Viral myocarditis (VMC) is a disease characterized by inflammation of myocardial cells caused by viral infection. Since the pathogenesis mechanism of VMC has not been fully elucidated, the diagnosis and treatment of this disease remains extremely challenging. Non-coding RNAs (ncRNAs) are a class of RNAs that do not encode proteins. An increasing number of studies have shown that ncRNAs are involved in regulating the occurrence and development of VMC, thus providing potential new targets for the treatment and diagnosis of VMC. This review summarizes the possible roles of ncRNAs in the pathogenesis and diagnosis of VMC revealed recently.
Coxsackievirus Infections ; Enterovirus B, Human ; Humans ; Inflammation ; Myocarditis/genetics* ; Virus Diseases/genetics*

OBJECTIVE: To report on a child with B-cell-negative severe combined immunodeficiency (B-SCID) manifesting as fulminant myocarditis and carry out genetic testing for her. METHODS: A child with B-SCID who presented at Fujian Maternity and Child Health Care Hospital on January 31, 2021 was selected as the subject. Whole exome sequencing was carried out for her. Candidate variant was verified by Sanger sequencing. RESULTS: The female infant had developed recurrent skin and lung infections soon after birth, and was admitted due to fulminant myocarditis. Serological examination has disclosed a remarkable reduction in immunoglobulins. Flow cytometric analysis revealed that her peripheral blood T and B lymphocytes and NK cells were significantly reduced. Whole exome sequencing revealed that she has harbored a homozygous c.C3007T (p.Q1003X) nonsense variant of the RAG1 gene, for which both of her parents were heterozygous carriers. The variant has not been recorded in normal population databases. Based on the guidelines from the American College of Medical Genetics and Genomics, the variant was predicted to be pathogenic. CONCLUSION A case of RAG1 gene associated B-SCID has been diagnosed. Above finding has enriched the spectrum of RAG1 gene variants and enabled early diagnosis and intervention of the disease.
Female ; Humans ; Pregnancy ; Genetic Testing ; Homeodomain Proteins/genetics* ; Mutation ; Myocarditis/genetics* ; Phenotype ; Severe Combined Immunodeficiency/diagnosis* ; Infant

OBJECTIVE: To detect the Coxsackie virus B3(CVB3) gene in myocardium and spleen tissues in viral myocarditis(VMC) with sudden death and to explore the diagnostic method for VMC by means of seeking pathogene. METHODS: By in situ RT-PCR, the detection of CVB3 gene in myocardium and spleen sections were performed in sudden death group caused by VMC and non-cardiac death group. RESULTS: In VMC group, CVB3 gene-positive signals were seen in myocardium sections(3 out of total 8 cases, No. 1, 4, 7 cases) and spleen sections(4 out of total 8 cases, No. 2, 4, 6, 7 cases). In non-cardiac death group, no positive signals were detected in both myocardium and spleen tissues. CONCLUSION Positive detection of CVB3 gene in both myocardium and spleen maybe an important character of VMC and can improve the detecting pathogene in diagnosing VMC.
Death, Sudden ; Enterovirus B, Human/genetics* ; Heart/virology* ; Humans ; Myocarditis/virology* ; Reverse Transcriptase Polymerase Chain Reaction ; Spleen/virology*

OBJECTIVETo construct the recombinant plasmid of human cardiac C protein (CCP) peptide with immunogenicity and to express, purification and renature fusion protein. The fusion protein was injected to Lewis rats to establish experimental autoimmune myocarditis (EAM) model.

METHODSTotal RNA was extracted from human heart and used as the template for reverse transcriptase-directed cDNA synthesis. The cDNA was then amplified by polymerase chain reaction (PCR) using oligonucleotide primers specific for CCP peptide with immunogenicity. Subsequently, the purified CCP peptide gene was cloned into PEASY-T1 vector and the ligated product was identified by PCR and DNA sequence analysis. Then the CCP target gene of positive clone was inserted into the pQE30, a prokaryotic expression vector, and the inserting plasmid was transformed into Escherichia coli. host M15. The positive clone extracted from the bacterium liquid was sieved by insertional inactivation sieve method and identified by PCR of bacterium liquid, CCP immunological peptide was purified and renatured in semipermeable membrane. EAM model in Lewis rats was induced by injection of mixture of 100 µg CCP fusion protein immunological peptide and 2.5 g/L completed Freund adjuvant from two double foot pad and subsequent abdominal injection of 0.5 µg pertussis toxin. Two, four, six, and eight weeks after immunization, hemodynamic evaluation was made and hearts underwent histological examination.

RESULTSThe DNA sequence analysis for cloning vector extraction revealed that the CCP target gene was cloned into pQE30 exactly. The DNA of 1000 bp length was obtained by PCR examination of bacterium liquid with transformation of express recombinants which were consistent with the expected size. Purified fusion protein in vertical slab gel electrophoresis showed 35 000 as expected. The recombinant CCP fusion protein existed in inclusion bodies of E. coli and amounted to 80% - 90% of the total protein. Hemodynamic and histological evaluations showed typical acute inflammatory responses at 2 weeks, subacute inflammatory and fibrosis changes at 4 weeks after injection, and signs of chronic dilated cardiomyopathy at 6 weeks post injection.

CONCLUSIONCombination of gene clone technique and histidine tag protein purification technique can be used to synthesize human cardiac C protein to induce EAM model in Lewis rat.

Animals ; Carrier Proteins ; biosynthesis ; genetics ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Humans ; Myocarditis ; Nervous System Autoimmune Disease, Experimental ; Plasmids ; Rats ; Recombinant Fusion Proteins ; biosynthesis ; genetics

OBJECTIVETo investigate the role and significance of cardiac mast cells and Toll-like receptor 4 (TLR4) in the development and progression of viral myocarditis (VMC).

METHODSForty-eight Balb/c mice were randomly divided into a control group (n=24) and a model group (n=24). Coxsackievirus B3 was intraperitoneally injected into the model group mice to establish a VMC model. In each group, cardiac tissues were collected from 8 mice at 7, 14 and 28 days after the model was established. The cardiac tissues were stained with hematoxylin and eosin as well as Masson trichrome to observe pathological changes in cardiac tissues. The number and degranulation of cardiac mast cells at each time point were measured and evaluated by toluidine blue staining and transmission electron microscopy. The mRNA and protein expression of TLR4 in cardiac tissues was measured by RT-PCR and immunohistochemistry. In the model group, the correlation between number of cardiac mast cells and mRNA expression of TLR4 at all time points was analyzed.

RESULTSThe model group had significantly higher pathological scores of cardiac tissues than the control group at all time points (P<0.05). The myocardial collagen volume fraction in the model group at 28 days was significantly higher than in the control group at all time points and higher than in the model group at 7 and 14 days (P<0.05). At each time point, the model group had a significantly increased number of mast cells (P<0.05), and significantly increased mRNA and protein expression of TLR4 (P<0.05) compared with the control group. In the model group, the number of cardiac mast cells was positively correlated with the mRNA expression of TLR4 at all time points (R²=0.877, P<0.05).

CONCLUSIONSMice with VMC have significantly increased numbers of cardiac mast cells and expression of TLR4 compared with control mice at all time points, suggesting that mast cells and TLR4 may play important roles in the inflammatory response and fibrosis of VMC.

Animals ; Coxsackievirus Infections ; immunology ; Enterovirus B, Human ; Female ; Mast Cells ; physiology ; Mice ; Mice, Inbred BALB C ; Myocarditis ; immunology ; Myocytes, Cardiac ; pathology ; Toll-Like Receptor 4 ; analysis ; genetics ; physiology

BACKGROUNDExtracellular matrix (ECM) orchestrates cell behaviour including growth, death, apoptosis, adhesion, migration, and invasion by activating several signalling pathways. Certain components of ECM, such as integrins, may act as receptors or co-receptors of enterovirus. ECM-activated gene expressions in myocardium of viral heart disease including myocarditis and partial cardiomyopathy remain elusive. This study was to investigate the expression of ECM-activated genes in myocardium of mouse with viral myocarditis.

METHODSBALB/c mice were infected with Coxsackie virus B3 (CVB3) to establish an animal model of myocarditis. Uninfected mice were also prepared and served as controls. Specific mRNA expression pattern in myocarditic mouse heart was analysed by an in-house cDNA microarray containing 8,192 genes. Overexpressed ECM genes were selected and subsequently confirmed by Northern blot analysis.

RESULTSNine ECM genes were isolated, from the array of 8,192 genes, as overexpressed genes in hearts of myocarditic mice in comparison with controls. Subsequent Northern blot analysis confirmed that four of the nine genes were highly expressed. Expression of these four genes, Fin15, ILk, Lamr1 and ADAMTS-1, has not been reported previously to be induced by Coxsackie virus.

CONCLUSIONCVB3-induced myocarditis is associated with gene expression profiles of certain ECM components.

Animals ; Blotting, Northern ; Enterovirus B, Human ; Enterovirus Infections ; metabolism ; Extracellular Matrix Proteins ; genetics ; Male ; Mice ; Mice, Inbred BALB C ; Myocarditis ; metabolism ; Myocardium ; metabolism ; Oligonucleotide Array Sequence Analysis

OBJECTIVETo investigate the CVB3 RNA concentration in myocardial tissues and inhibitory effect of Chinese herbal medicine Xin-Kang oral liquid on viral RNA replication in Coxsackievirus B3 infected myocardium.

METHODSThe total RNA was extracted from the infected murine myocardium, the reverse transcription-polymerase chain reaction (RT-PCR) was performed to detect enterovirus RNA concentration in infected mice with Coxsackievirus B3 gene band. We used "Gel works system" to scan the electrophoresis image to detect CVB3 gene band.

RESULTSThe mean concentration of myocardial CVB3 RNA of Xin-Kang oral liquid treated groups was markedly lower than that of virus control group (P>0.01), but the CVB3 RNA in myocardial tissues has not been destroyed by Xin-Kang oral liquid feed in different phase.

CONCLUSIONChinese herbal medicine Xin-Kang oral liquid could inhibit CVB3 RNA replication in myocardial tissue, but it did not destruct the virus.

Animals ; Coxsackievirus Infections ; drug therapy ; Drugs, Chinese Herbal ; pharmacology ; Enterovirus B, Human ; drug effects ; genetics ; Mice ; Myocarditis ; drug therapy ; virology ; Phytotherapy ; RNA, Viral ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Treatment Outcome ; Virus Replication ; drug effects ; genetics

To evaluate the effects of lentivirus-delivered short hairpin RNA (shRNA) on CVB3 infection in an animal model by RNA interference technique, we constructed a recombinant lentivirus expressing shRNA-3753 against the viral genome region 3753-3771, then transduced Lenti-sh3753 into mice infected with CVB3. We evaluated the antiviral ability of lenti-sh3753 by cytopathic effect (CPE), viral plaque assay and histological analysis of mice hearts. The results showed that Lenti-sh3753 exhibited a significant protective effect on cell viability and reduction of viral titers in supernatant of cell culture by specific inhibition on viral replication. Lenti-sh3753 also prolonged the mice survival and limited the viral production in mice hearts. These data proposed that Lenti-sh3753 can effectively inhibit CVB3 infection in a coxsackievirus-induced myocarditis model, suggesting its potential role in prevention and therapy of viral diseases.
Animals ; Coxsackievirus Infections ; drug therapy ; virology ; Down-Regulation ; Enterovirus B, Human ; genetics ; physiology ; Humans ; Male ; Mice ; Mice, Inbred BALB C ; Myocarditis ; drug therapy ; virology ; RNA Interference ; RNA, Small Interfering ; genetics ; therapeutic use ; RNA, Viral ; genetics ; Virus Replication

OBJECTIVE: To study the mechanism of osteopontin (OPN) in viral myocarditis by observing the expression of OPN and collagen I (Col I) in mice myocardium. METHODS: The viral myocarditis models were achieved by infection with myocarditic coxsackievirus B3 (CVB3). The myocardium of mice was stained by HE and Masson staining, and the pathological scores and the collagen volume fraction (CVF )of myocardium were tabulated. The expression of Col I mRNA was measured by RT-PCR. The expression of OPN was detected by RT-PCR and ELISA. RESULTS: The histopathological examination revealed a prevalence of myocardial cell necrosis and obvious inflammation changes at the 7th day post-infection. Subsequently the inflammatory lesions were gradually absorbed. At the 28th day, the inflammatory cells had almost disappeared and obvious fibrosis occurred. The pathological scores and the expression of OPN mRNA were higher than those of the control group (P<0.05), and reached the highest level at the 7th day (P<0.05). From the 14th day, these parameters decreased,reflected also in the ELISA results. At the 7th day and the 14th day, the Col I expression was similar to that of control. Col I expression at the 21th and 28th days was higher than those of the control (P<0.05), and correlated positively to the CVF results. CONCLUSION The OPN mRNA expression increased in acute stage of VMC, and higher than that of the control group when in recovery stage, suggesting that OPN might be related to the inflammatory response in acute stage of, and promote the collagen synthesis of recovery stage.
Animals ; Collagen Type I ; genetics ; metabolism ; Coxsackievirus Infections ; metabolism ; Enterovirus B, Human ; isolation & purification ; Male ; Mice ; Mice, Inbred BALB C ; Myocarditis ; metabolism ; virology ; Osteopontin ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism

OBJECTIVETo explore the role of interleukin-17 (IL-17) in the evolution of viral myocarditis (VMC) into dilated cardiomyopathy (DCM).

METHODSA mouse model of VMC was established in 100 male Balb/c mice by intraperitoneal injection of coxsackievirus B3. The expression of IL-17 protein in the cardiac tissue of the mice was detected immunohistochemically, and IL-17 mRNA in the splenocytes was examined by reverse transcription-polymerase chain reaction (RT-PCR). IL-17 levels in the plasma, peripheral blood mononuclear cell (PBMC) culture supernatants, and phytohemagglutinin (PHA)-stimulated PBMC culture supernatants were measured in 30 DCM patients, 26 non-DCM patients and 20 normal adults using enzyme-linked immunosorbent assay (ELISA), and IL-17 mRNA expression in the PBMCs was detected using RT-PCR.

RESULTSThe levels of IL-17 mRNA in the splenocytes of the mice with VMC were significantly higher at 4 and 6 weeks than those at 8 weeks (P<0.01), but not detected at 2 weeks. No IL-17 expression was found in the ventricular tissue of the mice at 2 weeks, but peaked at 4 weeks followed by gradual decrease (P<0.01). IL-17 level in PHA-stimulated PBMC culture supernatants but not the plasma, and its mRNA level in PHA-stimulated PBMCs but not the PBMC culture supernatants, were significantly elevated in DCM patients as compared with those in non-DCM patients and normal control subjects.

CONCLUSIONSThe mouse model of VMC in the chronic phase and DCM patients express high levels of IL-17, which may contribute to the transition from VMC to DCM.

Adult ; Animals ; Cardiomyopathy, Dilated ; etiology ; metabolism ; pathology ; Coxsackievirus Infections ; complications ; metabolism ; Enterovirus B, Human ; Female ; Humans ; Interleukin-17 ; genetics ; metabolism ; Male ; Mice ; Mice, Inbred BALB C ; Middle Aged ; Myocarditis ; complications ; metabolism ; virology ; RNA, Messenger ; genetics ; metabolism