CVB3 Replication Research Articles

N6-Methyladenosine Positively Regulates Coxsackievirus B3 Replication.

Enteroviruses such as coxsackievirus B3 are identified as a common cause of viral myocarditis, but the potential mechanism of its replication and pathogenesis are largely unknown. The genomes of a variety of viruses contain N6-methyladenosine (m6A), which plays important roles in virus replication. Here, by using the online bioinformatics tools SRAMP and indirect immunofluorescence assay (IFA), we predict that the CVB3 genome contains m6A sites and found that CVB3 infection could alter the expression and cellular localization of m6A-related proteins. Moreover, we found that 3-deazaadenosine (3-DAA), an m6A modification inhibitor, significantly decreased CVB3 replication. We also observed that the m6A methyltransferases methyltransferase-like protein 3 (METTL3) and METTL14 play positive roles in CVB3 replication, whereas m6A demethylases fat mass and obesity-associated protein (FTO) or AlkB homolog 5 (ALKBH5) have opposite effects. Knockdown of the m6A binding proteins YTH domain family protein 1 (YTHDF1), YTHDF2 and YTHDF3 strikingly decreased CVB3 replication. Finally, the m6A site mutation in the CVB3 genome decreased the replication of CVB3 compared with that in the CVB3 wild-type (WT) strain. Taken together, our results demonstrated that CVB3 could exploit m6A modification to promote viral replication, which provides new insights into the mechanism of the interaction between CVB3 and the host.

Antiviral Activity of trans-Hexenoic Acid against Coxsackievirus B and Enterovirus A71.

Enterovirus infections are life-threatening viral infections which occur mainly among children and are possible causes of viral outbreak. Until now, treatment and management of infections caused by members of the genus Enterovirus largely depended on supportive care, and no antiviral medications are currently approved for the treatment of most of these infections. The urgency of discovering new therapeutic options for the treatment of enterovirus infection is increasing. In the present study, we identified that trans-2-hexenoic acid (THA), a natural product from a dietary source, possesses antiviral activity against coxsackievirus B (CVB) and enterovirus A71 (EV-A71) in a dose-dependent manner. We found that THA possesses antiviral activity at 50% effective concentrations (EC50) of 2.9 μM and 3.21 μM against CVB3 and EV-A71 infections, respectively. The time of addition assay revealed that THA inhibits both CVB3 and EV-A71 replication at the entry stage of infection. Additional results from this study further suggest that THA inhibits viral replication by blocking viral entry. Given that THA has received approval as a food additive, treatment of enterovirus infections with THA might be a safe therapeutic option or could pave the way for semisynthetic manufacturing of more antiviral drugs in the future.

Inhibition of Calpain Alleviates Apoptosis in Coxsackievirus B3-induced Acute Virus Myocarditis Through Suppressing Endoplasmic Reticulum Stress.

Virus myocarditis (VMC) is a common cardiovascular disease and a major cause of sudden death in young adults. However, there is still a lack of effective treatments. Our previous studies found that calpain activation was involved in VMC pathogenesis. This study aims to explore the underlying mechanisms further. Neonatal rat cardiomyocytes (NRCMs) and transgenic mice overexpressing calpastatin (Tg-CAST), the endogenous calpain inhibitor, were used to establish VMC model. Hematoxylin and eosin and Masson staining revealed inflammatory cell infiltration and fibrosis. An ELISA array detected myocardial injury. Cardiac function was measured using echocardiography. CVB3 replication was assessed by capsid protein VP1. Apoptosis was measured by TUNEL staining, flow cytometry, and western blot. The endoplasmic reticulum (ER) stress-related proteins were detected by western blot. Our data showed that CVB3 infection resulted in cardiac injury, as evidenced by increased inflammatory responses and fibrosis, which induced myocardial apoptosis. Inhibiting calpain, both by PD150606 and calpastatin overexpression, could attenuate these effects. Furthermore, ER stress was activated during CVB3 infection. However, calpain inhibition could downregulate some ER stress-associated protein levels such as GRP78, pancreatic ER kinase-like ER kinase (PERK), and inositol-requiring enzyme-1α (IRE-1α), and ER stress-related apoptotic factors, during CVB3 infection. In conclusion, calpain inhibition attenuated CVB3-induced myocarditis by suppressing ER stress, thereby inhibiting cardiomyocyte apoptosis.

The regulatory role of miR-107 in Coxsackie B3 virus replication.

Coxsackie B3 virus (CVB3) is a member of small RNA viruses that belongs to the genus Enterovirus of the family Picornaviridae and CVB3 is the main pathogen of acute and chronic viral myocarditis. In this study RT-qPCR was used to determine the expression of miR-107 in CVB3-infected and uninfected HeLa cells. The experimental results show that the level of miR-107 began to rise at 4 h after the infection, and significantly boosted at 6 h. Based on the results of this experiment, we consider that miR-107 expression is related to CVB3 infection. In order to further clarify the effect of miR-107 in the process of CVB3 infection, we studied the effect of miR-107 upstream and downstream target genes on CVB3 replication. Levels of the target RNAs were detected by RT-qPCR after CVB3 infection, and the expression of CVB3 capsid protein VP1 by western blot analysis. Then the virus in the supernatant was quantitated via a viral plaque assay, reflecting the release of the virus. The experimental results showed that miRNA-107 expression is associated with CVB3 replication and proliferation, while KLF4 and BACE1 as the downstream of miR-107 weakened CVB3 replication. Overexpressions of KLF4 and BACE1 negatively regulated CVB3 replication, this effect on CVB3 was completely opposite to that of miR-107. Further experiments revealed that the upstream lncRNA004787, a new lncRNA that had not been reported, was located on chromosome 5, strand - from 37073250 to 37070908 (genome assembly: hg19). We sequenced and studied lncRNA004787 and found that it partially inhibited CVB3 replication. This prompted us to speculate that lncRNA004787 probably impacted the replication by other means. In conclusion, miR-107 interfered with CVB3 replication and release.

Lithium chloride confers protection against viral myocarditis via suppression of coxsackievirus B3 virus replication

Viral myocarditis (VMC) is a type of inflammation affecting myocardial cells caused by viral infection and has been an important cause of dilated cardiomyopathy (DCM) worldwide. Type B3 coxsackievirus (CVB3), a non-enveloped positive-strand RNA virus of the Enterovirus genus, is one of most common agent of viral myocarditis. Till now, effective treatments for VMC are lacking due to lack of drugs or vaccine. Lithium chloride (LiCl) is applied in the clinical management of manic depressive disorders. Accumulating evidence have demonstrated that LiCl, also as an effective antiviral drug, exhibited antiviral effects for specific viruses. However, there are few reports of evaluating LiCl's antiviral effect in mice model. Here, we investigated the inhibitory influence of LiCl on the CVB3 replication in vitro and in vivo and the development of CVB3-induced VMC. We found that LiCl significantly suppressed CVB3 replication in HeLa via inhibiting virus-induced cell apoptosis. Moreover, LiCl treatment in vivo obviously inhibited virus replication within the myocardium and alleviated CVB3-induced acute myocarditis. Collectively, our data demonstrated that LiCl inhibited CVB3 replication and negatively regulated virus-triggered inflammatory responses. Our finding further expands the antiviral targets of LiCl and provides an alternative agent for viral myocarditis.

Baicalin Inhibits Coxsackievirus B3 Replication by Reducing Cellular Lipid Synthesis.

Baicalin is a flavonoid extracted from Scutellariae Radix and shows a variety of biological activities as reducing lipids, diminishing inflammation, and inhibiting bacterial infection. However, there is no report of baicalin against CVB3 infection. In this study, we found that baicalin can reduce viral titer in a dose-dependent manner in vitro at a dose with no direct virucidal effect. Moreover, we revealed that baicalin can also improve survival rate, reduce heart weight/body weight ratio, prevent virus replication, and relieve myocardial inflammation in the acute viral myocarditis mouse model induced by CVB3. Then, in order to explore the mechanism of baicalin inhibiting CVB3 replication, we respectively examined the expression of autophagosome marker LC3-II by Western blot, tested the concentration of free fatty acid (FFA) and cholesterol (CHO) by commercial kits, detected the mRNA levels of fatty acid synthase (Fasn) and acetyl coenzyme a carboxylase (ACC) by RT-PCR, and observed the lipid content of cells by fluorescence staining. The results showed that CVB3 infection increased autophagosome formation and lipid content in HeLa cells, but these changes were significantly blocked by baicalin. Finally, in order to confirm that baicalin inhibits viral replication and reduces autophagosome formation by reducing cellular lipids, we added exogenous palmitate to cell culture supernatants to promote intracellular lipid synthesis and found that palmitate did not alter LC3-II and CVB3/VP1 expression in HeLa cells with or without CVB3 infection. Interestingly, palmitate can reverse the inhibitory effect of baicalin on autophagosome formation and viral replication. In conclusion, our results indicated that lipids play an important role in CVB3 replication, and the effect of baicalin against CVB3 was associated with its ability to reduce cellular lipid synthesis to limit autophagosome formation.

Synthesis and biological evaluation of berberine derivatives as a new class of broad-spectrum antiviral agents against Coxsackievirus B

A series of novel berberine (BBR) analogues were prepared and tested for their antiviral potencies against six different genotype Coxsackievirus B (CVB1–6) strains, taking BBR core for structural modification. Structure-activity relationship (SAR) research revealed that introduction of a primary amine through a linker at position 3 might be beneficial for both antiviral activity and safety. Compound 14c displayed most promising inhibitory potency with IC50 values of 3.08–9.94 µM against tested CVBs 2–6 strains and satisfactory SI value of 34.3 on CVB3, better than that of BBR. Also, 14c could inhibit CVB3 replication through down-regulating the expression of VP1 protein and VP1 RNA. The mechanism revealed that 14c could suppress host components JNK-MAPK, ERK-MAPK and p38-MAPK activation. Therefore, BBR derivatives were considered to be a new class of anti-CVB agents with an advantage of broad-spectrum anti-CVB potency.

The protective role of microRNA-21 against coxsackievirus B3 infection through targeting the MAP2K3/P38 MAPK signaling pathway

BackgroundThe P38 mitogen-activated protein kinase (MAPK) pathway plays an essential role in CVB3-induced diseases. We previously demonstrated microRNA-21 has potential inhibitory effect on the MAP2K3 which locates upstream of P38 MAPK and was upregulated in mouse hearts upon CVB3 infection. However, the effect and underlying mechanism of miRNA-21 on CVB3 infection remain unclear.MethodsWe detected continuous changes of cellular miRNA-21 and P38 MAPK proteins expression profiling post CVB3 infection in vitro within 12 h. P38 MAPK signaling was inhibited by the specific inhibitor, small interfering RNA and miRNA-21 mimic in vitro, CVB3 replication, cell apoptosis rate and proliferation were detected. Viral load in the mice heart, cardiomyocyte apoptosis rate and histological of the heart were also detected in the mice model of viral myocarditis pretreated with miRNA-21-lentivirus.ResultsWe observed significant upregulation of miRNA-21 expression followed by suppression of the MAP2K3/P38 MAPK signaling in CVB3-infected Hela cells. The inactivation of the MAP2K3/P38 MAPK signaling by P38 MAPK specific inhibitor, small interfering RNA against MAP2K3, or miRNA-21 overexpression significantly inhibited viral progeny release from CVB3-infected cells. Mechanistically, when compared with control miRNA, miRNA-21 showed no effect on capsid protein VP1 expression and viral load within host cells, while significantly reversing CVB3-induced caspase-3 activation and cell apoptosis rate, further promoting proliferation of infected cells, which indicates the inhibitory effect of miRNA-21 on CVB3 progeny release. In the in vivo study, when compared with control miRNA, miRNA-21 pretreatment remarkably inactivated the MAP2K3/P38 MAPK signaling in mice and protected them against CVB3 infection as evidenced by significantly alleviated cell apoptosis rate, reduced viral titers, necrosis in the heart as well as by remarkably prolonged survival time.ConclusionsmiRNA-21 were reverse correlated with P38 MAPK activation post CVB3 infection, miRNA-21 overexpression significantly inhibited viral progeny release and decreased myocytes apoptosis rate in vitro and in vivo, suggesting that miRNA-21 may serve as a potential therapeutic agent against CVB3 infection through targeting the MAP2K3/P38 MAPK signaling.

Biphasic and cardiomyocyte-specific IFIT activity protects cardiomyocytes from enteroviral infection.

Viral myocarditis is a serious disease, commonly caused by type B coxsackieviruses (CVB). Here we show that innate immune protection against CVB3 myocarditis requires the IFIT (IFN-induced with tetratricopeptide) locus, which acts in a biphasic manner. Using IFIT locus knockout (IFITKO) cardiomyocytes we show that, in the absence of the IFIT locus, viral replication is dramatically increased, indicating that constitutive IFIT expression suppresses CVB replication in this cell type. IFNβ pre-treatment strongly suppresses CVB3 replication in wild type (wt) cardiomyocytes, but not in IFITKO cardiomyocytes, indicating that other interferon-stimulated genes (ISGs) cannot compensate for the loss of IFITs in this cell type. Thus, in isolated wt cardiomyocytes, the anti-CVB3 activity of IFITs is biphasic, being required for protection both before and after T1IFN signaling. These in vitro findings are replicated in vivo. Using novel IFITKO mice we demonstrate accelerated CVB3 replication in pancreas, liver and heart in the hours following infection. This early increase in virus load in IFITKO animals accelerates the induction of other ISGs in several tissues, enhancing virus clearance from some tissues, indicating that–in contrast to cardiomyocytes–other ISGs can offset the loss of IFITs from those cell types. In contrast, CVB3 persists in IFITKO hearts, and myocarditis occurs. Thus, cardiomyocytes have a specific, biphasic, and near-absolute requirement for IFITs to control CVB infection.

Nicotinic Agonist Inhibits Cardiomyocyte Apoptosis in CVB3-Induced Myocarditis via α3β4-nAChR/PI3K/Akt-Dependent Survivin Upregulation.

Background Cardiomyocyte apoptosis is critical for the development of coxsackievirus B3- (CVB3-) induced myocarditis, which is a common cardiac disease that may result in heart failure or even sudden death. Previous studies have associated CVB3-induced apoptosis with the downregulation of antiapoptotic proteins. Here, attempts were made to examine whether nicotinic acetylcholine receptors (nAChRs), especially α3β4-nAChRs, were a novel therapeutic antiapoptotic target via the activation of survivin, a strong antiapoptotic protein, in viral myocarditis (VMC). Methods and Results In the present study, we demonstrated that nAChRs, α3β4-nAChR subunits in particular, were present and upregulated in CVB3-infected neonatal rat cardiomyocytes (NRC) and H9c2 cells by RT-qPCR. The function of α3β4-nAChRs was next examined using its specific blocker α-CTX AuIB in vitro. The results of the TUNEL assay and western blot experiments showed that the block of α3β4-nAChRs abrogated nicotine-mediated protection of NRC from CVB3-induced apoptosis, and this effect displayed a substantial correlation with the protein expressions of pAkt, survivin, and Cleaved Caspase-3. Hence, the involvement of the PI3K/Akt pathway was further verified by LY294002, a selective inhibitor of PI3K. As a result, nicotine-mediated induction of pAkt and survivin was abolished by LY294002; meanwhile, apoptotic NRC were increased accompanied by an increase of Cleaved Caspase-3 expression. Regarding CVB3-infected BALB/c mice, the α-CTX AuIB- and LY294002-treated groups had a lower survival rate, deteriorative ventricular systolic function, and more severe inflammation than the nicotine-treated group and the modulation of pAkt, survivin, and Cleaved Caspase-3 protein expressions was similar to that in CVB3-infected NRC. In addition, we found that a nicotinic agonist reduced CVB3 replication in a dose-dependent manner in vitro, which indicates that nAChR activation may serve as a possible protection mechanism of CVB3-induced myocarditis. Conclusions Our study demonstrated that α3β4-nAChR subunits are essential in the nicotine-mediated antiapoptotic effect of protecting cardiomyocytes from CVB3-induced apoptosis in vivo and in vitro. This protection correlated with the PI3K/Akt pathway and the inducement of the antiapoptotic protein survivin. A combination of these mechanisms serves as a novel protective response to treat viral myocarditis.

Antiviral effect of compounds derived from Angelica archangelica L. on Herpes simplex virus-1 and Coxsackievirus B3 infections

The dichloromethane extract from fruits of Angelica archangelica L. was separated by the modern high-performance countercurrent chromatography (HPCCC). The extract and five pure compounds: xanthotoxin, bergapten, imperatorin, phellopterin and isoimperatorin, and the mixture of imperatorin and phellopterin, have been studied as the potential antiviral agents against Herpes simplex virus type l and Coxsackievirus B3. The cytotoxicity was measured using the MTT method. Compounds were tested for the in vitro antiviral activity using the cytopathic effect (CPE) inhibitory assay and by the virus titre reduction assay. Real-time PCR was used to quantify the relative inhibition of the HSV-1 replication. The results indicate that the highest activity was demonstrated by the extract, imperatorin, phellopterin and the mixture of imperatorin and phellopterin, reducing the HSV-1 replication by 5.61 log, 4.7 log, 3.01 log and 3.73 log, respectively. The influence of isolated compounds on the CVB3 replication was not significant. Only the extract caused the decrease in the titre of virus in relation to the virus control. Our results show that coumarins of A. archangelica L. might be a potential candidate for the development of the alternative natural anti- HSV-1 compound. Moreover, the presence of isopentenyloxy moiety at C-8 position significantly improves their activity.

Berberine Restricts Coxsackievirus B Type 3 Replication via Inhibition of c-Jun N-Terminal Kinase (JNK) and p38 MAPK Activation In Vitro.

BackgroundAt present, the treatment of coxsackievirus-induced myocarditis remains difficult. Berberine (BBR), an isoquinoline alkaloid isolated from traditional medicine herbs, exhibits significant anti-viral efficacy against various viruses. However, the underlying mechanism by which BBR controls CVB3 infection has not yet been reported. The purpose of this study was to investigate the anti-viral efficacy of BBR against CVB3 infection and its mechanism.Material/MethodsIn our experiments, the protein levels of VP1 and MAPKs signal pathway were measured by Western blot. The mRNA level of VP1 was measured by RT-PCR. The virus titers were determined by TCID50 assay.ResultsWe found that BBR treatment significantly decreased CVB3 replication in HeLa cells. In addition, the BBR treatment reduced the phosphorylation levels of JNK and p38 MAPK upon CVB3 infection in both HeLa cells and primary rat myocardial cells.ConclusionsTaken together, these results suggest that BBR inhibits CVB3 replication through the suppression of JNK and p38 MAPK activation, shedding new light on the investigation of therapeutic strategies against CVB3-induced viral myocarditis.

Melittin ameliorates CVB3-induced myocarditis via activation of the HDAC2-mediated GSK-3β/Nrf2/ARE signaling pathway

Viral myocarditis (VMC) is characterized as an inflammatory process of the myocardium and can be fatal in infants and children. Melittin is a major polypeptide in honey bee venom that has been traditionally used against inflammation. However, its effect on VMC and the underlying molecular mechanism has not been reported. In this study, BALB/c mice were intraperitoneally injected with CVB3 to build a VMC model and treated with melittin. The results showed that melittin increased the mice's body weight and inhibited CVB3 replication. HE staining also showed that melittin alleviated myocardial injury in the VMC model. Flow cytometry showed that melittin inhibited myocardial cell apoptosis; in addition, real-time PCR showed that melittin decreased the expression of bax and caspase-3, and increased the expression of bcl-2. The results of echocardiographic examination showed that melittin improved cardiac function. Moreover, melittin decreased the activity of AST, CK, HBDH and LDH, and decreased the production of IL-1β, IL-6, TNF-α and MCP-1 in CVB3-induced myocardial tissues. Finally, we also found that melittin increased the expression of HDAC2 and activated the GSK-3β/Nrf2/ARE signaling pathway, whereas these changes were reversed by inhibition of HDAC2 in VMC model mice. In conclusion, our results suggested that melittin ameliorates CVB3-induced myocarditis via activation of the HDAC2-mediated GSK-3β/Nrf2/ARE signaling pathway.

Emodin inhibits coxsackievirus B3 replication via multiple signalling cascades leading to suppression of translation.

CVB3 (coxsackievirus 3) is a primary causal agent of viral myocarditis. Emodin is a natural compound isolated from certain plant roots. In the present study, we found that emodin inhibited CVB3 replication in vitro and in mice, and now we report an unrecognized mechanism by which emodin inhibits CVB3 replication through suppression of viral protein translation via multiple pathways. On one hand, emodin treatment inhibited Akt/mTOR (mammalian target of rapamycin) signalling and activated 4EBP1 (eukaryotic initiation factor 4R-binding protein 1), leading to suppression of translation initiation of ribosomal protein L32 encoded by a 5'-TOP (terminal oligopyrimidine) mRNA. On the other hand, emodin treatment differentially regulated multiple signal cascades, including Akt/mTORC1/p70(S6K) (p70 S6 kinase), ERK1/2 (extracellular-signal-regulated kinase 1/2)/p90(RSK) (p90 ribosomal S6 kinase) and Ca(2+)/calmodulin, leading to activation of eEF2K (eukaryotic elongation factor 2 kinase) and subsequent inactivation of eEF2 (eukaryotic elongation factor 2), resulting in inhibition of CVB3 VP1 (viral protein 1) synthesis. These data imply that eEF2K is a major factor mediating cross-talk of different arms of signalling cascades in this signal network. This notion was verified by either overexpressing eEF2K or treating the cells with siRNAs or eEF2K inhibitor A484954. We showed further that the emodin-induced decrease in p70(S6K) phosphorylation plays a dominant positive role in activation of eEF2K and in turn in conferring the antiviral effect of emodin. This finding was further solidified by expressing constitutively active and dominant-negative Akt. Collectively, our data reveal that emodin inhibits viral replication through impairing translational machinery and suppression of viral translation elongation.

Construction of a subgenomic CV-B3 replicon expressing emerald green fluorescent protein to assess viral replication of a cardiotropic enterovirus strain in cultured human cells

Coxsackieviruses B (CV-B) (Picornaviridae) are a common infectious cause of acute myocarditis in children and young adults, a disease, which is a precursor to 10–20% of chronic myocarditis and dilated cardiomyopathy (DCM) cases. The mechanisms involved in the disease progression from acute to chronic myocarditis phase and toward the DCM clinical stage are not fully understood but are influenced by both viral and host factors. Subgenomic replicons of CV-B can be used to assess viral replication mechanisms in human cardiac cells and evaluate the effects of potential antiviral drugs on viral replication activities. Our objectives were to generate a reporter replicon from a cardiotropic prototype CV-B3/28 strain and to characterize its replication properties into human cardiac primary cells. To obtain this replicon, a cDNA plasmid containing the full CV-B3/28 genome flanked by a hammerhead ribozyme sequence and an MluI restriction site was generated and used as a platform for the insertion of sequences encoding emerald green fluorescent protein (EmGFP) in place of those encoding VP3. In vitro transcribed RNA from this plasmid was transfected into HeLa cells and human primary cardiac cells and was able to produce EmGFP and VP1-containing polypeptides. Moreover, non-structural protein biological activity was assessed by the specific cleavage of eIF4G1 by viral 2Apro. Viral RNA replication was indirectly demonstrated by inhibition assays, fluoxetine was added to cell culture and prevented the EmGFP synthesis. Our results indicated that the EmGFP CV-B3 replicon was able to replicate and translate as well as the CV-B3/28 prototype strain. Our EmGFP CV-B3 replicon will be a valuable tool to readily investigate CV-B3 replication activities in human target cell models.

Identification of the interaction of VP1 with GM130 which may implicate in the pathogenesis of CVB3-induced acute pancreatitis.

Coxsackievirus B3 (CVB3) is a causative agent of viral myocarditis, pancreatitis, and meningitis in humans. Although the susceptibility of CVB3-induced acute pancreatitis is age-dependent, the underlying mechanisms remain unclear. Here we identified the host factor Golgi matrix protein 130 (GM130) as a novel target of CVB3 during CVB3-induced acute pancreatitis. The viral protein VP1 interacted with GM130, disrupted GM130-GRASP65 complexes, and caused GM130 degradation, which may lead to disruption of the Golgi ribbon and development of acute pancreatitis in mice. Interestingly, the expression level of GM130 in mouse pancreas was age-dependent, which was nicely correlated with the age-associated susceptibility of CVB3-induced acute pancreatitis. Furthermore, interference RNA-mediated knockdown of GM130 significantly reduced CVB3 replication in HeLa cells. Taken together, the study identified GM130 as a novel target of CVB3, which may implicate in the pathogenesis of CVB3-induced acute pancreatitis.

Liver X receptor activation enhances CVB3 viral replication during myocarditis by stimulating lipogenesis.

Viral myocarditis (VM) is severe cardiac inflammation that can result in sudden death or congestive heart failure in previously healthy adults, with no effective therapy. Liver X receptor (LXR) agonists have both anti-inflammatory and lipid-lowering properties. This study investigates whether LXR agonist T0901317 may modulate viral replication and cardiac inflammation during VM. (i) Adult mice were administered T0901317 or vehicle with the onset of inflammation during CVB3 virus myocarditis or (ii) treated 2 days prior to CVB3 infection. Against what we expected, T0901317 treatment did not alter leucocyte infiltration after CVB3 infection; yet pre-administration with T0901317 resulted in increased mortality upon CVB3 infection, higher cardiac viral presence, and increased cardiomyocyte damage when compared with the vehicle. Furthermore, we show a correlation of fatty acid synthase (FAS) and sterol regulatory element-binding protein 1c (SREBP-1c) with CVB3 viral load in the heart and that T0901317 is able to enhance the cardiac expression of FAS and SREBP-1c. Finally, we show in vitro that T0901317 is able to exaggerate CVB3-mediated damage of Vero cells, whereas inhibitors of FAS and the SREBP-1c reduce the viral presence of CVB3 in neonatal cardiomyocytes. LXR agonism does not modulate cardiac inflammation, but exacerbates virus-mediated myocardial damage during VM by stimulating lipid biosynthesis and enhancing CVB3 replication.

Coxsackievirus B3 engineered to contain microRNA targets for muscle-specific microRNAs displays attenuated cardiotropic virulence in mice.

Coxsackievirus B3 (CVB3) is trophic for cardiac tissue and is a major causative agent for viral myocarditis, where local viral replication in the heart may lead to heart failure or even death. Recent studies show that inserting microRNA target sequences into the genomes of certain viruses can eradicate these viruses within local host tissues that specifically express the cognate microRNA. Here, we demonstrated both in vitro and in vivo that incorporating target sequences for miRNA-133 and -206 into the 5' untranslated region of the CVB3 genome ameliorated CVB3 virulence in skeletal muscle and myocardial cells that specifically expressed the cognate cellular microRNAs. Compared to wild-type CVB3, viral replication of the engineered CVB3 was attenuated in human TE671 (rhabdomyosarcoma) and L6 (skeletal muscle) cell lines in vitro that expressed high levels of miRNA-206. In the in vivo murine CVB3-infection model, viral replication of the engineered CVB3 was attenuated specifically in the heart that expressed high levels of both miRNAs, but not in certain tissues, which allowed the host to retain the ability to induce a strong and protective humoral immune response against CVB3. The results of this study suggest that a microRNA-targeting strategy to control CVB3 tissue tropism and pathogenesis may be useful for viral attenuation and vaccine development. Coxsackievirus B3 (CVB3) is a major causative agent for viral myocarditis, and viral replication in the heart may lead to heart failure or even death. Limiting CVB3 replication within the heart may be a promising strategy to decrease CVB3 pathogenicity. miRNAs are ∼21-nucleotide-long, tissue-specific endogenous small RNA molecules that posttranscriptionally regulate gene expression by imperfectly binding to the 3' untranslated region (UTR), the 5' UTR, or the coding region within a gene. In our study, muscle-specific miRNA targets (miRT) were incorporated into the CVB3 genome. Replication of the engineered viruses was restricted in the important heart tissue of infected mice, which reduced cardiac pathology and increased mouse survival. Meanwhile, replication ability was retained in other tissues, thus inducing a strong humoral immune response and providing long-term protection against CVB3 rechallenge. This study suggests that a microRNA-targeting strategy can potentially control CVB3 tissue tropism and pathogenesis and may be useful for viral attenuation and vaccine development.

Effect of lovastatin on coxsackievirus B3 infection in human endothelial cells

The coxsackie and adenovirus receptor (CAR) mediates the entry of coxsackievirus B (CVB) and adenovirus into host cells and is, therefore, a key determinant for the molecular pathogenesis of viral diseases such as myocarditis. The aim was to investigate the influence of HMG-CoA reductase inhibitor lovastatin on CAR expression in endothelial cells. Human umbilical vein endothelial cells (HUVECs) were exposed to different concentrations of lovastatin (0.05-5 μmol/l) for up to 48 h. Alterations in CAR expression were examined by quantitative real-time PCR (qRT-PCR) and flow cytometry. In addition, after treatment with 1 μmol/l lovastatin for 48 h, HUVECs were infected for 8 h with CVB3 and virus replication was detected by qRT-PCR using viral-specific TaqMan probes. We found that lovastatin decreases CAR mRNA expression by up to 80% (p < 0.01) and CAR protein expression by up to 19% (p < 0.01), in a concentration-dependent manner. Moreover, virus replication of CVB3 was significantly inhibited after lovastatin treatment (p < 0.05). The signaling mechanism of CAR down-regulation by lovastatin depends on the Rac1/Cdc42 pathway. This study shows for the first time that lovastatin reduces the expression of CAR and subsequently the replication of CVB3 in HUVECs.

Effect of prophylactically applied CpG ODN on the development of myocarditis in mice infected with Coxsackievirus B3

Coxsackievirus B3 was one of the major pathogens causing viral myocarditis. Toll-like receptor 9 activation contributed to the innate immune response in the process of CVB3-induced myocarditis. In order to find out how CpG oligodeoxynucleotide, known as a TLR-9 agonist, would affect the CVB3-induced myocarditis, we chose a C-type CpG oligodeoxynucleotide (YW002) injected to the mice one day before CVB3 challenge. On day 4 post CVB3 infection, 3 mice in each group were randomly sacrificed and their hearts were isolated to detect CVB3 replication. On day 10, the CVB3 neutralizing antibody and inflammatory change of the hearts were detected. The results indicated that the CVB3-induced myocarditis was aggravated with the declining body weight of mice, decreasing neutralizing antibody, and uncontrolling virus replication by injecting 20μg YW002 per mouse. When adjusted the amount at 10μg YW002 per mouse, there were no signs of aggravation in myocarditis. Plus, the mortality of the infected mice was reduced, the neutralizing antibody level was raised and the replication of virus was restrained. These results suggested that a proper amount of CpG oligodeoxynucleotide application could help to inhibit CVB3 infection.