Identification, Molecular Cloning, and Analysis of Full-Length Hepatitis C Virus Transmitted/Founder Genotypes 1, 3, and 4
ABSTRACT Hepatitis C virus (HCV) infection is characterized by persistent replication of a complex mixture of viruses termed a “quasispecies.” Transmission is generally associated with a stringent population bottleneck characterized by infection by limited numbers of “transmitted/founder” (T/F) viru...
| Main Authors: | , , , , , , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
American Society for Microbiology
2015-05-01
|
| Series: | mBio |
| Online Access: | https://journals.asm.org/doi/10.1128/mBio.02518-14 |
| _version_ | 1818856336357588992 |
|---|---|
| author | Mark B. Stoddard Hui Li Shuyi Wang Mohsan Saeed Linda Andrus Wenge Ding Xinpei Jiang Gerald H. Learn Markus von Schaewen Jessica Wen Paul A. Goepfert Beatrice H. Hahn Alexander Ploss Charles M. Rice George M. Shaw |
| author_facet | Mark B. Stoddard Hui Li Shuyi Wang Mohsan Saeed Linda Andrus Wenge Ding Xinpei Jiang Gerald H. Learn Markus von Schaewen Jessica Wen Paul A. Goepfert Beatrice H. Hahn Alexander Ploss Charles M. Rice George M. Shaw |
| author_sort | Mark B. Stoddard |
| collection | DOAJ |
| description | ABSTRACT Hepatitis C virus (HCV) infection is characterized by persistent replication of a complex mixture of viruses termed a “quasispecies.” Transmission is generally associated with a stringent population bottleneck characterized by infection by limited numbers of “transmitted/founder” (T/F) viruses. Characterization of T/F genomes of human immunodeficiency virus type 1 (HIV-1) has been integral to studies of transmission, immunopathogenesis, and vaccine development. Here, we describe the identification of complete T/F genomes of HCV by single-genome sequencing of plasma viral RNA from acutely infected subjects. A total of 2,739 single-genome-derived amplicons comprising 10,966,507 bp from 18 acute-phase and 11 chronically infected subjects were analyzed. Acute-phase sequences diversified essentially randomly, except for the poly(U/UC) tract, which was subject to polymerase slippage. Fourteen acute-phase subjects were productively infected by more than one genetically distinct virus, permitting assessment of recombination between replicating genomes. No evidence of recombination was found among 1,589 sequences analyzed. Envelope sequences of T/F genomes lacked transmission signatures that could distinguish them from chronic infection viruses. Among chronically infected subjects, higher nucleotide substitution rates were observed in the poly(U/UC) tract than in envelope hypervariable region 1. Fourteen full-length molecular clones with variable poly(U/UC) sequences corresponding to seven genotype 1a, 1b, 3a, and 4a T/F viruses were generated. Like most unadapted HCV clones, T/F genomes did not replicate efficiently in Huh 7.5 cells, indicating that additional cellular factors or viral adaptations are necessary for in vitro replication. Full-length T/F HCV genomes and their progeny provide unique insights into virus transmission, virus evolution, and virus-host interactions associated with immunopathogenesis. IMPORTANCE Hepatitis C virus (HCV) infects 2% to 3% of the world's population and exhibits extraordinary genetic diversity. This diversity is mirrored by HIV-1, where characterization of transmitted/founder (T/F) genomes has been instrumental in studies of virus transmission, immunopathogenesis, and vaccine development. Here, we show that despite major differences in genome organization, replication strategy, and natural history, HCV (like HIV-1) diversifies essentially randomly early in infection, and as a consequence, sequences of actual T/F viruses can be identified. This allowed us to capture by molecular cloning the full-length HCV genomes that are responsible for infecting the first hepatocytes and eliciting the initial immune responses, weeks before these events could be directly analyzed in human subjects. These findings represent an enabling experimental strategy, not only for HCV and HIV-1 research, but also for other RNA viruses of medical importance, including West Nile, chikungunya, dengue, Venezuelan encephalitis, and Ebola viruses. |
| first_indexed | 2024-12-19T08:22:53Z |
| format | Article |
| id | doaj.art-8b5e61bca3f945b18a7dd2612c61c17d |
| institution | Directory Open Access Journal |
| issn | 2150-7511 |
| language | English |
| last_indexed | 2024-12-19T08:22:53Z |
| publishDate | 2015-05-01 |
| publisher | American Society for Microbiology |
| record_format | Article |
| series | mBio |
| spelling | doaj.art-8b5e61bca3f945b18a7dd2612c61c17d2022-12-21T20:29:22ZengAmerican Society for MicrobiologymBio2150-75112015-05-016210.1128/mBio.02518-14Identification, Molecular Cloning, and Analysis of Full-Length Hepatitis C Virus Transmitted/Founder Genotypes 1, 3, and 4Mark B. Stoddard0Hui Li1Shuyi Wang2Mohsan Saeed3Linda Andrus4Wenge Ding5Xinpei Jiang6Gerald H. Learn7Markus von Schaewen8Jessica Wen9Paul A. Goepfert10Beatrice H. Hahn11Alexander Ploss12Charles M. Rice13George M. Shaw14Departments of Medicine and Microbiology, University of Pennsylvania, Philadelphia, Pennsylvania, USADepartments of Medicine and Microbiology, University of Pennsylvania, Philadelphia, Pennsylvania, USADepartments of Medicine and Microbiology, University of Pennsylvania, Philadelphia, Pennsylvania, USACenter for the Study of Hepatitis C, Laboratory of Virology and Infectious Disease, the Rockefeller University, New York, New York, USACenter for the Study of Hepatitis C, Laboratory of Virology and Infectious Disease, the Rockefeller University, New York, New York, USADepartments of Medicine and Microbiology, University of Pennsylvania, Philadelphia, Pennsylvania, USADepartments of Medicine and Microbiology, University of Pennsylvania, Philadelphia, Pennsylvania, USADepartments of Medicine and Microbiology, University of Pennsylvania, Philadelphia, Pennsylvania, USADepartment of Molecular Biology, Princeton University, Princeton, New Jersey, USADepartment of Pediatrics, Children's Hospital of Philadelphia, University of Pennsylvania, Philadelphia, Pennsylvania, USAUniversity of Alabama at Birmingham, Birmingham, Alabama, USADepartments of Medicine and Microbiology, University of Pennsylvania, Philadelphia, Pennsylvania, USADepartment of Molecular Biology, Princeton University, Princeton, New Jersey, USACenter for the Study of Hepatitis C, Laboratory of Virology and Infectious Disease, the Rockefeller University, New York, New York, USADepartments of Medicine and Microbiology, University of Pennsylvania, Philadelphia, Pennsylvania, USAABSTRACT Hepatitis C virus (HCV) infection is characterized by persistent replication of a complex mixture of viruses termed a “quasispecies.” Transmission is generally associated with a stringent population bottleneck characterized by infection by limited numbers of “transmitted/founder” (T/F) viruses. Characterization of T/F genomes of human immunodeficiency virus type 1 (HIV-1) has been integral to studies of transmission, immunopathogenesis, and vaccine development. Here, we describe the identification of complete T/F genomes of HCV by single-genome sequencing of plasma viral RNA from acutely infected subjects. A total of 2,739 single-genome-derived amplicons comprising 10,966,507 bp from 18 acute-phase and 11 chronically infected subjects were analyzed. Acute-phase sequences diversified essentially randomly, except for the poly(U/UC) tract, which was subject to polymerase slippage. Fourteen acute-phase subjects were productively infected by more than one genetically distinct virus, permitting assessment of recombination between replicating genomes. No evidence of recombination was found among 1,589 sequences analyzed. Envelope sequences of T/F genomes lacked transmission signatures that could distinguish them from chronic infection viruses. Among chronically infected subjects, higher nucleotide substitution rates were observed in the poly(U/UC) tract than in envelope hypervariable region 1. Fourteen full-length molecular clones with variable poly(U/UC) sequences corresponding to seven genotype 1a, 1b, 3a, and 4a T/F viruses were generated. Like most unadapted HCV clones, T/F genomes did not replicate efficiently in Huh 7.5 cells, indicating that additional cellular factors or viral adaptations are necessary for in vitro replication. Full-length T/F HCV genomes and their progeny provide unique insights into virus transmission, virus evolution, and virus-host interactions associated with immunopathogenesis. IMPORTANCE Hepatitis C virus (HCV) infects 2% to 3% of the world's population and exhibits extraordinary genetic diversity. This diversity is mirrored by HIV-1, where characterization of transmitted/founder (T/F) genomes has been instrumental in studies of virus transmission, immunopathogenesis, and vaccine development. Here, we show that despite major differences in genome organization, replication strategy, and natural history, HCV (like HIV-1) diversifies essentially randomly early in infection, and as a consequence, sequences of actual T/F viruses can be identified. This allowed us to capture by molecular cloning the full-length HCV genomes that are responsible for infecting the first hepatocytes and eliciting the initial immune responses, weeks before these events could be directly analyzed in human subjects. These findings represent an enabling experimental strategy, not only for HCV and HIV-1 research, but also for other RNA viruses of medical importance, including West Nile, chikungunya, dengue, Venezuelan encephalitis, and Ebola viruses.https://journals.asm.org/doi/10.1128/mBio.02518-14 |
| spellingShingle | Mark B. Stoddard Hui Li Shuyi Wang Mohsan Saeed Linda Andrus Wenge Ding Xinpei Jiang Gerald H. Learn Markus von Schaewen Jessica Wen Paul A. Goepfert Beatrice H. Hahn Alexander Ploss Charles M. Rice George M. Shaw Identification, Molecular Cloning, and Analysis of Full-Length Hepatitis C Virus Transmitted/Founder Genotypes 1, 3, and 4 mBio |
| title | Identification, Molecular Cloning, and Analysis of Full-Length Hepatitis C Virus Transmitted/Founder Genotypes 1, 3, and 4 |
| title_full | Identification, Molecular Cloning, and Analysis of Full-Length Hepatitis C Virus Transmitted/Founder Genotypes 1, 3, and 4 |
| title_fullStr | Identification, Molecular Cloning, and Analysis of Full-Length Hepatitis C Virus Transmitted/Founder Genotypes 1, 3, and 4 |
| title_full_unstemmed | Identification, Molecular Cloning, and Analysis of Full-Length Hepatitis C Virus Transmitted/Founder Genotypes 1, 3, and 4 |
| title_short | Identification, Molecular Cloning, and Analysis of Full-Length Hepatitis C Virus Transmitted/Founder Genotypes 1, 3, and 4 |
| title_sort | identification molecular cloning and analysis of full length hepatitis c virus transmitted founder genotypes 1 3 and 4 |
| url | https://journals.asm.org/doi/10.1128/mBio.02518-14 |
| work_keys_str_mv | AT markbstoddard identificationmolecularcloningandanalysisoffulllengthhepatitiscvirustransmittedfoundergenotypes13and4 AT huili identificationmolecularcloningandanalysisoffulllengthhepatitiscvirustransmittedfoundergenotypes13and4 AT shuyiwang identificationmolecularcloningandanalysisoffulllengthhepatitiscvirustransmittedfoundergenotypes13and4 AT mohsansaeed identificationmolecularcloningandanalysisoffulllengthhepatitiscvirustransmittedfoundergenotypes13and4 AT lindaandrus identificationmolecularcloningandanalysisoffulllengthhepatitiscvirustransmittedfoundergenotypes13and4 AT wengeding identificationmolecularcloningandanalysisoffulllengthhepatitiscvirustransmittedfoundergenotypes13and4 AT xinpeijiang identificationmolecularcloningandanalysisoffulllengthhepatitiscvirustransmittedfoundergenotypes13and4 AT geraldhlearn identificationmolecularcloningandanalysisoffulllengthhepatitiscvirustransmittedfoundergenotypes13and4 AT markusvonschaewen identificationmolecularcloningandanalysisoffulllengthhepatitiscvirustransmittedfoundergenotypes13and4 AT jessicawen identificationmolecularcloningandanalysisoffulllengthhepatitiscvirustransmittedfoundergenotypes13and4 AT paulagoepfert identificationmolecularcloningandanalysisoffulllengthhepatitiscvirustransmittedfoundergenotypes13and4 AT beatricehhahn identificationmolecularcloningandanalysisoffulllengthhepatitiscvirustransmittedfoundergenotypes13and4 AT alexanderploss identificationmolecularcloningandanalysisoffulllengthhepatitiscvirustransmittedfoundergenotypes13and4 AT charlesmrice identificationmolecularcloningandanalysisoffulllengthhepatitiscvirustransmittedfoundergenotypes13and4 AT georgemshaw identificationmolecularcloningandanalysisoffulllengthhepatitiscvirustransmittedfoundergenotypes13and4 |