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Potent and broad cellular immune responses against the nonstructural (NS) proteins of hepatitis C virus (HCV) are associated with spontaneous viral clearance. In this study, we have improved the immunogenicity of an adenovirus (Ad)-based HCV vaccine by fusing NS3 from HCV (Strain J4; Genotype 1b) to the MHC class II chaperone protein invariant chain (Ii). We found that, after a single vaccination of C57BL/6 or BALB/c mice with Ad-IiNS3, the HCV NS3-specific CD8+ T cell responses were significantly enhanced, accelerated, and prolonged compared with the vaccine encoding NS3 alone. The AdIiNS3 vaccination induced polyfunctional CD8+ T cells characterized by coproduction of IFN-γ, TNF-α and IL-2, and this cell phenotype is associated with good viral control. The memory CD8+ T cells also expressed high levels of CD27 and CD127, which are markers of long-term survival and maintenance of T cell memory. Functionally, the AdIiNS3-vaccinated mice had a significantly increased cytotoxic capacity compared with the AdNS3 group. The AdIiNS3-induced CD8+ T cells protected mice from infection with recombinant vaccinia virus expressing HCV NS3 of heterologous 1b strains, and studies in knockout mice demonstrated that this protection was mediated primarily through IFN-γ production. On the basis of these promising results, we suggest that this vaccination technology should be evaluated further in the chimpanzee HCV challenge model.

Hepatitis C virus (HCV) purification by ultracentrifugation is difficult because of the low and heterogeneous density of native and cultured viruses. It was recently shown that inserting flag tag into envelope protein 2 (E2) of HCV permitted virus purification by affinity chromatography. However, flag-tagged viruses had drastically altered properties, and purification yield was low. In this study, we found that insertion of flag tag at the N-terminus of E2 in HCV recombinant J6/JFH1 did not affect viability in Huh7.5 cells, and that flag-tagged virus had physiochemical properties similar to the original virus. Flag-tagged virus was susceptible to flag-specific antibody neutralization, and infected cells could be immuno-stained by anti-flag antibodies. Using affinity chromatography with anti-flag resin we repeatedly obtained ~30% recovery of infectious particles. The full viability and unaltered physiochemical properties of flag-tagged HCV is an important improvement for utilizing these viruses for imaging, virion composition analysis and possibly vaccine development.

Summary. Twenty-five per cent of individuals infected with hepatitis C virus (HCV) are able to clear HCV spontaneously. Differences in host genetics are believed to affect the outcome of HCV infection. We analysed an exonic, a promoter and an intronic single nucleotide polymorphism (SNP) of the interferon-λ3 coding interleukin (IL)-28B gene to study the relationship between IL28B SNPs and outcome of HCV infection. Among 206 HIV-1-infected Europeans with evidence of HCV infection, 47 (23%) individuals had cleared HCV and 159 (77%) had developed chronic infection. The exonic rs8103142 CT, the promoter rs12979860 CT and the intronic rs11881222 AG genotypes were associated with a decreased HCV clearance rate with adjusted odds ratios (aOR) of 0.3 (95% CI, 0.1–0.7), 0.4 (95% CI, 0.2–0.8) and 0.4 (95% CI, 0.2–0.8), respectively. The haplotype block TCG CTA was associated with a decreased HCV clearance rate (aOR 0.4, 95% CI, 0.2–0.8). Further, we found significant differences in HCV RNA levels among individuals chronically infected with HCV genotype 1 for rs8103142 and rs12979860 (P ≤ 0.05). Chronically infected individuals with HCV genotype 3 and with the favourable haplotype block CTA CTA had higher median HCV RNA levels than individuals with unfavourable haplotype blocks (P ≤ 0.05). Our findings suggest that IL28B may account for some differences in HCV outcome but that other factors including the viral genotype, host genetics and the host–virus interaction are likely to influence the outcome of HCV infection.

Background & Aims Chronic liver diseases caused by hepatitis C virus (HCV) genotype 6 are prevalent in Asia, and millions of people require treatment with direct-acting antiviral regimens, such as NS5A inhibitor velpatasvir combined with the NS5B polymerase inhibitor sofosbuvir. We developed infectious cell culture models of HCV genotype 6a infection to study the effects of these inhibitors and the development of resistance. Methods The consensus sequences of strains HK2 (MG717925) and HK6a (MG717928), originating from serum of patients with chronic HCV infection, were determined by Sanger sequencing of genomes amplified by reverse-transcription polymerase chain reaction. In vitro noninfectious full-length clones of these 6a strains were subsequently adapted in Huh7.5 cells, primarily by using substitutions identified in JFH1-based Core-NS5A and Core-NS5B genotype 6a recombinants. We studied the efficacy of NS5A and NS5B inhibitors in concentration-response assays. We examined the effects of long-term culture of Huh7.5 cells incubated with velpatasvir and sofosbuvir singly or combined following infection with passaged full-length HK2 or HK6a recombinant viruses. Resistance-associated substitutions (RAS) were identified by Sanger and next-generation sequencing, and their effects on viral fitness and in drug susceptibility were determined in reverse-genetic experiments. Results Adapted full-length HCV genotype 6a recombinants HK2cc and HK6acc had fast propagation kinetics and high infectivity titers. Compared with an HCV genotype 1a recombinant, HCV genotype 6a recombinants of strains HK2 and HK6a were equally sensitive to daclatasvir, elbasvir, velpatasvir, pibrentasvir, and sofosbuvir, but less sensitive to ledipasvir, ombitasvir, and dasabuvir. Long-term exposure of HCV genotype 6a-infected Huh7.5 cells with a combination of velpatasvir and sofosbuvir resulted in clearance of the virus, but the virus escaped the effects of single inhibitors via emergence of the RAS L31V in NS5A (conferring resistance to velpatasvir) and S282T in NS5B (conferring resistance to sofosbuvir). Engineered recombinant genotype 6a viruses with single RAS mediated resistance to velpatasvir or sofosbuvir. HCV genotype 6a viruses with RAS NS5A-L31V or NS5B-S282T were however, able to propagate and escape in Huh7.5 cells exposed to the combination of velpatasvir and sofosbuvir. Further, HCV genotype 6a with NS5A-L31V was able to propagate and escape in the presence of pibrentasvir with emergence of NS5A-L28S, conferring a high level of resistance to this inhibitor. Conclusions Strains of HCV genotype 6a isolated from patients can be adapted to propagate in cultured cells, permitting studies of the complete life cycle for this important genotype. The combination of velpatasvir and sofosbuvir is required to block propagation of original HCV genotype 6a, which quickly becomes resistant to single inhibitors via the rapid emergence and persistence of RAS. These features of HCV genotype 6a could compromise treatment.

Genetic polymorphisms seem to influence the response on antidepressant treatment and moderate the impact of stress on depression. The present study aimed to assess, whether allelic variants and stressful life events interact on the clinical outcome of depression. In a sample of 290 systematically recruited patients diagnosed with a single depressive episode according to ICD-10, we assessed the outcome of antidepressant treatment and the presence of stressful life events in a 6-month period preceding onset of depression by means of structured interviews. Further, we genotyped nine polymorphisms in the genes encoding the serotonin transporter, brain derived neurotrophic factor, catechol-O-methyltransferase, angiotensin converting enzyme, tryptophan hydroxylase, and the serotonin receptors 1A, 2A, and 2C. We found no evidence that the effects of the genetic polymorphisms on treatment outcome were dependent on stressful life events experienced by the individual prior to onset of depression.

Background: It has never been investigated whether comorbid personality disorder or neuroticism predicts a poor treatment outcome in first episode depression. Methods: Medically treated patients discharged with a diagnosis of a single depressive episode from a psychiatric in- or outpatient hospital setting were consecutively sampled from the Danish Psychiatric Central Research Register. The patients participated in an extensive interview including the Schedules for Clinical Assessment in Neuropsychiatry, the Structured Clinical Interview for DSM-IV Axis II Personality Disorders and a detailed assessment of medical treatment history using standardised procedures (Treatment Response to Antidepressants Questionnaire, TRAQ). Remission was defined as a score of ≤7 on the Hamilton Depression Rating Scale, 17 items, and a score of ≧4 on the TRAQ following (1) a first adequate trial of antidepressant treatment, and (2) 2 trials of antidepressant treatment. Further personality traits were assessed by means of the Eysenck Personality Questionnaire. Results: Among a total of 301 patients with a single depressive episode, 31.9% fulfilled diagnostic criteria for at least 1 personality disorder of any kind. Comorbid personality disorder was associated with a 2.2-times (95% CI: 1.1–4.2) increased risk of non-remission following the first antidepressant trial, whereas no effect was found following the second antidepressant trial (OR: 1.6; 95% CI: 0.8–3.4). A high level of neuroticism was associated with non-remission in first as well as second trials. Conclusion: Comorbid personality disorder and high levels of neuroticism in first episode depression predict an increased risk of non-remission from depression.

GB virus B (GBV-B) causes acute hepatitis in experimentally infected tamarins. We compared evolutionary features in acute resolving and persistent GBV-B infection. We detected no evidence of evolution in four animals with clearance during weeks 9-12, whereas three animals with clearance during weeks 13-26 had several substitutions in their polyprotein sequence. A single tamarin had long-term GBV-B viraemia; analysis of virus recovered at weeks 2, 5, 12, 20, 26, 52 and 104 demonstrated that mutations accumulated over time. Overall, the amino acid substitution rate was 3.5x10(-3) and 1.1x10(-3) substitutions per site year(-1) during weeks 1-52 and 53-104, respectively. Thus, there was a significant decrease in evolution over time, as found for hepatitis C virus. The rate of non-synonymous substitution per non-synonymous site compared with that of synonymous substitution per synonymous site decreased over time, suggesting reduction of positive selective pressure. These data demonstrate that prolonged GBV-B infection is associated with viral evolution.

Six major hepatitis C virus (HCV) genotypes and numerous subtypes have been described, and recently a seventh major genotype was discovered. Genotypes show significant molecular and clinical differences, such as differential response to combination therapy with interferon-α and ribavirin. Recently, HCV research has been accelerated by cell culture systems based on the unique growth capacity of strain JFH1 (genotype 2a). By development of JFH1-based intergenotypic recombinants containing Core, envelope protein 1 and 2 (E1, E2), p7, and nonstructural protein 2 (NS2) of genotype 6a and 7a strains, as well as subtype 1b and 2b strains, we have completed a panel of culture systems for all major HCV genotypes. Efficient growth in Huh7.5 cells depended on adaptive mutations for HK6a/JFH1 (6a/2a, in E1 and E2) and J4/JFH1 (1b/2a, in NS2 and NS3); viability of J8/JFH1 (2b/2a) and QC69/JFH1 (7a/2a) did not require adaptation. To facilitate comparative studies, we generated virus stocks of genotype 1–7 recombinants with infectivity titers of 103.7 to 105.2 50% tissue culture infectious dose/mL and HCV RNA titers of 107.0 to 107.9 IU/mL. Huh7.5 cultures infected with genotype 1–6 viruses had similar spread kinetics, intracellular Core, NS5A, and lipid amounts, and colocalization of Core and NS5A with lipids. Treatment with interferon-α2b but not ribavirin or amantadine showed a significant antiviral effect. Infection with all genotypes could be blocked by specific antibodies against the putative coreceptors CD81 and scavenger receptor class B type I in a dose-dependent manner. Finally, neutralizing antibodies in selected chronic phase HCV sera had differential effects against genotype 1–7 viruses. Conclusion: We completed and characterized a panel of JFH1-based cell culture systems of all seven major HCV genotypes and important subtypes and used these viruses in comparative studies of antivirals, HCV receptor interaction, and neutralizing antibodies. (HEPATOLOGY 2009.)

Protective immunity after resolved hepatitis C virus (HCV) infection has been reported. However, the breadth of this immunity has remained controversial, and the role of neutralizing antibodies has not been well-defined. In the present study, two chimpanzees (CH96A008 and CH1494) with resolved monoclonal H77C (genotype 1a) infection were rechallenged with low-dose homologous H77C virus about 12 months after viral clearance; CH96A008 became persistently infected, and CH1494 had transient viremia lasting 2 weeks. CH1494 was subsequently either partially or completely protected following five homologous rechallenges with monoclonal H77C or polyclonal H77 and after six heterologous rechallenges with HC-J4 (genotype 1b) or HC-J6 (genotype 2a) viruses. Subsequently, a final challenge with H77C resulted in persistent HCV infection. In both chimpanzees, serum neutralizing antibodies against retroviral pseudoparticles bearing the H77C envelope proteins were not detected during the initial infection or during rechallenge. However, anamnestic cellular immune responses developed during the initial homologous rechallenge, in particular in CH96A008, which developed a persistent infection. Polyprotein sequences of viruses recovered from CH1494 after the two homologous rechallenges that resulted in transient viremia were identical with the H77C virus. In contrast, the polyprotein sequences of viruses recovered from both chimpanzees after homologous rechallenge resulting in persistent infection had numerous changes. These findings have important implications for our understanding of immunity against HCV; even in the best-case scenario with autologous rechallenge, low-level viral persistence was seen in the presence of primed T-cell responses.

The full-length hepatitis C virus (HCV) JFH1 genome (genotype 2a) produces moderate titers of infectious particles in cell culture but the optimal determinants required for virion production are unclear. It has been shown that intragenotypic recombinants encoding core to NS2 from J6CF in the context of JFH1 are more robust in the release of viral particles. To understand the contributions of structural and nonstructural genes to HCV replication potential and infectivity, we have characterized intragenotypic recombinant genotype 2a viruses with different portions of the J6 isolate engineered into the JFH1 infectious clone. All genomes produced high levels of intracellular HCV RNA and NS3 protein in Huh-7.5 transfected cells. However, JFH1 genomes containing J6 sequences from C to E2 (CE2) or C to p7 (Cp7) secreted up to 100-fold more infectious HCV particles than the parental JFH1 clone. Subsequent infection of naive Huh-7.5 cells with each of the J6/JFH1 recombinants at a multiplicity of infection of 0.0003 resulted in high viral titers only for CE2 and Cp7 viruses. Comparison of virion production by the Cp7 J6/JFH1 recombinant to previously described J6/JFH1 recombinants showed flexibility of the chimeric junction. Moreover, NTRNS2 a chimeric virus equivalent to the previously reported FL-J6/JFH chimera, showed a 10-fold enhancement of virus titers compared to CNS2. NTRNS2 differs from CNS2 by three nucleotide differences residing in the 5′ NTR and core coding sequence and all three nucleotide changes were necessary for increased virion production. Importantly, cells producing Cp7 virus showed increased apoptosis compared with JFH1, an effect correlating with virion production. These studies begin to unravel requirements for robust virus replication and the relationship between increased virion production and host cell viability.

The JFH1 strain of hepatitis C virus (HCV) is unique among HCV isolates, in that the wild-type virus can traverse the entire replication cycle in cultured cells. However, without adaptive mutations, only low levels of infectious virus are produced. In the present study, the effects of five mutations that were selected during serial passage in Huh-7.5 cells were studied. Recombinant genomes containing all five mutations produced 3–4 logs more infectious virions than did wild type. Neither a coding mutation in NS5A nor a silent mutation in E2 was adaptive, whereas coding mutations in E2, p7, and NS2 all increased virus production. A single-cycle replication assay in CD81-deficient cells was developed to study more precisely the effect of the adaptive mutations. The E2 mutation had minimal effect on the amount of infectious virus released but probably enhanced entry into cells. In contrast, both the p7 and NS2 mutations independently increased the amount of virus released.

Efficient in vitro systems to study the life cycle of hepatitis C virus (HCV) were recently developed for JFH1 (genotype 2a), which has unique replication capacity in Huh7 cells. We developed 4a/JFH1 intergenotypic recombinants containing the structural genes (Core, E1, and E2), p7, and all or part of NS2 of the 4a prototype strain ED43 that, after transfection of Huh7.5 cells with RNA transcripts, produced infectious viruses. Compared with the J6/JFH control virus, production of viruses was delayed. However, efficient spread of infection and high HCV RNA and infectivity titers were obtained in serial passages. Sequence analysis of recovered viruses and subsequent reverse genetic studies revealed a vital dependence on one or two NS2 mutations, depending on the 4a/2a junction. Infectivity of ED43/JFH1 viruses was CD81 dependent. The genotype 4 cell culture systems permit functional analyses as well as drug and vaccine research on an increasingly important genotype in the Middle East, Africa, and Europe. We also developed genotype 1a intergenotypic recombinants from H77C with vital mutations in NS3. Using H77C/JFH1 and ED43/JFH1 viruses, we demonstrated high homologous neutralizing antibody titers in 1a and 4a patient sera, respectively. Furthermore, availability of JFH1 viruses with envelope proteins of the six major HCV genotypes permitted cross-neutralization studies; 1a and 4a serum cross-neutralized 1a, 4a, 5a, and 6a but not 2a and 3a viruses. Thus, the JFH1 intergenotypic recombinants will be of importance for future studies of HCV neutralization and accelerate the development of passive and active immunoprophylaxis.

Background & Aims: Recently, full viral life cycle hepatitis C virus (HCV) cell culture systems were developed for strain JFH1 (genotype 2a) and an intragenotypic 2a/2a genome (J6/JFH). We aimed at exploiting the unique JFH1 replication characteristics to develop culture systems for genotype 3a, which has a high prevalence worldwide. Methods: Huh7.5 cells were transfected with RNA transcripts of an intergenotypic 3a/JFH1 recombinant with core, E1, E2, p7, and NS2 of the 3a reference strain S52, and released viruses were passaged. Cultures were examined for HCV core and/or NS5A expression (immunostaining), HCV RNA titers (real-time PCR), and infectivity titers (50% tissue culture infectious dose). The role of mutations identified by sequencing of recovered S52/JFH1 viruses was analyzed by reverse genetics studies. Results: S52/JFH1 and J6/JFH viruses passaged in Huh7.5 cells showed comparable growth kinetics and similar peak HCV RNA and infectivity titers. However, analysis of S52/JFH1 viruses identified 9 putative adaptive mutations in core, E2, p7, NS3, and NS5A. All 7 S52/JFH1 recombinants with an amino acid change in p7 combined with a change in NS3 or NS5A, but only 2 of 9 recombinants with individual mutations (in p7 and NS3, respectively) were fully viable without the requirement for additional mutations. The biological relevance of our system was shown by studying dependence of 3a/JFH1 infection on CD81, and its impact on distribution of intracellular lipids. Conclusions: We developed a robust intergenotypic recombinant cell culture system for HCV genotype 3a, providing a valuable tool for studies of 3a core-NS2 and related therapeutics.

Using both a mass spectrometry-based method and the classical method of cloning and sequencing, we demonstrated weekly changes in the hypervariable region 1 quasispecies of a chimpanzee infected with an infectious clone, coinciding with neutralizing antibody emergence. We also used the mass spectrometry method in the clinical follow-up of a chronically infected patient over a 5-year period.

A quantitative real-time PCR assay was developed that detects genomic RNA from reference strains representing the six major genotypes of hepatitis C virus (HCV) with equal sensitivity and accurately measured HCV RNA in JFH1 HCV-infected Huh7.5 cells. The method is indirectly calibrated to the first international (WHO 96/790) HCV standard preparation and has a linear dynamic range of 102.6–106.5 IU/ml. In addition, the inter- and intra-assay precision were ∼3% CV and

Hepatitis C virus (HCV) NS3 protease inhibitors (PIs) are important components of novel HCV therapy regimens. Studies of PI resistance initially focused on genotype 1. Therefore, knowledge about the determinants of PI resistance for the highly prevalent genotypes 2 to 6 remains limited. Using Huh7.5 cell culture-infectious HCV recombinants with genotype 1 to 6 NS3 protease, we identified protease positions 54, 155, and 156 as hot spots for the selection of resistance substitutions under treatment with the first licensed PIs, telaprevir and boceprevir. Treatment of a genotype 2 isolate with the newer PIs vaniprevir, faldaprevir, simeprevir, grazoprevir, paritaprevir, and deldeprevir identified positions 156 and 168 as hot spots for resistance; the Y56H substitution emerged for three newer PIs. Substitution selection also depended on the specific recombinant. The substitutions identified conferred cross-resistance to several PIs; however, most substitutions selected under telaprevir or boceprevir treatment conferred less resistance to certain newer PIs. In a single-cycle production assay, across genotypes, PI treatment primarily decreased viral replication, which was rescued by PI resistance substitutions. The substitutions identified resulted in differential effects on viral fitness, depending on the original recombinant and the substitution. Across genotypes, fitness impairment induced by resistance substitutions was due primarily to decreased replication. Most combinations of substitutions that were identified increased resistance or fitness. Combinations of resistance substitutions with fitness-compensating substitutions either rescued replication or compensated for decreased replication by increasing assembly. This comprehensive study provides insight into the selection patterns and effects of PI resistance substitutions for HCV genotypes 1 to 6 in the context of the infectious viral life cycle, which is of interest for clinical and virological HCV research.

The importance of neutralizing antibodies (NAbs) in protection against hepatitis C virus (HCV) remains controversial. We infused a chimpanzee with H06 immunoglobulin from a genotype 1a HCV-infected patient and challenged with genotype strains efficiently neutralized by H06 in vitro . Genotype 1a NAbs afforded no protection against genotype 4a or 5a. Protection against homologous 1a lasted 18 weeks, but infection emerged when NAb titers waned. However, 6a infection was prevented. The differential in vivo neutralization patterns have implications for HCV vaccine development.

Objective To determine the impact of the personality traits neuroticism and extraversion as well as comorbid personality disorders on the rate of remission, recurrence, and conversion to bipolar disorder after the first lifetime episode of depression. Methods A total of 301 inpatients or outpatients aged 18-70 years with a validated diagnosis of a single depressive episode according to ICD-10 were assessed by the Structured Clinical Interview for DSM-IV Axis II Personality Disorders and the Eysenck Personality Questionnaire from 2005 through 2007. At 5-year follow-up, 262 patients were reassessed by means of the Life Chart Method and diagnostic interviews from 2011 through 2013. Cox regression analyses were used to estimate the effect of personality factors on the rates of remission, recurrence, and conversion to bipolar disorder, respectively. Results A comorbid cluster C personality disorder decreased the rate of remission by 30% (HR = 0.7; 95% CI, 0.5-0.9; P = .02) and increased the rate of recurrence after remission of the first depression by 80% (HR = 1.8; 95% CI, 1.0-3.0; P = .04). A higher neuroticism score at baseline decreased the rate of remission by 20% for each increase of 1 SD (HR = 0.8; 95% CI, 0.7-0.9; P = .002), and a higher level of extraversion increased the rate of conversion to bipolar disorder by 60% for each increase of 1 SD (HR = 1.6; 95% CI, 1.0-2.5; P = .05). Conclusions Comorbidity of cluster C personality disorders and the level of neuroticism and extraversion have significant impact on the long-term prognosis of depression. The identified predictors of the course of illness should guide patients and clinicians for individualized tailored treatment of comorbid conditions in depression.

Recombinant hepatitis C virus (HCV) clones propagated in human hepatoma cell cultures yield relatively low infectivity titers. Here, we adapted the JFH1-based Core-NS2 recombinant SA13/JFH1 C3405G,A3696G (termed SA13/JFH1 orig ), of the poorly characterized genotype 5a, to Huh7.5 cells, yielding a virus with greatly improved spread kinetics and an infectivity titer of 6.7 log 10 focus-forming units (FFU)/ml. We identified several putative adaptive amino acid changes. In head-to-head infections at fixed multiplicities of infection, one SA13/JFH1 orig mutant termed SA13/JFH1 Core-NS5B , containing 13 amino acid changes (R114W and V187A [Core]; V235L [E1]; T385P [E2]; L782V [p7]; Y900C [NS2]; N2034D, E2238G, V2252A, L2266P, and I2340T [NS5A]; A2500S and V2841A [NS5B]), displayed fitness comparable to that of the polyclonal high-titer adapted virus. Single-cycle virus production assays in CD81-deficient Huh7-derived cells demonstrated that these changes did not affect replication but increased HCV assembly and specific infectivity as early as 24 h posttransfection. Infectious coculture assays in Huh7.5 cells showed a significant increase in cell-to-cell transmission for SA13/JFH1 Core-NS5B viruses as well as viruses with only p7 and nonstructural protein mutations. Interestingly, the E2 hypervariable region 1 (HVR1) mutation T385P caused (i) increased sensitivity to neutralizing patient IgG and human monoclonal antibodies AR3A and AR4A and (ii) increased accessibility of the CD81 binding site without affecting the usage of CD81 and SR-BI. We finally demonstrated that SA13/JFH1 orig and SA13/JFH1 Core-NS5B , with and without the E2 mutation T385P, displayed similar biophysical properties following iodixanol gradient ultracentrifugation. This study has implications for investigations requiring high virus concentrations, such as studies of HCV particle composition and development of whole-virus vaccine antigens. IMPORTANCE Hepatitis C virus (HCV) is a major global health care burden, affecting more than 150 million people worldwide. These individuals are at high risk of developing severe end-stage liver diseases. No vaccine exists. While it is possible to produce HCV particles resembling isolates of all HCV genotypes in human hepatoma cells (HCVcc), production efficacy varies. Thus, for several important studies, including vaccine development, in vitro systems enabling high-titer production of diverse HCV strains would be advantageous. Our study offers important functional data on how cell culture-adaptive mutations identified in genotype 5a JFH1-based HCVcc permit high-titer culture by affecting HCV genesis through increasing virus assembly and HCV fitness by enhancing the virus specific infectivity and cell-to-cell transmission ability, without influencing the biophysical particle properties. High-titer HCVcc like the one described in this study may be pivotal in future vaccine-related studies where large quantities of infectious HCV particles are necessary.

Eysenck and Eysenck identified the two-factor structure of personality, namely neuroticism and extraversion which has been widely used in clinical psychiatry, and generated much research on the psychometric properties of the scales. Using a classical psychometric approach the neuroticism and extraversion scales have shown robust psychometric properties. The present study used both classical psychometric and item response theory (IRT) analyses to evaluate the neuroticism and extraversion scales and improve scalability of the instrument. A first time depressed sample completed the Eysenck Personality Questionnaire (EPQ) and Hamilton Depression Rating Scale (Ham-D 17-item) at baseline and at follow-up five years later. For the neuroticism scale a subscale containing depression-related symptoms, a subscale containing anxietyrelated symptoms, and a subscale containing symptoms related to interpersonal sensitivity were identified. For the extraversion scale a shorter and psychometrically more robust version was identified together with a short introversion scale. Clinically discriminant validity was analysed using correlations. The correlation between depression (Ham-D-17) and neuroticism-anxiety was below clinical significance, while the correlation of depression (Ham-D-17) was above clinical significance for both interpersonal sensitivity and neuroticism-depression. Clinically discriminant validity was modest for the extraversion/ introversion subscales however the introversion subscale approached clinical significant correlation with depression. The identified subscales of the EPQ, neuroticism-anxiety, interpersonal sensitivity, neuroticism-depression, extraversion and introversion are psychometrically valid measures of type and severity of the stress response. Using these subscales, it is possible to perform a quick and psychometrically valid evaluation of the type and severity of the stress response. The subscales may be useful in predicting types of psychopathology and in identifying underlying vulnerability that could serve as specific treatment targets in preventing as well as treating depression and anxiety

GB virus B (GBV-B), which infects tamarins, is the virus most closely related to hepatitis C virus (HCV). HCV has a protein (p7) that is believed to form an ion channel. It is critical for viability. In vitro studies suggest that GBV-B has an analogous but larger protein (p13). We found that substitutions of the −1 and/or −3 residues of the putative cleavage sites (amino acid 613/614 and 732/733) abolished processing in vitro and rendered an infectious GBV-B clone nonviable in tamarins. Internal cleavage was predicted at two sites (amino acid 669/670 and 681/682), and in vitro analysis indicated processing at both sites, suggesting that p13 is processed into two components (p6 and p7). Mutants with substitution at amino acid 669 or 681 were viable in vivo , but the recovered viruses had changes at amino acid 669 and 681, respectively, which restored cleavage. A mutant lacking amino acid 614–681 (p6 plus part of p7) was nonviable. However, a mutant lacking amino acid 614–669 (p6) produced high titer viremia and acute resolving hepatitis; viruses recovered from both animals lacked the deleted sequence and had no other mutations. Thus, p6 was dispensable but p7 was essential for infectivity. The availability of a recombinant GBV-B virus containing a p7 protein with similarities to the HCV p7 will enhance the relevance of this model and will be of importance for identifying compounds that inhibit p7 function as additional therapeutic agents.

GBV-B, a member of the Flaviviridae family of viruses, is the virus most closely related to HCV, and GBV-B infection in tamarin monkeys might represent a valuable surrogate animal model of HCV infection. In the current study, GBV-B was successfully transmitted to two marmosets (Callithrix jaccus). The infection resulted in viremia of 14- and 17-week duration, respectively, and was accompanied by elevation of isocitrate dehydrogenase activity. These data confirm that marmosets might represent an attractive model for GBV-B infection. The sequence of GBV-B NS5A, which was previously reported to have one of the highest mutation rates during infection in tamarins, was determined for viruses recovered from the inoculum and from marmoset blood samples obtained at weeks 1, 8, and 14 post inoculation in one marmoset and at weeks 2, 8, and 17 post inoculation in the other marmoset. In both animals, we detected four substitutions (R1945K, K2052G, F2196L, and G2268E), in the virus recovered immediately before viral clearance. Interestingly, two of these mutations (F2196L and G2268E) were described recently for viruses recovered from persistently infected tamarins. Appearance of these mutations presumably reflects a mechanism of immune escape rather than adaptation of the virus to a new host.

Chimpanzees remain the only recognized animal model for the study of hepatitis C virus (HCV). Studies performed in chimpanzees played a critical role in the discovery of HCV and are continuing to play an essential role in defining the natural history of this important human pathogen. In the absence of a reproducible cell culture system, the infectivity titer of HCV challenge pools can be determined only in chimpanzees. Recent studies in chimpanzees have provided new insight into the nature of host immune responses-particularly the intrahepatic responses-following primary and secondary experimental HCV infections. The immunogenicity and efficacy of vaccine candidates against HCV can be tested only in chimpanzees. Finally, it would not have been possible to demonstrate the infectivity of infectious clones of HCV without chimpanzees. Chimpanzees became infected when RNA transcripts from molecular clones were inoculated directly into the liver. The infection generated by such transfection did not differ significantly from that observed in animals infected intravenously with wild-type HCV. The RNA inoculated into chimpanzees originated from a single sequence, and the animals therefore had a monoclonal HCV infection. Monoclonal infection simplifies studies of HCV, because virus interaction with the host is not confounded by the quasispecies invariably present in a natural infection. It furthermore permits true homologous challenge in studies of protective immunity and in testing the efficacy of vaccine candidates. Finally, this in vivo transfection system has made it possible to test for the first time the importance of genetic elements for HCV infectivity.

Our understanding of the humoral immune response to hepatitis C virus (HCV) is limited because the virus can be studied only in humans and chimpanzees and because previously described neutralization assays have not been robust or simple to perform. Nevertheless, epidemiologic and laboratory studies suggested that neutralizing Ab to HCV might be important in preventing infection. We have recently described a neutralization assay based on the neutralization of pseudotyped murine retrovirus constructs bearing HCV envelope glycoproteins on their surface. We have applied the assay to well characterized clinical samples from HCV-infected patients and chimpanzees, confirmed the existence of neutralizing Ab to HCV, and validated most previously reported neutralizations of the virus. We did not find neutralizing anti-HCV in resolving infections but did find relatively high titers (>1:320) of such Ab in chronic infections. Neutralizing Ab was directed not only to epitope(s) in the hypervariable region of the E2 envelope protein but also to one or more epitopes elsewhere in the envelope of the virus. Neutralizing Ab was broadly reactive and could neutralize pseudotype particles bearing the envelope glycoproteins of two different subgenotypes (1a and 1b). The ability to assay neutralizing anti-HCV should permit an assessment of the prospects for successful Ab-mediated passive and active immunoprophylaxis against hepatitis C.

The development of a subgenomic replicon derived from the hepatitis C virus (HCV) strain Con1 enabled the study of viral RNA replication in Huh-7 cells. The level of replication of replicons, as well as full-length Con1 genomes, increased significantly by a combination of two adaptive mutations in NS3 (E1202G and T1280I) and a single mutation in NS5A (S2197P). However, these cell culture-adaptive mutations influenced in vivo infectivity. After intrahepatic transfection of chimpanzees, the wild-type Con1 genome was infectious and produced viral titers similar to those produced by other infectious HCV clones. Repeated independent transfections with RNA transcripts of a Con1 genome containing the three adaptive mutations failed to achieve active HCV infection. Furthermore, although a chimpanzee transfected with RNA transcripts of a Con1 genome with only the NS5A mutation became infected, this mutation was detected only in virus genomes recovered from serum at day 4; viruses recovered at day 7 had a reversion back to the original Con1 sequence. Our study demonstrates that mutations that are adaptive for replication of HCV in cell culture may be highly attenuating in vivo .

DNA-based immunization may be of prophylactic and therapeutic value for hepatitis C virus (HCV) infection. In efforts to improve the immunogenicity of a plasmid expressing the second envelope protein (E2) of HCV, we evaluated in mice the role of the antigen localization and demonstrated that membrane-bound and secreted forms induced higher titers of E2-specific antibodies, as well as earlier and higher seroconversion rates, than the intracellular form, but all three forms induced strong CTL. We also investigated whether E2-specific antibody responses could be enhanced by CpG optimization of the plasmid backbone and showed that removal of neutralizing CpG dinucleotides did not have a significant effect but addition of 64 immunostimulatory CpG motifs significantly enhanced anti-E2 titers. These results may have implications for the design and development of HCV DNA vaccines.

Hepatitis C virus (HCV) is a major cause of end-stage liver diseases. With 3–4 million new HCV infections yearly, a vaccine is urgently needed. A better understanding of virus escape from neutralizing antibodies and their corresponding epitopes are important for this effort. However, for viral isolates with high antibody resistance, or antibodies with moderate potency, it remains challenging to induce escape mutations in vitro. Here, as proof-of-concept, we used antibody-sensitive HVR1-deleted (ΔHVR1) viruses to generate escape mutants for a human monoclonal antibody, AR5A, targeting a rare cross-genotype conserved epitope. By analyzing the genotype 1a envelope proteins (E1/E2) of recovered Core-NS2 recombinant H77/JFH1ΔHVR1 and performing reverse genetic studies we found that resistance to AR5A was caused by substitution L665W, also conferring resistance to the parental H77/JFH1. The mutation did not induce viral fitness loss, but abrogated AR5A binding to HCV particles and intracellular E1/E2 complexes. Culturing J6/JFH1ΔHVR1 (genotype 2a), for which fitness was decreased by L665W, with AR5A generated AR5A-resistant viruses with the substitutions I345V, L665S, and S680T, which we introduced into J6/JFH1 and J6/JFH1ΔHVR1. I345V increased fitness but had no effect on AR5A resistance. L665S impaired fitness and decreased AR5A sensitivity, while S680T combined with L665S compensated for fitness loss and decreased AR5A sensitivity even further. Interestingly, S680T alone had no fitness effect but sensitized the virus to AR5A. Of note, H77/JFH1L665S was non-viable. The resistance mutations did not affect cell-to-cell spread or E1/E2 interactions. Finally, introducing L665W, identified in genotype 1, into genotypes 2–6 parental and HVR1-deleted variants (not available for genotype 4a) we observed diverse effects on viral fitness and a universally pronounced reduction in AR5A sensitivity. Thus, we were able to take advantage of the neutralization-sensitive HVR1-deleted viruses to rapidly generate escape viruses aiding our understanding of the divergent escape pathways used by HCV to evade AR5A., Author summary Worldwide hepatitis C virus (HCV) is one of the leading causes of chronic liver diseases, including cirrhosis and cancer. Treatment accessibility is limited and development of a preventive vaccine has proven difficult, partly due to the high mutation rate of the virus. Recent studies of HCV antibody neutralization resistance have revealed important information about escape pathways and barriers to escape for several clinically promising human monoclonal antibodies. However, due to the varying levels of antibody shielding between HCV isolates these studies have been mostly limited to a few neutralization-sensitive HCV isolates. Here, we took advantage of the fact that deletion of the hypervariable region 1 (HVR1) increased antibody sensitivity of HCV isolates by increasing the exposure of important epitopes, thus facilitating studies of antibody escape for neutralization resistant isolates. We identified escape mutations in the envelope glycoprotein E2, at amino acid position L665, which conferred antibody resistance in parental HCV viruses from genotypes 1–6. We found that antibody escape was associated with loss of binding to HCV particles and intracellular envelope protein complexes. We also identified escape substitutions at L665 that were isolate-specific. Thus, our data sheds new light on antibody resistance mechanisms across diverse HCV isolates.

The first discovered and sequenced hepatitis C virus (HCV) genome and the first in vivo infectious HCV clones originated from the HCV prototype strains HCV-1 and H77, respectively, both widely used in research of this important human pathogen. In the present study, we developed efficient infectious cell culture systems for these genotype 1a strains by using the HCV-1/SF9_A and H77C in vivo infectious clones. We initially adapted a genome with the HCV-1 5′UTR-NS5A (where UTR stands for untranslated region) and the JFH1 NS5B-3′UTR (5-5A recombinant), including the genotype 2a-derived mutations F1464L/A1672S/D2979G (LSG), to grow efficiently in Huh7.5 cells, thus identifying the E2 mutation S399F. The combination of LSG/S399F and reported TNcc(1a)-adaptive mutations A1226G/Q1773H/N1927T/Y2981F/F2994S promoted adaptation of the full-length HCV-1 clone. An HCV-1 recombinant with 17 mutations (HCV1cc) replicated efficiently in Huh7.5 cells and produced supernatant infectivity titers of 10 4.0 focus-forming units (FFU)/ml. Eight of these mutations were identified from passaged HCV-1 viruses, and the A970T/I1312V/C2419R/A2919T mutations were essential for infectious particle production. Using CD81-deficient Huh7 cells, we further demonstrated the importance of A970T/I1312V/A2919T or A970T/C2419R/A2919T for virus assembly and that the I1312V/C2419R combination played a major role in virus release. Using a similar approach, we found that NS5B mutation F2994R, identified here from culture-adapted full-length TN viruses and a common NS3 helicase mutation (S1368P) derived from viable H77C and HCV-1 5-5A recombinants, initiated replication and culture adaptation of H77C containing LSG and TNcc(1a)-adaptive mutations. An H77C recombinant harboring 19 mutations (H77Ccc) replicated and spread efficiently after transfection and subsequent infection of naive Huh7.5 cells, reaching titers of 10 3.5 and 10 4.4 FFU/ml, respectively. IMPORTANCE Hepatitis C virus (HCV) was discovered in 1989 with the cloning of the prototype strain HCV-1 genome. In 1997, two molecular clones of H77, the other HCV prototype strain, were shown to be infectious in chimpanzees, but not in vitro . HCV research was hampered by a lack of infectious cell culture systems, which became available only in 2005 with the discovery of JFH1 (genotype 2a), a genome that could establish infection in Huh7.5 cells. Recently, we developed in vitro infectious clones for genotype 1a (TN), 2a (J6), and 2b (J8, DH8, and DH10) strains by identifying key adaptive mutations. Globally, genotype 1 is the most prevalent. Studies using HCV-1 and H77 prototype sequences have generated important knowledge on HCV. Thus, the in vitro infectious clones developed here for these 1a strains will be of particular value in advancing HCV research. Moreover, our findings open new avenues for the culture adaptation of HCV isolates of different genotypes.

Human monoclonal antibodies (HMAbs) with neutralizing capabilities constitute potential immune-based treatments or prophylaxis against hepatitis C virus (HCV). However, lack of cell culture-derived HCV (HCVcc) harboring authentic envelope proteins (E1/E2) has hindered neutralization investigations across genotypes, subtypes, and isolates. We investigated the breadth of neutralization of 10 HMAbs with therapeutic potential against a panel of 16 JFH1-based HCVcc-expressing patient-derived Core-NS2 from genotypes 1a (strains H77, TN, and DH6), 1b (J4, DH1, and DH5), 2a (J6, JFH1, and T9), 2b (J8, DH8, and DH10), 2c (S83), and 3a (S52, DBN, and DH11). Virus stocks used for in vitro neutralization analysis contained authentic E1/E2, with the exception of full-length JFH1 that acquired the N417S substitution in E2. The 50% inhibition concentration (IC50) for each HMAb against the HCVcc panel was determined by dose-response neutralization assays in Huh7.5 cells with antibody concentrations ranging from 0.0012 to 100 μg/mL. Interestingly, IC50 values against the different HCVcc's exhibited large variations among the HMAbs, and only three HMAbs (HC-1AM, HC84.24, and AR4A) neutralized all 16 HCVcc recombinants. Furthermore, the IC50 values for a given HMAb varied greatly with the HCVcc strain, which supports the use of a diverse virus panel. In cooperation analyses, HMAbs HC84.24, AR3A, and, especially HC84.26, demonstrated synergistic effects towards the majority of the HCVcc's when combined individually with AR4A. Conclusion: Through a neutralization analysis of 10 clinically relevant HMAbs against 16 JFH1-based Core-NS2 recombinants from genotypes 1a, 1b, 2a, 2b, 2c, and 3a, we identified at least three HMAbs with potent and broad neutralization potential. The neutralization synergism obtained when pooling the most potent HMAbs could have significant implications for developing novel strategies to treat and control HCV. (Hepatology 2014;60:1551–1562)

Identification of previously unrecognized viral agents in serum or plasma samples is of great medical interest but remains a major challenge, primarily because of abundant host DNA. The current methods, library screening or representational difference analysis (RDA), are very laborious and require selected sample sets. We have developed a simple and reproducible method for discovering viruses in single serum samples that is based on DNase treatment of the serum followed by restriction enzyme digestion and sequence-independent single primer amplification (SISPA) of the fragments, and have evaluated its performance on known viruses. Both DNA viruses and RNA viruses at a concentration of ≈10 6 genome equivalents per ml were reproducibly identified in 50 μl of serum. While evaluating the method, two previously unknown parvoviruses were discovered in the bovine sera used as diluent. The near complete genome sequence of each virus was determined; their classification as two species (provisionally named bovine parvoviruses 2 and 3) was confirmed by phylogenetic analysis. Both viruses were found to be frequent contaminants of commercial bovine serum. DNase treatment of serum samples may prove to be a very useful tool for virus discovery. The DNase-SISPA method is suitable for screening of a large number of samples and also enables rapid sequence determination of high-titer viruses.

The chimpanzee is the only recognized animal model for the study of hepatitis C virus (HCV). However, recently it was reported that rhesus monkeys were susceptible to HCV and developed hepatitis during infection. In the present study, we inoculated two rhesus monkeys each with HCV strain H77 (genotype 1a), strain HC-J6 (genotype 2a) or strain S52 (genotype 3a). Weekly serum samples were tested for liver enzyme values, HCV antibodies and HCV RNA. We did not find evidence of HCV infection in any of the monkeys during 24 weeks of follow-up. Our study demonstrates that rhesus monkeys are not readily infected with HCV and apparently do not represent a useful animal model for the study of HCV.

Persistent infection with hepatitis C virus (HCV) is an important cause of chronic liver disease worldwide. Therefore, the development of vaccines to prevent HCV infection, or at least to prevent progression to chronicity, is a major goal. Potential HCV vaccine candidates include recombinant proteins, recombinant viruses, DNA constructs, synthetic peptides and virus-like particles. Various vaccine candidates have been shown to generate humoral and cellular immune responses in animals, primarily in mice. However, the efficacy of most vaccine candidates in protecting against HCV has not been tested because the chimpanzee, the only animal other than humans that is susceptible to HCV, is not readily available, requires special facilities, and is very expensive. The course of infection in chimpanzees is similar in its diversity to that in humans and detailed studies in this model are beginning to define the immune responses that can terminate HCV infection. Of relevance for vaccine evaluation was the titration in chimpanzees of different HCV variants to provide well-characterized challenge pools. In addition, monoclonal virus pools generated from chimpanzees infected with cloned viruses make it possible now to examine immunity to HCV without the confounding factor of antigenic diversity of the challenge virus (quasispecies). The vaccine trials performed in chimpanzees to date all have tested the efficacy of immunizations with various forms of the envelope proteins of HCV.

Persistent infection with hepatitis C virus (HCV) is among the leading causes of chronic liver disease. Previous studies suggested that genetic variation in hypervariable region 1 (HVR1) of the second envelope protein, possibly in response to host immune pressure, influences the outcome of HCV infection. In the present study, a chimpanzee transfected intrahepatically with RNA transcripts of an infectious HCV clone (pCV-H77C) from which HVR1 was deleted became infected; the ΔHVR1 virus was subsequently transmitted to a second chimpanzee. Infection with ΔHVR1 virus resulted in persistent infection in the former chimpanzee and in acute resolving infection in the latter chimpanzee. Both chimpanzees developed hepatitis. The ΔHVR1 virus initially replicated to low titers, but virus titer increased significantly after mutations appeared in the viral genome. Thus, wild-type HCV without HVR1 was apparently attenuated, suggesting a functional role of HVR1. However, our data indicate that HVR1 is not essential for the viability of HCV, the resolution of infection, or the progression to chronicity.

Hepatitis C virus (HCV) infection is a major health problem in Egypt, where the seroprevalence is 10-20-fold higher than that in the United States. To characterize the HCV genotype distribution and concordance of genotype assessments on the basis of multiple genomic regions, specimens were obtained from blood donors in 15 geographically diverse governorates throughout Egypt. The 5' noncoding, core/E1, and NS5B regions were amplified by reverse transcription-polymerase chain reaction and analyzed by both restriction fragment length polymorphism (RFLP) and phylogenetic tree construction. For the 5' noncoding region, 122 (64%) of 190 specimens were amplified and analyzed by RFLP: 111 (91%) were genotype 4, 1 (1%) was genotype 1a, 1 (1%) was genotype 1b, and 9 (7%) could not be typed. Phylogenetic analyses of the core/E1 and NS5B regions confirmed the genotype 4 preponderance and revealed evidence of 3 new subtypes. Analysis of genetic distance between isolates was consistent with the introduction of multiple virus strains 75-140 years ago, and no clustering was detected within geographic regions, suggesting widespread dispersion at some time since then.

Hepatitis C virus (HCV) is a member of the Flaviviridae family.124 The Flaviviridae family of viruses are associated with human and animal diseases and encompass at least three different genera: pestiviruses, such as bovine viral diarrhea virus and classic swine fever virus that cause disease in cattle and pigs, respectively; flaviviruses, the most important cause of arthropod-transmitted viral diseases, for example, dengue fever and yellow fever; and hepacivirus, whose sole member is HCV. The GB viruses, which are also members of the Flaviviridae, are most closely related to HCV and infect humans and monkeys.13,89,144,145 One of the signature characteristics of HCV is its genetic heterogeneity.16 Thus, HCV isolates from around the world have been divided into six major genotypes and more than 100 subtypes.16,45 Moreover, genetic heterogeneity of HCV within infected individuals is manifested as a mixture of closely related but distinct genomes called a quasispecies.16,100 More than 2% of the world population is chronically infected with HCV.66,120,170 In developed countries, HCV is the main cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma; HCV-related end-stage liver disease is now the leading reason for liver transplantation.109 Hepatitis C virus infection is also associated with a number of extrahepatic diseases, such as cryoglobulinemia and glomerulonephritis.65 The most important feature of HCV infection is its high rate of chronicity. More than 80% of HCV-infected individuals develop a chronic infection.66 Unfortunately, treatment of chronic hepatitis C with interferon (IFN), the principal recognized therapeutic agent, succeeds only in 20% of the cases.109 The response rate is even lower in patients infected with genotype 1 HCV. Recently, a combination treatment of IFN and ribavirin has achieved higher response rates both in patients infected with genotype 1 and in patients infected with other genotypes.101,118 Despite the heartening progress achieved in treating chronic hepatitis C with combination therapy, the overall response rate in these patients is still below 50%. A better understanding of the molecular biology of HCV has highlighted targets for new therapeutic strategies. The genetic heterogeneity of HCV is a major confounding factor; thus, the design of effective drugs might be facilitated by targeting conserved regions of HCV. Drugs directed against conserved sequences of the 5′ untranslated region (UTR)8,53,123,130 and the serine-protease domain within NS328,84 have already been tested in vitro. The lack of a reliable cell culture system or small animal model for HCV (the chimpanzee is the only recognized animal model) presents significant obstacles for the study of these new therapeutic agents. The recent availability of infectious HCV cDNA clones80,172,174 may facilitate the establishment of useful in vitro replication systems.

Recently, a novel DNA virus, TT virus (TTV), was identified in patients with post-transfusion non-A-G hepatitis. We analyzed the prevalence and clinical implications of TTV infection in a cohort of 96 Spanish patients on long-term hemodialysis. TTV DNA was detected by nested PCR in 51 (53%) of 96 patients, a prevalence significantly higher than that found in healthy blood donors. Persistent liver test abnormalities were found in only 2 (7.7%) of 26 patients infected with TTV alone, compared with 12 (75%) of 16 patients infected with hepatitis C or hepatitis B virus, or both (P < 0.01). Mixed infections with multiple strains of TTV, including different major genotypes, were common in patients on hemodialysis. These patients had received a significantly greater number of blood units (22.7 +/- 20) compared with patients apparently infected with a single strain of TTV (8.9 +/- 11) (P = 0.01). Phylogenetic analyses of TTV from infected patients identified strains of genotypes 1, 2, 3, and 4. In summary, TTV infection was common in patients on hemodialysis but was not associated with liver disease

GB virus-B (GBV-B) is a member of the Flaviviridae family of viruses. This RNA virus infects tamarins, but its natural host is not known. GBV-B has special interest because it is the virus that is most closely related to hepatitis C virus (HCV), an important human pathogen. In the present study, we identified a previously unrecognized sequence at the 3' end of the GBV-B genome. This new 3' terminal sequence can form several predicted stem-loop structures as is typical for other members of the Flaviviridae family. We constructed molecular clones and showed that the new 3' UTR sequence was critical for in vivo infectivity. After intrahepatic transfection of two tamarins with RNA transcripts of the full-length GBV-B clone, we detected high viral titers from Week 1 postinoculation with peak titers of approximately 10(8) genome equivalents/ml. The viremic pattern of GBV-B infection in the transfected animals was the same as in animals inoculated intravenously with the virus pool used as the cloning source. The sequence of the recombinant virus was recovered from one of the tamarins and shown to be identical to that of the infectious clone. The development of severe hepatitis in both tamarins infected with the recombinant GBV-B virus provides formal proof that GBV-B is a true hepatitis virus.

Large sections of the 3′ untranslated region (UTR) of hepatitis C virus (HCV) were deleted from an infectious cDNA clone, and the RNA transcripts from seven deletion mutants were tested sequentially for infectivity in a chimpanzee. Mutants lacking all or part of the 3′ terminal conserved region or the poly(U–UC) region were unable to infect the chimpanzee, indicating that both regions are critical for infectivity in vivo . However, the third region, the variable region, was able to tolerate a deletion that destroyed the two putative stem–loop structures within this region. Mutant VR-24 containing a deletion of the proximal 24 nt of the variable region of the 3′ UTR was viable in the chimpanzee and seemed to replicate as well as the undeleted parent virus. The chimpanzee became viremic 1 week after inoculation with mutant VR-24, and the HCV genome titer increased over time during the early acute infection. Therefore, the poly(U–UC) region and the conserved region, but not the variable region, of the 3′ UTR seem to be critical for in vivo infectivity of HCV.

Six major genotypes (genotypes 1-6) of hepatitis C virus (HCV) have been identified. These genetic variants are being transmitted to chimpanzees, the only recognized animal model for the study of HCV. Genotype 5a (strain SA13), a variant found primarily in South Africa, has been transmitted to chimpanzees for the first time. Experimental infection of 2 chimpanzees was characterized by early appearance of viremia and peak virus titers of 10(5)-10(6) genome equivalents/mL. The HCV infection was resolved by week 15 after inoculation in 1 chimpanzee and persisted in the other. Both chimpanzees became anti-HCV-positive by week 14 after inoculation. Both chimpanzees developed viral hepatitis. The infectivity titer of a genotype 5a challenge pool prepared from the first passage of HCV in a chimpanzee was approximately 10(4) infectious doses/mL. Finally, sequence analysis of strain SA13 confirmed that genotype 5a is genetically distinct from other genotypes of HCV.

We have succeeded in constructing a stable full-length cDNA clone of strain H77 (genotype 1a) of hepatitis C virus (HCV). We devised a cassette vector with fixed 5′ and 3′ termini and constructed multiple full-length cDNA clones of H77 in a single step by cloning of the entire ORF, which was amplified by long reverse transcriptase–PCR, directly into this vector. The infectivity of two complete full-length cDNA clones was tested by the direct intrahepatic injection of a chimpanzee with RNA transcripts. However, we found no evidence for HCV replication. Sequence analysis of these and 16 additional full-length clones revealed that seven clones were defective for polyprotein synthesis, and the remaining nine clones had 6–28 amino acid mutations in the predicted polyprotein compared with the consensus sequence of H77. Next, we constructed a consensus chimera from four of the full-length cDNA clones with just two ligation steps. Injection of RNA transcripts from this consensus clone into the liver of a chimpanzee resulted in viral replication. The sequence of the virus recovered from the chimpanzee was identical to that of the injected RNA transcripts. This stable infectious molecular clone should be an important tool for developing a better understanding of the molecular biology and pathogenesis of HCV.

Recently, cell culture systems producing hepatitis C virus particles (HCVcc) were developed. Establishment of serum-free culture conditions is expected to facilitate development of a whole-virus inactivated HCV vaccine. We describe generation of genotype 1–6 serum-free HCVcc (sf-HCVcc) from Huh7.5 hepatoma cells cultured in adenovirus expression medium. Compared to HCVcc, sf-HCVcc showed 0.6–2.1log10 higher infectivity titers (4.7–6.2log10Focus Forming Units/mL), possibly due to increased release and specific infectivity of sf-HCVcc. In contrast to HCVcc, sf-HCVcc had a homogeneous single-peak density profile. Entry of sf-HCVcc depended on HCV co-receptors CD81, LDLr, and SR-BI, and clathrin-mediated endocytosis. HCVcc and sf-HCVcc were neutralized similarly by chronic-phase patient sera and by human monoclonal antibodies targeting conformational epitopes. Thus, we developed serum-free culture systems producing high-titer single-density sf-HCVcc, showing similar biological properties as HCVcc. This methodology has the potential to advance HCV vaccine development and to facilitate biophysical studies of HCV.

Alpha interferon (IFN-α) is an essential component of innate antiviral immunity and of treatment regimens for chronic hepatitis C virus (HCV) infection. Resistance to IFN might be important for HCV persistence and failure of IFN-based therapies. Evidence for HCV genetic correlates of IFN resistance is limited. Experimental studies were hampered by lack of HCV culture systems. Using genotype (strain) 1a(H77) and 3a(S52) Core-NS2 JFH1-based recombinants, we aimed at identifying viral correlates of IFN-α resistance in vitro . Long-term culture with IFN-α2b in Huh7.5 cells resulted in viral spread with acquisition of putative escape mutations in HCV structural and nonstructural proteins. Reverse genetic studies showed that primarily amino acid changes I348T in 1a(H77) E1 and F345V/V414A in 3a(S52) E1/E2 increased viral fitness. Single-cycle assays revealed that I348T and F345V/V414A enhanced viral entry and release, respectively. In assays allowing viral spread, these mutations conferred a level of IFN-α resistance exceeding the observed fitness effect. The identified mutations acted in a subtype-specific manner but were not found in genotype 1a and 3a patients, who failed IFN-α therapy. Studies with HCV recombinants with different degrees of culture adaptation confirmed the correlation between viral fitness and IFN-α resistance. In conclusion, in vitro escape experiments led to identification of HCV envelope mutations resulting in increased viral fitness and conferring IFN-α resistance. While we established a close link between viral fitness and IFN-α resistance, identified mutations acted via different mechanisms and appeared to be relatively specific to the infecting virus, possibly explaining difficulties in identifying signature mutations for IFN resistance.

Pharmacological treatments for depression have insufficient efficacy in 30-40% of patients and fail to reverse cognitive deficits. Erythropoietin (EPO) has neurotrophic actions and aids neurocognitive function. The aim of this exploratory study was to determine whether recombinant human EPO improves mood and memory in treatment-resistant depression. Forty treatment-resistant depressed unipolar patients with Hamilton Depression Rating Scale-17 (HDRS-17) score ≥ 17 were randomized to eight weekly EPO (Eprex; 40,000 IU) or saline infusions in a double-blind, placebo-controlled, parallel-group design. Patients were assessed at baseline and at weeks 5, 9, and 14. Primary outcome was reduction in HDRS-17 score. Global assessment of function (GAF) was reported in addition. Secondary outcome was remission rate, and tertiary outcomes were changes in Rey Auditory Verbal Learning Test (RAVLT), Beck Depression Inventory-21 (BDI-21), and World Health Organization Quality of life-BREF (WHOQOL-BREF). Exploratory outcomes were depression and cognition composite scores. HDRS-17, GAF, and remission rates showed no effects of EPO over saline at week 9 (P-value ≥ 0.09). However, EPO improved BDI (P=0.02) and WHOQOL-BREF (P=0.01), and this was maintained at follow-up week 14 (P-values ≤ 0.04). EPO enhanced verbal recall (P=0.02) and recognition (P=0.03), which was sustained at follow-up (P-values ≤ 0.04). Exploratory analysis in patients fulfilling depression severity criteria at trial start revealed ameliorated HDRS-17 in EPO (N=14) vs saline groups (N=17), which was sustained at week 14 (P-values ≤ 0.05). Exploratory analysis in the complete cohort showed that EPO reduced depression composite at weeks 9 and 14 (P-values=0.02). The findings of this exploratory study highlight EPO as an interesting compound for treatment-resistant depression, which deserves further investigation.

Hepatitis C virus (HCV) is an important human pathogen infecting hepatocytes. With the advent of infectious cell culture systems, the HCV particle assembly and release processes are finally being uncovered. The HCV core and NS5A proteins co-localize on cytoplasmic lipid droplets (cLDs) or on the endoplasmic reticulum (ER) at different stages of particle assembly. Current knowledge on assembly and release is primarily based on studies in genotype 2a cell culture systems; however, given the high genetic heterogeneity of HCV, variations might exist among genotypes. Here, we developed novel HCV strain JFH1-based recombinants expressing core–NS2 and NS5A from genotypes 1–7, and analysed core and NS5A co-localization in infected cells. Huh7.5 cells were transfected with RNA of core–NS2/NS5A recombinants and putative adaptive mutations were analysed by reverse genetics. Adapted core–NS2/NS5A recombinants produced infectivity titres of 102.5–104.5 f.f.u. ml−1. Co-localization analysis demonstrated that the core and NS5A proteins from all genotypes co-localized extensively, and there was no significant difference in protein co-localization among genotypes. In addition, we found that the core and NS5A proteins were highly associated with cLDs at 12 h post-infection but became mostly ER associated at later stages. Finally, we found that different genotypes showed varying levels of core/cLD co-localization, with a possible effect on viral assembly/release. In summary, we developed a panel of HCV genotype 1–7 core–NS2/NS5A recombinants producing infectious virus, and an immunostaining protocol detecting the core and NS5A proteins from seven different genotypes. These systems will allow, for the first time, investigation of core/NS5A interactions during assembly and release of HCV particles of all major genotypes.

Background & Aims Hepatitis C virus (HCV) therapy will benefit from the preclinical evaluation of direct-acting antiviral (DAA) agents in infectious culture systems that test the effects on different virus genotypes. We developed HCV recombinants comprising the 5′ untranslated region−NS5A (5-5A) from genotypes 1−6 and 2a(JFH1) NS5B-3′ untranslated region, and tested the effects of NS3 protease and NS5A inhibitors on these recombinants. Methods The HCV 5-5A recombinants with previously identified mutations in the NS3-helicase (F1464L), NS4A (A1672S), and NS5B (D2979G) were adapted and improved, by incorporating additional recovered mutations that increased their propagation in Huh7.5 cells. Concentration−response profiles were determined for each DAA agent in replicate infected Huh7.5 cells. Results Developed efficient 1a(H77), 1a(TN), 3a(S52), 4a(ED43), 5a(SA13), and 6a(HK6a) 5-5A recombinants did not require mutations after viral passage in the NS3 protease or NS5A domain-I regions targeted by the drugs. They were inhibited in a concentration-dependent manner by the NS3 protease inhibitors telaprevir, boceprevir, asunaprevir, simeprevir, vaniprevir, faldaprevir, and MK-5172 and by the NS5A inhibitor daclatasvir. The 1a(TN) 5-5A and JFH1-independent full-length viruses had similar levels of sensitivity to the DAA agents, validating the 5-5A recombinants as surrogates for full-length viruses in DAA testing. Compared with the 1a(TN) full-length virus, the 3a(S52) 5-5A recombinant was highly resistant to all protease inhibitors, and the 4a(ED43) recombinant was highly resistant to telaprevir and boceprevir, but most sensitive to other protease inhibitors. Compared with other protease inhibitors, MK-5172 had exceptional potency against all HCV genotypes. The NS5A inhibitor daclatasvir had the highest potency observed, but with genotype-dependent activity. Conclusions The mutations F1464L, A1672S, and D2979G permitted the development of efficient HCV recombinants comprising genotype-specific 5′ untranslated region−NS5A (5-5A), which include the natural NS3 protease and NS5A domain-I drug targets. The robust replication of adapted 5-5A recombinants allowed for direct comparison of NS3 protease and NS5A inhibitors against HCV strains of genotypes 1−6.

With the development of directly acting antivirals, hepatitis C virus (HCV) therapy entered a new era. However, rapid selection of resistance mutations necessitates combination therapy. To study combination therapy in infectious culture systems, we aimed at developing HCV semi-full-length (semi-FL) recombinants relying only on the JFH1 NS3 helicase, NS5B, and the 3′ untranslated region. With identified adaptive mutations, semi-FL recombinants of genotypes(isolates) 1a(TN) and 3a(S52) produced supernatant infectivity titers of ∼4 log 10 focus-forming units/ml in Huh7.5 cells. Genotype 1a(TN) adaptive mutations allowed generation of 1a(H77) semi-FL virus. Concentration-response profiles revealed the higher efficacy of the NS3 protease inhibitor asunaprevir (BMS-650032) and the NS5A inhibitor daclatasvir (BMS-790052) against 1a(TN and H77) than 3a(S52) viruses. Asunaprevir had intermediate efficacy against previously developed 2a recombinants J6/JFH1 and J6cc. Daclatasvir had intermediate efficacy against J6/JFH1, while low sensitivity was confirmed against J6cc. Using a cross-titration scheme, infected cultures were treated until viral escape or on-treatment virologic suppression occurred. Compared to single-drug treatment, combination treatment with relatively low concentrations of asunaprevir and daclatasvir suppressed infection with all five recombinants. Escaped viruses primarily had substitutions at amino acids in the NS3 protease and NS5A domain I reported to be genotype 1 resistance mutations. Inhibitors showed synergism at drug concentrations reported in vivo . In summary, semi-FL HCV recombinants, including the most advanced reported genotype 3a infectious culture system, permitted genotype-specific analysis of combination treatment in the context of the complete viral life cycle. Despite differential sensitivity to lead compound NS3 protease and NS5A inhibitors, genotype 1a, 2a, and 3a viruses were suppressed by combination treatment with relatively low concentrations.

Genetic recombination is an important mechanism for increasing diversity of RNA viruses, and constitutes a viral escape mechanism to host immune responses and to treatment with antiviral compounds. Although rare, epidemiologically important hepatitis C virus (HCV) recombinants have been reported. In addition, recombination is an important regulatory mechanism of cytopathogenicity for the related pestiviruses. Here we describe recombination of HCV RNA in cell culture leading to production of infectious virus. Initially, hepatoma cells were co-transfected with a replicating JFH1ΔE1E2 genome (genotype 2a) lacking functional envelope genes and strain J6 (2a), which has functional envelope genes but does not replicate in culture. After an initial decrease in the number of HCV positive cells, infection spread after 13–36 days. Sequencing of recovered viruses revealed non-homologous recombinants with J6 sequence from the 5′ end to the NS2–NS3 region followed by JFH1 sequence from Core to the 3′ end. These recombinants carried duplicated sequence of up to 2400 nucleotides. HCV replication was not required for recombination, as recombinants were observed in most experiments even when two replication incompetent genomes were co-transfected. Reverse genetic studies verified the viability of representative recombinants. After serial passage, subsequent recombination events reducing or eliminating the duplicated region were observed for some but not all recombinants. Furthermore, we found that inter-genotypic recombination could occur, but at a lower frequency than intra-genotypic recombination. Productive recombination of attenuated HCV genomes depended on expression of all HCV proteins and tolerated duplicated sequence. In general, no strong site specificity was observed. Non-homologous recombination was observed in most cases, while few homologous events were identified. A better understanding of HCV recombination could help identification of natural recombinants and thereby lead to improved therapy. Our findings suggest mechanisms for occurrence of recombinants observed in patients., Author Summary Genetic recombination is the alternative joining of nucleic acids leading to novel combinations of genetic information. While DNA recombination in cells is of importance for evolution and adaptive immunity, RNA recombination often has only transient effects. However, RNA viruses are rapidly evolving and recombination can be an important evolutionary step in addition to mutations introduced by the viral polymerase. Recombination can allow escape from the host immune system and from antiviral treatment, and recombination of live attenuated viral vaccines has led to re-emergence of disease. Hepatitis C virus (HCV) is an important human pathogen that chronically infects more than 130 million worldwide and leads to serious liver disease. For HCV, naturally occurring recombinants are rare but clinically important. HCV recombination constitutes a challenge to antiviral treatment and can potentially provide an escape mechanism for the virus. In this study, we established an assay for HCV RNA recombination and characterized the emerging homologous and non-homologous recombinant viruses. Interestingly, recombination did not depend on viral replication, occurred most efficiently between isolates of the same genotype and did not occur with strong site-specificity. Better diagnosis of clinically important recombinants and an increased knowledge on viral recombination could strengthen antiviral and vaccine development.