Analysis of the 5'UTR of HCV genotype 3 grown in vitro in human B cells, T cells, and macrophages

<p>Abstract</p> <p>Background</p> <p>Previously, we have reported the isolation and molecular characterization of human Hepatitis C virus genotype 1 (HCV-1) from infected patients. We are now reporting an analysis of HCV obtained from patients infected with HCV genotype 3 (HCV-3) as diagnosed by clinical laboratories.</p> <p>Results</p> <p>HCV was cultured <it>in vitro </it>using our system. HCV RNA was isolated from patients' blood and from HCV cultured in various cell types for up to three months. The 5'UTR of these isolates were used for comparisons. Results revealed a number of sequence changes as compared to the serum RNA. The HCV RNA produced efficiently by infected macrophages, B-cells, and T-cells had sequences similar to HCV-1, which suggests that selection of the variants was performed at the level of macrophages. Virus with sequences similar to HCV-1 replicated better in macrophages than HCV having a 5'UTR similar to HCV-3.</p> <p>Conclusions</p> <p>Although HCV-3 replicates in cell types such as B-cells, T-cells, and macrophages, it may require a different primary cell type for the same purpose. Therefore, in our opinion, HCV-3 does not replicate efficiently in macrophages, and patients infected with HCV-3 may contain a population of HCV-1 in their blood.</p

Discovery of significant variants containing large deletions in the 5'UTR of human hepatitis C virus (HCV)

We recently reported the isolation and in vitro replication of hepatitis C virus. These isolates were termed CIMM-HCV and analyzed to establish genotypes and subtypes, which are reported elsewhere. During this analysis, an HCV isolated from a patient was discovered that had large deletions in the 5'UTR. 57% of the HCV RNA found in this patient's sera had 113 or 116 bp deletions. Sequence data showed that domains IIIa to IIIc were missing. Previous studies have suggested that these domains may be important for translation. In vitro replicated HCV from this patient did not contain these deletions, however, it contained a 148 bp deletion in the 5'UTR. Whereas the patient HCV lacked domains IIIa through IIIc, the isolate lacked domains IIIa through IIId. HCV from this patient continues to produce large deletions in vitro, suggesting that the deletion may not be important for the assembly or replication of the virus. This is the first report describing these large deletions

The simultaneous presence and expression of human hepatitis C virus (HCV), human herpesvirus-6 (HHV-6), and human immunodeficiency virus-1 (HIV-1) in a single human T-cell

We have developed a system that isolates and replicates HCV in vitro. These isolates are called CIMM-HCV. This system has made it possible to analyze the biology, nature, and extent of HCV variability, among other things. Individuals that are infected with HIV-1 are often also infected with HCV and HHV-6. In addition to HCV, our lab has systems for replicating HIV-1 and HHV-6. We asked whether all these viruses could infect the same cells. We report here the successful infection of a T-cell (CEM) by CIMM-HCV, HHV-6, and HIV-1. PCR analyses demonstrated that the CEM cells were productively infected by HHV-6A. RT-PCR showed that the same cell culture was positive for HCV and HIV-1. Co-infection of a T-cell by all three viruses was confirmed by transmission electron microscopy (TEM). All these viruses are highly cytolytic; therefore, triply-infected cells were short lived. However, HIV-1 and HCV co-infected cells unexpectedly lasted for several weeks. Viral replication was unhindered and the phenomenon of 'dominance' was not observed in our experiments. In addition, CIMM-HCV was present in the perinuclear space, suggesting their possible synthesis in the nucleus. This report is based entirely on viruses produced in vitro in our laboratories. As part of the determinations of host ranges of these viruses, studies were designed to demonstrate the infection of a single cell by these viruses and to study the consequences of this phenomenon. All measurements were made on cultured cells and cell culture supernatants

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

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

Transmission of human hepatitis C virus from patients in secondary cells for long term culture

Infection by human hepatitis C virus (HCV) is the principal cause of post-transfusion hepatitis and chronic liver diseases worldwide. A reliable in vitro culture system for the isolation and analysis of this virus is not currently available, and, as a consequence, HCV pathogenesis is poorly understood. We report here the first robust in vitro system for the isolation and propagation of HCV from infected donor blood. This system involves infecting freshly prepared macrophages with HCV and then transmission of macrophage-adapted virus into freshly immortalized B-cells from human fetal cord blood. Using this system, newly isolated HCV have been replicated in vitro in continuous cultures for over 130 weeks. These isolates were also transmitted by cell-free methods into different cell types, including B-cells, T-cells and neuronal precursor cells. These secondarily infected cells also produced in vitro transmissible infectious virus. Replication of HCV-RNA was validated by RT-PCR analysis and by in situ hybridization. Although nucleic acid sequencing of the HCV isolate reported here indicates that the isolate is probably of type 1a, other HCV types have also been isolated using this system. Western blot analysis shows the synthesis of major HCV structural proteins. We present here, for the first time, a method for productively growing HCV in vitro for prolonged periods of time. This method allows studies related to understanding the replication process, viral pathogenesis, and the development of anti-HCV drugs and vaccines

Ultra-structural cell distribution of the melanoma marker iodobenzamide: improved potentiality of SIMS imaging in life sciences

BACKGROUND: Analytical imaging by secondary ion mass spectrometry (SIMS) provides images representative of the distribution of a specific ion within a sample surface. For the last fifteen years, concerted collaborative research to design a new ion microprobe with high technical standards in both mass and lateral resolution as well as in sensitivity has led to the CAMECA NanoSims 50, recently introduced onto the market. This instrument has decisive capabilities, which allow biological applications of SIMS microscopy at a level previously inaccessible. Its potential is illustrated here by the demonstration of the specific affinity of a melanoma marker for melanin. This finding is of great importance for the diagnosis and/or treatment of malignant melanoma, a tumour whose worldwide incidence is continuously growing. METHODS: The characteristics of the instrument are briefly described and an example of application is given. This example deals with the intracellular localization of an iodo-benzamide used as a diagnostic tool for the scintigraphic detection of melanic cells (e.g. metastasis of malignant melanoma). B16 melanoma cells were injected intravenously to C(57)BL(6)/J(1)/co mice. Multiple B16 melanoma colonies developed in the lungs of treated animals within three weeks. Iodobenzamide was injected intravenously in tumour bearing mice six hours before sacrifice. Small pieces of lung were prepared for SIMS analysis. RESULTS: Mouse lung B16 melanoma colonies were observed with high lateral resolution. Cyanide ions gave "histological" images of the cell, representative of the distribution of C and N containing molecules (e.g. proteins, nucleic acids, melanin, etc.) while phosphorus ions are mainly produced by nucleic acids. Iodine was detected only in melanosomes, confirming the specific affinity of the drug for melanin. No drug was found in normal lung tissue. CONCLUSION: This study demonstrates the potential of SIMS microscopy, which allows the study of ultra structural distribution of a drug within a cell. On the basis of our observations, drug internalization via membrane sigma receptors can be excluded

The triple combination of tenofovir, emtricitabine and efavirenz shows synergistic anti-HIV-1 activity in vitro: a mechanism of action study

<p>Abstract</p> <p>Background</p> <p>Tenofovir disoproxil fumarate (TDF), emtricitabine (FTC), and efavirenz (EFV) are the three components of the once-daily, single tablet regimen (Atripla) for treatment of HIV-1 infection. Previous cell culture studies have demonstrated that the double combination of tenofovir (TFV), the parent drug of TDF, and FTC were additive to synergistic in their anti-HIV activity, which correlated with increased levels of intracellular phosphorylation of both compounds.</p> <p>Results</p> <p>In this study, we demonstrated the combinations of TFV+FTC, TFV+EFV, FTC+EFV, and TFV+FTC+EFV synergistically inhibit HIV replication in cell culture and synergistically inhibit HIV-1 reverse transcriptase (RT) catalyzed DNA synthesis in biochemical assays. Several different methods were applied to define synergy including median-effect analysis, MacSynergy^®II and quantitative isobologram analysis. We demonstrated that the enhanced formation of dead-end complexes (DEC) by HIV-1 RT and TFV-terminated DNA in the presence of FTC-triphosphate (TP) could contribute to the synergy observed for the combination of TFV+FTC, possibly through reduced terminal NRTI excision. Furthermore, we showed that EFV facilitated efficient formation of stable, DEC-like complexes by TFV- or FTC-monophosphate (MP)-terminated DNA and this can contribute to the synergistic inhibition of HIV-1 RT by TFV-diphosphate (DP)+EFV and FTC-TP+EFV combinations.</p> <p>Conclusion</p> <p>This study demonstrated a clear correlation between the synergistic antiviral activities of TFV+FTC, TFV+EFV, FTC+EFV, and TFV+FTC+EFV combinations and synergistic HIV-1 RT inhibition at the enzymatic level. We propose the molecular mechanisms for the TFV+FTC+EFV synergy to be a combination of increased levels of the active metabolites TFV-DP and FTC-TP and enhanced DEC formation by a chain-terminated DNA and HIV-1 RT in the presence of the second and the third drug in the combination. This study furthers the understanding of the longstanding observations of synergistic anti-HIV-1 effects of many NRTI+NNRTI and certain NRTI+NRTI combinations in cell culture, and provides biochemical evidence that combinations of anti-HIV agents can increase the intracellular drug efficacy, without increasing the extracellular drug concentrations.</p

The Political Economy of Tax Reform in Bangladesh: Political Settlements, Informal Institutions and the Negotiation of Reform

political economy, tax reform, political settlementsThis paper explores the political economy of tax reform in Bangladesh over several decades, shedding light on the complex factors that account for unusually effective and sustained resistance to significant reform. We contend that it is necessary to understand both deep-seated formal and informal institutions and the micro-level incentives that shape the negotiation of short-term reform in order to comprehend tax outcomes. We describe a tax system that is highly informal, largely manual and characterised by high levels of discretion and corruption. However, despite appearing highly dysfunctional on the surface, this system serves the core interests of powerful political, economic and administrative actors. Underpinned by robust informal institutions, the current system delivers low and predictable tax rates to businesses, provides extensive discretion and opportunities for corruption to the tax administration, and acts as an important vehicle for political elites to raise funds and distribute patronage and economic rents. While the tax system has not been without reform, individual reform efforts have been constrained by the parameters of this broader settlement, leaving competing interest groups to pursue strategic gains at the margins while seeking to satisfy external reform demands. This tax bargain reflects Bangladesh’s broader political economy, which is characterised by entrenched informal institutions underpinning the combination of generally weak governance and high levels of economic growth – the so-called ‘paradox of Bangladesh’.DfID, NORA

BIOlogical Factors that Limit sustAined Remission in rhEumatoid arthritis (the BIO-FLARE study): protocol for a non-randomised longitudinal cohort study

Background Our knowledge of immune-mediated inflammatory disease (IMID) aetiology and pathogenesis has improved greatly over recent years, however, very little is known of the factors that trigger disease relapses (flares), converting diseases from inactive to active states. Focussing on rheumatoid arthritis (RA), the challenge that we will address is why IMIDs remit and relapse. Extrapolating from pathogenetic factors involved in disease initiation, new episodes of inflammation could be triggered by recurrent systemic immune dysregulation or locally by factors within the joint, either of which could be endorsed by overarching epigenetic factors or changes in systemic or localised metabolism. Methods The BIO-FLARE study is a non-randomised longitudinal cohort study that aims to enrol 150 patients with RA in remission on a stable dose of non-biologic disease-modifying anti-rheumatic drugs (DMARDs), who consent to discontinue treatment. Participants stop their DMARDs at time 0 and are offered an optional ultrasound-guided synovial biopsy. They are studied intensively, with blood sampling and clinical evaluation at weeks 0, 2, 5, 8, 12 and 24. It is anticipated that 50% of participants will have a disease flare, whilst 50% remain in drug-free remission for the study duration (24 weeks). Flaring participants undergo an ultrasound-guided synovial biopsy before reinstatement of previous treatment. Blood samples will be used to investigate immune cell subsets, their activation status and their cytokine profile, autoantibody profiles and epigenetic profiles. Synovial biopsies will be examined to profile cell lineages and subtypes present at flare. Blood, urine and synovium will be examined to determine metabolic profiles. Taking into account all generated data, multivariate statistical techniques will be employed to develop a model to predict impending flare in RA, highlighting therapeutic pathways and informative biomarkers. Despite initial recruitment to time and target, the SARS-CoV-2 pandemic has impacted significantly, and a decision was taken to close recruitment at 118 participants with complete data. Discussion This study aims to investigate the pathogenesis of flare in rheumatoid arthritis, which is a significant knowledge gap in our understanding, addressing a major unmet patient need