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

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

Specificity, duplex degradation and subcellular localization of antagomirs

MicroRNAs (miRNAs) are an abundant class of 20–23-nt long regulators of gene expression. The study of miRNA function in mice and potential therapeutic approaches largely depend on modified oligonucleotides. We recently demonstrated silencing miRNA function in mice using chemically modified and cholesterol-conjugated RNAs termed ‘antagomirs’. Here, we further characterize the properties and function of antagomirs in mice. We demonstrate that antagomirs harbor optimized phosphorothioate modifications, require >19-nt length for highest efficiency and can discriminate between single nucleotide mismatches of the targeted miRNA. Degradation of different chemically protected miRNA/antagomir duplexes in mouse livers and localization of antagomirs in a cytosolic compartment that is distinct from processing (P)-bodies indicates a degradation mechanism independent of the RNA interference (RNAi) pathway. Finally, we show that antagomirs, although incapable of silencing miRNAs in the central nervous system (CNS) when injected systemically, efficiently target miRNAs when injected locally into the mouse cortex. Our data further validate the effectiveness of antagomirs in vivo and should facilitate future studies to silence miRNAs for functional analysis and in clinically relevant settings

Complexes Formed in Solution Between Vanadium(IV)/(V) and the Cyclic Dihydroxamic Acid Putrebactin or Linear Suberodihydroxamic Acid

The ability of microbial siderophores to coordinate metal ions, particularly Fe(III), continues to generate interest in the poten-tial applications of these bioligands in the environment and medicine.13 Siderophores produced by terrestrial and marin

Research priorities for global food security under extreme events

International audienc

DNA Extraction by XPCR

The extraction of DNA strands including a given sequence of bases is a crucial step in the Adleman-Lipton extract model of DNA computing. A special type of PCR is presented in this paper, with a related algorithm which performs a specified extraction from a given pool of DNA double stranded molecules. This kind of PCR, Cross Pairing PCR (or XPCR), was tested in several situations and used ina biotechnological procedure which implements the extraction algorithm

Temperature gradient-based DNA computing for graph problems with weighted edges

Abstract. We propose an encoding method of numerical data in DNA using temperature gradient. We introduce melting temperature (Tm) for this purpose. Melting temperature is a unique characteristic to manipulate the hybridization and denaturation processes that used in the key steps in DNA computing such as the solution generation step and the amplification step. DNA strands of lower melting temperature tend to denature with ease and also be easily amplified by slightly modified polymerase chain reaction, called denaturation temperature gradient polymerase chain reaction. Using these properties, we implement a local search molecular algorithm using temperature gradient, which is contrasted to conventional exhaustive search molecular algorithms. The proposed methods are verified by solving an instance of the travelling salesman problem. We could effectively amplify the correct solution and the use of temperature gradient made the detection of solutions easier.