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 develop RT-nPCR assays for amplifying partial and complete VP1 genes of human enteroviruses (HEVs) from clinical samples and to contribute to etiological surveillance of HEV-related diseases. Methods: A panel of RT-nPCR assays, consisting of published combined primer pairs for VP1 genes of HEV A-C and in-house designed primers for HEV-D, was established in this study. The sensitivity of each RT-nPCR assay was evaluated with serially diluted virus stocks of five serotypes expressed as CCID Results: The sensitivity of RT-nPCR assays for amplifying partial VP1 gene of HEVs was 0.1 CCID Conclusion This RT-nPCR system is capable of amplifying the partial and complete VP1 gene of HEV A-D, providing rapid, sensitive, and reliable options for molecular typing and molecular epidemiology of HEVs in clinical specimens.
Capsid Proteins/genetics* ; Enterovirus A, Human/genetics* ; Enterovirus B, Human/genetics* ; Enterovirus C, Human/genetics* ; Enterovirus D, Human/genetics* ; Humans ; Molecular Epidemiology/methods* ; Molecular Typing/methods* ; Reverse Transcriptase Polymerase Chain Reaction/methods*

Child ; Child, Preschool ; China ; epidemiology ; Enterovirus B, Human ; genetics ; Female ; Hand, Foot and Mouth Disease ; epidemiology ; Humans ; Infant ; Male ; Molecular Epidemiology ; Phylogeny

<b>OBJECTIVEb>To provide a feasible and cost-effective next-generation sequencing (NGS) method for accurate identification of viral pathogens in clinical specimens, because enormous limitations impede the clinical use of common NGS, such as high cost, complicated procedures, tremendous data analysis, and high background noise in clinical samples.

<b>METHODSb>Viruses from cell culture materials or clinical specimens were identified following an improved NGS procedure: reduction of background noise by sample preprocessing, viral enrichment by barcoded oligonucleotide (random hexamer or non-ribosomal hexanucleotide) primer-based amplification, fragmentation-free library construction and sequencing of one-tube mixtures, as well as rapid data analysis using an in-house pipeline.

<b>RESULTSb>NGS data demonstrated that both barcoded primer sets were useful to simultaneously capture multiple viral pathogens in cell culture materials or clinical specimens and verified that hexanucleotide primers captured as many viral sequences as hexamers did. Moreover, direct testing of clinical specimens using this improved hexanucleotide primer-based NGS approach provided further detailed genotypes of enteroviruses causing hand, foot, and mouth disease (HFMD) and identified other potential viruses or differentiated misdiagnosis events.

<b>CONCLUSIONb>The improved barcoded oligonucleotide primer-based NGS approach is simplified, time saving, cost effective, and appropriate for direct identification of viral pathogens in clinical practice.

Clinical Laboratory Techniques ; DNA Barcoding, Taxonomic ; DNA Primers ; Enterovirus ; classification ; genetics ; isolation & purification ; Herpesvirus 4, Human ; genetics ; isolation & purification ; Humans ; Influenza B virus ; genetics ; isolation & purification ; Real-Time Polymerase Chain Reaction ; Sequence Analysis, DNA ; methods ; Sequence Analysis, RNA ; methods

<b>OBJECTIVEb>To investigate the effect of tranilast on myocardial fibrosis in mice with viral myocarditis (VMC).

<b>METHODSb>Male balb/c mice (n=72) were randomly divided into control, VMC and tranilast groups (n=24 each). In the VMC and tranilast groups, the mice were infected with Coxsackie virus B3 (CVB3) to prepare VMC model, while the control group was treated with Eagle's medium. After modeling, the tranilast group was administrated with tranilast [200 mg/(kg.d)] until the day before sampling. On days 7, 14 and 28 after CVB3 or Eagle's medium infection, heart specimens (n=8) were taken and examined after Toluidine blue staining and Nissl staining for counts of mast cells (MC), hematoxylin-eosin staining for myocardial pathological changes, and Masson staining for myocardial fibrosis. The expression of CTGF and type I collagen (Col I) in the myocardial tissue was measured by RT-PCR and Western blot. The correlations of CTGF mRNA expression with MC counts and Col I expression were analyzed.

<b>RESULTSb>The myocardial pathological changes and collagen volume fraction in the VMC group were significantly higher than in the control group at all three time points (P<0.05). Tranilast treatment significantly decreased the myocardial pathological changes and collagen volume fraction compared with the VMC group (P<0.05). The mRNA and protein expression of CTGF and Col I increased in the VMC group compared with the control group, and the increases were reduced with tranilast treatment (P<0.05). The number of MC was positively correlated to CTGF mRNA expression on the 7th day and 14th day (r=0.439, P=0.049) in the VMC group. There were positive correlations between the mRNA expression of Col I and CTGF on the 7th day and 14th day (r=0.646, P=0.007) and the 28th day (r=0.326, P=0.031).

<b>CONCLUSIONSb>Tranilast may inhibit the aggregation of MC and down-regulate the expression of CTGF, relieving myocardial fibrosis of mice with VMC.

Animals ; Collagen Type I ; genetics ; Connective Tissue Growth Factor ; genetics ; Coxsackievirus Infections ; drug therapy ; Enterovirus B, Human ; Fibrosis ; Male ; Mice ; Mice, Inbred BALB C ; Myocarditis ; drug therapy ; Myocardium ; pathology ; RNA, Messenger ; analysis ; ortho-Aminobenzoates ; pharmacology

This study aimed to analyse the genetically characterize isolates of Echovirus 11 from Longyan City,Fujian Province,and to reveal their genetic relationships with other isolates from China and abroad. Cerebrospinal fluid specimens from patients diagnosed with viral encephalitis or central nervous system (CNS) infections were collected from Longyan First Hospital between January and December 2011. Seven Echo11 strains were isolated and identified using the RIMV serum panel. The entire VP1 coding regions of four strains were sequenced and typed as Echo11 by an online blast program and,subsequently, phylogenet- ic analyses of the VP1 sequences of these stains and others published on GenBank were conducted. There were 600 nucleotides (nt) in each complete VP1 coding region that encoded 200 amino acids (aa). Among those four Echo11 strains, the sequence identities of nt and aa were 100% and 99%-100% respectively. And phylogenetic analyses indicate belong to subtype DS, the homology compared with DS strain (GU393713) were 93% (nt) and 99% (aa). The sequence identities for the nt and aa were 75%-76% and 90%, respectively, between the current isolates from Longyan and the Gregory prototype strain found in 1953. The sequence identity of nt and aa between the Longyan virus strains and the domestic Shandong strains isolated in 2010 were lower, at 74% and 88%-89%, respectively. However,the highest level of ho- mology was found when the Longyan strains were compared with the Netherlands strain (GU393773) found in 2007 (nt and aa identity: 94%-95% and 98%-99%, respectively). The relatively low levels of similarity between domestic isolates suggest that different transmission routes exist for Echo11 in mainland China.
Adolescent ; Amino Acid Sequence ; Base Sequence ; Child ; Child, Preschool ; Chin ; China ; Encephalitis, Viral ; virology ; Enterovirus B, Human ; chemistry ; classification ; genetics ; isolation & purification ; Enterovirus Infections ; virology ; Female ; Genotype ; Humans ; Male ; Middle Aged ; Molecular Sequence Data ; Phylogeny ; Sequence Alignment ; Viral Proteins ; chemistry ; genetics

To study the effect of miR-490 on Coxsackievirus B3 (CVB3) replication, HeLa cells were trans- fected with miR-490 in vitro, and infected with a Renilla luciferase (RLuc)-expressing CVB3 variant (RLuc-CVB3). The activities of RLuc in these cells were measured at 8h intervals from 0 to 40 h post-infection (p.i.), and the effects of miR-490 on RLuc-CVB3 replication were observed. In a further study, HeLa cells were transfected with either miR-490 or antisense miR-490 (AMO-miR-490), and were then infected with an enhanced green fluorescence protein (EGFP)-expressing CVB3 variant (EGFP-CVB3). The replication of EGFP-CVB3 was then determined by detecting the expression of EGFP. We observed that miR-490 could significantly inhibit the expression of RLuc in infected cells at 32 h p. i. Furthermore, in HeLa cells infected with EGFP-CVB3 at 32 h p.i., EGFP expression was also significantly inhibited by the presence of mniR-490. The inhibitory effect of miR-490 on EGFP expression in EGFP-CVB3-infected cells could be reversed by tranfection with AMQ-miR-490. These results indicated that miR-490 significantly inhibits the replication and expression of QVB3.
Enterovirus B, Human ; genetics ; physiology ; Enterovirus Infections ; genetics ; metabolism ; virology ; HeLa Cells ; Humans ; MicroRNAs ; genetics ; metabolism ; Virus Replication

We wished to analyze the genetic characterization of echovirus 11 (Echo11) from samples of environmental sewage in Shandong Province (China). The VP1 coding region was typed as the strains were amplified. Phylogenetic analyses on the VP1 sequences from these isolates, strains isolated from AFP cases in the period 1994-2010 and others published in GenBank were conducted. From 2011 to 2012, 94 Echo11 strains were isolated from samples of environmental sewage in Jinan and Linyi City in Shandong Province. Numbers of Echo11 were seasonal and reached peaks in the summer and autumn in both cities; A- mong these isolates, nucleotide (nt) identities were 89.5%-100.0% whereas amino acid (aa) identities were 95.4%-100.0%. The nt and aa identities were 76.6%-79.7% and 90.4%-92.5% between those strains and the prototype (Gregory) strain of Echo11, respectively. All isolates from Shandong Province were the A genotype and the strains evolved very rapidly, which suggested that several transmission chains was co-circulating. We described the temporal fluctuation and genetic characterization of Echo11 isolates from surveillance of environmental sewage in Shandong Province, thereby providing important information for exploring the dynamic change and genetic variation of circulating human enteroviruses in this Province in China.
China ; Enterovirus B, Human ; classification ; genetics ; isolation & purification ; Phylogeny ; Sewage ; virology

PURPOSE: This study investigated the possible relationship between viral infection and first trimester pregnancy loss. MATERIALS AND METHODS: A prospective study was performed on 51 gravidas with missed abortion, fetal anomaly, pre-term delivery, and full-tem delivery at Hanyang University Hospital. Enteroviruses were detected by semi-nested reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry in abortive tissues and placentas. Enterovirus serotypes were confirmed by genome sequencing. Herpesviruses were detected by PCR. RESULTS: Coxsackievirus B3 (CVB3) was detected in 8 of 14 missed abortion cases, 1 of 27 full-term cases, and none of the 9 pre-term cases. Coxsackievirus B1 (CVB1) was detected in an encephalocele case. Herpes simplex virus type 1 was found in 4 full-term cases, 3 pre-term cases, and none of the missed abortion cases. CONCLUSION: The prevalence of CVB3 was significantly higher in missed abortion cases compared to full-term or pre-term delivery cases. CVB infection may therefore be an important etiological agent of missed abortion.
Abortion, Missed/*etiology ; Adult ; Coxsackievirus Infections/complications/*diagnosis/virology ; Enterovirus B, Human/genetics/*isolation & purification ; Female ; Humans ; Immunohistochemistry ; Placenta/virology ; Pregnancy ; Pregnancy Complications, Infectious/*virology ; Pregnancy Trimester, First ; Prevalence ; Prospective Studies ; Republic of Korea ; Reverse Transcriptase Polymerase Chain Reaction ; Sequence Analysis, DNA ; Uterus/*virology

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

<b>METHODSb>Forty-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.

<b>RESULTSb>The 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).

<b>CONCLUSIONSb>Mice 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