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) 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.

Innate and Adaptive Immune Responses in Chronic HCV Infection

“…The resulting pool of novel viral RNA sequences provides the raw material for natural selection and rapid evolution under pressures that include immune recognition. HCV must evolve within each new host to maximize its potential to replicate and minimize immune-mediated clearance (203–209). As HCV infects a new host, viral sequence diversity may initially be limited by a founder effect.…”

Exposure to hepatitis C virus in homeless men in Central Brazil: a cross-sectional study

“…It is estimated that 2.8% of the world population, which corresponds to 185 million people, is living with the chronic form of hepatitis C, and that each year about 350 thousand people die as a result of its complications, such as liver cirrhosis and hepatocellular carcinoma [2, 3]. In Brazil, the prevalence of HCV infection in the general population is 1.3%, with differences among Brazilian regions (from 0.68% in the Northeast to 2.1% in the North); in the Midwest, a prevalence of 1.6% is estimated among individuals over 20 years-old [4].…”

Pathogen-Associated Molecular Pattern Recognition of Hepatitis C Virus Transmitted/Founder Variants by RIG-I Is Dependent on U-Core Length

“…In order to study the molecular, biological, and immunopathogenic properties of HCV T/F viruses and their contributions to early virus-host interactions, single-genome sequence (SGS) analyses were conducted to include full-length T/F genomic analyses of HCV, including the 5= and 3= UTR (44). Six subjects from whom a total of 7 unique full-length T/F genomes were inferred on the basis of SGS analyses of the 5= UTR, all structural and regulatory genes, and the X region were previously reported (44). The sequences of these T/F genomes, exclusive of polyU/UC sequences, were unambiguous because, in each case, the plasma vRNA/cDNA sequences conformed to a model of random variation from which a coalescent T/F genome could be inferred.…”

“…Extensive analysis of polyU/UC sequences from the six acute-infection subjects included sequential sampling and analysis of plasma vRNA throughout the acuteinfection period. Results from these analyses indicated that MuLV reverse transcriptase and, to a lesser extent, Taq polymerase were associated with artificially shortened polyU/UC sequences of T/F genomes (44). HCV RNA-dependent RNA polymerase slippage could also occur but was assumed to contribute less given the known structure-function relationships of this polymerase and its processivity.…”