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

Building blocks for DNA selfassembly

Abstract. DNA complexes, like the double crossover, are used as building blocks for the assembly of higher-order structures. Currently, the number of experimentally proven reliable complexes is small. We have begun work on expanding the collection of such complexes. Here we report on our design concepts and initial experiments. In particular, we present experimental evidence of two new complexes: quadruple crossovers and triangles. In principle, quadruple crossovers can be extended to three-dimensional, spacefilling lego brick complexes, while triangles are capable of hexagonally tiling the plane.

Solution of a 20-variable 3-SAT Problem on a DNA computer

A 20-variable instance of the NP-complete three-satisfiability (3-SAT) problem was solved on a simple DNA computer. The unique answer was found after an exhaustive search of more than 1 million (2 20) possibilities. This computational problem may be the largest yet solved by nonelectronic means. Problems of this size appear to be beyond the normal range of unaided human computation. The vast parallelism, exceptional energy efficiency, and extraordinary information density inherent in molecular computation have raised the possibility that molecular computers might some day prove capable of attacking problems that have resisted conventional methods (1–6). Numerous architectures for molecular computing have been proposed (1, 2, 7–17), and of these, several have been explored experimentall

The lipid–teichoic acid complex in the cytoplasmic membrane of Streptococcus faecalis N.C.I.B. 8191

1. A lipid–teichoic acid complex was isolated from Streptococcus faecalis N.C.I.B. 8191. The covalent nature of the linkage between teichoic acid and lipid was established. 2. The complex exhibits macromolecular properties in solution, and ultracentrifugation studies show that these are due to micelle formation. 3. From chemical studies it is concluded that the teichoic acid is a poly(glycerol phosphate) in which some of the glycerol hydroxyl groups possess kojibiosyl [2-O-α-d-glucopyranosyl-(1→2)-α-d- glucopyranosyl] substituents, together with d-alanine ester residues. 4. The lipid is 1-kojibiosyl diglyceride, already known as a membrane component of this organism, with probably a phosphatidyl substituent. The phosphatidyl kojibiosyl diglyceride is attached to the teichoic acid through a phosphodiester linkage, and the chain of the teichoic acid contains 28–35 units. 5. Although the complex represents the whole of the membrane teichoic acid in this organism, only about 12% of the membrane glycolipid is associated with teichoic acid. 6. Two phosphatidyl glycolipids, closely resembling that bearing the teichoic acid, were isolated from the lipids of the organism and were partly characterized