A Novel Cis-Acting RNA Structural Element Embedded in the Core Coding Region of the Hepatitis C Virus Genome Directs Internal Translation Initiation of the Overlapping Core+1 ORF.

Hepatitis C virus (HCV) genome translation is initiated via an internal ribosome entry site (IRES) embedded in the 5'-untranslated region (5'UTR). We have earlier shown that the conserved RNA stem-loops (SL) SL47 and SL87 of the HCV core-encoding region are important for viral genome translation in cell culture and in vivo. Moreover, we have reported that an open reading frame overlapping the core gene in the +1 frame (core+1 ORF) encodes alternative translation products, including a protein initiated at the internal AUG codons 85/87 of this frame (nt 597-599 and 603-605), downstream of SL87, which is designated core+1/Short (core+1/S). Here, we provide evidence for SL47 and SL87 possessing a novel cis-acting element that directs the internal translation initiation of core+1/S. Firstly, using a bicistronic dual luciferase reporter system and RNA-transfection experiments, we found that nucleotides 344-596 of the HCV genotype-1a and -2a genomes support translation initiation at the core+1 frame AUG codons 85/87, when present in the sense but not the opposite orientation. Secondly, site-directed mutagenesis combined with an analysis of ribosome-HCV RNA association elucidated that SL47 and SL87 are essential for this alternative translation mechanism. Finally, experiments using cells transfected with JFH1 replicons or infected with virus-like particles showed that core+1/S expression is independent from the 5'UTR IRES and does not utilize the polyprotein initiation codon, but it requires intact SL47 and SL87 structures. Thus, SL47 and SL87, apart from their role in viral polyprotein translation, are necessary elements for mediating the internal translation initiation of the alternative core+1/S ORF.

Hepatitis C Virus core+1/ARF Protein Modulates the Cyclin D1/pRb Pathway and Promotes Carcinogenesis.

Viruses often encompass overlapping reading frames and unconventional translation mechanisms in order to maximize the output from a minimum genome and to orchestrate their timely gene expression. Hepatitis C virus (HCV) possesses such an unconventional open reading frame (ORF) within the core-coding region, encoding an additional protein, initially designated ARFP, F, or core+1. Two predominant isoforms of core+1/ARFP have been reported, core+1/L, initiating from codon 26, and core+1/S, initiating from codons 85/87 of the polyprotein coding region. The biological significance of core+1/ARFP expression remains elusive. The aim of the present study was to gain insight into the functional and pathological properties of core+1/ARFP through its interaction with the host cell, combining in vitro and in vivo approaches. Our data provide strong evidence that the core+1/ARFP of HCV-1a stimulates cell proliferation in Huh7-based cell lines expressing either core+1/S or core+1/L isoforms and in transgenic liver disease mouse models expressing core+1/S protein in a liver-specific manner. Both isoforms of core+1/ARFP increase the levels of cyclin D1 and phosphorylated Rb, thus promoting the cell cycle. In addition, core+1/S was found to enhance liver regeneration and oncogenesis in transgenic mice. The induction of the cell cycle together with increased mRNA levels of cell proliferation-related oncogenes in cells expressing the core+1/ARFP proteins argue for an oncogenic potential of these proteins and an important role in HCV-associated pathogenesis.IMPORTANCE This study sheds light on the biological importance of a unique HCV protein. We show here that core+1/ARFP of HCV-1a interacts with the host machinery, leading to acceleration of the cell cycle and enhancement of liver carcinogenesis. This pathological mechanism(s) may complement the action of other viral proteins with oncogenic properties, leading to the development of hepatocellular carcinoma. In addition, given that immunological responses to core+1/ARFP have been correlated with liver disease severity in chronic HCV patients, we expect that the present work will assist in clarifying the pathophysiological relevance of this protein as a biomarker of disease progression.

High prevalence of antibodies to core+1/ARF protein in HCV-infected patients with advanced cirrhosis.

Hepatitis C virus (HCV) possesses a second open reading frame (ORF) within the core gene encoding an additional protein, known as the alternative reading frame protein (ARFP), F or core+1. The biological significance of the core+1/ARF protein remains elusive. However, several independent studies have shown the presence of core+1/ARFP antibodies in chronically HCV-infected patients. Furthermore, a higher prevalence of core+1/ARFP antibodies was detected in patients with HCV-associated hepatocellular carcinoma (HCC). Here, we investigated the incidence of core+1/ARFPantibodies in chronically HCV-infected patients at different stages of cirrhosis in comparison to chronically HCV-infected patients at earlier stages of disease. Using ELISA, we assessed the prevalence of anti-core+1 antibodies in 30 patients with advanced cirrhosis [model for end-stage liver disease (MELD) ≥15] in comparison with 50 patients with mild cirrhosis (MELD <15) and 164 chronic HCV patients without cirrhosis. 28.7 % of HCV patients with cirrhosis were positive for anti-core+1 antibodies, in contrast with 16.5 % of non-cirrhotic HCV patients. Moreover, there was significantly higher positivity for anti-core+1 antibodies in HCV patients with advanced cirrhosis (36.7 %) compared to those with early cirrhosis (24 %) (P<0.05). These findings, together with the high prevalence of anti-core+1 antibodies in HCV patients with HCC, suggest that core+1 protein may have a role in virus-associated pathogenesis, and provide evidence to suggest that the levels of anti-core+1 antibodies may serve as a marker for disease progression.

Early Transcriptome Signatures from Immunized Mouse Dendritic Cells Predict Late Vaccine-Induced T-Cell Responses.

Systems biology offers promising approaches for identifying response-specific signatures to vaccination and assessing their predictive value. Here, we designed a modelling strategy aiming to predict the quality of late T-cell responses after vaccination from early transcriptome analysis of dendritic cells. Using standardized staining with tetramer, we first quantified antigen-specific T-cell expansion 5 to 10 days after vaccination with one of a set of 41 different vaccine vectors all expressing the same antigen. Hierarchical clustering of the responses defined sets of high and low T cell response inducers. We then compared these responses with the transcriptome of splenic dendritic cells obtained 6 hours after vaccination with the same vectors and produced a random forest model capable of predicting the quality of the later antigen-specific T-cell expansion. The model also successfully predicted vector classification as low or strong T-cell response inducers of a novel set of vaccine vectors, based on the early transcriptome results obtained from spleen dendritic cells, whole spleen and even peripheral blood mononuclear cells. Finally, our model developed with mouse datasets also accurately predicted vaccine efficacy from literature-mined human datasets.

Expression of the novel hepatitis C virus core+1/ARF protein in the context of JFH1-based replicons.

Hepatitis C virus contains a second open reading frame within the core gene, designated core+1/ARF. Here we demonstrate for the first time expression of core+1/ARF protein in the context of a bicistronic JFH1-based replicon and report the production of two isoforms, core+1/L (long) and core+1/S (short), with different kinetics.

Persistence of HCV in Acutely-Infected Patients Depletes C24-Ceramide and Upregulates Sphingosine and Sphinganine Serum Levels.

Hepatitis C virus (HCV) substantially affects lipid metabolism, and remodeling of sphingolipids appears to be essential for HCV persistence in vitro. The aim of the current study is the evaluation of serum sphingolipid variations during acute HCV infection. We enrolled prospectively 60 consecutive patients with acute HCV infection, most of them already infected with human immunodeficiency virus (HIV), and serum was collected at the time of diagnosis and longitudinally over a six-month period until initiation of antiviral therapy or confirmed spontaneous clearance. Quantification of serum sphingolipids was performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Spontaneous clearance was observed in 11 out of 60 patients (18.3%), a sustained viral response (SVR) in 43 out of 45 patients (95.5%) receiving an antiviral treatment after follow-up, whereas persistence of HCV occurred in six out of 60 patients (10%). C24-ceramide (C24-Cer)-levels increased at follow-up in patients with spontaneous HCV eradication (p < 0.01), as compared to baseline. Sphingosine and sphinganine values were significantly upregulated in patients unable to clear HCV over time compared to patients with spontaneous clearance of HCV infection on follow-up (p = 0.013 and 0.006, respectively). In summary, the persistence of HCV after acute infection induces a downregulation of C24Cer and a simultaneous elevation of serum sphingosine and sphinganine concentrations.

Hepatitis C virus modulates lipid regulatory factor Angiopoietin-like 3 gene expression by repressing HNF-1α activity.

BACKGROUND & AIMS: HCV relies on host lipid metabolism to complete its life cycle and HCV core is crucial to this interaction. Liver secreted ANGPTL-3 is an LXR- and HNF-1α-regulated protein, which plays a key role in lipid metabolism by increasing plasma lipids via inhibition of lipase enzymes. Here we aimed to investigate the modulation of ANGPTL-3 by HCV core and identify the molecular mechanisms involved. METHODS: qRT-PCR and ELISA were used to assess ANGPTL-3 mRNA and protein levels in HCV patients, the JFH-1 infectious system and liver cell lines. Transfections, chromatin immunoprecipitation and immunofluorescence delineated parts of the molecular mechanisms implicated in the core-mediated regulation of ANGPTL-3 gene expression. RESULTS: ANGPTL-3 gene expression was decreased in HCV-infected patients and the JFH-1 infectious system. mRNA and promoter activity levels were down-regulated by core. The response was lost when an HNF-1α element in ANGPTL-3 promoter was mutated, while loss of HNF-1α DNA binding to this site was recorded in the presence of HCV core. HNF-1α mRNA and protein levels were not altered by core. However, trafficking between nucleus and cytoplasm was observed and then blocked by an inhibitor of the HNF-1α-specific kinase Mirk/Dyrk1B. Transactivation of LXR/RXR signalling could not restore core-mediated down-regulation of ANGPTL-3 promoter activity. CONCLUSIONS: ANGPTL-3 is negatively regulated by HCV in vivo and in vitro. HCV core represses ANGPTL-3 expression through loss of HNF-1α binding activity and blockage of LXR/RXR transactivation. The putative ensuing increase in serum lipid clearance and uptake by the liver may sustain HCV virus replication and persistence.

Hepatitis C virus core+1/ARF protein decreases hepcidin transcription through an AP1 binding site.

Chronic viral hepatitis C is characterized by iron accumulation in the liver, and hepcidin regulates iron absorption. Hepatitis C virus (HCV) core+1/ARFP is a novel protein produced by a second functional ORF within the core gene. Here, using reporter assays and HCV bicistronic replicons, we show that, similarly to core, core+1/ARFP decreases hepcidin expression in hepatoma cells. The activator protein 1 (AP1) binding site of the human hepcidin promoter, shown here to be relevant to basal promoter activity and to the repression by core, is essential for the downregulation by core+1/ARFP while the previously described C/EBP (CCAAT/enhancer binding protein) and STAT (signal transducer and activator of transcription) sites are not. Consistently, expression of the AP1 components c-jun and c-fos obliterated the repressive effect of core and core+1/ARFP. In conclusion, we provide evidence that core+1/ARFP downregulates AP1-mediated transcription, providing new insights into the biological role of core+1/ARFP, as well as the transcriptional modulation of hepcidin, the main regulator of iron metabolism.

Modulation of IL-2 expression after uptake of hepatitis C virus non-enveloped capsid-like particles: the role of p38 kinase.

Hepatitis C virus (HCV) has been shown to actively replicate in cells of the immune system, altering both their function and cytokine expression. Naked nucleocapsids have been reported in the serum of infected patients. We investigated interference of recombinant non-enveloped capsid-like particles with signaling pathways in T cells. HCV non-enveloped particles (HCVne) internalization was verified in Jurkat and Hut 78 T cells, as well as primary human peripheral blood and intrahepatic mononuclear cells. HCVne uptake leads to activation of the MAPKs-p38 signaling pathway. Using specific phosphoantibodies, signaling pathways inhibitors, and chemical agents, it was demonstrated that p38 activation in T cells correlated with IL-2 transcriptional activation and was accompanied by a parallel increase of IL-2 cytokine secretion. c-fos and egr-1, two transcription factors, essential for IL-2 promoter activity, were also found to be elevated. We propose that HCVne uptake by T lymphocytes results in increased MAPKs-p38 activity and IL-2 expression, thus altering the host immune response.

A Proteomic Approach to Study the Biological Role of Hepatitis C Virus Protein Core+1/ARFP.

Hepatitis C virus is the major cause of chronic liver diseases and the only cytoplasmic RNA virus known to be oncogenic in humans. The viral genome gives rise to ten mature proteins and to additional proteins, which are the products of alternative translation initiation mechanisms. A protein-known as ARFP (alternative reading frame protein) or Core+1 protein-is synthesized by an open reading frame overlapping the HCV Core coding region in the (+1) frame of genotype 1a. Almost 20 years after its discovery, we still know little of the biological role of the ARFP/Core+1 protein. Here, our differential proteomic analysis of stable hepatoma cell lines expressing the Core+1/Long isoform of HCV-1a relates the expression of the Core+1/Long isoform with the progression of the pathology of HCV liver disease to cancer.

Hepatitis C Core Protein Induces a Genotype-Specific Susceptibility of Hepatocytes to TNF-Induced Death In Vitro and In Vivo.

Hepatitis C virus (HCV) core protein is a multifunctional protein that is involved in the proliferation, inflammation, and apoptosis mechanism of hepatocytes. HCV core protein genetic variability has been implicated in various outcomes of HCV pathology and treatment. In the present study, we aimed to analyze the role of the HCV core protein in tumor necrosis factor α (TNFα)-induced death under the viewpoint of HCV genetic variability. Immortalized hepatocytes (IHH), and not the Huh 7.5 hepatoma cell line, stably expressing HCV subtype 4a and HCV subtype 4f core proteins showed that only the HCV 4a core protein could increase sensitivity to TNFα-induced death. Development of two transgenic mice expressing the two different core proteins under the liver-specific promoter of transthyretin (TTR) allowed for the in vivo assessment of the role of the core in TNFα-induced death. Using the TNFα-dependent model of lipopolysaccharide/D-galactosamine (LPS/Dgal), we were able to recapitulate the in vitro results in IHH cells in vivo. Transgenic mice expressing the HCV 4a core protein were more susceptible to the LPS/Dgal model, while mice expressing the HCV 4f core protein had the same susceptibility as their littermate controls. Transcriptome analysis in liver biopsies from these transgenic mice gave insights into HCV core molecular pathogenesis while linking HCV core protein genetic variability to differential pathology in vivo.

Association between vitamin D receptor gene polymorphisms and fibrosis susceptibility in Greek patients with HCV infection.

Introduction: Hepatitis C virus (HCV) infection is a prime cause of chronic hepatitis worldwide, that often silently progresses to fibrosis, cirrhosis and hepatocellular carcinoma (HCC). Notably, the majority of individuals infected with HCV develop symptoms at late stages, often associated with liver damage that cannot revert after virus clearance. Thus, current antiviral therapy alone is rather insufficient to eliminate the global burden of HCV in the near future.During the past few years, vitamin D deficiency as well as certain single nucleotide polymorphisms in the vitamin D receptor (VDR) gene have been associated with liver fibrosis. Therefore, the aim of the present study was to investigate the possible correlation between VDR polymorphisms ApaI (rs7975232) and TaqI (rs731236) and the fibrosis stage of patients with HCV infection from Thrace, Greece. Methods: Eighty-one patients with HCV infection underwent transient elastography for the assessment of their fibrosis stage, and PCR-restriction fragment length polymorphism (RFLP) genotyping for VDR ApaI and TaqI polymorphisms. VDR genotypes were then statistically associated with the patients' fibrosis stage using ordinal regression models. Results: Non-cirrhotic stages were positively correlated with TaqI TT genotype (p=0.003) and negatively correlated with TaqI TC genotype (p=0.007). In the presence of Hardy-Weinberg equilibrium and linkage disequilibrium between the two VDR polymorphisms, mild fibrosis stages (F0-2) were correlated with ApaI/TaqI GG/TT (p=0.002) and TG/TT (p=0.008) genotypes, while cirrhotic stage F4 was associated with ApaI/TaqI TG/TC genotype (p=0.038). Conclusions: TaqI TT and ApaI/TaqI GG/TT, TG/TT and TG/TC genotypes could be explored as prognostic genetic markers for fibrosis susceptibility in HCV patients.

Endocytosis of hepatitis C virus non-enveloped capsid-like particles induces MAPK-ERK1/2 signaling events.

Although HCV is an enveloped virus, naked nucleocapsids have been reported in the serum of infected patients. The HCV core particle serves as a protective capsid shell for the viral genome and recombinant in vitro assembled HCV core particles induce strong specific immunity. We investigated the post-binding mechanism of recombinant core particle uptake and its intracellular fate. In hepatic cells, these particles are internalized, most likely in a clathrin-dependent pathway, reaching early to late endosomes and finally lysosomes. The endocytic acidic milieu is implicated in trafficking process. Using specific phosphoantibodies, signaling pathway inhibitors and chemical agents, ERK(1/2) was found to be activated in a sustained way after endocytosis, followed by downstream immediate early genes (c-fos and egr-1) modulation. We propose that the intriguing properties of cellular internalization of HCV non-enveloped particles can induce specific ERK(1/2)-MAPKs events that could be important in HCV life cycle and pathogenesis of HCV infection.

ER targeting and retention of the HCV NS4B protein relies on the concerted action of multiple structural features including its transmembrane domains.

The Hepatitis C virus (HCV) NS4B protein, a multispanning endoplasmic reticulum (ER) membrane protein, generates intracellular rearrangements of ER-derived membranes, essential for HCV replication. In this study, we characterized NS4B elements involved in the process of targeting, association and retention in the ER membrane. We investigated the localization and membrane association of a number of C- or N-terminal NS4B deletions expressed as GFP chimeras by biochemical and fluorescence microscopy techniques. A second set of GFP-NS4B chimeras containing the plasma membrane ecto-ATPase CD39 at the C-terminus of each NS4B deletion mutant was used to further examine the role of N-terminal NS4B sequences in ER retention. Several structural elements, besides the first two transmembrane domains (TMs), within the NS4B N-terminal half (residues 1-130) were found to mediate association of the NS4B-GFP chimeras with ER membranes. Both TM1 and TM2 are required for ER anchoring and retention but are not sufficient for ER retention. Sequences upstream of TM1 are also required. These include two putative amphipathic alpha-helices and a Leucine Rich Repeat-like motif, a sequence highly conserved in all HCV genotypes. The N-terminal 55peptidic sequence, containing the 1st amphipathic helix, mediates association of the 55N-GFP chimera with cellular membranes including the ER, but is dispensable for ER targeting of the entire NS4B molecule. Importantly, the C-terminal 70peptidic sequence can associate with membranes positive for ER markers in the absence of any predicted TMs. In conclusion, HCV NS4B targeting and retention in the ER results from the concerted action of several NS4B structural elements.

Novel tumour-specific promoters for transcriptional targeting of hepatocellular carcinoma by herpes simplex virus vectors.

BACKGROUND: Hepatocellular carcinoma (HCC) is a cancer of poor prognosis, with limited success in patient treatment, which it makes an excellent target for gene therapy and viral oncolysis. Accordingly, herpes virus simplex type-1 (HSV-1) is one of the most promising viral platforms for transferring therapeutic genes and the development of oncolytic vectors that can target, multiply in, and eradicate hepatoma cells via their lytic cycle. Enhanced efficacy and specificity of HSV-1-based vectors towards HCC may be achieved by using HCC-specific gene promoters to drive selective viral gene expression and accomplish conditional replication and/or to control the expression of therapeutic genes. However, careful verification of promoter function in the context of the replication-competent HSV-1 vectors is required. The present study aimed to identify novel HCC-specific promoters that could efficiently direct transgene expression to HCC cells and maintain their activity during active viral replication. METHODS: Publicly available microarray data from human HCC biopsies were analysed in order to detect novel candidate genes induced primarily in HCC compared to normal liver. HCC specificity and promoter activity were evaluated by RT-PCR and chromatin immunoprecipitation. Additionally, transcriptional activity of promoters was further evaluated in the context of HSV-1 genome, using luciferase assays in cultured cells and animal models. RESULTS: Eight HCC-specific genes were characterised in this study: Angiopoietin-like-3, Cytochrome P450, family 2, subfamily C, polypeptide 8, Vitronectin, Alcohol dehydrogenase 6-class V, Apolipoprotein B, Fibrinogen beta chain, Inter-alpha-globulin-inhibitor H3 and Inter-alpha-globulin-inhibitor H1. Specific HCC expression and active gene transcription were confirmed in human liver and non-liver cell lines and further evaluated in primary neoplastic cells from hepatitis C and B virus (HCV- and HBV)-associated HCC patients. High promoter activity and specificity in the presence of HSV-1 infection and from within the viral genome, was validated, both in vitro and in vivo. CONCLUSIONS: We identified and experimentally characterized novel hepatoma-specific promoters, which were valuable for cancer-specific gene therapy, using HSV-1 vectors.

Role of the hepatitis C virus core+1 open reading frame and core cis-acting RNA elements in viral RNA translation and replication.

Four conserved RNA stem-loop structures designated SL47, SL87, SL248, and SL443 have been predicted in the hepatitis C virus (HCV) core encoding region. Moreover, alternative translation products have been detected from a reading frame overlapping the core gene (core+1/ARFP/F). To study the importance of the core+1 frame and core-RNA structures for HCV replication in cell culture and in vivo, a panel of core gene silent mutations predicted to abolish core+1 translation and affecting core-RNA stem-loops were introduced into infectious-HCV genomes of the isolate JFH1. A mutation disrupting translation of all known forms of core+1 and affecting SL248 did not alter virus production in Huh7 cells and in mice xenografted with human liver tissue. However, a combination of mutations affecting core+1 at multiple codons and at the same time, SL47, SL87, and SL248, delayed RNA replication kinetics and substantially reduced virus titers. The in vivo infectivity of this mutant was impaired, and in virus genomes recovered from inoculated mice, SL87 was restored by reversion and pseudoreversion. Mutations disrupting the integrity of this stem-loop, as well as that of SL47, were detrimental for virus viability, whereas mutations disrupting SL248 and SL443 had no effect. This phenotype was not due to impaired RNA stability but to reduced RNA translation. Thus, SL47 and SL87 are important RNA elements contributing to HCV genome translation and robust replication in cell culture and in vivo.

The HCV ARFP/F/core+1 protein: production and functional analysis of an unconventional viral product.

Hepatitis C virus (HCV) is an enveloped positive-strand RNA virus of the Flaviviridae family. It has a genome of about 9,600 nucleotides encoding a large polyprotein (about 3,000 amino acids) that is processed by cellular and viral proteases into at least 10 structural and nonstructural viral proteins. A novel HCV protein has also been identified by our laboratory and others. This protein--known as ARFP (alternative reading frame protein), F (for frameshift) or core+1 (to indicate the position) protein--is synthesized by an open reading frame overlapping the core gene at nucleotide +1 (core+1 ORF). However, almost 10 years after its discovery, we still know little of the biological role of the ARFP/F/core+1 protein. Abolishing core+1 protein production has no affect on HCV replication in cell culture or uPA-SCID mice, suggesting that core+1 protein is probably not important for the HCV reproductive cycle. However, the detection of specific anti-core+1 antibodies and T-cell responses in HCV-infected patients, as reported by many independent laboratories, provides strong evidence that this protein is produced in vivo. Furthermore, analyses of the HCV sequences isolated from patients with hepatocellular carcinoma and in vitro studies have provided strong preliminary evidence to suggest that core+1 protein plays a role in advanced liver disease and liver cancer. The available in vitro data also suggest that certain core function proteins may depend on production of the core+1 protein. We describe here the discovery of the various forms of the core+1 protein and what is currently known about the mechanisms of their production and their biochemical and functional properties. We also provide a detailed summary of the results of patient-based research.

Comparative methods for genotyping hepatitis C virus isolates from Romania.

Accurate genotyping of hepatitis C virus (HCV) has clinical implications for treatment orientation and epidemiological impact in tracing the contamination sources. The aim of the study was to compare a genotyping assay by restriction fragment length polymorphism (RFLP) in the HCV 5'untranslated region (5'UTR) with sequencing in the 5'untranslated and NS5B regions. One hundred and three samples, collected between 2004 and 2006 from chronically infected patients with HCV, were tested with the 5'UTR and NS5B protocols. Of the total number of the samples tested by the 5'UTR-RFLP assay (n=103) the HCV subtype could be inferred by this method for 92 samples, by 5'UTR sequencing for 16 samples out of 23 tested (n=23) and by using the NS5B sequencing for all the samples tested (n=34). Our results showed that the HCV genotype distribution in Romania is: 1b--86.4%, 1a--10.7% and 4a--2.9%. In conclusion, RFLP screening in the 5'UTR is a convenient method for HCV genotyping and discrimination between 1b and non-1b genotypes but has a poor resolving power for subtyping and evaluation of the transmission routes. Sequencing in NS5B region is more adapted than RFLP and sequencing in 5'UTR for subtyping and epidemiological investigation.

Intergenotypic 2k/1b hepatitis C virus recombinants in the East Macedonia and Thrace region of Greece.

BACKGROUND: Intergenotypic recombinant hepatitis C virus (HCV) strains emerge rarely during coinfection of the same individual with two HCV genotypes. Few recombinant HCV strains have been identified to date and only one, CRF01 2k/1b, has become a worldwide concern. This study reevaluated the genotyping of three HCV genotype 2 strains from a group of patients with an unusually low rate of sustained virological response after pegylated interferon/ribavirin treatment. In addition, genetic determinants of host interferon resistance were evaluated. METHODS: The HCV type 2 strains from the patients' serum were subjected to partial sequencing of the core-E1, NS2, NS5A and NS5B regions by reverse transcription polymerase chain reaction. Furthermore, the IFNL3 rs12979860 and the IFNL4 rs368234815 single nucleotide polymorphisms were defined in two of the three patients. RESULTS: All three strains were phylogenetically related to the Russia-derived CRF01 2k/1b while they encompassed the exact same 2k/1b junction site within NS2. CONCLUSION: This is the first report of HCV 2k/1b recombinants in Greece and the greater area of the Balkans.

Comparison of Dendritic Cell Activation by Virus-Based Vaccine Delivery Vectors Emphasizes the Transcriptional Downregulation of the Oxidative Phosphorylation Pathway.

Antigen delivery platforms based on engineered viruses or virus-like particles are currently developed as vaccines against infectious diseases. As the interaction of vaccines with dendritic cells (DCs) shapes the immunological response, we compared the interaction of a range of virus-based vectors and virus-like particles with DCs in a murine model of systemic administration and transcriptome analyses of splenic DCs. The transcriptome profiles of DCs separated the vaccine vectors into two distinct groups characterized by high- and low-magnitude differential gene expression, which strongly correlated with (1) the surface expression of costimulatory molecules CD40, CD83, and CD86 on DCs, and (2) antigen-specific T-cell responses. Pathway analysis using PANOGA (Pathway and Network-Oriented GWAS Analysis) revealed that the JAK/STAT pathway was significantly activated by both groups of vaccines. In contrast, the oxidative phosphorylation pathway was significantly downregulated only by the high-magnitude DC-stimulating vectors. A gene signature including exclusively chemokine-, cytokine-, and receptor-related genes revealed a vector-specific pattern. Overall, this in vivo DC stimulation model demonstrated a strong relationship between the levels of induced DC maturation and the intensity of T-cell-specific immune responses with a distinct cytokine/chemokine profile, metabolic shifting, and cell surface expression of maturation markers. It could represent an important tool for vaccine design.