Objective To investigate the role and mechanism of RNA-Specific adenosine deaminase 3 (Adar3) in regulating macrophage polarization during Coxsackievirus B3(CVB3)-induced viral myocarditis (VM). Methods Bone marrow-derived macrophages (BMDM) from mice were cultured in vitro and induced into M1/M2 macrophages using interferon-gamma (IFN-γ)/lipopolysaccharide (LPS) or interleukin 4 (IL-4), respectively. The mRNA expression levels of Adar1, Adar2, and Adar3 in each group of cells were assessed by real-time quantitative PCR (qRT-PCR). Specific siRNAs targeting the Adar3 gene were designed, synthesized, and transiently transfected into M2 macrophages. The mRNA levels of M2 polarization-related marker genes-including arginase 1 (Arg1), chitinase 3-like molecule 3 (YM1/Chi3l3), and resistin-like molecule alpha (RELMα/FIZZ1)-were detected by qRT-PCR. RNA sequencing was performed to analyze the signaling pathways affected by Adar3. The expression levels of Wnt/β-catenin signaling pathway were further validated using qRT-PCR and Western blot. The adeno-associated virus overexpressing Adar3 was designed, synthesized, and injected into mice via tail vein. Three weeks later, a myocarditis mouse model was established. After an additional week, the phenotype and function of cardiac macrophages, as well as multiple indicators of VM (including echocardiography, body weight, histopathology and serology) were examined. Additionally, the protein levels of the Wnt/β-catenin signaling pathway were assessed. Results Compared to M0-type macrophages, the expression level of Adar3 was significantly increased in M2-type macrophages. After transfection of Adar3 siRNA, the mRNA levels of Arg1, YM1 and FIZZ1 in M2 macrophages were downregulated. RNA sequencing revealed 149 upregulated genes and 349 downregulated genes. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis and subsequent validation experiments indicated that Adar3 modulated the Wnt/β-catenin signaling pathway. In vivo experiments demonstrated that Adar3 overexpression alleviated the cardiac dysfunction of VM mice. The proportion of M1 macrophages in the heart decreased, while the proportion of M2 macrophages increased. At the same time, the Adar3 overexpression activated the Wnt/β-catenin signaling pathway. Conclusion Adar3 promotes macrophage polarization toward the M2 phenotype by activating the Wnt/β-catenin signaling pathway, thereby alleviating VM.
Animals ; Adenosine Deaminase/metabolism* ; Macrophages/immunology* ; Wnt Signaling Pathway/genetics* ; Myocarditis/immunology* ; Mice ; Coxsackievirus Infections/metabolism* ; Male ; Mice, Inbred BALB C ; Enterovirus B, Human/physiology* ; beta Catenin/genetics*

The bioactivity-guided isolation of potentially active natural products has been widely utilized in pharmaceutical discovery. In this study, by screening fungal extracts against coxsackievirus B3 (CVB3), three new aspochalasins, templichalasins A‒C (1‒3), along with six known aspochalasins (4‒9) were isolated from an active extract derived from the endophytic fungus Aspergillus templicola LHWf045. Compound 1 features a unique 5/6/5/7/5 pentacyclic ring system, while compounds 2 and 3 possess unusual 5/6/6/7 tetracyclic skeletons. Their structures were characterized through extensive spectroscopic analyses, electronic circular dichroism (ECD) calculations, and single-crystal X-ray diffraction analysis. Additionally, we demonstrated that compound 4 can be readily converted into compounds 1‒3 under mild acidic conditions and proposed a plausible mechanism for this conversion. Bioactivity evaluation of compounds 1‒9 against CVB3 revealed the inhibitory effects of all compounds against the virus. Notably, compound 9 exhibited superior antiviral activity, surpassing the commercial drug ribavirin in selectivity index (SI) value.
Antiviral Agents/isolation & purification* ; Aspergillus/chemistry* ; Molecular Structure ; Enterovirus B, Human/drug effects* ; Endophytes/chemistry* ; Cytochalasins/isolation & purification* ; Drug Discovery ; Humans

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*

Echovirus 30 is one of the major causes of meningitis in children and adults. The purpose of our current study was to investigate whether selected antiviral drugs could provide antiviral activity against echovirus 30. Using RD cells, we assessed the cytopathic effect of echovirus 30, including viral RNA levels as indicators of viral replication. The effects of gemcitabine were compared to rupintrivir, a well-known antiviral drug. To understand the activity gemcitabine exerts on the viral life cycle, time course and time-of-addition assays were implemented. The most effective compounds against echovirus 30 were gemcitabine and rupintrivir, as demonstrated by their concentration-dependent activity. Gemcitabine affects the early stages of echovirus 30 infection by disrupting viral replication. However, gemcitabine failed to directly inactivate echovirus 30 particles or impede viral uptake into the RD cells. Gemcitabine can be considered as a lead candidate in the development of echovirus 30 antiviral drugs, specifically in the early stages of echovirus 30 replication.
Adult ; Antiviral Agents ; Child ; Enterovirus B, Human ; Humans ; In Vitro Techniques ; Life Cycle Stages ; Meningitis ; RNA, Viral

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

OBJECTIVE: Autophagy is a highly conserved intracellular degradation pathway. Many picornaviruses induce autophagy to benefit viral replication, but an understanding of how autophagy occurs remains incomplete. In this study, we explored whether coxsackievirus B3 (CVB3) infection induced autophagy through endoplasmic reticulum (ER) stress. METHODS: In CVB3-infected HeLa cells, the specific molecules of ER stress and autophagy were detected using Western blotting, reverse transcription polymerase chain reaction (RT-PCR), and confocal microscopy. Then PKR-like ER protein kinase (PERK) inhibitor, inositol-requiring protein-1 (IRE1) inhibitor, or activating transcription factor-6 (ATF6) inhibitor worked on CVB3-infected cells, their effect on autophagy was assessed by Western blotting for detecting microtubule-associated protein light chain 3 (LC3). RESULTS: CVB3 infection induced ER stress, and ER stress sensors PERK/eIF2α, IRE1/XBP1, and ATF6 were activated. CVB3 infection increased the accumulation of green fluorescent protein (GFP)-LC3 punctuation and induced the conversion from LC3-I to phosphatidylethanolamine-conjugated LC3-1 (LC3-II). CVB3 infection still decreased the expression of mammalian target of rapamycin (mTOR) and p-mTOR. Inhibition of PERK, IRE1, or ATF6 significantly decreased the ratio of LC3-II to LC3-I in CVB3-infected HeLa cells. CONCLUSION CVB3 infection induced autophagy through ER stress in HeLa cells, and PERK, IRE1, and ATF6a pathways participated in the regulation of autophagy. Our data suggested that ER stress may inhibit mTOR signaling pathway to induce autophagy during CVB3 infection.
Activating Transcription Factor 6 ; metabolism ; Autophagy ; Coxsackievirus Infections ; metabolism ; Endoplasmic Reticulum Stress ; Endoribonucleases ; metabolism ; Enterovirus B, Human ; HeLa Cells ; Humans ; Protein-Serine-Threonine Kinases ; metabolism ; Signal Transduction ; eIF-2 Kinase ; metabolism

<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

Swine vesicular disease (SVD) is a highly contagious viral disease that causes vesicular disease in pigs. The importance of the disease is due to its indistinguishable clinical signs from those of foot-and-mouth disease, which prevents international trade of swine and related products. SVD-specific antibody detection via an enzyme-linked immunosorbent assay (ELISA) is the most versatile and commonly used method for SVD surveillance and export certification. Inactivated SVD virus is the commonly used antigen in SVD-related ELISA. A recombinant SVD virus-like particle (VLP) was generated by using a Bac-to-Bac baculovirus expression system. Results of SVD-VLP analyses from electron microscopy, western blotting, immunofluorescent assay, and mass spectrometry showed that the recombinant SVD-VLP morphologically resemble authentic SVD viruses. The SVD-VLP was evaluated as a replacement for inactivated whole SVD virus in competitive and isotype-specific ELISAs for the detection of antibodies against SVD virus. The recombinant SVD-VLP assay produced results similar to those from inactivated whole virus antigen ELISA. The VLP-based ELISA results were comparable to those from the virus neutralization test for antibody detection in pigs experimentally inoculated with SVD virus. Use of the recombinant SVD-VLP is a safe and valuable alternative to using SVD virus antigen in diagnostic assays.
Animals ; Antibodies ; Baculoviridae ; Blotting, Western ; Certification ; Enterovirus B, Human ; Enzyme-Linked Immunosorbent Assay ; Foot-and-Mouth Disease ; Mass Spectrometry ; Methods ; Microscopy, Electron ; Neutralization Tests ; Serologic Tests* ; Swine Vesicular Disease* ; Swine* ; Virus Diseases

PURPOSE: Enterovirus infection in children can manifest various disease and enterovirus have many serotypes. This study was aimed to investigate neurologic manifestations according to serotypes of enterovirus in pediatric inpatients in Incheon. METHODS: We collected the stool samples from the admitted pediatric patients in Inha University Hospital from January 2015 to September 2016. Enterovirus detection and serotypes identification were performed by real-time reverse transcriptase polymerase chain reaction (RT-PCR) and semi-nested RT-PCR. RESULTS: A total of 527 samples were collected during study period and 170 patients (32.2%) were diagnosed with enterovirus infections. Genetic sequences of enteroviruses were identified: echovirus 18 (50, 40.5%), enterovirus 71 (12, 9.6%), coxakievirus A10 (10, 8.0%), echovirus 6 (7, 5.6%). Virus in patient with meningitis were identified: echovirus 18 (15, 75%), coxakievirus B5 (2, 10%), enterovirus 71 (2, 10%), and echovirus 6 (1, 5%). Neurologic manifestations of echovirus 18 are headache (15, 30%), vomiting (17, 34%), meningeal irritation sign (10, 20.0%). And enterovirus 71 have headache (3, 25%), vomiting (3, 25%), meningeal irritation sign (2, 16.0%), seizure (1, 8.3%), neurologic sequelae (1, 8.3%). Echovirus 18 and neurologic manifestation have a statistically significant correlation with other serotypes (r=0.701, P < 0.01) CONCLUSION: Echovirus 18 infection was more prominent in neurological symptoms than in other serotypes. The major serotype of meningitis was echovirus 18 but there was no reported neurologic sequelae. Enterovirus infection has different neurological symptoms, depending on the serotypes.
Child ; Echovirus 6, Human ; Enterovirus B, Human ; Enterovirus Infections ; Enterovirus* ; Headache ; Humans ; Incheon* ; Inpatients* ; Meningitis ; Neurologic Manifestations* ; Reverse Transcriptase Polymerase Chain Reaction ; Seizures ; Serogroup* ; Vomiting