The Experts below are selected from a list of 273 Experts worldwide ranked by ideXlab platform
Hans J Eggers - One of the best experts on this subject based on the ideXlab platform.
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Echovirus 9 protein 2c binds single stranded rna unspecifically
Journal of General Virology, 2000Co-Authors: Marcus Klein, Hans J Eggers, Birgit NelsensalzAbstract:Polypeptide 2C is essential for picornavirus replication. Although many data on multiple functions of this highly conserved protein are available, the mechanism of RNA binding is still obscure. In this work, protein 2C of Echovirus-9 strain Barty was expressed as a histidine-tagged protein in E. coli followed by nondenaturing purification to homogeneity. After incubation of 2C protein with different kinds of RNA fragments, binding was shown in gel retardation assays. Competition experiments revealed that 2C targets linear RNA unspecifically; however, single-stranded linear DNA does not react with this protein. In contrast to poliovirus, protein 2C of Echovirus-9 only recognizes RNA with a low content of secondary structures. This may be a first hint of a different binding specificity of 2C in echo- and polioviruses.
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the picornavirus replication inhibitors hbb and guanidine in the Echovirus 9 system the significance of viral protein 2c
Journal of General Virology, 2000Co-Authors: Marcus Klein, Hans J Eggers, Holger Zimmermann, Dirk Hadaschik, Birgit NelsensalzAbstract:HBB [2-(α-hydroxybenzyl)-benzimidazole] and guanidine are potent inhibitors of picornavirus replication. Among other evidence, limited cross-resistance and a synergistic effect of both inhibitors suggest similar but not identical mechanisms of antiviral action. Echovirus-9 variants resistant to each of these drugs were characterized and sequenced. Complete resistance to HBB or guanidine was shown to be due to single but different point mutations in the non-structural protein 2C. Protein 2C was expressed as GST fusion and His-tagged proteins for the wild-type and various mutants. Although three mutations were located in or near conserved NTP binding motifs, NTPase activity was not altered in the presence of HBB or guanidine.
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Echovirus 9 strain barty non structural protein 2c has ntpase activity
Virus Research, 1999Co-Authors: Marcus Klein, Hans J Eggers, Birgit NelsensalzAbstract:Abstract Non-structural protein 2C is known to play a fundamental role in the replication of picornaviruses. Sequence analyses revealed that 2C belongs to a rapidly expanding group of proteins containing a consensus sequence for nucleotide binding (NTB). We report that Echovirus 9 polypeptide 2C displays NTPase activity in vitro. In our experiments, several P2 genes were expressed in Escherichia coli as fusion proteins linked to glutathione S-transferase (GST) prior to purification close to homogeneity. In contrast to GST-2B, both GST-2C and GST-2BC showed ATPase as well as GTPase activity indicating that the site for NTB binding and splitting is located in 2C.
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dependence of Echovirus 9 on the enterovirus rna replication inhibitor 2 α hydroxybenzyl benzimidazole maps to nonstructural protein 2c
Journal of Virology, 1999Co-Authors: Dirk Hadaschik, Hans J Eggers, Marcus Klein, Holger Zimmermann, Birgit NelsensalzAbstract:HBB [2-(α-hydroxybenzyl)-benzimidazole] selectively inhibits RNA synthesis of most enteroviruses. However, isolation of HBB-dependent variants is possible. Sequence analysis and characterization of recombinant viruses revealed that HBB dependence maps to the nonstructural protein 2C. A single point mutation at position C4782U is sufficient to establish the HBB-dependent phenotype in our Echovirus 9 model.
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integrin αvβ3 vitronectin receptor is a candidate receptor for the virulent Echovirus 9 strain barty
Journal of General Virology, 1999Co-Authors: Birgit Nelsensalz, Hans J Eggers, Holger ZimmermannAbstract:The enterovirus Echovirus 9 strain Barty (E9/Barty) is pathogenic for newborn mice as well as for humans. In contrast to the apathogenic prototype strain Hill, strain Barty encodes an RGD motif in the C-terminal part of the structural protein VP1. Data are presented that show that E9/Barty binds its target cells via contact of the RGD motif to the αvβ3 integrin (vitronectin receptor), whereas prototype Hill uses a different, still unidentified receptor site. Furthermore, virus titres of murine muscle tissue were compared after infection of newborn and 1-, 2-, 3- and 12-week-old mice. The replication capacity of the virus decreased dramatically with age of the infected mice. Since E9/Barty does not replicate or replicates only poorly in mice older than about 5 days, and expression of the vitronectin receptor is reported to be down-regulated in striated muscle tissue during development, it is suggested that susceptibility of mice to this Echovirus infection is controlled by the availability of αvβ3 integrin.
Birgit Nelsensalz - One of the best experts on this subject based on the ideXlab platform.
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Echovirus 9 protein 2c binds single stranded rna unspecifically
Journal of General Virology, 2000Co-Authors: Marcus Klein, Hans J Eggers, Birgit NelsensalzAbstract:Polypeptide 2C is essential for picornavirus replication. Although many data on multiple functions of this highly conserved protein are available, the mechanism of RNA binding is still obscure. In this work, protein 2C of Echovirus-9 strain Barty was expressed as a histidine-tagged protein in E. coli followed by nondenaturing purification to homogeneity. After incubation of 2C protein with different kinds of RNA fragments, binding was shown in gel retardation assays. Competition experiments revealed that 2C targets linear RNA unspecifically; however, single-stranded linear DNA does not react with this protein. In contrast to poliovirus, protein 2C of Echovirus-9 only recognizes RNA with a low content of secondary structures. This may be a first hint of a different binding specificity of 2C in echo- and polioviruses.
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the picornavirus replication inhibitors hbb and guanidine in the Echovirus 9 system the significance of viral protein 2c
Journal of General Virology, 2000Co-Authors: Marcus Klein, Hans J Eggers, Holger Zimmermann, Dirk Hadaschik, Birgit NelsensalzAbstract:HBB [2-(α-hydroxybenzyl)-benzimidazole] and guanidine are potent inhibitors of picornavirus replication. Among other evidence, limited cross-resistance and a synergistic effect of both inhibitors suggest similar but not identical mechanisms of antiviral action. Echovirus-9 variants resistant to each of these drugs were characterized and sequenced. Complete resistance to HBB or guanidine was shown to be due to single but different point mutations in the non-structural protein 2C. Protein 2C was expressed as GST fusion and His-tagged proteins for the wild-type and various mutants. Although three mutations were located in or near conserved NTP binding motifs, NTPase activity was not altered in the presence of HBB or guanidine.
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Echovirus 9 strain barty non structural protein 2c has ntpase activity
Virus Research, 1999Co-Authors: Marcus Klein, Hans J Eggers, Birgit NelsensalzAbstract:Abstract Non-structural protein 2C is known to play a fundamental role in the replication of picornaviruses. Sequence analyses revealed that 2C belongs to a rapidly expanding group of proteins containing a consensus sequence for nucleotide binding (NTB). We report that Echovirus 9 polypeptide 2C displays NTPase activity in vitro. In our experiments, several P2 genes were expressed in Escherichia coli as fusion proteins linked to glutathione S-transferase (GST) prior to purification close to homogeneity. In contrast to GST-2B, both GST-2C and GST-2BC showed ATPase as well as GTPase activity indicating that the site for NTB binding and splitting is located in 2C.
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dependence of Echovirus 9 on the enterovirus rna replication inhibitor 2 α hydroxybenzyl benzimidazole maps to nonstructural protein 2c
Journal of Virology, 1999Co-Authors: Dirk Hadaschik, Hans J Eggers, Marcus Klein, Holger Zimmermann, Birgit NelsensalzAbstract:HBB [2-(α-hydroxybenzyl)-benzimidazole] selectively inhibits RNA synthesis of most enteroviruses. However, isolation of HBB-dependent variants is possible. Sequence analysis and characterization of recombinant viruses revealed that HBB dependence maps to the nonstructural protein 2C. A single point mutation at position C4782U is sufficient to establish the HBB-dependent phenotype in our Echovirus 9 model.
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integrin αvβ3 vitronectin receptor is a candidate receptor for the virulent Echovirus 9 strain barty
Journal of General Virology, 1999Co-Authors: Birgit Nelsensalz, Hans J Eggers, Holger ZimmermannAbstract:The enterovirus Echovirus 9 strain Barty (E9/Barty) is pathogenic for newborn mice as well as for humans. In contrast to the apathogenic prototype strain Hill, strain Barty encodes an RGD motif in the C-terminal part of the structural protein VP1. Data are presented that show that E9/Barty binds its target cells via contact of the RGD motif to the αvβ3 integrin (vitronectin receptor), whereas prototype Hill uses a different, still unidentified receptor site. Furthermore, virus titres of murine muscle tissue were compared after infection of newborn and 1-, 2-, 3- and 12-week-old mice. The replication capacity of the virus decreased dramatically with age of the infected mice. Since E9/Barty does not replicate or replicates only poorly in mice older than about 5 days, and expression of the vitronectin receptor is reported to be down-regulated in striated muscle tissue during development, it is suggested that susceptibility of mice to this Echovirus infection is controlled by the availability of αvβ3 integrin.
Diana Hardie - One of the best experts on this subject based on the ideXlab platform.
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Molecular characterization of an outbreak of enterovirus-associated meningitis in Mossel Bay, South Africa, December 2015–January 2016
BMC Infectious Diseases, 2018Co-Authors: Heidi Smuts, Sarah Cronje, Juno Thomas, Delene Brink, Stephen Korsman, Diana HardieAbstract:Background Human enteroviruses (HEVs) are common causal agents of aseptic meningitis in young children. Laboratory and syndromic surveillance during December 2015 and January 2016 noted an unusually high number of paediatric aseptic meningitis cases at a hospital in Mossel Bay, Western Cape Province, South Africa. HEV was detected in clinical samples, prompting an outbreak investigation. Methods Epidemiological investigations were conducted to ascertain possible linkage between cases. Amplification, sequencing and phylogenetic analysis of the 5’UTR and VP1 regions was undertaken to determine the HEV serotype associated with the outbreak as well as other cases of aseptic meningitis in the area in the preceding 6 weeks. Results Over the 2-month period, 63 CSF samples were available for testing. A total of 43 outbreak cases (68.3%) were observed, and the 26 (60.5%) that could be typed were coxsackie virus A9 (CVA9). Children attending three crèche facilities were epidemiologically linked, accounting for 60.5% (26/43) of the CVA9 cases. The majority of patients were under 10 years of age (55/63, 87.3%) and there was a male predominance (66%). Nucleotide sequence analysis of the 5’UTR and VP1 regions identified 2 lineages of CVA9 co-circulating during the outbreak, although the VP1 capsid protein sequence was identical as all nucleotide differences were synonymous. There was a unique isoleucine at position 64 and all outbreak viruses had a valine to threonine change in the hypervariable BC loop of VP1. Other HEV types circulating in the preceding period were Echovirus 30 ( n = 4), Echovirus 5 ( n = 3) and 1 each of Echovirus 6, Echovirus 9 and Echovirus 15. Conclusion CVA9 was identified as the pathogen responsible for the large outbreak of aseptic meningitis, with 2 distinct co-circulating lineages.
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Molecular characterization of an outbreak of enterovirus-associated meningitis in Mossel Bay, South Africa, December 2015–January 2016
BMC Infectious Diseases, 2018Co-Authors: Heidi Smuts, Sarah Cronje, Juno Thomas, Delene Brink, Stephen Korsman, Diana HardieAbstract:Human enteroviruses (HEVs) are common causal agents of aseptic meningitis in young children. Laboratory and syndromic surveillance during December 2015 and January 2016 noted an unusually high number of paediatric aseptic meningitis cases at a hospital in Mossel Bay, Western Cape Province, South Africa. HEV was detected in clinical samples, prompting an outbreak investigation. Epidemiological investigations were conducted to ascertain possible linkage between cases. Amplification, sequencing and phylogenetic analysis of the 5’UTR and VP1 regions was undertaken to determine the HEV serotype associated with the outbreak as well as other cases of aseptic meningitis in the area in the preceding 6 weeks. Over the 2-month period, 63 CSF samples were available for testing. A total of 43 outbreak cases (68.3%) were observed, and the 26 (60.5%) that could be typed were coxsackie virus A9 (CVA9). Children attending three creche facilities were epidemiologically linked, accounting for 60.5% (26/43) of the CVA9 cases. The majority of patients were under 10 years of age (55/63, 87.3%) and there was a male predominance (66%). Nucleotide sequence analysis of the 5’UTR and VP1 regions identified 2 lineages of CVA9 co-circulating during the outbreak, although the VP1 capsid protein sequence was identical as all nucleotide differences were synonymous. There was a unique isoleucine at position 64 and all outbreak viruses had a valine to threonine change in the hypervariable BC loop of VP1. Other HEV types circulating in the preceding period were Echovirus 30 (n = 4), Echovirus 5 (n = 3) and 1 each of Echovirus 6, Echovirus 9 and Echovirus 15. CVA9 was identified as the pathogen responsible for the large outbreak of aseptic meningitis, with 2 distinct co-circulating lineages.
Holger Zimmermann - One of the best experts on this subject based on the ideXlab platform.
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the picornavirus replication inhibitors hbb and guanidine in the Echovirus 9 system the significance of viral protein 2c
Journal of General Virology, 2000Co-Authors: Marcus Klein, Hans J Eggers, Holger Zimmermann, Dirk Hadaschik, Birgit NelsensalzAbstract:HBB [2-(α-hydroxybenzyl)-benzimidazole] and guanidine are potent inhibitors of picornavirus replication. Among other evidence, limited cross-resistance and a synergistic effect of both inhibitors suggest similar but not identical mechanisms of antiviral action. Echovirus-9 variants resistant to each of these drugs were characterized and sequenced. Complete resistance to HBB or guanidine was shown to be due to single but different point mutations in the non-structural protein 2C. Protein 2C was expressed as GST fusion and His-tagged proteins for the wild-type and various mutants. Although three mutations were located in or near conserved NTP binding motifs, NTPase activity was not altered in the presence of HBB or guanidine.
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dependence of Echovirus 9 on the enterovirus rna replication inhibitor 2 α hydroxybenzyl benzimidazole maps to nonstructural protein 2c
Journal of Virology, 1999Co-Authors: Dirk Hadaschik, Hans J Eggers, Marcus Klein, Holger Zimmermann, Birgit NelsensalzAbstract:HBB [2-(α-hydroxybenzyl)-benzimidazole] selectively inhibits RNA synthesis of most enteroviruses. However, isolation of HBB-dependent variants is possible. Sequence analysis and characterization of recombinant viruses revealed that HBB dependence maps to the nonstructural protein 2C. A single point mutation at position C4782U is sufficient to establish the HBB-dependent phenotype in our Echovirus 9 model.
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integrin αvβ3 vitronectin receptor is a candidate receptor for the virulent Echovirus 9 strain barty
Journal of General Virology, 1999Co-Authors: Birgit Nelsensalz, Hans J Eggers, Holger ZimmermannAbstract:The enterovirus Echovirus 9 strain Barty (E9/Barty) is pathogenic for newborn mice as well as for humans. In contrast to the apathogenic prototype strain Hill, strain Barty encodes an RGD motif in the C-terminal part of the structural protein VP1. Data are presented that show that E9/Barty binds its target cells via contact of the RGD motif to the αvβ3 integrin (vitronectin receptor), whereas prototype Hill uses a different, still unidentified receptor site. Furthermore, virus titres of murine muscle tissue were compared after infection of newborn and 1-, 2-, 3- and 12-week-old mice. The replication capacity of the virus decreased dramatically with age of the infected mice. Since E9/Barty does not replicate or replicates only poorly in mice older than about 5 days, and expression of the vitronectin receptor is reported to be down-regulated in striated muscle tissue during development, it is suggested that susceptibility of mice to this Echovirus infection is controlled by the availability of αvβ3 integrin.
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determinants of pathogenicity of Echovirus 9 in men significance of a functional rgd motif
Zentralblatt Fur Bakteriologie-international Journal of Medical Microbiology Virology Parasitology and Infectious Diseases, 1999Co-Authors: Birgit Nelsensalz, Hans J Eggers, Marcus Klein, Oliver Schildgen, Dirk Hadaschik, Holger ZimmermannAbstract:Summary In this study, we investigated nine independent Echovirus 9 isolates obtained from sick children in 1995. It is discovered that these isolates differ in respect to their pathogenicity for newborn mice indicating that the degree of human pathogenicity of an Echovirus 9 variant does not necessarily correlate with mouse pathogenicity. Nevertheless, all virus variants are found to code for an RGD-motif within their VP1 protein. Hence, the RGD-motif and its highly conserved flanking regions are the conditio sine qua non , but, as expected, not sufficient for the mouse-pathogenic character.
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Integrin alpha(v)beta3 (vitronectin receptor) is a candidate receptor for the virulent Echovirus 9 strain Barty.
The Journal of general virology, 1999Co-Authors: Birgit Nelsen-salz, Hans J Eggers, Holger ZimmermannAbstract:The enterovirus Echovirus 9 strain Barty (E9/Barty) is pathogenic for newborn mice as well as for humans. In contrast to the apathogenic prototype strain Hill, strain Barty encodes an RGD motif in the C-terminal part of the structural protein VP1. Data are presented that show that E9/Barty binds its target cells via contact of the RGD motif to the alpha(v)beta3 integrin (vitronectin receptor), whereas prototype Hill uses a different, still unidentified receptor site. Furthermore, virus titres of murine muscle tissue were compared after infection of newborn and 1-, 2-, 3- and 12-week-old mice. The replication capacity of the virus decreased dramatically with age of the infected mice. Since E9/Barty does not replicate or replicates only poorly in mice older than about 5 days, and expression of the vitronectin receptor is reported to be down-regulated in striated muscle tissue during development, it is suggested that susceptibility of mice to this Echovirus infection is controlled by the availability of alpha(v)beta3 integrin.
Merja Roivainen - One of the best experts on this subject based on the ideXlab platform.
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a single amino acid substitution in viral vp1 protein alters the lytic potential of clone derived variants of Echovirus 9 dm strain in human pancreatic islets
Journal of Medical Virology, 2013Co-Authors: Anja Paananen, Petri Ylipaasto, Jochem M D Galama, Teemu Smura, Marko Lempinen, Merja RoivainenAbstract:In vitro studies with primary human pancreatic islets suggest that several enterovirus serotypes are able to infect and replicate in beta cells. Some enterovirus strains are highly cytolytic in vitro whereas others show virus replication with no apparent islet destruction. The capability to induce islet destruction is determined only partially by the virus serotype, since strain specific differences have been detected within some serotypes including Echovirus 9 (E-9). In this study, the viral genetic factors determining the outcome of islet infection (i.e., destructive vs. benign) were investigated by constructing parallel infectious clones of lytic E-9-DM strain that was isolated from a small child at the clinical onset of type 1 diabetes. The capabilities of these clone-derived viruses to induce islet destruction were monitored and the lytic potential of clones was modified by site-directed mutagenesis. The lytic capabilities of these clone-derived viruses in human pancreatic islets were modified by a single amino acid substitution (T81A) in the capsid protein VP1. The data presented outline the importance of amino acid point mutations in the pathogenetic process leading to islet necrosis. However, although the amino acid substitution (T81A) modifies the lytic capabilities of E-9-DM strain-derived microvariant strains, it is likely that additional viral genetic determinants of pancreatic islet pathogenicity exist in other E-9 strains. J. Med. Virol. 85:1267-1273, 2013. © 2013 Wiley Periodicals, Inc.
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The Association of Recombination Events in the Founding and Emergence of Subgenogroup Evolutionary Lineages of Human Enterovirus 71
Journal of Virology, 2011Co-Authors: E. C. Mcwilliam Leitch, María Cabrerizo, Jane Cardosa, Heli Harvala, Ivanova Oe, Satoshi Koike, Aloys C. M. Kroes, Alexander N. Lukashev, David Perera, Merja RoivainenAbstract:Enterovirus 71 (EV71) is responsible for frequent large-scale outbreaks of hand, foot, and mouth disease worldwide and represent a major etiological agent of severe, sometimes fatal neurological disease. EV71 variants have been classified into three genogroups (GgA, GgB, and GgC), and the latter two are further subdivided into subgenogroups B1 to B5 and C1 to C5. To investigate the dual roles of recombination and evolution in the epidemiology and transmission of EV71 worldwide, we performed a large-scale genetic analysis of isolates (n = 308) collected from 19 countries worldwide over a 40-year period. A series of recombination events occurred over this period, which have been identified through incongruities in sequence grouping between the VP1 and 3Dpol regions. Eleven 3Dpol clades were identified, each specific to EV71 and associated with specific subgenogroups but interspersed phylogenetically with clades of coxsackievirus A16 and other EV species A serotypes. The likelihood of recombination increased with VP1 sequence divergence; mean half-lives for EV71 recombinant forms (RFs) of 6 and 9 years for GgB and GgC overlapped with those observed for the EV-B serotypes, Echovirus 9 (E9), E30, and E11, respectively (1.3 to 9.8 years). Furthermore, within genogroups, sporadic recombination events occurred, such as the linkage of two B4 variants to RF-W instead of RF-A and of two C4 variants to RF-H. Intriguingly, recombination events occurred as a founding event of most subgenogroups immediately preceding their lineage expansion and global emergence. The possibility that recombination contributed to their subsequent spread through improved fitness requires further biological and immunological characterization.
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Vitronectin receptors, alpha v integrins, are recognized by several non-RGD-containing Echoviruses in a continuous laboratory cell line and also in primary human Langerhans' islets and endothelial cells.
The Journal of general virology, 2009Co-Authors: Petri Ylipaasto, Tapani Hovi, Mervi Eskelinen, Kaija Salmela, Merja RoivainenAbstract:Previously published data suggest that the RGD-recognizing integrin, alphavbeta3, known as the vitronectin receptor, acts as a cellular receptor for RGD-containing enteroviruses, coxsackievirus A9 (CAV-9) and Echovirus 9 (E-9), in several continuous cell lines as well as in primary human Langerhans' islets. As this receptor is also capable of binding the ligands by a non-RGD-dependent mechanism, we investigated whether vitronectin receptors, alpha v integrins, might act as receptors for other Echoviruses that do not have the RGD motif. Blocking experiments with polyclonal anti-alphavbeta3 antibody showed that both primary human islets and a continuous laboratory cell line of green monkey kidney origin (GMK) are protected similarly from the adverse effects of several non-RGD-containing Echovirus (E-7, -11, -25, -30, -32) infections. In contrast, corresponding studies on primary human endothelial cells showed that the receptor works only for E-25, E-30, E-32 and CAV-9. The inhibitory effect of the antibody was not restricted to prototype strains of Echoviruses, as GMK cells infected with several field isolates of the corresponding serotypes were also protected from virus-induced cytopathic effects. Co-localization of virus particles with the receptor molecules in both GMK and primary human endothelial cells was demonstrated by live-cell stainings and confocal microscopy. Remarkably, in spite of similar virus-receptor co-localization and a comparable protective effect of the alphavbeta3 antibody, the entry pathways of the studied virus strains seemed to be divergent.
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Vitronectin receptors, αv-integrins, are recognized by several non-RGD containing Echoviruses in a continuous laboratory cell line and also in Primary human Langerhans' islets and endothelial cells
Journal of General Virology, 2009Co-Authors: Petri Ylipaasto, Tapani Hovi, Mervi Eskelinen, Kaija Salmela, Merja RoivainenAbstract:Previously published data suggest that the RGD-recognizing integrin, αvβ3, known as the vitronectin receptor, acts as a cellular receptor for RGD-containing enteroviruses, coxsackievirus A9 (CAV-9) and Echovirus 9 (E-9), in several continuous cell lines as well as in primary human Langerhans' islets. As this receptor is also capable of binding the ligands by a non-RGD-dependent mechanism, we investigated whether vitronectin receptors, αv integrins, might act as receptors for other Echoviruses that do not have the RGD motif. Blocking experiments with polyclonal anti-αvβ3 antibody showed that both primary human islets and a continuous laboratory cell line of green monkey kidney origin (GMK) are protected similarly from the adverse effects of several non-RGD-containing Echovirus (E-7, -11, -25, -30, -32) infections. In contrast, corresponding studies on primary human endothelial cells showed that the receptor works only for E-25, E-30, E-32 and CAV-9. The inhibitory effect of the antibody was not restricted to prototype strains of Echoviruses, as GMK cells infected with several field isolates of the corresponding serotypes were also protected from virus-induced cytopathic effects. Co-localization of virus particles with the receptor molecules in both GMK and primary human endothelial cells was demonstrated by live-cell stainings and confocal microscopy. Remarkably, in spite of similar virus–receptor co-localization and a comparable protective effect of the αvβ3 antibody, the entry pathways of the studied virus strains seemed to be divergent.
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molecular and biological analysis of Echovirus 9 strain isolated from a diabetic child
Journal of Medical Virology, 2003Co-Authors: Anja Paananen, Petri Ylipaasto, Elizabeth Rieder, Tapani Hovi, Jochem M D Galama, Merja RoivainenAbstract:The full-length infectious cDNA clone was constructed and sequenced from the strain DM of Echovirus 9, which was recently isolated from a 6-week-old child at the clinical onset of type 1 diabetes. Parallel with the isolate DM, the full-length infectious cDNA clone of the prototype strain Echovirus 9 Barty (Barty-INF), was constructed and sequenced. Genetic relationships of the sequenced echo 9 viruses to the other members of the human enterovirus type B species were studied by phylogenetic analyses. Comparison of capsid protein sequences showed that the isolate DM was closely related to both prototype strains: Hill and Barty-INF. The only exception was the inner capsid protein VP4 where serotype specificity was not evident and the isolate DM clustered with the strain Hill and the strain Barty-INF with Echovirus 30 Bastianni. Likewise, the nonstructural protein coding region, P2P3, of isolate DM was more similar to strain Hill than to strain Barty-INF. However, like Echovirus 9 Barty, the isolate DM contained the RGD-motif in the carboxy terminus of capsid protein VP1. By blocking experiments using an RGD-containing peptide and a polyclonal rabbit antiserum to the alpha(v)beta(3)-integrin, it was shown that this molecule works as a cellular receptor for isolate DM. By using primary human islets, it was shown that the isolate DM is capable of infecting insulin-producing beta-cells like the corresponding prototype strains did. However, only isolate DM was clearly cytolytic for beta-cells. The infectious clones that were made allow further investigations of the molecular features responsible for the diabetogenicity of the isolate DM.
