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Detection of Low Copy Number Integrated Viral DNA Formed by In Vitro Hepatitis B Infection
Published on: November 7, 2018
Identification, molecular cloning, and analysis of full-length hepatitis C virus transmitted/founder genotypes 1, 3,
Mark B Stoddard1, Hui Li1, Shuyi Wang1
1Departments of Medicine and Microbiology, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
Insights
Researchers identified complete Hepatitis C virus (HCV) transmitted/founder (T/F) genomes, revealing random early diversification similar to HIV-1. This breakthrough enables new insights into HCV transmission and evolution.
Area of Science:
- Virology
- Molecular Biology
- Genetics
Background:
- Hepatitis C virus (HCV) infection presents significant global health challenges due to its high genetic diversity.
- Understanding transmitted/founder (T/F) viruses is crucial for studying viral transmission and pathogenesis, as demonstrated by HIV-1 research.
- Previous research lacked complete T/F genome characterization for HCV, hindering in-depth analysis of early infection dynamics.
Purpose of the Study:
- To identify and characterize complete transmitted/founder (T/F) genomes of Hepatitis C virus (HCV).
- To investigate the early diversification patterns and potential recombination events of HCV upon transmission.
- To establish a foundation for studying HCV transmission, evolution, and host interactions through molecular cloning of T/F genomes.
Main Methods:
- Single-genome sequencing of plasma viral RNA from acutely and chronically infected subjects.
- Analysis of over 2,700 single-genome-derived amplicons (10.9 million base pairs).
- Molecular cloning of full-length T/F HCV genomes from diverse genotypes (1a, 1b, 3a, 4a).
Main Results:
- Complete T/F genomes of HCV were successfully identified using single-genome sequencing.
- Early HCV diversification was largely random, with exceptions in the poly(U/UC) tract due to polymerase slippage.
- No evidence of recombination was detected among the analyzed sequences; T/F envelope sequences lacked distinct transmission signatures.
- Generated 14 full-length molecular clones of T/F viruses, which showed limited replication in vitro.
Conclusions:
- The study successfully identified complete T/F HCV genomes, enabling detailed analysis of early viral evolution.
- HCV transmission involves a limited bottleneck, with early diversification patterns resembling those of HIV-1.
- These findings provide critical insights into HCV transmission, evolution, and host-virus interactions, paving the way for future research on RNA viruses.
Unlabelled:
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.
