay of more than 48 h.If the biting animal is a cat or dog and the gender of the victim is female, as well as biting place is rural area, 15.3% will have a delay of more than 48 h.If the biting animal is a cat or dog and gender of the victim is male, as well as animal status is escaped or dead, 9.1% will have a delay of more than 48 h.Post-exposure vaccination against rabies is essential for prevention of this fatal disease. Obviously, several essential factors in the proper implementation of PEP, are available. Many factors influence timely access to PEP and its administration. This study investigated the factors causing delay in receiving anti-rabies PEP.In our study, 8.5% of the victims did not receive timely PEP treatment. The WHO advises that immediate washing and flushing of wounds with soap and water for at least 15 minutes, or water alone, and disinfection with substances with anti-viral activity is essential after exposure to rabies virus . Taking The present study found a higher rate of delay among women compared to men victims of animal bite (11.4% compared to 7.8%). A study conducted in shiraz province, Iran , which hAs demonstrated in a previous study, some people may think that domestic animals are less dangerous than roaming ones . On the On the presence of watchdogs in most rural households in the study area, appropriate education programs should be provided for the teaching of behavioral skills in high-risk groups. Similarly, those who live far from health centers, and are in lower socioeconomic classes undergo longer delays in receiving PEP, which increases the risk of developing rabies .In our study, delay more than 48 h belonged to the rustic region. Because of development of the primary health care (PHC) in Iran in past decades, and availability of health houses and health centers in almost all villages, it seems that there is no physical barrier to access the care animal bite victims . It is rBased on various variables that affect the initiation of PEPbeing bitten by a carnivorous, being bitten in the rural places, and especially being a female. Development of educational programs for dog owners, especially in rural areas, and increasing public awareness may help us prevent the delay in initiation of PEP for animal bite victims. First, given the limitations of the cross-sectional study design, it is not possible to claim causal effect. Due to the study’s retrospective design, authors were unable to collect other variables such as education level, past history of rabies vaccination, socioeconomic status, and other variables related to delay in receiving PEP. Possible recall bias by the animal bite victims in remembering the time of bite may result in information bias in calculation of delay of treatments.This study was supported by the Tabriz University of Medical sciences, Tabriz, Iran. And the fund number was 60198.


We investigated the prevalence of retinal vascular occlusion and intraocular bleeding and compare their risks in patients undergoing anticoagulant therapy, either with non-vitamin K-antagonist oral anticoagulants (NOAC) or warfarin. We performed a cohort study (January 2015 to April 2018) in 281,970 patients with nonvalvular atrial fibrillation (AF) using health claims in the nationwide database of the Health Insurance Review and Assessment service of Korea. A Cox-proportional hazard regression was used to calculate the hazard ratio (HR) for retinal vascular occlusion or intraocular bleeding. The HR of retinal vascular occlusion was estimated to 1.59  for NOAC users compared to that with warfarin users. Among the various types of NOACs, all NOACs showed higher risk of retinal vascular occlusion than did warfarin. For intraocular bleeding, the HR was estimated to be 0.86  for NOAC users compared with that with warfarin users. The risk of retinal vascular occlusion was higher in NOAC users than in warfarin users, while the risk of intraocular bleeding was lower with NOAC therapy. NOACs were not found to be as effective as warfarin for retinal vascular occlusion, but safe in terms of intraocular bleeding. Warfarin can effectively prevent stroke and systemic embolism in patients with AF but increases the incidence of major bleeding such as intracranial hemorrhage2. Non-vitamin K-antagonist oral anticoagulants (NOACs) have been recently approved for prevention of stroke in patients with non-valvular AF, and do not require anticoagulation monitoring3. Since NOACs are at least as effective and safe compared to warfarin, they have become essential for patients with non-valvular AF4.Non-valvular atrial fibrillation (AF) is a global health burden and the prevention of AF-related thromboembolic events is a major concern5; retinal vein occlusion (RVO) is associated with thromboembolism due to degeneration of vascular walls and compression or vasospasm6. Retinal vascular occlusion is not only associated with predisposing factors for atheroembolic diseases7, but also with the risk of stroke, myocardial infarction, and total mortality8.Retinal vascular occlusion is a major vascular disease of the retina that can lead to severe visual impairment. Retinal artery occlusion (RAO) results from retinal thromboembolic events originating in the ipsilateral carotid artery, aortic arch, or cardiac origin9. Hence, several studies on ocular diseases have examined the impact of anticoagulant therapy with NOAC, with a focus on ocular bleeding as a major safety issue11. While NOACs benefit patients with stroke and systemic thromboembolisms12, their efficacy on microvascular conditions, such as retinal vascular occlusion, requires further evaluation.In terms of the safety in patients taking anticoagulants, intraocular bleeding is not a life-threatening ‘major’ complication, but is critical and can lead to severe visual loss and decrease vision-related quality of life. Previous studies have reported that there is an increased risk of intraocular bleeding with warfarin or other oral antithrombotics, raising concerns over the safety of these medicationsThis study is to verify the efficacy and safety of NOACs in retinal disorders in terms of the incidence of retinal vascular occlusion and intraocular bleeding. We compared these incidences in NOAC and warfarin users by using a Korean cohort study.The patients were initially categorized according to anticoagulant treatment. Among patients with non-valvular AF, 93,691 used NOAC and 27,496 used warfarin , and lowest with apixaban (2.66).The average follow-up period was 2.68 ± 1.33 years for warfarin users and 1.24 ± 0.84 years for NOAC users. The baseline characteristics are listed in Table P for interaction = 0.0017). Among the different types of NOAC, when compared with warfarin, the HR for retinal vascular occlusion was 1.49, 1.47, 1.66, and 1.64 for dabigatran, rivaroxaban, apixaban, and edoxaban, respectively  for warfarin users and 0.82%  for NOAC users. Person-years were taken into account as the follow-up period differed by patient, and the NOAC users showed higher incidence rate (IR) compared to warfarin users (IR 6.03 vs. 3.33 per 1000 person-years). The NOAC group had a higher risk of retinal vascular occlusion than the warfarin group (hazard ratio (HR), 1.61; 95% confidence interval (CI) 1.37–1.91), which was consistent after adjusting multiple covariates  but not for RAO with NOAC . These were also consistent after adjusting for multiple covariates . There were no differences for other retinal diseases, including age-related macular degeneration, choroidal neovascularization, and retinal vascular occlusion.In patients with underlying retinal vascular disorders related to intraocular bleeding, more patients with diabetic retinopathy were administered warfarin than NOAC . Among the different types of NOAC, the HR of intraocular bleeding was 0.84, 0.85, 0.80, and 0.81 for dabigatran, rivaroxaban, apixaban, and edoxaban respectively when compared with warfarin 13. A cohort study reported that the incidence of intraocular bleeding was less in NOAC (dabigatran and rivaroxaban) users compared to that of warfarin users9. Other meta-analyses also revealed a lower risk of intraocular bleeding with NOAC use15, suggesting the safety of NOAC for intraocular bleeding.NOACs are known to be non-inferior or even superior to warfarin for prevention of stroke and systemic embolism in patients with non-valvular AF, and also associated with lower rates of bleeding, especially life-threatening hemorrhage , so that NOACs may preserve hemostatic mechanisms by selectively targeting thrombin and not interfering tissue factor-VIIa complexes16. Warfarin has limitations related with the diet and drug interaction and the need for anticoagulation monitoring, and the pharmacokinetics of warfarin is less predictable due to the polymorphisms in the genes 19. Accordingly, the lower risk of bleeding with NOACs may be associated with the selective actions on coagulation pathway compared to warfarin3.Our study also showed that NOAC users have a lower association with intraocular bleeding than did warfarin users. The mechanism for lower bleeding risk is not fully understood, but several points suggested in studies on major bleeding might be applied. Warfarin inhibits vitamin K-dependent proteins , thereby suppressing production of factor VIIa and formation of tissue factor-VIIa complexes21. There are also cohort studies which have reported that retinal vascular occlusion increases the risk of stroke24. Interestingly, our study found that the risk of retinal vascular occlusion was higher in NOAC users than in warfarin users in Korea. Although this study did not investigate mechanisms associated with the higher risk of retinal vascular occlusion in NOAC users, the selective inhibition of specific coagulation factors by NOACs might be responsible4. NOACs are safe in terms of bleeding complications by preserving hemostatic mechanism3, while this would work the other way around as more retinal vascular occlusion in NOAC users. The lower trough level of NOACs compared to that of warfarin might be another factor for the inefficacy of NOACs25. It should be noted that patients with newly diagnosed retinal vascular occlusions are often referred to internists in clinical practice, who then evaluate systemic risk factors for future cardiovascular events and often prescribe anticoagulants26. However, there is a lack of high-quality evidence to support the routine use of anticoagulants or antithrombotic drugs for retinal vascular occlusions, and the benefits and risks of this therapy need to be considered in clinical settings27. Based on our findings, the appropriate anticoagulant should be selected in each patient based on the risk of intraocular bleeding and retinal vascular occlusions.Retinal vascular occlusion is often associated with cardiovascular disease and have many of the same risk factors28. On the other hand, the precipitating factors for RVO are more variable: vasospasm, inflammation, compression, and localized thrombosis6. Typical atherosclerosis risk factors are commonly associated with all types of RVO, while hyperviscosity and hypercoagulability are also related risk factors26. Second, RAO is relatively uncommon as compared to RVO20, which may affect statistical significance. Finally, the exact doses for anticoagulants were not investigated in this study. There is a tendency in Asian countries to use the lower doses of NOACs in clinical practice30, which may have affected the results of this study.The risk of RAO was comparable between the NOAC and warfarin groups, while the risk of RVO was higher in the NOAC group compared to the warfarin group. There are possible reasons for this difference in efficacy of NOACs, besides the pharmacokinetics of NOACs acting on specific factors in coagulation pathway. First, RAO is related with atherosclerotic thromboembolism similar to stroke, and retinal artery emboli are commonly associated with AF, hypertension, and coronary artery disease31. Most studies showed comparable major bleeding rate in patients without significant interaction with diabetes31, while there was also a report that diabetic patients showed a reduced rate of intracranial hemorrhage when treated with dabigatran than with warfarin32. The mechanisms for this lower risk of bleeding in diabetic patients are not clarified, but less diet and drug interactions with NOACs might explain one of possible reasons for the safety of NOACs in diabetic patients3. In terms of retinal vascular occlusion, female patients showed a higher risk of retinal vascular occlusion with NOACs than male patients. Our study did not investigate the dosage by sex, while more female patients are known to be prescribed lower dose of NOACs in Korea30. This may result in a higher risk of retinal vascular occlusion with lower efficacy. Similarly, off-label low dose of NOAC was associated with increased risk of cerebral ischemic events33, and these tendencies of ischemic event need further investigation along with the gender difference.There were some interesting points in subgroup analyses. Diabetic patients had a remarkably lower risk of intraocular bleeding with NOACs than the opposite group. Diabetes is a well-known risk factor for ischemic stroke, while there were controversies on hemorrhagic risk associated with NOAC treatment in diabetic patients with AFi.e. intraocular bleeding, all the NOACs showed a lower risk of bleeding than did warfarin. This is similar to previous cohort studies which reported a reduced risk for intraocular bleeding15. Few guidelines have addressed the issue of anticoagulant use for treatment of retinal vascular occlusions and the possible risk for intraocular bleeding26. No specific medication has been established to directly improve perfusion, and current ophthalmic treatments focus on the complications of retinal vascular occlusions such as macular edema, ocular neovascularization, and intraocular bleeding7. All NOACs have a merit in terms of safety for intraocular bleeding; however, efficacy for the prevention of retinal vascular occlusion was not clarified in this study. As the incidence and prevalence of AF increases with advancing age, further studies are needed to investigate the efficacy of NOACs for preventing microvascular disease.There was no definite superiority among the different type of NOACs. All the NOACs showed a higher risk for retinal vascular occlusion and a comparable risk of intraocular bleeding than did warfarin. Regarding ocular safety in the present study, 29. We could not classify the patients according to dose as many patients changed the dose during the study period. Third, detailed information on patients’ history of socioeconomic status, smoking, or alcohol consumption, and laboratory profiles including international normalized ratio (INR) of prothrombin time were not available from the HIRA database. Some of the patients on warfarin could be in supratherapeutic range of INR, but this is less likely as the proportion of warfarin users in supratherapeutic range is lower than those in subtherapeutic range in Asia34. Although we adjusted for various confounding factors using diagnostic and procedure codes from the HIRA database, more studies in real clinical practice are needed to confirm the effects of NOACs in retinal microvascular diseases. Lastly, the findings in this study need cautious interpretation due to the retrospective nature. Although the association of NOACs and higher incidence rate of retinal vascular occlusion was found in this study, further studies prospective in nature are needed to verify this association.This cohort study has some limitations. First, the presence of retinal vascular occlusion was defined by the presence of diagnostic codes from HIRA database. Although the diagnostic codes are mandatory for any patient, the accuracy of diagnoses was not confirmed by the medical records. However, this limitation might affect both NOAC and warfarin groups and therefore, a one-sided application might be prevented. Second, we could not investigate the difference between standard-dose NOAC users and low-dose NOAC users. As mentioned above, Asia shows a tendency for prescribing lower doses than do Western countriesIn conclusion, the present study showed that use of a NOAC was not superior to warfarin in terms of retinal vascular occlusions, unlike its effect on the treatment of stroke or systemic embolism. However, NOACs were safe in terms of intraocular bleeding. Further studies in clinical practice may help to identify the most appropriate anticoagulants for patients with microvascular diseases.This retrospective cohort study protocol was reviewed and approved by the Institutional Review Board of Ajou University Hospital (AJIRB-MED-EXP-18-380). The requirement for informed consent was waived by the Institutional Review Board of Ajou University Hospital as the data in this database were de-identified. All methods were performed in accordance with relevant guidelines and regulations.th revision (ICD-10). The inclusion criteria were: (i) patients aged ≥20 years, (ii) ≥ 1 criteria for non-valvular AF  in the primary or within 3rd order of secondary diagnosis, and (iii) ≥1 prescription of NOACs or warfarin between January 2015 and April 2018. The exclusion criteria were patients who had: (i) been diagnosed with AF in 2014 to select the patients who were newly diagnosed with AF from 2015, (ii) both warfarin and NOAC during the study period, (iii) mitral stenosis and/or prosthetic heart valve, (iv) prior diagnosis of retinal vascular occlusion, and (v) aged <20 years at the time of AF diagnosis. Those who were diagnosed with AF prior to 2014 but received a prescription between January 2015 and April 2018 were excluded. These criteria are summarized in Fig. The Health Insurance Review and Assessment (HIRA) service reviews all of the health claims in Korea, including those submitted through the Korean National Health Insurance service which covers 97% of the population. Diagnoses are coded using the International Classification of Diseases, 10Each participant’s index date was set to the date of earliest prescription. The follow-up for each event extended from the index date to the earliest incidence of retinal vascular occlusion, or intraocular bleeding, or the end of the observation date , if events did not occur.The presence of retinal vascular occlusion was determined on the basis of diagnostic codes of retinal vascular occlusions in the primary or within 3rd order of secondary diagnosis, including RAO , RVO , and other retinal vascular occlusions . Similarly, the presence of intraocular bleeding was determined using the diagnostic codes for non-traumatic hyphema , vitreous hemorrhage , retinal hemorrhage , and choroidal hemorrhage (H31.3). For retinal disorders leading to intraocular bleeding, the presence of the previously stated retinal vascular diseases was investigated. The NOACs included those available in Korea and covered by the national insurance service such as dabigatran, rivaroxaban, apixaban, and edoxaban.2DS2-VASc score, and calendar index year. The CHA2DS2-VASc score, which is used in guidelines for stroke prevention in AF implying comorbid diseases , was used in this study including AF patients35. A list of variables with corresponding ICD-10 codes, procedures, operation codes, and anatomical therapeutic chemical codes is provided in Supplementary Table The following covariates were included for baseline adjustments to minimize confounding factors: age at diagnosis of AF, sex, hypertension, dyslipidemia, chronic kidney disease, diabetes mellitus, coronary heart disease, stroke, venous thromboembolism, CHAt-test for continuous variables.The baseline characteristics were summarized and compared for the patients with NOAC and warfarin using a chi-square test of homogeneity for categorical variables and a two sample 2DS2-VASc score, calendar index year, and comorbidities . The time from initiation of NOAC or warfarin to primary events was assessed using the Kaplan-Meier survival curve and compared using the log-rank test. Additionally, the unadjusted cumulative incidence was calculated according to the type of medication.The IRs were stratified by each medication and estimated with the number of retinal vascular occlusion or intraocular bleeding events per 1,000 person-years, and the 95% CI was calculated by the Mid-P exact test. The cox proportional-hazards regression model was used to calculate the HR and 95% CI. The HR with 95% CI was presented with separate statistical models; (i) unadjusted, (ii) adjusted with age and sex, and (iii) adjusted with age, sex, CHAP value of <0.05 was considered significant.In the secondary analysis, the same analysis was performed with different types of NOACs, such as dabigatran, rivaroxaban, apixaban, and edoxaban, and compared with warfarin. Those who switched medications (within the NOAC group) at any point in the time were excluded in the secondary analysis. Additionally, the unadjusted HR was presented by subgroups according to age ≥65, sex, diabetes, and stroke. All statistical analyses were performed using SAS software . A Supplementary information.


As neuronal activity can be achieved using non-invasive measures, it may be of interest to utilize the innate ability of neuronal activity to instruct myelination as a novel strategy for myelin repair in demyelinating disorders such as multiple sclerosis (MS). Preclinical studies indicate that stimulation of neuronal activity in demyelinated lesions indeed has the potential to improve remyelination and that the stimulation paradigm is an important determinant of success. However, future studies will need to reveal the most efficient stimulation protocols as well as the biological mechanisms implicated. Nonetheless, clinical studies have already explored non-invasive brain stimulation as an attractive therapeutic approach that ameliorates MS symptomatology. However, whether symptom improvement is due to improved myelin repair remains unclear. In this mini-review, we discuss the neurobiological basis and potential of enhancing neuronal activity as a novel therapeutic approach in MS.Enhanced neuronal activity in the healthy brain can induce  The most accepted mechanism of remyelination consists of both recruitment and proliferation of oligodendrocyte precursor cells (OPCs) as well as the differentiation of these new OPCs into mature myelinating oligodendrocytes (OLs) . Howeverin vivo, it became clear that activation of neurons can stimulate the proliferation of OPCs as well as their differentiation into myelinating OLs. For example, optogenetic stimulation of layer V projection neurons leads to increased OPC proliferation and differentiation and an increase in myelin thickness (in vivo has different effects on OPC proliferation and differentiation (Initial studies in the 90’s demonstrated that neurons communicate with OL lineage cells at different stages of OL maturation, thereby modulating the myelination process . From rehickness . Moreovehickness . When pahickness . Notablyhickness . In thishickness . Taken tntiation . Indirecntiation . This isntiation . It shountiation . As suchvia mechanotransduction (de novo myelination resulting in behavioral changes. Consequently, this makes stimulation of neuronal activity an interesting approach for myelin repair in demyelinating disorders such as MS.How electrically active axons communicate with OPCs and OLs to promote myelination remains unclear. However, there are multiple synaptic and non-synaptic signaling mechanisms that may play a role . Electrisduction . Taken t+ channel blocker in demyelinated lesions induced by ethidium bromide showed a decreased differentiation of OPCs into myelinating OLs, an increased OL apoptosis and a reduced remyelination (in vivo (While activity-driven myelination is accepted as a key mechanism in the CNS, literature on the effect of neuronal activity on myelin repair is still scarce. Nevertheless, a limited number of studies have reported on the potency of manipulating neuronal activity as a remyelination strategy in animal models. The intracerebral infusion of AMPA/kainate receptor antagonists and a voltage-dependent Nalination . These rlination . Several(in vivo . Distinc(in vivo . This stRecent studies in animal models have addressed the effects of indirect methods of neuronal stimulation on remyelination. As indirect neuronal stimulation methods such as TMS, tDCS and behavioral training are readily applicable in the clinic, it is worthwhile to investigate different stimulation methods in rodents. Sixty Hz electromagnetic stimulation increases remyelination in toxin demyelinated lesions of the rat corpus callosum  and low Behavioral paradigms recruiting remyelinating brain regions might be able to accelerate the remyelination process, likely through increased neuronal activity. A recent study reported increased neuronal firing rates in the motor cortex following cuprizone demyelination that recovered to normal during the same period in which remyelination was completed, suggesting a correlation between the level of neuronal activity and remyelination . Behavio2+ and Ca2+ channel blocking agents have been shown to reduce damage to neurons in demyelinating lesions by inhibiting a neuronal cell membrane depolarizing mechanism (via neuronal activity (Although neuronal activity can be enhanced in a controlled manner to limit deleterious effects such as excitotoxicity, it cannot be excluded that apart from benefits for remyelination, enhanced neuronal activity may negatively affect the demyelinated neurons themselves. Indeed, Naechanism . Furtherechanism . Future activity .in vitro study has shown that magnetic field stimulation induces calcium influx in OPCs and enhances OPC differentiation and OL myelination (per se may also induce direct effects on oligodendroglia in the absence of neuronal activity. Taken together, these findings suggest that complex mechanisms implicating calcium signals in OL lineage cells may be involved in neuronal activity-induced remyelination and that further research is necessary to unravel the associated signaling pathways.Although emerging lines of evidence point to a role of neuronal activity in modulating oligodendroglia dynamics in lesions, few papers have investigated the signaling mechanisms that underlie the communication between electrically active axons and oligodendroglia during remyelination. Since the frequency of calcium transients is increased during remyelination , it is llination , suggestAs emerging evidence suggests that increased neuronal activity can aid remyelination, it is worthwhile to examine studies in which MS patients with demyelinating lesions undergo neuronal stimulation. Deep brain stimulation is the only direct stimulation method that has been tried in MS patients. Several studies have shown that thalamic deep brain stimulation significantly reduces tremor and improves quality of life in MS patients . HoweverOther than non-invasive brain stimulation, exercise and behavioral paradigms have also been shown to positively affect MS symptoms and their effects might be mediated by enhanced neuronal activity. For example, 8 weeks of high intensity exercise improve clinical outcome  and bothde novo myelination in the healthy brain. The facts that neuronal activity can initiate myelination and that neuronal stimulation can be achieved relatively easily make activity-dependent myelination an attractive strategy for myelin repair in demyelinating disorders such as MS. Research into neuronal activity-dependent myelin repair is still scarce, however, the few in vivo preclinical studies that have been conducted suggest that enhanced neuronal activity in a demyelinated lesion indeed has the potential to improve remyelination (see Neuronal activity-dependent myelination can take place regardless of neuronal identity, but the effects of neuronal activity on oligodendroglial cells likely depend on the cumulative pattern of activity of a neuronal network. This means that both direct stimulation of neurons by for example optogenetics and indirect stimulation via non-invasive methods such as TMS and behavioral training can achieve tion see . In rodetion see .It is known that electrically active axons can communicate with oligodendroglia cells via synaptic and non-synaptic sites involving a number of receptors and messenger molecules that often lead to a depolarization of oligodendroglia and subsequent intracellular calcium signals. The nature of the communication from neurons to oligodendroglia and the frequency and amplitude of intracellular calcium increases in oligodendroglia are thought to determine the OL response to neuronal activity. Future studies need to reveal the molecular pathways involved in, and the calcium response characteristics of oligodendroglia to neuronal electrical activity.In conclusion, the stimulation of neuronal activity is a promising strategy for myelin repair in demyelinating disorders such as MS and future research into the exact neurobiological mechanisms as well as clinical evaluation of changes in the brain after neuronal stimulation in patients will aid the development of this novel therapeutic approach to myelin repair.DM and MCA wrote and corrected the manuscript. Both authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.