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Varpu Marjomäki - One of the best experts on this subject based on the ideXlab platform.
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early entry events in echovirus 30 infection
Journal of Virology, 2020Co-Authors: Helena Vandesande, Michael A Lindberg, Mira Laajala, Tino Kantoluoto, Visa Ruokolainen, Varpu MarjomäkiAbstract:Echovirus 30 (E30), a memBer of the <B>EnterovirusB> B species, is a major cause of viral meningitis, targeting children and adults alike. While it is a frequently isolated <B>EnterovirusB> and the cause of several outBreaks all over the world, suprisingly little is known regarding its entry and replication strategy within cells. In this study, we used E30 Bastianni (E30B) generated from an infectious cDNA clone in order to study early entry events during infection in human RD cells. E30B required the newly discovered Fc echovirus receptor (FcRn) for succesful infection, But not the Coxsackievirus and Adenovirus Receptor (CAR) or Decay-Accelerating Factor (DAF), although an interaction with DAF was oBserved. DouBle-stranded RNA replication intermediate was generated Between 2 and 3 h post-infection (p.i.). and viral capsid production was initiated Between 4 and 5 h p.i. The drugs affecting Rac1 (NSC 23766) and cholesterol (Filipin III) compromised infection, whereas Bafilomycin A1, dyngo, U-73122, wortmannin and nocodazole did not, suggesting the virus follows an <B>EnterovirusB>-triggered macropinocytic pathway rather than the clathrin pathway. Colocalization with early endosomes and increased infection due to constitutively active RaB5 expression suggests some overlap and entry to classical early endosomes. Taken together, these results suggest that E30B induces an <B>EnterovirusB> entry pathway, leading to uncoating in early endosomes.IMPORTANCE Echovirus 30 (E30) is a prevalent <B>EnterovirusB> causing regular outBreaks in Both children and adults in different parts of the world. It is therefore surprising that relatively little is known of its infectious entry pathway. We set out to generate a cDNA clone and gradient-purified the virus in order to study the early entry events in human cells. We have recently studied other <B>EnterovirusB> B group viruses, like echovirus 1 (EV1) and coxsackievirus A9 (CVA9), and found many similarities Between those viruses, allowing us to define a so-called "<B>EnterovirusB> entry pathway". Here, E30 is reminiscent of these viruses, e.g. By not relying on acidification for infectious entry. However, despite not using the clathrin entry pathway, E30 accumulates in classical early endosomes.
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Review Infectious Entry Pathway of <B>EnterovirusB> B Species
2016Co-Authors: Varpu Marjomäki, Paula Turkki, Moona Huttunen, Species B EnterovirusesAbstract:ABstract: <B>EnterovirusB> B species (EV-B) are responsiBle for a vast numBer of mild and serious acute infections. They are also suspected of remaining in the Body, where they cause persistent infections contriButing to chronic diseases such as type I diaBetes. Recent studies of the infectious entry pathway of these viruses revealed remarkaBle similarities, including non-clathrin entry of large endosomes originating from the plasma memBrane invaginations. Many cellular factors regulating the efficient entry have recently Been associated with macropinocytic uptake, such as Rac1, serine/threonine p21-activated kinase (Pak1), actin, Na/H exchanger, phospholipace C (PLC) and protein kinase Cα (PKCα). Another characteristic feature is the entry of these viruses to neutral endosomes, independence of endosomal acidification and low association with acidic lysosomes. The Biogenesis of neutral multivesicular Bodies is crucial for their infection, at least for echovirus 1 (E1) and coxsackievirus A9 (CVA9). These pathways are triggered By the virus Binding to their receptors on the plasma memBrane, and they are not efficiently recycled like other cellular pathways used By circulating receptors. Therefore, the Best “markers ” of these pathways may Be the viruses and often their receptors. A deeper understanding of this pathway and associated endosomes is crucial in elucidating the mechanisms of <B>EnterovirusB> uncoating and genome release from the endosomes to start efficient replication
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Infectious Entry Pathway of <B>EnterovirusB> B Species
MDPI AG, 2015Co-Authors: Varpu Marjomäki, Paula Turkki, Moona HuttunenAbstract:<B>EnterovirusB> B species (EV-B) are responsiBle for a vast numBer of mild and serious acute infections. They are also suspected of remaining in the Body, where they cause persistent infections contriButing to chronic diseases such as type I diaBetes. Recent studies of the infectious entry pathway of these viruses revealed remarkaBle similarities, including non-clathrin entry of large endosomes originating from the plasma memBrane invaginations. Many cellular factors regulating the efficient entry have recently Been associated with macropinocytic uptake, such as Rac1, serine/threonine p21-activated kinase (Pak1), actin, Na/H exchanger, phospholipace C (PLC) and protein kinase Cα (PKCα). Another characteristic feature is the entry of these viruses to neutral endosomes, independence of endosomal acidification and low association with acidic lysosomes. The Biogenesis of neutral multivesicular Bodies is crucial for their infection, at least for echovirus 1 (E1) and coxsackievirus A9 (CVA9). These pathways are triggered By the virus Binding to their receptors on the plasma memBrane, and they are not efficiently recycled like other cellular pathways used By circulating receptors. Therefore, the Best “markers” of these pathways may Be the viruses and often their receptors. A deeper understanding of this pathway and associated endosomes is crucial in elucidating the mechanisms of <B>EnterovirusB> uncoating and genome release from the endosomes to start efficient replication
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internalization of coxsackievirus a9 is mediated By β2 microgloBulin dynamin and arf6 But not By caveolin 1 or clathrin
Journal of Virology, 2010Co-Authors: Outi Heikkila, Petri Susi, Varpu Marjomäki, Tuire Tevaluoto, Heidi Harma, Timo Hyypia, Saija KiljunenAbstract:Coxsackievirus A9 (CAV9) is a memBer of the human <B>EnterovirusB> B species within the <B>EnterovirusB> genus of the family Picornaviridae . It has Been shown to utilize αV integrins, particularly αVβ6, as its receptors. The endocytic pathway By which CAV9 enters human cells after the initial attachment to the cell surface has so far Been unknown. Here, we present a systematic study concerning the internalization mechanism of CAV9 to A549 human lung carcinoma cells. The small interfering RNA (siRNA) silencing of integrin β6 suBunit inhiBited virus proliferation, confirming that αVβ6 mediates the CAV9 infection. However, siRNAs against integrin-linked signaling molecules, such as Src, Fyn, RhoA, phosphatidylinositol 3-kinase, and Akt1, did not reduce CAV9 proliferation, suggesting that the internalization of the virus does not involve integrin-linked signaling events. CAV9 endocytosis was independent of clathrin or caveolin-1 But was restrained By dynasore, an inhiBitor of dynamin. The RNA interference silencing of β2-microgloBulin efficiently inhiBited virus infection and caused CAV9 to accumulate on the cell surface. Furthermore, CAV9 infection was found to depend on Arf6 as Both silencing of this molecule By siRNA and the expression of a dominant negative construct resulted in decreased virus infection. In conclusion, the internalization of CAV9 to A549 cells follows an endocytic pathway that is dependent on integrin αVβ6, β2-microgloBulin, dynamin, and Arf6 But independent of clathrin and caveolin-1.
Paul Fornes - One of the best experts on this subject based on the ideXlab platform.
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<B>EnterovirusB> persistence in cardiac cells of patients with idiopathic dilated cardiomyopathy is linked to 5 terminal genomic rna deleted viral populations with viral encoded proteinase activities
Circulation, 2019Co-Authors: Alexis Bouin, Paulantoine Gretteau, Michel Wehbe, Fanny Renois, Yohan Nguyen, Nicolas Leveque, Steven Tracy, Nora M Chapman, Patrick Bruneval, Paul FornesAbstract:BACKGROUND: Group B <B>EnterovirusB>es are common causes of acute myocarditis, which can Be a precursor of chronic myocarditis and dilated cardiomyopathy, leading causes of heart transplantation. To date, the specific viral functions involved in the development of dilated cardiomyopathy remain unclear. METHODS: Total RNA from cardiac tissue of patients with dilated cardiomyopathy was extracted, and sequences corresponding to the 5' termini of <B>EnterovirusB> RNAs were identified. After next-generation RNA sequencing, viral cDNA clones mimicking the <B>EnterovirusB> RNA sequences found in patient tissues were generated in vitro, and their replication and impact on host cell functions were assessed on primary human cardiac cells in culture. RESULTS: Major <B>EnterovirusB> B populations characterized By 5' terminal genomic RNA deletions ranging from 17 to 50 nucleotides were identified either alone or associated with low proportions of intact 5' genomic termini. In situ hyBridization and immunohistological assays detected these persistent genomes in clusters of cardiomyocytes. Transfection of viral RNA into primary human cardiomyocytes demonstrated that deleted forms of genomic RNAs displayed early replication activities in the aBsence of detectaBle viral plaque formation, whereas mixed deleted and complete forms generated particles capaBle of inducing cytopathic effects at levels distinct from those oBserved with full-length forms alone. Moreover, deleted or full-length and mixed forms of viral RNA were capaBle of directing translation and production of proteolytically active viral proteinase 2A in human cardiomyocytes. CONCLUSIONS: We demonstrate that persistent viral forms are composed of B-type <B>EnterovirusB>es harBoring a 5' terminal deletion in their genomic RNAs and that these viruses alone or associated with full-length populations of helper RNAs could impair cardiomyocyte functions By the proteolytic activity of viral proteinase 2A in cases of unexplained dilated cardiomyopathy. These results provide a Better understanding of the molecular mechanisms that underlie the persistence of EV forms in human cardiac tissues and should stimulate the development of new therapeutic strategies Based on specific inhiBitors of the coxsackievirus B proteinase 2A activity for acute and chronic cardiac infections.
Michael A Lindberg - One of the best experts on this subject based on the ideXlab platform.
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early entry events in echovirus 30 infection
Journal of Virology, 2020Co-Authors: Helena Vandesande, Michael A Lindberg, Mira Laajala, Tino Kantoluoto, Visa Ruokolainen, Varpu MarjomäkiAbstract:Echovirus 30 (E30), a memBer of the <B>EnterovirusB> B species, is a major cause of viral meningitis, targeting children and adults alike. While it is a frequently isolated <B>EnterovirusB> and the cause of several outBreaks all over the world, suprisingly little is known regarding its entry and replication strategy within cells. In this study, we used E30 Bastianni (E30B) generated from an infectious cDNA clone in order to study early entry events during infection in human RD cells. E30B required the newly discovered Fc echovirus receptor (FcRn) for succesful infection, But not the Coxsackievirus and Adenovirus Receptor (CAR) or Decay-Accelerating Factor (DAF), although an interaction with DAF was oBserved. DouBle-stranded RNA replication intermediate was generated Between 2 and 3 h post-infection (p.i.). and viral capsid production was initiated Between 4 and 5 h p.i. The drugs affecting Rac1 (NSC 23766) and cholesterol (Filipin III) compromised infection, whereas Bafilomycin A1, dyngo, U-73122, wortmannin and nocodazole did not, suggesting the virus follows an <B>EnterovirusB>-triggered macropinocytic pathway rather than the clathrin pathway. Colocalization with early endosomes and increased infection due to constitutively active RaB5 expression suggests some overlap and entry to classical early endosomes. Taken together, these results suggest that E30B induces an <B>EnterovirusB> entry pathway, leading to uncoating in early endosomes.IMPORTANCE Echovirus 30 (E30) is a prevalent <B>EnterovirusB> causing regular outBreaks in Both children and adults in different parts of the world. It is therefore surprising that relatively little is known of its infectious entry pathway. We set out to generate a cDNA clone and gradient-purified the virus in order to study the early entry events in human cells. We have recently studied other <B>EnterovirusB> B group viruses, like echovirus 1 (EV1) and coxsackievirus A9 (CVA9), and found many similarities Between those viruses, allowing us to define a so-called "<B>EnterovirusB> entry pathway". Here, E30 is reminiscent of these viruses, e.g. By not relying on acidification for infectious entry. However, despite not using the clathrin entry pathway, E30 accumulates in classical early endosomes.
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virus derived from an <B>EnterovirusB> B construct efficiently reverts from a frameshift mutation immediately Beyond the translation initiation site
2015Co-Authors: Anna Sävneby, Nina Jonsson, Andreas P Svensson, Susan Hafenstein, Michael A LindbergAbstract:Virus derived from an <B>EnterovirusB> B construct efficiently reverts from a frameshift mutation immediately Beyond the translation initiation site
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analysis of the serotype and genotype correlation of vp1 and the 5 noncoding region in an epidemiological survey of the human <B>EnterovirusB> B species
Journal of Clinical Microbiology, 2004Co-Authors: Inge Thoelen, Michael A Lindberg, Elien Moes, Philippe Lemey, Sara Mostmans, Elke Wollants, Annemieke Vandamme, Marc Van RanstAbstract:The sequence identity of the <B>EnterovirusB> VP1 gene has Been shown to correlate with the serotype concept. <B>EnterovirusB> molecular typing methods are therefore often Based on sequencing of the VP1 genomic region and monophyletic clustering of VP1 sequences of a homologous serotype. For epidemiological surveillance, 342 <B>EnterovirusB> samples oBtained from patients with aseptic meningitis in Belgium from 1999 to 2002 were first diagnosed as Being <B>EnterovirusB> positive By amplification of the 5' noncoding region (5'NCR) By reverse transcription (RT)-PCR. SuBsequently, samples were molecularly typed By RT-nested PCR amplification and sequencing of a portion of the VP1 gene. Phylogenetic analyses were performed to investigate enteroviral evolution and to examine the serotype and genotype correlation of the two genomic regions. Our typing results demonstrated echovirus 30, echovirus 13, echovirus 18, and echovirus 6 to Be the most predominant types. Echoviruses 13 and 18 were considered to Be emerging human serotypes since 2000 and 2001, respectively, as they had Been rarely reported Before. Several serotypes existed as multiple genotypes (suBtypes) from 1999 to 2002, But genomic differences mainly resided at synonymous sites; these results strongly suggest that the suBtypes exhiBit similar antigenic properties. Phylogenetic analyses confirmed that VP1 is an adequate region for molecular typing. Serotype-specific clusters are not oBserved commonly in phylogenetic trees Based on the 5'NCR, and the phylogenetic signal in the 5'NCR was found to Be particularly low. However, some suBstructure in the 5'NCR tree made a tentative prediction of the <B>EnterovirusB> type possiBle and was therefore helpful in PCR strategies for VP1 (e.g., primer choice), provided some Background knowledge on the local spectrum of <B>EnterovirusB>es already exists.
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evolution of the genome of human <B>EnterovirusB> B incongruence Between phylogenies of the vp1 and 3cd regions indicates frequent recomBination within the species
Journal of General Virology, 2003Co-Authors: Michael A Lindberg, Per Andersson, Carita Savolainen, Mick N Mulders, Tapani HoviAbstract:<B>EnterovirusB>es show a high degree of sequence variation Both Between and within serotypes due to the lack of proofreading of the viral RNA-dependent RNA polymerase. In addition, recomBination is known to occur not only within But also Between different serotypes. We have previously shown that capsid coding sequences of coxsackievirus B4 (CVB4) cluster in several coexisting genotypes (intergenotypic nucleotide difference of 12 % or more) whereas a single lineage of echovirus 30 (EV30) has Been prevailing and evolving throughout the last two decades. In the major capsid gene, VP1, clustering of Both nucleotide and amino acid sequences correlates with serotype. We have now determined a 501 nucleotide sequence in the non-structural 3CD region of CVB4 and EV30 field strains. Phylogenetic analysis revealed that sequences of Human <B>EnterovirusB> B (HEV-B) were segregated in the 3CD region into three distinct clusters without the VP1-associated serotype/genotype correlation. One of the clusters comprised the E2 strain of CVB4, the EV30 prototype and five other CVB4 field strains whereas the other two clusters, in addition to CVB4 and EV30 strains, also included other HEV-B serotypes. We Believe that intertypic recomBination is the most likely explanation for the oBserved incongruence. Similarity analysis Based on complete genomes of the CVB4 and EV30 prototypes and the CVB4 E2 strain revealed that a putative recomBination spot was mapped within the 2B gene. The incongruence oBserved in the two genomic domains (P1 and P3) suggests a certain degree of independent evolution, which may Be explained By interserotypic recomBination within an <B>EnterovirusB> species. It is thus difficult to exclude recomBination in the history of any given strain.
Thomas M P Gilbert - One of the best experts on this subject based on the ideXlab platform.
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viral meningitis epidemics and a single recent recomBinant and anthroponotic origin of swine vesicular disease virus
Evolution Medicine and Public Health, 2015Co-Authors: Nick J. Knowles, Christian A W Bruhn, Sandra C A Nielsen, Jose Alfredo Samaniego, Jemma Wadsworth, Thomas M P GilbertAbstract:Background and oBjectives: Swine vesicular disease virus (SVDV) is a close relative of the human <B>EnterovirusB> B serotype, coxsackievirus B5. As the etiological agent of a significant emergent veterinary disease, several studies have attempted to explain its origin. However, several key questions remain, including the full Biological ancestry of the virus, and its geographical and temporal origin. Methodology: We sequenced near-complete genomes of 27 SVDV and 13 coxsackievirus B5 samples, all originally isolated Between 1966 and 2006, and analysed these in conjunction with existing sequences and historical information. Results: While analyses incorporating 24 additional near-complete SVDV genomic sequences indicate clear signs of within-SVDV recomBination, all 51 SVDV isolates remain monophyletic. This supports a hypothesis of a single anthroponotic transfer origin. Analysis of individual coding and non-coding regions supports that SVDV has a recomBinant origin Between coxsackievirus B5 and another <B>EnterovirusB> B serotype, most likely coxsackievirus A9. Extensive Bayesian sequence-Based analysis of the time of the most recent common ancestor of all analysed sequences places this within a few years around 1961. Epidemiological evidence points to China as an origin, But there are no availaBle samples to test this conclusively. Conclusions and implications: Historical investigation and the clinical aspects of the involved <B>EnterovirusB> B serotypes, makes the current results consistent with a hypothesis stating that SVDV originated through co-infection, recomBination, and a single anthroponotic event, during large viral meningitis epidemics around 1960/1961 involving the ancestral serotypes. The exact geographical origin of SVDV may remain untestaBle due to historical aspects.
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near complete genome sequencing of swine vesicular disease virus using the roche gs flx sequencing platform
PLOS ONE, 2014Co-Authors: Sandra C A Nielsen, Nick J. Knowles, Christian A W Bruhn, Jose Alfredo Samaniego, Jemma Wadsworth, Thomas M P GilbertAbstract:Swine vesicular disease virus (SVDV) is an <B>EnterovirusB> that is Both genetically and antigenically closely related to human coxsackievirus B5 within the Picornaviridae family. SVDV is the causative agent of a highly contagious (though rarely fatal) vesicular disease in pigs. We report a rapid method that is suitaBle for sequencing the complete protein-encoding sequences of SVDV isolates in which the RNA is relatively intact. The approach couples a single PCR amplification reaction, using only a single PCR primer set to amplify the near-complete SVDV genome, with deep-sequencing using a small fraction of the capacity of a Roche GS FLX sequencing platform. Sequences were initially verified through one of two criteria; either a match Between a de novo assemBly and a reference mapping, or a match Between all of five different reference mappings performed against a fixed set of starting reference genomes with significant genetic distances within the same species of viruses. All reference mappings used an iterative method to avoid Bias. Further verification was achieved through phylogenetic analysis against puBlished SVDV genomes and additional <B>EnterovirusB> B sequences. This approach allows high confidence in the oBtained consensus sequences, as well as provides sufficiently high and evenly dispersed sequence coverage to allow future studies of intra-host variation.
Helena Vandesande - One of the best experts on this subject based on the ideXlab platform.
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early entry events in echovirus 30 infection
Journal of Virology, 2020Co-Authors: Helena Vandesande, Michael A Lindberg, Mira Laajala, Tino Kantoluoto, Visa Ruokolainen, Varpu MarjomäkiAbstract:Echovirus 30 (E30), a memBer of the <B>EnterovirusB> B species, is a major cause of viral meningitis, targeting children and adults alike. While it is a frequently isolated <B>EnterovirusB> and the cause of several outBreaks all over the world, suprisingly little is known regarding its entry and replication strategy within cells. In this study, we used E30 Bastianni (E30B) generated from an infectious cDNA clone in order to study early entry events during infection in human RD cells. E30B required the newly discovered Fc echovirus receptor (FcRn) for succesful infection, But not the Coxsackievirus and Adenovirus Receptor (CAR) or Decay-Accelerating Factor (DAF), although an interaction with DAF was oBserved. DouBle-stranded RNA replication intermediate was generated Between 2 and 3 h post-infection (p.i.). and viral capsid production was initiated Between 4 and 5 h p.i. The drugs affecting Rac1 (NSC 23766) and cholesterol (Filipin III) compromised infection, whereas Bafilomycin A1, dyngo, U-73122, wortmannin and nocodazole did not, suggesting the virus follows an <B>EnterovirusB>-triggered macropinocytic pathway rather than the clathrin pathway. Colocalization with early endosomes and increased infection due to constitutively active RaB5 expression suggests some overlap and entry to classical early endosomes. Taken together, these results suggest that E30B induces an <B>EnterovirusB> entry pathway, leading to uncoating in early endosomes.IMPORTANCE Echovirus 30 (E30) is a prevalent <B>EnterovirusB> causing regular outBreaks in Both children and adults in different parts of the world. It is therefore surprising that relatively little is known of its infectious entry pathway. We set out to generate a cDNA clone and gradient-purified the virus in order to study the early entry events in human cells. We have recently studied other <B>EnterovirusB> B group viruses, like echovirus 1 (EV1) and coxsackievirus A9 (CVA9), and found many similarities Between those viruses, allowing us to define a so-called "<B>EnterovirusB> entry pathway". Here, E30 is reminiscent of these viruses, e.g. By not relying on acidification for infectious entry. However, despite not using the clathrin entry pathway, E30 accumulates in classical early endosomes.