Oncolytic Newcastle disease virus induced degradation of YAP through E3 ubiquitin ligase PRKN to exacerbate ferroptosis in tumor cells.

Ferroptosis, a form of programmed cell death characterized by iron-dependent lipid peroxidation, has recently gained considerable attention in the field of cancer therapy. There is significant crosstalk between ferroptosis and several classical signaling pathways, such as the Hippo pathway, which suppresses abnormal growth and is frequently aberrant in tumor tissues. Yes-associated protein 1 (YAP), the core effector molecule of the Hippo pathway, is abnormally expressed and activated in a variety of malignant tumor tissues. We previously proved that the oncolytic Newcastle disease virus (NDV) activated ferroptosis to kill tumor cells. NDV has been used in tumor therapy; however, its oncolytic mechanism is not completely understood. In this study, we demonstrated that NDV exacerbated ferroptosis in tumor cells by inducing ubiquitin-mediated degradation of YAP at Lys90 through E3 ubiquitin ligase parkin (PRKN). Blocking YAP degradation suppressed NDV-induced ferroptosis by suppressing the expression of Zrt/Irt-like protein 14 (ZIP14), a metal ion transporter that regulates iron uptake. These findings demonstrate that NDV exacerbated ferroptosis in tumor cells by inducing YAP degradation. Our study provides new insights into the mechanism of NDV-induced ferroptosis and highlights the critical role that oncolytic viruses play in the treatment of drug-resistant cancers.IMPORTANCEThe oncolytic Newcastle disease virus (NDV) is being developed for use in cancer treatment; however, its oncolytic mechanism is still not completely understood. The Hippo pathway, which is a tumor suppressor pathway, is frequently dysregulated in tumor tissues due to aberrant yes-associated protein 1 (YAP) activation. In this study, we have demonstrated that NDV degrades YAP to induce ferroptosis and promote virus replication in tumor cells. Notably, NDV was found to induce ubiquitin-mediated degradation of YAP at Lys90 through E3 ubiquitin ligase parkin (PRKN). Our study reveals a new mechanism by which NDV induces ferroptosis and provides new insights into NDV as an oncolytic agent for cancer treatment.

The Ca2+-dependent phosphatase calcineurin dephosphorylates TBK1 to suppress antiviral innate immunity.

Tumor necrosis factor receptor-associated factor family member-associated NF-κB activator-binding kinase 1 (TBK1) plays a key role in the induction of the type 1 interferon (IFN-I) response, which is an important component of innate antiviral defense. Viruses target calcium (Ca2+) signaling networks, which participate in the regulation of the viral life cycle, as well as mediate the host antiviral response. Although many studies have focused on the role of Ca2+ signaling in the regulation of IFN-I, the relationship between Ca2+ and TBK1 in different infection models requires further elucidation. Here, we examined the effects of the Newcastle disease virus (NDV)-induced increase in intracellular Ca2+ levels on the suppression of host antiviral responses. We demonstrated that intracellular Ca2+ increased significantly during NDV infection, leading to impaired IFN-I production and antiviral immunity through the activation of calcineurin (CaN). Depletion of Ca²+ was found to lead to a significant increase in virus-induced IFN-I production resulting in the inhibition of viral replication. Mechanistically, the accumulation of Ca2+ in response to viral infection increases the phosphatase activity of CaN, which in turn dephosphorylates and inactivates TBK1 in a Ca2+-dependent manner. Furthermore, the inhibition of CaN on viral replication was counteracted in TBK1 knockout cells. Together, our data demonstrate that NDV hijacks Ca2+ signaling networks to negatively regulate innate immunity via the CaN-TBK1 signaling axis. Thus, our findings not only identify the mechanism by which viruses exploit Ca2+ signaling to evade the host antiviral response but also, more importantly, highlight the potential role of Ca2+ homeostasis in the viral innate immune response.IMPORTANCEViral infections disrupt intracellular Ca2+ homeostasis, which affects the regulation of various host processes to create conditions that are conducive for their own proliferation, including the host immune response. The mechanism by which viruses trigger TBK1 activation and IFN-I induction through viral pathogen-associated molecular patterns has been well defined. However, the effects of virus-mediated Ca2+ imbalance on the IFN-I pathway requires further elucidation, especially with respect to TBK1 activation. Herein, we report that NDV infection causes an increase in intracellular free Ca2+ that leads to activation of the serine/threonine phosphatase CaN, which subsequently dephosphorylates TBK1 and negatively regulates IFN-I production. Furthermore, depletion of Ca2+ or inhibition of CaN activity exerts antiviral effects by promoting the production of IFN-I and inhibiting viral replication. Thus, our results reveal the potential role of Ca2+ in the innate immune response to viruses and provide a theoretical reference for the treatment of viral infectious diseases.

Newcastle Disease Virus Manipulates Mitochondrial MTHFD2-Mediated Nucleotide Metabolism for Virus Replication.

Viruses require host cell metabolic reprogramming to satisfy their replication demands; however, the mechanism by which the Newcastle disease virus (NDV) remodels nucleotide metabolism to support self-replication remains unknown. In this study, we demonstrate that NDV relies on the oxidative pentose phosphate pathway (oxPPP) and the folate-mediated one-carbon metabolic pathway to support replication. In concert with [1,2-13C2] glucose metabolic flow, NDV used oxPPP to promote pentose phosphate synthesis and to increase antioxidant NADPH production. Metabolic flux experiments using [2,3,3-2H] serine revealed that NDV increased one-carbon (1C) unit synthesis flux through the mitochondrial 1C pathway. Interestingly, methylenetetrahydrofolate dehydrogenase (MTHFD2) was upregulated as a compensatory mechanism for insufficient serine availability. Unexpectedly, direct knockdown of enzymes in the one-carbon metabolic pathway, except for cytosolic MTHFD1, significantly inhibited NDV replication. Specific complementation rescue experiments on small interfering RNA (siRNA)-mediated knockdown further revealed that only a knockdown of MTHFD2 strongly restrained NDV replication and was rescued by formate and extracellular nucleotides. These findings indicated that NDV replication relies on MTHFD2 to maintain nucleotide availability. Notably, nuclear MTHFD2 expression was increased during NDV infection and could represent a pathway by which NDV steals nucleotides from the nucleus. Collectively, these data reveal that NDV replication is regulated by the c-Myc-mediated 1C metabolic pathway and that the mechanism of nucleotide synthesis for viral replication is regulated by MTHFD2. IMPORTANCE Newcastle disease virus (NDV) is a dominant vector for vaccine and gene therapy that accommodates foreign genes well but can only infect mammalian cells that have undergone cancerous transformation. Understanding the remodeling of nucleotide metabolic pathways in host cells by NDV proliferation provides a new perspective for the precise use of NDV as a vector or in antiviral research. In this study, we demonstrated that NDV replication is strictly dependent on pathways involved in redox homeostasis in the nucleotide synthesis pathway, including the oxPPP and the mitochondrial one-carbon pathway. Further investigation revealed the potential involvement of NDV replication-dependent nucleotide availability in promoting MTHFD2 nuclear localization. Our findings highlight the differential dependence of NDV on enzymes for one-carbon metabolism, and the unique mechanism of action of MTHFD2 in viral replication, thereby providing a novel target for antiviral or oncolytic virus therapy.

Ubiquitination on Lysine 247 of Newcastle Disease Virus Matrix Protein Enhances Viral Replication and Virulence by Driving Nuclear-Cytoplasmic Trafficking.

The Newcastle disease virus (NDV) matrix (M) protein is the pivotal element for viral assembly, budding, and proliferation. It traffics through the cellular nucleus but performs its primary function in the cytoplasm. To investigate the biological importance of M protein nuclear-cytoplasmic trafficking and the mechanism involved, the regulatory motif nuclear export signal (NES) and nuclear localization signal (NLS) were analyzed. Here, two types of combined NLSs and NESs were identified within the NDV-M protein. The Herts/33-type M protein was found to mediate efficient nuclear export and stable virus-like particle (VLP) release, while the LaSota-type M protein was retained mostly in the nuclei and showed retarded VLP production. Two critical residues, namely, 247 and 263, within the motif were identified and associated with nuclear export efficiency. We identified, for the first time, residue 247 as an important monoubiquitination site, of which its modification regulates the nuclear-cytoplasmic trafficking of NDV-M. Subsequently, mutant LaSota strains were rescued via reverse genetics, which contained either single or double amino acid substitutions that were similar to the M of Herts/33. The rescued LaSota (rLaSota) strains rLaSota-R247K, -S263R, and -double mutation (DM) showed about 2-fold higher hemagglutination (HA) titers and 10-fold higher 50% egg infective dose (EID50) titers than wild-type (wt) rLaSota. Furthermore, the mean death time (MDT) and intracerebral pathogenicity index (ICPI) values of those recombinant viruses were slightly higher than those of wt rLaSota probably due to their higher proliferation rates. Our findings contribute to a better understanding of the molecular mechanism of the replication and pathogenicity of NDV and even those of all other paramyxoviruses. This information is beneficial for the development of vaccines and therapies for paramyxoviruses. IMPORTANCE Newcastle disease virus (NDV) is a pathogen that is lethal to birds and causes heavy losses in the poultry industry worldwide. The World Organization for Animal Health (OIE) ranked Newcastle disease (ND) as the third most significant poultry disease and the eighth most important wildlife disease in the World Livestock Disease Atlas in 2011. The matrix (M) protein of NDV is very important for viral assembly and maturation. It is interesting that M proteins enter the cellular nucleus before performing their primary function in the cytoplasm. We found that NDV-M has a combined nuclear import and export signal. The ubiquitin modification of a lysine residue within this signal is critical for quick, efficient nuclear export and subsequent viral production. Our findings shed new light on viral replication and open up new possibilities for therapeutics against NDV and other paramyxoviruses; furthermore, we demonstrate a novel approach for improving paramyxovirus vaccines.

Inhibition of anti-viral stress granule formation by coronavirus endoribonuclease nsp15 ensures efficient virus replication.

Cytoplasmic stress granules (SGs) are generally triggered by stress-induced translation arrest for storing mRNAs. Recently, it has been shown that SGs exert anti-viral functions due to their involvement in protein synthesis shut off and recruitment of innate immune signaling intermediates. The largest RNA viruses, coronaviruses, impose great threat to public safety and animal health; however, the significance of SGs in coronavirus infection is largely unknown. Infectious Bronchitis Virus (IBV) is the first identified coronavirus in 1930s and has been prevalent in poultry farm for many years. In this study, we provided evidence that IBV overcomes the host antiviral response by inhibiting SGs formation via the virus-encoded endoribonuclease nsp15. By immunofluorescence analysis, we observed that IBV infection not only did not trigger SGs formation in approximately 80% of the infected cells, but also impaired the formation of SGs triggered by heat shock, sodium arsenite, or NaCl stimuli. We further demonstrated that the intrinsic endoribonuclease activity of nsp15 was responsible for the interference of SGs formation. In fact, nsp15-defective recombinant IBV (rIBV-nsp15-H238A) greatly induced the formation of SGs, along with accumulation of dsRNA and activation of PKR, whereas wild type IBV failed to do so. Consequently, infection with rIBV-nsp15-H238A strongly triggered transcription of IFN-ß which in turn greatly affected rIBV-nsp15-H238A replication. Further analysis showed that SGs function as an antiviral hub, as demonstrated by the attenuated IRF3-IFN response and increased production of IBV in SG-defective cells. Additional evidence includes the aggregation of pattern recognition receptors (PRRs) and signaling intermediates to the IBV-induced SGs. Collectively, our data demonstrate that the endoribonuclease nsp15 of IBV interferes with the formation of antiviral hub SGs by regulating the accumulation of viral dsRNA and by antagonizing the activation of PKR, eventually ensuring productive virus replication. We further demonstrated that nsp15s from PEDV, TGEV, SARS-CoV, and SARS-CoV-2 harbor the conserved function to interfere with the formation of chemically-induced SGs. Thus, we speculate that coronaviruses employ similar nsp15-mediated mechanisms to antagonize the host anti-viral SGs formation to ensure efficient virus replication.

Peptide-mediated Aqueous Synthesis of NIR-II Emitting Ag2 S Quantum Dots for Rapid Photocatalytic Bacteria Disinfection.

Pathogenic microorganisms in the environment are a great threat to global human health. The development of disinfection method with rapid and effective antibacterial properties is urgently needed. In this study, a biomimetic silver binding peptide AgBP2 was introduced to develop a facile synthesis of biocompatible Ag2 S quantum dots (QDs). The AgBP2 capped Ag2 S QDs exhibited excellent fluorescent emission in the second near-infrared (NIR-II) window, with physical stability and photostability in the aqueous phase. Under 808 nm NIR laser irradiation, AgBP2-Ag2 S QDs can serve not only as a photothermal agent to realize NIR photothermal conversion but also as a photocatalyst to generate reactive oxygen species (ROS). The obtained AgBP2-Ag2 S QDs achieved a highly effective disinfection efficacy of 99.06 % against Escherichia coli within 25 min of NIR irradiation, which was ascribed to the synergistic effects of photogenerated ROS during photocatalysis and hyperthermia. Our work demonstrated a promising strategy for efficient bacterial disinfection.

Newcastle Disease virus infection activates PI3K/Akt/mTOR and p38 MAPK/Mnk1 pathways to benefit viral mRNA translation via interaction of the viral NP protein and host eIF4E.

Newcastle disease virus (NDV), a member of the Paramyxoviridae family, can activate PKR/eIF2α signaling cascade to shutoff host and facilitate viral mRNA translation during infection, however, the mechanism remains unclear. In this study, we revealed that NDV infection up-regulated host cap-dependent translation machinery by activating PI3K/Akt/mTOR and p38 MAPK/Mnk1 pathways. In addition, NDV infection induced p38 MAPK/Mnk1 signaling participated 4E-BP1 hyperphosphorylation for efficient viral protein synthesis when mTOR signaling is inhibited. Furthermore, NDV NP protein was found to be important for selective cap-dependent translation of viral mRNAs through binding to eIF4E during NDV infection. Taken together, NDV infection activated multiple signaling pathways for selective viral protein synthesis in infected cells, via interaction between viral NP protein and host translation machinery. Our results may help to design novel targets for therapeutic intervention against NDV infection and to understand the NDV anti-oncolytic mechanism.

External fixation with bone cement implantation for open calcaneal fractures: A case series.

OBJECTIVE: The purpose of this study was to retrospectively evaluate patients who had open reduction, external fixation and bone cement implantation of open calcaneal fractures. METHODS: The records of 14 patients with open calcaneus fractures from January 2015 to January 2019 were reviewed retrospectively. Clinical evaluations consisting of AOFAS, MFS and EQ-5D VAS scores and radiological evaluations consisting of the height, width and length of the calcaneus as well as Bohler's and Gissane angle performed at 3 months, 1 year and the last follow-up postoperatively. Time to surgery, wound complications were recorded. RESULTS: Our study sample consisted of 9 males and 5 females with a mean age of 38.5 ± 9.8 years and a mean follow-up of 31.4 ± 7.7 months. The mean period from injury to surgery was 5.4 ± 1.9 days and the mean duration of hospitalization was 13.2 ± 4.5 days. The AOFAS, MFS and EQ-5D VAS scores were 92.5 ± 10.3, 84.1 ± 9.7 and 86.4 ± 15.1 respectively at the final follow-up. The Bohler's angle increased from (12.9 ± 3.1)° preoperatively to (28.5 ± 6.3)° at the final follow-up (P < 0.001), with the Gissane's angle from (104.5 ± 9.7)° to (116.4 ± 8.9)° (P < 0.001). One patients (7.1%) developed pin infections and one patient (7.1%) suffered from dorso-lateral hindfoot hypoaesthesia. There was complete fracture healing without secondary loss of reduction in all cases. CONCLUSION: External fixation with bone cement implantation is a valid alternative treatment for the management of displaced open calcaneal fractures with a low rate of complications. LEVEL OF EVIDENCE: IV, retrospective case series.

Upregulation of DUSP6 impairs infectious bronchitis virus replication by negatively regulating ERK pathway and promoting apoptosis.

Elucidating virus-cell interactions is fundamental to understanding viral replication and identifying targets for therapeutic control of viral infection. The extracellular signal-regulated kinase (ERK) pathway has been shown to regulate pathogenesis during many viral infections, but its role during coronavirus infection is undetermined. Infectious bronchitis virus is the representative strain of Gammacoronavirus, which causes acute and highly contagious diseases in the poultry farm. In this study, we investigated the role of ERK1/2 signaling pathway in IBV infection. We found that IBV infection activated ERK1/2 signaling and the up-regulation of phosphatase DUSP6 formed a negative regulation loop. Pharmacological inhibition of MEK1/2-ERK1/2 signaling suppressed the expression of DUSP6, promoted cell death, and restricted virus replication. In contrast, suppression of DUSP6 by chemical inhibitor or siRNA increased the phosphorylation of ERK1/2, protected cells from apoptosis, and facilitated IBV replication. Overexpression of DUSP6 decreased the level of phospho-ERK1/2, promoted apoptosis, while dominant negative mutant DUSP6-DN lost the regulation function on ERK1/2 signaling and apoptosis. In conclusion, these data suggest that MEK-ERK1/2 signaling pathway facilitates IBV infection, probably by promoting cell survival; meanwhile, induction of DUSP6 forms a negative regulation loop to restrict ERK1/2 signaling, correlated with increased apoptosis and reduced viral load. Consequently, components of the ERK pathway, such as MEK1/2 and DUSP6, represent excellent targets for the development of antiviral drugs.

A modified intrafocal pinning technique with three-dimensional planning to facilitate volar plating in dorsally comminuted AO/OTA C2 and C3 distal radius fractures.

BACKGROUNDS: Theaim of this study was to assess the efficacy of a modified intrafocal pinningtechnique with three-dimensional (3D) planning to facilitate volar plating in dorsally comminuted intra-articular distal radius fractures. METHODS: Intotal 35 AO/OTA type C2 and C3 fractures were finally included.The 3D digital model of the fracture was reconstructed based on preoperative computedtomographic (CT) images, with the displacement of the comminuted dorsalfragment and the intra-articular fragment analyzed for preoperative planning. During operation, amodified intrafocal pinning technique was applied percutaneously from thedorsal aspect of the radius to reduce the collapsed intra-articular fragmentfollowing volar plating. Adequate reduction was confirmed in all of patientsconsidering radial height, radial inclination and volar tilt in postoperativeradiographs. RESULTS: No significant fracture re-displacement wasobserved in most of the cases during a mean follow-up period of 17.4 months, exceptfor two patients withthe C3 fracture. All of the patients achieved adequate clinicalROMs at 12 months postoperatively, with a mean DASH score of 12.0. Most of the patients achievedan excellent (n = 21) or good (n = 12) Gartland and Werley wrist score. CONCLUSIONS: Ourmodified intrafocal pinning technique with 3D planning contributes to a satisfactoryclinical and radiological outcome in dorsally comminuted intra-articular distalradius fractures fixed with a volar locking plate. TRIALREGISTRATION: Notapplicable because the design of the study is retrospective.

BDNF promoted osteoblast migration and fracture healing by up-regulating integrin ß1 via TrkB-mediated ERK1/2 and AKT signalling.

Brain-derived neurotrophic factor (BDNF) has been reported to participate in fracture healing, whereas the mechanism is still unclear. Since osteoblast migration is important for fracture healing, investigating effects of BDNF on osteoblasts migration may help to reveal its mechanism. Here, MC3T3-E1 cells were used in vitro while closed femur fracture mice were applied in vivo. Cells migration was assessed with Transwell assay. The protein expression was analysed by immunoblotting. X-ray and Micro-CT were performed at different time after fracture. Our results showed that BDNF promoted MC3T3-E1 cells migration, integrin ß1 expression and ERK1/2 and AKT phosphorylation. K252a, a specific inhibitor for TrkB, suppressed BDNF-induced migration, integrin ß1 expression and activation of ERK1/2 and AKT. PD98059 (an ERK1/2 inhibitor) and LY294002 (an AKT inhibitor) both inhibited BDNF-induced migration and integrin ß1 expression while integrin ß1 blocking antibody only suppressed cell migration. X-ray and Micro-CT analyses showed that the adenoviral carried integrin ß1 shRNA group had slower fracture healing at 7 and 21 days, but not 35 days compared to the control group. Thus, we proposed that BDNF stimulated MC3T3-E1 cells migration by up-regulating integrin ß1 via TrkB mediated ERK1/2 and AKT signalling, and this may help to enhance the fracture healing.

CD4 Expression and Env Conformation Are Critical for HIV-1 Restriction by SERINC5.

Serine incorporator 5 (SERINC5) is a recently identified restriction factor that strongly blocks HIV-1 entry but is counteracted by Nef. Notably, tier 1 HIV-1 Env proteins are sensitive to SERINC5, whereas the majority of tier 2/3 Env proteins are resistant to SERINC5, when viruses are produced from CD4-negative cells and tested by a single-round replication assay. Here, we investigated the Env-dependent SERINC5 antiviral mechanism by comparing tier 1 NL Env with tier 3 AD8 Env proteins. We found that when NL and AD8 viruses were inoculated into CD4+ T cells and human peripheral blood mononuclear cells (PBMCs), the propagation of the two viruses was restricted to a similar level when Nef was not expressed. Using a bimolecular fluorescence complementation (BiFC) assay, we detected Env-Env association and Env-SERINC5 interactions. A much greater level of NL Env-SERINC5 interactions was detected than was AD8 Env-SERINC5 interactions, which was further validated by immunoprecipitation assays. In addition, SERINC5 dissociated the NL Env trimeric complex more effectively than the AD8 Env trimeric complex when CD4 was not expressed. However, when CD4 was expressed, SERINC5 became more capable of interacting with AD8 Env and dissociating its trimeric complex. Moreover, AD8 and several other tier 2/3 viruses produced in the presence of CD4 became sensitive to SERINC5 when measured by the single-round replication assay. Because tier 1 and tier 2/3 Env trimers have open and closed conformations, respectively, and CD4 opens the closed conformation, we conclude that SERINC5 selectively dissociates Env trimers with an open conformation to restrict HIV-1 replication.IMPORTANCE Restriction factors provide the first line of defense against retrovirus infection by posing several blocks to the viral replication cycle. SERINC5 is a novel restriction factor that strongly blocks HIV-1 entry, although it is counteracted by Nef. Currently, it is still unclear how HIV-1 entry is blocked by SERINC5. Notably, this entry block is dependent on viral Env proteins. Laboratory-adapted HIV-1 strains are sensitive, whereas primary isolates are highly resistant to SERINC5. Env proteins mediate virus entry via extensive conformational rearrangements from a closed ground state to a CD4-bound open state. We detected Env-Env associations and Env-SERINC5 interactions in live cells by a novel bimolecular fluorescence assay. We demonstrate that CD4 expression increases the Env sensitivity to SERINC5 and allows SERINC5 to dissociate the Env complex, suggesting that SERINC5 restriction is dependent on Env conformation. Our results provide new insights into the poorly defined Env-dependent SERINC5 antiviral mechanism.

Newcastle Disease Virus V Protein Degrades Mitochondrial Antiviral Signaling Protein To Inhibit Host Type I Interferon Production via E3 Ubiquitin Ligase RNF5.

Paramyxovirus establishes an intimate and complex interaction with the host cell to counteract the antiviral responses elicited by the cell. Of the various pattern recognition receptors in the host, the cytosolic RNA helicases interact with viral RNA to activate the mitochondrial antiviral signaling protein (MAVS) and subsequent cellular interferon (IFN) response. On the other hand, viruses explore multiple strategies to resist host immunity. In this study, we found that Newcastle disease virus (NDV) infection induced MAVS degradation. Further analysis showed that NDV V protein degraded MAVS through the ubiquitin-proteasome pathway to inhibit IFN-ß production. Moreover, NDV V protein led to proteasomal degradation of MAVS through Lys362 and Lys461 ubiquitin to prevent IFN production. Further studies showed that NDV V protein recruited E3 ubiquitin ligase RNF5 to polyubiquitinate and degrade MAVS. Compared with levels for wild-type NDV infection, V-deficient NDV induced attenuated MAVS degradation and enhanced IFN-ß production at the late stage of infection. Several other paramyxovirus V proteins showed activities of degrading MAVS and blocking IFN production similar to those of NDV V protein. The present study revealed a novel role of NDV V protein in targeting MAVS to inhibit cellular IFN production, which reinforces the fact that the virus orchestrates the cellular antiviral response to its own benefit.IMPORTANCE Host anti-RNA virus innate immunity relies mainly on the recognition by retinoic acid-inducible gene I and melanoma differentiation-associated protein 5 and subsequently initiates downstream signaling through interaction with MAVS. On the other hand, viruses have developed various strategies to counteract MAVS-mediated signaling. The mechanism for paramyxoviruses regulating MAVS to benefit their infection remains unknown. In this article, we demonstrate that the V proteins of NDV and several other paramyxoviruses target MAVS for ubiquitin-mediated degradation through E3 ubiquitin ligase RING-finger protein 5 (RNF5). MAVS degradation leads to the inhibition of the downstream IFN-ß pathway and therefore benefits virus proliferation. Our study reveals a novel mechanism of NDV evading host innate immunity and provides insight into the therapeutic strategies for the control of paramyxovirus infection.

What Is the Most Reliable Classification System to Assess Tibial Pilon Fractures?

The aim of this study was to assess inter- and intraobserver agreement of the traditional systems (Ruedi-Allgower, AO [Arbeitsgemeinschaft für Osteosynthesefragen], and Topliss) and the newly proposed Leonetti classification system of pilon fractures. We studied all patients at our center who underwent pilon fracture surgery over a 2-year period: 68 patients (70 legs) were included. Four observers independently classified each pilon fracture according to the Ruedi-Allgower, AO, Topliss, and Leonetti systems by evaluating radiographs and computed tomography images on 2 occasions. The inter- and intraobserver agreements were calculated using the Fleiss kappa test. Interobserver reliability was good for AO types (A, B, and C) and Ruedi-Allgower (κ = 0.71 and 0.61, respectively), whereas the interobserver reliability was moderate for AO groups (A1, A2, A3, B1, B2, B3, C1, C2, and C3), Topliss families, Topliss subfamilies, Leonetti types, and Leonetti subtypes. Intraobserver reproducibility was excellent for the Ruedi-Allgower classification, AO types, and Topliss families and good for AO groups, Topliss subfamilies, and Leonetti types and subtypes. Ruedi-Allgower and AO classification systems are the most reliable among those currently used for pilon fractures, but with lower agreement at the AO group level. The use of Topliss and Leonetti classification systems is not recommended because of less favorable results.

HIV-1 Nef Antagonizes SERINC5 Restriction by Downregulation of SERINC5 via the Endosome/Lysosome System.

The primate lentiviral accessory protein Nef downregulates CD4 and major histocompatibility complex class I (MHC-I) from the cell surface via independent endosomal trafficking pathways to promote viral pathogenesis. In addition, Nef antagonizes a novel restriction factor, SERINC5 (Ser5), to increase viral infectivity. To explore the molecular mechanism of Ser5 antagonism by Nef, we determined how Nef affects Ser5 expression and intracellular trafficking in comparison to CD4 and MHC-I. We confirm that Nef excludes Ser5 from human immunodeficiency virus type 1 (HIV-1) virions by downregulating its cell surface expression via similar functional motifs required for CD4 downregulation. We find that Nef decreases both Ser5 and CD4 expression at steady-state levels, which are rescued by NH4Cl or bafilomycin A1 treatment. Nef binding to Ser5 was detected in living cells using a bimolecular fluorescence complementation assay, where Nef membrane association is required for interaction. In addition, Nef triggers rapid Ser5 internalization via receptor-mediated endocytosis and relocalizes Ser5 to Rab5+ early, Rab7+ late, and Rab11+ recycling endosomes. Manipulation of AP-2, Rab5, Rab7, and Rab11 expression levels affects the Nef-dependent Ser5 and CD4 downregulation. Moreover, although Nef does not promote Ser5 polyubiquitination, Ser5 downregulation relies on the ubiquitination pathway, and both K48- and K63-specific ubiquitin linkages are required for the downregulation. Finally, Nef promotes Ser5 colocalization with LAMP1, which is enhanced by bafilomycin A1 treatment, suggesting that Ser5 is targeted to lysosomes for destruction. We conclude that Nef uses a similar mechanism to downregulate Ser5 and CD4, which sorts Ser5 into a point-of-no-return degradative pathway to counteract its restriction.IMPORTANCE Human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) express an accessory protein called Nef to promote viral pathogenesis. Nef drives immune escape in vivo through downregulation of CD4 and MHC-I from the host cell surface. Recently, Nef was reported to counteract a novel host restriction factor, Ser5, to increase viral infectivity. Nef downregulates cell surface Ser5, thus preventing its incorporation into virus particles, resulting in disruption of its antiviral activity. Here, we report mechanistic studies of Nef-mediated Ser5 downregulation in comparison to CD4 and MHC-I. We demonstrate that Nef binds directly to Ser5 in living cells and that Nef-Ser5 interaction requires Nef association with the plasma membrane. Subsequently, Nef internalizes Ser5 from the plasma membrane via receptor-mediated endocytosis, and targets ubiquitinated Ser5 to endosomes and lysosomes for destruction. Collectively, these results provide new insights into our ongoing understanding of the Nef-Ser5 arms race in HIV-1 infection.

Supplementation of Vitamin E Protects Chickens from Newcastle Disease Virus-Mediated Exacerbation of Intestinal Oxidative Stress and Tissue Damage.

BACKGROUND/AIMS: Newcastle disease virus (NDV) causes a highly devastating and contagious disease in poultry, which is mainly attributed to extensive tissue damages in the digestive, respiratory and nervous systems. However, nature and dynamics of NDV-induced oxidative stresses in the intestine of chickens remain elusive. METHODS: In this study, we examined the magnitude of intestinal oxidative stress and histopathological changes caused by the virulent NDV infection, and explored the protective roles of vitamin E (vit. E) in ameliorating these pathological changes. For these purposes, chickens were divided into four groups namely i) non supplemented and non-challenged (negative control, CON); ii) no supplementation of vit. E but challenged with ZJ1 (positive control, NS+CHA); iii) vit. E supplementation at the dose of 50 IU/day/Kg body weight and ZJ1 challenge (VE50+CHA); and 4) vit. E supplementation at the dose of 100 IU/day/Kg body weight and ZJ1 challenge (VE100+CHA). In all groups, we analyzed concentrations of glutathione (GSH), malondialdehyde (MDA), nitric oxide (NO), total antioxidant capacity (T-AOC), and activity of glutathione S-transferase (GST), superoxide dismutase (SOD), catalase (CAT) using biochemical methods. The virus loads were determined by quantitative RT-PCR and antibody titers by hemagglutination inhibition assays. We also examined the histopathological changes in the duodenal and jejunal mucosa at 3 and 5-day post infection (dpi) with NDV. RESULTS: A significant elevation in the NO level was observed in NDV challenged chickens compared to the CON chickens at 2 dpi. The MDA contents were significantly increased whereas GSH was significantly decreased in NDV-challenged chickens compared to control. Furthermore, activities of GST, CAT, SOD, as well as the TOAC were markedly decreased in challenged chickens in comparison with control. Virus copy numbers were higher in NDV infected NS+CHA group compared to other groups. Severe histopathological changes including inflammation, degeneration and broken villi were observed in the intestine of NDV challenged chickens. However, all these malfunctions of antioxidant system and pathological changes in the intestine were partially or completely reversed by the vit. E supplementation. CONCLUSIONS: Our results suggest that NDV infection causes oxidative stress and histopathological changes in the duodenum and jejunum of chickens, which can be partially or fully ameliorated by supplementation of vit. E. Additionally, these findings suggest that oxidative stress contributes to the intestinal damages in NDV infected chickens. These findings will help to understand the pathogenesis of NDV and further investigation of therapeutic agents for control of Newcastle disease.

Newcastle disease virus induces stable formation of bona fide stress granules to facilitate viral replication through manipulating host protein translation.

Mammalian cells respond to various environmental stressors to form stress granules (SGs) by arresting cytoplasmic mRNA, protein translation element, and RNA binding proteins. Virus-induced SGs function in different ways, depending on the species of virus; however, the mechanism of SG regulation of virus replication is not well understood. In this study, Newcastle disease virus (NDV) triggered stable formation of bona fide SGs on HeLa cells through activating the protein kinase R (PKR)/eIF2α pathway. NDV-induced SGs contained classic SG markers T-cell internal antigen (TIA)-1, Ras GTPase-activating protein-binding protein (G3BP)-1, eukaryotic initiation factors, and small ribosomal subunit, which could be disassembled in the presence of cycloheximide. Treatment with nocodazole, a microtubule disruption drug, led to the formation of relatively small and circular granules, indicating that NDV infection induces canonical SGs. Furthermore, the role of SGs on NDV replication was investigated by knockdown of TIA-1 and TIA-1-related (TIAR) protein, the 2 critical components involved in SG formation from the HeLa cells, followed by NDV infection. Results showed that depletion of TIA-1 or TIAR inhibited viral protein synthesis, reduced extracellular virus yields, but increased global protein translation. FISH revealed that NDV-induced SGs contained predominantly cellular mRNA rather than viral mRNA. Deletion of TIA-1 or TIAR reduced NP mRNA levels in polysomes. These results demonstrate that NDV triggers stable formation of bona fide SGs, which benefit viral protein translation and virus replication by arresting cellular mRNA.-Sun, Y., Dong, L., Yu, S., Wang, X., Zheng, H., Zhang, P., Meng, C., Zhan, Y., Tan, L., Song, C., Qiu, X., Wang, G., Liao, Y., Ding, C. Newcastle disease virus induces stable formation of bona fide stress granules to facilitate viral replication through manipulating host protein translation.

Coating the plate with antibiotic cement to treat early infection after fracture fixation with retention of the implants: a technical note.

BACKGROUND: Local antibiotic therapy has gained increasing attraction in the prevention and treatment of fracture infection. However, no reports have used local antibiotic therapy in the management of early infection after fracture fixation with retention of implants. METHODS: The present surgical technique report the use of antibiotic impregnated bone cement in the management of early infection after fracture fixation. Initially, the fractures were fixed with plates. The average time from initial procedure to debridement was15 days (range 9 to 25 days). The infections were treated with irrigation, debridement, and retention of the implant. The lateral surface of the plates was coated with antibiotic cement and the bone defect was filled with antibiotic cement spacer after thorough debridement. RESULTS: Ten patients underwent this technique. The mean follow-up was 2.0 years (range 6 months to 4 years). The bone union rate was 100%, and the average time to bone healing was5.5 months.There was recurrence of infection in one patient before bone healing, but the implants were left in place until bone healed, and the infection was eradicated after implant removal. CONCLUSION: Coating the plate with antibiotic cement is a simple technique which may play a role in the management of early infection after fracture fixation.

Evolution of Newcastle Disease Virus Quasispecies Diversity and Enhanced Virulence after Passage through Chicken Air Sacs.

UNLABELLED: It has been reported that lentogenic Newcastle disease virus (NDV) isolates have the potential to become velogenic after their transmission and circulation in chickens, but the underlying mechanism is unclear. In this study, a highly velogenic NDV variant, JS10-A10, was generated from the duck-origin lentogenic isolate JS10 through 10 consecutive passages in chicken air sacs. The velogenic properties of this selected variant were determined using mean death time (MDT) assays, intracerebral pathogenicity index (ICPI), the intravenous pathogenicity index (IVPI), histopathology, and the analysis of host tissue tropism. In contrast, JS10 remained lentogenic after 20 serial passages in chicken eggs (JS10-E20). The JS10, JS10-A10, and JS10-E20 genomes were sequenced and found to be nearly identical, suggesting that both JS10-A10 and JS10-E20 were directly generated from JS10. To investigate the mechanism for virulence enhancement, the partial genome covering the F0 cleavage site of JS10 and its variants were analyzed using ultradeep pyrosequencing (UDPS) and the proportions of virulence-related genomes in the quasispecies were calculated. Velogenic NDV genomes accumulated as a function of JS10 passaging through chicken air sacs. Our data suggest that lentogenic NDV strains circulating among poultry might be a risk factor to future potential velogenic NDV outbreaks in chickens. IMPORTANCE: An avirulent isolate, JS10, was passaged through chicken air sacs and embryos, and the pathogenicity of the variants was assessed. A virulent variant, JS10-A10, was generated from consecutive passage in air sacs. We developed a deep-sequencing approach to detect low-frequency viral variants across the NDV genome. We observed that virulence enhancement of JS10 was due to the selective accumulation of velogenic quasispecies and the concomitant disappearance of lentogenic quasispecies. Our results suggest that because it is difficult to avoid contact between natural waterfowl reservoirs and sensitive poultry operations, circulating lentogenic NDV strains may represent a potential reservoir for emergent velogenic NDV strains that could cause outbreaks in chickens.

Phylogenetic, antigenic and biological characterization of pigeon paramyxovirus type 1 circulating in China.

BACKGROUND: For many years, ND has been one of the most important infectious pigeon diseases in China. In recent years, a high mortality has been observed in ND-infected pigeons in China. Mortality is from 40% to 80% or 100% in some cases. METHODS: The full-length genomes of four pigeon paramyxovirus type 1 (PPMV-1) strains, which were isolated from infected pigeons in China in 2012 and 2013, were sequenced and analyzed to determine the phylogenetic characteristics of PPMV-1 circulating in pigeons of China in recent years. Furthermore, cross hemagglutination inhibition and cross virus neutralization assays, as well as animal experiments were conducted to determine the antigenicity and pathogenicity of those viruses. Proteolytic cleavage sites (residues 112-117) of the F proteins were identified as the typical virulence motif, 112RRQKR↓F117 for all four PPMV-1 strains investigated. RESULTS: Phylogenetic analysis based on sequences of complete genomes and F gene revealed that the four PPMV-1 isolates and most of recent isolates in China were highly homologous to European isolates from 1998 to 2011. All those isolates were clustered in one clade of genotype VI NDV, termed as subgroup 4bii f. The R value was calculated based on cross hemagglutination inhibition and cross virus neutralization results, and confirmed antigenic difference of the PPMV-1 strains isolated in 2013 from the LaSota vaccine strain. Several mutations were identified in the surface glycoproteins F and HN, which probably gave rise to those antigenic differences. CONCLUSION: Our result suggested that the PPMV-1 strain prevailing in China in the last decade diverged from a common ancestor and was presumably transmitted from Europe. PPMV-1 isolates displayed obvious antigenic differences from vaccine strain LaSota. Even though PPMV-1 did not cause high mortality in experimental pigeons, the infected pigeons were exhibiting viral shedding for 3 weeks after infection, suggesting PPMV-1 is a potential threat to NDV control worldwide.