Human Coxsackievirus B3 Research Articles

Genome-Driven Discovery of Antiviral Atralabdans A-C from the Soil-Dwelling Streptomyces atratus.

Heterologous expression of an atr terpenoid gene cluster derived from Streptomyces atratus Gö66 in S. albus J1074 led to the discovery of three novel labdane diterpenoids featuring an unprecedented 6/6/5-fused tricyclic skeleton, designated as atralabdans A-C (1-3), along with a known compound, labdanmycin A. Compounds 1-3 were identified through extensive spectroscopic analysis, including NMR calculations with DP4+ probability analysis, and a comparative assessment of experimental and theoretical electronic circular dichroism (ECD) spectra. A plausible biosynthetic pathway for these compounds was proposed. Compounds 1-3 exhibited inhibitory activity against the human neurotropic coxsackievirus B3 (CVB3); 1 was the most potent, surpassing the positive control ribavirin with a higher therapeutic index.

Aichivirus A1 replicates in human intestinal epithelium and bronchial tissue: Lung–gut axis?

The role of aichivirus A1 (AiV-A1) in acute gastroenteritis remains controversial and in vitro data illustrating its pathogenesis in suitable human models are scarce. Here, we demonstrate that AiV-A1 isolate A846/88 replicates in ApoA1- (absorptive) and Ki-67-positive (proliferative) enterocytes in stem cell-derived human small intestinal epithelium (HIE) as well as in patient biopsy samples, but not in any of the tested human cell lines. The infection did not result in tissue damage and did not trigger type I and type III interferon (IFN) signalling, whereas the control, human coxsackievirus B3 (strain Nancy), triggered both IFNs. To investigate the tissue tropism, we infected a human tracheal/bronchial epithelium model (HTBE) with AiV-A1 isolates A846/88 and kvgh99012632/2010 and, as a control, with rhinovirus A2 (RV-A2). AiV-A1 isolate kvgh99012632/2010, but not isolate A846/88, replicated in HTBE and induced type III IFN and ISGs signalling. By using various pharmacological inhibitors, we elaborated that cellular entry of AiV-A1 depends on clathrin, dynamin, and lipid rafts and is strongly reliant on endosome acidification. Viral particles co-localised with Rab5a-positive endosomes and promoted leakage of endosomal content. Our data shed light on the early events of AiV-A1 infection and reveal that different isolates exhibit distinct tissue tropism. This supports its clinical importance as a human pathogen with the potential to evolve toward broader tissue specificity.

A Short 5'triphosphate RNA nCoV-L Induces a Broad-Spectrum Antiviral Response by Activating RIG-I.

Small molecular nucleic acid drugs produce antiviral effects by activating pattern recognition receptors (PRRs). In this study, a small molecular nucleotide containing 5'triphosphoric acid (5'PPP) and possessing a double-stranded structure was designed and named nCoV-L. nCoV-L was found to specifically activate RIG-I, induce interferon responses, and inhibit duplication of four RNA viruses (Human enterovirus 71, Human poliovirus 1, Human coxsackievirus B5 and Influenza A virus) in cells. In vivo, nCoV-L quickly induced interferon responses and protected BALB/c suckling mice from a lethal dose of the enterovirus 71. Additionally, prophylactic administration of nCoV-L was found to reduce mouse death and relieve morbidity symptoms in a K18-hACE2 mouse lethal model of SARS-CoV-2. In summary, these findings indicate that nCoV-L activates RIG-I and quickly induces effective antiviral signals. Thus, it has potential as a broad-spectrum antiviral drug.

Jessel Josephs

<h3>ABSTRACT</h3> ISG15 is an interferon-stimulated, ubiquitin-like protein that can conjugate to substrate proteins (ISGylation) to counteract microbial infection, but the underlying mechanisms remain elusive. Here, we used a viral-like particle...

Stabilin-1 mediates beneficial monocyte recruitment and tolerogenic macrophage programming during CVB3-induced viral myocarditis

Pathological innate and adaptive immune response upon viral infection may lead to cardiac injury and dysfunction. Stabilin-1 is a scavenger receptor that regulates several aspects of the innate immunity. Whether stabilin-1 affects the inflammatory response during viral myocarditis (VM) is entirely unknown. Here, we assess the role of stabilin-1 in the pathogenesis of VM and its suitability as a therapeutic target. Genetic loss of stabilin-1 increased mortality and cardiac necrosis in a mouse model of human Coxsackievirus B3 (CVB3)-induced myocarditis. Absence of stabilin-1 significantly reduced monocyte recruitment and strongly reduced the number of alternatively activated anti-inflammatory macrophages in the heart, enhancing a pro-inflammatory cardiac niche with a detrimental T lymphocyte response during VM. Yeast two-hybrid screening, confirmed by affinity chromatography, identified fibronectin as a stabilin-1 interacting partner. Absence of stabilin-1 specifically decreased monocyte adhesion on extracellular fibronectin in vitro. Loss of Type III repeats Extra Domain A (EDA) of fibronectin during VM also increased the mortality and cardiac necrosis as in stabilin-1 knockout mice, with reduced monocytic cardiac recruitment and increased T lymphocyte response.Collectively, stabilin-1 has an immune-suppressive role of limiting myocardial damage during VM, regulating anti-inflammatory monocyte-recruitment to the site of inflammation.

Ring finger protein 213 assembles into a sensor for ISGylated proteins with antimicrobial activity

ISG15 is an interferon-stimulated, ubiquitin-like protein that can conjugate to substrate proteins (ISGylation) to counteract microbial infection, but the underlying mechanisms remain elusive. Here, we use a virus-like particle trapping technology to identify ISG15-binding proteins and discover Ring Finger Protein 213 (RNF213) as an ISG15 interactor and cellular sensor of ISGylated proteins. RNF213 is a poorly characterized, interferon-induced megaprotein that is frequently mutated in Moyamoya disease, a rare cerebrovascular disorder. We report that interferon induces ISGylation and oligomerization of RNF213 on lipid droplets, where it acts as a sensor for ISGylated proteins. We show that RNF213 has broad antimicrobial activity in vitro and in vivo, counteracting infection with Listeria monocytogenes, herpes simplex virus 1, human respiratory syncytial virus and coxsackievirus B3, and we observe a striking co-localization of RNF213 with intracellular bacteria. Together, our findings provide molecular insights into the ISGylation pathway and reveal RNF213 as a key antimicrobial effector.

Cucurbit[7]uril as a Broad-Spectrum Antiviral Agent against Diverse RNA Viruses.

The emergence and re-emergence of RNA virus outbreaks highlight the urgent need for the development of broad-spectrum antivirals. Polyamines are positively-charged small molecules required for the infectivity of a wide range of RNA viruses, therefore may become good antiviral targets. Cucurbit[7]uril (CB[7]), a synthetic macrocyclic molecule, which can bind with amine-based organic compounds with high affinity, has been shown to regulate bioactive molecules through competitive binding. In this study, we tested the antiviral activity of CB[7] against diverse RNA viruses, including a panel of enteroviruses (i.e. human enterovirus A71, coxsackievirus A16, coxsackievirus B3, and echovirus 11), some flaviviruses (i.e. dengue virus and Zika virus), and an alphavirus representative Semliki forest virus. CB[7] can inhibit virus replications in a variety of cell lines, and its mechanism of action is through the competitive binding with polyamines. Our findings not only for the first time provide evidence that CB[7] can be a promising broad-spectrum antiviral agent, but more importantly, offer a novel therapeutic strategy to fight against RNA viruses by supramolecular sequestration of polyamines.

P5565S100A8/A9 serum levels as a diagnostic biomarker and its potential connection to NOD2-NLRP3 in patients with a recent onset of myocarditis

Abstract Background The alarmin S100A8/A9 has been shown to be of importance in several inflammatory cardiovascular disorders. We recently demonstrated the pivotal role of cardiac S100A8/A9 in human and experimental Coxsackievirus B3 (CVB3)-induced myocarditis (MC). Purpose We aimed to evaluate whether serum S100A8/A9 levels are a marker in patients with a recent onset of MC Methods Serum S100A8/A9, hsCRP, and NT pro-BNP levels were analyzed in patients with a recent onset of MC (≤30 days (d), n=29; ejection fraction (EF): 44.3%±13%), dilated cardiomyopathy patients with inflammation (DCMi: n=112; EF: 28.8%±12%) or without inflammation (DCM: n=58; EF: 26.7%±9%), and controls (co: n=25; EF: 68.5%±5%). Blood samples and endomyocardial biopsies (EMB) were collected at time point (T1). In a subgroup, S100A8/A9 serum levels and EMB were available at T1 (n=10) and follow-up (T2, n=10, mean follow-up 8 months). Results MC ≤30 d patients showed a 4.5-fold (p&lt;0.0001), 19.3-fold (p&lt;0.0001), and 4.0-fold (p&lt;0.0001) increase in S100A8/A9, hsCRP, NT pro-BNP levels vs co, respectively. S100A8/A9 levels correlated with the disease activity, displayed by EMB counts of inflammatory cells (CD3: r=0.464, p=0.0128, XY pairs=28, LFA-1: r=0.551, p=0.002, XY pairs=28, Mac-1: r=0.418, p=0.026, XY pairs=28), and the EF (r=0.545, p=0.0027, XY pairs=28). MC ≤30 d patients showed an association between serum S100A8/A9 levels and EMB S100A8 (r=0.482, p=0.060, XY pairs=16), S100A9 (r=0.441, p=0.088, XY pairs=16), nucleotide-binding oligomerization domain containing-protein 2 (NOD2, r=0.55, p=0.035, XY=15), and Nod-like receptor family, pyrin domain-containing 3 protein (NLRP3, r=0.52, p=0.048, XY=15) mRNA levels. Also EMB S100A8 and S100A9 mRNA levels showed a significant correlation with EMB NOD2 and NLRP3 mRNA expression. Serum S100A8/A9 levels were increased by 3.0-fold (p&lt;0.0001) and 1.8-fold (p=0.0005) in DCMi (n=112), and DCM (n=58) patients vs co, respectively. However, the S100A8/A9 levels of DCMi and DCM patients were 1.5-fold (p=0.07) and 2.5-fold (p&lt;0.0001) lower vs MC ≤30 d patients. ROC analyses of S100A8/A9 in MC ≤30 d provided a cut-off of 583 ng/ml with a specificity=92%, sensitivity=86.2%, a PPV=92.6%, a NPV=85.2%, and an AUC=0.934 vs co, which was superior to hsCRP (cut-off=5 mg/l): specificity=95.8%, sensitivity=58.6%, a PPV=94.4%, a NPV=65.7%, AUC=0.885. In the subgroup, S100A8/A9 levels decreased after heart failure medication (T1: 2454±1931 ng/ml vs T2: 934.4±552 ng/ml; p=0.002), reflected by a decrease of EMB inflammatory cells. Baseline serum S100A8/A9 levels predicted the change in EMB CD3 and Mac-1. Conclusions These results support an additional value for S100A8/A9 serum levels as a potential diagnostic biomarker and as a tool to monitor the course of the disease. We provide first evidence that S100A8/A9 is connected to the NOD2-NLRP3 pathway in these patients. Acknowledgement/Funding Novartis

Enterovirus inhibitory activity of C-8-tert-butyl substituted 4-aryl-6,7,8,9-tetrahydrobenzo[4,5]thieno[3,2-e][1,2,4]triazolo[4,3-a]pyrimidin-5(4H)-ones

Members of a series of 4-aryl-6,7,8,9-tetrahydrobenzo[4,5]thieno[3,2-e][1,2,4]triazolo[4,3-a]pyrimidin-5(4H)-ones (1, Fig. 2) were prepared and tested against representative enteroviruses including Human Coxsackievirus B1 (Cox B1), Human Coxsackievirus B3 (Cox B3), human Poliovirus 3 (PV3), human Rhinovirus 14 (HRV14), human Rhinovirus 21 (HRV 21) and human Rhinovirus 71 (HRV 71). The C-8-tert-butyl group on the tetrahydrobenzene ring in these substances was found to be crucial for their enterovirus activity. One member of this group, 1e, showed single digit micromolar activities (1.6–8.85μM) against a spectrum of viruses screened, and the highest selectivity index (SI) values for Cox B1 (>11.2), for Cox B3 (>11.5), and for PV3 (>51.2), respectively. In contrast, 1p, was the most active analog against the selected HRVs (1.8–2.6μM), and showed the highest selectivity indices among the group of compounds tested. The SI values for 1p were 11.5 for HRV14, 8.4 for HRV21, and 12.1 for HRV71, respectively.

2,3,4-Trihydroxybenzyl-hydrazide analogues as novel potent coxsackievirus B3 3C protease inhibitors

Human coxsackievirus B3 (CVB3) 3C protease plays an essential role in the viral replication of CVB3, which is a non-enveloped and positive single-stranded RNA virus belonging to Picornaviridae family, causing acute viral myocarditis mainly in children. During optimization based on SAR studies of benserazide (3), which was reported as a novel anti-CVB3 3Cpro agent from a screening of compound libraries, the 2,3,4-trihydroxybenzyl moiety of 3 was identified as a key pharmacophore for inhibitory activity against CVB3 3Cpro. Further optimization was performed by the introduction of various aryl-alkyl substituted hydrazide moieties instead of the serine moiety of 3. Among the optimized compounds, 11Q, a 4-hydroxyphenylpentanehydrazide derivative, showed the most potent inhibitory activity (IC50 = 0.07 μM). Enzyme kinetics studies indicated that 11Q exhibited a mixed inhibitory mechanism of action. The antiviral activity against CVB3 was confirmed using the further derived analogue (14b) with more cell permeable valeryl ester group at the 2,3,4-trihydroxy moiety.

Near-complete genome sequencing of swine vesicular disease virus using the Roche GS FLX sequencing platform.

Swine vesicular disease virus (SVDV) is an enterovirus that is both genetically and antigenically closely related to human coxsackievirus B5 within the Picornaviridae family. SVDV is the causative agent of a highly contagious (though rarely fatal) vesicular disease in pigs. We report a rapid method that is suitable for sequencing the complete protein-encoding sequences of SVDV isolates in which the RNA is relatively intact. The approach couples a single PCR amplification reaction, using only a single PCR primer set to amplify the near-complete SVDV genome, with deep-sequencing using a small fraction of the capacity of a Roche GS FLX sequencing platform. Sequences were initially verified through one of two criteria; either a match between a de novo assembly and a reference mapping, or a match between all of five different reference mappings performed against a fixed set of starting reference genomes with significant genetic distances within the same species of viruses. All reference mappings used an iterative method to avoid bias. Further verification was achieved through phylogenetic analysis against published SVDV genomes and additional Enterovirus B sequences. This approach allows high confidence in the obtained consensus sequences, as well as provides sufficiently high and evenly dispersed sequence coverage to allow future studies of intra-host variation.

Phylodynamics of Human Coxsackievirus B5

ABSTRACT: Aim: Coxsackievirus B5 is recognized as an important pathogen in aseptic meningitis and hand, foot and mouth disease of children. A new distinctive sublineage of Coxsackievirus B5 associated with outbreak of neurological hand, foot and mouth disease in China was recently reported. Materials &amp; methods: We employed a molecular evolution method to study the genetic variation and evolutionary history of Coxsackievirus B5 in China. Coxsackievirus B5 isolates from China can be divided into four major groups with obvious temporal evolution routes. Results: A 'mutation box' covering amino acids 75, 85, 90 and 95 of VP1 protein was observed to be unique in the isolates group from Henan and Shandong province. The temporal evolution routes of the amino acids changes in the 'mutation box' were studied. Conclusion: Our results provide primary insight into the relationship between genetic variation and epidemic behavior of Coxsackievirus B5.

Complete nucleotide sequence of a human coxsackievirus B3 strain A103/KM/09 isolated in Yunnan province, 2009

To analyze the genetic characterization of the complete genome from a human coxsackievirus B3 strain A103/KM/09 isolated in Yunnan province, 2009. By using RT-PCR, all the eight fragments which containing about 1000 nucleotides and covering full viral genome, were sequenced. By using Mega 5.05,Geneious, RDP 3 and SimPlot 3.5.1 software, sequences were aligned with other enterovirus reference sequences. Phylogenetic and recombination analysis were also carried out. The A103/KM/09 isolate genome showed 7389 nucleotides in length , encoding for 2185 amino acids. In the complete genome, the homology of nucleotide and amino acid among the seven coxsackievirus B3 isolates were 81.0%-88.0% and 95.7%-98.0%, respectively. There appeared 81.0% and 95.7% homology when compared with that of Nancy prototype strain. Results from the Phylogenetic analysis showed that the coxsackievirus B3 formed five distinct clades, I-V. Nucleotide divergence rates between clades were 16.2%-24.3% . The A103/KM/09 strain belonged to clade V. Clade V was further divided into four sub-clades,A-D. The nucleotide divergence between sub-clades was 4.3%-11.4%. Putative recombinant event for A103/ KM/09 was detected. All coxsackievirus B3 isolates could be divided into five clades, with A103/KM/09 strain belonged to Clade V-D. Evolution of coxsackievirus B3 had occurred in China.

Phylogenetic Patterns of Human Coxsackievirus B5 Arise from Population Dynamics between Two Genogroups and Reveal Evolutionary Factors of Molecular Adaptation and Transmission

The aim of this study was to gain insights into the tempo and mode of the evolutionary processes that sustain genetic diversity in coxsackievirus B5 (CVB5) and into the interplay with virus transmission. We estimated phylodynamic patterns with a large sample of virus strains collected in Europe by Bayesian statistical methods, reconstructed the ancestral states of genealogical nodes, and tested for selection. The genealogies estimated with the structural one-dimensional gene encoding the VP1 protein and nonstructural 3CD locus allowed the precise description of lineages over time and cocirculating virus populations within the two CVB5 clades, genogroups A and B. Strong negative selection shaped the evolution of both loci, but compelling phylogenetic data suggested that immune selection pressure resulted in the emergence of the two genogroups with opposed evolutionary pathways. The genogroups also differed in the temporal occurrence of the amino acid changes. The virus strains of genogroup A were characterized by sequential acquisition of nonsynonymous changes in residues exposed at the virus 5-fold axis. The genogroup B viruses were marked by selection of three changes in a different domain (VP1 C terminus) during its early emergence. These external changes resulted in a selective sweep, which was followed by an evolutionary stasis that is still ongoing after 50 years. The inferred population history of CVB5 showed an alternation of the prevailing genogroup during meningitis epidemics across Europe and is interpreted to be a consequence of partial cross-immunity.

Recombinant Human Coxsackievirus B3 from Children with Acute Myocarditis in China

Recombination events were found in two human coxsackievirus B3 strains, Beijing0811 and SD2012CHN. The strains were isolated separately from five newborns diagnosed with severe hospital-acquired acute myocarditis in Beijing in 2008 and from two children diagnosed with hand, foot, and mouth disease with concurrent acute myocarditis in Shandong in 2012.

Recombination in human coxsackievirus B5 strains that caused an outbreak of viral encephalitis in Henan, China

In 2011, human coxsackievirus B5 (CVB5) caused an outbreak of viral encephalitis in Henan, China. Complete genome sequence analysis based on two isolates showed that the 5' half of the genome (nt 1-4540) had high similarity (>97%) to that of CVB5 strain GU376747, and the 3' half (nt 4700-7402) showed high similarity (>96%) to that of human coxsackievirus B3 (CVB3) strain GQ141875. These isolates had the highest similarity (97.7%) to the Changchun strain, based on the complete genome, rather than to other CVB5 strains isolated from Henan in recent years. There were therefore at least two groups of CVB5 circulating in Henan Province, which evolved at different rates.

The flame-retardant BDE-99 dose-dependently affects viral replication in CVB3-infected mice

The flame retardant component 2,2′,4,4′,5-penta-BDE (BDE-99) is found in the environment and in human tissues and fluids. In mice the common human coxsackievirus B3 (CVB3) infection has been shown to change the tissue distribution of BDE-99. We now investigate how CVB3 infection in mice affects liver uptake of 14C at two doses of radiolabelled BDE-99, and whether increased tissue levels are related to changed virus replication and gene expression of the proinflammatory chemokine monocyte chemoattractant protein-1 (MCP-1). Mice were infected on day 0, orally treated either with 200μg or 20mg 14C-BDE-99/kgbw on day 1, and euthanised on day 3. Serum and liver levels of 14C-BDE-99, as well as virus levels and gene expressions of MCP-1 in the liver, were measured. In non-infected mice, there was a dose-dependent uptake of BDE-99 in both liver and serum, and in infected animals the liver BDE-99 levels was further increased. When comparing infected mice exposed to the two BDE-99 doses, the higher BDE dose resulted in increased virus amounts in the liver, and decreased infection-induced expression of MCP-1. Consequently, a high enough dose/tissue concentration of BDE-99 may result in a disturbed mobilisation of immune cells into infected tissues that could explain higher virus titres and a possibly altered clinical course of the disease. Moreover, the fact that CVB3 infection increased the BDE-99 levels in liver but not in serum may impair the risk assessment of polybrominated diphenyl ethers (PBDEs) in subclinical and clinically infected individuals, as serum levels is the common marker of exposure.

Complete Genome Sequence of a Human Coxsackievirus B3 from a Child with Myocarditis in Beijing, China

A human coxsackievirus B3 (CVB3), designated strain Beijing0811, was isolated from a child diagnosed with hospital-acquired infectious acute myocarditis in Beijing, China, and propagated in human rhabdomyosarcoma cells. The complete genome sequence of this virus was 7,402 nucleotides, excluding the 3′ poly(A) tail, which encoded a large polyprotein with 2,185 amino acids. This report will help us to analyze the evolutionary and epidemic characteristics of CVB3.

The matricellular proteins thrombospondin‐2, osteonectin and osteoglycin modulate cardiac inflammation, injury and function during viral myocarditis.

Non‐structural matri‐cellular proteins (MCPs) modulate the process of heart failure (HF) by acting on different cardiac cellular compartments. Here, we investigated whether MCPs, including TSP2, SPARC or osteoglycin (OGN), may affect cardiac inflammation. Therefore, TSP‐2, SPARC and OGN‐null animals and their WT littermates were submitted to human Coxsackievirus B3 (CVB3)‐induced myocarditis. Whereas the absence of TSP‐2 or SPARC increased cardiac inflammation, necrosis and overall mortality as compared to WT at 14 days, resulting in systolic dysfunction at 35 days, lack of OGN was protective. Increased cardiac inflammation and injury was caused by decreased activation of the anti‐inflammatory T‐regulatory cells (TSP‐2‐null) and increased trans‐endothelial leukocyte migration (SPARC‐null). Decreased cytokine activation caused less cardiac inflammation in OGN‐null. Finally, over expression of TSP‐2 or SPARC in WT hearts with AAV9‐TSP2 or AdVSPARC significantly reduced cardiac inflammation and injury. In conclusion, whereas increased SPARC or TSP‐2 protect against cardiac necrosis and failure during viral myocarditis, OGN is detrimental.

Thrombospondin-2 prevents cardiac injury and dysfunction in viral myocarditis through the activation of regulatory T-cells

Thrombospondin-2 (TSP-2) modulates matrix integrity and myocyte survival in the hypertensive or ageing heart. Whether TSP-2 may affect cardiac inflammation and injury, in particular during acute viral myocarditis, is completely unknown. Therefore, mortality, cardiac inflammation, and function were assessed in TSP-2-null (KO) and wild-type (WT) mice in human Coxsackie virus B3 (CVB3)-induced myocarditis. TSP-2 KO had an increased mortality when compared with WT mice during viral myocarditis. The absence of TSP-2 resulted in increased cardiac inflammation and injury at 14 days, which resulted in depressed systolic function [fractional shortening (FS); 34 ± 2.6 in WT vs. 24 ± 1.8 in KO mice, P< 0.05] and increased cardiac dilatation (end-diastolic dimensions, mm; 3.7 ± 0.09 in WT vs. 4.8 ± 0.06 in KO mice, P< 0.05) 35 days post-infection. Lack of TSP-2 resulted in a decreased activation of the anti-inflammatory T-regulatory cells, as indicated by a lower number of CD25-positive T-cells, and significantly decreased gene expression of regulatory T-cell markers, Foxp3 and CTLA-4. Finally, overexpression of TSP-2 in WT hearts using cardiotropic vectors derived from adeno-associated virus serotype 9 (AAV9) inhibited cardiac inflammation and injury at 14 days and improved cardiac function at 35 days post-CVB3 infection when compared with control AAV9. TSP-2 has a protective role against cardiac inflammation, injury, and dysfunction in acute viral myocarditis.