Coxsackievirus B3 Replication Research Articles

Miltefosine reduces coxsackievirus B3 lethality of mice with enhanced STAT3 activation

Coxsackievirus B3 (CVB3), one serotype of enteroviruses, can induce fatal myocarditis and hepatitis in neonates, but both treatment and vaccine are unavailable. Few reports tested antivirals to reduce CVB3. Several antivirals were developed against other enterovirus serotypes, but these antivirals failed in clinical trials due to side effects and drug resistance. Repurposing of clinical drugs targeting cellular factors, which enhance viral replication, may be another option. Parasite and cancer studies showed that the cellular protein kinase B (Akt) decreases interferon (IFN), apoptosis, and interleukin (IL)-6-induced STAT3 responses, which suppress CVB3 replication. Furthermore, miltefosine, the Akt inhibitor used in the clinic for parasite infections, enhances IL-6, IFN, and apoptosis responses in treated patients, suggesting that miltefosine could be the potential antiviral for CVB3. This study was therefore designated to test the antiviral effects of miltefosine against CVB3 in vitro and especially, in mice, as few studies test miltefosine in vitro, but not in vivo. In vitro results showed that miltefosine inhibited viral replication with enhanced activation of the cellular transcription factor, STAT3, which is reported to reduce CVB3 both in vitro and in mice. Notably, STAT3 knockdown abolished the anti-CVB3 activity of miltefosine in vitro. Mouse studies demonstrated that miltefosine pretreatment reduced CVB3 lethality of mice with decreased virus loads, organ damage, and apoptosis, but enhanced STAT3 activation. Miltefosine could be prophylaxis for CVB3 by targeting Akt to enhance STAT3 activation in the mechanism, which is independent of IFN responses and hardly reported in pathogen infections.

Tribbles Pseudokinase 3 promotes enterovirus A71 infection via dual mechanisms

Enterovirus A71 (EV-A71) is the main pathogen causing hand, foot and mouth disease (HFMD) in children and occasionally associated with neurological diseases such as aseptic meningitis, brainstem encephalitis (BE) and acute flaccid paralysis. We report here that cellular pseudokinase tribbles 3 (TRIB3) facilitates the infection of EV-A71 via dual mechanisms. In one hand, TRIB3 maintains the metabolic stability of scavenger receptor class B member 2 (SCARB2), the bona fide receptor of EV-A71, to enhance the infectious entry and spreading of the virus. On the other hand, TRIB3 facilitates the replication of EV-A71 RNA in a SCARB2-independent manner. The critical role of TRIB3 in EV-A71 infection and pathogenesis was further demonstrated in vivo in mice. In comparison to wild-type C57BL/6 mice, EV-A71 infection in TRIB3 knockdown mice (Trib3 +/-) resulted in significantly lower viral loads in muscular tissues and reduced lethality and severity of clinical scores and tissue pathology. In addition, TRIB3 also promoted the replication of coxsackievirus B3 (CVB3) and coxsackievirus A16 (CVA16) in vitro. In conclusion, our results suggest that TRIB3 is one of key host cellular proteins required for the infection and pathogenesis of EV-A71 and some other human enteroviruses and may thus be a potential therapeutic target for combating the infection of those viruses.

Activation of cGAS-STING suppresses coxsackievirus replication via interferon-dependent signaling

Coxsackievirus B3 (CVB3) is a non-enveloped, single-stranded, positive RNA virus known for its role in provoking inflammatory diseases that affect the heart, pancreas, and brain, leading to conditions such as myocarditis, pancreatitis, and meningitis. Currently, there are no FDA-approved drugs treating CVB3 infection; therefore, identifying potential molecular targets for antiviral drug development is imperative. In this study, we examined the possibility of activating the cyclic GMP–AMP (cGAMP) synthase (cGAS)–stimulator of interferon genes (STING) pathway, a cytosolic DNA-sensing pathway that triggers a type-I interferon (IFN) response, in inhibiting CVB3 infection. We found that activation of the cGAS-STING pathway through the application of cGAS (poly dA:dT and herring testes DNA) or STING agonists (2′3′-cGAMP and diamidobenzimidazole), or the overexpression of STING, significantly suppresses CVB3 replication. Conversely, gene-silencing of STING enhances viral replication. Mechanistically, we demonstrated that cGAS-STING activation combats CVB3 infection by inducing IFN response. Notably, we discovered that knockdown of IFN-α/β receptor, a key membrane receptor in type-I IFN signaling, or inhibition of the downstream JAK1/2 signaling with ruxolitinib, mitigates the effects of STING activation, resulting in increased viral protein production. Furthermore, we investigated the interplay between CVB3 and the cGAS-STING pathway. We showed that CVB3 does not trigger cGAS-STING activation; instead, it antagonizes STING and the downstream TBK1 activation induced by cGAMP. In summary, our results provide insights into the interaction of an RNA virus and the DNA-sensing pathway, highlighting the potential for agonist activation of the cGAS-STING pathway in the development of anti-CVB3 drugs.

Antiviral Mechanisms of Saucerneol from Saururus chinensis against Enterovirus A71, Coxsackievirus A16, and Coxsackievirus B3: Role of Mitochondrial ROS and the STING/TKB-1/IRF3 Pathway.

Enterovirus A71 (EV71), coxsackievirus A16 (CVA16), and coxsackievirus B3 (CVB3) are pathogenic members of the Picornaviridae family that cause a range of diseases, including severe central nervous system complications, myocarditis, and pancreatitis. Despite the considerable public health impact of these viruses, no approved antiviral treatments are currently available. In the present study, we confirmed the potential of saucerneol, a compound derived from Saururus chinensis, as an antiviral agent against EV71, CVA16, and CVB3. In the in vivo model, saucerneol effectively suppressed CVB3 replication in the pancreas and alleviated virus-induced pancreatitis. The antiviral activity of saucerneol is associated with increased mitochondrial ROS (mROS) production. In vitro inhibition of mROS generation diminishes the antiviral efficacy of saucerneol. Moreover, saucerneol treatment enhanced the phosphorylation of STING, TBK-1, and IRF3 in EV71- and CVA16-infected cells, indicating that its antiviral effects were mediated through the STING/TBK-1/IRF3 antiviral pathway, which was activated by increased mROS production. Saucerneol is a promising natural antiviral agent against EV71, CVA16, and CVB3 and has potential against virus-induced pancreatitis and myocarditis. Further studies are required to assess its safety and efficacy, which is essential for the development of effective antiviral strategies against these viruses.

Ruxolitinib ameliorated coxsackievirus B3-induced acute viral myocarditis by suppressing the JAK-STAT pathway

BackgroundAccumulating evidences have demonstrated that overwhelming inflammation occurs in the process of Coxsackievirus B3 (CVB3)-induced acute viral myocarditis (AVM). No specific therapy is available. More than an effective Janus-associated kinase (JAK) inhibiter, ruxolitinib exerts a critical role in the inflammatory diseases. In this study, we investigated the potential effect of ruxolitinib on CVB3-induced acute viral myocarditis. MethodIn vivo, BALB/c mice were intraperitoneally injected of CVB3, treated of a successive gavage of ruxolitinib for seven days, and subjected to a series of analysis. In vitro, primary bone marrow-derived macrophages (BMDMs) and cardiac fibroblasts were isolated, cultured, treated, harvested and finally detected. ResultsIn vivo, acute viral myocarditis was successfully induced by the injection of CVB3 characterized by impaired cardiac function, predominant infiltration of inflammatory cells, necroptosis of myocardium, great increase of cardiac troponin I (cTnI) and cytokine levels, replication of CVB3, and excessive activation of JAK-STAT pathways. Oral administration of ruxolitinib suppressed the activation of JAK-STAT pathway in a dosage-dependent way, lessened the infiltration of inflammatory cells and necroptosis of myocardium, reduced the levels of cTnI and cytokines, and finally alleviated CVB3-induced cardiac dysfunction, with the reduced production of type I interferon and no promising effect on the replication of CVB3. In vitro, the treatment of ruxolitinib inhibited the activation of JAK-STAT pathway and increase of multiple cytokines mRNA levels in BMDMs and had no protective effect against CVB3 replication in cardiac fibroblasts. ConclusionsOur study suggested that ruxolitinib ameliorated CVB3-induced AVM by inhibiting the activation of JAK-STAT pathway, infiltration of inflammatory cells and necroptosis of myocardium, which may provide a novel strategy for AVM therapy.

Cardiac miR-19a/19b was induced and hijacked by CVB3 to facilitate virus replication via targeting viral genomic RdRp-encoding region

Coxsackievirus B3 (CVB3) is one of the major pathogens of viral myocarditis, lacking specific anti-virus therapeutic options. Increasing evidence has shown an important involvement of the miR-17-92 cluster both in virus infection and cardiovascular development and diseases, while its role in CVB3-induced viral myocarditis remains unclear. In this study, we found that miR-19a and miR-19b were significantly up-regulated in heart tissues of CVB3-infected mice and exerted a significant facilitatory impact on CVB3 biosynthesis and replication, with a more pronounced effect observed in miR-19b, by targeting the encoding region of viral RNA-dependent RNA polymerase 3D (RdRp, 3Dpol) to increase viral genomic RNA stability. The virus-promoting effects were nullified by the synonymous mutations in the viral 3Dpol-encoding region, which corresponded to the seed sequence shared by miR-19a and miR-19b. In parallel, treatment with miR-19b antagomir not only resulted in a noteworthy suppression of CVB3 replication and infection in infected cells, but also demonstrated a significant reduction in the cardiac viral load of CVB3-infected mice, resulting in a considerable alleviation of myocarditis. Collectively, our study showed that CVB3-induced cardiac miR-19a/19b contributed to viral myocarditis via facilitating virus biosynthesis and replication, and targeting miR-19a/19b might represent a novel therapeutic target for CVB3-induced viral myocarditis.

Comprehensive Metabolite Profiling of Chemlali Olive Tree Root Extracts Using LC-ESI-QTOF-MS/MS, Their Cytotoxicity, and Antiviral Assessment.

The large quantity of olive roots resulting from a large number of old and unfruitful trees encouraged us to look for ways of adding value to these roots. For this reason, the current research work is devoted to the valorization of olive roots by identifying active phytochemicals and assessing their biological activities, including the cytotoxicity and antiviral potential of different extracts from the Olea europaea Chemlali cultivar. The extract, obtained by ultrasonic extraction, was analyzed using the liquid chromatography-mass spectrometry technique (LC-MS). The cytotoxicity was evaluated through the use of the microculture tetrazolium assay (MTT) against VERO cells. Subsequently, the antiviral activity was determined for HHV-1 (Human Herpesvirus type 1) and CVB3 (Coxsackievirus B3) replication in the infected VERO cells. LC-MS analysis allowed the identification of 40 compounds, classified as secoiridoids (53%), organic acids (13%), iridoids (10%), lignans (8%), caffeoylphenylethanoid (5%), phenylethanoids (5%),sugars and derivatives (2%), phenolic acids (2%), and flavonoids (2%). It was found that extracts were not toxic to the VERO cells. Moreover, the extracts did not influence the appearance of HHV-1 or CVB3 cytopathic effects in the infected VERO cells and failed to decrease the viral infectious titer.

Cathelicidins Target HSP60 To Restrict CVB3 Transmission via Disrupting the Exosome and Reducing Cardiomyocyte Apoptosis.

Cathelicidin antimicrobial peptides (mouse, CRAMP; human, LL-37) have broad-spectrum antiviral activities against enveloped viruses, but their mechanisms of action against nonenveloped viruses remain to be elucidated. Coxsackievirus B3 (CVB3), a member of nonenveloped virus belonging to the Enterovirus genus of Picornaviridae, is an important pathogen of viral myocarditis and dilated cardiomyopathy. Here, we observed that cardiac CRAMP expression was significantly upregulated in mice after CVB3 infection. The administration of CRAMP or LL-37 markedly suppressed CVB3 infection in mice, and CRAMP deficiency increased the susceptibility of mice to CVB3. CRAMP and LL-37 inhibited CVB3 replication in primary cardiomyocytes. However, they did not inactivate CVB3 particles and did not regulate the response of cardiomyocytes against CVB3 infection. Intriguingly, they inhibited CVB3 transmission through the exosome, but not virus receptor. In detail, CRAMP and LL-37 directly induced the lysis of exosomes by interfering with exosomal heat shock protein 60 (HSP60) and then blocked the diffusion of exosomes to recipient cells and inhibited the establishment of productive infection by exosomes. In addition, the interaction of CRAMP and LL-37 with HSP60 simultaneously inhibited HSP60-induced apoptosis in cardiomyocytes and reduced HSP60-enhanced CVB3 replication. Our findings reveal a novel mechanism of cathelicidins against viral infection and provide a new therapeutic strategy for CVB3-induced viral myocarditis. IMPORTANCE The relative mechanisms that cathelicidin antimicrobial peptides use to influence nonenveloped virus infection are unclear. We show here that cathelicidin antimicrobial peptides (CRAMP and LL-37) directly target exosomal HSP60 to destroy exosomes, which in turn block the diffusion of exosomes to recipient cardiomyocytes and reduced HSP60-induced apoptosis, thus restricting coxsackievirus B3 infection. Our results provide new insights into the mechanisms cathelicidin antimicrobial peptides use against viral infection.

Stress Granules Inhibit Coxsackievirus B3-Mediated Cell Death via Reduction of Mitochondrial Reactive Oxygen Species and Viral Extracellular Release.

Stress granules (SGs) are cytoplasmic aggregates of RNA-protein complexes that form in response to various cellular stresses and are known to restrict viral access to host translational machinery. However, the underlying molecular mechanisms of SGs during viral infections require further exploration. In this study, we evaluated the effect of SG formation on cellular responses to coxsackievirus B3 (CVB3) infection. Sodium arsenite (AS)-mediated SG formation suppressed cell death induced by tumor necrosis factor-alpha (TNF-a)/cycloheximide (CHX) treatment in HeLa cells, during which G3BP1, an essential SG component, contributed to the modulation of apoptosis pathways. SG formation in response to AS treatment blocked CVB3-mediated cell death, possibly via the reduction of mitochondrial reactive oxygen species. Furthermore, we examined whether AS treatment would affect small extracellular vesicle (sEV) formation and secretion during CVB3 infection and modulate human monocytic cell (THP-1) response. CVB3-enriched sEVs isolated from HeLa cells were able to infect and replicate THP-1 cells without causing cytotoxicity. Interestingly, sEVs from AS-treated HeLa cells inhibited CVB3 replication in THP-1 cells. These findings suggest that SG formation during CVB3 infection modulates cellular response by inhibiting the release of CVB3-enriched sEVs.

Coxsackievirus B3 infection induces glycolysis to facilitate viral replication.

Coxsackievirus B3 (CVB3) is a leading cause of viral myocarditis, but no effective treatment strategy against CVB3 is available. Viruses lack an inherent metabolic system and thus depend on host cellular metabolism for their benefit. In this study, we observed that CVB3 enhanced glycolysis in H9c2 rat cardiomyocytes and HL-1 mouse cardiomyocytes. Therefore, three key glycolytic enzymes, namely, hexokinase 2 (HK2), muscle phosphofructokinase (PFKM), and pyruvate kinase M2 (PKM2), were measured in CVB3-infected H9c2 and HL-1 cells. Expression levels of HK2 and PFKM, but not PKM2, were increased in CVB3-infected H9c2 cells. All three key glycolytic enzymes showed elevated expression in CVB3-infected HL-1 cells. To further investigate this, we used 2 deoxyglucose, sodium citrate, and shikonin as glycolysis inhibitors for HK2, PFKM, and PKM2, respectively. Glycolysis inhibitors significantly reduced CVB3 replication, while the glycolysis enhancer dramatically promoted it. In addition, glycolysis inhibitors decreased autophagy and accelerated autophagosome degradation. The autophagy inducer eliminated partial inhibition effects of glycolysis inhibitors on CVB3 replication. These results demonstrate that CVB3 infection enhances glycolysis and thus benefits viral replication.

CircDDX17 enhances coxsackievirus B3 replication through regulating miR-1248/NOTCH receptor 2 axis.

Coxsackievirus B3 (CVB3) was one of the most common pathogens to cause viral myocarditis. Circular RNAs as novel non-coding RNAs with a closed loop molecular structure have been confirmed to be involved in virus infectious diseases, but the function in CVB3 infection was not systematically studied. In this study, we identified that hsa_circ_0063331 (circDDX17) was drastically decreased after CVB3 infection by circRNA microarray. In vivo and in vitro, when cells or mice were infected with CVB3, the expression of circDDX17 was significantly reduced, as demonstrated by quantitative real-time PCR assays. Additionally, circDDX17 enhanced CVB3 replication by downregulating the expression of miR-1248 in HeLa and HL-1 cells, and miR-1248 regulated CVB3 replication through interacting with the gene coding for NOTCH Receptor 2 (NOTCH2), and NOTCH2 could upregulate methyltransferase-like protein 3 (METTL3). Taken together, this study suggested that circDDX17 promoted CVB3 replication and regulated NOTCH2 by targeting miR-1248 as a miRNAs sponge.

Cleavage of AUF1 by Coxsackievirus B Affects DDX5 Regulatory on Viral Replication through iTRAQ Proteomics Analysis.

Coxsackievirus B (CVB) 3C protease (3Cpro) plays a specific cleavage role on AU-rich binding factor (AUF1, also called hnRNP D), which consequently disputes the regulation of AUF1 on downstream molecules. In our study, the iTRAQ approach was first used to quantify the differentially expressed cellular proteins in AUF1-overexpressing HeLa cells, which provides straightforward insight into the role of AUF1 during viral infection. A total of 1,290 differentially expressed proteins (DEPs), including 882 upregulated and 408 downregulated proteins, were identified. The DEPs are involved in a variety of cellular processes via GO terms, protein–protein interactions, and a series of further bioinformatics analyses. Among the DEPs, some demonstrated important roles in cellular metabolism. In particular, DDX5 was further verified to be negatively regulated by AUF1 and increased in CVB-infected cells, which in turn promoted CVB replication. These findings provide potential novel ideas for exploring new antiviral therapy targets.

Testosterone Promotes the Intestinal Replication and Dissemination of Coxsackievirus B3 in an Oral Inoculation Mouse Model.

Enteroviruses initiate infection in the gastrointestinal tract, and sex is often a biological variable that impacts pathogenesis. Previous data suggest that sex hormones can influence the intestinal replication of Coxsackievirus B3 (CVB3), an enterovirus in the Picornaviridae family. However, the specific sex hormone(s) that regulates intestinal CVB3 replication is poorly understood. To determine if testosterone promotes intestinal CVB3 replication, we orally inoculated male and female Ifnar-/- mice that were treated with either placebo or testosterone-filled capsules. Following oral inoculation, we found that the testosterone-treated male and female mice shed significantly more CVB3 in their feces than did the placebo-treated mice, indicating that testosterone enhances intestinal replication. Similarly, testosterone enhanced viral dissemination in both sexes, as we observed higher viral loads in peripheral tissues following infection. Further, the testosterone-treated male mice also had a higher mortality rate than did the testosterone-depleted male mice. Finally, we observed that testosterone significantly affected the immune response to CVB3. We found that testosterone broadly increased proinflammatory cytokines and chemokines while decreasing the number of splenic B cells and dendritic cells following CVB3 infection. Moreover, while testosterone did not affect the early CD4 T cell response to CVB3, testosterone reduced the activation of CD8 T cells. These data indicate that testosterone can promote intestinal CVB3 replication and dissemination while also impacting the subsequent viral immune response. IMPORTANCE Biological sex plays a significant role in the outcomes of various infections and diseases. The impact of sex hormones on the intestinal replication and dissemination of Coxsackievirus B3 remains poorly understood. Using an oral inoculation model, we found that testosterone enhances CVB3 shedding and dissemination in male and female mice. Further, testosterone can alter the immune response to CVB3. This work highlights the role of testosterone in CVB3 pathogenesis and suggests that sex hormones can impact the replication and dissemination of enteric viruses.

A Single Mutation in the Cryptic AUG (cAUG) Affects In Vitro Translation and Replication Efficiencies and In Vivo Virulence of Coxsackievirus B3 (CVB3).

The 5'UTR of the genomic RNA of CVB3, unusually long and rich on highly structured secondary structure, contains a conserved cis acting RNA element named the cryptic AUG (cAUG), where the cellular 48S complex is formed. In this study, we investigate the role of this cAUG in CVB3 translation, replication, and virulence. Mutant viral sub-genomic replicon RNA was constructed by site-directed mutagenesis. We characterize in vitro translation and replication efficiencies and in vivo virulence of a cAUG mutant in comparison with wild-type strain. UV-cross-linking assay and Real-Time PCR were used, respectively, to detect binding host proteins and to quantify viral production. Secondary structures of domain containing the cAUG site were studied and compared. The results suggest that introduced mutation in the CVB3 5'UTR affects in vitro and ex vivo viral translation which cannot be rescued by compensatory mutations. A reduced interaction of the La and PCBP2 translation initiation factors with cAUG residue of mutant was revealed. Decreasing production of viral mutant RNA was also demonstrated. Furthermore, secondary structure prediction reveals changes in the ribosome binding sites of the cAUG moiety of mutant sense strand RNA and no alterations in the structure of wild type, suggesting that cAUG mutation specifically affects the secondary structure of the sense RNA strand. Taken together, AUG integrity influences the efficiency of ribosome recruitment through IRES element and the capacity of replication.

Poly(rC) binding protein 1 benefits coxsackievirus B3 infection via suppressing the translation of p62/SQSTM1

Coxsackievirus B3 (CVB3) is a positive single-strand RNA virus causing myocarditis, pancreatitis and meningitis. During CVB3 infection, various host cellular components, including proteins and non-coding RNAs, interact with the virus and affect viral infection. Poly(rC) binding protein 1 (PCBP1) is a multifunctional RNA binding protein regulating transcription, translation and mRNA stability of a variety of genes. In this study, we observed a significant reduction of PCBP1 protein during CVB3 infection. By bioinformatic prediction and luciferase-assay verification, we confirmed that the expression of PCBP1 was directly inhibited by miR-21, a microRNA upregulated during CVB3 infection. Furthermore, we found that overexpression of PCBP1 promoted CVB3 infection and knocking down of PCBP1 inhibited it. In the subsequent mechanism study, our results revealed that PCBP1 blocked the translation of p62/SQSTM1 (sequestosome 1), an autophagy-receptor protein suppressing CVB3 replication, by interacting with the cis-element in the 5′ untranslational region (5′ UTR) of p62/SQSTM1. In summary, our studies have identified PCBP1 as a beneficial factor for CVB3 infection. These findings may deepen the understanding of host-virus interactions and provide a potential target for intervention of CVB3 infection.

Combination of dasabuvir and PSI-6206 for the treatment of coxsackievirus B3 infection

Coxsackievirus B3 (CVB3) infections may cause life-threatening diseases and have no approved specific treatment. Some promising approaches to treat viral diseases include drug repurposing and combination therapy. We have selected in this study dasabuvir, an approved antiviral drug, and PSI-6206, an experimental drug and determined their individual and combined antiviral activity against CVB3 replication in vitro. Our results show that the individual drugs inhibited CVB3 infection in a dose-dependent manner, at a selective index >10 with a strong synergetic antiviral effect of the two compounds. Given that dasabuvir has already been approved for the treatment of hepatitis C virus infection, treatment of CVB3-related disease with this drug may represent a promising treatment strategy.

The Nuclear Pore Complex 62 Suppression Inhibits Coxsackievirus B Replication and Inflammatory Response.

Coxsackievirus B3 (CVB3) is one of the major viruses associated with human viral myocarditis, in members of the Picornaviridae order. Cellular localization depends on the activity of nuclear pore complexes, which are composed of nucleoporins (Nups), including Nup62. To better understand interactions between Nup62 and CVB3, we investigated the impact of CVB3 infection on Nup62 levels and the impact of Nup62 production on CVB3 replication in cultured cells. We found that CVB3 infection correlated with decreased Nup62 expression in vitro and that lower levels of Nup62 led to inhibition of CVB3 replication and to decreased activation of AKT and extracellular signal-related kinase. Our study reveals that Nup62 regulates the CVB3 replication during infection.

The circRNA circSIAE Inhibits Replication of Coxsackie Virus B3 by Targeting miR-331-3p and Thousand and One Amino-Acid Kinase 2.

Coxsackie virus B3 (CVB3), an enterovirus, is the main pathogen causing viral myocarditis, pericarditis, hepatitis and other inflammation-related diseases. Non-coding RNAs with a closed loop molecular structure, called circular RNAs (circRNAs), have been shown to be involved in multiple virus-related processes, but roles and mechanisms in CVB3 infection have not been systematically studied. In this study, when HeLa cells were infected with CVB3, the expression of hsa_circ_0000367 (circSIAE) was significantly decreased as demonstrated by real-time quantitative PCR assays. We found that circSIAE downregulated the expression of miR-331-3p through direct binding and inhibited the replication of CVB3 in HeLa and 293T cells. The analysis of signals downstream of miR-331-3p suggested that miR-331-3p promotes CVB3 replication, viral plaque formation and fluorescent virus cell production through interactions with the gene coding for thousand and one amino-acid kinase 2 (TAOK2). In conclusion, this study found that circSIAE can target TAOK2 through sponge adsorption of miR-331-3p to inhibit the replication and proliferation of CVB3 virus, providing an early molecular target for the diagnosis of CVB3 infection.

Obesity Exacerbates Coxsackievirus Infection via Lipid-Induced Mitochondrial Reactive Oxygen Species Generation

Coxsackievirus B3 (CVB3) infection causes acute pancreatitis and myocarditis. However, its pathophysiological mechanism is unclear. Here, we investigated how lipid metabolism is associated with exacerbation of CVB3 pathology using high-fat diet (HFD)-induced obese mice. Mice were intraperitoneally inoculated with 1×106 pfu/mouse of CVB3 after being fed a control or HFD to induce obesity. Mice were treated with mitoquinone (MitoQ) to reduce the level of mitochondrial ROS (mtROS). In obese mice, lipotoxicity of white adipose tissue-induced inflammation caused increased replication of CVB3 and mortality. The coxsackievirus adenovirus receptor increased under obese conditions, facilitating CVB3 replication in vitro. However, lipid-treated cells with receptor-specific inhibitors did not reduce CVB3 replication. In addition, lipid treatment increased mitochondria-derived vesicle formation and the number of multivesicular bodies. Alternatively, we found that inhibition of lipid-induced mtROS decreased viral replication. Notably, HFD-fed mice were more susceptible to CVB3-induced mortality in association with increased levels of CVB3 replication in adipose tissue, which was ameliorated by administration of the mtROS inhibitor, MitoQ. These results suggest that mtROS inhibitors can be used as potential treatments for CVB3 infection.

Neutralization of interleukin-38 exacerbates coxsackievirus B3-induced acute myocarditis in mice

BackgroundInterleukin (IL)-38, a novel member of the IL-1 family, has been reported to be involved in several diseases associated with viral infection. However, the expression and functional role of IL-38 in acute viral myocarditis (AVMC) have not been investigated.MethodsMale BALB/c mice were treated with intraperitoneal (i.p.) injection of coxsackievirus B3 (CVB3) for establishing AVMC models. On day 7 post-injection, the expression of IL-38 and IL-36R (IL-36 receptor) were measured. Mice were then treated with i.p. injection of mouse Anti-IL-38 Antibodies (Abs) for neutralization of IL-38. The survival, bodyweight loss, cardiac function, and myocarditis severity of mice were recorded. The percentages of splenic Th1 and Th17 cells, the expression levels of Th1/Th17-related master transcription factors (T-bet and RORγt) and cytokines were determined by flow cytometry, RT-qPCR, and ELISA, respectively. Cardiac viral replication was further detected.ResultsThe mRNA and protein expression levels of IL-38 in myocardium and serum, as well as cardiac IL-36R mRNA levels were significantly elevated in mice with AVMC. Increased IL-38 levels were negatively correlated with the severity of AVMC. Neutralization of IL-38 exacerbated CVB3-induced AVMC, as verified by the lower survival rate, impaired cardiac function, continuous bodyweight loss, and higher values of HW/BW and cardiac pathological scores. In addition, neutralization of IL-38 suppressed Th1 cells differentiation while promoted Th17 cells differentiation, accompanied by decreased T-bet mRNA expression and increased RORγt expression. Down-regulation of IFN-γ and up-regulation of IL-17, TNF-α, and IL-6 mRNA and protein expression levels in myocardium and serum were also observed in the IL-38 neutralization group. Furthermore, neutralization of IL-38 markedly promoted cardiac viral replication.ConclusionsNeutralization of IL-38 exacerbates CVB3-induced AVMC in mice, which may be attributable to the imbalance of Th1/Th17 cells and increased CVB3 replication. Thus, IL-38 can be considered as a potential therapeutic target for AVMC.