A rat model of Parkinson's disease induced by Japanese encephalitis virus.

In Fischer rats infected with Japanese encephalitis virus (JEV) at 13 days after birth and sacrificed 12 weeks later, the major pathological changes resembled those found in Parkinson's disease. Specifically there was neuronal loss with gliosis which was confined mainly to the zona compacta of the substantia nigra, with a notable absence of lesions in the cerebral cortex and cerebellum. Changes were bilateral being most severe in the central part of the zona compacta. Immunohistochemical studies with anti-tyrosine hydroxylase (TH) demonstrated that the number of TH-positive neurons was significantly decreased in the substantia nigra compared to controls, while comparable numbers of TH-positive neurons were found in the basal ganglia in both JEV-treated rats and age-matched controls. JEV-infected rats showed marked bradykinesia, with significant behavioral improvement being observed following administration of L-DOPA. Immunohistochemical studies failed to detect JEV antigens in any region of the rat brain and the JEV genome was undetectable in the substantia nigra and the cerebral cortex using the reverse transcription-polymerase chain reaction (RT-PCR). The findings suggest that JEV infection of rats under the conditions described may serve as a model of virus induced Parkinson's Disease.

Assembly of JC virus-like particles in COS7 cells.

JC virus lacks an appropriate cell line to support virus replication. The establishment of a JC pseudovirus assembly system could play an alternative role for a virus culture system. COS7 cells and a transfer vector, pcDL-SR alpha 296, were used to express JC viral structural genes. VP231-SR alpha, which encodes VP2/VP3 and VP1, but lacks 137 bp of the 5'-terminus of agnogene, showed both efficient nuclear migration and quantitative expression of the major capsid protein VP1. JC pseudovirus assembly was observed in the nucleus of VP231-SR alpha transfected cells. Evidence of JC pseudovirus assembly is presented. The further utilization of this system, which includes a study for the viral morphogenesis, serological diagnosis, as well as the potential application for gene transfer vector, is discussed.

Apoptotic process of cerebellar degeneration in experimental methylmercury intoxication of rats.

This report deals with the mechanism involved in the cerebellar degeneration following experimental methylmercury poisoning of male Wistar rats. The cerebellar granule cells of animals that exhibited typical hind leg paresis were shrunken and displayed marked nuclear pyknosis. At the ultrastructural level, the nuclei of these cells were condensed and fragmented, features which are characteristic of apoptosis. In situ staining for DNA strand breaks revealed that the pyknotic nuclei were positively labeled. DNA fragmentation was confirmed by agarose gel electrophoresis; a ladder pattern of multiples of approximately 200-base pair fragments, typical of apoptosis, was observed with the cerebellar DNA of the methylmercury-treated animals. These observations suggest that the degeneration of cerebellar granule cells by alkyl mercury compounds involves an apoptotic process.

Establishment and characterization of a carrier cell culture producing high titres of polyoma JC virus.

This report concerns a carrier cell culture (designated JCI) infected persistently with JC virus (JCV). Immunostaining with an anti-JCV antiserum revealed that JCI was a carrier culture in which only a small fraction of the cells (approximately 1.5%) produced the virus. The JCV titre was increased strikingly by incubating confluent JCI cells for 4-6 days in medium containing a low concentration of fetal bovine serum (2%). Viral genomes cloned from the persistently infected JCI cells were heterogeneous with respect to size, but most clones had an alteration of the same regulatory region (designated CR-JCI). Transfection experiments with a chimeric JCV DNA (Mad-1/CR-JCI), in which the regulatory region was CR-JCI and the other region was derived from an infectious JCV (Mad-1) DNA, showed that CR-JCI was less efficient in inducing viral growth than the regulatory regions of IMR-32-adapted JCVs. The transfected cells could be readily subcultured, and they continued to produce JCV. It is concluded that a decrease in the activity of the JCV regulatory region is of importance for the maintenance of the carrier state of JCI cells.

[Establishment and analysis of cell culture infected persistently with JC virus].

JC polyomavirus (JCV) is the causative agent of a demyelinating disease in the central nervous system, known as progressive multifocal leukoencephalopathy. Since it is difficult to propagate JCV in vitro, many laboratories have attempted to develop cell lines permissive for replication of JCV. It was recently reported that serial passage of JCV (strain Mad-1) in a human neuroblastoma cell line (IMR-32 cells) generated JCV variants (M1-IMRa, -IMRb, -IMRc) adapted to the growth in IMR-32 cells. We obtained a persistently-infected cell culture, designated JCI cells, by continued subcultivation of IMR-32 cells which had been infected with these adapted variants. To understand the mechanism by which the carrier state was maintained, we molecularly cloned multiple JCV DNAs from JCI cells. Sequencing of the cloned DNAs revealed that a majority of the clones had the same altered regulatory sequence which was probably generated from M1-IMRa. A DNA transfection experiment using a chimeric JCV DNA, in which the regulatory region was derived from JCI and the other region from Mad-1, showed that the JCI regulatory region was less efficient in inducing virus production in IMR-32 cells. Therefore, it could be concluded that the regulatory region is primarily important for maintaining the carrier state in JCI cells. Although JCI cells contained a significant amount of JCV, the yield of JCV could remarkably be increased by incubating a confluent JCI culture in a medium with a low serum concentration. Thus, JCI cells should greatly facilitate the production of JCV for future studies of the virus.