Development and validation of a "capture-fusion" model to study drug sensitivity of patient-derived hepatitis C.

UNLABELLED: Emerging therapies for chronic hepatitis C viral (HCV) infection involve inhibition of viral enzymes with drug combinations. Natural, or treatment-induced, enzyme polymorphisms reduce efficacy. We developed a phenotyping assay to aid drug selection based on viral transfer from monocytes to hepatocytes. We studied HCV in monocytes from infected patients and developed a model in which patient-derived HCV is "captured" by the cell line THP-1 and replication assessed after fusion to hepatoma cells. We found that monocytes from HCV-infected patients harbor virus that replicates when cells are fused to hepatocytes. THP-1 cells incubated with infected sera capture HCV, which replicates when fused to hepatocytes. Inhibitable replication of all HCV genotypes was achieved (42 of 52 isolates). We measured sensitivity of telaprevir (TVR) and alisporivir (AVR) in different genotypes, and showed differences in 50% inhibitory concentration (IC50 ) correlating with clinical response (TVR IC50 for genotype (G)1 was 0.042 ± 0.003 vs. 0.117 ± 0.015 µM for G3, whereas AVR IC50 for G1 was 0.139 ± 0.013 vs. 0.044 ± 0.007 µM for G3). We tested TVR-resistant viral isolates and identified changes in IC50 . One patient with a poor clinical response to TVR and wild-type viral sequence showed reduced TVR sensitivity in our assay. We studied samples from a 2-week TVR monotherapy study in which 5 of 8 patients with G3 HCV did not respond whereas 3 of 8 patients did. The "capture-fusion" assay correctly identified responders. CONCLUSION: The capture-fusion model represents a promising new technique that may help identify appropriate treatment strategies for patients with chronic HCV infection.

Detection and significance of a G1862T variant of hepatitis B virus in Chinese patients with fulminant hepatitis.

The prevalence of a G1862T variant of hepatitis B virus (HBV) has been investigated in patients with fulminant hepatitis and chronic liver disease, using primer mismatch amplification, followed by restriction fragment length polymorphism analysis. This variant was five times more common in patients with fulminant hepatitis (13.7%, 7 of 52) than in chronic carriers (2.5%, 2 of 81). The G-->T substitution at position 1862 leads to an amino acid change in codon 17 of the precore protein of the virus, which is part of a signal peptidase recognition motif. Variants with this mutation were only seen in patients infected with genotype B. In vitro translation experiments showed that this variant has greatly reduced capacity to produce hepatitis B e antigen (HBeAg) from its precore protein precursor. Furthermore, 88.5% of patients with fulminant hepatitis had mutations that are known to be associated with abrogated or reduced production of HBeAg. This suggests that, following HBV infection, the absence or reduced amounts of HBeAg may be a contributing factor in fulminant disease.