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Generation of virus from cDNA clones in RD cells. (A) Flow cytometric analysis of CAR and DAF expression on RD cells. For detection of CAR and DAF, anti-CAR (RmcB) and anti- DAF (BRIC110) monoclonal antibodies were used (black histograms). A mouse IgG1 antibody was used as a negative control (gray histograms ). (B) Total virus yield (extracellular and intracellular) determined for indicated viral variants by a TCID 50 assay in GMK cells. Viruses were generated from infectious cDNA clones by transfection and one passage in RD cells (96 h p.i.). Error bars represent mean SEM of triplicate samples.
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Coxsackievirus B2 (CVB2), one of six human pathogens of the group B coxsackieviruses within the enterovirus genus of Picornaviridae, causes a wide spectrum of human diseases ranging from mild upper respiratory illnesses to myocarditis and meningitis. The
CVB2 prototype strain Ohio-1 (CVB2O) was originally isolated from a patient with summer grippe...
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Context 1
... CVB2O vari- ants were constructed by employing a reverse genetics ap- proach (Fig. 1A). The substitutions in the VP1 protein (I118F and Q164K) and 2C protein (K185R) where introduced indi- vidually into a full-length CVB2O cDNA clone. Viruses were derived from cDNA clones in RD cells that expressed DAF but without evident expression of CAR ( Fig. 2A). Sequencing of progeny virus populations showed that the mutations were individually tolerated and also that no other substitutions or silent mutations were introduced during viral replication. In order to determine the yield of infectious viral particles pro- duced in RD cells after transfection, the different CVB2O variants were ...
Context 2
... of progeny virus populations showed that the mutations were individually tolerated and also that no other substitutions or silent mutations were introduced during viral replication. In order to determine the yield of infectious viral particles pro- duced in RD cells after transfection, the different CVB2O variants were titrated on GMK cells (Fig. 2B), a cell line where both the parental and the RD variant of CVB2O cause cytol- ysis. Similar virus titers (10 5.0 to 10 5.9 TCID 50 s/ml) were ob- tained for CVB2Owt and the three CVB2O variants (96 h p.i.). Hence, both the wild-type CVB2O virus and the CVB2O single mutants were able to replicate with similar efficiency in RD ...
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Citations
... It was shown that both non-structural and structural (protein-encoding) regions affect the infectivity of enteroviruses. For coxsackievirus B2 and EV-A71, pathogenicity determinants were shown to reside in VP1 capsid protein-encoding regions, respectively [34][35][36][37]. While serving as a cell surface entry receptor for several picornaviruses, DAF receptor is also broadly expressed in malignant tumors and plays a major role in promoting tumorigenesis [38]. ...
Rigvir® is a cell-adapted, oncolytic virotherapy enterovirus, which derives from an echovirus 7 (E7) isolate. While it is claimed that Rigvir® causes cytolytic infection in several cancer cell lines, there is little molecular evidence for its oncolytic and oncotropic potential. Previously, we genome-sequenced Rigvir® and five echovirus 7 isolates, and those sequences are further analyzed in this paper. A phylogenetic analysis of the full-length data suggested that Rigvir® was most distant from the other E7 isolates used in this study, placing Rigvir® in its own clade at the root of the phylogeny. Rigvir® contained nine unique mutations in the viral capsid proteins VP1-VP4 across the whole data set, with a structural analysis showing six of the mutations concerning residues with surface exposure on the cytoplasmic side of the viral capsid. One of these mutations, E/Q/N162G, was located in the region that forms the contact interface between decay-accelerating factor (DAF) and E7. Rigvir® and five other isolates were also subjected to cell infectivity assays performed on eight different cell lines. The used cell lines contained both cancer and non-cancer cell lines for observing Rigvir®’s claimed properties of being both oncolytic and oncotropic. Infectivity assays showed that Rigvir® had no discernable difference in the viruses’ oncolytic effect when compared to the Wallace prototype or the four other E7 isolates. Rigvir® was also seen infecting non-cancer cell lines, bringing its claimed effect of being oncotropic into question. Thus, we conclude that Rigvir®’s claim of being an effective treatment against multiple different cancers is not warranted under the evidence presented here. Bioinformatic analyses do not reveal a clear mechanism that could elucidate Rigvir®’s function at a molecular level, and cell infectivity tests do not show a discernable difference in either the oncolytic or oncotropic effect between Rigvir® and other clinical E7 isolates used in the study.
... The various clinical symptoms of coxsackievirus B2 seem to be based on genetic diversity, as they are rapidly evolving viruses. 25 Triantafyllopoulou and coautors associated on the coxsackievirus B4 as a possible trigger in the development of Sjogren's syndrome. 5 Other researchers tested patients with autoimmune diseases and demonstrated that autoantibodies recognized coxsackieviral peptides in 37% of patients with Sjogren's syndrome and in 28% of patients with systemic lupus. ...
Objective. Coxsackievirus B (1-6) infections are the common infections of children and adults. Clinical manifestations include fever, aseptic meningitis, pleurodinia, myocarditis, gastroenterocolitis, maculous exanthem. The clinical course of the infection is influenced by the characteristics of the host, as well as the virus serotype. The pathogenesis of the diseases is explained by the immune mediated mechanism and the direct cytotoxic effect of the virus. Methods. Retrospectively analyzed virus serotype, clinical and biochemical data in patients with coxsackievirus B (1-6) infection. Patients who had an unclear febrile condition for more than six months were tested for autoantibodies. Results. We examined a total of 378 patients with coxsackievirus B (1-6) infection (302 women, 76 men), age 19 to 79 years. The dominant symptoms were weakness, elevated body temperature, fatigue and muscle aches. In 55% the clinical course was fever of unknown origin, in 13% myalgia/pleurodinia, 9% acute gastroenterocolitis and acute myocarditis/ pericarditis, 2% aseptic meningitis, 2.4% respiratory disease, 3% acute pancreatitis and 1% diabetes mellitus. Autoantibodies were detected in 69% of patients with fever of unknown origin. Antinuclear antibodies were most common, in 67%. Serotype B2 had 36% of these patients. Serotype B2 had 36% of these patients and serotype B4 had 14%. Conclusion. The most common clinical form of coxsackievirus B (1-6) infection is an fever of unknown origin caused by a B2 serotype of the virus. In most of these patients, an elevated titre of antinuclear antibodies can be detected.
... They also found extensive virus-induced neuronal death in the lesion area, consistent with the observed apoptosis in CVB3-induced diseases, including a murine model of myocarditis, depending on the mouse strain and the virus variants used (36-40). Gullberg et al described cytolytic replication in rhabdomyosarcoma cells resulting in apoptosis when a single amino acid was changed within the VP1 protein of the Ohio-1 strain of CVB2 (41). We confirmed that this virus induces apoptosis in neurons and astroglia, which was seen around the replication sites after intracerebral inoculation. ...
Coxsackievirus B (CVB) causes severe morbidity and mortality in neonates and is sometimes associated with severe brain damage resulting from acute severe viral encephalomyelitis. However, the neuropathology of CVB infection remains unclear. A prototype strain of coxsackievirus B2 (Ohio-1) induces brain lesions in neonatal mice, resulting in dome-shaped heads, ventriculomegaly, and loss of the cerebral cortex. Here, we characterized the glial pathology in this mouse model. Magnetic resonance imaging revealed an absence of the cerebral cortex within 2 weeks after inoculation. Histopathology showed that virus replication triggered activation of microglia and astrocytes, and induced apoptosis in the cortex, with severe necrosis and lateral ventricular dilation. In contrast, the brainstem and cerebellum remained morphologically intact. Immunohistochemistry revealed high expression of the coxsackievirus and adenovirus receptor (a primary receptor for CVB) in mature neurons of the cortex, hippocampus, thalamus, and midbrain, demonstrating CVB2 infection of mature neurons in these areas. However, apoptosis and neuroinflammation from activated microglia and astrocytes differed in thalamic and cortical areas. Viral antigens were retained in the brains of animals in the convalescence phase with seroconversion. This animal model will contribute to a better understanding of the neuropathology of CVB infection.
... Our data here support this hypothesis, since only a subpopulation of the cells was infected, and clear signs of cytotoxicity and cytolysis were observed in these infected cells (data not shown), but the cell culture in general remained viable. In addition to persistent infections, it seems that enteroviruses cultured in RD cells obtain adaptations that lead to increased CPE within the culture and eventually to total cytolysis of the culture (26,27). After nine passages of CVB2 Ohio strain (CVB2O) in RD cells, Polacek et al. (25) reported a phenotype of CVB2O that caused cytolysis of the culture. ...
... Both the parental and the adapted strains led to similar titers of cytolytic viruses in HeLa cells, while the parental strain showed a decreased replication level in RD cells. This cytolytic trait was identified to be due to a single-amino-acid change within the exposed region of VP1 capsid protein (26), suggesting a role in receptor usage. By using a high MOI, it was, however, possible to adapt the persistent CVB2O infection to a cytolytic one, and the single mutation needed was a single mutation in VP1 (26). ...
... This cytolytic trait was identified to be due to a single-amino-acid change within the exposed region of VP1 capsid protein (26), suggesting a role in receptor usage. By using a high MOI, it was, however, possible to adapt the persistent CVB2O infection to a cytolytic one, and the single mutation needed was a single mutation in VP1 (26). CVB3 has also been previously shown to adapt to RD cells by serial passaging, causing increased CPE in these cells. ...
Enteroviruses cause several severe diseases, with lytic infections that lead to rapid cell death but also persistent infections that are more silent and lead to chronic states of infection. Our study compared a cytolytic echovirus 1 infection to persistent coxsackievirus B5 infection by making a chimera with the structural proteins of echovirus 1 and the nonstructural proteins of coxsackievirus B5. Coxsackievirus B5 infection was found to lead to the production of a high number of empty viruses (empty capsids) that do not contain genetic material and are unable to continue the infection. Coinciding with the high number of empty capsids, the amount of infective virions decreased. This characteristic property was not observed in the constructed chimera virus, suggesting that structural proteins are in charge of these phenomena. These results shed light on the mechanisms that may cause persistent infections. Understanding events leading to efficient or inefficient infections is essential in understanding virus-caused pathologies.
... Reports show that the interaction between the virus and host cell apoptotic mechanisms may be a determinant of whether the outcome of an infection is cytolytic or not [11][12][13]. Several picornaviral proteins, for example VP1, VP2 and VP3 are known to induce apoptotic responses [14][15][16][17]. ...
... It was also shown that the change to a cytolytic infection was due to a single mutation in the VP1 protein, a glutamine (Q) to a lysine (K), and that the cytolytic virus triggered apoptotic responses. Furthermore, it was shown that the two virus variants, despite the difference in replication strategy, had very similar growth kinetics, and the infections produced approximately the same amount of infectious units [17]. ...
... The conditions were: infection with the variant vVP1Q164K and incubation for either 24 hours or 48 hours; infection with CVB2Owt and incubation for 48 hours; and mock-infection (treated identically but without the addition of viruses) and incubation for 48 hours. As in the previous study [17], infections were carried out with multiplicity of infection (MOI) 1, calculated based on tissue culture infectivity dose 50% (TCID 50 ) performed on green monkey kidney (GMK) cells. The cells were infected through incubation with the virus, diluted in a serum-free version of the cell culture medium described above, at room temperature for one hour. ...
The transcriptomes of cells infected with lytic and non-lytic variants of coxsackievirus B2 Ohio-1 (CVB2O) were analyzed using next generation sequencing. This approach was selected with the purpose of elucidating the effects of lytic and non-lytic viruses on host cell transcription. Total RNA was extracted from infected cells and sequenced. The resulting reads were subsequently mapped against the human and CVB2O genomes. The amount of intracellular RNA was measured, indicating lower proportions of human RNA in the cells infected with the lytic virus compared to the non-lytic virus after 48 hours. This may be explained by reduced activity of the cellular transcription/translation machinery in lytic enteroviral replication due to activities of the enteroviral proteases 2A and/or 3C. Furthermore, differential expression in the cells infected with the two virus variants was identified and a number of transcripts were singled out as possible answers to the question of how the viruses interact with the host cells, resulting in lytic or non-lytic infections.
... The linearized template was then subjected to the in vitro transcription reaction, and the RNA transcripts were transfected into mammalian cells from which infectious enterovirus particles were recovered. Mutagenesis of enterovirus cDNA clones was typically carried out in a time-consuming process including subcloning of the target region into a propagation plasmid, PCR of the target region by mutagenic primers and subcloning of the target back to the viral cDNA backbone using compatible restriction enzyme-cutting sites [8,10,11,12,13]. Interestingly enough, such methods are still being used [12,13] even if high fidelity, long-range PCR enzymes have already been used to amplify long genomic fragments or even full-length genomes in a single step 20 years ago [14,15,16,17]. ...
... Mutagenesis of enterovirus cDNA clones was typically carried out in a time-consuming process including subcloning of the target region into a propagation plasmid, PCR of the target region by mutagenic primers and subcloning of the target back to the viral cDNA backbone using compatible restriction enzyme-cutting sites [8,10,11,12,13]. Interestingly enough, such methods are still being used [12,13] even if high fidelity, long-range PCR enzymes have already been used to amplify long genomic fragments or even full-length genomes in a single step 20 years ago [14,15,16,17]. More recent studies have included long PCR protocols for cDNA cloning [18,19,20,21]. ...
... The linearized template was then subjected to the in vitro transcription reaction, and the RNA transcripts were transfected into mammalian cells from which infectious enterovirus particles were recovered. Mutagenesis of enterovirus cDNA clones was typically carried out in a time-consuming process including subcloning of the target region into a propagation plasmid, PCR of the target region by mutagenic primers and subcloning of the target back to the viral cDNA backbone using compatible restriction enzyme-cutting sites [8,[10][11][12][13]. Interestingly enough, such methods are still being used [12,13] even if high fidelity, long-range PCR enzymes have already been used to amplify long genomic fragments or even full-length genomes in a single step 20 years ago [14][15][16][17]. ...
Research on human enteroviruses has resulted in the identification of more than 100 enterovirus types, which use more than 10 protein receptors and/or attachment factors required in cell binding and initiation of the replication cycle. Many of these “viral” receptors are overexpressed in cancer cells. Receptor binding and the ability to replicate in specific target cells define the tropism and pathogenesis of enterovirus types, because cellular infection often results in cytolytic response, i.e., disruption of the cells. Viral tropism and cytolytic properties thus make native enteroviruses prime candidates for oncolytic virotherapy. Copy DNA cloning and modification of enterovirus genomes have resulted in the generation of enterovirus vectors with properties that are useful in therapy or in vaccine trials where foreign antigenic epitopes are expressed from or on the surface of the vector virus. The small genome size and compact particle structure, however, set limits to enterovirus genome modifications. This review focuses on the therapeutic use of native and recombinant enteroviruses and the methods that have been applied to modify enterovirus genomes for therapy.
... Intriguingly, despite the phenotypic similarities, the amino acids that mediate DAF-binding of CV-B3-RD strain (Pan et al., 2011; Yoder et al., 2012) or CV-B3-PD strain (Schmidtke et al., 2000) are not homologous to the CV-B6 VP1 K257E substitution identified as a putative DAF-binding determinant of CV-B6-DUCT strain in this study. The identification of a single CV-B6 amino acid that is most likely responsible for the lytic phenotype, more efficient progeny production and DAF-usage is in line with previous reports that have shown strong effects for single or few amino acid substitutions on the receptor usage, cell and tissue tropism and pathogenesis (Al-Hello et al., 2005 Caggana et al., 1993; Chua et al., 2008; Cifuente et al., 2011; Gullberg et al., 2010; Halim and Ramsingh, 2000; Kim and Racaniello, 2007; Knowlton et al., 1996; Novoselov et al., 2012; Paananen et al., 2013; Pan et al., 2011; Pelletier et al., 1998; Polacek et al., 2005; Ramsingh and Collins, 1995; Ramsingh et al., 1997; Schmidtke et al., 2000). A phenomenon similar to our CV-B6 model has been described previously using CV-B2-Ohio strain that induces persistent non-cytolytic infection in CAR-deficient RD-cells. ...
... A phenomenon similar to our CV-B6 model has been described previously using CV-B2-Ohio strain that induces persistent non-cytolytic infection in CAR-deficient RD-cells. A single amino acid substitution in VP1 protein transforms the virus phenotype from non-cytolytic to cytolytic (Gullberg et al., 2010; Polacek et al., 2005). However, this adapted CV-B2 strain does not bind to DAF (i.e., uses yet un-indentified receptor but retains CAR-binding) and the amino acid substitution responsible for the cytolytic phenotype of CV-B2 (Q164K at VP1) is not homologous to the amino acid responsible for CV-B6-DUCT adaptation (although both map to the adjacent regions in capsid surface). ...
... However, this adapted CV-B2 strain does not bind to DAF (i.e., uses yet un-indentified receptor but retains CAR-binding) and the amino acid substitution responsible for the cytolytic phenotype of CV-B2 (Q164K at VP1) is not homologous to the amino acid responsible for CV-B6-DUCT adaptation (although both map to the adjacent regions in capsid surface). Intriguingly, CV-B2-Ohio strain induces lytic infection in the HPDE-cells although it does not have DAF-binding capacity (Bergelson et al., 1997b; Gullberg et al., 2010; Shafren et al., 1995). Therefore, the ability to use DAF as a receptor is, most likely, not the sole determinant of lytic infection in pancreatic ductal cells. ...
Enterovirus infections have been suspected to be involved in the development of type 1 diabetes. However, the pathogenetic mechanism of enterovirus-induced type 1 diabetes is not known. Pancreatic ductal cells are closely associated with pancreatic islets. Therefore, enterovirus infections in ductal cells may also affect beta-cells and be involved in the induction of type 1 diabetes. The aim of this study was to assess the ability of different enterovirus strains to infect, replicate and produce cytopathic effect in human pancreatic ductal cells. Furthermore, the viral factors that affect these capabilities were studied.
... The mechanism(s) by which VP2 binding to SIVA enhances its apoptotic function is not fully understood. Additionally, a single point mutation in the VP1 capsid protein of a strain of CVB2 that does not normally cause cell death in rhabdomyosarcoma cells was sufficient to cause the strain to begin inducing cell death (101). Viral capsid protein may or may not be necessary to induce the JNK signaling pathway, depending on the EV. ...
Enteroviruses (EVs) are the most common human viral pathogens. They cause a variety of pathologies, including myocarditis and meningoencephalopathies, and have been linked to the onset of type I diabetes. These pathologies result from the death of cells in the myocardium, central nervous system, and pancreas, respectively. Understanding the role of EVs in inducing cell death is crucial to understanding the etiologies of these diverse pathologies. EVs both induce and delay host cell death, and their exquisite control of this balance is crucial for their success as human viral pathogens. Thus, EVs are tightly involved with cell death signaling pathways and interact with host cell signaling at multiple points Here, we review the literature detailing the mechanisms of EV-induced cell death. We discuss the mechanisms by which EVs induce cell death, the signaling pathways involved in these pathways, and the strategies by which EVs antagonize cell death pathways. We also discuss the role of cell death in both the resulting pathology in the host and in the facilitation of viral spread.
... The VP3 of avian encephalomyelitis virus was localized to mitochondria and induced apoptosis (Liu et al., 2002). Lastly, a single amino acid substitution in the VP1 capsid protein of Coxsackie virus B2 (CVB2) transformed a noncytolytic virus to a cytolytic one, causing apoptotic responses that included caspase activation and DNA fragmentation (Gullberg et al., 2010). ...
Viral capsid proteins (CPs) are characterized by their role in forming protective shells around viral genomes. However, CPs have additional and important roles in the virus infection cycles and in the cellular responses to infection. These activities involve CP binding to RNAs in both sequence-specific and nonspecific manners as well as association with other proteins. This review focuses on CPs of both plant and animal-infecting viruses with positive-strand RNA genomes. We summarize the structural features of CPs and describe their modulatory roles in viral translation, RNA-dependent RNA synthesis, and host defense responses.
... Trends in Microbiology December 2012, Vol. 20,No. 12 proapoptotic and antiapoptotic host factors. Alterations in favor of proapoptotic proteins are sufficient to elicit suicidal reaction [9]. Such a switch can be caused by activation of unspecific innate immunity mechanisms through sensors of viral infection as well as by interaction of viral proteins with components of the host apoptotic ...
... Although other examples of the dependence of the apoptosis-triggering capacity of picornaviruses on the properties of their capsid proteins are known [12], it is unclear whether this dependence is linked to the activation of the extrinsic apoptotic pathway or to the viral competence to efficiently infect the cells. In most known cases apoptotic response is caused by replicating picornaviruses, although modulations of the apoptotic program through poliovirus interaction with its receptor was documented [13]. ...
... Competition between death programs of virus-infected cells Different cellular antiviral defensive death programs compete with each other. For example, at early stages of poliovirus infection, HeLa cells appear to become commit-Opinion Trends in Microbiology December 2012, Vol. 20,No. 12 ted to apoptosis but later on the necrotic program prevails [39] (Figure 1). Molecular mechanisms underlying this competition are not well elucidated. ...
The capacity to injure infected cells is a widespread property of viruses. Usually, this cytopathic effect (CPE) is ascribed to viral hijacking of cellular resources to fulfill viral needs. However, evidence is accumulating that CPE is not necessarily directly coupled to viral reproduction but may largely be due to host defensive and viral antidefensive activities. A major part in this virus-cell interaction appears to be played by a putative host-encoded program with multiple competing branches, leading to necrotic, apoptotic, and, possibly, other types of cell suicide. Manifestations of this program are controlled and modulated by host, viral, and environmental factors.
