Enterovirus-71 utilizes small extracellular vesicles to cross the blood-brain barrier for infecting the central nervous system via transcytosis.

BACKGROUND: Central nervous system (CNS) infections caused by Enterovirus 71 (EV71) pose a serious threat to children, causing severe neurogenic complications and even fatality in some patients. However, the pathogenesis of EV71 infections in the CNS remains unclear. METHODS: An in vitro blood-brain barrier (BBB) model was constructed by coculturing brain microvascular endothelial cells (BMECs) and astrocytes in transwell inserts for simulating CNS infections. EV71 virions and small extracellular vesicles (sEVs) derived from EV71-infected cells (EV71-sEVs) were isolated from the cell culture supernatant by density gradient centrifugation. The BBB model was separately infected with EV71 virions and EV71-sEVs. The mechanism of crossing the BBB was determined by inhibiting the different endocytic modes. A murine model of EV71 infection was constructed for confirming the results of in vitro experiments. RESULTS: The EV71-sEVs containing viral components were endocytosed by BMECs and released on the abluminal side of the BBB model, where they infected the astrocytes without disrupting the BBB in the early stages of infection. The integrity of the tight junctions (TJs) between BMECs was breached via downregulation of PI3K/Akt signaling in the late stages of infection. CONCLUSIONS: EV71 utilized the circulating sEVs for infecting the CNS by crossing the BBB.

Roles and mechanisms of exosomal microRNAs in viral infections.

Exosomes are small extracellular vesicles with a diameter of 30-150 nm that originate from endosomes and fuse with the plasma membrane. They are secreted by almost all kinds of cells and can stably transfer different kinds of cargo from donor to recipient cells, thereby altering cellular functions for assisting cell-to-cell communication. Exosomes derived from virus-infected cells during viral infections are likely to contain different microRNAs (miRNAs) that can be transferred to recipient cells. Exosomes can either promote or suppress viral infections and therefore play a dual role in viral infection. In this review, we summarize the current knowledge about the role of exosomal miRNAs during infection by six important viruses (hepatitis C virus, enterovirus A71, Epstein-Barr virus, human immunodeficiency virus, severe acute respiratory syndrome coronavirus 2, and Zika virus), each of which causes a significant global public health problem. We describe how these exosomal miRNAs, including both donor-cell-derived and virus-encoded miRNAs, modulate the functions of the recipient cell. Lastly, we briefly discuss their potential value for the diagnosis and treatment of viral infections.

New method for emergency decision making with an integrated regret theory-EDAS method in 2-tuple spherical linguistic environment.

With the frequent occurrence of various emergency events, emergency decision making (EDM) has become an important research focus recently and many studies have been conducted to decrease the negative impact of emergencies. Normally, it is essential for decision makers to make satisfactory and reasonable emergency decisions in the shortest possible time as inappropriate decisions may result in enormous economic losses and serious social consequences. To ensure that an emergency response can be made efficiently, we propose a new EDM method by integrating regret theory and evaluation based on distance from average solution (EDAS) method within the 2-tuple spherical linguistic environment. First, the 2-tuple spherical linguistic term sets (TSLTSs) are employed by decision makers to express their uncertain and vague evaluation information on emergency alternatives. Then, an integrated EDM method based on regret theory and EDAS method is proposed to rank emergency alternatives and find out the optimal one. Besides, the criteria importance through inter-criteria correlation (CRITIC) method is used to determine criteria weights objectively in the EDM process. Finally, the proposed regret theory-EDAS method is applied to select the optimal response solution for a public health emergency in China. The superiority and practicality of the designed method are further justified through a comparative analysis with other EDM methods.

DNA aptamer against EV-A71 VP1 protein: selection and application.

BACKGROUND: Enterovirus 71 (EV-A71) is a highly infectious pathogen associated with hand, foot and mouth disease, herpangina, and various neurological complications, so it is important for the early detection and treatment of EV-A71. An aptamer is a nucleotide sequence that screened in vitro by the technology named systematic evolution of ligands by exponential enrichment technology (SELEX). Similar to antibodies, aptamers can bind to the targets with high specificity and affinity. Besides, emerging aptamers have many advantages comparing with antibodies, such as ease of synthesis and modification, having a wide variety of target materials, low manufacturing cost and easy flexibility in amending. Therefore, aptamers are promising in virus detection and anti-virus therapy. METHODS: Aptamers were selected by SELEX. Specificity, affinity and second structure were used to characterize the selected aptamers. Chemiluminescence was adopted to build an aptamer-based detection method for EV-A71. Cytopathogenic effects trial, the level of intracellular EV-A71 RNA and protein expression were used to evaluate the antiviral effect of the selected aptamers. RESULTS: Three DNA aptamers with high specificity and affinity for EV-A71structual protein VP1 were screened out. A rapid chemiluminutesescence aptamer biosensor for EV-A71 detection was designed out. The selected aptamers could inhibit the RNA replication and protein expression of EV-A71 in RD cells and ameliorate the cytopathogenic effects. CONCLUSIONS: The aptamers against EV-A71 have the potentiality to be applied as attractive candidates used for EV-A71 detection and treatment in the future.

The role of exosomal PD-L1 in tumor progression and immunotherapy.

Programmed death ligand 1 (PD-L1), a type I transmembrane protein, binds to its receptor PD-1 to suppress the activation of T cells, thereby maintaining immunological homeostasis. In contrast, tumor cells highly express PD-L1, which binds to receptor PD-1 expressed on activated T cells, leading to immune escape. Anti-PD-1/PD-L1 immune checkpoint therapy blocks the binding of PD-1/PD-L1 to reinvigorate the exhausted T cells, thereby inhibiting tumor growth. Exosomes are biologically active lipid-bilayer nanovesicles secreted by various cell types that mediate intercellular signal communication. Numerous studies have shown that tumor cells are able to promote tumor epithelial-mesenchymal transition, angiogenesis, and immune escape by releasing exosomes. Recent studies imply that tumor-derived exosomes could carry PD-L1 in the same membrane topology as the cell surface, thereby resisting immune checkpoint therapy. In this review, we mainly discuss the role of exosomes in the regulation of tumor progression and the potential resistance mechanism to immunotherapy via exosomal PD-L1. In addition, we propose that exosomal PD-L1 may have the potential to be a target to overcome resistance to anti-PD-1/PD-L1 antibody therapy.

Long non-coding RNA RUNXOR accelerates MDSC-mediated immunosuppression in lung cancer.

BACKGROUND: RUNX1 overlapping RNA (RUNXOR) is a long non-coding RNA that has been indicated as a key regulator in the development of myeloid cells by targeting runt-related transcription factor 1 (RUNX1). Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of cells consisting of immature granulocytes and monocytes with immunosuppression. However, the impact of lncRNA RUNXOR on the development of MDSCs remains unknown. METHODS: Both the expressions of RUNXOR and RUNX1 in the peripheral blood were measured by qRT-PCR. Human MDSCs used in this study were isolated from tumor tissue of patients with lung cancer by FCM or induced from PBMCs of healthy donors with IL-1ß + GM-CSF. Specific siRNA was used to knockdown the expression of RUNXOR in MDSCs. RESULTS: In this study, we found that the lncRNA RUNXOR was upregulated in the peripheral blood of lung cancer patients. In addition, as a target gene of RUNXOR, the expression of RUNX1 was downregulated in lung cancer patients. Finally, the expression of RUNXOR was higher in MDSCs isolated from the tumor tissues of lung cancer patients compared with cells from adjacent tissue. In addition, RUNXOR knockdown decreased Arg1 expression in MDSCs. CONCLUSIONS: Based on our findings, it is illustrated that RUNXOR is significantly associated with the immunosuppression induced by MDSCs in lung cancer patients and may be a target of anti-tumor therapy.

Subtype distribution of hepatitis C virus in Jiangsu, China.

Hepatitis C virus (HCV) genotype distribution varied by regions and transmission modes. In this study, we investigated HCV genotype distribution in five cities of Jiangsu, China, all of which are located in the Yangtze River Delta Region. A total of 363 samples were collected during 2011-2012. C/E2 and NS5B fragments of HCV were amplified using a multiple RT-nested PCR strategy and subjected to sequencing. Phylogenetic analysis was performed for HCV genotyping. Among 106 PCR positive cases, HCV subtypes 1a (0.9%), 1b (61.3%), 2a (15.1%), 3a (4.7%), 3b (9.4%), 6a (6.6%), and 6n (1.9%) were detected. Together with our previous data, we found that HCV subtypes were more among injection drug users (IDUs) (nine) than among general population (GP) (six), and the most common subtype among GP was 1b (73.9%), followed by 2a (14.5%), while the top four common subtypes among IDUs were 3a, 1b, 3b, and 6a, with similar prevalence rates (24.4%, 22.7%, 20.9%, and 17.4%, respectively). There were nine HCV subtypes prevalent among IDUs in Jiangsu, more than those in Xinjiang, Hubei, Yunnan, Guangxi, Guangdong, and Hong Kong. The top four common subtypes among IDUs in Jiangsu covered all the two most common HCV subtypes (except 6n subtype) observed in six targeted provinces/region. These results suggested that Jiangsu may be an important gathering place for various HCV subtypes and the gathering may be involved in the large scale of population migration from other regions of China to Eastern China.

Enterovirus 71 transmission by exosomes establishes a productive infection in human neuroblastoma cells.

Exosomes are small secreted cellular vesicles for intercellular communications which contain proteins, mRNAs, and microRNAs (miRNAs). Recent studies have shown that exosomes play an important role in the transmission of infectious agents including hepatitis C virus, human immunodeficiency virus, and so on. However, the role of exosomes in the transfer of enterovirus 71 (EV71) between host cells remains unknown. In this study, we show that the exosomes derived from EV71-infected rhabdomyosarcoma cells contain EV71 RNA and capsid protein VP1, determined by quantitative reverse transcription-PCR (QRT-PCR) and Western blot analysis. The shedding of exosomes containing virus can establish a productive infection in human neuroblastoma cell line (SK-N-SH). A comparative analysis of neutralization by EV71-specific immunoglobulins showed different levels of neutralization of exosomes-mediated infection compared with free virus. In conclusion, exosomes from EV71-infected cells may play an important role in virus dissemination and are partially resisted to antibody neutralization. Our results suggest that there is an exosomal route of EV71 transmission infection.

Mechanism and complex roles of HSC70/HSPA8 in viral entry.

The process of viruses entering host cells is complex, involving multiple aspects of the molecular organization of the cell membrane, viral proteins, the interaction of receptor molecules, and cellular signaling. Most viruses depend on endocytosis for uptake, when viruses reach the appropriate location, they are released from the vesicles, undergo uncoating, and release their genomes. Heat shock cognate protein 70(HSC70): also known as HSPA8, a protein involved in mediating clathrin-mediated endocytosis (CME), is involved in various viral entry processes. In this mini-review, our goal is to provide a summary of the function of HSC70 in viral entry. Understanding the interaction networks of HSC70 with viral proteins helps to provide new directions for targeted therapeutic strategies against viral infections.

Molecular characteristics of the structure protein VP1 in Coxsackievirus A10 Isolates from China.

INTRODUCTION: Coxsackievirus A10 (CVA10) is a non-enveloped, positive-sense single-stranded RNA virus classified within the Enterovirus genus in the Picornaviridae family. It is among the pathogens that can cause hand, foot and mouth disease. This study aimed to analyze the temporal and spatial distribution of CVA10 in China to understand its epidemiological characteristics of CVA10. METHODOLOGY: We collected the VP1 sequences of CVA10 from January 1, 2004, to December 31, 2019, from the GenBank database and created the global map using MapChart. We selected 56 known CVA10 genotype sequences. Then, MEGA6.06 was used to construct a phylogenetic tree with the collected gene sequences and the known reference sequences for comparative analysis to assess the distribution of CVA10 genotypes in different countries between 2004 and 2019. RESULTS: CVA10 has been widely detected or reported globally. In China, the prevalent genotype of CVA10 was mainly genotype B before 2008 and genotype C after 2009. In other countries, the prevalence of genotype D was dominant, followed by genotypes C and F, and the prevalence of CVA10 varied from continent to continent. CONCLUSIONS: Monitoring CVA10 genotypes or evolutionary branches should be strengthened, and the study of epidemic genotype characteristics should be enhanced. This will serve as a basis for further research and development of monovalent CVA10 or polyvalent vaccines designed for effective disease prevention.

Extracellular vesicles (EVs)' journey in recipient cells: from recognition to cargo release. / èƒžå¤–å›Šæ³¡åœ¨å—ä½“ç»†èƒžä¸­çš„æ— ç¨‹ï¼šä»Žè¯†åˆ«åˆ°è´§ç‰©é‡Šæ”¾.

Extracellular vesicles (EVs) are nano-sized bilayer vesicles that are shed or secreted by virtually every cell type. A variety of biomolecules, including proteins, lipids, coding and non-coding RNAs, and mitochondrial DNA, can be selectively encapsulated into EVs and delivered to nearby and distant recipient cells, leading to alterations in the recipient cells, suggesting that EVs play an important role in intercellular communication. EVs play effective roles in physiology and pathology and could be used as diagnostic and therapeutic tools. At present, although the mechanisms of exosome biogenesis and secretion in donor cells are well understood, the molecular mechanism of EV recognition and uptake by recipient cells is still unclear. This review summarizes the current understanding of the molecular mechanisms of EVs' biological journey in recipient cells, from recognition to uptake and cargo release. Furthermore, we highlight how EVs escape endolysosomal degradation after uptake and thus release cargo, which is crucial for studies applying EVs as drug-targeted delivery vehicles. Knowledge of the cellular processes that govern EV uptake is important to shed light on the functions of EVs as well as on related clinical applications.

Research Note: Synergistic effect of isopropoxy benzene guanidine and colistin against mcr-1-positive Escherichia coli in vitro and in duck intestine infection models.

Colistin (CST) is considered as "agent of last resort" against gram-negative bacteria as feed additive. Its clinical effectiveness has reduced since the emergence of mcr-1 gene in ducks. Isopropoxy benzene guanidine (IBG), a new guanidine derivative, showed positive effects on improving animal weights and alleviating intestinal pathogens, therefore, the objective of this study was to evaluate the effect of this compound supplement with CST in ducks and explore the possibilities in feed additive. A total of fifteen duck-origin Escherichia coli carrying the mcr-1 gene were included in this study. A checkerboard microdilution assay was used to evaluate the in vitro antibacterial activity of IBG combined with CST against mcr-1-positive E. coli. A 3-by-2 time-kill array of IBG (16, 32, and 64 µg/mL) and CST (1/2 MIC and 1/4 MIC) over 24 hours was utilized to characterize the activity of the agents alone and in combination against E. coli strain 1 in vitro. The intestinal colonization model was used to evaluate the in vivo effect of IBG combined with CST. These results indicated that the combination of IBG plus CST showed a synergistic effect against all clinical isolates (FICI < 0.5). The bacterial burden was reduced by more than 2 log10 CFU/mL when E. coli strain 1 was tested with 1/2 MIC CST plus 64 µg/mL IBG for 24 h. Further experiments in vivo demonstrated that the CST combined with IBG was able to increase duck weights, reduced intestinal pathogenic E. coli and showed a synergistic antibacterial effect. Combination of CST (4 mg/kg b.w.) plus IBG (32 or 64 mg/kg b.w.) achieved 1.84 to 3.29 log10 CFU/g killing after 7 d of therapy, which was significantly different from that in the challenge control group (p<0.05). In summary, our study demonstrated the potential use of IBG as feed additive for veterinary purposes in ducks and provided new insights into overcoming resistance in the future.

Targeting HNRNPA2B1 inhibits enterovirus 71 replication in SK-N-SH cells.

OBJECTIVE: To investigate the effect of heterogeneous nuclear ribonucleoprotein A2B1 (HNRNPA2B1) on the replication of enterovirus 71 (EV-71) in SK-N-SH cells. METHODS: The mRNA and protein expression of HNRNPA2B1 in SK-N-SH cells were detected by real-time quantitative PCR (qRT-PCR) and western blotting (WB), respectively. WB was used to detect HNRNPA2B1 protein expression in the nucleus and cytosol. The localization of HNRNPA2B1 protein in the nucleus and cytosol was detected by immunofluorescence (IF). The expression of HNRNPA2B1 was inhibited by small interfering RNA (si-HNRNPA2B1). Viral RNA, viral structural protein VP1, and viral titer were detected by qRT-PCR, WB, and viral dilution counting, respectively. RESULTS: EV-71 infection significantly upregulates the expression of HNRNPA2B1 in SK-N-SH cells. EV-71 infection promotes HNRNPA2B1 nucleus-cytoplasm redistribution. Down-regulation of HNRNPA2B1 expression significantly inhibited EV-71 replication. CONCLUSION: HNRNPA2B1 protein redistributed from nucleus to cytoplasm and is highly expressed in the cytoplasm during EV-71 infection. Inhibition of HNRNPA2B1 levels effectively inhibits EV-71 replication in SK-N-SH cells.

Enterovirus A71 Promotes Exosome Secretion by the Nonstructural Protein 3A Interacting with Rab27a.

Exosomes are small membrane-bound vesicles which are intraluminal vesicles (ILVs) secreted to the extracellular space after multivesicular bodies (MVBs) fuse with the plasma membrane. Although it is known that exosomes play a multitude of roles during viral infection, the mechanism that regulates their secretion during viral infection is unknown. Here, we found that enterovirus A71 (EV-A71) infection increased exosome secretion both in vivo and in vitro. Importantly, the expression of nonstructural protein 3A was sufficient to promote exosome secretion, while a mutation affecting the amino acid 18 position abrogated this effect, without changing the size of exosomes in vivo or in vitro. Transmission electron microscopy (TEM) analysis revealed that 3A decreases the number of MVBs and ILVs in vivo and in vitro, which suggested 3A may boost the fusion between MVBs and the plasma membrane. Furthermore, we demonstrated that an interaction between 3A and the small GTPase protein, Rab27a, protected Rab27a from ubiquitination, resulted in increasing exosome release. Data indicated a novel mechanism by which EV-A71 3A modifies exosome secretion during viral infection. IMPORTANCE Research has shown that viral infection impacts exosome secretion, but its regulation mechanisms remain poorly understood. Nonstructural protein 3A of EV-A71 interacts with many host factors and is involved in the remodeling of cellular membranes. In this investigation, we applied exogenous expression of 3A protein for exploring its regulation on exosome secretion and utilized immunoprecipitation combined with proteomics approaches to identify 3A-interacting factors. Our results demonstrate that 3A protein upregulates the release of the exosomes and that the 3A mutant strain of EV-A71 induce less exosome release compared with the EV-A71 wild type. Viral 3A protein interacts with the host factor Rab27a to prevent it from being ubiquitinated, which in turn improves exosome secretion both in vitro and in vivo. EV-A71 3A protein is a novel viral factor in the control of exosome production.

A Review of Labeling Approaches Used in Small Extracellular Vesicles Tracing and Imaging.

Small extracellular vesicles (sEVs), a subset of extracellular vesicles (EVs) originating from the endosomal compartment, are a kind of lipid bilayer vesicles released by almost all types of cells, serving as natural carriers of nucleic acids, proteins, and lipids for intercellular communication and transfer of bioactive molecules. The current findings suggest their vital role in physiological and pathological processes. Various sEVs labeling techniques have been developed for the more advanced study of the function, mode of action, bio-distribution, and related information of sEVs. In this review, we summarize the existing and emerging sEVs labeling techniques, including fluorescent labeling, radioisotope labeling, nanoparticle labeling, chemical contrast agents labeling, and label-free technique. These approaches will pave the way for an in-depth study of sEVs. We present a systematic and comprehensive review of the principles, advantages, disadvantages, and applications of these techniques, to help promote applications of these labeling approaches in future research on sEVs.

Dual roles and potential applications of exosomes in HCV infections.

The hepatitis C virus (HCV) causes severe liver diseases, including hepatitis, liver cirrhosis, and hepatocellular carcinoma, which have high morbidity and mortality. Antibody targeting receptor-mediated HCV infections have limited therapeutic benefits, suggesting that the transmission of HCV infections is possibly mediated via receptor-independent mechanisms. Exosomes are membrane-enclosed vesicles with a diameter of 30-200 nm, which originate from the fusion of endosomal multivesicular bodies with the plasma membrane. Accumulating evidence suggests that exosomes have a pivotal role in HCV infections. Exosomes can transfer viral and cellular bioactive substances, including nucleic acids and proteins, to uninfected cells, thus spreading the infection by masking these materials from immunological recognition. In addition, exosomes originating from some cells can deliver antiviral molecules or prompt the immune response to inhibit HCV infection. Exosomes can be used for the diagnosis of HCV-related diseases, and are being presently evaluated as therapeutic tools for anti-HCV drug delivery. This review summarizes the current knowledge on the dual roles and potential clinical applications of exosomes in HCV infections.

The functional roles of m6A modification in T lymphocyte responses and autoimmune diseases.

RNA N6-methyladenosine (m6A) modification is abundant in eukaryotes, bacteria and archaea. It is an RNA modification mainly existing in messenger RNA (mRNAs) and has a significant effect on the metabolism and function of mRNAs. m6A modification is controlled by three types of proteins, namely methyltransferase as the "writers", demethylase as the "erasers", and specific m6A recognized protein (YTHDF1-3) as the "readers". Recent studies have shown that m6A modification plays an important role in cancer, viral infection and autoimmune diseases. In this review, we will elaborate on the m6A modifications in the homeostasis and differentiation of T cells. Then we will further summarize the effects of m6A modification on the T cell responses and T cell-mediated autoimmune diseases. This will advance T cell epigenetics research and provide potential biomarkers and therapeutic targets for autoimmune diseases.

Revisiting PD-1/PD-L pathway in T and B cell response: Beyond immunosuppression.

The regulation of T cell response depends on co-inhibitory pathways that serve to control immune-mediated tissue damage and resolve inflammation by modulating the magnitude and duration of immune response. In this process, the axis of T-cell-expressed programmed death-1 (PD-1) and its ligands (PD-L1 and PD-L2) play a key role. While the PD-1/PD-L pathway has received considerable attention for its role in the maintenance of T cell exhaustion in cancer and chronic infection, the PD-1/PD-L pathway also plays diverse roles in regulating host immunity beyond T cell exhaustion. In this review, we will discuss emerging concepts in co-stimulatory functions of PD-1/PD-L pathway on T cell- and B cell response and explore the potential underlying mechanisms. In addition, based on the elevated expression of PD-1 and its ligands in local inflamed tissues, we further discussed the role of PD-1/PD-L pathway in autoimmune diseases.

The Role of Viral Proteins in the Regulation of Exosomes Biogenesis.

Exosomes are membrane-bound vesicles of endocytic origin, secreted into the extracellular milieu, in which various biological components such as proteins, nucleic acids, and lipids reside. A variety of external stimuli can regulate the formation and secretion of exosomes, including viruses. Viruses have evolved clever strategies to establish effective infections by employing exosomes to cloak their viral genomes and gain entry into uninfected cells. While most recent exosomal studies have focused on clarifying the effect of these bioactive vesicles on viral infection, the mechanisms by which the virus regulates exosomes are still unclear and deserve further attention. This article is devoted to studying how viral components regulate exosomes biogenesis, composition, and secretion.

Mechanism of cargo sorting into small extracellular vesicles.

Extracellular vesicles (EVs) are special membranous structures released by almost every cell type that carry and protect some biomolecules from being degraded. They transport important signaling molecules involved in cell communication, migration, and numerous physiological processes. EVs can be categorized into two main types according to their size: i) small extracellular vesicles (sEVs), such as exosomes (30-150 nm), released from the fusion of multivesicular bodies (MVBs) with the plasma membrane, and ii) large EVs, such as microvesicles (100-1000 nm). These are no longer considered a waste product of cells, but regulators of intercellular communication, as they can transport specific repertoires of cargos, such as proteins, lipids, and nucleic acids to receptor cells to achieve cell-to-cell communication. This indicates the existence of different mechanisms, which controls the cargos sorting into EVs. This review mainly gives a description about the biological roles of the cargo and the sorting mechanisms of sEVs, especially exosomes.