Multifunctional Flexible Ferroelectric Thin Films with Large Electrocaloric Effect and High Energy Storage Performance.

Flexible ferroelectric films with high polarization hold great promise for energy storage and electrocaloric (EC) refrigeration. Herein, we fabricate a lead-free Mn-modified 0.75 Bi(Mg0.5Ti0.5)O3-0.25 BaTiO3 (BMT-BTO) thin film based on a flexible mica substrate. Excellent EC performance with maximum adiabatic temperature change (ΔT ∼23.5 K) and isothermal entropy change (ΔS ∼33.1 J K-1 kg-1) is achieved in the flexible BMT-BTO film, which is attributed to the local structural transition and lattice disorder near 90 °C. Meanwhile, a good energy storage density of ∼70.6 J cm-3 and a quite high efficiency of ∼82% are realized in the same ferroelectric film, accompanied by excellent stability of frequency and electric fatigue (500-10 kHz and 108 cycles). Furthermore, there is no apparent variation in performance under different bending strains. These prominent properties indicate that the multifunctional BMT-BTO ferroelectric film is a promising candidate for applications of flexible energy storage and EC refrigeration.

Drawing Dispersion Curves: Band Structure Customization via Nonlocal Phononic Crystals.

Dispersion relations govern wave behaviors, and tailoring them is a grand challenge in wave manipulation. We demonstrate the inverse design of phononic dispersion using nonlocal interactions on one-dimensional spring-mass chains. For both single-band and double-band cases, we can achieve any valid dispersion curves with analytical precision. We further employ our method to design phononic crystals with multiple ordinary (roton or maxon) and higher-order (undulation) critical points and investigate their wave packet dynamics.

A novel live attenuated duck Tembusu virus vaccine targeting N7 methyltransferase protects ducklings against pathogenic strains.

Duck Tembusu virus (DTMUV), an emerging pathogenic flavivirus, causes markedly decreased egg production in laying duck and neurological dysfunction and death in ducklings. Vaccination is currently the most effective means for prevention and control of DTMUV. In previous study, we have found that methyltransferase (MTase) defective DTMUV is attenuated and induces a higher innate immunity. However, it is not clear whether MTase-deficient DTMUV can be used as a live attenuated vaccine (LAV). In this study, we investigated the immunogenicity and immunoprotection of N7-MTase defective recombinant DTMUV K61A, K182A and E218A in ducklings. These three mutants were highly attenuated in both virulence and proliferation in ducklings but still immunogenic. Furthermore, a single-dose immunization with K61A, K182A or E218A could induce robust T cell responses and humoral immune responses, which could protect ducks from the challenge of a lethal-dose of DTMUV-CQW1. Together, this study provides an ideal strategy to design LAVs for DTMUV by targeting N7-MTase without changing the antigen composition. This attenuated strategy targeting N7-MTase may apply to other flaviviruses.

Periodic Ferroelectric Stripe Domains in α-In2Se3 Nanoflakes Grown via Reverse-Flow Chemical Vapor Deposition.

The two-dimensional (2D) layered semiconductor α-In2Se3 has aroused great interest in atomic-scale ferroelectric transistors, artificial synapses, and nonvolatile memory devices due to its distinguished 2D ferroelectric properties. We have synthesized α-In2Se3 nanosheets with rare in-plane ferroelectric stripe domains at room temperature on mica substrates using a reverse flow chemical vapor deposition (RFCVD) method and optimized growth parameters. This stripe domain contrast is found to be strongly correlated to the stacking of layers, and the interrelated out-of-plane (OOP) and in-plane (IP) polarization can be manipulated by mapping the artificial domain structure. The acquisition of amplitude and phase hysteresis loops confirms the OOP polarization ferroelectric property. The emergence of striped domains enriches the variety of the ferroelectric structure types and novel properties of 2D In2Se3. This work paves a new way for the controllable growth of van der Waals ferroelectrics and facilitates the development of novel ferroelectric memory device applications.

ANXA1-derived peptide for targeting PD-L1 degradation inhibits tumor immune evasion in multiple cancers.

BACKGROUND: Immune checkpoint inhibitors (ICIs) therapy targeting programmed cell death 1 (PD-1)/programmed cell death ligand 1 (PD-L1) shows promising clinical benefits. However, the relatively low response rate highlights the need to develop an alternative strategy to target PD-1/PD-L1 immune checkpoint. Our study focuses on the role and mechanism of annexin A1 (ANXA1)-derived peptide A11 degrading PD-L1 and the effect of A11 on tumor immune evasion in multiple cancers. METHODS: Binding of A11 to PD-L1 was identified by biotin pull-down coupled with mass spectrometry analysis. USP7 as PD-L1's deubiquitinase was found by screening a human deubiquitinase cDNA library. The role and mechanism of A11 competing with USP7 to degrade PD-L1 were analyzed. The capability to enhance the T cell-mediated tumor cell killing activity and antitumor effect of A11 via suppressing tumor immune evasion were investigated. The synergistic antitumor effect of A11 and PD-L1 mAb (monoclonal antibody) via suppressing tumor immune evasion were also studied in mice. The expression and clinical significance of USP7 and PD-L1 in cancer tissues were evaluated by immunohistochemistry. RESULTS: A11 decreases PD-L1 protein stability and levels by ubiquitin proteasome pathway in breast cancer, lung cancer and melanoma cells. Mechanistically, A11 competes with PD-L1's deubiquitinase USP7 for binding PD-L1, and then degrades PD-L1 by inhibiting USP7-mediated PD-L1 deubiquitination. Functionally, A11 promotes T cell ability of killing cancer cells in vitro, inhibits tumor immune evasion in mice via increasing the population and activation of CD8+ T cells in tumor microenvironment, and A11 and PD-1 mAb possess synergistic antitumor effect in mice. Moreover, expression levels of both USP7 and PD-L1 are significantly higher in breast cancer, non-small cell lung cancer and skin melanoma tissues than those in their corresponding normal tissues and are positively correlated in cancer tissues, and both proteins for predicting efficacy of PD-1 mAb immunotherapy and patient prognosis are superior to individual protein. CONCLUSION: Our results reveal that A11 competes with USP7 to bind and degrade PD-L1 in cancer cells, A11 exhibits obvious antitumor effects and synergistic antitumor activity with PD-1 mAb via inhibiting tumor immune evasion and A11 can serve as an alternative strategy for ICIs therapy in multiple cancers.

Interlayer Coupling Enhanced Energy Storage Performance in a Flexible BMT-BTO/BMT Multilayer Ferroelectric Film Capacitor.

Flexible ferroelectric capacitors with high energy density and storage efficiency are highly desirable in the next generation of flexible electronic devices. To develop high-performance ferroelectric capacitors, a conventional approach is chemical modification. Here, a novel approach of interlayer coupling is proposed to achieve high energy storage performance in BiMg0.5Ti0.5O3-BaTiO3/BiMg0.5Ti0.5O3 (BMT-BTO/BMT)N multilayer ferroelectric films fabricated on flexible mica substrates via a sol-gel coating method. The interlayer electrostatic coupling between the ferroelectric BMT and relaxor ferroelectric BMT-BTO layers leads to small remnant polarization and large breakdown field strength, resulting in an outstanding energy storage density of ∼106.8 J cm-3 and a good efficiency of ∼75.6% in the multilayer thin films. Further, the energy storage performance remains stable in a wide range of temperatures (25-200 °C) and frequencies (500 Hz to 10 kHz) after 108 electrical loading cycles. The energy storage performance also has no obvious deterioration when the multilayer film experiences 104 mechanical bending cycles with a bending radius of 4 mm. The approach proposed in the present work should be generally implementable in other multilayer flexible ferroelectric capacitors and offers a novel avenue to enhance energy storage performance by tuning the interlayer coupling.

Toll-like receptor 4 and lipopolysaccharide from commensal microbes regulate Tembusu virus infection.

Unlike most flaviviruses transmitted by arthropods, Tembusu virus (TMUV) is still active during winter and causes outbreaks in some areas, indicating vector-independent spread of the virus. Gastrointestinal transmission might be one of the possible routes of vector-free transmission, which also means that the virus has to interact with more intestinal bacteria. Here, we found evidence that TMUV indeed can transmit through the digestive tract. Interestingly, using an established TMUV disease model by oral gavage combined with an antibiotic treatment, we revealed that a decrease in intestinal bacteria significantly reduced local TMUV proliferation in the intestine, revealing that the bacterial microbiome is important in TMUV infection. We found that lipopolysaccharide (LPS) present in the outer membrane of Gram-negative bacteria enhanced TMUV proliferation by promoting its attachment. Toll-like receptor 4 (TLR4), a cell surface receptor, can transmit signal from LPS. We confirmed colocalization of TLR4 with TMUV envelope (E) protein as well as their interaction in infected cells. Coherently, TMUV infection of susceptible cells was inhibited by an anti-TLR4 antibody, purified soluble TLR4 protein, and knockdown of TLR4 expression. LPS-enhanced TMUV proliferation could also be blocked by a TLR4 inhibitor. Meanwhile, pretreatment of duck primary cells with TMUV significantly impaired LPS-induced interleukin 6 production. Collectively, our study provides first insights into vector-free transmission mechanisms of flaviviruses.

Duck Tembusu Virus Inhibits Type I Interferon Production through the JOSD1-SOCS1-IRF7 Negative-Feedback Regulation Pathway.

Duck Tembusu virus (DTMUV) is an emerging pathogenic flavivirus that mainly causes a decrease in egg production in infected waterfowl. Similar to other members of the Flaviviridae family, it can proliferate in most mammalian cells and may also pose a potential threat to nonavian animals. In previous studies, we found that DTMUV infection can upregulate suppressor of cytokine signaling 1 (SOCS1) to inhibit type I interferon (IFN) production and promote virus replication, but the specific mechanism is unclear. Furthermore, little is known about the regulatory role of ubiquitination during flavivirus infection. In this study, we found that activation of Toll-like receptor 3 (TLR3) signaling rather than type I IFN stimulation led to the upregulation of SOCS1 during DTMUV infection. Further studies revealed that JOSD1 stabilized SOCS1 expression by binding to the SH2 domain of SOCS1 and mediating its deubiquitination. In addition, JOSD1 also inhibited type I IFN production through SOCS1. Finally, SOCS1 acts as an E3 ubiquitin ligase that binds to IFN regulatory factor 7 (IRF7) through its SH2 domain and mediates K48-linked ubiquitination and proteasomal degradation of IRF7, ultimately inhibiting type I IFN production mediated by IRF7 and promoting viral proliferation. These results will enrich and deepen our understanding of the mechanism by which DTMUV antagonizes the host interferon system. IMPORTANCE DTMUV is a newly discovered flavivirus that seriously harms the poultry industry. In recent years, there have been numerous studies on the involvement of ubiquitination in the regulation of innate immunity. However, little is known about the involvement of ubiquitination in the regulation of flavivirus-induced type I IFN signaling. In this study, we found that SOCS1 was induced by TLR3 signaling during DTMUV infection. Furthermore, we found for the first time that duck SOCS1 protein was also modified by K48-linked polyubiquitination, whereas our previous study found that SOCS1 was upregulated during DTMUV infection. Further studies showed that JOSD1 stabilized SOCS1 expression by mediating the deubiquitination of SOCS1. While SOCS1 acts as a negative regulator of cytokines, we found that DTMUV utilized SOCS1 to mediate the ubiquitination and proteasomal degradation of IRF7 and ultimately inhibit type I IFN production, thereby promoting its proliferation.

The DHAV-1 protein VP1 interacts with PI3KC3 to induce autophagy through the PI3KC3 complex.

Duck hepatitis A virus type 1 (DHAV-1) is one of the main pathogens responsible for death in ducklings. Autophagy is a catabolic process that maintains cellular homeostasis, and the PI3KC3 protein plays an important role in the initiation of autophagy. DHAV-1 infection induces autophagy in duck embryo fibroblasts (DEFs) but the molecular mechanism between it and autophagy has not been reported. First, we determined that DHAV-1 infection induces autophagy in DEFs and that autophagy induction is dependent on the integrity of viral proteins by infecting DEFs with UV-inactivated or heat-inactivated DHAV-1. Then, in experiments using the pharmacological autophagy inducer rapamycin and the autophagy inhibitor chloroquine, autophagy inhibition was shown to reduce intracellular and extracellular DHAV-1 genome copies and viral titres. These results suggest that autophagy activated by DHAV-1 infection in DEFs affects DHAV-1 proliferation and extracellular release. Next, we screened the autophagy-inducing effects of the DHAV-1 structural proteins VP0, VP3, and VP1 and found that all DHAV-1 structural proteins could induce autophagy in DEFs but not the full autophagic flux. Finally, we found that VP1 promotes protein expression of PI3KC3 and Beclin1 by western blot experiments and that VP1 interacts with PI3KC3 by co-immunoprecipitation experiments; moreover, 3-MA-induced knockdown of PI3KC3 inhibited VP1 protein-induced autophagy in DEFs. In conclusion, the DHAV-1 structural protein VP1 regulates the PI3KC3 complex by interacting with PI3KC3 to induce autophagy in DEFs.

A proposed disease classification system for duck viral hepatitis.

The nomenclature of duck viral hepatitis (DVH) was historically not a problem. However, 14 hepatotropic viruses among 10 different genera are associated with the same disease name, DVH. Therefore, the disease name increasingly lacks clarity and may no longer fit the scientific description of the disease. Because one disease should not be attributed to 10 genera of viruses, this almost certainly causes misunderstanding regarding the disease-virus relationship. Herein, we revisited the problem and proposed an update to DVH disease classification. This classification is based on the nomenclature of human viral hepatitis and the key principle of Koch's postulates ("one microbe and one disease"). In total, 10 types of disease names have been proposed. These names were literately matched with hepatitis-related viruses. We envision that this intuitive nomenclature system will facilitate scientific communication and consistent interpretation in this field, especially in the Asian veterinary community, where these diseases are most commonly reported.

The substitution at residue 218 of the NS5 protein methyltransferase domain of Tembusu virus impairs viral replication and translation and may triggers RIG-I-like receptor signaling.

Flavivirus RNA cap-methylation plays an important role in viral infection, proliferation, and escape from innate immunity. The methyltransferase (MTase) of the flavivirus NS5 protein catalyzes viral RNA methylation. The E218 amino acid of the NS5 protein MTase domain is one of the active sites of flavivirus methyltransferase. In flaviviruses, the E218A mutation abolished 2'-O methylation activity and significantly reduced N-7 methylation activity. Tembusu virus (TMUV, genus Flavivirus) was a pathogen that caused neurological symptoms in ducklings and decreased egg production in laying ducks. In this study, we focused on a comprehensive understanding of the effects of the E218A mutation on TMUV characteristics and the host immune response. E218A mutation reduced TMUV replication and proliferation, but did not affect viral adsorption and entry. Based on a TMUV replicon system, we found that the E218A mutation impaired viral translation. In addition, E218A mutant virus might be more readily recognized by RIG-I-like receptors to activate the corresponding antiviral immune signaling than WT virus. Together, our data suggest that the E218A mutation of TMUV MTase domain impairs viral replication and translation and may activates RIG-I-like receptor signaling, ultimately leading to a reduction in viral proliferation.

Simultaneous determination of 13 nitrosamine impurities in biological medicines using salting-out liquid-liquid extraction coupled with liquid chromatography tandem mass spectrometry.

Nitrosamine impurities are being detected in various pharmaceutical products recently. However, no analytical method is provided for biopharmaceuticals. In present work, a salting-out liquid-liquid extraction (SALLE) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for quantification of thirteen nitrosamine contaminations in antibody drugs. The method showed excellent linearity over the range of 0.5-5.0 µg/L with LOQ (limit of quantitation) of 0.5 µg/L for targeted nitrosamines. The method was demonstrated to be accurate (recovery in a range of 75.4-114.7 %) and precise (RSD ≤ 13.2 %) for all nitrosamines using spiked samples. Especially, we found that the satisfactory recoveries for N-nitrosomethyl-4-aminobutyric acid (NMBA, 78.0-96.0 %) and 1-methyl-4-nitrosopiperazine (MeNP, 90.0-109.0 %) were just obtained in the opposite condition (with and without formic acid, respectively). In conclusion, we provide a sensitive and reliable method for nitrosamine estimations to ensure the safety of biological medications.

Duck plague virus UL41 protein inhibits RIG-I/MDA5-mediated duck IFN-ß production via mRNA degradation activity.

Retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) are cytosolic pattern recognition receptors that initiate innate antiviral immunity. Recent reports found that duck RLRs significantly restrict duck plague virus (DPV) infection. However, the molecular mechanism by which DPV evades immune responses is unknown. In this study, we first found that the DPV UL41 protein inhibited duck interferon-ß (IFN-ß) production mediated by RIG-I and melanoma differentiation-associated gene 5 (MDA5) by broadly downregulating the mRNA levels of important adaptor molecules, such as RIG-I, MDA5, mitochondrial antiviral signalling protein (MAVS), stimulator of interferon gene (STING), TANK-binding kinase 1 (TBK1), and interferon regulatory factor (IRF) 7. The conserved sites of the UL41 protein, E229, D231, and D232, were responsible for this activity. Furthermore, the DPV CHv-BAC-ΔUL41 mutant virus induced more duck IFN-ß and IFN-stimulated genes (Mx, OASL) production in duck embryo fibroblasts (DEFs) than DPV CHv-BAC parent virus. Our findings provide insights into the molecular mechanism underlying DPV immune evasion.

Tracing genetic signatures of bat-to-human coronaviruses and early transmission of North American SARS-CoV-2.

Highly pathogenic coronaviruses, including SARS-CoV-2, SARS-CoV and MERS-CoV, are thought to be transmitted from bats to humans, but the viral genetic signatures that contribute to bat-to-human transmission remain largely obscure. In this study, we identified an identical ribosomal frameshift motif among the three bat-human pairs of viruses and strong purifying selection after jumping from bats to humans. This represents genetic signatures of coronaviruses that are related to bat-to-human transmission. To further trace the early human-to-human transmission of SARS-CoV-2 in North America, a geographically stratified genome-wide association study (North American isolates and the remaining isolates) and a retrospective study were conducted. We determined that the single nucleotide polymorphisms (SNPs) 1,059.C > T and 25,563.G > T were significantly associated with approximately half of the North American SARS-CoV-2 isolates that accumulated largely during March 2020. Retrospectively tracing isolates with these two SNPs was used to reconstruct the early, reliable transmission history of North American SARS-CoV-2, and European isolates (February 26, 2020) showed transmission 3 days earlier than North American isolates and 17 days earlier than Asian isolates. Collectively, we identified the genetic signatures of the three pairs of coronaviruses and reconstructed an early transmission history of North American SARS-CoV-2. We envision that these genetic signatures are possibly diagnosable and predic markers for public health surveillance.

The lysine at position 151 of the duck hepatitis A virus 1 2C protein is critical for its NTPase activities.

The duck hepatitis A virus 1 (DHAV-1) 2C protein was predicted to be a superfamily III helicase member and includes nucleotide binding (NTB) and putative RNA helicase activity motifs. To study whether DHAV-1 2C protein has NTB activity, we expressed DHAV-1 2C protein with maltose binding protein (MBP) to solve its poor solubility in a prokaryotic expression system. We showed that the DHAV-1 2C protein has nucleoside triphosphatase (NTPase) activity by measuring the released phosphate. The NTPase of the DHAV-1 2C protein is Mg2+ indispensable and affected by other biochemical characteristics such as Mn2+, Ca2+, Zn2+, Na+ and pH. Guanidine hydrochloride (GdnHCl), a potent inhibitor of viral RNA replication, inhibited ATPase activity of the DHAV-1 2C protein in a dose-dependent manner. Finally, we constructed three mutants to identify the key site for the ATPase activity of the DHAV-1 2C protein. These results indicate that lysine at position 151 of the DHAV-1 2C protein is very important for NTPase activity. Here, we demonstrated and partially characterized that the DHAV-1 2C protein has NTPase activity and showed that mutation of the lysine in the conserved Walker A impairs that activity. The results serve to confirm what is readily predicted from previous work on picornavirus 2C proteins. It also provides a basis for further study of the 2C protein and the function of NTPase activity on the viral life cycle.

Decreased virulence of duck Tembusu virus harboring a mutant NS2A with impaired interaction with STING and IFNß induction.

Our previous studies revealed that duck Tembusu virus (DTMUV) NS2A inhibited IFNß signaling pathway by competitively binding to STING with TBK1, leading to reducing the phosphorylation of TBK1. Herein, we found that the 114-143 aa region of NS2A is critical for its interaction with STING and suppression of STING-mediated IFNß signaling. We further identified the amino acids at positions L129, N130, L139, R140 and F143 of NS2A critical for NS2A-STING interaction. Subsequently, single residue substitution in the NS2A protein was introduced into the DTMUV replicon and infectious clone. The replicons with NS2A L129A and L130A mutations significantly inhibited viral genome RNA replication. The rDTMUV NS2A L129A, L139A and R140A mutant viruses yielded significantly lower titer levels than WT in both BHK-21 and DEF cells, with much more obvious effect on the viral genome level, and infectious virions formed outside of infected cells. Especially, the rDTMUV L129A mutant showed a significantly lower mortality in both embryos and ducks than WT. All NS2A-mutants decreased the weight gain of infected ducklings and reduced the viral loads in the spleen relative to WT. However, no significant differences of viral loads were observed in the blood, thymus, or liver. Our findings extend our previous study on the immune evasion role of flavivirus NS2A protein. The targeted therapy of disabling the viral strategies developed for evading innate defense can be applied to the development of attenuated flaviviruses.

DHAV-1 Blocks the Signaling Pathway Upstream of Type I Interferon by Inhibiting the Interferon Regulatory Factor 7 Protein.

Duck hepatitis A virus (DHAV), which mainly infects 1- to 4-week-old ducklings, has a fatality rate of 95% and poses a huge economic threat to the duck industry. However, the mechanism by which DHAV-1 regulates the immune response of host cells is rarely reported. This study examined whether DHAV-1 contains a viral protein that can regulate the innate immunity of host cells and its specific regulatory mechanism, further exploring the mechanism by which DHAV-1 resists the host immune response. In the study, the dual-luciferase reporter gene system was used to screen the viral protein that regulates the host innate immunity and the target of this viral protein. The results indicate that the DHAV-1 3C protein inhibits the pathway upstream of interferon (IFN)-ß by targeting the interferon regulatory factor 7 (IRF7) protein. In addition, we found that the 3C protein inhibits the nuclear translocation of the IRF7 protein. Further experiments showed that the 3C protein interacts with the IRF7 protein through its N-terminus and that the 3C protein degrades the IRF7 protein in a caspase 3-dependent manner, thereby inhibiting the IFN-ß-mediated antiviral response to promote the replication of DHAV-1. The results of this study are expected to serve as a reference for elucidating the mechanisms of DHAV-1 infection and pathogenicity.

The LORF5 Gene Is Non-essential for Replication but Important for Duck Plague Virus Cell-to-Cell Spread Efficiently in Host Cells.

Duck plague virus (DPV) can cause high morbidity and mortality in many waterfowl species within the order Anseriformes. The DPV genome contains 78 open reading frames (ORFs), among which the LORF2, LORF3, LORF4, LORF5, and SORF3 genes are unique genes of avian herpesvirus. In this study, to investigate the role of this unique LORF5 gene in DPV proliferation, we generated a recombinant virus that lacks the LORF5 gene by a two-step red recombination system, which cloned the DPV Chinese virulent strain (DPV CHv) genome into a bacterial artificial chromosome (DPV CHv-BAC); the proliferation law of LORF5-deleted mutant virus on DEF cells and the effect of LORF5 gene on the life cycle stages of DPV compared with the parent strain were tested. Our data revealed that the LORF5 gene contributes to the cell-to-cell transmission of DPV but is not relevant to virus invasion, replication, assembly, and release formation. Taken together, this study sheds light on the role of the avian herpesvirus-specific gene LORF5 in the DPV proliferation life cycle. These findings lay the foundation for in-depth functional studies of the LORF5 gene in DPV or other avian herpesviruses.

Methyltransferase-Deficient Avian Flaviviruses Are Attenuated Due to Suppression of Viral RNA Translation and Induction of a Higher Innate Immunity.

The 5' end of the flavivirus genome contains a type 1 cap structure formed by sequential N-7 and 2'-O methylations by viral methyltransferase (MTase). Cap methylation of flavivirus genome is an essential structural modification to ensure the normal proliferation of the virus. Tembusu virus (TMUV) (genus Flavivirus) is a causative agent of duck egg drop syndrome and has zoonotic potential. Here, we identified the in vitro activity of TMUV MTase and determined the effect of K61-D146-K182-E218 enzymatic tetrad on N-7 and 2'-O methylation. The entire K61-D146-K182-E218 motif is essential for 2'-O MTase activity, whereas N-7 MTase activity requires only D146. To investigate its phenotype, the single point mutation (K61A, D146A, K182A or E218A) was introduced into TMUV replicon (pCMV-Rep-NanoLuc) and TMUV infectious cDNA clone (pACYC-TMUV). K-D-K-E mutations reduced the replication ability of replicon. K61A, K182A and E218A viruses were genetically stable, whereas D146A virus was unstable and reverted to WT virus. Mutant viruses were replication and virulence impaired, showing reduced growth and attenuated cytopathic effects and reduced mortality of duck embryos. Molecular mechanism studies showed that the translation efficiency of mutant viruses was inhibited and a higher host innate immunity was induced. Furthermore, we found that the translation inhibition of MTase-deficient viruses was caused by a defect in N-7 methylation, whereas the absence of 2'-O methylation did not affect viral translation. Taken together, our data validate the debilitating mechanism of MTase-deficient avian flavivirus and reveal an important role for cap-methylation in viral translation, proliferation, and escape from innate immunity.