Nonstructural 5B Research Articles

Ledipasvir-sofosbuvir: interferon-/ribavirin-free regimen for chronic hepatitis C virus infection.

To review the pharmacology, efficacy, and safety of ledipasvir-sofosbuvir for the treatment of chronic hepatitis C virus (HCV). A literature search through clinicaltrials.gov, EMBASE, and PubMed was conducted (January 1966 to October 2014) using the terms ledipasvir, sofosbuvir, GS-5885, and GS-7977. References from retrieved articles and abstracts presented at recent meetings were reviewed for any additional material. The prescribing information was also reviewed. Phase 1, 2, and 3 human and animal studies describing the pharmacology, pharmacokinetics, efficacy, and safety of ledipasvir and sofosbuvir for HCV were identified. Ledipasvir-sofosbuvir, a fixed-dose combination (FDC) tablet inhibiting nonstructural (NS) 5A and 5B proteins, without peginterferon and ribavirin is indicated for adult patients with genotype 1 HCV infection who are treatment naïve or experienced, with or without cirrhosis. Pivotal trials (n = 1952) have demonstrated that once-daily administration of ledipasvir-sofosbuvir for 12 or 24 weeks is effective at achieving sustained virological response (SVR) rates (94%-99%) in treatment-naïve patients (12 weeks), treatment-experienced patients without cirrhosis (12 weeks), and treatment-experienced patients with cirrhosis (24 weeks). Treatment-naïve patients without cirrhosis and baseline viral levels of less than 6 million IU/mL may be considered for 8 weeks of treatment. The most common adverse drug events (ADEs) associated with ledipasvir-sofosbuvir include headache, fatigue, insomnia, nausea, and diarrhea. Ledipasvir-sofosbuvir is the first interferon- and ribavirin-free FDC agent that has SVR rates much greater than 94%, with minimal ADEs, for the treatment of chronic HCV genotype 1 in naïve and treatment-experienced patients.

Characterization of hepatitis C virus intergenotypic recombinant strains and associated virological response to sofosbuvir/ribavirin.

To date, intergenotypic recombinant hepatitis C viruses (HCVs) and their treatment outcomes have not been well characterized. This study characterized 12 novel HCV recombinant strains and their response to sofosbuvir in combination with ribavirin (SOF/RBV) treatment. Across the phase II/III studies of SOF, HCV samples were genotyped using both the Siemens VERSANT HCV Genotype INNO-LiPA 2.0 Assay (Innogenetics, Ghent, Belgium) and nonstructural (NS)5B sequencing. Among these patient samples, genotype assignment discordance between the two methods was found in 0.5% of all cases (12 of 2,363), of which all were identified as genotype 2 by INNO-LiPA (12 of 487; 2.5%). HCV full-genome sequences were obtained for these 12 samples by a sequence-independent amplification method coupled with next-generation sequencing. HCV full-genome sequencing revealed that these viruses were recombinant HCV strains, with the 5' part corresponding to genotype 2 and the 3' part corresponding to genotype 1. The recombination breakpoint between genotypes 2 and 1 was consistently located within 80 amino acids of the NS2/NS3 junction. Interestingly, one of the recombinant viruses had a 34-amino-acid duplication at the location of the recombination breakpoint. Eleven of these twelve patients were treated with a regimen for genotype 2 HCV infection, but responded as if they had genotype 1 infection; 1 patient had received placebo. Twelve new HCV intergenotypic recombinant genotype 2/1 viruses have been characterized. The antiviral response to a 12- to 16-week course of SOF/RBV treatment in these patients was more similar to responses among genotype 1 patients than genotype 2 patients, consistent with their genotype 1 NS5B gene.

Clinical and In Vitro Resistance to GS-9669, a Thumb Site II Nonnucleoside Inhibitor of the Hepatitis C Virus NS5B Polymerase

Treatment with GS-9669, a novel nonnucleoside inhibitor (site II) of hepatitis C virus (HCV) nonstructural 5B (NS5B) polymerase, resulted in significant antiviral activity in HCV genotype (GT) 1 patients dosed at 50 and 500 mg once daily (QD) and at 50, 100, and 500 mg twice daily (BID) for 3 days. This report characterizes the virologic resistance to GS-9669 in vitro and in GT1 HCV-infected patients from a phase I clinical study. An in vitro resistance selection study with GS-9669 revealed substitutions at several NS5B residues that conferred resistance. The M423 variants were selected at low drug concentrations (5× the 50% effective concentration [EC50]), and the L419, R422, and I482 variants were selected at higher drug concentrations (20× the EC50). During the phase I clinical study, substitutions at NS5B residues 419, 422, and 486 were the predominant changes associated with GS-9669 monotherapy. Substitutions at position 423 were observed only in GT1a patients in the low-dose groups (50 and 100 mg BID). Interestingly, four HCV patients had substitutions at position 423 at baseline. Consistent with the low resistance level at this position, three patients with M423I or M423V at baseline achieved >2-log10 reductions of HCV RNA when treated with 100 mg BID or with 500 mg QD or BID of GS-9669. The fourth patient, who had the M423V substitution at baseline, had a 4.4-log10 reduction of HCV RNA with 500 mg BID of GS-9669. Phenotypic analyses demonstrated that the viral isolates with multiple GS-9669 resistance-associated variants have reduced susceptibility to GS-9669 and lomibuvir (VX-222) but are not cross-resistant to other classes of HCV inhibitors. (This study has been registered at ClinicalTrials.gov under registration no. NCT01431898.).

Naturally Occurring Mutations in the Nonstructural Region 5B of Hepatitis C Virus (HCV) from Treatment-Naïve Korean Patients Chronically Infected with HCV Genotype 1b

The nonstructural 5B (NS5B) protein of the hepatitis C virus (HCV) with RNA-dependent RNA polymerase (RdRp) activity plays a pivotal role in viral replication. Therefore, monitoring of its naturally occurring mutations is very important for the development of antiviral therapies and vaccines. In the present study, mutations in the partial NS5B gene (492 bp) from 166 quasispecies of 15 genotype-1b (GT) treatment-naïve Korean chronic patients were determined and mutation patterns and frequencies mainly focusing on the T cell epitope regions were evaluated. The mutation frequency within the CD8+ T cell epitopes was significantly higher than those outside the CD8+ T cell epitopes. Of note, the mutation frequency within predicted CD4+ T cell epitopes, a particular mutational hotspot in Korean patients was significantly higher than it was in patients from other areas, suggesting distinctive CD4+ T cell-mediated immune pressure against HCV infection in the Korean population. The mutation frequency in the NS5B region was positively correlated with patients with carrier-stage rather than progressive liver disease (chronic hepatitis, liver cirrhosis and hepatocellular carcinoma). Furthermore, the mutation frequency in four codons (Q309, A333, V338 and Q355) known to be related to the sustained virological response (SVR) and end-of treatment response (ETR) was also significantly higher in Korean patients than in patients from other areas. In conclusion, a high degree of mutation frequency in the HCV GT-1b NS5B region, particularly in the predicted CD4+ T cell epitopes, was found in Korean patients, suggesting the presence of distinctive CD4+ T cell pressure in the Korean population. This provides a likely explanation of why relatively high levels of SVR after a combined therapy of pegylated interferon (PEG-IFN) and ribavirin (RBV) in Korean chronic patients with GT-1b infections are observed.

Naturally Occurring Mutations of Hepatitis B virus and Hepatitis C Virus in Korean Chronic Patients by Distinct CD4 T Cell Responses

Hepatitis B virus (HBV) and hepatitis C virus (HCV) infections are among the most common causes of chronic liver disease worldwide. The host immune pressure against hepatitis viruses during the chronic infection has led to mutations in their coding genes, which could play a pivotal role in the clinical outcomes of chronic patients. Our recent molecular epidemiologic studies regarding the HBV precore/core (preC/C) regions and HCV nonstructural 5B (NS5B) protein suggest the presence of distinct CD4 T cell immune pressure against HBV and HCV in Korean chronic patients. However, induced HBV and HCV mutations seem to exert an opposite effect on Korean chronic hepatitis B (CHB) and chronic hepatitis C (CHC) patients, respectively. On the basis of two of our recent papers, we focused in this review on the relationships between the mutation patterns of HBV preC/C and HCV NS5B, which were presumed to be caused by distinct CD4 T cell pressure in the Korean population and their effect on the clinical outcomes and liver disease progression of CHB and CHC patients.

Molecular Modeling Comparison of the Performance of NS5b Polymerase Inhibitor (PSI-7977) on Prevalent HCV Genotypes

The current available treatment for hepatitis C virus (HCV)-the causative of liver cirrhosis and development of liver cancer-is a dual therapy using modified interferon and ribavirin. While this regimen increases the sustained viral response rate up to 40-80% in different genotypes, unfortunately, it is poorly tolerated by patients. PSI-7977, a prodrug for PSI-7409, is a Non-Structural 5b (NS5b) polymerase nucleoside inhibitor that is currently in phase III clinical trials. The activated PSI-7977 is a direct acting antiviral (DAA) drug that acts on NS5b polymerase of HCV through a coordination bond with the two Mg(+2) present at the GDD active site motif. The present work utilizes a molecular modeling approach for studying the interaction between the activated PSI-7977 and the 12 amino acids constituting a 5Å region surrounding the GDD active triad motif for HCV genotypes 1a, 2b, 3b and 4a. The analysis of the interaction parameters suggests that PSI-7977 is probably a better DAA drug for HCV genotypes 1a and 3b rather than genotypes 2b and 4a.

Self-Enhancement of Hepatitis C Virus Replication by Promotion of Specific Sphingolipid Biosynthesis

Lipids are key components in the viral life cycle that affect host-pathogen interactions. In this study, we investigated the effect of HCV infection on sphingolipid metabolism, especially on endogenous SM levels, and the relationship between HCV replication and endogenous SM molecular species. We demonstrated that HCV induces the expression of the genes (SGMS1 and 2) encoding human SM synthases 1 and 2. We observed associated increases of both total and individual sphingolipid molecular species, as assessed in human hepatocytes and in the detergent-resistant membrane (DRM) fraction in which HCV replicates. SGMS1 expression had a correlation with HCV replication. Inhibition of sphingolipid biosynthesis with a hepatotropic serine palmitoyltransferase (SPT) inhibitor, NA808, suppressed HCV-RNA production while also interfering with sphingolipid metabolism. Further, we identified the SM molecular species that comprise the DRM fraction and demonstrated that these endogenous SM species interacted with HCV nonstructural 5B polymerase to enhance viral replication. Our results reveal that HCV alters sphingolipid metabolism to promote viral replication, providing new insights into the formation of the HCV replication complex and the involvement of host lipids in the HCV life cycle.

Hepatitis C virus non-structural 5B protein interacts with cyclin A2 and regulates viral propagation

Hepatitis C virus (HCV) requires host cellular proteins for its own propagation. To identify the cellular factors necessary for HCV propagation, we have recently screened the small interfering RNA (siRNA) library targeting cell cycle genes using cell culture grown HCV (HCVcc)-infected cells. In the current study, we have selected and characterized the gene encoding Cyclin A2 (CycA2). Deregulation of CycA2 has been implicated in many types of cancers, including hepatocellular carcinoma. The effects of CycA2 on HCV propagation were investigated by siRNA-mediated knockdown assay, in vitro and in vivo protein binding assays, luciferase reporter gene assay, and immunoblot assay. We showed that siRNA-mediated depletion of CycA2 significantly inhibited HCV replication in both HCV subgenomic replicon cells and HCVcc-infected cells. Furthermore, HCV non-structural 5B (NS5B) specifically interacted with CycA2 in vitro and in vivo. Protein interaction was mediated through the cyclin box of CycA2 and the palm domain of NS5B. We further showed that R/HxL motif in the palm domain of HCV NS5B mediated protein interaction with CycA2 and this interaction was necessary for HCV replication. Moreover, we demonstrated that tylophorine, the natural plant product exerting a CycA2 inhibitory function, abrogated HCV replication. HCV regulates CycA2 via NS5B protein for its own propagation. In addition, tylophorine may be a potential therapeutic agent for HCV.

HCV RNA levels in a multiethnic cohort of injection drug users: Human genetic, viral and demographic associations

In patients with chronic hepatitis C, the hepatitis C virus (HCV) RNA level is an important predictor of treatment response. To explore the relationship of HCV RNA with viral and demographic factors, as well as IL28B genotype, we examined viral levels in an ethnically diverse group of injection drug users (IDUs). Between 1998 and 2000, the Urban Health Study (UHS) recruited IDUs from street settings in San Francisco Bay area neighborhoods. Participants who were positive by HCV enzyme immunoassay were tested for HCV viremia by a branched-chain DNA assay. HCV genotype was determined by sequencing the HCV nonstructural 5B protein region. For a subset of participants, IL28B rs12979860 genotype was determined by Taqman. Among 1,701 participants with HCV viremia, median age was 46 years and median duration of injection drug use was 26 years; 56.0% were African American and 34.0% were of European ancestry (non-Hispanic). Human immunodeficiency virus type 1 (HIV-1) prevalence was 13.9%. The overall median HCV RNA level was 6.45 log(10) copies/mL. In unadjusted analyses, higher levels were found with older age, male gender, African-American ancestry, hepatitis B virus infection, HIV-1 infection, and IL28B rs12979860-CC genotype; compared to participants infected with HCV genotype 1, HCV RNA was lower in participants with genotypes 3 or 4. In an adjusted analysis, age, gender, racial ancestry, HIV-1 infection, HCV genotype, and IL28B rs12979860 genotype were all independently associated with HCV RNA. The level of HCV viremia is influenced by a large number of demographic, viral, and human genetic factors.

Dengue hemorrhagic fever-associated immunomediators induced via maturation of dengue virus nonstructural 4B protein in monocytes modulate endothelial cell adhesion molecules and human microvascular endothelial cells permeability

We previously demonstrated that dengue virus (DENV) nonstructural 4B protein (NS4B) induced dengue hemorrhagic fever (DHF)-associated immunomediators in THP-1 monocytes. Moreover, cleavage of NS4AB polyprotein by the NS2B3 protease, significantly increased immunomediator production to levels found after DENV infection. In this report using primary human microvascular endothelial cells (HMVEC) transwell permeability model and HMVEC monolayer, we demonstrate that the immunomediators secreted in the supernatants of DENV-infected monocytes increase HMVEC permeability and expression of ICAM-1, VCAM-1 and E-selectin. Moreover, maturation of NS4B via cleavage of 2KNS4B is sufficient to induce immunomediators that cause HMVEC phenotypic changes, which appear to be synergistically induced by TNFα and IL-8. These data suggest that therapies targeting the maturation steps of NS4B, particularly 2KNS4B processing, may reduce overall DHF-associated immunomediator levels, thereby reducing DHF-associated morbidity and mortality. Alternatively, TNFα inhibitors may be a valid intervention strategy during the later stages of infection to prevent DHF progression.

Antiviral activity of the hepatitis C virus polymerase inhibitor filibuvir in genotype 1–infected patients

More effective and better-tolerated therapies are needed for chronic hepatitis C virus (HCV) infection. Among the direct-acting anti-HCV agents in development is the nonstructural 5B protein (NS5B polymerase) non-nucleoside inhibitor filibuvir. We investigated the antiviral activity, pharmacokinetics, safety, and tolerability of multiple doses of filibuvir in treatment-naive and treatment-experienced patients who were chronically infected with HCV genotype 1 in two phase 1b clinical studies (study 1 was a randomized, placebo-controlled dose escalation study and study 2 was a nonrandomized, open-label study). The filibuvir doses evaluated ranged from 200-1400 mg daily, and the duration of dosing ranged from 3-10 days. Genotypic changes in the NS5B nucleotide sequence following short-term filibuvir therapy were also assessed. Filibuvir potently inhibited viral replication in a dose-dependent manner. Mean maximum HCV RNA change from baseline ranged from -0.97 log(10) IU/mL with filibuvir given at 100 mg twice daily to -2.30 log(10) IU/mL with filibuvir given at 700 mg twice daily in treatment-naive patients. In treatment-experienced patients, an HCV RNA reduction of 2.20 log(10) IU/mL was achieved with filibuvir given at 450 mg twice daily. Filibuvir was well tolerated in both studies. Adverse events were mild or moderate in severity. No discontinuations, serious adverse events, or deaths were reported. NS5B sequencing identified residue 423 as the predominant site of mutation after filibuvir dosing. Filibuvir administration resulted in significant reductions in HCV RNA concentrations at doses that were well tolerated in patients infected with HCV genotype 1. Filibuvir is currently being evaluated in combination with pegylated interferon alfa 2a plus ribavirin in treatment-naive patients.

Charged Residues in Hepatitis C Virus NS4B Are Critical for Multiple NS4B Functions in RNA Replication

The nonstructural 4B (NS4B) protein of hepatitis C virus (HCV) plays a central role in the formation of the HCV replication complex. To gain insight into the role of charged residues for NS4B function in HCV RNA replication, alanine substitutions were engineered in place of 28 charged residues residing in the N- and C-terminal cytoplasmic domains of the NS4B protein of the HCV genotype 1b strain Con1. Eleven single charged-to-alanine mutants were not viable, while the remaining mutants were replication competent, albeit to differing degrees. By selecting revertants, second-site mutations were identified for one of the lethal NS4B mutations. Second-site mutations mapped to NS4B and partially suppressed the lethal replication phenotype. Further analyses showed that three NS4B mutations disrupted the formation of putative replication complexes, one mutation altered the stability of the NS4B protein, and cleavage at the NS4B/5A junction was significantly delayed by another mutation. Individual charged-to-alanine mutations did not affect interactions between the NS4B and NS3-4A proteins. A triple charged-to-alanine mutation produced a temperature-sensitive replication phenotype with no detectable RNA replication at 39°C, demonstrating that conditional mutations can be obtained by altering the charge characteristics of NS4B. Finally, NS4B mutations dispensable for efficient Con1 RNA replication were tested in the context of the chimeric genotype 2a virus, but significant defects in infectious-virus production were not detected. Taken together, these findings highlight the importance of charged residues for multiple NS4B functions in HCV RNA replication, including the formation of a functional replication complex.

Molecular characterization of hepatitis C virus genotype 4 sequences in HIV‐coinfected patients from Argentina

The prevalence of hepatitis C virus genotype 4 (HCV-4) is increasing in different parts of the World but in Latin America the data are still scarce. We aimed to characterize HCV-4 isolates from 383 HIV-coinfected patients in Argentina. Sequence analyses were based on the non-structural 5B region of HCV. Results from 18 patients indicated a genetic heterogeneity that involved three genotype 4 subtypes. Sequences were ascribed to subtype 4d (67%), 4a (22%), and 4m (11%). In spite of different sources of transmission were defined among patients, no statistical association was found with the genotype 4 subtype. The scenario is also compatible with multiple importation of the epidemic and there is no evidence for transmission-specific clusters or network-like transmission of HCV-4. This HCV-4 does not represent a recent introduction in Argentina, it circulates in all transmission groups and its presence is increasing among HIV-infected patients.

The synthesis of novel heteroaryl-fused 7,8,9,10-tetrahydro-6H-azepino[1,2-a]indoles, 4-oxo-2,3-dihydro-1H-[1,4]diazepino[1,7-a]indoles and 1,2,4,5-tetrahydro-[1,4]oxazepino[4,5-a]indoles. Effective inhibitors of HCV NS5B polymerase

Three synthetic approaches have been developed that allow efficient access to novel heteroaryl fused indole ring systems, including: 7,8,9,10-tetrahydro-6H-azepino[1,2-a]indoles, 4-oxo-2,3-dihydro-1H-[1,4]diazepino[1,7-a]indoles and 1,2,4,5-tetrahydro-[1,4]oxazepino[4,5-a]indoles. Each strategy is fully exemplified and the relative merits and limitations of the approaches are discussed. The hepatitis C virus (HCV) non-structural 5B (NS5B) polymerase inhibitory activities of select examples from each molecular class are briefly presented.

The Future of HCV Therapy: NS4B as an Antiviral Target

Chronic hepatitis C virus (HCV) infection is a major worldwide cause of liver disease, including cirrhosis and hepatocellular carcinoma. It is estimated that more than 170 million individuals are infected with HCV, with three to four million new cases each year. The current standard of care, combination treatment with interferon and ribavirin, eradicates the virus in only about 50% of chronically infected patients. Notably, neither of these drugs directly target HCV. Many new antiviral therapies that specifically target hepatitis C (e.g. NS3 protease or NS5B polymerase inhibitors) are therefore in development, with a significant number having advanced into clinical trials. The nonstructural 4B (NS4B) protein, is among the least characterized of the HCV structural and nonstructural proteins and has been subjected to few pharmacological studies. NS4B is an integral membrane protein with at least four predicted transmembrane (TM) domains. A variety of functions have been postulated for NS4B, such as the ability to induce the membranous web replication platform, RNA binding and NTPase activity. This review summarizes potential targets within the nonstructural protein NS4B, with a focus on novel classes of NS4B inhibitors.

Natural intragenotypic and intergenotypic HCV recombinants are rare in southwest China even among patients with multiple exposures

Background Both intragenotypic and intergenotypic hepatitis C virus (HCV) recombinants were recently identified, whereas the frequency of HCV recombinants in circulation in certain parts of the world remains uncertain. Objectives To assess the frequency of natural intragenotypic and intergenotypic HCV recombinants in southwest China and that of multitypic HCV infection, which is the prerequisite factor for recombination. Study design Comparison of the genotyping results based on core-envelope 1 and non-structural 5B sequence phylogenetic analyses to detect the possible recombinants. Multitypic HCV infection was detected by colony analysis of the core-envelope 1 region. Results There was no discrepancy between genotyping results based on the core-envelope 1 and non-structural 5B regions in 243 sera from 222 patients, including 107 samples from 92 intravenous drug users, 26 samples from 20 hemodialysis patients, and 110 samples from 110 other patients. Multitypic HCV carriage was identified in 9 (47.4%) of the 19 patients with multiple exposures who were randomly selected, and 11 (45.8%) of the 24 specimens. There were 26 repeatedly exposed patients who had more than one HCV RNA positive sera at different time points, but neither of their HCV subtypes had switched over time. Conclusion Although the high frequency of multitypic HCV infection was identified in patients with multiple exposures, intergenotypic or intragenotypic recombination remains an infrequent event in southwest China; thus, routine genotyping with more than one subgenomic region for clinical use is not warranted.

Evaluation of core and NS4B synthetic peptide-based immunoassays for the detection of hepatitis C virus antibodies in clinical samples from Cameroon, Central Africa

Background According to previous data, the antibodies produced during natural hepatitis C virus (HCV) infection frequently recognize amino acids 10–43 in the core protein and 1689–1740 or 1921–1940 in the non-structural 4B (NS4B) protein. The reactivity of these peptides with the corresponding antibodies has mainly been evaluated using serum samples from Western countries where HCV genotype 1 (HCV-1) is predominant, and no information is available concerning samples from sub-Saharan countries where high HCV variability has been reported. Objective of this study To evaluate the performance of HCV core and NS4B peptide-based immunoassays in the serodiagnosis of HCV infection in Cameroon subjects. Study design Three core and four NS4B-based synthetic peptides derived from HCV genotypes 1b and 2a were designed and tested against a panel of 151 serum samples from Cameroon (40 positive for HCV-1, 32 for HCV-2, 39 HCV-4, and 40 HCV-negative). Results The three core peptides all demonstrated strong immunoreactivity, regardless of the HCV genotype from which they were derived, with greater than 90% and 92% sensitivity and specificity. In contrast, the NS4B-derived peptides exhibited lower sensitivities (24.3–65.8% depending on the HCV genotype) but higher specificities (100% for all four peptides tested). Conclusions Our findings indicate that an HCV core peptide could be used for the diagnosis of chronic HCV infection. Among the NS4B peptides tested, a chimeric NS4B peptide encompassing both N- and C-terminal portions of the NS4B protein gave a much better performance than the two component N- and C-terminal peptides used individually.

A small molecule inhibits HCV replication and alters NS4B's subcellular distribution

Hepatitis C Virus (HCV) is a leading cause of liver disease and represents a significant public health challenge. Treatments for this disease are inadequate and improved antiviral therapies are necessary. Several such antivirals are in development, most of which target the well-characterized NS3 protease or the NS5B polymerase. In contrast, the nonstructural 4B (NS4B) protein, though essential for HCV RNA replication, has been the subject of few pharmacological studies. One of the functions ascribed to this protein is the ability to form intracellular membrane-associated foci (MAF), which are believed to be related to the sites of viral replication. Here, we report the identification of a small molecule that inhibits HCV replication and disrupts the organization of these MAF. Genetic analysis links the compound's mode of action to the NS4B gene product, and transient transfections of NS4B-GFP demonstrate that treatment with this compound can lead to the formation of novel elongated assemblies of NS4B. Furthermore, an in vitro dynamic light scattering assay provides evidence that the second amphipathic helix of NS4B may be the target of the drug. Our results demonstrate that this molecule represents a new potential class of HCV inhibitors and also provides us with a useful tool for studying the HCV life cycle.

Hepatitis C virus genotypes in hemodialysis patients in the Federal District, Brazil

Hepatitis C virus (HCV) genotypes and subtypes were determined in hemodialysis patients in the Federal District, Brazil, by sequencing of the 5' noncoding (NC) and nonstructural 5B (NS5B) regions. From 761 patients, 66 anti-HCV-positive samples were tested for HCV RNA. All 51 HCV RNA-positive samples by PCR of the 5' NC region were genotyped as genotypes 1 (90.2%) and 3 (9.8%). Subtype 1a (82.3%) was the most prevalent, followed by subtypes 3a (9.8%), 1b (5.9%) and 1a/1b (2.0%). Forty-two samples could be amplified and genotyped in the NS5B region: 38 (90.5%) as genotype 1, subtypes 1a, and 8 (9.5%) as genotype 3, subtype 3a. For the 42 samples sequenced in both regions, the genotypes and subtypes determined were concordant in 100% and 95.2% of cases, respectively. Two samples presented discrepant results, with the 5' NC region not distinguishing correctly the subtypes 1a and 1b. These findings indicate that the HCV genotype 1, subtype 1a, is the most prevalent among hemodialysis patients in the Federal District, Brazil.

Monitoring hepatitis C infection in a major Swedish nephrology unit and molecular resolution of a new case of nosocomial transmission

Hepatitis C virus (HCV) infection is a frequent problem in hemodialysis units. The prevalence and incidence of HCV infection over a decade were studied in a nephrology unit affected by previous nosocomial HCV transmission. The HCV non-structural 5B protein gene was sequenced to achieve phylogenetic analysis of a new (incident) case of infection. Proportions of patients who were and were not infected with HCV remained similar over the period, as did the inflow and outflow of patients infected previously. In 1997, 12/157 (8%) of patients at the unit (treatment: hemodialysis, peritoneal dialysis, and renal transplant recipients) were positive in HCV RNA, whereas in 2007 the overall number was 9/239 (4%). One patient acquired an HCV infection, and the NS5B sequence in that case clustered with genotype 2b sequences found in patients from an earlier outbreak. Comparing the HCV from the incident patient with several stored longitudinal samples and cloned PCR products from the most likely source patient revealed close phylogenetic relationship with an HCV quasispecies member from the possible source. The source patient and the incident newly infected patient were not scheduled on the same dialysis shift, although the records showed that simultaneous treatment occurred on two occasions during the months preceding transmission. In conclusion, over the 10-year period, the proportion of HCV-infected patients at the unit was unchanged. Only one new infection occurred, which originated from a fellow patient's quasispecies. This establishes phylogenetic analysis as a valuable tool for tracing patient sources of HCV transmission.