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

OBJECTIVE: To study the association of interleukin-10 (IL-10) -1082A/G, -819C/T, and -592C/A polymorphisms with IL-10 level and the severity of enterovirus 71 (EV71) infection in children. METHODS: A total of 137 children with hand-foot-mouth disease due to EV71 infection were enrolled as EV71 infection group, which was further divided into mild group with 91 children and severe group with 46 children, and 122 healthy children who underwent physical examination were enrolled as healthy control group. Related clinical data were collected. ELISA was used to measure the serum level of IL-10, and polymerase chain reaction-restriction fragment length polymorphism was used to analyze IL-10 -1082A/G, -819C/T and -592C/A polymorphisms. RESULTS: Compared with the healthy control group, the children with EV71 infection had significantly higher frequency of -1082 AA genotype and A allele (P<0.05). Among the children with EV71 infection, the severe group had significantly higher frequency of -1082 AA genotype and A allele than the mild group (P<0.05), while there was no significant difference in the distribution of IL-10 -819C/T and IL-10 -592C/A polymorphisms between the two groups (P>0.05). The severe group had a significantly higher serum level of IL-10 than the mild group and the healthy control group. IL-10 -1082 AA genotype, -819 TT genotype, and -592 AA genotype were associated with the low expression of IL-10 (P<0.05). As for haplotype, the EV71 infection group had a significantly lower frequency of GCC haplotype than the healthy control group (P<0.05). In the severe group, the children with ATA haplotype had a significantly lower IL-10 level than those with other haplotypes, and the children with GCC haplotype had a significantly higher IL-10 level than those with other haplotypes (P<0.05). There was no significant difference in IL-10 level between children with different haplotypes in the mild group and the healthy control group (P>0.05). CONCLUSIONS IL-10 gene polymorphisms are associated with IL-10 expression and the severity of EV71 infection in children.
Child ; Enterovirus A, Human ; Enterovirus Infections ; Gene Frequency ; Genetic Predisposition to Disease ; Genotype ; Haplotypes ; Humans ; Interleukin-10 ; genetics ; Polymorphism, Single Nucleotide

OBJECTIVETo eliminate the side effects of aluminum adjuvant and His-tag, we constructed chimeric VLPs displaying the epitope of EV71 (SP70) without His-tagged. Then evaluating whether the VLPs could efficiently evoke not only humoral but also cellular immune responses against EV71 without adjuvant.

METHODSThe fusion protein was constructed by inserting SP70 into the MIR of truncated HBcAg sequence, expressed in E. Coli, and purified through ion exchange chromatography and density gradient centrifugation. Mice were immunized with the VLPs and sera were collected afterwards. The specific antibody titers, IgG subtypes and neutralizing efficacy were detected by ELISA, neutralization assay, and EV71 lethal challenge. IFN-γ and IL-4 secreted by splenocytes were tested by ELISPOT assay.

RESULTSHBc-SP70 proteins can self-assemble into empty VLPs. After immunization with HBc-SP70 VLPs, the detectable anti-EV71 antibodies were effective in neutralizing EV71 and protected newborn mice from EV71 lethal challenge. There was no significant difference for the immune efficacy whether the aluminum adjuvant was added or not. The specific IgG subtypes were mainly IgG1 and IgG2b and splenocytes from the mice immunized produced high levels of IFN-γ and IL-4.

CONCLUSIONThe fusion proteins without His-tagged was expressed and purified as soluble chimeric HBc-SP70 VLPs without renaturation. In the absence of adjuvant, they were efficient to elicit high levels of Th1/Th2 mixed immune response as well as assisted by aluminum adjuvant. Furthermore, the chimeric VLPs have potential to prevent HBV and EV71 infection simultaneously.

Adjuvants, Immunologic ; Animals ; Antibodies, Neutralizing ; Antibodies, Viral ; blood ; Enterovirus A, Human ; genetics ; Enterovirus Infections ; immunology ; virology ; Epitopes ; immunology ; metabolism ; Escherichia coli ; metabolism ; Female ; Immunity, Cellular ; Immunity, Humoral ; Mice ; Recombinant Fusion Proteins ; immunology

Enterovirus 71 is a neuroinvasive virus that is associated with severe neurological complications. We had earlier suggested that the replication capacity of a severe strain was higher than that of a mild strain. The recombinant 3CRV and 3CDRV virus strains were successfully rescued in our previous study. In the present study, we found no difference in virulence between 3CRV and severe strains. However, the capacity of replication and to cause cell injury of 3CDRV strain decreased in vitro, especially at 39.5 °C. Replacement of 3CD region in the severe strain led to milder symptoms, less body weight loss, and lower viral load in ICR mice. Histopathological findings indicated less severe injury in mice infected with 3CDRV strain. This study suggests that the 3CD region contributes to the attenuation of the severe strain, including its replication capacity and temperature sensitivity.
Animals ; Cytopathogenic Effect, Viral ; Enterovirus A, Human ; genetics ; pathogenicity ; Enterovirus Infections ; pathology ; virology ; Gene Expression Regulation, Viral ; Mice ; Mice, Inbred ICR ; Mutation ; Viral Load ; Viral Proteins ; genetics ; metabolism ; Virulence ; Virus Replication

OBJECTIVEKnowledge of an enterovirus genome sequence is very important in epidemiological investigation to identify transmission patterns and ascertain the extent of an outbreak. The MinION sequencer is increasingly used to sequence various viral pathogens in many clinical situations because of its long reads, portability, real-time accessibility of sequenced data, and very low initial costs. However, information is lacking on MinION sequencing of enterovirus genomes.

METHODSIn this proof-of-concept study using Enterovirus 71 (EV71) and Coxsackievirus A16 (CA16) strains as examples, we established an amplicon-based whole genome sequencing method using MinION. We explored the accuracy, minimum sequencing time, discrimination and high-throughput sequencing ability of MinION, and compared its performance with Sanger sequencing.

RESULTSWithin the first minute (min) of sequencing, the accuracy of MinION was 98.5% for the single EV71 strain and 94.12%-97.33% for 10 genetically-related CA16 strains. In as little as 14 min, 99% identity was reached for the single EV71 strain, and in 17 min (on average), 99% identity was achieved for 10 CA16 strains in a single run.

CONCLUSIONMinION is suitable for whole genome sequencing of enteroviruses with sufficient accuracy and fine discrimination and has the potential as a fast, reliable and convenient method for routine use.

Child, Preschool ; Enterovirus ; genetics ; Enterovirus A, Human ; genetics ; Enterovirus Infections ; virology ; Feces ; Genome, Viral ; Hand, Foot and Mouth Disease ; virology ; Humans ; Nucleic Acid Amplification Techniques ; instrumentation ; methods

OBJECTIVETo 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.

METHODSViruses 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.

RESULTSNGS 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.

CONCLUSIONThe 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

OBJECTIVETo investigate the association between rs9722 polymorphisms in the S100B gene and hand, foot and mouth disease (HFMD) caused by enterovirus 71.

METHODSA total of 124 HFMD children with enterovirus 71 infection were enrolled as subjects, and 56 healthy children were enrolled as control group. The rs9722 polymorphisms in the S100B gene were detected for both groups, and the serum level of S100B protein was measured for 74 HFMD children.

RESULTSThe rs9722 locus of the S100B gene had three genotypes, CC, CT, and TT, and the genotype frequencies were in accordance with Hardy-Weinberg equilibrium. Compared with the control group, the HFMD group had significant increases in the frequencies of TT genotype and T allele (P<0.01). Children with severe HFMD caused by enterovirus 71 infection had significantly higher frequencies of TT genotype and T allele than those with moderate or mild HFMD (P<0.05). Compared with the cured patients, the patients with poor prognosis had significant increases in the frequencies of TT genotype and T allele in the rs9722 locus of the S100B gene (P<0.05). Among the 74 children with HFMD, the children with TT genotype had the highest serum level of S100B protein, and those with CC genotype had the lowest level (P<0.01).

CONCLUSIONST allele in the rs9722 locus of the S100B gene might be a risk factor for severe HFMD caused by enterovirus 71 infection.

Child, Preschool ; Enterovirus A, Human ; Enterovirus Infections ; complications ; Female ; Genotype ; Hand, Foot and Mouth Disease ; etiology ; genetics ; Humans ; Infant ; Male ; Polymorphism, Genetic ; S100 Calcium Binding Protein beta Subunit ; genetics

OBJECTIVETo investigate the association of gene polymorphisms of Toll-like receptor 3 (TLR3)-1377C/T and expression of TLR3 with the susceptibility to enterovirus 71 (EV71) encephalitis in children.

METHODSA total of 187 children with EV71 infection (59 children in the encephalitis group and 128 in the non-encephalitis group) and 232 children who underwent physical examination were enrolled in the case-control study. Polymerase chain reaction-restriction fragment length polymorphism was used to detect the TLR3-1377C/T gene polymorphisms. ELISA was used to measure the serum level of TLR3.

RESULTSThere were no significant differences in the genotype and allele frequencies of TLR3-1377C/T between the non-encephalitis group and the encephalitis group. Compared with the control group, the encephalitis group and the non-encephalitis group had significant increases in the serum level of TLR3 (P<0.05), and the non-encephalitis group had the highest level (P<0.05). The encephalitis group had a significantly higher EV71 viral load than the non-encephalitis group (P<0.01). The children aged <1 year or ≥1 year in the encephalitis group and the non-encephalitis group had significant increases in the serum level of TLR3 compared with their counterparts in the control group (P<0.05), and the children aged <1 year or ≥1 year in the non-encephalitis group had a significantly higher serum level of TLR3 than those in the encephalitis group (P<0.05). In the encephalitis group, the children aged ≥1 year had a significantly higher TLR3 concentration than those aged <1 year (P<0.05), and there were no significant differences in the TLR3 concentration between the children aged ≥1 year and <1 year in the non-encephalitis group and the control group. In the encephalitis group, the proportion of children aged <1 year was significantly higher than those aged ≥1 year (P<0.05).

CONCLUSIONSThe TLR3-1377C/T gene polymorphisms are not significantly associated with the development of EV71 encephalitis. Low expression of TLR3 might weaken the inhibitory effect on virus replication and promote the development of EV71 encephalitis. The deficiency in the expression of TLR3 in serum after EV71 infection might be an important factor for the development of encephalitis in infants.

Child, Preschool ; Encephalitis, Viral ; genetics ; Enterovirus A, Human ; Enterovirus Infections ; genetics ; Female ; Genetic Predisposition to Disease ; Humans ; Infant ; Male ; Polymorphism, Single Nucleotide ; Toll-Like Receptor 3 ; genetics