Search

Coxsackievirus B (CVB) is a significant pathogen of neonatal diseases with severe systemic involvement and high mortality. Hence, it is essential to develop a CVB-induced acute systemic disease model on newborn mouse and study the injury at the onset phase. In this work, a clinical strain of CVB3, Nancy, and its variant strain, Macocy, were adopted in 24 hour old neonates by oral infection. The pathological changes in the heart, liver and lung tissues were analyzed by pathology assays. In situ end labeling assay for programmed cell death was carried out for liver tissues. The data on fatality and infection rates and pathology scores were analyzed statistically. The genomic sequences of the two strains were aligned. The model represented the manifest clinical syndromes of hepatitis, pneumonia and myocardial injury at the onset phase, in which massive numbers of hepatocytes had undergone programmed cell death. Statistical and pathological analysis indicated that the myocardial injury was mild, whereas the liver and lung were more severe. The fatality rate, infection and pathology of the two CVB strains were the same. Therefore, two nucleotide mutations in the 5' UTR and four amino acid mutations in polyprotein, which did not alter virulence, were shown. By peroral CVB infection of neonatal mice, we developed an acute systemic disease model for studying visceral pathology and systemic disease. At the onset of acute neonatal systemic disease, the hepatitis and pneumonia may be the dominant reason of death, as the injury of liver and lung is more severe than that of heart.

To successfully complete their replication cycles, picornaviruses modify several host proteins to alter the cellular environment to favor virus production. One such target of viral proteinase cleavage is AU-rich binding factor 1 (AUF1), a cellular protein that binds to AU-rich elements, or AREs, in the 3' noncoding regions (NCRs) of mRNAs to affect the stability of the RNA. Previous studies found that, during poliovirus or human rhinovirus infection, AUF1 is cleaved by the viral proteinase 3CD and that AUF1 can interact with the long 5' NCR of these viruses in vitro. Here, we expand on these initial findings to demonstrate that all four isoforms of AUF1 bind directly to stem-loop IV of the poliovirus 5' NCR, an interaction that is inhibited through proteolytic cleavage of AUF1 by the viral proteinase 3CD. Endogenous AUF1 was observed to relocalize to the cytoplasm of infected cells in a viral protein 2A-driven manner and to partially colocalize with the viral protein 3CD. We identify a negative role for AUF1 in poliovirus infection, as AUF1 inhibited viral translation and, ultimately, overall viral titers. Our findings also demonstrate that AUF1 functions as an antiviral factor during infection by coxsackievirus or human rhinovirus, suggesting a common mechanism that targets these related picornaviruses.

Viral myocarditis (VMC) is a common cardiovascular disease, and microRNAs (miRNAs) have been postulated to be involved in its pathology. Using microarrays, we observed that miRNA-21 and -146b were upregulated in a murine model of VMC. We also found that miRNA-451 was downregulated. In vivo silencing of miRNA-21 and -146b resulted in less-severe VMC. Overexpression of miRNA-451 did not ameliorate the severity of VMC. Further work revealed that inhibition of miRNA-21 and -146b decreased the expression levels of Th17 and RORγt . Overexpression of miRNA-451 had no effect on IL-17 and RORγt expression. Inhibition of miRNA-21 and -146b might ameliorate myocardium inflammation by mediating downregulation of RORγt expression, indicating that these miRNAs are involved in the pathogenesis of murine VMC.

Recent findings indicate that TLR3 polymorphisms increase susceptibility to enteroviral myocarditis and inflammatory dilated cardiomyopathy (iDCM) in patients. TLR3 signaling has been found to inhibit coxsackievirus B3 (CVB3) replication and acute myocarditis in mouse models, but its role in the progression from myocarditis to iDCM has not been previously investigated. In this study we found that TLR3 deficiency increased acute (P = 5.9 × 10(-9)) and chronic (P = 6.0 × 10(-7)) myocarditis compared with WT B6.129, a mouse strain that is resistant to chronic myocarditis and iDCM. Using left ventricular in vivo hemodynamic assessment, we found that TLR3-deficient mice developed progressively worse chronic cardiomyopathy. TLR3 deficiency significantly increased viral replication in the heart during acute myocarditis from day 3 through day 12 after infection, but infectious virus was not detected in the heart during chronic disease. TLR3 deficiency increased cytokines associated with a T helper (Th)2 response, including IL-4 (P = 0.03), IL-10 (P = 0.008), IL-13 (P = 0.002), and TGF-β(1) (P = 0.005), and induced a shift to an immunoregulatory phenotype in the heart. However, IL-4-deficient mice had improved heart function during acute CVB3 myocarditis by echocardiography and in vivo hemodynamic assessment compared with wild-type mice, indicating that IL-4 impairs cardiac function during myocarditis. IL-4 deficiency increased regulatory T-cell and macrophage populations, including FoxP3(+) T cells (P = 0.005) and Tim-3(+) macrophages (P = 0.004). Thus, TLR3 prevents the progression from myocarditis to iDCM following CVB3 infection by reducing acute viral replication and IL-4 levels in the heart.

Coxsackievirus B3 (CVB3) is the primary causal agent of viral myocarditis. During infection, it hijacks host genes to favour its own replication. However, the underlying mechanism is still unclear. Although the viral receptor is an important factor for viral infectivity, other factors such as microRNAs (miRNA) may also play an essential role in its replication after host cell entry. miRNAs are post-transcriptional gene regulators involved in various fundamental biological processes as well as in diseases. To identify miRNAs involved in CVB3 pathogenesis, we performed microarray analysis of miRNAs using CVB3-infected murine hearts and identified miR-203 as one of the most upregulated candidates. We found that miR-203 upregulation is through the activation of protein kinase C/transcription factor AP-1 pathway. We further identified zinc finger protein-148 (ZFP-148), a transcription factor, as a novel target of miR-203. Ectopic expression of miR-203 downregulated ZFP-148 translation, increased cell viability and subsequently enhanced CVB3 replication. Silencing of ZFP-148 by siRNA showed similar effects on CVB3 replication. Finally, analyses of the signalling cascade downstream of ZFP-148 revealed that miR-203-induced suppression of ZFP-148 differentially regulated the expression of prosurvival and proapoptotic genes of the Bcl-2 family proteins as well as the cell cycle regulators. This altered gene expression promoted cell survival and growth, which provided a favourable environment for CVB3 replication, contributing to the further damage of the infected cells. Taken together, this study identified a novel target of miR-203 and revealed, for the first time, the molecular link between miR-203/ZFP-148 and the pathogenesis of CVB3.

Enterovirus 71 (EV71) and coxsackievirus (CVA) are the most common causative factors for hand, foot, and mouth disease (HFMD) and neurological disorders in children. Lack of a reliable animal model is an issue in investigating EV71-induced disease manifestation in humans, and the current clinical therapies are symptomatic. We generated a novel EV71-infectious model with hSCARB2-transgenic mice expressing the discovered receptor human SCARB2 (hSCARB2). The challenge of hSCARB2-transgenic mice with clinical isolates of EV71 and CVA16 resulted in HFMD-like and neurological syndromes caused by E59 (B4) and N2838 (B5) strains, and lethal paralysis caused by 5746 (C2), N3340 (C4), and CVA16. EV71 viral loads were evident in the tissues and CNS accompanied the upregulated pro-inflammatory mediators (CXCL10, CCL3, TNF-α, and IL-6), correlating to recruitment of the infiltrated T lymphocytes that result in severe diseases. Transgenic mice pre-immunized with live E59 or the FI-E59 vaccine was able to resist the subsequent lethal challenge with EV71. These results indicate that hSCARB2-transgenic mice are a useful model for assessing anti-EV71 medications and for studying the pathogenesis induced by EV71.

Viral infections of host cells cause multiple changes of cellular metabolism including immediate defense mechanisms as well as processes to support viral replication. Coxsackievirus B3 (CVB3) is a member of the Picornavirus family and is responsible for a wide variety of mild or severe infections including acute and chronic inflammations. Thereby, intracellular signaling can be changed very comprehensively. In order to compare the influence of CVB3 replication on gene expression pattern of two different cell lines, DNA microarray systems were used to study a set of 780 genes related to inflammation. Expression analysis of HeLa cells and HepG2 cells infected with CVB3 identified 34 genes whose mRNA levels were altered significantly upon infection. The expression of additional 16 genes in HepG2 cells and 31 genes in HeLa cells were found to be influenced during CVB3 replication as well. All genes expressed differentially were sorted with regard to their functions and interpreted in view of known contributors to the infection process. The activation of the tumor necrosis factor pathways by CVB3 represents one peculiar observation, including apoptosis, stress, and inflammation responses., (Copyright © 2012 Elsevier B.V. All rights reserved.)

As a result of 4 year monitoring the landscape of enteroviruses circulating in European territory of Russia was established to be presented by at least 50 serologic types. Phylogenetic analysis of ECHO30, ECHO9, Coxsackie A9, ECHO6 virus strains that had caused a seasonal increase of aseptic meningitis morbidity in 2008 - 2011 was carried out.

Background: Recent studies have revealed that Mitochondrial Antiviral Signaling (MAVS) protein plays an essential role in the inhibition of viral infection through type I interferon (IFN) pathway. It has been shown that 3C (pro) cysteine protease of coxsackievirus B3 (CVB3) cleaves MAVS to inhibit type I IFNs induction. Other workers also found that MAVS knock-out mice suffered CVB3 susceptibility and severe histopathological change. Accordingly,our experiments were designed to explore the protection of over-expressing MAVS against CVB3 infection and the possible mechanism., Results: In this study, HeLa cells (transfected with MAVS constructs pre- or post- exposure to CVB3) were used to analyze the function of exogenous MAVS on CVB3 infection. The results revealed that though CVB3 infection induced production of type I IFNs, viral replication and cell death were not effectively inhibited. Similarly, exogenous MAVS increased type I IFNs moderately. Morever, we observed robust production of type I IFNs in CVB3 post-infected HeLa cells thereby successfully inhibiting CVB3 infection, as well formation of cytopathic effect (CPE) and cell death. Finally, introduction of exogenous MAVS into CVB3 pre-infected cells also restricted viral infection efficiently by greatly up-regulating IFNs., Conclusions: In summary, exogenous MAVS effectively prevents and controls CVB3 infection by modulating and promoting the production of type I IFNs. The IFNs level in MAVS over-expressing cells is still tightly regulated by CVB3 infection. Thus, the factors that up-regulate MAVS might be an alternative prescription in CVB3-related syndromes by enhancing IFNs production.

Epithelial integrity is essential for homeostasis and poses a formidable barrier to pathogen entry. Major factors for viral entry into epithelial cells are the localization and abundance of the primary receptor. The coxsackievirus and adenovirus receptor (CAR) is a primary receptor for these two pathogenic groups of viruses. In polarized epithelia, a low-abundance, alternatively spliced eight-exon isoform of CAR, CAR(Ex8), is localized apically where it can support viral infection from the air-exposed surface. Using biochemical, cell biology, genetic, and spectroscopic approaches, we show that the levels of apical CAR(Ex8) are negatively regulated by the PDZ domain-containing protein MAGI-1 (membrane-associated guanylate kinase with inverted orientation protein-1) and that two MAGI-1 PDZ domains, PDZ1 and PDZ3, regulate CAR(Ex8) levels in opposing ways. Similar to full-length MAGI-1, expression of the isolated PDZ3 domain significantly reduces cell surface CAR(Ex8) abundance and adenovirus infection. In contrast, the PDZ1 domain is able to rescue CAR(Ex8) and adenovirus infection from MAGI-1-mediated suppression. These data suggest a novel cell-based strategy to either suppress viral infection or augment adenovirus-based gene therapy.

Rationale: Viral myocarditis results from an adverse immune response to cardiotropic viruses, which causes irreversible myocyte destruction and heart failure in previously healthy people. The involvement of microRNAs and their usefulness as therapeutic targets in this process are unknown., Objective: To identify microRNAs involved in viral myocarditis pathogenesis and susceptibility., Methods and Results: Cardiac microRNAs were profiled in both human myocarditis and in Coxsackievirus B3-injected mice, comparing myocarditis-susceptible with nonsusceptible mouse strains longitudinally. MicroRNA responses diverged depending on the susceptibility to myocarditis after viral infection in mice. MicroRNA-155, -146b, and -21 were consistently and strongly upregulated during acute myocarditis in both humans and susceptible mice. We found that microRNA-155 expression during myocarditis was localized primarily in infiltrating macrophages and T lymphocytes. Inhibition of microRNA-155 by a systemically delivered LNA-anti-miR attenuated cardiac infiltration by monocyte-macrophages, decreased T lymphocyte activation, and reduced myocardial damage during acute myocarditis in mice. These changes were accompanied by the derepression of the direct microRNA-155 target PU.1 in cardiac inflammatory cells. Beyond the acute phase, microRNA-155 inhibition reduced mortality and improved cardiac function during 7 weeks of follow-up., Conclusions: Our data show that cardiac microRNA dysregulation is a characteristic of both human and mouse viral myocarditis. The inflammatory microRNA-155 is upregulated during acute myocarditis, contributes to the adverse inflammatory response to viral infection of the heart, and is a potential therapeutic target for viral myocarditis.

Coxsackievirus B (CVB) can cause aseptic meningitis, myocarditis and respiratory disease, especially in newborn infants. To compare the susceptibility to CVB infection of fetal and adult mice, we prepared primary alveolar epithelial cells (AECs) from lungs of BALB/c mice. In contrast to fetal mouse AECs, those of adults were less susceptible to CVB3 infection, as indicated by decreased cytopathic effects, and reduced levels of viral particles bound at the cell surface. In adult mouse AECs, amplification of the viral genome and virus capsid protein VP1 synthesis were concomitantly reduced. In addition, the cell-surface expression of coxsackievirus and adenovirus receptor (CAR), which plays a key role in the initiation of CVB and pulmonary infection, was downregulated in adult mouse AECs. These findings demonstrate that adult mouse AECs are less susceptible to CVB3 due to decreased CAR levels. Thus, these findings strongly indicate that the level of virus receptors on AECs is one of the crucial determinants for the age-dependence of CVB virulence in the mouse lung.

IL-21 is a multi-functional cytokine which can promote survival, proliferation and activation of T and B lymphocytes including CD8 T cells. Previous studies have shown that autoimmune CD8+ T cells are the primary pathogenic effector cell in coxsackievirus B3 (CVB3) induced myocarditis in C57Bl/6 mice. To evaluate the role of IL-21 in promoting CD8+ T cell mediated cardiac injury in myocarditis, C57Bl/6 and IL-21RKO mice were infected with CVB3. IL-21RKO mice developed significantly less myocarditis than C57Bl/6 animals although cardiac virus titers were equivalent between the mouse strains. Numbers of CD8+IFNγ+ cells were decreased in IL-21RKO mice but numbers of either CD4+IFNγ+ or CD4+IL-4+ cells were not significantly different from C57Bl/6 animals indicating a selective effect of IL-21 signaling on the CD8+ T cell response. To confirm that IL-21 signaling exclusively functions at the level of the CD8+ T cell in CVB3 induced myocarditis, purified CD8+ cells were isolated from either C57Bl/6 or IL-21RKO donors and adoptively transferred into CD8KO recipients prior to CVB3 infection. CD8KO recipients given either C57Bl/6 or IL-21RKO CD8+ cells showed equivalent reconstitution of the CD8+ cells in the spleen but the recipients given C57Bl/6 CD8+ cells showed significantly greater myocarditis than recipients of IL-21RKO CD8+ cells. These data demonstrate that IL-21 signaling directly in the CD8+ cell population is required for CVB3-induced myocarditis., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The Th17/interleukin (IL)-17 axis controls inflammation and might be important in the pathogenesis of experimental autoimmune myocarditis (EAM) and other autoimmune diseases. However, the mechanism underlying the increased Th17 cell response in coxsackievirus-induced myocarditis remains unclear. This study aimed to elucidate the regulatory mechanisms affected by blocking IL-17A responses in acute virus-induced myocarditis (AVMC) mice. The results showed that IL-17A and COX-2 proteins were significantly increased in the cardiac tissue of acute myocarditis, as were Th17 cells in the spleen. Using anti-mouse IL-17Ab to block IL-17A on day 7 of the viral myocarditis led to decreased expressions of cardiac tumor-necrosis factor alpha, IL-17A and transforming growth factor beta in AVMC mice compared to isotype control mice. COX-2 and prostaglandin E2 proteins were dramatically elevated, followed by marked reductions in CVB3 replication and myocardial injury. These results hint that the Th17/IL-17 axis is intimately associated with viral replication in acute myocarditis via induction of COX-2 and prostaglandin E2., (© 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

MicroRNAs (miRNAs) are increasingly reported to have important roles in diverse biological and pathological processes. Changes in abundance of muscle-specific microRNA, miR-1, have been implicated in cardiac disease, including arrhythmia and heart failure. However, the specific molecular targets and cellular mechanisms involved in the miR-1 function in the heart are only beginning to emerge. In this study, we investigated miR-1 expression and its potential role in the mouse model of viral myocarditis (VMC). The expression levels of miR-1 and its target gene Connexin 43 (Cx43) were measured by real-time PCR and western blotting, respectively. The miR-1 expression levels were significantly increased in cardiac myocytes from VMC mice in comparison with control samples (relative expression: 10 ± 2.5 vs. 31 ± 7.6, P < 0.05). Among the target genes of miR-1, the expression Cx43 protein was significantly reduced in such mice while there was no significant difference in the its mRNA levels. Our results revealed an inverse correlation between miR-1 levels and Cx43 protein expression in VMC samples. Using a bioinformatics-based approach, we found two identical potential binding sites were found in mouse miR-1 and Cx43 3'- untranslated region, this confirms a possible regulatory role of miR-1. In cultured, miRNA transfected myocardial cells, we show overexpression of miR-1 accompanied by a decrease in Cx43 protein's expression. There was only a slight (not statistically significant) drop in Cx43 mRNA levels. Our results indicate that miR-1 is involved in VMC via post-transcriptional repression of Cx43, and might constitute potentially valuable data for the development of a new approach in the treatment of this disease.

Coxsackievirus B type 3 (CVB3) is one of the major pathogens associated with human heart disease. miRNAs are a class of short, noncoding RNA that can post-transcriptionally modulate gene expression. By comparing the CVB3 genome and miR-342-5p sequences, we found there were potential miR-342-5p targets in the CVB3 genome. To verify the effect of miR-342-5p on CVB3 biosynthesis, HeLa cells were infected with a Renilla luciferase (RLuc)-expressing CVB3 variant (RLuc-CVB3). We observed that miR-342-5p could significantly inhibit the expression of RLuc in infected cells. In HeLa cells infected with an enhanced green fluorescence protein (EGFP)-expressing CVB3 variant (EGFP-CVB3), EGFP expression was also significantly inhibited by miR-342-5p. The inhibitory effect of miR-342-5p on EGFP expression in EGFP-CVB3-infected cells could be reversed by transfection with anti-miR-342-5p oligonucleotide (AMO-miR-342-5p). Moreover, RNA and protein biosynthesis in wild-type CVB3 was significantly inhibited by miR-342-5p. By mutating the putative targets of miR-342-5p in the 2C-coding region, a sequence, nt4989-nt5015, was identified as the miR-342-5p target. The conserved nt4989-nt5015 sequences of CVB type 1-5 suggest miR-342-5p may exert its inhibitory effect in other types of coxsackievirus besides CVB3. Western blotting indicated that miR-342-5p could indeed suppress protein expression in CVB type 1 and 5. There was a moderate abundance of miR-342-5p in the gut, heart, and brain of Balb/c mice, suggesting that miR-342-5p may interact with CVB3 in vivo. Taken together, these results indicate that miR-342-5p can inhibit CVB3 biosynthesis by targeting its 2C-coding region and therefore may be a potential therapeutic agent in the treatment of CVB3 infection., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Viral infections are able to induce autoimmune inflammation in the heart. Here, we investigated the role of virus-activated Toll-like receptor (TLR)3 and its adaptor TRIF on the development of autoimmune coxsackievirus B3 (CVB3) myocarditis in mice. Although TLR3- or TRIF-deficient mice developed similarly worse acute CVB3 myocarditis and viral replication compared to control mice, disease was significantly worse in TRIF compared to TLR3-deficient mice. Interestingly, TLR3-deficient mice developed an interleukin (IL)-4-dominant T helper (Th)2 response during acute CVB3 myocarditis with elevated markers of alternative activation, while TRIF-deficient mice elevated the Th2-associated cytokine IL-33. Treatment of TLR3-deficient mice with recombinant IL-33 improved heart function indicating that elevated IL-33 in the context of a classic Th2-driven response protects against autoimmune heart disease. We show for the first time that TLR3 versus TRIF deficiency results in different Th2 responses that uniquely influence the progression to chronic myocarditis.

IL-17-producing (Th17) and regulatory T (Treg) cells have been well established in the pathogenesis of many inflammatory diseases. To assess whether Th17 and Treg were altered in acute virus-induced myocarditis (AVMC) mice, we assessed Th17/Treg functions on different levels in AVMC. It was shown that the expression of splenic Th17 cells and Th17-related cytokines (IL-17A, IL-21) markedly increased. Interestingly, the expression of splenic Treg cells and Treg-related cytokines (TGF-β, IL-10) also significantly increased. Using neutralization of IL-17 in the AVMC, we found that Treg cells roughly decreased compared with isotype control mice. However, T cells and perforin dramatically increased, followed by a marked reduction in CVB3 replication. The results suggested that Th17 cells possibly contributed to viral replication through the action of Treg cells in mediating T cells and perforin response in AVMC., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Enterovirus 71 (EV71) and Coxsackievirus A16 (CA16) are the main etiological agents of Hand, Foot and Mouth Disease (HFMD), a common disease among children and had caused several outbreaks in the Asia-Pacific region. Although being genetically close to each other, EV71 infection can cause serious and fatal neurological complications like encephalitis, myocarditis, acute flaccid paralysis (AFP) and aseptic meningitis, but not in CA16 infections. In this study, the cellular response of host cells infected with EV71 and CA16 was characterized and compared by 2-dimensional proteome analyses. A total of 16 proteins were identified to be differentially expressed in EV71 and CA16-infected host cells. Desmin and HSP27, both indirectly regulate the contraction of muscle cells, were significantly downregulated as a result of EV71 infection, suggesting a link to acute flaccid paralysis. The ability of EV71 to evade host immune system may be due to the downregulation of MHC-I synthesis proteins like protein disulfide isomerase A3 and calreticulin. Proteins such as nucleophosmin, nuclear ribonucleoprotein C, and eukaryotic translation initiation factor 2 were all downregulated significantly, suggesting the rapid shutting down of host translation machinery by EV71. These findings provide insight into the nature of high virulent EV71 infection as compared to CA16., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Adiponectin (APN), a cytokine constitutively produced in fat tissue, has been shown to exert anti-inflammatory effects in various disease models. While the influence of APN on monocytic cells has been extensively studied in vitro, little is known about its role in T cells. In this study, we show that while <10% of human peripheral blood T cells express adiponectin receptors (AdipoRs) on their surface, most T cells store AdipoRs in intracellular compartments. AdipoRs colocalized with immune regulatory molecules CTLA-4 and TIRC7 within clathrin-coated vesicles. After stimulation, the expression of adiponectin receptor 1 (AdipoR1) and AdipoR2 was upregulated on the surface of antigen-specific T cells, as determined by tetramer or CD137 staining, and AdipoR1 and AdipoR2 coexpressed with CTLA-4. Addition of APN resulted in a significant diminution of antigen-specific T-cell expansion. Mechanistically, APN enhanced apoptosis and inhibited proliferation of antigen-specific T-cell lines. Further, APN directly inhibited cytokine production in response to antigen stimulation. In line with the in vitro data, APN-deficient (knockout, KO) mice had higher frequencies of CD137(+) T cells upon Coxsackie B virus infection. Altogether, our data suggest that APN is a novel negative T-cell regulator. In contrast to the CTLA-4 ligand B7 only expressed on APCs, APN is abundant in human plasma., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Among Coxsackie B viruses, Coxsckievirus B5 is one of the most predominant serotypes in human, it is frequently associated with cases of neurological diseases, epidemics of meningitis and is a common cause of cardiomyopathy and diabetes. In the present study 27 isolates of Coxsackievirus B5 from North Africa, obtained from cerebrospinal fluid and stool samples of healthy individuals, patients with acute flaccid paralysis or aseptic meningitis were investigated by partial sequencing in the 5' half of the VP1 region and compared to the up-to-date published Coxsackievirus B5 sequences in the same genomic region. Four distinct genomic groups and ten different clusters were individualized. Most of the isolates from Algeria and Tunisia belonged to two clusters. For both, the sequences from North Africa clustered mainly with sequences from European countries, the majority isolated recently during the 2000s. The analysis of the alignment of amino-acids sequences in the VP1 gene revealed four major substitutions in strains from different clusters, we also noticed changes in the BC-loop region; this region is associated with viral antigenicity. This study permit to better identify circulating Coxsackievirus B5 strains throughout the world and their genetic relationship. The protein analysis showed changes that could imply some antigenic significance. J. Med. Virol. 83:1247-1254, 2011. © 2011 Wiley-Liss, Inc., (Copyright © 2011 Wiley-Liss, Inc.)

Coxsackievirus B3 (CVB3) infection is the most common cause of viral myocarditis. The pathogenesis of viral myocarditis is strongly controlled by host genetic factors. Although certain indispensable components of immunity have been identified, the genes and pathways underlying natural variation between individuals remain unclear. Previously, we isolated the viral myocarditis susceptibility 1 (Vms1) locus on chromosome 3, which influences pathogenesis. We hypothesized that confirmation and further study of Vms1 controlling CVB3-mediated pathology, combined with pathway analysis and consomic mapping approaches, would elucidate both pathological and protective mechanisms accounting for natural variation in response to CVB3 infection. Vms1 was originally mapped to chromosome 3 using a segregating cross between susceptible A/J and resistant B10.A mice. To validate Vms1, C57BL/6J-Chr 3(A)/NaJ (a chromosome substitution strain that carries a diploid A/J chromosome 3) were used to replicate susceptibility compared with resistant C57BL/6J (B6). A second segregating F2 cross was generated between these, confirming both the localization and effects of Vms1. Microarray analysis of the four strains (A/J, B10.A, C57BL/6J, and C57BL/6J-Chr 3(A)/NaJ) illuminated a core program of response to CVB3 in all strains that is comprised mainly of IFN-stimulated genes. Microarray analysis also revealed strain-specific differential expression programs and genes that may be prognostic or diagnostic of susceptibility to CVB3 infection. A combination of analyses revealed very strong evidence for the existence and location of Vms1. Differentially expressed pathways were identified by microarray, and candidate gene analysis revealed Fpgt, H28, and Tnni3k as likely candidates for Vms1.

Purpose: Acute hemorrhagic conjunctivitis (AHC), a highly contagious eye disease, is caused primarily by either enterovirus 70 (EV70) or coxsackievirus A24 (CVA24) infection. Yet methods to prevent or cure AHC are not available. Recent evidence has shown that small-interfering RNAs (siRNAs), mediators of posttranscriptional gene knockdown, can act as effective antiviral agents. Thus, the authors attempted to develop a novel siRNA-based anti-AHC agent effective against both EV70 and CVA24., Methods: Concurrent screening of the entire viral genome sequences of EV70 and CVA24 using the CAPSID program identified five different siRNA candidates complementary to genome regions of both viruses. The antiviral potentials of these siRNAs were assessed by treating MRC5 and primary human conjunctival cells with the siRNAs and following this with viral challenge., Results: Among the five siRNAs, AHCe-3D-3 siRNA showed excellent cytoprotective effects and dramatic decreases in virus replication and virus protein synthesis. This siRNA, targeting the virus polymerase 3D gene, also induced similar antiviral effects in primary human conjunctival cells., Conclusions: These findings strongly suggest that the AHCe-3D-3 siRNA, homologous to two different AHC-associated enteroviruses, can provide equivalent antiviral activities against both AHC-causing enteroviruses. Such an siRNA may be developed as a clinically valuable AHC control agent.

We studied the susceptibility of human embryonic stem cells and derived contractile embryoid bodies from WAO9, HUES-5 and HUES-16 cell lines to Coxsackievirus B infection. After validating stem cell-like properties and cardiac phenotype, Coxsackievirus B receptors CAR and DAF, as well as type I interferon receptors were detected in all cell lines and differentiation stages studied. Real-time PCR analysis showed that CAR mRNA levels were 3.4-fold higher in undifferentiated cells, while DAF transcript levels were 2.78-fold more abundant in differentiated cultures (P<0.05). All cell lines were susceptible to Coxsackievirus serotypes B1-5 infection as shown by RT-PCR detection of viral RNA, immunofluorescence detection of viral protein and infectivity titration of cell culture supernatants resulting in cell death. Supernatants infectivity titers 24-48 h post-infection ranged from 10⁵-10⁶ plaque forming units (PFU)/ml, the highest titers were detected in undifferentiated cells. Cell viability detected by a colorimetric assay, showed inverse correlation with infectivity titers of cell culture supernatants. Treatment with 100 U of interferon Iβ significantly reduced viral replication and associated cell death during a 24-48 h observation period, as detected by reduced infectivity titers in the supernatants and increased cell viability by a colorimetric assay, respectively. We propose human embryonic stem cell and derived contractile embryoid bodies as a valid model to study cardiac Coxsackievirus B infection., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Human Enteroviruses (HEV) (picornaviridae) are considered as one the major viral causes of childhood acute respiratory wheezing illnesses including bronchiolitis and asthma exacerbation. To identify the mechanisms that can regulate the development of airway mucosa inflammation during HEV respiratory lower tract infection, we investigated the profile and the levels of mRNA and protein secretion for CC and CXC human chemokines by HEV-infected primary human bronchial epithelial cells (SAE cells) using RT-PCR array and Bio-Plex assay. Cultures of SAE cells were infected by reference and wild-type HEV respiratory strains, demonstrating a replicative and productive viral infection. We observed that the replicative infection of the SAE cells by reference and wild-type HEV strains induced specific dose and time-dependent increases in mRNA and protein secretion only for RANTES, MCP-1 and IL-8 and not for all other CC and CXC human chemokines tested. The protein secretion of these chemokines appeared to be significantly increased at 48 or 72h post-infection in cultures treated by low-doses of IFN-gamma comparatively to mock-infected cells (P<0.001), and was correlated to the viral replication activity. In conclusion, our findings demonstrated a selective production of RANTES, IL-8 and MCP-1 released by HEV-infected epithelial cells of the small bronchioles along with mechanisms of amplification mediated by IFN-gamma., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)

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, p58(IPK), 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 p58(IPK) was associated with the activation of PKR (PERK) and the phosphorylation of eIF2alpha, suggesting that p58(IPK), 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 P58(IPK) and induction/activation of CHOP, SREBP1, and caspase-12.

The innate antiviral response is mediated, at least in part, by Toll-like receptors (TLRs). TLR3 signaling is activated in response to viral infection, and the absence of TLR3 in mice significantly increases mortality after infection with enteroviruses that cause myocarditis and/or dilated cardiomyopathy. We screened TLR3 in patients diagnosed with enteroviral myocarditis/cardiomyopathy and identified a rare variant in one patient as well as a significantly increased occurrence of a common polymorphism compared with controls. Expression of either variant resulted in significantly reduced TLR3-mediated signaling after stimulation with synthetic double-stranded RNA. Furthermore, Coxsackievirus B3 infection of cell lines expressing mutated TLR3 abrogated activation of the type I interferon pathway, leading to increased viral replication. TLR3-mediated type I interferon signaling required cellular autophagy and was suppressed by 3-methyladenine and bafilomycin A1, by inhibitors of lysosomal proteolysis, and by reduced expression of Beclin 1, Atg5, or microtubule-associated protein 1 light chain 3beta (MAP1LC3beta). However, TLR3-mediated signaling was restored upon exogenous expression of Beclin 1 or a variant MAP1LC3beta fusion protein refractory to RNA interference. These data suggest that individuals harboring these variants may have a blunted innate immune response to enteroviral infection, leading to reduced viral clearance and an increased risk of cardiac pathology.

Background: Group B coxsackieviruses (CVBs) are the prototypical agents of acute myocarditis and chronic dilated cardiomyopathy, but an effective targeted therapy is still not available. Here, we analyze the therapeutic potential of a soluble (s) virus receptor molecule against CVB3 myocarditis using a gene therapy approach., Methods and Results: We generated an inducible adenoviral vector (AdG12) for strict drug-dependent delivery of sCAR-Fc, a fusion protein composed of the coxsackievirus-adenovirus receptor (CAR) extracellular domains and the carboxyl terminus of human IgG1-Fc. Decoy receptor expression was strictly doxycycline dependent, with no expression in the absence of an inducer. CVB3 infection of HeLa cells was efficiently blocked by supernatant from AdG12-transduced cells, but only in the presence of doxycycline. After liver-specific transfer, AdG12 (plus doxycycline) significantly improved cardiac contractility and diastolic relaxation compared with a control vector in CVB3-infected mice if sCAR-Fc was induced before infection (left ventricular pressure 59+/-3.8 versus 45.4+/-2.7 mm Hg, median 59 versus 45.8 mm Hg, P<0.01; dP/dt(max) 3645.1+/-443.6 versus 2057.9+/-490.2 mm Hg/s, median 3526.6 versus 2072 mm Hg/s, P<0.01; and dP/dt(min) -2125.5+/-330.5 versus -1310.2+/-330.3 mm Hg/s, median -2083.7 versus -1295.9 mm Hg/s, P<0.01) and improved contractility if induced concomitantly with infection (left ventricular pressure 76.4+/-19.2 versus 56.8+/-10.3 mm Hg, median 74.8 versus 54.4 mm Hg, P<0.05; dP/dt(max) 5214.2+/-1786.2 versus 3011.6+/-918.3 mm Hg/s, median 5182.1 versus 3106.6 mm Hg/s, P<0.05), respectively. Importantly, hemodynamics of animals treated with AdG12 (plus doxycycline) were similar to uninfected controls. Preinfection induction of sCAR-Fc completely blocked and concomitant induction strongly reduced cardiac CVB3 infection, myocardial injury, and inflammation., Conclusions: AdG12-mediated sCAR-Fc delivery prevents cardiac dysfunction in CVB3 myocarditis under prophylactic and therapeutic conditions.

Type 1 diabetes (T1D) is an autoimmune disease that is caused by the destruction of insulin-producing beta cells. Viral infections induce immune responses that can damage beta cells and promote T1D or on the other hand prevent the development of the disease. However, the opposing roles of viral infections in T1D are not understood mechanistically. We report here that viruses that do not inflict damage on beta cells provided protection from T1D by triggering immunoregulatory mechanisms. Infection of prediabetic NOD mice with Coxsackie virus B3 or lymphocytic choriomeningitis virus (LCMV) delayed diabetes onset and reduced disease incidence. Delayed T1D onset was due to transient upregulation of programmed cell death-1 ligand 1 (PD-L1) on lymphoid cells, which prevented the expansion of diabetogenic CD8+ T cells expressing programmed cell death-1 (PD-1). Reduced T1D incidence was caused by increased numbers of invigorated CD4+CD25+ Tregs, which produced TGF-beta and maintained long-term tolerance. Full protection from T1D resulted from synergy between PD-L1 and CD4+CD25+ Tregs. Our results provide what we believe to be novel mechanistic insight into the role of viruses in T1D and should be valuable for prospective studies in humans.

Livers and hearts from mice deficient in glycerol-3-phosphate acyltransferase 1 (GPAT1; Gpat1(-/-)) have a decreased content of glycerolipid intermediates and triacylglycerol, an altered composition of liver phospholipids, and elevated markers of oxidative stress. Compared with control C57BL/6 mice, infection of Gpat1(-/-) mice with coxsackievirus B3 (CVB3) resulted in higher mortality, an approximately 50% increase in heart pathology, a significant increase in liver viral titers, and a 100-fold increase in heart viral titers. Moreover, heart mRNA levels for proinflammatory cytokines tumor necrosis factor-alpha, interleukin (IL)-6, and IL-1B were increased in the Gpat1(-/-) mice. Loss of Gpat1 also resulted in dysregulation of specific immune cells. Splenic dendritic cells from Gpat1(-/-) mice were fully capable of stimulating T cells from control mice; however, splenic T cells from Gpat1(-/-) mice were defective in their response to CVB3 antigen. Our data indicate that a lack of GPAT1 activity affects both innate and adaptive immune mechanisms. Innate mechanisms may be affected by altered membrane composition or host redox status, whereas the adaptive response may require GPAT1 activity itself.

Dilated cardiomyopathy as a consequence of myocarditis induced by Coxsackieviruses B3 (CVB3) is a worldwide cause of morbidity and death for which current therapies have shown only modest effectiveness. In the present study we tested the efficacy of a novel CVB3 small interfering RNA (siRNA), directed against the protease 2A genome region (siRNA 3626), alone or in combination with another recently described protease 2A siRNA (siRNA 3541). siRNAs (400 nM) were delivered by lipofection to HeLa cells, which were then infected by CVB3 and analyzed. Under optimized transfection conditions, siRNA 3626 markedly improved the viability of CVB3-infected HeLa cells. This was due to a drastic reduction in the amounts of CVB3 genome, viral capsid protein VP1 and vital virions. Finally, the combination of siRNA 3626/3541 did not potentiate the anti-CVB3 effect compared with an equimolar concentration of either siRNA. In conclusion, these data show the potency of siRNA 3626 in downmodulating CVB3 infection together with the lack of any significant transfection-related toxicity. In addition to placing the selected siRNA 3626 among the most effective anti-CVB3 agents so far described, these data fully justify future attempts to further optimize the siRNA-mediated approach to CVB3-induced myocarditis., (Copyright 2009 Prous Science, S.A.U. or its licensors. All rights reserved.)

Objective: To investigate the genetic pathogenesis of dilated cardiomyopathy (DCM) by examining the heterogeneity of Coxsackievirus binding domain (exon 4) of Adenovirus receptor (CAR) in DCM patients and healthy adults, and to detect possible mutation site in exon 4., Methods: Using polymerase chain reaction (PCR), we amplified exon 4 of CAR DNA extracted from blood samples obtained from 50 DCM patients and 40 healthy adults. The PCR products were screened with single-strand conformation polymorphism (SSCP) and then sequenced alternatively based on the SSCP results., Results: The segment of CAR exon 4 was successfully amplified and there was no single strand conformational disparity in all samples examined by SSCP. Sequence analysis demonstrated that all amplified sequences of CAR exon 4 from samples of the two groups were identical and there was no genetic heterogeneity of CAR exon 4 between the two groups., Conclusion: The genetic heterogeneity of CAR exon 4 might not be responsible for the pathogenesis of DCM.

The coxsackievirus-adenovirus receptor (CAR) was originally described as a transmembrane protein involved in viral infection and was later found to be required for normal heart development. However, the role of CAR in virus-induced myocarditis has not been investigated so far. The purpose of this study was to assess myocardial CAR expression in response to cytokine-induced inflammatory reactions during the course of coxsackievirus-induced myocarditis. In Balb/c mice intraperitoneally infected with either 2x10(4) plaque-forming units (PFUs) of coxsackie B3 virus (CVB3) or 10(2) PFUs CVB3, CAR expression and tyrosine phosphorylation of signal transducer and activator of transcription 1 (STAT1), a known cytokine-inducible transcription factor involved in viral defense, were determined. Our results demonstrated that within the first 7 days after virus inoculation, when the viral replication and STAT1 activation in the heart tissue was most prominent, the expression of CAR did not surpass that of uninfected controls. However, the up-regulation of CAR was observed 9 weeks later, when enteroviral RNA was no longer detectable and activation of STAT1 had already ceased. In contrast to the STAT1 target genes Mig and Irf1, interferon gamma stimulation failed to up-regulate Car expression in isolated cardiomyocytes. In human endomyocardial biopsies, Car expression was found to be elevated in approximately one third of patients with dilated cardiomyopathy (9 of 30 patients) as compared with controls. Thus, activation of STAT1 clearly precedes the enhanced CAR expression observed during tissue reorganization, suggesting an essential role of STAT1 transcription factors in orchestrating the sequential actions involved in adaptive immune response.

While infections with the group B coxsackieviruses are generally asymptomatic, these viruses occasionally cause acute and chronic inflammatory diseases of the pancreas and heart. Chronic inflammatory diseases are of major clinical concern, since they predispose affected individuals to cancer. For example, chronic pancreatitis, which can develop from acute pancreatitis, is a major risk factor for pancreatic cancer, a disease with an exceedingly poor prognosis. It is crucial that we understand the mechanisms underlying the development of acute pancreatitis and the progression to chronic disease, if we are to identify new therapeutic targets to prevent the deterioration of the exocrine pancreas. We have developed a mouse model that allows us to explore the mechanisms by which CVB4 causes acute and chronic pancreatitis. The present review develops the idea that disease progression is a continuum, from acute inflammatory disease to chronic inflammatory disease to cancer, and that accompanying changes in gene expression are both quantitative and qualitative.

Common viral infections have been shown to change the tissue distribution of xenobiotics, including polybrominated diphenyl ethers (PBDEs). In previous studies, it has been shown that CYP2B gene expression is induced after PBDE exposure whereas coxsackievirus B3 (CBV3) infection suppresses the expression of CYP-gene expression in the liver. In the present study, CVB3 adapted to Balb/c mice was used to study the combined effects of infection and exposure to pure BDE-99 or the commercial mixture Bromkal on CYP1A1 and CYP2B expression in the lungs and pancreas on day 3 of the infection. The quantitative gene expression of virus, CYP1A1 and CYP2B was measured by real-time polymerase chain reaction (RT-PCR). PBDE exposure in the non-infected mice tended to increase CYP2B expression in the lungs but not in the pancreas. Infection in both non-exposed and PBDE-exposed mice increased CYP2B expression in the lungs but was non-detectable in the pancreas. In the non-infected mice PBDE exposure left the CYP1A1 expression unaltered in both the lungs and pancreas. Infection in both non-exposed and PBDE-exposed mice tended to decrease the gene expression of CYP1A1 in the lungs but to induce it in the pancreas. A correlation between the amount of virus and the gene expression of CYP2B was found in the lungs. However, no effects of PBDE on virus replication were observed in any organ. In conclusion, viral infection affects CYP-gene expression differently in the pancreas and lungs whereas PBDE-induced effects were not obvious. The organ-specific change in gene expression could explain a changed tissue distribution of xenobiotics during infection.

In the present study coxsackievirus B3 (CVB3) adapted to Balb/c mice was used to examine whether infection affects xenobiotic-metabolising CYP1A1 and CYP2B gene expression (measured by RT-PCR) and the corresponding enzyme activities of ethoxyresorufin-O-deethylase (EROD) and pentoxyresorufin-O-depentylase (PROD), as observed on day 3 of infection. To study the simultaneous effects of xenobiotic exposure, mice were administered the polybrominated diphenyl ether (PBDE) compounds BDE-99 (single congener) and Bromkal 70-5 DE (commercial mixture). Serum thyroxine levels were also measured. High numbers of CVB3 were found in the livers of infected mice but no significant effects of PBDE on virus replication were observed. In infected mice gene expression and CYP activities were decreased in comparison with non-infected mice, especially for CYP2B. PBDE exposure in the non-infected mice was characterised by an increase in both CYP2B and PROD levels/activities, whereas CYP1A levels increased and EROD activity decreased. In general, PBDE exposure in the infected mice did not increase EROD and PROD activities to the same extent as in the non-infected exposed mice. Infected mice exposed to BDE-99 showed significantly higher CYP2B and PROD levels than both the infected non-exposed and Bromkal-exposed groups. T(4) levels were greatly decreased by infection and a tendency of reduced T(4) levels after PBDE exposure could be observed in non-infected mice. In conclusion, infection reduced the detoxifying capacity of the liver and the serum T(4) levels. PBDE exposure can modify these effects. Notably, in the infected mice differences between BDE-99 and Bromkal were observed on CYP2B gene expression and PROD activity.

VAP-1, an ecto-enzyme expressed on the surface of endothelial cells, is involved in leukocyte trafficking between the blood and tissues under physiological and pathological conditions. In this study, we used VAP-1-deficient mice to elucidate whether absence of VAP-1 alters the immune system under normal conditions and upon immunization and microbial challenge. We found that VAP-1-deficient mice display age-dependent paucity of lymphocytes, in the Peyer's patches of the gut. IgA concentration in serum was also found to be lower in VAP-1(-/-) animals than in wild-type mice. Although there were slightly less CD11a on B and T cells isolated from VAP-1-deficient mice than on those from wild-type mice, there were no differences in the expression of gut-homing-associated adhesion molecules or chemokine receptors. Because anti-VAP-1 therapies are being developed for clinical use to treat inflammation, we determined the effect of VAP-1 deletion on useful immune responses. Oral immunization with OVA showed defective T and B cell responses in VAP-1-deficient mice. Antimicrobial immune responses against Staphylococcus aureus and coxsackie B4 virus were also affected by the absence of VAP-1. Importantly, when the function of VAP-1 was acutely neutralized using small molecule enzyme inhibitors and anti-VAP-1 Abs rather than by gene deletion, no significant impairment in antimicrobial control was detected. In conclusion, VAP-1-deficient mice have mild deviations in the mucosal immune system and therapeutic targeting of VAP-1 does not appear to cause a generalized increase in the risk of infection.

Cardiac fibrosis and inflammation are major pathologic conditions that result from viral myocarditis. Connective tissue growth factor (CTGF) stimulates fibroblast proliferation and induces production of extracellular matrix molecules. We studied the correlation between CTGF and cardiac fibrosis in an acute Coxsackievirus B3 (CVB3) myocarditis animal model. Eight-week-old BALB/c mice were infected intraperitoneally with 10(4) plaque forming units (PFU) of CVB3. Myocardial inflammation peaked on day 7 and decreased markedly by day 14 post-infection (pi); cardiac fibrosis was noted from day 7 and peaked on day 14. By contrast, CTGF was weakly expressed by the interstitial cells in uninfected control hearts and also in the hearts of day 3 pi. CTGF expression measured by real-time PCR was elevated on day 3 and peaked on day 7 pi. TGF-beta expression peaked at day 7 pi. The cell type of CTGF expression changed from interstitial cells to myocytes after virus infection. On day 7, CTGF was strongly expressed by myocytes and inflammatory cells surrounding calcified necrotic areas. In addition, cardiac myocytes expressed CTGF on day 14. Our results, based on an acute CVB3 model of myocarditis, provide evidence that CTGF may mediate the development of fibrosis after viral myocarditis, and that the cells expressed CTGF changes during the course of viral myocarditis.

The pathogenesis of viral myocarditis is a multifactorial process involving host genetics, viral genetics and the environment in which they interact. We have used a model of infection with coxsackievirus B3 (CVB3) to characterize the contribution of host genetics to viral myocarditis in mice of different genetic backgrounds but with a common H2 haplotype: A/J and B10.A-H2(a). Here we have used Evans blue dye as a quantitative biomarker for susceptibility to CVB3-induced myocarditis in addition to histopathological semiquantitative measures. We have found evidence of linkage between susceptibility to viral myocarditis and three loci. A locus on chromosome 1 centered on D1Mit200 was linked to sarcolemmal disruption in males (P=0.00005), a second locus on chromosome 4 centered on D4Mit81 was also linked to sarcolemmal disruption in males (P=0.0022). A third locus on distal chromosome 3 centered on D3Mit19 was linked to myocardial infiltration, with a logarithm of odds (LOD) score of 4.7 (P=0.0045), as well as sarcolemmal disruption in females (P=0.0015). These results provide strong evidence for the presence of loci contributing to the susceptibility of mice to viral myocarditis.

The coxsackievirus group B (CVB) of the genus Enterovirus and the species human enterovirus B is a nonenveloped virus containing a single-stranded positive-sense RNA genome. Coxsackievirus has icosahedral symmetry and four capsid proteins, VP1, VP2, VP3, and VP4. Specific antibodies against each viral protein are prerequisites for various studies. In this study, we developed seven peptide-derived antibodies directed against coxsackievirus VP1 (NO1-NO5), VP2 (B3), and VP3 (GL3). We developed a type-specific antibody (NO1) and broadly cross-reactive antibodies (NO3 and NO5) to VP1. Anti-VP2 and anti-VP3 antibodies (B3 and GL3, respectively) are also cross-reactive to human enterovirus B such as CVB and echoviruses. Their sensitivities and reactivities are likely to be better than those of the commercial VP1 monoclonal antibody (MAb). The dot-blot analysis also showed that NO5 against VP1 is able to detect less than 1 microg [2x10(6) plaque-forming unit (pfu) of CVB3] of viruses, suggesting that it could be used to develop a diagnostic kit that can directly detect human enterovirus B. The antibodies produced here may allow us to undertake several studies, such as those involving viral trafficking, expression kinetics, and the roles of viral proteins in infection, and the development of diagnostic kits.

Cardiovascular disease is the number one killer of men and women in North America. Male BALB/c mice infected with coxsackievirus B3 (CVB3) develop more severe inflammatory heart disease compared to female mice, similar to the increased heart disease that occurs in men. We show here that increased inflammation in male mice is not due to increased viral replication in the heart, but associated with increased proinflammatory cytokines IL-1beta, IL-18 and IFN-gamma. We have previously reported that IL-12Rbeta1 signaling increases CVB3-induced myocarditis and IL-1beta/IL-18 levels in males, while IL-12(p35)/STAT4-induced IFN-gamma does not alter the severity of acute disease. However, whether differences exist between males and females in these two cytokine signaling pathways is unknown. In this study, we examined sex differences in 1) IL-12Rbeta1 signaling or 2) STAT4/IFN-gamma pathways following CVB3 infection in BALB/c mice. We found that male and female mice deficient in IL-12Rbeta1 had decreased inflammation and viral replication in the heart, indicating that IL-12Rbeta1 signaling increases myocarditis in both sexes. In contrast, STAT4 deficiency did not alter the sex difference in myocarditis, with males maintaining increased inflammation over females. IFN-gamma deficient males, however, had decreased myocarditis and viral replication compared to females. Thus, IFN-gamma increases inflammation in males independent from STAT4. These results demonstrate that sex differences greatly influence viral replication and the severity of acute CVB3-induced myocarditis.

Eukaryotic cells utilize a cycle of ribosome trafficking on the endoplasmic reticulum (ER) to partition mRNAs between the cytosol and ER compartments. In this process, ribosomes engaged in the synthesis of signal sequence-bearing proteins are trafficked to the endoplasmic reticulum via the signal-recognition particle pathway and are released from the ER upon translation termination. Though the processes governing ribosome trafficking to the ER are well understood, little is known regarding the complementary ribosome release process. In this study, Coxsackie B virus (CBV) infection was used to inactivate the initiation stage of protein synthesis, thereby limiting translation to the elongation and termination stages. Ribosome partitioning between the cytosol and ER compartments was examined to determine the role of termination in ribosome release from the ER. CBV infection resulted in efficient cleavage of eIF4G and PABP, coincident with polyribosome breakdown in the cytosol and ER compartments. Termination resulted in the continued association of ribosomes with the ER compartment, rather than the expected process of ribosome release. Analyses of ribosome/mRNA loading patterns in the cytosol and ER revealed that CBV infection was accompanied by a suppression of mRNA translation in the cytosol and the sustained, although reduced, translation in the ER compartment. Direct biosynthetic labeling experiments demonstrated that protein synthesis on the ER was enhanced relative to the cytosol following CBV infection. In total, these data demonstrate that ribosome and mRNA release from the ER is regulated independent of translation termination and identify the ER as a privileged site for protein synthesis.

Objective: To analyze the T cell receptor (TCR) Vbeta gene usage of the cytolytic T-lymphocytes which result in immune-mediated myocyte injury in Coxsackievirus B3-induced myocarditis., Methods: An experimental murine model with a myocarditic variant of Coxsackievirus B, type 3 has been developed with newborn Balb/c male mice. Mice were killed on day 7 after virus inoculated, which was the time when T cells show significant differentiation. With immunoadsorption, ACTLs, VCTLs, MCTLs are respectively isolated from mesenteric lymph nodes of Balb/c mice suffered from viral myocarditis. T-lymphocytes prepared from mesenteric lymph nodes of uninfected Balb/c mice served as control group. RT-PCR was performed with standard method. The Vbeta gene usage was analyzed by running the electophoresis of PCR products on agarose gel., Results: T cells of control group expressed all Vbeta genes of 20 families. But the TCR VP gene usage of CTLs in experimental groups was markedly restricted. ACTL expressed predominantly Vbeta6, Vbeta8.1, Vbeta8.2, Vbeta8.3, MCTL expressed predominantly Vbeta5.1, Vbeta8.1, Vbeta8.2, Vbeta8.3 and VCTL expressed predominantly Vbeta7, Vbeta8.1, Vbeta8.2, Vbeta8.3., Conclusions: The TCR Vbeta gene usage of CTLs which result in immune-mediated myocyte injury in Coxsackievirus B3-induced myocarditis is markedly restricted in cells stimulated by specific antigen.

Viruses rely on attachment to specific cell surface receptors to infect host cells. Selective expression of viral receptors has the potential to attenuate infection of susceptible tissues by redirecting virus to cells that cannot support viral replication. We propose that erythrocytes are an ideal instrument for this strategy, because they are present in vast numbers, permeate every organ, and cannot serve as hosts for viral propagation. To test this hypothesis, we generated a transgenic mouse, termed globin transcription factor 1 (GATA1)-coxsackie and adenovirus receptor (CAR), that expressed the CAR on erythrocytes. Coxsackievirus group B (CVB) adhered to the surface of CAR-expressing erythrocytes and was rendered noninfectious. Upon infection with CVB, GATA1-CAR mice had diminished viremia and reduced viral replication in heart, brain, and liver. Furthermore, when faced with a CVB challenge that was lethal to WT littermates, the survival of GATA1-CAR mice was prolonged, and their ultimate mortality was reduced. The GATA1-CAR mouse model presented here demonstrates that erythrocyte expression of CAR limits CVB pathogenesis. Erythrocytes also may be coated with a variety of receptors by nontransgenic methods, making this a very flexible model for the treatment of infectious diseases in humans.

Aims/hypothesis: It is thought that enterovirus infections initiate or facilitate the pathogenetic processes leading to type 1 diabetes. Exposure of cultured human islets to cytolytic enterovirus strains kills beta cells after a protracted period, suggesting a role for secondary virus-induced factors such as cytokines., Methods: To clarify the molecular mechanisms involved in virus-induced beta cell destruction, we analysed the global pattern of gene expression in human islets. After 48 h, RNA was extracted from three independent human islet preparations infected with coxsackievirus B5 or exposed to interleukin 1beta (50 U/ml) plus interferon gamma (1,000 U/ml), and gene expression profiles were analysed using Affymetrix HG-U133A gene chips, which enable simultaneous analysis of 22,000 probe sets., Results: As many as 13,077 genes were detected in control human islets, and 945 and 1293 single genes were found to be modified by exposure to viral infection and the indicated cytokines, respectively. Four hundred and eighty-four genes were similarly modified by the cytokines and viral infection., Conclusions/interpretation: The large number of modified genes observed emphasises the complex responses of human islet cells to agents potentially involved in insulitis. Notably, both cytokines and viral infection significantly (p<0.02) increased the expression of several chemokines, the cytokine IL-15 and the intercellular adhesion molecule ICAM-1, which might contribute to the homing and activation of mononuclear cells in the islets during infection and/or an early autoimmune response. The present results provide novel insights into the molecular mechanisms involved in viral- and cytokine-induced human beta cell dysfunction and death.

Acrylamide (AA) has been shown to cause neurotoxic effects in humans and neurotoxic, genotoxic, reproductive, and carcinogenic effects in laboratory animals. Infection with the human coxsackievirus B3 (CB3) in the murine model results in changed uptake and tissue distribution of several environmental pollutants, which may result in aggravated disease. In the present study female Balb/c mice were infected with CB3, and on day 1 of the infection, dosed orally with approximately 50 microg/kg bw of [(14)C]acrylamide ((14)AA) and subsequently sacrificed on day 3 of the infection for studies of the distribution of radioactivity and genotoxic effects in terms of the frequency of micronucleated erythrocytes. Infected mice developed an expected clinical signs of disease. The infection decreased the radioactivity by 45% (p<0.05) in the pancreas but increased it by 70% (p<0.05) in the blood and more than two-fold in the thymus (p<0.01). However, the infection caused no changes in the radioactivity in the brain, heart, liver, lungs, spleen, or kidneys. As a response to the infection the proportion of young red blood cells (PCE) decreased to about a third (p<0.001) of that in the control mice, but no genotoxic effects were observed. Thus, the tissue radioactivity after (14)AA administration indicated an infection-induced change in the metabolism of AA, the exact pathogenic interpretation of which warrants further studies.

Modern molecular approaches in Human Enterovirus detection rely on the designing of generic and often degenerate primers in order to amplify specific sequences within the enterovirus genome. In the present study a comparative application of primer sets targeting 5'UTR, the VP1 region, the 3D region as well as a long genomic fragment including the 3'end of VP1, the full length of 2A and 2B, and the 5' moiety of the 2C-coding region was attempted, in order to evaluate their specificity and suitability. The best amplification results from the investigation of 21 CAV reference strains, all six CBV reference strains and 44 clinical strains varying in origin and time of isolation, arose using primer sets 292-222 and UC53-UG52. Based on the above results we conclude that some of the published protocols need to be improved so as to fulfill the demands of an accurate detection and typing of Coxsackie A and B viruses. Contrarily, two of the protocols applied were proved to be more accurate in terms of specificity and general applicability, suggesting that RT-PCR followed by a simple RFLP assay in the case of primer pair UC53-UG52 or by sequencing and sequence analysis in the case of primer set 292-222 should constitute alternative means of modern typing and diagnostics against conventional immunological classification methods.