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Mark Harris - One of the best experts on this subject based on the ideXlab platform.
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Structure–function analysis of the equine Hepacivirus 5′ untranslated region highlights the conservation of translational mechanisms across the Hepaciviruses
Journal of General Virology, 2019Co-Authors: Joseph Lattimer, Hazel Stewart, Nicolas Locker, Andrew Tuplin, Nicola J Stonehouse, Mark HarrisAbstract:Equine Hepacivirus (EHcV) (now also classified as Hepacivirus A) is the closest genetic relative to hepatitis C virus (HCV) and is proposed to have diverged from HCV within the last 1000 years. The 5′ untranslated regions (UTRs) of both HCV and EHcV exhibit internal ribosome entry site (IRES) activity, allowing cap-independent translational initiation, yet only the HCV 5′UTR has been systematically analysed. Here, we report a detailed structural and functional analysis of the EHcV 5′UTR. The secondary structure was determined using selective 2′ hydroxyl acylation analysed by primer extension (SHAPE), revealing four stem–loops, termed SLI, SLIA, SLII and SLIII, by analogy to HCV. This guided a mutational analysis of the EHcV 5′UTR, allowing us to investigate the roles of the stem–loops in IRES function. This approach revealed that SLI was not required for EHcV IRES-mediated translation. Conversely, SLIII was essential, specifically SLIIIb, SLIIId and a GGG motif that is conserved across the Hepaciviridae. Further SHAPE analysis provided evidence that this GGG motif mediated interaction with the 40S ribosomal subunit, whilst a CUU sequence in the apical loop of SLIIIb mediated an interaction with eIF3. In addition, we showed that a microRNA122 target sequence located between SLIA and SLII mediated an enhancement of translation in the context of a subgenomic replicon. Taken together, these results highlight the conservation of hepaciviral translation mechanisms, despite divergent primary sequences.
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structure function analysis of the equine Hepacivirus 5 untranslated region highlights the conservation of translational mechanisms across the Hepaciviruses
Journal of General Virology, 2019Co-Authors: Joseph Lattimer, Hazel Stewart, Nicolas Locker, Andrew Tuplin, Nicola J Stonehouse, Mark HarrisAbstract:Equine Hepacivirus (EHcV) (now also classified as Hepacivirus A) is the closest genetic relative to hepatitis C virus (HCV) and is proposed to have diverged from HCV within the last 1000 years. The 5′ untranslated regions (UTRs) of both HCV and EHcV exhibit internal ribosome entry site (IRES) activity, allowing cap-independent translational initiation, yet only the HCV 5′UTR has been systematically analysed. Here, we report a detailed structural and functional analysis of the EHcV 5′UTR. The secondary structure was determined using selective 2′ hydroxyl acylation analysed by primer extension (SHAPE), revealing four stem–loops, termed SLI, SLIA, SLII and SLIII, by analogy to HCV. This guided a mutational analysis of the EHcV 5′UTR, allowing us to investigate the roles of the stem–loops in IRES function. This approach revealed that SLI was not required for EHcV IRES-mediated translation. Conversely, SLIII was essential, specifically SLIIIb, SLIIId and a GGG motif that is conserved across the Hepaciviridae. Further SHAPE analysis provided evidence that this GGG motif mediated interaction with the 40S ribosomal subunit, whilst a CUU sequence in the apical loop of SLIIIb mediated an interaction with eIF3. In addition, we showed that a microRNA122 target sequence located between SLIA and SLII mediated an enhancement of translation in the context of a subgenomic replicon. Taken together, these results highlight the conservation of hepaciviral translation mechanisms, despite divergent primary sequences.
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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, Peter Simmonds, Sinead Lyons, Cheryl T Walter, Dale Jones, 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.
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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, Peter Simmonds, Sinead Lyons, Cheryl T Walter, Dale Jones, 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.
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short communication the non primate Hepacivirus 59 untranslated region possesses internal ribosomal entry site activity
2013Co-Authors: Hazel Stewart, Peter Simmonds, Sinead Lyons, Cheryl T Walter, Dale Jones, Mark HarrisAbstract:The 59 untranslated region (59UTR) 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 59UTR 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 59UTR within the HCV subgenomic replicon significantly impaired RNA replication, indicating that longrange interactions between the 59UTR 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 59UTR appear functionally distinct from those of HCV.
Hazel Stewart - One of the best experts on this subject based on the ideXlab platform.
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Structure–function analysis of the equine Hepacivirus 5′ untranslated region highlights the conservation of translational mechanisms across the Hepaciviruses
Journal of General Virology, 2019Co-Authors: Joseph Lattimer, Hazel Stewart, Nicolas Locker, Andrew Tuplin, Nicola J Stonehouse, Mark HarrisAbstract:Equine Hepacivirus (EHcV) (now also classified as Hepacivirus A) is the closest genetic relative to hepatitis C virus (HCV) and is proposed to have diverged from HCV within the last 1000 years. The 5′ untranslated regions (UTRs) of both HCV and EHcV exhibit internal ribosome entry site (IRES) activity, allowing cap-independent translational initiation, yet only the HCV 5′UTR has been systematically analysed. Here, we report a detailed structural and functional analysis of the EHcV 5′UTR. The secondary structure was determined using selective 2′ hydroxyl acylation analysed by primer extension (SHAPE), revealing four stem–loops, termed SLI, SLIA, SLII and SLIII, by analogy to HCV. This guided a mutational analysis of the EHcV 5′UTR, allowing us to investigate the roles of the stem–loops in IRES function. This approach revealed that SLI was not required for EHcV IRES-mediated translation. Conversely, SLIII was essential, specifically SLIIIb, SLIIId and a GGG motif that is conserved across the Hepaciviridae. Further SHAPE analysis provided evidence that this GGG motif mediated interaction with the 40S ribosomal subunit, whilst a CUU sequence in the apical loop of SLIIIb mediated an interaction with eIF3. In addition, we showed that a microRNA122 target sequence located between SLIA and SLII mediated an enhancement of translation in the context of a subgenomic replicon. Taken together, these results highlight the conservation of hepaciviral translation mechanisms, despite divergent primary sequences.
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structure function analysis of the equine Hepacivirus 5 untranslated region highlights the conservation of translational mechanisms across the Hepaciviruses
Journal of General Virology, 2019Co-Authors: Joseph Lattimer, Hazel Stewart, Nicolas Locker, Andrew Tuplin, Nicola J Stonehouse, Mark HarrisAbstract:Equine Hepacivirus (EHcV) (now also classified as Hepacivirus A) is the closest genetic relative to hepatitis C virus (HCV) and is proposed to have diverged from HCV within the last 1000 years. The 5′ untranslated regions (UTRs) of both HCV and EHcV exhibit internal ribosome entry site (IRES) activity, allowing cap-independent translational initiation, yet only the HCV 5′UTR has been systematically analysed. Here, we report a detailed structural and functional analysis of the EHcV 5′UTR. The secondary structure was determined using selective 2′ hydroxyl acylation analysed by primer extension (SHAPE), revealing four stem–loops, termed SLI, SLIA, SLII and SLIII, by analogy to HCV. This guided a mutational analysis of the EHcV 5′UTR, allowing us to investigate the roles of the stem–loops in IRES function. This approach revealed that SLI was not required for EHcV IRES-mediated translation. Conversely, SLIII was essential, specifically SLIIIb, SLIIId and a GGG motif that is conserved across the Hepaciviridae. Further SHAPE analysis provided evidence that this GGG motif mediated interaction with the 40S ribosomal subunit, whilst a CUU sequence in the apical loop of SLIIIb mediated an interaction with eIF3. In addition, we showed that a microRNA122 target sequence located between SLIA and SLII mediated an enhancement of translation in the context of a subgenomic replicon. Taken together, these results highlight the conservation of hepaciviral translation mechanisms, despite divergent primary sequences.
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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, Peter Simmonds, Sinead Lyons, Cheryl T Walter, Dale Jones, 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.
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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, Peter Simmonds, Sinead Lyons, Cheryl T Walter, Dale Jones, 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.
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short communication the non primate Hepacivirus 59 untranslated region possesses internal ribosomal entry site activity
2013Co-Authors: Hazel Stewart, Peter Simmonds, Sinead Lyons, Cheryl T Walter, Dale Jones, Mark HarrisAbstract:The 59 untranslated region (59UTR) 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 59UTR 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 59UTR within the HCV subgenomic replicon significantly impaired RNA replication, indicating that longrange interactions between the 59UTR 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 59UTR appear functionally distinct from those of HCV.
Peter Simmonds - One of the best experts on this subject based on the ideXlab platform.
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ICTV Virus Taxonomy Profile: Flaviviridae.
Journal of General Virology, 2017Co-Authors: Peter Simmonds, Paul Becher, Jens Bukh, Ernest A Gould, Gregor Meyers, Thomas P Monath, Alexander G Pletnev, Scott Muerhoff, Rebecca Rico-hesse, Donald B SmithAbstract:The Flaviviridae is a family of small enveloped viruses with RNA genomes of 9000–13 000 bases. Most infect mammals and birds. Many flaviviruses are host-specific and pathogenic, such as hepatitis C virus in the genus Hepacivirus. The majority of known members in the genus Flavivirus are arthropod borne, and many are important human and veterinary pathogens (e.g. yellow fever virus, dengue virus). This is a summary of the current International Committee on Taxonomy of Viruses (ICTV) report on the taxonomy of the Flaviviridae, which is available at www.ictv.global/report/flaviviridae.
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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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viraemic frequencies and seroprevalence of non primate Hepacivirus and equine pegiviruses in horses and other mammalian species
Journal of General Virology, 2014Co-Authors: Sinead Lyons, Amit Kapoor, B Schneider, Nathan D Wolfe, Geoff Culshaw, Brendan Corcoran, Andy E Durham, Faith Burden, Bruce Mcgorum, Peter SimmondsAbstract:Non-primate Hepacivirus (NPHV), equine pegivirus (EPgV) and Theiler’s disease associated virus (TDAV) are newly discovered members of two genera in the Flaviviridae family, Hepacivirus and Pegivirus respectively, that include human hepatitis C virus (HCV) and human pegivirus (HPgV). To investigate their epidemiology, persistence and clinical features of infection, large cohorts of horses and other mammalian species were screened for NPHV, EPgV and TDAV viraemia and for past exposure through serological assays for NPHV and EPgV-specific antibodies. NPHV antibodies were detected in 43 % of 328 horses screened for antibodies to NS3 and core antibodies, of which three were viraemic by PCR. All five horses that were stablemates of a viraemic horse were seropositive, as was a dog on the same farm. With this single exception, all other species were negative for NPHV antibodies and viraemia: donkeys (n = 100), dogs (n = 112), cats (n = 131), non-human primates (n = 164) and humans (n = 362). EPgV antibodies to NS3 were detected in 66.5 % of horses, including 10 of the 12 horses that had EPgV viraemia. All donkey samples were negative for EPgV antibody and RNA. All horse and donkey samples were negative for TDAV RNA. By comparing viraemia frequencies in horses with and without liver disease, no evidence was obtained that supported an association between active NPHV and EPgV infections with hepatopathy. The study demonstrates that NPHV and EPgV infections are widespread and enzootic in the study horse population and confirms that NPHV and potentially EPgV have higher frequencies of viral clearance than HCV and HPgV infections in humans.
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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, Peter Simmonds, Sinead Lyons, Cheryl T Walter, Dale Jones, 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.
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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, Peter Simmonds, Sinead Lyons, Cheryl T Walter, Dale Jones, 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.
Troels K. H. Scheel - One of the best experts on this subject based on the ideXlab platform.
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Correction: miRNA independent Hepacivirus variants suggest a strong evolutionary pressure to maintain miR-122 dependence.
PLoS pathogens, 2018Co-Authors: Yingpu Yu, Amit Kapoor, Troels K. H. Scheel, Eiko Nishiuchi, Joseph M Luna, Hachung Chung, Margaret A Scull, Natalia Echeverria, Inna Ricardo-lax, W Ian LipkinAbstract:[This corrects the article DOI: 10.1371/journal.ppat.1006694.].
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mirna independent Hepacivirus variants suggest a strong evolutionary pressure to maintain mir 122 dependence
PLOS Pathogens, 2017Co-Authors: Yingpu Yu, Troels K. H. Scheel, Eiko Nishiuchi, Joseph M Luna, Hachung Chung, Margaret A Scull, Natalia Echeverria, Inna Ricardolax, 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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characterization of nonprimate Hepacivirus and construction of a functional molecular clone
Proceedings of the National Academy of Sciences of the United States of America, 2015Co-Authors: Amit Kapoor, Troels K. H. Scheel, Eiko Nishiuchi, Kenny V Brock, Yingpu Yu, Linda Andrus, Meigang Gu, Randall W RenshawAbstract:Nonprimate Hepacivirus (NPHV) is the closest known relative of hepatitis C virus (HCV) and its study could enrich our understanding of HCV evolution, immunity, and pathogenesis. High seropositivity is found in horses worldwide with ∼3% viremic. NPHV natural history and molecular virology remain largely unexplored, however. Here, we show that NPHV, like HCV, can cause persistent infection for over a decade, with high titers and negative strand RNA in the liver. NPHV is a near-universal contaminant of commercial horse sera for cell culture. The complete NPHV 3′-UTR was determined and consists of interspersed homopolymer tracts and an HCV-like 3′-terminal poly(U)-X-tail. NPHV translation is stimulated by miR-122 and the 3′-UTR and, similar to HCV, the NPHV NS3-4A protease can cleave mitochondrial antiviral-signaling protein to inactivate the retinoic acid-inducible gene I pathway. Using an NPHV consensus cDNA clone, replication was not observed in primary equine fetal liver cultures or after electroporation of selectable replicons. However, intrahepatic RNA inoculation of a horse initiated infection, yielding high RNA titers in the serum and liver. Delayed seroconversion, slightly elevated circulating liver enzymes and mild hepatitis was observed, followed by viral clearance. This establishes the molecular components of a functional NPHV genome. Thus, NPHV appears to resemble HCV not only in genome structure but also in its ability to establish chronic infection with delayed seroconversion and hepatitis. This NPHV infectious clone and resulting acute phase sera will facilitate more detailed studies on the natural history, pathogenesis, and immunity of this novel Hepacivirus in its natural host.
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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.”
Amit Kapoor - One of the best experts on this subject based on the ideXlab platform.
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vaccination to prevent t cell subversion can protect against persistent Hepacivirus infection
Nature Communications, 2019Co-Authors: Alex S Hartlage, Satyapramod Murthy, Arvind Kumar, Sheetal Trivedi, Piyush Dravid, Himanshu Sharma, Christopher M Walker, Amit KapoorAbstract:Efforts to develop an effective vaccine against the hepatitis C virus (HCV; human Hepacivirus) have been stymied by a lack of small animal models. Here, we describe an experimental rat model of chronic HCV-related Hepacivirus infection and its response to T cell immunization. Immune-competent rats challenged with a rodent Hepacivirus (RHV) develop chronic viremia characterized by expansion of non-functional CD8+ T cells. Single-dose vaccination with a recombinant adenovirus vector expressing Hepacivirus non-structural proteins induces effective immunity in majority of rats. Resolution of infection coincides with a vigorous recall of intrahepatic cellular responses. Host selection of viral CD8 escape variants can subvert vaccine-conferred immunity. Transient depletion of CD8+ cells from vaccinated rats prolongs infection, while CD4+ cell depletion results in chronic viremia. These results provide direct evidence that co-operation between CD4+ and CD8+ T cells is important for Hepacivirus immunity, and that subversion of responses can be prevented by prophylactic vaccination. Development of a HCV vaccine is hampered by a lack of appropriate small animal models. Here, Hartlage et al. describe a rat model of Hepacivirus persistence and show that persistence can be prevented by vaccination with viral non-structural proteins.
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Correction: miRNA independent Hepacivirus variants suggest a strong evolutionary pressure to maintain miR-122 dependence.
PLoS pathogens, 2018Co-Authors: Yingpu Yu, Amit Kapoor, Troels K. H. Scheel, Eiko Nishiuchi, Joseph M Luna, Hachung Chung, Margaret A Scull, Natalia Echeverria, Inna Ricardo-lax, W Ian LipkinAbstract:[This corrects the article DOI: 10.1371/journal.ppat.1006694.].
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mirna independent Hepacivirus variants suggest a strong evolutionary pressure to maintain mir 122 dependence
PLOS Pathogens, 2017Co-Authors: Yingpu Yu, Troels K. H. Scheel, Eiko Nishiuchi, Joseph M Luna, Hachung Chung, Margaret A Scull, Natalia Echeverria, Inna Ricardolax, 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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characterization of nonprimate Hepacivirus and construction of a functional molecular clone
Proceedings of the National Academy of Sciences of the United States of America, 2015Co-Authors: Amit Kapoor, Troels K. H. Scheel, Eiko Nishiuchi, Kenny V Brock, Yingpu Yu, Linda Andrus, Meigang Gu, Randall W RenshawAbstract:Nonprimate Hepacivirus (NPHV) is the closest known relative of hepatitis C virus (HCV) and its study could enrich our understanding of HCV evolution, immunity, and pathogenesis. High seropositivity is found in horses worldwide with ∼3% viremic. NPHV natural history and molecular virology remain largely unexplored, however. Here, we show that NPHV, like HCV, can cause persistent infection for over a decade, with high titers and negative strand RNA in the liver. NPHV is a near-universal contaminant of commercial horse sera for cell culture. The complete NPHV 3′-UTR was determined and consists of interspersed homopolymer tracts and an HCV-like 3′-terminal poly(U)-X-tail. NPHV translation is stimulated by miR-122 and the 3′-UTR and, similar to HCV, the NPHV NS3-4A protease can cleave mitochondrial antiviral-signaling protein to inactivate the retinoic acid-inducible gene I pathway. Using an NPHV consensus cDNA clone, replication was not observed in primary equine fetal liver cultures or after electroporation of selectable replicons. However, intrahepatic RNA inoculation of a horse initiated infection, yielding high RNA titers in the serum and liver. Delayed seroconversion, slightly elevated circulating liver enzymes and mild hepatitis was observed, followed by viral clearance. This establishes the molecular components of a functional NPHV genome. Thus, NPHV appears to resemble HCV not only in genome structure but also in its ability to establish chronic infection with delayed seroconversion and hepatitis. This NPHV infectious clone and resulting acute phase sera will facilitate more detailed studies on the natural history, pathogenesis, and immunity of this novel Hepacivirus in its natural host.
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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.”