The Experts below are selected from a list of 474 Experts worldwide ranked by ideXlab platform
Peter Simmonds - One of the best experts on this subject based on the ideXlab platform.
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viral persistence liver disease and host response in a hepatitis c like virus rat model
Hepatology, 2018Co-Authors: Sheetal Trivedi, Troels K H Scheel, Peter Simmonds, Arvind Kumar, Lokendra V. Chauhan, Alex S Hartlage, Satyapramod Murthy, Himanshu Sharma, Sashi V Gadi, Eva BillerbeckAbstract:The lack of a relevant, tractable, and immunocompetent animal model for hepatitis C virus (HCV) has severely impeded investigations of viral persistence, immunity and pathogenesis. In the absence of immunocompetent models with robust HCV infection, homolog Hepaciviruses in their natural host could potentially provide useful surrogate models. We isolated a rodent hepacivirus (RHV) from wild rats (Rattus norvegicus), RHV-rn1, acquired the complete viral genome sequence and developed an infectious reverse genetics system. RHV-rn1 resembles HCV in genomic features including the pattern of polyprotein cleavage sites and secondary structures in the viral 5' and 3' UTRs. We used site-directed and random mutagenesis to determine that only the first of the two miR-122 seed sites in viral 5'UTR is required for viral replication and persistence in rats. Next, we used the clone derived virus progeny to infect several inbred and outbred rat strains. Our results determined that RHV-rn1 possesses several HCV-defining hallmarks: hepatotropism, propensity to persist, and the ability of induce gradual liver damage. Histological examination of liver samples revealed the presence of lymphoid aggregates, parenchymal inflammation and macro/micro vesicular steatosis in chronically infected rats. Gene expression analysis demonstrated that the intrahepatic response during RHV-rn1 infection in rats mirrors that of HCV infection, including persistent activation of interferon signaling pathways. Finally, we determined that the backbone drug of HCV direct acting antiviral (DAA) therapy, Sofosbuvir, effectively suppresses chronic RHV-rn1 infection in rats. Taken together, we developed RHV-rn1 infected rats as a fully immunocompetent and informative surrogate model to delineate the mechanisms of HCV-related viral persistence, immunity and pathogenesis. This article is protected by copyright. All rights reserved.
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Virome Analysis of Transfusion Recipients Reveals a Novel Human Virus That Shares Genomic Features with Hepaciviruses and Pegiviruses
mBio, 2015Co-Authors: Amit Kapoor, Peter Simmonds, Arvind Kumar, Nishit Bhuva, Lokendra V. Chauhan, Bohyun Lee, Amadou A. Sall, Zhezhen Jin, Stephen S. Morse, Beth H. ShazAbstract:To investigate the transmission of novel infectious agents by blood transfusion, we studied changes in the virome composition of blood transfusion recipients pre- and posttransfusion. Using this approach, we detected and genetically charac- terized a novel human virus, human hepegivirus 1 (HHpgV-1), that shares features with hepatitis C virus (HCV) and human pegivirus (HPgV; formerly called GB virus C or hepatitis G virus). HCV and HPgV belong to the genera Hepacivirus andPegivi- rusof the familyFlaviviridae. HHpgV-1 was found in serum samples from two blood transfusion recipients and two hemophilia patients who had received plasma-derived clotting factor concentrates. In the former, the virus was detected only in the post- transfusion samples, indicating blood-borne transmission. Both hemophiliacs were persistently viremic over periods of at least 201 and 1,981 days. The 5=untranslated region (UTR) of HHpgV-1 contained a type IV internal ribosome entry site (IRES), structurally similar to although highly divergent in sequence from that of HCV and other Hepaciviruses. However, phylogenetic analysis of nonstructural genes (NS3 and NS5B) showed that HHpgV-1 forms a branch within the pegivirus clade distinct from HPgV and homologs infecting other mammalian species. In common with some pegivirus variants infecting rodents and bats, the HHpgV-1 genome encodes a short, highly basic protein upstream of E1, potentially possessing a core-like function in packag- ing RNA during assembly. Identification of this new human virus, HHpgV-1, expands our knowledge of the range of genome configurations of these viruses and may lead to a reevaluation of the original criteria by which the genera Hepacivirus andPegivi- rusare defined. IMPORTANCE More than 30 million blood components are transfused annually in the United States alone. Surveillance for infec- tious agents in the blood supply is key to ensuring the safety of this critical resource for medicine and public health. Here, we report the identification of a new and highly diverse HCV/GB virus (GBV)-like virus from human serum samples. This new vi- rus, human hepegivirus 1 (HHpgV-1), was found in serum samples from blood transfusion recipients, indicating its potential for transmission via transfusion products. We also found persistent long-term HHpgV-1 viremia in two hemophilia patients. HHpgV-1 is unique because it shares genetic similarity with both highly pathogenic HCV and the apparently nonpathogenic HPgV (GBV-C). Our results add to the list of human viruses and provide data to develop reagents to study virus transmission and disease association and for interrupting virus transmission and new human infections.
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Surveying the global virome: Identification and characterization of HCV-related animal Hepaciviruses
Antiviral Research, 2014Co-Authors: Troels K H Scheel, Peter Simmonds, Amit KapoorAbstract:Abstract Recent advances in sequencing technologies have greatly enhanced our abilities to identify novel microbial sequences. Thus, our understanding of the global virome and the virome of specific host species in particular is rapidly expanding. Identification of animal viruses is important for understanding animal disease, the origin and evolution of human viruses, as well as zoonotic reservoirs for emerging infections. Although the human hepacivirus, hepatitis C virus (HCV), was identified 25 years ago, its origin has remained elusive. In 2011, the first HCV homolog was reported in dogs but subsequent studies showed the virus to be widely distributed in horses. This indicated a wider hepacivirus host range and paved the way for identification of rodent, bat and non-human primate Hepaciviruses. The equine non-primate hepacivirus (NPHV) remains the closest relative of HCV and is so far the best characterized. Identification and characterization of novel Hepaciviruses may in addition lead to development of tractable animal models to study HCV persistence, immune responses and pathogenesis. This could be particular important, given the current shortage of immunocompetent models for robust HCV infection. Much remains to be learned on the novel Hepaciviruses, including their association with disease, and thereby how relevant they will become as HCV model systems and for studies of animal disease. This review discusses how virome analysis led to identification of novel hepaci- and pegiviruses, their genetic relationship and characterization and the potential use of animal Hepaciviruses as models to study hepaciviral infection, immunity and pathogenesis. This article forms part of a symposium in Antiviral Research on “Hepatitis C: Next steps toward global eradication.”
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detection of zoonotic pathogens and characterization of novel viruses carried by commensal rattus norvegicus in new york city
Mbio, 2014Co-Authors: Cadhla Firth, Simon H. Williams, Peter Simmonds, Juliette M Conte, Meera Bhat, Matthew A Firth, Matthew J Frye, James Ng, Joel A Garcia, Nishit BhuvaAbstract:Norway rats (Rattus norvegicus) are globally distributed and concentrate in urban environments, where they live and feed in closer proximity to human populations than most other mammals. Despite the potential role of rats as reservoirs of zoo- notic diseases, the microbial diversity present in urban rat populations remains unexplored. In this study, we used targeted mo- lecular assays to detect known bacterial, viral, and protozoan human pathogens and unbiased high-throughput sequencing to identify novel viruses related to agents of human disease in commensal Norway rats in New York City. We found that these rats are infected with bacterial pathogens known to cause acute or mild gastroenteritis in people, including atypical enteropatho- genic Escherichia coli, Clostridium difficile, and Salmonella enterica, as well as infectious agents that have been associated with undifferentiated febrile illnesses, including Bartonella spp., Streptobacillus moniliformis, Leptospira interrogans, and Seoul han- tavirus. We also identified a wide range of known and novel viruses from groups that contain important human pathogens, in- cluding sapoviruses, cardioviruses, kobuviruses, parechoviruses, rotaviruses, and Hepaciviruses. The two novel Hepaciviruses discovered in this study replicate in the liver of Norway rats and may have utility in establishing a small animal model of human hepatitis C virus infection. The results of this study demonstrate the diversity of microbes carried by commensal rodent species and highlight the need for improved pathogen surveillance and disease monitoring in urban environments. IMPORTANCE The observation that most emerging infectious diseases of humans originate in animal reservoirs has led to wide- scale microbial surveillance and discovery programs in wildlife, particularly in the developing world. Strikingly, less attention has been focused on commensal animals like rats, despite their abundance in urban centers and close proximity to human popu- lations. To begin to explore the zoonotic disease risk posed by urban rat populations, we trapped and surveyed Norway rats col- lected in New York City over a 1-year period. This analysis revealed a striking diversity of known pathogens and novel viruses in our study population, including multiple agents associated with acute gastroenteritis or febrile illnesses in people. Ourfindings indicate that urban rats are reservoirs for a vast diversity of microbes that may affect human health and indicate a need for in- creased surveillance and awareness of the disease risks associated with urban rodent infestation.
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the non primate hepacivirus 5 untranslated region possesses internal ribosomal entry site activity
Journal of General Virology, 2013Co-Authors: Hazel Stewart, Cheryl T Walter, Dale Jones, Peter Simmonds, Sinead Lyons, Mark HarrisAbstract:The 5′ untranslated region (5′UTR) of the recently described non-primate hepacivirus (NPHV) contains a region with sequence homology to the internal ribosomal entry site (IRES) of hepatitis C virus (HCV) and GB virus B (GBV-B). Here, we demonstrated internal translation initiation by the NPHV 5′UTR in a bicistronic vector. An RNA stem–loop upstream of the NPHV IRES was structurally distinct from corresponding regions in HCV and GBV-B, and was not required for IRES function. Insertion of the NPHV stem–loop into the corresponding region of the HCV 5′UTR within the HCV subgenomic replicon significantly impaired RNA replication, indicating that long-range interactions between the 5′UTR and cis-acting downstream elements within the NPHV genome are not interchangeable with those of HCV. Despite similarities in IRES structure and function between Hepaciviruses, replication elements in the NPHV 5′UTR appear functionally distinct from those of HCV.
Annette Martin - One of the best experts on this subject based on the ideXlab platform.
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NS2 proteases from hepatitis C virus and related Hepaciviruses share composite active sites and previously unrecognized intrinsic proteolytic activities
PLOS Pathogens, 2018Co-Authors: Célia Boukadida, Brigitte Blumen, Matthieu Fritz, Marie-laure Fogeron, Francois Penin, Annette MartinAbstract:Over the recent years, several homologues with varying degrees of genetic relatedness to hepatitis C virus (HCV) have been identified in a wide range of mammalian species. HCV infectious life cycle relies on a first critical proteolytic event of its single polyprotein, which is carried out by nonstructural protein 2 (NS2) and allows replicase assembly and genome replication. In this study, we characterized and evaluated the conservation of the proteolytic mode of action and regulatory mechanisms of NS2 across HCV and animal Hepaciviruses. We first demonstrated that NS2 from equine, bat, rodent, New and Old World primate Hepaciviruses also are cysteine proteases. Using tagged viral protein precursors and catalytic triad mutants, NS2 of equine NPHV and simian GBV-B, which are the most closely and distantly related viruses to HCV, respectively, were shown to function, like HCV NS2 as dimeric proteases with two composite active sites. Consistent with the reported essential role for NS3 N-terminal domain (NS3N) as HCV NS2 protease cofactor via NS3N key hydrophobic surface patch, we showed by gain/loss of function mutagenesis studies that some heterologous hepacivirus NS3N may act as cofactors for HCV NS2 provided that HCV-like hydrophobic residues are conserved. Unprecedently, however, we also observed efficient intrinsic proteolytic activity of NS2 protease in the absence of NS3 moiety in the context of C-terminal tag fusions via flexible linkers both in transiently transfected cells for all Hepaciviruses studied and in the context of HCV dicistronic full-length genomes. These findings suggest that NS3N acts as a regulatory rather than essential cofactor for hepacivirus NS2 protease. Overall, unique features of NS2 including enzymatic function as dimers with two composite active sites and additional NS3-independent proteolytic activity are conserved across Hepaciviruses regardless of their genetic distances, highlighting their functional significance in hepacivirus life cycle.
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Summary of NS2 protease characteristics across various mammalian Hepaciviruses.
2018Co-Authors: Célia Boukadida, Brigitte Blumen, Matthieu Fritz, Marie-laure Fogeron, Francois Penin, Annette MartinAbstract:Summary of NS2 protease characteristics across various mammalian Hepaciviruses.
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Functionality of NS2 proteases from various Hepaciviruses in the absence of NS3N domain.
2018Co-Authors: Célia Boukadida, Brigitte Blumen, Matthieu Fritz, Marie-laure Fogeron, Francois Penin, Annette MartinAbstract:Cells were transfected with pCMV/NS2hepaci-4GS-GFP DNAs (A), allowing the transient expression of NS2 from the indicated HCV strains (subtypes in parentheses) (B) or Hepaciviruses (C). NS2 proteases comprised either the native catalytic triad (wt) or an Ala substitution of the catalytic Cys residue (CA). Uncleaved precursors and cleaved products were immunodetected with anti-GFP antibodies following SDS-PAGE separation of transfected cell extracts and are indicated by closed and open arrowheads, respectively.
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Role and virus specificity of HCV NS3N domain as NS2 protease cofactor.
2018Co-Authors: Célia Boukadida, Brigitte Blumen, Matthieu Fritz, Marie-laure Fogeron, Francois Penin, Annette MartinAbstract:(A) Schematic representation of the chimeric NS2HCV-NS3Nhepaci-ST precursors. Precursors spanning NS2 from HCV-JFH1 and NS3N from NPHV, BHV, GHV, RHV or GBV-B (hepaci) were expressed downstream of a heterologous signal peptide (sp) and C-terminally fused to Strep-tag (ST). The sequence alignment of NS3 N-terminal sequences from the various Hepaciviruses is depicted in the blown-up scheme with respect to HCV corresponding sequence, where identical residues are indicated by dots. (B) Extracts from cells transfected with pCMV/NS2HCV(wt)-NS3Nhepaci-ST or pCMV/NS2HCV(CA)-NS3Nhepaci-ST DNAs encoding NS2 protease with either native (wt) or mutated (CA) catalytic triad, respectively, were probed with anti-ST antibodies. Uncleaved precursors and cleaved products are indicated by closed and open arrowheads, respectively. The decreasing overall sequence similarity of hepacivirus NS3N with respect to HCV NS3N is represented by a grey triangle.
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NS2 Proteins of GB Virus B and Hepatitis C Virus Share Common Protease Activities and Membrane Topologies.
Journal of Virology, 2014Co-Authors: Célia Boukadida, Brigitte Blumen, Jerome Gouttenoire, Francois Penin, Darius Moradpour, Roland Montserret, Caroline Marnata, Lisette Cohen, Annette MartinAbstract:: GB virus B (GBV-B), which is hepatotropic in experimentally infected small New World primates, is a member of the Hepacivirus genus but phylogenetically relatively distant from hepatitis C virus (HCV). To gain insights into the role and specificity of hepaciviral nonstructural protein 2 (NS2), which is required for HCV polyprotein processing and particle morphogenesis, we investigated whether NS2 structural and functional features are conserved between HCV and GBV-B. We found that GBV-B NS2, like HCV NS2, has cysteine protease activity responsible for cleavage at the NS2/NS3 junction, and we experimentally confirmed the location of this junction within the viral polyprotein. A model for GBV-B NS2 membrane topology was experimentally established by determining the membrane association properties of NS2 segments fused to green fluorescent protein (GFP) and their nuclear magnetic resonance structures using synthetic peptides as well as by applying an N-glycosylation scanning approach. Similar glycosylation studies confirmed the HCV NS2 organization. Together, our data show that despite limited amino acid sequence similarity, GBV-B and HCV NS2 proteins share a membrane topology with 3 N-terminal transmembrane segments, which is also predicted to apply to other recently discovered Hepaciviruses. Based on these data and using trans-complementation systems, we found that intragenotypic hybrid NS2 proteins with heterologous N-terminal membrane segments were able to efficiently trans-complement an assembly-deficient HCV mutant with a point mutation in the NS2 C-terminal domain, while GBV-B/HCV or intergenotypic NS2 chimeras were not. These studies indicate that virus- and genotype-specific intramolecular interactions between N- and C-terminal domains of NS2 are critically involved in HCV morphogenesis. IMPORTANCE: Nonstructural protein 2 (NS2) of hepatitis C virus (HCV) is a multifunctional protein critically involved in polyprotein processing and virion morphogenesis. To gain insights into NS2 mechanisms of action, we investigated whether NS2 structural and functional features are conserved between HCV and GB virus B (GBV-B), a phylogenetically relatively distant primate hepacivirus. We showed that GBV-B NS2, like HCV NS2, carries cysteine protease activity. We experimentally established a model for GBV-B NS2 membrane topology and demonstrated that despite limited sequence similarity, GBV-B and HCV NS2 share an organization with three N-terminal transmembrane segments. We found that the role of HCV NS2 in particle assembly is genotype specific and relies on critical interactions between its N- and C-terminal domains. This first comparative analysis of NS2 proteins from two Hepaciviruses and our structural predictions of NS2 from other newly identified mammal Hepaciviruses highlight conserved key features of the hepaciviral life cycle.
Hans C Van Leeuwen - One of the best experts on this subject based on the ideXlab platform.
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hepatitis c virus ns4b carboxy terminal domain is a membrane binding domain
Virology Journal, 2009Co-Authors: Jolanda M P Liefhebber, Bernd W Brandt, Rene Broer, Willy J M Spaan, Hans C Van LeeuwenAbstract:Background Hepatitis C virus (HCV) induces membrane rearrangements during replication. All HCV proteins are associated to membranes, pointing out the importance of membranes for HCV. Non structural protein 4B (NS4B) has been reported to induce cellular membrane alterations like the membranous web. Four transmembrane segments in the middle of the protein anchor NS4B to membranes. An amphipatic helix at the amino-terminus attaches to membranes as well. The carboxy-terminal domain (CTD) of NS4B is highly conserved in Hepaciviruses, though its function remains unknown.
Francois Penin - One of the best experts on this subject based on the ideXlab platform.
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NS2 proteases from hepatitis C virus and related Hepaciviruses share composite active sites and previously unrecognized intrinsic proteolytic activities
PLOS Pathogens, 2018Co-Authors: Célia Boukadida, Brigitte Blumen, Matthieu Fritz, Marie-laure Fogeron, Francois Penin, Annette MartinAbstract:Over the recent years, several homologues with varying degrees of genetic relatedness to hepatitis C virus (HCV) have been identified in a wide range of mammalian species. HCV infectious life cycle relies on a first critical proteolytic event of its single polyprotein, which is carried out by nonstructural protein 2 (NS2) and allows replicase assembly and genome replication. In this study, we characterized and evaluated the conservation of the proteolytic mode of action and regulatory mechanisms of NS2 across HCV and animal Hepaciviruses. We first demonstrated that NS2 from equine, bat, rodent, New and Old World primate Hepaciviruses also are cysteine proteases. Using tagged viral protein precursors and catalytic triad mutants, NS2 of equine NPHV and simian GBV-B, which are the most closely and distantly related viruses to HCV, respectively, were shown to function, like HCV NS2 as dimeric proteases with two composite active sites. Consistent with the reported essential role for NS3 N-terminal domain (NS3N) as HCV NS2 protease cofactor via NS3N key hydrophobic surface patch, we showed by gain/loss of function mutagenesis studies that some heterologous hepacivirus NS3N may act as cofactors for HCV NS2 provided that HCV-like hydrophobic residues are conserved. Unprecedently, however, we also observed efficient intrinsic proteolytic activity of NS2 protease in the absence of NS3 moiety in the context of C-terminal tag fusions via flexible linkers both in transiently transfected cells for all Hepaciviruses studied and in the context of HCV dicistronic full-length genomes. These findings suggest that NS3N acts as a regulatory rather than essential cofactor for hepacivirus NS2 protease. Overall, unique features of NS2 including enzymatic function as dimers with two composite active sites and additional NS3-independent proteolytic activity are conserved across Hepaciviruses regardless of their genetic distances, highlighting their functional significance in hepacivirus life cycle.
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Summary of NS2 protease characteristics across various mammalian Hepaciviruses.
2018Co-Authors: Célia Boukadida, Brigitte Blumen, Matthieu Fritz, Marie-laure Fogeron, Francois Penin, Annette MartinAbstract:Summary of NS2 protease characteristics across various mammalian Hepaciviruses.
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Functionality of NS2 proteases from various Hepaciviruses in the absence of NS3N domain.
2018Co-Authors: Célia Boukadida, Brigitte Blumen, Matthieu Fritz, Marie-laure Fogeron, Francois Penin, Annette MartinAbstract:Cells were transfected with pCMV/NS2hepaci-4GS-GFP DNAs (A), allowing the transient expression of NS2 from the indicated HCV strains (subtypes in parentheses) (B) or Hepaciviruses (C). NS2 proteases comprised either the native catalytic triad (wt) or an Ala substitution of the catalytic Cys residue (CA). Uncleaved precursors and cleaved products were immunodetected with anti-GFP antibodies following SDS-PAGE separation of transfected cell extracts and are indicated by closed and open arrowheads, respectively.
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Role and virus specificity of HCV NS3N domain as NS2 protease cofactor.
2018Co-Authors: Célia Boukadida, Brigitte Blumen, Matthieu Fritz, Marie-laure Fogeron, Francois Penin, Annette MartinAbstract:(A) Schematic representation of the chimeric NS2HCV-NS3Nhepaci-ST precursors. Precursors spanning NS2 from HCV-JFH1 and NS3N from NPHV, BHV, GHV, RHV or GBV-B (hepaci) were expressed downstream of a heterologous signal peptide (sp) and C-terminally fused to Strep-tag (ST). The sequence alignment of NS3 N-terminal sequences from the various Hepaciviruses is depicted in the blown-up scheme with respect to HCV corresponding sequence, where identical residues are indicated by dots. (B) Extracts from cells transfected with pCMV/NS2HCV(wt)-NS3Nhepaci-ST or pCMV/NS2HCV(CA)-NS3Nhepaci-ST DNAs encoding NS2 protease with either native (wt) or mutated (CA) catalytic triad, respectively, were probed with anti-ST antibodies. Uncleaved precursors and cleaved products are indicated by closed and open arrowheads, respectively. The decreasing overall sequence similarity of hepacivirus NS3N with respect to HCV NS3N is represented by a grey triangle.
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Aminoterminal amphipathic α-helix AH1 of hepatitis C virus nonstructural protein 4B possesses a dual role in RNA replication and virus production.
PLoS pathogens, 2014Co-Authors: Jerome Gouttenoire, Ralf Bartenschlager, Francois Penin, Roland Montserret, David Paul, Rosa Castillo, Simon Meister, Darius MoradpourAbstract:Nonstructural protein 4B (NS4B) is a key organizer of hepatitis C virus (HCV) replication complex formation. In concert with other nonstructural proteins, it induces a specific membrane rearrangement, designated as membranous web, which serves as a scaffold for the HCV replicase. The N-terminal part of NS4B comprises a predicted and a structurally resolved amphipathic α-helix, designated as AH1 and AH2, respectively. Here, we report a detailed structure-function analysis of NS4B AH1. Circular dichroism and nuclear magnetic resonance structural analyses revealed that AH1 folds into an amphipathic α-helix extending from NS4B amino acid 4 to 32, with positively charged residues flanking the helix. These residues are conserved among Hepaciviruses. Mutagenesis and selection of pseudorevertants revealed an important role of these residues in RNA replication by affecting the biogenesis of double-membrane vesicles making up the membranous web. Moreover, alanine substitution of conserved acidic residues on the hydrophilic side of the helix reduced infectivity without significantly affecting RNA replication, indicating that AH1 is also involved in virus production. Selective membrane permeabilization and immunofluorescence microscopy analyses of a functional replicon harboring an epitope tag between NS4B AH1 and AH2 revealed a dual membrane topology of the N-terminal part of NS4B during HCV RNA replication. Luminal translocation was unaffected by the mutations introduced into AH1, but was abrogated by mutations introduced into AH2. In conclusion, our study reports the three-dimensional structure of AH1 from HCV NS4B, and highlights the importance of positively charged amino acid residues flanking this amphipathic α-helix in membranous web formation and RNA replication. In addition, we demonstrate that AH1 possesses a dual role in RNA replication and virus production, potentially governed by different topologies of the N-terminal part of NS4B.
Amit Kapoor - One of the best experts on this subject based on the ideXlab platform.
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mirna independent hepacivirus variants suggest a strong evolutionary pressure to maintain mir 122 dependence
PLOS Pathogens, 2017Co-Authors: Yingpu Yu, Eiko Nishiuchi, Joseph M Luna, Hachung Chung, Margaret A Scull, Natalia Echeverria, Inna Ricardolax, Troels K H Scheel, Amit KapoorAbstract:Hepatitis C virus (HCV) requires the liver specific micro-RNA (miRNA), miR-122, to replicate. This was considered unique among RNA viruses until recent discoveries of HCV-related Hepaciviruses prompting the question of a more general miR-122 dependence. Among Hepaciviruses, the closest known HCV relative is the equine non-primate hepacivirus (NPHV). Here, we used Argonaute cross-linking immunoprecipitation (AGO-CLIP) to confirm AGO binding to the single predicted miR-122 site in the NPHV 5’UTR in vivo. To study miR-122 requirements in the absence of NPHV-permissive cell culture systems, we generated infectious NPHV/HCV chimeric viruses with the 5’ end of NPHV replacing orthologous HCV sequences. These chimeras were viable even in cells lacking miR-122, although miR-122 presence enhanced virus production. No other miRNAs bound this region. By random mutagenesis, we isolated HCV variants partially dependent on miR-122 as well as robustly replicating NPHV/HCV variants completely independent of any miRNAs. These miRNA independent variants even replicate and produce infectious particles in non-hepatic cells after exogenous delivery of apolipoprotein E (ApoE). Our findings suggest that miR-122 independent HCV and NPHV variants have arisen and been sampled during evolution, yet miR-122 dependence has prevailed. We propose that Hepaciviruses may use this mechanism to guarantee liver tropism and exploit the tolerogenic liver environment to avoid clearance and promote chronicity.
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the strange expanding world of animal Hepaciviruses
Annual Review of Virology, 2016Co-Authors: Alex S Hartlage, John M. Cullen, Amit KapoorAbstract:Hepaciviruses and pegiviruses constitute two closely related sister genera of the family Flaviviridae. In the past five years, the known phylogenetic diversity of the hepacivirus genera has absolutely exploded. What was once an isolated infection in humans (and possibly other primates) has now expanded to include horses, rodents, bats, colobus monkeys, cows, and, most recently, catsharks, shedding new light on the genetic diversity and host range of Hepaciviruses. Interestingly, despite the identification of these many animal and primate Hepaciviruses, the equine Hepaciviruses remain the closest genetic relatives of the human Hepaciviruses, providing an intriguing clue to the zoonotic source of hepatitis C virus. This review summarizes the significance of these studies and discusses current thinking about the origin and evolution of the animal Hepaciviruses as well as their potential usage as surrogate models for the study of hepatitis C virus.
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Virome Analysis of Transfusion Recipients Reveals a Novel Human Virus That Shares Genomic Features with Hepaciviruses and Pegiviruses
mBio, 2015Co-Authors: Amit Kapoor, Peter Simmonds, Arvind Kumar, Nishit Bhuva, Lokendra V. Chauhan, Bohyun Lee, Amadou A. Sall, Zhezhen Jin, Stephen S. Morse, Beth H. ShazAbstract:To investigate the transmission of novel infectious agents by blood transfusion, we studied changes in the virome composition of blood transfusion recipients pre- and posttransfusion. Using this approach, we detected and genetically charac- terized a novel human virus, human hepegivirus 1 (HHpgV-1), that shares features with hepatitis C virus (HCV) and human pegivirus (HPgV; formerly called GB virus C or hepatitis G virus). HCV and HPgV belong to the genera Hepacivirus andPegivi- rusof the familyFlaviviridae. HHpgV-1 was found in serum samples from two blood transfusion recipients and two hemophilia patients who had received plasma-derived clotting factor concentrates. In the former, the virus was detected only in the post- transfusion samples, indicating blood-borne transmission. Both hemophiliacs were persistently viremic over periods of at least 201 and 1,981 days. The 5=untranslated region (UTR) of HHpgV-1 contained a type IV internal ribosome entry site (IRES), structurally similar to although highly divergent in sequence from that of HCV and other Hepaciviruses. However, phylogenetic analysis of nonstructural genes (NS3 and NS5B) showed that HHpgV-1 forms a branch within the pegivirus clade distinct from HPgV and homologs infecting other mammalian species. In common with some pegivirus variants infecting rodents and bats, the HHpgV-1 genome encodes a short, highly basic protein upstream of E1, potentially possessing a core-like function in packag- ing RNA during assembly. Identification of this new human virus, HHpgV-1, expands our knowledge of the range of genome configurations of these viruses and may lead to a reevaluation of the original criteria by which the genera Hepacivirus andPegivi- rusare defined. IMPORTANCE More than 30 million blood components are transfused annually in the United States alone. Surveillance for infec- tious agents in the blood supply is key to ensuring the safety of this critical resource for medicine and public health. Here, we report the identification of a new and highly diverse HCV/GB virus (GBV)-like virus from human serum samples. This new vi- rus, human hepegivirus 1 (HHpgV-1), was found in serum samples from blood transfusion recipients, indicating its potential for transmission via transfusion products. We also found persistent long-term HHpgV-1 viremia in two hemophilia patients. HHpgV-1 is unique because it shares genetic similarity with both highly pathogenic HCV and the apparently nonpathogenic HPgV (GBV-C). Our results add to the list of human viruses and provide data to develop reagents to study virus transmission and disease association and for interrupting virus transmission and new human infections.
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Surveying the global virome: Identification and characterization of HCV-related animal Hepaciviruses
Antiviral Research, 2014Co-Authors: Troels K H Scheel, Peter Simmonds, Amit KapoorAbstract:Abstract Recent advances in sequencing technologies have greatly enhanced our abilities to identify novel microbial sequences. Thus, our understanding of the global virome and the virome of specific host species in particular is rapidly expanding. Identification of animal viruses is important for understanding animal disease, the origin and evolution of human viruses, as well as zoonotic reservoirs for emerging infections. Although the human hepacivirus, hepatitis C virus (HCV), was identified 25 years ago, its origin has remained elusive. In 2011, the first HCV homolog was reported in dogs but subsequent studies showed the virus to be widely distributed in horses. This indicated a wider hepacivirus host range and paved the way for identification of rodent, bat and non-human primate Hepaciviruses. The equine non-primate hepacivirus (NPHV) remains the closest relative of HCV and is so far the best characterized. Identification and characterization of novel Hepaciviruses may in addition lead to development of tractable animal models to study HCV persistence, immune responses and pathogenesis. This could be particular important, given the current shortage of immunocompetent models for robust HCV infection. Much remains to be learned on the novel Hepaciviruses, including their association with disease, and thereby how relevant they will become as HCV model systems and for studies of animal disease. This review discusses how virome analysis led to identification of novel hepaci- and pegiviruses, their genetic relationship and characterization and the potential use of animal Hepaciviruses as models to study hepaciviral infection, immunity and pathogenesis. This article forms part of a symposium in Antiviral Research on “Hepatitis C: Next steps toward global eradication.”
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Identification of a Pegivirus (GB Virus-Like Virus) That Infects Horses
Journal of virology, 2013Co-Authors: Amit Kapoor, J. L. Medina, Eiko Nishiuchi, Troels K H Scheel, Peter Simmonds, Peter D. Burbelo, John M. Cullen, Federico Giannitti, Kenny V. Brock, Charles M. RiceAbstract:The recent identification of nonprimate Hepaciviruses in dogs and then in horses prompted us to look for pegiviruses (GB virus-like viruses) in these species. Although none were detected in canines, we found widespread natural infection of horses by a novel pegivirus. Unique genomic features and phylogenetic analyses confirmed that the tentatively named equine pegivirus (EPgV) represents a novel species within the Pegivirus genus. We also determined that EPgV causes persistent viremia whereas its clinical significance is undetermined.