Elucidation of Hepatitis C Virus Transmission and Early Diversification by Single Genome Sequencing

Abstract

A precise molecular identification of transmitted hepatitis C virus (HCV) genomes could illuminate key aspects of transmission biology, immunopathogenesis and natural history. We used single genome sequencing of 2,922 half or quarter genomes from plasma viral RNA to identify transmitted/founder (T/F) viruses in 17 subjects with acute community-acquired HCV infection. Sequences from 13 of 17 acute subjects, but none of 14 chronic controls, exhibited one or more discrete low diversity viral lineages. Sequences within each lineage generally revealed a star-like phylogeny of mutations that coalesced to unambiguous T/F viral genomes. Numbers of transmitted viruses leading to productive clinical infection were estimated to range from 1 to 37 or more (median = 4). Four acutely infected subjects showed a distinctly different pattern of virus diversity that deviated from a star-like phylogeny. In these cases, empirical analysis and mathematical modeling suggested high multiplicity virus transmission from individuals who themselves were acutely infected or had experienced a virus population bottleneck due to antiviral drug therapy. These results provide new quantitative and qualitative insights into HCV transmission, revealing for the first time virus-host interactions that successful vaccines or treatment interventions will need to overcome. Our findings further suggest a novel experimental strategy for identifying full-length T/F genomes for proteome-wide analyses of HCV biology and adaptation to antiviral drug or immune pressures.

Broadly neutralizing antibodies with few somatic mutations and hepatitis C virus clearance

“…HCV hemigenomes from plasma virus were amplified by RT-PCR after limiting dilution to ensure single-genome amplification, using previously described methods (53). PCR products were gel extracted and directly Sanger sequenced.…”

“…med.som.jhmi.edu), with 20-codon windows and 1-codon steps. T/F genomes were inferred as previously described (53,55). Highlighter plots were generated using aligned E1E2 amino acid sequences and the Highlighter tool at the Los Alamos HIV database (http://www.lanl.gov/).…”

Sequence and Functional Analysis of the Envelope Glycoproteins of Hepatitis C Virus Variants Selectively Transmitted to a New Host

“…This results in the generation of a group of related, but genetically different, viral variants within each infected individual (9). The genetic bottleneck generally observed after transmission indicates that productive infections may be initiated by one or a small number of viral variants (10)(11)(12)(13). These variants are then subjected to constant immune pressure, and the role of the immune response in the clearance of HCV infection or the establishment of chronic hepatitis C has been thoroughly investigated.…”

“…However, little is known about the transmitted variants responsible for the spread of the disease, principally because it is difficult to recruit patients early enough in acute infection for such studies. Moreover, studies of the viruses transmitted in humans have essentially focused on genetic identification of the transmitted/ founder (T/F) variants and their early diversification, through phylogenetic and mathematical approaches, without direct comparison with donor virus populations (10)(11)(12). A key question in the rational design of strategies for preventing HCV infection concerns the phenotypic determinants conferring fitness for transmission in T/F viruses.…”

“…We mapped the genetic bottleneck leading to productive clinical infection by single-genome amplification of viral envelope glycoprotein E1E2 sequences, direct amplicon sequencing, and phylogenetic analyses (12,14). The recovery of full-length E1E2 sequences from donors and recipients made it possible to investigate the phenotypic properties of these proteins potentially relevant to transmission.…”