Neutralizing Activity Induced by the Attenuated Coxsackievirus B3 Sabin3-like Strain Against CVB3 Infection

3C protease of Coxsackievirus B3 (CVB3) plays an essential role in the viral replication cycle, and therefore, emerged as an attractive therapeutic target for the treatment of human diseases caused by CVB3 infection.

Download Full-text

In Vitro Model Systems of Coxsackievirus B3-Induced Myocarditis: Comparison of Commonly Used Cell Lines and Characterization of CVB3-Infected iCell® Cardiomyocytes

Viruses ◽

10.3390/v13091835 ◽

2021 ◽

Vol 13 (9) ◽

pp. 1835

Author(s):

Lisa Kraft ◽

Martina Sauter ◽

Guiscard Seebohm ◽

Karin Klingel

Keyword(s):

Cell Death ◽

Viral Load ◽

Cell Lines ◽

Hela Cells ◽

Coxsackievirus B3 ◽

Model System ◽

Model Systems ◽

H9c2 Cells ◽

Cvb3 Infection

Coxsackievirus B3 (CVB3) belongs to the enteroviruses, which are a well-known cause of acute and chronic myocarditis, primarily infecting cardiac myocytes. As primary human cardiomyocytes are difficult to obtain, viral myocarditis is quite frequently studied in vitro in different non-cardiac and cardiac-like cell lines. Recently, cardiomyocytes that have been differentiated from human-induced pluripotent stem cells have been described as a new model system to study CVB3 infection. Here, we compared iCell® Cardiomyocytes with other cell lines that are commonly used to study CVB3 infection regarding their susceptibility and patterns of infection and the mode of cell death. iCell® Cardiomyocytes, HeLa cells, HL-1 cells and H9c2 cells were infected with CVB3 (Nancy strain). The viral load, CVB3 RNA genome localization, VP1 expression (including the intracellular localization), cellular morphology and the expression of cell death markers were compared. The various cell lines clearly differed in their permissiveness to CVB3 infection, patterns of infection, viral load, and mode of cell death. When studying the mode of cell death of CVB3-infected iCell® Cardiomyocytes in more detail, especially regarding the necroptosis key players RIPK1 and RIPK3, we found that RIPK1 is cleaved during CVB3 infection. iCell® Cardiomyocytes represent well the natural host of CVB3 in the heart and are thus the most appropriate model system to study molecular mechanisms of CVB3-induced myocarditis in vitro. Doubts are raised about the suitability of commonly used cell lines such as HeLa cells, HL-1 cells and H9c2 cells to evaluate molecular pathways and processes occurring in vivo in enteroviral myocarditis.

Download Full-text

Interaction between coxsackievirus B3 infection and α-synuclein in models of Parkinson’s disease

PLoS Pathogens ◽

10.1371/journal.ppat.1010018 ◽

2021 ◽

Vol 17 (10) ◽

pp. e1010018

Author(s):

Soo Jin Park ◽

Uram Jin ◽

Sang Myun Park

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Inclusion Body ◽

Dopaminergic Neurons ◽

Inclusion Bodies ◽

Lewy Bodies ◽

Coxsackievirus B3 ◽

Body Formation ◽

Inclusion Body Formation ◽

Cvb3 Infection

Parkinson’s disease (PD) is one of the most common neurodegenerative diseases. PD is pathologically characterized by the death of midbrain dopaminergic neurons and the accumulation of intracellular protein inclusions called Lewy bodies or Lewy neurites. The major component of Lewy bodies is α-synuclein (α-syn). Prion-like propagation of α-syn has emerged as a novel mechanism in the progression of PD. This mechanism has been investigated to reveal factors that initiate Lewy pathology with the aim of preventing further progression of PD. Here, we demonstrate that coxsackievirus B3 (CVB3) infection can induce α-syn-associated inclusion body formation in neurons which might act as a trigger for PD. The inclusion bodies contained clustered organelles, including damaged mitochondria with α-syn fibrils. α-Syn overexpression accelerated inclusion body formation and induced more concentric inclusion bodies. In CVB3-infected mice brains, α-syn aggregates were observed in the cell body of midbrain neurons. Additionally, α-syn overexpression favored CVB3 replication and related cytotoxicity. α-Syn transgenic mice had a low survival rate, enhanced CVB3 replication, and exhibited neuronal cell death, including that of dopaminergic neurons in the substantia nigra. These results may be attributed to distinct autophagy-related pathways engaged by CVB3 and α-syn. This study elucidated the mechanism of Lewy body formation and the pathogenesis of PD associated with CVB3 infection.

Download Full-text

Coxsackievirus B3 Replication Is Reduced by Inhibition of the Extracellular Signal-Regulated Kinase (ERK) Signaling Pathway

Journal of Virology ◽

10.1128/jvi.76.7.3365-3373.2002 ◽

2002 ◽

Vol 76 (7) ◽

pp. 3365-3373 ◽

Cited By ~ 149

Author(s):

Honglin Luo ◽

Bobby Yanagawa ◽

Jingchun Zhang ◽

Zongshu Luo ◽

Mary Zhang ◽

...

Keyword(s):

Signaling Pathways ◽

Intracellular Signaling ◽

Late Phase ◽

Viral Myocarditis ◽

Coxsackievirus B3 ◽

Host Cells ◽

Virus Protein ◽

Signaling Mechanism ◽

Intracellular Signaling Pathways ◽

Cvb3 Infection

ABSTRACT Coxsackievirus B3 (CVB3) is the most common human pathogen for viral myocarditis. We have previously shown that the signaling protein p21 ras GTPase-activating protein (RasGAP) is cleaved and that mitogen-activated protein kinases (MAPKs) ERK1/2 are activated in the late phase of CVB3 infection. However, the role of intracellular signaling pathways in CVB3-mediated myocarditis and the relative advantages of such pathways to host or virus remain largely unclear. In this study we extended our prior studies by examining the interaction between CVB3 replication and intracellular signaling pathways in HeLa cells. We observed that CVB3 infection induced a biphasic activation of ERK1/2, early transient activation versus late sustained activation, which were regulated by different mechanisms. Infection by UV-irradiated, inactivated virus capable of receptor binding and endocytosis triggered early ERK1/2 activation, but was insufficient to trigger late ERK1/2 activation. By using a general caspase inhibitor (zVAD.fmk) we further demonstrated that late ERK1/2 activation was not a result of CVB3-mediated caspase cleavage. Treatment of cells with U0126, a selective inhibitor of MAPK kinase (MEK), significantly inhibited CVB3 progeny release and decreased virus protein production. Furthermore, inhibition of ERK1/2 activation circumvented CVB3-induced apoptosis and viral protease-mediated RasGAP cleavage. Taken together, these data suggest that ERK1/2 activation is important for CVB3 replication and contributes to virus-mediated changes in host cells. Our findings demonstrate coxsackievirus takeover of a particular host signaling mechanism and uncover a prospective approach to stymie virus spread and preserve myocardial integrity.

Download Full-text

Inoculation of the attenuated Coxsackievirus B3 Sabin3-like strain induces a protection against virulent CVB3 Nancy and CVB4 E2 strains in Swiss mice by both oral and intraperitoneal routes

Biologia ◽

10.2478/s11756-013-0292-1 ◽

2014 ◽

Vol 69 (1) ◽

Author(s):

Nadia Jrad-Battikh ◽

Amira Souii ◽

Rym Hadhri ◽

Mahjoub Aouni ◽

Jawhar Gharbi ◽

...

Keyword(s):

Coxsackievirus B3 ◽

Oral Route ◽

Post Inoculation ◽

Translation Efficiency ◽

Post Challenge ◽

Genomic Context ◽

Swiss Mice ◽

Cvb3 Infection ◽

Wild Strains

AbstractWe have previously addressed the question of whether the attenuating mutations of domain V of the Poliovirus IRES were specific for a given genomic context or whether they could be extrapolated to a genomic related virus, the Coxsackievirus B3 (CVB3). Accordingly, we have described that Sabin3-like mutation (U473→C) introduced in the CVB3 genome led to a defective mutant with a serious reduction in translation efficiency. In this study, we assessed the protection provided by the Sabin3-like mutant against CVB3 infection. For this purpose, we analyzed, in vivo, the Sabin3-like phenotype in Swiss mice inoculated with CVB3 and CVB4 E2 prototype strains either by oral or intraperitoneal (i.p) routes and explored the capacity of this mutant to act as a vaccine vector after the challenge. The Sabin3-like RNA was detected by semi-nested PCR in different organs: heart, pancreas and intestine at 10 days post-inoculation with both oral and i.p routes. Additionally, we did not observe any histological alterations in heart and intestine tissues. RNA was detected in the different organs of all mice immunized with the Sabin3-like strain and challenged with either CVB3 or CVB4 E2 by oral route at 7 days post-challenge. In contrast, no histological alteration of heart or pancreas tissues was observed after challenge with both wild-strains. Interestingly, the detection of viral RNA in heart, pancreas and intestine of mice immunized by i.p route was negative at 7 days post-challenge with CVB3 and CVB4 E2, and mice were protected from myocarditis and pancreatitis.

Download Full-text

Coxsackievirus B3 Infection Activates the Unfolded Protein Response and Induces Apoptosis through Downregulation of p58IPK and Activation of CHOP and SREBP1

Journal of Virology ◽

10.1128/jvi.01416-09 ◽

2010 ◽

Vol 84 (17) ◽

pp. 8446-8459 ◽

Cited By ~ 56

Author(s):

Huifang M. Zhang ◽

Xin Ye ◽

Yue Su ◽

Ji Yuan ◽

Zhen Liu ◽

...

Keyword(s):

Transcription Factor ◽

Er Stress ◽

Unfolded Protein Response ◽

Hela Cells ◽

Coxsackievirus B3 ◽

Unfolded Protein ◽

Caspase 12 ◽

Cvb3 Infection ◽

The Unfolded Protein Response ◽

Protein Response

ABSTRACT Cardiomyocyte apoptosis is a hallmark of coxsackievirus B3 (CVB3)-induced myocarditis. We used cardiomyocytes and HeLa cells to explore the cellular response to CVB3 infection, with a focus on pathways leading to apoptosis. CVB3 infection triggered endoplasmic reticulum (ER) stress and differentially regulated the three arms of the unfolded protein response (UPR) initiated by the proximal ER stress sensors ATF6a (activating transcription factor 6a), IRE1-XBP1 (X box binding protein 1), and PERK (PKR-like ER protein kinase). Upon CVB3 infection, glucose-regulated protein 78 expression was upregulated, and in turn ATF6a and XBP1 were activated via protein cleavage and mRNA splicing, respectively. UPR activity was further confirmed by the enhanced expression of UPR target genes ERdj4 and EDEM1. Surprisingly, another UPR-associated gene, p58IPK, which often is upregulated during infections with other types of viruses, was downregulated at both mRNA and protein levels after CVB3 infection. These findings were observed similarly for uninfected Tet-On HeLa cells induced to overexpress ATF6a or XBP1. In exploring potential connections between the three UPR pathways, we found that the ATF6a-induced downregulation of p58IPK was associated with the activation of PKR (PERK) and the phosphorylation of eIF2α, suggesting that p58IPK, a negative regulator of PERK and PKR, mediates cross-talk between the ATF6a/IRE1-XBP1 and PERK arms. Finally, we found that CVB3 infection eventually produced the induction of the proapoptoic transcription factor CHOP and the activation of SREBP1 and caspase-12. Taken together, these data suggest that CVB3 infection activates UPR pathways and induces ER stress-mediated apoptosis through the suppression of P58IPK and induction/activation of CHOP, SREBP1, and caspase-12.

Download Full-text

Using Recombinant Coxsackievirus B3 To Evaluate the Induction and Protective Efficacy of CD8+ T Cells during Picornavirus Infection

Journal of Virology ◽

10.1128/jvi.75.5.2377-2387.2001 ◽

2001 ◽

Vol 75 (5) ◽

pp. 2377-2387 ◽

Cited By ~ 67

Author(s):

Mark K. Slifka ◽

Robb Pagarigan ◽

Ignacio Mena ◽

Ralph Feuer ◽

J. Lindsay Whitton

Keyword(s):

T Cells ◽

Coxsackievirus B3 ◽

T Cell Response ◽

Wild Type Virus ◽

Foreign Protein ◽

Target Cells ◽

Viral Pathogen ◽

Ctl Epitopes ◽

Cvb3 Infection

ABSTRACT Coxsackievirus B3 (CVB3) is a common human pathogen that has been associated with serious diseases including myocarditis and pancreatitis. To better understand the effect of cytotoxic T-lymphocyte (CTL) responses in controlling CVB3 infection, we have inserted well-characterized CTL epitopes into the CVB3 genome. Constructs were made by placing the epitope of interest upstream of the open reading frame encoding the CVB3 polyprotein, separated by a poly-glycine linker and an artificial 3Cpro/3CDpro cleavage site. This strategy results in the foreign protein being translated at the amino- terminus of the viral polyprotein, from which it is cleaved prior to viral assembly. In this study, we cloned major histocompatibility complex class I-restricted CTL epitopes from lymphocytic choriomeningitis virus (LCMV) into recombinant CVB3 (rCVB3). In vitro, rCVB3 growth kinetics showed a 1- to 2-h lag period before exponential growth was initiated, and peak titers were ∼1 log unit lower than for wild-type virus. rCVB3 replicated to high titers in vivo and caused severe pancreatitis but minimal myocarditis. Despite the high virus titers, rCVB3 infection of naive mice failed to induce a strong CD8+ T-cell response to the encoded epitope; this has implications for the proposed role of “cross-priming” during virus infection and for the utility of recombinant picornaviruses as vaccine vectors. In contrast, rCVB3 infection of LCMV-immune mice resulted in direct ex vivo cytotoxic activity against target cells coated with the epitope peptide, demonstrating that the rCVB3-encoded LCMV-specific epitope was expressed and presented in vivo. The preexisting CD8+memory T cells could limit rCVB replication; compared to naive mice, infection of LCMV-immune mice with rCVB3 resulted in ∼50-fold-lower virus titers in the heart and ∼6-fold-lower virus titers in the pancreas. Although the inserted CTL epitope was retained by rCVB3 through several passages in tissue culture, it was lost in an organ-specific manner in vivo; a substantial proportion of viruses from the pancreas retained the insert, compared to only 0 to 1.8% of myocardial viruses. Together, these results show that expression of heterologous viral proteins by recombinant CVB3 provides a useful model for determining the mechanisms underlying the immune response to this viral pathogen.

Download Full-text

Inhibition of Coxsackievirus B3 in Cell Cultures and in Mice by Peptide-Conjugated Morpholino Oligomers Targeting the Internal Ribosome Entry Site

Journal of Virology ◽

10.1128/jvi.00900-06 ◽

2006 ◽

Vol 80 (23) ◽

pp. 11510-11519 ◽

Cited By ~ 45

Author(s):

Ji Yuan ◽

David A. Stein ◽

Travis Lim ◽

Dexin Qiu ◽

Shaun Coughlin ◽

...

Keyword(s):

Cell Cultures ◽

Internal Ribosomal Entry Site ◽

Viral Myocarditis ◽

Coxsackievirus B3 ◽

Infection Model ◽

Viral Titer ◽

Entry Site ◽

Infection Period ◽

Cvb3 Infection

ABSTRACT Coxsackievirus B3 (CVB3) is a primary cause of viral myocarditis, yet no effective therapeutic against CVB3 is available. Nucleic acid-based interventional strategies against various viruses, including CVB3, have shown promise experimentally, but limited stability and inefficient delivery in vivo remain as obstacles to their potential as therapeutics. We employed phosphorodiamidate morpholino oligomers (PMO) conjugated to a cell-penetrating arginine-rich peptide, P007 (to form PPMO), to address these issues. Eight CVB3-specific PPMO were evaluated with HeLa cells and HL-1 cardiomyocytes in culture and in a murine infection model. One of the PPMO (PPMO-6), designed to target a sequence in the 3′ portion of the CVB3 internal ribosomal entry site, was found to be especially potent against CVB3. Treatment of cells with PPMO-6 prior to CVB3 infection produced an approximately 3-log10 decrease in viral titer and largely protected cells from a virus-induced cytopathic effect. A similar antiviral effect was observed when PPMO-6 treatment began shortly after the virus infection period. A/J mice receiving intravenous administration of PPMO-6 once prior to and once after CVB3 infection showed an ∼2-log10-decreased viral titer in the myocardium at 7 days postinfection and a significantly decreased level of cardiac tissue damage, compared to the controls. Thus, PPMO-6 provided potent inhibition of CVB3 amplification both in cell cultures and in vivo and appears worthy of further evaluation as a candidate for clinical development.

Download Full-text

Transfection of prion protein gene suppresses coxsackievirus B3 replication in prion protein gene-deficient cells

Journal of General Virology ◽

10.1099/vir.0.19222-0 ◽

2003 ◽

Vol 84 (12) ◽

pp. 3495-3502 ◽

Cited By ~ 16

Author(s):

Yuko Nakamura ◽

Akikazu Sakudo ◽

Keiichi Saeki ◽

Tomomi Kaneko ◽

Yoshitsugu Matsumoto ◽

...

Keyword(s):

Cell Line ◽

Prion Protein ◽

Protein Gene ◽

Primary Cultures ◽

Neuronal Cell ◽

Coxsackievirus B3 ◽

Type I ◽

Prion Protein Gene ◽

Transfected Cell Line ◽

Cvb3 Infection

The susceptibility of prion protein gene (Prnp)-null cells to coxsackievirus B3 (CVB3) was investigated. Primary cultures of murine Prnp −/− brain cells were more sensitive to CVBs than corresponding cells from wild-type mice. The viral susceptibility of a Prnp-null cell line (HpL3-4) derived from the murine hippocampus was compared with that of two established cell lines (HeLa and HEp-2) that are widely employed for CVB3 studies. After infection with CVB3, HpL3-4 cells showed a very rapid and complete cytopathic effect (CPE). CPE developed earlier and viruses replicated at higher titres in HpL3-4 cells compared with HeLa and HEp-2 cells. Under a semi-solid medium, plaques developed rapidly in CVB3-infected HpL3-4 cells. To confirm the effect of Prnp on virus infection, a Prnp −/− cell line and a Prnp-transfected neuronal cell line were analysed. The replication and release of infectious particles of CVB3 in Prnp −/− cells were significantly more effective than those of the Prnp-transfected cell line. Levels of type I interferon (IFN) after CVB3 infection were higher in the Prnp-transfected cell line than in Prnp −/− cells, whereas apoptotic cells were more obvious in the Prnp −/− cells than in those of the Prnp-transfected cell line. These findings suggest that the absence of Prnp retards the induction of CVB3-induced IFNs, resulting in an enhanced CVB3 production and apoptotic cell death. Furthermore, our data indicate that the HpL3-4 cell line may provide a novel and sensitive system for isolation of CVB3 from clinical specimens.

Download Full-text

CD11b is involved in coxsackievirus B3-induced viral myocarditis in mice by inducing Th17 cells

Open Life Sciences ◽

10.1515/biol-2020-0085 ◽

2020 ◽

Vol 15 (1) ◽

pp. 1024-1032

Author(s):

Heng Wei ◽

Chong-Kai Lin ◽

Sheng-Jian Lu ◽

Yu-Xin Wen ◽

Shuai Yuan ◽

...

Keyword(s):

Immune Responses ◽

Th17 Cells ◽

Cytotoxic Effect ◽

Viral Myocarditis ◽

Coxsackievirus B3 ◽

Rt Pcr ◽

Cardiac Damage ◽

Cvb3 Infection ◽

Life Threatening

AbstractViral myocarditis (VMC) caused by coxsackievirus B3 (CVB3) infection is a life-threatening disease. The cardiac damage during VMC is not mainly due to the direct cytotoxic effect of the virus on cardiomyocytes but mostly involves the induction of immune responses. Integrin CD11b plays an important role in immune response, for instance, in the induction of Th17 cells. However, the role of CD11b in the pathogenesis of VMC remains largely unknown. In the present study, a mouse model of VMC was established by CVB3 infection and CD11b was knocked down in the VMC mice by transfection with siRNA-CD11b. The expression of CD11b and IL-17 in heart tissues, frequency of Th17 cells in spleen tissues and serum IL-17 levels were measured using quantitative RT-PCR, Western blot, immunohistochemistry, flow cytometry and ELISA. Results showed that CVB3 infection caused the pathological changes in heart tissues with the increases in the following indexes: expression of CD11b and IL-17 in heart tissues, frequency of Th17 cells in spleen tissues and serum IL-17 levels. The expression of CD11b was positively correlated with IL-17 expression in heart tissues. Depletion of CD11b attenuated the damage caused by CVB3 and decreased the frequency of Th17 cells in spleen tissues as well as in IL-17, IL-23 and STAT3 expression in heart tissues. In summary, our findings reveal that disruption of CD11b function reduced CVB3-induced myocarditis, suggesting that CD11b may be a novel therapeutic target for VMC.

Download Full-text