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Sample records for virus hcv rna

  1. Hepatitis C virus (HCV) RNA profiles among chronic HIV/HCV-coinfected individuals in ESPRIT; spontaneous HCV RNA clearance observed in nine individuals.

    Science.gov (United States)

    Grint, D; Tedaldi, E; Peters, L; Mocroft, A; Edlin, B; Gallien, S; Klinker, H; Boesecke, C; Kokordelis, P; Rockstroh, J K

    2017-07-01

    Studies have shown that hepatitis C virus (HCV) RNA levels remain stable over time in HIV/HCV-coinfected individuals taking combination antiretroviral therapy (cART), while spontaneous clearance of HCV RNA during the persistent infection phase has been documented only rarely among those with the CC interleukin (IL)-28B genotype. This study describes HCV RNA profiles and factors associated with changes over time in HCV RNA levels in the ESPRIT study. HIV/HCV-coinfected individuals positive for HCV RNA were included in the study. Follow-up was counted from the first HCV RNA positive test and censored at the initiation of interferon-based treatment. HCV RNA and IL-28B measurements were performed in the same reference laboratory. Random effects mixed models were used to analyse changes over time in HCV RNA. A total of 312 ESPRIT patients were included in the study (151 in the arm receiving subcutaneous recombinant IL-2 and 161 in the control arm). Most of the patients were white (89%) and male (76%), and they had a median of 5 HCV RNA measurements per person [interquartile range (IQR) 3-6; range 1-9]. Median follow-up was 5 years (IQR: 2-6 years). At baseline, 96% of patients were taking cART and 93% had undetectable HIV RNA. Mean HCV RNA levels decreased by 13% per year over the study period [95% confidence interval (CI) 8-18%; P < 0.0001]. Baseline HCV RNA levels and the change over time in HCV RNA did not differ by randomization arm (P = 0.16 and P = 0.56, respectively). Nine individuals spontaneously cleared HCV RNA during follow-up [IL-28B genotypes: CC, five patients (56%); CT, four patients (44%)]. HCV RNA levels decreased over time in this population with well-controlled HIV infection. Spontaneous clearance of HCV RNA was documented in five individuals with IL-28B genotype CC and four with the CT genotype. © 2016 British HIV Association.

  2. Stability of hepatitis C virus (HCV) RNA levels among interferon-naïve HIV/HCV-coinfected individuals treated with combination antiretroviral therapy

    DEFF Research Database (Denmark)

    Grint, D; Peters, L; Reekie, J

    2013-01-01

    Infection with hepatitis C virus (HCV) is a major cause of chronic liver disease. High HCV RNA levels have been associated with poor treatment response. This study aimed to examine the natural history of HCV RNA in chronically HCV/HIV-coinfected individuals....

  3. Dynamics of HCV RNA levels during acute hepatitis C virus infection

    Science.gov (United States)

    Hajarizadeh, Behzad; Grebely, Jason; Applegate, Tanya; Matthews, Gail V; Amin, Janaki; Petoumenos, Kathy; Hellard, Margaret; Rawlinson, William; Lloyd, Andrew; Kaldor, John; Dore, Gregory J

    2014-01-01

    Understanding viral dynamics during acute hepatitis C virus (HCV) infection can provide important insights into immunopathogenesis and guide early treatment. The aim of this study was investigating the dynamics of HCV RNA and alanine transaminase (ALT) levels during recent HCV infection in the Australian Trial in Acute Hepatitis C (ATAHC). ATAHC was a prospective study of the natural history of recently acquired HCV infection. Longitudinal HCV RNA and ALT levels were compared among individuals with ultimately persistent infection and spontaneous clearance outcomes. Among those with HCV persistence (n=104) and HCV clearance (n=30), median HCV RNA (5.2 vs. 4.1 log IU/mL, respectively) and ALT levels (779 vs. 1765 IU/L, respectively) were high during month two following infection, and then declined during months three and four in both groups. Among those with HCV persistence, median HCV RNA was 2.9 log IU/mL during months four, increased to 5.5 log IU/mL during month five, and remained subsequently relatively stable. Among those with HCV clearance, median HCV RNA was undetectable by month five. Median HCV RNA levels were comparable between individuals with HCV persistence and HCV clearance during month three following infection (3.2 vs. 3.5 log IU/mL, respectively; P=0.935), but markedly different during month five (5.5 vs. 1.0 log IU/mL, respectively; P<0.001). In conclusion, dynamics of HCV RNA levels in those with HCV clearance and HCV persistence diverged between months three and five following infection, with the latter time-point being potentially useful for commencing early treatment. PMID:25042465

  4. Hepatitis C virus (HCV) RNA profiles among chronic HIV/HCV-coinfected individuals in ESPRIT; spontaneous HCV RNA clearance observed in nine individuals

    DEFF Research Database (Denmark)

    Grint, D; Tedaldi, Ellen; Peters, L

    2017-01-01

    with the CC interleukin (IL)-28B genotype. This study describes HCV RNA profiles and factors associated with changes over time in HCV RNA levels in the ESPRIT study. METHODS: HIV/HCV-coinfected individuals positive for HCV RNA were included in the study. Follow-up was counted from the first HCV RNA positive...... test and censored at the initiation of interferon-based treatment. HCV RNA and IL-28B measurements were performed in the same reference laboratory. Random effects mixed models were used to analyse changes over time in HCV RNA. RESULTS: A total of 312 ESPRIT patients were included in the study (151...

  5. High rate of hepatitis C virus (HCV) recurrence in HIV-infected individuals with spontaneous HCV RNA clearance

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    Peters, L; Mocroft, A; Soriano, V

    2014-01-01

    OBJECTIVES: Following resolution of hepatitis C virus (HCV) infection, recurrence has been shown to occur in some persons with repeated exposure to HCV. We aimed to investigate the rate and factors associated with HCV RNA recurrence among HIV-1-infected patients with prior spontaneous HCV RNA...... clearance in the EuroSIDA cohort. METHODS: All HIV-infected patients with documented prior spontaneous HCV clearance, and at least one subsequently collected plasma sample, were examined. The last sample was tested for HCV RNA and those with HCV RNA ≥ 615 IU/mL were defined as having HCV recurrence...... (IDUs). The median time between the first and last samples was 3.6 years (interquartile range 2.0-5.8 years). After adjustment, those on combination antiretroviral therapy [odds ratio (OR) 0.44; 95% CI 0.20-0.99; P = 0.046] and older persons (OR 0.51 per 10 years older; 95% CI 0.28-0.95; P = 0.033) were...

  6. Antiretroviral Effects on Host Lipoproteins Are Associated With Changes in Hepatitis C Virus (HCV) RNA Levels in Human Immunodeficiency Virus/HCV Coinfected Individuals

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    Naggie, Susanna; Patel, Keyur; Yang, Lan-Yan; Chow, Shein-Chung; Johnson, Victoria; Guyton, John R.; Muir, Andrew J.; Sulkowski, Mark; Hicks, Charles

    2015-01-01

    We evaluated the impact of antiretroviral-induced dyslipidemia on hepatitis C virus (HCV) biogenesis in human immunodeficiency virus (HIV)/HCV coinfected patients. This study used serum samples from antiretroviral-naive HIV/HCV patients initiating their first regimen as part of AIDS Clinical Trials Group study protocols (A5142, A5202). Initiation of antiretrovirals increased most lipoproteins and apolipoproteins. In the multivariable model, changes in apolipoproteins were associated with changes in log10 HCV RNA from baseline to week-24 of therapy. Off-target lipogenic changes need to be considered in the context of liver and other metabolic disease in HIV/HCV patients. PMID:26110167

  7. Undetectable hepatitis C virus RNA during syphilis infection in two HIV/HCV-co-infected patients

    DEFF Research Database (Denmark)

    Salado-Rasmussen, Kirsten; Knudsen, Andreas; Krarup, Henrik Bygum

    2014-01-01

    BACKGROUND: Treponema pallidum, the causative agent of syphilis, elicits a vigorous immune response in the infected host. This study sought to describe the impact of syphilis infection on hepatitis C virus (HCV) RNA levels in patients with HIV and chronic HCV infection. METHODS: Patients...... with chronic HIV/HCV and syphilis co-infection were identified by their treating physicians from 1 October 2010 to 31 December 2013. Stored plasma samples obtained before, during, and after syphilis infection were analysed for interleukin (IL)-2, IL-4, IL-6, IL-8, IL-10, tumour necrosis factor alpha (TNF......-α), interferon gamma (IFN-γ), and IFN-γ-inducible protein 10 kDa (IP-10). RESULTS: Undetectable HCV RNA at the time of early latent syphilis infection was observed in 2 patients with HIV and chronic HCV infection. After treatment of the syphilis infection, HCV RNA levels increased again in patient 1, whereas...

  8. Presence of hepatitis C virus (HCV)-RNA in peripheral blood mononuclear cells in HCV serum negative patients during interferon and ribavirin therapy.

    Science.gov (United States)

    Januszkiewicz-Lewandowska, Danuta; Wysocki, Jacek; Pernak, Monika; Nowicka, Karina; Zawada, Mariola; Rembowska, Jolanta; Lewandowski, Krzysztof; Mańkowski, Przemysław; Nowak, Jerzy

    2007-02-01

    Identification of hepatitis C virus (HCV)-RNA in blood serum is crucial for hepatitis C diagnosis and for appropriate treatment. Detection of HCV-RNA in blood serum is used for therapy monitoring of patients with hepatitis C. Despite HCV-RNA elimination from blood serum during treatment in some patients, HCV viremia appears again after the completion of therapy. The aim of this study was to assess HCV-RNA in peripheral blood mononuclear cells (PBMCs) of hepatitis C patients in relation to HCV-RNA and antibodies to HCV in the serum. The study involved 71 patients undergoing anti-viral therapy (interferon and ribavirin). RNA isolated from serum and PBMCs was examined for the presence of HCV-RNA by an RT-PCR technique using specific oligonucleotide primers or by commercially available kits. In order to show the possible presence of HCV sequences in PBMCs, molecular DNA probes were constructed with a PCR amplicon and biotin-labelled by nick translation, and FISH and extended chromatin fibers in situ hybridization (ECFs-FISH) techniques were used. A 24-month follow-up study revealed that 34 out of 59 patients (58%) eliminated HCV-RNA from their sera. In the serum negative group, HCV-RNA was detected in PBMCs of 2 patients. The presence of HCV-RNA in PBMCs was confirmed by the FISH technique. In the ECFs-FISH procedure, no signal was found in all examined patients. Our data suggest that PBMCs infected with HCV can serve as a virus reservoir. HCV-RNA serum negative patients who have HCV-RNA in their leukocytes after completion of anti-viral therapy would be at great risk of hepatitis C recurrence. These HCV-RNA serum negative but PBMCs positive patients would be a potential source of HCV spread.

  9. Stability of hepatitis C virus (HCV) RNA levels among interferon-naïve HIV/HCV-coinfected individuals treated with combination antiretroviral therapy.

    Science.gov (United States)

    Grint, D; Peters, L; Reekie, J; Soriano, V; Kirk, O; Knysz, B; Suetnov, O; Lazzarin, A; Ledergerber, B; Rockstroh, J K; Mocroft, A

    2013-07-01

    Infection with hepatitis C virus (HCV) is a major cause of chronic liver disease. High HCV RNA levels have been associated with poor treatment response. This study aimed to examine the natural history of HCV RNA in chronically HCV/HIV-coinfected individuals. Mixed models were used to analyse the natural history of HCV RNA changes over time in HIV-positive patients with chronic HCV infection. A total of 1541 individuals, predominantly White (91%), male (73%), from southern (35%) and western central Europe (23%) and with HCV genotype 1 (58%), were included in the analysis. The median follow-up time was 5.0 years [interquartile range (IQR) 2.8 to 8.3 years]. Among patients not on combination antiretroviral therapy (cART), HCV RNA levels increased by a mean 27.6% per year [95% confidence interval (CI) 6.1-53.5%; P = 0.0098]. Among patients receiving cART, HCV RNA levels were stable, increasing by a mean 2.6% per year (95% CI -1.1 to 6.5%; P = 0.17). Baseline HCV RNA levels were 25.5% higher (95% CI 8.8 to 39.1%; P = 0.0044) in individuals with HCV genotype 1 compared with HCV genotypes 2, 3 and 4. A 1 log HIV-1 RNA copies/mL increase in HIV RNA was associated with a 10.9% increase (95% CI 2.3 to 20.2%; P = 0.012) in HCV RNA. While HCV RNA levels increased significantly in patients prior to receiving cART, among those treated with cART HCV RNA levels remained stable over time. © 2013 British HIV Association.

  10. Pretransplant interferon prevents hepatitis C virus-associated glomerulonephritis in renal allografts by HCV-RNA clearance.

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    Cruzado, Josep M; Casanovas-Taltavull, Teresa; Torras, Joan; Baliellas, Carme; Gil-Vernet, Salvador; Grinyó, Josep M

    2003-03-01

    The purpose of this study was to examine the effect of pretransplant interferon administration on the occurrence of post-transplant de novo glomerulonephritis in hepatitis C virus (HCV)-positive renal allografts. From December 1992 to December 2000, 78 HCV-positive patients received a renal allograft in our unit. Fifteen out of 78 received pretransplant interferon for 1 year. Hepatitis C virus was investigated by serology and qualitative polymerase chain reaction (PCR). Hepatitis C virus-related de novo glomerulonephritis (membranoproliferative or membranous) was suggested by proteinuria (>1.5 g/24 h) and/or microhematuria and always diagnosed by renal biopsy. Of 15 HCV-positive recipients who received pretransplant interferon, 10 (67%) became HCV-RNA negative at the time of transplantation and only one out of the 15 (6.7%) developed de novo glomerulonephritis (this patient was HCV-RNA positive at transplantation). Among non-interferon-treated allograft recipients, 28.7% had negative HCV-RNA and 12 out of 63 (19%) developed de novo glomerulonephritis (9, membranoproliferative; 3 membranous), all 12 having positive HCV-RNA at transplantation (p < 0.0001). In conclusion, pretransplant interferon may reduce the occurrence of post-transplant HCV-related de novo glomerulonephritis. Our results suggest that the indication for pretransplant interferon should be extended to treat all HCV-RNA positive candidates for renal transplantation.

  11. HCV Virus and Lymphoid Neoplasms

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    Yutaka Tsutsumi

    2011-01-01

    Full Text Available Hepatitis C virus (HCV is one of the viruses known to cause hepatic cancer. HCV is also believed to be involved in malignant lymphoma. In this paper, we investigated characteristics of malignant lymphoma cases that were anti-HCV antibody (HCV-Ab positive. We were able to perform pathological examinations on 13 out of 14 HCV-positive cases. Of these, lymphoid tissues of 10 stained positive for HCV-Ab. There was no significant correlation between the degree of HCV staining and the rate of recurrence or resistance to treatment. However, there did appear to be a consistent decrease in the amount of HCV-RNA between pre- and posttreatment among HCV-Ab-positive cases; that is, treatment-resistant cases that exhibited resistance from the first treatment and recurrent cases more frequently had a higher HCV level at treatment termination compared to the pretreatment level. This suggests that the HCV virus either accelerates oncogenesis by direct interaction with B cells or indirectly affects lymphoma prognosis.

  12. Interferon (IFN and Cellular Immune Response Evoked in RNA-Pattern Sensing During Infection with Hepatitis C Virus (HCV

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    Masato Nakai

    2015-10-01

    Full Text Available Hepatitis C virus (HCV infects hepatocytes but not dendritic cells (DCs, but DCs effectively mature in response to HCV-infected hepatocytes. Using gene-disrupted mice and hydrodynamic injection strategy, we found the MAVS pathway to be crucial for induction of type III interferons (IFNs in response to HCV in mouse. Human hepatocytes barely express TLR3 under non-infectious states, but frequently express it in HCV infection. Type I and III IFNs are induced upon stimulation with polyI:C, an analog of double-stranded (dsRNA. Activation of TLR3 and the TICAM-1 pathway, followed by DC-mediated activation of cellular immunity, is augmented during exposure to viral RNA. Although type III IFNs are released from replication-competent human hepatocytes, DC-mediated CTL proliferation and NK cell activation hardly occur in response to the released type III IFNs. Yet, type I IFNs and HCV-infected hepatocytes can induce maturation of DCs in either human or mouse origin. In addition, mouse CD8+ DCs mature in response to HCV-infected hepatocytes unless the TLR3/TICAM-1 pathway is blocked. We found the exosomes containing HCV RNA in the supernatant of the HCV-infected hepatocytes act as a source of TLR3-mediated DC maturation. Here we summarize our view on the mechanism by which DCs mature to induce NK and CTL in a status of HCV infection.

  13. Label Free Inhibitor Screening of Hepatitis C Virus (HCV NS5B Viral Protein Using RNA Oligonucleotide

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    Sang Eun Kim

    2011-06-01

    Full Text Available Globally, over 170 million people (ca. 3% of the World’s population are infected with the hepatitis C virus (HCV, which can cause serious liver diseases such as chronic hepatitis, evolving into subsequent health problems. Driven by the need to detect the presence of HCV, as an essential factor in diagnostic medicine, the monitoring of viral protein has been of great interest in developing simple and reliable HCV detection methods. Despite considerable advances in viral protein detection as an HCV disease marker, the current enzyme linked immunosorbent assay (ELISA based detection methods using antibody treatment have several drawbacks. To overcome this bottleneck, an RNA aptamer become to be emerged as an antibody substitute in the application of biosensor for detection of viral protein. In this study, we demonstrated a streptavidin-biotin conjugation method, namely, the RNA aptamer sensor system that can quantify viral protein with detection level of 700 pg mL−1 using a biotinylated RNA oligonucleotide on an Octet optical biosensor. Also, we showed this method can be used to screen inhibitors of viral protein rapidly and simply on a biotinylated RNA oligonucleotide biosensor. Among the inhibitors screened, (−-Epigallocatechin gallate showed high binding inhibition effect on HCV NS5B viral protein. The proposed method can be considered a real-time monitoring method for inhibitor screening of HCV viral protein and is expected to be applicable to other types of diseases.

  14. [Analyses of anti-hCV detected by ELISA and HCV RNA detected by RT-nPCR in chronic hepatitis C virus infectors].

    Science.gov (United States)

    Gao, Qiuju; Liu, Dianwu; Zhang, Shiyong; Tong, Lixin

    2007-01-01

    In order to provide the basis for the clinical test and the blood station screening the health donator, the results of anti-HCV tested by ELISA (enzyme-linked-immuno-absorbed assay ) and HCV RNA tested by RT-nPCR (reverse-transcript-nested-polymerase-chain-reaction) were compared in the chronic hepatitis C virus infectors. Venous blood samples of 133 chronic hepatitis C virus infectors, 52 health controls were collected in May 2005. These infectors were infected with HCV nearly in 1990 through plasma donator and diagnosed in 1993 in a rural area of Zhao County in Hebei Province, which remained the same diagnosis as HCV infectors in 2002 Hebei Province. The anti-HCV was tested by ELISA and HCV RNA was tested by RT-nPCR. (1) In 185 cases, the positive rates of both anti-HCV and HCV RNA were 49.73% (92/185). The rate of anti-HCV negative but HCV RNA positive was 9.73% (18/185). The rate of anti-HCV positive but HCV RNA negative was 11.89% (22/185). The negative rate of both anti-HCV and HCV RNA tested was 28.65% (53/185). The result-agreement rate of ELISA and RT-nPCR methods were 78.38% [(92 + 53)/185]. The disagreement rate between ELISA and RT-nPCR methods was not obviously different (paired chi2 = 0.40, P > 0.05). (2) In the chronic HCV infectors, the sensitivity of anti-HCV tested by ELISA was 82.71%, the specificity was 92.31%, and the omitting rate was 17.29%. The sensitivity of HCV RNA tested by RT-nPCR was 81.20%, the specificity was 96.15%, and the omitting rate was 18.80%. The sensitivity between ELISA and RT-nPCR was not obviously different (chi2 = 0.102, P > 0.05). (3) The sensitivity tested by ELISA combined with RT-nPCR was 96.75%, which was evidently higher than that of single ELISA (82.71%) (chi2 = 9.62, P < 0.01). The false negative rate was nearly 17% when anti-HCV was tested with single ELISA in HCV infectors. The positive testing rate of HCV infection was increased remarkably when ELISA and RT-nPCR were tested simultaneously.

  15. Detection of hepatitis C virus RNA in saliva samples from patients with seric anti-HCV antibodies

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    Patrícia L. Gonçalves

    Full Text Available We examined the frequency of HCV-RNA in saliva samples from anti-HCV positive patients. Both plasma and saliva samples from 39 HCV patients (13 with normal liver enzymes, 19 with abnormal liver enzymes and 13 with cirrhosis were investigated. Stimulated saliva and fresh plasma were centrifuged (900 x g,10 min and stored at -70ºC, after the addition of guanidine isothiocyanate RNA extraction buffer. HCV-RNA was detected by RT- nested-PCR (amplification of HCV-cDNA for two rounds, using HCV primers 939/209 and 940/211. HCV genotyping was carried out by RFLP (using Mva I and Hinf 1 or Hae III and Rsa I restriction enzymes. Thirty-two out of 39 (82%; 95% CI=70-94% anti-HCV-positive patients had HCV-RNA in plasma samples. Eight out of 39 (20.5%; 95% CI=6.6-34.4% had HCV-RNA in the saliva. The HCV genotype in saliva samples from these patients matched the genotype found for plasma HCV-RNA. No significant correlation between the presence of HCV and either age, gender, HCV genotype or any risk factor for HCV infection was found. The observed prevalence (20.5% of anti HCV positive patients or 25% of the patients with HCV-RNA in plasma was lower than that previously reported from other countries. The low frequency of HCV-RNA in saliva samples observed in our study may be due to the use of cell-free saliva. Other authors reporting higher frequencies of HCV-RNA in saliva used whole saliva, without centrifugation.

  16. Preclinical evaluation of an anti-HCV miRNA cluster for treatment of HCV infection.

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    Yang, Xiao; Marcucci, Katherine; Anguela, Xavier; Couto, Linda B

    2013-03-01

    We developed a strategy to treat hepatitis C virus (HCV) infection by replacing five endogenous microRNA (miRNA) sequences of a natural miRNA cluster (miR-17-92) with sequences that are complementary to the HCV genome. This miRNA cluster (HCV-miR-Cluster 5) is delivered to cells using adeno-associated virus (AAV) vectors and the miRNAs are expressed in the liver, the site of HCV replication and assembly. AAV-HCV-miR-Cluster 5 inhibited bona fide HCV replication in vitro by up to 95% within 2 days, and the spread of HCV to uninfected cells was prevented by continuous expression of the anti-HCV miRNAs. Furthermore, the number of cells harboring HCV RNA replicons decreased dramatically by sustained expression of the anti-HCV miRNAs, suggesting that the vector is capable of curing cells of HCV. Delivery of AAV-HCV-miR-Cluster 5 to mice resulted in efficient transfer of the miRNA gene cluster and expression of all five miRNAs in liver tissue, at levels up to 1,300 copies/cell. These levels achieved up to 98% gene silencing of cognate HCV sequences, and no liver toxicity was observed, supporting the safety of this approach. Therefore, AAV-HCV-miR-Cluster 5 represents a different paradigm for the treatment of HCV infection.

  17. HCV RNA in peripheral blood mononuclear cells (PBMCs) as a ...

    African Journals Online (AJOL)

    Abdel Fatah Fahmy Hanno

    2013-06-27

    Jun 27, 2013 ... Objectives: To study hepatitis virus C (HCV) RNA in peripheral blood mononuclear cells. (PBMCs) of patients with chronic HCV infection, and explore the relationship between the HCV. RNA in the PBMCs and response to interferon (IFN) therapy. Methods: Twenty-five patients with chronic viral hepatitis C ...

  18. Interferon Response in Hepatitis C Virus (HCV Infection: Lessons from Cell Culture Systems of HCV Infection

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    Pil Soo Sung

    2015-10-01

    Full Text Available Hepatitis C virus (HCV is a positive-stranded RNA virus that infects approximately 130–170 million people worldwide. In 2005, the first HCV infection system in cell culture was established using clone JFH-1, which was isolated from a Japanese patient with fulminant HCV infection. JFH-1 replicates efficiently in hepatoma cells and infectious virion particles are released into the culture supernatant. The development of cell culture-derived HCV (HCVcc systems has allowed us to understand how hosts respond to HCV infection and how HCV evades host responses. Although the mechanisms underlying the different outcomes of HCV infection are not fully understood, innate immune responses seem to have a critical impact on the outcome of HCV infection, as demonstrated by the prognostic value of IFN-λ gene polymorphisms among patients with chronic HCV infection. Herein, we review recent research on interferon response in HCV infection, particularly studies using HCVcc infection systems.

  19. HCV RNA in peripheral blood mononuclear cells (PBMCs) as a ...

    African Journals Online (AJOL)

    Abdel Fatah Fahmy Hanno

    2013-06-27

    Jun 27, 2013 ... Abstract Background: Hepatitis C virus (HCV) has been found to infect peripheral blood mono- nuclear cells (PBMCs), using them as a reservoir, which might contribute to the development of resistance to treatment. Objectives: To study hepatitis virus C (HCV) RNA in peripheral blood mononuclear cells.

  20. HCV RNA in peripheral blood mononuclear cells (PBMCs) as a ...

    African Journals Online (AJOL)

    Background: Hepatitis C virus (HCV) has been found to infect peripheral blood mononuclear cells (PBMCs), using them as a reservoir, which might contribute to the development of resistance to treatment. Objectives: To study hepatitis virus C (HCV) RNA in peripheral blood mononuclear cells (PBMCs) of patients with ...

  1. Packaging of HCV-RNA into lentiviral vector

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    Caval, Vincent [INSERM U966, Universite Francois Rabelais de Tours, Faculte de Medecine, 10 Bd. Tonnelle, 37000 Tours (France); Piver, Eric [INSERM U966, Universite Francois Rabelais de Tours, Faculte de Medecine, 10 Bd. Tonnelle, 37000 Tours (France); Service de Biochimie et Biologie Moleculaire, CHRU de Tours (France); Ivanyi-Nagy, Roland; Darlix, Jean-Luc [LaboRetro, ENS-Lyon INSERM, U758, 46 Allee d' Italie, 69364 Lyon (France); Pages, Jean-Christophe, E-mail: jean-christophe.pages@univ-tours.fr [INSERM U966, Universite Francois Rabelais de Tours, Faculte de Medecine, 10 Bd. Tonnelle, 37000 Tours (France); Service de Biochimie et Biologie Moleculaire, CHRU de Tours (France)

    2011-11-04

    Highlights: Black-Right-Pointing-Pointer Description of HCV-RNA Core-D1 interactions. Black-Right-Pointing-Pointer In vivo evaluation of the packaging of HCV genome. Black-Right-Pointing-Pointer Determination of the role of the three basic sub-domains of D1. Black-Right-Pointing-Pointer Heterologous system involving HIV-1 vector particles to mobilise HCV genome. Black-Right-Pointing-Pointer Full length mobilisation of HCV genome and HCV-receptor-independent entry. -- Abstract: The advent of infectious molecular clones of Hepatitis C virus (HCV) has unlocked the understanding of HCV life cycle. However, packaging of the genomic RNA, which is crucial to generate infectious viral particles, remains poorly understood. Molecular interactions of the domain 1 (D1) of HCV Core protein and HCV RNA have been described in vitro. Since compaction of genetic information within HCV genome has hampered conventional mutational approach to study packaging in vivo, we developed a novel heterologous system to evaluate the interactions between HCV RNA and Core D1. For this, we took advantage of the recruitment of Vpr fusion-proteins into HIV-1 particles. By fusing HCV Core D1 to Vpr we were able to package and transfer a HCV subgenomic replicon into a HIV-1 based lentiviral vector. We next examined how deletion mutants of basic sub-domains of Core D1 influenced HCV RNA recruitment. The results emphasized the crucial role of the first and third basic regions of D1 in packaging. Interestingly, the system described here allowed us to mobilise full-length JFH1 genome in CD81 defective cells, which are normally refractory to HCV infection. This finding paves the way to an evaluation of the replication capability of HCV in various cell types.

  2. HCV-induced autophagosomes are generated via homotypic fusion of phagophores that mediate HCV RNA replication.

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    Linya Wang

    2017-09-01

    Full Text Available Hepatitis C virus (HCV induces autophagy to promote its replication, including its RNA replication, which can take place on double-membrane vesicles known as autophagosomes. However, how HCV induces the biogenesis of autophagosomes and how HCV RNA replication complex may be assembled on autophagosomes were largely unknown. During autophagy, crescent membrane structures known as phagophores first appear in the cytoplasm, which then progress to become autophagosomes. By conducting electron microscopy and in vitro membrane fusion assay, we found that phagophores induced by HCV underwent homotypic fusion to generate autophagosomes in a process dependent on the SNARE protein syntaxin 7 (STX7. Further analyses by live-cell imaging and fluorescence microscopy indicated that HCV-induced phagophores originated from the endoplasmic reticulum (ER. Interestingly, comparing with autophagy induced by nutrient starvation, the progression of phagophores to autophagosomes induced by HCV took significantly longer time, indicating fundamental differences in the biogenesis of autophagosomes induced by these two different stimuli. As the knockdown of STX7 to inhibit the formation of autophagosomes did not affect HCV RNA replication, and purified phagophores could mediate HCV RNA replication, the assembly of the HCV RNA replication complex on autophagosomes apparently took place during the formative stage of phagophores. These findings provided important information for understanding how HCV controlled and modified this important cellular pathway for its own replication.

  3. Correlation between alanine aminotransferase level, HCV-RNA titer ...

    African Journals Online (AJOL)

    Reham Al Swaff

    2012-04-04

    Apr 4, 2012 ... Abstract The relationship of serum alanine aminotransferase (ALT) level and viral replication to liver damage in chronic hepatitis C virus (HCV) patients remains unclear. The aim of the present study was to determine whether the stage of fibrosis correlates with HCV-. RNA titer and/or serum ALT level in ...

  4. Treatment of HCV Infection by Targeting MicroRNA

    NARCIS (Netherlands)

    Janssen, Harry L. A.; Reesink, Hendrik W.; Lawitz, Eric J.; Zeuzem, Stefan; Rodriguez-Torres, Maribel; Patel, Keyur; van der Meer, Adriaan J.; Patick, Amy K.; Chen, Alice; Zhou, Yi; Persson, Robert; King, Barney D.; Kauppinen, Sakari; Levin, Arthur A.; Hodges, Michael R.

    2013-01-01

    BACKGROUND The stability and propagation of hepatitis C virus (HCV) is dependent on a functional interaction between the HCV genome and liver-expressed microRNA-122 (miR-122). Miravirsen is a locked nucleic acid-modified DNA phosphorothioate antisense oligonucleotide that sequesters mature miR-122

  5. IP-10 predicts the first phase decline of HCV RNA and overall viral response to therapy in patients co-infected with chronic hepatitis C virus infection and HIV

    DEFF Research Database (Denmark)

    Falconer, Karolin; Askarieh, Galia; Weis, Nina Margrethe

    2010-01-01

    The aim of this study was to investigate the utility of baseline plasma interferon-gamma inducible protein-10 (IP-10) levels in human immunodeficiency virus (HIV)-hepatitis C virus (HCV) co-infected patients. Baseline IP-10 was monitored during HCV combination therapy in 21 HIV-HCV co-infected pa......The aim of this study was to investigate the utility of baseline plasma interferon-gamma inducible protein-10 (IP-10) levels in human immunodeficiency virus (HIV)-hepatitis C virus (HCV) co-infected patients. Baseline IP-10 was monitored during HCV combination therapy in 21 HIV-HCV co......-infected patients (HCV genotype 1 (n = 16), 2 (n = 2), and 3 (n = 3)). Lower baseline IP-10 was significantly associated with a rapid decline in HCV RNA, in particular with the first phase reduction, and similar cut-off levels ( 600 pg/ml) as in HCV mono-infected patients apply. In conclusion, baseline IP......-10 therapy in HIV-HCV co-infected patients, and may thus be useful in encouraging such difficult-to-treat patients to initiate therapy....

  6. Hepatitis C virus (HCV) status in newborns born to HCV positive ...

    African Journals Online (AJOL)

    Group one: 30 women HCV antibody (Ab) positive/HCV RNA negative. Group two: 30 women HCV Ab positive/ HCV RNA positive. Newborn sera were subjected to HCV antibody testing, and detection of HCV viral RNA by PCR. Results: None of the newborns born to PCR negative females undergoing ICSI cycles showed ...

  7. Ongoing risk behavior and the presence of HCV-RNA affect the hepatitis C virus (HCV)-Specific CD4(+) T cell response

    NARCIS (Netherlands)

    van den Berg, Charlotte H S B; Nanlohy, Nening M; van de Laar, Thijs J W; Prins, Maria; van Baarle, Debbie

    The largest population of people at risk for HCV-infection is injecting drug users (DU). We hypothesize that recurrent exposure to HCV, by continuing risk behavior, influences the development of an HCV-specific T-cell response. Therefore, we studied the association between repeated exposure to and

  8. HCV RNA traffic and association with NS5A in living cells

    Energy Technology Data Exchange (ETDEWEB)

    Fiches, Guillaume N.; Eyre, Nicholas S.; Aloia, Amanda L.; Van Der Hoek, Kylie [Department of Molecular and Cellular Biology, Research Centre for Infectious Diseases, University of Adelaide, Adelaide and Centre for Cancer Biology, SA Pathology, Adelaide, SA (Australia); Betz-Stablein, Brigit; Luciani, Fabio [Systems Immunology, School of Medical Sciences, University of New South Wales, Sydney, NSW (Australia); Chopra, Abha [Institute for Immunology and infectious diseases (IIID), Murdoch University, Perth, WA (Australia); Beard, Michael R., E-mail: michael.beard@adelaide.edu.au [Department of Molecular and Cellular Biology, Research Centre for Infectious Diseases, University of Adelaide, Adelaide and Centre for Cancer Biology, SA Pathology, Adelaide, SA (Australia)

    2016-06-15

    The spatiotemporal dynamics of Hepatitis C Virus (HCV) RNA localisation are poorly understood. To address this we engineered HCV genomes harbouring MS2 bacteriophage RNA stem-loops within the 3′-untranslated region to allow tracking of HCV RNA via specific interaction with a MS2-Coat-mCherry fusion protein. Despite the impact of these insertions on viral fitness, live imaging revealed that replication of tagged-HCV genomes induced specific redistribution of the mCherry-tagged-MS2-Coat protein to motile and static foci. Further analysis showed that HCV RNA was associated with NS5A in both static and motile structures while a subset of motile NS5A structures was devoid of HCV RNA. Further investigation of viral RNA traffic with respect to lipid droplets (LDs) revealed HCV RNA-positive structures in close association with LDs. These studies provide new insights into the dynamics of HCV RNA traffic with NS5A and LDs and provide a platform for future investigations of HCV replication and assembly. - Highlights: • HCV can tolerate can bacteriophage MS2 stem-loop insertions within the 3′ UTR. • MS2 stem-loop containing HCV genomes allow for real-time imaging of HCV RNA. • HCV RNA is both static and motile and associates with NS5A and lipid droplets.

  9. HCV core-antigen assay as an alternative to HCV RNA quantification: A correlation study for the assessment of HCV viremia.

    Science.gov (United States)

    Alonso, Roberto; Pérez-García, Felipe; López-Roa, Paula; Alcalá, Luis; Rodeño, Pilar; Bouza, Emilio

    2017-02-25

    Detection of hepatitis C virus (HCV) RNA and the HCV core antigen assay (HCV-Ag) are reliable techniques for the diagnosis of active and chronic HCV infection. Our aim was to evaluate the HCV-Ag assay as an alternative to quantification of HVC RNA. A comparison was made of the sensitivity and specificity of an HCV-Ag assay (204 serum samples) with those of a PCR assay, and the correlation between the two techniques was determined. The sensitivity and specificity of HCV-Ag was 76.6% and 100%, respectively. Both assays were extremely well correlated (Pearson coefficient=0.951). The formula (LogCV=1.15*LogAg+2.26) was obtained to calculate the viral load by PCR from HCV-Ag values. HCV-Ag was unable to detect viral loads below 5000IU/mL. Although the HCV-Ag assay was less sensitive than the PCR assay, the correlation between both assays was excellent. HCV-Ag can be useful as a first step in the diagnosis of acute or chronic HCV infection and in emergency situations. Copyright © 2016 Elsevier España, S.L.U. and Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.

  10. The history of hepatitis C virus (HCV)

    DEFF Research Database (Denmark)

    Bukh, Jens

    2016-01-01

    The discovery of hepatitis C virus (HCV) in 1989 permitted basic research to unravel critical components of a complex life cycle for this important human pathogen. HCV is a highly divergent group of viruses classified in 7 major genotypes and a great number of subtypes, and circulating in infected...

  11. Detection limit of architect hepatitis C core antigen assay in correlation with HCV RNA, and renewed confirmation algorithm for reactive anti-HCV samples.

    Science.gov (United States)

    Ottiger, Cornelia; Gygli, Nicole; Huber, Andreas R

    2013-11-01

    hepatitis C infections are detected by anti-HCV screening tests. Reactive anti-HCV results give no information about the presence or absence of hepatitis C viruses, or of unspecific reactivity. To obtain information about the viral load, HCV RNA measurements, following a reactive anti-HCV result, are performed in well equipped and specialised laboratories. Anti-HCV immunoblots are the only means to exclude non specific reactivity. The measurement of HCV core antigen (HCV-Ag), as an alternative to HCV RNA, is discussed, as it can be analysed on the same instrument as anti-HCV. The detection limit of HCV-Ag is crucial to use it in lieu of HCV RNA, in regard of the different genotypes. A renewed algorithm is proposed to exclude unspecific reactivity of anti-HCV. Samples were tested on Architect i2000SR (Abbott) for anti-HCV and HCV-Ag. HCV RNA measurements were obtained by Cobas Ampliprep/Taqman (Roche) or m2000rt(®) (Abbott). Comparison between HCV-Ag and HCV RNA from 126 samples of 101 patients with chronic hepatitis C gave linear regression R(2) 0.89, slope 0.885 and intercept -2.258, which were independent of the genotypes. The detection limit of HCV-Ag was between 2.4 and 4.5 Log(10)IU/mL. A renewed algorithm for confirmation of reactive anti-HCV results is proposed: active or resolved hepatitis C infections or false reactivity can be differentiated by sequenced reflex testing due to HCV-Ag, anti-HCV immunoblot and HCV RNA. Copyright © 2013 Elsevier B.V. All rights reserved.

  12. Spontaneous viral clearance, viral load, and genotype distribution of hepatitis C virus (HCV) in HIV-infected patients with anti-HCV antibodies in Europe

    DEFF Research Database (Denmark)

    Soriano, Vincent; Mocroft, Amanda; Rockstroh, Juergen

    2008-01-01

    BACKGROUND: Variables influencing serum hepatitis C virus (HCV) RNA levels and genotype distribution in individuals with human immunodeficiency virus (HIV) infection are not well known, nor are factors determining spontaneous clearance after exposure to HCV in this population. METHODS: All HCV...

  13. MicroRNA-122 antagonism against hepatitis C virus genotypes 1-6 and reduced efficacy by host RNA insertion or mutations in the HCV 5' UTR

    DEFF Research Database (Denmark)

    Li, Yiping; Gottwein, Judith; Scheel, Troels Kasper Høyer

    2011-01-01

    , 1b, 2a, 2b, 3a, 4a, 5a, and 6a with efficient growth in Huh7.5 cells. Deletion mutagenesis studies demonstrated that the 5' UTR SLI was essential for genotypes 1-6 infection. However, lack of SLI could be compensated for by insertion of other structured HCV or host RNA sequences, including U3 small......; this was attributed to the lack of an intact S1 by reverse genetics studies. Therefore, we engineered the corresponding U3 RNA sequences into S1 and demonstrated that HCV recombinants with wild-type SLI and single or combined mutations at four of eight nucleotides of S1 were viable in Huh7.5 cells. These mutations...

  14. Peripheral blood mononuclear cells of HIV- and HCV-antibody-positive individuals contain HCV RNA but No HCV DNA despite evidence for reverse transcription of HIV RNA into DNA

    NARCIS (Netherlands)

    Penning, M.; Beld, M.; Goudsmit, J.

    2000-01-01

    Following reports of the finding of cDNA of RNA viruses in cells containing an endogenous retrovirus-encoded reverse transcriptase, we looked for the presence of hepatitis C virus (HCV) DNA in peripheral blood mononuclear cells (PBMC) of injecting drug users seropositive for both HCV and human

  15. Prevalence of mixed hepatitis C virus (HCV genotypes among recently diagnosed dialysis patients with HCV infection

    Directory of Open Access Journals (Sweden)

    Mohammed A Al Balwi

    2011-01-01

    Full Text Available Hepatitis C virus (HCV infection is considered a major health problem recognized globally. HCV is a major cause of chronic liver disease that may lead to cirrhosis and hepatocellular carcinoma. The aim of this study was to investigate the prevalence of multiple (mixed HCV genotypes in Saudi patients recently diagnosed with HCV infection and their association with various clinical risk factors. We examined a total of 1,292 newly diagnosed HCV-positive cases between January 2006 and July 2009 at the Molecular Pathology Laboratory, King Abdulaziz Medical City, Riyadh. The clinical and laboratory data of the study patients were collected. The HCV-RNA viral load and its genotyping were carried out with RT-PCR technology to assist in the follow-up and management of HCV-infected patients undergoing antiviral therapy. Twenty-two patients (1.7% were found to have mixed HCV genotypes; of them, mixed genotypes associated with genotype-4 were seen in 19 patients (86%, mixed genotypes associated with genotype-1 were found in 68.4%, with genotype-3 in 26.3% and with genotype-2 in 5.3%. Additionally, mixed genotypes associated with genotype-1 were seen in three cases (13.6%; they were associated with genotype-2 in two (66.7% and with genotype-5 in one patient (33.3%. In conclusion, the prevalence rate of mixed HCV genotypes in the cohort of the newly infected Saudi patients was 1.7%, with genotype-4 being the most frequent genotype encountered.

  16. Differences between quantification of genotype 3 hepatitis C virus RNA by Versions 1.0 and 2.0 of the COBAS AmpliPrep/COBAS TaqMan HCV Test.

    Science.gov (United States)

    Pierce, Virginia M; Eversley, Jacqueline S; Tran, Thuy K; Rosenberg, Eric S

    2017-06-27

    Differences between the designs of hepatitis C virus (HCV) viral load assays can result in genotype-related variability in RNA quantification. We tested paired aliquots of plasma specimens from HCV-infected individuals using two versions (v1.0 and v2.0) of the Roche COBAS AmpliPrep/COBAS TaqMan HCV Test (CAP/CTM HCV) and noted variability between results for a subset of specimens; we then sought to determine whether discrepant results were more prevalent among specific HCV genotypes. Archived and prospectively-collected plasma samples from 114 unique patients were tested using CAP/CTM HCV v1.0 and v2.0. The HCV genotype result for each patient was determined by retrospectively reviewing laboratory records. All (46/46) specimens with quantifiable viral loads from patients with genotype 1 or 2 infection had CAP/CTM HCV v1.0 and v2.0 results that were within 0.5 log10 IU/mL; in contrast, only 3/11 (27.3%) from patients with HCV genotype 3 (mean difference, 0.56 log10 IU/mL higher with v2.0) and 0/3 (0%) from patients with HCV genotype 4 (mean difference, 0.91 log10 IU/mL higher with v2.0) had results within 0.5 log10 IU/mL. Among specimens with detectable HCV RNA below the lower limit of quantification with v1.0, greater proportions of genotype 3 (4/7, 57.1%) and genotype 4 (3/4, 75.0%) specimens than genotype 1 or 2 specimens (6/30, 20.0%) had v2.0 results within the quantifiable range. In patients infected with HCV genotype 3, sequential CAP/CTM HCV viral load results should be compared with caution and interpreted in the context of the specific assay version used.

  17. Molecular Epidemiology of Hepatitis C Virus (HCV) in Kadun State ...

    African Journals Online (AJOL)

    Objective: To determine the distribution of hepatitis C virus (HCV) genotypes and subtypes among blood donors and outpatients attendees positive for antibody to HCV (anti-HCV). Justification: Hepatitis C virus (HCV) continues to be a major disease burden on the world and Man is the only known natural host of Hepatitis C ...

  18. hcv

    African Journals Online (AJOL)

    boaz

    ABSTRACT. Background: Hepatitis C virus (HCV) infection is a major public health concern. The aim of this study was to ascertain the seroprevalence and risk factors of HCV antibodies among pregnant women in Anyigba, Kogi State North Central Nigeria. Materials and methods:Blood samples (5mls) were collected from ...

  19. Anti-HCV RNA Aptamers Targeting the Genomic cis-Acting Replication Element

    Directory of Open Access Journals (Sweden)

    Alfredo Berzal-Herranz

    2011-12-01

    Full Text Available Hepatitis C virus (HCV replication is dependent on the existence of several highly conserved functional genomic RNA domains. The cis-acting replication element (CRE, located within the 3' end of the NS5B coding region of the HCV genome, has been shown essential for efficient viral replication. Its sequence and structural features determine its involvement in functional interactions with viral RNA-dependent RNA polymerase and distant RNA domains of the viral genome. This work reports the use of an in vitro selection strategy to select aptamer RNA molecules against the complete HCV-CRE. After six selection cycles, five potential target sites were identified within this domain. Inhibition assays using a sample of representative aptamers showed that the selected RNAs significantly inhibit the replication (>80% of a subgenomic HCV replicon in Huh-7 cell cultures. These results highlight the potential of aptamer RNA molecules as therapeutic antiviral agents.

  20. Correlation between alanine aminotransferase level, HCV-RNA titer ...

    African Journals Online (AJOL)

    Quantitative HCV-RNA level measurement, HCV genotyping, and abdominal ultrasonography were investigated in all patients. Liver biopsy was done for 80 patients and the remaining 58 patients were examined using Fibroscan. Highly significant higher percentage of cases with high level of HCV viremia was found among ...

  1. Mechanism of cell infection with hepatitis C virus (HCV)--a new paradigm in virus-cell interaction.

    Science.gov (United States)

    Budkowska, Agata

    2009-01-01

    Hepatitis C virus (HCV) is an enveloped, single-stranded RNA virus, belonging to the Flaviviridae family. HCV infection is a major cause of chronic hepatitis worldwide, leading to steatosis, liver cirrosis and hepatocellular carcinoma. Significant advances in understanding the mechanisms of HCV infection have been made since the development of a cell culture system reproducing the complete HCV cell cycle in vitro. HCV represents a new paradigm in interactions between the virus and its target cell, the human hepatocyte, due to the central role of lipoproteins in the HCV life cycle. Very low density lipoproteins are required for virus particle assembly and secretion. Upon the release, the infectious virus circulates in the blood as triglyceride-rich particles and infects cells using lipoprotein-receptor dependent mechanisms. HCV cell entry is a multi-step process: heparan sulphate and/or low-density lipoprotein receptor are cell surface factors mediating an initial virus attachment; subsequent virus interaction with tetraspanin CD81 and the human scavenger receptor SR-BI, the main HCV receptors, triggers virus movement to the tight junctions and its uptake via Claudin-1 and occludin. Another originality of HCV is that initiation of productive infection requires dynamic microtubules. Whereas other viruses use kinesin or dynein-dependent transport, HCV exploits mechanisms driven by microtubule polymerization to efficiently infect its target cell, in which virus nucleocapsid protein might play a particular role. An improved of understanding of the cellular events involved in HCV cell entry and transport, leading to the initiation of productive HCV infection, may reveal novel targets for anti-viral interventions.

  2. IP-10 predicts the first phase decline of HCV RNA and overall viral response to therapy in patients co-infected with chronic hepatitis C virus infection and HIV

    DEFF Research Database (Denmark)

    Falconer, Karolin; Askarieh, Galia; Weis, Nina Margrethe

    2010-01-01

    The aim of this study was to investigate the utility of baseline plasma interferon-gamma inducible protein-10 (IP-10) levels in human immunodeficiency virus (HIV)-hepatitis C virus (HCV) co-infected patients. Baseline IP-10 was monitored during HCV combination therapy in 21 HIV-HCV co......-10 HIV-HCV co-infected patients, and may thus be useful in encouraging such difficult-to-treat patients to initiate therapy....

  3. Genotyping of HCV RNA Reveals That 3a Is the Most Prevalent Genotype in Mardan, Pakistan

    Directory of Open Access Journals (Sweden)

    Sajid Ali

    2014-01-01

    Full Text Available The clinical outcomes of patients infected with hepatitis C virus (HCV range from acute resolving hepatitis to chronic liver diseases such as liver cirrhosis or hepatocellular carcinoma. Identification of the infecting virus genotype is indispensable for the exploration of many aspects of HCV infection, including epidemiology, pathogenesis, and response to antiviral therapy. 1419 individuals were screened for anti-HCV in this study, of which 166 (11.7% were found reactive by ICT (Immunochromatographic test. These 166 anti-HCV positive and 26 normal individuals were further analyzed. RNA was extracted from serum and reverse-transcribed to cDNA and the core region of HCV genome was targeted and amplified by multiplex PCR. HCV RNA was detected in 121 individuals, of which 87 were male and 34 were female. Genotype 3a was the most prevalent among all the genotypes observed followed by 3b. Genotypes 1a, 2a, and 2b were found in 10.89%, 13.22%, and 6.61% patients, respectively. 25.41% of the HCV RNA positive samples were not typed. 6.05% of patients were found having mixed genotypes. These findings will not only help the physicians to prescribe more appropriate treatment for the HCV infection but will also draw the attention of health-related policy makers to devise strategies to curb the disease more effectively.

  4. Comparison of HCV core antigen and anti-HCV with HCV RNA results

    African Journals Online (AJOL)

    Background: The measurement of anti-HCV antibodies using immunological methods and the confirmation of viral nuclear acid based on molecular methods is important in diagnosis and follow-up of the HCV infection. Objectives: In this study, we aimed to analyse HCV core Antigen positivity among anti-HCV antibody ...

  5. Detection of HCV-RNA in saliva of patients with chronic hepatitis C.

    OpenAIRE

    Couzigou, P; Richard, L; Dumas, F; Schouler, L; Fleury, H

    1993-01-01

    Previous studies have provided conflicting results on the presence of hepatitis C virus-RNA in saliva. In this study, 23 (62%) of 37 patients tested positive for hepatitis C virus-RNA in saliva, using polymerase chain reaction analysis. A slightly greater proportion had a sporadic rather than a parenteral origin of chronic hepatitis C. These results provide a biological basis for saliva as a possible source of hepatitis C virus (HCV) infection, but do not necessarily imply transmission by thi...

  6. Hepatitis C virus (HCV) interaction with astrocytes: nonproductive infection and induction of IL-18.

    Science.gov (United States)

    Liu, Ziqing; Zhao, Fang; He, Johnny J

    2014-06-01

    Hepatitis C virus (HCV) infection causes the central nervous system (CNS) abnormalities in more than 50 % of chronically infected subjects. However, the underlying mechanisms are largely unknown. In this study, we characterized the HCV interactions with astrocytes, one of the putative HCV target cells in the brain. We demonstrated that primary human astrocytes (PHA) were very inefficiently infected by HCV, either in the cell-free form or through cell-cell contact. We then determined the potential restriction steps of HCV infection and replication in these cells. PHA expressed all known HCV receptors but failed to support HCV entry. HCV IRES-mediated RNA translation was functional in PHA and further enhanced by miR122 expression. Nevertheless, PHA did not support HCV replication regardless of miR122 expression. To our great surprise, we found that HCV exposure induced robust IL-18 expression in PHA and exhibited direct neurotoxicity. Taken together, these results showed that astrocytes did not support productive HCV infection and replication, but HCV interactions with astrocytes and neurons alone might be sufficient to cause CNS dysfunction.

  7. Evaluation of a total hepatitis C virus (HCV) core antigen assay for the detection of antigenaemia in anti-HCV positive individuals.

    Science.gov (United States)

    Valcavi, Pierpaolo; Medici, Maria Cristina; Casula, Francesca; Arcangeletti, Maria Cristina; De Conto, Flora; Pinardi, Federica; Calderaro, Adriana; Chezzi, Carlo; Dettori, Giuseppe

    2004-07-01

    A new, sensitive enzyme immunoassay has been developed for detecting and quantifying total hepatitis C virus (HCV) core antigen in anti-HCV positive or negative sera ("trak-C", Ortho Clinical Diagnostics, Raritan, NJ). The purpose of this study was to evaluate the performance of trak-C as an additional laboratory diagnostic marker of viraemia. The performance was compared to HCV-RNA detection in the "screening" of sera from a large heterogeneous population of hospitalised patients and outpatients. Six hundred and eighteen anti-HCV negative sera, 405 anti-HCV positive/HCV-RNA negative sera, 604 anti-HCV positive/HCV-RNA positive sera and 67 anti-HCV negative sera containing antigens or antibodies potentially interfering with the performance of the assay were analysed. Supplemental HCV antibody testing was performed using a commercial strip immunoblot assay. HCV-RNA was investigated using a qualitative commercial assay. A quantitative commercial RT-PCR was used for the analysis of selected samples. Sensitivity and specificity values were 94.7 and 100%, respectively. The latter was also confirmed when anti-HCV negative samples containing potentially interfering antigens/antibodies were examined. Sensitivity below 100% was probably due to an antigenaemia below the detection limit of trak-C. Besides, because 65.6% of HCV-RNA positive/trak-C negative samples presented specific antibodies against all four RIBA antigens, the hypothesis was raised that, in some cases, the dissociation step efficiency could be sub-optimal. In conclusion, trak-C seems suitable for identifying HCV infection on large based populations. It is a rapid to perform, reliable and specific assay that can be adapted to any laboratory setting. Copyright 2004 Wiley-Liss, Inc.

  8. Detection of HCV RNA in saliva does not correlate with salivary flow or xerostomia in patients with chronic hepatitis C.

    Science.gov (United States)

    de Mattos Camargo Grossmann, Soraya; Teixeira, Rosângela; de Oliveira, Guilherme Corrêa; do Carmo, Maria Auxiliadora Vieira

    2010-06-01

    The objective of this study was to investigate the prevalence of hepatitis C virus (HCV) RNA in saliva and its possible association with xerostomia and hyposalivation in patients with chronic hepatitis C. One hundred and thirty-six patients with confirmed diagnosis of chronic hepatitis C were prospectively analyzed before HCV treatment. The prevalence of xerostomia and hyposalivation was clinically evaluated. HCV RNA was investigated in saliva samples by qualitative PCR test. Univariate and multivariate analyses were used to verify associations. Xerostomia was reported by 48 (35.3%) patients, whereas hyposalivation was observed in 26 (19.1%). HCV RNA was positive in the saliva of 53 (39.0%) patients. An association among HCV RNA-positive saliva with xerostomia or hyposalivation was not observed. Our results demonstrate that the detection of HCV in saliva does not correlate with salivary flow or xerostomia in patients with chronic hepatitis C. Copyright 2010 Mosby, Inc. All rights reserved.

  9. Liver histology in co-infection of hepatitis C virus (HCV and Hepatitis G virus (HGV

    Directory of Open Access Journals (Sweden)

    STRAUSS Edna

    2002-01-01

    Full Text Available As little is known about liver histology in the co-infection of hepatitis C virus (HCV and hepatitis G virus (HGV, HGV RNA was investigated in 46 blood donors with hepatitis C, 22 of them with liver biopsy: co-infection HCV / HGV (n = 6 and HCV isolated infection (n = 16. Besides staging and grading of inflammation at portal, peri-portal and lobular areas (Brazilian Consensus, the fibrosis progression index was also calculated. All patients had no symptoms or signs of liver disease and prevalence of HGV / HCV co-infection was 15.2%. Most patients had mild liver disease and fibrosis progression index, calculated only in patients with known duration of infection, was 0.110 for co-infection and 0.130 for isolated HCV infection, characterizing these patients as "slow fibrosers". No statistical differences could be found between the groups, although a lesser degree of inflammation was always present in co-infection. In conclusion co-infection HCV / HGV does not induce a more aggressive liver disease, supporting the hypothesis that HGV is not pathogenic.

  10. Changes in HIV RNA and CD4 cell count after acute HCV infection in chronically HIV-infected individuals

    NARCIS (Netherlands)

    Gras, Luuk; de Wolf, Frank; Smit, Colette; Prins, Maria; van der Meer, Jan T. M.; Vanhommerig, Joost W.; Zwinderman, Aeilko H.; Schinkel, Janke; Geskus, Ronald B.; Kuijpers, T. W.; Scherpbier, H. J.; Godfried, M. H.; Reiss, P.; van der Poll, T.; Nellen, F. J. B.; Lange, J. M. A.; Geerlings, S. E.; van Vugt, M.; Pajkrt, D.; Bos, J. C.; van der Valk, M.; Wiersinga, W. J.; Goorhuis, A.; Hovius, J. W. R.; Lowe, S.; Oude Lashof, A.; Posthouwer, D.; Pronk, M. J. H.; Ammerlaan, H. S. M.; van der Ende, M. E.; de Vries-Sluijs, T. E. M. S.; Schurink, C. A. M.; Nouwen, J. L.; Verbon, A.; Rijnders, B. J. A.; van Gorp, E. C. M.; van der Feltz, M.; Driessen, G. J. A.; van Rossum, A. M. C.; Branger, J.; Schippers, E. F.; van Nieuwkoop, C.; van Elzakker, E. P.; Groeneveld, H. P.; Bouwhuis, J. W.; Soetekouw, R.; ten Kate, R. W.; Kroon, F. P.; van Dissel, J. T.; Arend, S. M.; de Boer, M. G. J.; Jolink, H.; Vollaard, A. M.; Bauer, M. P.; den Hollander, J. G.; Pogany, K.; van Twillert, G.; Kortmann, W.; Cohen Stuart, J. W. T.; Diederen, B. M. W.; Leyten, E. M. S.; Gelinck, L. B. S.; Kootstra, G. J.; Delsing, C. E.; Brinkman, K.; Blok, W. L.; Frissen, P. H. J.; Schouten, W. E. M.; van den Berk, G. E. L.; van Kasteren, M. E. E.; Brouwer, A. E.; Veenstra, J.; Lettinga, K. D.; Mulder, J. W.; Vrouenraets, S. M. E.; Lauw, F. N.; van Eeden, A.; Verhagen, D. W. M.; Sprenger, H. G.; Scholvinck, E. H.; van Assen, S.; Bierman, W. F. W.; Wilting, K. R.; Stienstra, Y.; Koopmans, P. P.; Keuter, M.; van der Ven, A. J. A. M.; ter Hofstede, H. J. M.; Dofferhoff, A. S. M.; Warris, A.; van Crevel, R.; Hoepelman, A. I. M.; Mudrikova, T.; Schneider, M. M. E.; Ellerbroek, P. M.; Oosterheert, J. J.; Arends, J. E.; Wassenberg, W. W. M.; Barth, R. E.; van Agtmael, M. A.; Perenboom, R. M.; Claessen, F. A. P.; Bomers, M.; Peters, E. J. G.; Geelen, S. P. M.; Wolfs, T. F. W.; Bont, L. J.; Richter, C.; van der Berg, J. P.; Gisolf, E. H.; van den Berge, M.; Stegeman, A.; van Vonderen, M. G. A.; van Houte, D. P. F.; Weijer, S.; el Moussaoui, R.; Winkel, C.; Muskiet, F.; Durand, N. N.; Voigt, R.

    2015-01-01

    Little is known about the impact of acute hepatitis C virus (HCV) co-infection on HIV-1 disease progression. We investigated CD4 cell count and HIV RNA concentration changes after HCV infection in individuals chronically infected with HIV-1. We selected individuals that had the last negative and

  11. Efficient infectious cell culture systems of the hepatitis C virus (HCV) prototype strains HCV-1 and H77

    DEFF Research Database (Denmark)

    Li, Yi-Ping; Ramirez, Santseharay; Mikkelsen, Lotte

    2015-01-01

    UNLABELLED: 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...... 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...

  12. Immune Responses to HCV and Other Hepatitis Viruses

    Science.gov (United States)

    Park, Su-Hyung; Rehermann, Barbara

    2014-01-01

    Summary Five human hepatitis viruses cause most acute and chronic liver disease worldwide. Over the past 25 years hepatitis C virus (HCV) in particular has received much interest because of its ability to persist in most immunocompetent adults and the lack of a protective vaccine. Here we examine innate and adaptive immune responses to HCV infection. Although HCV activates an innate immune response, it employs an elaborate set of mechanisms to evade interferon (IFN)-based antiviral immunity. By comparing innate and adaptive immune responses to HCV with those to hepatitis A and B viruses, we suggest that prolonged innate immune activation impairs the development of successful adaptive immune responses. Comparative immunology furthermore provides insights into the maintenance of immune protection. We conclude by discussing prospects for an HCV vaccine and future research needs for the hepatitis viruses. PMID:24439265

  13. GB virus C (GBV-C) infection in hepatitis C virus (HCV)/HIV-coinfected patients receiving HCV treatment: importance of the GBV-C genotype.

    Science.gov (United States)

    Schwarze-Zander, Carolynne; Blackard, Jason T; Zheng, Hui; Addo, Marylyn M; Lin, Wenyu; Robbins, Gregory K; Sherman, Kenneth E; Zdunek, Dietmar; Hess, Georg; Chung, Raymond T

    2006-08-15

    Persistent GB virus C (GBV-C) coinfection leads to slower human immunodeficiency virus (HIV) progression. Despite the existence of multiple GBV-C genotypes, their relevance to the progression of HIV disease is unknown. We therefore investigated (1) the prevalence and genotype of GBV-C in hepatitis C virus (HCV)/HIV-coinfected patients and (2) the impact of HCV treatment on GBV-C RNA clearance. We retrospectively studied 130 HCV/HIV-coinfected patients initiating HCV therapy. Anti-E2 enzyme-linked immunosorbent assay, reverse-transcription polymerase chain reaction (PCR), and real-time PCR were used to detect and quantify GBV-C infection. GBV-C genotype was determined by sequencing the 5' untranslated region. GBV-C infection (past or current) was identified in 111 (85%) of the patients. Ongoing GBV-C replication was detected in 40 patients. Coinfection with GBV-C genotype 2 was associated with significantly higher CD4(+) cell counts. After 24 weeks of HCV therapy, GBV-C RNA clearance was observed in 50% of patients, although this was not associated with changes in HIV load or with CD4(+) cell counts. Sustained GBV-C RNA clearance was observed in 31% of patients with GBV-C RNA detected at baseline. GBV-C coinfection was extremely common. GBV-C RNA clearance with HCV therapy was associated with neither short-term loss of HIV control nor impaired immune status. The association of GBV-C genotype 2 with higher CD4(+) cell counts merits further study.

  14. Seroprevalence of Hepatitis C Virus (HCV) antibodies in pregnant ...

    African Journals Online (AJOL)

    Background: Hepatitis C virus (HCV) infection is a major public health concern. The aim of this study was to ascertain the seroprevalence and risk factors of HCV antibodies among pregnant women in Anyigba, Kogi State North Central Nigeria. Materials and methods:Blood samples (5mls) were collected from one hundred ...

  15. Historical epidemiology of hepatitis C virus (HCV) in selected countries

    DEFF Research Database (Denmark)

    Bruggmann, P; Berg, T; Øvrehus, A L H

    2014-01-01

    Chronic infection with hepatitis C virus (HCV) is a leading indicator for liver disease. New treatment options are becoming available, and there is a need to characterize the epidemiology and disease burden of HCV. Data for prevalence, viremia, genotype, diagnosis and treatment were obtained thro...

  16. Analysis of in vitro replicated human hepatitis C virus (HCV for the determination of genotypes and quasispecies

    Directory of Open Access Journals (Sweden)

    Chelyapov Nickolas

    2006-09-01

    Full Text Available Abstract Isolation and self-replication of infectious HCV has been a difficult task. However, this is needed for the purposes of developing rational drugs and for the analysis of the natural virus. Our recent report of an in vitro system for the isolation of human HCV from infected patients and their replication in tissue culture addresses this challenge. At California Institute of Molecular Medicine several isolates of HCV, called CIMM-HCV, were grown for over three years in cell culture. This is a report of the analysis of CIMM-HCV isolates for subtypes and quasispecies using a 269 bp segment of the 5'UTR. HCV RNA from three patients and eleven CIMM-HCV were analyzed for this purpose. All isolates were essentially identical. Isolates of HCV from one patient were serially transmitted into fresh cells up to eight times and the progeny viruses from each transmission were compared to each other and also to the primary isolates from the patient's serum. Some isolates were also transmitted to different cell types, while others were cultured continuously without retransmission for over three years. We noted minor sequence changes when HCV was cultured for extended periods of time. HCV in T-cells and non-committed lymphoid cells showed a few differences when compared to isolates obtained from immortalized B-cells. These viruses maintained close similarity despite repeated transmissions and passage of time. There were no subtypes or quasispecies noted in CIMM-HCV.

  17. Hepatitis C virus (HCV): ever in reliable partnerships?

    African Journals Online (AJOL)

    GRACE

    2006-06-16

    Jun 16, 2006 ... hemophiliacs, multiple changes in HCV genotypes were observed in 58 % of the subjects, over a 3–15- ..... increase in HCV RNA levels in hemophiliacs (Eyster et. Amoador-Canizares and Duenas-Carrera .... and colleagues results showed that, among women who continued to receive protease inhibitors ...

  18. HCV-RNA quantification in liver bioptic samples and extrahepatic compartments, using the abbott RealTime HCV assay.

    Science.gov (United States)

    Antonucci, FrancescoPaolo; Cento, Valeria; Sorbo, Maria Chiara; Manuelli, Matteo Ciancio; Lenci, Ilaria; Sforza, Daniele; Di Carlo, Domenico; Milana, Martina; Manzia, Tommaso Maria; Angelico, Mario; Tisone, Giuseppe; Perno, Carlo Federico; Ceccherini-Silberstein, Francesca

    2017-08-01

    We evaluated the performance of a rapid method to quantify HCV-RNA in the hepatic and extrahepatic compartments, by using for the first time the Abbott RealTime HCV-assay. Non-tumoral (NT), tumoral (TT) liver samples, lymph nodes and ascitic fluid from patients undergoing orthotopic-liver-transplantation (N=18) or liver resection (N=4) were used for the HCV-RNA quantification; 5/22 patients were tested after or during direct acting antivirals (DAA) treatment. Total RNA and DNA quantification from tissue-biopsies allowed normalization of HCV-RNA concentrations in IU/μg of total RNA and IU/10 6 liver-cells, respectively. HCV-RNA was successfully quantified with high reliability in liver biopsies, lymph nodes and ascitic fluid samples. Among the 17 untreated patients, a positive and significant HCV-RNA correlation between serum and NT liver-samples was observed (Pearson: rho=0.544, p=0.024). Three DAA-treated patients were HCV-RNA "undetectable" in serum, but still "detectable" in all tested liver-tissues. Differently, only one DAA-treated patient, tested after sustained-virological-response, showed HCV-RNA "undetectability" in liver-tissue. HCV-RNA was successfully quantified with high reliability in liver bioptic samples and extrahepatic compartments, even when HCV-RNA was "undetectable" in serum. Abbott RealTime HCV-assay is a good diagnostic tool for HCV quantification in intra- and extra-hepatic compartments, whenever a bioptic sample is available. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Prevalence of Hepatitis C Virus (HCV) and Human ...

    African Journals Online (AJOL)

    Prevalence of Hepatitis C Virus (HCV) and Human Immunodeficiency Virus (HIV) Co-Infection Among Pregnant Women Attending Antenatal Clinics in Abuja, Nigeria. ... with this virus complicates issues related to diagnosis, clinical disease progression, monitoring disease activity, treatment options and basic immunology.

  20. Several Human Liver Cell Expressed Apolipoproteins Complement HCV Virus Production with Varying Efficacy Conferring Differential Specific Infectivity to Released Viruses.

    Science.gov (United States)

    Hueging, Kathrin; Weller, Romy; Doepke, Mandy; Vieyres, Gabrielle; Todt, Daniel; Wölk, Benno; Vondran, Florian W R; Geffers, Robert; Lauber, Chris; Kaderali, Lars; Penin, François; Pietschmann, Thomas

    2015-01-01

    Apolipoprotein E (ApoE), an exchangeable apolipoprotein, is necessary for production of infectious Hepatitis C virus (HCV) particles. However, ApoE is not the only liver-expressed apolipoprotein and the role of other apolipoproteins for production of infectious HCV progeny is incompletely defined. Therefore, we quantified mRNA expression of human apolipoproteins in primary human hepatocytes. Subsequently, cDNAs encoding apolipoproteins were expressed in 293T/miR-122 cells to explore if they complement HCV virus production in cells that are non-permissive due to limiting endogenous levels of human apolipoproteins. Primary human hepatocytes expressed high mRNA levels of ApoA1, A2, C1, C3, E, and H. ApoA4, A5, B, D, F, J, L1, L2, L3, L4, L6, M, and O were expressed at intermediate levels, and C2, C4, and L5 were not detected. All members of the ApoA and ApoC family of lipoproteins complemented HCV virus production in HCV transfected 293T/miR-122 cells, albeit with significantly lower efficacy compared with ApoE. In contrast, ApoD expression did not support production of infectious HCV. Specific infectivity of released particles complemented with ApoA family members was significantly lower compared with ApoE. Moreover, the ratio of extracellular to intracellular infectious virus was significantly higher for ApoE compared to ApoA2 and ApoC3. Since apolipoproteins complementing HCV virus production share amphipathic alpha helices as common structural features we altered the two alpha helices of ApoC1. Helix breaking mutations in both ApoC1 helices impaired virus assembly highlighting a critical role of alpha helices in apolipoproteins supporting HCV assembly. In summary, various liver expressed apolipoproteins with amphipathic alpha helices complement HCV virus production in human non liver cells. Differences in the efficiency of virus assembly, the specific infectivity of released particles, and the ratio between extracellular and intracellular infectivity point to

  1. Etiology of hepatitis G virus (HGV) and hepatitis type C virus (HCV ...

    African Journals Online (AJOL)

    Hepatitis G virus (HGV) and hepatitis type C virus (HCV) may implicate malignant lymphoma including non-Hodgkin's lymphoma (NHL) for inducing the proliferative process of lymphocytes. In this study, the molecular and serologic prevalence of HGV and HCV infections was evaluated in patients with NHL and compared ...

  2. 21 CFR 610.47 - Hepatitis C virus (HCV) “lookback” requirements.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 7 2010-04-01 2010-04-01 false Hepatitis C virus (HCV) âlookbackâ requirements... Disease Agents § 610.47 Hepatitis C virus (HCV) “lookback” requirements. (a) If you are an establishment... after a donor tests reactive for evidence of hepatitis C virus (HCV) infection when tested under § 610...

  3. Hepatitis C virus RNA: molecular switches mediated by long-range RNA-RNA interactions?

    Science.gov (United States)

    Shetty, Sumangala; Stefanovic, Snezana; Mihailescu, Mihaela Rita

    2013-02-01

    Multiple conserved structural cis-acting regulatory elements have been recognized both in the coding and untranslated regions (UTRs) of the hepatitis C virus (HCV) genome. For example, the cis-element 5BSL3.2 in the HCV-coding region has been predicted to use both its apical and internal loops to interact with the X RNA in the 3'-UTR, with the IIId domain in the 5'-UTR and with the Alt sequence in the coding region. Additionally, the X RNA region uses a palindromic sequence that overlaps the sequence required for the interaction with 5BSL3.2, to dimerize with another HCV genome. The ability of the 5BSL3.2 and X RNA regions to engage in multi-interactions suggests the existence of one or more molecular RNA switches which may regulate different steps of the HCV life cycle. In this study, we used biophysical methods to characterize the essential interactions of these HCV cis-elements at the molecular level. Our results indicate that X RNA interacts with 5BSL3.2 and another X RNA molecule by adopting two different conformations and that 5BSL3.2 engages simultaneously in kissing interactions using its apical and internal loops. Based on these results, we propose a mode of action for possible molecular switches involving the HCV RNA.

  4. Il controllo di qualità nell’impiego della PCR applicata alla determinazione qualitativa dell’HCV-RNA

    Directory of Open Access Journals (Sweden)

    Giuseppe Giuliani

    2004-03-01

    Full Text Available Detection of hepatitis C virus (HCV RNA in samples of plasma/serum has become an essential part of the diagnosis and management of HCV-infected patients. Qualitative HCV-RNA tests are used to identify acute HCV infections as well as chronic HCV carriers.In recent years,a variety of commercial and non commercial test systems have been developed for this purpose. Each of these methods is calibrate with proprietary standards and exhibits its own sensitivity (detection limit and specificity. Obviously, laboratories performing HCV-RNA test should report accurate and reliable results regardless of the type of assay used.Where commercial kit are used for part of or the complete analytical procedure, documented validation points already covered by the kit manufacturer can substitute for the validation by the user.Nevertheless, the performance of the kit with respect to its intended use has to be demonstrated by the user. One of the best ways to assess the performance of individual laboratories for validation of qualitative HCV-RNA test is determine: 1. Specificity. In order to validate the specificity of the analytical procedure, at least 100 HCV-RNA-negative plasma pools should be tested and shown to be non-reactive. 2. Positive cut-off point (detection limit/sensitivity.The positive cut-off point (as defined in the Ph Eur General Method 2. 6. 21 is the minimum number of the target sequences per volume sample which can be detected in 95% of test runs.A dilution series of a working reagent or reference material, which has been calibrated against the WHO HCV International Standard (96/790, should be tested on different days to examine variation between test runs.At least 3 independent dilution series should be tested with a sufficient number of replicates at each dilution to give a total number of 24 test results for each dilution to enable a statistical analysis of the results; 3. Robustness.To demonstrate robustness, at least 20 HCV-RNA negative plasma

  5. SEN virus co-infection among HCV-RNA-positive mothers, risk of transmission to the offspring and outcome of child infection during a 1-year follow-up.

    Science.gov (United States)

    Moriondo, M; Resti, M; Betti, L; Indolfi, G; Poggi, G M; de Martino, M; Vierucci, A; Azzari, C

    2007-05-01

    SEN is a newly discovered blood-transmissible virus. Among its variants, SENV-D and -H are most often associated with non-A, -E hepatitis. Very little is known about the risk of vertical transmission of the virus. By using polymerase chain reaction with specific primers for SENV-D and -H, we investigated the prevalence of SENV-H and -D infection, the transmission rate of SENV infection and clinical features of SENV-infected children in 89 hepatitis C virus (HCV)-positive human immunodeficiency virus type 1-negative mothers. SENV infection was found in 36 (40%) mothers, and SENV-D was more frequent than SENV-H infection (34/36, 94%vs 5/36, 14%, P < 0.01). No difference in SENV infection rates was found between injection drug user (IDU) mothers (17/51, 33%) and mothers with no risk for bloodborne infection (19/38, 50%, P = ns). SENV-H infection was found only in IDU mothers and mothers with HCV genotype1b. Both SENV-D and -H can be transmitted to the offspring with an overall rate of 47%. Vertical transmission of HCV does not facilitate SENV infection of the offspring. Among 17 SENV-infected children, none was co-infected with HCV. Maternal HCV genotype or viral load does not interfere with mother-to-infant transmission of SENV. Persistence of SENV infection was demonstrated in 100% of infected children after 1-year follow-up, but none had clinical evidence of liver disease.

  6. Strategies to manage hepatitis C virus (HCV) disease burden

    DEFF Research Database (Denmark)

    Wedemeyer, H; Duberg, A S; Buti, M

    2014-01-01

    and (ii) increasing efficacy and treatment rate. This analysis suggests that successful diagnosis and treatment of a small proportion of patients can contribute significantly to the reduction of disease burden in the countries studied. The largest reduction in HCV-related morbidity and mortality occurs......The number of hepatitis C virus (HCV) infections is projected to decline while those with advanced liver disease will increase. A modeling approach was used to forecast two treatment scenarios: (i) the impact of increased treatment efficacy while keeping the number of treated patients constant...

  7. Impaired Hepatitis C Virus (HCV)–Specific Interferon-γ Responses in Individuals With HIV Who Acquire HCV Infection: Correlation With CD4+ T-Cell Counts

    Science.gov (United States)

    Flynn, Jacqueline K.; Dore, Gregory J.; Matthews, Gail; Hellard, Margaret; Yeung, Barbara; Rawlinson, William D.; White, Peter A.; Kaldor, John M.; Lloyd, Andrew R.; Ffrench, Rosemary A.

    2012-01-01

    Studies examining the effect of coinfection with human immunodeficiency virus (HIV) and hepatitis C virus (HCV) on the HCV-specific immune response in acute HCV infection are limited. This study directly compared acute HCV-specific T-cell responses and cytokine profiles between 20 HIV/HCV-coinfected and 20 HCV-monoinfected subjects, enrolled in the Australian Trial in Acute Hepatitis C (ATAHC), using HCV peptide enzyme-linked immunospot (ELISPOT) and multiplex in vitro cytokine production assays. HIV/HCV coinfection had a detrimental effect on the HCV-specific cytokine production in acute HCV infection, particularly on HCV-specific interferon γ (IFN-γ) production (magnitude P = .004; breadth P = .046), which correlated with peripheral CD4+ T-cell counts (ρ = 0.605; P = .005) but not with detectable HIV viremia (ρ = 0.152; P = .534). PMID:22949308

  8. Impaired hepatitis C virus (HCV)-specific interferon-γ responses in individuals with HIV who acquire HCV infection: correlation with CD4(+) T-cell counts.

    Science.gov (United States)

    Flynn, Jacqueline K; Dore, Gregory J; Matthews, Gail; Hellard, Margaret; Yeung, Barbara; Rawlinson, William D; White, Peter A; Kaldor, John M; Lloyd, Andrew R; Ffrench, Rosemary A

    2012-11-15

    Studies examining the effect of coinfection with human immunodeficiency virus (HIV) and hepatitis C virus (HCV) on the HCV-specific immune response in acute HCV infection are limited. This study directly compared acute HCV-specific T-cell responses and cytokine profiles between 20 HIV/HCV-coinfected and 20 HCV-monoinfected subjects, enrolled in the Australian Trial in Acute Hepatitis C (ATAHC), using HCV peptide enzyme-linked immunospot (ELISPOT) and multiplex in vitro cytokine production assays. HIV/HCV coinfection had a detrimental effect on the HCV-specific cytokine production in acute HCV infection, particularly on HCV-specific interferon γ (IFN-γ) production (magnitude P = .004; breadth P = .046), which correlated with peripheral CD4(+) T-cell counts (ρ = 0.605; P = .005) but not with detectable HIV viremia (ρ = 0.152; P = .534).

  9. Lipoprotein lipase inhibits hepatitis C virus (HCV infection by blocking virus cell entry.

    Directory of Open Access Journals (Sweden)

    Patrick Maillard

    Full Text Available A distinctive feature of HCV is that its life cycle depends on lipoprotein metabolism. Viral morphogenesis and secretion follow the very low-density lipoprotein (VLDL biogenesis pathway and, consequently, infectious HCV in the serum is associated with triglyceride-rich lipoproteins (TRL. Lipoprotein lipase (LPL hydrolyzes TRL within chylomicrons and VLDL but, independently of its catalytic activity, it has a bridging activity, mediating the hepatic uptake of chylomicrons and VLDL remnants. We previously showed that exogenously added LPL increases HCV binding to hepatoma cells by acting as a bridge between virus-associated lipoproteins and cell surface heparan sulfate, while simultaneously decreasing infection levels. We show here that LPL efficiently inhibits cell infection with two HCV strains produced in hepatoma cells or in primary human hepatocytes transplanted into uPA-SCID mice with fully functional human ApoB-lipoprotein profiles. Viruses produced in vitro or in vivo were separated on iodixanol gradients into low and higher density populations, and the infection of Huh 7.5 cells by both virus populations was inhibited by LPL. The effect of LPL depended on its enzymatic activity. However, the lipase inhibitor tetrahydrolipstatin restored only a minor part of HCV infectivity, suggesting an important role of the LPL bridging function in the inhibition of infection. We followed HCV cell entry by immunoelectron microscopy with anti-envelope and anti-core antibodies. These analyses demonstrated the internalization of virus particles into hepatoma cells and their presence in intracellular vesicles and associated with lipid droplets. In the presence of LPL, HCV was retained at the cell surface. We conclude that LPL efficiently inhibits HCV infection by acting on TRL associated with HCV particles through mechanisms involving its lipolytic function, but mostly its bridging function. These mechanisms lead to immobilization of the virus at the cell

  10. Hepatitis C virus recurrence after liver transplantation: relationship to anti-HCV core IgM, genotype, and level of viremia.

    Science.gov (United States)

    Crespo, J; Carte, B; Lozano, J L; Casafont, F; Rivero, M; de la Cruz, F; Pons-Romero, F

    1997-09-01

    Factors that determine the severity of hepatitis C virus (HCV)-recurrent disease in patients undergoing orthotopic liver transplantation (OLT) for HCV cirrhosis have not been clearly identified. To address this issue, we evaluated the histological and virological outcome in 25 patients who underwent OLT for HCV cirrhosis. HCV-RNA was detected by qualitative and quantitative polymerase chain reaction. The HCV genotype also was determined by polymerase chain reaction. Anti-HCV core IgM was tested by ELISA. Disease severity was expressed as a histological score. Sixteen patients had evidence of HCV-recurrent disease. HCV-RNA levels before transplantation (p = 0.029) and after transplantation (15 days, p = 0.004; 90 days, p = 0.040; 360 days, p = 0.010) were significantly higher among patients who subsequently developed recurrent hepatitis than among those who did not. The presence of anti-HCV core IgM before (p = 0.044) and after OLT (15 days, p = 0.017; 90 days, p = 0.037; and 360 days, p = 0.040) was significantly related to recurrence of hepatitis. The genotype was not related to the level of viremia, to the prevalence of recurrent hepatitis, to the presence of anti-HCV core IgM, or to disease severity. The recurrence of HCV hepatitis in patients undergoing OLT for HCV cirrhosis is related to higher levels of viremia and the presence of anti-HCV core IgM, but not to the HCV genotype. However, disease severity is not related to viremia levels, HCV genotype, or positivity of anti-HCV core IgM.

  11. Hepatitis C virus (HCV genotype 1 subtype identification in new HCV drug development and future clinical practice.

    Directory of Open Access Journals (Sweden)

    Stéphane Chevaliez

    Full Text Available BACKGROUND: With the development of new specific inhibitors of hepatitis C virus (HCV enzymes and functions that may yield different antiviral responses and resistance profiles according to the HCV subtype, correct HCV genotype 1 subtype identification is mandatory in clinical trials for stratification and interpretation purposes and will likely become necessary in future clinical practice. The goal of this study was to identify the appropriate molecular tool(s for accurate HCV genotype 1 subtype determination. METHODOLOGY/PRINCIPAL FINDINGS: A large cohort of 500 treatment-naïve patients eligible for HCV drug trials and infected with either subtype 1a or 1b was studied. Methods based on the sole analysis of the 5' non-coding region (5'NCR by sequence analysis or reverse hybridization failed to correctly identify HCV subtype 1a in 22.8%-29.5% of cases, and HCV subtype 1b in 9.5%-8.7% of cases. Natural polymorphisms at positions 107, 204 and/or 243 were responsible for mis-subtyping with these methods. A real-time PCR method using genotype- and subtype-specific primers and probes located in both the 5'NCR and the NS5B-coding region failed to correctly identify HCV genotype 1 subtype in approximately 10% of cases. The second-generation line probe assay, a reverse hybridization assay that uses probes targeting both the 5'NCR and core-coding region, correctly identified HCV subtypes 1a and 1b in more than 99% of cases. CONCLUSIONS/SIGNIFICANCE: In the context of new HCV drug development, HCV genotyping methods based on the exclusive analysis of the 5'NCR should be avoided. The second-generation line probe assay is currently the best commercial assay for determination of HCV genotype 1 subtypes 1a and 1b in clinical trials and practice.

  12. Inhibition of viral RNA polymerases by nucleoside and nucleotide analogs: therapeutic applications against positive-strand RNA viruses beyond hepatitis C virus.

    Science.gov (United States)

    Deval, Jerome; Symons, Julian A; Beigelman, Leo

    2014-12-01

    A number of important human infections are caused by positive-strand RNA viruses, yet almost none can be treated with small molecule antiviral therapeutics. One exception is the chronic infection caused by hepatitis C virus (HCV), against which new generations of potent inhibitors are being developed. One of the main molecular targets for anti-HCV drugs is the viral RNA-dependent RNA polymerase, NS5B. This review summarizes the search for nucleoside and nucleotide analogs that inhibit HCV NS5B, which led to the FDA approval of sofosbuvir in 2013. Advances in anti-HCV therapeutics have also stimulated efforts to develop nucleoside analogs against other positive-strand RNA viruses. Although it remains to be validated in the clinic, the prospect of using nucleoside analogs to treat acute infections caused by RNA viruses represents an important paradigm shift and a new frontier for future antiviral therapies. Copyright © 2014 Elsevier B.V. All rights reserved.

  13. Hepatitis C Virus (HCV) Registry Veterans in VHA Care in 2015, for the Nation, by VISN and by Station

    Data.gov (United States)

    Department of Veterans Affairs — This report describes the number of Hepatitis C Virus (HCV) registry Veterans in VHA care in 2015 based on serologic evidence of HCV infection status (HCV Positive)...

  14. 77 FR 30293 - Recommendations for the Identification of Hepatitis C Virus (HCV) Chronic Infection

    Science.gov (United States)

    2012-05-22

    ... Hepatitis C Virus (HCV) Chronic Infection AGENCY: Centers for Disease Control and Prevention (CDC... an email to [email protected] . SUPPLEMENTARY INFORMATION: Hepatitis C virus infection is a contagious... illness. It results from infection with the hepatitis C virus (HCV), which is spread primarily through...

  15. Hepatitis C virus RNA functionally sequesters miR-122

    DEFF Research Database (Denmark)

    Luna, Joseph M; Scheel, Troels K H; Danino, Tal

    2015-01-01

    Hepatitis C virus (HCV) uniquely requires the liver-specific microRNA-122 for replication, yet global effects on endogenous miRNA targets during infection are unexplored. Here, high-throughput sequencing and crosslinking immunoprecipitation (HITS-CLIP) experiments of human Argonaute (AGO) during...

  16. Large scale screening of human sera for HCV RNA and GBV-C RNA.

    Science.gov (United States)

    Keys, Jessica R; Leone, Peter A; Eron, Joseph J; Alexander, Kelcie; Brinson, Myra; Swanstrom, Ronald

    2014-03-01

    North Carolina locates acute HIV cases by pooled nucleic acid testing of HIV-antibody negative serum samples. Here, 224 pools of 80 HIV-negative samples (N = 17,920) were screened for viral RNA from HCV, GBV-C, and influenza A. No evidence of influenza A was found, but HCV and GBV-C were common (1.2% and 1.7% prevalence, respectively), demonstrating the utility of pooled testing in locating individuals that may remain undiagnosed otherwise. By sequencing positive pools, potential transmission clusters may be located as well. © 2013 Wiley Periodicals, Inc.

  17. Xerostomia, hyposalivation and sialadenitis in patients with chronic hepatitis C are not associated with the detection of HCV RNA in saliva or salivary glands.

    Science.gov (United States)

    Grossmann, Soraya de Mattos Camargo; Teixeira, Rosângela; Oliveira, Guilherme Corrêa de; Gleber-Netto, Frederico Omar; Araújo, Flávio Marcos Gomes; Araújo, Filipe Maia; Carmo, Maria Auxiliadora Vieira do

    2010-11-01

    Salivary gland disorders in patients with chronic hepatitis C (CHC) have been considered oral extrahepatic manifestations, reinforcing the hepatitis C virus (HCV) as a sialotropic virus. Hence, the authors investigated the prevalence of HCV RNA in saliva and salivary glands and its possible association with xerostomia, hyposalivation and sialadenitis in patients with CHC. In 65 patients with confirmed CHC, the HCV RNA was investigated by nested RT-PCR in saliva samples and minor salivary glands. Xerostomia, hyposalivation, clinical and histopathological evidence of sialadenitis were also evaluated. Univariate and multivariate analyses were employed to verify associations. HCV RNA was detected in the saliva of 26/65 (40.0%) patients and in 12/65 (18.5%) salivary glands. Xerostomia was reported by 23/65 (35.4%) patients, and hyposalivation was diagnosed in 13/65 (20.0%) patients. Sialadenitis was confirmed by histopathological features in 31/65 (47.7%) patients. Twelve (38.7%) of the 31 patients with sialadenitis presented HCV RNA in saliva and 2/31 (6.5%) in salivary glands. No associations were found between xerostomia, hyposalivation or sialadenitis and the detection of HCV RNA in saliva or in salivary glands. Although xerostomia, hyposalivation and sialadenitis are frequent findings in CHC patients, our study did not confirm the association between the detection of HCV RNA in saliva or salivary glands with these salivary gland disorders. However, an indirect role of HCV by immune-mediated virus mechanisms in the pathogenesis of salivary gland disorders in this group of patients cannot be ruled out.

  18. PCBP2 enhances the antiviral activity of IFN-α against HCV by stabilizing the mRNA of STAT1 and STAT2.

    Directory of Open Access Journals (Sweden)

    Zhongshuai Xin

    Full Text Available Interferon-α (IFN-α is a natural choice for the treatment of hepatitis C, but half of the chronically infected individuals do not achieve sustained clearance of hepatitis C virus (HCV during treatment with IFN-α alone. The virus can impair IFN-α signaling and cellular factors that have an effect on the viral life cycles. We found that the protein PCBP2 is down-regulated in HCV-replicon containing cells (R1b. However, the effects and mechanisms of PCBP2 on HCV are unclear. To determine the effect of PCBP2 on HCV, overexpression and knockdown of PCBP2 were performed in R1b cells. Interestingly, we found that PCBP2 can facilitate the antiviral activity of IFN-α against HCV, although the RNA level of HCV was unaffected by either the overexpression or absence of PCBP2 in R1b cells. RIP-qRT-PCR and RNA half-life further revealed that PCBP2 stabilizes the mRNA of STAT1 and STAT2 through binding the 3'Untranslated Region (UTR of these two molecules, which are pivotal for the IFN-α anti-HCV effect. RNA pull-down assay confirmed that there were binding sites located in the C-rich tracts in the 3'UTR of their mRNAs. Stabilization of mRNA by PCBP2 leads to the increased protein expression of STAT1 and STAT2 and a consistent increase of phosphorylated STAT1 and STAT2. These effects, in turn, enhance the antiviral effect of IFN-α. These findings indicate that PCBP2 may play an important role in the IFN-α response against HCV and may benefit the HCV clinical therapy.

  19. cis-Acting RNA elements in the hepatitis C virus RNA genome.

    Science.gov (United States)

    Sagan, Selena M; Chahal, Jasmin; Sarnow, Peter

    2015-08-03

    Hepatitis C virus (HCV) infection is a rapidly increasing global health problem with an estimated 170 million people infected worldwide. HCV is a hepatotropic, positive-sense RNA virus of the family Flaviviridae. As a positive-sense RNA virus, the HCV genome itself must serve as a template for translation, replication and packaging. The viral RNA must therefore be a dynamic structure that is able to readily accommodate structural changes to expose different regions of the genome to viral and cellular proteins to carry out the HCV life cycle. The ∼ 9600 nucleotide viral genome contains a single long open reading frame flanked by 5' and 3' non-coding regions that contain cis-acting RNA elements important for viral translation, replication and stability. Additional cis-acting RNA elements have also been identified in the coding sequences as well as in the 3' end of the negative-strand replicative intermediate. Herein, we provide an overview of the importance of these cis-acting RNA elements in the HCV life cycle. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. Identification of Variants of Hepatitis C Virus (HCV) Entry Factors in Patients Highly Exposed to HCV but Remaining Uninfected: An ANRS Case-Control Study.

    Science.gov (United States)

    Fouquet, Baptiste; Ghosn, Jade; Quertainmont, Yann; Salmon, Dominique; Rioux, Christophe; Duvivier, Claudine; Delfraissy, Jean-François; Misrahi, Micheline

    2015-01-01

    Hepatitis C virus (HCV) causes persistent infection in 75% of cases and is a major public health problem worldwide. More than 92% of intravenous drug users (IDU) infected by human immunodeficiency virus type 1 (HIV-1) are seropositive for HCV, and it is conceivable that some HIV-1-infected IDU who remain uninfected by HCV may be genetically resistant.Here we conducted a case-control study to identify mutations in HCV entry coreceptors in HIV-infected IDU who remained uninfected by HCV. We recruited 138 patients, comprising 22 HIV+ HCV- case IDU and 116 HIV+ HCV+ control IDU. We focused on coreceptors in which point mutations are known to abolish HCV infectivity in vitro. Our previous study of the Claudin-1 gene revealed no specific variants in the same case population. Here we performed direct genomic sequencing of the Claudin-6, Claudin-9, Occludin and Scavenger receptor-B1 (SCARB1) gene coding regions. Most HIV+ HCV- IDU had no mutations in HCV coreceptors. However, two HIV+ HCV- patients harbored a total of four specific mutations/variants of HCV entry factors that were not found in the HIV+ HCV+ controls. One case patient harbored heterozygous variants of both Claudin-6 and Occludin, and the other case patient harbored two heterozygous variants of SCARB1. This suggests that HCV resistance might involve complex genetic events and factors other than coreceptors, a situation similar to that reported for HIV-1 resistance.

  1. Identification of Variants of Hepatitis C Virus (HCV Entry Factors in Patients Highly Exposed to HCV but Remaining Uninfected: An ANRS Case-Control Study.

    Directory of Open Access Journals (Sweden)

    Baptiste Fouquet

    Full Text Available Hepatitis C virus (HCV causes persistent infection in 75% of cases and is a major public health problem worldwide. More than 92% of intravenous drug users (IDU infected by human immunodeficiency virus type 1 (HIV-1 are seropositive for HCV, and it is conceivable that some HIV-1-infected IDU who remain uninfected by HCV may be genetically resistant.Here we conducted a case-control study to identify mutations in HCV entry coreceptors in HIV-infected IDU who remained uninfected by HCV. We recruited 138 patients, comprising 22 HIV+ HCV- case IDU and 116 HIV+ HCV+ control IDU. We focused on coreceptors in which point mutations are known to abolish HCV infectivity in vitro. Our previous study of the Claudin-1 gene revealed no specific variants in the same case population. Here we performed direct genomic sequencing of the Claudin-6, Claudin-9, Occludin and Scavenger receptor-B1 (SCARB1 gene coding regions. Most HIV+ HCV- IDU had no mutations in HCV coreceptors. However, two HIV+ HCV- patients harbored a total of four specific mutations/variants of HCV entry factors that were not found in the HIV+ HCV+ controls. One case patient harbored heterozygous variants of both Claudin-6 and Occludin, and the other case patient harbored two heterozygous variants of SCARB1. This suggests that HCV resistance might involve complex genetic events and factors other than coreceptors, a situation similar to that reported for HIV-1 resistance.

  2. Pharmacokinetics of a Cholesterol-conjugated Aptamer Against the Hepatitis C Virus (HCV) NS5B Protein

    Science.gov (United States)

    Lee, Chang Ho; Lee, Soo-Han; Kim, Ji Hyun; Noh, Yook-Hwan; Noh, Gyu-Jeong; Lee, Seong-Wook

    2015-01-01

    Hepatitis C virus (HCV) is the major cause of progressive liver disease such as chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Previously, we reported that a 29 nucleotide-long 2'-F pyrimidine modified RNA aptamer against the HCV nonstructural protein 5B efficiently inhibited HCV replication and suppressed HCV infectious virus particle formation in a cell culture system. In this study, we modified this aptamer through conjugation of cholesterol for in vivo availability. This cholesterol-conjugated aptamer (chol-aptamer) efficiently entered the cell and inhibited HCV RNA replication, without any alteration in gene expression profiling including innate immune response-related genes. Moreover, systemic administration of the chol-aptamer was well tolerated without any abnormalities in mice. To evaluate the pharmacokinetics of the chol-aptamer in vivo, dose proportionality, bioavailability, and pharmacokinetic parameters were evaluated by noncompartmental analyses in normal BALB/c mice. Population analysis was performed using nonlinear mixed effects modeling. Moreover, the pharmacokinetics of two different routes (intravenous, IV, versus intraperitoneal, IP) were compared. Cholesterol conjugation showed dose proportionality, extended the time that the aptamer was in the plasma, and enhanced aptamer exposure to the body. Noticeably, the IV route was more suitable than the IP route due to the chol-aptamer remaining in the plasma for a longer period of time. PMID:26440598

  3. High awareness of hepatitis C virus (HCV) but limited knowledge of HCV complications among HIV-positive and HIV-negative men who have sex with men

    NARCIS (Netherlands)

    Lambers, Femke A. E.; Prins, Maria; Davidovich, Udi; Stolte, Ineke G.

    2014-01-01

    Hepatitis C virus (HCV) has emerged as a sexually transmitted infection among HIV-positive men who have sex with men (MSM) in high-income countries. Little is reported about HCV awareness among MSM, although this is essential for developing targeted prevention strategies. We, therefore, studied HCV

  4. Hepatitis G virus co-infection may affect the elimination of hepatitis C virus RNA from the peripheral blood of hemodialysis patients.

    Science.gov (United States)

    Januszkiewicz-Lewandowska, D; Wysocki, J; Rembowska, J; Lewandowski, K; Nowak, T; Pernak, M; Nowak, J

    2001-01-01

    Hemodialysis patients are at risk for hepatitis C virus (HCV) and hepatitis G virus (HGV) infection. The aim of this study was to investigate the possible influence of HGV co-infection on HCV RNA elimination from the peripheral blood of hemodialysis patients. The study involved 144 persons, all with HCV antibodies and HCV RNA. Among 144 patients 24 (16.7%) were positive for HGV RNA. After 2.5 years of observation 80 patients (55.6%) were still HCV RNA-positive. In the latter group 18 patients were co-infected with HGV and 62 were HGV RNA-negative. During 2.5 years of the follow-up study 64 patients eliminated HCV RNA from the serum. In this group only 6 patients were HGV co-infected. None of the HGV-positive patients eliminated HGV RNA from the serum. The higher incidence of HGV co-infection in the group of patients who remained HCV RNA-positive (18/80, 22.5%), in comparison to the group of HCV antibodies-positive patients who lost HCV in the blood (6/64, 9.4%, P < 0.0001) suggests, that the co-infection with HGV may delay the spontaneous elimination of HCV RNA from the blood.

  5. Hepatitis C virus genomic RNA dimerization is mediated via a kissing complex intermediate.

    Science.gov (United States)

    Shetty, Sumangala; Kim, Seungtaek; Shimakami, Tetsuro; Lemon, Stanley M; Mihailescu, Mihaela-Rita

    2010-05-01

    With over 200 million people infected with hepatitis C virus (HCV) worldwide, there is a need for more effective and better-tolerated therapeutic strategies. The HCV genome is a positive-sense; single-stranded RNA encoding a large polyprotein cleaved at multiple sites to produce at least ten proteins, among them an error-prone RNA polymerase that confers a high mutation rate. Despite considerable overall sequence diversity, in the 3'-untranslated region of the HCV genomic RNA there is a 98-nucleotide (nt) sequence named X RNA, the first 55 nt of which (X55 RNA) are 100% conserved among all HCV strains. The X55 region has been suggested to be responsible for in vitro dimerization of the genomic RNA in the presence of the viral core protein, although the mechanism by which this occurs is unknown. In this study, we analyzed the X55 region and characterized the mechanism by which it mediates HCV genomic RNA dimerization. Similar to a mechanism proposed previously for the human immunodeficiency 1 virus (HIV-1) genome, we show that dimerization of the HCV genome involves formation of a kissing complex intermediate, which is converted to a more stable extended duplex conformation in the presence of the core protein. Mutations in the dimer linkage sequence loop sequence that prevent RNA dimerization in vitro significantly reduced but did not completely ablate the ability of HCV RNA to replicate or produce infectious virus in transfected cells.

  6. The Rationale for a Preventative HCV Virus-Like Particle (VLP Vaccine

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    Joseph Torresi

    2017-11-01

    Full Text Available HCV represents a global health problem with ~200 million individuals currently infected, worldwide. With the high cost of antiviral therapies, the global burden of chronic hepatitis C infection (CHCV infection will be substantially reduced by the development of an effective vaccine for HCV. The field of HCV vaccines is generally divided into proponents of strategies to induce neutralizing antibodies (NAb and those who propose to elicit cell mediated immunity (CMI. However, for a hepatitis C virus (HCV vaccine to be effective in preventing infection, it must be capable of generating cross-reactive CD4+, CD8+ T cell, and NAb responses that will cover the major viral genotypes. Simulation models of hepatitis C have predicted that a vaccine of even modest efficacy and coverage will significantly reduce the incidence of hepatitis C. A HCV virus like particle (VLP based vaccine would fulfill the requirement of delivering critical conformational neutralizing epitopes in addition to providing HCV specific CD4+ and CD8+ epitopes. Several approaches have been reported including insect cell-derived genotype 1b HCV VLPs; a human liver-derived quadrivalent genotype 1a, 1b, 2, and 3a vaccine; a genotype 1a HCV E1 and E2 glycoprotein/MLV Gag pseudotype VLP vaccine; and chimeric HBs-HCV VLP vaccines. All to result in the production of cross-NAb and/or T cell responses against HCV. This paper summarizes the evidence supporting the development of a HCV VLP based vaccine.

  7. Claudin-6 and Occludin Natural Variants Found in a Patient Highly Exposed but Not Infected with Hepatitis C Virus (HCV Do Not Confer HCV Resistance In Vitro.

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    Lucie Fénéant

    Full Text Available The clinical course of Hepatitis C Virus (HCV infection is highly variable between infected individual hosts: up to 80% of acutely HCV infected patients develop a chronic infection while 20% clear infection spontaneously. Spontaneous clearance of HCV infection can be predicted by several factors, including symptomatic acute infection, favorable IFNL3 polymorphisms and gender. In our study, we explored the possibility that variants in HCV cell entry factors might be involved in resistance to HCV infection. In a same case patient highly exposed but not infected by HCV, we previously identified one mutation in claudin-6 (CLDN6 and a rare variant in occludin (OCLN, two tight junction proteins involved in HCV entry into hepatocytes. Here, we conducted an extensive functional study to characterize the ability of these two natural variants to prevent HCV entry. We used lentiviral vectors to express Wildtype or mutated CLDN6 and OCLN in different cell lines and primary human hepatocytes. HCV infection was then investigated using cell culture produced HCV particles (HCVcc as well as HCV pseudoparticles (HCVpp expressing envelope proteins from different genotypes. Our results show that variants of CLDN6 and OCLN expressed separately or in combination did not affect HCV infection nor cell-to-cell transmission. Hence, our study highlights the complexity of HCV resistance mechanisms supporting the fact that this process probably not primarily involves HCV entry factors and that other unknown host factors may be implicated.

  8. Claudin-6 and Occludin Natural Variants Found in a Patient Highly Exposed but Not Infected with Hepatitis C Virus (HCV) Do Not Confer HCV Resistance In Vitro.

    Science.gov (United States)

    Fénéant, Lucie; Ghosn, Jade; Fouquet, Baptiste; Helle, François; Belouzard, Sandrine; Vausselin, Thibaut; Séron, Karin; Delfraissy, Jean-François; Dubuisson, Jean; Misrahi, Micheline; Cocquerel, Laurence

    2015-01-01

    The clinical course of Hepatitis C Virus (HCV) infection is highly variable between infected individual hosts: up to 80% of acutely HCV infected patients develop a chronic infection while 20% clear infection spontaneously. Spontaneous clearance of HCV infection can be predicted by several factors, including symptomatic acute infection, favorable IFNL3 polymorphisms and gender. In our study, we explored the possibility that variants in HCV cell entry factors might be involved in resistance to HCV infection. In a same case patient highly exposed but not infected by HCV, we previously identified one mutation in claudin-6 (CLDN6) and a rare variant in occludin (OCLN), two tight junction proteins involved in HCV entry into hepatocytes. Here, we conducted an extensive functional study to characterize the ability of these two natural variants to prevent HCV entry. We used lentiviral vectors to express Wildtype or mutated CLDN6 and OCLN in different cell lines and primary human hepatocytes. HCV infection was then investigated using cell culture produced HCV particles (HCVcc) as well as HCV pseudoparticles (HCVpp) expressing envelope proteins from different genotypes. Our results show that variants of CLDN6 and OCLN expressed separately or in combination did not affect HCV infection nor cell-to-cell transmission. Hence, our study highlights the complexity of HCV resistance mechanisms supporting the fact that this process probably not primarily involves HCV entry factors and that other unknown host factors may be implicated.

  9. Sensing of RNA viruses

    DEFF Research Database (Denmark)

    Jensen, Søren; Thomsen, Allan Randrup

    2012-01-01

    Our knowledge regarding the contribution of the innate immune system in recognizing and subsequently initiating a host response to an invasion of RNA virus has been rapidly growing over the last decade. Descriptions of the receptors involved and the molecular mechanisms they employ to sense viral...

  10. HEPATITIS C VIRUS (HCV) SEROPREVALENCE, ANTIGENAEMIA AND ASSOCIATED RISK FACTORS AMONG PREGNANT WOMEN IN NIGERIA.

    Science.gov (United States)

    Owolabi, Omolola Beatrice; Adesina, Kikelomo Temilola; Fadeyi, Abayomi; Popoola, Gbenga

    2015-10-01

    Hepatitis C viral infection is a significant public health challenge with potential risk of progressing to liver cirrhosis and hepatocellular carcinoma (HCC). Actively infected mothers can transmit the virus to their babies who may develop liver cirrhosis and HCC as young adults. We determined the seroprevalence of HCV, its antigenaemia and associated risk factors among pregnant women. We recruited 400 pregnant women and tested their serum for HCV antibodies using immune-chromatographic test and determined the HCV core antigenaemia among HCV sero-positives by enzyme-immunoassay (EIA). The bio-socio-demographic variables of the participants were statistically correlated to the test results. Seroprevalence of HCV was 5.8% (23/400) and the prevalence of HCV core antigenaemia was 73.9% (17/23). None of the bio-socio-demographic variables of the participants and other known risk factors evaluated had. significant influence on either seroprevalence of HCV or its antigenaemia. Only the employment status of the participants' husbands (p = 0.01) significantly affected seropositivity of HCV. HCV core antigenaemia is high among pregnant women who have antibodies to HCV in our environment and this signifies an active hepatitis C virus infection.

  11. Performance of ARCHITECT HCV core antigen test with specimens from US plasma donors and injecting drug users.

    Science.gov (United States)

    Mixson-Hayden, Tonya; Dawson, George J; Teshale, Eyasu; Le, Thao; Cheng, Kevin; Drobeniuc, Jan; Ward, John; Kamili, Saleem

    2015-05-01

    Hepatitis C virus (HCV) core antigen is a serological marker of current HCV infection. The aim of this study was mainly to evaluate the performance characteristics of the ARCHITECT HCV core antigen assay with specimens from US plasma donors and injecting drug users. A total of 551 serum and plasma samples with known anti-HCV and HCV RNA status were tested for HCV core antigen using the Abbott ARCHITECT HCV core antigen test. HCV core antigen was detectable in 100% of US plasma donor samples collected during the pre-seroconversion phase of infection (anti-HCV negative/HCV RNA positive). Overall sensitivity of the HCV core antigen assay was 88.9-94.3% in samples collected after seroconversion. The correlation between HCV core antigen and HCV RNA titers was 0.959. HCV core antigen testing may be reliably used to identify current HCV infection. Published by Elsevier B.V.

  12. Active RNA replication of hepatitis C virus downregulates CD81 expression.

    Directory of Open Access Journals (Sweden)

    Po-Yuan Ke

    Full Text Available So far how hepatitis C virus (HCV replication modulates subsequent virus growth and propagation still remains largely unknown. Here we determine the impact of HCV replication status on the consequential virus growth by comparing normal and high levels of HCV RNA expression. We first engineered a full-length, HCV genotype 2a JFH1 genome containing a blasticidin-resistant cassette inserted at amino acid residue of 420 in nonstructural (NS protein 5A, which allowed selection of human hepatoma Huh7 cells stably-expressing HCV. Short-term establishment of HCV stable cells attained a highly-replicating status, judged by higher expressions of viral RNA and protein as well as higher titer of viral infectivity as opposed to cells harboring the same genome without selection. Interestingly, maintenance of highly-replicating HCV stable cells led to decreased susceptibility to HCV pseudotyped particle (HCVpp infection and downregulated cell surface level of CD81, a critical HCV entry (coreceptor. The decreased CD81 cell surface expression occurred through reduced total expression and cytoplasmic retention of CD81 within an endoplasmic reticulum -associated compartment. Moreover, productive viral RNA replication in cells harboring a JFH1 subgenomic replicon containing a similar blasticidin resistance gene cassette in NS5A and in cells robustly replicating full-length infectious genome also reduced permissiveness to HCVpp infection through decreasing the surface expression of CD81. The downregulation of CD81 surface level in HCV RNA highly-replicating cells thus interfered with reinfection and led to attenuated viral amplification. These findings together indicate that the HCV RNA replication status plays a crucial determinant in HCV growth by modulating the expression and intracellular localization of CD81.

  13. Structural basis of hepatitis C virus neutralization by broadly neutralizing antibody HCV1

    Energy Technology Data Exchange (ETDEWEB)

    Kong, Leopold; Giang, Erick; Robbins, Justin B.; Stanfield, Robyn L.; Burton, Dennis R.; Wilson, Ian A.; Law, Mansun (Scripps)

    2012-10-29

    Hepatitis C virus (HCV) infects more than 2% of the global population and is a leading cause of liver cirrhosis, hepatocellular carcinoma, and end-stage liver diseases. Circulating HCV is genetically diverse, and therefore a broadly effective vaccine must target conserved T- and B-cell epitopes of the virus. Human mAb HCV1 has broad neutralizing activity against HCV isolates from at least four major genotypes and protects in the chimpanzee model from primary HCV challenge. The antibody targets a conserved antigenic site (residues 412-423) on the virus E2 envelope glycoprotein. Two crystal structures of HCV1 Fab in complex with an epitope peptide at 1.8-{angstrom} resolution reveal that the epitope is a {beta}-hairpin displaying a hydrophilic face and a hydrophobic face on opposing sides of the hairpin. The antibody predominantly interacts with E2 residues Leu{sup 413} and Trp{sup 420} on the hydrophobic face of the epitope, thus providing an explanation for how HCV isolates bearing mutations at Asn{sup 415} on the same binding face escape neutralization by this antibody. The results provide structural information for a neutralizing epitope on the HCV E2 glycoprotein and should help guide rational design of HCV immunogens to elicit similar broadly neutralizing antibodies through vaccination.

  14. Interference of hepatitis C virus RNA replication by short interfering RNAs

    Science.gov (United States)

    Kapadia, Sharookh B.; Brideau-Andersen, Amy; Chisari, Francis V.

    2003-02-01

    Hepatitis C virus (HCV) infection is a major cause of chronic liver disease, which can lead to the development of liver cirrhosis and hepatocellular carcinoma. Current therapy of patients with chronic HCV infection includes treatment with IFN in combination with ribavirin. Because most treated patients do not resolve the infection, alternative treatment is essential. RNA interference (RNAi) is a recently discovered antiviral mechanism present in plants and animals that induces double-stranded RNA degradation. Using a selectable subgenomic HCV replicon cell culture system, we have shown that RNAi can specifically inhibit HCV RNA replication and protein expression in Huh-7 cells that stably replicate the HCV genome, and that this antiviral effect is independent of IFN. These results suggest that RNAi may represent a new approach for the treatment of persistent HCV infection.

  15. HCV and Oxidative Stress: Implications for HCV Life Cycle and HCV-Associated Pathogenesis

    Directory of Open Access Journals (Sweden)

    Regina Medvedev

    2016-01-01

    Full Text Available HCV (hepatitis C virus is a member of the Flaviviridae family that contains a single-stranded positive-sense RNA genome of approximately 9600 bases. HCV is a major causative agent for chronic liver diseases such as steatosis, fibrosis, cirrhosis, and hepatocellular carcinoma which are caused by multifactorial processes. Elevated levels of reactive oxygen species (ROS are considered as a major factor contributing to HCV-associated pathogenesis. This review summarizes the mechanisms involved in formation of ROS in HCV replicating cells and describes the interference of HCV with ROS detoxifying systems. The relevance of ROS for HCV-associated pathogenesis is reviewed with a focus on the interference of elevated ROS levels with processes controlling liver regeneration. The overview about the impact of ROS for the viral life cycle is focused on the relevance of autophagy for the HCV life cycle and the crosstalk between HCV, elevated ROS levels, and the induction of autophagy.

  16. Can Hepatitis C Virus (HCV) Direct-Acting Antiviral Treatment as Prevention Reverse the HCV Epidemic Among Men Who Have Sex With Men in the United Kingdom? Epidemiological and Modeling Insights

    Science.gov (United States)

    Martin, Natasha K.; Thornton, Alicia; Hickman, Matthew; Sabin, Caroline; Nelson, Mark; Cooke, Graham S.; Martin, Thomas C. S.; Delpech, Valerie; Ruf, Murad; Price, Huw; Azad, Yusef; Thomson, Emma C.; Vickerman, Peter

    2016-01-01

    Background. We report on the hepatitis C virus (HCV) epidemic among human immunodeficiency virus (HIV)-positive men who have sex with men (MSM) in the United Kingdom and model its trajectory with or without scaled-up HCV direct-acting antivirals (DAAs). Methods. A dynamic HCV transmission model among HIV–diagnosed MSM in the United Kingdom was calibrated to HCV prevalence (antibody [Ab] or RNA positive), incidence, and treatment from 2004 to 2011 among HIV-diagnosed MSM in the UK Collaborative HIV Cohort (UK CHIC). The epidemic was projected with current or scaled-up HCV treatment, with or without a 20% behavioral risk reduction. Results. HCV prevalence among HIV-positive MSM in UK CHIC increased from 7.3% in 2004 to 9.9% in 2011, whereas primary incidence was flat (1.02–1.38 per 100 person-years). Over the next decade, modeling suggests 94% of infections are attributable to high-risk individuals, comprising 7% of the population. Without treatment, HCV chronic prevalence could have been 38% higher in 2015 (11.9% vs 8.6%). With current treatment and sustained virological response rates (status quo), chronic prevalence is likely to increase to 11% by 2025, but stabilize with DAA introduction in 2015. With DAA scale-up to 80% within 1 year of diagnosis (regardless of disease stage), and 20% per year thereafter, chronic prevalence could decline by 71% (to 3.2%) compared to status quo in 2025. With additional behavioral interventions, chronic prevalence could decline further to <2.5% by 2025. Conclusions. Epidemiological data and modeling suggest a continuing HCV epidemic among HIV-diagnosed MSM in the United Kingdom driven by high-risk individuals, despite high treatment rates. Substantial reductions in HCV transmission could be achieved through scale-up of DAAs and moderately effective behavioral interventions. PMID:26908813

  17. A cell-based assay for RNA synthesis by the HCV polymerase reveals new insights on mechanism of polymerase inhibitors and modulation by NS5A.

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    C T Ranjith-Kumar

    Full Text Available RNA synthesis by the genotype 1b hepatitis C virus (HCV polymerase (NS5B transiently expressed in Human embryonic kidney 293T cells or liver hepatocytes was found to robustly stimulate RIG-I-dependent luciferase production from the interferon β promoter in the absence of exogenously provided ligand. This cell-based assay, henceforth named the 5BR assay, could be used to examine HCV polymerase activity in the absence of other HCV proteins. Mutations that decreased de novo initiated RNA synthesis in biochemical assays decreased activation of RIG-I signaling. In addition, NS5B that lacks the C-terminal transmembrane helix but remains competent for RNA synthesis could activate RIG-I signaling. The addition of cyclosporine A to the cells reduced luciferase levels without affecting agonist-induced RIG-I signaling. Furthermore, non-nucleoside inhibitor benzothiadiazines (BTDs that bind within the template channel of the 1b NS5B were found to inhibit the readout from the 5BR assay. Mutation M414T in NS5B that rendered the HCV replicon resistant to BTD was also resistant to BTDs in the 5BR assay. Co-expression of the HCV NS5A protein along with NS5B and RIG-I was found to inhibit the readout from the 5BR assay. The inhibition by NS5A was decreased with the removal of the transmembrane helix in NS5B. Lastly, NS5B from all six major HCV genotypes showed robust activation of RIG-I in the 5BR assay. In summary, the 5BR assay could be used to validate inhibitors of the HCV polymerase as well as to elucidate requirements for HCV-dependent RNA synthesis.

  18. Modulation of GB Virus B RNA Abundance by MicroRNA-122: Dependence on and Escape from MicroRNA-122 Restriction

    Science.gov (United States)

    Sarnow, Peter

    2013-01-01

    Hepatitis C virus (HCV) RNA forms an unusual interaction with human microRNA-122 (miR-122) that promotes viral RNA accumulation in cultured human liver cells and in the livers of infected chimpanzees. GB virus B (GBV-B) is a hepatotropic virus and close relative of HCV. Thus, GBV-B has been used as a surrogate system to study HCV amplification in cultured cells and in infected tamarins. It was discovered that the 5′-terminal sequences of GBV-B RNA, like HCV RNA, forms an Argonaute 2-mediated complex with two miR-122 molecules that are essential for accumulation of GBV-B subgenomic replicon RNA. However, sequences in miR-122 that anneal to each viral RNA genome were different, suggesting distinct overall structural features in HCV:miR-122 and GBV-B:miR-122 complexes. Surprisingly, a deletion that removed both miR-122 binding sites from the subgenomic GBV-B RNAs rendered viral RNA amplification independent from miR-122 and Argonaute 2. This finding suggests that structural features at the end of the viral genome dictate whether miR-122 is required to aid in maintaining viral RNA abundance. PMID:23616647

  19. Effects of HCV proteins in current HCV transgenic models.

    Science.gov (United States)

    Jiao, Jian; Wang, Jiangbin; Sallberg, Matii

    2010-02-01

    Hepatits C virus (HCV) is an enveloped virus with positive-sense single-stranded RNA genome that causes both acute and persistent infections associated with chronic hepatitis, cirrhosis and hepatocellular carcinoma, which needs fully functional human hepatocytes for its development. Due to the strict human tropism of HCV, only human and higher primates such as chimpanzees have been receptive to HCV infection and development, cognition about pathophysiololgy and host immune responses of HCV infection is limited by lacking of simple laboratory models of infection for a long time. During the past decade, gene transfer approaches have been helpful to the understanding of the molecular basis of human disease. Transgenic cell lines, chimeric and transgenic animal models were developed and had been demonstrated their invaluable benefits. This review focuses on the existing HCV transgenic models and summarize the relative results about probable pathophysical changes induced by HCV proteins.

  20. Post-transcriptional inhibition of hepatitis C virus replication through small interference RNA

    Directory of Open Access Journals (Sweden)

    Rehman Sidra

    2011-03-01

    Full Text Available Abstract Background Hepatitis C Virus (HCV infection is a major health problem throughout world that causes acute and chronic infection which resulted in liver fibrosis, hepatocellular carcinoma and death. The only therapy currently available for HCV infection is the combination of pegylated interferon alpha (PEG-IFN α and ribavirin. This therapy can effectively clear the virus infection in only 50% of infected individuals. Hence, there is a dire need to develop antiviral agents against HCV. Results This study was design to examine the ability of exogenous small interfering RNAs (siRNAs to block the replication of HCV in human liver cells. In the present study six 21-bp siRNAs were designed against different regions of HCV non-structural genes (NS2, NS3 serine protease/helicase, NS4Band NS5B RNA dependent RNA polymerase. siRNAs were labeled as NS2si241, NS3si-229, NS3si-858, NS4Bsi-166, NS5Bsi-241 and NS5Bsi-1064. We found that siRNAs against HCV NS2- NS5B efficiently inhibit HCV replication in Huh-7 cells. Our results demonstrated that siRNAs directed against HCV NS3 (NS3si-229 and NS3si-858 showed 58% and 88% reduction in viral titer respectively. Moreover, NS4Bsi-166 and NS5Bsi-1064 exhibited a dramatic reduction in HCV viral RNA and resulted in greater than 90% inhibition at a 20 μM concentration, while NS2si-241 showed 27% reduction in viral titer. No significant inhibition was detected in cells transfected with the negative control siRNA. Conclusion Our results suggest that siRNAs targeting against HCV non-structural genes (NS2-NS5B efficiently inhibit HCV replication and combination of these siRNAs of different targets and interferon will be better option to treat HCV infection throughout the world.

  1. HCV core protein-induced down-regulation of microRNA-152 promoted aberrant proliferation by regulating Wnt1 in HepG2 cells.

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    Shifeng Huang

    Full Text Available BACKGROUND: Hepatitis C virus (HCV has been reported to regulate cellular microRNAs (miRNAs. The HCV core protein is considered to be a potential oncoprotein in HCV-related hepatocellular carcinoma (HCV-HCC, but HCV core-regulated miRNAs are largely unknown. Our preliminary experiments revealed significant down-regulation of microRNA-152 (miR-152 by HCV core protein in HepG2 cells. Through target gene prediction softwares, Wnt1 was predicted to be a potential target of miR-152. The present study was initiated to investigate whether miR-152 is aberrantly regulated by the HCV core protein, and involved in the regulation of the aberrant proliferation of HCV-HCC cells. METHODS: MiR-152 levels were examined by stem-loop real-time RT-PCR (SLqRT-PCR. Cell proliferation was analyzed by MTT and colony formation assay. Cell cycle analysis was performed by flow cytometry. Luciferase reporter assay was conducted to confirm miRNA-target association. Wnt1 expression was determined by real-time qPCR and Western blotting. RESULTS: HCV core protein significantly suppressed miR-152 expression, and led to significant Wnt1 up-regulation with a concomitant aberrantly promoted proliferation. Moreover, we validated that miR-152 inhibition promoted, while miR-152 mimics inhibited cell proliferation. Using, qRT-PCR and western blot, Wnt1 was demonstrated to be regulated by miR-152. Luciferase activity assay showed that while miR-152 mimics significantly reduced the luciferase activity by 83.76% (P<0.0001, miR-152 inhibitor showed no effect on luciferase reporter. Most notably, salvage expression of miR-152 after Ad-HCV core infection for 24 h almost totally reversed the proliferation-promoting effect of the HCV core protein, and meanwhile, reduced the expression of both Wnt1 mRNA and protein to basal levels. CONCLUSION: These findings provide important evidence that the reduced miR-152 expression by HCV core protein can indirectly lose an inhibitory effect on Wnt1

  2. Hepatitis C virus (HCV): ever in reliable partnerships? | Yalena ...

    African Journals Online (AJOL)

    There is no preventive vaccine against HCV and treatment, consisting of interferon alpha plus Ribavirin, is generally effective in less than 50% of cases. HCV has evolved ... Issues regarding tropism, disease progression and antiviral treatment response, among other aspects, are discussed. Data accumulated reveal that ...

  3. microRNA-122 abundance in hepatocellular carcinoma and non-tumor liver tissue from Japanese patients with persistent HCV versus HBV infection.

    Science.gov (United States)

    Spaniel, Carolyn; Honda, Masao; Selitsky, Sara R; Yamane, Daisuke; Shimakami, Tetsuro; Kaneko, Shuichi; Lanford, Robert E; Lemon, Stanley M

    2013-01-01

    Mechanisms of hepatic carcinogenesis in chronic hepatitis B and hepatitis C are incompletely defined but often assumed to be similar and related to immune-mediated inflammation. Despite this, several studies hint at differences in expression of miR-122, a liver-specific microRNA with tumor suppressor properties, in hepatocellular carcinoma (HCC) associated with hepatitis B virus (HBV) versus hepatitis C virus (HCV) infection. Differences in the expression of miR-122 in these cancers would be of interest, as miR-122 is an essential host factor for HCV but not HBV replication. To determine whether the abundance of miR-122 in cancer tissue is influenced by the nature of the underlying virus infection, we measured miR-122 by qRT-PCR in paired tumor and non-tumor tissues from cohorts of HBV- and HCV-infected Japanese patients. miR-122 abundance was significantly reduced from normal in HBV-associated HCC, but not in liver cancer associated with HCV infection. This difference was independent of the degree of differentiation of the liver cancer. Surprisingly, we also found significant differences in miR-122 expression in non-tumor tissue, with miR-122 abundance reduced from normal in HCV- but not HBV-infected liver. Similar differences were observed in HCV- vs. HBV-infected chimpanzees. Among HCV-infected Japanese subjects, reductions in miR-122 abundance in non-tumor tissue were associated with a single nucleotide polymorphism near the IL28B gene that predicts poor response to interferon-based therapy (TG vs. TT genotype at rs8099917), and correlated negatively with the abundance of multiple interferon-stimulated gene transcripts. Reduced levels of miR-122 in chronic hepatitis C thus appear to be associated with endogenous interferon responses to the virus, while differences in miR-122 expression in HCV- versus HBV-associated HCC likely reflect virus-specific mechanisms contributing to carcinogenesis. The continued expression of miR-122 in HCV-associated HCC may signify

  4. microRNA-122 abundance in hepatocellular carcinoma and non-tumor liver tissue from Japanese patients with persistent HCV versus HBV infection.

    Directory of Open Access Journals (Sweden)

    Carolyn Spaniel

    Full Text Available Mechanisms of hepatic carcinogenesis in chronic hepatitis B and hepatitis C are incompletely defined but often assumed to be similar and related to immune-mediated inflammation. Despite this, several studies hint at differences in expression of miR-122, a liver-specific microRNA with tumor suppressor properties, in hepatocellular carcinoma (HCC associated with hepatitis B virus (HBV versus hepatitis C virus (HCV infection. Differences in the expression of miR-122 in these cancers would be of interest, as miR-122 is an essential host factor for HCV but not HBV replication. To determine whether the abundance of miR-122 in cancer tissue is influenced by the nature of the underlying virus infection, we measured miR-122 by qRT-PCR in paired tumor and non-tumor tissues from cohorts of HBV- and HCV-infected Japanese patients. miR-122 abundance was significantly reduced from normal in HBV-associated HCC, but not in liver cancer associated with HCV infection. This difference was independent of the degree of differentiation of the liver cancer. Surprisingly, we also found significant differences in miR-122 expression in non-tumor tissue, with miR-122 abundance reduced from normal in HCV- but not HBV-infected liver. Similar differences were observed in HCV- vs. HBV-infected chimpanzees. Among HCV-infected Japanese subjects, reductions in miR-122 abundance in non-tumor tissue were associated with a single nucleotide polymorphism near the IL28B gene that predicts poor response to interferon-based therapy (TG vs. TT genotype at rs8099917, and correlated negatively with the abundance of multiple interferon-stimulated gene transcripts. Reduced levels of miR-122 in chronic hepatitis C thus appear to be associated with endogenous interferon responses to the virus, while differences in miR-122 expression in HCV- versus HBV-associated HCC likely reflect virus-specific mechanisms contributing to carcinogenesis. The continued expression of miR-122 in HCV

  5. Cellular DEAD-box RNA helicase DDX6 modulates interaction of miR-122 with the 5' untranslated region of hepatitis C virus RNA.

    Science.gov (United States)

    Biegel, Jason M; Henderson, Eric; Cox, Erica M; Bonenfant, Gaston; Netzband, Rachel; Kahn, Samantha; Eager, Rachel; Pager, Cara T

    2017-07-01

    Hepatitis C virus (HCV) subverts the cellular DEAD-box RNA helicase DDX6 to promote virus infection. Using polysome gradient analysis and the subgenomic HCV Renilla reporter replicon genome, we determined that DDX6 does not affect HCV translation. Rather expression of the subgenomic HCV Renilla luciferase reporter at late times, as well as labeling of newly synthesized viral RNA with 4-thiouridine showed that DDX6 modulates replication. Because DDX6 is an effector protein of the microRNA pathway, we also investigated its role in miR-122-directed HCV gene expression. Similar to sequestering miR-122, depletion of DDX6 modulated HCV RNA stability. Interestingly, miR-122-HCV RNA interaction assays with mutant HCV genomes sites and compensatory exogenous miR-122 showed that DDX6 affects the function of miR-122 at one particular binding site. We propose that DDX6 facilitates the miR-122 interaction with HCV 5' UTR, which is necessary for stabilizing the viral genome and the switch between translation and replication. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. 21 CFR 610.48 - Hepatitis C virus (HCV) “lookback” requirements based on review of historical testing records.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 7 2010-04-01 2010-04-01 false Hepatitis C virus (HCV) âlookbackâ requirements... STANDARDS Testing Requirements for Communicable Disease Agents § 610.48 Hepatitis C virus (HCV) “lookback... the following actions: (1) You must: (i) Review all records of donor testing for hepatitis C virus...

  7. Discovery of SCH446211 (SCH6): A New Ketoamide Inhibitor of the HCV NS3 Serine Protease and HCV Subgenomic RNA Replication

    Energy Technology Data Exchange (ETDEWEB)

    Bogen, Stephane L.; Arasappan, Ashok; Bennett, Frank; Chen, Kevin; Jao, Edwin; Liu, Yi-Tsung; Lovey, Raymond G.; Venkatraman, Srikanth; Pan, Weidong; Parekh, Tajel; Pike, Russel E.; Ruan, Sumei; Liu, Rong; Baroudy, Bahige; Agrawal, Sony; Chase, Robert; Ingravallo, Paul; Pichardo, John; Prongay, Andrew; Brisson, Jean-Marc; Hsieh, Tony Y.; Cheng, Kuo-Chi; Kemp, Scott J.; Levy, Odile E.; Lim-Wilby, Marguerita; Tamura, Susan Y.; Saksena, Anil K.; Girijavallabhan, Viyyoor; Njoroge, F. George (SPRI)

    2008-06-30

    Introduction of various modified prolines at P{sub 2} and optimization of the P{sub 1} side chain led to the discovery of SCH6 (24, Table 2), a potent ketoamide inhibitor of the HCV NS3 serine protease. In addition to excellent enzyme potency (K*{sub i} = 3.8 nM), 24 was also found to be a potent inhibitor of HCV subgenomic RNA replication with IC{sub 50} and IC{sub 90} of 40 and 100 nM, respectively. Recently, antiviral activity of 24 was demonstrated with inhibition of the full-length genotype 2a HCV genome. In addition, 24 was found to restore the responsiveness of the interferon regulatory factor 3 (IRF-3) in cells containing HCV RNA replicons.

  8. Discovery of SCH446211 (SCH6): a new ketoamide inhibitor of the HCV NS3 serine protease and HCV subgenomic RNA replication.

    Science.gov (United States)

    Bogen, Stéphane L; Arasappan, Ashok; Bennett, Frank; Chen, Kevin; Jao, Edwin; Liu, Yi-Tsung; Lovey, Raymond G; Venkatraman, Srikanth; Pan, Weidong; Parekh, Tajel; Pike, Russel E; Ruan, Sumei; Liu, Rong; Baroudy, Bahige; Agrawal, Sony; Chase, Robert; Ingravallo, Paul; Pichardo, John; Prongay, Andrew; Brisson, Jean-Marc; Hsieh, Tony Y; Cheng, Kuo-Chi; Kemp, Scott J; Levy, Odile E; Lim-Wilby, Marguerita; Tamura, Susan Y; Saksena, Anil K; Girijavallabhan, Viyyoor; Njoroge, F George

    2006-05-04

    Introduction of various modified prolines at P(2) and optimization of the P(1) side chain led to the discovery of SCH6 (24, Table 2), a potent ketoamide inhibitor of the HCV NS3 serine protease. In addition to excellent enzyme potency (K(i)*= 3.8 nM), 24 was also found to be a potent inhibitor of HCV subgenomic RNA replication with IC(50) and IC(90) of 40 and 100 nM, respectively. Recently, antiviral activity of 24 was demonstrated with inhibition of the full-length genotype 2a HCV genome. In addition, 24 was found to restore the responsiveness of the interferon regulatory factor 3 (IRF-3) in cells containing HCV RNA replicons.

  9. Novel perspectives for hepatitis A virus therapy revealed by comparative analysis of hepatitis C virus and hepatitis A virus RNA replication.

    Science.gov (United States)

    Esser-Nobis, Katharina; Harak, Christian; Schult, Philipp; Kusov, Yuri; Lohmann, Volker

    2015-08-01

    Hepatitis A virus (HAV) and hepatitis C virus (HCV) are two positive-strand RNA viruses sharing a similar biology, but causing opposing infection outcomes, with HAV always being cleared and HCV establishing persistence in the majority of infections. To gain deeper insight into determinants of replication, persistence, and treatment, we established a homogenous cell-culture model allowing a thorough comparison of RNA replication of both viruses. By screening different human liver-derived cell lines with subgenomic reporter replicons of HAV as well as of different HCV genotypes, we found that Huh7-Lunet cells supported HAV- and HCV-RNA replication with similar efficiency and limited interference between both replicases. HAV and HCV replicons were similarly sensitive to interferon (IFN), but differed in their ability to establish persistent replication in cell culture. In contrast to HCV, HAV replicated independently from microRNA-122 and phosphatidylinositol 4-kinase IIIα and β (PI4KIII). Both viruses were efficiently inhibited by cyclosporin A and NIM811, a nonimmunosuppressive analog thereof, suggesting an overlapping dependency on cyclophilins for replication. However, analysis of a broader set of inhibitors revealed that, in contrast to HCV, HAV does not depend on cyclophilin A, but rather on adenosine-triphosphate-binding cassette transporters and FK506-binding proteins. Finally, silibinin, but not its modified intravenous formulation, efficiently inhibited HAV genome replication in vitro, suggesting oral silibinin as a potential therapeutic option for HAV infections. We established a cell-culture model enabling comparative studies on RNA replication of HAV and HCV in a homogenous cellular background with comparable replication efficiency. We thereby identified new host cell targets and potential treatment options for HAV and set the ground for future studies to unravel determinants of clearance and persistence. © 2015 by the American Association for the

  10. Structure of Hepatitis C Virus Polymerase in Complex with Primer-Template RNA

    Energy Technology Data Exchange (ETDEWEB)

    Mosley, Ralph T.; Edwards, Thomas E.; Murakami, Eisuke; Lam, Angela M.; Grice, Rena L.; Du, Jinfa; Sofia, Michael J.; Furman, Philip A.; Otto, Michael J. (Pharmasset); (Emerald)

    2012-08-01

    The replication of the hepatitis C viral (HCV) genome is accomplished by the NS5B RNA-dependent RNA polymerase (RdRp), for which mechanistic understanding and structure-guided drug design efforts have been hampered by its propensity to crystallize in a closed, polymerization-incompetent state. The removal of an autoinhibitory {beta}-hairpin loop from genotype 2a HCV NS5B increases de novo RNA synthesis by >100-fold, promotes RNA binding, and facilitated the determination of the first crystallographic structures of HCV polymerase in complex with RNA primer-template pairs. These crystal structures demonstrate the structural realignment required for primer-template recognition and elongation, provide new insights into HCV RNA synthesis at the molecular level, and may prove useful in the structure-based design of novel antiviral compounds. Additionally, our approach for obtaining the RNA primer-template-bound structure of HCV polymerase may be generally applicable to solving RNA-bound complexes for other viral RdRps that contain similar regulatory {beta}-hairpin loops, including bovine viral diarrhea virus, dengue virus, and West Nile virus.

  11. High awareness of hepatitis C virus (HCV) but limited knowledge of HCV complications among HIV-positive and HIV-negative men who have sex with men.

    Science.gov (United States)

    Lambers, Femke A E; Prins, Maria; Davidovich, Udi; Stolte, Ineke G

    2014-04-01

    Hepatitis C virus (HCV) has emerged as a sexually transmitted infection among HIV-positive men who have sex with men (MSM) in high-income countries. Little is reported about HCV awareness among MSM, although this is essential for developing targeted prevention strategies. We, therefore, studied HCV awareness and knowledge among HIV-positive and HIV-negative MSM from the Amsterdam Cohort Studies (ACS). During two visits, 1 year apart and starting in October 2007, MSM from the ACS answered questions regarding HCV awareness, knowledge of HCV transmission (7 items), complications (8 items) and sexual risk behaviour. We examined the percentage of HCV awareness and correctly answered knowledge items, and whether awareness and knowledge improved significantly over time. Using logistic regression, we studied whether HIV status and sexual risk behaviour were associated with awareness. Seventy percent (312/444) of HIV-negative and 80% (74/92) of HIV-positive MSM reported to have ever heard of HCV on the first visit. Overall, awareness increased with 9% between the first and second visit (p awareness was borderline significant (OR 1.49, 95% CI 0.97-2.30). Compared with knowledge of transmission routes, knowledge of complications appeared to be limited. In the ACS, awareness of HCV is high, particularly among those reporting group sex, an important risk factor for HCV transmission. The majority of participants had good knowledge of transmission routes, but limited knowledge of complications of chronic HCV infection. HCV prevention messages could be strengthened, therefore, by further addressing the complications of HCV infection.

  12. On-treatment HCV RNA as a predictor of sustained virological response in HCV genotype 3-infected patients treated with daclatasvir and sofosbuvir.

    Science.gov (United States)

    Kowdley, Kris V; Nelson, David R; Lalezari, Jacob P; Box, Terry; Gitlin, Norman; Poleynard, Gary; Rabinovitz, Mordechai; Ravendhran, Natarajan; Sheikh, Aasim M; Siddique, Asma; Bhore, Rafia; Noviello, Stephanie; Rana, Khurram

    2016-11-01

    Many currently available direct-acting antiviral (DAA) regimens are less effective against HCV genotype 3 than against other HCV genotypes. The all-oral, pangenotypic DAA combination of daclatasvir (NS5A inhibitor) + sofosbuvir (nucleotide NS5B inhibitor) was studied in genotype 3-infected treatment-naive and -experienced patients (ALLY-3) who achieved rates of sustained virological response at post-treatment Week 12 (SVR12) of 90 and 86% respectively. In this analysis, we assessed whether on-treatment responses to daclatasvir + sofosbuvir in genotype 3-infected patients could predict treatment outcome. In ALLY-3, treatment-naive and -experienced patients, with or without cirrhosis, were treated with daclatasvir + sofosbuvir for 12 weeks. HCV RNA kinetics and categorical virological responses on treatment were assessed. The proportions of responders and nonresponders by study week, and time to first undetectable HCV RNA, were analysed for utility in predicting treatment outcome. Overall, HCV RNA levels declined rapidly during Week 1 of treatment in both treatment-naive and -experienced cohorts. Although patients with cirrhosis had a slower initial virological response as measured by the proportion of patients with HCV RNA below the lower limit of quantification at Week 1, responses converged thereafter. Positive and negative predictive values calculated for on-treatment responses were generally comparable with the overall SVR12 rate and were therefore limited indicators of outcome. SVR12 rates were not impacted by time to first undetectable HCV RNA. On-treatment responses are not useful predictors of ultimate virological response to the daclatasvir + sofosbuvir regimen. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  13. Inhibition of full length Hepatitis C Virus particles of 1a genotype through small interference RNA

    Directory of Open Access Journals (Sweden)

    Rehman Sidra

    2011-05-01

    Full Text Available Abstract Background Hepatitis C virus (HCV, a member of the Flaviviridae family of viruses, is a major cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. Currently, the only treatment available consists of a combination of Pegylated interferon alpha (INF-α and ribavirin, but only half of the patients treated show a sufficient antiviral response. Thus there is a great need for the development of new treatments for HCV infections. RNA interference (RNAi represents a new promising approach to develop effective antiviral drugs and has been extremely effective against HCV infection. Results This study was design to assess or explore the silencing effect of small interference RNAs (siRNAs against full length HCV particles of genotype 1a. In the present study six 21-bp siRNAs were designed against different regions of HCV structural genes (Core, E1 and E2. Selected siRNAs were labeled as Csi 301, Csi 29, E1si 52, E1si 192, E2si 86 and E2si 493. Our results demonstrated that siRNAs directed against HCV core gene showed 70% reduction in viral titer in HCV infected liver cells. Moreover, siRNAs against E1 and E2 envelop genes showed a dramatic reduction in HCV viral RNA, E2si 86 exhibited 93% inhibition, while E1si 192, E2si 493 and E1si 52 showed 87%, 80%, and 66% inhibition respectively. No significant inhibition was detected in cells transfected with the negative control siRNA. Conclusion Our results suggested that siRNAs targeted against HCV structural genes efficiently silence full length HCV particles and provide an effective therapeutic option against HCV infection.

  14. Multicenter evaluation of the Elecsys® anti-HCV II assay for the diagnosis of hepatitis C virus infection.

    Science.gov (United States)

    Esteban, Juan I; van Helden, Josef; Alborino, Flora; Bürgisser, Philippe; Cellerai, Cristina; Pantaleo, Giuseppe; Eiras, Adolfo; Rodriguez, Maria I; Ghisetti, Valeria; Gleich, Michael; Imdahl, Roland; Kaiser, Claudia; Möller, Petra; Wetlitzky, Olaf; Segovia, Manuel; Schennach, Harald; Mühlbacher, Annelies

    2013-08-01

    Routine screening of patients at risk of hepatitis C virus (HCV) infection has become a priority given recent improvements in therapeutic options and the asymptomatic nature of most chronic infections. The aim of this study was to evaluate the performance of the Elecsys® Anti-HCV II assay, a new qualitative antibody immunoassay, compared with currently available assays, and assess its suitability for routine diagnostic testing. The sensitivity of the Elecsys® Anti-HCV II, ARCHITECT® Anti-HCV, AxSYM® HCV 3.0, PRISM® HCV, Vitros® ECi Anti-HCV, Elecsys® Anti-HCV, and ADVIA Centaur® HCV assays was compared using commercially available seroconversion panels and samples from patients known to be HCV positive and infected with HCV genotypes 1-6. Specificity was investigated using samples from blood donors, unselected hospitalized patients, and patients with potential cross-reacting factors or from high-risk groups. The Elecsys® Anti-HCV II assay detected more positive bleeds than the comparator assays, was more sensitive in recognizing early HCV infection, and correctly identified all 765 samples known to be HCV positive, regardless of genotype. The overall specificity of the Elecsys(®) Anti-HCV II assay was 99.84% (n=6,850) using blood donor samples, 99.66% (n=3,922) using samples from unselected hospitalized patients, and 99.66% (n=2,397) using samples from patients with potentially cross-reacting factors or from high-risk groups. The specificity of the Elecsys® Anti-HCV II assay was superior or equal to the comparator assays. In conclusion, the Elecsys® Anti-HCV II assay is a sensitive and specific assay suitable for routine use in the reliable detection of anti-HCV antibodies. Copyright © 2013 Wiley Periodicals, Inc.

  15. Comparison of Elecsys Anti-HCV II Assay With Other HCV Screening Assays.

    Science.gov (United States)

    Li, Dongdong; Zhu, Siyuan; Wang, Tingting; An, Jingna; Wang, Lanlan; Tao, Chuanmin

    2016-09-01

    Early detection of hepatitis C virus (HCV) is an important step in preventing progression to cirrhosis and hepatocellular carcinoma. Serologic assays for anti-HCV antibody are valuable first-line tests in the screening and diagnosis of HCV infection. This study's aim was to evaluate the sensitivity and specificity of Elecsys Anti-HCV II assay for HCV screening. A total of 1,044 routine sera, 20 known HCV-positive samples, plus 54 preselected weakly positive samples were tested for anti-HCV with Elecsys Anti-HCV II assay, Elecsys Anti-HCV assays, InTec HCV enzymoimmunoassay (EIA), and Livzon Anti-HCV EIA. Interference test was assessed with additional 423 specimens without clinical evidence of HCV infection: preselected HCV weak reactive samples; dialysis samples; anti-HBc (antibody to HBV core antigen) (+), anti-Treponema pallidum (+), and anti-HIV (+) sera; and samples form autoimmune/alcoholic hepatitis or systemic Lupus erythematosus (SLE). Discrepant results were evaluated with recombinant immunoblot assay. The seroconversion panels were evaluated to assess how early each assay could detect HCV infection. The specificity (99.81%) of the Elecsys Anti-HCV II assay was less than that with the two EIA comparison methods. However, false-negative results were easily seen in the EIA assays. When serial bleeds of HCV panels were compared with the above-mentioned methods, the assay detected acute HCV infection only 3.5 days after a positive HCV-RNA nucleic acid test and earlier than the comparator assays. Sensitivities and specificities of the anti-HCV assays were sufficiently high for use in this study. The Elecsys Anti-HCV II assay is suitable for screening and reliable early detection of HCV infection. © 2015 Wiley Periodicals, Inc.

  16. Phosphorylation of NS5A Serine-235 is essential to hepatitis C virus RNA replication and normal replication compartment formation

    Energy Technology Data Exchange (ETDEWEB)

    Eyre, Nicholas S., E-mail: nicholas.eyre@adelaide.edu.au [School of Biological Sciences and Research Centre for Infectious Diseases, University of Adelaide, Adelaide (Australia); Centre for Cancer Biology, SA Pathology, Adelaide (Australia); Hampton-Smith, Rachel J.; Aloia, Amanda L. [School of Biological Sciences and Research Centre for Infectious Diseases, University of Adelaide, Adelaide (Australia); Centre for Cancer Biology, SA Pathology, Adelaide (Australia); Eddes, James S. [Adelaide Proteomics Centre, School of Biological Sciences, University of Adelaide, Adelaide (Australia); Simpson, Kaylene J. [Victorian Centre for Functional Genomics, Peter MacCallum Cancer Centre, East Melbourne (Australia); The Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville (Australia); Hoffmann, Peter [Adelaide Proteomics Centre, School of Biological Sciences, University of Adelaide, Adelaide (Australia); Institute for Photonics and Advanced Sensing (IPAS), University of Adelaide, Adelaide (Australia); Beard, Michael R. [School of Biological Sciences and Research Centre for Infectious Diseases, University of Adelaide, Adelaide (Australia); Centre for Cancer Biology, SA Pathology, Adelaide (Australia)

    2016-04-15

    Hepatitis C virus (HCV) NS5A protein is essential for HCV RNA replication and virus assembly. Here we report the identification of NS5A phosphorylation sites Ser-222, Ser-235 and Thr-348 during an infectious HCV replication cycle and demonstrate that Ser-235 phosphorylation is essential for HCV RNA replication. Confocal microscopy revealed that both phosphoablatant (S235A) and phosphomimetic (S235D) mutants redistribute NS5A to large juxta-nuclear foci that display altered colocalization with known replication complex components. Using electron microscopy (EM) we found that S235D alters virus-induced membrane rearrangements while EM using ‘APEX2’-tagged viruses demonstrated S235D-mediated enrichment of NS5A in irregular membranous foci. Finally, using a customized siRNA screen of candidate NS5A kinases and subsequent analysis using a phospho-specific antibody, we show that phosphatidylinositol-4 kinase III alpha (PI4KIIIα) is important for Ser-235 phosphorylation. We conclude that Ser-235 phosphorylation of NS5A is essential for HCV RNA replication and normal replication complex formation and is regulated by PI4KIIIα. - Highlights: • NS5A residues Ser-222, Ser-235 and Thr-348 are phosphorylated during HCV infection. • Phosphorylation of Ser-235 is essential to HCV RNA replication. • Mutation of Ser-235 alters replication compartment localization and morphology. • Phosphatidylinositol-4 kinase III alpha is important for Ser-235 phosphorylation.

  17. [Capping strategies in RNA viruses].

    Science.gov (United States)

    Bouvet, Mickaël; Ferron, François; Imbert, Isabelle; Gluais, Laure; Selisko, Barbara; Coutard, Bruno; Canard, Bruno; Decroly, Etienne

    2012-04-01

    Most viruses use the mRNA-cap dependent cellular translation machinery to translate their mRNAs into proteins. The addition of a cap structure at the 5' end of mRNA is therefore an essential step for the replication of many virus families. Additionally, the cap protects the viral RNA from degradation by cellular nucleases and prevents viral RNA recognition by innate immunity mechanisms. Viral RNAs acquire their cap structure either by using cellular capping enzymes, by stealing the cap of cellular mRNA in a process named "cap snatching", or using virus-encoded capping enzymes. Many viral enzymes involved in this process have recently been structurally and functionally characterized. These studies have revealed original cap synthesis mechanisms and pave the way towards the development of specific inhibitors bearing antiviral drug potential. © 2012 médecine/sciences – Inserm / SRMS.

  18. Determinazione quantitativa di HCV-RNA: valutazione comparativa dei saggi Abbott Real-Time e Versant bDNA v.3

    Directory of Open Access Journals (Sweden)

    Aldo Manzin

    2007-06-01

    Full Text Available Hepatitis C virus (HCV RNA measurement before, during and after antiviral therapy has become an essential tool in the management of interferon-based treatment of HCV-related infections. Conventional Polymerase Chain Reaction (PCR has been largely used to obtain quantitative data, but laborious, time-consuming post-PCR handling steps are required to gain valuable results. Real time (RT PCR now provides advantages over end-point (EP PCR due to its improved rapidity, sensitivity, reproducibility and the reduced risk of carry-over contamination, and has now proven itself to be valuable for the more precise monitoring of viral load kinetics and assessing antiviral response.The Abbott Real-Time HCV-RNA is a recently introduced assay for the automated processing of clinical samples and HCV-RNA quantitation: its basic technology relies on use of fluorescent linear probes (dynamic range using 0.5 ml as input target= 12-108 IU/mL and a hybridization/detection step at low temperature (35°C, which allows target mismatches to be tolerated. To determine the clinical application of the Abbott Real-Time assay and defining its correlation with the Bayer Versant bDNA v.3 assay, 68 consecutive samples from unselected HCV-infected patients were retrospectively analysed with RT and the results obtained using the two tests compared.A good correlation was found between RT-PCR and bDNA: 97% of samples tested had a result within a 0.5 log HCV IU/mL difference (bias=0.15 log, whereas 6 samples negative with bDNA gave positive results with Abbott RT (range, 1.89-3.07 log IU/mL and “in-house” qualitative RT-PCR assays.

  19. The Combination of Grazoprevir, a Hepatitis C Virus (HCV) NS3/4A Protease Inhibitor, and Elbasvir, an HCV NS5A Inhibitor, Demonstrates a High Genetic Barrier to Resistance in HCV Genotype 1a Replicons.

    Science.gov (United States)

    Lahser, Frederick C; Bystol, Karin; Curry, Stephanie; McMonagle, Patricia; Xia, Ellen; Ingravallo, Paul; Chase, Robert; Liu, Rong; Black, Todd; Hazuda, Daria; Howe, Anita Y M; Asante-Appiah, Ernest

    2016-05-01

    The selection of resistance-associated variants (RAVs) against single agents administered to patients chronically infected with hepatitis C virus (HCV) necessitates that direct-acting antiviral agents (DAAs) targeting multiple viral proteins be developed to overcome failure resulting from emergence of resistance. The combination of grazoprevir (formerly MK-5172), an NS3/4A protease inhibitor, and elbasvir (formerly MK-8742), an NS5A inhibitor, was therefore studied in genotype 1a (GT1a) replicon cells. Both compounds were independently highly potent in GT1a wild-type replicon cells, with 90% effective concentration (EC90) values of 0.9 nM and 0.006 nM for grazoprevir and elbasvir, respectively. No cross-resistance was observed when clinically relevant NS5A and NS3 RAVs were profiled against grazoprevir and elbasvir, respectively. Kinetic analyses of HCV RNA reduction over 14 days showed that grazoprevir and elbasvir inhibited prototypic NS5A Y93H and NS3 R155K RAVs, respectively, with kinetics comparable to those for the wild-type GT1a replicon. In combination, grazoprevir and elbasvir interacted additively in GT1a replicon cells. Colony formation assays with a 10-fold multiple of the EC90 values of the grazoprevir-elbasvir inhibitor combination suppressed emergence of resistant colonies, compared to a 100-fold multiple for the independent agents. The selected resistant colonies with the combination harbored RAVs that required two or more nucleotide changes in the codons. Mutations in the cognate gene caused greater potency losses for elbasvir than for grazoprevir. Replicons bearing RAVs identified from resistant colonies showed reduced fitness for several cell lines and may contribute to the activity of the combination. These studies demonstrate that the combination of grazoprevir and elbasvir exerts a potent effect on HCV RNA replication and presents a high genetic barrier to resistance. The combination of grazoprevir and elbasvir is currently approved for

  20. Plant RNA binding proteins for control of RNA virus infection

    Directory of Open Access Journals (Sweden)

    Sung Un eHuh

    2013-12-01

    Full Text Available Plant RNA viruses have effective strategies to infect host plants through either direct or indirect interactions with various host proteins, thus suppressing the host immune system. When plant RNA viruses enter host cells exposed RNAs of viruses are recognized by the host immune system through processes such as siRNA-dependent silencing. Interestingly, some host RNA binding proteins have been involved in the inhibition of RNA virus replication, movement, and translation through RNA-specific binding. Host plants intensively use RNA binding proteins for defense against viral infections in nature. In this mini review, we will summarize the function of some host RNA binding proteins which act in a sequence-specific binding manner to the infecting virus RNA. It is important to understand how plants effectively suppresses RNA virus infections via RNA binding proteins, and this defense system can be potentially developed as a synthetic virus defense strategy for use in crop engineering.

  1. The present and future disease burden of hepatitis C virus (HCV) infection with today's treatment paradigm

    DEFF Research Database (Denmark)

    Razavi, H; Waked, I; Sarrazin, C

    2014-01-01

    The disease burden of hepatitis C virus (HCV) is expected to increase as the infected population ages. A modelling approach was used to estimate the total number of viremic infections, diagnosed, treated and new infections in 2013. In addition, the model was used to estimate the change in the tot...

  2. A Review of Hepatitis C Virus (HCV) and the Current Management ...

    African Journals Online (AJOL)

    Background: Chronic Hepatitis C virus (HCV) is the primary cause of cirrhosis, hepatocellular carcinoma (HCC), and end- stage liver disease. The addition of protease inhibitor with peginterferon alfa and ribavirin (triple therapy) for genotype 1 infected patients, are the current standard of care. Method: Data was sourced ...

  3. Uridine composition of the poly-U/UC tract of HCV RNA defines non-self recognition by RIG-I.

    Directory of Open Access Journals (Sweden)

    Gretja Schnell

    Full Text Available Viral infection of mammalian cells triggers the innate immune response through non-self recognition of pathogen associated molecular patterns (PAMPs in viral nucleic acid. Accurate PAMP discrimination is essential to avoid self recognition that can generate autoimmunity, and therefore should be facilitated by the presence of multiple motifs in a PAMP that mark it as non-self. Hepatitis C virus (HCV RNA is recognized as non-self by RIG-I through the presence of a 5'-triphosphate (5'-ppp on the viral RNA in association with a 3' poly-U/UC tract. Here we define the HCV PAMP and the criteria for RIG-I non-self discrimination of HCV by examining the RNA structure-function attributes that impart PAMP function to the poly-U/UC tract. We found that the 34 nucleotide poly-uridine "core" of this sequence tract was essential for RIG-I activation, and that interspersed ribocytosine nucleotides between poly-U sequences in the RNA were required to achieve optimal RIG-I signal induction. 5'-ppp poly-U/UC RNA variants that stimulated strong RIG-I activation efficiently bound purified RIG-I protein in vitro, and RNA interaction with both the repressor domain and helicase domain of RIG-I was required to activate signaling. When appended to 5'-ppp RNA that lacks PAMP activity, the poly-U/UC U-core sequence conferred non-self recognition of the RNA and innate immune signaling by RIG-I. Importantly, HCV poly-U/UC RNA variants that strongly activated RIG-I signaling triggered potent anti-HCV responses in vitro and hepatic innate immune responses in vivo using a mouse model of PAMP signaling. These studies define a multi-motif PAMP signature of non-self recognition by RIG-I that incorporates a 5'-ppp with poly-uridine sequence composition and length. This HCV PAMP motif drives potent RIG-I signaling to induce the innate immune response to infection. Our studies define a basis of non-self discrimination by RIG-I and offer insights into the antiviral therapeutic

  4. Virion-Independent Transfer of Replication-Competent Hepatitis C Virus RNA between Permissive Cells

    OpenAIRE

    Longatti, Andrea; Boyd, Bryan; Chisari, Francis V.

    2014-01-01

    In this study, we show that replication-competent subgenomic hepatitis C virus (HCV) RNA can be transferred to permissive Huh7 cells, leading to the establishment of viral RNA replication. Further, we show that these events are mediated by exosomes rather than infectious virus particles. If similar events occur in vivo, this could represent a novel, albeit inefficient, mechanism of viral spread and immune escape.

  5. Virion-independent transfer of replication-competent hepatitis C virus RNA between permissive cells.

    Science.gov (United States)

    Longatti, Andrea; Boyd, Bryan; Chisari, Francis V

    2015-03-01

    In this study, we show that replication-competent subgenomic hepatitis C virus (HCV) RNA can be transferred to permissive Huh7 cells, leading to the establishment of viral RNA replication. Further, we show that these events are mediated by exosomes rather than infectious virus particles. If similar events occur in vivo, this could represent a novel, albeit inefficient, mechanism of viral spread and immune escape. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  6. Risk of transmission associated with sharing drug injecting paraphernalia: analysis of recent hepatitis C virus (HCV) infection using cross-sectional survey data.

    Science.gov (United States)

    Palmateer, N; Hutchinson, S; McAllister, G; Munro, A; Cameron, S; Goldberg, D; Taylor, A

    2014-01-01

    Sharing injecting paraphernalia (containers, filters and water) poses a risk of transmitting the hepatitis C virus (HCV). The prevalence of, and risk of HCV from, such behaviour has not been extensively reported in Europe. People who inject drugs (PWID) were recruited in cross-sectional surveys from services providing sterile injecting equipment across Scotland between 2008 and 2010. Participants completed a questionnaire and provided a blood spot for anonymous testing. Logistic regression was used to examine the association between recent HCV infection (anti-HCV negative and HCV-RNA positive) and self-reported measures of injecting equipment sharing in the 6 months preceding interview. Twelve per cent of the sample reported sharing needles/syringes, and 40% reported sharing paraphernalia in the previous 6 months. The adjusted odds ratios (AOR) for sharing needles/syringes (+/- paraphernalia), and sharing only paraphernalia in the last 6 months were 6.7 (95% CI 2.6-17.1) and 3.0 (95% CI 1.2-7.5), respectively. Among those who reported not sharing needles/syringes, sharing containers and filters were both significantly associated with recent HCV infection (AOR 3.1, 95% CI 1.3-7.8 and 3.1, 95% CI 1.3-7.5, respectively); sharing water was not. We present the first study to apply a cross-sectional approach to the analysis of the association between sharing paraphernalia and incident HCV infection and demonstrate consistent results with previous longitudinal studies. The prevalence of paraphernalia sharing in our study population is high, representing significant potential for HCV transmission. © 2013 John Wiley & Sons Ltd.

  7. Detection of HCV core antigen and its diagnostic significance

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    YANG Jie

    2013-02-01

    Full Text Available ObjectiveTo compare the abilities of the hepatitis C virus (HCV core antigen (cAg test and the HCV RNA assay for confirming anti-HCV presence in order to determine the clinical utility of the HCV-cAg as an alternative or confirmatory diagnostic tool. MethodsSerum samples collected from 158 patients diagnosed with HCV infection were subjected to the enzyme-linked immunosorbent assay-based HCV-cAg test. The optical density (OD measured values were used to calculate the ratio of specimen absorbance to the cutoff value (S/CO. Simultaneously, the serum samples were subjected to PCR-based nucleic acid amplification quantitative fluorescence detection of HCV RNA. ResultsNone of the serum samples had a S/CO value <1 for the HCV-cAg test (100% negative, but all of the samples had a S/CO value >5 (100% positive. The HCV-cAg test sensitivity was 87.05%, specificity was 76.67%, positive predictive value was 9653%, and negative predictive value was 44.23%. As the S/CO value gradually increased, the significantly higher positive coincident rate of the HCV RNA test decreased. The HCV RNA negative coincident rate was significantly higher than that of the HCV-cAg test. HCV-cAg S/CO values between 1 and 2 corresponded to an HCV RNA values between 1.0×103 copies/ml and 1.0×104 copies/ml. The highest S/CO value obtained was 1.992. ConclusionThe HCV-cAg test is comparable to the HCV RNA assay for diagnosing HCV infection.

  8. Prophylactic and Therapeutic Vaccination against Hepatitis C Virus (HCV: Developments and Future Perspectives

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    Marian E. Major

    2009-08-01

    Full Text Available Studies in patients and chimpanzees that spontaneously clear Hepatitis C Virus (HCV have demonstrated that natural immunity to the virus is induced during primary infections and that this immunity can be cross protective. These discoveries led to optimism regarding prophylactic HCV vaccines and a number of studies in the chimpanzee model have been performed, all of which resulted in modified infections after challenge but did not always prevent persistence of the virus. Therapeutic vaccine strategies have also been pursued in an effort to reduce the costs and side effects associated with anti-viral drug treatment. This review summarizes the studies performed thus far in both patients and chimpanzees for prophylactic and therapeutic vaccination, assesses the progress made and future perspectives.

  9. Exosomes from Hepatitis C Infected Patients Transmit HCV Infection and Contain Replication Competent Viral RNA in Complex with Ago2-miR122-HSP90

    Science.gov (United States)

    Kodys, Karen; Bala, Shashi; Szabo, Gyongyi

    2014-01-01

    Antibodies targeting receptor-mediated entry of HCV into hepatocytes confer limited therapeutic benefits. Evidence suggests that exosomes can transfer genetic materials between cells; however, their role in HCV infection remains obscure. Here, we show that exosomes isolated from sera of chronic HCV infected patients or supernatants of J6/JFH1-HCV-infected Huh7.5 cells contained HCV RNA. These exosomes could mediate viral receptor-independent transmission of HCV to hepatocytes. Negative sense HCV RNA, indicative of replication competent viral RNA, was present in exosomes of all HCV infected treatment non-responders and some treatment-naïve individuals. Remarkably, HCV RNA was associated with Ago2, HSP90 and miR-122 in exosomes isolated from HCV-infected individuals or HCV-infected Huh7.5 cell supernatants. Exosome-loading with a miR-122 inhibitor, or inhibition of HSP90, vacuolar H+-ATPases, and proton pumps, significantly suppressed exosome-mediated HCV transmission to naïve cells. Our findings provide mechanistic evidence for HCV transmission by blood-derived exosomes and highlight potential therapeutic strategies. PMID:25275643

  10. Exosomes from hepatitis C infected patients transmit HCV infection and contain replication competent viral RNA in complex with Ago2-miR122-HSP90.

    Science.gov (United States)

    Bukong, Terence N; Momen-Heravi, Fatemeh; Kodys, Karen; Bala, Shashi; Szabo, Gyongyi

    2014-10-01

    Antibodies targeting receptor-mediated entry of HCV into hepatocytes confer limited therapeutic benefits. Evidence suggests that exosomes can transfer genetic materials between cells; however, their role in HCV infection remains obscure. Here, we show that exosomes isolated from sera of chronic HCV infected patients or supernatants of J6/JFH1-HCV-infected Huh7.5 cells contained HCV RNA. These exosomes could mediate viral receptor-independent transmission of HCV to hepatocytes. Negative sense HCV RNA, indicative of replication competent viral RNA, was present in exosomes of all HCV infected treatment non-responders and some treatment-naïve individuals. Remarkably, HCV RNA was associated with Ago2, HSP90 and miR-122 in exosomes isolated from HCV-infected individuals or HCV-infected Huh7.5 cell supernatants. Exosome-loading with a miR-122 inhibitor, or inhibition of HSP90, vacuolar H+-ATPases, and proton pumps, significantly suppressed exosome-mediated HCV transmission to naïve cells. Our findings provide mechanistic evidence for HCV transmission by blood-derived exosomes and highlight potential therapeutic strategies.

  11. Prevalence and Factors Associated with HCV (Hepatitis C Virus Seropositivity in Islamabad, Pakistan

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    Hammad Ali Qazi

    2010-12-01

    Full Text Available An estimated 150-200 million people worldwide are infected with hepatitis C. Prevalence is higher in some countries in Asia and Africa. Only limited information about the epidemiology of Hepatitis C Virus (HCV infection especially in females is available. The aim of this study is to determine the prevalence of anti-HCV antibodies and the possible factors for transmission in the female population of a largely urban city Islamabad. A cross sectional study was conducted from May 2006 to August 2006 in Islamabad. We select 252 female households (n=252 following the selection criteria. The primary outcome variables were HCV seropositivity and factors like history of major surgical procedure, blood transfusion, Intravenous drug use etc. The results showed mean age of the sample was 33.21 (±9.95 years and HCV seropositivity was present in 62 (24.6% females. Final Forward Stepwise multiple logistic regression showed blood transfusion [OR, 10.094 95% CI 1.950-52.257], dental procedure [OR, 5.381 95% CI 2.315-12.507] and dilation and curettage [OR, 3.869 95% 1.867-8.015] were significantly associated with HCV seropositivity in females. The study highlights poor quality of care provided and a massive need to educate general population including patients as well as health professionals and allied health workers.

  12. Prevalence and Factors Associated with HCV (Hepatitis C Virus Seropositivity in Islamabad, Pakistan

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    Hammad Ali Qazi

    2010-11-01

    Full Text Available "nAn estimated 150-200 million people worldwide are infected with hepatitis C. Prevalence is higher in some countries in Asia and Africa. Only limited information about the epidemiology of Hepatitis C Virus (HCV infection especially in females is available. The aim of this study is to determine the prevalence of anti-HCV antibodies and the possible factors for transmission in the female population of a largely urban city Islamabad. A cross sectional study was conducted from May 2006 to August 2006 in Islamabad. We select 252 female households (n=252 following the selection criteria. The primary outcome variables were HCV seropositivity and factors like history of major surgical procedure, blood transfusion, Intravenous drug use etc. The results showed mean age of the sample was 33.21 (±9.95 years and HCV seropositivity was present in 62 (24.6% females. Final Forward Stepwise multiple logistic regression showed blood transfusion [OR, 10.094 95% CI 1.950-52.257], dental procedure [OR, 5.381 95% CI 2.315-12.507] and dilation and curettage [OR, 3.869 95% 1.867-8.015] were significantly associated with HCV seropositivity in females. The study highlights poor quality of care provided and a massive need to educate general population including patients as well as health professionals and allied health workers.

  13. Targeting microRNA-122 to Treat Hepatitis C Virus Infection

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    Catherine L. Jopling

    2010-07-01

    Full Text Available An important host factor for hepatitis C virus (HCV is microRNA-122 (miR-122. miR-122 is a liver-specific member of a family of small, non-coding RNA molecules known as microRNAs that play major roles in the regulation of gene expression by direct interaction with RNA targets. miR-122 binds directly to two sites in the 5′ untranslated region (UTR of HCV RNA and positively regulates the viral life cycle. The mechanism by which this regulation occurs is still not fully understood. There has been a great deal of interest in potential therapeutics based on small RNAs, and targeting miR-122 to combat HCV is one of the furthest advanced. Chemical inhibitors of miR-122 can be introduced into mammals intravenously and result in potent and specific knockdown of the microRNA, with no detectable adverse effects on liver physiology. This strategy was recently applied to chimpanzees chronically infected with HCV and resulted in a sustained reduction in viral load in the animals. Inhibition of miR-122 therefore presents a very attractive novel approach to treating HCV, a virus for which improved therapeutics are urgently needed.

  14. Adenovirus vectors lacking virus-associated RNA expression enhance shRNA activity to suppress hepatitis C virus replication

    Science.gov (United States)

    Pei, Zheng; Shi, Guoli; Kondo, Saki; Ito, Masahiko; Maekawa, Aya; Suzuki, Mariko; Saito, Izumu; Suzuki, Tetsuro; Kanegae, Yumi

    2013-12-01

    First-generation adenovirus vectors (FG AdVs) expressing short-hairpin RNA (shRNA) effectively downregulate the expressions of target genes. However, this vector, in fact, expresses not only the transgene product, but also virus-associated RNAs (VA RNAs) that disturb cellular RNAi machinery. We have established a production method for VA-deleted AdVs lacking expression of VA RNAs. Here, we showed that the highest shRNA activity was obtained when the shRNA was inserted not at the popularly used E1 site, but at the E4 site. We then compared the activities of shRNAs against hepatitis C virus (HCV) expressed from VA-deleted AdVs or conventional AdVs. The VA-deleted AdVs inhibited HCV production much more efficiently. Therefore, VA-deleted AdVs were more effective than the currently used AdVs for shRNA downregulation, probably because of the lack of competition between VA RNAs and the shRNAs. These VA-deleted AdVs might enable more effective gene therapies for chronic hepatitis C.

  15. Primuline Derivatives That Mimic RNA to Stimulate Hepatitis C Virus NS3 Helicase-catalyzed ATP Hydrolysis*

    Science.gov (United States)

    Sweeney, Noreena L.; Shadrick, William R.; Mukherjee, Sourav; Li, Kelin; Frankowski, Kevin J.; Schoenen, Frank J.; Frick, David N.

    2013-01-01

    ATP hydrolysis fuels the ability of helicases and related proteins to translocate on nucleic acids and separate base pairs. As a consequence, nucleic acid binding stimulates the rate at which a helicase catalyzes ATP hydrolysis. In this study, we searched a library of small molecule helicase inhibitors for compounds that stimulate ATP hydrolysis catalyzed by the hepatitis C virus (HCV) NS3 helicase, which is an important antiviral drug target. Two compounds were found that stimulate HCV helicase-catalyzed ATP hydrolysis, both of which are amide derivatives synthesized from the main component of the yellow dye primuline. Both compounds possess a terminal pyridine moiety, which was critical for stimulation. Analogs lacking a terminal pyridine inhibited HCV helicase catalyzed ATP hydrolysis. Unlike other HCV helicase inhibitors, the stimulatory compounds differentiate between helicases isolated from various HCV genotypes and related viruses. The compounds only stimulated ATP hydrolysis catalyzed by NS3 purified from HCV genotype 1b. They inhibited helicases from other HCV genotypes (e.g. 1a and 2a) or related flaviviruses (e.g. Dengue virus). The stimulatory compounds interacted with HCV helicase in the absence of ATP with dissociation constants of about 2 μm. Molecular modeling and site-directed mutagenesis studies suggest that the stimulatory compounds bind in the HCV helicase RNA-binding cleft near key residues Arg-393, Glu-493, and Ser-231. PMID:23703611

  16. [Antigenicity of hepatitis C virus F protein and serum prevalence of anti-F in HCV-infected patients].

    Science.gov (United States)

    Shao, Sheng-Wen; Wu, Wen-Bin; Yu, Jian-Guo; Zhao, Ping; Qi, Zhong-Tian

    2006-12-01

    To examine the antigenicity of hepatitis C virus (HCV) F protein and investigate serum prevalence of anti-F in HCV-infected patients. Eleven pairs of overlapping primers were used to synthesize the full-length HCV f gene, from which the truncated HCV f65 gene fragment was amplified by PCR. HCV f65 gene was then cloned into pET32a(+), and transformed into E. coli strain Plyss (DE3). This recombinant E.coli was induced by IPTG for the production of HCV F65 protein. The expressed HCV F65 protein, purified by Ni-NTA agarose, was further used in ELISA to detect serum anti-F, and to immunize rabbits for making polyclonal anti-F. The rabbit polyclonal anti-F was purified by Staphylococcus aureus protein A agarose. After recombinant pET32a(+)-f65 was constructed successfully, HCV F65 protein was expressed and purified. The purified HCV F65 protein was used as a capture antigen in ELISA to detect serum anti-F in HCV infected patients (n = 30). The result showed that the mean A450 value and the positive rate of serum anti-F were 0.125+/-0.061 and 63.3%, respectively. The rabbit-derived polyclonal anti-F reacted specifically with HCV F65 protein, of which the titer was 1:30,000. Our expressed HCV F65 protein is of antigenicity, and can be used to determine serum anti-F. Anti-F IgG does exist in the sera of the HCV-infected patients. Moreover, the rabbit-derived polyclonal anti-F can be used to detect HCV F protein.

  17. Focal adhesion kinase (FAK mediates the induction of pro-oncogenic and fibrogenic phenotypes in hepatitis C virus (HCV-infected cells.

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    Anna Alisi

    Full Text Available Hepatitis C Virus (HCV infection is one of the most common etiological factors involved in fibrosis development and its progression to hepatocellular carcinoma (HCC. The pivotal role of hepatic stellate cells (HCSs and extracellular matrix (ECM in fibrogenesis is now certainly accepted, while the network of molecular interactions connecting HCV is emerging as a master regulator of several biological processes including proliferation, inflammation, cytoskeleton and ECM remodeling. In this study, the effects of HCV proteins expression on liver cancer cells, both pro-invasive and pro-fibrogenic phenotypes were explored. As a model of HCV infection, we used permissive Huh7.5.1 hepatoma cells infected with JFH1-derived ccHCV. Conditioned medium from these cells was used to stimulate LX-2 cells, a line of HSCs. We found that the HCV infection of Huh7.5.1 cells decreased adhesion, increased migration and caused the delocalization of alpha-actinin from plasma membrane to cytoplasm and increased expression levels of paxillin. The treatment of LX-2 cells, with conditioned medium from HCV-infected Huh7.5.1 cells, caused an increase in cell proliferation, expression of alpha-smooth muscle actin, hyaluronic acid release and apoptosis rate measured as cleaved poly ADP-ribose polymerase (PARP. These effects were accompanied in Huh7.5.1 cells by an HCV-dependent increasing of FAK activation that physically interacts with phosphorylated paxillin and alpha-actinin, and a rising of tumor necrosis factor alpha production/release. Silencing of FAK by siRNA reverted all effects of HCV infection, both those directed on Huh7.5.1 cells, and those indirect effects on the LX-2 cells. Moreover and interestingly, FAK inhibition enhances apoptosis in HCV-conditioned LX-2 cells. In conclusion, our findings demonstrate that HCV, through FAK activation, may promote cytoskeletal reorganization and a pro-oncogenic phenotype in hepatocyte-like cells, and a fibrogenic phenotype in

  18. New Insights in Recurrent HCV Infection after Liver Transplantation

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    Shih-Hsien Hsu

    2013-01-01

    Full Text Available Hepatitis C virus (HCV is a small-enveloped RNA virus belonging to the Flaviviridae family. Since first identified in 1989, HCV has been estimated to infect 170 million people worldwide. Mostly chronic hepatitis C virus has a uniform natural history, from liver cirrhosis to the development of hepatocellular carcinoma. The current therapy for HCV infection consists of a combination of Pegylated interferon and ribavirin. On the other hand, HCV-related liver disease is also the leading indication for liver transplantation. However, posttransplant HCV re-infection of the graft has been reported to be universal. Furthermore, the graft after HCV re-infection often results in accelerated progression to liver failure. In addition, treatment of recurrent HCV infection after liver transplantation is often compromised by enhanced adverse effects and limited efficacy of interferon-based therapies. Taken together, poor outcome after HCV re-infection, regardless of grafts or recipients, poses a major issue for the hepatologists and transplant surgeons. The aim of this paper is to review several specific aspects regarding HCV re-infection after transplant: risk factors, current therapeutics for HCV in different stages of liver transplantation, cellular function of HCV proteins, and molecular mechanisms of HCV entry. Hopefully, this paper will inspire new strategies and novel inhibitors against recurrent HCV infection after liver transplantation and greatly improve its overall outcome.

  19. Sofosbuvir and Ledipasvir for 8 Weeks for the Treatment of Chronic Hepatitis C Virus (HCV) Infection in HCV-Monoinfected and HIV-HCV-Coinfected Individuals: Results From the German Hepatitis C Cohort (GECCO-01).

    Science.gov (United States)

    Ingiliz, Patrick; Christensen, Stefan; Kimhofer, Torben; Hueppe, Dietrich; Lutz, Thomas; Schewe, Knud; Busch, Heiner; Schmutz, Günther; Wehmeyer, Malte H; Boesecke, Christoph; Simon, Karl-Georg; Berger, Florian; Rockstroh, Jürgen K; Schulze zur Wiesch, Julian; Baumgarten, Axel; Mauss, Stefan

    2016-11-15

    Shortening the duration of treatment with HCV direct-acting antivirals (DAAs) leads to substantial cost reductions. According to the label, sofosbuvir and ledipasvir can be prescribed for 8 weeks (SL8) in noncirrhotic women or men with HCV genotype 1 and low viral loads. However, real-world data about the efficacy and safety of SL8 are largely missing. Interim results from an ongoing prospective, multicenter cohort of 9 treatment centers in Germany (GECCO). All patients started on treatment with HCV DAAs since January 2014 were included. This report describes safety and efficacy outcomes in 210 patients with HCV monoinfection and 35 with human immunodeficiency virus (HIV)-HCV coinfection given SL8 in a real-world setting. Of 1353 patients included into the GECCO cohort until December 2015, a total of 1287 had complete data sets for this analysis; 337 (26.2%) fulfilled the criteria for SL8 according to the package insert, but only 193 (57.2%) were eventually treated for 8 weeks. Another 52 patients did not fulfill the criteria but were treated for 8 weeks. SL8 was generally well tolerated. The overall sustained virologic response rate 12 weeks after the end of treatment was 93.5% (186 of 199). The on-treatment response rate was 99.4% (159 of 160) in HCV-monoinfected and 96.4% (27 of 28) in HIV-HCV-coinfected patients. Ten patients were lost to follow-up. SL8 seems highly effective and safe in well-selected HCV-monoinfected and HIV-HCV-coinfected patients in a real-world setting. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

  20. A miRNA-responsive cell-free translation system facilitates isolation of hepatitis C virus miRNP complexes

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    Bradrick, Shelton S.; Nagyal, Simardeep; Novatt, Hilary

    2013-01-01

    Micro(mi)RNAs are 21- to 23-nt RNAs that regulate multiple biological processes. In association with Argonaute (Ago) proteins and other factors that form the RNA-induced silencing complex (RISC), miRNAs typically bind mRNA 3′ untranslated regions (UTRs) and repress protein production through antagonizing translation and transcript stability. For a given mRNA–miRNA interaction, cis-acting RNA elements and trans-acting RNA-binding proteins (RBPs) may influence mRNA fate. This is particularly true of the hepatitis C virus (HCV) genome which interacts with miR-122, an abundant liver miRNA. miR-122 binding to HCV RNA considerably stimulates virus replication in cultured cells and primates, but the mechanism(s) and associated host factors required for enhancement of HCV replication have not been fully elucidated. We recapitulated miR-122–HCV RNA interactions in a cell-free translation system derived from cells that express miR-122. Specifically, lysates produced from HEK-293 cells that inducibly transcribe and process pri-miR-122 were characterized alongside those from isogenic cells lacking miR-122 expression. We observed a stimulatory effect of miR-122 on HCV reporter mRNAs in a manner that depended on expression of miR-122 and intact target sites within the HCV 5′ UTR. We took advantage of this system to affinity-purify miR-122-HCV RNP complexes. Similar to functional assays, we found that association of immobilized HCV internal ribosome entry site (IRES) RNA with endogenous Ago2 requires both miR-122 expression and intact miR-122 target sites in cis. This combined approach may be generalizable to affinity purification of miRNP complexes for selected target mRNAs, allowing identification of miRNP components and RBPs that may contribute to regulation. PMID:23793894

  1. New pandemics: HIV and AIDS, HCV and chronic hepatitis, Influenza virus and flu

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    Cohen Éric A

    2007-02-01

    Full Text Available Abstract New pandemics are a serious threat to the health of the entire world. They are essentially of viral origin and spread at large speed. A meeting on this topic was held in Lyon, France, within the XIXth Jacques Cartier Symposia, a series of France-Québec meetings held every year. New findings on HIV and AIDS, on HCV and chronic hepatitis, and an update on influenza virus and flu were covered during this meeting on December 4 and 5, 2006. Aspects of viral structure, virus-host interactions, antiviral defenses, drugs and vaccinations, and epidemiological aspects were discussed for HIV and HCV. Old and recent data on the flu epidemics ended this meeting.

  2. A magnesium-induced RNA conformational switch at the internal ribosome entry site of hepatitis C virus genome visualized by atomic force microscopy

    OpenAIRE

    García-Sacristán, Ana; Moreno Molina, Miguel; Ariza-Mateos, Ascensión; López-Camacho, Elena; Jáudenes, Rosa M.; Vázquez, Luis; Gómez, Jordi; Martín-Gago, José A.; Briones, Carlos

    2014-01-01

    The 5? untranslated region of hepatitis C virus (HCV) genomic RNA contains an internal ribosome entry site (IRES) element, composed of domains II?IV, which is required for cap-independent translation initiation. Little information on the 3D structure of the whole functional HCV IRES is still available. Here, we use atomic force microscopy to visualize the HCV IRES conformation in its natural sequence context, which includes the upstream domain I and the essential, downstream domains V and VI....

  3. Point -of -care testing (POCT) in molecular diagnostics: Performance evaluation of GeneXpert HCV RNA test in diagnosing and monitoring of HCV infection.

    Science.gov (United States)

    Gupta, Ekta; Agarwala, Pragya; Kumar, Guresh; Maiwall, Rakhi; Sarin, Shiv Kumar

    2017-03-01

    Molecular testing at the point-of-care may turn out to be game changer for HCV diagnosis and treatment monitoring, through increased sensitivity, reduced turnaround time, and ease of performance. One such assay GeneXpert(®) has recently been released. Comparative analysis between performances of GeneXpert(®) and Abbott HCV-RNA was done. 174 HCV infected patients were recruited and, one time plasma samples from 154 patients and repeated samples from 20 patients, obtained at specific treatment time-points (0, 4, 12 and 24) weeks were serially re-tested on Xpert(®). Genotype 3 was the commonest, seen in 80 (66%) of the cases, genotype 1 in 34 (28.3%), genotype 4 in 4 (3.3%) and genotypes 2 and 5 in 1 (0.8%) each. Median HCV RNA load was 4.69 log10 (range: 0-6.98log10) IU/ml. Overall a very good correlation was seen between the two assays (R(2)=0.985), concordance of the results between the assays was seen in 138 samples (89.6%). High and low positive standards were tested ten times on Xpert(®) to evaluate the precision and the coefficient of variation was 0.01 for HPC and 0.07 for the LPC. Monitoring of patients on two different regimes of treatment, pegylated interferon plus ribavirin and sofosbuvir plus ribavirin was done by both the systems at baseline, 4, 12 and 24 weeks. Perfect correlation between the assays in the course of therapy at different treatment time- point in genotypes 3 and 1 was seen. The study demonstrates excellent performance of the Xpert(®) HCV assay in viral load assessment and in treatment course monitoring consistency. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Quantification of C4d deposition and hepatitis C virus RNA in tissue in cases of graft rejection and hepatitis C recurrence after liver transplantation

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    Alice Tung Wan Song

    2015-02-01

    Full Text Available Histology is the gold standard for diagnosing acute rejection and hepatitis C recurrence after liver transplantation. However, differential diagnosis between the two can be difficult. We evaluated the role of C4d staining and quantification of hepatitis C virus (HCV RNA levels in liver tissue. This was a retrospective study of 98 liver biopsy samples divided into four groups by histological diagnosis: acute rejection in patients undergoing liver transplant for hepatitis C (RejHCV+, HCV recurrence in patients undergoing liver transplant for hepatitis C (HCVTx+, acute rejection in patients undergoing liver transplant for reasons other than hepatitis C and chronic hepatitis C not transplanted (HCVTx-. All samples were submitted for immunohistochemical staining for C4d and HCV RNA quantification. Immunoexpression of C4d was observed in the portal vessels and was highest in the HCVTx- group. There was no difference in C4d expression between the RejHCV+ and HCVTx+ groups. However, tissue HCV RNA levels were higher in the HCVTx+ group samples than in the RejHCV+ group samples. Additionally, there was a significant correlation between tissue and serum levels of HCV RNA. The quantification of HCV RNA in liver tissue might prove to be an efficient diagnostic test for the recurrence of HCV infection.

  5. Quantitation of HCV RNA in liver of patients with chronic hepatitis C Quantificação do RNA-HCV no fígado de pacientes com hepatite C crônica

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    Ana de Lôurdes Candolo MARTINELLI

    2000-10-01

    Full Text Available Background/Aims - Liver HCV RNA has been quantitated in few studies and the feasibility and the role of this parameter in the evaluation of patients with chronic HCV hepatitis still warrant study. Our aim was to determine the concentrations of HCV RNA in the liver of chronic HCV patients and to correlate the results with serum viral load. We also studied the relation of levels of HCV RNA in the liver with serum aminotransferases levels and with the presence of cirrhosis. Methods - Twenty patients (14 males, aged 28 to 61 years were studied. Twelve were infected by HCV type 1, six by type 3 and one by type 5. Percutaneous liver biopsy samples were obtained from 14 patients, and the remainder from liver explant in patients undergoing OLT. Twelve had chronic hepatitis and eight cirrhosis. HCV RNA levels were determined by bDNA. Results - HCV RNA levels below the detection limit were found in one liver and in five serum samples. HCV RNA (mean ± SD was 2.1 x 10(8 ± 2.2 x 10(8Eq/gm in the liver and 94 x 10(5 ± 93 x 10(5Eq/mL in serum, with a significant correlation between these values (r = 0.89; P Introdução/Objetivos - Poucos estudos avaliam a quantificação do RNA-HCV no fígado, portanto a praticabilidade e a aplicação desse parâmetro na avaliação de pacientes com hepatite C crônica ainda não estão definidas. O objetivo foi determinar as concentrações do RNA-HCV no fígado de pacientes com infecção crônica pelo vírus C da hepatite e correlacionar os resultados com a carga viral do soro. Foram também estudadas a relação dos níveis de RNA-HCV no fígado com os de aminotransferases no soro e com a presença de cirrose. Métodos - Foram estudados 20 pacientes (14 homens, 28 a 61 anos. A genotipagem do vírus da hepatite C revelou: tipo 1 (12 pacientes, tipo 3 (6 pacientes , tipo 5 (1 paciente. Amostras de fígado foram obtidas por via percutânea em 14 pacientes e de explantes de fígado de pacientes submetidos a transplante em

  6. Challenges in RNA virus bioinformatics.

    Science.gov (United States)

    Marz, Manja; Beerenwinkel, Niko; Drosten, Christian; Fricke, Markus; Frishman, Dmitrij; Hofacker, Ivo L; Hoffmann, Dieter; Middendorf, Martin; Rattei, Thomas; Stadler, Peter F; Töpfer, Armin

    2014-07-01

    Computer-assisted studies of structure, function and evolution of viruses remains a neglected area of research. The attention of bioinformaticians to this interesting and challenging field is far from commensurate with its medical and biotechnological importance. It is telling that out of >200 talks held at ISMB 2013, the largest international bioinformatics conference, only one presentation explicitly dealt with viruses. In contrast to many broad, established and well-organized bioinformatics communities (e.g. structural genomics, ontologies, next-generation sequencing, expression analysis), research groups focusing on viruses can probably be counted on the fingers of two hands. The purpose of this review is to increase awareness among bioinformatics researchers about the pressing needs and unsolved problems of computational virology. We focus primarily on RNA viruses that pose problems to many standard bioinformatics analyses owing to their compact genome organization, fast mutation rate and low evolutionary conservation. We provide an overview of tools and algorithms for handling viral sequencing data, detecting functionally important RNA structures, classifying viral proteins into families and investigating the origin and evolution of viruses. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  7. Evolving strategy for HCV testing in an Italian tertiary care hospital.

    Science.gov (United States)

    Medici, Maria Cristina; Chezzi, Carlo; De Conto, Flora; Ferraglia, Francesca; Pinardi, Federica; Arcangeletti, Maria Cristina; Bernasconi, Daniela; Galli, Claudio; Calderaro, Adriana

    2016-04-01

    Diagnostic tests for hepatitis C virus (HCV) infection should be adapted according to the clinical status of the patient. We exploited the application of different HCV diagnostic algorithms in a tertiary care hospital practice. The laboratory clinical reports to the medical orders for HCV testing during three years were clustered by different combinations of assays for anti-HCV antibodies (HCV Ab) (screening and confirmatory), HCV nucleic acid (HCV-RNA), HCV core antigen (HCV Ag). The latter was the first-line assay in acute HCV infections requiring a rapid assessment of the infectious state. The majority (91.9%) of the 2726 subjects whose samples were analyzed were inpatients. Most of the patients/subjects were tested for clinical suspicion of viral hepatitis (49.2%), or occupational accident to health care professionals (20.0%). On 66% of samples HCV Ag test alone was performed and resulted positive in 116 cases (6%), while it was detected in 50.3% of anti-HCV positive samples. The agreement between HCV Ag and HCV-RNA was very high (k=0.97); HCV Ag positivity rates increased according to the signal of the HCV Ab screening test. The use of different testing strategies according to the patients' history and clinical status allowed a significant reduction of the number of tests performed and the time needed to provide a diagnostic response useful for patients' management without compromising the overall diagnostic accuracy for HCV infection. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Preclinical evaluation of multi antigenic HCV DNA vaccine for the prevention of Hepatitis C virus infection.

    Science.gov (United States)

    Lee, Hyojin; Jeong, Moonsup; Oh, Jooyeon; Cho, Youngran; Shen, Xuefei; Stone, John; Yan, Jian; Rothkopf, Zachary; Khan, Amir S; Cho, Byung Mun; Park, Young K; Weiner, David B; Son, Woo-Chan; Maslow, Joel N

    2017-03-07

    Direct-acting antiviral treatment for hepatitis C virus (HCV) infection is costly and does not protect from re-infection. For human and chimpanzees, recovery from acute HCV infection correlates with host CD4+ and CD8+ T cell responses. DNA plasmids targeting the HCV non-structural antigens NS3, NS4, and NS5, were previously reported to induce robust and sustained T cell responses in mice and primates. These plasmids were combined with a plasmid encoding cytokine IL-28B, together named as VGX-6150. The dose-dependent T cell response and safety of VGX-6150 administered intramuscularly and followed by electroporation was assessed in mice. Immune responses plateaued at 20 μg/dose with IL-28B demonstrating significant immunoadjuvant activity. Mice administered VGX-6150 at 40, 400, and 800 μg given either as a single injection or as 14 injections given bi-weekly over 26 weeks showed no vaccine related changes in any clinical parameter compared to placebo recipients. There was no evidence of VGX-6150 accumulation at the injection site or in any organ 1 month following the 14th vaccination. Based on these studies, the approximate lethal dose (ALD) exceeds 800 μg/dose and the NOAEL was 800 μg/dose in mouse. In conclusion, VGX-6150 appears safe and a promising preventive vaccine candidate for HCV infection.

  9. Claudin-1 required for HCV virus entry has high potential for phosphorylation and O-glycosylation

    Directory of Open Access Journals (Sweden)

    Fouzia Kiran

    2011-05-01

    Full Text Available Abstract HCV is a leading cause of hepatocellular carcinoma and cirrhosis all over the world. Claudins belong to family of tight junction's proteins that are responsible for establishing barriers for controlling the flow of molecules around cells. For therapeutic strategies, regulation of viral entry into the host cells holds a lot of promise. During HCV infection claudin-1 is highly expressed in liver and believed to be associated with HCV virus entry after HCV binding with or without co-receptor CD81. The claudin-1 assembly with tight junctions is regulated by post translational modifications. During claudins assembly and disassembly with tight junctions, phosphorylation is required at C-terminal tail. In cellular proteins, interplay between phosphorylation and O-β-GlcNAc modification is believed to be functional switch, but it is very difficult to monitor these functional and vibrant changes in vivo. Netphos 2.0 and Disphos 1.3 programs were used for potential phosphorylation; NetPhosK 1.0 and KinasePhos for kinase prediction; and YinOYang 1.2 and OGPET to predict possible O-glycosylation sites. We also identified Yin Yang sites that may have potential for O-β-GlcNAc and phosphorylation interplay at same Ser/Thr residues. We for the first time proposed that alternate phosphorylation and O-β-GlcNAc modification on Ser 192, Ser 205, Ser 206; and Thr 191 may provide an on/off switch to regulate assembly of claudin-1 at tight junctions. In addition these phosphorylation sites may be targeted by novel chemotherapeutic agents to prevent phosphorylation lead by HCV viral entry complex.

  10. Inhibition of Hepatitis C Virus in Mice by a Small Interfering RNA Targeting a Highly Conserved Sequence in Viral IRES Pseudoknot.

    Directory of Open Access Journals (Sweden)

    Jae-Su Moon

    Full Text Available The hepatitis C virus (HCV internal ribosome entry site (IRES that directs cap-independent viral translation is a primary target for small interfering RNA (siRNA-based HCV antiviral therapy. However, identification of potent siRNAs against HCV IRES by bioinformatics-based siRNA design is a challenging task given the complexity of HCV IRES secondary and tertiary structures and association with multiple proteins, which can also dynamically change the structure of this cis-acting RNA element. In this work, we utilized siRNA tiling approach whereby siRNAs were tiled with overlapping sequences that were shifted by one or two nucleotides over the HCV IRES stem-loop structures III and IV spanning nucleotides (nts 277-343. Based on their antiviral activity, we mapped a druggable region (nts 313-343 where the targets of potent siRNAs were enriched. siIE22, which showed the greatest anti-HCV potency, targeted a highly conserved sequence across diverse HCV genotypes, locating within the IRES subdomain IIIf involved in pseudoknot formation. Stepwise target shifting toward the 5' or 3' direction by 1 or 2 nucleotides reduced the antiviral potency of siIE22, demonstrating the importance of siRNA accessibility to this highly structured and sequence-conserved region of HCV IRES for RNA interference. Nanoparticle-mediated systemic delivery of the stability-improved siIE22 derivative gs_PS1 siIE22, which contains a single phosphorothioate linkage on the guide strand, reduced the serum HCV genome titer by more than 4 log10 in a xenograft mouse model for HCV replication without generation of resistant variants. Our results provide a strategy for identifying potent siRNA species against a highly structured RNA target and offer a potential pan-HCV genotypic siRNA therapy that might be beneficial for patients resistant to current treatment regimens.

  11. Differential efficacy of protease inhibitors against HCV genotypes 2a, 3a, 5a, and 6a NS3/4A protease recombinant viruses

    DEFF Research Database (Denmark)

    Gottwein, Judith M; Scheel, Troels K H; Jensen, Tanja B

    2011-01-01

    The hepatitis C virus (HCV) genotype influences efficacy of interferon (IFN)-based therapy. HCV protease inhibitors are being licensed for treatment of genotype 1 infection. Because there are limited or no data on efficacy against HCV genotypes 2-7, we aimed at developing recombinant infectious c...... cell culture systems expressing genotype-specific nonstructural (NS) protein 3 protease (NS3P)....

  12. Initiation of RNA Synthesis by the Hepatitis C Virus RNA-Dependent RNA Polymerase Is Affected by the Structure of the RNA Template

    Science.gov (United States)

    2015-01-01

    The hepatitis C virus (HCV) RNA-dependent RNA polymerase NS5B is a central enzyme of the intracellular replication of the viral (+)RNA genome. Here, we studied the individual steps of NS5B-catalyzed RNA synthesis by a combination of biophysical methods, including real-time 1D 1H NMR spectroscopy. NS5B was found to bind to a nonstructured and a structured RNA template in different modes. Following NTP binding and conversion to the catalysis-competent ternary complex, the polymerase revealed an improved affinity for the template. By monitoring the folding/unfolding of 3′(−)SL by 1H NMR, the base pair at the stem’s edge was identified as the most stable component of the structure. 1H NMR real-time analysis of NS5B-catalyzed RNA synthesis on 3′(−)SL showed that a pronounced lag phase preceded the processive polymerization reaction. The presence of the double-stranded stem with the edge base pair acting as the main energy barrier impaired RNA synthesis catalyzed by NS5B. Our observations suggest a crucial role of RNA-modulating factors in the HCV replication process. PMID:25310724

  13. Exploiting hepatitis C virus activation of NFkappaB to deliver HCV-responsive expression of interferons alpha and gamma.

    Science.gov (United States)

    Matskevich, A A; Strayer, D S

    2003-10-01

    Chronic infection with hepatitis C virus (HCV) may lead to liver failure and hepatocellular carcinoma. Current treatment for HCV includes high systemic doses of interferonalpha (IFNalpha), which is effective in less than half of patients and may have severe side effects. We designed conditional IFNalpha and IFNgamma expression constructs to be triggered by HCV-induced activation of NFkappaB, and delivered these using highly efficient recombinant Tag-deleted SV40-derived vectors. NFkappaB activates the HIV-1NL4-3 long terminal repeat (HIVLTR) as a promoter, which accounts for the conditional transgene expression. Human hepatocyte lines and primary rat hepatocytes (PRH) were transduced with SV[HIVLTR](IFN) vectors, and transfected with HCV cDNA. Production of human and murine IFNalpha and IFNgamma in cytosol and culture supernatants was measured. HCV activated the HIVLTR to produce and secrete IFNs, and did so largely through the NFkappaB binding sites of the HIVLTR. Levels of IFNs secreted, and the magnitude of induction in response to HCV, were greater in hepatocyte lines than in primary cultured hepatocytes. However, even in the latter, supernatant IFNalpha concentrations achieved by this approach were similar to therapeutic serum concentrations sought in systemic IFNalpha-treated patients. In coculture studies, secreted IFNalpha activated its cognate response elements in untransduced cells, suggesting that its potential inhibitory effects on HCV may not be limited to transduced cells. Although HCV replication in culture is difficult to assess, HCV-induced IFNalpha production demonstrably reduced HCV transcription. Conditional expression of IFNs within the liver may represent an attractive approach to therapy of severe chronic HCV infection that could avoid the side effects of systemic treatment regimens.

  14. GB Virus C (GBV-C Infection in Hepatitis C Virus (HCV Seropositive Women with or at Risk for HIV Infection.

    Directory of Open Access Journals (Sweden)

    Jason T Blackard

    Full Text Available GB virus C (GBV-C may have a beneficial impact on HIV disease progression; however, the epidemiologic characteristics of this virus are not well characterized. Behavioral factors and gender may lead to differential rates of GBV-C infection; yet, studies have rarely addressed GBV-C infections in women or racial/ethnic minorities. Therefore, we evaluated GBV-C RNA prevalence and genotype distribution in a large prospective study of high-risk women in the US.438 hepatitis C virus (HCV seropositive women, including 306 HIV-infected and 132 HIV-uninfected women, from the HIV Epidemiologic Research Study were evaluated for GBV-C RNA. 347 (79.2% women were GBV-C RNA negative, while 91 (20.8% were GBV-C RNA positive. GBV-C positive women were younger than GBV-C negative women. Among 306 HIV-infected women, 70 (22.9% women were HIV/GBV-C co-infected. Among HIV-infected women, the only significant difference between GBV-negative and GBV-positive women was age (mean 38.4 vs. 35.1 years; p<0.001. Median baseline CD4 cell counts and plasma HIV RNA levels were similar. The GBV-C genotypes were 1 (n = 31; 44.3%, 2 (n = 36; 51.4%, and 3 (n = 3; 4.3%. The distribution of GBV-C genotypes in co-infected women differed significantly by race/ethnicity. However, median CD4 cell counts and log10 HIV RNA levels did not differ by GBV-C genotype. GBV-C incidence was 2.7% over a median follow-up of 2.9 (IQR: 1.5, 4.9 years, while GBV-C clearance was 35.7% over a median follow-up of 2.44 (1.4, 3.5 years. 4 women switched genotypes.Age, injection drug use, a history of sex for money or drugs, and number of recent male sex partners were associated with GBV-C infection among all women in this analysis. However, CD4 cell count and HIV viral load of HIV/HCV/GBV-C co-infected women were not different although race was associated with GBV-C genotype.

  15. Stabilization of hepatitis C virus RNA by an Ago2–miR-122 complex

    Science.gov (United States)

    Shimakami, Tetsuro; Yamane, Daisuke; Jangra, Rohit K.; Kempf, Brian J.; Spaniel, Carolyn; Barton, David J.; Lemon, Stanley M.

    2012-01-01

    MicroRNAs (miRNAs) are small noncoding RNAs that regulate eukaryotic gene expression by binding to regions of imperfect complementarity in mRNAs, typically in the 3′ UTR, recruiting an Argonaute (Ago) protein complex that usually results in translational repression or destabilization of the target RNA. The translation and decay of mRNAs are closely linked, competing processes, and whether the miRNA-induced silencing complex (RISC) acts primarily to reduce translation or stability of the mRNA remains controversial. miR-122 is an abundant, liver-specific miRNA that is an unusual host factor for hepatitis C virus (HCV), an important cause of liver disease in humans. Prior studies show that it binds the 5′ UTR of the messenger-sense HCV RNA genome, stimulating translation and promoting genome replication by an unknown mechanism. Here we show that miR-122 binds HCV RNA in association with Ago2 and that this slows decay of the viral genome in infected cells. The stabilizing action of miR-122 does not require the viral RNA to be translationally active nor engaged in replication, and can be functionally substituted by a nonmethylated 5′ cap. Our data demonstrate that a RISC-like complex mediates the stability of HCV RNA and suggest that Ago2 and miR-122 act coordinately to protect the viral genome from 5′ exonuclease activity of the host mRNA decay machinery. miR-122 thus acts in an unconventional fashion to stabilize HCV RNA and slow its decay, expanding the repertoire of mechanisms by which miRNAs modulate gene expression. PMID:22215596

  16. Transmission of HCV infection among long-term hospitalized onco-haematological patients.

    Science.gov (United States)

    Januszkiewicz-Lewandowska, D; Wysocki, J; Rembowska, J; Pernak, M; Lewandowski, K; Nowak, T; Nowicka-Kujawska, K; Nowak, J

    2003-02-01

    Hepatitis C virus (HCV) infection is becoming a substantial problem in long-term hospitalized patients. Onco-haematological patients undergoing chemotherapy are especially prone to HCV infection. These patients are usually immunosuppressed and therefore antibodies to HCV are not produced despite the presence of HCV RNA in peripheral blood. The aim of the study was to see how often long-term hospitalized patients acquired HCV infection, and what were the possible sources and routes of virus transmission. The study involved 129 children with lymphoproliferative diseases, 36 patients with solid tumours, and 61 healthcare workers from onco-haematological wards. All were HCV RNA and anti-HCV negative at the time of first hospitalization. During a two and a half-year follow-up study among 165 onco-haematological patients, HCV RNA appeared in 87 in subsequent hospitalizations. The majority of infections were (82/87) were 1a genotype, 2 were 1b, 1 was 1a + 1b and 1 was 1a + 3a. In an attempt to establish the origin of HCV infection, healthcare workers were screened for HCV genotyping. All HCV-infected staff working on wards had the same genotype (1a). None of the staff was infected with 1b genotype. As the most prevalent genotype in Polish blood donors is 1b, HCV infection in onco-haematological patients is most likely due to horizontal transmission, probably involving genotype 1a, and potential horizontal transmission of HCV is implied by the presence of 1a genotype of HCV in saliva and urine of selected patients. Spread of hospital HCV infection among children may be facilitated by micro-injury of the skin and mucosa. Early detection of HCV RNA is important in such immunosuppressed patients, as they are not able to produce anti-HCV antibodies. This may enable the introduction of prophylactic steps to prevent the spread of HCV infection by horizontal transmission. Copyright 2003 The Hospital Infection Society

  17. GB Virus C (GBV-C) Infection in Hepatitis C Virus (HCV) Seropositive Women with or at Risk for HIV Infection.

    Science.gov (United States)

    Blackard, Jason T; Ma, Gang; Welge, Jeffrey A; King, Caroline C; Taylor, Lynn E; Mayer, Kenneth H; Klein, Robert S; Celentano, David D; Sobel, Jack D; Jamieson, Denise J; Gardner, Lytt

    2014-01-01

    GB virus C (GBV-C) may have a beneficial impact on HIV disease progression; however, the epidemiologic characteristics of this virus are not well characterized. Behavioral factors and gender may lead to differential rates of GBV-C infection; yet, studies have rarely addressed GBV-C infections in women or racial/ethnic minorities. Therefore, we evaluated GBV-C RNA prevalence and genotype distribution in a large prospective study of high-risk women in the US. 438 hepatitis C virus (HCV) seropositive women, including 306 HIV-infected and 132 HIV-uninfected women, from the HIV Epidemiologic Research Study were evaluated for GBV-C RNA. 347 (79.2%) women were GBV-C RNA negative, while 91 (20.8%) were GBV-C RNA positive. GBV-C positive women were younger than GBV-C negative women. Among 306 HIV-infected women, 70 (22.9%) women were HIV/GBV-C co-infected. Among HIV-infected women, the only significant difference between GBV-negative and GBV-positive women was age (mean 38.4 vs. 35.1 years; pGBV-C genotypes were 1 (n = 31; 44.3%), 2 (n = 36; 51.4%), and 3 (n = 3; 4.3%). The distribution of GBV-C genotypes in co-infected women differed significantly by race/ethnicity. However, median CD4 cell counts and log10 HIV RNA levels did not differ by GBV-C genotype. GBV-C incidence was 2.7% over a median follow-up of 2.9 (IQR: 1.5, 4.9) years, while GBV-C clearance was 35.7% over a median follow-up of 2.44 (1.4, 3.5) years. 4 women switched genotypes. Age, injection drug use, a history of sex for money or drugs, and number of recent male sex partners were associated with GBV-C infection among all women in this analysis. However, CD4 cell count and HIV viral load of HIV/HCV/GBV-C co-infected women were not different although race was associated with GBV-C genotype.

  18. The modeled structure of the RNA dependent RNA polymerase of GBV-C virus suggests a role for motif E in Flaviviridae RNA polymerases.

    Science.gov (United States)

    Ferron, François; Bussetta, Cécile; Dutartre, Hélène; Canard, Bruno

    2005-10-14

    The Flaviviridae virus family includes major human and animal pathogens. The RNA dependent RNA polymerase (RdRp) plays a central role in the replication process, and thus is a validated target for antiviral drugs. Despite the increasing structural and enzymatic characterization of viral RdRps, detailed molecular replication mechanisms remain unclear. The hepatitis C virus (HCV) is a major human pathogen difficult to study in cultured cells. The bovine viral diarrhea virus (BVDV) is often used as a surrogate model to screen antiviral drugs against HCV. The structure of BVDV RdRp has been recently published. It presents several differences relative to HCV RdRp. These differences raise questions about the relevance of BVDV as a surrogate model, and cast novel interest on the "GB" virus C (GBV-C). Indeed, GBV-C is genetically closer to HCV than BVDV, and can lead to productive infection of cultured cells. There is no structural data for the GBV-C RdRp yet. We show in this study that the GBV-C RdRp is closest to the HCV RdRp. We report a 3D model of the GBV-C RdRp, developed using sequence-to-structure threading and comparative modeling based on the atomic coordinates of the HCV RdRp structure. Analysis of the predicted structural features in the phylogenetic context of the RNA polymerase family allows rationalizing most of the experimental data available. Both available structures and our model are explored to examine the catalytic cleft, allosteric and substrate binding sites. Computational methods were used to infer evolutionary relationships and to predict the structure of a viral RNA polymerase. Docking a GTP molecule into the structure allows defining a GTP binding pocket in the GBV-C RdRp, such as that of BVDV. The resulting model suggests a new proposition for the mechanism of RNA synthesis, and may prove useful to design new experiments to implement our knowledge on the initiation mechanism of RNA polymerases.

  19. The modeled structure of the RNA dependent RNA polymerase of GBV-C Virus suggests a role for motif E in Flaviviridae RNA polymerases

    Directory of Open Access Journals (Sweden)

    Dutartre Hélène

    2005-10-01

    Full Text Available Abstract Background The Flaviviridae virus family includes major human and animal pathogens. The RNA dependent RNA polymerase (RdRp plays a central role in the replication process, and thus is a validated target for antiviral drugs. Despite the increasing structural and enzymatic characterization of viral RdRps, detailed molecular replication mechanisms remain unclear. The hepatitis C virus (HCV is a major human pathogen difficult to study in cultured cells. The bovine viral diarrhea virus (BVDV is often used as a surrogate model to screen antiviral drugs against HCV. The structure of BVDV RdRp has been recently published. It presents several differences relative to HCV RdRp. These differences raise questions about the relevance of BVDV as a surrogate model, and cast novel interest on the "GB" virus C (GBV-C. Indeed, GBV-C is genetically closer to HCV than BVDV, and can lead to productive infection of cultured cells. There is no structural data for the GBV-C RdRp yet. Results We show in this study that the GBV-C RdRp is closest to the HCV RdRp. We report a 3D model of the GBV-C RdRp, developed using sequence-to-structure threading and comparative modeling based on the atomic coordinates of the HCV RdRp structure. Analysis of the predicted structural features in the phylogenetic context of the RNA polymerase family allows rationalizing most of the experimental data available. Both available structures and our model are explored to examine the catalytic cleft, allosteric and substrate binding sites. Conclusion Computational methods were used to infer evolutionary relationships and to predict the structure of a viral RNA polymerase. Docking a GTP molecule into the structure allows defining a GTP binding pocket in the GBV-C RdRp, such as that of BVDV. The resulting model suggests a new proposition for the mechanism of RNA synthesis, and may prove useful to design new experiments to implement our knowledge on the initiation mechanism of RNA

  20. Maintenance of Th1 hepatitis C virus (HCV)-specific responses in individuals with acute HCV who achieve sustained virological clearance after treatment.

    Science.gov (United States)

    Flynn, Jacqueline K; Dore, Gregory J; Hellard, Margaret; Yeung, Barbara; Rawlinson, William D; White, Peter A; Kaldor, John M; Lloyd, Andrew R; Ffrench, Rosemary A

    2013-11-01

    T-cell responses against hepatitis C are believed to be critical in achieving both natural and treatment-induced clearance. However, rapid clearance of antigen with early treatment of primary infection may result in reduced or poorly sustained cellular immunity. This study longitudinally examined Th1 and Th2 hepatitis C virus (HCV)-specific cytokine production and T-cell effector function from subjects enrolled in the Australian Trial in Acute Hepatitis C comparing three groups: treatment-induced clearance (sustained virological response [SVR]), treatment non-response, and untreated spontaneous clearance. HCV-specific T-cell responses were characterized by HCV peptide ELISpot, in vitro cytokine production, and T-cell flow cytometry assays. Treated subjects with a sustained virological response (SVR) displayed a better maintenance of HCV-specific Th1 responses compared to treatment non-responders (higher interferon [IFN]-γ and interleukin (IL)-2 magnitude at week 24, broader IFN-γ responses at weeks 24 and 48, P < 0.05) and significantly increased IFN-γ responses between screening and week 48 (magnitude P = 0.026, breadth P = 0.009). Treatment-induced viral clearance was also associated with a trend toward decreased IL-10 responses (screening to week 48, P = 0.070), higher expression of CD45RO (P = 0.042) and CD38 (P = 0.088) on CD4+ T cells, and higher IFN-γR expression (CD56+ IFN-γR+ P = 0.033) compared to treatment non-responders. Untreated subjects with viral clearance also displayed high magnitude and broad HCV-specific IFN-γ and IL-2 responses early in infection; however, IFN-γ responses were not as well maintained compared to treated subjects with a SVR (week 48 magnitude, breadth P = 0.064). Treatment-induced viral clearance of recent HCV infection is associated with maintenance of HCV-specific Th1 responses. © 2013 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd.

  1. Strategies underlying RNA silencing suppression by negative strand RNA viruses

    NARCIS (Netherlands)

    Hemmes, J.C.

    2007-01-01

    The research described in this thesis focused on the strategies of negative strand RNA viruses to counteract antiviral RNA silencing. In plants and insects, RNA silencing has been shown to act as a sequence specific antiviral defence mechanism that is characterised by the processing of double

  2. Downregulation of viral RNA translation by hepatitis C virus non-structural protein NS5A requires the poly(U/UC) sequence in the 3' UTR.

    Science.gov (United States)

    Hoffman, Brett; Li, Zhubing; Liu, Qiang

    2015-08-01

    Hepatitis C virus (HCV) non-structural protein 5A (NS5A) is essential for viral replication; however, its effect on HCV RNA translation remains controversial partially due to the use of reporters lacking the 3' UTR, where NS5A binds to the poly(U/UC) sequence. We investigated the role of NS5A in HCV translation using a monocistronic RNA containing a Renilla luciferase gene flanked by the HCV UTRs. We found that NS5A downregulated viral RNA translation in a dose-dependent manner. This downregulation required both the 5' and 3' UTRs of HCV because substitution of either sequence with the 5' and 3' UTRs of enterovirus 71 or a cap structure at the 5' end eliminated the effects of NS5A on translation. Translation of the HCV genomic RNA was also downregulated by NS5A. The inhibition of HCV translation by NS5A required the poly(U/UC) sequence in the 3' UTR as NS5A did not affect translation when it was deleted. In addition, we showed that, whilst the amphipathic α-helix of NS5A has no effect on viral translation, the three domains of NS5A can inhibit translation independently, also dependent on the presence of the poly(U/UC) sequence in the 3' UTR. These results suggested that NS5A downregulated HCV RNA translation through a mechanism involving the poly(U/UC) sequence in the 3' UTR.

  3. Assessment of immunological changes in Epstein-Barr virus co-infection in Egyptian chronic HCV patients

    Science.gov (United States)

    Shoman, Sahar; Nabil, Mohamed; Tabl, Ashraf; Ghanem, Hussam; kafrawy, Sherif El

    2014-01-01

    Epstein-Barr virus (EBV) plays a major role in liver pathology. Similar to other members of the herpesvirus family, EBV establishes a persistent infection in more than 90% of adults. The aim of this study was to evaluate the impact of EBV and chronic hepatitis C co-infection (HCV) on biochemical and immunological responses in patients. The study was conducted in 62 patients and 33 apparently healthy controls. Patients were divided into three groups: group I, consisting of 31 patients with chronic hepatitis C infection (CHC), group II, consisting of eight patients with EBV infection and without HCV infection and group III, consisting of 23 patients with EBV and chronic HCV. The percentage of CD3+ cells, helper CD4+ cells and CD19+ B-cells was measured by flow cytometry. Human interferon-γ (IFN-γ) and interleukin (IL)-15 levels were measured by an ELISA. The levels of liver alanine aminotransferase and aspartate aminotransferase enzymes were higher in EBV/HCV patients compared to that in EBV and HCV mono-infected patients. EBV/HCV patients had significantly reduced percentages of CD3+ and CD4+ cells compared to EBV patients. Serum IFN-γ levels were significantly reduced in EBV/HCV patients (3.86 pg/mL) compared to CHC patients (6.76 pg/mL) and normal controls (4.69 pg/mL). A significant increase in serum IL-15 levels was observed in EBV/HCV patients (67.7 pg/mL) compared to EBV patients (29.3 pg/mL). Taken together, these observations suggest that HCV and EBV co-infection can potentiate immune response dampening in patients. PMID:25317700

  4. Assessment of immunological changes in Epstein-Barr virus co-infection in Egyptian chronic HCV patients

    Directory of Open Access Journals (Sweden)

    Sahar Shoman

    2014-09-01

    Full Text Available Epstein-Barr virus (EBV plays a major role in liver pathology. Similar to other members of the herpesvirus family, EBV establishes a persistent infection in more than 90% of adults. The aim of this study was to evaluate the impact of EBV and chronic hepatitis C co-infection (HCV on biochemical and immunological responses in patients. The study was conducted in 62 patients and 33 apparently healthy controls. Patients were divided into three groups: group I, consisting of 31 patients with chronic hepatitis C infection (CHC, group II, consisting of eight patients with EBV infection and without HCV infection and group III, consisting of 23 patients with EBV and chronic HCV. The percentage of CD3+ cells, helper CD4+ cells and CD19+ B-cells was measured by flow cytometry. Human interferon-γ (IFN-γ and interleukin (IL-15 levels were measured by an ELISA. The levels of liver alanine aminotransferase and aspartate aminotransferase enzymes were higher in EBV/HCV patients compared to that in EBV and HCV mono-infected patients. EBV/HCV patients had significantly reduced percentages of CD3+ and CD4+ cells compared to EBV patients. Serum IFN-γ levels were significantly reduced in EBV/HCV patients (3.86 pg/mL compared to CHC patients (6.76 pg/mL and normal controls (4.69 pg/mL. A significant increase in serum IL-15 levels was observed in EBV/HCV patients (67.7 pg/mL compared to EBV patients (29.3 pg/mL. Taken together, these observations suggest that HCV and EBV co-infection can potentiate immune response dampening in patients.

  5. Translation-competent 48S complex formation on HCV IRES requires the RNA-binding protein NSAP1.

    Science.gov (United States)

    Park, Sung Mi; Paek, Ki Young; Hong, Ka Young; Jang, Christopher J; Cho, Sungchan; Park, Ji Hoon; Kim, Jong Heon; Jan, Eric; Jang, Sung Key

    2011-09-01

    Translation of many cellular and viral mRNAs is directed by internal ribosomal entry sites (IRESs). Several proteins that enhance IRES activity through interactions with IRES elements have been discovered. However, the molecular basis for the IRES-activating function of the IRES-binding proteins remains unknown. Here, we report that NS1-associated protein 1 (NSAP1), which augments several cellular and viral IRES activities, enhances hepatitis C viral (HCV) IRES function by facilitating the formation of translation-competent 48S ribosome-mRNA complex. NSAP1, which is associated with the solvent side of the 40S ribosomal subunit, enhances 80S complex formation through correct positioning of HCV mRNA on the 40S ribosomal subunit. NSAP1 seems to accomplish this positioning function by directly binding to both a specific site in the mRNA downstream of the initiation codon and a 40S ribosomal protein (or proteins).

  6. Seroprevalence of HCV among Cairo University students in Egypt.

    Science.gov (United States)

    Esmat, Gamal; Raziky, Maissa El; Nabeel, Mohammed M; Maher, Rabab; Zakaria, Zeinab

    2016-08-01

    Hepatitis C virus (HCV) is highly prevalent in Egypt. This work aimed at determining the seroprevalence of HCV among Cairo University students. The present study included 3,000 students from Cairo University, Egypt. Blood sample was obtained from each participant to be tested for HCV seromarker. HCV RNA detection by polymerase chain reaction (PCR) was carried out for those with positive anti-HCV. Overall prevalence rate of HCV antibody (anti-HCV) was 4.6%. It showed that the prevalence was relatively higher among females (86/1660; 5.2%) while males (51/1340; 3.8%) with no significant difference. PCR for HCV RNA was detected in 31.4% of the HCV antibody positive subjects (43/137). Which showed statistical significant difference between males (29/51) and females (14/86) at P = 0.001. Despite the prevalence rate reported in the present study was similar to anti-HCV prevalence among persons in the same age group, confirmed that HCV infection is detected among Cairo University students. J. Med. Virol. 88:1384-1387, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  7. The molecular biology of hepatitis C virus. Genotypes and quasispecies.

    Science.gov (United States)

    Forns, X; Bukh, J

    1999-11-01

    Hepatitis C virus (HCV) is an important cause of chronic liver disease worldwide. HCV is a positive-strand genotype RNA virus with extensive genetic heterogeneity; HCV isolates define 6 major genotypes, and HCV circulates within an infected individual as a number of closely related but distinct species, termed a quasispecies. This article reviews characteristic aspects of HCV molecular biology and their implications for treatment and vaccine development.

  8. RNA topology remolds electrostatic stabilization of viruses

    OpenAIRE

    Erdemci-Tandogan, G; Wagner, J.; Schoot, van der, PPAM Paul; Podgornik, R.; Zandi, R

    2013-01-01

    Simple RNA viruses efficiently encapsulate their genome into a nano-sized protein shell: the capsid. Spontaneous coassembly of the genome and the capsid proteins is driven predominantly by electrostatic interactions between the negatively charged RNA and the positively charged inner capsid wall. Using field theoretic formulation we show that the inherently branched RNA secondary structure allows viruses to maximize the amount of encapsulated genome and make assembly more efficient, allowing v...

  9. Bioinformatic analysis of the neutrality of RNA secondary structure elements across genotypes reveals evidence for direct evolution of genetic robustness in HCV.

    Science.gov (United States)

    Churkin, Alexander; Cohen, Moriah; Shemer-Avni, Yonat; Barash, Danny

    2010-12-01

    The properties and origin of genetic robustness have recently been investigated in several works that examined microRNA stem-loop structures, and a variety of conclusions have been reached without agreement. Considering that this is a universal phenomenon that is not restricted to miRNAs, we recall the original work on this topic that began from looking at viral RNAs of several types. We provide a link to this work by examining the neutrality of HCV structural elements, performing a detailed bioinformatic analysis using RNA secondary structure predictions across genotypes. This study provides supporting evidence for direct evolution of genetic robustness that is not limited to noncoding RNAs participating in gene regulation, but includes functionally important structural elements of the hepatitis C virus (HCV) that show excess of robustness beyond the intrinsic robustness of their stem-loop structure. These findings further support the adaptive behavior of genetic robustness in functional RNAs of various types that seem to have evolved with selection pressure towards increased robustness.

  10. Detection of HGV/GBV-C RNA in Healthy Individuals non Carrier of HBV, HIV-1/2 and HCV Detecção do RNA HGV/GBV-C em Indivíduos Saudáveis não Portadores de HBV, HIV-1/2 e HCV

    Directory of Open Access Journals (Sweden)

    Maria Angelica Ehara Watanabe

    2004-10-01

    Full Text Available The GB virus C (GBV-C/hepatitis G virus (HGV is a member of the Flaviviridae family. Based on the clinical and epidemiological profiles, this virus can be acquired mainly by parenteral transmission through contaminated blood. It was therefore investigated the presence of GBV-C/HGV RNA in the peripheral blood from healthy blood donors in the abscence of virus markers including HBV surface antigen (HBsAg, HBV core antibody (anti-HBc and HCV antibody. HIV-1, HIV-2, were also investigated. GBVC RNA was detected by reverse transcriptase and polymerase chain reactions (RT-PCR. It was detected GBV-C RNA in 6/50 (12 % blood donors. The presence of GBV-C RNA in the abscence of hepatitis B and C infection in young patients and healthy donors could indicate that this virus is capable of independent transmission and does not contribute to liver disease. O vírus da hepatite G (HGV ou GBV-C é um membro da família Flaviviridae. Baseado no perfil clínico e epidemiológico, este vírus pode ser adquirido principalmente por transmissão parenteral, por meio de sangue contaminado. Nós investigamos a presença do RNA do GBV-C/HGV em amostras de sangue periférico de doadores normais na ausência de marcadores como antígenos de superfície do HBV (HBsAg, anticorpos anti-HBc (anti-HBc, anticorpos anti-HCV e anticorpos anti-HIV-1/HIV-2. O RNA GBV-C foi detectado por reação de transcriptase reversa e reação em cadeia catalisada pela polimerase (RT-PCR. Foram analisadas 50 amostras de plasma e identificados 6 (12% amostras positivas para o RNA do GBVC. A presença de RNA GBV-C na ausência de hepatite B e C em doadores saudáveis pode indicar que este vírus é capaz de transmissão independente e não contribui para doença hepática.  

  11. Survey of both hepatitis B virus (HBsAg and hepatitis C virus (HCV-Ab coinfection among HIV positive patients

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    Pournia Yadollah

    2009-11-01

    Full Text Available Abstract Background HIV, HBVand HCV is major public health concerns. Because of shared routes of transmission, HIV-HCV coinfection and HIV-HBV coinfection are common. HIV-positive individuals are at risk of coinfection with HBV and HCV infections. The prevalence rates of coinfection with HBV and HCV in HIV-patients have been variable worldwide depending on the geographic regions, and the type of exposure. Aim This study aimed to examine HBV and HCV coinfection serologically and determine the shared and significant factors in the coinfection of HIV-positive patients. Methods This descriptive, cross-sectional study was carried out on 391 HIV-positive patients including 358 males and 33 females in Lorestan province, west Iran, to survey coinfection with HBsAg and anti-HCV. The retrospective demographic data of the subjects was collected and the patients' serums were analyzed by ELISA kits including HBsAg and anti-HCV. The collected data was analyzed with SPSS software (15 and Chi-square. Fisher's exact test with 5% error intervals was used to measure the correlation of variables and infection rates. Results The results of the study indicated that the prevalence of coinfection in HIV-positive patients with hepatitis viruses was 94.4% (370 in 391, out of whom 57 (14.5% cases were HBsAg positive, 282 (72% cases were anti-HCV positive, and 31 (7.9% cases were both HBsAg and anti-HCV positive. Conclusion There was a significant correlation between coinfection with HCV and HBV and/or both among HIV-positive patients depending on different variables including sex, age, occupation, marital status, exposure to risk factors.(p

  12. A peptide derived from hepatitis C virus E2 envelope protein inhibits a post-binding step in HCV entry.

    Science.gov (United States)

    Liu, R; Tewari, M; Kong, R; Zhang, R; Ingravallo, P; Ralston, R

    2010-05-01

    The HCV envelope proteins E1 and E2 are required for virus binding to cellular receptors and pH-dependent fusion with endosomal membranes. Envelope protein interactions within this multistep process may provide novel targets for development of antiviral agents. To identify E1 and E2 regions involved in critical steps of HCV entry, we screened an E1E2 overlapping peptide library for inhibition of infection using a lentiviral reporter vector pseudotyped with E1E2 envelope proteins. A 16-residue polypeptide containing a portion of the E2 transmembrane domain (Peptide 75) inhibited HCV pseudoparticle infection with an IC50 of approximately 0.3microM and did not inhibit infection by VSV-g pseudoparticles at concentrations up to 50microM. Structure-activity analysis of Peptide 75 showed that antiviral activity was dependent upon L-configuration and hydrophobic character, and that the native sequence was required for maximal activity. Peptide 75 did not show virocidal activity against HCV pseudoparticles or other viruses. Temperature-shift experiments showed that the peptide acted at a post-binding step and that inhibition was further increased when used in combination with an anti-CD81 antibody previously shown to inhibit pseudoparticle entry at a post-binding step. These data suggest that interactions involving the C terminal region of E2 may play an important role in the HCV entry process.

  13. Prevalence of Hepatitis C Virus (HCV) in healthy adults and Human ...

    African Journals Online (AJOL)

    The prevalence of HCV infection in Abuja, FCT, Nigeria was determined among healthy adults and HIV infected persons. A total of n=520 apparently healthy HIV negative persons and n=1,200 infected persons were tested for antibodies against HCV by rapid chromatographic immunoassay HCV kit (Acon, ACON ...

  14. The Shift in Emphasis From Risk-Based to Age-Based Hepatitis C Virus (HCV) Testing in the US Tends to Remove Injection Drug Use From Discourse on HCV.

    Science.gov (United States)

    Jordan, Ashly E; Perlman, David C

    2017-02-23

    Hepatitis C virus (HCV) infection is hyperendemic among people who inject drugs; nonsterile drug injection is the principle risk for HCV acquisition. Due to gaps in the HCV care continuum, there have been recommendations in the United States emphasizing age-rather than risk-based testing strategies. The central research focus of this project is to explore the meanings and implications of the shift in emphasis from risk-based to age-based HCV testing with regard to people who use drugs. Content analysis and critical discourse analysis, informed by eco-social theory, were used to examine relevant documents. Fifteen documents were assessed for eligibility; 6 documents comprised the final set reviewed. In content analysis, age-based testing was both mentioned more frequently and was supported more strongly than risk-based testing. Risk-based testing was frequently mentioned in terms minimizing its use and drug use was often mentioned only euphemistically. The reframed emphasis largely removed discussion of injection drug use from discussion of HCV risks. Shifting the emphasis of HCV testing from testing based on specific routes of transmission and risk to testing based on age removes injection drug use from HCV discourse. This has the potential to either facilitate HCV care for drug users or to further stigmatize and marginalize drug use and people who use drugs. The potential implications of this shift in testing emphasis for public health merit further investigation.

  15. Functional RNA during Zika virus infection

    NARCIS (Netherlands)

    Göertz, Giel P.; Abbo, Sandra R.; Fros, Jelke J.; Pijlman, Gorben P.

    2017-01-01

    Zika virus (ZIKV; family Flaviviridae; genus Flavivirus) is a pathogenic mosquito-borne RNA virus that currently threatens human health in the Americas, large parts of Asia and occasionally elsewhere in the world. ZIKV infection is often asymptomatic but can cause severe symptoms including

  16. The Tat protein of human immunodeficiency virus-1 enhances hepatitis C virus replication through interferon gamma-inducible protein-10

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    Qu Jing

    2012-04-01

    Full Text Available Abstract Background Co-infection with human immunodeficiency virus-1 (HIV-1 and hepatitis C virus (HCV is associated with faster progression of liver disease and an increase in HCV persistence. However, the mechanism by which HIV-1 accelerates the progression of HCV liver disease remains unknown. Results HIV-1/HCV co-infection is associated with increased expression of interferon gamma-induced protein-10 (IP-10 mRNA in peripheral blood mononuclear cells (PBMCs. HCV RNA levels were higher in PBMCs of patients with HIV-1/HCV co-infection than in patients with HCV mono-infection. HIV-1 Tat and IP-10 activated HCV replication in a time-dependent manner, and HIV-1 Tat induced IP-10 production. In addition, the effect of HIV-1 Tat on HCV replication was blocked by anti-IP-10 monoclonal antibody, demonstrating that the effect of HIV-1 Tat on HCV replication depends on IP-10. Taken together, these results suggest that HIV-1 Tat protein activates HCV replication by upregulating IP-10 production. Conclusions HIV-1/HCV co-infection is associated with increased expression of IP-10 mRNA and replication of HCV RNA. Furthermore, both HIV-1 Tat and IP-10 activate HCV replication. HIV-1 Tat activates HCV replication by upregulating IP-10 production. These results expand our understanding of HIV-1 in HCV replication and the mechanism involved in the regulation of HCV replication mediated by HIV-1 during co-infection.

  17. CXCL10 Decreases GP73 Expression in Hepatoma Cells at the Early Stage of Hepatitis C Virus (HCV Infection

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    Yuan Liu

    2013-12-01

    Full Text Available Golgi protein 73 (GP73, which is up-regulated in hepatocellular carcinoma (HCC, has recently been identified as a novel serum marker for HCC diagnosis. Several reports also noted the increased levels of GP73 expression in chronic liver disease in patients with acute hepatitis of various etiologies, chronic Hepatitis C virus (HCV infection and alcoholic liver disease. The molecular mechanisms of GP73 expression in HCV related liver disease still need to be determined. In this study, we aimed to evaluate the effect of HCV infection on GP73 expression. GP73 was highly expressed in Huh7, Hep3B, 293T and HUVEC cells, and was low-expressed in HepG2 cells. HCV infection led to down-regulation of GP73 in Huh7 and HepG2/CD81 cells at the early stage of infection. CXCL10 decreased GP73 expression in Huh7 and HepG2 cells. Up-regulation of GP73 was noted in hepatocytes with cytopathic effect at advanced stage of HCV infection, and further research is needed to determine the unknown factors affecting GP73 expression. In conclusion, our study provided additional evidence for the roles of GP73 in liver disease.

  18. Performance characteristics of a combined hepatitis C virus core antigen and anti–hepatitis C virus antibody test in different patient groups

    Directory of Open Access Journals (Sweden)

    Jeng-Fu Yang

    2011-07-01

    Full Text Available We evaluated the performance of a hepatitis C virus (HCV antigen/antibody combination test [Murex HCV Antigen/Antibody Combination Test (Murex Ag/Ab test] by comparing it with the current third-generation HCV antibody enzyme immunoassay (anti-HCV. A total of 403 serum samples were consecutively collected from four patient groups: healthy controls (n=100; HCV-infected patients (HCV group, n=102; Human immunodeficiency virus (HIV/HCV-infected patients (HIV/HCV group, n=100; and patients with uremia (uremia group, n=101. Performances were evaluated for the Murex Ag/Ab, anti-HCV, and HCV RNA in the HIV/HCV and uremia patient groups. In the HCV group, all 102 samples showed concordant positive and negative results for anti-HCV, Murex Ag/Ab, and HCV RNA tests. In the HIV/HCV group, all 100 samples were positive for both anti-HCV and Murex Ag/Ab tests, whereas 88 patients (88% were HCV RNA positive. In the uremia group, 14 (69.0% of the 23 anti-HCV-positive patients were HCV RNA positive, whereas 14 (77.8% of the 18 Murex Ag/Ab–positive patients were HCV RNA positive. None of anti-HCV-negative or Murex Ag/Ab–negative patients were HCV RNA positive. Based on the HCV RNA assay, the sensitivities for both anti-HCV and Murex Ag/Ab assays were 100%, whereas the specificities of these two assays were 89.7% and 95.4%, respectively. With good sensitivity and specificity, the Murex Ag/Ab assay could be a useful alternative diagnostic tool, especially in immunocompromised populations, such as patients with uremia or those infected with HIV.

  19. RNA topology remolds electrostatic stabilization of viruses

    NARCIS (Netherlands)

    Erdemci-Tandogan, Gonca; Wagner, Jef; Van Der Schoot, Paul; Podgornik, Rudolf; Zandi, Roya

    2014-01-01

    Simple RNA viruses efficiently encapsulate their genome into a nano-sized protein shell: the capsid. Spontaneous coassembly of the genome and the capsid proteins is driven predominantly by electrostatic interactions between the negatively charged RNA and the positively charged inner capsid wall.

  20. The RNA synthesis machinery of negative-stranded RNA viruses

    Energy Technology Data Exchange (ETDEWEB)

    Ortín, Juan, E-mail: jortin@cnb.csic.es [Department of Molecular and Cellular Biology, Centro Nacional de Biotecnología (CSIC) and CIBER de Enfermedades Respiratorias (ISCIII), Madrid (Spain); Martín-Benito, Jaime, E-mail: jmartinb@cnb.csic.es [Department of Macromolecular Structures, Centro Nacional de Biotecnología (CSIC), Madrid (Spain)

    2015-05-15

    The group of Negative-Stranded RNA Viruses (NSVs) includes many human pathogens, like the influenza, measles, mumps, respiratory syncytial or Ebola viruses, which produce frequent epidemics of disease and occasional, high mortality outbreaks by transmission from animal reservoirs. The genome of NSVs consists of one to several single-stranded, negative-polarity RNA molecules that are always assembled into mega Dalton-sized complexes by association to many nucleoprotein monomers. These RNA-protein complexes or ribonucleoproteins function as templates for transcription and replication by action of the viral RNA polymerase and accessory proteins. Here we review our knowledge on these large RNA-synthesis machines, including the structure of their components, the interactions among them and their enzymatic activities, and we discuss models showing how they perform the virus transcription and replication programmes. - Highlights: • Overall organisation of NSV RNA synthesis machines. • Structure and function of the ribonucleoprotein components: Atomic structure of the RNA polymerase complex. • Commonalities and differences between segmented- and non-segmented NSVs. • Transcription versus replication programmes.

  1. RNA topology remolds electrostatic stabilization of viruses.

    Science.gov (United States)

    Erdemci-Tandogan, Gonca; Wagner, Jef; van der Schoot, Paul; Podgornik, Rudolf; Zandi, Roya

    2014-03-01

    Simple RNA viruses efficiently encapsulate their genome into a nano-sized protein shell: the capsid. Spontaneous coassembly of the genome and the capsid proteins is driven predominantly by electrostatic interactions between the negatively charged RNA and the positively charged inner capsid wall. Using field theoretic formulation we show that the inherently branched RNA secondary structure allows viruses to maximize the amount of encapsulated genome and make assembly more efficient, allowing viral RNAs to out-compete cellular RNAs during replication in infected host cells.

  2. Supporting Role for GTPase Rab27a in Hepatitis C Virus RNA Replication through a Novel miR-122-Mediated Effect.

    Directory of Open Access Journals (Sweden)

    Tzu-Chun Chen

    2015-08-01

    Full Text Available The small GTPase Rab27a has been shown to control membrane trafficking and microvesicle transport pathways, in particular the secretion of exosomes. In the liver, high expression of Rab27a correlates with the development of hepatocellular carcinoma. We discovered that low abundance of Rab27a resulted in decreased hepatitis C virus (HCV RNA and protein abundances in virus-infected cells. Curiously, both cell-associated and extracellular virus yield decreased in Rab27a depleted cells, suggesting that reduced exosome secretion did not cause the observed effect. Instead, Rab27a enhanced viral RNA replication by a mechanism that involves the liver-specific microRNA miR-122. Rab27a surrounded lipid droplets and was enriched in membrane fractions that harbor viral replication proteins, suggesting a supporting role for Rab27a in viral gene expression. Curiously, Rab27a depletion decreased the abundance of miR-122, whereas overexpression of miR-122 in Rab27a-depleted cells rescued HCV RNA abundance. Because intracellular HCV RNA abundance is enhanced by the binding of two miR-122 molecules to the extreme 5' end of the HCV RNA genome, the diminished amounts of miR-122 in Rab27a-depleted cells could have caused destabilization of HCV RNA. However, the abundance of HCV RNA carrying mutations on both miR-122-binding sites and whose stability was supported by ectopically expressed miR-122 mimetics with compensatory mutations also decreased in Rab27a-depleted cells. This result indicates that the effect of Rab27a depletion on HCV RNA abundance does not depend on the formation of 5' terminal HCV/miR-122 RNA complexes, but that miR-122 has a Rab27a-dependent function in the HCV lifecycle, likely the downregulation of a cellular inhibitor of HCV gene expression. These findings suggest that the absence of miR-122 results in a vulnerability not only to exoribonucleases that attack the viral genome, but also to upregulation of one more cellular factor that inhibit

  3. Occult HCV infection: an unexpected finding in a population unselected for hepatic disease.

    Directory of Open Access Journals (Sweden)

    Laura De Marco

    Full Text Available BACKGROUND: Occult Hepatitis C virus (HCV infection is a new pathological entity characterized by presence of liver disease and absence or very low levels of detectable HCV-RNA in serum. Abnormal values of liver enzymes and presence of replicative HCV-RNA in peripheral blood mononuclear cells are also observed. Aim of the study was to evaluate occult HCV occurrence in a population unselected for hepatic disease. METHODOLOGY/PRINCIPAL FINDINGS: We chose from previous epidemiological studies three series of subjects (n = 276, age range 40-65 years unselected for hepatic disease. These subjects were tested for the presence of HCV antibodies and HCV-RNA in plasma and in the peripheral blood mononuclear cells (PBMCs by using commercial systems. All subjects tested negative for HCV antibodies and plasma HCV-RNA and showed normal levels of liver enzymes; 9/276 patients (3.3% were positive for HCV-RNA in PBMCs, identifying a subset of subjects with potential occult HCV infection. We could determine the HCV type for 8 of the 9 patients finding type 1a (3 patients, type 1b (2 patients, and type 2a (3 patients. CONCLUSIONS: The results of this study show evidence that occult HCV infection may occur in a population unselected for hepatic disease. A potential risk of HCV infection spread by subjects harbouring occult HCV infection should be considered. Design of prospective studies focusing on the frequency of infection in the general population and on the clinical evolution of occult HCV infection will be needed to verify this unexpected finding.

  4. The roles of endoplasmic reticulum overload response induced by HCV and NS4B protein in human hepatocyte viability and virus replication.

    Directory of Open Access Journals (Sweden)

    Lingbao Kong

    Full Text Available Hepatitis C virus (HCV replication is associated with endoplasmic reticulum (ER and its infection triggers ER stress. In response to ER stress, ER overload response (EOR can be activated, which involves the release of Ca2+ from ER, production of reactive oxygen species (ROS and activation of nuclear factor κB (NF-κB. We have previously reported that HCV NS4B expression activates NF-κB via EOR-Ca2+-ROS pathway. Here, we showed that NS4B expression and HCV infection activated cancer-related NF-κB signaling pathway and induced the expression of cancer-related NF-κB target genes via EOR-Ca2+-ROS pathway. Moreover, we found that HCV-activated EOR-Ca2+-ROS pathway had profound effects on host cell viability and HCV replication. HCV infection induced human hepatocyte death by EOR-Ca2+-ROS pathway, whereas activation of EOR-Ca2+-ROS-NF-κB pathway increased the cell viability. Meanwhile, EOR-Ca2+-ROS-NF-κB pathway inhibited acute HCV replication, which could alleviate the detrimental effect of HCV on cell viability and enhance chronic HCV infection. Together, our findings provide new insights into the functions of EOR-Ca2+-ROS-NF-κB pathway in natural HCV replication and pathogenesis.

  5. Variation of transaminases, HCV-RNA levels and Th1/Th2 cytokine production during the post-partum period in pregnant women with chronic hepatitis C.

    Directory of Open Access Journals (Sweden)

    Angeles Ruiz-Extremera

    Full Text Available This study analyses the evolution of liver disease in women with chronic hepatitis C during the third trimester of pregnancy and the post-partum period, as a natural model of immune modulation and reconstitution. Of the 122 mothers recruited to this study, 89 were HCV-RNA+ve/HIV-ve and 33 were HCV-RNA-ve/HIV-ve/HCVantibody+ve and all were tested during the third trimester of pregnancy, at delivery and post-delivery. The HCV-RNA+ve mothers were categorized as either Type-A (66%, with an increase in ALT levels in the post-partum period (>40 U/L; P<0.001 or as Type-B (34%, with no variation in ALT values. The Type-A mothers also presented a significant decrease in serum HCV-RNA levels in the post-delivery period (P<0.001 and this event was concomitant with an increase in Th1 cytokine levels (INFγ, P = 0.04; IL12, P = 0.01 and IL2, P = 0.01. On the other hand, the Type-B mothers and the HCV-RNA-ve women presented no variations in either of these parameters. However, they did present higher Th1 cytokine levels in the partum period (INFγ and IL2, P<0.05 than both the Type-A and the HCV-RNA-ve women. Cytokine levels at the moment of delivery do not constitute a risk factor associated with HCV vertical transmission. It is concluded that differences in the ALT and HCV-RNA values observed in HCV-RNA+ve women in the postpartum period might be due to different ratios of Th1 cytokine production. In the Type-B women, the high partum levels of Th1 cytokines and the absence of post-partum variation in ALT and HCV-RNA levels may be related to permanent Th1 cytokine stimulation.

  6. Men who have sex with men starting pre-exposure prophylaxis (PrEP) are at risk of HCV infection: evidence from the Amsterdam PrEP study.

    Science.gov (United States)

    Hoornenborg, Elske; Achterbergh, Roel C A; Schim Van Der Loeff, Maarten F; Davidovich, Udi; Hogewoning, Arjan; Vries, Henry J C de; Schinkel, Janke; Prins, Maria; Laar, Thijs J W van de

    2017-05-01

    Hepatitis C virus (HCV) has been recognised as an emerging sexually transmitted infection (STI) among HIV-positive men who have sex with men (MSM). However, HIV-negative MSM at high risk for HIV might also be at increased risk for HCV. We studied the HCV prevalence in HIV-negative MSM who start pre-exposure prophylaxis (PrEP) in Amsterdam. Phylogenetic analysis was used to compare HCV strains obtained from HIV-negative and HIV-positive MSM. At enrolment in the Amsterdam PrEP (AMPrEP) demonstration project, HIV-negative MSM were tested for the presence of HCV antibodies and HCV RNA. If positive for HCV RNA, an HCV NS5B gene fragment (709 bp) was sequenced and compared with HCV isolates from HIV-positive MSM (n = 223) and risk groups other than MSM (n = 153), using phylogenetic analysis. Of 375 HIV-negative MSM enrolled in AMPrEP, 18 (4.8%, 95%CI 2.9%-7.5%) of participants were anti-HCV and/or HCV RNA positive at enrolment; 15/18 (83%) had detectable HCV RNA. HCV genotyping showed genotype 1a (73%), 4d (20%) and 2b (7%). All HCV-positive MSM starting PrEP were part of MSM-specific HCV clusters containing MSM with and without HIV. HCV prevalence among HIV-negative MSM who started PrEP was higher than previously reported. All HIV-negative HCV-positive MSM were infected with HCV strains already circulating among HIV-positive MSM. The increasing overlap between sexual networks of HIV-positive and HIV-negative MSM might result in an expanding HCV-epidemic irrespective of HIV-status. Hence, routine HCV testing should be offered to MSM at high risk for HIV, especially for those enrolling in PrEP programs.

  7. DNA vaccine expressing the non-structural proteins of hepatitis C virus diminishes the expression of HCV proteins in a mouse model.

    Science.gov (United States)

    Wada, Takeshi; Kohara, Michinori; Yasutomi, Yasuhiro

    2013-12-05

    Most of the people infected with hepatitis C virus (HCV) develop chronic hepatitis, which in some cases progresses to cirrhosis and ultimately to hepatocellular carcinoma. Although various immunotherapies against the progressive disease status of HCV infection have been studied, a preventive or therapeutic vaccine against this pathogen is still not available. In this study, we constructed a DNA vaccine expressing an HCV structural protein (CN2), non-structural protein (N25) or the empty plasmid DNA as a control and evaluated their efficacy as a candidate HCV vaccine in C57BL/6 and novel genetically modified HCV infection model (HCV-Tg) mice. Strong cellular immune responses to several HCV structural and non-structural proteins, characterized by cytotoxicity and interferon-gamma (IFN-γ) production, were observed in CN2 or N25 DNA vaccine-immunized C57BL/6 mice but not in empty plasmid DNA-administered mice. The therapeutic effects of these DNA vaccines were also examined in HCV-Tg mice that conditionally express HCV proteins in their liver. Though a reduction in cellular immune responses was observed in HCV-Tg mice, there was a significant decrease in the expression of HCV protein in mice administered the N25 DNA vaccine but not in mice administered the empty plasmid DNA. Moreover, both CD8(+) and CD4(+) T cells were required for the decrease of HCV protein in the liver. We found that the N25 DNA vaccine improved pathological changes in the liver compared to the empty plasmid DNA. Thus, these DNA vaccines, especially that expressing the non-structural protein gene, may be an alternative approach for treatment of individuals chronically infected with HCV. Copyright © 2013 Elsevier Ltd. All rights reserved.

  8. How RNA viruses maintain their genome integrity.

    Science.gov (United States)

    Barr, John N; Fearns, Rachel

    2010-06-01

    RNA genomes are vulnerable to corruption by a range of activities, including inaccurate replication by the error-prone replicase, damage from environmental factors, and attack by nucleases and other RNA-modifying enzymes that comprise the cellular intrinsic or innate immune response. Damage to coding regions and loss of critical cis-acting signals inevitably impair genome fitness; as a consequence, RNA viruses have evolved a variety of mechanisms to protect their genome integrity. These include mechanisms to promote replicase fidelity, recombination activities that allow exchange of sequences between different RNA templates, and mechanisms to repair the genome termini. In this article, we review examples of these processes from a range of RNA viruses to showcase the diverse approaches that viruses have evolved to maintain their genome sequence integrity, focusing first on mechanisms that viruses use to protect their entire genome, and then concentrating on mechanisms that allow protection of the genome termini, which are especially vulnerable. In addition, we discuss examples in which it might be beneficial for a virus to 'lose' its genomic termini and reduce its replication efficiency.

  9. Effects of short RNA structural analogues against hepatitis C virus genotypes 2, 3 and 4 in replicon cells.

    Science.gov (United States)

    Elshaffei, Ismail M; Gupta, Nidhi; Wu, Catherine H; Wu, David C; Hammad, Lamiaa N; Abo-Elmatty, Dina M; Mesbah, Noha M; Wu, George Y

    2015-08-01

    To determine whether computer-predicted short RNA structural analogues could inhibit hepatitis C virus (HCV) genotype 2a, 3a and 4a replication in cultured cells. Short RNA sequences, X12, X12a and X12b, designed to be identical in secondary structure to the X region in the 3'-untranslated region (3'-UTR) of the HCV 1b genome, as well as shorter stem-loop components of X region, were inserted into a plasmid and transfected into separate Huh7.5 human hepatoma cells stably transfected with subgenomic replicons for genotypes 2a, 3a and 4a. All replicons included a firefly luciferase reporter gene. After 48 h of plasmid transfection, the inhibition of HCV replication was determined by HCV RNA isolation and quantification by real-time polymerase chain reaction and luciferase assays. All the secondary structural analogues to genotype 1b X region cross-inhibited genotype 2a, 3a and 4a replicons. The maximum inhibition by genotype 1b X region structural analogues was obtained against genotype 2a cells in which X12, X12a and X12b inhibited replication by 30%, 63% and 72%, respectively (P < 0.05 for all), compared to an unrelated hepatitis B viral analogue. Despite substantial sequence dissimilarity, HCV RNA genotype 1b X region analogues cross-inhibited the replication of HCV genotypes 2a, 3a and 4a. Particular conformations and not the sequence of the stem-loops of the X region are involved in HCV replication. © 2015 Chinese Medical Association Shanghai Branch, Chinese Society of Gastroenterology, Renji Hospital Affiliated to Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd.

  10. The Irish paradigm on the natural progression of hepatitis C virus infection: an investigation in a homogeneous patient population infected with HCV 1b (review).

    LENUS (Irish Health Repository)

    Fanning, Liam J

    2012-02-03

    The aetiological agent of chronic hepatitis C is the hepatitis C virus. The hepatitis C virus is spread by parenteral transmission of body fluids, primarily blood or blood products. In 1989, after more than a decade of research, HCV was isolated and characterised. The hepatitis C viral genome is a positive-sense, single-stranded RNA molecule approximately 9.4 kb in length, which encodes a polyprotein of about 3100 amino acids. There are 6 main genotypes of HCV, each further stratified by subtype. In 1994, a cohort of women was identified in Ireland as having been iatrogenically exposed to the hepatitis C virus. The women were all young and exposed as a consequence of the receipt of HCV 1b contaminated anti-D immunoglobulin. The source of the infection was identified as an acutely infected female. As part of a voluntary serological screening programme involving 62,667 people, 704 individuals were identified as seropositive for exposure to the hepatitis C virus; 55.4% were found to be positive for the viral genome 17 years after exposure. Of these women 98% had evidence of inflammation, but surprisingly, a remarkable 49% showed no evidence of fibrosis. Clinicopathology and virological analysis has identified associations between viral load and the histological activity index for inflammation, and, between inflammation and levels of the liver enzyme alanine aminotransferase. Infection at a younger age appears to protect individuals from progression to advanced liver disease. Molecular analyses of host immunogenetic elements shows that particular class II human leukocyte associated antigen alleles are associated with clearance of the hepatitis C virus. Additional class II alleles have been identified that are associated with stable viraemia over an extended period of patient follow-up. Although, investigation of large untreated homogeneous cohorts is likely to become more difficult, as the efficacy of anti-viral therapy improves, further investigation of host and viral

  11. Look-back of anti-HCV ELISA-positive, HCV-RNA PCR-negative donors and recipients of their blood products

    NARCIS (Netherlands)

    Vrielink, H.; Reésink, H. W.; Zaaijer, H. L.; Scholten, E.; Kremer, L. C.; Cuypers, H. T.; Lelie, P. N.; van Oers, M. H.; van der Poel, C. L.

    1997-01-01

    BACKGROUND AND OBJECTIVES: To establish the infectivity of anti-HCV ELISA-positive, but cDNA-PCR-negative blood components transfused before the introduction of routine anti-HCV blood donor screening, we enrolled recipients of such blood products in a look-back programme. MATERIALS AND METHODS: The

  12. Psychiatric and substance use disorders in HIV/hepatitis C virus (HCV)-coinfected patients: does HCV clearance matter? [Agence Nationale de Recherche sur le SIDA et les Hépatites Virales (ANRS) HEPAVIH CO13 cohort].

    Science.gov (United States)

    Michel, L; Lions, C; Winnock, M; Lang, J-P; Loko, M-A; Rosenthal, E; Marchou, B; Valantin, M-A; Morlat, P; Roux, P; Sogni, P; Spire, B; Poizot-Martin, I; Lacombe, K; Lascoux-Combe, C; Duvivier, C; Neau, D; Dabis, F; Salmon-Ceron, D; Carrieri, M P

    2016-11-01

    The objective of this nested study was to assess the prevalence of psychiatric disorders in a sample of HIV/hepatitis C virus (HCV)-coinfected patients according to their HCV status. The nested cross-sectional study, untitled HEPAVIH-Psy survey, was performed in a subset of HIV/HCV-coinfected patients enrolled in the French Agence Nationale de Recherche sur le SIDA et les Hépatites Virales (ANRS) CO13 HEPAVIH cohort. Psychiatric disorders were screened for using the Mini International Neuropsychiatric Interview (MINI 5.0.0). Among the 286 patients enrolled in the study, 68 (24%) had never received HCV treatment, 87 (30%) were treatment nonresponders, 44 (15%) were currently being treated and 87 (30%) had a sustained virological response (SVR). Of the 286 patients enrolled, 121 patients (42%) screened positive for a psychiatric disorder other than suicidality and alcohol/drug abuse/dependence, 40 (14%) screened positive for alcohol abuse/dependence, 50 (18%) screened positive for drug abuse/dependence, 50 (17.5%) were receiving an antidepressant treatment and 69 (24%) were receiving an anxiolytic. Patients with an SVR did not significantly differ from the other groups in terms of psychiatric disorders. Patients receiving HCV treatment screened positive less often for an anxiety disorder. The highest rate of drug dependence/abuse was among HCV treatment-naïve patients. Psychiatric disorders were frequent in HIV/HCV-coinfected patients and their rates were comparable between groups, even for patients achieving an SVR. Our results emphasize the need for continuous assessment and care of coinfected patients, even after HCV clearance. Drug addiction remains an obstacle to access to HCV treatment. Despite the recent advent and continued development of directly acting antiviral agents (DAAs), it is still crucial to offer screening and comprehensive care for psychiatric and addictive disorders. © 2016 British HIV Association.

  13. RNA polymerase activity of Ustilago maydis virus

    Energy Technology Data Exchange (ETDEWEB)

    Yie, S.W.

    1986-01-01

    Ustilago maydis virus has an RNA polymerase enzyme which is associated with virion capsids. In the presence of Mg/sup 2 +/ ion and ribonucleotide triphosphate, the enzyme catalyzes the in vitro synthesis of mRNA by using dsRNA as a template. The products of the UmV RNA polymerase were both ssRNA and dsRNA. The dsRNA was determined by characteristic mobilities in gel electrophoresis, lack of sensitivity to RNase, and specific hybridization tests. The ssRNAs were identified by elution from a CF-11 column and by their RNase sensitivity. On the basis of the size of ssRNAs, it was concluded that partial transcripts were produced from H dsRNA segments, and full length transcripts were produced from M and L dsRNA segments. The following observations indicates that transcription occurs by strand displacement; (1) Only the positive strand of M2 dsRNA was labeled by the in vitro reaction. (2) The M2 dsRNA which had been labeled with /sup 32/''P-UTP in vitro could be chased from dsRNA with unlabeled UTP. The transcription products of three UmV strains were compared, and the overall pattern of transcription was very similar among them.

  14. Internal control for real-time polymerase chain reaction based on MS2 bacteriophage for RNA viruses diagnostics

    Directory of Open Access Journals (Sweden)

    Miriam Ribas Zambenedetti

    Full Text Available BACKGROUND Real-time reverse transcription polymerase chain reaction (RT-PCR is routinely used to detect viral infections. In Brazil, it is mandatory the use of nucleic acid tests to detect hepatitis C virus (HCV, hepatitis B virus and human immunodeficiency virus in blood banks because of the immunological window. The use of an internal control (IC is necessary to differentiate the true negative results from those consequent from a failure in some step of the nucleic acid test. OBJECTIVES The aim of this study was the construction of virus-modified particles, based on MS2 bacteriophage, to be used as IC for the diagnosis of RNA viruses. METHODS The MS2 genome was cloned into the pET47b(+ plasmid, generating pET47b(+-MS2. MS2-like particles were produced through the synthesis of MS2 RNA genome by T7 RNA polymerase. These particles were used as non-competitive IC in assays for RNA virus diagnostics. In addition, a competitive control for HCV diagnosis was developed by cloning a mutated HCV sequence into the MS2 replicase gene of pET47b(+-MS2, which produces a non-propagating MS2 particle. The utility of MS2-like particles as IC was evaluated in a one-step format multiplex real-time RT-PCR for HCV detection. FINDINGS We demonstrated that both competitive and non-competitive IC could be successfully used to monitor the HCV amplification performance, including the extraction, reverse transcription, amplification and detection steps, without compromising the detection of samples with low target concentrations. In conclusion, MS2-like particles generated by this strategy proved to be useful IC for RNA virus diagnosis, with advantage that they are produced by a low cost protocol. An attractive feature of this system is that it allows the construction of a multicontrol by the insertion of sequences from more than one pathogen, increasing its applicability for diagnosing different RNA viruses.

  15. Correlation of autoimmune reactivity with hepatitis B and C virus (HBV and HCV infection in histologically proven chronic liver diseases

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    Shantha S

    2002-01-01

    Full Text Available PURPOSE: To comprehensively study the possibility of autoimmune reactivity by hepatitis viruses B and C (HBV & HCV in Indian chronic liver disease (CLD patients. METHODS: One hundred and sixty histopathologically proven CLD cases and 100 matched controls were analysed for viral serology for HBV and HCV and autoimmune serology for antinuclear antibody (ANA, anti smooth muscle antibody (ASMA and Liver kidney microsomal antibody (LKM using standard immunofluorescence technique. RESULTS: 43.7% of cases were chronic hepatitis B while 16.2% were positive for HCV. CLD-B cases showed ANA positivity in 27.1% and ASMA positivity in 25.7%. CLD-C cases revealed 26.9%, 46.1% and 11.1% positivity for ANA, ASMA and LKM antibodies respectively. These rates and titres of autoantibodies were statistically significant (p=<0.02 when compared with that of controls. Conclusions: Based on the pattern of autoantibody positivity, it could be concluded that chronic HBV infection may induce autoimmune hepatitis (AIH type I and chronic HCV infection might trigger AIH - Type II in Indian CLD cases.

  16. HBV And HCV Molecular Evolution

    Directory of Open Access Journals (Sweden)

    Flor H. Pujol

    2007-02-01

    hepatitis C virus (HCV. Six genotypes and a large number of subtypes in each genotype have been described for this member of the Flaviviridae family. Infections with HCV genotype 1 are associated with the lowest therapeutic success. HCV genotype 1b has also been more frequently associated with a more severe liver disease. However, this association seems to be due to the fact that individuals infected with this genotype have a longer mean duration of infection. HCV genotypes 1, 2, and 3 have a worldwide distribution and display an apidemic pattern of distribution. HCV subtypes 1a and 1b are the most common genotypes in the United States and are also are predominant in Europe, while in Japan, subtype 1b is predominant. Although HCV subtypes 2a and 2b are relatively common in America, Europe, and Japan, subtype 2c is found commonly in northern Italy. HCV genotype 3a is frequent in intravenous drug abusers in Europe and the United States. HCV genotype 4 appears to be prevalent in Africa and theMiddle East, and genotypes 5 and 6 seem to be confined to South Africa and Asia, respectively. These last genotypes display an endemic pattern of distribution. In addition, a change in the frequency of the prevailing genotypes has been described in several countries: in general, HCV genotype 1b is being displaced by genotypes 3a and/or 2. Coalescent studies have allowed to describe the epidemic pattern of dissemination of some HCV subtypes in specific countries, generally around 100 years ago. The origin of this virus is still an open question, but several studies traces it diversification only around 1,000 years ago.

    The replication of HCV is dependent on a RNA-polymerase RNA dependent which lacks proofreading activity, which confers to this virus a high rate of variability. This virus circulates as a quasispecies. This population dynamic inside a single strain confers to this virus the ability to

  17. Hepatitis B Virus (HBV), Hepatitis C Virus (HCV) and Syphilis Co ...

    African Journals Online (AJOL)

    Background: Victims of HIV infection may suffer a co-infection with hepatitis B and C viruses that share similar mode of transmission. Syphilis, a major cause of Sexually Transmitted Infections, is known to predispose to the transmission of these infections. Aim: The objective of this study is to determine the seroprevalence of ...

  18. Isolation as a strategy for controlling the transmission of hepatitis C virus (HCV) infection in haemodialysis units.

    Science.gov (United States)

    Bravo Zuñiga, Jessica I; Loza Munárriz, César; López-Alcalde, Jesús

    2016-08-11

    The hepatitis C virus (HCV) infection affects about 2% of the world's population and can cause chronic liver infection and persistent long-term sequelae such as cirrhosis and liver cancer.The prevalence of HCV infection among people on haemodialysis is often higher than the general population. The virus is easily transmitted parenterally, and blood transfusions have previously played a significant role in transmission; however, erythropoietin therapy has reduced the need for transfusions, and coupled with improved screening of donated blood, has significantly decreased transmission by transfusion. Although control of hospital-acquired infection has improved with the advent of biosafety measures, stopping HCV transmission in haemodialysis units remains challenging.Isolating people infected with HCV involves physical separation from others to limit direct or indirect transmission and includes a number of strategies during dialysis. The evidence for isolating people infected with HCV during haemodialysis is sparse with some inconsistencies. To evaluate the benefits and harms of isolation of HCV-infected patients during haemodialysis on the transmission of HCV to other patients. We searched the Cochrane Kidney and Transplant Specialised Register to 26 November 2015 through contact with the Information Specialist using search terms relevant to this review. We also searched the Latin American and Caribbean Health Sciences Literature Database (LILACS) (1982 to 2015), Web of Science Conference Proceedings Citation Index-Science (CPCI-S, 1990 to 2015), ProQuest Dissertations & Theses Database (1990 to 2015), and Open Grey (1990 to 2015). We included randomised controlled trials (RCTs), quasi-RCTs and cluster RCTs evaluating the clinical benefits and harms of isolating HCV-infected patients during haemodialysis on the transmission of HCV to other patients. We considered incidence of dialysis-acquired HCV infection, all-cause mortality, and adverse effects associated with

  19. HCV clearance patterns in saliva and serum of patients with chronic HCV infection under interferon plus ribavirin therapy.

    Science.gov (United States)

    Diz Dios, P; Castro, A; Rodríguez, I; Reforma, N G; Castro, M; Eirea, M; Hermida, M

    2005-05-01

    Hepatitis C virus (HCV)-RNA is often present in saliva of HCV-infected patients, with plasma viral load being the only known predictable factor. Interferon plus ribavirin therapy yields a sustained reduction in HCV viremia. This study aimed to assess the presence of HCV in saliva and serum specimens from patients undergoing this combination therapy (CT). Paired serum and saliva specimens were collected from 44 chronic HCV-infected patients at basal time, 4 and 12 weeks after CT onset, at the end of treatment and 6 months latter. Serum HCV-RNA levels were determined by the polymerase chain reaction (PCR) Amplicor system. Presence of HCV-RNA in saliva was tested by a highly sensitive non-commercialized nested-PCR. The HCV-RNA was detected in 26 saliva specimens at basal time (59.1%). In 34.1% of cases, a concordance viral clearance pattern in serum and saliva was observed in both responders (pattern 1a) and non-responders (pattern 1b). In pattern 2 (13.6% of cases), HCV was detected longer during CT in serum than in saliva (pattern 2a) or in saliva than in serum (pattern 2b). In 11.3% of patients, viral clearance was corroborated either in their serum (pattern 3a) or in their saliva (pattern 3b), but not in both fluids. Of the eight primary responders with 1a clearance pattern, seven were sustained responders. None of the patients with 2a clearance pattern was a sustained responder. Of the two primary responders showing the 3b salivary pattern, one had already relapsed in the first 6 months of follow up. The present results suggest that the monitoring of salivary levels of HCV would be a helpful means of determining sustained antiviral effects of interferon and ribavirin in the treatment of HCV disease.

  20. Treatment for hepatitis B virus (HBV) and hepatitis C virus (HCV) infection - Danish national guidelines 2011.

    Science.gov (United States)

    Christensen, Peer Brehm; Clausen, Mette Rye; Krarup, Henrik; Laursen, Alex Lund; Schlichting, Poul; Weis, Nina

    2012-06-01

    The Danish Society of Infectious Diseases and Danish Society of Gastroenterology and Hepatology set up a committee in 2007 to produce national guidelines for treatment of viral hepatitis B and C. The 2011 version of the guidelines have been endorsed by the scientific societies and are presented below. Annual updates will be available at the websites of the societies. As this present English version has been written six months after the Danish 2011 version, it contains minor changes that will be integrated in the Danish 2012 version, available at the end this year. Viral hepatitis is not common in Denmark. The prevalence has not been determined by national surveys, but it is estimated that 10,000-15,000 patients are chronically infected with hepatitis B and 15,000-20,000 with chronic hepatitis C. The majority of patients with HBV infection in Denmark are emigrants from high endemic countries, probably infected at birth or early childhood in their country of origin, while the majority of patients with HCV infection have been infected by drug use. For both groups it is estimated that only half of the patients have been diagnosed, of whom only 20% attends specialized care for their chronic viral hepatitis. According to the Danish National Board of Health, patients with chronic viral hepatitis should be followed with regular intervals, at clinics specialized in either infectious diseases or gastroenterology/hepatology. The primary aim is to identify patients with significant liver disease to initiate treatment in order to prevent development of cirrhosis and death. This is primarily done by liver biopsy, but screening for fibrosis with non-invasive methods such as elastography may be sufficient in some patients. Patients with established cirrhosis should enter screening programs for complications such as esophageal varices and hepatocellular carcinoma.

  1. A Review of Hepatitis C Virus (HCV) and the Current Management ...

    African Journals Online (AJOL)

    DATONYE ALASIA

    deliveries from HCV infected women . The incidence is higher (14%-17%) in patients co- infected ... from women of childbearing age before initiation of HCV treatment. Women who are pregnant or attempting to ... infection; persons with hemophilia who received clotting factor concentrate prior to 1987, persons who have ...

  2. Exosomes and Their Role in the Life Cycle and Pathogenesis of RNA Viruses

    Science.gov (United States)

    Chahar, Harendra Singh; Bao, Xiaoyong; Casola, Antonella

    2015-01-01

    Exosomes are membrane-enclosed vesicles actively released into the extracellular space, whose content reflect the physiological/pathological state of the cells they originate from. These vesicles participate in cell-to-cell communication and transfer of biologically active proteins, lipids, and RNAs. Their role in viral infections is just beginning to be appreciated. RNA viruses are an important class of pathogens and affect millions of people worldwide. Recent studies on Human Immunodeficiency Virus (HIV), Hepatitis C Virus (HCV), human T-cell lymphotropic virus (HTLV), and Dengue Virus (DENV) have demonstrated that exosomes released from infected cells harbor and deliver many regulatory factors including viral RNA and proteins, viral and cellular miRNA, and other host functional genetic elements to neighboring cells, helping to establish productive infections and modulating cellular responses. Exosomes can either spread or limit an infection depending on the type of pathogen and target cells, and can be exploited as candidates for development of antiviral or vaccine treatments. This review summarizes recent progress made in understanding the role of exosomes in RNA virus infections with an emphasis on their potential contribution to pathogenesis. PMID:26102580

  3. Inactivation of RNA viruses by gamma irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Nonomiya, Takashi; Morimoto, Akinori; Iwatsuki, Kazuo; Tsutsumi, Takamasa (Ministry of Agriculture, Forestry and fisheries, Yokohama, Kanagawa (Japan). Animal Quarantine Service); Ito, Hitoshi; Yamashiro, Tomio; Ishigaki, Isao

    1992-09-01

    Four kinds of RNA viruses, Bluetongue virus (BT), Bovine Virus Diarrhea-Mucosal Disease virus (BVD[center dot]MD), Bovine Respiratory Syncytial virus (RS), Vesicular Stmatitis virus (VS), were subjected to various doses of gamma irradiation to determine the lethal doses. The D[sub 10] values, which are the dose necessary to decimally reduce infectivity, ranged from 1.5 to 3.4 kGy under frozen condition at dry-ice temperature, and they increased to 2.6 to 5.0 kGy under frozen condition at dry-ice temperature. Serum neutralzing antibody titer of Infectious Bovine Rhinotracheitis (IBR) was not adversely changed by the exposure to 36 kGy of gamma-rays under frozen condition. Analysis of electrophoresis patterns of the bovine serum also reveales that the serum proteins were not remarkably affected, even when exposed to 36 kGy of gamma radiation under frozen condition. The results suggested that gamma irradiation under frozen condition is an effective means for inactivating both DNA and RNA viruses without adversely affecting serum proteins and neutralizing antibody titer. (author).

  4. The negative impact of HBV/HCV coinfection on cirrhosis and its consequences.

    Science.gov (United States)

    Pol, S; Haour, G; Fontaine, H; Dorival, C; Petrov-Sanchez, V; Bourliere, M; Capeau, J; Carrieri, P; Larrey, D; Larsen, C; Marcellin, P; Pawlostky, J-M; Nahon, P; Zoulim, F; Cacoub, P; de Ledinghen, V; Mathurin, P; Negro, F; Pageaux, G-P; Yazdanpanah, Y; Wittkop, L; Zarski, J-P; Carrat, F

    2017-10-09

    Hepatitis B virus (HBV)/hepatitis C virus (HCV) confection has been rarely studied in nonasian series. To compare the characteristics of HBV/HCV coinfected patients to those of HBV- or HCV-monoinfected patients in the ANRS CO22 HEPATHER cohort study. Of the 20 936 included patients, 95 had HBV/HCV coinfection (hepatitis B surface antigen, anti-HCV antibody and HCV RNA positive) and were matched with 375 HBV- and 380 HCV-monoinfected patients on age, gender and time since HBV or HCV diagnosis. F3-F4 fibrosis was more frequent in coinfected patients (58%) than in HBV- (32%, P HBV- (2%, P = .0002) or HCV- (4%, P = .0275) monoinfected patients. Past excessive alcohol use was more frequent in coinfected patients (26%) than in HBV (12%, P = .0011), but similar in HCV monoinfected patients (32%, P = .2868). Coinfected patients had a higher proportion with arterial hypertension (42%) than HBV- (26%) or HCV-monoinfected patients (25%) (P HBV-infected patients (OR = 3.84, 95% CI 1.99-7.43) and the association between decompensated cirrhosis and coinfection in HBV infected (OR = 5.58, 95% CI 1.42-22.0) or HCV infected patients (OR = 3.02, 95% CI 1.22-7.44). HCV coinfection harmfully affects liver fibrosis in HBV patients, while decompensated cirrhosis is increased in coinfected patients compared with HBV- or HCV-monoinfected patients. HCV treatment is as safe and effective in coinfected as monoinfected patients and should be considered following the same rules as HCV monoinfected patients. © 2017 John Wiley & Sons Ltd.

  5. Hepatitis C Virus and Hepatocellular Carcinoma

    Directory of Open Access Journals (Sweden)

    Masao Omata

    2013-01-01

    Full Text Available Hepatitis C virus (HCV, a hepatotropic virus, is a single stranded-positive RNA virus of ~9,600 nt. length belonging to the Flaviviridae family. HCV infection causes acute hepatitis, chronic hepatitis, cirrhosis and hepatocellular carcinoma (HCC. It has been reported that HCV-coding proteins interact with host-cell factors that are involved in cell cycle regulation, transcriptional regulation, cell proliferation and apoptosis. Severe inflammation and advanced liver fibrosis in the liver background are also associated with the incidence of HCV-related HCC. In this review, we discuss the mechanism of hepatocarcinogenesis in HCV-related liver diseases.

  6. Role of HCV Core gene of genotype 1a and 3a and host gene Cox-2 in HCV-induced pathogenesis

    Directory of Open Access Journals (Sweden)

    Ahmad Waqar

    2011-04-01

    Full Text Available Abstract Background Hepatitis C virus (HCV Core protein is thought to trigger activation of multiple signaling pathways and play a significant role in the alteration of cellular gene expression responsible for HCV pathogenesis leading to hepatocellular carcinoma (HCC. However, the exact molecular mechanism of HCV genome specific pathogenesis remains unclear. We examined the in vitro effects of HCV Core protein of HCV genotype 3a and 1a on the cellular genes involved in oxidative stress and angiogenesis. We also studied the ability of HCV Core and Cox-2 siRNA either alone or in combination to inhibit viral replication and cell proliferation in HCV serum infected Huh-7 cells. Results Over expression of Core gene of HCV 3a genotype showed stronger effect in regulating RNA and protein levels of Cox-2, iNOS, VEGF, p-Akt as compared to HCV-1a Core in hepatocellular carcinoma cell line Huh-7 accompanied by enhanced PGE2 release and cell proliferation. We also observed higher expression levels of above genes in HCV 3a patient's blood and biopsy samples. Interestingly, the Core and Cox-2-specific siRNAs down regulated the Core 3a-enhanced expression of Cox-2, iNOS, VEGF, p-Akt. Furthermore, the combined siRNA treatment also showed a dramatic reduction in viral titer and expression of these genes in HCV serum-infected Huh-7 cells. Taken together, these results demonstrated a differential response by HCV 3a genotype in HCV-induced pathogenesis, which may be due to Core and host factor Cox-2 individually or in combination. Conclusions Collectively, these studies not only suggest a genotype-specific interaction between key players of HCV pathogenesis but also may represent combined viral and host gene silencing as a potential therapeutic strategy.

  7. Genetic History of Hepatitis C Virus in Venezuela: High Diversity and Long Time of Evolution of HCV Genotype 2

    Science.gov (United States)

    Sulbarán, Maria Z.; Di Lello, Federico A.; Sulbarán, Yoneira; Cosson, Clarisa; Loureiro, Carmen L.; Rangel, Héctor R.; Cantaloube, Jean F.; Campos, Rodolfo H.; Moratorio, Gonzalo; Cristina, Juan; Pujol, Flor H.

    2010-01-01

    Background The subtype diversity of the hepatitis C virus (HCV) genotypes is unknown in Venezuela. Methodology/Principal Findings Partial sequencing of the NS5B region was performed in 310 isolates circulating in patients from 1995 to 2007. In the samples collected between 2005 and 2007, HCV genotype 1 (G1) was the most common genotype (63%), composed as expected of mainly G1a and G1b. G2 was the second most common genotype (33%), being G2a almost absent and G2j the most frequent subtype. Sequence analysis of the core region confirmed the subtype assignment performed within the NS5b region in 63 isolates. The complete genome sequence of G2j was obtained. G2j has been described in France, Canada and Burkina Fasso, but it was not found in Martinique, where several subtypes of G2 circulate in the general population. Bayesian coalescence analysis indicated a most recent common ancestor (MRCA) of G2j around 1785, before the introduction of G1b (1869) and G1a (1922). While HCV G1a and G1b experienced a growth reduction since 1990, coincident with the time when blood testing was implemented in Venezuela, HCV G2j did not seem to reach growth equilibrium during this period. Conclusions/Significance Assuming the introduction of G2j from Africa during the slave trade, the high frequency of G2j found in Venezuela could suggest: 1- the introduction of African ethnic groups different from the ones introduced to Martinique or 2- the occurrence of a founder effect. This study represents an in-depth analysis of the subtype diversity of HCV in Venezuela, which is still unexplored in the Americas and deserves further studies. PMID:21179440

  8. Inhibition of virus replication by RNA interference

    NARCIS (Netherlands)

    Haasnoot, P. C. Joost; Cupac, Daniel; Berkhout, Ben

    2003-01-01

    RNA interference (RNAi) is a sequence-specific gene-silencing mechanism in eukaryotes, which is believed to function as a defence against viruses and transposons. Since its discovery, RNAi has been developed into a widely used technique for generating genetic knock-outs and for studying gene

  9. Visualization and measurement of ATP levels in living cells replicating hepatitis C virus genome RNA.

    Directory of Open Access Journals (Sweden)

    Tomomi Ando

    Full Text Available Adenosine 5'-triphosphate (ATP is the primary energy currency of all living organisms and participates in a variety of cellular processes. Although ATP requirements during viral lifecycles have been examined in a number of studies, a method by which ATP production can be monitored in real-time, and by which ATP can be quantified in individual cells and subcellular compartments, is lacking, thereby hindering studies aimed at elucidating the precise mechanisms by which viral replication energized by ATP is controlled. In this study, we investigated the fluctuation and distribution of ATP in cells during RNA replication of the hepatitis C virus (HCV, a member of the Flaviviridae family. We demonstrated that cells involved in viral RNA replication actively consumed ATP, thereby reducing cytoplasmic ATP levels. Subsequently, a method to measure ATP levels at putative subcellular sites of HCV RNA replication in living cells was developed by introducing a recently-established Förster resonance energy transfer (FRET-based ATP indicator, called ATeam, into the NS5A coding region of the HCV replicon. Using this method, we were able to observe the formation of ATP-enriched dot-like structures, which co-localize with non-structural viral proteins, within the cytoplasm of HCV-replicating cells but not in non-replicating cells. The obtained FRET signals allowed us to estimate ATP concentrations within HCV replicating cells as ∼5 mM at possible replicating sites and ∼1 mM at peripheral sites that did not appear to be involved in HCV replication. In contrast, cytoplasmic ATP levels in non-replicating Huh-7 cells were estimated as ∼2 mM. To our knowledge, this is the first study to demonstrate changes in ATP concentration within cells during replication of the HCV genome and increased ATP levels at distinct sites within replicating cells. ATeam may be a powerful tool for the study of energy metabolism during replication of the viral genome.

  10. Intracellular expression of IRF9 Stat fusion protein overcomes the defective Jak-Stat signaling and inhibits HCV RNA replication

    Directory of Open Access Journals (Sweden)

    Balart Luis A

    2010-10-01

    Full Text Available Abstract Interferon alpha (IFN-α binds to a cell surface receptor that activates the Jak-Stat signaling pathway. A critical component of this pathway is the translocation of interferon stimulated gene factor 3 (a complex of three proteins Stat1, Stat2 and IRF9 to the nucleus to activate antiviral genes. A stable sub-genomic replicon cell line resistant to IFN-α was developed in which the nuclear translocation of Stat1 and Stat2 proteins was prevented due to the lack of phosphorylation; whereas the nuclear translocation of IRF9 protein was not affected. In this study, we sought to overcome defective Jak-Stat signaling and to induce an antiviral state in the IFN-α resistant replicon cell line by developing a chimera IRF9 protein fused with the trans activating domain (TAD of either a Stat1 (IRF9-S1C or Stat2 (IRF9-S2C protein. We show here that intracellular expression of fusion proteins using the plasmid constructs of either IRF9-S1C or IRF9-S2C, in the IFN-α resistant cells, resulted in an increase in Interferon Stimulated Response Element (ISRE luciferase promoter activity and significantly induced HLA-1 surface expression. Moreover, we show that transient transfection of IRF9-S1C or IRF9-S2C plasmid constructs into IFN-α resistant replicon cells containing sub-genomic HCV1b and HCV2a viruses resulted in an inhibition of viral replication and viral protein expression independent of IFN-α treatment. The results of this study indicate that the recombinant fusion proteins of IRF9-S1C, IRF9-S2C alone, or in combination, have potent antiviral properties against the HCV in an IFN-α resistant cell line with a defective Jak-Stat signaling.

  11. Clearance of low levels of HCV viremia in the absence of a strong adaptive immune response

    Directory of Open Access Journals (Sweden)

    Manns Michael P

    2007-06-01

    Full Text Available Abstract Spontaneous clearance of hepatitis C virus (HCV has frequently been associated with the presence of HCV-specific cellular immunity. However, there had been also reports in chimpanzees demonstrating clearance of HCV-viremia in the absence of significant levels of detectable HCV-specific cellular immune responses. We here report seven asymptomatic acute hepatitis C cases with peak HCV-RNA levels between 300 and 100.000 copies/ml who all cleared HCV-RNA spontaneously. Patients were identified by a systematic screening of 1176 consecutive new incoming offenders in a German young offender institution. Four of the seven patients never developed anti-HCV antibodies and had normal ALT levels throughout follow-up. Transient weak HCV-specific CD4+ T cell responses were detectable in five individuals which did not differ in strength and breadth from age- and sex-matched patients with chronic hepatitis C and long-term recovered patients. In contrast, HCV-specific MHC-class-I-tetramer-positive cells were found in 3 of 4 HLA-A2-positive patients. Thus, these cases highlight that clearance of low levels of HCV viremia is possible in the absence of a strong adaptive immune response which might explain the low seroconversion rate after occupational exposure to HCV.

  12. Hepatitis C virus internal ribosome entry site RNA contains a tertiary structural element in a functional domain of stem–loop II

    Science.gov (United States)

    Lyons, Alita J.; Lytle, J. Robin; Gomez, Jordi; Robertson, Hugh D.

    2001-01-01

    The internal ribosome entry site (IRES) of hepatitis C virus (HCV) RNA contains >300 bases of highly conserved 5′-terminal sequence, most of it in the uncapped 5′-untranslated region (5′-UTR) upstream from the single AUG initiator triplet at which translation of the HCV polyprotein begins. Although progress has been made in defining singularities like the RNA pseudoknot near this AUG, the sequence and structural features of the HCV IRES which stimulate accurate and efficient initiation of protein synthesis are only partially defined. Here we report that a region further upstream from the AUG, stem–loop II of the HCV IRES, also contains an element of local tertiary structure which we have detected using RNase H cleavage and have mapped using the singular ability of two bases therein to undergo covalent intra-chain crosslinking stimulated by UV light. This pre-existing element maps to two non-contiguous stretches of the HCV IRES sequence, residues 53–68 and 103–117. Several earlier studies have shown that the correct sequence between bases 45 and 70 of the HCV IRES stem–loop II domain is required for initiation of protein synthesis. Because features of local tertiary structure like the one we report here are often associated with protein binding, we propose that the HCV stem–loop II element is directly involved in IRES action. PMID:11410661

  13. Cyclosporin A associated helicase-like protein facilitates the association of hepatitis C virus RNA polymerase with its cellular cyclophilin B.

    Directory of Open Access Journals (Sweden)

    Kengo Morohashi

    Full Text Available BACKGROUND: Cyclosporin A (CsA is well known as an immunosuppressive drug useful for allogeneic transplantation. It has been reported that CsA inhibits hepatitis C virus (HCV genome replication, which indicates that cellular targets of CsA regulate the viral replication. However, the regulation mechanisms of HCV replication governed by CsA target proteins have not been fully understood. PRINCIPAL FINDINGS: Here we show a chemical biology approach that elucidates a novel mechanism of HCV replication. We developed a phage display screening to investigate compound-peptide interaction and identified a novel cellular target molecule of CsA. This protein, named CsA associated helicase-like protein (CAHL, possessed RNA-dependent ATPase activity that was negated by treatment with CsA. The downregulation of CAHL in the cells resulted in a decrease of HCV genome replication. CAHL formed a complex with HCV-derived RNA polymerase NS5B and host-derived cyclophilin B (CyPB, known as a cellular cofactor for HCV replication, to regulate NS5B-CyPB interaction. CONCLUSIONS: We found a cellular factor, CAHL, as CsA associated helicase-like protein, which would form trimer complex with CyPB and NS5B of HCV. The strategy using a chemical compound and identifying its target molecule by our phage display analysis is useful to reveal a novel mechanism underlying cellular and viral physiology.

  14. Psychiatric barriers to readiness for treatment for hepatitis C Virus (HCV) infection among injection drug users: clinical experience of an addiction psychiatrist in the HIV-HCV coinfection clinic of a public health hospital.

    Science.gov (United States)

    Scheft, Harriet; Fontenette, Dominique C

    2005-04-15

    Among injection drug users, psychological and psychiatric barriers to readiness for treatment for hepatitis C virus (HCV) infection include mood and anxiety disorders, cognitive deficits, temperament disorders, and personality vulnerabilities, as well as ongoing drug use. Many aspects of these barriers can be overcome with direct treatment or social support. To establish effective treatment for HCV infection in this population of patients, it is essential that the patient and providers develop a rapport that allows for active communication. It is also important that the patient make an effort to adhere to the treatment requirements and that the patient receive the appropriate evaluation and management of treatable barriers.

  15. Analytical variables influencing the HCV RNA determination by TaqMan real-time PCR in routine clinical laboratory practice.

    Science.gov (United States)

    Raza, Abida; Ali, Zameer; Irfan, Javaid; Murtaza, Shahnaz; Shakeel, Samina

    2012-07-01

    Hepatitis C virus (HCV) quantification is used as a prognostic marker for treatment success. In a routine clinical laboratory some infinitesimal sample handling factors can contribute to variability and loss of precision in HCV quantification. This may include blood collection tubes, blood drawing procedure, sample processing and storage temperatures. In current study blood was collected in tubes with different anticoagulant type (spray vs. liquid), group 1, blood was drawn with possible suck of methylated spirit through needle (experimental group) while avoiding the methylated spirit suck (control group) group 2, plasma separation was delayed from 0 to 60 min for group 3, plasma storage at different temperatures group 4. All samples were analyzed using Corbett research real time PCR system using AJ Roboscreen Kit. Mean viral load difference between spray vs. liquid was found 3.6 × 10(5) IU/ml (p spirit inhibited the viral load quantification with a value of 4.8 × 10(5) IU/ml (p levels (p > 0.05). In conclusion blood collection tubes and procedures can be a key factor in variability of results, that might affect the treatment response decision.

  16. Historical Trends in the Hepatitis C Virus Epidemics in North America and Australia

    OpenAIRE

    Rodrigo, Chaturaka; Eltahla, Auda A.; Bull, Rowena A.; Grebely, Jason; Dore, Gregory J.; Applegate, Tanya; Page, Kimberly; Bruneau, Julie; Morris, Meghan D.; Cox, Andrea L.; Osburn, William; Kim, Arthur Y.; Schinkel, Janke; Shoukry, Naglaa H.; Lauer, Georg M.

    2016-01-01

    Background. Bayesian evolutionary analysis (coalescent analysis) based on genetic sequences has been used to describe the origins and spread of rapidly mutating RNA viruses, such as influenza, Ebola, human immunodeficiency virus (HIV), and hepatitis C virus (HCV).

  17. Prevalence of HCV infection and associated factors among illicit drug users in Breves, State of Pará, northern Brazil

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    Suzy Danielly Barbosa Pacheco

    2014-06-01

    Full Text Available Introduction: Illicit drug users (DUs are vulnerable to hepatitis C virus (HCV infection. The shared use of illicit drugs is the main method of HCV transmission. Methods: A cross-sectional study was conducted in Breves, in northern Brazil. We surveyed 187 DUs to determine the prevalence of and factors associated with HCV infection. Results: The prevalence of anti-HCV antibodies was 36.9%, and the prevalence of hepatitis C virus-ribonucleic acid (HCV-RNA was 31%. Hepatitis C virus infection was associated with tattoos, intravenous drug use, shared use of equipment for drug use, drug use for longer than 3 years, and daily drug use. Conclusions: Strategies for preventing and controlling HCV transmission should be implemented among DUs.

  18. RIG-I in RNA virus recognition

    Science.gov (United States)

    Kell, Alison M.; Gale, Michael

    2015-01-01

    Antiviral immunity is initiated upon host recognition of viral products via non-self molecular patterns known as pathogen-associated molecular patterns (PAMPs). Such recognition initiates signaling cascades that induce intracellular innate immune defenses and an inflammatory response that facilitates development of the acquired immune response. The retinoic acid-inducible gene I (RIG-I) and the RIG-I-like receptor (RLR) protein family are key cytoplasmic pathogen recognition receptors that are implicated in the recognition of viruses across genera and virus families, including functioning as major sensors of RNA viruses, and promoting recognition of some DNA viruses. RIG-I, the charter member of the RLR family, is activated upon binding to PAMP RNA. Activated RIG-I signals by interacting with the adapter protein MAVS leading to a signaling cascade that activates the transcription factors IRF3 and NF-κB. These actions induce the expression of antiviral gene products and the production of type I and III interferons that lead to an antiviral state in the infected cell and surrounding tissue. RIG-I signaling is essential for the control of infection by many RNA viruses. Recently, RIG-I crosstalk with other pathogen recognition receptors and components of the inflammasome has been described. In this review, we discuss the current knowledge regarding the role of RIG-I in recognition of a variety of virus families and its role in programming the adaptive immune response through cross-talk with parallel arms of the innate immune system, including how RIG-I can be leveraged for antiviral therapy. PMID:25749629

  19. Hepatitis C virus (HCV) E1 and E2 protein regions that specifically bind to HepG2 cells.

    Science.gov (United States)

    Garcia, Javier Eduardo; Puentes, Alvaro; Súarez, Jorge; López, Ramses; Vera, Ricardo; Rodríguez, Luis Eduardo; Ocampo, Marisol; Curtidor, Hernando; Guzman, Fanny; Urquiza, Mauricio; Patarroyo, Manuel Elkin

    2002-02-01

    Identify hepatitis C virus (HCV) sequences in E1 and E2 protein binding to HepG2. Synthetic 20-mer long, ten-residue overlapped peptides, from E1 and E2 proteins, were tested in HepG2 or Raji cell-binding assays. Affinity constants, binding site number per cell and Hill coefficients were determined by saturation assay for high activity binding peptides (HABPs). Receptors for HepG2 cell were determined by cross-linking and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Twelve HABPs were found in HCV genotype 1a, allowing six hepatocyte-binding sequences (HBSs) to be defined: two peptide-binding regions in E1 HABPs 4913 (YQVRNSTGLYHVTNDCPNSS) and 4918 (MTPTVATRDGKLPATQLRRHY). Four hepatocyte-binding regions were defined in E2: region-I, peptide 4931 (ETHVTGGSAGHTVSGFVSLLY); region-II, 4937-4939 (HHKFNSSGCPERLASCRPLTDFDQGWGPISYANGSGPDQR); region-III, 4943-4945 (PVYCFTPSPVVVGTTDRSGAPTYSWGENDTDVFVLNNTR) and region-IV, 4949-4952 (CGAPPCVIGGAGNNTLHCPTDCFRKHPDATYSRCGSGPWITPRCLVDYPY). The underlined sequences are most relevant in the binding process. HABPs 4913 and 4938 also bind to CD81 positive Raji cells. Region-II 4938 HABPs bind to 50 and 60kDa HepG2 cell membrane surface proteins. Six HVRs to the HepG2 were identified. Some HABPs have been previously found to be antigenic and immunogenic. HABPs, 4918 (from E1), 4938, 4949, 4950, 4951 and 4952 (from E2) have not been previously recognised. These HABPs could be relevant to HCV invasion of hepatocytes.

  20. Impedimetric genosensor for detection of hepatitis C virus (HCV1) DNA using viral probe on methylene blue doped silica nanoparticles.

    Science.gov (United States)

    Singhal, Chaitali; Ingle, Aviraj; Chakraborty, Dhritiman; Pn, Anoop Krishna; Pundir, C S; Narang, Jagriti

    2017-05-01

    An impedimetric genosensor was fabricated for detection of hepatitis C virus (HCV) genotype 1 in serum, based on hybridization of the probe with complementary target cDNA from sample. To achieve it, probe DNA complementary to HCVgene was immobilized on the surface of methylene blue (MB) doped silica nanoparticles MB@SiNPs) modified fluorine doped tin oxide (FTO) electrode. The synthesized MB@SiNPs was characterized using scanning electron microscopy (SEM), high resolution transmission electron microscopy (HRTEM) and X-ray diffraction (XRD) pattern. This modified electrode (ssDNA/MB@SiNPs/FTO) served both as a signal amplification platform (due to silica nanoparticles (SiNPs) as well as an electrochemical indicator (due to methylene blue (MB)) for the detection of the HCV DNA in patient serum sample. The genosensor was optimized and evaluated. The sensor showed a dynamic linear range 100-10 6 copies/mL, with a detection limit of 90 copies/mL. The sensor was applied for detection of HCV in sera of hepatitis patient and could be renewed. The half life of the sensor was 4 weeks. The MB@SiNPs/FTO electrode could be used for preparation of other gensensors also. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Inhibitory effect of theobromine on induction of angiogenesis and VEGF mRNA expression in v-raf transfectants of human urothelial cells HCV-29.

    Science.gov (United States)

    Skopinska-Rózewska, E; Janik, P; Przybyszewska, M; Sommer, E; Bialas-Chromiec, B

    1998-12-01

    Neovascularisation plays a crucial role in solid tumor growth and metastasis formation. Our previous studies showed that theophylline and theobromine suppressed cutaneous neovascular reaction induced in mice by human blood leukocytes, and lung as well as ovarian cancer cells. Here, we investigated the in vivo effect of theobromine on angiogenic activity of human urothelial cell line HCV-29, v-raf transfected (mouse cutaneous assay), and the in vitro effect of this drug on VEGF, tPA, uPA and PAI mRNA expression in these cells (RT-PCR method). Theobromine suppressed angiogenesis induced in mice by HCV-29-v-raf cells, inhibited VEGF mRNA expression, and had no effect on transcription of uPA and tPA in these cells. HCV-29-v-raf transfectants do not display transcripts of PAI, in the presence or the absence of theobromine.

  2. Mutual Interference between Genomic RNA Replication and Subgenomic mRNA Transcription in Brome Mosaic Virus

    OpenAIRE

    Grdzelishvili, Valery Z.; Garcia-Ruiz, Hernan; Watanabe, Tokiko; Ahlquist, Paul

    2005-01-01

    Replication by many positive-strand RNA viruses includes genomic RNA amplification and subgenomic mRNA (sgRNA) transcription. For brome mosaic virus (BMV), both processes occur in virus-induced, membrane-associated compartments, require BMV replication factors 1a and 2a, and use negative-strand RNA3 as a template for genomic RNA3 and sgRNA syntheses. To begin elucidating their relations, we examined the interaction of RNA3 replication and sgRNA transcription in Saccharomyces cerevisiae expres...

  3. The 5BSL3.2 Functional RNA Domain Connects Distant Regions in the Hepatitis C Virus Genome

    Directory of Open Access Journals (Sweden)

    Cristina Romero-López

    2017-10-01

    Full Text Available Viral genomes are complexly folded entities that carry all the information required for the infective cycle. The nucleotide sequence of the RNA virus genome encodes proteins and functional information contained in discrete, highly conserved structural units. These so-called functional RNA domains play essential roles in the progression of infection, which requires their preservation from one generation to the next. Numerous functional RNA domains exist in the genome of the hepatitis C virus (HCV. Among them, the 5BSL3.2 domain in the cis-acting replication element (CRE at the 3′ end of the viral open reading frame has become of particular interest given its role in HCV RNA replication and as a regulator of viral protein synthesis. These functionalities are achieved via the establishment of a complex network of long-distance RNA–RNA contacts involving (at least as known to date the highly conserved 3′X tail, the apical loop of domain IIId in the internal ribosome entry site, and/or the so-called Alt region upstream of the CRE. Changing contacts promotes the execution of different stages of the viral cycle. The 5BSL3.2 domain thus operates at the core of a system that governs the progression of HCV infection. This review summarizes our knowledge of the long-range RNA–RNA interaction network in the HCV genome, with special attention paid to the structural and functional consequences derived from the establishment of different contacts. The potential implications of such interactions in switching between the different stages of the viral cycle are discussed.

  4. Neonatal Exposure to Hepatitis C Virus Antigens in Uninfected Children Born to Infected Mothers.

    Science.gov (United States)

    Psaros Einberg, Afrodite; Brenndörfer, Erwin Daniel; Frelin, Lars; Hallberg, Lena; Sällberg, Matti; Fischler, Björn

    2017-09-26

    Vertical transmission of hepatitis C virus (HCV) infection is uncommon and occurs in around 5% of births from HCV infected mothers. The reason for the low transmission rate is unclear. We aimed to investigate if there is evidence of HCV exposure also in the non-infected children born to HCV infected mothers by the presence of a detectable immune response. Serum and peripheral blood mononuclear cells from 9 HCV vertically infected children, 32 uninfected children born to HCV infected mothers, and 15 HCV chronically infected mothers, were analyzed. HCV-RNA negative adults and children were used as controls. HCV specific T cell responses were analyzed by interferon gamma (IFN-γ) using an enzyme-linked immunospot (ELISpot) assay and 3H-thymidine incorporation assay. HCV antibodies were also analyzed. An HCV specific T cell response was detected in 73% (11/15) of the HCV infected mothers, 67% (6/9) of the vertically infected children, 56% (18/32) of the exposed but uninfected children and in 10% and 20% of the control groups, respectively. The two groups of HCV exposed children both had a significantly higher proportion of HCV specific T cell responders compared to pediatric controls (p = 0.01 and p = 0.02). HCV specific immune responses were more common in children born to HCV infected mothers, regardless of the presence of HCV RNA. We conclude that non-infected children born to HCV infected mothers may have been exposed to HCV antigens.

  5. Nuclear proteins hijacked by mammalian cytoplasmic plus strand RNA viruses

    Energy Technology Data Exchange (ETDEWEB)

    Lloyd, Richard E., E-mail: rlloyd@bcm.edu

    2015-05-15

    Plus strand RNA viruses that replicate in the cytoplasm face challenges in supporting the numerous biosynthetic functions required for replication and propagation. Most of these viruses are genetically simple and rely heavily on co-opting cellular proteins, particularly cellular RNA-binding proteins, into new roles for support of virus infection at the level of virus-specific translation, and building RNA replication complexes. In the course of infectious cycles many nuclear-cytoplasmic shuttling proteins of mostly nuclear distribution are detained in the cytoplasm by viruses and re-purposed for their own gain. Many mammalian viruses hijack a common group of the same factors. This review summarizes recent gains in our knowledge of how cytoplasmic RNA viruses use these co-opted host nuclear factors in new functional roles supporting virus translation and virus RNA replication and common themes employed between different virus groups. - Highlights: • Nuclear shuttling host proteins are commonly hijacked by RNA viruses to support replication. • A limited group of ubiquitous RNA binding proteins are commonly hijacked by a broad range of viruses. • Key virus proteins alter roles of RNA binding proteins in different stages of virus replication.

  6. Invertebrate RNA virus diversity from a taxonomic point of view.

    Science.gov (United States)

    Ryabov, Eugene V

    2017-07-01

    Invertebrates are hosts to diverse RNA viruses that have all possible types of encapsidated genomes (positive, negative and ambisense single stranded RNA genomes, or a double stranded RNA genome). These viruses also differ markedly in virion morphology and genome structure. Invertebrate RNA viruses are present in three out of four currently recognized orders of RNA viruses: Mononegavirales, Nidovirales, and Picornavirales, and 10 out of 37 RNA virus families that have yet to be assigned to an order. This mini-review describes general properties of the taxonomic groups, which include invertebrate RNA viruses on the basis of their current classification by the International Committee on Taxonomy of Viruses (ICTV). Copyright © 2016 Elsevier Inc. All rights reserved.

  7. ER stress, autophagy, and RNA viruses

    Directory of Open Access Journals (Sweden)

    Jia-Rong eJheng

    2014-08-01

    Full Text Available Endoplasmic reticulum (ER stress is a general term for representing the pathway by which various stimuli affect ER functions. ER stress induces the evolutionarily conserved signaling pathways, called the unfolded protein response (UPR, which compromises the stimulus and then determines whether the cell survives or dies. In recent years, ongoing research has suggested that these pathways may be linked to the autophagic response, which plays a key role in the cell’s response to various stressors. Autophagy performs a self-digestion function, and its activation protects cells against certain pathogens. However, the link between the UPR and autophagy may be more complicated. These two systems may act dependently, or the induction of one system may interfere with the other. Experimental studies have found that different viruses modulate these mechanisms to allow them to escape the host immune response or, worse, to exploit the host’s defense to their advantage; thus, this topic is a critical area in antiviral research. In this review, we summarize the current knowledge about how RNA viruses, including influenza virus, poliovirus, coxsackievirus, enterovirus 71, Japanese encephalitis virus, hepatitis C virus, and dengue virus, regulate these processes. We also discuss recent discoveries and how these will produce novel strategies for antiviral treatment.

  8. [Cognitive performances in workers with chronic virus hepatitis (HBV-HCV)].

    Science.gov (United States)

    Micali, E; Squadrito, G; Abbate, C; Raimondo, G; Trimarchi, F; Barbaro, M

    2007-01-01

    Chronic condition in subjects with chronic viral hepatitis determines issues neuropsychic. The sample of 21 workers suffering from chronic viral hepatitis in drug treatment has been studied with a battery of standardized tests to assess the cognitive performance, the neurobehavioral effects and psychological disorders that interfere with quality of life, comparing the results of subjects with HBV with those of subjects suffering from HCV. The results showed that both subjects with chronic HBV and HCV have relational-work restrictions that determine long periods of absence from the workplace, with the depression, anxiety, irritability and dysphoria. It is that in patients with chronic HCV physical functioning is significantly impaired with clinical manifestations of the disease that lead to major depression and deficit cognitive function.

  9. Frequency and genotypic distribution of GB virus C (GBV-C) among Colombian population with Hepatitis B (HBV) or Hepatitis C (HCV) infection.

    Science.gov (United States)

    Alvarado-Mora, Mónica V; Botelho, Livia; Nishiya, Anna; Neto, Raymundo A; Gomes-Gouvêa, Michele S; Gutierrez, Maria F; Carrilho, Flair J; Pinho, João Rr

    2011-07-11

    GB virus C (GBV-C) is an enveloped positive-sense ssRNA virus belonging to the Flaviviridae family. Studies on the genetic variability of the GBV-C reveals the existence of six genotypes: genotype 1 predominates in West Africa, genotype 2 in Europe and America, genotype 3 in Asia, genotype 4 in Southwest Asia, genotype 5 in South Africa and genotype 6 in Indonesia. The aim of this study was to determine the frequency and genotypic distribution of GBV-C in the Colombian population. Two groups were analyzed: i) 408 Colombian blood donors infected with HCV (n = 250) and HBV (n = 158) from Bogotá and ii) 99 indigenous people with HBV infection from Leticia, Amazonas. A fragment of 344 bp from the 5' untranslated region (5' UTR) was amplified by nested RT PCR. Viral sequences were genotyped by phylogenetic analysis using reference sequences from each genotype obtained from GenBank (n = 160). Bayesian phylogenetic analyses were conducted using Markov chain Monte Carlo (MCMC) approach to obtain the MCC tree using BEAST v.1.5.3. Among blood donors, from 158 HBsAg positive samples, eight 5.06% (n = 8) were positive for GBV-C and from 250 anti-HCV positive samples, 3.2%(n = 8) were positive for GBV-C. Also, 7.7% (n = 7) GBV-C positive samples were found among indigenous people from Leticia. A phylogenetic analysis revealed the presence of the following GBV-C genotypes among blood donors: 2a (41.6%), 1 (33.3%), 3 (16.6%) and 2b (8.3%). All genotype 1 sequences were found in co-infection with HBV and 4/5 sequences genotype 2a were found in co-infection with HCV. All sequences from indigenous people from Leticia were classified as genotype 3. The presence of GBV-C infection was not correlated with the sex (p = 0.43), age (p = 0.38) or origin (p = 0.17). It was found a high frequency of GBV-C genotype 1 and 2 in blood donors. The presence of genotype 3 in indigenous population was previously reported from Santa Marta region in Colombia and in native people from Venezuela and

  10. Frequency and genotypic distribution of GB virus C (GBV-C among Colombian population with Hepatitis B (HBV or Hepatitis C (HCV infection

    Directory of Open Access Journals (Sweden)

    Carrilho Flair J

    2011-07-01

    Full Text Available Abstract Background GB virus C (GBV-C is an enveloped positive-sense ssRNA virus belonging to the Flaviviridae family. Studies on the genetic variability of the GBV-C reveals the existence of six genotypes: genotype 1 predominates in West Africa, genotype 2 in Europe and America, genotype 3 in Asia, genotype 4 in Southwest Asia, genotype 5 in South Africa and genotype 6 in Indonesia. The aim of this study was to determine the frequency and genotypic distribution of GBV-C in the Colombian population. Methods Two groups were analyzed: i 408 Colombian blood donors infected with HCV (n = 250 and HBV (n = 158 from Bogotá and ii 99 indigenous people with HBV infection from Leticia, Amazonas. A fragment of 344 bp from the 5' untranslated region (5' UTR was amplified by nested RT PCR. Viral sequences were genotyped by phylogenetic analysis using reference sequences from each genotype obtained from GenBank (n = 160. Bayesian phylogenetic analyses were conducted using Markov chain Monte Carlo (MCMC approach to obtain the MCC tree using BEAST v.1.5.3. Results Among blood donors, from 158 HBsAg positive samples, eight 5.06% (n = 8 were positive for GBV-C and from 250 anti-HCV positive samples, 3.2%(n = 8 were positive for GBV-C. Also, 7.7% (n = 7 GBV-C positive samples were found among indigenous people from Leticia. A phylogenetic analysis revealed the presence of the following GBV-C genotypes among blood donors: 2a (41.6%, 1 (33.3%, 3 (16.6% and 2b (8.3%. All genotype 1 sequences were found in co-infection with HBV and 4/5 sequences genotype 2a were found in co-infection with HCV. All sequences from indigenous people from Leticia were classified as genotype 3. The presence of GBV-C infection was not correlated with the sex (p = 0.43, age (p = 0.38 or origin (p = 0.17. Conclusions It was found a high frequency of GBV-C genotype 1 and 2 in blood donors. The presence of genotype 3 in indigenous population was previously reported from Santa Marta region in

  11. Analytical and clinical performance of the Hologic Aptima HCV Quant Dx Assay for the quantification of HCV RNA in plasma samples

    DEFF Research Database (Denmark)

    Schønning, Kristian; Pedersen, Martin Schou; Johansen, Kim

    2017-01-01

    dilution series of four HCV genotypes (slope of the regression line: 1.00-1.02). The Aptima assay detected significantly more replicates below targeted 2 Log IU/mL than the CAPCTMv2 test, and yielded clearly interpretable results when used to analyze samples from patients treated with DAAs. CONCLUSIONS...

  12. In vitro transcription of Sonchus yellow net virus RNA by a virus-associated RNA-dependent RNA polymerase

    NARCIS (Netherlands)

    Flore, P.H.

    1986-01-01

    The aim of the investigation presented in this thesis was to elucidate the nature of the RNA- dependent RNA polymerase, thought to be associated with Sonchus yellow net virus (SYNV), a rhabdovirus infecting plants. This research was initiated to shed light on the

  13. Hepatitis C virus RNA replication depends on specific cis- and trans-acting activities of viral nonstructural proteins.

    Directory of Open Access Journals (Sweden)

    Teymur Kazakov

    2015-04-01

    Full Text Available Many positive-strand RNA viruses encode genes that can function in trans, whereas other genes are required in cis for genome replication. The mechanisms underlying trans- and cis-preferences are not fully understood. Here, we evaluate this concept for hepatitis C virus (HCV, an important cause of chronic liver disease and member of the Flaviviridae family. HCV encodes five nonstructural (NS genes that are required for RNA replication. To date, only two of these genes, NS4B and NS5A, have been trans-complemented, leading to suggestions that other replicase genes work only in cis. We describe a new quantitative system to measure the cis- and trans-requirements for HCV NS gene function in RNA replication and identify several lethal mutations in the NS3, NS4A, NS4B, NS5A, and NS5B genes that can be complemented in trans, alone or in combination, by expressing the NS3-5B polyprotein from a synthetic mRNA. Although NS5B RNA binding and polymerase activities can be supplied in trans, NS5B protein expression was required in cis, indicating that NS5B has a cis-acting role in replicase assembly distinct from its known enzymatic activity. Furthermore, the RNA binding and NTPase activities of the NS3 helicase domain were required in cis, suggesting that these activities play an essential role in RNA template selection. A comprehensive complementation group analysis revealed functional linkages between NS3-4A and NS4B and between NS5B and the upstream NS3-5A genes. Finally, NS5B polymerase activity segregated with a daclatasvir-sensitive NS5A activity, which could explain the synergy of this antiviral compound with nucleoside analogs in patients. Together, these studies define several new aspects of HCV replicase structure-function, help to explain the potency of HCV-specific combination therapies, and provide an experimental framework for the study of cis- and trans-acting activities in positive-strand RNA virus replication more generally.

  14. Establishment of a Novel Permissive Cell Line for the Propagation of Hepatitis C Virus by Expression of MicroRNA miR122

    Science.gov (United States)

    Kambara, Hiroto; Fukuhara, Takasuke; Shiokawa, Mai; Ono, Chikako; Ohara, Yuri; Kamitani, Wataru

    2012-01-01

    The robust cell culture systems for hepatitis C virus (HCV) are limited to those using cell culture-adapted clones (HCV in cell culture [HCVcc]) and cells derived from the human hepatoma cell line Huh7. However, accumulating data suggest that host factors, including innate immunity and gene polymorphisms, contribute to the variation in host response to HCV infection. Therefore, the existing in vitro systems for HCV propagation are not sufficient to elucidate the life cycle of HCV. A liver-specific microRNA, miR122, has been shown to participate in the efficient replication of HCV. In this study, we examined the possibility of establishing a new permissive cell line for HCV propagation by the expression of miR122. A high level of miR122 was expressed by a lentiviral vector placed into human liver cell lines at a level comparable to the endogenous level in Huh7 cells. Among the cell lines that we examined, Hep3B cells stably expressing miR122 (Hep3B/miR122) exhibited a significant enhancement of HCVcc propagation. Surprisingly, the levels of production of infectious particles in Hep3B/miR122 cells upon infection with HCVcc were comparable to those in Huh7 cells. Furthermore, a line of “cured” cells, established by elimination of HCV RNA from the Hep3B/miR122 replicon cells, exhibited an enhanced expression of miR122 and a continuous increase of infectious titers of HCVcc in every passage. The establishment of the new permissive cell line for HCVcc will have significant implications not only for basic HCV research but also for the development of new therapeutics. PMID:22114337

  15. The polymerase of negative-stranded RNA viruses.

    Science.gov (United States)

    Morin, Benjamin; Kranzusch, Philip J; Rahmeh, Amal A; Whelan, Sean P J

    2013-04-01

    Negative-sense (NS) RNA viruses deliver into cells a mega-dalton RNA-protein complex competent for transcription. Within this complex, the RNA is protected in a nucleocapsid protein (NP) sheath which the viral polymerase negotiates during RNA synthesis. The NP-RNA templates come as nonsegmented (NNS) or segmented (SNS), necessitating distinct strategies for transcription by their polymerases. Atomic-level understanding of the NP-RNA of both NNS and SNS RNA viruses show that the RNA must be transiently dissociated from NP during RNA synthesis. Here we summarize and compare the polymerases of NNS and SNS RNA viruses, and the current structural data on the polymerases. Those comparisons inform us on the evolution of related RNA synthesis machines which use two distinct mechanisms for mRNA cap formation. Copyright © 2013. Published by Elsevier B.V.

  16. Bim-mediated apoptosis and PD-1/PD-L1 pathway impair reactivity of PD1(+)/CD127(-) HCV-specific CD8(+) cells targeting the virus in chronic hepatitis C virus infection.

    Science.gov (United States)

    Larrubia, Juan R; Benito-Martínez, Selma; Miquel, Joaquín; Calvino, Miryam; Sanz-de-Villalobos, Eduardo; González-Praetorius, Alejandro; Albertos, Sonia; García-Garzón, Silvia; Lokhande, Megha; Parra-Cid, Trinidad

    2011-01-01

    PD-1 molecule promotes anergy and IL-7 receptor (CD127) induces an anti-apoptotic effect on T cells. Correlation between PD-1/CD127 phenotype and hepatitis C virus (HCV)-specific CD8(+) cell reactivity in resolved infection (RI) after treatment and persistent HCV-infection (PI) was analysed. Directly ex vivo, PD-1 and CD127 expression on HCV-specific CD8(+) cells displayed a positive and negative correlation, respectively with viraemia. Proliferation after stimulation on PD-1(-)/CD127(+) cells from RI cases was preserved, while it was impaired on PD-1(+)/CD127(-) cells from PI patients. PD1(+)/CD127(+) population was observed in PI, and these maintained expansion ability but they did not target the virus. Frequency of PI cases with HCV-specific CD8(+) cell proliferation increased after anti-PD-L1 and anti-apoptotic treatment. Bim expression on HCV-specific CD8(+) cells from PI patients was enhanced. In conclusion, during chronic HCV infection non-reactive HCV-specific CD8(+) cells targeting the virus are PD-1(+)/CD127(-)/Bim(+) and, blocking apoptosis and PD-1/PD-L1 pathway on them enhances in vitro reactivity. Copyright © 2011 Elsevier Inc. All rights reserved.

  17. Adherence to treatment for recently acquired hepatitis C virus (HCV) infection among injecting drug users.

    Science.gov (United States)

    Grebely, Jason; Matthews, Gail V; Hellard, Margaret; Shaw, David; van Beek, Ingrid; Petoumenos, Kathy; Alavi, Maryam; Yeung, Barbara; Haber, Paul S; Lloyd, Andrew R; Kaldor, John M; Dore, Gregory J

    2011-07-01

    Adherence to HCV therapy impacts sustained virological response (SVR) but there are limited data on adherence, particularly among injecting drug users (IDUs). We assessed 80/80 adherence (≥80% of PEG-IFN doses, ≥80% treatment), on-treatment adherence, and treatment completion in a study of treatment of recent HCV infection (ATAHC). Participants with HCV received pegylated interferon (PEG-IFN) alfa-2a (180μg/week, n=74) and those with HCV/HIV received PEG-IFN alfa-2a with ribavirin (n=35), for a planned 24 weeks. Logistic regression analyses were used to identify predictors of PEG-IFN 80/80 adherence. A total of 109 out of 163 patients received treatment (HCV, n=74; HCV/HIV, n=35), with 75% ever reporting IDU. The proportion with 80/80 PEG-IFN adherence was 82% (n=89). During treatment, 14% missed ≥1 dose (on-treatment adherence=99%). Completion of 0-4, 5-19, 20-23, and all 24 weeks of PEG-IFN therapy occurred in 10% (n=11), 14% (n=15), 6% (n=7) and 70% (n=76) of cases, respectively. Participants with no tertiary education were less likely to have 80/80 PEG-IFN adherence (AOR 0.29, p=0.045). IDU prior to or during treatment did not impact 80/80 PEG-IFN adherence. SVR was higher among those patients with ≥80/80 PEG-IFN adherence (67% vs. 35%, p=0.007), but similar among those with and without missed doses during therapy (73% vs. 60%, p=0.309). SVR in those patients discontinuing therapy between 0-4, 5-19, 20-23, and 24 weeks was 9%, 33%, 43%, and 76%, respectively (p<0.001). High adherence to treatment for recent HCV was observed, irrespective of IDU prior to, or during, therapy. Sub-optimal PEG-IFN exposure was mainly driven by early treatment discontinuation rather than missed doses during therapy. Copyright © 2010 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

  18. Is there a need to include HIV, HBV and HCV viruses in the Saudi premarital screening program on the basis of their prevalence and transmission risk factors?

    Science.gov (United States)

    Alswaidi, F M; O'Brien, S J

    2010-11-01

    In January 2008, the Saudi Arabian health authority included mandatory testing for HIV, HBV and HCV viruses in the premarital screening program. Epidemiologically, there were few justifications for their inclusion as disease prevalences and distributions are poorly understood in the population. This study aims to provide information about HBV, HCV and HIV prevalences and risk factors for disease transmission and so produce evidence for informed decision-making on the inclusion of these infectious diseases in the screening program. This is a cross-sectional descriptive study embedded in the existing national premarital screening program for thalassaemia and sickle cell disease to estimate the prevalence of HIV, HBV and HCV infections (n=74,662 individuals), followed by a case-control study to identify risk factors responsible for infection transmission (n=540). The average HIV prevalence is 0.03%, 1.31% for HBV and 0.33% for HCV. Sharing personal belongings particularly razors, blood transfusions, cuts at barbershops and extramarital relationships showed the highest significant associations with the transmission of these viruses. The prevalences of HIV, HBV and HCV in Saudi Arabia are among the lowest worldwide. However, all the important risk factors associated with transmitting these viruses are significantly present in the Saudi community. Saudi Arabia is financially capable of screening for these infections in the mandatory premarital program and of providing medical care for the discovered cases, but focusing on the health education programs may offset the need to mandatory testing.

  19. HCV antibody quantitative levels in liver transplant patients: do they have any relevance in clinical practice?

    Science.gov (United States)

    Jain, Ashok; Menegus, Marilyn; Mohanka, Ravi; Orloff, Mark; Abt, Peter; Mantry, Parvez; Bozorgzadeh, Adel

    2006-06-01

    Hepatitis C virus (HCV) is not directly cytopathic to the hepatocytes; however, host immune response against the virus does cause hepatic injury. Production of the HCV antibody is a host immune response to a viral antigen. The currently used HCV antibody assay is a qualitative, not quantitative, assessment. In this study, we sought to quantitatively estimate HCV antibody levels in patients who had undergone liver transplantations at the University of Rochester Medical Center, Rochester, New York, and correlate these levels with HCV RNA viral load, genotype, severity of recurrence, and anti-HCV treatment. From 39 liver transplantation patients, we obtained 141 blood samples for quantitative HCV RNA to measure HCV antibody levels quantitatively. Most antibody levels were within a narrow range with a mean of 32.9+/-5.1. Samples with undetectable RNA had a mean antibody level of 31.4+/-8.0, and samples with a positive RNA had mean level of 33.0+/-4.6. The mean antibody levels were significantly higher for patients with genotype 1 (n=33) compared with those with genotype 2 (n=5) (33.2 vs 29.1; P=.007). No correlation was found between antibody levels and severity of hepatic injury with regard to hepatitis activity index or fibrosis score. Six patients with no response to anti-HCV treatment had no change in their mean antibody levels (33.7 vs 34.5). Ten patients who responded to anti-HCV therapy had lower mean levels after therapy, but the changes were not significant (34.2 vs 30.4). Antibody levels in this study did not correlate with viral load or hepatic injury. However, genotype-2 patients had significantly lower levels compared with genotype-1 patients, and patients who responded to anti-HCV therapy demonstrated decreased antibody levels.

  20. Performance Comparison of the Versant HCV Genotype 2.0 Assay (LiPA) and the Abbott Realtime HCV Genotype II Assay for Detecting Hepatitis C Virus Genotype 6

    Science.gov (United States)

    Yang, Ruifeng; Cong, Xu; Du, Shaocai; Fei, Ran; Rao, Huiying

    2014-01-01

    The Versant HCV genotype 2.0 assay (line probe assay [LiPA] 2.0), based on reverse hybridization, and the Abbott Realtime HCV genotype II assay (Realtime II), based on genotype-specific real-time PCR, have been widely used to analyze hepatitis C virus (HCV) genotypes. However, their performances for detecting HCV genotype 6 infections have not been well studied. Here, we analyzed genotype 6 in 63 samples from the China HCV Genotyping Study that were originally identified as genotype 6 using the LiPA 2.0. The genotyping results were confirmed by nonstructural 5B (NS5B) or core sequence phylogenetic analysis. A total of 57 samples were confirmed to be genotype 6 (51 genotype 6a, 5 genotype 6n, and 1 genotype 6e). Four samples identified as a mixture of genotypes 6 and 4 by the LiPA 2.0 were confirmed to be genotype 3b. The remaining two samples classified as genotype 6 by the LiPA 2.0 were confirmed to be genotype 1b, which were intergenotypic recombinants and excluded from further comparison. In 57 genotype 6 samples detected using the Realtime II version 2.00 assay, 47 genotype 6a samples were identified as genotype 6, one 6e sample was misclassified as genotype 1, and four 6a and five 6n samples yielded indeterminate results. Nine nucleotide profiles in the 5′ untranslated region affected the performances of both assays. Therefore, our analysis shows that both assays have limitations in identifying HCV genotype 6. The LiPA 2.0 cannot distinguish some 3b samples from genotype 6 samples. The Realtime II assay fails to identify some 6a and all non-6a subtypes, and it misclassifies genotype 6e as genotype 1. PMID:25100817

  1. The polymerase of negative-stranded RNA viruses

    OpenAIRE

    Morin, Benjamin; Kranzusch, Philip J.; Rahmeh, Amal A.; Whelan, Sean P. J.

    2013-01-01

    Negative-sense (NS) RNA viruses deliver into cells a mega-dalton RNA-protein complex competent for transcription. Within this complex, the RNA is protected in a nucleocapsid protein (NP) sheath which the viral polymerase negotiates during RNA synthesis. The NP-RNA templates come as nonsegemented (NNS) or segmented (SNS), necessitating distinct strategies for transcription by their polymerases. Atomic-level understanding of the NP-RNA of both NNS and SNS RNA viruses show that the RNA must be t...

  2. Resistance to HCV nucleoside analogue inhibitors of hepatitis C virus RNA-dependent RNA polymerase.

    Science.gov (United States)

    Najera, Isabel

    2013-10-01

    Despite the approval of protease inhibitors Telaprevir and Boceprevir and the increased SVR rates observed in GT 1 infected patients, new compounds are needed to achieve higher cure rates, shorten duration of treatment and reduce side-effects. Emergence of resistance continues to be a key factor limiting antiviral efficacy and is therefore an important parameter to control for in clinical trials. Understanding the nature of the resistance mechanism(s), natural prevalence of resistant mutants and their fitness and persistence will allow a better design of treatment options. Nucleos(t)ide analogues present a high barrier to resistance, pangenotypic activity and are not cross resistant to other direct acting antivirals which make them good candidates for combination therapy for the treatment of hepatitis. Copyright © 2013 Elsevier B.V. All rights reserved.

  3. A novel simple assay system to quantify the percent HCV-RNA levels of NS5A Y93H mutant strains and Y93 wild-type strains relative to the total HCV-RNA levels to determine the indication for antiviral therapy with NS5A inhibitors.

    Directory of Open Access Journals (Sweden)

    Yoshihito Uchida

    Full Text Available Oral treatment with asunaprevir and daclatasvir has been reported to yield a SVR ratio of 80% in patients with genotype 1b HCV infection, however, treatment failure has been reported, especially in patients with HCV strains showing the NS5A-Y93H mutation at baseline. An assay system to detect such strains was established to facilitate selection of appropriate candidates for this antiviral therapy.Primer sets and 2 types of cycling probe mixtures were designed, and real-time PCR was performed with HCV-RNA purified from 332 patients with genotype 1b HCV infection, and the results were compared with those obtained by direct sequencing.Both the wild-type and mutant strains were quantified, with a threshold of 4.0 Log copies/mL, in 295 of the 332 patients (88.9%, and the percentage of the mutant strains relative to the total HCV-RNA level in the serum was calculated. The percentage was 0% in 237 patients (80.3% and 100% in 23 patients (7.8%, identical to the results of direct sequencing. Both wild-type and mutant strains were detected in the remaining 35 patients (11.9%, at levels between 1% and 99%, despite the mutant strains having been undetectable by direct sequencing in 11 patients with percentages of these strains of less than 25%.A novel assay system to quantify the percent RNA of Y93H mutant strains relative to the total HCV-RNA level was established. This system may be useful to determine the indication for treatment with NA5A inhibitors in patients with HCV.

  4. Inhibition of human immunodeficiency virus type 1 by RNA interference using long-hairpin RNA

    NARCIS (Netherlands)

    Konstantinova, P.; de Vries, W.; Haasnoot, J.; ter Brake, O.; de Haan, P.; Berkhout, B.

    2006-01-01

    Inhibition of virus replication by means of RNA interference has been reported for several important human pathogens, including human immunodeficiency virus type 1 (HIV-1). RNA interference against these pathogens has been accomplished by introduction of virus-specific synthetic small interfering

  5. Different Culture Metabolites of the Red Sea Fungus Fusarium equiseti Optimize the Inhibition of Hepatitis C Virus NS3/4A Protease (HCV PR).

    Science.gov (United States)

    Hawas, Usama W; Al-Farawati, Radwan; Abou El-Kassem, Lamia T; Turki, Adnan J

    2016-10-20

    The endophytic fungus Fusarium equiseti was isolated from the brown alga Padina pavonica, collected from the Red Sea. The fungus was identified by its morphology and 18S rDNA. Cultivation of this fungal strain in biomalt-peptone medium led to isolation of 12 known metabolites of diketopeprazines and anthraquinones. The organic extract and isolated compounds were screened for their inhibition of hepatitis C virus NS3/4A protease (HCV PR). As a result, the fungal metabolites showed inhibition of HCV protease (IC50 from 19 to 77 μM), and the fungus was subjected to culture on Czapek's (Cz) media, with a yield of nine metabolites with potent HCV protease inhibition ranging from IC50 10 to 37 μM. The Cz culture extract exhibited high-level inhibition of HCV protease (IC50 27.6 μg/mL) compared to the biomalt culture extract (IC50 56 μg/mL), and the most potent HCV PR isolated compound (Griseoxanthone C, IC50 19.8 μM) from the bio-malt culture extract showed less of an inhibitory effect compared to isolated ω-hydroxyemodin (IC50 10.7 μM) from the optimized Cz culture extract. Both HCV PR active inhibitors ω-hydroxyemodin and griseoxanthone C were considered as the lowest selective safe constituents against Trypsin inhibitory effect with IC50 48.5 and 51.3 μM, respectively.

  6. RNA-dependent RNA polymerases from cowpea mosaic virus-infected cowpea leaves

    NARCIS (Netherlands)

    Dorssers, L.

    1983-01-01

    The aim of the research described in this thesis was the purification and identification of the RNA-dependent RNA polymerase engaged in replicating viral RNA in cowpea mosaic virus (CPMV)- infected cowpea leaves.

    Previously, an RNA-dependent RNA polymerase produced upon infection of

  7. Hepatitis C virus genotype and subtype distribution in Chinese chronic hepatitis C patients: nationwide spread of HCV genotypes 3 and 6.

    Science.gov (United States)

    Ju, Wei; Yang, Song; Feng, Shenghu; Wang, Qi; Liu, Shunai; Xing, Huichun; Xie, Wen; Zhu, Liying; Cheng, Jun

    2015-07-25

    Hepatitis C virus (HCV) genotype and subtype are related to disease progression and response to antiviral therapy. Current HCV genotype and subtype distribution data, especially for genotypes 3 and 6, are limited in China. Our purpose was to investigate the current HCV genotype and subtype distributions in chronic hepatitis C patients in China. Chronic hepatitis C patients (n = 1012) were enrolled, and demographic information and possible transmission risk factors were collected. Serum samples were subjected to reverse-transcription polymerase chain reaction, followed by direct DNA sequencing and phylogenetic analysis of the NS5B and core/E1 regions to determine HCV genotypes/subtypes. The geographical distributions of HCV genotypes/subtypes were analyzed. Demographic information and transmission risk factors were compared between different HCV genotypes/subtypes. Four genotypes and seven subtypes of HCV were detected in 970 patients. Subtypes 1b, 2a, 3a, 6a, 3b, 6n, and 1a were detected at frequencies of 71.96%, 19.90%, 3.20%, 2.16%, 1.96%, 0.41%, and 0.41%, respectively. Genotypes 3 and 6 showed an increasingly wide geographic distribution over time. Patients with subtypes 1b and 2a were older than those with 3a, 3b, 6a, and 6n subtypes (p genotype 1 and 2 patients underwent blood transfusion than those with genotype 3 (all p genotype 3 and 6 patients had a history of intravenous drug use than those with genotypes 1 and 2 (all p genotype 3 and 6 HCV infections have already spread nationwide from southern and western China.

  8. The International development of PROQOL-HCV: An instrument to assess the health-related quality of life of patients treated for Hepatitis C virus.

    Science.gov (United States)

    Armstrong, Andrew Richard; Herrmann, Susan Elizabeth; Chassany, Olivier; Lalanne, Christophe; Da Silva, Mariliza Henrique; Galano, Eliana; Carrieri, Patrizia M; Estellon, Vincent; Sogni, Philippe; Duracinsky, Martin

    2016-08-23

    Hepatitis C virus (HCV) compromises Health-related Quality of Life (HRQL) with detriments to Physical, Mental and Social health domains. Treatment with interferon and ribavirin is associated with side effects which further impair HRQL. New treatments appear potent, effective and tolerable. However, Patient Reported Outcomes instruments that capture the impact on HRQL for people with hepatitis C are largely non-specific and will be needed in the new treatment era. Therefore, we developed a conceptually valid multidimensional model of HCV-specific quality of life and pilot survey instrument, the Patient Reported Outcome Quality of Life survey for HCV (PROQOL-HCV). HCV patients from France (n = 30), Brazil (n = 20) and Australia (n = 20) were interviewed to investigate HCV-HRQL issues raised in the scientific literature and by treatment specialists. Interviews were recorded, transcribed and translated into English and French. Fifteen content dimensions were derived from the qualitative analysis, refined and fitted to four domains: (1) Physical Health included: fatigue, pain, sleep, sexual impairment and physical activity; (2) Mental Health: psychological distress, psychosocial impact, and cognition; (3) Social Health: support, stigma, social activity, substance use; (4) TREATMENT: management, side effects, and fear of treatment failure. The impact of some dimensions extended beyond their primary domain including: physical activity, cognition, sleep, sexual impairment, and the three treatment dimensions. A bank of 300 items was constructed to reflect patient reports and, following expert review, reduced to a 72-item pilot questionnaire. We present a conceptually valid multidimensional model of HCV-specific quality of life and the pilot survey instrument, PROQOL-HCV. The model is widely inclusive of the experience of hepatitis C and the first to include the treatment dimension.

  9. RNA Replicons - A New Approach for Influenza Virus Immunoprophylaxis

    Directory of Open Access Journals (Sweden)

    Gert Zimmer

    2010-01-01

    Full Text Available RNA replicons are derived from either positive- or negative-strand RNA viruses. They represent disabled virus vectors that are not only avirulent, but also unable to revert to virulence. Due to autonomous RNA replication, RNA replicons are able to drive high level, cytosolic expression of recombinant antigens stimulating both the humoral and the cellular branch of the immune system. This review provides an update on the available literature covering influenza virus vaccines based on RNA replicons. The pros and cons of these vaccine strategies will be discussed and future perspectives disclosed.

  10. Strategic Approach To Produce Low-Cost, Efficient, and Stable Competitive Internal Controls for Detection of RNA Viruses by Use of Reverse Transcription-PCR▿

    Science.gov (United States)

    Villanova, Gabriela V.; Gardiol, Daniela; Taborda, Miguel A.; Reggiardo, Virginia; Tanno, Hugo; Rivadeneira, Emilia D.; Perez, Germán R.; Giri, Adriana A.

    2007-01-01

    Molecular diagnostics based on reverse transcription (RT)-PCR are routinely complicated by the lack of stable internal controls, leading to falsely negative results. We describe a strategy to produce a stable competitive internal control (CIC) based on a Qβ phage derivative (recombinant Qβ [rQβ]) bearing primers KY78 and KY80, which are widely used in the detection of hepatitis C virus (HCV). rQβ was RNase resistant and stable at 4°C for 452 days in SM medium (0.1 M NaCl, 8 mM MgSO4·7H2O, 50 mM Tris HCl [pH 7.5], 2% gelatin) and for 125 days after lyophilization and reconstitution. rQβ performance as a CIC was evaluated. rQβ was added to HCV-positive samples, followed by RNA extraction and a CIC-HCV RT-PCR assay. This method combines RT-PCR, liquid hybridization with nonradioactive probes, and enzyme immunoanalysis. No influence of the CIC on qualitative HCV detection was observed independently of viral load, and results had high concordance with those of commercial kits. In conclusion, we describe a versatile, low-cost alternative strategy to armored RNA technology that can be adapted for detection or real-time applications of any RNA target. Moreover, the CIC reported here is an essential reagent for HCV screening in blood banks in resource-limited settings. PMID:17699653

  11. Intrathecal humoral immunity to encephalitic RNA viruses.

    Science.gov (United States)

    Phares, Timothy W; Stohlman, Stephen A; Bergmann, Cornelia C

    2013-02-15

    The nervous system is the target for acute encephalitic viral infections, as well as a reservoir for persisting viruses. Intrathecal antibody (Ab) synthesis is well documented in humans afflicted by infections associated with neurological complications, as well as the demyelinating disease, multiple sclerosis. This review focuses on the origin, recruitment, maintenance, and biological relevance of Ab-secreting cells (ASC) found in the central nervous system (CNS) following experimental neurotropic RNA virus infections. We will summarize evidence for a highly dynamic, evolving humoral response characterized by temporal alterations in B cell subsets, proliferation, and differentiation. Overall local Ab plays a beneficial role via complement-independent control of virus replication, although cross or self-reactive Ab to CNS antigens may contribute to immune-mediated pathogenesis during some infections. Importantly, protective Ab exert anti-viral activity not only by direct neutralization, but also by binding to cell surface-expressed viral glycoproteins. Ab engagement of viral glycoproteins blocks budding and mediates intracellular signaling leading to restored homeostatic and innate functions. The sustained Ab production by local ASC, as well as chemokines and cytokines associated with ASC recruitment and retention, are highlighted as critical components of immune control.

  12. Birth-death skyline plot reveals temporal changes of epidemic spread in HIV and hepatitis C virus (HCV).

    Science.gov (United States)

    Stadler, Tanja; Kühnert, Denise; Bonhoeffer, Sebastian; Drummond, Alexei J

    2013-01-02

    Phylogenetic trees can be used to infer the processes that generated them. Here, we introduce a model, the bayesian birth-death skyline plot, which explicitly estimates the rate of transmission, recovery, and sampling and thus allows inference of the effective reproductive number directly from genetic data. Our method allows these parameters to vary through time in a piecewise fashion and is implemented within the BEAST2 software framework. The method is a powerful alternative to the existing coalescent skyline plot, providing insight into the differing roles of incidence and prevalence in an epidemic. We apply this method to data from the United Kingdom HIV-1 epidemic and Egyptian hepatitis C virus (HCV) epidemic. The analysis reveals temporal changes of the effective reproductive number that highlight the effect of past public health interventions.

  13. The dsRNA Virus Papaya Meleira Virus and an ssRNA Virus Are Associated with Papaya Sticky Disease.

    Directory of Open Access Journals (Sweden)

    Tathiana Ferreira Sá Antunes

    Full Text Available Papaya sticky disease, or "meleira", is one of the major diseases of papaya in Brazil and Mexico, capable of causing complete crop loss. The causal agent of sticky disease was identified as an isometric virus with a double stranded RNA (dsRNA genome, named papaya meleira virus (PMeV. In the present study, PMeV dsRNA and a second RNA band of approximately 4.5 kb, both isolated from latex of papaya plants with severe symptoms of sticky disease, were deep-sequenced. The nearly complete sequence obtained for PMeV dsRNA is 8,814 nucleotides long and contains two putative ORFs; the predicted ORF1 and ORF2 display similarity to capsid proteins and RdRp's, respectively, from mycoviruses tentatively classified in the family Totiviridae. The sequence obtained for the second RNA is 4,515 nucleotides long and contains two putative ORFs. The predicted ORFs 1 and 2 display 48% and 73% sequence identity, respectively, with the corresponding proteins of papaya virus Q, an umbravirus recently described infecting papaya in Ecuador. Viral purification in a sucrose gradient allowed separation of particles containing each RNA. Mass spectrometry analysis indicated that both PMeV and the second RNA virus (named papaya meleira virus 2, PMeV2 were encapsidated in particles formed by the protein encoded by PMeV ORF1. The presence of both PMeV and PMeV2 was confirmed in field plants showing typical symptoms of sticky disease. Interestingly, PMeV was detected alone in asymptomatic plants. Together, our results indicate that sticky disease is associated with double infection by PMeV and PMeV2.

  14. The dsRNA Virus Papaya Meleira Virus and an ssRNA Virus Are Associated with Papaya Sticky Disease

    Science.gov (United States)

    Sá Antunes, Tathiana Ferreira; Amaral, Raquel J. Vionette; Ventura, José Aires; Godinho, Marcio Tadeu; Amaral, Josiane G.; Souza, Flávia O.; Zerbini, Poliane Alfenas; Zerbini, Francisco Murilo

    2016-01-01

    Papaya sticky disease, or “meleira”, is one of the major diseases of papaya in Brazil and Mexico, capable of causing complete crop loss. The causal agent of sticky disease was identified as an isometric virus with a double stranded RNA (dsRNA) genome, named papaya meleira virus (PMeV). In the present study, PMeV dsRNA and a second RNA band of approximately 4.5 kb, both isolated from latex of papaya plants with severe symptoms of sticky disease, were deep-sequenced. The nearly complete sequence obtained for PMeV dsRNA is 8,814 nucleotides long and contains two putative ORFs; the predicted ORF1 and ORF2 display similarity to capsid proteins and RdRp's, respectively, from mycoviruses tentatively classified in the family Totiviridae. The sequence obtained for the second RNA is 4,515 nucleotides long and contains two putative ORFs. The predicted ORFs 1 and 2 display 48% and 73% sequence identity, respectively, with the corresponding proteins of papaya virus Q, an umbravirus recently described infecting papaya in Ecuador. Viral purification in a sucrose gradient allowed separation of particles containing each RNA. Mass spectrometry analysis indicated that both PMeV and the second RNA virus (named papaya meleira virus 2, PMeV2) were encapsidated in particles formed by the protein encoded by PMeV ORF1. The presence of both PMeV and PMeV2 was confirmed in field plants showing typical symptoms of sticky disease. Interestingly, PMeV was detected alone in asymptomatic plants. Together, our results indicate that sticky disease is associated with double infection by PMeV and PMeV2. PMID:27166626

  15. Gaining greater insight into HCV emergence in HIV-infected men who have sex with men: the HEPAIG Study.

    Directory of Open Access Journals (Sweden)

    Christine Larsen

    Full Text Available OBJECTIVES: The HEPAIG study was conducted to better understand Hepatitis C virus (HCV transmission among human immuno-deficiency (HIV-infected men who have sex with men (MSM and assess incidence of HCV infection among this population in France. METHODS AND RESULTS: Acute HCV infection defined by anti-HCV or HCV ribonucleic acid (RNA positivity within one year of documented anti-HCV negativity was notified among HIV-infected MSM followed up in HIV/AIDS clinics from a nationwide sampling frame. HIV and HCV infection characteristics, HCV potential exposures and sexual behaviour were collected by the physicians and via self-administered questionnaires. Phylogenetic analysis of the HCV-NS5B region was conducted. HCV incidence was 48/10 000 [95% Confidence Interval (CI:43-54] and 36/10 000 [95% CI: 30-42] in 2006 and 2007, respectively. Among the 80 men enrolled (median age: 40 years, 55% were HIV-diagnosed before 2000, 56% had at least one sexually transmitted infection in the year before HCV diagnosis; 55% were HCV-infected with genotype 4 (15 men in one 4d-cluster, 32.5% with genotype 1 (three 1a-clusters; five men were HCV re-infected; in the six-month preceding HCV diagnosis, 92% reported having casual sexual partners sought online (75.5% and at sex venues (79%, unprotected anal sex (90% and fisting (65%; using recreational drugs (62% and bleeding during sex (55%. CONCLUSIONS: This study emphasizes the role of multiple unprotected sexual practices and recreational drugs use during sex in the HCV emergence in HIV-infected MSM. It becomes essential to adapt prevention strategies and inform HIV-infected MSM with recent acute HCV infection on risk of re-infection and on risk-reduction strategies.

  16. Phomopsis longicolla RNA virus 1 - Novel virus at the edge of myco- and plant viruses.

    Science.gov (United States)

    Hrabáková, Lenka; Koloniuk, Igor; Petrzik, Karel

    2017-06-01

    The complete nucleotide sequence of a new RNA mycovirus in the KY isolate of Phomopsis longicolla Hobbs 1985 and its protoplasts subcultures p5, p9, and ME711 was discovered. The virus, provisionally named Phomopsis longicolla RNA virus 1 (PlRV1), was localized in mitochondria and was determined to have a genome 2822 nucleotides long. A single open reading frame could be translated in silico by both standard and mitochondrial genetic codes into a product featuring conservative domains for an RNA-dependent RNA polymerase (RdRp). The RdRp of PlRV1 has no counterpart among mycoviruses, but it is about 30% identical with the RdRp of plant ourmiaviruses. Recently, new mycoviruses related to plant ourmiaviruses and forming one clade with PlRV1 have been discovered. This separate clade could represent the crucial link between plant and fungal viruses. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. A cooperative interaction between nontranslated RNA sequences and NS5A protein promotes in vivo fitness of a chimeric hepatitis C/GB virus B.

    Directory of Open Access Journals (Sweden)

    Lucile Warter

    Full Text Available GB virus B (GBV-B is closely related to hepatitis C virus (HCV, infects small non-human primates, and is thus a valuable surrogate for studying HCV. Despite significant differences, the 5' nontranslated RNAs (NTRs of these viruses fold into four similar structured domains (I-IV, with domains II-III-IV comprising the viral internal ribosomal entry site (IRES. We previously reported the in vivo rescue of a chimeric GBV-B (vGB/III(HC containing HCV sequence in domain III, an essential segment of the IRES. We show here that three mutations identified within the vGB/III(HC genome (within the 3'NTR, upstream of the poly(U tract, and NS5A coding sequence are necessary and sufficient for production of this chimeric virus following intrahepatic inoculation of synthetic RNA in tamarins, and thus apparently compensate for the presence of HCV sequence in domain III. To assess the mechanism(s underlying these compensatory mutations, and to determine whether 5'NTR subdomains participating in genome replication do so in a virus-specific fashion, we constructed and evaluated a series of chimeric subgenomic GBV-B replicons in which various 5'NTR subdomains were substituted with their HCV homologs. Domains I and II of the GBV-B 5'NTR could not be replaced with HCV sequence, indicating that they contain essential, virus-specific RNA replication elements. In contrast, domain III could be swapped with minimal loss of genome replication capacity in cell culture. The 3'NTR and NS5A mutations required for rescue of the related chimeric virus in vivo had no effect on replication of the subgenomic GBneoD/III(HC RNA in vitro. The data suggest that in vivo fitness of the domain III chimeric virus is dependent on a cooperative interaction between the 5'NTR, 3'NTR and NS5A at a step in the viral life cycle subsequent to genome replication, most likely during particle assembly. Such a mechanism may be common to all hepaciviruses.

  18. A dsRNA virus with filamentous viral particles.

    Science.gov (United States)

    Jia, Hengxia; Dong, Kaili; Zhou, Lingling; Wang, Guoping; Hong, Ni; Jiang, Daohong; Xu, Wenxing

    2017-08-01

    Viruses with double-stranded RNA genomes form isometric particles or are capsidless. Here we report a double-stranded RNA virus, Colletotrichum camelliae filamentous virus 1 (CcFV-1) isolated from a fungal pathogen, that forms filamentous particles. CcFV-1 has eight genomic double-stranded RNAs, ranging from 990 to 2444 bp, encoding 10 putative open reading frames, of which open reading frame 1 encodes an RNA-dependent RNA polymerase and open reading frame 4 a capsid protein. When inoculated, the naked CcFV-1 double-stranded RNAs are infectious and induce the accumulation of the filamentous particles in vivo. CcFV-1 is phylogenetically related to Aspergillus fumigatus tetramycovirus-1 and Beauveria bassiana polymycovirus-1, but differs in morphology and in the number of genomic components. CcFV-1 might be an intermediate virus related to truly capsidated viruses, or might represent a distinct encapsidating strategy. In terms of genome and particle architecture, our findings are a significant addition to the knowledge of the virosphere diversity.Viruses with double-stranded RNA (dsRNA) genomes form typically isometric particles or are capsid-less. Here, the authors identify a mycovirus with an eight-segmented dsRNA genome that forms exceptionally long filamentous particles and could represent an evolutionary link between ssRNA and dsRNA viruses.

  19. Toll-like receptor 3-activated macrophages confer anti-HCV activity to hepatocytes through exosomes.

    Science.gov (United States)

    Zhou, Yu; Wang, Xu; Sun, Li; Zhou, Li; Ma, Tong-Cui; Song, Li; Wu, Jian-Guo; Li, Jie-Liang; Ho, Wen-Zhe

    2016-12-01

    Exosomes are a class of cell-released small vesicles that mediate intercellular communication by delivering functional factors to recipient cells. During hepatitis C virus (HCV) infection, the interaction between liver resident macrophages and hepatocytes is a key component in liver innate immunity. In this study, we explored the role of exosomes in the delivery of innate anti-HCV factors to hepatocytes from macrophages. We showed that supernatant from TLR3-activated macrophage cultures could efficiently inhibit HCV replication in Huh7 cells. This macrophage-mediated anti-HCV activity was through exosomes because inhibiting exosomes could abrogate the action of macrophages. Further analyses demonstrated that TLR3-activated macrophages release exosomes that contain anti-HCV microRNA (miRNA)-29 family members. Inhibiting miRNA29 could restore HCV replication. These findings suggest a novel antiviral mechanism in liver innate immunity against HCV infection and provide insights to support further studies on developing exosome-based delivery system for disease treatment.-Zhou, Y., Wang, X., Sun, L., Zhou, L., Ma, T.-C., Song, L., Wu, J.-G., Li, J.-L., Ho, W.-Z. Toll-like receptor 3-activated macrophages confer anti-HCV activity to hepatocytes through exosomes. © FASEB.

  20. seroprevalence of hepatitis c virus antibodies amongst blood donors

    African Journals Online (AJOL)

    Dr Oboro VO

    HCV is a single stranded RNA virus which until. 1989 was named non A, non B hepatitis virus, was responsible for 80% of post transfusion hepatitis (1,2,3). The modes of transmission are sexual intercourse, accidental inoculation (as in intravenous drug use, tattooing, acupuncture) with HCV- contaminated instruments, ...

  1. Modulation of monocyte/macrophage-derived cytokine and chemokine profile by persistent Hepatitis C virus (HCV infection leads to chronic inflammation

    Directory of Open Access Journals (Sweden)

    Penelope Mavromara

    2012-02-01

    Full Text Available HCV infection presents a major public health problem, with more than 170 million people infected worldwide. Chronicity and persistence of infection constitute the hallmark of the disease. Although HCV is a hepatotropic virus, subsets of immune cells have been found to be permissive to infection and viral replication. Peripheral blood monocytes, attracted to the site of infection and differentiated into macrophages, and resident hepatic macrophages, known as Kupffer cells, are important mediators of innate immunity, through production of several chemokines and cytokines in addition to their phagocytic activity. HCV proteins have been shown to modulate the cytokine and chemokine production profile of monocytes/macrophages, as it is suggested by both in vitro and clinical studies. This modified expression profile appears crucial for the establishment of aberrant inflammation that leads to liver cirrhosis and hepatocellular carcinoma.

  2. Potyvirus virion structure shows conserved protein fold and RNA binding site in ssRNA viruses.

    Science.gov (United States)

    Zamora, Miguel; Méndez-López, Eduardo; Agirrezabala, Xabier; Cuesta, Rebeca; Lavín, José L; Sánchez-Pina, M Amelia; Aranda, Miguel A; Valle, Mikel

    2017-09-01

    Potyviruses constitute the second largest genus of plant viruses and cause important economic losses in a large variety of crops; however, the atomic structure of their particles remains unknown. Infective potyvirus virions are long flexuous filaments where coat protein (CP) subunits assemble in helical mode bound to a monopartite positive-sense single-stranded RNA [(+)ssRNA] genome. We present the cryo-electron microscopy (cryoEM) structure of the potyvirus watermelon mosaic virus at a resolution of 4.0 Å. The atomic model shows a conserved fold for the CPs of flexible filamentous plant viruses, including a universally conserved RNA binding pocket, which is a potential target for antiviral compounds. This conserved fold of the CP is widely distributed in eukaryotic viruses and is also shared by nucleoproteins of enveloped viruses with segmented (-)ssRNA (negative-sense ssRNA) genomes, including influenza viruses.

  3. Naturally Occurring Resistance-Associated Variants to Hepatitis C Virus Direct-Acting Antiviral Agents in Treatment-Naive HCV Genotype 6a-Infected Patients

    Directory of Open Access Journals (Sweden)

    Zhanyi Li

    2017-01-01

    Full Text Available Background and Objective. The direct-acting antiviral agents (DAAs antiviral therapy has drastically improved the prognosis of hepatitis C virus (HCV patients. However, the viral drug resistance-associated variants (RAVs can limit the efficacy of DAAs. For the HCV-6a is not the predominant prevalent genotype; the data on the prevalence of naturally occurring RAVs in it is scarce. Our study aims to assess the prevalence of RAVs in treatment-naive HCV-6a patients. Methods. Nested PCR assays were performed on 95 HCV-6a patients to amplify HCV viral regions of NS3, NS5A, and NS5B. Results. In NS3/4A region, we detected Q80K in 95.5% isolates (84/88 and D168E in 2.3% isolates (2/88. In NS5A region, we detected Q30R in 93.2% isolates (82/88, L31M in 4.6% isolates (4/88, and H58P in 6.8% isolates (6/88. In NS5B region, we detected A15G in 2.3% isolates (2/88, S96T in 1.1% isolates (1/88, and S282T in 20.7% isolates (17/88 and we detected I482L in 100% isolates (4/4, V494A in 50% isolates (2/4, and V499A in 100% isolates (4/4. Conclusions. RAVs to DAAs preexist in treatment-naive HCV-6a patients. Further studies should address the issue of the impact of RAVs in response to DAA therapies for HCV-6a patients.

  4. Prevalence of Occult Hepatitis C Virus Infection among Blood Donors in Jiangsu, China.

    Science.gov (United States)

    Lin, Hong; Chen, Xian; Zhu, Shaowen; Mao, Ping; Zhu, Shanshan; Liu, Yanchun; Huang, Chengyin; Sun, Jun; Zhu, Jin

    2016-01-01

    In 2010, only 1 donor blood sample was found to be anti-hepatitis C virus (HCV) negative and HCV RNA positive, as detected by nucleic acid testing. In occult HCV infection (OCI), HCV RNA is found in peripheral blood mononuclear cells (PBMCs). We investigated the prevalence of OCI among blood donors. We collected 513 samples from 334 eligible and 179 deferred donors, including 55 anti-HCV-positive, 113 alanine aminotransferase (ALT)-elevated, and 11 hepatitis B virus surface antigen (HBsAg)-positive blood donors. PBMCs were isolated, the 5'-untranslated region of HCV RNA was amplified by reverse transcription nested PCR, and the genotype of the core region was determined. No HCV RNA was detected among the eligible samples. Among the deferred donors, 15 (27.2%) had detectable HCV RNA in 55 anti-HCV PBMC specimens. HCV RNA was detected in 1 (9.1%) HBsAg-positive and 9 (8%) ALT-elevated samples. The prevalence of OCI in the blood donors was 2.2% (10/458). HCV genotypes were determined in 10 subjects, indicating that 2 (20.0%) were subtype 2a, 7 (70.0%) were 1b, and 1 (10%) was 6a. This study showed that OCI does exist among Chinese blood donors. However, to determine the epidemiology and outcome of this HCV infection, further follow-up with more participants and patients receiving blood components with OCI is needed. © 2017 S. Karger AG, Basel.

  5. Viral counterdefense on RNA silencing : analysis of RNA silencing suppressors from arthropod-borne negative strand RNA plant viruses

    NARCIS (Netherlands)

    Schnettler, E.

    2010-01-01

    This thesis describes that RNA silencing suppressor (RSS) proteins encoded by negative-stranded RNA plant viruses are able to interfere with different RNA silencing pathways in a variety of organisms by interacting with double stranded (ds)RNA molecules. These RSS proteins are able to counteract the

  6. RNA induced polymerization of the Borna disease virus nucleoprotein.

    Science.gov (United States)

    Hock, Miriam; Kraus, Ina; Schoehn, Guy; Jamin, Marc; Andrei-Selmer, Cornelia; Garten, Wolfgang; Weissenhorn, Winfried

    2010-02-05

    The Borna disease virus (BDV) nucleoprotein (N) monomer resembles the nucleoprotein structures from rabies virus (RABV) and vesicular stomatitis virus (VSV). We show that BDV N assembles into ring- and string-like structures in the presence of 5' genomic BDV RNA. RNA induced polymerization is partly RNA-specific since polymerization is inefficient in the presence of 3' genomic BDV RNA or E. coli RNA. Mutagenesis of basic residues located in the cleft made up by the N- and C-terminal domains of N abrogate RNA-induced polymerization indicating that BDV N binds RNA similarly as observed in case of RABV and VSV N-RNA complexes. Bound RNA is not protected and sensitive to degradation. N-RNA polymers form complexes with the phosphoprotein P as required for functional transcription or replication units. Our data indicate that BDV N utilizes similar structural principles for N-RNA and N-P-RNA complex formation as observed for related negative strand RNA viruses. Copyright 2009 Elsevier Inc. All rights reserved.

  7. Effect of HCV Core Antigen and RNA Clearance during Therapy with Direct Acting Antivirals on Hepatic Stiffness Measured with Shear Wave Elastography in Patients with Chronic Viral Hepatitis C

    Directory of Open Access Journals (Sweden)

    Mariusz Łucejko

    2018-01-01

    Full Text Available To assess a combination of novel measures of therapeutic success in the treatment of chronic hepatitis C (CHC infection, we evaluated liver stiffness (LS with shear wave elastography and hepatitis C virus core antigen (HCVcAg concentrations. We followed 34 patients during and after treatment with direct acting antivirals. All patients achieved a sustained virologic and serologic response and a significant increase of albumin levels. Decreases of alanine aminotransferase (ALT activity and alpha-fetoprotein (AFP level were observed during the treatment and follow-up period. A significant decrease in LS was observed between baseline, end of treatment (EOT, and at 24- and 96-week post-treatment follow-up. LS decline between EOT and 96-week follow-up (FU96 was observed in 79% of patients. Significant LS changes were seen in patients with advanced fibrosis, particularly in cirrhotics and in patients with ALT exceeding 100 IU/mL. There was a positive correlation between ALT activity and LS changes at the baseline versus FU96. A negative correlation was demonstrated between individual HCVcAg baseline concentrations and reduction of LS at the baseline versus FU96. In conclusion, we observed that LS significantly declined during and after antiviral treatment. It was accompanied by improvement in some liver function measures, and disappearance of both HCVcAg and HCV ribonucleic acid (HCV RNA.

  8. Verification of an Assay for Quantification of Hepatitis C Virus RNA by Use of an Analyte-Specific Reagent and Two Different Extraction Methods

    Science.gov (United States)

    Forman, Michael S.; Valsamakis, Alexandra

    2004-01-01

    Protocols were designed for quantification and detection of hepatitis C virus (HCV) RNA by the use of an analyte-specific reagent (ASR) (Roche COBAS TaqMan48 [CTM48] HCV) after manual and automated RNA extraction. The purposes were to determine (i) assay performance characteristics using manual and automated RNA extraction methods, (ii) whether measurable range and limit of detection (LOD) of the ASR assay were influenced by genotype, and (iii) correlation of quantification by CTM48 HCV ASR and COBAS Monitor HCV v. 2.0. For HCV genotype 1 (Gt1), the lower limits of quantification after manual extraction were slightly lower than those for automated extraction (1.0 versus 1.5 log10 IU/ml). Results were linear up to the highest concentration tested after extraction by both methods (manual, 6.1 log10; automated, 6.4 log10). Similar results were obtained for Gt2 (1.8 to 6.8 log10 IU/ml) and Gt3 (1.6 to 6.8 log10 IU/ml) after automated extraction. The LOD of Gt1 virus was 10 IU/ml after manual extraction and between 25 and 37.5 IU/ml after automated extraction. Results with Gt2 and Gt3 viruses were similar after automated extraction (Gt2, between 25 and 50 IU/ml; Gt3, 25 IU/ml). Variability (intrarun and interrun, at concentrations throughout the range of quantification) was ≤13% for both extraction methods. Clinical specimens tested by Monitor were quantified using the CTM48 HCV ASR assay. Characteristics of the regression line included a slope of 0.98 and y intercept of −0.23. Quantification by the two methods was correlated (r = 0.97). CTM48 HCV ASR assay values were on average twofold lower than those for Monitor HCV v. 2.0. These data suggest that our assay combines the characteristics of qualitative and quantitative PCR platforms into a single test. PMID:15297501

  9. Daclatasvir-like inhibitors of NS5A block early biogenesis of hepatitis C virus-induced membranous replication factories, independent of RNA replication.

    Science.gov (United States)

    Berger, Carola; Romero-Brey, Inés; Radujkovic, Danijela; Terreux, Raphael; Zayas, Margarita; Paul, David; Harak, Christian; Hoppe, Simone; Gao, Min; Penin, Francois; Lohmann, Volker; Bartenschlager, Ralf

    2014-11-01

    Direct-acting antivirals that target nonstructural protein 5A (NS5A), such as daclatasvir, have high potency against the hepatitis C virus (HCV). They are promising clinical candidates, yet little is known about their antiviral mechanisms. We investigated the mechanisms of daclatasvir derivatives. We used a combination of biochemical assays, in silico docking models, and high-resolution imaging to investigate inhibitor-induced changes in properties of NS5A, including its interaction with phosphatidylinositol-4 kinase IIIα and induction of the membranous web, which is the site of HCV replication. Analyses were conducted with replicons, infectious virus, and human hepatoma cells that express a HCV polyprotein. Studies included a set of daclatasvir derivatives and HCV variants with the NS5A inhibitor class-defining resistance mutation Y93H. NS5A inhibitors did not affect NS5A stability or dimerization. A daclatasvir derivative interacted with NS5A and molecular docking studies revealed a plausible mode by which the inhibitor bound to NS5A dimers. This interaction was impaired in mutant forms of NS5A that are resistant to daclatavir, providing a possible explanation for the reduced sensitivity of the HCV variants to this drug. Potent NS5A inhibitors were found to block HCV replication by preventing formation of the membranous web, which was not linked to an inhibition of phosphatidylinositol-4 kinase IIIα. Correlative light-electron microscopy revealed unequivocally that NS5A inhibitors had no overall effect on the subcellular distribution of NS5A, but completely prevented biogenesis of the membranous web. Highly potent inhibitors of NS5A, such as daclatasvir, block replication of HCV RNA at the stage of membranous web biogenesis-a new paradigm in antiviral therapy. Copyright © 2014 AGA Institute. Published by Elsevier Inc. All rights reserved.

  10. miRNA independent hepacivirus variants suggest a strong evolutionary pressure to maintain miR-122 dependence

    DEFF Research Database (Denmark)

    Yu, Yingpu; Scheel, Troels K H; Luna, Joseph M

    2017-01-01

    Hepatitis C virus (HCV) requires the liver specific micro-RNA (miRNA), miR-122, to replicate. This was considered unique among RNA viruses until recent discoveries of HCV-related hepaciviruses prompting the question of a more general miR-122 dependence. Among hepaciviruses, the closest known HCV ...... independent HCV and NPHV variants have arisen and been sampled during evolution, yet miR-122 dependence has prevailed. We propose that hepaciviruses may use this mechanism to guarantee liver tropism and exploit the tolerogenic liver environment to avoid clearance and promote chronicity....

  11. External quality assessment on detection of hepatitis C virus RNA in clinical laboratories of China.

    Science.gov (United States)

    Wang, Lu-nan; Zhang, Rui; Shen, Zi-yu; Chen, Wen-xiang; Li, Jin-ming

    2008-06-05

    As with many studies carried out in European countries, a quality assurance program has been established by the National Center for Clinical Laboratories in China (NCCL). The results showed that the external quality assessment significantly improves laboratory performance for quantitative evaluation of hepatitis C virus (HCV) RNA. Serum panels were delivered twice annually to the clinical laboratories which performed HCV RNA detection in China. Each panel made up of 5 coded samples. All laboratories were requested to carry out the detection within the required time period and report on testing results which contained qualitative and/or quantitative test findings, reagents used and relevant information about apparatus. All the positive samples were calibrated against the first International Standard for HCV RNA in a collaborative study and the range of comparison target value (TG) designated as +/- 0.5 log. The numbers of laboratories reporting on qualitative testing results for the first and second time external quality assessment were 168 and 167 in the year of 2003 and increased to 209 and 233 in 2007; the numbers of laboratories reporting on quantitative testing results were 134 and 147 in 2003 and rose to 340 and 339 in 2007. Deviation between the mean value for quantitative results at home in 2003 and the target value was above 0.5 log, which was comparatively high. By 2007, the target value was close to the national average except for the low concentrated specimens (10(3) IU/ml). The percentage of results within the range of GM +/- 0.5 log(10) varied from 8.2% to 93.5%. Some laboratories had some difficulties in the exact quantification of the lowest (3.00 log IU/ml) as well as of the highest viral levels (6.37 log IU/ml) values, very near to the limits of the dynamic range of the assays. The comparison of these results with the previous study confirms that a regular participation in external quality assessment (EQA) assures the achievement of a high

  12. RNA-virus proteases counteracting host innate immunity.

    Science.gov (United States)

    Lei, Jian; Hilgenfeld, Rolf

    2017-10-01

    Virus invasion triggers host immune responses, in particular, innate immune responses. Pathogen-associated molecular patterns of viruses (such as dsRNA, ssRNA, or viral proteins) released during virus replication are detected by the corresponding pattern-recognition receptors of the host, and innate immune responses are induced. Through production of type-I and type-III interferons as well as various other cytokines, the host innate immune system forms the frontline to protect host cells and inhibit virus infection. Not surprisingly, viruses have evolved diverse strategies to counter this antiviral system. In this review, we discuss the multiple strategies used by proteases of positive-sense single-stranded RNA viruses of the families Picornaviridae, Coronaviridae, and Flaviviridae, when counteracting host innate immune responses. © 2017 Federation of European Biochemical Societies.

  13. Geno2pheno[HCV] - A Web-based Interpretation System to Support Hepatitis C Treatment Decisions in the Era of Direct-Acting Antiviral Agents.

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    Prabhav Kalaghatgi

    Full Text Available The face of hepatitis C virus (HCV therapy is changing dramatically. Direct-acting antiviral agents (DAAs specifically targeting HCV proteins have been developed and entered clinical practice in 2011. However, despite high sustained viral response (SVR rates of more than 90%, a fraction of patients do not eliminate the virus and in these cases treatment failure has been associated with the selection of drug resistance mutations (RAMs. RAMs may be prevalent prior to the start of treatment, or can be selected under therapy, and furthermore they can persist after cessation of treatment. Additionally, certain DAAs have been approved only for distinct HCV genotypes and may even have subtype specificity. Thus, sequence analysis before start of therapy is instrumental for managing DAA-based treatment strategies. We have created the interpretation system geno2pheno[HCV] (g2p[HCV] to analyse HCV sequence data with respect to viral subtype and to predict drug resistance. Extensive reviewing and weighting of literature related to HCV drug resistance was performed to create a comprehensive list of drug resistance rules for inhibitors of the HCV protease in non-structural protein 3 (NS3-protease: Boceprevir, Paritaprevir, Simeprevir, Asunaprevir, Grazoprevir and Telaprevir, the NS5A replicase factor (Daclatasvir, Ledipasvir, Elbasvir and Ombitasvir, and the NS5B RNA-dependent RNA polymerase (Dasabuvir and Sofosbuvir. Upon submission of up to eight sequences, g2p[HCV] aligns the input sequences, identifies the genomic region(s, predicts the HCV geno- and subtypes, and generates for each DAA a drug resistance prediction report. g2p[HCV] offers easy-to-use and fast subtype and resistance analysis of HCV sequences, is continuously updated and freely accessible under http://hcv.geno2pheno.org/index.php. The system was partially validated with respect to the NS3-protease inhibitors Boceprevir, Telaprevir and Simeprevir by using data generated with recombinant

  14. [Prevalence of response to anti-HBV infection in patients on maintenance hemodialysis infected with hepatitis C virus (HCV)].

    Science.gov (United States)

    Muszytowski, M; Manitius, J; Ruszkiewicz-Fołda, M

    1996-01-01

    We have studied the antibody response to hepatitis B vaccine in 42 hemodialysis patients; 8 of them were diagnosed as having HCV infection before vaccination. Hemodialysis patients received four doses of recombinant HB vaccine (Engerix-B, SKB, 40 micrograms per dose). Seroconversion occurred in 71.4% of all hemodialysis patients; in 79.4% of HCV-negative and in 37.5% of HCV-positive patients. Effective immunity (anti-HBs titer higher than 100 m IU/ml) was observed in 12.5% of HCV positive and in 35.3% of HCV-negative patients. We conclude that HCV infection may modify or postpone the response to hepatitis B vaccine.

  15. Arthropods as a source of new RNA viruses.

    Science.gov (United States)

    Bichaud, L; de Lamballerie, X; Alkan, C; Izri, A; Gould, E A; Charrel, R N

    2014-12-01

    The discovery and development of methods for isolation, characterisation and taxonomy of viruses represents an important milestone in the study, treatment and control of virus diseases during the 20th century. Indeed, by the late-1950s, it was becoming common belief that most human and veterinary pathogenic viruses had been discovered. However, at that time, knowledge of the impact of improved commercial transportation, urbanisation and deforestation, on disease emergence, was in its infancy. From the late 1960s onwards viruses, such as hepatitis virus (A, B and C) hantavirus, HIV, Marburg virus, Ebola virus and many others began to emerge and it became apparent that the world was changing, at least in terms of virus epidemiology, largely due to the influence of anthropological activities. Subsequently, with the improvement of molecular biotechnologies, for amplification of viral RNA, genome sequencing and proteomic analysis the arsenal of available tools for virus discovery and genetic characterization opened up new and exciting possibilities for virological discovery. Many recently identified but "unclassified" viruses are now being allocated to existing genera or families based on whole genome sequencing, bioinformatic and phylogenetic analysis. New species, genera and families are also being created following the guidelines of the International Committee for the Taxonomy of Viruses. Many of these newly discovered viruses are vectored by arthropods (arboviruses) and possess an RNA genome. This brief review will focus largely on the discovery of new arthropod-borne viruses. Copyright © 2014 Elsevier Ltd. All rights reserved.

  16. Interactions between the Structural Domains of the RNA Replication Proteins of Plant-Infecting RNA Viruses

    OpenAIRE

    O’Reilly, Erin K.; Wang, Zhaohui; French, Roy; Kao, C. Cheng

    1998-01-01

    Brome mosaic virus (BMV), a positive-strand RNA virus, encodes two replication proteins: the 2a protein, which contains polymerase-like sequences, and the 1a protein, with N-terminal putative capping and C-terminal helicase-like sequences. These two proteins are part of a multisubunit complex which is necessary for viral RNA replication. We have previously shown that the yeast two-hybrid assay consistently duplicated results obtained from in vivo RNA replication assays and biochemical assays ...

  17. The International development of PROQOL-HCV: An instrument to assess the health-related quality of life of patients treated for Hepatitis C virus

    OpenAIRE

    Armstrong, Andrew Richard; Herrmann, Susan Elizabeth; Chassany, Olivier; Lalanne, Christophe; Da Silva, Mariliza Henrique; Galano,Eliana; Carrieri, Patrizia M.; Estellon, Vincent; Sogni, Philippe; Duracinsky, Martin

    2016-01-01

    Background Hepatitis C virus (HCV) compromises Health-related Quality of Life (HRQL) with detriments to Physical, Mental and Social health domains. Treatment with interferon and ribavirin is associated with side effects which further impair HRQL. New treatments appear potent, effective and tolerable. However, Patient Reported Outcomes instruments that capture the impact on HRQL for people with hepatitis C are largely non-specific and will be needed in the new treatment era. Therefore, we deve...

  18. Effects of RNA branching on the electrostatic stabilization of viruses

    OpenAIRE

    Erdemci-Tandogan, Gonca; Wagner, Jef; van der Schoot, Paul; Podgornik, Rudolf; Zandi, Roya

    2016-01-01

    Many single-stranded (ss) RNA viruses self assemble from capsid protein subunits and the nucleic acid to form an infectious virion. It is believed that the electrostatic interactions between the negatively charged RNA and the positively charged viral capsid proteins drive the encapsidation, although there is growing evidence that the sequence of the viral RNA also plays a role in packaging. In particular the sequence will determine the possible secondary structures that the ssRNA will take in...

  19. The effect of HIV infection and HCV viremia on inflammatory mediators and hepatic injury-The Women's Interagency HIV Study.

    Directory of Open Access Journals (Sweden)

    Sheila M Keating

    Full Text Available Hepatitis C virus infection induces inflammation and while it is believed that HIV co-infection enhances this response, HIV control may reduce inflammation and liver fibrosis in resolved or viremic HCV infection. Measurement of systemic biomarkers in co-infection could help define the mechanism of inflammation on fibrosis and determine if HIV control reduces liver pathology. A nested case-control study was performed to explore the relationship of systemic biomarkers of inflammation with liver fibrosis in HCV viremic and/or seropositive women with and without HIV infection. Serum cytokines, chemokines, growth factors and cell adhesion molecules were measured in HIV uninfected (HIV-, n = 18, ART-treated HIV-controlled (ARTc, n = 20, uncontrolled on anti-retroviral therapy (ARTuc, n = 21 and elite HIV controllers (Elite, n = 20. All were HCV seroreactive and had either resolved (HCV RNA-; <50IU/mL or had chronic HCV infection (HCV RNA+. In HCV and HIV groups, aspartate aminotransferase to platelet ratio (APRI was measured and compared to serum cytokines, chemokines, growth factors and cell adhesion molecules. APRI correlated with sVCAM, sICAM, IL-10, and IP-10 levels and inversely correlated with EGF, IL-17, TGF-α and MMP-9 levels. Collectively, all HCV RNA+ subjects had higher sVCAM, sICAM and IP-10 compared to HCV RNA-. In the ART-treated HCV RNA+ groups, TNF-α, GRO, IP-10, MCP-1 and MDC were higher than HIV-, Elite or both. In ARTuc, FGF-2, MPO, soluble E-selectin, MMP-9, IL-17, GM-CSF and TGF-α are lower than HIV-, Elite or both. Differential expression of soluble markers may reveal mechanisms of pathogenesis or possibly reduction of fibrosis in HCV/HIV co-infection.

  20. The ESCRT system is required for hepatitis C virus production.

    Directory of Open Access Journals (Sweden)

    Yasuo Ariumi

    Full Text Available BACKGROUND: Recently, lipid droplets have been found to be involved in an important cytoplasmic organelle for hepatitis C virus (HCV production. However, the mechanisms of HCV assembly, budding, and release remain poorly understood. Retroviruses and some other enveloped viruses require an endosomal sorting complex required for transport (ESCRT components and their associated proteins for their budding process. METHODOLOGY/PRINCIPAL FINDINGS: To determine whether or not the ESCRT system is needed for HCV production, we examined the infectivity of HCV or the Core levels in culture supernatants as well as HCV RNA levels in HuH-7-derived RSc cells, in which HCV-JFH1 can infect and efficiently replicate, expressing short hairpin RNA or siRNA targeted to tumor susceptibility gene 101 (TSG101, apoptosis-linked gene 2 interacting protein X (Alix, Vps4B, charged multivesicular body protein 4b (CHMP4b, or Brox, all of which are components of the ESCRT system. We found that the infectivity of HCV in the supernatants was significantly suppressed in these knockdown cells. Consequently, the release of the HCV Core into the culture supernatants was significantly suppressed in these knockdown cells after HCV-JFH1 infection, while the intracellular infectivity and the RNA replication of HCV-JFH1 were not significantly affected. Furthermore, the HCV Core mostly colocalized with CHMP4b, a component of ESCRT-III. In this context, HCV Core could bind to CHMP4b. Nevertheless, we failed to find the conserved viral late domain motif, which is required for interaction with the ESCRT component, in the HCV-JFH1 Core, suggesting that HCV Core has a novel motif required for HCV production. CONCLUSIONS/SIGNIFICANCE: These results suggest that the ESCRT system is required for infectious HCV production.

  1. Implementation of a Large System-Wide Hepatitis C Virus Screening and Linkage to Care Program for Baby Boomers.

    Science.gov (United States)

    Castrejón, Mariana; Chew, Kara W; Javanbakht, Marjan; Humphries, Romney; Saab, Sammy; Klausner, Jeffrey D

    2017-01-01

    We implemented and evaluated a large health system-wide hepatitis C virus (HCV) screening and linkage to care program for persons born between 1945 and 1965 ("baby boomers"). An electronic health record (EHR) clinical decision support (CDS) tool for HCV screening for baby boomers was introduced in August 2015 for patients seen in the outpatient University of California, Los Angeles healthcare system setting. An HCV care coordinator was introduced in January 2016 to facilitate linkage to HCV care. We compared HCV testing in the year prior (August 2014-July 2015) to the year after (August 2015-July 2016) implementation of the CDS tool. Among patients with reactive HCV antibody testing, we compared outcomes related to the care cascade including HCV ribonucleic acid (RNA) testing, HCV RNA positivity, and linkage to HCV specialty care. During the study period, 19606 participants were screened for HCV antibody. Hepatitis C virus antibody screening increased 145% (from 5676 patients tested to 13930 tested) after introduction of the CDS intervention. Screening increased across all demographic groups including age, sex, and race/ethnicity, with the greatest increases among those in the older age groups. The addition of an HCV care coordinator increased follow-up HCV RNA testing for HCV antibody positive patients from 83% to 95%. Ninety-four percent of HCV RNA positive patients were linked to care postimplementation. Introduction of an EHR CDS tool and care coordination markedly increased the number of baby boomers screened for HCV, rates of follow-up HCV RNA testing, and linkage to specialty HCV care for patients with chronic HCV infection.

  2. Comparative interactomics for virus-human protein-protein interactions: DNA viruses versus RNA viruses.

    Science.gov (United States)

    Durmuş, Saliha; Ülgen, Kutlu Ö

    2017-01-01

    Viruses are obligatory intracellular pathogens and completely depend on their hosts for survival and reproduction. The strategies adopted by viruses to exploit host cell processes and to evade host immune systems during infections may differ largely with the type of the viral genetic material. An improved understanding of these viral infection mechanisms is only possible through a better understanding of the pathogen-host interactions (PHIs) that enable viruses to enter into the host cells and manipulate the cellular mechanisms to their own advantage. Experimentally-verified protein-protein interaction (PPI) data of pathogen-host systems only became available at large scale within the last decade. In this study, we comparatively analyzed the current PHI networks belonging to DNA and RNA viruses and their human host, to get insights into the infection strategies used by these viral groups. We investigated the functional properties of human proteins in the PHI networks, to observe and compare the attack strategies of DNA and RNA viruses. We observed that DNA viruses are able to attack both human cellular and metabolic processes simultaneously during infections. On the other hand, RNA viruses preferentially interact with human proteins functioning in specific cellular processes as well as in intracellular transport and localization within the cell. Observing virus-targeted human proteins, we propose heterogeneous nuclear ribonucleoproteins and transporter proteins as potential antiviral therapeutic targets. The observed common and specific infection mechanisms in terms of viral strategies to attack human proteins may provide crucial information for further design of broad and specific next-generation antiviral therapeutics.

  3. Analysis of GB virus C infection among HIV-HCV coinfected patients Análise da infecção pelo vírus GB-C em pacientes com coinfecção VIH-VHC

    Directory of Open Access Journals (Sweden)

    Aline de Jesus Barbosa

    2009-10-01

    Full Text Available The aim of this study was to evaluate the effect of GB virus C on laboratory markers and histological parameters among HIV-seropositive patients coinfected with HCV. Lower degrees of hepatic lesions were observed in the triple-infected patients, in comparison with HIV-HCV coinfected patients who were negative for GBV-C RNA.O objetivo do estudo foi avaliar o efeito da infecção pelo vírus GB-C em marcadores laboratoriais e parâmetros histológicos em pacientes HIV soropositivos coinfectados com VHC. Menor grau de lesão hepática foi observado nos pacientes com tripla infecção em comparação aos pacientes coinfectados com VIH-VHC negativos para GBV-C RNA.

  4. RNA Base Pairing Determines the Conformations of RNA Inside Spherical Viruses

    Science.gov (United States)

    Erdemci-Tandogan, Gonca; Orland, Henri; Zandi, Roya

    2017-11-01

    Many simple RNA viruses enclose their genetic material by a protein shell called the capsid. While the capsid structures are well characterized for most viruses, the structure of RNA inside the shells and the factors contributing to it remain poorly understood. We study the impact of base pairing on the conformations of RNA and find that it undergoes a swollen coil to globule continuous transition as a function of the strength of the pairing interaction. We also observe a first order transition and kink profile as a function of RNA length. All these transitions could explain the different RNA profiles observed inside viral shells.

  5. Evolution of mutational robustness in an RNA virus.

    Directory of Open Access Journals (Sweden)

    Rebecca Montville

    2005-11-01

    Full Text Available Mutational (genetic robustness is phenotypic constancy in the face of mutational changes to the genome. Robustness is critical to the understanding of evolution because phenotypically expressed genetic variation is the fuel of natural selection. Nonetheless, the evidence for adaptive evolution of mutational robustness in biological populations is controversial. Robustness should be selectively favored when mutation rates are high, a common feature of RNA viruses. However, selection for robustness may be relaxed under virus co-infection because complementation between virus genotypes can buffer mutational effects. We therefore hypothesized that selection for genetic robustness in viruses will be weakened with increasing frequency of co-infection. To test this idea, we used populations of RNA phage phi6 that were experimentally evolved at low and high levels of co-infection and subjected lineages of these viruses to mutation accumulation through population bottlenecking. The data demonstrate that viruses evolved under high co-infection show relatively greater mean magnitude and variance in the fitness changes generated by addition of random mutations, confirming our hypothesis that they experience weakened selection for robustness. Our study further suggests that co-infection of host cells may be advantageous to RNA viruses only in the short term. In addition, we observed higher mutation frequencies in the more robust viruses, indicating that evolution of robustness might foster less-accurate genome replication in RNA viruses.

  6. Metagenomics reshapes the concepts of RNA virus evolution by revealing extensive horizontal virus transfer.

    Science.gov (United States)

    Dolja, Valerian V; Koonin, Eugene V

    2017-11-08

    Virus metagenomics is a young research filed but it has already transformed our understanding of virus diversity and evolution, and illuminated at a new level the connections between virus evolution and the evolution and ecology of the hosts. In this review article, we examine the new picture of the evolution of RNA viruses, the dominant component of the eukaryotic virome, that is emerging from metagenomic data analysis. The major expansion of many groups of RNA viruses through metagenomics allowed the construction of substantially improved phylogenetic trees for the conserved virus genes, primarily, the RNA-dependent RNA polymerases (RdRp). In particular, a new superfamily of widespread, small positive-strand RNA viruses was delineated that unites tombus-like and noda-like viruses. Comparison of the genome architectures of RNA viruses discovered by metagenomics and by traditional methods reveals an extent of gene module shuffling among diverse virus genomes that far exceeds the previous appreciation of this evolutionary phenomenon. Most dramatically, inclusion of the metagenomic data in phylogenetic analyses of the RdRp resulted in the identification of numerous, strongly supported groups that encompass RNA viruses from diverse hosts including different groups of protists, animals and plants. Notwithstanding potential caveats, in particular, incomplete and uneven sampling of eukaryotic taxa, these highly unexpected findings reveal horizontal virus transfer (HVT) between diverse hosts as the central aspect of RNA virus evolution. The vast and diverse virome of invertebrates, particularly nematodes and arthropods, appears to be the reservoir, from which the viromes of plants and vertebrates evolved via multiple HVT events. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  7. Analysis of RNA binding by the dengue virus NS5 RNA capping enzyme.

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    Brittney R Henderson

    Full Text Available Flaviviruses are small, capped positive sense RNA viruses that replicate in the cytoplasm of infected cells. Dengue virus and other related flaviviruses have evolved RNA capping enzymes to form the viral RNA cap structure that protects the viral genome and directs efficient viral polyprotein translation. The N-terminal domain of NS5 possesses the methyltransferase and guanylyltransferase activities necessary for forming mature RNA cap structures. The mechanism for flavivirus guanylyltransferase activity is currently unknown, and how the capping enzyme binds its diphosphorylated RNA substrate is important for deciphering how the flavivirus guanylyltransferase functions. In this report we examine how flavivirus NS5 N-terminal capping enzymes bind to the 5' end of the viral RNA using a fluorescence polarization-based RNA binding assay. We observed that the K(D for RNA binding is approximately 200 nM Dengue, Yellow Fever, and West Nile virus capping enzymes. Removal of one or both of the 5' phosphates reduces binding affinity, indicating that the terminal phosphates contribute significantly to binding. RNA binding affinity is negatively affected by the presence of GTP or ATP and positively affected by S-adensyl methoninine (SAM. Structural superpositioning of the dengue virus capping enzyme with the Vaccinia virus VP39 protein bound to RNA suggests how the flavivirus capping enzyme may bind RNA, and mutagenesis analysis of residues in the putative RNA binding site demonstrate that several basic residues are critical for RNA binding. Several mutants show differential binding to 5' di-, mono-, and un-phosphorylated RNAs. The mode of RNA binding appears similar to that found with other methyltransferase enzymes, and a discussion of diphosphorylated RNA binding is presented.

  8. Molecular architecture of the vesicular stomatitis virus RNA polymerase.

    Science.gov (United States)

    Rahmeh, Amal A; Schenk, Andreas D; Danek, Eric I; Kranzusch, Philip J; Liang, Bo; Walz, Thomas; Whelan, Sean P J

    2010-11-16

    Nonsegmented negative-strand (NNS) RNA viruses initiate infection by delivering into the host cell a highly specialized RNA synthesis machine comprising the genomic RNA completely encapsidated by the viral nucleocapsid protein and associated with the viral polymerase. The catalytic core of this protein-RNA complex is a 250-kDa multifunctional large (L) polymerase protein that contains enzymatic activities for nucleotide polymerization as well as for each step of mRNA cap formation. Working with vesicular stomatitis virus (VSV), a prototype of NNS RNA viruses, we used negative stain electron microscopy (EM) to obtain a molecular view of L, alone and in complex with the viral phosphoprotein (P) cofactor. EM analysis, combined with proteolytic digestion and deletion mapping, revealed the organization of L into a ring domain containing the RNA polymerase and an appendage of three globular domains containing the cap-forming activities. The capping enzyme maps to a globular domain, which is juxtaposed to the ring, and the cap methyltransferase maps to a more distal and flexibly connected globule. Upon P binding, L undergoes a significant rearrangement that may reflect an optimal positioning of its functional domains for transcription. The structural map of L provides new insights into the interrelationship of its various domains, and their rearrangement on P binding that is likely important for RNA synthesis. Because the arrangement of conserved regions involved in catalysis is homologous, the structural insights obtained for VSV L likely extend to all NNS RNA viruses.

  9. The reverse genetics applied to fish RNA viruses

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    Biacchesi Stéphane

    2011-01-01

    Full Text Available Abstract Aquaculture has expanded rapidly to become a major economic and food-producing sector worldwide these last 30 years. In parallel, viral diseases have emerged and rapidly spread from farm to farm causing enormous economic losses. The most problematic viruses encountered in the field are mainly, but not exclusively, RNA viruses belonging to the Novirhabdovirus, Aquabirnavirus, Alphavirus and Betanodavirus genera. The recent establishment of reverse genetics systems to recover infectious fish RNA viruses entirely from cDNA has made possible to genetically manipulate the viral genome. These systems have provided powerful tools to study all aspects of the virus biology and virus-host interactions but also gave the opportunity to use these viruses as live vaccines or as gene vectors. This review provides an overview on the recent breakthroughs achieved by using these reverse genetics systems in terms of viral protein function, virulence and host-specificity factor, vaccine development and vector design.

  10. Negative-strand RNA viruses: The plant-infecting counterparts

    NARCIS (Netherlands)

    Kormelink, R.J.M.; Garcia, M.L.; Goodin, M.; Sasaya, T.; Haenni, A.L.

    2011-01-01

    While a large number of negative-strand (-)RNA viruses infect animals and humans, a relative small number have plants as their primary host. Some of these have been classified within families together with animal/human infecting viruses due to similarities in particle morphology and genome

  11. Inhibition of core gene of HCV 3a genotype using synthetic and vector derived siRNAs.

    Science.gov (United States)

    Khaliq, Saba; Jahan, Shah; Ijaz, Bushra; Ahmad, Waqar; Asad, Sultan; Pervaiz, Asim; Samreen, Baila; Khan, Mahwish; Hassan, Sajida

    2010-11-13

    Hepatitis C virus (HCV) is a major causative agent of liver associated diseases throughout the world, with genotype 3a responsible for most of the cases in Pakistan. Due to the limited efficiency of current therapy, RNA interference (RNAi) a novel regulatory and powerful silencing approach for molecular therapeutics through a sequence-specific RNA degradation process represents an alternative option. The current study was purposed to assess and explore the possibility of RNAi to silence the HCV-3a Core gene expression, which play complex role in regulation of cell growth and host genes expression essential for infectivity and disease progression. To identify the potent siRNA target sites, 5 small interfering RNAs (siRNAs) against Core gene were designed and in vitro transcribed after consensus sequence analysis of different HCV-3a isolates. Antiviral effects of siRNAs showed upto 80% inhibition of Core gene expression by different siRNAs into Huh-7 cells as compared with Mock transfected and control siRNAs treated cells. For long lasting effect of siRNAs, vector based short hairpin siRNAs (shRNAs) were designed and tested against HCV-3a Core which resulted in a similar pattern of inhibition on RNA and protein expression of HCV Core as synthetic siRNAs. Furthermore, the efficacy of cell culture tested siRNA and shRNA, were evaluated for inhibition of HCV replication in HCV infected serum inoculated Huh-7 cells and a significant decrease in HCV viral copy number was observed. Our results support the possibility of using consensus siRNA and shRNA-based molecular therapy as a promising strategy in effective inhibition of HCV-3a genotype.

  12. Stearoyl coenzyme A desaturase 1 is associated with hepatitis C virus replication complex and regulates viral replication

    DEFF Research Database (Denmark)

    Nguyen, LN; Lim, YS; Pham, Long

    2014-01-01

    The hepatitis C virus (HCV) life cycle is tightly regulated by lipid metabolism of host cells. In order to identify host factors involved in HCV propagation, we have recently screened a small interfering RNA (siRNA) library targeting host genes that control lipid metabolism and lipid droplet...... formation using cell culture-grown HCV (HCVcc)-infected cells. We selected and characterized the gene encoding stearoyl coenzyme A (CoA) desaturase 1 (SCD1). siRNA-mediated knockdown or pharmacological inhibition of SCD1 abrogated HCV replication in both subgenomic replicon and Jc1-infected cells, while...... exogenous supplementation of either oleate or palmitoleate, products of SCD1 activity, resurrected HCV replication in SCD1 knockdown cells. SCD1 was coimmunoprecipitated with HCV nonstructural proteins and colocalized with both double-stranded RNA (dsRNA) and HCV nonstructural proteins, indicating that SCD1...

  13. Roles of the Coding and Noncoding Regions of Rift Valley Fever Virus RNA Genome Segments in Viral RNA Packaging

    OpenAIRE

    Murakami, Shin; Terasaki, Kaori; Narayanan, Krishna; Makino, Shinji

    2012-01-01

    We characterized the RNA elements involved in the packaging of Rift Valley fever virus RNA genome segments, L, M, and S. The 5′-terminal 25 nucleotides of each RNA segment were equally competent for RNA packaging and carried an RNA packaging signal, which overlapped with the RNA replication signal. Only the deletion mutants of L RNA, but not full-length L RNA, were efficiently packaged, implying the possible requirement of RNA compaction for L RNA packaging.

  14. Identification and Analysis of Novel Inhibitors against NS3 Helicase and NS5B RNA-Dependent RNA Polymerase from Hepatitis C Virus 1b (Con1

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    Na Yang

    2017-11-01

    Full Text Available Hepatitis C virus (HCV leads to severe liver diseases, including liver fibrosis, cirrhosis and hepatocellular carcinoma. Non-structural protein 3 helicase (NS3h and non-structural protein 5B RNA-dependent RNA polymerase (NS5B are involved in the replication of HCV RNA genome, and have been proved to be excellent targets for discovery of direct-acting antivirals. In this study, two high-throughput screening systems, fluorescence polarization (FP-based ssDNA binding assay and fluorescence intensity (FI-based dsRNA formation assay, were constructed to identify candidate NS3h and NS5B inhibitors, respectively. A library of approximately 800 small molecules and crude extracts, derived from marine microorganisms or purchased from the National Compound Resource Center, China, were screened, with three hits selected for further study. Natural compound No.3A5, isolated from marine fungi, inhibited NS3h activity with an IC50 value of 2.8 μM. We further demonstrated that compound No.3A5 inhibited the abilities of NS3h to bind ssDNA in electrophoretic mobility shift assay and to hydrolyze ATP. The NS3h-inhibitory activity of compound No.3A5 was reversible in our dilution assay, which indicated there was no stable NS3h-No.3A5 complex formed. Additionally, compound No.3A5 exhibited no binding selectivity on NS3h or single strand binding protein of Escherichia coli. In NS5B assays, commercial compounds No.39 and No.94 previously reported as kinase inhibitors were found to disrupt dsRNA formation, and their IC50 values were 62.9 and 18.8 μM, respectively. These results highlight how identifying new uses for existing drugs is an effective method for discovering novel HCV inhibitors. To our knowledge, all inhibitors reported in this study were originally discovered with HCV anti-non-structural protein activities in vitro.

  15. Influenza A virus targets a cGAS-independent STING pathway that controls enveloped RNA viruses

    NARCIS (Netherlands)

    Holm, Christian K; Rahbek, Stine H; Gad, Hans Henrik; Bak, Rasmus O; Jakobsen, Martin R; Jiang, Zhaozaho; Hansen, Anne Louise; Jensen, Simon K; Sun, Chenglong; Thomsen, Martin K; Laustsen, Anders; Nielsen, Camilla G; Severinsen, Kasper; Xiong, Yingluo; Burdette, Dara L; Hornung, Veit; Lebbink, Robert Jan; Duch, Mogens; Fitzgerald, Katherine A; Bahrami, Shervin; Mikkelsen, Jakob Giehm; Hartmann, Rune; Paludan, Søren R

    2016-01-01

    Stimulator of interferon genes (STING) is known be involved in control of DNA viruses but has an unexplored role in control of RNA viruses. During infection with DNA viruses STING is activated downstream of cGAMP synthase (cGAS) to induce type I interferon. Here we identify a STING-dependent,

  16. Effects of RNA branching on the electrostatic stabilization of viruses

    NARCIS (Netherlands)

    Erdemci-Tandogan, Gonca; Wagner, Jef; Schoot, Paul van der; Podgornik, Rudolf; Zandi, Roya

    2016-01-01

    Many single-stranded (ss) RNA viruses self assemble from capsid protein subunits and the nucleic acid to form an infectious virion. It is believed that the electrostatic interactions between the negatively charged RNA and the positively charged viral capsid proteins drive the encapsidation, although

  17. Suppression of RNA interference by adenovirus virus-associated RNA

    NARCIS (Netherlands)

    Andersson, M. Gunnar; Haasnoot, P. C. Joost; Xu, Ning; Berenjian, Saideh; Berkhout, Ben; Akusjärvi, Göran

    2005-01-01

    We show that human adenovirus inhibits RNA interference (RNAi) at late times of infection by suppressing the activity of two key enzyme systems involved, Dicer and RNA-induced silencing complex (RISC). To define the mechanisms by which adenovirus blocks RNAi, we used a panel of mutant adenoviruses

  18. Mechanisms of human immunodeficiency virus type 2 RNA packaging

    DEFF Research Database (Denmark)

    Ni, Na; Nikolaitchik, Olga A; Dilley, Kari A

    2011-01-01

    Human immunodeficiency virus type 2 (HIV-2) has been reported to have a distinct RNA packaging mechanism, referred to as cis packaging, in which Gag proteins package the RNA from which they were translated. We examined the progeny generated from dually infected cell lines that contain two HIV-2...... that specifically recognize stem-loop motifs in the viral genomes, an assay termed single virion analysis. These studies revealed that >90% of the HIV-2 particles contained viral RNAs and that RNAs derived from different viruses were copackaged frequently. Furthermore, the frequencies of heterozygous particles...... do not support the cis-packaging hypothesis but instead indicate that trans packaging is the major mechanism of HIV-2 RNA packaging. To further characterize the mechanisms of HIV-2 RNA packaging, we visualized HIV-2 RNA in individual particles by using fluorescent protein-tagged RNA-binding proteins...

  19. [siRNAs targeting La, hVAP-33, eIF2Bgamma, and HCV IRES inhibit the replication and expression of HCV in Huh7 cells].

    Science.gov (United States)

    Wang, Mei-xia; Xu, Bin; Duan, Jin; Fu, Xiao-qing; Jin, Ming

    2012-10-01

    To investigate the in vivo functional roles of the La autoantigen (La), the human homologue of the 33-kDa vesicle-associated membrane protein-associated protein (hVAP-33), and the subunit gamma of the human eukaryotic initiation factors 2B (eIF2Bgamma) as co-infection factors supporting chronic infection with hepatitis C virus (HCV). Small interfering (si)RNAs were designed against the HCV internal ribosome entry site (IRES) and transfected into Huh7 cells chronically infected with the HCV pseudovirus (designated as Huh7-HCV cells). The IRES siRNA producing the most effective silencing was selected for further analysis by fluorescence quantitative polymerase chain reaction (qPCR). siRNAs designed against La, hVAP-33, and eIF2Bgamma and the IRES-specific siRNA were then transfected, respectively or in various combinations, into the Huh7-HCV cell line for 48 h. The delta CT values were calculated and used to compare the HCV inhibitive efficacies of the siRNAs in isolation or in combination. Western blotting analysis was used to compare the quantity of core protein expression in each group. The four gene-specific siRNAs, in isolation or in combination, caused inhibition of HCV replication and gene and protein expressions to varying degrees. The combination of La + IRES siRNAs produced the strongest inhibition of HCV core antigen expression. The combinations of hVAP-33 + IRES siRNAs and eIF2Bgamma + IRES siRNAs produced stronger inhibitions of HCV replication and gene and protein expressions than either hVAP-33 siRNA or eIF2Bgamma siRNA alone. La, hVAP-33, and eIF2Bgamma act as co-infection factors of HCV chronic infection in vivo. HCV replication and gene and protein expression can be inhibited significantly by RNA interference of these co-infection factors and/or HCV IRES.

  20. Hepatitis C virus (HCV evades NKG2D-dependent NK cell responses through NS5A-mediated imbalance of inflammatory cytokines.

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    Damien Sène

    Full Text Available Understanding how hepatitis C virus (HCV induces and circumvents the host's natural killer (NK cell-mediated immunity is of critical importance in efforts to design effective therapeutics. We report here the decreased expression of the NKG2D activating receptor as a novel strategy adopted by HCV to evade NK-cell mediated responses. We show that chronic HCV infection is associated with expression of ligands for NKG2D, the MHC class I-related Chain (MIC molecules, on hepatocytes. However, NKG2D expression is downmodulated on circulating NK cells, and consequently NK cell-mediated cytotoxic capacity and interferon-γ production are impaired. Using an endotoxin-free recombinant NS5A protein, we show that NS5A stimulation of monocytes through Toll-like Receptor 4 (TLR4 promotes p38- and PI3 kinase-dependent IL-10 production, while inhibiting IL-12 production. In turn, IL-10 triggers secretion of TGFβ which downmodulates NKG2D expression on NK cells, leading to their impaired effector functions. Moreover, culture supernatants of HCV JFH1 replicating Huh-7.5.1 cells reproduce the effect of recombinant NS5A on NKG2D downmodulation. Exogenous IL-15 can antagonize the TGFβ effect and restore normal NKG2D expression on NK cells. We conclude that NKG2D-dependent NK cell functions are modulated during chronic HCV infection, and demonstrate that this alteration can be prevented by exogenous IL-15, which could represent a meaningful adjuvant for therapeutic intervention.

  1. Low prevalence of HCV infection with predominance of genotype 4 among HIV patients living in Libreville, Gabon.

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    Angélique Ndjoyi-Mbiguino

    Full Text Available Gabon is an endemic area for human immunodeficiency virus (HIV and hepatitis C virus (HCV and the risk of co-infection is high.Between November 2015 and April 2016, we conducted retrospective study on HCV infection among people living with HIV/AIDS (PLHA. A total of 491 PLHA were included in this study and tested for the presence of HCV infection. HIV viral loads were obtained using the Generic HIV viral Load® assay and the CD4+ T cells count was performed using BD FACSCount™ CD4 reagents. HCV screening was performed using the MP Diagnostics HCV ELISA 4.0 kit. HCV genotypes were determined by sequence analysis of NS5B and Core regions. The Mann-Whitney test was used to compare the groups. Chi-2 test and Fisher's Exact Test were used to compare prevalence.HCV seroprevalence was 2.9% (14/491, (95% confidence interval (CI:1.4-4.3%. The percentage of HCV viremic patients, defined by the detection of HCV RNA in plasma, was 57% (8/14, representing 1.6% of the total population. HCV seroprevalence and replicative infection were not statistically differ with gender. The percentage of co-infection increased with age. No correlation with CD4+ T cells count and HIV viral load level was registered in this study. Identified HCV strains were predominantly of genotype 4 (87.5% including 4k, 4e, 4g, 4p, 4f and 4c subtypes. Only one strain belonged to genotype 2 (subtype 2q. Analysis of the NS5B region did not reveal the presence of resistance-associated substitutions for sofosbuvir.A systematic screening of hepatitis C is therefore strongly recommended as well as genotyping of HCV strains in order to adapt treatments for the specific case of people living with HIV/AIDS in Central Africa.

  2. The effect of HIV infection and HCV viremia on inflammatory mediators and hepatic injury-The Women's Interagency HIV Study.

    Science.gov (United States)

    Keating, Sheila M; Dodge, Jennifer L; Norris, Philip J; Heitman, John; Gange, Stephen J; French, Audrey L; Glesby, Marshall J; Edlin, Brian R; Latham, Patricia S; Villacres, Maria C; Greenblatt, Ruth M; Peters, Marion G

    2017-01-01

    Hepatitis C virus infection induces inflammation and while it is believed that HIV co-infection enhances this response, HIV control may reduce inflammation and liver fibrosis in resolved or viremic HCV infection. Measurement of systemic biomarkers in co-infection could help define the mechanism of inflammation on fibrosis and determine if HIV control reduces liver pathology. A nested case-control study was performed to explore the relationship of systemic biomarkers of inflammation with liver fibrosis in HCV viremic and/or seropositive women with and without HIV infection. Serum cytokines, chemokines, growth factors and cell adhesion molecules were measured in HIV uninfected (HIV-, n = 18), ART-treated HIV-controlled (ARTc, n = 20), uncontrolled on anti-retroviral therapy (ARTuc, n = 21) and elite HIV controllers (Elite, n = 20). All were HCV seroreactive and had either resolved (HCV RNA-; infection (HCV RNA+). In HCV and HIV groups, aspartate aminotransferase to platelet ratio (APRI) was measured and compared to serum cytokines, chemokines, growth factors and cell adhesion molecules. APRI correlated with sVCAM, sICAM, IL-10, and IP-10 levels and inversely correlated with EGF, IL-17, TGF-α and MMP-9 levels. Collectively, all HCV RNA+ subjects had higher sVCAM, sICAM and IP-10 compared to HCV RNA-. In the ART-treated HCV RNA+ groups, TNF-α, GRO, IP-10, MCP-1 and MDC were higher than HIV-, Elite or both. In ARTuc, FGF-2, MPO, soluble E-selectin, MMP-9, IL-17, GM-CSF and TGF-α are lower than HIV-, Elite or both. Differential expression of soluble markers may reveal mechanisms of pathogenesis or possibly reduction of fibrosis in HCV/HIV co-infection.

  3. Metagenomic analysis of RNA viruses in a fresh water lake.

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    Appolinaire Djikeng

    Full Text Available Freshwater lakes and ponds present an ecological interface between humans and a variety of host organisms. They are a habitat for the larval stage of many insects and may serve as a medium for intraspecies and interspecies transmission of viruses such as avian influenza A virus. Furthermore, freshwater bodies are already known repositories for disease-causing viruses such as Norwalk Virus, Coxsackievirus, Echovirus, and Adenovirus. While RNA virus populations have been studied in marine environments, to this date there has been very limited analysis of the viral community in freshwater. Here we present a survey of RNA viruses in Lake Needwood, a freshwater lake in Maryland, USA. Our results indicate that just as in studies of other aquatic environments, the majority of nucleic acid sequences recovered did not show any significant similarity to known sequences. The remaining sequences are mainly from viral types with significant similarity to approximately 30 viral families. We speculate that these novel viruses may infect a variety of hosts including plants, insects, fish, domestic animals and humans. Among these viruses we have discovered a previously unknown dsRNA virus closely related to Banna Virus which is responsible for a febrile illness and is endemic to Southeast Asia. Moreover we found multiple viral sequences distantly related to Israeli Acute Paralysis virus which has been implicated in honeybee colony collapse disorder. Our data suggests that due to their direct contact with humans, domestic and wild animals, freshwater ecosystems might serve as repositories of a wide range of viruses (both pathogenic and non-pathogenic and possibly be involved in the spread of emerging and pandemic diseases.

  4. Metagenomic analysis of RNA viruses in a fresh water lake.

    Science.gov (United States)

    Djikeng, Appolinaire; Kuzmickas, Ryan; Anderson, Norman G; Spiro, David J

    2009-09-29

    Freshwater lakes and ponds present an ecological interface between humans and a variety of host organisms. They are a habitat for the larval stage of many insects and may serve as a medium for intraspecies and interspecies transmission of viruses such as avian influenza A virus. Furthermore, freshwater bodies are already known repositories for disease-causing viruses such as Norwalk Virus, Coxsackievirus, Echovirus, and Adenovirus. While RNA virus populations have been studied in marine environments, to this date there has been very limited analysis of the viral community in freshwater. Here we present a survey of RNA viruses in Lake Needwood, a freshwater lake in Maryland, USA. Our results indicate that just as in studies of other aquatic environments, the majority of nucleic acid sequences recovered did not show any significant similarity to known sequences. The remaining sequences are mainly from viral types with significant similarity to approximately 30 viral families. We speculate that these novel viruses may infect a variety of hosts including plants, insects, fish, domestic animals and humans. Among these viruses we have discovered a previously unknown dsRNA virus closely related to Banna Virus which is responsible for a febrile illness and is endemic to Southeast Asia. Moreover we found multiple viral sequences distantly related to Israeli Acute Paralysis virus which has been implicated in honeybee colony collapse disorder. Our data suggests that due to their direct contact with humans, domestic and wild animals, freshwater ecosystems might serve as repositories of a wide range of viruses (both pathogenic and non-pathogenic) and possibly be involved in the spread of emerging and pandemic diseases.

  5. Interleukin-28B polymorphisms are associated with hepatitis C virus clearance and viral load in a HIV-1-infected cohort

    DEFF Research Database (Denmark)

    Clausen, L N; Weis, N; Astvad, K

    2011-01-01

    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......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...

  6. Cloning and expression of NS3 helicase fragment of hepatitis C virus and the study of its immunoreactivity in HCV infected patients

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    Mahrou Sadri

    2015-02-01

    Full Text Available Objective(s: Hepatitis C is a major cause of liver failure worldwide. Current therapies applied for this disease are not fully effective and produce side effects in most cases. Non-structural protein 3 helicase (NS3 of HCV is one of the key enzymes in viral replication and infection. Therefore, this region is a promising target to design new drugs and therapies against HCV infection. The aim of this study was cloning and expression of HCV NS3 helicase fragment in Escherichia coli BL21 (DE3 using pET102/D-TOPO expression vector and studying immunoreactivity of the expressed antigen in Iranian infected with hepatitis C. Materials and Methods: The viral RNA was extracted from the serum of HCV infected patient. The NS3 helicase region was amplified by RT-PCR. The PCR product was directionally cloned into the expression vector pET102/D-TOPO and transformed into the BL21 strain of E. coli (DE3. The transformed bacteria were then induced by adding 1mM isopropyl-β-D-thiogalactopyranoside (IPTG into the culture medium to enhance the protein expression. SDS-PAGE and western blotting were carried out to identify the protein under investigation, and finally purified recombinant fusion protein was used as the antigen for ELISA method. Results: Theinsertion of theDNA fragment of the NS3 regioninto the expression vectorwas further confirmed by PCR and sequencing. SDS-PAGE analysis showed the successful expression of the recombinant protein of interest. Furthermore, immunoreactivity of fusion NS3 helicase was confirmed by ELISA and western blotting. Conclusion: It seems that this recombinant protein could be a useful source of antigen for future studies on HCV diagnosis and therapy.

  7. Prevalence of hepatitis C virus and human immunodeficiency virus in a group of patients newly diagnosed with active tuberculosis in Porto Alegre, Southern Brazil.

    Science.gov (United States)

    Costi, Cintia; Grandi, Tarciana; Halon, Maria Laura; Silva, Márcia Susana Nunes; Silva, Cláudia Maria Dornelles da; Gregianini, Tatiana Schäffer; Possuelo, Lia Gonçalves; Jarczewski, Carla Adriane; Niel, Christian; Rossetti, Maria Lucia Rosa

    2017-04-01

    Porto Alegre is the Brazilian state capital with second highest incidence of tuberculosis (TB) and the highest proportion of people infected with human immunodeficiency virus (HIV) among patients with TB. Hepatitis C virus (HCV) infection increases the risk of anti-TB drug-induced hepatotoxicity, which may result in discontinuation of the therapy. The aim of this study was (i) to estimate prevalence of HCV and HIV in a group of patients newly diagnosed with active TB in a public reference hospital in Porto Alegre and (ii) to compare demographic, behavioural, and clinical characteristics of patients in relation to their HCV infection status. One hundred and thirty-eight patients with TB were tested for anti-HCV antibody, HCV RNA, and anti-HIV1/2 antibody markers. HCV RNA from real-time polymerase chain reaction (PCR)-positive samples was submitted to reverse transcription and PCR amplification. The 5' non-coding region of the HCV genome was sequenced, and genotypes of HCV isolates were determined. Anti-HCV antibody, HCV RNA, and anti-HIV antibodies were detected in 27 [20%; 95% confidence interval (CI), 13-26%], 17 (12%; 95% CI, 7-18%), and 34 (25%; 95% CI, 17-32%) patients, respectively. HCV isolates belonged to genotypes 1 (n = 12) and 3 (n = 4). Some characteristics were significantly more frequent in patients infected with HCV. Among them, non-white individuals, alcoholics, users of illicit drugs, imprisoned individuals, and those with history of previous TB episode were more commonly infected with HCV (p < 0.05). HCV screening, including detection of anti-HCV antibody and HCV RNA, will be important to improving the management of co-infected patients, given their increased risk of developing TB treatment-related hepatotoxicity.

  8. Viral genome imaging of hepatitis C virus to probe heterogeneous viral infection and responses to antiviral therapies

    NARCIS (Netherlands)

    Ramanan, Vyas; Trehan, Kartik; Ong, Mei Lyn; Luna, Joseph M.; Hoffmann, Hans Heinrich; Espiritu, Christine; Sheahan, Timothy P.; Chandrasekar, Hamsika; Schwartz, Robert E.; Christine, Kathleen S.; Rice, Charles M.; van Oudenaarden, Alexander; Bhatia, Sangeeta N.

    2016-01-01

    Hepatitis C virus (HCV) is a positive single-stranded RNA virus of enormous global health importance, with direct-acting antiviral therapies replacing an immunostimulatory interferon-based regimen. The dynamics of HCV positive and negative-strand viral RNAs (vRNAs) under antiviral perturbations have

  9. Viral genome imaging of hepatitis C virus to probe heterogeneous viral infection and responses to antiviral therapies

    NARCIS (Netherlands)

    Ramanan, Vyas; Trehan, Kartik; Ong, Mei-Lyn; Luna, Joseph M; Hoffmann, Hans-Heinrich; Espiritu, Christine; Sheahan, Timothy P; Chandrasekar, Hamsika; Schwartz, Robert E; Christine, Kathleen S; Rice, Charles M; van Oudenaarden, Alexander; Bhatia, Sangeeta N

    Hepatitis C virus (HCV) is a positive single-stranded RNA virus of enormous global health importance, with direct-acting antiviral therapies replacing an immunostimulatory interferon-based regimen. The dynamics of HCV positive and negative-strand viral RNAs (vRNAs) under antiviral perturbations have

  10. [Recurrence of hepatitis caused by hepatitis c virus in patients receiving a liver transplant].

    Science.gov (United States)

    Crespo, J; Casafont, F; Carte, B; Lozano, J L; Fábrega, E; Sánchez-Antolín, G; Dueñas, C; Pons Romero, F

    1997-01-25

    Liver disease due to hepatitis C virus (HCV) is an increasingly frequent indication for liver transplantation. We performed a clinical and virological study of 20 HCV-infected liver transplant recipients to correlate virological markers with histological recurrence of disease. In ninety-four patients who were given transplants for end-stage cirrhosis, IgG and IgM antibodies to HCV and IgM to HCV tested by ELISA; all samples were further examined in a four-antigen recombinant immunoblot assay (2-RIBA). HCV viremia was measured by the conventional nested PCR, HCV genotype was determined by PCR amplification using type-specific primers. We have analyzed de novo infection by HCV, HCV recurrence and the influence of genotype in these recurrence. Nineteen of 20 antibody-positive patients (95%) had HCV RNA before transplantation. All 19 patients who were viremic before transplantation had persistent infection after LT. HCV genotype 1b was the predominant type before and after LT (75%). Ten of the 20 (50%) patients developed histological findings of chronic hepatitis (CH) in liver allografts. HCV recurrent liver disease after LT was not related with HCV genotype. Of 4 deaths after transplant in hepatitis C group, only one was related to recurrent disease. We have not found de novo hepatitis C. Our results indicate the general persistence of hepatitis C virus infection and the excellent short-term prognosis after liver transplantation. Chronic hepatitis by HCV in liver transplant was not related with HCV genotype.

  11. Detection of HIV and HCV RNA in semen from Brazilian coinfected men using multiplex PCR before and after semen washing

    OpenAIRE

    Cynthia Liliane Motta do Canto; Segurado, Aluisio C; Cláudio Pannut; Agnaldo Cedenho; Miguel Srougi; Deborah Spaine; Silvana Fernandes; Nadily Carretiero; Maria Carolina Bernal; José Eduardo Levi

    2006-01-01

    INTRODUCTION: Prolonged survival of patients under HAART has resulted in new demands for assisted reproductive technologies. HIV serodiscordant couples wish to make use of assisted reproduction techniques in order to avoid viral transmission to the partner or to the newborn. It is therefore essential to test the effectiveness of techniques aimed at reducing HIV and HCV loads in infected semen using molecular biology tests. METHODS: After seminal analysis, semen samples from 20 coinfected pati...

  12. [Diverse double-stranded RNA viruses infecting fungi].

    Science.gov (United States)

    Chiba, Sotaro; Suzuki, Nobuhiro

    2014-01-01

    Most of reported fungal viruses (mycoviruses) have double-stranded RNA (dsRNA) genomes. This may reflect the simple, easy method for mycovirus hunting that entails detection of dsRNAs as a sign of viral infections. There are an increasing number of screens of various fungi, particularly phytopathogenic fungi for viruses pathogenic to host fungi or able to confer hypovirulence to them. This bases on an attractive research field of biological control of fungal plant diseases using viruses (virocontrol), mainly targeting important phytopathogenic fungi. While isolated viruses usually induce asymptomatic symptoms, they show a considerably high level of diversity. As of 2014, fungal dsRNA viruses are classified into six families: Reoviridae, Totiviridae, Chrysoviridae, Partitiviridae, Megabirnaviridae and Quadriviridae. These exclude unassigned mycoviruses which will definitely be placed into distinct families and/or genera. In this review article, dsRNA viruses isolated from the kingdom Fungi including as-yet-unclassified taxa are overviewed. Some recent achievements in the related field are briefly introduced as well.

  13. Transient elastography: A non-invasive tool for assessing liver fibrosis in HIV/HCV patients

    Science.gov (United States)

    Li Vecchi, Valentina; Soresi, Maurizio; Colomba, Claudia; Mazzola, Giovanni; Colletti, Pietro; Mineo, Maurizio; Di Carlo, Paola; La Spada, Emanuele; Vizzini, Giovanni; Montalto, Giuseppe

    2010-01-01

    AIM: To assess the prevalence of advanced liver fibrosis (ALF) in human immunodeficiency virus (HIV), hepatitis C virus (HCV) and HIV/HCV patients using transient elastography, and to identify factors associated with ALF. METHODS: Between September 2008 and October 2009, 71 HIV mono-infected, 57 HIV/HCV co-infected and 53 HCV mono-infected patients on regular follow-up at our Center were enrolled in this study. Alcohol intake, the main parameters of liver function, presence of HCV-RNA, HIV-RNA, duration of highly active anti-retroviral therapy (HAART) and CD4 cell count were recorded. ALF was defined as liver stiffness (LS) ≥ 9.5 kPa. To estimate liver fibrosis (LF) a further 2 reliable biochemical scores, aspartate aminotransferase platelet ratio index (APRI) and FIB-4, were also used. RESULTS: LS values of co-infected patients were higher than in either HIV or HCV mono-infected patients (χ2MH = 4, P 9.5 kPa. There was no significant correlation between extent of LF and HAART exposure or duration of HAART exposure, in particular with specific dideoxynucleoside analogues. CONCLUSION: ALF was more frequent in co-infected than mono-infected patients. This result correlated with lower CD4 levels. Protective immunological effects of HAART on LF progression outweigh its hepatotoxic effects. PMID:21049556

  14. Phosphatidic acid produced by phospholipase D promotes RNA replication of a plant RNA virus.

    Directory of Open Access Journals (Sweden)

    Kiwamu Hyodo

    2015-05-01

    Full Text Available Eukaryotic positive-strand RNA [(+RNA] viruses are intracellular obligate parasites replicate using the membrane-bound replicase complexes that contain multiple viral and host components. To replicate, (+RNA viruses exploit host resources and modify host metabolism and membrane organization. Phospholipase D (PLD is a phosphatidylcholine- and phosphatidylethanolamine-hydrolyzing enzyme that catalyzes the production of phosphatidic acid (PA, a lipid second messenger that modulates diverse intracellular signaling in various organisms. PA is normally present in small amounts (less than 1% of total phospholipids, but rapidly and transiently accumulates in lipid bilayers in response to different environmental cues such as biotic and abiotic stresses in plants. However, the precise functions of PLD and PA remain unknown. Here, we report the roles of PLD and PA in genomic RNA replication of a plant (+RNA virus, Red clover necrotic mosaic virus (RCNMV. We found that RCNMV RNA replication complexes formed in Nicotiana benthamiana contained PLDα and PLDβ. Gene-silencing and pharmacological inhibition approaches showed that PLDs and PLDs-derived PA are required for viral RNA replication. Consistent with this, exogenous application of PA enhanced viral RNA replication in plant cells and plant-derived cell-free extracts. We also found that a viral auxiliary replication protein bound to PA in vitro, and that the amount of PA increased in RCNMV-infected plant leaves. Together, our findings suggest that RCNMV hijacks host PA-producing enzymes to replicate.

  15. A Broad RNA Virus Survey Reveals Both miRNA Dependence and Functional Sequestration

    DEFF Research Database (Denmark)

    Scheel, Troels K H; Luna, Joseph M; Liniger, Matthias

    2016-01-01

    immunoprecipitation (CLIP) of the Argonaute (AGO) proteins to characterize strengths and specificities of miRNA interactions in the context of 15 different RNA virus infections, including several clinically relevant pathogens. Notably, replication of pestiviruses, a major threat to milk and meat industries...

  16. seroprevalence of hav, hbv, hcv, and hev among acute hepatitis ...

    African Journals Online (AJOL)

    2013-07-30

    00100, Nairobi, J. Ngaira, Jomo Kenyatta. University of ... (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV) and hepatitis E virus .... analysis was carried out using SPSS 11.5 computer software.

  17. Effects of RNA branching on the electrostatic stabilization of viruses.

    Science.gov (United States)

    Erdemci-Tandogan, Gonca; Wagner, Jef; van der Schoot, Paul; Podgornik, Rudolf; Zandi, Roya

    2016-08-01

    Many single-stranded (ss) ribonucleic acid (RNA) viruses self-assemble from capsid protein subunits and the nucleic acid to form an infectious virion. It is believed that the electrostatic interactions between the negatively charged RNA and the positively charged viral capsid proteins drive the encapsidation, although there is growing evidence that the sequence of the viral RNA also plays a role in packaging. In particular, the sequence will determine the possible secondary structures that the ssRNA will take in solution. In this work, we use a mean-field theory to investigate how the secondary structure of the RNA combined with electrostatic interactions affects the efficiency of assembly and stability of the assembled virions. We show that the secondary structure of RNA may result in negative osmotic pressures while a linear polymer causes positive osmotic pressures for the same conditions. This may suggest that the branched structure makes the RNA more effectively packaged and the virion more stable.

  18. Molecular Assay and Genotyping of Hepatitis C Virus among Infected Egyptian and Saudi Arabian Patients

    Directory of Open Access Journals (Sweden)

    Mohamed M.S. Farag

    2015-01-01

    Full Text Available Hepatitis C virus (HCV infection is a major health problem recognized globally. HCV is a common cause of liver fibrosis that may lead to liver cirrhosis or hepatocellular carcinoma. The aim of this study was to estimate the prevalence of HCV infection and genotyping among Egyptian and Saudi Arabian chronic patients using different molecular techniques. HCV RNA viral load was assessed by real-time polymerase chain reaction (RT-PCR technology. For HCV genotyping, RT-PCR hybridization fluorescence-based method and reverse hybridization line probe assay (INNO-LiPA were used. A total of 40 anti-HCV-positive patients with chronic hepatitis C were examined for HCV RNA, genotyping, and different laboratory investigations. In the present study, HCV genotypes 4, mixed 4.1b, and 1 were detected in patients of both countries, while genotype 2 was only detected in Saudi Arabian patients. Genotyping methods for HCV showed no difference in the classification at the genotype level. With regard to HCV subtypes, INNO-LiPA assay was a reliable test in HCV genotyping for the detection of major genotypes and subtypes, while RT-PCR-based assay was a good test at the genotype level only. HCV genotype 4 was found to be the predominant genotype among Egyptian and Saudi Arabian chronic patients. In conclusion, data analysis for detecting and genotyping HCV was an important factor for understanding the epidemiology and treatment strategies of HCV among Egyptian and Saudi Arabian chronic patients.

  19. Prevalence of HIV-1/2, HTLV-I/II, hepatitis B virus (HBV, hepatitis C virus (HCV, Treponema pallidum and Trypanosoma cruzi among prison inmates at Manhuaçu, Minas Gerais State, Brazil

    Directory of Open Access Journals (Sweden)

    Catalan-Soares Bernadette Corrêa

    2000-01-01

    Full Text Available The purpose of this study was to determine the seroprevalence of human immunodeficiency virus (HIV-1/2, human T-cell lymphotropic virus (HTLV-I/II, hepatitis B virus (HBV, hepatitis C virus (HCV, Treponema pallidum and Trypanosoma cruzi among 63 male prisoners in Manhuaçu, Minas Gerais, Brazil and to compare this with data from eligible blood donors. The positive results were as follows: 11/63 (17.5% for HBV, 5/63 (7.4% for syphilis, 4/63 (6.3% for HCV, 3/63 (4.8% for Chagas' disease, 2/63 (3.2% for HIV-1/2 and 1/63 (1.6% for HTLV-I/II. The seroprevalence in prisoners was higher than among blood donors, mainly for antibodies to HIV-1/2, HCV and HBV. This is probably due to low social economic level, illiteracy, higher proportion with a prior history of intravenous drug use and/or unsafe sexual behavior. Therefore, these prisoners constitute a high risk group and routine screening and counseling are recommended.

  20. Proteome analysis of liver cells expressing a full-length hepatitis C virus (HCV) replicon and biopsy specimens of posttransplantation liver from HCV-infected patients

    Czech Academy of Sciences Publication Activity Database

    Jacobs, J. M.; Diamond, D. L.; Chan, E. Y.; Gritsenko, M. A.; Qian, W.; Šťastná, Miroslava; Baas, T.; Camp II, D. G .H.; Carithers Jr., R. L.; Smith, R. D.; Katze, M. G.

    2005-01-01

    Roč. 79, č. 12 (2005), s. 7558-7569 ISSN 0022-538X Institutional research plan: CEZ:AV0Z40310501 Keywords : proteome analysis * hepatitis C Virus * mass spectrometry Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 5.178, year: 2005

  1. 5' cap-independent translation of dengue virus genomic RNA

    OpenAIRE

    Pérez Dominguez, Mariela; Facultad de Odontología. Universidad de Carabobo. Valencia, Venezuela. Instituto de Investigaciones Biomédicas “Dr. Francisco J. Triana Alonso”, Facultad de Ciencias de la Salud, Universidad de Carabobo. Sede Aragua, Maracay, Venezuela.; Rojas, Roselbis; Instituto de Investigaciones Biomédicas “Dr. Francisco J. Triana Alonso”, Facultad de Ciencias de la Salud, Universidad de Carabobo. Sede Aragua, Maracay, Venezuela.; Requena, Dayana; Instituto de Investigaciones Biomédicas “Dr. Francisco J. Triana Alonso”, Facultad de Ciencias de la Salud, Universidad de Carabobo. Sede Aragua, Maracay, Venezuela.; Ferreras, Ana C.; Instituto de Investigaciones Biomédicas “Dr. Francisco J. Triana Alonso”, Facultad de Ciencias de la Salud, Universidad de Carabobo. Sede Aragua, Maracay, Venezuela.; Triana, Juana L.; Instituto de Investigaciones Biomédicas “Dr. Francisco J. Triana Alonso”, Facultad de Ciencias de la Salud, Universidad de Carabobo. Sede Aragua, Maracay, Venezuela.; Triana-Alonso, Francisco; Instituto de Investigaciones Biomédicas “Dr. Francisco J. Triana Alonso”, Facultad de Ciencias de la Salud, Universidad de Carabobo. Sede Aragua, Maracay, Venezuela.

    2015-01-01

    Objetives. To analyze the involvement of methyl guanosine triphosphate cap (5’cap) and the start site of the genomic RNA ofDengue virus serotype 2 (DENV-2) American genotype in translation, using a cell-free system prepared from human placenta.Materials and methods. The recombinant plasmid pTZ18R-D2 was prepared containing DNA encoding the 5’UTR and thefirst 201 nucleotides of the viral capsid. This plasmid was used to transcribe the corresponding RNA (RNA-D2) without the 5’cap. The RNA-D2 wa...

  2. Prevalence and impact of GBV-C, SEN-V and HBV occult infections in HIV-HCV co-infected patients on HCV therapy.

    Science.gov (United States)

    Piroth, Lionel; Carrat, Fabrice; Larrat, Sylvie; Goderel, Isabelle; Martha, Benoit; Payan, Christopher; Lunel-Fabiani, Françoise; Bani-Sadr, Firouze; Perronne, Christian; Cacoub, Patrice; Pol, Stanislas; Morand, Patrice

    2008-12-01

    It has been suggested that, in HIV-HCV co-infected patients, co-infections with other viruses may affect the response to HCV therapy. We aimed to assess the prevalence of GBV-C, SEN-V and occult HBV infections, their impact on HCV and HIV infections and on the response to HCV therapy in HIV-HCV co-infected patients. Three-hundred and sixty eight patients were tested before starting interferon-ribavirin for the presence of occult hepatitis B DNA, GBV-C RNA and SEN-V DNA by using real time PCR. Clinical, immunological, virological, histological characteristics and response to HCV therapy were compared according to the presence or not of each viral co-infection. HBV DNA, GBV-C RNA and SEN-V DNA were found in 5 (1.4%, CI95%: 0.2-2.4%), 104 (29.9%, CI95%: 25.1-34.7%) and 209 patients (57.9%, CI95%: 52.8-63.0%), respectively. GBV-C positive patients had significantly higher CD4 count at baseline, during and after HCV therapy, even after stratification on antiretroviral treatment. No other significant difference was observed according to the presence or not of GBV-C or SEN-V co-infection, in particular regarding virological responses to HCV combination therapy. There is no reason to withhold HCV therapy in HIV infected patients who have access to HAART, because of occult HBV, GBV-C or SEN-V co-infections.

  3. RNA interference against viruses: strike and counterstrike

    NARCIS (Netherlands)

    Haasnoot, Joost; Westerhout, Ellen M.; Berkhout, Ben

    2007-01-01

    RNA interference (RNAi) is a conserved sequence-specific, gene-silencing mechanism that is induced by double-stranded RNA. RNAi holds great promise as a novel nucleic acid-based therapeutic against a wide variety of diseases, including cancer, infectious diseases and genetic disorders. Antiviral

  4. Down-regulation of IRES containing 5'UTR of HCV genotype 3a using siRNAs

    Directory of Open Access Journals (Sweden)

    Ali Ashfaq Usman

    2011-05-01

    Full Text Available Abstract Background Hepatitis C virus (HCV is a major causative agent of liver associated diseases leading to the development of hepatocellular carcinoma (HCC all over the world and genotype-3a responsible for most of the cases in Pakistan. Due to the limited efficiency of current chemotherapy of interferon-α (IFN-α and ribavirin against HCV infection alternative options are desperately needed out of which the recently discovered RNAi represent a powerful silencing approach for molecular therapeutics through a sequence-specific RNA degradation process to silence virus infection or replication. HCV translation is mediated by a highly conserved internal ribosome entry site (IRES within the 5'UTR region making it a relevant target for new drug development. Materials and methods The present study was proposed to assess and explore the possibility of HCV silencing using siRNA targeting 5'UTR. For this analysis full length HCV 5'UTR of HCV-3a (pCR3.1/5'UTR was tagged with GFP protein for in vitro analysis in Huh-7 cells. siRNA targeting 5'UTR were designed, and tested against constructed vector in Huh-7 cell line both at RNA and Protein levels. Furthermore, the effect of these siRNAs was confirmed in HCV-3a serum infected Huh-7 cell line. Results The expression of 5'UTR-GFP was dramatically reduced both at mRNA and protein levels as compared with Mock transfected and control siRNAs treated cells using siRNAs against IRES of HCV-3a genotype. The potential of siRNAs specificity to inhibit HCV-3a replication in serum-infected Huh-7 cells was also investigated; upon treatment with siRNAs a significant decrease in HCV viral copy number and protein expression was observed. Conclusions Overall, the present work of siRNAs against HCV 5'UTR inhibits HCV-3a expression and represents effective future therapeutic opportunities against HCV-3a genotype.

  5. Completion of the Entire Hepatitis C Virus Life Cycle in Vero Cells Derived from Monkey Kidney.

    Science.gov (United States)

    Murayama, Asako; Sugiyama, Nao; Wakita, Takaji; Kato, Takanobu

    2016-06-14

    A hepatitis C virus (HCV) cell culture system incorporating the JFH-1 strain and the human hepatoma cell line HuH-7 enabled the production of infectious HCV particles. Several host factors were identified as essential for HCV replication. Supplementation of these factors in nonhepatic human cell lines enabled HCV replication and particle production. Vero cells established from monkey kidney are commonly used for the production of vaccines against a variety of viruses. In this study, we aimed to establish a novel Vero cell line to reconstruct the HCV life cycle. Unmodified Vero cells did not allow HCV infection or replication. The expression of microRNA 122 (miR-122), an essential factor for HCV replication, is notably low in Vero cells. Therefore, we supplemented Vero cells with miR-122 and found that HCV replication was enhanced. However, Vero cells that expressed miR-122 still did not allow HCV infection. We supplemented HCV receptor molecules and found that scavenger receptor class B type I (SRBI) was essential for HCV infection in Vero cells. The supplementation of apolipoprotein E (ApoE), a host factor important for virus production, enabled the production of infectious virus in Vero cells. Finally, we created a Vero cell line that expressed the essential factors miR-122, SRBI, and ApoE; the entire HCV life cycle, including infection, replication, and infectious virus production, was completed in these cells. In conclusion, we demonstrated that miR-122, SRBI, and ApoE were necessary and sufficient for the completion of the entire HCV life cycle in nonhuman, nonhepatic Vero cells. HCV is a major cause of chronic liver diseases worldwide, and an effective prophylactic HCV vaccine is needed. For safety reasons, the current HCV cell culture system using HuH-7 cells, which was established from a hepatocellular carcinoma, is not suitable for the production of a vaccine against HCV. A robust HCV production system using non-cancer-derived cells is indispensable for

  6. Buprenorphine for human immunodeficiency virus/hepatitis C virus-coinfected patients: does it serve as a bridge to hepatitis C virus therapy?

    Science.gov (United States)

    Taylor, Lynn E; Maynard, Michaela A; Friedmann, Peter D; Macleod, Cynthia J; Rich, Josiah D; Flanigan, Timothy P; Sylvestre, Diana L

    2012-09-01

    Buprenorphine is associated with enhanced human immunodeficiency virus (HIV) treatment outcomes including increased antiretroviral therapy initiation rates, adherence, and CD4 cell counts among HIV-infected opioid-dependent individuals. Buprenorphine facilitates hepatitis C virus (HCV) treatment in opioid-dependent patients with HCV monoinfection. Less is known about buprenorphine's role in HIV/HCV coinfection. We conducted a retrospective chart review to evaluate HCV care for HIV-infected buprenorphine patients in the first 4 years of buprenorphine's integration into a Rhode Island HIV clinic. Sixty-one patients initiated buprenorphine. All had HCV antibody testing; 57 (93%) were antibody-positive. All antibody-positive patients underwent HCV RNA testing; 48 (84%) were RNA-positive. Of these, 15 (31%) were not referred to HCV care. Among chronically infected patients, 3 received HCV treatment after buprenorphine; all had cirrhosis and none achieved viral eradication. At buprenorphine induction, most patients had inadequately controlled HIV infection, with detectable HIV RNA (59%) or CD4 cell count less than or equal to 350/μL (38%). Buprenorphine has shown limited success to date as a bridge to HCV treatment within an HIV clinic. Buprenorphine's stabilization of opioid dependence and HIV disease may permit the use of HCV therapy over time.

  7. Hepatitis C virus: an important occupational hazard?

    Science.gov (United States)

    Stevens, A B; Coyle, P V

    2000-08-01

    Infection with Hepatitis C virus (HCV) is estimated to affect 3% of the world's population and is an important cause of liver disease. It is most commonly transmitted by percutaneous exposure. Although current evidence does not suggest an increased prevalence of HCV infection among healthcare workers, transmission of infection following occupational exposure has been demonstrated. An average transmission rate of 1.8%, following percutaneous injury, has been reported. The risk of transmission is higher from patients with viraemia, as measured by a positive polymerase chain reaction for HCV RNA. After exposure to HCV, healthcare workers should be actively followed up, initially using a test to detect viral RNA. This may facilitate earlier diagnosis and treatment. Recent reports in the UK, of transmission of infection to patients from HCV infected healthcare workers, have prompted a review of the appropriateness of HCV infected individuals undertaking exposure prone procedures.

  8. Infection of hepatitis C virus genotypes in hepatocellular carcinoma ...

    African Journals Online (AJOL)

    The aim of this retrospective study was to investigate the infection of hepatitis C virus (HCV) genotypes in hepatocellular carcinoma (HCC) patients from rural areas of Faisalabad region. Among 179 HCC subjects, men and women were 51 and 49%, respectively. All samples positive for HCV RNA by qualitative PCR were ...

  9. Hepatitis C virus genotypes in Tirana, Albania.

    Science.gov (United States)

    Haldeda, Migena; Baume, Julien; Tamalet, Catherine; Bizhga, Melpomeni; Colson, Philippe

    2014-01-01

    Hepatitis C virus (HCV) infection is a worldwide concern. Knowledge of the HCV genotype is clinically important because it predicts the rate of response to therapy and guides the treatment duration. Moreover, it allows molecular epidemiology to be performed. To our knowledge, the prevalence of HCV genotypes has been assessed only once in Albania, using a line probe genotyping assay. We determined HCV genotypes by population sequencing of HCV-infected patients in Tirana, Albania. HCV genotype and sequence analyses were performed for serum samples collected from January 2011 through May 2012 from 61 HCV-seropositive patients using population sequencing of the NS3 protease gene and alternatively the NS5b gene and the 5' untranslated region (UTR). HCV RNA was retrieved from the blood samples of 50 patients. The HCV NS3 protease gene was sequenced for 28 patients and NS5b and/or 5'UTR fragments were sequenced for an additional 22 patients. The predominant genotype was 1b in 25 patients (50%), followed by genotypes 2c, 4a, 3a, and 1a in 18%, 14%, 8%, and 6% of cases, respectively. Best matches for these HCV RNAs in GenBank were obtained in different countries worldwide. One NS3 protease naturally harbored an amino acid conferring minor drug resistance to newly available HCV protease inhibitors. In conclusion, HCV-1b was predominant in the present Albanian population, as in southeastern Europe. Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.

  10. Hepatitis C virus-induced innate immune responses in human iPS cell-derived hepatocyte-like cells.

    Science.gov (United States)

    Sakurai, Fuminori; Kunito, Takemaru; Takayama, Kazuo; Hashimoto, Rina; Tachibana, Masashi; Sakamoto, Naoya; Wakita, Takaji; Mizuguchi, Hiroyuki

    2017-10-15

    Hepatitis C virus (HCV) infection is a major cause of liver-related morbidity and mortality. In order to develop effective remedies for hepatitis C, it is important to understand the HCV infection profile and host-HCV interaction. HCV-induced innate immune responses play a crucial role in spontaneous HCV clearance; however, HCV-induced innate immune responses have not been fully evaluated in hepatocytes, partly because there are few in vitro models of HCV-induced innate immunity. Recently, human induced pluripotent stem (iPS) cells have received much attention as an in vitro model of infection with various pathogens, including HCV. We previously established highly functional hepatocyte-like cells differentiated from human iPS cells (iPS-HLCs). Here, we examined the potential of iPS-HLCs as an in vitro HCV infection model, especially for evaluation of the relationship between HCV infection levels and HCV-induced innate immunity. Significant expressions of type I and III interferons (IFNs) and IFN-stimulated genes (ISGs) were induced following transfection with HCV genomic replicon RNA in iPS-HLCs. Following inoculation with the HCV JFH-1 strain in iPS-HLCs, peaks of HCV genome replication and HCV protein expression were observed on day 2, and then both the HCV genome and protein levels gradually declined, while the mRNA levels of type III IFNs and ISGs peaked at day 2 following inoculation. These results suggest that the HCV genome efficiently replicates in iPS-HLCs, resulting in HCV genome-induced up-regulation of IFNs and ISGs, and thereafter, HCV genome-induced up-regulation of IFNs and ISGs mediates a reduction in the HCV genome and protein levels in iPS-HLCs. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Family level phylogenies reveal modes of macroevolution in RNA viruses.

    Science.gov (United States)

    Kitchen, Andrew; Shackelton, Laura A; Holmes, Edward C

    2011-01-04

    Despite advances in understanding the patterns and processes of microevolution in RNA viruses, little is known about the determinants of viral diversification at the macroevolutionary scale. In particular, the processes by which viral lineages assigned as different "species" are generated remain largely uncharacterized. To address this issue, we use a robust phylogenetic approach to analyze patterns of lineage diversification in five representative families of RNA viruses. We ask whether the process of lineage diversification primarily occurs when viruses infect new host species, either through cross-species transmission or codivergence, and which are defined here as analogous to allopatric speciation in animals, or by acquiring new niches within the same host species, analogous to sympatric speciation. By mapping probable primary host species onto family level viral phylogenies, we reveal a strong clustering among viral lineages that infect groups of closely related host species. Although this is consistent with lineage diversification within individual hosts, we argue that this pattern more likely represents strong biases in our knowledge of viral biodiversity, because we also find that better-sampled human viruses rarely cluster together. Hence, although closely related viruses tend to infect related host species, it is unlikely that they often infect the same host species, such that evolutionary constraints hinder lineage diversification within individual host species. We conclude that the colonization of new but related host species may represent the principle mode of macroevolution in RNA viruses.

  12. Comparative analysis of measles virus RNA by oligonucleotide fingerprinting

    Energy Technology Data Exchange (ETDEWEB)

    Stephenson, J.R.; Meulen, V. ter (Wuerzburg Univ. (Germany, F.R.))

    1982-01-01

    Isolates from two cases of acute measles, one case of acute measles encephalitis and three patients with subacute sclerosing panencephalitis were compared. This comparison was based upon the electrophoretic analysis of T/sub 1/ oligonucleotides from single-stranded, full-length RNA isolated from cytoplasmic nucleocapsids. Although all viruses have oligonucleotides in common, each isolate generated a unique pattern of oligonucleotides. However, no group of oligonucleotides was observed which would allow differentiation between viruses isolated from acute infections and those isolated from CNS diseases; indicating that probably all measles viruses differ in their nucleotide sequence, regardless of origin.

  13. Assessment of the RNASound RNA Sampling Card for the preservation of influenza virus RNA

    Directory of Open Access Journals (Sweden)

    Hilda Lau

    2016-11-01

    Full Text Available Shipping influenza virus specimens, isolates or purified RNA is normally conducted at ultra-low temperatures using dry ice to ensure minimal degradation of the samples but this is expensive and requires special packaging and shipping conditions. Therefore, alternative methods for shipping influenza viruses or RNA at ambient temperatures would be desirable.The RNASound RNA Sampling Card (FortiusBio LLC, CA, USA is a device that enables specimens or isolates to be applied to a card, whereby viruses are inactivated, while RNA is preserved and purified RNA can also easily be eluted. To evaluate this card, we applied influenza virus cell culture isolate supernatants to either the RNASound card or Whatman Grade No. 1 filter paper (GE Healthcare, NSW, Australia and compared the preservation to that of material stored in liquid form. Preservation was tested using influenza A and B viruses at two different storage temperatures (cool 2-8oC or room temperature 18-22oC and these were compared with control material stored at -80°C, for 7, 14 or 28 days. The quality of the RNA recovered was assessed using real time RT-PCR and Sanger sequencing. The RNASound card was effective in preserving influenza RNA at room temperature for up to 28 days, with only a minor change in real-time RT-PCR cycle threshold values for selected gene targets when comparing between viruses applied to the card or stored at -80°C. Similar results were obtained with filter paper, whilst virus in liquid form performed the worst. Nevertheless, as the RNASound card also has the capability to inactivate viruses in addition to preserving RNA at room temperature for many weeks, this makes it feasible to send samples to laboratories using regular mail, and thus avoid the need for expensive shipping conditions requiring biohazard containers and dry ice. Moreover, the quick and simple RNA recovery from the RNASound card allows recipient labs to obtain RNA without the need for special reagents

  14. RNA Elements in Open Reading Frames of the Bluetongue Virus Genome Are Essential for Virus Replication

    NARCIS (Netherlands)

    Feenstra, F.; Gennip, van H.G.P.; Water, van de S.G.P.; Rijn, van P.A.

    2014-01-01

    Members of the Reoviridae family are non-enveloped multi-layered viruses with a double stranded RNA genome consisting of 9 to 12 genome segments. Bluetongue virus is the prototype orbivirus (family Reoviridae, genus Orbivirus), causing disease in ruminants, and is spread by Culicoides biting midges.

  15. Metagenomic analysis of the turkey gut RNA virus community

    Directory of Open Access Journals (Sweden)

    Scheffler Brian E

    2010-11-01

    Full Text Available Abstract Viral enteric disease is an ongoing economic burden to poultry producers worldwide, and despite considerable research, no single virus has emerged as a likely causative agent and target for prevention and control efforts. Historically, electron microscopy has been used to identify suspect viruses, with many small, round viruses eluding classification based solely on morphology. National and regional surveys using molecular diagnostics have revealed that suspect viruses continuously circulate in United States poultry, with many viruses appearing concomitantly and in healthy birds. High-throughput nucleic acid pyrosequencing is a powerful diagnostic technology capable of determining the full genomic repertoire present in a complex environmental sample. We utilized the Roche/454 Life Sciences GS-FLX platform to compile an RNA virus metagenome from turkey flocks experiencing enteric disease. This approach yielded numerous sequences homologous to viruses in the BLAST nr protein database, many of which have not been described in turkeys. Our analysis of this turkey gut RNA metagenome focuses in particular on the turkey-origin members of the Picornavirales, the Caliciviridae, and the turkey Picobirnaviruses.

  16. NS4A protein as a marker of HCV history suggests that different HCV genotypes originally evolved from genotype 1b

    Science.gov (United States)

    2011-01-01

    Background The 9.6 kb long RNA genome of Hepatitis C virus (HCV) is under the control of RNA dependent RNA polymerase, an error-prone enzyme, for its transcription and replication. A high rate of mutation has been found to be associated with RNA viruses like HCV. Based on genetic variability, HCV has been classified into 6 different major genotypes and 11 different subtypes. However this classification system does not provide significant information about the origin of the virus, primarily due to high mutation rate at nucleotide level. HCV genome codes for a single polyprotein of about 3011 amino acids which is processed into structural and non-structural proteins inside host cell by viral and cellular proteases. Results We have identified a conserved NS4A protein sequence for HCV genotype 3a reported from four different continents of the world i.e. Europe, America, Australia and Asia. We investigated 346 sequences and compared amino acid composition of NS4A protein of different HCV genotypes through Multiple Sequence Alignment and observed amino acid substitutions C22, V29, V30, V38, Q46 and Q47 in NS4A protein of genotype 1b. Furthermore, we observed C22 and V30 as more consistent members of NS4A protein of genotype 1a. Similarly Q46 and Q47 in genotype 5, V29, V30, Q46 and Q47 in genotype 4, C22, Q46 and Q47 in genotype 6, C22, V38, Q46 and Q47 in genotype 3 and C22 in genotype 2 as more consistent members of NS4A protein of these genotypes. So the different amino acids that were introduced as substitutions in NS4A protein of genotype 1 subtype 1b have been retained as consistent members of the NS4A protein of other known genotypes. Conclusion These observations indicate that NS4A protein of different HCV genotypes originally evolved from NS4A protein of genotype 1 subtype 1b, which in turn indicate that HCV genotype 1 subtype 1b established itself earlier in human population and all other known genotypes evolved later as a result of mutations in HCV genotype 1b

  17. NS4A protein as a marker of HCV history suggests that different HCV genotypes originally evolved from genotype 1b

    Directory of Open Access Journals (Sweden)

    Asad Sultan

    2011-06-01

    Full Text Available Abstract Background The 9.6 kb long RNA genome of Hepatitis C virus (HCV is under the control of RNA dependent RNA polymerase, an error-prone enzyme, for its transcription and replication. A high rate of mutation has been found to be associated with RNA viruses like HCV. Based on genetic variability, HCV has been classified into 6 different major genotypes and 11 different subtypes. However this classification system does not provide significant information about the origin of the virus, primarily due to high mutation rate at nucleotide level. HCV genome codes for a single polyprotein of about 3011 amino acids which is processed into structural and non-structural proteins inside host cell by viral and cellular proteases. Results We have identified a conserved NS4A protein sequence for HCV genotype 3a reported from four different continents of the world i.e. Europe, America, Australia and Asia. We investigated 346 sequences and compared amino acid composition of NS4A protein of different HCV genotypes through Multiple Sequence Alignment and observed amino acid substitutions C22, V29, V30, V38, Q46 and Q47 in NS4A protein of genotype 1b. Furthermore, we observed C22 and V30 as more consistent members of NS4A protein of genotype 1a. Similarly Q46 and Q47 in genotype 5, V29, V30, Q46 and Q47 in genotype 4, C22, Q46 and Q47 in genotype 6, C22, V38, Q46 and Q47 in genotype 3 and C22 in genotype 2 as more consistent members of NS4A protein of these genotypes. So the different amino acids that were introduced as substitutions in NS4A protein of genotype 1 subtype 1b have been retained as consistent members of the NS4A protein of other known genotypes. Conclusion These observations indicate that NS4A protein of different HCV genotypes originally evolved from NS4A protein of genotype 1 subtype 1b, which in turn indicate that HCV genotype 1 subtype 1b established itself earlier in human population and all other known genotypes evolved later as a result of

  18. HCV Infection and B-Cell Lymphomagenesis

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    Masahiko Ito

    2011-01-01

    Full Text Available Hepatitis C virus (HCV has been recognized as a major cause of chronic liver diseases worldwide. It has been suggested that HCV infects not only hepatocytes but also mononuclear lymphocytes including B cells that express the CD81 molecule, a putative HCV receptor. HCV infection of B cells is the likely cause of B-cell dysregulation disorders such as mixed cryoglobulinemia, rheumatoid factor production, and B-cell lymphoproliferative disorders that may evolve into non-Hodgkin's lymphoma (NHL. Epidemiological data indicate an association between HCV chronic infection and the occurrence of B-cell NHL, suggesting that chronic HCV infection is associated at least in part with B-cell lymphomagenesis. In this paper, we aim to provide an overview of recent literature, including our own, to elucidate a possible role of HCV chronic infection in B-cell lymphomagenesis.

  19. Inhibition of Monkeypox virus replication by RNA interference

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    Jahrling Peter B

    2009-11-01

    Full Text Available Abstract The Orthopoxvirus genus of Poxviridae family is comprised of several human pathogens, including cowpox (CPXV, Vaccinia (VACV, monkeypox (MPV and Variola (VARV viruses. Species of this virus genus cause human diseases with various severities and outcome ranging from mild conditions to death in fulminating cases. Currently, vaccination is the only protective measure against infection with these viruses and no licensed antiviral drug therapy is available. In this study, we investigated the potential of RNA interference pathway (RNAi as a therapeutic approach for orthopox virus infections using MPV as a model. Based on genome-wide expression studies and bioinformatic analysis, we selected 12 viral genes and targeted them by small interference RNA (siRNA. Forty-eight siRNA constructs were developed and evaluated in vitro for their ability to inhibit viral replication. Two genes, each targeted with four different siRNA constructs in one pool, were limiting to viral replication. Seven siRNA constructs from these two pools, targeting either an essential gene for viral replication (A6R or an important gene in viral entry (E8L, inhibited viral replication in cell culture by 65-95% with no apparent cytotoxicity. Further analysis with wild-type and recombinant MPV expressing green fluorescence protein demonstrated that one of these constructs, siA6-a, was the most potent and inhibited viral replication for up to 7 days at a concentration of 10 nM. These results emphasis the essential role of A6R gene in viral replication, and demonstrate the potential of RNAi as a therapeutic approach for developing oligonucleotide-based drug therapy for MPV and other orthopox viruses.

  20. Genome rearrangement affects RNA virus adaptability on prostate cancer cells

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    Kendra ePesko

    2015-04-01

    Full Text Available Gene order is often highly conserved within taxonomic groups, such that organisms with rearranged genomes tend to be less fit than wildtype gene orders, and suggesting natural selection favors genome architectures that maximize fitness. But it is unclear whether rearranged genomes hinder adaptability: capacity to evolutionarily improve in a new environment. Negative-sense nonsegmented RNA viruses (order Mononegavirales have specific genome architecture: 3′ UTR – core protein genes – envelope protein genes – RNA-dependent RNA-polymerase gene – 5′ UTR. To test how genome architecture affects RNA virus evolution, we examined vesicular stomatitis virus (VSV variants with the nucleocapsid (N gene moved sequentially downstream in the genome. Because RNA polymerase stuttering in VSV replication causes greater mRNA production in upstream genes, N-gene translocation towards the 5’ end leads to stepwise decreases in N transcription, viral replication and progeny production, and also impacts the activation of type 1 interferon mediated antiviral responses. We evolved VSV gene-order variants in two prostate cancer cell lines: LNCap cells deficient in innate immune response to viral infection, and PC3 cells that mount an IFN stimulated anti-viral response to infection. We observed that gene order affects phenotypic adaptability (reproductive growth; viral suppression of immune function, especially on PC3 cells that strongly select against virus infection. Overall, populations derived from the least-fit ancestor (most-altered N position architecture adapted fastest, consistent with theory predicting populations with low initial fitness should improve faster in evolutionary time. Also, we observed correlated responses to selection, where viruses improved across both hosts, rather than suffer fitness trade-offs on unselected hosts. Whole genomics revealed multiple mutations in evolved variants, some of which were conserved across selective

  1. Beet yellows virus replicase and replicative compartments: parallels with other RNA viruses.

    Science.gov (United States)

    Gushchin, Vladimir A; Solovyev, Andrey G; Erokhina, Tatyana N; Morozov, Sergey Y; Agranovsky, Alexey A

    2013-01-01

    In eukaryotic virus systems, infection leads to induction of membranous compartments in which replication occurs. Virus-encoded subunits of the replication complex mediate its interaction with membranes. As replication platforms, RNA viruses use the cytoplasmic surfaces of different membrane compartments, e.g., endoplasmic reticulum (ER), Golgi, endo/lysosomes, mitochondria, chloroplasts, and peroxisomes. Closterovirus infections are accompanied by formation of multivesicular complexes from cell membranes of ER or mitochondrial origin. So far the mechanisms for vesicles formation have been obscure. In the replication-associated 1a polyprotein of Beet yellows virus (BYV) and other closteroviruses, the region between the methyltransferase and helicase domains (1a central region (CR), 1a CR) is marginally conserved. Computer-assisted analysis predicts several putative membrane-binding domains in the BYV 1a CR. Transient expression of a hydrophobic segment (referred to here as CR-2) of the BYV 1a in Nicotiana benthamiana led to reorganization of the ER and formation of ~1-μm mobile globules. We propose that the CR-2 may be involved in the formation of multivesicular complexes in BYV-infected cells. This provides analogy with membrane-associated proteins mediating the build-up of "virus factories" in cells infected with diverse positive-strand RNA viruses (alpha-like viruses, picorna-like viruses, flaviviruses, and nidoviruses) and negative-strand RNA viruses (bunyaviruses).

  2. Hepatitis C Virus RNA-Dependent RNA Polymerase Interacts with the Akt/PKB Kinase and Induces Its Subcellular Relocalization

    Science.gov (United States)

    Valero, María Llanos; Sabariegos, Rosario; Cimas, Francisco J.; Perales, Celia; Domingo, Esteban; Sánchez-Prieto, Ricardo

    2016-01-01

    Hepatitis C virus (HCV) interacts with cellular components and modulates their activities for its own benefit. These interactions have been postulated as a target for antiviral treatment, and some candidate molecules are currently in clinical trials. The multifunctional cellular kinase Akt/protein kinase B (PKB) must be activated to increase the efficacy of HCV entry but is rapidly inactivated as the viral replication cycle progresses. Viral components have been postulated to be responsible for Akt/PKB inactivation, but the underlying mechanism remained elusive. In this study, we show that HCV polymerase NS5B interacts with Akt/PKB. In the presence of transiently expressed NS5B or in replicon- or virus-infected cells, NS5B changes the cellular localization of Akt/PKB from the cytoplasm to the perinuclear region. Sequestration of Akt/PKB by NS5B could explain its exclusion from its participation in early Akt/PKB inactivation. The NS5B-Akt/PKB interaction represents a new regulatory step in the HCV infection cycle, opening possibilities for new therapeutic options. PMID:27021315

  3. Statin (3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor-based therapy for hepatitis C virus (HCV infection-related diseases in the era of direct-acting antiviral agents [version 3; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Sara Sobhy Kishta

    2017-01-01

    Full Text Available Recent improvements have been made in the treatment of hepatitis C virus (HCV infection with the introduction of direct-acting antiviral agents (DAAs. However, despite successful viral clearance, many patients continue to have HCV-related disease progression. Therefore, new treatments must be developed to achieve viral clearance and prevent the risk of HCV-related diseases. In particular, the use of pitavastatin together with DAAs may improve the antiviral efficacy as well as decrease the progression of liver fibrosis and the incidence of HCV-related hepatocellular carcinoma. To investigate the management methods for HCV-related diseases using pitavastatin and DAAs, clinical trials should be undertaken. However, concerns have been raised about potential drug interactions between statins and DAAs. Therefore, pre-clinical trials using a replicon system, human hepatocyte-like cells, human neurons and human cardiomyocytes from human-induced pluripotent stem cells should be conducted. Based on these pre-clinical trials, an optimal direct-acting antiviral agent could be selected for combination with pitavastatin and DAAs. Following the pre-clinical trial, the combination of pitavastatin and the optimal direct-acting antiviral agent should be compared to other combinations of DAAs (e.g., sofosbuvir and velpatasvir according to the antiviral effect on HCV infection, HCV-related diseases and cost-effectiveness.

  4. Replication Inhibition of Hepatitis B Virus and Hepatitis C Virus in Co-Infected Patients in Chinese Population.

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    Ge Yu

    Full Text Available Hepatitis B virus (HBV and hepatitis C virus (HCV co-infections contributes to a substantial proportion of liver disease worldwide. The aim of this study was to assess the clinical and virological features of HBV-HCV co-infection.Demographic data were collected for 3238 high-risk people from an HCV-endemic region in China. Laboratory tests included HCV antibody and HBV serological markers, liver function tests, and routine blood analysis. Anti-HCV positive samples were analyzed for HCV RNA levels and subgenotypes. HBsAg-positive samples were tested for HBV DNA.A total of 1468 patients had chronic HCV and/or HBV infections. Among them, 1200 individuals were classified as HCV mono-infected, 161 were classified as HBV mono-infected, and 107 were classified as co-infected. The HBV-HCV co-infected patients not only had a lower HBV DNA positive rate compared to HBV mono-infected patients (84.1% versus 94.4%, respectively; P < 0.001. The median HCV RNA levels in HBV-HCV co-infected patients were significantly lower than those in the HCV mono-infected patients (1.18[Interquartile range (IQR 0-5.57] versus 5.87[IQR, 3.54-6.71] Log10 IU/mL, respectively; P < 0.001. Furthermore, co-infected patients were less likely to have detectable HCV RNA levels than HCV mono-infected patients (23.4% versus 56.5%, respectively; P < 0.001. Those HBV-HCV co-infected patients had significantly lower median HBV DNA levels than those mono-infected with HBV (1.97[IQR, 1.3-3.43] versus 3.06[IQR, 2-4.28] Log10 IU/mL, respectively; P < 0.001. The HBV-HCV co-infection group had higher ALT, AST, ALP, GGT, APRI and FIB-4 levels, but lower ALB and total platelet compared to the HBV mono-infection group, and similar to that of the HCV mono-infected group.These results suggest that co-infection with HCV and HBV inhibits the replication of both viruses. The serologic results of HBV-HCV co-infection in patients suggests more liver injury compared to HBV mono-infected patients, but is

  5. Noncoding subgenomic flavivirus RNA: multiple functions in West Nile virus pathogenesis and modulation of host responses

    NARCIS (Netherlands)

    Roby, J.A.; Pijlman, G.P.; Wilusz, J.; Khromykh, A.A.

    2014-01-01

    Flaviviruses are a large group of positive strand RNA viruses transmitted by arthropods that include many human pathogens such as West Nile virus (WNV), Japanese encephalitis virus (JEV), yellow fever virus, dengue virus, and tick-borne encephalitis virus. All members in this genus tested so far are

  6. Hepatitis delta virus and GBV-C infection in two neighboring hepatitis B virus and hepatitis C virus-endemic villages in Taiwan.

    Science.gov (United States)

    Chang, Chang-Jung; Chiang, Jui-Chin; Lu, Sheng-Nan; Wang, Jing-Houng

    2010-01-01

    Previous reports in Taiwan have shown that the hepatitis B virus (HBV)- and hepatitis C virus (HCV)-endemic areas are also endemic for hepatitis D virus (HDV), GBV-C and TT virus. This study aimed to elucidate the epidemiology of HDV and GBV-C infection in two neighboring HBV- and HCV-endemic villages, to deduce the epidemiological characteristics of multiple viral infections in communities. A total of 74 adult residents of Wukwai (W) village and 95 adults residents of Haipu (H) village were studied. Laboratory tests for all subjects included alanine transaminase (ALT), hepatitis B surface antigen (HBsAg), anti-HCV, HCV RNA, genotype of HCV, GBV-C RNA, and anti-GBV-C E2. Anti- HDV was checked only in HBsAg-positive subjects. Subjects from W village were older than those from H village (61.7 +/- 11.8 vs 56.6 +/- 16.4 years, p = 0.02). The prevalence of ALT elevation (37.8% vs 15.8%, p = 0.006), anti-HCV (67.6% vs 34.7%, p GBV-C infection (39.2% vs 24.2%, p = 0.054), and the distribution of HCV genotype 1b (37.8% vs 70.4%, p = 0.01) were different in W and H villages, respectively. Among anti-HCV-positive subjects, HCV RNA-positive rates were 75.9% (63/83), and were higher for men (88.2%) than women (67.3%). Only one HBsAg-positive subject was positive for anti-HDV, and one anti-HCVnegative subject was positive for HCV RNA. In multivariate analyses, GBVC infection correlated with HCV infection or HCV endemicity, and HCV RNA was the only determining factor in ALT elevation. In HBV and HCV-endemic areas, GBV-C was more prevalent in areas with a higher prevalence of anti-HCV. Positive HCV-RNA, but not GBV-C infection, was associated with ALT elevation.

  7. Dinucleotide Composition in Animal RNA Viruses Is Shaped More by Virus Family than by Host Species.

    Science.gov (United States)

    Di Giallonardo, Francesca; Schlub, Timothy E; Shi, Mang; Holmes, Edward C

    2017-04-15

    Viruses use the cellular machinery of their hosts for replication. It has therefore been proposed that the nucleotide and dinucleotide compositions of viruses should match those of their host species. If this is upheld, it may then be possible to use dinucleotide composition to predict the true host species of viruses sampled in metagenomic surveys. However, it is also clear that different taxonomic groups of viruses tend to have distinctive patterns of dinucleotide composition that may be independent of host species. To determine the relative strength of the effect of host versus virus family in shaping dinucleotide composition, we performed a comparative analysis of 20 RNA virus families from 15 host groupings, spanning two animal phyla and more than 900 virus species. In particular, we determined the odds ratios for the 16 possible dinucleotides and performed a discriminant analysis to evaluate the capability of virus dinucleotide composition to predict the correct virus family or host taxon from which it was isolated. Notably, while 81% of the data analyzed here were predicted to the correct virus family, only 62% of these data were predicted to their correct subphylum/class host and a mere 32% to their correct mammalian order. Similarly, dinucleotide composition has a weak predictive power for different hosts within individual virus families. We therefore conclude that dinucleotide composition is generally uniform within a virus family but less well reflects that of its host species. This has obvious implications for attempts to accurately predict host species from virus genome sequences alone. IMPORTANCE Determining the processes that shape virus genomes is central to understanding virus evolution and emergence. One question of particular importance is why nucleotide and dinucleotide frequencies differ so markedly between viruses. In particular, it is currently unclear whether host species or virus family has the biggest impact on dinucleotide frequencies and

  8. Interleukin-21 mRNA expression during virus infections

    DEFF Research Database (Denmark)

    Holm, Christian; Nyvold, Charlotte Guldborg; Paludan, Søren Riis

    2006-01-01

    and activational effects of IL-21 on different leukocytes come into play in vivo in an immune response has so far not been fully investigated. We show here for the first time in vivo, that IL-21 mRNA is produced in the spleen when mice are challenged with herpes simplex virus type 2 (HSV-2) or lymphocytic...... choriomeningitis virus (LCMV). We show in HSV-2 challenged mice that this production takes place in CD4+ T cell fractions and is absent in CD4+ T cell-depleted fractions. We also show that the peak of IL-21 mRNA production in both the HSV-2 and LCMV-challenged mice coincides with the onset of the adaptive immune...... response. Thus, our data suggest a role for IL-21 in the early stages of adaptive immune response against virus infections....

  9. Interferon Induction by RNA Viruses and Antagonism by Viral Pathogens

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    Yuchen Nan

    2014-12-01

    Full Text Available Interferons are a group of small proteins that play key roles in host antiviral innate immunity. Their induction mainly relies on host pattern recognition receptors (PRR. Host PRR for RNA viruses include Toll-like receptors (TLR and retinoic acid-inducible gene I (RIG-I like receptors (RLR. Activation of both TLR and RLR pathways can eventually lead to the secretion of type I IFNs, which can modulate both innate and adaptive immune responses against viral pathogens. Because of the important roles of interferons, viruses have evolved multiple strategies to evade host TLR and RLR mediated signaling. This review focuses on the mechanisms of interferon induction and antagonism of the antiviral strategy by RNA viruses.

  10. Antiviral activity of the dichloromethane extracts from Artocarpus heterophyllus leaves against hepatitis C virus

    OpenAIRE

    Achmad Fuad Hafid; Chie Aoki-Utsubo; Adita Ayu Permanasari; Myrna Adianti; Lydia Tumewu; Aty Widyawaruyanti; Sri Puji Astuti Wahyuningsih; Tutik Sri Wahyuni; Maria Inge Lusida; Soetjipto; Hak Hotta

    2017-01-01

    Objective: To determine anti-viral activities of three Artocarpus species: Artocarpus altilis, Artocarpus camansi, and Artocarpus heterophyllus (A. heterophyllus) against Hepatitis C Virus (HCV). Methods: Antiviral activities of the crude extracts were examined by cell culture method using Huh7it-1 cells and HCV genotype 2a strain JFH1. The mode of action for anti-HCV activities was determined by time-of-addition experiments. The effect on HCV RNA replication and HCV accumulation in cells ...

  11. Prevalence of hepatitis C Virus infection among hemophiliacs in Central Brazil

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    Adriana P Barbosa

    2002-07-01

    Full Text Available In order to investigate the hepatitis C virus (HCV infection prevalence and risk factors in hemophiliacs in Central Brazil, 90 patients were interviewed and serum samples tested for HCV RNA and anti-HCV antibodies. An overall prevalence of 63.3% (CI 95%: 53.0-72.7 was found. Multivariate analysis of risk factors showed that number of blood transfusions was significantly associated with this infection. Most hemophiliacs received locally produced cryoprecipitate. All infected patients were transfused before the screening of blood units for anti-HCV. However, hemophiliacs who received exclusively screened cryoprecipitate were HCV negative. It confirms the expected decline in transfusion-acquired hepatitis C.

  12. Similarities between Human Immunodeficiency Virus Type 1 and Hepatitis C Virus Genetic and Phenotypic Protease Quasispecies Diversity.

    Science.gov (United States)

    Martinez, Miguel Angel; Nevot, Maria; Jordan-Paiz, Ana; Franco, Sandra

    2015-10-01

    Human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) are two highly variable RNA viruses that cause chronic infections in humans. Although HCV likely preceded the AIDS epidemic by some decades, the global spread of both viruses is a relatively recent event. Nevertheless, HCV global diversity is higher than that of HIV-1. To identify differences in mutant diversity, we compared the HIV-1 protease and HCV NS3 protease quasispecies. Three protease gene quasispecies samples per virus, isolated from a total of six infected patients, were genetically and phenotypically analyzed at high resolution (HIV-1, 308 individual clones; HCV, 299 clones). Single-nucleotide variant frequency did not differ between quasispecies from the two viruses (HIV-1, 2.4 × 10(-3) ± 0.4 × 10(-3); HCV, 2.1 × 10(-3) ± 0.5 × 10(-3)) (P = 0.1680). The proportion of synonymous substitutions to potential synonymous sites was similar (3.667 ± 0.6667 and 2.183 ± 0.9048, respectively) (P = 0.2573), and Shannon's entropy values did not differ between HIV-1 and HCV (0.84 ± 0.02 and 0.83 ± 0.12, respectively) (P = 0.9408). Of note, 65% (HIV-1) and 67% (HCV) of the analyzed enzymes displayed detectable protease activity, suggesting that both proteases have a similar mutational robustness. In both viruses, there was a rugged protease enzymatic activity landscape characterized by a sharp peak, representing the master sequence, surrounded by a collection of diverse variants present at lower frequencies. These results indicate that nucleotide quasispecies diversification during chronic infection is not responsible for the higher worldwide genetic diversity observed in HCV. HCV global diversity is higher than that of HIV-1. We asked whether HCV genetic diversification during infection is responsible for the higher worldwide genetic diversity observed in HCV. To this end, we analyzed and compared the genotype and enzymatic activities of HIV-1 and HCV protease quasispecies existing in

  13. Host RNA circles and the origin of hepatitis delta virus.

    Science.gov (United States)

    Taylor, John M

    2014-03-21

    Recent reports show that many cellular RNAs are processed to form circular species that are relatively abundant and resistant to host nucleases. In some cases, such circles actually bind host microRNAs. Such depletion of available microRNAs appears to have biological roles; for instance, in homeostasis and disease. These findings regarding host RNA circles support a speculative reappraisal of the origin and mode of replication of hepatitis delta virus, hepatitis delta virus (HDV), an agent with a small circular RNA genome; specifically, it is proposed that in hepatocytes infected with hepatitis B virus (HBV), some viral RNA species are processed to circular forms, which by a series of chance events lead to an RNA that can be both replicated by host enzymes and assembled, using HBV envelope proteins, to form particles some of which are infectious. Such a model also may provide some new insights into the potential pathogenic potential of HDV infections. In return, new insights into HDV might provide information leading to a better understanding of the roles of the host RNA circles.

  14. The effect of HCV Core protein on the expression of miR-150

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    Sayad Khanizadeh

    2016-09-01

    Full Text Available Background : Hepatitis C virus (HCV is considered as one of the major pathogenic agents of chronic liver diseases. Previous studies have shown that HCV proteins can interaction with gene regulatory networks such as microRNAs. The aim of this study was to investigate the effect of HCV core protein on the expression of miR-150 in a cell culture model. Materials and Methods: Plasmids expressing full HCV core protein was transfected into Huh7 cell lines while a GFP expressing plasmid employed as negative control. Subsequently, total RNA extracted and Real-Time PCR performed to measure the expression level of miR-150 expression. Moreover, trypan blue exclusion assay was performed to investigate the effect of core protein on cell viability. Results: The gene expression analysis of miR-150 in Huh7 cells showed that endogenous HCV core protein could significantly down regulation of miR-150 when compared to GFP control plasmid and normal cells (P<0.01. Beside, core protein induced no significant proliferative or cytotoxic effects on hepatic cells as determined by trypan blue exclusion assay (P<0.05. Conclusion: Our study suggests that HCV core protein can led to down regulation of miR-150 expression. This data revealed that HCV protein interactions with cell regulatory machinery may contribute to pathogenesis of chronic liver diseases.

  15. Animal models for HCV and HBV studies

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    Isabelle Chemin

    2007-02-01

    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. 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.

    Although chimpanzees are the only animals fully permissive for HBV infection, their use for research purpose is severely limited by the high costs and strong ethical constrains. The only alternative source of HBV-permissive hepatocytes is the Asian tree shrew Tupaia belangeri. Though experimental infection of these squirrel-like mammals, phylogenetically related to primates, results only in a mild, transient replication, primary hepatocytes isolated from T. belangeri turned out to be a reliable tool for in vitro HBV infection experiments.

    Along with invaluable infection studies in chimpanzees, avian and mammalian HBV-related viruses continue to offer ample opportunities for studies in naturally occurring hosts. In general, most of our progresses in hepatitis B virus research are based on infection studies with two HBV-related animal viruses: the woodchuck HBV (WHV, which infects the Eastern American woodchuck (Marmota monax, and the duck HBV (DHBV, which infects Peking ducks. Both animal models have been essential for understanding various steps of viral life-cycle and factors involved in establishment of virus

  16. Initiation of RNA Polymerization and Polymerase Encapsidation by a Small dsRNA Virus.

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    Aaron M Collier

    2016-04-01

    Full Text Available During the replication cycle of double-stranded (ds RNA viruses, the viral RNA-dependent RNA polymerase (RdRP replicates and transcribes the viral genome from within the viral capsid. How the RdRP molecules are packaged within the virion and how they function within the confines of an intact capsid are intriguing questions with answers that most likely vary across the different dsRNA virus families. In this study, we have determined a 2.4 Å resolution structure of an RdRP from the human picobirnavirus (hPBV. In addition to the conserved polymerase fold, the hPBV RdRP possesses a highly flexible 24 amino acid loop structure located near the C-terminus of the protein that is inserted into its active site. In vitro RNA polymerization assays and site-directed mutagenesis showed that: (1 the hPBV RdRP is fully active using both ssRNA and dsRNA templates; (2 the insertion loop likely functions as an assembly platform for the priming nucleotide to allow de novo initiation; (3 RNA transcription by the hPBV RdRP proceeds in a semi-conservative manner; and (4 the preference of virus-specific RNA during transcription is dictated by the lower melting temperature associated with the terminal sequences. Co-expression of the hPBV RdRP and the capsid protein (CP indicated that, under the conditions used, the RdRP could not be incorporated into the recombinant capsids in the absence of the viral genome. Additionally, the hPBV RdRP exhibited higher affinity towards the conserved 5'-terminal sequence of the viral RNA, suggesting that the RdRP molecules may be encapsidated through their specific binding to the viral RNAs during assembly.

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

    DEFF Research Database (Denmark)

    Pham, Long; Ngo, HT; Lim, YS

    2012-01-01

    Background & Aims Hepatitis C virus (HCV) requires host cellular proteins for its own propagation. To identify the cellular factors necessary for HCV propagation, we have recently screened the small interfering RNA (siRNA) library targeting cell cycle genes using cell culture grown HCV (HCVcc......)-infected cells. In the current study, we have selected and characterized the gene encoding Cyclin A2 (CycA2). Deregulation of CycA2 has been implicated in many types of cancers, including hepatocellular carcinoma. Methods The effects of CycA2 on HCV propagation were investigated by siRNA-mediated knockdown assay...

  18. Modulations of cell cycle checkpoints during HCV associated disease

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    Jafri Wasim

    2009-08-01

    Full Text Available Abstract Background Impaired proliferation of hepatocytes has been reported in chronic Hepatitis C virus infection. Considering the fundamental role played by cell cycle proteins in controlling cell proliferation, altered regulation of these proteins could significantly contribute to HCV disease progression and subsequent hepatocellular carcinoma (HCC. This study aimed to identify the alterations in cell cycle genes expression with respect to early and advanced disease of chronic HCV infection. Methods Using freshly frozen liver biopsies, mRNA levels of 84 cell cycle genes in pooled RNA samples from patients with early or advanced fibrosis of chronic HCV infection were studied. To associate mRNA levels with respective protein levels, four genes (p27, p15, KNTC1 and MAD2L1 with significant changes in mRNA levels (> 2-fold, p-value Results In the early fibrosis group, increased mRNA levels of cell proliferation genes as well as cell cycle inhibitor genes were observed. In the advanced fibrosis group, DNA damage response genes were up-regulated while those associated with chromosomal stability were down-regulated. Increased expression of CDK inhibitor protein p27 was consistent with its mRNA level detected in early group while the same was found to be negatively associated with liver fibrosis. CDK inhibitor protein p15 was highly expressed in both early and advanced group, but showed no correlation with fibrosis. Among the mitotic checkpoint regulators, expression of KNTC1 was significantly reduced in advanced group while MAD2L1 showed a non-significant decrease. Conclusion Collectively these results are suggestive of a disrupted cell cycle regulation in HCV-infected liver. The information presented here highlights the potential of identified proteins as predictive factors to identify patients with high risk of cell transformation and HCC development.

  19. The Acyclic Retinoid Peretinoin Inhibits Hepatitis C Virus Replication and Infectious Virus Release in Vitro

    Science.gov (United States)

    Shimakami, Tetsuro; Honda, Masao; Shirasaki, Takayoshi; Takabatake, Riuta; Liu, Fanwei; Murai, Kazuhisa; Shiomoto, Takayuki; Funaki, Masaya; Yamane, Daisuke; Murakami, Seishi; Lemon, Stanley M.; Kaneko, Shuichi

    2014-04-01

    Clinical studies suggest that the oral acyclic retinoid Peretinoin may reduce the recurrence of hepatocellular carcinoma (HCC) following surgical ablation of primary tumours. Since hepatitis C virus (HCV) infection is a major cause of HCC, we assessed whether Peretinoin and other retinoids have any effect on HCV infection. For this purpose, we measured the effects of several retinoids on the replication of genotype 1a, 1b, and 2a HCV in vitro. Peretinoin inhibited RNA replication for all genotypes and showed the strongest antiviral effect among the retinoids tested. Furthermore, it reduced infectious virus release by 80-90% without affecting virus assembly. These effects could be due to reduced signalling from lipid droplets, triglyceride abundance, and the expression of mature sterol regulatory element-binding protein 1c and fatty acid synthase. These negative effects of Peretinoin on HCV infection may be beneficial in addition to its potential for HCC chemoprevention in HCV-infected patients.

  20. Hepatitis C Virus Replication.

    Science.gov (United States)

    Suzuki, Tetsuro

    2017-01-01

    Viruses use synthetic mechanism and organelles of the host cells to facilitate their replication and make new viruses. Host's ATP provides necessary energy. Hepatitis C virus (HCV) is a major cause of liver disease. Like other positive-strand RNA viruses, the HCV genome is thought to be synthesized by the replication complex, which consists of viral- and host cell-derived factors, in tight association with structurally rearranged vesicle-like cytoplasmic membranes. The virus-induced remodeling of subcellular membranes, which protect the viral RNA from nucleases in the cytoplasm, promotes efficient replication of HCV genome. The assembly of HCV particle involves interactions between viral structural and nonstructural proteins and pathways related to lipid metabolisms in a concerted fashion. Association of viral core protein, which forms the capsid, with lipid droplets appears to be a prerequisite for early steps of the assembly, which are closely linked with the viral genome replication. This review presents the recent progress in understanding the mechanisms for replication and assembly of HCV through its interactions with organelles or distinct organelle-like structures.

  1. Potent inhibitors of HCV-NS3 protease derived from boronic acids

    Energy Technology Data Exchange (ETDEWEB)

    Venkatraman, Srikanth; Wu, Wanli; Prongay, Andrew; Girijavallabhan, Viyyoor; Njoroge, F. George; (SPRI)

    2009-07-23

    Chronic hepatitis C infection is the leading causes for cirrhosis of the liver and hepatocellular carcinoma, leading to liver failure and liver transplantation. The etiological agent, HCV virus produces a single positive strand of RNA that is processed with the help of serine protease NS3 to produce mature virus. Inhibition of NS3 protease can be potentially used to develop effective drugs for HCV infections. Numerous efforts are now underway to develop potent inhibitors of HCV protease that contain ketoamides as serine traps. Herein we report the synthesis of a series of potent inhibitors that contain a boronic acid as a serine trap. The activity of these compounds were optimized to 200 pM. X-ray structure of compound 17 bound to NS3 protease is also discussed.

  2. A single vertebrate DNA virus protein disarms invertebrate immunity to RNA virus infection

    Science.gov (United States)

    Gammon, Don B; Duraffour, Sophie; Rozelle, Daniel K; Hehnly, Heidi; Sharma, Rita; Sparks, Michael E; West, Cara C; Chen, Ying; Moresco, James J; Andrei, Graciela; Connor, John H; Conte, Darryl; Gundersen-Rindal, Dawn E; Marshall, William L; Yates, John R; Silverman, Neal; Mello, Craig C

    2014-01-01

    Virus-host interactions drive a remarkable diversity of immune responses and countermeasures. We found that two RNA viruses with broad host ranges, vesicular stomatitis virus (VSV) and Sindbis virus (SINV), are completely restricted in their replication after entry into Lepidopteran cells. This restriction is overcome when cells are co-infected with vaccinia virus (VACV), a vertebrate DNA virus. Using RNAi screening, we show that Lepidopteran RNAi, Nuclear Factor-κB, and ubiquitin-proteasome pathways restrict RNA virus infection. Surprisingly, a highly conserved, uncharacterized VACV protein, A51R, can partially overcome this virus restriction. We show that A51R is also critical for VACV replication in vertebrate cells and for pathogenesis in mice. Interestingly, A51R colocalizes with, and stabilizes, host microtubules and also associates with ubiquitin. We show that A51R promotes viral protein stability, possibly by preventing ubiquitin-dependent targeting of viral proteins for destruction. Importantly, our studies reveal exciting new opportunities to study virus-host interactions in experimentally-tractable Lepidopteran systems. DOI: http://dx.doi.org/10.7554/eLife.02910.001 PMID:24966209

  3. Interactions between the structural domains of the RNA replication proteins of plant-infecting RNA viruses.

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    O'Reilly, E K; Wang, Z; French, R; Kao, C C

    1998-09-01

    Brome mosaic virus (BMV), a positive-strand RNA virus, encodes two replication proteins: the 2a protein, which contains polymerase-like sequences, and the 1a protein, with N-terminal putative capping and C-terminal helicase-like sequences. These two proteins are part of a multisubunit complex which is necessary for viral RNA replication. We have previously shown that the yeast two-hybrid assay consistently duplicated results obtained from in vivo RNA replication assays and biochemical assays of protein-protein interaction, thus permitting the identification of additional interacting domains. We now map an interaction found to take place between two 1a proteins. Using previously characterized 1a mutants, a perfect correlation was found between the in vivo phenotypes of these mutants and their abilities to interact with wild-type 1a (wt1a) and each other. Western blot analysis revealed that the stabilities of many of the noninteracting mutant proteins were similar to that of wt1a. Deletion analysis of 1a revealed that the N-terminal 515 residues of the 1a protein are required and sufficient for 1a-1a interaction. This intermolecular interaction between the putative capping domain and itself was detected in another tripartite RNA virus, cucumber mosaic virus (CMV), suggesting that the 1a-1a interaction is a feature necessary for the replication of tripartite RNA viruses. The boundaries for various activities are placed in the context of the predicted secondary structures of several 1a-like proteins of members of the alphavirus-like superfamily. Additionally, we found a novel interaction between the putative capping and helicase-like portions of the BMV and CMV 1a proteins. Our cumulative data suggest a working model for the assembly of the BMV RNA replicase.

  4. Rewriting nature's assembly manual for a ssRNA virus.

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    Patel, Nikesh; Wroblewski, Emma; Leonov, German; Phillips, Simon E V; Tuma, Roman; Twarock, Reidun; Stockley, Peter G

    2017-11-14

    Satellite tobacco necrosis virus (STNV) is one of the smallest viruses known. Its genome encodes only its coat protein (CP) subunit, relying on the polymerase of its helper virus TNV for replication. The genome has been shown to contain a cryptic set of dispersed assembly signals in the form of stem-loops that each present a minimal CP-binding motif AXXA in the loops. The genomic fragment encompassing nucleotides 1-127 is predicted to contain five such packaging signals (PSs). We have used mutagenesis to determine the critical assembly features in this region. These include the CP-binding motif, the relative placement of PS stem-loops, their number, and their folding propensity. CP binding has an electrostatic contribution, but assembly nucleation is dominated by the recognition of the folded PSs in the RNA fragment. Mutation to remove all AXXA motifs in PSs throughout the genome yields an RNA that is unable to assemble efficiently. In contrast, when a synthetic 127-nt fragment encompassing improved PSs is swapped onto the RNA otherwise lacking CP recognition motifs, assembly is partially restored, although the virus-like particles created are incomplete, implying that PSs outside this region are required for correct assembly. Swapping this improved region into the wild-type STNV1 sequence results in a better assembly substrate than the viral RNA, producing complete capsids and outcompeting the wild-type genome in head-to-head competition. These data confirm details of the PS-mediated assembly mechanism for STNV and identify an efficient approach for production of stable virus-like particles encapsidating nonnative RNAs or other cargoes. Copyright © 2017 the Author(s). Published by PNAS.

  5. Psychometric evaluation of the hepatitis C virus patient-reported outcomes (HCV-PRO) instrument: validity, responsiveness, and identification of the minimally important difference in a phase 2 clinical trial.

    Science.gov (United States)

    Anderson, Roger T; Baran, Robert W; Erickson, Pennifer; Revicki, Dennis A; Dietz, Birgitta; Gooch, Katherine

    2014-04-01

    To describe the psychometric properties and identify the minimally important difference (MID) of the hepatitis C virus patient-reported outcomes (HCV-PRO) instrument. Chronic HCV infection and associated treatments negatively affect PROs of function and well-being. In a phase 2 trial, HCV-infected patients received direct-acting antivirals (DAAs) for 12 weeks with peg-interferon/ribavirin (peg-IFN/RBV) for 48 weeks, or placebo plus peg-IFN/RBV. The HCV-PRO total score, SF-36 PCS and MCS scores, EQ-5D-3L, and EQ VAS were measured at baseline, week 8, end of DAA treatment (EODT), end of peg-IFN/RBV treatment (EOT), and posttreatment week 24 (SVR24). Convergent validity of the HCV-PRO was assessed by Pearson's correlation coefficients. Discriminant validity was assessed by analyzing mean HCV-PRO total scores by EQ-5D anxiety/depression and pain/discomfort domain scores (none vs. some) and presence/absence of depression or fatigue adverse events. MID was identified through effect size (ES) and receiver-operating characteristic (ROC) curve analyses (HCV-PRO response vs. SF-36 PCS/MCS and EQ VAS MID thresholds). In 74 patients (22 % female; 81 % White; 51 % ≥50 years), correlations (0.64-0.96) between HCV-PRO total scores, SF-36 PCS/MCS scores, and EQ VAS scores at all time points supported convergent validity. HCV-PRO total scores were reduced to 10-30 points in patients impaired by depression, pain, or fatigue symptoms. Impact of peg-IFN/RBV regimen on HCV-PRO ES increased over time (EODT -0.76; EOT -0.93). ES and ROC curve analyses indicated an MID of -10 points. The HCV-PRO was valid and responsive in the population studied. An MID of -10 points represented a threshold of clinical significance for the HCV-PRO.

  6. RNA-dependent RNA polymerase 6 of rice (Oryza sativa) plays role in host defense against negative-strand RNA virus, Rice stripe virus.

    Science.gov (United States)

    Jiang, Lin; Qian, Dan; Zheng, Hong; Meng, Lin-Yan; Chen, Jie; Le, Wen-Jing; Zhou, Tong; Zhou, Yi-Jun; Wei, Chun-Hong; Li, Yi

    2012-02-01

    RNA-dependent RNA polymerases (RDRs) from fungi, plants and some invertebrate animals play fundamental roles in antiviral defense. Here, we investigated the role of RDR6 in the defense of economically important rice plants against a negative-strand RNA virus (Rice stripe virus, RSV) that causes enormous crop damage. In three independent transgenic lines (OsRDR6AS line A, B and C) in which OsRDR6 transcription levels were reduced by 70-80% through antisense silencing, the infection and disease symptoms of RSV were shown to be significantly enhanced. The hypersusceptibilities of the OsRDR6AS plants were attributed not to enhanced insect infestation but to enhanced virus infection. The rise in symptoms was associated with the increased accumulation of RSV genomic RNA in the OsRDR6AS plants. The deep sequencing data showed reduced RSV-derived siRNA accumulation in the OsRDR6AS plants compared with the wild type plants. This is the first report of the antiviral role of a RDR in a monocot crop plant in the defense against a negative-strand RNA virus and significantly expands upon the current knowledge of the antiviral roles of RDRs in the defense against different types of viral genomes in numerous groups of plants. Copyright © 2011 Elsevier B.V. All rights reserved.

  7. An overview on hepatitis C virus genotypes and its control

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    Faisal Nouroz

    2015-10-01

    Full Text Available Hepatitis C virus (HCV is a blood borne, circular and positive single stranded virus with high spread rates. With the passage of time the frequency of HCV is increasing in different parts of the world. HCV is a major cause, which may end in liver cirrhosis and hepatocellular carcinoma. HCV has six main genotypes with many subtypes, which have variable sequence homology with each other. Symptoms can appear anytime from 2 weeks to 6 months, which include jaundice, fatigue, gray-colored stool, joint pain, belly pain, weakness, anorexia, itchy skin and dark urine. Genotyping is more significant for planning of HCV treatment period and helps to cure HCV infections. For the quantification and identification of hepatitis C virus-ribonucleic acid, many molecular techniques are performed; the most significant are HCV ELISA, quantitative HCV-RNA PCR and recombinant immunoblot assay. PCR is the major technique targeting 5′ untranslated region (UTR. HCV can be transmitted by contaminated blood, ear and nose piercing and contaminated medical instruments. To overcome the rate of HCV, guidance should be provided to make aware the persons about risk factors, transmission and prevention. Discovery and designing of new therapies and vaccines to overcome this disease are the necessity of the present era. Four types of vaccines such as vector vaccines, peptide vaccines, DNA vaccines and recombinant protein vaccines are available in clinical trials.

  8. Association of IL28B SNPs rs12979860 and rs8099917 on Hepatitis C Virus-RNA Status in Donors/Recipients of Living Donor Liver Transplantation.

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    King-Wah Chiu

    Full Text Available To investigate the effect of IL28B single nucleotide polymorphisms (SNPs (rs8099917 and rs12979860 in the donors and recipients on the outcome of Hepatitis C virus-RNA clearance after living donor liver transplantation (LDLT. The rs8099917 and rs12979860 genotypes in 50 donor and recipients pairs were explored on the pre-operative day (POD and post-operative day 30 (POD30. There was a significant difference in HCV-RNA clearance before (12%, 6/50 and after (48%, 24/50 liver transplantation (P < 0.001. The rs8099917 genotype TT was dominant in both the recipients (82%, 41/50 and donors (86%, 43/50, but had no significant effect on HCV-RNA clearance (87.5%, 21/24 and recurrence (76.9%, 20/26 after LDLT. One recipient was detected with genotype GG on POD, which changed to genotype GT on POD30. Prevalence of rs12979860 genotype CT was 98% (49/50 recipient and 92% (46/50 donor and prevalence of genotype CC was 2% (1/50 recipient and 8% (4/50 donor on POD and POD30, respectively. Of the 4 recipients with rs12979860 genotype CC on POD30, 3 recipients (12.5%, 3/24 exhibited HCV clearance and 1 experienced recurrence (3.9%, 1/26, however, this was not statistically significant. In conclusion, alterations in IL28B SNP genotype may occur after LDLT, leading to modifications in the host genome or donor proteome by HCV. This predicted mechanism will need to be investigated further.

  9. Aeginetia indica Decoction Inhibits Hepatitis C Virus Life Cycle.

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    Lin, Cheng-Wei; Lo, Chieh-Wen; Tsai, Chia-Ni; Pan, Ting-Chun; Chen, Pin-Yin; Yu, Ming-Jiun

    2018-01-09

    Chronic hepatitis C virus (HCV) infection is still a global epidemic despite the introduction of several highly effective direct-acting antivirals that are tagged with sky-high prices. The present study aimed to identify an herbal decoction that ameliorates HCV infection. Among six herbal decoctions tested, the Aeginetia indica decoction had the most profound effect on the HCV reporter activity in infected Huh7.5.1 liver cells in a dose- and time-dependent manner. The Aeginetia indica decoction exerted multiple inhibitory effects on the HCV life cycle. Pretreatment of the cells with the Aeginetia indica decoction prior to HCV infection reduced the HCV RNA and non-structural protein 3 (NS3) protein levels in the infected cells. The Aeginetia indica decoction reduced HCV internal ribosome entry site-mediated protein translation activity. It also reduced the HCV RNA level in the infected cells in association with reduced NS5A phosphorylation at serine 235, a predominant phosphorylation event indispensable to HCV replication. Thus, the Aeginetia indica decoction inhibits HCV infection, translation, and replication. Mechanistically, the Aeginetia indica decoction probably reduced HCV replication via reducing NS5A phosphorylation at serine 235.

  10. Genetic analysis of the function of the plum pox virus CI RNA helicase in virus movement.

    Science.gov (United States)

    Gómez de Cedrón, Marta; Osaba, Lourdes; López, Lissett; García, Juan Antonio

    2006-03-01

    The CI protein forms the cylindrical inclusions typical of potyviral infections and is involved in genome replication and virus movement. In this work, we have analyzed the effect of a series of point mutations at the N-terminal region of the CI protein of Plum pox virus (PPV) on the enzymatic activities and the self-interaction ability of the protein, and on virus replication and movement. DD3,4AA mutation, which had no apparent effects on ATPase and RNA helicase activities in vitro, and on virus replication in protoplasts, drastically impaired cell-to-cell spread of the virus. The effect of KK101,102AA mutation was host-specific. While no signals of virus infection were detected in Chenopodium foetidum inoculated with PPV KK101,102AA, the mutation caused a moderate effect on short distance movement in Nicotiana benthamiana and N. clevelandii, which resulted in a more drastic disturbance of systemic spread. None of the mutations analyzed abolished PPV CI self-interaction in the yeast Two-Hybrid system, but they caused a notable reduction in the binding strength, which appears to positively correlate with their effect on virus movement, suggesting that CI-CI interactions required for RNA replication and virus movement could be rather different.

  11. QSAR studies of the bioactivity of hepatitis C virus (HCV) NS3/4A protease inhibitors by multiple linear regression (MLR) and support vector machine (SVM).

    Science.gov (United States)

    Qin, Zijian; Wang, Maolin; Yan, Aixia

    2017-07-01

    In this study, quantitative structure-activity relationship (QSAR) models using various descriptor sets and training/test set selection methods were explored to predict the bioactivity of hepatitis C virus (HCV) NS3/4A protease inhibitors by using a multiple linear regression (MLR) and a support vector machine (SVM) method. 512 HCV NS3/4A protease inhibitors and their IC 50 values which were determined by the same FRET assay were collected from the reported literature to build a dataset. All the inhibitors were represented with selected nine global and 12 2D property-weighted autocorrelation descriptors calculated from the program CORINA Symphony. The dataset was divided into a training set and a test set by a random and a Kohonen's self-organizing map (SOM) method. The correlation coefficients (r 2 ) of training sets and test sets were 0.75 and 0.72 for the best MLR model, 0.87 and 0.85 for the best SVM model, respectively. In addition, a series of sub-dataset models were also developed. The performances of all the best sub-dataset models were better than those of the whole dataset models. We believe that the combination of the best sub- and whole dataset SVM models can be used as reliable lead designing tools for new NS3/4A protease inhibitors scaffolds in a drug discovery pipeline. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. [Differential display of messenger RNA and identification of selenocysteine lyase gene in hepatocellular carcinoma cells transiently expressing hepatitis C virus core protein].

    Science.gov (United States)

    Yepes, Jesús Orlando; Luz Gunturiz, María; Henao, Luis Felipe; Navas, María Cristina; Balcázar, Norman; Gómez, Luis Alberto

    2006-06-01

    Hepatitis C virus is associated with diverse liver diseases including acute and chronic hepatitis, steatosis, cirrhosis and hepatocellular carcinoma. Several studies have explored viral mechanisms involved in the establishment of persistent infection and oncogenic Hepatitis C virus. Expression assays of Hepatitis C virus core protein suggest that this protein has transforming and carcinogenic properties with multifunctional activities in host cells. Characterization of expressed genes in cells expressing Core protein is important in order to identify candidate genes responsible for these pathogenic alterations. To compare and identify gene expression profiles in the human hepatocarcinoma derived cell line, HepG2, with transient expression of Hepatitis C virus Core protein. We have used comparative PCR-mediated differential display of mRNA from HepG2 hepatocarcinoma with and without transient expression of HCV Core protein or green fluorescent protein, previously obtained using the Semliki Forest Virus-based expression, through transduction of recombinant particles, rSFV-Core and rSFV-GFP, respectively. We observed differences in band intensities of mRNA in HepG2 cells transduced with rSFV-Core compared with those detected in cells without transduction, and transduced with rSFV-GFP. Cloning and sequencing of a gene fragment (258 bp) that was expressed differentially in HepG2 cells transduced with rSFV-Core, was identified as selenocystein lyase. The results confirm that HCV Core protein expressed in HepG2 is associated with specific changes in mRNA expression, including the gene for selenocystein lyase. This gene may be involved in the pathophysiology of hepatocellular carcinoma.

  13. HCV viremia is associated with drug use in young HIV-1 and HCV coinfected pregnant and non-pregnant women*

    Science.gov (United States)

    Nikolopoulou, Georgia B.; Nowicki, Marek J.; Du, Wenbo; Homans, James; Stek, Alice; Kramer, Francoise; Kovacs, Andrea

    2011-01-01

    Aims Vertical transmission of HCV is increased among HIV-1/HCV coinfected women and is related to HCV viral load. In this study we assessed clinical and demographic factors associated with HCV viremia in a cohort of young pregnant and non-pregnant mothers coinfected with HIV-1. Design A cross-sectional clinic-based study nested within a prospective cohort study. Methods From 1988 to 2000, HIV-1 + pregnant and non-pregnant women with children followed in a large maternal, child and adolescent HIV-1 clinic were evaluated for HCV infection using EIA 3.0. HCV RNA levels were determined for HCV antibody + women using polymerase chain reaction. Demographic and clinical characteristics between HCV-RNA(+) and HCV-RNA(−) women and between pregnant and non-pregnant HIV-1/HCV coinfected women were compared using univariate and multivariate analyses. Findings Among 359 HIV-1(+) women, 84 (23%) were HCV-ab + and 49/84 (58%) had detectable HCV-RNA in plasma. Median age was 31. CD4 counts, HIV-1 RNA levels and demographic characteristics were similar for viremic and non-viremic women and pregnant and non-pregnant women. However, viremic women were more likely to report a history of (88% versus 43%; P < 0.001) or active injection drug use (AIDU) (83% versus 29%; P < 0.001). Logistic regression analysis showed that HCV viremia was associated significantly with AIDU (adjusted OR: 15.17; 95% CI: 3.56, 64.56) after adjusting for age, race, number of sexual partners, pregnancy status, CD4 counts and HIV-1 viral load. Conclusion In this cohort of young HIV-1 and HCV coinfected women, HCV viremia was associated strongly with active injection drug use, perhaps due to reinfection or reactivation of HCV. Thus, careful evaluation for HCV infection and counseling related to drug use may be necessary. PMID:15847620

  14. A Sendai virus-derived RNA agonist of RIG-I as a virus vaccine adjuvant.

    Science.gov (United States)

    Martínez-Gil, L; Goff, P H; Hai, R; García-Sastre, A; Shaw, M L; Palese, P

    2013-02-01

    The innate immune system is responsible for recognizing invading pathogens and initiating a protective response. In particular, the retinoic acid-inducible gene 1 protein (RIG-I) participates in the recognition of single- and double-stranded RNA viruses. RIG-I activation leads to the production of an appropriate cytokine and chemokine cocktail that stimulates an antiviral state and drives the adaptive immune system toward an efficient and specific response against the ongoing infection. One of the best-characterized natural RIG-I agonists is the defective interfering (DI) RNA produced by Sendai virus strain Cantell. This 546-nucleotide RNA is a well-known activator of the innate immune system and an extremely potent inducer of type I interferon. We designed an in vitro-transcribed RNA that retains the type I interferon stimulatory properties, and the RIG-I affinity of the Sendai virus produced DI RNA both in vitro and in vivo. This in vitro-synthesized RNA is capable of enhancing the production of anti-influenza virus hemagglutinin (HA)-specific IgG after intramuscular or intranasal coadministration with inactivated H1N1 2009 pandemic vaccine. Furthermore, our adjuvant is equally effective at increasing the efficiency of an influenza A/Puerto Rico/8/34 virus inactivated vaccine as a poly(I·C)- or a squalene-based adjuvant. Our in vitro-transcribed DI RNA represents an excellent tool for the study of RIG-I agonists as vaccine adjuvants and a starting point in the development of such a vaccine.

  15. Modulation of mitogen-activated protein kinase-activated protein kinase 3 by hepatitis C virus core protein

    DEFF Research Database (Denmark)

    Ngo, HT; Pham, Long; Kim, JW

    2013-01-01

    Hepatitis C virus (HCV) is highly dependent on cellular proteins for its own propagation. In order to identify the cellular factors involved in HCV propagation, we performed protein microarray assays using the HCV core protein as a probe. Of ~9,000 host proteins immobilized in a microarray...... inducers. Binding of HCV core to MAPKAPK3 was confirmed by in vitro pulldown assay and further verified by coimmunoprecipitation assay. HCV core protein interacted with MAPKAPK3 through amino acid residues 41 to 75 of core and the N-terminal half of kinase domain of MAPKAPK3. In addition, both RNA...... increased HCV IRES-mediated translation and MAPKAPK3-dependent HCV IRES activity was further increased by core protein. These data suggest that HCV core may modulate MAPKAPK3 to facilitate its own propagation....

  16. A comparative method for finding and folding RNA secondary structures within protein-coding regions

    DEFF Research Database (Denmark)

    Pedersen, Jakob Skou; Meyer, Irmtraud Margret; Forsberg, Roald

    2004-01-01

    Existing computational methods for RNA secondary-structure prediction tacitly assume RNA to only encode functional RNA structures. However, experimental studies have revealed that some RNA sequences, e.g. compact viral genomes, can simultaneously encode functional RNA structures as well as proteins...... that RNA-DECODER's parameters can be automatically trained to successfully fold known secondary structures within the HCV genome. We scan the genomes of HCV and polio virus for conserved secondary-structure elements, and analyze performance as a function of available evolutionary information. On known...... secondary structures, RNA-DECODER shows a sensitivity similar to the programs MFOLD, PFOLD and RNAALIFOLD. When scanning the entire genomes of HCV and polio virus for structure elements, RNA-DECODER's results indicate a markedly higher specificity than MFOLD, PFOLD and RNAALIFOLD....

  17. Hepatitis C virus infection of a Vero cell clone displaying efficient virus-cell binding.

    Science.gov (United States)

    Valli, M B; Carloni, G; Manzin, A; Nasorri, F; Ponzetto, A; Clementi, M

    1997-01-01

    The susceptibility of Vero cells and derivative cell clones to hepatitis C virus (HCV) infection was assayed by qualitative and quantitative polymerase chain reaction (PCR)-based methods. Cell extracts from Vero cells inoculated with HCV were tested for the presence of both positive and negative strands of HCV RNA; in parallel, cell-free HCV genomes were assayed in culture supernatant fluids. Quantitation of genomic HCV RNA molecules in infected cells by competitive reverse transcription PCR (cRT-PCR) indicated that HCV replication was more efficient in a derivative clone (named clone 10) than in parental Vero cells or other clones under study. Analysis of HCV-binding to cell receptors, performed by cRT-PCR quantitation of viral particles adsorbed to the cell surface, demonstrated a 10-fold higher virus-binding level of clone 10 than that of parental Vero cells. The results shown here indicate that the Vero clone 10 may constitute an efficient model system for analysing early events in HCV infection as well as a source of virus for diagnostic and biotechnological applications.

  18. Liver fibrosis progression in hepatitis C virus infection after seroconversion.

    Science.gov (United States)

    Butt, Adeel A; Yan, Peng; Lo Re, Vincent; Rimland, David; Goetz, Matthew B; Leaf, David; Freiberg, Matthew S; Klein, Marina B; Justice, Amy C; Sherman, Kenneth E

    2015-02-01

    Knowing the rate of liver fibrosis progression in hepatitis C virus (HCV)-infected persons can help inform patients and providers (clinicians, medical institutions or organizations, and third-party payers) in making treatment decisions. To determine the rate and factors associated with liver fibrosis progression and hepatic decompensation in persons after acquiring HCV infection. Secondary data analysis of persons in the Electronically Retrieved Cohort of HCV Infected Veterans (ERCHIVES), a national Veterans Affairs (VA) database, between 2002 and 2012. Among 610 514 persons in ERCHIVES (half were HCV positive), we identified those with an initial negative and subsequent positive test result for HCV antibody and positive HCV RNA test result (HCV+). Controls had 2 negative HCV antibody test results (HCV-) in a comparable time frame and were matched 1:1 on age (in 5-year blocks), race, and sex. We excluded persons with human immunodeficiency virus, hepatitis B, less than 24 months of follow-up, hepatocellular carcinoma, and cirrhosis at baseline. Progression of liver fibrosis as estimated by the Fibrosis-4 (FIB-4) index; development of cirrhosis, defined by a FIB-4 score greater than 3.5; and development of hepatic decompensation. The evaluable data set consisted of 1840 persons who were HCV+ and 1840 HCV- controls. The HCV+ persons were younger and had a lower mean (SD) body mass index (27.39 [5.51] vs 29.49 [6.16]; P vs 6.1%). Nine years after diagnosis of cirrhosis, hepatic decompensation events were uncommon but had a higher rate in the HCV+ group (1.79% vs 0.33%). Persons who seroconverted for HCV have a more rapid progression of liver fibrosis and accelerated time to development of cirrhosis after seroconversion compared with HCV- controls. Fibrosis progression occurs early after infection; however, hepatic decompensation is uncommon after diagnosis of cirrhosis.

  19. Apoptosis and clinical severity in patients with psoriasis and HCV infection

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    Sami A Gabr

    2014-01-01

    Full Text Available Background: It has been proposed that hepatitis C virus (HCV antigens are involved in the pathogenesis of psoriasis and may contribute to severity of the disease. Increased expression of the apoptosis-regulating proteins p53 and tTG and decreased levels of bcl-2 in the keratinocytes of the skin of psoriatic patients have been reported. Aim: This study aims to identify the serum levels of apoptosis-regulating proteins in patients with psoriasis and without HCV infection and to study the relation between clinical severity of psoriasis and the presence of HCV infection. Materials and Methods: Disease severity was assessed by psoriasis area severity index score (PASI of 90 patients with psoriasis grouped as mild (n = 30, moderate (n = 30 and severe (n = 30; 20 healthy individuals were used as controls. All groups were subjected for complete history taking, clinical examination, and tests for liver function and HCV infection. The serum levels of apoptosis related proteins: p53, tTG and bcl-2 were estimated by enzyme linked immune sorbent assay (ELISA. Results: There was a statistically significant (P < 0.001 correlation between clinical severity of psoriasis and presence of HCV antibodies and HCV-mRNA. In addition, significantly (P < 0.001 raised serum p53 and tTG, and reduced bcl-2 were observed among HCV-positive patients as compared to HCV-negative patients and control patients. Conclusion: These results conclude that clinical severity of psoriasis is affected by the presence of HCV antibodies and overexpression of apoptotic related proteins. In addition, altered serum levels of apoptosis-regulating proteins could be useful prognostic markers and therapeutic targets of psoriatic disease.

  20. Detecting RNA viruses in living mammalian cells by fluorescence microscopy.

    Science.gov (United States)

    Sivaraman, Divya; Biswas, Payal; Cella, Lakshmi N; Yates, Marylynn V; Chen, Wilfred

    2011-07-01

    Traditional methods that rely on viral isolation and culture techniques continue to be the gold standards used for detection of infectious viral particles. However, new techniques that rely on visualization of live cells can shed light on understanding virus-host interaction for early stage detection and potential drug discovery. Live-cell imaging techniques that incorporate fluorescent probes into viral components provide opportunities for understanding mRNA expression, interaction, and virus movement and localization. Other viral replication events inside a host cell can be exploited for non-invasive detection, such as single-virus tracking, which does not inhibit viral infectivity or cellular function. This review highlights some of the recent advances made using these novel approaches for visualization of viral entry and replication in live cells. Copyright © 2011 Elsevier Ltd. All rights reserved.

  1. [Prevalence change of HGV(GBV-C) infection and its coinfection with HBV a HCV infections in haemodialysis patients].

    Science.gov (United States)

    Klusonová, Hana; Stepánová, Vlasta; Plísková, Lenka; Stilec, Roman

    2003-01-01

    Prevalence of HGV(GBV-C) infection and its coinfection with HBV a HCV infections were studied in group of 82 haemodialysis patients. This study was realized 20 months latter again -- 16 patients from 82 were running in dialysis, 17 patients were transplanted and 49 patients died (non of this viruses was cause of their death). HGV(GBV-C) RNA was detected in serum of 22 patients, 20 months latter it was detected in serum of 3 patients; one positive was new. 20 months latter any HGV(GBV-C) RNA was not detected in serum of 4 originally positive patients. Three of ten HBsAg positive patients were coinfected by HGV(GBV-C) RNA; 20 months latter any coinfection was found. In the first we found HGV(GBV-C) RNA in serum of 5 anti-HCV positive patients and in serum of 1 HCV RNA positive patient; 20 months latter it was in serum of 1 and 1 respectively. Elevation of ALT and AST levels were found in serum of 3 from 82 patients; two patients were coinfected with HBV or HCV. Any from 2 running dialysis patients with elevation of ALT and AST levels was not HGV(GBV-C) RNA positive. This virus is not probably frequent cause of liver disease in dialysis patients and it is not necessary to routinely screen for HGV(GBV-C) infection in this group of patients.

  2. Usefulness of the hepatitis C virus core antigen assay for screening of a population undergoing routine medical checkup.

    Science.gov (United States)

    Gaudy, Catherine; Thevenas, Catherine; Tichet, Jean; Mariotte, Nicole; Goudeau, Alain; Dubois, Frédéric

    2005-04-01

    We studied the usefulness of the recently designed Trak-C assay for the detection and quantification of the hepatitis C virus (HCV) core antigen (Ag) for the screening of HCV infection in 4,201 subjects selected from 74,150 consecutive volunteers undergoing routine medical checkups. Subjects were selected for screening because they had risk factors (group II, n = 321) and/or elevated alanine transaminase activity (group I, n = 3847). Initially, the anti-HCV antibody assay and the Trak-C assay were performed on each patient. Subsequently, the Trak-C assay was performed only when the anti-HCV enzyme immune assay (EIA) was positive. Positive samples were further evaluated for anti-HCV antibodies by a third-generation strip immunoblot assay and for HCV RNA. Four samples (1.2%) from group II and 113 (2.9%) from group I were anti-HCV EIA positive. We also tested 33 subjects who previously tested positive for anti-HCV in our medical center. Among the 150 anti-HCV EIA-positive samples, the HCV core Ag result was in accord with the HCV RNA result in 146 cases (97.3%). When the EIA result was positive, the HCV core Ag concentration and the HCV RNA load were correlated (r(2) = 0.78; P < 0.001). Four samples with low viral loads were Trak-C negative but HCV RNA positive. Among the 2,395 anti-HCV EIA-negative serum samples collected during the first part of the study, 17 (0.7%) were found to contain very low levels of HCV core Ag (<8.5 pg/ml, the cutoff value being 1.5 pg/ml). All these samples were HCV RNA negative and considered to be false positives. This was confirmed by HCV core Ag neutralization analysis. The HCV core Ag assay is a useful method in the screening strategy of HCV infection and provides a reliable means of distinguishing between current and cleared HCV infections that is well correlated with HCV RNA testing.

  3. HCV and HCC molecular epidemiology

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    Flor H. Pujol

    2007-02-01

    Full Text Available

    iHepatitis C virus (HCV is a member of the family Flaviviridae, responsible for the majority of the non-A non-B post-transfusion hepatitis before 1990. Around 170 millions persons in the world are thought to be infected with this virus. A high number of HCV-infected people develop cirrhosis and from these, a significant proportion progresses to hepatocellular carcinoma (HCC. Six HCV genotypes and a large number of subtypes in each genotype have been described. Infections with HCV genotype 1 are associated with the lowest therapeutic success. HCV genotypes 1, 2, and 3 have a worldwide distribution. HCV subtypes 1a and 1b are the most common genotypes in the United States and are also are predominant in Europe, while in Japan, subtype 1b is predominant. Although HCV subtypes 2a and 2b are relatively common in America, Europe, and Japan, subtype 2c is found commonly in northern Italy. HCV genotype 3a is frequent in intravenous drug abusers in Europe and the United States. HCV genotype 4 appears to be prevalent in Africa and the Middle East, and genotypes 5 and 6 seem to be confined to South Africa and Asia, respectively. HCC accounts for approximately 6% of all human cancers. Around 500,000 to 1 million cases occur annually worldwide, with HCC being the fifth common malignancy in men and the ninth in women. HCC is frequently a consequence of infection by HBV and HCV. The first line of evidences comes from epidemiologic studies. While HBV is the most frequent cause of HCC in many countries of Asia and South America, both HBV and HCV are found at similar frequencies, and eventually HCV at a higher frequency than HBV, among HCC patients in Europe, North America, and Japan. The cumulative appearance rate of HCC might be higher for HCV

  4. Simple genomes, complex interactions: Epistasis in RNA virus

    Science.gov (United States)

    Elena, Santiago F.; Solé, Ricard V.; Sardanyés, Josep

    2010-06-01

    Owed to their reduced size and low number of proteins encoded, RNA viruses and other subviral pathogens are often considered as being genetically too simple. However, this structural simplicity also creates the necessity for viral RNA sequences to encode for more than one protein and for proteins to carry out multiple functions, all together resulting in complex patterns of genetic interactions. In this work we will first review the experimental studies revealing that the architecture of viral genomes is dominated by antagonistic interactions among loci. Second, we will also review mathematical models and provide a description of computational tools for the study of RNA virus dynamics and evolution. As an application of these tools, we will finish this review article by analyzing a stochastic bit-string model of in silico virus replication. This model analyzes the interplay between epistasis and the mode of replication on determining the population load of deleterious mutations. The model suggests that, for a given mutation rate, the deleterious mutational load is always larger when epistasis is predominantly antagonistic than when synergism is the rule. However, the magnitude of this effect is larger if replication occurs geometrically than if it proceeds linearly.

  5. Inhibition of RNA recruitment and replication of an RNA virus by acridine derivatives with known anti-prion activities.

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    Zsuzsanna Sasvari

    Full Text Available BACKGROUND: Small molecule inhibitors of RNA virus replication are potent antiviral drugs and useful to dissect selected steps in the replication process. To identify antiviral compounds against Tomato bushy stunt virus (TBSV, a model positive stranded RNA virus, we tested acridine derivatives, such as chlorpromazine (CPZ and quinacrine (QC, which are active against prion-based diseases. METHODOLOGY/PRINCIPAL FINDINGS: Here, we report that CPZ and QC compounds inhibited TBSV RNA accumulation in plants and in protoplasts. In vitro assays revealed that the inhibitory effects of these compounds were manifested at different steps of TBSV replication. QC was shown to have an effect on multiple steps, including: (i inhibition of the selective binding of the p33 replication protein to the viral RNA template, which is required for recruitment of viral RNA for replication; (ii reduction of minus-strand synthesis by the tombusvirus replicase; and (iii inhibition of translation of the uncapped TBSV genomic RNA. In contrast, CPZ was shown to inhibit the in vitro assembly of the TBSV replicase, likely due to binding of CPZ to intracellular membranes, which are important for RNA virus replication. CONCLUSION/SIGNIFICANCE: Since we found that CPZ was also an effective inhibitor of other plant viruses, including Tobacco mosaic virus and Turnip crinkle virus, it seems likely that CPZ has a broad range of antiviral activity. Thus, these inhibitors constitute effective tools to study similarities in replication strategies of various RNA viruses.

  6. Inhibition of RNA Recruitment and Replication of an RNA Virus by Acridine Derivatives with Known Anti-Prion Activities

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    Sasvari, Zsuzsanna; Bach, Stéphane; Blondel, Marc; Nagy, Peter D.

    2009-01-01

    Background Small molecule inhibitors of RNA virus replication are potent antiviral drugs and useful to dissect selected steps in the replication process. To identify antiviral compounds against Tomato bushy stunt virus (TBSV), a model positive stranded RNA virus, we tested acridine derivatives, such as chlorpromazine (CPZ) and quinacrine (QC), which are active against prion-based diseases. Methodology/Principal Findings Here, we report that CPZ and QC compounds inhibited TBSV RNA accumulation in plants and in protoplasts. In vitro assays revealed that the inhibitory effects of these compounds were manifested at different steps of TBSV replication. QC was shown to have an effect on multiple steps, including: (i) inhibition of the selective binding of the p33 replication protein to the viral RNA template, which is required for recruitment of viral RNA for replication; (ii) reduction of minus-strand synthesis by the tombusvirus replicase; and (iii) inhibition of translation of the uncapped TBSV genomic RNA. In contrast, CPZ was shown to inhibit the in vitro assembly of the TBSV replicase, likely due to binding of CPZ to intracellular membranes, which are important for RNA virus replication. Conclusion/Significance Since we found that CPZ was also an effective inhibitor of other plant viruses, including Tobacco mosaic virus and Turnip crinkle virus, it seems likely that CPZ has a broad range of antiviral activity. Thus, these inhibitors constitute effective tools to study similarities in replication strategies of various RNA viruses. PMID:19823675

  7. Detection of high biliary and fecal viral loads in patients with chronic hepatitis C virus infection.

    Science.gov (United States)

    Monrroy, Hugo; Angulo, Jenniffer; Pino, Karla; Labbé, Pilar; Miquel, Juan Francisco; López-Lastra, Marcelo; Soza, Alejandro

    2017-05-01

    The life cycle of the hepatitis C virus (HCV) is closely associated with lipid metabolism. Recently, NPC1L1 (a cholesterol transporter) has been reported to function as an HCV receptor. This receptor is expressed in the hepatocyte canalicular membrane and in the intestine; serving as a key transporter for the cholesterol enterohepatic cycle. We hypothesized that HCV might have a similar cycle, so we aimed to study the presence of HCV in bile and stools of infected patients. Blood, feces, and duodenal bile samples were collected from patients infected with HCV. The biliary viral load was normalized to the bile salt concentration of each sample and the presence of HCV core protein was also evaluated. A total of 12 patients were recruited. HCV RNA was detected in the bile from ten patients. The mean viral load was 2.5log10IU/60mg bile salt. In the stool samples, HCV RNA was detected in ten patients (mean concentration 2.7log10IU/g of feces). HCV RNA is readily detectable and is present at relatively high concentrations in the bile and stool samples of infected patients. This may be relevant as a source of infection in men who have sex with men. Biliary HCV secretion may perhaps play a role in the persistence of viral infection via an enterohepatic cycle of the virus or intrahepatic spread. Copyright © 2017 Elsevier España, S.L.U., AEEH y AEG. All rights reserved.

  8. MicroRNAs, HIV and HCV: a complex relation towards pathology.

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    Piedade, Diogo; Azevedo-Pereira, José Miguel

    2016-05-01

    MicroRNAs are small non-coding RNAs that modulate protein production by post-transcriptional gene regulation. They impose gene expression control by interfering with mRNA translation and stability in cell cytoplasm through a mechanism involving specific binding to mRNA based on base pair complementarity. Because of their intracellular replication cycle it is no surprise that viruses evolved in a way that allows them to use microRNAs to infect, replicate and persist in host cells. Several ways of interference between virus and host-cell microRNA machinery have been described. Most of the time, viruses drastically alter host-cell microRNA expression or synthesize their own microRNA to facilitate infection and pathogenesis. HIV and HCV are two prominent examples of this complex interplay revealing how fine-tuning of microRNA expression is crucial for controlling key host pathways that allow viral infection and replication, immune escape and persistence. In this review we delve into the mechanisms underlying cellular and viral-encoded microRNA functions in the context of HIV and HCV infections. We focus on which microRNAs are differently expressed and deregulated upon viral infection and how these alterations dictate the fate of virus and cell. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  9. Noncoding subgenomic flavivirus RNA is processed by the mosquito RNA interference machinery and determines West Nile virus transmission by Culex pipiens mosquitoes

    NARCIS (Netherlands)

    Göertz, G.P.; Fros, J.J.; Miesen, P.; Vogels, C.B.F.; Bent, van der M.L.; Geertsema, C.; Koenraadt, C.J.M.; Rij, van R.P.; Oers, van M.M.; Pijlman, G.P.

    2016-01-01

    Flaviviruses, such as Zika virus, yellow fever virus, dengue virus, and West Nile virus (WNV), are a serious concern for human health. Flaviviruses produce an abundant noncoding subgenomic flavivirus RNA (sfRNA) in infected cells. sfRNA results from stalling of the host 5=-3= exoribonuclease

  10. Noncoding Subgenomic Flavivirus RNA Is Processed by the Mosquito RNA Interference Machinery and Determines West Nile Virus Transmission by Culex pipiens Mosquitoes

    NARCIS (Netherlands)

    Goertz, G.P.; Fros, J.J.; Miesen, P.; Vogels, C.B.F.; Bent, M.L. van der; Geertsema, C.; Koenraadt, C.J.M.; Rij, R.P. van; Oers, M.M. van; Pijlman, G.P.

    2016-01-01

    Flaviviruses, such as Zika virus, yellow fever virus, dengue virus, and West Nile virus (WNV), are a serious concern for human health. Flaviviruses produce an abundant noncoding subgenomic flavivirus RNA (sfRNA) in infected cells. sfRNA results from stalling of the host 5'-3' exoribonuclease

  11. Variety of genotypes of a HCV virus and outcomes of chronic hepatitis C: results 5 summer supervision in the territory of the Kirov region

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    S. V. Baramzina

    2013-01-01

    Full Text Available The analysis of the epidemiological situation in respect of chronic hepatitis C in the Russian Federation and the Kirov region for the period 1998–2012 yy. The data on the characteristics and frequency of outcomes of chronic hepatitis C, according to the 5-year observation of patients Kirov branch of viral hepatitis infectious diseases hospital. The results of genotyping HCV-virus in 730 patients with chronic hepatitis C in the dynamics from 2006–2010, and in comparison with other regions of Russia. Dominant in the region are the genotypes 1b and 3a, minor – 2 and 1a. During the analyzed period, there was a trend to a decrease in the proportion of genotypes 1b and 1a, and increase the proportion of subtype 3a and 2.

  12. In Silico Design and Experimental Validation of siRNAs Targeting Conserved Regions of Multiple Hepatitis C Virus Genotypes.

    Science.gov (United States)

    ElHefnawi, Mahmoud; Kim, TaeKyu; Kamar, Mona A; Min, Saehong; Hassan, Nafisa M; El-Ahwany, Eman; Kim, Heeyoung; Zada, Suher; Amer, Marwa; Windisch, Marc P

    2016-01-01

    RNA interference (RNAi) is a post-transcriptional gene silencing mechanism that mediates the sequence-specific degradation of targeted RNA and thus provides a tremendous opportunity for development of oligonucleotide-based drugs. Here, we report on the design and validation of small interfering RNAs (siRNAs) targeting highly conserved regions of the hepatitis C virus (HCV) genome. To aim for therapeutic applications by optimizing the RNAi efficacy and reducing potential side effects, we considered different factors such as target RNA variations, thermodynamics and accessibility of the siRNA and target RNA, and off-target effects. This aim was achieved using an in silico design and selection protocol complemented by an automated MysiRNA-Designer pipeline. The protocol included the design and filtration of siRNAs targeting highly conserved and accessible regions within the HCV internal ribosome entry site, and adjacent core sequences of the viral genome with high-ranking efficacy scores. Off-target analysis excluded siRNAs with potential binding to human mRNAs. Under this strict selection process, two siRNAs (HCV353 and HCV258) were selected based on their predicted high specificity and potency. These siRNAs were tested for antiviral efficacy in HCV genotype 1 and 2 replicon cell lines. Both in silico-designed siRNAs efficiently inhibited HCV RNA replication, even at low concentrations and for short exposure times (24h); they also exceeded the antiviral potencies of reference siRNAs targeting HCV. Furthermore, HCV353 and HCV258 siRNAs also inhibited replication of patient-derived HCV genotype 4 isolates in infected Huh-7 cells. Prolonged treatment of HCV replicon cells with HCV353 did not result in the appearance of escape mutant viruses. Taken together, these results reveal the accuracy and strength of our integrated siRNA design and selection protocols. These protocols could be used to design highly potent and specific RNAi-based therapeutic oligonucleotide

  13. Hepatitis C virus infection and increased risk of cerebrovascular disease.

    Science.gov (United States)

    Lee, Mei-Hsuan; Yang, Hwai-I; Wang, Chih-Hao; Jen, Chin-Lan; Yeh, Shiou-Hwei; Liu, Chun-Jen; You, San-Lin; Chen, Wei J; Chen, Chien-Jen

    2010-12-01

    The association between hepatitis C virus (HCV) infection and cerebrovascular disease remains controversial. This study aimed to assess the risk of lethal cerebrovascular diseases associated with chronic HCV infection. In this community-based prospective cohort study, 23 665 residents (aged 30 to 65 years) were enrolled in 1991 to 1992. They were personally interviewed using structured questionnaires and provided blood samples for various serological and biochemical tests at study entry. Serum HCV RNA level and HCV genotype were tested for participants seropositive for antibodies against HCV (anti-HCV). Deaths from cerebrovascular disease during follow-up were ascertained by computerized linkage with National Death Certification profiles from 1991 to 2008 (International Classification of Diseases, 9th Revision 430 to 438). Multivariate-adjusted hazard ratio with 95% CI was estimated for each risk predictor. There were 255 cerebrovascular deaths during 382 011 person-years of follow-up. The cumulative risk of cerebrovascular deaths was 1.0% and 2.7% for seronegatives and seropositives of anti-HCV, respectively (P<0.001). The hazard ratio (95% CI) of cerebrovascular death was 2.18 (1.50 to 3.16) for anti-HCV seropositives after adjustment for several conventional risk factors of cerebrovascular disease. Compared with participants seronegative for anti-HCV as the referent, the multivariate-adjusted hazard ratio (95% CI) was 1.40 (0.62 to 3.16), 2.36 (1.42 to 3.93), and 2.82 (1.25 to 6.37), respectively, for anti-HCV-seropositive participants with undetectable, low, and high serum levels of HCV RNA (P<0.001 for trend). However, no significant association was observed between HCV genotype and cerebrovascular death. Chronic HCV infection is an independent risk predictor of cerebrovascular deaths showing a biological gradient of cerebrovascular mortality with increasing serum HCV RNA level.

  14. Ebola Virus GP Gene Polyadenylation Versus RNA Editing.

    Science.gov (United States)

    Volchkova, Valentina A; Vorac, Jaroslav; Repiquet-Paire, Laurie; Lawrence, Philip; Volchkov, Viktor E

    2015-10-01

    Synthesis of Ebola virus (EBOV) surface glycoprotein (GP) is dependent on transcriptional RNA editing. Northern blot analysis of EBOV-infected cells using GP-gene-specific probes reveals that, in addition to full-length GP messenger RNAs (mRNAs), a shorter RNA is also synthesized, representing >40% of the total amount of GP mRNA. Sequence analysis demonstrates that this RNA is a truncated version of the full-length GP mRNA that is polyadenylated at the editing site and thus lacks a stop codon. An absence of detectable levels of protein synthesis in cellulo is consistent with the existence of tight regulation of the translation of such mRNA. However, nonstop GP mRNA was shown to be only slightly less stable than the same mRNA containing a stop codon, against the general belief in nonstop decay mechanisms aimed at detecting and destroying mRNAs lacking a stop codon. In conclusion, we demonstrate that the editing site indeed serves as a cryptic transcription termination/polyadenylation site, which rarely also functions to edit GP mRNA for expression of surface GP. This new data suggest that the downregulation of surface GP expression is even more dramatic than previously thought, reinforcing the importance of the GP gene editing site for EBOV replication and pathogenicity. © The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  15. Microarray analysis identifies a common set of cellular genes modulated by different HCV replicon clones

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    Gerosolimo Germano

    2008-06-01

    Full Text Available Abstract Background Hepatitis C virus (HCV RNA synthesis and protein expression affect cell homeostasis by modulation of gene expression. The impact of HCV replication on global cell transcription has not been fully evaluated. Thus, we analysed the expression profiles of different clones of human hepatoma-derived Huh-7 cells carrying a self-replicating HCV RNA which express all viral proteins (HCV replicon system. Results First, we compared the expression profile of HCV replicon clone 21-5 with both the Huh-7 parental cells and the 21-5 cured (21-5c cells. In these latter, the HCV RNA has been eliminated by IFN-α treatment. To confirm data, we also analyzed microarray results from both the 21-5 and two other HCV replicon clones, 22-6 and 21-7, compared to the Huh-7 cells. The study was carried out by using the Applied Biosystems (AB Human Genome Survey Microarray v1.0 which provides 31,700 probes that correspond to 27,868 human genes. Microarray analysis revealed a specific transcriptional program induced by HCV in replicon cells respect to both IFN-α-cured and Huh-7 cells. From the original datasets of differentially expressed genes, we selected by Venn diagrams a final list of 38 genes modulated by HCV in all clones. Most of the 38 genes have never been described before and showed high fold-change associated with significant p-value, strongly supporting data reliability. Classification of the 38 genes by Panther System identified functional categories that were significantly enriched in this gene set, such as histones and ribosomal proteins as well as extracellular matrix and intracellular protein traffic. The dataset also included new genes involved in lipid metabolism, extracellular matrix and cytoskeletal network, which may be critical for HCV replication and pathogenesis. Conclusion Our data provide a comprehensive analysis of alterations in gene expression induced by HCV replication and reveal modulation of new genes potentially useful

  16. Multicenter evaluation of the new Abbott RealTime assays for quantitative detection of human immunodeficiency virus type 1 and hepatitis C virus RNA

    NARCIS (Netherlands)

    Schutten, Martin; Peters, D; Back, N K T; Beld, M; Beuselinck, K; Foulongne, V; Geretti, A-M; Pandiani, L; Tiemann, C; Niesters, H G M

    The analytical performances of the new Abbott RealTime hepatitis C virus (HCV) and human immunodeficiency virus type 1 viral load assays were compared at nine laboratories with different competitor assays. These included the Abbott LcX, Bayer Versant bDNA, Roche COBAS Amplicor, and Roche COBAS

  17. Genotyping and infection rate of GBV-C among Iranian HCV- infected patients.

    Science.gov (United States)

    Ghanbari, Reza; Ravanshad, Mehrdad; Hosseini, Seyed Younes; Yaghobi, Ramin; Shahzamani, Kiana

    2010-01-01

    Hepatitis G virus/GB virus-C (HGV/GBV-C) is a newly identified member of the Flaviviridae family. Its clinical significance in chronic hepatitis C infection remains controversial. There is a geographical difference in the distribution of GBV-C in the world. The frequency of GBV-C infection among hepatitis C virus (HCV) infected patients varies. The aim of the current study was to determine the prevalence and genotypes of GBV-C among Iranian patients infected with chronic HCV. Infection with GBV-C was surveyed in 71 chronic confirmed hepatitis C infected patients. These samples were collected at the Digestive Disease Research Center (DDRC) of Shariati Hospital, Tehran, Iran from January to October 2007. The 5'-UTR region of GBV-C RNA was detected using a novel in-house touchdown nested reverse transcription polymerase chain reaction (RT-PCR), the products were sequenced and the results were aligned and phylogenically analyzed. Of the 71 HCV-infected patients, 31 (43.6%) were found positive for GBV-C RNA. Sequencing and phylogenic analysis showed that the samples were Genotype 2 of GBV-C. It seems that there is a high rate of GBV-C infection among Iranian patients infected with chronic HCV. In comparison with the six reference genotypes, it was observed that all the samples were categorized in Genotype 2 of GBV-C, prevalent in North America, Africa and in European countries.

  18. The internal initiation of translation in bovine viral diarrhea virus RNA depends on the presence of an RNA pseudoknot upstream of the initiation codon

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    Moes Lorin

    2007-11-01

    Full Text Available Abstract Background Bovine viral diarrhea virus (BVDV is the prototype representative of the pestivirus genus in the Flaviviridae family. It has been shown that the initiation of translation of BVDV RNA occurs by an internal ribosome entry mechanism mediated by the 5' untranslated region of the viral RNA 1. The 5' and 3' boundaries of the IRES of the cytopathic BVDV NADL have been mapped and it has been suggested that the IRES extends into the coding of the BVDV polyprotein 2. A putative pseudoknot structure has been recognized in the BVDV 5'UTR in close proximity to the AUG start codon. A pseudoknot structure is characteristic for flavivirus IRESes and in the case of the closely related classical swine fever virus (CSFV and the more distantly related Hepatitis C virus (HCV pseudoknot function in translation has been demonstrated. Results To characterize the BVDV IRESes in detail, we studied the BVDV translational initiation by transfection of dicistronic expression plasmids into mammalian cells. A region coding for the amino terminus of the BVDV SD-1 polyprotein contributes considerably to efficient initiation of translation. The translation efficiency mediated by the IRES of BVDV strains NADL and SD-1 approximates the poliovirus type I IRES directed translation in BHK cells. Compared to the poliovirus IRES increased expression levels are mediated by the BVDV IRES of strain SD-1 in murine cell lines, while lower levels are observed in human cell lines. Site directed mutagenesis revealed that a RNA pseudoknot upstream of the initiator AUG is an important structural element for IRES function. Mutants with impaired ability to base pair in stem I or II lost their translational activity. In mutants with repaired base pairing either in stem 1 or in stem 2 full translational activity was restored. Thus, the BVDV IRES translation is dependent on the pseudoknot integrity. These features of the pestivirus IRES are reminiscent of those of the classical

  19. Global organization of a positive-strand RNA virus genome.

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    Baodong Wu

    Full Text Available The genomes of plus-strand RNA viruses contain many regulatory sequences and structures that direct different viral processes. The traditional view of these RNA elements are as local structures present in non-coding regions. However, this view is changing due to the discovery of regulatory elements in coding regions and functional long-range intra-genomic base pairing interactions. The ∼4.8 kb long RNA genome of the tombusvirus tomato bushy stunt virus (TBSV contains these types of structural features, including six different functional long-distance interactions. We hypothesized that to achieve these multiple interactions this viral genome must utilize a large-scale organizational strategy and, accordingly, we sought to assess the global conformation of the entire TBSV genome. Atomic force micrographs of the genome indicated a mostly condensed structure composed of interconnected protrusions extending from a central hub. This configuration was consistent with the genomic secondary structure model generated using high-throughput selective 2'-hydroxyl acylation analysed by primer extension (i.e. SHAPE, which predicted different sized RNA domains originating from a central region. Known RNA elements were identified in both domain and inter-domain regions, and novel structural features were predicted and functionally confirmed. Interestingly, only two of the six long-range interactions known to form were present in the structural model. However, for those interactions that did not form, complementary partner sequences were positioned relatively close to each other in the structure, suggesting that the secondary structure level of viral genome structure could provide a basic scaffold for the formation of different long-range interactions. The higher-order structural model for the TBSV RNA genome provides a snapshot of the complex framework that allows multiple functional components to operate in concert within a confined context.

  20. Use of Cellular Decapping Activators by Positive-Strand RNA Viruses

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    Jennifer Jungfleisch

    2016-12-01

    Full Text Available Positive-strand RNA viruses have evolved multiple strategies to not only circumvent the hostile decay machinery but to trick it into being a priceless collaborator supporting viral RNA translation and replication. In this review, we describe the versatile interaction of positive-strand RNA viruses and the 5′-3′ mRNA decay machinery with a focus on the viral subversion of decapping activators. This highly conserved viral trickery is exemplified with the plant Brome mosaic virus, the animal Flock house virus and the human hepatitis C virus.

  1. Influence of GB virus C on IFN-γ and IL-2 production and CD38 expression in T lymphocytes from chronically HIV-infected and HIV-HCV-co-infected patients

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    Giovana Lotici Baggio-Zappia

    2011-09-01

    Full Text Available This study was designed to assess the effect of GB virus (GBV-C on the immune response to human immunodeficiency virus (HIV in chronically HIV-infected and HIV- hepatitis C virus (HCV-co-infected patients undergoing antiretroviral therapy. A cohort of 159 HIV-seropositive patients, of whom 52 were HCV-co-infected, was included. Epidemiological data were collected and virological and immunological markers, including the production of interferon gamma (IFN-γ and interleukin (IL-2 by CD4, CD8 and Tγδ cells and the expression of the activation marker, CD38, were assessed. A total of 65 patients (40.8% presented markers of GBV-C infection. The presence of GBV-C did not influence HIV and HCV replication or TCD4 and TCD8 cell counts. Immune responses, defined by IFN-γ and IL-2 production and CD38 expression did not differ among the groups. Our results suggest that neither GBV-C viremia nor the presence of E2 antibodies influence HIV and HCV viral replication or CD4 T cell counts in chronically infected patients. Furthermore, GBV-C did not influence cytokine production or CD38-driven immune activation among these patients. Although our results do not exclude a protective effect of GBV-C in early HIV disease, they demonstrate that this effect may not be present in chronically infected patients, who represent the majority of patients in outpatient clinics.

  2. Convergent evolution of argonaute-2 slicer antagonism in two distinct insect RNA viruses.

    NARCIS (Netherlands)

    Mierlo, J.T. van; Bronkhorst, A.W.; Overheul, G.J.; Sadanandan, S.A.; Ekstrom, J.O.; Heestermans, M.; Hultmark, D.; Antoniewski, C.; Rij, R.P. van

    2012-01-01

    RNA interference (RNAi) is a major antiviral pathway that shapes evolution of RNA viruses. We show here that Nora virus, a natural Drosophila pathogen, is both a target and suppressor of RNAi. We detected viral small RNAs with a signature of Dicer-2 dependent small interfering RNAs in Nora virus

  3. Virus-derived transgenes expressing hairpin RNA give immunity to Tobacco mosaic virus and Cucumber mosaic virus

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    Liu Yong

    2011-01-01

    Full Text Available Abstract Background An effective method for obtaining resistant transgenic plants is to induce RNA silencing by expressing virus-derived dsRNA in plants and this method has been successfully implemented for the generation of different plant lines resistant to many plant viruses. Results Inverted repeats of the partial Tobacco mosaic virus (TMV movement protein (MP gene and the partial Cucumber mosaic virus (CMV replication protein (Rep gene were introduced into the plant expression vector and the recombinant plasmids were transformed into Agrobacterium tumefaciens. Agrobacterium-mediated transformation was carried out and three transgenic tobacco lines (MP16-17-3, MP16-17-29 and MP16-17-58 immune to TMV infection and three transgenic tobacco lines (Rep15-1-1, Rep15-1-7 and Rep15-1-32 immune to CMV infection were obtained. Virus inoculation assays showed that the resistance of these transgenic plants could inherit and keep stable in T4 progeny. The low temperature (15℃ did not influence the resistance of transgenic plants. There was no significant correlation between the resistance and the copy number of the transgene. CMV infection could not break the resistance to TMV in the transgenic tobacco plants expressing TMV hairpin MP RNA. Conclusions We have demonstrated that transgenic tobacco plants expressed partial TMV movement gene and partial CMV replicase gene in the form of an intermolecular intron-hairpin RNA exhibited complete resistance to TMV or CMV infection.

  4. Expression of interferon receptor genes (IFNAR1 and IFNAR2 mRNA) in the liver may predict outcome after interferon therapy in patients with chronic genotype 2a or 2b hepatitis C virus infection.

    Science.gov (United States)

    Morita, K; Tanaka, K; Saito, S; Kitamura, T; Kondo, M; Sakaguchi, T; Morimoto, M; Sekihara, H

    1998-03-01

    Hepatitis C virus (HCV) genotype 2a or 2b is associated with a favorable outcome after interferon therapy. However, 19% to 33% of patients do not respond to therapy. We investigated whether interferon receptor gene (IFNAR1 and IFNAR2 mRNA) expression in the liver before interferon therapy predicts long-term response to therapy in patients with genotype 2a or 2b HCV infection. Twenty-seven patients who subsequently received interferon-alpha therapy underwent liver biopsies before interferon therapy. Hepatic IFNAR1 and IFNAR2 mRNA were determined using a reverse transcription-polymerase chain reaction assay. Twenty (74%) patients responded to interferon therapy, while the remaining seven (26%) patients were nonresponders. The expression rates of IFNAR1 and IFNAR2 mRNA in the liver were significantly higher in responders than nonresponders (p IFNAR1 or IFNAR2 mRNA predicted complete response to interferon treatment, with a positive predictive value of 100%. Multiple logistic regression analysis showed that IFNAR1 and/or IFNAR2 mRNA expression was the only significant predictor of the effectiveness of IFN therapy (p = 0.0002). We conclude that expression of interferon receptor genes in the liver is a useful index for predicting the long-term efficacy of interferon therapy in patients with chronic genotype 2a or 2b HCV infection.

  5. Hepatitis C virus coinfection does not influence the CD4 cell recovery in HIV-1-infected patients with maximum virologic suppression

    DEFF Research Database (Denmark)

    Peters, Lars; Mocroft, Amanda; Soriano, Vincent

    2009-01-01

    BACKGROUND: Conflicting data exist whether hepatitis C virus (HCV) affects the CD4 cell recovery in patients with HIV starting antiretroviral treatment. OBJECTIVE: To investigate the influence of HCV coinfection on the CD4 recovery in patients with maximum virologic suppression within the Euro...... for HCV treatment and HCV-RNA VL did not change the findings. CONCLUSIONS: HCV serostatus did not influence the CD4 recovery in patients with HIV with maximum virologic suppression after starting combination antiretroviral therapy. Furthermore, no difference in CD4 gain was found when comparing distinct...

  6. Detection of hepatitis C viral RNA sequences in fresh and paraffin-embedded liver biopsy specimens of non-A, non-B hepatitis patients

    NARCIS (Netherlands)

    Bresters, D.; Cuypers, H. T.; Reesink, H. W.; Chamuleau, R. A.; Schipper, M. E.; Boeser-Nunnink, B. D.; Lelie, P. N.; Jansen, P. L.

    1992-01-01

    In this study methods of HCV-RNA detection in fresh frozen and formalin-fixed, paraffin-embedded liver biopsies are described. Of 22 untreated chronic non-A, non-B hepatitis patients and 6 control patients, a plasma sample and part of a liver biopsy were freshly frozen for hepatitis C virus (HCV)

  7. The efficiency of RNA interference for conferring stable resistance to Plum Pox Virus

    Science.gov (United States)

    Plum transformed with an intron hairpin RNA CP (ihRNA-CP) were resistant to PPV infection through the specific process of RNA silencing involving both small interfering -RNA interfering (siRNA) and a methylated virus transgene. This recognition process specifically targeted the triggered PPV genome...

  8. PEG-Interferon-α ribavirin-induced HCV viral clearance: a pharmacogenetic multicenter Spanish study

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    Javier Milara

    2015-01-01

    Full Text Available Objective: Dual PEGylated interferon-(PEG-IFN and ribavirin therapy has been the main hepatitis C virus (HCV treatment of the last decade. Current direct-acting antiviral agents have improved the outcome of therapy but also have increased the cost and management complexity of treatment. The current study analyzes host genetics, viral and clinical predictors of sustained viral response (SVR to dual PEG-IFN and ribavirin therapy in a representative Spanish population. Methods: Observational prospective multicentre pharmacogenetic cohort study conducted in 12 different hospitals of 12 different Spanish regions. A total of 98 patients with SVR and 106 with non-SVR in response to PEG-IFN and ribavirin therapy were included. 33 single nucleotide polymorphisms located in 24 different genes related with inflammatory, immune and virus response were selected. Clinical and viral data were also analyzed as candidate of SVR predictors. Results: IL-28B (rs12979860, rs7248668, rs8105790, rs8099917 and TNFRSF1B (rs1061622 genotypes, as well as TNFRSF1B/IL-10/TNF(-308 non-TTG and TNFRSF1B/IL- 10/IL-4 non-TTC haplotypes together with lower age, lower basal HCV RNA load, higher basal serum LDL cholesterol values, VHC genotypes 2 and 3 and basal low grade fibrosis 0-2 were associated with a SVR in the univariate analysis. Independent predictors of SVR in the multivariate analysis were IL-28B rs12979860 CC, TNFRSF1B/IL-10/IL-4 non-TTC along with low baseline HCV RNA load and HCV genotypes 2 and 3. Conclusions: IL-28B rs12979860 CC, TNFRSF1B/ IL-10/ IL-4 non-TTC haplotype, low baseline HCV RNA load and HCV genotypes 2 and 3 may help to predict successful outcome to PEG-IFN/ribavirin therapy in Spanish population

  9. Hepatitis C virus: virology and life cycle

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    Chang Wook Kim

    2013-03-01

    Full Text Available Hepatitis C virus (HCV is a positive sense, single-stranded RNA virus in the Flaviviridae family. It causes acute hepatitis with a high propensity for chronic infection. Chronic HCV infection can progress to severe liver disease including cirrhosis and hepatocellular carcinoma. In the last decade, our basic understanding of HCV virology and life cycle has advanced greatly with the development of HCV cell culture and replication systems. Our ability to treat HCV infection has also been improved with the combined use of interferon, ribavirin and small molecule inhibitors of the virally encoded NS3/4A protease, although better therapeutic options are needed with greater antiviral efficacy and less toxicity. In this article, we review various aspects of HCV life cycle including viral attachment, entry, fusion, viral RNA translation, posttranslational processing, HCV replication, viral assembly and release. Each of these steps provides potential targets for novel antiviral therapeutics to cure HCV infection and prevent the adverse consequences of progressive liver disease.

  10. Elimination of HCV via a non-ISG-mediated mechanism by vaniprevir and BMS-788329 combination therapy in human hepatocyte chimeric mice.

    Science.gov (United States)

    Uchida, Takuro; Hiraga, Nobuhiko; Imamura, Michio; Yoshimi, Satoshi; Kan, Hiromi; Miyaki, Eisuke; Tsuge, Masataka; Abe, Hiromi; Hayes, C Nelson; Aikata, Hiroshi; Ishida, Yuji; Tateno, Chise; Ellis, Joan D; Chayama, Kazuaki

    2016-02-02

    We previously reported that interferon (IFN)-free direct-acting antiviral combination treatment succeeded in eradicating genotype 1b hepatitis C virus (HCV) in human hepatocyte chimeric mice. In this study, we examined the effect of vaniprevir (MK7009, NS3/4A protease inhibitor) and BMS-788329 (NS5A inhibitor) combination treatment on HCV genotype 1b and the expression of IFN-stimulated genes (ISGs) using a subgenomic replicon system and the same animal model. Combination treatment with vaniprevir and BMS-788329 significantly reduced HCV replication compared to vaniprevir monotherapy in HCV replicon cells (Huh7/Rep-Feo cells). HCV genotype 1b-infected human hepatocyte chimeric mice were treated with vaniprevir alone or in combination with BMS-788329 for four weeks. Vaniprevir monotherapy reduced serum HCV RNA titers in mice, but viral breakthrough was observed in mice with high HCV titers. Ultra-deep sequence analysis revealed a predominant replacement by drug-resistant substitutions at 168 in HCV NS3 region in these mice. Conversely, in mice with low HCV titers, HCV was eradicated by vaniprevir monotherapy without viral breakthrough. In contrast to monotherapy, combination treatment with vaniprevir and BMS-788329 succeeded in completely eradicating HCV regardless of serum viral titer. IFN-alpha treatment significantly increased ISG expression; however, vaniprevir and BMS-788329 combination treatment caused no increase in ISG expression both in cultured cells and in mouse livers. Therefore, combination treatment with vaniprevir and BMS-788329 eliminated HCV via a non-ISG-mediated mechanism. This oral treatment might offer an alternative DAA combination therapy for patients with chronic hepatitis C. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. Genetic Polymorphism of Epidermal Growth Factor rs4444903 Influences Susceptibility to HCV-Related Liver Cirrhosis and Hepatocellular Carcinoma in a Chinese Han Population.

    Science.gov (United States)

    Zhang, Shitian; Qiao, Kunyan; Trieu, Congdoanh; Huo, Zhixiao; Dai, Qinghai; Du, Yanan; Lu, Wei; Hou, Wei

    2017-04-01

    Genetic polymorphism in the epidermal growth factor (EGF, rs4444903) gene has been demonstrated to be associated with the clinical deterioration in hepatitis C virus (HCV)-related liver cirrhosis (LC) and the development of hepatocellular carcinoma (HCC). Whether this single nucleotide polymorphism (SNP) influences susceptibility to HCV-related LC and HCC in the Chinese Han population is largely unknown. In this case-control study, a total of 187 Chinese Han patients with chronic HCV infection were enrolled, including 62 HCV-related LC patients, 46 HCV-related HCC patients, and 79 chronic hepatitis C (CHC) patients without LC and HCC, and the genetic polymorphism was genotyped via a matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) assay. The logistical regression analysis was employed to determine the correlation between the genetic polymorphism and risk of HCV-related LC and HCC. The distribution of EGF rs4444903 genotypes and alleles significantly differed between LC patients and CHC subjects (p = 0.045, p = 0.043, respectively). Under the recessive model, the GG genotype was significantly associated with a two-fold risk of HCV-related LC compared to the AA+AG genotype after an adjustment for age, gender, body mass index (BMI), duration of HCV infection, and HCV RNA level (OR = 2.188; 95% CI = 1.072 - 4.465; p = 0.031). Significant association was observed as well between the GG genotype and increased HCV-related HCC risk (OR = 3.104; 95% CI = 1.319 - 7.307; p = 0.010). The EGF rs4444903 GG genotype is associated with higher susceptibility to HCV-related LC and HCC in the Chinese Han population. Screening of host genetic polymorphisms might be helpful in designing effective and efficient LC and HCC surveillance programs for chronic HCV-infected patients.

  12. Discovery of Chromane Containing Hepatitis C Virus (HCV) NS5A Inhibitors with Improved Potency against Resistance-Associated Variants.

    Science.gov (United States)

    Yu, Wensheng; Tong, Ling; Hu, Bin; Zhong, Bin; Hao, Jinglai; Ji, Tao; Zan, Shuai; Coburn, Craig A; Selyutin, Oleg; Chen, Lei; Rokosz, Laura; Agrawal, Sony; Liu, Rong; Curry, Stephanie; McMonagle, Patricia; Ingravallo, Paul; Asante-Appiah, Ernest; Chen, Shiying; Kozlowski, Joseph A

    2016-11-23

    The discovery of potent and pan-genotypic HCV NS5A inhibitors faces many challenges including the significant diversity among genotypes, substantial potency shift conferred on some key resistance-associated variants, inconsistent SARs between different genotypes and mutants, and the lacking of models of inhibitor/protein complexes for rational inhibitor design. As part of ongoing efforts on HCV NS5A inhibition at Merck, we now describe the discovery of a novel series of chromane containing NS5A inhibitors. SAR studies around the "Z" group of the tetracyclic indole scaffold explored fused bicyclic rings as alternates to the phenyl group of elbasvir (1, MK-8742) and identified novel chromane and 2,3-dihydrobenzofuran derivatives as "Z" group replacements offered good potency across all genotypes. This effort, incorporating the C-1 fluoro substitution at the tetracyclic indole core, led to the discovery of a new series of NS5A inhibitors, such as compounds 14 and 25-28, with significantly improved potency against resistance-associated variants, such as GT2b, GT1a Y93H, and GT1a L31V. Compound 14 also showed reasonable PK exposures in preclinical species (rat and dog).

  13. Promotion of Hendra virus replication by microRNA 146a.

    Science.gov (United States)

    Stewart, Cameron R; Marsh, Glenn A; Jenkins, Kristie A; Gantier, Michael P; Tizard, Mark L; Middleton, Deborah; Lowenthal, John W; Haining, Jessica; Izzard, Leonard; Gough, Tamara J; Deffrasnes, Celine; Stambas, John; Robinson, Rachel; Heine, Hans G; Pallister, Jackie A; Foord, Adam J; Bean, Andrew G; Wang, Lin-Fa

    2013-04-01

    Hendra virus is a highly pathogenic zoonotic paramyxovirus in the genus Henipavirus. Thirty-nine outbreaks of Hendra virus have been reported since its initial identification in Queensland, Australia, resulting in seven human infections and four fatalities. Little is known about cellular host factors impacting Hendra virus replication. In this work, we demonstrate that Hendra virus makes use of a microRNA (miRNA) designated miR-146a, an NF-κB-responsive miRNA upregulated by several innate immune ligands, to favor its replication. miR-146a is elevated in the blood of ferrets and horses infected with Hendra virus and is upregulated by Hendra virus in human cells in vitro. Blocking miR-146a reduces Hendra virus replication in vitro, suggesting a role for this miRNA in Hendra virus replication. In silico analysis of miR-146a targets identified ring finger protein (RNF)11, a member of the A20 ubiquitin editing complex that negatively regulates NF-κB activity, as a novel component of Hendra virus replication. RNA interference-mediated silencing of RNF11 promotes Hendra virus replication in vitro, suggesting that increased NF-κB activity aids Hendra virus replication. Furthermore, overexpression of the IκB superrepressor inhibits Hendra virus replication. These studies are the first to demonstrate a host miRNA response to Hendra virus infection and suggest an important role for host miRNAs in Hendra virus disease.

  14. Promotion of Hendra Virus Replication by MicroRNA 146a

    Science.gov (United States)

    Marsh, Glenn A.; Jenkins, Kristie A.; Gantier, Michael P.; Tizard, Mark L.; Middleton, Deborah; Lowenthal, John W.; Haining, Jessica; Izzard, Leonard; Gough, Tamara J.; Deffrasnes, Celine; Stambas, John; Robinson, Rachel; Heine, Hans G.; Pallister, Jackie A.; Foord, Adam J.; Bean, Andrew G.; Wang, Lin-Fa

    2013-01-01

    Hendra virus is a highly pathogenic zoonotic paramyxovirus in the genus Henipavirus. Thirty-nine outbreaks of Hendra virus have been reported since its initial identification in Queensland, Australia, resulting in seven human infections and four fatalities. Little is known about cellular host factors impacting Hendra virus replication. In this work, we demonstrate that Hendra virus makes use of a microRNA (miRNA) designated miR-146a, an NF-κB-responsive miRNA upregulated by several innate immune ligands, to favor its replication. miR-146a is elevated in the blood of ferrets and horses infected with Hendra virus and is upregulated by Hendra virus in human cells in vitro. Blocking miR-146a reduces Hendra virus replication in vitro, suggesting a role for this miRNA in Hendra virus replication. In silico analysis of miR-146a targets identified ring finger protein (RNF)11, a member of the A20 ubiquitin editing complex that negatively regulates NF-κB activity, as a novel component of Hendra virus replication. RNA interference-mediated silencing of RNF11 promotes Hendra virus replication in vitro, suggesting that increased NF-κB activity aids Hendra virus replication. Furthermore, overexpression of the IκB superrepressor inhibits Hendra virus replication. These studies are the first to demonstrate a host miRNA response to Hendra virus infection and suggest an important role for host miRNAs in Hendra virus disease. PMID:23345523

  15. Direct anti-HCV agents

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    Xingquan Zhang

    2016-01-01

    Full Text Available Unlike human immunodeficiency virus (HIV and hepatitis B virus (HBV, hepatitis C virus (HCV infection is a curable disease. Current direct antiviral agent (DAA targets are focused on HCV NS3/4A protein (protease, NS5B protein (polymerase and NS5A protein. The first generation of DAAs includes boceprevir and telaprevir, which are protease inhibitors and were approved for clinical use in 2011. The cure rate for genotype 1 patients increased from 45% to 70% when boceprevir or telaprevir was added to standard PEG-IFN/ribavirin. More effective and less toxic second generation DAAs supplanted these drugs by 2013. The second generation of DAAs includes sofosbuvir (Sovaldi, simeprevir (Olysio, and fixed combination medicines Harvoni and Viekira Pak. These drugs increase cure rates to over 90% without the need for interferon and effectively treat all HCV genotypes. With these drugs the “cure HCV” goal has become a reality. Concerns remain about drug resistance mutations and the high cost of these drugs. The investigation of new HCV drugs is progressing rapidly; fixed dose combination medicines in phase III clinical trials include Viekirax, asunaprevir+daclatasvir+beclabuvir, grazoprevir+elbasvir and others.

  16. Nucleoproteins of Negative Strand RNA Viruses; RNA Binding, Oligomerisation and Binding to Polymerase Co-Factor

    Directory of Open Access Journals (Sweden)

    Thibaut Crépin

    2010-01-01

    Full Text Available Commentary on Tawar, R.G.; Duquerroy, S.; Vonrhein, C.; Varela, P.F.; Damier-Piolle, L.; Castagné, N.; MacLellan, K.; Bedouelle, H.; Bricogne, G.; Bhella, D.; Eléouët, J.-F.; Rey, F.A. Crystal structure of a nucleocapsid-like nucleoprotein-RNA complex of respiratory syncytial virus. Science 2009, 326, 1279-1283.

  17. Sexual transmission of hepatitis C virus

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    Fátima Mitiko TENGAN

    2001-06-01

    Full Text Available In order to contribute to a better understanding of the forms of acquisition of hepatitis C virus (HCV in Brazil, with special emphasis on sexual transmission, we determined the presence of HCV infection in regular partners and in non-sexual home communicants of blood donors seen at Fundação Pró-Sangue Hemocentro de São Paulo from January 1992 to July 1996. Of 154 blood donors with HCV infection (index cases, 111 had had regular partners for at least 6 months. Sixty-eight of 111 partners were evaluated for HCV infection. Of these, 8 (11.76% were considered to have current or previous HCV infection; a history of sexually transmissible diseases and index cases with a positive HCV-RNA test were more prevalent among partners with HCV infection. Of the 68 index cases whose partners were studied, 56 had non-sexual home communicants. Of the total of 81 home communicants, 66 accepted to be evaluated for HCV infection. None of them was HCV-positive, suggesting that the high prevalence of HCV infection among partners may be attributed at least partially to sexual transmission.

  18. Virological Mechanisms in the Coinfection between HIV and HCV

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    Maria Carla Liberto

    2015-01-01

    Full Text Available Due to shared transmission routes, coinfection with Hepatitis C Virus (HCV is common in patients infected by Human Immunodeficiency Virus (HIV. The immune-pathogenesis of liver disease in HIV/HCV coinfected patients is a multifactorial process. Several studies demonstrated that HIV worsens the course of HCV infection, increasing the risk of cirrhosis and hepatocellular carcinoma. Also, HCV might increase immunological defects due to HIV and risk of comorbidities. A specific cross-talk among HIV and HCV proteins in coinfected patients modulates the natural history, the immune responses, and the life cycle of both viruses. These effects are mediated by immune mechanisms and by a cross-talk between the two viruses which could interfere with host defense mechanisms. In this review, we focus on some virological/immunological mechanisms of the pathogenetic interactions between HIV and HCV in the human host.

  19. Humoral Immune Response in Japanese Acute Hepatitis Patients with Hepatitis C Virus Infection

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    N Yamaguchi

    2000-01-01

    Full Text Available The humoral immune response to acute infection by hepatitis C virus (HCV is not yet perfectly clear in terms of immunoglobulin (Ig response, diversity of HCV antigen, and the relation with hepatitis severity and antibody response.  Serum IgM and IgG anti-HCV levels in patients with HCV and either acute hepatitis (AH or fulminant hepatitis (FH were investigated; the diversity of HCV antigen was investigated by RIBA test III.  Of 22 AH patients, 12 (54.5% were positive for IgM anti-HCV, mainly reacting to HCV core protein. The mean interval until the appearance of IgM anti-HCV after onset was 24.1±26.2 days. IgG anti-HCV mainly reacted to both core and NS-3 antigen, appearing 42.6±42.1 days after onset.  From a serial study of 15 AH patients, it was considered that in seven AH patients (46.7%, the IgM response would precede the IgG response. In another two AH patients, IgM anti-HCV was not detected during the acute disease phase. Of 48 chronic hepatitis patients with HCV-RNA, 40 patients were positive for IgM anti-HCV.  Therefore, IgM anti-HCV was useful for diagnosis in some of the AH patients, but it was difficult to use for distinguishing between acute and chronic infection. All four FH patients with HCV-RNA were positive for both IgM and IgG antibody to HCV at onset. Their antibody titres were higher than those of AH patients. These results suggested that, as in FH due to HBV, FH due to HCV could induce strong and rapid humoral immunity.

  20. Biochemical characterization of a recombinant Japanese encephalitis virus RNA-dependent RNA polymerase

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    Kim Chan-Mi

    2007-07-01

    Full Text Available Abstract Background Japanese encephalitis virus (JEV NS5 is a viral nonstructural protein that carries both methyltransferase and RNA-dependent RNA polymerase (RdRp domains. It is a key component of the viral RNA replicase complex that presumably includes other viral nonstructural and cellular proteins. The biochemical properties of JEV NS5 have not been characterized due to the lack of a robust in vitro RdRp assay system, and the molecular mechanisms for the initiation of RNA synthesis by JEV NS5 remain to be elucidated. Results To characterize the biochemical properties of JEV RdRp, we expressed in Escherichia coli and purified an enzymatically active full-length recombinant JEV NS5 protein with a hexahistidine tag at the N-terminus. The purified NS5 protein, but not the mutant NS5 protein with an Ala substitution at the first Asp of the RdRp-conserved GDD motif, exhibited template- and primer-dependent RNA synthesis activity using a poly(A RNA template. The NS5 protein was able to use both plus- and minus-strand 3'-untranslated regions of the JEV genome as templates in the absence of a primer, with the latter RNA being a better template. Analysis of the RNA synthesis initiation site using the 3'-end 83 nucleotides of the JEV genome as a minimal RNA template revealed that the NS5 protein specifically initiates RNA synthesis from an internal site, U81, at the two nucleotides upstream of the 3'-end of the template. Conclusion As a first step toward the understanding of the molecular mechanisms for JEV RNA replication and ultimately for the in vitro reconstitution of viral RNA replicase complex, we for the first time established an in vitro JEV RdRp assay system with a functional full-length recombinant JEV NS5 protein and characterized the mechanisms of RNA synthesis from nonviral and viral RNA templates. The full-length recombinant JEV NS5 will be useful for the elucidation of the structure-function relationship of this enzyme and for the

  1. Completion of the Entire Hepatitis C Virus Life Cycle in Vero Cells Derived from Monkey Kidney

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    Asako Murayama

    2016-06-01

    Full Text Available A hepatitis C virus (HCV cell culture system incorporating the JFH-1 strain and the human hepatoma cell line HuH-7 enabled the production of infectious HCV particles. Several host factors were identified as essential for HCV replication. Supplementation of these factors in nonhepatic human cell lines enabled HCV replication and particle production. Vero cells established from monkey kidney are commonly used for the production of vaccines against a variety of viruses. In this study, we aimed to establish a novel Vero cell line to reconstruct the HCV life cycle. Unmodified Vero cells did not allow HCV infection or replication. The expression of microRNA 122 (miR-122, an essential factor for HCV replication, is notably low in Vero cells. Therefore, we supplemented Vero cells with miR-122 and found that HCV replication was enhanced. However, Vero cells that expressed miR-122 still did not allow HCV infection. We supplemented HCV receptor molecules and found that scavenger receptor class B type I (SRBI was essential for HCV infection in Vero cells. The supplementation of apolipoprotein E (ApoE, a host factor important for virus production, enabled the production of infectious virus in Vero cells. Finally, we created a Vero cell line that expressed the essential factors miR-122, SRBI, and ApoE; the entire HCV life cycle, including infection, replication, and infectious virus production, was completed in these cells. In conclusion, we demonstrated that miR-122, SRBI, and ApoE were necessary and sufficient for the completion of the entire HCV life cycle in nonhuman, nonhepatic Vero cells.

  2. Circulating ECV-Associated miRNAs as Potential Clinical Biomarkers in Early Stage HBV and HCV Induced Liver Fibrosis.

    Science.gov (United States)

    Lambrecht, Joeri; Jan Poortmans, Pieter; Verhulst, Stefaan; Reynaert, Hendrik; Mannaerts, Inge; van Grunsven, Leo A

    2017-01-01

    Introduction: Chronic hepatitis B (HBV) and C (HCV) virus infection is associated with the activation of hepatic stellate cells (HSCs) toward a myofibroblastic phenotype, resulting in excessive deposition of extracellular matrix, the development of liver fibrosis, and its progression toward cirrhosis. The gold standard for the detection and staging of liver fibrosis remains the liver biopsy, which is, however, associated with some mild and severe drawbacks. Other non-invasive techniques evade these drawbacks, but lack inter-stage specificity and are unable to detect early stages of fibrosis. We investigated whether circulating vesicle-associated miRNAs can be used in the diagnosis and staging of liver fibrosis in HBV and HCV patients. Methods: Plasma samples were obtained from 14 healthy individuals and 39 early stage fibrotic patients (F0-F2) with chronic HBV or HCV infection who underwent transient elastography (Fibroscan). Extracellular vesicles were extracted from the plasma and the level of miRNA-122, -150, -192, -21, -200b, and -92a was analyzed by qRT-PCR in total plasma and circulating vesicles. Finally, these same miRNAs were also quantified in vesicles extracted from in vitro activating primary HSCs. Results: In total plasma samples, only miRNA-200b (HBV: p = 0.0384; HCV: p = 0.0069) and miRNA-122 (HBV: p < 0.0001; HCV: p = 0.0007) were significantly up-regulated during early fibrosis. In circulating vesicles, miRNA-192 (HBV: p < 0.0001; HCV: p < 0.0001), -200b (HBV: p < 0.0001; HCV: p < 0.0001), -92a (HBV: p < 0.0001; HCV: p < 0.0001), and -150 (HBV: p = 0.0016; HCV: p = 0.004) displayed a significant down-regulation in both HBV and HCV patients. MiRNA expression profiles in vesicles isolated from in vitro activating primary mouse HSCs resembled the miRNA expression profile in circulating vesicles. Conclusion: Our analysis revealed a distinct miRNA expression pattern in total plasma and its circulating vesicles. The expression profile of miRNAs in

  3. Inhibition of Bovine Viral Diarrhea Virus RNA Synthesis by Thiosemicarbazone Derived from 5,6-Dimethoxy-1-Indanone▿

    Science.gov (United States)

    Castro, Eliana F.; Fabian, Lucas E.; Caputto, María E.; Gagey, Dolores; Finkielsztein, Liliana M.; Moltrasio, Graciela Y.; Moglioni, Albertina G.; Campos, Rodolfo H.; Cavallaro, Lucía V.

    2011-01-01

    In the present work, we described the activity of the thiosemicarbazone derived from 5,6-dimethoxy-1-indanone (TSC), which we previously characterized as a new compound that inhibits bovine viral diarrhea virus (BVDV) infection. We showed that TSC acts at a point of time that coincides with the onset of viral RNA synthesis and that it inhibits the activity of BVDV replication complexes (RCs). Moreover, we have selected five BVDV mutants that turned out to be highly resistant to TSC but still susceptible to ribavirin (RBV). Four of these resistant mutants carried an N264D mutation in the viral RNA-dependent RNA polymerase (RdRp). The remaining mutant showed an A392E mutation within the same protein. Some of these mutants replicated slower than the wild-type (wt) virus in the absence of TSC, whereas others showed a partial reversion to the wt phenotype over several passages in the absence of the compound. The docking of TSC in the crystal structure of the BVDV RdRp revealed a close contact between the indane ring of the compound and several residues within the fingers domain of the enzyme, some hydrophobic contacts, and hydrogen bonds with the thiosemicarbazone group. Finally, in the mutated RdRp from resistant BVDV, these interactions with TSC could not be achieved. Interestingly, TSC inhibited BVDV replication in cell culture synergistically with RBV. In conclusion, TSC emerges as a new nonnucleoside inhibitor of BVDV RdRp that is synergistic with RBV, a feature that turns it into a potential compound to be evaluated against hepatitis C virus (HCV). PMID:21430053

  4. Variation in RNA virus mutation rates across host cells.

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    Marine Combe

    2014-01-01

    Full Text Available It is well established that RNA viruses exhibit higher rates of spontaneous mutation than DNA viruses and microorganisms. However, their mutation rates vary amply, from 10(-6 to 10(-4 substitutions per nucleotide per round of copying (s/n/r and the causes of this variability remain poorly understood. In addition to differences in intrinsic fidelity or error correction capability, viral mutation rates may be dependent on host factors. Here, we assessed the effect of the cellular environment on the rate of spontaneous mutation of the vesicular stomatitis virus (VSV, which has a broad host range and cell tropism. Luria-Delbrück fluctuation tests and sequencing showed that VSV mutated similarly in baby hamster kidney, murine embryonic fibroblasts, colon cancer, and neuroblastoma cells (approx. 10(-5 s/n/r. Cell immortalization through p53 inactivation and oxygen levels (1-21% did not have a significant impact on viral replication fidelity. This shows that previously published mutation rates can be considered reliable despite being based on a narrow and artificial set of laboratory conditions. Interestingly, we also found that VSV mutated approximately four times more slowly in various insect cells compared with mammalian cells. This may contribute to explaining the relatively slow evolution of VSV and other arthropod-borne viruses in nature.

  5. Influence of HIV and HCV on T cell antigen presentation and challenges in the development of vaccines.

    Science.gov (United States)

    John, Mina; Gaudieri, Silvana

    2014-01-01

    Some of the central challenges for developing effective vaccines against HIV and hepatitis C virus (HCV) are similar. Both infections are caused by small, highly mutable, rapidly replicating RNA viruses with the ability to establish long-term chronic pathogenic infection in human hosts. HIV has caused 60 million infections globally and HCV 180 million and both viruses may co-exist among certain populations by virtue of common blood-borne, sexual, or vertical transmission. Persistence of both pathogens is achieved by evasion of intrinsic, innate, and adaptive immune defenses but with some distinct mechanisms reflecting their differences in evolutionary history, replication characteristics, cell tropism, and visibility to mucosal versus systemic and hepatic immune responses. A potent and durable antibody and T cell response is a likely requirement of future HIV and HCV vaccines. Perhaps the single biggest difference between the two vaccine design challenges is that in HCV, a natural model of protective immunity can be found in those who resolve acute infection spontaneously. Such spontaneous resolvers exhibit durable and functional CD4(+) and CD8(+) T cell responses (Diepolder et al., 1995; Cooper et al., 1999; Thimme et al., 2001; Grakoui et al., 2003; Lauer et al., 2004; Schulze Zur Wiesch et al., 2012). However, frequent re-infection suggests partial or lack of protective immunity against heterologous HCV strains, possibly indicative of the degree of genetic diversity of circulating HCV genotypes and subtypes. There is no natural model of protective immunity in HIV, however, studies of "elite controllers," or individuals who have durably suppressed levels of plasma HIV RNA without antiretroviral therapy, has provided the strongest evidence for CD8(+) T cell responses in controlling viremia and limiting reservoir burden in established infection. Here we compare and contrast the specific mechanisms of immune evasion used by HIV and HCV, which subvert adaptive human

  6. Convergent evolution of argonaute-2 slicer antagonism in two distinct insect RNA viruses.

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    Joël T van Mierlo

    Full Text Available RNA interference (RNAi is a major antiviral pathway that shapes evolution of RNA viruses. We show here that Nora virus, a natural Drosophila pathogen, is both a target and suppressor of RNAi. We detected viral small RNAs with a signature of Dicer-2 dependent small interfering RNAs in Nora virus infected Drosophila. Furthermore, we demonstrate that the Nora virus VP1 protein contains RNAi suppressive activity in vitro and in vivo that enhances pathogenicity of recombinant Sindbis virus in an RNAi dependent manner. Nora virus VP1 and the viral suppressor of RNAi of Cricket paralysis virus (1A antagonized Argonaute-2 (AGO2 Slicer activity of RNA induced silencing complexes pre-loaded with a methylated single-stranded guide strand. The convergent evolution of AGO2 suppression in two unrelated insect RNA viruses highlights the importance of AGO2 in antiviral defense.

  7. Structural features of NS3 of Dengue virus serotypes 2 and 4 in solution and insight into RNA binding and the inhibitory role of quercetin.

    Science.gov (United States)

    Pan, Ankita; Saw, Wuan Geok; Subramanian Manimekalai, Malathy Sony; Grüber, Ardina; Joon, Shin; Matsui, Tsutomu; Weiss, Thomas M; Grüber, Gerhard

    2017-05-01

    Dengue virus (DENV), which has four serotypes (DENV-1 to DENV-4), is the causative agent of the viral infection dengue. DENV nonstructural protein 3 (NS3) comprises a serine protease domain and an RNA helicase domain which has nucleotide triphosphatase activities that are essential for RNA replication and viral assembly. Here, solution X-ray scattering was used to provide insight into the overall structure and flexibility of the entire NS3 and its recombinant helicase and protease domains for Dengue virus serotypes 2 and 4 in solution. The DENV-2 and DENV-4 NS3 forms are elongated and flexible in solution. The importance of the linker residues in flexibility and domain-domain arrangement was shown by the compactness of the individual protease and helicase domains. Swapping of the 174 PPAVP 179 linker stretch of the related Hepatitis C virus (HCV) NS3 into DENV-2 NS3 did not alter the elongated shape of the engineered mutant. Conformational alterations owing to RNA binding are described in the protease domain, which undergoes substantial conformational alterations that are required for the optimal catalysis of bound RNA. Finally, the effects of ATPase inhibitors on the enzymatically active DENV-2 and DENV-4 NS3 and the individual helicases are presented, and insight into the allosteric effect of the inhibitor quercetin is provided.

  8. In depth analysis on the binding sites of adamantane derivatives in HCV (hepatitis C virus p7 channel based on the NMR structure.

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    Qi-Shi Du

    Full Text Available BACKGROUND: The recently solved solution structure of HCV (hepatitis C virus p7 ion channel provides a solid structure basis for drug design against HCV infection. In the p7 channel the ligand amantadine (or rimantadine was determined in a hydrophobic pocket. However the pharmocophore (-NH2 of the ligand was not assigned a specific binding site. RESULTS: The possible binding sites for amino group of adamantane derivatives is studied based on the NMR structure of p7 channel using QM calculation and molecular modeling. In the hydrophobic cavity and nearby three possible binding sites are proposed: His17, Phe20, and Trp21. The ligand binding energies at the three binding sites are studied using high level QM method CCSD(T/6-311+G(d,p and AutoDock calculations, and the interaction details are analyzed. The potential application of the binding sites for rational inhibitor design are discussed. CONCLUSIONS: Some useful viewpoints are concluded as follows. (1 The amino group (-NH2 of adamantane derivatives is protonated (-NH3+, and the positively charged cation may form cation-π interactions with aromatic amino acids. (2 The aromatic amino acids (His17, Phe20, and Trp21 are the possible binding sites for the protonated amino group (-NH3+ of adamantane derivatives, and the cation-π bond energies are 3 to 5 times stronger than the energies of common hydrogen bonds. (3 The higher inhibition potent of rimantadine than amantadine probably because of its higher pKa value (pKa = 10.40 and the higher positive charge in the amino group. The potential application of p7 channel structure for inhibitor design is discussed.

  9. Sequence analysis of the medium RNA segment of three Simbu serogroup viruses, Akabane, Aino, and Peaton viruses.

    Science.gov (United States)

    Yanase, Tohru; Yoshida, Kazuo; Ohashi, Seiichi; Kato, Tomoko; Tsuda, Tomoyuki

    2003-05-01

    The sequence analysis was carried out for the medium (M) RNA segment of the Akabane virus (AKAV), Aino virus (AINV), and Peaton virus (PEAV) of the Simbu serogroup of the genus Orthobunyavirus of the family Bunyaviridae. The complementary sequences of the M RNA segments of AKAV, AINV, and PEAV contain a single large open reading frame (ORF), like other orthobunyaviruses. The ORFs potentially encode 1401 amino acids (aa), 1404 aa, and 1400 aa polypeptides, respectively. The identity of the M segment among these viruses is remarkably low, although previous researchers reported that the small RNA segments are highly conserved. Because the M segment codes for the viral surface glycoproteins G1 and G2, the variability of the M segment may affect the antigenicity of these viruses. Phylogenetic studies based on the M and S segment sequences suggested that genetic reassortment has been occurring among ancestral viruses of the three Simbu serogroup viruses throughout their evolution.

  10. Detection and molecular characterization of double-stranded RNA viruses in Philippine Trichomonas vaginalis isolates

    OpenAIRE

    Rivera, Windell L.; Christine Aubrey C. Justo; Relucio-San Diego, Mary Ann Cielo V.; Loyola, Lorenz M.

    2017-01-01

    Background/Purpose: The flagellated protozoon Trichomonas vaginalis that parasitizes the urogenital tract of humans was reported to harbor double-stranded RNA (dsRNA) viruses. These viruses, identified as Trichomonas vaginalis virus (TVV), belong to the genus Trichomonasvirus of the family Totiviridae. Four species, formally recognized by the International Committee on Taxonomy of Viruses (ICTV), have been reported and distinguished by pairwise comparisons of the sequences of genes coding for...

  11. Historical Trends in the Hepatitis C Virus Epidemics in North America and Australia

    NARCIS (Netherlands)

    Rodrigo, Chaturaka; Eltahla, Auda A.; Bull, Rowena A.; Grebely, Jason; Dore, Gregory J.; Applegate, Tanya; Page, Kimberly; Bruneau, Julie; Morris, Meghan D.; Cox, Andrea L.; Osburn, William; Kim, Arthur Y.; Schinkel, Janke; Shoukry, Naglaa H.; Lauer, Georg M.; Maher, Lisa; Hellard, Margaret; Prins, Maria; Estes, Chris; Razavi, Homie; Lloyd, Andrew R.; Luciani, Fabio

    2016-01-01

    Bayesian evolutionary analysis (coalescent analysis) based on genetic sequences has been used to describe the origins and spread of rapidly mutating RNA viruses, such as influenza, Ebola, human immunodeficiency virus (HIV), and hepatitis C virus (HCV).  Full-length subtype 1a and 3a sequences from

  12. Ethanol and reactive species increase basal sequence heterogeneity of hepatitis C virus and produce variants with reduced susceptibility to antivirals.

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    Scott Seronello

    Full Text Available Hepatitis C virus (HCV exhibits a high level of genetic variability, and variants with reduced susceptibility to antivirals can occur even before treatment begins. In addition, alcohol decreases efficacy of antiviral therapy and increases sequence heterogeneity of HCV RNA but how ethanol affects HCV sequence is unknown. Ethanol metabolism and HCV infection increase the level of reactive species that can alter cell metabolism, modify signaling, and potentially act as mutagen to the viral RNA. Therefore, we investigated whether ethanol and reactive species affected the basal sequence variability of HCV RNA in hepatocytes. Human hepatoma cells supporting a continuous replication of genotype 1b HCV RNA (Con1, AJ242652 were exposed to ethanol, acetaldehyde, hydrogen peroxide, or L-buthionine-S,R-sulfoximine (BSO that decreases intracellular glutathione as seen in patients. Then, NS5A region was sequenced and compared with genotype 1b HCV sequences in the database. Ethanol and BSO elevated nucleotide and amino acid substitution rates of HCV RNA by 4-18 folds within 48 hrs which were accompanied by oxidative RNA damage. Iron chelator and glutathione ester decreased both RNA damage and mutation rates. Furthermore, infectious HCV and HCV core gene were sufficient to induce oxidative RNA damage even in the absence of ethanol or BSO. Interestingly, the dn/ds ratio and percentage of sites undergoing positive selection increased with ethanol and BSO, resulting in an increased detection of NS5A variants with reduced susceptibility to interferon alpha, cyclosporine, and ribavirin and others implicated in immune tolerance and modulation of viral replication. Therefore, alcohol is likely to synergize with virus-induced oxidative/nitrosative stress to modulate the basal mutation rate of HCV. Positive selection induced by alcohol and reactive species may contribute to antiviral resistance.

  13. Zebrafish as a potential model organism for drug test against hepatitis C virus.

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    Cun-Bao Ding

    Full Text Available Screening and evaluating anti- hepatitis C virus (HCV drugs in vivo is difficult worldwide, mainly because of the lack of suitable small animal models. We investigate whether zebrafish could be a model organism for HCV replication. To achieve NS5B-dependent replication an HCV sub-replicon was designed and created with two vectors, one with HCV ns5b and fluorescent rfp genes, and the other containing HCV's 5'UTR, core, 3'UTR and fluorescent gfp genes. The vectors containing sub-replicons were co-injected into zebrafish zygotes. The sub-replicon amplified in liver showing a significant expression of HCV core RNA and protein. The sub-replicon amplification caused no abnormality in development and growth of zebrafish larvae, but induced gene expression change similar to that in human hepatocytes. As the amplified core fluorescence in live zebrafish was detectable microscopically, it rendered us an advantage to select those with replicating sub-replicon for drug experiments. Ribavirin and oxymatrine, two known anti-HCV drugs, inhibited sub-replicon amplification in this model showing reduced levels of HCV core RNA and protein. Technically, this method had a good reproducibility and is easy to operate. Thus, zebrafish might be a model organism to host HCV, and this zebrafish/HCV (sub-replicon system could be an animal model for anti-HCV drug screening and evaluation.

  14. Low prevalence of HCV, HIV, and HTLV-I/II infection markers in northwestern Greece: results of a 3-year prospective donor study (1995-1997).

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    Zervou, E K.; Boumba, D S.; Liaskos, Ch; Georgiadou, S; Tsianos, E V.; Dalekos, G N.

    2003-02-01

    Background: The risk of infection with transfusion-transmitted viruses has been reduced remarkably. A zero-risk blood supply, however, remains a popular goal. A 3-year prospective donor study was conducted in the Epirus region of Greece to determine the prevalence of human immunodeficiency virus (HIV), human T-lymphotropic virus (HTLV), hepatitis B virus, and hepatitis C virus (HCV). Herein, we report the prevalence of HIV, HTLV, and HCV infection markers in this area. Methodology: Between January 1, 1995 and December 31, 1997, 6696 donors were investigated for the presence of anti-HIV, anti-HTLV, and anti-HCV antibodies using standard enzyme immunoassays (EIA). Every sample with anti-HCV reactivity by third-generation EIA was further investigated using a third-generation recombinant immunoblot assay (RIBA 3.0) and HCV-RNA by a combination of polymerase chain reaction (PCR) and DNA EIA. Results: None of the donors tested positive for anti-HIV or anti-HTLV antibodies. In contrast, anti-HCV was detected in 41 donors (0.61%). Using a RIBA 3.0 test, eight donors tested positive and eight had indeterminate results, while 25 tested negative. Seven of the eight donors with both EIA and RIBA 3.0 reactivity had increased levels of aminotransferases and detectable serum HCV-RNA. The remaining 34 donors had repeatedly normal aminotransferases and three times negative HCV-RNA. Liver biopsy was performed in anti-HCV/HCV-RNA-positive donors (7/41). The lesions were compatible with chronic hepatitis C in all of them. Conclusion: A zero prevalence of HIV and HTLV infection markers was found. Although the number of annual donations in this study was relatively low, the negative data for HIV and HTLV clearly indicate that rates of these infections are low in our region and that infected donors will be seen infrequently. HCV infection in blood donors remains very low in our region and is similar to the data reported in other industrialized countries. In fact, the prevalence of

  15. Plasma interferon-gamma-inducible protein-10 levels are associated with early, but not sustained virological response during treatment of acute or early chronic HCV infection.

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    Jordan J Feld

    Full Text Available BACKGROUND: High plasma levels of interferon-gamma inducible protein-10 (IP-10 have been shown to be associated with impaired treatment response in chronic hepatitis C virus (HCV infection. Whether IP-10 levels predict treatment in acute HCV infection is unknown. METHODS: Patients with acute or early chronic HCV infection from the Australian Trial in Acute Hepatitis C (ATAHC cohort were evaluated. Baseline and on-treatment plasma IP-10 levels were measured by ELISA. IL28B genotype was determined by sequencing. RESULTS: Overall, 74 HCV mono-infected and 35 HIV/HCV co-infected patients were treated in ATAHC, of whom 89 were adherent to therapy and were included for analysis. IP-10 levels correlated with HCV RNA levels at baseline (r = 0.48, P600 pg/mL achieved RVR. There was no association with IP-10 levels and early virological response (EVR or sustained virological response (SVR. CONCLUSIONS: Baseline IP-10 levels are associated with early viral kinetics but not ultimate treatment outcome in acute HCV infection. Given previous data showing that patients with high baseline IP-10 are unlikely to spontaneously clear acute HCV infection, they should be prioritized for early antiviral therapy.

  16. Lipoprotein Receptors and Lipid Enzymes in Hepatitis C Virus Entry and Early Steps of Infection

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    Eve-Isabelle Pécheur

    2012-01-01

    Full Text Available Viruses are obligate intracellular agents that depend on host cells for successful propagation, hijacking cellular machineries to their own profit. The molecular interplay between host factors and invading viruses is a continuous coevolutionary process that determines viral host range and pathogenesis. The hepatitis C virus (HCV is a strictly human pathogen, causing chronic liver injuries accompanied by lipid disorders. Upon infection, in addition to protein-protein and protein-RNA interactions usual for such a positive-strand RNA virus, HCV relies on protein-lipid interactions at multiple steps of its life cycle to establish persistent infection, making use of hepatic lipid pathways. This paper focuses on lipoproteins in HCV entry and on receptors and enzymes involved in lipid metabolism that HCV exploits to enter hepatocytes.

  17. HIV infection is associated with higher levels of monocyte chemoattractant protein-1 and eotaxin among people with recent hepatitis C virus infection.

    Science.gov (United States)

    Lamoury, François M J; Hajarizadeh, Behzad; Keoshkerian, Elizabeth; Feld, Jordan J; Amin, Janaki; Teutsch, Suzy; Matthews, Gail V; Hellard, Margaret; Dore, Gregory J; Lloyd, Andrew R; Applegate, Tanya L; Grebely, Jason

    2016-06-01

    Human immunodeficiency virus (HIV) infection leads to more rapid progression of hepatitis C virus (HCV)-related liver fibrosis, which could be linked to differences in the severity of liver inflammation among HIV/HCV co-infected individuals compared to HCV mono-infected individuals. This study assessed the association of HIV co-infection with pro-inflammatory and pro-fibrogenic cytokines and chemokines during recent HCV infection. Participants from the ATAHC study, a prospective cohort of recent HCV infection, with detectable HCV RNA at the time of acute HCV detection were included. Concentrations of 27 plasma cytokines and chemokines were measured by multiplex immunoassays and compared between those with, and without, HIV co-infection. Out of 117 individuals with recent HCV infection included in analysis, 73 had HCV mono-infection and 44 had HIV/HCV co-infection. Individuals with HIV/HCV co-infection had significantly higher mean levels of eotaxin (1.79 vs. 1.62 log pg/mL; P HIV/HCV co-infection (adjusted β: 0.12; 95%CI: 0.01, 0.24; P = 0.039). Higher MCP-1 levels were also independently associated with HIV/HCV co-infection in adjusted analysis (adjusted β: 0.11; 95%CI: 0.03, 0.18; P = 0.009). During recent HCV, those with HIV/HCV co-infection had a stronger pro-fibrogenic mediator profile compared to those with HCV mono-infection. These findings may provide a potential explanation for accelerated liver fibrosis in HIV/HCV co-infection. Australian Trial in Acute Hepatitis C (ATAHC) study was registered with ClinicalTrials.gov registry on September 11, 2005. NCT00192569 .

  18. External Quality Assessment for Rubella Virus RNA Detection Using Armored RNA in China.

    Science.gov (United States)

    Zhang, D; Lin, G; Yi, L; Hao, M; Fan, G; Yang, X; Peng, R; Ding, J; Zhang, K; Zhang, R; Li, J

    2017-02-01

    Although tremendous efforts have been made to reduce rubella incidence, there are still 300 new cases of congenital rubella syndrome daily; thus, rubella infections remain one of the leading causes of preventable congenital birth defects. An effective surveillance system, which could be achieved and maintained by using an external quality assessment program, is critical for prevention and control of this disease. Armored RNAs, which are noninfectious and RNase-resistant, were used for encapsulation of the E1 gene of rubella virus and for preparation of a 10-specimen panel for external quality assessment. Thirty-two laboratories across mainland China that used nucleic acid tests for rubella virus RNA detection were included in the external quality assessment program organized by the National Center for Clinical Laboratories of China. Different kinds of commercial kits were used by the laboratories for nucleic acid extraction and TaqMan real-time reverse-transcription PCR for rubella virus RNA detection; 99.2% sensitivity and 100% specificity were achieved in this external quality assessment program. Most of the participating laboratories obtained accurate results for rubella nucleic acid tests, thereby achieving the quality required for regional rubella and congenital rubella syndrome elimination.

  19. [Presence of autocomplementary RNA with viral specificity in cells infected with herpes virus].

    Science.gov (United States)

    Béchet, J M; Montagnier, L; Latarjet, R

    1975-01-13

    RNA from cells infected with Herpes simplex virus contain a higher percentage of double-stranded RNA than non-infected cells. This percentage increases three-fold upon self-annealing. The complementary RNA sequences were shown to be virus-specific by the following criteria: (1) high melting temperature than double-stranded RNA from non infected cells; (2) higher density in caesium sulphate; (3) specific hybridization with viral DNA.

  20. Nicotiana small RNA sequences support a host genome origin of cucumber mosaic virus satellite RNA.

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    Kiran Zahid

    2015-01-01

    Full Text Available Satellite RNAs (satRNAs are small noncoding subviral RNA pathogens in plants that depend on helper viruses for replication and spread. Despite many decades of research, the origin of satRNAs remains unknown. In this study we show that a β-glucuronidase (GUS transgene fused with a Cucumber mosaic virus (CMV Y satellite RNA (Y-Sat sequence (35S-GUS:Sat was transcriptionally repressed in N. tabacum in comparison to a 35S-GUS transgene that did not contain the Y-Sat sequence. This repression was not due to DNA methylation at the 35S promoter, but was associated with specific DNA methylation at the Y-Sat sequence. Both northern blot hybridization and small RNA deep sequencing detected 24-nt siRNAs in wild-type Nicotiana plants with sequence homology to Y-Sat, suggesting that the N. tabacum genome contains Y-Sat-like sequences that give rise to 24-nt sRNAs capable of guiding RNA-directed DNA methylation (RdDM to the Y-Sat sequence in the 35S-GUS:Sat transgene. Consistent with this, Southern blot hybridization detected multiple DNA bands in Nicotiana plants that had sequence homology to Y-Sat, suggesting that Y-Sat-like sequences exist in the Nicotiana genome as repetitive DNA, a DNA feature associated with 24-nt sRNAs. Our results point to a host genome origin for CMV satRNAs, and suggest novel approach of using small RNA sequences for finding the origin of other satRNAs.

  1. Genetic recombination in plant-infecting messenger-sense RNA viruses: overview and research perspectives

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    Jozef Julian Bujarski

    2013-03-01

    Full Text Available RNA recombination is one of the driving forces of genetic variability in (+-strand RNA viruses. Various types of RNA-RNA crossovers were described including crosses between the same or different viral RNAs or between viral and cellular RNAs. Likewise, a variety of molecular mechanisms are known to support RNA recombination, such as replicative events (based on internal or end-to-end replicase switchings along with nonreplicative joining among RNA fragments of viral and/or cellular origin. Such mechanisms as RNA decay or RNA interference are responsible for RNA fragmentation and trans-esterification reactions which are likely accountable for ligation of RNA fragments. Numerous host factors were found to affect the profiles of viral RNA recombinants and significant differences in recombination frequency were observed among various RNA viruses. Comparative analyses of viral sequences allowed for the development of evolutionary models in order to explain adaptive phenotypic changes and co-evolving sites. Many questions remain to be answered by forthcoming RNA recombination research. (i How various factors modulate the ability of viral replicase to switch templates, (ii What is the intracellular location of RNA-RNA template switchings, (iii Mechanisms and factors responsible for non-replicative RNA recombination, (iv Mechanisms of integration of RNA viral sequences with cellular genomic DNA, and (v What is the role of RNA splicing and ribozyme activity. From an evolutionary stand point, it is not known how RNA viruses parasitize new host species via recombination, nor is it obvious what the contribution of RNA recombination is among other RNA modification pathways. We do not understand why the frequency of RNA recombination varies so much among RNA viruses and the status of RNA recombination as a form of sex is not well documented.

  2. Injection drug use facilitates hepatitis C virus infection of peripheral blood mononuclear cells.

    Science.gov (United States)

    Resti, Massimo; Azzari, Chiara; Moriondo, Maria; Betti, Letizia; Sforzi, Idanna; Novembre, Elio; Vierucci, Alberto

    2002-08-01

    Infection of peripheral blood mononuclear cells (PBMCs) with hepatitis C virus (HCV) has been demonstrated and has been found to play a role in relapse of HCV disease and vertical transmission of HCV. Injection drug use is thought to impair function of the immune system and induce tolerance to viruses; therefore, HCV infection of PBMCs could be more likely to occur in injection drug users (IDUs) with HCV infection. Of 108 women who tested negative for human immunodeficiency virus type 1 and positive for HCV RNA, 51 had a history of injection drug use and 57 had no known risk factor for HCV infection. HCV infection was found, by nested reverse-transcription polymerase chain reaction analysis, in the PBMCs of 33 IDUs and of 13 non-IDUs (P=.00003). No correlation was found between infection of the PBMCs and HCV genotype or virus load. Route of transmission and viral factors, as well as immunologic dysfunction, may play a role in viral tropism.

  3. Structural comparisons of the nucleoprotein from three negative strand RNA virus families

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    Tsao Jun

    2007-07-01

    Full Text Available Abstract Structures of the nucleoprotein of three negative strand RNA virus families, borna disease virus, rhabdovirus and influenza A virus, are now available. Structural comparisons showed that the topology of the RNA binding region from the three proteins is very similar. The RNA was shown to fit into a cavity formed by the two distinct domains of the RNA binding region in the rhabdovirus nucleoprotein. Two helices connecting the two domains characterize the center of the cavity. The nucleoproteins contain at least 5 conserved helices in the N-terminal domain and 3 conserved helices in the C-terminal domain. Since all negative strand RNA viruses are required to have the ribonucleoprotein complex as their active genomic templates, it is perceivable that the (5H+3H structure is a common motif in the nucleoprotein of negative strand RNA viruses.

  4. Leishmania RNA virus: when the host pays the toll

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    Hartley, Mary-Anne; Ronet, Catherine; Zangger, Haroun; Beverley, Stephen M.; Fasel, Nicolas

    2012-01-01

    The presence of an RNA virus in a South American subgenus of the Leishmania parasite, L. (Viannia), was detected several decades ago but its role in leishmanial virulence and metastasis was only recently described. In Leishmania guyanensis, the nucleic acid of Leishmania RNA virus (LRV1) acts as a potent innate immunogen, eliciting a hyper-inflammatory immune response through toll-like receptor 3 (TLR3). The resultant inflammatory cascade has been shown to increase disease severity, parasite persistence, and perhaps even resistance to anti-leishmanial drugs. Curiously, LRVs were found mostly in clinical isolates prone to infectious metastasis in both their human source and experimental animal model, suggesting an association between the viral hyperpathogen and metastatic complications such as mucocutaneous leishmaniasis (MCL). MCL presents as chronic secondary lesions in the mucosa of the mouth and nose, debilitatingly inflamed and notoriously refractory to treatment. Immunologically, this outcome has many of the same hallmarks associated with the reaction to LRV: production of type 1 interferons, bias toward a chronic Th1 inflammatory state and an impaired ability of host cells to eliminate parasites through oxidative stress. More intriguing, is that the risk of developing MCL is found almost exclusively in infections of the L. (Viannia) subtype, further indication that leishmanial metastasis is caused, at least in part, by a parasitic component. LRV present in this subgenus may contribute to the destructive inflammation of metastatic disease either by acting in concert with other intrinsic “metastatic factors” or by independently preying on host TLR3 hypersensitivity. Because LRV amplifies parasite virulence, its presence may provide a unique target for diagnostic and clinical intervention of metastatic leishmaniasis. Taking examples from other members of the Totiviridae virus family, this paper reviews the benefits and costs of endosymbiosis, specifically

  5. In routine clinical practice, few physicians use early viral kinetics to guide HCV dual therapy treatment decisions.

    Science.gov (United States)

    Mangia, Alessandra; Bányai, Tivadar; De Bartolomeo, Giuseppe; Gervain, Judit; Habersetzer, François; Mulkay, Jean-Pierre; Ouzan, Denis; Parruti, Giustino; Passariello, Nicola; Remy, Andre-Jean; Rizzetto, Mario; Shiffman, Mitchell L; Tice, Alan D; Schmitz, Manuela; Tatsch, Fernando; Rodriguez-Torres, Maribel

    2014-08-01

    PROPHESYS is a large, multinational, non-interventional prospective cohort study of chronic hepatitis C patients treated with peginterferon alfa/ribavirin. This subanalysis assesses rates of premature treatment discontinuation stratified by on-treatment virological response (VR). This PROPHESYS subanalysis is restricted to treatment-naive, hepatitis C virus (HCV) genotype (G)1/2/3 mono-infected patients who received peginterferon alfa-2a (40KD)/ribavirin with intended treatment duration of 48 (G1) or 24 weeks (G2/3). Early virological responses were classified into four mutually exclusive categories [rapid VR (RVR), complete early VR (cEVR), partial EVR (pEVR), no RVR/EVR], using standard criteria. The likelihood for shortening treatment owing to good efficacy was highest among patients with an RVR and HCV RNA≤400 000 IU/ml (G1 10.0%; G2/3 5.8%) whereas for poor efficacy, it was highest in G1 non-RVR/EVR patients with HCV RNA>400 000 IU/ml (56.6%). Factors significantly associated with early treatment discontinuation as a result of good efficacy in G1 patients included RVR vs. no RVR/EVR and, at baseline, lower HCV RNA, lower FIB-4 score, HCV infection via injection drug use. For G2/3 patients, factors included lower baseline HCV RNA and G2 vs. G3 infection. Most patients started with the recommended peginterferon alfa-2a dose, but a high proportion received a higher-than-recommended ribavirin dose. Despite international guidelines, few physicians used early viral kinetics to abbreviate treatment. Therefore, relatively few patients with an RVR and low baseline HCV RNA abbreviated treatment. In addition, there were deviations in ribavirin starting doses, suggesting that physicians tailor treatment according to local guidelines or previous experience. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  6. Factors associated with uptake of treatment for recent hepatitis C virus infection in a predominantly injecting drug user cohort: The ATAHC Study.

    Science.gov (United States)

    Grebely, J; Petoumenos, K; Matthews, G V; Haber, P; Marks, P; Lloyd, A R; Kaldor, J M; Dore, G J; Hellard, M

    2010-03-01

    Despite that the majority of hepatitis C virus (HCV) infection occurs among injection drug users (IDUs), little is known about HCV treatment uptake in this group, particularly during recent infection. We evaluated uptake of treatment for recent HCV infection, including associated factors, within a population predominantly made up of IDUs. The Australian Trial in Acute Hepatitis C was a study of the natural history and treatment of recent HCV infection. All participants with detectable HCV RNA at screening were offered HCV treatment, assessed for eligibility and those initiating treatment were identified. Logistic regression analyses were used to identify predictors of HCV treatment uptake. Between June 2004 and February 2008, 163 were enrolled, with 146 positive for HCV RNA at enrolment. The mean age was 35 years, 77% (n=113) participants had ever injected illicit drugs and 23% (n=34) reported having ever received methadone or buprenorphine treatment. The uptake of HCV treatment was 76% (111 of 146) among those who were eligible on the basis of positive HCV RNA. Estimated duration of HCV infection (OR=1.03 per week, 95% CI=1.00-1.06, P=0.035) and log(10) HCV RNA (OR=1.92 per log(10) increase, 95% CI=1.36-2.73, P<0.001) were independently associated with treatment uptake whereas injection drug use was not. This study demonstrates that a high uptake of HCV treatment can be achieved among participants with recently acquired HCV infection. Decisions about whether to initiate treatment for recently acquired HCV were mainly driven by clinical factors, rather than factors related to sociodemographics or injecting behaviors. C