Dendritic cell-based biomimetic nanoparticles for foot-and-mouth disease induce robust cellular immunity.

Foot-and-mouth disease (FMD) is a highly contagious and economically devastating viral disease of ruminants and swine, badly affecting the livestock industry worldwide. In clinical practice, vaccination is a frequently employed strategy to prevent foot-and-mouth disease (FMDV). However, commercial inactivated vaccines for FMD mainly rely on humoral immunity, exhibiting poor cellular immune responses and causing adverse reactions. Here, we use the double emulsion method to prepare poly (lactic-co-glycolic acid) nanoparticles (PLGA-NP) encapsulated with IL-2 cytokines, wrap the dendritic cell (DC) membrane carrying FMDV antigen information on the surface of the nanoparticles, obtaining a biomimetic nanoparticle vaccine Biom@DC with uniform size. This vaccine can effortlessly move through lymph nodes due to its nanoscale size advantage. It also possesses DC ability to present antigens, and antigen presentation can be made more effective with high biocompatibility. The sustained release of IL-2 encapsulated in the core of PLGA-NP in vivo can effectively promote the body's cellular immune response. Immune tests on mice have shown that Biom@DC may greatly increase T cell activation and proliferation both in vivo and in vitro, while also significantly reducing the fraction of inhibitory Treg cells. Furthermore, in the micro serum neutralization assay for FMDV, it has been demonstrated that the group vaccinated with Biom@DC exhibits a clear neutralizing effect. Given its strong immunogenicity, Biom@DC has the potential to develop into a novel, potent anti-FMDV vaccination.

FGFR1-mediated enhancement of foot-and-mouth disease virus entry.

Foot-and-mouth disease virus (FMDV), a member of picornavirus, can enter into host cell via macropinocytosis. Although it is known that receptor tyrosine kinases (RTKs) play a crucial role in FMDV macropinocytic entry, the specific RTK responsible for regulating this process and the intricacies of RTK-mediated downstream signaling remain to be elucidated. Here, we conducted a screening of RTK inhibitors to assess their efficacy against FMDV. Our findings revealed that two compounds specifically targeting fibroblast growth factor receptor 1 (FGFR1) and FMS-like tyrosine kinase 3 (FLT3) significantly disrupted FMDV entry. Furthermore, additional evaluation through gene knockdown and overexpression confirmed the promotion effect of FGFR1 and FLT3 on FMDV entry. Interestingly, we discovered that the increasement of FMDV entry facilitated by FGFR1 and FLT3 can be ascribed to increased macropinocytic uptake. Additionally, in-depth mechanistic study demonstrated that FGFR1 interacts with FMDV VP3 and undergoes phosphorylation during FMDV entry. Furthermore, the FGFR1 inhibitor inhibited FMDV-induced activation of p21-activated kinase 1 (PAK1) on Thr212 and Thr423 sites. Consistent with these findings, the ectopic expression of FGFR1 resulted in a concomitant increase in phosphorylation level of PAK1 on Thr212 and Thr423 sites. Taken together, our findings represent the initial exploration of FGFR1's involvement in FMDV macropinocytic entry, providing novel insights with potential implications for the development of antiviral strategies.

Nuclear ribonucleoprotein RALY downregulates foot-and-mouth disease virus replication but antagonized by viral 3C protease.

The internal ribosome entry site (IRES) element constitutes a cis-acting RNA regulatory sequence that recruits the ribosomal initiation complex in a cap-independent manner, assisted by various RNA-binding proteins and IRES trans-acting factors. Foot-and-mouth disease virus (FMDV) contains a functional IRES element and takes advantage of this element to subvert host translation machinery. Our study identified a novel mechanism wherein RALY, a member of the heterogeneous nuclear ribonucleoproteins (hnRNP) family belonging to RNA-binding proteins, binds to the domain 3 of FMDV IRES via its RNA recognition motif residue. This interaction results in the downregulation of FMDV replication by inhibiting IRES-driven translation. Furthermore, our findings reveal that the inhibitory effect exerted by RALY on FMDV replication is not attributed to the FMDV IRES-mediated assembly of translation initiation complexes but rather to the impediment of 80S ribosome complex formation after binding with 40S ribosomes. Conversely, 3Cpro of FMDV counteracts RALY-mediated inhibition by the ubiquitin-proteasome pathway. Therefore, these results indicate that RALY, as a novel critical IRES-binding protein, inhibits FMDV replication by blocking the formation of 80S ribosome, providing a deeper understanding of how viruses recruit and manipulate host factors. IMPORTANCE: The translation of FMDV genomic RNA driven by IRES element is a crucial step for virus infections. Many host proteins are hijacked to regulate FMDV IRES-dependent translation, but the regulatory mechanism remains unknown. Here, we report for the first time that cellular RALY specifically interacts with the IRES of FMDV and negatively regulates viral replication by blocking 80S ribosome assembly on FMDV IRES. Conversely, RALY-mediated inhibition is antagonized by the viral 3C protease by the ubiquitin-proteasome pathway. These results would facilitate further understanding of virus-host interactions and translational control during viral infection.

Foot-and-mouth disease virus downregulates vacuolar protein sorting 28 to promote viral replication.

Vacuolar protein sorting 28 (Vps28), a component of the ESCRT-I (endosomal sorting complex required for transport I), plays an important role in the pathogen life cycle. Here, we investigated the reciprocal regulation between Vps28 and the foot-and-mouth disease virus (FMDV). Overexpression of Vps28 decreased FMDV replication. On the contrary, the knockdown of Vps28 increased viral replication. Subsequently, the mechanistic study showed that Vps28 destabilized the replication complex (RC) by associating with 3A rather than 2C protein. In addition, Vps28 targeted FMDV VP0, VP1, and VP3 for degradation to inhibit viral replication. To counteract this, FMDV utilized tactics to restrict Vps28 to promote viral replication. FMDV degraded Vps28 mainly through the ubiquitin-proteasome pathway. Additional data demonstrated that 2B and 3A proteins recruited E3 ubiquitin ligase tripartite motif-containing protein 21 to degrade Vps28 at Lys58 and Lys25, respectively, and FMDV 3Cpro degraded Vps28 through autophagy and its protease activity. Meantime, the 3Cpro-mediated Vps28 degradation principally alleviated the ability to inhibit viral propagation. Intriguingly, we also demonstrated that the N-terminal and C-terminal domains of Vps28 were responsible for the suppression of FMDV replication, which suggested the elaborated counteraction between FMDV and Vps28. Collectively, our results first investigate the role of ESCRTs in host defense against picornavirus and unveil underlying strategies utilized by FMDV to evade degradation machinery for triumphant propagation. IMPORTANCE ESCRT machinery plays positive roles in virus entry, replication, and budding. However, little has been reported on its negative regulation effects during viral infection. Here, we uncovered the novel roles of ESCRT-I subunit Vps28 on FMDV replication. The data indicated that Vps28 destabilized the RC and impaired viral structural proteins VP0, VP1, and VP3 to inhibit viral replication. To counteract this, FMDV hijacked intracellular protein degradation pathways to downregulate Vps28 expression and thus promoted viral replication. Our findings provide insights into how ESCRT regulates pathogen life cycles and elucidate additional information regarding FMDV counteraction of host antiviral activity.

Advances and Breakthroughs in IRES-Directed Translation and Replication of Picornaviruses.

Viruses lack the properties to replicate independently due to the limited resources encoded in their genome; therefore, they hijack the host cell machinery to replicate and survive. Picornaviruses get the prerequisite for effective protein synthesis through specific sequences known as internal ribosome entry sites (IRESs). In the past 2 decades, significant progress has been made in identifying different types of IRESs in picornaviruses. This review will discuss the past and current findings related to the five different types of IRESs and various internal ribosome entry site trans-acting factors (ITAFs) that either promote or suppress picornavirus translation and replication. Some IRESs are inefficient and thus require ITAFs. To achieve their full efficiency, they recruit various ITAFs, which enable them to translate more effectively and efficiently, except type IV IRES, which does not require any ITAFs. Although there are two kinds of ITAFs, one promotes viral IRES-dependent translation, and the second type restricts. Picornaviruses IRESs are classified into five types based on their use of sequence, ITAFs, and initiation factors. Some ITAFs regulate IRES activity by localizing to the viral replication factories in the cytoplasm. Also, some drugs, chemicals, and herbal extracts also regulate viral IRES-dependent translation and replication. Altogether, this review will elaborate on our understanding of the past and recent advancements in the IRES-dependent translation and replication of picornaviruses. IMPORTANCE The family Picornaviridae is divided into 68 genera and 158 species. The viruses belonging to this family range from public health importance, such as poliovirus, enterovirus A71, and hepatitis A virus, to animal viruses of great economic importance, such as foot-and-mouth disease virus. The genomes of picornaviruses contain 5' untranslated regions (5' UTRs), which possess crucial and highly structured stem-loops known as IRESs. IRES assemble the ribosomes and facilitate the cap-independent translation. Virus-host interaction is a hot spot for researchers, which warrants deep insight into understanding viral pathogenesis better and discovering new tools and ways for viral restriction to improve human and animal health. The cap-independent translation in the majority of picornaviruses is modulated by ITAFs, which bind to various IRES regions to initiate the translation. The discoveries of ITAFs substantially contributed to understanding viral replication behavior and enhanced our knowledge about virus-host interaction more effectively than ever before. This review discussed the various types of IRESs found in Picornaviridae, past and present discoveries regarding ITAFs, and their mechanism of action. The herbal extracts, drugs, and chemicals, which indicated their importance in controlling viruses, were also summarized. In addition, we discussed the movement of ITAFs from the nucleus to viral replication factories. We believe this review will stimulate researchers to search for more novel ITAFs, drugs, herbal extracts, and chemicals, enhancing the understanding of virus-host interaction.

Poly(I:C) and CpG improve the assembly of foot-and-mouth disease virus-like particles and immune response in mice.

Virus-like particles (VLPs) are extremely potent, safe, and serviceable vaccine platforms. Good assembly efficiency of VLPs is the key to reducing vaccine production costs and eliciting a robust immune response. This study adopted CpG and Poly (I:C) as scaffolds to facilitate the assembly of foot-and-mouth disease virus (FMDV) VLPs in vitro. The VLPs and the co-assembly products were characterized by particle size, zeta potential, gel retardation measurement, nuclease digestion experiments, size-exclusion chromatography, transmission electron microscopy and circular dichroism analysis. Our results indicated the successful encapsulation of CpG and Poly (I:C) inside VLPs without any effect on shape or size. Vaccination in mice also elicited a robust immune response. This study demonstrated that CpG and Poly (I:C) improved the efficiency of FMDV VLPs assembly and enhanced immune response, further proposing a new idea for improving the efficiency of VLPs assembly and enriching the in vitro VLPs assembly strategies.

Heat Shock Protein 60 Is Involved in Viral Replication Complex Formation and Facilitates Foot and Mouth Virus Replication by Stabilizing Viral Nonstructural Proteins 3A and 2C.

The maintenance of viral protein homeostasis depends on the machinery of the infected host cells, giving us an insight into the interplay between host and virus. Accumulating evidence suggests that heat shock protein 60 (HSP60), as one molecular chaperone, is involved in regulating virus infection. However, the role of HSP60 during foot-and-mouth disease virus (FMDV) replication and its specific mechanisms have not been reported. We demonstrate that HSP60 modulates the FMDV life cycle. HSP60 plays a role at the postentry stage of the viral life cycle, including RNA replication and mRNA translation; however, HSP60 does not affect viral replication of Seneca Valley virus (SVA) or encephalomyocarditis virus (EMCV). We found that HSP60 is involved in FMDV replication complex (RC) formation. Furthermore, our results indicate that HSP60 interacts with FMDV nonstructural proteins 3A and 2C, key elements of the viral replication complex. We also show that HSP60 regulates the stability of 3A and 2C via caspase-dependent and autophagy-lysosome-dependent degradation, thereby promoting FMDV RNA synthesis and mRNA translation mediated by the RC. Additionally, we determined that the apical domain of HSP60 is responsible for interacting with 3A and 2C. The N terminus of 3A and ATPase domain of 2C are involved in binding to HSP60. Importantly, HSP60 depletion potently reduced FMDV pathogenicity in infected mice. Altogether, this study demonstrates a specific role of HSP60 in promoting FMDV replication. Furthermore, targeting host HSP60 will help us design the FMDV-specific antiviral drugs. IMPORTANCE FMDV is the leading cause of the foot-and-mouth disease (FMD), affecting cloven-hoofed animals with high morbidity and mortality. We determined that HSP60 is required for efficient viral RNA replication and mRNA translation during FMDV infection. Furthermore, we demonstrate that HSP60 interacts with FMDV nonstructural proteins 3A and 2C, the elements of the RC; HSP60 contributes to the stability of 3A and 2C to affect the formation and function of the RC. We also validated the potential role of HSP60 as the antiviral target in vivo using small interfering RNA. These findings deepen the understanding of the host-virus interaction and provide information supporting the design of novel therapeutics for FMDV infection.

Bio-mineralization of virus-like particles by metal-organic framework nanoparticles enhances the thermostability and immune responses of the vaccines.

Virus-like particle (VLPs) vaccines have been extensively studied due to their good immunogenicity and safety; however, they highly rely on cold-chain storage and transportation. Nanotechnology of bio-mineralization as a useful strategy has been employed to improve the thermal stability and immunogenicity of VLPs. A zeolitic imidazole framework (ZIF-8), a core-shell structured nanocomposite, was applied to encapsulate foot-and-mouth disease virus (FMDV) VLPs. It was found that the ZIF-8 shell enhanced the heat resistance of VLPs and promoted their ability to be taken up by cells and escape from lysosomes. The VLPs-ZIF-8 easily activated antigen-presenting cells (APCs), triggered higher secretion levels of cytokines, and elicited stronger immune responses than VLPs alone even after being treated at 37 °C for 7 days. This platform has good potential in the development of VLP-based vaccine products without transportation.

DDX21, a Host Restriction Factor of FMDV IRES-Dependent Translation and Replication.

In cells, the contributions of DEAD-box helicases (DDXs), without which cellular life is impossible, are of utmost importance. The extremely diverse roles of the nucleolar helicase DDX21, ranging from fundamental cellular processes such as cell growth, ribosome biogenesis, protein translation, protein-protein interaction, mediating and sensing transcription, and gene regulation to viral manipulation, drew our attention. We designed this project to study virus-host interactions and viral pathogenesis. A pulldown assay was used to investigate the association between foot-and-mouth disease virus (FMDV) and DDX21. Further insight into the DDX21-FMDV interaction was obtained through dual-luciferase, knockdown, overexpression, qPCR, and confocal microscopy assays. Our results highlight the antagonistic feature of DDX21 against FMDV, as it progressively inhibited FMDV internal ribosome entry site (IRES) -dependent translation through association with FMDV IRES domains 2, 3, and 4. To subvert this host helicase antagonism, FMDV degraded DDX21 through its non-structural proteins 2B, 2C, and 3C protease (3Cpro). Our results suggest that DDX21 is degraded during 2B and 2C overexpression and FMDV infection through the caspase pathway; however, DDX21 is degraded through the lysosomal pathway during 3Cpro overexpression. Further investigation showed that DDX21 enhanced interferon-beta and interleukin-8 production to restrict viral replication. Together, our results demonstrate that DDX21 is a novel FMDV IRES trans-acting factor, which negatively regulates FMDV IRES-dependent translation and replication.

Sec62 Regulates Endoplasmic Reticulum Stress and Autophagy Balance to Affect Foot-and-Mouth Disease Virus Replication.

Endoplasmic reticulum (ER) stress-induced autophagy is closely associated with viral infection and propagation. However, the intrinsic link between ER stress, autophagy, and viral replication during foot-and-mouth disease virus (FMDV) infection is not fully elucidated. Our previous studies demonstrated that FMDV infection activated the ER stress-associated UPR of the PERK-eIF2a and ATF6 signaling pathway, whereas the IRE1a signaling was suppressed. We found that the activated-ATF6 pathway participated in FMDV-induced autophagy and FMDV replication, while the IRE1α pathway only affected FMDV replication. Further studies indicated that Sec62 was greatly reduced in the later stages of FMDV infection and blocked the activation of the autophagy-related IRE1α-JNK pathway. Moreover, it was also found that Sec62 promoted IRE1a phosphorylation and negatively regulated FMDV proliferation. Importantly, Sec62 may interact with LC3 to regulate ER stress and autophagy balance and eventually contribute to FMDV clearance via fusing with lysosomes. Altogether, these results suggest that Sec62 is a critical molecule in maintaining and recovering ER homeostasis by activating the IRE1α-JNK pathway and delivering autophagosome into the lysosome, thus providing new insights on FMDV-host interactions and novel antiviral therapies.

Ribosomal Protein L13 Participates in Innate Immune Response Induced by Foot-and-Mouth Disease Virus.

In addition to ribosomal protein synthesis and protein translation, ribosomal proteins also participate in tumorigenesis and tumor progression, immune responses, and viral replication. Here, we show that ribosomal protein L13 (RPL13) participates in the antiviral immune response induced by foot-and-mouth disease virus (FMDV), inhibiting FMDV replication. The overexpression of RPL13 promoted the induction and activation of the promoters of the nuclear factor-κB (NF-κB) and interferon-ß (IFN-ß) genes, and the expression and protein secretion of the antiviral factor IFN-ß and proinflammatory cytokine interleukin-6 (IL-6). The knockdown of RPL13 had the opposite effects. We also found that the FMDV 3Cpro protease interacts with RPL13, and that its activity reduces the expression of RPL13, thus antagonizing the RPL13-mediated antiviral activity. This study extends our knowledge of the extraribosomal functions of ribosomal proteins and provides new scientific information on cellular antiviral defenses and virus-antagonizing mechanisms.

Nucleolin Promotes IRES-Driven Translation of Foot-and-Mouth Disease Virus by Supporting the Assembly of Translation Initiation Complexes.

Nucleolin (NCL), a stress-responsive RNA-binding protein, has been implicated in the translation of internal ribosome entry site (IRES)-containing mRNAs, which encode proteins involved in cell proliferation, carcinogenesis, and viral infection (type I IRESs). However, the details of the mechanisms by which NCL participates in IRES-driven translation have not hitherto been described. Here, we identified NCL as a protein that interacts with the IRES of foot-and-mouth disease virus (FMDV), which is a type II IRES. We also mapped the interactive regions within FMDV IRES and NCL in vitro. We found that NCL serves as a substantial regulator of FMDV IRES-driven translation but not of bulk cellular or vesicular stomatitis virus cap-dependent translation. NCL also modulates the translation of and infection by Seneca Valley virus (type III-like IRES) and classical swine fever virus (type III IRES), which suggests that its function is conserved in unrelated IRES-containing viruses. We also show that NCL affects viral replication by directly regulating the production of viral proteins and indirectly regulating FMDV RNA synthesis. Importantly, we observed that the cytoplasmic relocalization of NCL during FMDV infection is a substantial step for viral IRES-driven translation and that NCL specifically promotes the initiation phase of the translation process by recruiting translation initiation complexes to viral IRES. Finally, the functional importance of NCL in FMDV pathogenicity was confirmed in vivo. Taken together, our findings demonstrate a specific function for NCL in selective mRNA translation and identify a target for the development of a broad-spectrum class of antiviral interventions. IMPORTANCE FMDV usurps the cellular translation machinery to initiate viral protein synthesis via a mechanism driven by IRES elements. It allows the virus to shut down bulk cellular translation, while providing an advantage for its own gene expression. With limited coding capacity in its own genome, FMDV has evolved a mechanism to hijack host proteins to promote the recruitment of the host translation machinery, a process that is still not well understood. Here, we identified nucleolin (NCL) as a positive regulator of the IRES-driven translation of FMDV. Our study supports a model in which NCL relocalizes from the nucleus to the cytoplasm during the course of FMDV infection, where the cytoplasmic NCL promotes FMDV IRES-driven translation by bridging the translation initiation complexes with viral IRES. Our study demonstrates a previously uncharacterized role of NCL in the translation initiation of IRES-containing viruses, with important implications for the development of broad antiviral interventions.

Identification of Different Extracellular Vesicles in the Hydatid Fluid of Echinococcus granulosus and Immunomodulatory Effects of 110 K EVs on Sheep PBMCs.

Echinococcosis, mainly caused by Echinococcus granulosus, is one of the 17 neglected tropical diseases. Extracellular vesicles (EVs) play an essential role in the host-parasite interplay. However, the EVs in the hydatid fluid (HF) of E. granulosus are not fully characterized. Herein, three different types of HF EVs, designated as 2 K, 10 K, and 110 K EVs based on the centrifugal force used, were morphologically identified. A total of 97, 80, and 581 proteins were identified in 2 K, 10 K, and 110 K EVs, respectively, 39 of which were commonly shared. Moreover, 11, 8, and 25 miRNAs were detected, respectively, and all of the 7 selected miRNAs were validated by qPCR to be significantly lower abundant than that in protoscoleces. It was further deemed that 110 K EVs were internalized by sheep peripheral blood mononuclear cells (PBMCs) in a time-dependent manner and thus induced interleukin (IL)-10, tumor necrosis factor-α (TNF-α), and IRF5 were significantly upregulated and IL-1ß, IL-17, and CD14 were significantly downregulated (p < 0.05). These data demonstrate the physical discrepancy of three HF EVs and an immunomodulatory effect of 110 K EVs on sheep PMBCs, suggesting a role in immune responses during E. granulosus infection.

Gut bacteria formation and influencing factors.

The gut microbiota plays an important role in human health. In modern life, with the improvement of living conditions, the intake of high-sugar and high-fat diets as well as the large-scale use of antibacterial drugs have an extensive impact on the gut microbiota, even leading to gut microbiota-orchestrating disorders. This review discusses the effects of various factors, including geographic location, age, diet, antibacterial drugs, psychological situation and exercise on gut bacteria, which helps us profoundly to understand the significance of gut bacteria to human health and to find effective solutions to prevent or treat related diseases.

Comparative analysis of different extracellular vesicles secreted by Echinococcus granulosus protoscoleces.

Extracellular vesicles (EVs) are heterogeneous populations of different membrane-wrapped vesicles in size and encapsulated cargo and have recently emerged as a crucial carrier with the functions in intercellular communication, being involved in host-parasite interactions. However, Echinococcus granulosus EVs are not fully described. To separate EVs with a different size, the culture supernatant of E. granulosus protoscoleces (PSCs) was sequentially centrifuged at 2,000g, 10,000g and 110,000g, and the resulting precipitates were accordingly named as 2K, 10K and 110K EVs, respectively. The size and morphology of three different EVs were identified using ZETASIZER NANO and transmission electron microscopy (TEM), respectively. Then mass spectrometry was applied to define protein cargo of EVs and EV internalization was assessed using fluorescent microscopy and flow cytometry. The results showed that 2K EVs mainly ranged from 450 to 950 nm in diameter, 10K EVs ranged from 220 to 390 nm and 110K EVs from 60 to 150 nm. A total of 901 EV proteins were identified, 328 of which were commonly found in the three types of EVs. GO analysis revealed that these proteins were mainly involved in binding (44%) and catalytic activity (44%). Three types of EVs were different in biomarkers (Enolase and 14-3-3) and in reactivity with anti-echinococcosis positive serum. Moreover, 110K EVs were more easily internalized by hepatic cells than 10K EVs as well as 2K EVs (p < 0.0001). These results reveal the physical and biological discrepancy among 2K, 10K and 110K EVs, suggesting a distinct role in host-parasite interactions.

The DDX23 Negatively Regulates Translation and Replication of Foot-and-Mouth Disease Virus and Is Degraded by 3C Proteinase.

DEAD-box helicase 23 (DDX23) is a host nuclear helicase, which is a part of the spliceosomal complex and involved in pre-mRNA splicing. To investigate whether DDX23, an internal ribosomal entry sites transacting factor (ITAF) affects foot-and-mouth disease virus (FMDV) replication and translation through internal ribosome entry site (IRES)-dependent manner. For this, we utilized a pull-down assay, Western blotting, quantitative real-time PCR, confocal microscopy, overexpression and small interfering RNA knockdown, as well as the median tissue culture infective dose. Our findings showed that FMDV infection inhibited DDX23 expression and the overexpression of DDX23 reduced viral replication, however, CRISPR Cas9 knockout/small interfering RNA knockdown increased FMDV replication. FMDV IRES domain III and IV interacted with DDX23, whereas DDX23 interacted with FMDV 3C proteinase and significantly degraded. The enzymatic activity of FMDV 3C proteinase degraded DDX23, whereas FMDV degraded DDX23 via the lysosomal pathway. Additionally, IRES-driven translation was suppressed in DDX23-overexpressing cells, and was enhanced in DDX23 knocked down. Collectively, our results demonstrated that DDX23 negatively affects FMDV IRES-dependent translation, which could be a useful target for the design of antiviral drugs.

Argonaute proteins: Structural features, functions and emerging roles.

Argonaute proteins are highly conserved in almost all organisms. They not only involve in the biogenesis of small regulatory RNAs, but also regulate gene expression and defend against foreign pathogen invasion via small RNA-mediated gene silencing pathways. As a key player in these pathways, the abnormal expression and/or mis-modifications of Argonaute proteins lead to the disorder of small RNA biogenesis and functions, thus influencing multiply biological processes and disease development, especially cancer. In this review, we focus on the post-translational modifications and novel functions of Argonaute proteins in alternative splicing, host defense and genome editing.

miRNA-seq of Echinococcus multilocularis Extracellular Vesicles and Immunomodulatory Effects of miR-4989.

Alveolar echinococcosis caused by Echinococcus multilocularis is an important zoonotic disease. In the infected mice, emu-miR-4989-3p is present in sera, but its role remains unknown. Using high-throughput sequencing and qPCR, emu-miR-4989-3p was herein confirmed to be encapsulated into E. multilocularis extracellular vesicles. In the transfected macrophages, emu-miR-4989-3p was demonstrated to significantly inhibit NO production compared to the control (p < 0.05). Moreover, transfection of emu-miR-4989-3p also gave rise to the increased expression of TNF-α (p < 0.01). Furthermore, emu-miR-4989-3p induced the dysregulation of several key components in the LPS/TLR4 signaling pathway compared with the control, especially TLR4 and NF-κB that both were upregulated. Conversely, the NO production and the expression of TNF-α, TLR4 and NF-κB tended to be increased and decreased in the mimics-transfected cells upon emu-miR-4989-3p low expression, respectively. These results suggest that emu-miR-4989-3p is one of 'virulence' factors encapsulated into the extracellular vesicles, potentially playing a role in the pathogenesis of E. multilocularis.

Comparative analysis of miRNAs in exosomes released by sheeppox virus-infected ovine testicular cells.

Exosomes, secreted by various cells, are nanometer-scale vesicles with the functions in intercellular communication. To understand a role of exosomal miRNAs in the sheeppox virus infection, exosomes were isolated from sheeppox virus-infected sheep testicular cells 0 h, 24 h and 72 h post infection. The results of transmission electron microscopy and size distribution showed that all three exosome samples were spherical particles with negatively-stained membrane, ranging from 39 nm to 127 nm in diameter. A total of 106 known and 279 novel miRNAs were identified, and 78 known and 54 novel miRNAs were commonly detected in three exosome samples. Compared with the exosomes by the uninfected controls, a total of 34 known miRNAs were aberrantly expressed in the exosomes from infected cells. In agreement with the sequencing data, the expression of oar-miR-21 and oar-miR-10b was shown to be the highest in exosomes at 24 h after SPPV-infected, and the expression of oar-let-7f was the highest in exosomes at 72 h. Conversely, the expression of oar-let-7b and oar-miR-221 was significantly decreased 24 h and 72 h post infection compared with 0 h. The analysis results also revealed that differentially expressed miRNAs were mostly involved in an immune system process and stimulus response. These results provide rich data to further investigate a role of exosomal miRNAs in SPPV-host interactions.

High-throughput identification of microRNAs in Taenia hydatigena, a cestode threatening livestock breeding industry.

Infection of Cysticercus tenuicollis, the larval stage of Taenia hydatigena, is extensively found in sheep and pigs and jeopardizes the breeding and meat industry. miRNAs are a subclass of small noncoding regulatory RNAs and closely associated with the pathogenesis and biology of parasites. Here, using HiSeq sequencing we identified 49 known and 2 potential novel miRNAs in C. tenuicollis, of which both thy-miR-71 and -87 were predominant. Using RT-qPCR, 6 selected miRNAs were validated, and thy-miR-71 and -miR-87 were confirmed to be highly expressed, with the copy number of approximately 82,340 ±â€¯2079 and 19,580 ±â€¯609 per 1 ng total RNA, respectively. Similar to other cestodes, T. hydatigena was predicted to have two conserved miRNA clusters thy-miR-71/2c/2b and thy-miR-4989/277, and three members of the former were confirmed to reside sequentially within the genomic region of 253 bp by PCR. The current data provide us a valuable resource for further studies of a role of miRNAs in T. hydatigena biology and infection.