Proteome-wide copy-number estimation from transcriptomics.

Protein copy numbers constrain systems-level properties of regulatory networks, but proportional proteomic data remain scarce compared to RNA-seq. We related mRNA to protein statistically using best-available data from quantitative proteomics and transcriptomics for 4366 genes in 369 cell lines. The approach starts with a protein's median copy number and hierarchically appends mRNA-protein and mRNA-mRNA dependencies to define an optimal gene-specific model linking mRNAs to protein. For dozens of cell lines and primary samples, these protein inferences from mRNA outmatch stringent null models, a count-based protein-abundance repository, empirical mRNA-to-protein ratios, and a proteogenomic DREAM challenge winner. The optimal mRNA-to-protein relationships capture biological processes along with hundreds of known protein-protein complexes, suggesting mechanistic relationships. We use the method to identify a viral-receptor abundance threshold for coxsackievirus B3 susceptibility from 1489 systems-biology infection models parameterized by protein inference. When applied to 796 RNA-seq profiles of breast cancer, inferred copy-number estimates collectively re-classify 26-29% of luminal tumors. By adopting a gene-centered perspective of mRNA-protein covariation across different biological contexts, we achieve accuracies comparable to the technical reproducibility of contemporary proteomics.

An intranasal attenuated Coxsackievirus B3 vaccine induces strong systemic and mucosal immunity against CVB3 lethal challenge.

Coxsackievirus B3 (CVB3) triggers viral myocarditis, with no effective vaccine yet. This fecal-oral transmitted pathogen has prompted interest in mucosal immunization strategies to impede CVB3 spread. We developed a new attenuated vaccine strain, named CVB3(mu). The potential of CVB3(mu) to stimulate mucosal immune protection remains to be elucidated. This study evaluates the attenuation characteristics of CVB3(mu) via a rapid evolution cellular model and RNA sequencing. Its temperature sensitivity and safety were evaluated through in vitro and in vivo experiments. The mucosal immunity protection of CVB3(mu) was assessed via intranasal immunization in Balb/c mice. The results indicate that CVB3(mu) exhibits temperature sensitivity and forms smaller plaques. It sustains fewer genetic mutations and still possesses certain attenuated traits up to the 25th passage, in comparison to CVB3(WT). Intranasal immunization elicited a significant serum neutralizing antibodies, and a substantial sIgA response in nasal washes. In vivo trials revealed CVB3(mu) protection in adult mice and passive protection in suckling mice against lethal CVB3(WT) challenges. In conclusion, CVB3(mu), a live attenuated intranasal vaccine, provides protection involving humoral and mucosal immunity, making it a promising candidate to control CVB3 spread and infection.

miR-29b-3p regulates cardiomyocytes pyroptosis in CVB3-induced myocarditis through targeting DNMT3A.

BACKGROUND: Viral myocarditis (VMC) is a disease resulting from viral infection, which manifests as inflammation of myocardial cells. Until now, the treatment of VMC is still a great challenge for clinicians. Increasing studies indicate the participation of miR-29b-3p in various diseases. According to the transcriptome sequencing analysis, miR-29b-3p was markedly upregulated in the viral myocarditis model. The purpose of this study was to investigate the role of miR-29b-3p in the progression of VMC. METHODS: We used CVB3 to induce primary cardiomyocytes and mice to establish a model of viral myocarditis. The purity of primary cardiomyocytes was identified by immunofluorescence. The cardiac function of mice was detected by Vevo770 imaging system. The area of inflammatory infiltration in heart tissue was shown by hematoxylin and eosin (H&E) staining. The expression of miR-29b-3p and DNMT3A was detected by quantitative real time polymerase chain reaction (qRT-PCR). The expression of a series of pyroptosis-related proteins was detected by western blot. The role of miR-29b-3p/DNMT3A in CVB3-induced pyroptosis of cardiomyocytes was studied in this research. RESULTS: Our data showed that the expression of miR-29b-3p was upregulated in CVB3-induced cardiomyocytes and heart tissues in mice. To explore the function of miR-29b-3p in CVB3-induced VMC, we conducted in vivo experiments by knocking down the expression of miR-29b-3p using antagomir. We then assessed the effects on mice body weight, histopathology changes, myocardial function, and cell pyroptosis in heart tissues. Additionally, we performed gain/loss-of-function experiments in vitro to measure the levels of pyroptosis in primary cardiomyocytes. Through bioinformatic analysis, we identified DNA methyltransferases 3A (DNMT3A) as a potential target gene of miR-29b-3p. Furthermore, we found that the expression of DNMT3A can be modulated by miR-29b-3p during CVB3 infection. CONCLUSIONS: Our results demonstrate a correlation between the expression of DNMT3A and CVB3-induced pyroptosis in cardiomyocytes. These findings unveil a previously unidentified mechanism by which CVB3 induces cardiac injury through the regulation of miR-29b-3p/DNMT3A-mediated pyroptosis.

STAT3 Increases CVB3 Replication and Acute Pancreatitis and Myocarditis Pathology via Impeding Nuclear Translocation of STAT1 and Interferon-Stimulated Gene Expression.

Acute pancreatitis (AP) is an inflammatory disease initiated by the death of exocrine acinar cells, but its pathogenesis remains unclear. Signal transducer and activator of transcription 3 (STAT3) is a multifunctional factor that regulates immunity and the inflammatory response. The protective role of STAT3 is reported in Coxsackievirus B3 (CVB3)-induced cardiac fibrosis, yet the exact role of STAT3 in modulating viral-induced STAT1 activation and type I interferon (IFN)-stimulated gene (ISG) transcription in the pancreas remains unclarified. In this study, we tested whether STAT3 regulated viral-induced STAT1 translocation. We found that CVB3, particularly capsid VP1 protein, markedly upregulated the phosphorylation and nuclear import of STAT3 (p-STAT3) while it significantly impeded the nuclear translocation of p-STAT1 in the pancreases and hearts of mice on day 3 postinfection (p.i.). Immunoblotting and an immunofluorescent assay demonstrated the increased expression and nuclear translocation of p-STAT3 but a blunted p-STAT1 nuclear translocation in CVB3-infected acinar 266-6 cells. STAT3 shRNA knockdown or STAT3 inhibitors reduced viral replication via the rescue of STAT1 nuclear translocation and increasing the ISRE activity and ISG transcription in vitro. The knockdown of STAT1 blocked the antiviral effect of the STAT3 inhibitor. STAT3 inhibits STAT1 activation by virally inducing a potent inhibitor of IFN signaling, the suppressor of cytokine signaling-3 ((SOCS)-3). Sustained pSTAT1 and the elevated expression of ISGs were induced in SOCS3 knockdown cells. The in vivo administration of HJC0152, a pharmaceutical STAT3 inhibitor, mitigated the viral-induced AP and myocarditis pathology via increasing the IFNß as well as ISG expression on day 3 p.i. and reducing the viral load in multi-organs. These findings define STAT3 as a negative regulator of the type I IFN response via impeding the nuclear STAT1 translocation that otherwise triggers ISG induction in infected pancreases and hearts. Our findings identify STAT3 as an antagonizing factor of the IFN-STAT1 signaling pathway and provide a potential therapeutic target for viral-induced AP and myocarditis.

Sex Differences in Expression of Pro-Inflammatory Markers and miRNAs in a Mouse Model of CVB3 Myocarditis.

Myocarditis is an inflammatory disease that may lead to dilated cardiomyopathy. Viral infection of the myocardium triggers immune responses, which involve, among others, macrophage infiltration, oxidative stress, expression of pro-inflammatory cytokines, and microRNAs (miRNAs). The cardioprotective role of estrogen in myocarditis is well documented; however, sex differences in the miRNA expression in chronic myocarditis are still poorly understood, and studying them further was the aim of the present study. Male and female ABY/SnJ mice were infected with CVB3. Twenty-eight days later, cardiac tissue from both infected and control mice was used for real-time PCR and Western blot analysis. NFκB, IL-6, iNOS, TNF-α, IL-1ß, MCP-1, c-fos, and osteopontin (OPN) were used to examine the inflammatory state in the heart. Furthermore, the expression of several inflammation- and remodeling-related miRNAs was analyzed. NFκB, IL-6, TNF-α, IL-1ß, iNOS, and MCP-1 were significantly upregulated in male mice with CVB3-induced chronic myocarditis, whereas OPN mRNA expression was increased only in females. Further analysis revealed downregulation of some anti-inflammatory miRNA in male hearts (let7a), with upregulation in female hearts (let7b). In addition, dysregulation of remodeling-related miRNAs (miR27b and mir199a) in a sex-dependent manner was observed. Taken together, the results of the present study suggest a sex-specific expression of pro-inflammatory markers as well as inflammation- and remodeling-related miRNAs, with a higher pro-inflammatory response in male CVB3 myocarditis mice.

Role of Coxsackievirus B3-Induced Immune Responses in the Transition from Myocarditis to Dilated Cardiomyopathy and Heart Failure.

Dilated cardiomyopathy (DCM) is a cardiac disease marked by the stretching and thinning of the heart muscle and impaired left ventricular contractile function. While most patients do not develop significant cardiac diseases from myocarditis, disparate immune responses can affect pathological outcomes, including DCM progression. These altered immune responses, which may be caused by genetic variance, can prolong cytotoxicity, induce direct cleavage of host protein, or encourage atypical wound healing responses that result in tissue scarring and impaired mechanical and electrical heart function. However, it is unclear which alterations within host immune profiles are crucial to dictating the outcomes of myocarditis. Coxsackievirus B3 (CVB3) is a well-studied virus that has been identified as a causal agent of myocarditis in various models, along with other viruses such as adenovirus, parvovirus B19, and SARS-CoV-2. This paper takes CVB3 as a pathogenic example to review the recent advances in understanding virus-induced immune responses and differential gene expression that regulates iron, lipid, and glucose metabolic remodeling, the severity of cardiac tissue damage, and the development of DCM and heart failure.

Effect of FoxO1 on Cardiomyocyte Apoptosis and Inflammation in Viral Myocarditis via Modultion of the TLR4/NF-κB Signaling Pathway.

To investigate the possible effect of FoxO on coxsackievirus B3 (CVB3) -induced cardiomyocyte inflammation and apoptosis via modulation of the TLR4/NF-κB signaling pathway.Viral myocarditis (VMC) models were establied via CVB3 infection both in vivo and in vitro. Western blotting was adopted to detect FoxO1 and TLR4 expressions in myocardial tissues and cells. Cardiomyocytes of suckling mouse were divided into the control, CVB3, CVB3 + pcDNA, CVB3 + pcDNA-FoxO1, CVB3 + TLR4 siRNA, and CVB3 + pcDNA-FoxO1 + TLR4 siRNA groups. Flow cytometry was employed to evaluate cell apoptosis. The expressions of inflammatory factors including TNF-α, IL-1ß, and IL-6 were detected via quantitative reverse transcriptase polymerase chain reaction and enzyme-linked immunosorbent assay. Then, TLR4/NF-κB pathway-related proteins were determined via Western blotting.VMC mice had increased FoxO1 and TLR4 expressions in myocardial tissues. Cardiomyocytes with CVB3 infection also had upregulated protein expressions of p-FoxO1/FoxO1 and TLR4. Compared with those in the control group, the cardiomyocytes in the CVB3 group were increased in LDH and CK-MB levels, cell apoptosis rate and inflammatory factors (TNF-α, IL-1ß and IL-6), as well as protein expressions of TLR4 and p-p65/p65. Compared with those in the CVB3 group, the cardiomyocytes in the CVB3 + pcDNA-FoxO1 group were further upregulated whereas those in the CVB3 +TLR4 siRNA group were downregulated in the aforementioned indicators. Furthermore, TLR4 siRNA can reverse the effect of pcDNA-FoxO1 on the aggravation of cardiomyocyte injury induced by CVB3 infection.FoxO1 can upregulate the TLR4/NF-κB signaling pathway to promote cardiomyocyte apoptosis and inflammatory injury in CVB3-induced VMC.

Stem cell factor/mast cell/CCL2/monocyte/macrophage axis promotes Coxsackievirus B3 myocarditis and cardiac fibrosis by increasing Ly6Chigh monocyte influx and fibrogenic mediators production.

Mast cells (MCs), central players in allergy and parasitic infections, play key roles in inflammation and fibrosis. Here, the impact of MCs on the progression of Coxsackievirus B3 (CVB3)-induced viral myocarditis (VMC) and fibrosis was investigated using MC-deficient KitW-sh mice. Viral titres, cellular infiltrates and heart pathologies were evaluated and compared with wild-type (WT) mice during acute CVB3 infection of C57BL/6 mice. CVB3 infection induced an increased accumulation and degranulation of MCs in the hearts of mice during acute infection. MC-deficient KitW-sh mice had slightly higher viral titres, decreased VMC and cardiac fibrosis and improved cardiac dysfunction compared to WT mice via decreasing cardiac influx of Ly6Chigh monocytes/macrophages (Mo/Mφ). While bone marrow-derived MC reconstitution decreased viral titre and worsened improved survival and VMC severity in Wsh mice. MC-fibroblasts co-culture revealed a cardiac MC-fibroblasts crosstalk during early infection: fibroblasts trigger MC degranulation and secretion of CCL2 and tumour necrosis factor alpha (TNF-α) via producing early stem cell factor (SCF); while MCs-fibrogenic mediators (TNF-α) stimulate fibroblasts to increase CCL2, α-smooth muscle actin (SMA), collagen and transforming growth factor beta（TGFß) expression, thus aggravating cardiac fibrosis. MCs and fibroblast-derived CCL2s are both essential for cardiac Ly6Chigh Mo/Mφ influx. Administration of recombinant mouse SCF to CVB3-infected mice aggravates VMC via accelerating MCs accumulation and cardiac influx of Ly6Chi Mo/Mφ. Collectively, our data highlight an early MC-fibroblast crosstalk and SCF/MC/CCL2/Mo/Mφ axis as important mechanisms required for triggering VMC and myocardial fibrosis. This finding indicates critical roles of MCs in initiating and modulating cardiac innate response to CVB3 and has an implication in developing new and more effective treatments for VMC.

Whole-genome analysis of coxsackievirus B3 reflects its genetic diversity in China and worldwide.

BACKGROUND: Coxsackievirus B3 (CVB3) has emerged as an active pathogen in myocarditis, aseptic meningitis, hand, foot, and mouth disease (HFMD), and pancreatitis, and is a heavy burden on public health. However, CVB3 has not been systematically analyzed with regard to whole-genome diversity and recombination. Therefore, this study was undertaken to systematically examine the genetic characteristics of CVB3 based on its whole genome. METHODS: We combined CVB3 isolates from our national HFMD surveillance and global sequences retrieved from GenBank. Phylogenetic analysis was performed to examine the whole genome variety and recombination forms of CVB3 in China and worldwide. RESULTS: Phylogenetic analysis showed that CVB3 strains isolated worldwide could be classified into clusters A-E based on the sequence of the entire VP1 region. The predominant CVB3 strains in China belonged to cluster D, whereas cluster E CVB3 might be circulated globally compared to other clusters. The average nucleotide substitution rate in the P1 region of CVB3 was 4.82 × 10-3 substitutions/site/year. Myocarditis was more common with cluster A. Clusters C and D presented more cases of acute flaccid paralysis, and cluster D may be more likely to cause HFMD. Multiple recombination events were detected among CVB3 variants, and there were twenty-three recombinant lineages of CVB3 circulating worldwide. CONCLUSIONS: Overall, this study provides full-length genomic sequences of CVB3 isolates with a wide geographic distribution over a long-term time scale in China, which will be helpful for understanding the evolution of this pathogen. Simultaneously, continuous surveillance of CVB3 is indispensable to determine its genetic diversity in China as well as worldwide.

Bioguided fractionation of procyanidin B2 as potent anti coxsackie virus B and Herpes simplex from cypress (Cupressus sempervirens L.).

The methanolic extracts of cypress (Cupressus sempervirens L.) collected at three phenological stages were evaluated for their cytotoxicity on Vero cells by MTT test as well as on Herpes simplex (HSV-2) and coxsackie (CVB-3) viruses by plaque reduction assay. The methanolic extract exhibited the highest cytotoxicity against HSV-2 (IC50 = 20.40 µg/mL) and CVB-3 (IC50 = 47.50 µg/mL) at the flowering stage. This extract also exhibited a virucidal action both during the entry of viruses and the release of newly formed virions. The methanolic extract bioguided purification showed that the ethyl-acetate fraction was responsible for virucidal activity. This fraction was endowed with more important selectivity index of 8.15 for HSV-2 and 4.40 for CVB-3. The ethyl acetate fraction was subjected to thin layer chromatography fractionation and identification by HPLC-DAD-ESI-MSn. Results showed that the condensed tannin procyanidin B2 was identified for the first time responsible of the antiviral activity of cypress.

Berberine impairs coxsackievirus B3-induced myocarditis through the inhibition of virus replication and host pro-inflammatory response.

Berberine (BBR), an isoquinoline alkaloid isolated from Rhizoma coptidis, is reported to possess antiviral activity. Our previous study has shown that BBR alleviates coxsackievirus B3 (CVB3) replication in HeLa cells. However, the anti-CVB3 activity of BBR is still unclear in vivo. In this study, we explored the effect of BBR on CVB3-induced viral myocarditis in mice. These results demonstrated the beneficial effect of BBR on alleviating CVB3-induced myocarditis in vivo, which sheds new light on the utility of BBR as a therapeutic strategy against CVB3-induced viral myocarditis.

Vascular Endothelial Integrity Affects the Severity of Enterovirus-Mediated Cardiomyopathy.

Coxsackievirus and adenovirus receptor (CAR) is present in epithelial and vascular endothelial cell junctions. We have previously shown a hemorrhagic phenotype in germ-line CAR knock-out mouse embryos; we have also found that CAR interacts with ZO-1 and ß-catenin. However, the role of CAR in vascular endothelial junction permeability has not been proven. To understand the roles of CAR in the vascular endothelial junctions, we generated endothelium-specific CAR knockout (CAR-eKO) mice. In the absence of CAR, the endothelial cell layer showed an increase in transmembrane electrical resistance (TER, Ω) and coxsackievirus permeability. Evans blue dye and 70 kDa dextran-FITC were delivered by tail vein injection. We observed increased vascular permeability in the hearts of adult CAR-eKO mice compare with wild-type (WT) mice. There was a marked increase in monocyte and macrophage penetration into the peritoneal cavity caused by thioglycolate-induced peritonitis. We found that CAR ablation in endothelial cells was not significantly increased coxsackievirus B3 (CVB3) induced myocarditis in murine model. However, tissue virus titers were significantly higher in CAR-eKO mice compared with WT. Moreover, CVB3 was detected in the brain of CAR-eKO mice. Endothelial CAR deletion affects the expression of major endothelial junction proteins, such as cadherin and platelet endothelial cell adhesion molecule-1 (PECAM-1) in the cultured endothelial cells as well as liver vessel. We suggest that CAR expression is required for normal vascular permeability and endothelial tight junction homeostasis. Furthermore, CVB3 organ penetration and myocarditis severities were dependent on the endothelial CAR level.

Synthesis and anti-CVB3 activity of 4-amino acid derivative substituted pyrimidine nucleoside analogues.

Seven novel 4-amino acid derivative substituted pyrimidine nucleoside analogues were designed, synthesized, and tested for their anti-CVB3 activity. Initial biological studies indicated that among these 4-amino acid derivative substituted pyrimidine nucleoside analogues, 4-N-(2'-amino-glutaric acid-1'-methylester)-1-(2'- deoxy-2'-ß-fluoro-4'-azido)-furanosyl-cytosine 2 exhibited the most potent anti-CVB activity (IC50 = 9.3 µM). The cytotoxicity of these compounds has also been assessed. The toxicity of compound 2 was similar to that of ribavirin.

Antiviral Effects of Novel 2-Benzoxyl-Phenylpyridine Derivatives.

Coxsackievirus B3 (CVB3) is the most common cause of acute and chronic viral myocarditis, primarily in children, while human adenovirus infections represent a significant cause of morbidity and mortality worldwide, in people of all ages. A series of novel 2-benzoxyl-phenylpyridine derivatives were evaluated for their potential antiviral activities against CVB3 and adenovirus type 7 (ADV7). Preliminary assays indicated that some of these compounds exhibited excellent antiviral effects on both CVB3 and ADV7 viruses; they could effectively inhibit virus-induced cytopathic effects, reduce viral progeny yields, and had similar or superior antiviral activities compared with the control drug, ribavirin. Further, these compounds targeted the early stages of CVB3 replication in cells, including viral RNA replication and protein synthesis, rather than inactivating the virus directly, inhibiting virus adsorption/entry, or affecting viral release from cells. Our data demonstrate that the tested 2-benzoxyl-phenylpyridine derivatives are effective inhibitors of CVB3 and ADV7, raising the possibility that these compounds might be feasible candidates for anti-viral agents.

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

BACKGROUND: The 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. METHODS: We 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. RESULTS: We 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. CONCLUSIONS: miRNA-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.

TRAF6: A player in CVB3-induced myocarditis?

Coxsackievirus B3 (CVB3) is an important inducer of myocarditis, which, in susceptible individuals, can chronify and eventually lead to the development of dilated cardiomyopathy and heart failure. The respective mechanisms are not completely understood. Here, we analyzed expression of the TRAF6 gene, encoding TNF receptor-associated factor 6 (TRAF6), a signal transduction scaffold protein that acts downstream of cytokine receptors, in heart tissue of susceptible and non-susceptible mouse strains. We found that after infection, TRAF6 expression was upregulated in both non-susceptible C57BL/6 wildtype and susceptible A.BY/SnJ and C57BL/6-TLR3 (-/-) mice, however, to different degrees. In infected HeLa cells, we also found moderately elevated TRAF6 levels after infection, in addition, activity of the transcription factor nuclear factor kappa B (NFκB), which can be activated downstream of TRAF6, was strongly enhanced in infected cells. To functionally analyze the role of TRAF6 with regard to infection progression, TRAF6 expression was knocked down in cultured HeLa cells using specific siRNAs. We found that reduction of TRAF6 expression had no effect on NFκB activation in response to infection. Taken together, our data suggest that CVB3 infection enhances TRAF6 levels, however, this induction might not be necessary for infection-induced NFκB activation.

Spontaneous C-cleavage of a truncated intein as fusion tag to produce tag-free VP1 inclusion body nanoparticle vaccine against CVB3-induced viral myocarditis by the oral route.

BACKGROUND: Oral vaccine is highly desired for infectious disease which is caused by pathogens infection through the mucosal surface. The design of suitable vaccine delivery system is ongoing for the antigen protection from the harsh gastric environment and target to the Peyer's patches to induce sufficient mucosal immune responses. Among various potential delivery systems, bacterial inclusion bodies have been widely used as delivery systems in the field of nanobiomedicine. However, a large number of heterologous complex proteins could be difficult to propagate in E. coli and fusion partners are often used to enhance target protein expression. As a safety concern the fusion protein need to be removed from the target protein to get tag-free protein, especially for the production of protein antigen in vaccinology. Until now, there is no report on how to remove fusion tag from inclusion body particles in vitro and in vivo. Coxsackievirus B3 (CVB3) is a leading causative agent of viral myocarditis and orally protein vaccine is high desired for CVB3-induced myocarditis. In this context, we explored a tag-free VP1 inclusion body nanoparticles production protocol though a truncated Ssp DnaX mini-intein spontaneous C-cleavage in vivo and also exploited the VP1 inclusion bodies as an oral protein nanoparticle vaccine to protect mice against CVB3-induced myocarditis. RESULTS: We successfully produced the tag-free VP1 inclusion body nanoparticle antigen of CVB3 and orally administrated to mice. The results showed that the tag-free VP1 inclusion body nanoparticles as an effective antigen delivery system targeting to the Peyer's patches had the capacity to induce mucosal immunity as well as to efficiently protect mice from CVB3 induce myocarditis without any adjuvant. Then, we proposed the use of VP1 inclusion body nanoparticles as good candidate for oral vaccine to against CVB3-induced myocarditis. CONCLUSIONS: Our tag-free inclusion body nanoparticles production procedure is easy and low cost and may have universal applicability to produce a variety of tag-free inclusion body nanoparticles for oral vaccine.

Role of neutrophils in CVB3 infection and viral myocarditis.

Coxsackievirus B3 (CVB3) is a globally prevalent enterovirus of the Picornaviridae family that is frequently associated with viral myocarditis (VM). Neutrophils, as first responders, may be key cells in determining viral disease outcomes; however, neutrophils have been poorly studied with respect to viral infection. Although neutrophils have been ascribed a relevant role in early cardiac inflammation, their precise role in CVB3 infection has not yet been evaluated. In this study, we aimed to determine if the interaction between human neutrophils and CVB3 could lead to viral replication and/or modulation of neutrophil survival and biological functions, and whether neutrophil depletion in a murine model has a beneficial or harmful effect on CVB3 infection. Our results show that CVB3 interacted with but did not replicate in human neutrophils. Neutrophils recognized CVB3 mainly through endosomal TLR-8, and infection triggered NFκB activation. Virus internalization resulted in increased cell survival, up-regulation of CD11b, enhanced adhesion to fibrinogen and fibronectin, and the secretion of IL-6, IL-1ß, TNF-α, and IL-8. Supernatants from infected neutrophils exerted chemotactic activity partly mediated by IL-8. The infected neutrophils released myeloperoxidase and triggered neutrophil extracellular trap formation in the presence of TNF-α. In mice infected with CVB3, viral RNA was detected in neutrophils as well as in mononuclear cells. After neutrophil depletion, mice showed reduced VM reflected by a reduction in viral titers, cell exudates, and CCL-2 mRNA levels, as well as the abrogation of reactive cardiomyocyte hypertrophy. Our results indicate that neutrophils have relevant direct and indirect roles in the pathogenesis of CVB3-induced VM.

Semaphorin7A aggravates coxsackievirusB3-induced viral myocarditis by increasing α1ß1-integrin macrophages and subsequent enhanced inflammatory response.

Semaphorin7A (Sema7A) has been reported to play various roles in nerve axon growth, tumor suppression, and tissue remodeling, as well as regulation of intestinal inflammation diseases. Viral myocarditis (VMC) characterized by viral-myocardial-cell necrosis and inflammatory cell infiltration is a common clinical disease of the cardiovascular system. However, the role of Sema7A in coxsackievirus B3 (CVB3)-induced VMC has not been reported. In this study, we generated an acute VMC mouse model by CVB3 infection, and manipulated Sema7A expression by in vivo polyethyleneimine-mediated Sema7A down-regulation. Our results indicated that Sema7A was up-regulated in cardiomyocytes during VMC, and that Sema7A down-regulation following short hairpin RNA interference or mAb neutralization effectively protected mice from VMC. Additionally, reduced inflammatory responses were observed along with Sema7A down-regulation. Furthermore, adoptive transfer of α1ß1-integrin macrophages exacerbated CVB3-induced myocarditis, suggesting the significance of α1ß1-integrin macrophages in response to VMC. We observed that co-culture of neonatal myocardiocytes with macrophages increased the percentage of α1ß1-integrin macrophages, while Sema7A neutralization reduced α1ß1-integrin macrophages in heart tissue of VMC mice. These results demonstrated that Sema7A, as an inflammation regulator in CVB3-induced VMC, might interact with α1ß1-integrin in macrophages to enhance the inflammatory response and aggravate disease severity. Our findings provided insight into the potential role of Sema7A as a therapeutic treatment for VMC.

Nox4-dependent ROS production is involved in CVB3-induced myocardial apoptosis.

Viral myocarditis is a cardiovascular disease that seriously affects human health. Its mechanism is not clear. Coxsackievirus B3 (CVB3) is a member of the picornavirus family and is the leading cause of viral myocarditis. Our group tested the genes in a mouse model of CVB3 virus infection and confirmed that the NADPH oxidase gene had a high expression trend in the acute phase of infection. Whether Nox4, the homologue of NADPH oxidase, participates in the process of viral myocarditis has not been reported. In this study, we found increased expression of Nox4 in viral myocarditis in vivo and in vitro. DPI is a non-specific inhibitor of Nox4 that improved CVB3-induced myocarditis after injection in vivo. DPI also inhibited intracellular ROS release and apoptosis in vitro. Our data indicated that Nox4-dependent ROS production was involved in CVB3-induced myocardial apoptosis.