Two novel compounds inhibit Flavivirus infection in vitro and in vivo by targeting lipid metabolism.

Flavivirus infection capitalizes on cellular lipid metabolism to remodel the cellular intima, creating a specialized lipid environment conducive to viral replication, assembly, and release. The Japanese encephalitis virus (JEV), a member of the Flavivirus genus, is responsible for significant morbidity and mortality in both humans and animals. Currently, there are no effective antiviral drugs available to combat JEV infection. In this study, we embarked on a quest to identify anti-JEV compounds within a lipid compound library. Our research led to the discovery of two novel compounds, isobavachalcone (IBC) and corosolic acid (CA), which exhibit dose-dependent inhibition of JEV proliferation. Time-of-addition assays indicated that IBC and CA predominantly target the late stage of the viral replication cycle. Mechanistically, JEV nonstructural proteins 1 and 2A (NS1 and NS2A) impede 5'-adenosine monophosphate (AMP)-activated protein kinase (AMPK) activation by obstructing the liver kinase B1 (LKB1)-AMPK interaction, resulting in decreased p-AMPK expression and a consequent upsurge in lipid synthesis. In contrast, IBC and CA may stimulate AMPK by binding to its active allosteric site, thereby inhibiting lipid synthesis essential for JEV replication and ultimately curtailing viral infection. Most importantly, in vivo experiments demonstrated that IBC and CA protected mice from JEV-induced mortality, significantly reducing viral loads in the brain and mitigating histopathological alterations. Overall, IBC and CA demonstrate significant potential as effective anti-JEV agents by precisely targeting AMPK-associated signaling pathways. These findings open new therapeutic avenues for addressing infections caused by Flaviviruses. IMPORTANCE: This study is the inaugural utilization of a lipid compound library in antiviral drug screening. Two lipid compounds, isobavachalcone (IBC) and corosolic acid (CA), emerged from the screening, exhibiting substantial inhibitory effects on the Japanese encephalitis virus (JEV) proliferation in vitro. In vivo experiments underscored their efficacy, with IBC and CA reducing viral loads in the brain and mitigating JEV-induced histopathological changes, effectively shielding mice from fatal JEV infection. Intriguingly, IBC and CA may activate 5'-adenosine monophosphate (AMP)-activated protein kinase (AMPK) by binding to its active site, curtailing the synthesis of lipid substances, and thus suppressing JEV proliferation. This indicates AMPK as a potential antiviral target. Remarkably, IBC and CA demonstrated suppression of multiple viruses, including Flaviviruses (JEV and Zika virus), porcine herpesvirus (pseudorabies virus), and coronaviruses (porcine deltacoronavirus and porcine epidemic diarrhea virus), suggesting their potential as broad-spectrum antiviral agents. These findings shed new light on the potential applications of these compounds in antiviral research.

Kinesin-1 Regulates Endocytic Trafficking of Classical Swine Fever Virus along Acetylated Microtubules.

Classical swine fever (CSF), caused by classical swine fever virus (CSFV), is an important and highly infectious pig disease worldwide. Kinesin-1, a molecular motor responsible for transporting cargo along the microtubule, has been demonstrated to be involved in the infections of diverse viruses. However, the role of kinesin-1 in the CSFV life cycle remains unknown. Here, we first found that Kif5B played a positive role in CSFV entry by knockdown or overexpression of Kif5B. Subsequently, we showed that Kif5B was associated with the endosomal and lysosomal trafficking of CSFV in the early stage of CSFV infection, which was reflected by the colocalization of Kif5B and Rab7, Rab11, or Lamp1. Interestingly, trichostatin A (TSA) treatment promoted CSFV proliferation, suggesting that microtubule acetylation facilitated CSFV endocytosis. The results of chemical inhibitors and RNA interference showed that Rac1 and Cdc42 induced microtubule acetylation after CSFV infection. Furthermore, confocal microscopy revealed that cooperation between Kif5B and dynein help CSFV particles move in both directions along microtubules. Collectively, our study shed light on the role of kinesin motor Kif5B in CSFV endocytic trafficking, indicating the dynein/kinesin-mediated bidirectional CSFV movement. The elucidation of this study provides the foundation for developing CSFV antiviral drugs. IMPORTANCE The minus end-directed cytoplasmic dynein and the plus end-directed kinesin-1 are the molecular motors that transport cargo on microtubules in intracellular trafficking, which plays a notable role in the life cycles of diverse viruses. Our previous studies have reported that the CSFV entry host cell is dependent on the microtubule-based motor dynein. However, little is known about the involvement of kinesin-1 in CSFV infection. Here, we revealed the critical role of kinesin-1 that regulated the viral endocytosis along acetylated microtubules induced by Cdc42 and Rac1 after CSFV entry. Mechanistically, once CSFV transported by dynein met an obstacle, it recruited kinesin-1 to move in reverse to the anchor position. This study extends the theoretical basis of intracellular transport of CSFV and provides a potential target for the control and treatment of CSFV infection.

The Small GTPase Rab14 Regulates the Trafficking of Ceramide from Endoplasmic Reticulum to Golgi Apparatus and Facilitates Classical Swine Fever Virus Assembly.

Classical swine fever virus (CSFV) is a highly pathogenic RNA virus belonging to the Flaviviridae family that can cause deadly classical swine fever (CSF) in pigs. However, the molecular details of virus replication in the host are still unclear. Our previous studies have reported that several Rab proteins mediate CSFV entry into host cells, but it is unknown whether CSFV hijacks other Rab proteins for effective viral infection. Here, we systematically studied the role of Rab14 protein in regulating lipid metabolism for promoting viral assembly. First, Rab14 knockdown and overexpression significantly affected CSFV replication, indicating the essential role of Rab14 in CSFV infection. Interestingly, Rab14 could significantly affect virus replication in the late stage of infection. Mechanistically, CSFV NS5A recruited Rab14 to the ER, followed by ceramide transportation to the Golgi apparatus, where sphingomyelin was synthesized. The experimental data of small molecule inhibitors, RNA interference, and replenishment assay showed that the phosphatidylinositol-3-kinase (PI3K)/AKT/AS160 signaling pathway regulated the function of Rab14 to affect the transport of ceramide. More importantly, sphingomyelin on the Golgi apparatus contributed to the assembly of viral particles. Blockage of the Rab14 regulatory pathway induced the reduction of the content of sphingomyelin on the Golgi apparatus, impairing the assembly of virus particles. Our study clarifies that Rab14 regulates lipid metabolism and promotes CSFV replication, which provides insight into a novel function of Rab14 in regulating vesicles to transport lipids to the viral assembly factory. IMPORTANCE The Rab protein family members participate in the viral replication of multiple viruses and play important roles in the virus infection cycle. Our previous research focused on Rab5/7/11, which regulated the trafficking of vesicles in the early stage of CSFV infection, especially in viral endocytosis. However, the role of other Rab proteins in CSFV replication is unclear and needs further clarification. Strikingly, we screened some Rabs and found the important role of Rab14 in CSFV infection. Virus infection mobilized Rab14 to regulate the vesicle to transport ceramide from the ER to the Golgi apparatus, further promoting the synthesis of sphingomyelin and facilitating virus assembly. The treatment of inhibitors showed that the lipid transport mediated by Rab14 was regulated by the PI3K/AKT/AS160 signaling pathway. Knockdown of Rab14 or the treatment with PI3K/AKT/AS160 inhibitors reduced the ceramide content in infected cells and hindered virus assembly. Our study is the first to explain that vesicular lipid transport regulated by Rab promotes CSFV assembly, which is conducive to the development of antiviral drugs.

Intracellular Vimentin Regulates the Formation of Classical Swine Fever Virus Replication Complex through Interaction with NS5A Protein.

Vimentin (VIM), an indispensable protein, is responsible for the formation of intermediate filament structures within cells and plays a crucial role in viral infections. However, the precise role of VIM in classical swine fever virus (CSFV) infection remains unclear. Herein, we systematically investigated the function of VIM in CSFV replication. We demonstrated that both knockdown and overexpression of VIM affected CSFV replication. Furthermore, we observed by confocal microscopy the rearrangement of cellular VIM into a cage-like structure during CSFV infection. Three-dimensional (3D) imaging indicated that the cage-like structures were localized in the endoplasmic reticulum (ER) and ringed around the double-stranded RNA (dsRNA), thereby suggesting that VIM was associated with the formation of the viral replication complex (VRC). Mechanistically, phosphorylation of VIM at serine 72 (Ser72), regulated by the RhoA/ROCK signaling pathway, induced VIM rearrangement upon CSFV infection. Confocal microscopy and coimmunoprecipitation assays revealed that VIM colocalized and interacted with CSFV NS5A. Structurally, it was determined that amino acids 96 to 407 of VIM and amino acids 251 to 416 of NS5A were the respective important domains for this interaction. Importantly, both VIM knockdown and disruption of VIM rearrangement inhibited the localization of NS5A in the ER, implying that VIM rearrangement recruited NS5A to the ER for VRC formation. Collectively, our results suggest that VIM recruits NS5A to form a stable VRC that is protected by the cage-like structure formed by VIM rearrangement, ultimately leading to enhanced virus replication. These findings highlight the critical role of VIM in the formation and stabilization of VRC, which provides alternative strategies for the development of antiviral drugs. IMPORTANCE Classical swine fever (CSF), caused by classical swine fever virus (CSFV), is a highly infectious disease that poses a significant threat to the global pig industry. Therefore, gaining insights into the virus and its interaction with host cells is crucial for developing effective antiviral measures and controlling the spread of CSF. Previous studies have shown that CSFV infection induces rearrangement of the endoplasmic reticulum, leading to the formation of small vesicular organelles containing nonstructural protein and double-stranded RNA of CSFV, as well as some host factors. These organelles then assemble into viral replication complexes (VRCs). In this study, we have discovered that VIM recruited CSFV NS5A to form a stable VRC that was protected by a cage-like structure formed by rearranged VIM. This enhanced viral replication. Our findings not only shed light on the molecular mechanism of CSFV replication but also offer new insights into the development of antiviral strategies for controlling CSFV.

Cellular ESCRT components are recruited to regulate the endocytic trafficking and RNA replication compartment assembly during classical swine fever virus infection.

As the important molecular machinery for membrane protein sorting in eukaryotic cells, the endosomal sorting and transport complexes (ESCRT-0/I/II/III and VPS4) usually participate in various replication stages of enveloped viruses, such as endocytosis and budding. The main subunit of ESCRT-I, Tsg101, has been previously revealed to play a role in the entry and replication of classical swine fever virus (CSFV). However, the effect of the whole ESCRT machinery during CSFV infection has not yet been well defined. Here, we systematically determine the effects of subunits of ESCRT on entry, replication, and budding of CSFV by genetic analysis. We show that EAP20 (VPS25) (ESCRT-II), CHMP4B and CHMP7 (ESCRT-III) regulate CSFV entry and assist vesicles in transporting CSFV from Clathrin, early endosomes, late endosomes to lysosomes. Importantly, we first demonstrate that HRS (ESCRT-0), VPS28 (ESCRT-I), VPS25 (ESCRT-II) and adaptor protein ALIX play important roles in the formation of virus replication complexes (VRC) together with CHMP2B/4B/7 (ESCRT-III), and VPS4A. Further analyses reveal these subunits interact with CSFV nonstructural proteins (NS) and locate in the endoplasmic reticulum, but not Golgi, suggesting the role of ESCRT in regulating VRC assembly. In addition, we demonstrate that VPS4A is close to lipid droplets (LDs), indicating the importance of lipid metabolism in the formation of VRC and nucleic acid production. Altogether, we draw a new picture of cellular ESCRT machinery in CSFV entry and VRC formation, which could provide alternative strategies for preventing and controlling the diseases caused by CSFV or other Pestivirus.

Fatty Acid Synthase Is Involved in Classical Swine Fever Virus Replication by Interaction with NS4B.

Classical swine fever virus (CSFV), a member of the genus Pestivirus of the family Flaviviridae, relies on host machinery to complete its life cycle. Previous studies have shown a close connection between virus infection and fatty acid biosynthesis, mainly regulated by fatty acid synthase (FASN). However, the molecular action of how FASN participates in CSFV replication remains to be elucidated. In this study, two chemical inhibitors of the fatty acid synthesis pathway [5-(tetradecyloxy)-2-furoic acid (TOFA) and tetrahydro-4-methylene-2R-octyl-5-oxo-3S-furancarboxylic acid (C75)] significantly impaired the late stage of viral propagation, suggesting CSFV replication required fatty acid synthesis. We next found that CSFV infection stimulated the expression of FASN, whereas knockdown of FASN inhibited CSFV replication. Furthermore, confocal microscopy showed that FASN participated in the formation of replication complex (RC), which was associated with the endoplasmic reticulum (ER). Interestingly, CSFV NS4B interacted with FASN and promoted overexpression of FASN, which is regulated by functional Rab18. Moreover, we found that FASN regulated the formation of lipid droplets (LDs) upon CSFV infection, promoting virus proliferation. Taken together, our work provides mechanistic insight into the role of FASN in the viral life of CSFV, and it highlights the potential antiviral target for the development of therapeutics against pestiviruses. IMPORTANCE Classical swine fever, caused by classical swine fever virus (CSFV), is one of the notifiable diseases by the World Organization for Animal Health (OIE) and causes significant financial losses to the pig industry globally. CSFV, like other (+)-strand RNA viruses, requires lipid and sterol biosynthesis for efficient replication. However, the role of lipid metabolism in CSFV replication remains unknown. Here, we found that fatty acid synthase (FASN) was involved in viral propagation. Moreover, FASN is recruited to CSFV replication sites in the endoplasmic reticulum (ER) and interacts with NS4B to regulate CSFV replication that requires Rab18. Furthermore, we speculated that lipid droplet (LD) biosynthesis, indirectly regulated by FASN, ultimately promotes CSFV replication. Our results highlight a critical role for de novo fatty acid synthesis in CSFV infection, which might help control this devastating virus.

The ESCRT-I Subunit Tsg101 Plays Novel Dual Roles in Entry and Replication of Classical Swine Fever Virus.

Classical swine fever (CSF), caused by classical swine fever virus (CSFV), is a highly contagious disease of swine with high morbidity and mortality that negatively affects the pig industry worldwide, in particular in China. Soon after the endocytosis of CSFV, the virus makes full use of the components of host cells to complete its life cycle. The endocytosis sorting complex required for transport (ESCRT) system is a central molecular machine for membrane protein sorting and scission in eukaryotic cells that plays an essential role in many physiological metabolic processes, including invasion and egress of envelope viruses. However, the molecular mechanism that ESCRT uses to regulate the replication of CSFV is unknown. In this study, we demonstrated that the ESCRT-I complex Tsg101 protein participates in clathrin-mediated endocytosis of CSFV and is also involved in CSFV trafficking. Tsg101 assists the virus in entering the host cell through the late endosome (Rab7 and Rab9) and finally reaching the lysosome (Lamp-1). Interestingly, Tsg101 is also involved in the viral replication process by interacting with nonstructural proteins 4B and 5B of CSFV. Finally, confocal microscopy showed that the replication complex of Tsg101 and double-stranded RNA (dsRNA) or NS4B and NS5B protein was close to the endoplasmic reticulum (ER), not the Golgi, in the cytoplasm. Collectively, our finding highlights that Tsg101 regulates the process of CSFV entry and replication, indicating that the ESCRT plays an important role in the life cycle of CSFV. Thus, ESCRT molecules could serve as therapeutic targets to combat CSFV infection.IMPORTANCE CSF, caused by CSFV, is a World Organization for Animal Health (OIE) notifiable disease and causes significant financial losses to the pig industry globally. The ESCRT machinery plays an important regulatory role in several members of the genera Flavivirus and Hepacivirus within the family Flaviviridae, such as hepatitis C virus, Japanese encephalitis virus, and dengue virus. Previous reports have shown that assembling and budding of these viruses require ESCRT. However, the role of ESCRT in Pestivirus infection remains to be elucidated. We determined the molecular mechanisms of the regulation of CSFV infection by the major subunit Tsg101 of ESCRT-I. Interestingly, Tsg101 plays an essential regulatory role in both clathrin-mediated endocytosis and genome replication of CSFV. Overall, the results of this study provide further insights into the molecular function of ESCRT-I complex protein Tsg101 during CSFV infection, which may serve as a molecular target for pestivirus inhibitors.

Microfilaments and microtubules alternately coordinate the multi-step endosomal trafficking of Classical Swine Fever Virus.

Cytoskeleton, as a ubiquitous structure in the cells, plays an important role in the process of virus entry, replication, and survival. However, the action mechanism of cytoskeleton in the invasion of Pestivirus into host cells remains unclear. In this study, we systematically dissected the key roles of the main cytoskeleton components, microfilaments and microtubules in the endocytosis of porcine Pestivirus, Classical swine fever virus (CSFV). We observed the dynamic changes of actin filaments in CSFV entry. Confocal microscopy showed that CSFV invasion induced the dissolution and aggregation of stress fibers, resulting in the formation of lamellipodia and filopodia. Chemical inhibitors and RNA interference were used to find that the dynamic changes of actin were caused by EGFR-PI3K/MAPK-RhoA/Rac1/Cdc42-cofilin signaling pathway, which regulates the microfilaments to help CSFV entry. Furthermore, co-localization of the microfilaments with clathrin and Rab5 (early endosome), as well as microtubules with Rab7 (late endosome) and Lamp1 (lysosome) revealed that microfilaments were activated and rearranged to help CSFV trafficking to early endosome after endocytosis. Subsequently, recruitment of microtubules by CSFV also assisted membrane fusion of the virions from late endosome to lysosome with the help of a molecular motor, dynein. Unexpectedly, vimentin, which is an intermediate filament, had no effect on CSFV entry. Taken together, our findings comprehensively revealed the molecular mechanisms of cytoskeletal components that regulated CSFV endocytosis and facilitated further understanding of Pestivirus entry, which would be conducive to explore antiviral molecules to control classical swine fever.IMPORTANCEEndocytosis, an essential biological process mediating cellular internalization events, is often exploited by pathogens for their entry into target cells. Previously, we have reported different mechanisms of CSFV endocytosis into the porcine epithelial cells (PK-15) and macrophages (3D4/21); however, the details of microfilaments/microtubules mediated virus migration within the host cells remained to be elucidated. In this study, we found that CSFV infection induced rearrangement of actin filaments regulated by cofilin through EGFR-PI3K/MAPK-RhoA/Rac1/Cdc42 pathway. Furthermore, we found that CSFV particles were trafficked along actin filaments in early and late endosomes, and through microtubules in lysosomes after entry. Here, we provide for the first time a comprehensive description of the cytoskeleton that facilitates entry and intracellular transport of highly pathogenic swine virus. Results from this study will greatly contribute to the understanding of virus-induced early and complex changes in host cells that are important in CSFV pathogenesis.

Activation of macrophage TBK1-HIF-1α-mediated IL-17/IL-10 signaling by hyperglycemia aggravates the complexity of coronary atherosclerosis: An in vivo and in vitro study.

Our purpose was to study the effect of hyperglycemia on macrophage TBK1-HIF-1α-mediated IL-17/IL-10 signaling and its correlation with coronary atherosclerosis. A total of 135 patients with coronary heart disease (CHD) were divided into a stable CHD (SCHD) group (n = 30) and an acute myocardial infarction (AMI) group (n = 105) [nondiabetes mellitus (non-DM)-AMI, n = 60; DM-AMI, n = 45] from January to September 2020. The SYNTAX score and metabolic and inflammatory markers were quantified and compared. THP-1 cell studies and an animal study of coronary intimal hyperplasia were also carried out. We found that the DM-AMI group showed a higher SYNTAX score than the non-DM-AMI group (P < .05). The DM-AMI group showed the highest expression levels of TANK-binding kinase 1 (TBK1), hypoxia-inducible factor 1α (HIF-1α), and interleukin (IL)-17 and the lowest expression level of IL-10, followed by the non-DM-AMI group and the SCHD group (P < .05). THP-1 cell studies showed that BAY87-2243 (a HIF-1α inhibitor) reversed the increase in IL-17 and decrease in IL-10 expression induced by hyperglycemia. Amlexanox (a TBK1 inhibitor) reversed the increase in HIF-1α expression induced by hyperglycemia. Amlexanox treatment resulted in lower coronary artery intimal hyperplasia and a larger lumen area in a diabetic swine model. We conclude that hyperglycemia might aggravate the complexity of coronary atherosclerosis through activation of TBK1-HIF-1α-mediated IL-17/IL-10 signaling. Thus, TBK1 may be a novel drug therapy target for CHD complicated with DM.

Cancer Cell Membrane Vesicle for Multiplex MicroRNA Imaging in Living Cells.

Highly efficient cellular transfection and intracellular signal amplification is a prerequisite for low-abundant microRNA (miRNA) imaging and biomedical application. Herein, we report a functional cancer cell membrane (CM) vesicle, Au-P/DSN@CM (DSN, double-specific nucleases), which consists of Au nanoparticles modified with three types of fluorescent miRNA detection probes (Au-P) and DSN that simultaneously encapsulate in cancer CM. We find that the Au-P/DSN@CM could specifically target the cancer cell and transfect the cell with higher efficiency than Au nanoparticles. The internalized Au-P/DSN@CM could further specifically recognize the target miRNA and induce DSN-assisted target recycle signal amplification, leading to multiple miRNA simultaneous detection with high sensitivity. It successfully detects oncogenic miRNAs in MCF-7 cells with high sensitivity and is amenable to monitor the dynamic expression change of oncogenic miRNAs in cancer cells. Our study represents a promising gene delivery vector for cancer diagnosis and potential therapy.

Serum HBV RNA Dynamic and Drug Withdrawal Predictor Value in Patients With Chronic HBV Infection on Long-term Nucleos(t)ide Analogue (NA) Therapy.

AIMS: This study aimed to investigate the dynamic pattern of serum hepatitis B virus (HBV) RNA in chronic hepatitis B (CHB) patients on long-term nucleos(t)ide analogue (NA) therapy and evaluate predictor value of end-of-treatment (EOT) serum HBV RNA status on drug-withdrawal durability. METHODS: We carried out a real-life cohort study of 326 CHB patients on NA treatment between February 12, 2016 and February 21, 2018. Thirty of these patients discontinued NA treatment after enrollment, and were included in 2-year off-therapy follow-up. Serum HBV RNA levels were determined using the RNA simultaneous amplification testing method. RESULTS: Both serum HBV RNA and DNA levels declined significantly in long-term antiviral progress. When the treatment duration was longer than 3 years, the undetectable rates of HBV RNA and DNA were 55.10% and 97.0%, respectively. The serum HBV RNA-negative rate was 39.5%. The cumulative 2-year off-therapy viral and clinical relapse rate was 40.56%; 95% confidence interval (95% CI), 21.51-59.61 and 31.31%; 95% CI, 11.32-51.29 in all patients, respectively. Patients with EOT hepatitis B surface antigen (HBsAg)≤1000 IU/mL plus HBV RNA negativity had a relatively lower cumulative 2-year off-therapy viral relapse rate (23.01%; 95% CI, 0.17-45.99). EOT HBsAg≤1000 IU/mL plus HBV RNA negativity showed obvious superiority for the EOT HBsAg≤1000 IU/mL single in drug withdrawal durability prediction, with better specificity (18.18% vs. 72.73%, P=0.03), and the positive predictive value and negative predictive value were 76.92% and 47.06%, respectively. CONCLUSIONS: In the long-term antiviral process, both serum HBV RNA and DNA levels declined significantly. EOT serum HBV RNA negativity was not an independent drug withdrawal marker, but can complement the HBsAg titer to monitor drug withdrawal in CHB patients on long-term NA therapy.

Rab5, Rab7, and Rab11 Are Required for Caveola-Dependent Endocytosis of Classical Swine Fever Virus in Porcine Alveolar Macrophages.

The members of Flaviviridae utilize several endocytic pathways to enter a variety of host cells. Our previous work showed that classical swine fever virus (CSFV) enters porcine kidney (PK-15) cells through a clathrin-dependent pathway that requires Rab5 and Rab7. The entry mechanism for CSFV into other cell lines remains unclear, for instance, porcine alveolar macrophages (3D4/21 cells). More importantly, the trafficking of CSFV within endosomes controlled by Rab GTPases is unknown in 3D4/21 cells. In this study, entry and postinternalization of CSFV were analyzed using chemical inhibitors, RNA interference, and dominant-negative (DN) mutants. Our data demonstrated that CSFV entry into 3D4/21 cells depends on caveolae, dynamin, and cholesterol but not clathrin or macropinocytosis. The effects of DN mutants and knockdown of four Rab proteins that regulate endosomal trafficking were examined on CSFV infection, respectively. The results showed that Rab5, Rab7, and Rab11, but not Rab9, regulate CSFV endocytosis. Confocal microscopy showed that virus particles colocalize with Rab5, Rab7, or Rab11 within 30 min after virus entry and further with lysosomes, suggesting that after internalization CSFV moves to early, late, and recycling endosomes and then into lysosomes before the release of the viral genome. Our findings provide insights into the life cycle of pestiviruses in macrophages.IMPORTANCE Classical swine fever, is caused by classical swine fever virus (CSFV). The disease is notifiable to World Organisation for Animal Health (OIE) in most countries and causes significant financial losses to the pig industry globally. Understanding the processes of CSFV endocytosis and postinternalization will advance our knowledge of the disease and provide potential novel drug targets against CSFV. With this objective, we used systematic approaches to dissect these processes in CSFV-infected 3D4/21 cells. The data presented here demonstrate for the first time to our knowledge that CSFV is able to enter cells via caveola-mediated endocytosis that requires Rab5, Rab7 and Rab11, in addition to the previously described classical clathrin-dependent pathway that requires Rab5 and Rab7. The characterization of CSFV entry will further promote our current understanding of Pestivirus cellular entry pathways and provide novel targets for antiviral drug development.

Serum HBV RNA quantification: useful for monitoring natural history of chronic hepatitis B infection.

BACKGROUND: As an alternative biomarker of intrahepatic covalently closed circular DNA (cccDNA) transcriptional activity, hepatitis B virus (HBV) RNA may evolve during long-lasting virus-host interactions during chronic hepatitis B viral infection. The distribution pattern of serum HBV RNA levels in the natural course of chronic HBV infection remains unclear. The aim of this study was to evaluate the levels of HBV RNA during the natural course of CHB and the role in distinguishing the natural history of HBV infection. METHODS: A total of 291 treatment-naïve chronic HBV carriers were enrolled. Based on the clinical, biochemical, serological, and histological data as well as HBV DNA levels, patients were classified into the following four categories: the immune-tolerant phase (IT,n = 35), HBeAg-positive immune-active phase (EPIA,n = 121), inactive chronic hepatitis B(ICH,n = 77) and HBeAg-negative immune reactive hepatitis (ENH,n = 58) [corrected]. The parameters and distribution patterns of serum HBV RNA were evaluated in relation to viral replication status, immune phase, disease category and Child-Pugh class. The relationships between serum HBV RNA and other serum hepatitis B viral markers were also analyzed. RESULTS: Serum HBV RNA levels were significantly lower in the HBeAg-negative patients compared to those in the HBeAg-positive patients, with the lowest levels seen in inactive carriers. In HBeAg-negative patients, serum HBV RNA levels increased if there is reactivation to active hepatitis and showed obvious superiority for the combination of serum HBV DNA (cutoff>3.39 Log copies/mL) and HBsAg (cutoff>2.74 Log IU/mL) in discriminating between 'HBeAg-negative immune reactive' phase and inactive chronic hepatitis B phases of HBeAg-negative chronic HBV infection. Serum HBV RNA levels were positively correlated with serum HBV DNA and HBsAg levels in all chronic HBV-infected patients. A stratified analysis revealed that a correlation between serum HBV RNA and HBV DNA or HBsAg was present in HBeAg-positive patients; however, in HBeAg-negative patients, serum HBV RNA was positively correlated with HBV DNA only. CONCLUSION: During the natural course of chronic HBV infection, serum HBV RNA levels vary. Serum HBV RNA can act as a biomarker to predict the natural history of disease in chronic hepatitis B patients. In treatment-naïve HBeAg-negative chronic HBV-infected individuals, serum HBV RNA shows superiority in differentiating the 'HBeAg-negative reactive' phase.

Correction to: Serum HBV RNA quantification: useful for monitoring natural history of chronic hepatitis B infection.

Following publication of the original article [1].

Five-pointed star lattice sutures for fixation of patella transverse fractures: a clinical study.

INTRODUCTION: Fixation of displaced patella fractures with metal implants may be associated with implant failure, post-operative pain, and high re-operation rate. This study reports preliminary clinical results of using five-pointed star lattice sutures for the management of patella transverse fractures. METHODS: A five-pointed star lattice suture configuration was produced intraoperatively, and 25 patients with patella transverse fractures were treated with this newly designed sutures fixation. All patients were followed up until union of the fractures or until further surgical intervention. At a mean of 1.6 years (range 0.8-2.5 years) of follow-up, the notes and plain radiographs of the 25 patients were reviewed. Bostman score was used to evaluate the therapeutic effects. RESULTS: All 25 patients experienced union of the patella fractures, with excellent knee function in 19 patients and good in 6 patients evaluated with Bostman score. CONCLUSION: The newly designed five-pointed star lattice sutures fixation may be a feasible alternative to metal implants fixation in the management of patella transverse fracture. LEVEL OF EVIDENCE: Level IV case series.

Structural, vibrational and thermal expansion properties of Sc2W4O15.

A novel oxide material with the formula of Sc2W4O15 and orthorhombic symmetry is synthesized by solid state reactions and its structure, composition, vibrational properties and thermal expansion are investigated and identified by temperature-dependent X-ray diffraction (XRD), scanning electron microscopy (SEM), energy dispersive spectrometry (EDS), X-ray photoelectron spectrometry (XPS) and dilatometry. It is shown that the oxide material with an orthorhombic symmetry shows a similar structure to that of Sc2W3O12, but with W partially occupying the position of Sc, leading to not only the corner-sharing ScO6-WO4 connections but also the corner-sharing WO6-WO4 connections. Raman spectroscopic studies show that compared to Sc2W3O12, the FWHMs of most Raman modes in Sc2W4O15 increase due to the occupation of W6+ in the Sc3+ position. Besides, the W-O bonds in Sc2W4O15 are slightly harder than those in Sc2W3O12. An intrinsic thermal contraction in a wide range of temperatures (93-572 K) is demonstrated, which is attributed to the librational and translational vibrations of the corner-sharing polyhedra as well as the transverse vibrations of the bridging O atoms in the Sc-O-W and W-O-W linkages.

Continuously Tunable Wettability by Using Surface Patterned Shape Memory Polymers with Giant Deformability.

Designing smart surfaces with tunable wettability has drawn much attention in recent years for academic research and practical applications. Most of the previous methods to achieve such surfaces demand some particular materials that inherently have special features or complicated structures which are usually not easy to obtain. A novel strategy to achieve such smart surfaces is proposed by using the surface patterned shape memory polymers of chemically crosslinked polycyclooctene which shows a giant deformability of up to ≈730% strain. The smart surfaces possess the ability to continuously tune the wettability by controlling the recovery temperature and/or time. Coating the modified titanium dioxide nanoparticles onto such surfaces renders the surface superhydrophobicity and expands the tunable range of contact angles (CAs). Theoretical calculations of the CAs at different strains via modified Cassie model well explain the tunable wettability behaviors of such smart surfaces.

Copper nanoclusters trigger muscle cell apoptosis and atrophy in vitro and in vivo.

Copper nanoclusters (CuNCs) are increasingly being used in nanomedicine owing to their utility in cellular imaging and as catalysts. Additionally, nanotoxicology research of CuNCs is gaining attention. We report here the synthesis and characterization of CuNCs and their cytotoxic impact on muscle cells. A simple protein-directed synthesis of stable CuNCs was prepared, using bovine serum albumin as the stabling agent. Physicochemical characterization of the synthesized CuNCs was performed using transmission electron microscopy. To evaluate the in vitro cytotoxicity, C2C12 cells were exposed to increasing doses (from 0.1 to 50 µg ml(-1)) of CuNCs. CuNCs affected the viability of C2C12 cells in a dose-dependent manner, as detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and a lactate dehydrogenase release assay. Further studies indicated that CuNCs induced the formation of reactive oxygen species and decreased the activities of catalase and glutathione. CuNC treatment decreased the mitochondrial membrane potential and induced apoptosis, accompanied by an increase in the protein expression ratio of Bax/Bcl-2 and caspase-3/9 activity in C2C12 cells. CuNCs treatment resulted in atrophy of the C2C12 myotubes, which was characterized by the increased expression of atrophy-related genes, such as atrogin-1 and MuRF1. Finally, CuNCs induce morphological atrophy of primary muscle cells and mouse gastrocnemius muscle. Taken together, these results suggest that exposure to CuNCs may be a risk factor for the skeletal muscle system.

A novel surgical method for treating fractures of the middle third of the clavicle.

BACKGROUND: The clavicle is recognized as the bone most vulnerable to fractures. Moreover, approximately 80% of fractures occur in the middle third of the clavicle. Conservative treatment is associated with a higher rate of nonunion, while surgical treatment of fracture via internal fixation may have a variety of postoperative complications. Therefore, to improve patient satisfaction and reduce the complications related to internal fixation techniques, we modified the surgical approach to external fixation. OBJECTIVE: The purpose of this study was to assess the modified intervention's prospects for clinical application. METHODS: A total of 36 patients with middle clavicle fractures were treated with screw-rod external fixation between April 2015 and October 2019. We observed the operative time, intraoperative blood loss, length of hospital stay, and fracture healing time. The patients were followed up regularly, and the clinical efficacy of the modified intervention was evaluated. Finally, the patients' shoulder function was assessed based on the disabilities of the arm, shoulder, and hand (DASH) score. RESULTS: For the screw-rod external fixation, the mean operative time was found to be 48.6 ± 6.8 min, the intraoperative blood loss was 30.6 ± 17.2 mL, the length of hospital stay was 4.5 ± 1.5 days, and the fracture healing time was 2.8 ± 0.4 months. Eventually, all the patients healed well, with the combined "excellent" and "good" rate of shoulder function being assessed to be as high as 94.44%. Furthermore, the DASH scores were all less than 10, with the average score being 4.65 ± 3.34. CONCLUSIONS: The screw-rod external fixation technique offers the advantages of convenience, reliability, and good aesthetics, suggesting that it could be used as an alternative treatment method for fractures of the middle third of the clavicle.

Osteoblast-derived exosomes promote osteogenic differentiation of osteosarcoma cells via URG4/Wnt signaling pathway.

Osteosarcoma is a primary malignant bone tumor. Although surgery and chemotherapy are the main treatment methods, the overall curative effect remains unsatisfactory. Therefore, there is an urgent need to develop new therapeutic options for osteosarcoma. In this study, the effect and molecular mechanism of osteoblast-derived exosomes on the treatment of osteosarcoma were evaluated. Human primary osteoblasts were cultured to observe the effects of osteoblast-derived exosomes on the osteogenic differentiation of osteosarcoma cells both in vitro and in vivo. Alizarin red staining and alkaline phosphatase detection were used to evaluate the degree of osteogenic differentiation, and immunofluorescence and Western blotting were used to detect protein expression. The results showed that osteoblast-derived exosomes effectively inhibited the proliferation of osteosarcoma cells and promoted their mineralization in vitro. The exosomes also significantly inhibited tumor growth and promoted tumor tissue mineralization in vivo. Osteoblast-derived exosomes upregulated the expression of bone sialoprotein, osteonectin, osteopontin, runt-related transcription factor 2, and Wnt inhibitory factor 1, downregulated the expression of cyclin D1, and suppressed the nuclear accumulation of ß-catenin and promoted its phosphorylation in vitro and in vivo. However, these effects were significantly reversed by upregulated gene (URG) 4 overexpression. These findings suggest that osteoblast-derived exosomes could activate the osteogenic differentiation process in osteosarcoma cells and promote their differentiation by targeting the URG4/Wnt signaling pathway.