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.

Hepatitis C virus (HCV) is present in the host with multiple variants generated by its error prone RNA-dependent RNA polymerase. Little is known about the initial viral diversification and the viral life cycle processes that influence diversity. We studied the diversification of HCV during acute infection in 17 plasma donors, with frequent sampling early in infection. To analyze these data, we developed a new stochastic model of the HCV life cycle. We found that the accumulation of mutations is surprisingly slow: at 30 days, the viral population on average is still 46% identical to its transmitted viral genome. Fitting the model to the sequence data, we estimate the median in vivo viral mutation rate is 2.5×10−5 mutations per nucleotide per genome replication (range 1.6–6.2×10−5), about 5-fold lower than previous estimates. To confirm these results we analyzed the frequency of stop codons (N = 10) among all possible non-sense mutation targets (M = 898,335), and found a mutation rate of 2.8–3.2×10−5, consistent with the estimate from the dynamical model. The slow accumulation of mutations is consistent with slow turnover of infected cells and replication complexes within infected cells. This slow turnover is also inferred from the viral load kinetics. Our estimated mutation rate, which is similar to that of other RNA viruses (e.g., HIV and influenza), is also compatible with the accumulation of substitutions seen in HCV at the population level. Our model identifies the relevant processes (long-lived cells and slow turnover of replication complexes) and parameters involved in determining the rate of HCV diversification.

Background & Aims Liver sinusoidal endothelial cells (LSECs) make up a large proportion of the non-parenchymal cells in the liver. LSECs are involved in induction of immune tolerance, but little is known about their functions during hepatitis C virus (HCV) infection. Methods Primary human LSECs (HLSECs) and immortalized liver endothelial cells (TMNK-1) were exposed to various forms of HCV, including full-length transmitted/founder virus, sucrose-purified Japanese Fulminant Hepatitis-1 (JFH-1), a virus encoding a luciferase reporter, and the HCV-specific pathogen-associated molecular pattern molecules. Cells were analyzed by confocal immunofluorescence, immunohistochemical, and PCR assays. Results HLSECs internalized HCV, independent of cell–cell contacts; HCV RNA was translated but not replicated. Through pattern recognition receptors (TLR7 and retinoic acid inducible gene 1), HCV RNA induced consistent and broad transcription of multiple interferons (IFNs); supernatants from primary HLSECs transfected with HCV-specific pathogen-associated molecular pattern molecules increased induction of IFNs and IFN-stimulated genes in HLSECs. Recombinant type I and type III IFNs strongly up-regulated HLSEC transcription of interferon λ 3 (IFNL3) and viperin (RSAD2), which inhibit replication of HCV. Compared to CD8+ T cells, HLSECs suppressed HCV replication within Huh7.5.1 cells, also inducing IFN-stimulated genes in co-culture. Conditioned media from IFN-stimulated HLSECs induced expression of antiviral genes by uninfected primary human hepatocytes. Exosomes, derived from HLSECs following stimulation with either type I or type III IFNs, controlled HCV replication in a dose-dependent manner. Conclusions Cultured HLSECs produce factors that mediate immunity against HCV. HLSECs induce self-amplifying IFN-mediated responses and release of exosomes with antiviral activity.

No abstract

Bacterial endotoxin interacts with the human immune system via complex immunological pathways. The evaluation of endotoxicity is important in the development of safe vaccines and immunomodulatory therapeutics. The Limulus amebocyte lysate (LAL) assay is generally accepted by the FDA for use for the quantification of lipopolysaccharide (LPS), while the rabbit pyrogen test (RPT) is used to estimate pyrogenicity during early development and production. Other in vitro assays, such as cytokine release assays with human whole blood (WB) or peripheral blood mononuclear cells (PBMCs), have also been used and may better estimate the human immunological response to products containing novel LPS molecules. In this study, WB and PBMC interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-␣) release assays were used to estimate the endotoxic activities of purified LPS and native outer membrane vesicle (NOMV) vaccines derived from wild-type (hexa-acylated lipid A) and genetically detoxified (penta-and tetra-acylated lipid A) group B Neisseria meningitidis. A method for quantification of the differences in endotoxicity observed in the WB and PBMC assays is elucidated. The LAL assay was shown to be relatively insensitive to lipid A variations, and the RPT was less sensitive than the cytokine release assay with WB. The IL-6 and TNF-␣ assays with WB but not the assays with PBMCs distinguished between vaccines containing LPS from penta-and tetra-acylated strains. The high degree of sensitivity of the WB system to LPS variations and the presumed relevance of the use of human tissues to predict toxicity in humans suggest that this assay may be particularly well suited for the safety evaluation of vaccines and therapeutics containing acylation variants of LPS.