Evelyne Schvoerer

Scavenger receptor class B type I is a key host factor for hepatitis C virus infection required for an entry step closely linked to CD81.

Hepatitis C virus (HCV) is a major cause of chronic hepatitis worldwide. Scavenger receptor class B type I (SR‐BI) has been shown to bind HCV envelope glycoprotein E2, participate in entry of HCV pseudotype particles, and modulate HCV infection. However, the functional role of SR‐BI for productive HCV infection remains unclear. In this study, we investigated the role of SR‐BI as an entry factor for infection of human hepatoma cells using cell culture–derived HCV (HCVcc). Anti–SR‐BI antibodies directed against epitopes of the human SR‐BI extracellular loop specifically inhibited HCVcc infection in a dose‐dependent manner. Down‐regulation of SR‐BI expression by SR‐BI–specific short interfering RNAs (siRNAs) markedly reduced the susceptibility of human hepatoma cells to HCVcc infection. Kinetic studies demonstrated that SR‐BI acts predominately after binding of HCV at an entry step occurring at a similar time point as CD81–HCV interaction. Although the addition of high‐density lipoprotein (HDL) enhanced the efficiency of HCVcc infection, anti–SR‐BI antibodies and SR‐BI–specific siRNA efficiently inhibited HCV infection independent of lipoprotein. Conclusion: Our data suggest that SR‐BI (i) represents a key host factor for HCV entry, (ii) is implicated in the same HCV entry pathway as CD81, and (iii) targets an entry step closely linked to HCV–CD81 interaction. (HEPATOLOGY 2007.)

Influence of the HCV subtype on the virological response to pegylated interferon and ribavirin therapy.

The hepatitis C virus genotype is considered to be the most important baseline predictor of a sustained virological response in patients with chronic hepatitis C treated with pegylated interferon and ribavirin. The influence of the subtype on the sustained virological response was investigated in patients infected with genotypes 1, 4, 5, or 6. This study was done on 597 patients with chronic hepatitis C who were given pegylated interferon and ribavirin for 48 weeks. The overall rate of sustained virological response in the 597 patients was 37.8%. Univariate analysis indicated that the sustained virological response of patients infected with subtype 1b (39%) tended to be higher than that of patients infected with subtype 1a (30.6%; P = 0.06) and it was similar to those patients infected with subtypes 4a (51.3%; P = 0.12) or 4d (51.7%; P = 0.16). Multivariate analysis indicated that five factors were independently associated with sustained virological response: the age (OR 0.97; 95% CI = 0.95–0.99), absence of cirrhosis (OR: 2.92; 95% CI = 1.7–5.0; P < 0.01), absence of HIV co‐infection (OR: 2.08; 95% CI = 1.2–3.5; P < 0.01), low baseline plasma HCV RNA concentration (OR: 1.74; 95% CI = 1.2–2.6; P < 0.01), and the subtype 1b (OR: 1.61; 95% CI = 1.0–2.5; P = 0.04) or subtypes 4a and 4d (OR: 2.03; 95% CI = 1.1–3.8; P = 0.03). In conclusion, among difficult‐to‐treat genotypes, the subtype 1a is associated with a lower response to anti‐HCV therapy than subtypes 1b, 4a, and 4d. J. Med. Virol. 81:2029–2035, 2009.

Qualitative and quantitative molecular detection of enteroviruses in water from bathing areas and from a sewage treatment plant.

Pathogenic enteric viruses can be introduced into the environment as a result of human activities. Enteroviruses are regularly detected in environmental waters or shellfish and can provoke potentially serious diseases. Some authors believe that enteroviruses could represent an interesting indicator of viral contamination in the environment. Since molecular approaches seem to be promising for the detection of these viruses, we developed a simple qualitative RT-PCR procedure for enteroviruses, together with a quantitative RT-PCR assay using RNA internal standard. After one-tube-RT-PCR, this standard and wild enterovirus RNA were detected by differential hybridization with specific probes and a fluorimetric reaction. The quantification of enteroviruses, conducted in a sewage treatment plant, showed a decreasing number of genomic copies from the entrance to the exit (from 3.8 x 10(5) to 5.4 x 10(4) RNA copies/mL) but indicated the presence of enterovirus RNA in the neighboring river (2.2 x 10(3) RNA copies/mL). In bathing areas, enterovirus RNA was detected in 16 out of 226 samples, with copies numbers ranging from 3.7 x 10(2) RNA copies/mL to 7 x 10(4) RNA copies/mL.

PCR detection of human enteric viruses in bathing areas, waste waters and human stools in southwestern France

Detection of human pathogenic viruses by molecular techniques might be suitable for identifying viral pollution in environmental waters and for improving diagnosis in patients. Environmental samples were taken from bathing areas and sewage treatment plants in southwestern France. Small volume samples (50 microL) were tested. Five groups of enteric pathogenic viruses were studied: enteroviruses, Norwalk-like viruses (NLVs), hepatitis A virus, rotaviruses and adenoviruses. Moreover, human samples were tested for NLV. After extraction of viral nucleic acids (Booms procedure), a nested polymerase chain reaction was conducted before hybridization. Five bathing waters out of 26 were positive for one viral group, without systematic association with bacterial contamination. Eight sewage plant samples out of 13 were positive for at least one viral group. Seven patients out of 45 were NLV-positive. Molecular techniques allow efficient screening of viral contamination in environmental waters and the study of NLV molecular epidemiology.

Early Evolution of Hepatitis C Virus (HCV) Quasispecies after Liver Transplant for HCV-Related Disease

BACKGROUND End-stage liver disease as a result of chronic hepatitis C virus (HCV) infection is the main indication for liver transplant (LT), but allografts are systematically infected with HCV soon after transplant. Viral quasispecies are poorly described during the early posttransplant period. METHODS For 17 patients who received an LT for HCV disease, plasma viral quasispecies evolution was determined by sequence analysis of hypervariable region 1 of the E2 envelope gene before transplant (BT), after 7 days (D7), and after 1 month (M1). T helper (Th)1/Th2 cytokine levels were determined concomitantly. RESULTS HCV quasispecies showed a significant decrease in amino acid diversity at D7 and M1, compared with BT (P<.05). A correlation was observed between low plasma tumor necrosis factor-alpha levels at D7 and decreased quasispecies amino acid complexity at the same date. Nucleic acid diversity was lower for genotype 1 than for genotype 3 infection (P<.05). The complexity and diversity of amino acids were lower in patients with hepatocellular carcinoma (HCC) BT than in those without HCC (P<.05). Conserved amino acid residues within quasispecies were shared by the whole cohort before and after LT. CONCLUSION Viral structural and/or host immunological features could favor the emergence of fitter HCV strains after LT.

Multicenter Quality Control for the Detection of Hepatitis C Virus RNA in Seminal Plasma Specimens

ABSTRACT The discrepant results available in the literature about the presence of hepatitis C virus (HCV) RNA in seminal plasma of men chronically infected by this agent are related, at least in part, to the molecular techniques used and particularly to the wide range of protocols dedicated to RNA extraction. In order to evaluate these protocols and to standardize the method of detection of HCV RNA in this fluid, a panel of coded specimens was tested blindly in 12 French laboratories; it included 14 seminal plasma specimens and four water controls spiked with HCV RNA ranging from 10 to 20,000 IU/ml and two HCV-negative seminal plasma specimens. The extraction step was performed according to methods using either silica beads (NucliSens [Organon Teknika S.A., Fresnes, France]; RNA viral kit [Qiagen, Courtaboeuf, France]) or guanidinium thiocyanate (Amplicor HCV assay; Roche Diagnostics, Meylan, France), preceded or not by a centrifugation of the seminal plasma. For the amplification step, all the laboratories performed the same reverse transcription-PCR technique (Amplicor HCV Cobas assay). The percentage of correct results ranged from 53.3 to 100, the poorest results being obtained when no centrifugation step preceded the Amplicor extraction protocol. The rate of correct results was significantly higher in laboratories using a preliminary centrifugation of the specimen (P = 0.034 by chi-square test). By contrast, the overall number of correct results was not correlated to the initial volume of sample used for the test. These results allowed us to validate standardized techniques adapted to the performance of this test on a routine basis, especially in men infected with HCV and involved in programs of medically assisted reproduction.

Impact of protease inhibitors on intrahepatic hepatitis C virus viral load.

During chronic hepatitis C, hepatitis C virus (HCV) load in plasma was shown to be higher in HIV-co-infected than in immunocompetent patients [1]. The reason for this increased HCV replication is not known. It may be as a result of HIV-induced immune deficiency [2], although some authors did not find any correlation with the CD4 cell count [3]. A direct interaction between HCV and HIV was also hypothesized [4]. Protease inhibitors (PI) used in highly active antiretroviral therapy (HAART) have no HCV reduction effect during the first months of treatment [5-8]. However, a decrease in HCV plasma load was recently described in patients treated with HAART for a year [9,10]. We therefore investigated the potential impact of HAART on intrahepatic HCV load.

Frequent Compartmentalization of Hepatitis C Virus with Leukocyte-Related Amino Acids in the Setting of Liver Transplantation

BACKGROUND Nonrandom distribution of hepatitis C virus (HCV) quasispecies (compartmentalization between blood plasma and leukocytes) suggests the presence of HCV leukotropic variants. HCV compartmentalization in the setting of liver transplantation (LT) is poorly understood. To study HCV leukotropic variants, we investigated the evolution of HCV compartmentalization after immunosuppression in liver transplant recipients. METHODS Plasma and peripheral blood mononuclear cell (PBMC) samples were collected from 5 liver transplant recipients before and after LT. We used clone sequencing to analyze the hypervariable region 1 (HVR1)-E2(384-419) region, which plays a key role in HCV entry and the induction of neutralizing responses, and assessed compartmentalization through phylogenetic analyses and Mantels test. RESULTS Compartmentalization was frequent in the LT setting. HCV quasispecies were more homogeneous after LT in both the plasma and PBMC compartments, with a significant decrease in quasispecies complexity (P = .003) and genetic distances (P = .004) after transplantation. Our analysis identified 8 PBMC-related amino acid residues in HVR1. CONCLUSIONS HCV compartmentalization between plasma and PBMCs and the emergence of leukotropic variants could be potentiated by immunosuppression in liver transplant recipients. The identification of defined leukotropic variants may contribute to the understanding of virus-host interactions after transplantation.

Sequence analysis of env C2/V3, gag p17/p24, and pol protease regions of 25 HIV type 1 isolates from Ho Chi Minh City, Vietnam.

Env C2/V3, gag p17/p24, pol protease, and RT regions of HIV-1 isolates recently obtained from 25 HIV-1 seropositive individuals from Ho Chi Minh City (Vietnam) were studied, and genes subtypes were determined by DNA sequence analyses. Twenty-three isolates out of 25 were identified as belonging to subtype E, now recognized as circulating recombinant form 1 (CRF01_AE). The motif at the top of the V3 loop (generally GPGQ) was then preceded by an isoleucine or a methionine (M) residue; the M residue might be a local signature of Vietnamese E isolates compared to Thai E viruses. Two isolates (8%) were shown to be intersubtype recombinants: one E/B and one CRF02_AG(IBNG)/D. The polymorphism of pol protease was considered only for CRF01_AE isolates and is clearly different from that recorded for B viruses with substitutions at positions 13, 35, 36, 41, 69, and 89.

Naturally Occurring Resistance-Associated Variants of Hepatitis C Virus Protease Inhibitors in Poor Responders to Pegylated Interferon-Ribavirin

ABSTRACT The pretherapeutic presence of protease inhibitor (PI) resistance-associated variants (RAVs) has not been shown to be predictive of triple-therapy outcomes in treatment-naive patients. However, they may influence the outcome in patients with less effective pegylated interferon (pegIFN)-ribavirin (RBV) backbones. Using hepatitis C virus (HCV) population sequence analysis, we retrospectively investigated the prevalence of baseline nonstructural 3 (NS3) RAVs in a multicenter cohort of poor IFN-RBV responders (i.e., prior null responders or patients with a viral load decrease of <1 log IU/ml during the pegIFN-RBV lead-in phase). The impact of the presence of these RAVs on the outcome of triple therapy was studied. Among 282 patients, the prevalances (95% confidence intervals) of baseline RAVs ranged from 5.7% (3.3% to 9.0%) to 22.0% (17.3% to 27.3%), depending to the algorithm used. Among mutations conferring a >3-fold shift in 50% inhibitory concentration (IC50) for telaprevir or boceprevir, T54S was the most frequently detected mutation (3.9%), followed by A156T, R155K (0.7%), V36M, and V55A (0.35%). Mutations were more frequently found in patients infected with genotype 1a (7.5 to 23.6%) than 1b (3.3 to 19.8%) (P = 0.03). No other sociodemographic or viroclinical characteristic was significantly associated with a higher prevalence of RAVs. No obvious effect of baseline RAVs on viral load was observed. In this cohort of poor responders to IFN-RBV, no link was found with a sustained virological response to triple therapy, regardless of the algorithm used for the detection of mutations. Based on a cross-study comparison, baseline RAVs are not more frequent in poor IFN-RBV responders than in treatment-naive patients and, even in these difficult-to-treat patients, this study demonstrates no impact on treatment outcome, arguing against resistance analysis prior to treatment.