Generation of a Cell Culture-Adapted Hepatitis C Virus with Longer Half Life at Physiological Temperature

BACKGROUND: We previously reported infectious HCV clones that contain the convenient reporters, green fluorescent protein (GFP) and Renilla luciferase (Rluc), in the NS5a-coding sequence. Although these viruses were useful in monitoring viral proliferation and screening of anti-HCV drugs, the infectivity and yield of the viruses were low. METHODOLOGY/PRINCIPAL FINDINGS: In order to obtain a highly efficient HCV cultivation system, we transfected Huh7.5.1 cells [1] with JFH 5a-GFP RNA and then cultivated cells for 20 days. We found a highly infectious HCV clone containing two cell culture-adapted mutations. Two cell culture-adapted mutations which were responsible for the increased viral infectivity were located in E2 and p7 protein coding regions. The viral titer of the variant was ∼100-fold higher than that of the parental virus. The mutation in the E2 protein increased the viability of virus at 37°C by acquiring prolonged interaction capability with a HCV receptor CD81. The wild-type and p7-mutated virus had a half-life of ∼2.5 to 3 hours at 37°C. In contrast, the half-life of viruses, which contained E2 mutation singly and combination with the p7 mutation, was 5 to 6 hours at 37°C. The mutation in the p7 protein, either singly or in combination with the E2 mutation, enhanced infectious virus production about 10-50-fold by facilitating an early step of virion production. CONCLUSION/SIGNIFICANCE: The mutation in the E2 protein generated by the culture system increases virion viability at 37°C. The adaptive mutation in the p7 protein facilitates an earlier stage of virus production, such as virus assembly and/or morphogenesis. These reporter-containing HCV viruses harboring adaptive mutations are useful in investigations of the viral life cycle and for developing anti-viral agents against HCV

Progress in gene therapy for neurological disorders

Diseases of the nervous system have devastating effects and are widely distributed among the population, being especially prevalent in the elderly. These diseases are often caused by inherited genetic mutations that result in abnormal nervous system development, neurodegeneration, or impaired neuronal function. Other causes of neurological diseases include genetic and epigenetic changes induced by environmental insults, injury, disease-related events or inflammatory processes. Standard medical and surgical practice has not proved effective in curing or treating these diseases, and appropriate pharmaceuticals do not exist or are insufficient to slow disease progression. Gene therapy is emerging as a powerful approach with potential to treat and even cure some of the most common diseases of the nervous system. Gene therapy for neurological diseases has been made possible through progress in understanding the underlying disease mechanisms, particularly those involving sensory neurons, and also by improvement of gene vector design, therapeutic gene selection, and methods of delivery. Progress in the field has renewed our optimism for gene therapy as a treatment modality that can be used by neurologists, ophthalmologists and neurosurgeons. In this Review, we describe the promising gene therapy strategies that have the potential to treat patients with neurological diseases and discuss prospects for future development of gene therapy

Cryopreservation of Seeds and Seed Embryos in Orthodox-, Intermediate-, and Recalcitrant-Seeded Species

Seeds are one of the preferable and most used sources of germplasm for the ex situ preservation of plant genetic resources. They are generally stored dry at -20 °C in seed banks following international standards. However, some seeds do not tolerate drying and/or storage at -20 °C, or present short lifespans at these conditions. For them cryopreservation is indicated for long-term preservation. When seeds tolerate desiccation (i.e., orthodox seeds), they can be dried at about 32 ± 3% relative humidity at 18 °C and stored in the vapor phase of liquid nitrogen. This is the method followed in the Millennium Seed Bank of the Royal Botanic Gardens, Kew, for wild species with short lifespans in the standard conditions of seed banks. When seeds do not tolerate desiccation (i.e., recalcitrant seeds) or their tolerance to desiccation and/or -20 °C storage is limited (i.e., intermediate seeds), drying and cooling procedures must be adjusted, and often, cryoprotection is also required. Some methods are detailed for diverse species of temperate and tropical origin