JC virus infection of meningeal and choroid plexus cells in patients with progressive multifocal leukoencephalopathy.

JC virus (JCV) can cause a lytic infection of oligodendrocytes and astrocytes in the central nervous system (CNS) leading to progressive multifocal leukoencephalopathy (PML). JCV can also infect meningeal and choroid plexus cells causing JCV meningitis (JCVM). Whether JCV also infects meningeal and choroid plexus cells in PML patients and other immunosuppressed individuals with no overt symptoms of meningitis remains unknown. We therefore analyzed archival formalin-fixed, paraffin-embedded brain samples from PML patients, and HIV-seropositive and seronegative control subjects by immunohistochemistry for the presence of JCV early regulatory T Ag and JCV VP1 late capsid protein. In meninges, we detected JCV T Ag in 11/48 (22.9%) and JCV VP1 protein in 8/48 (16.7%) PML patients. In choroid plexi, we detected JCV T Ag in 1/7 (14.2%) and JCV VP1 protein in 1/8 (12.5%) PML patients. Neither JCV T Ag nor VP1 protein could be detected in meninges or choroid plexus of HIV-seropositive and HIV-seronegative control subjects without PML. In addition, examination of underlying cerebellar cortex of PML patients revealed JCV-infected cells in the molecular layer, including GAD 67+ interneurons, but not in HIV-seropositive and HIV-seronegative control subjects without PML. Our findings suggest that productive JCV infection of meningeal cells and choroid plexus cells also occurs in PML patients without signs or symptoms of meningitis. The phenotypic characterization of JCV-infected neurons in the molecular layer deserves further study. This data provides new insight into JCV pathogenesis in the CNS.

Retrograde infection of precerebellar nuclei neurons by injection of a recombinant adenovirus into the cerebellar cortex of normal and reeler mice.

The reeler mouse is an autosomal recessive mutant mouse caused by mutation of the reelin gene and characterized by cerebellar ataxia. To determine whether the distribution pattern of precerebellar nuclei neurons in the brainstem of the reeler mouse changes, we injected a small volume of a replication-defective recombinant adenovirus carrying E. coli beta-galactosidase (lacZ) into the cerebellar cortex of normal and reeler mice. Five days later, the mice were transcardially perfused by a fixative solution. X-gal staining of coronal or sagittal sections of the brainstem revealed that many origins for reticulocerebellar, cuneocerebellar, trigeminocerebellar, and pontocerebellar projections were retrogradely labeled, but only a few olivocerebellar neurons were labeled. Retrogradely labeled neurons in the lateral reticular nucleus tended to locate more laterally and be more condensed into a small compartment in the reeler compared with their normal counterparts. Retrogradely labeled neurons in the external cuneate nucleus were more dorsally shifted in the reeler mice compared with their normal counterparts. We could not find any differences between the normal and reeler mice in the distribution patterns of their trigeminocerebellar projection neurons. Retrogradely labeled pontocerebellar neurons in the basilar pons of the reeler mouse were reduced in number compared with their normal counterparts in addition to being more ventrally and laterally shifted. These findings strongly suggest that the migration of some precerebellar nuclei neurons from the rhombic lip to their final loci may be obstructed in the reeler mice.

Tioman virus infection in experimentally infected mouse brain and its association with apoptosis.

Tioman virus is a newly described bat-urine derived paramyxovirus isolated in Tioman Island, Malaysia in 2001. Hitherto, neither human nor animal infection by this virus has been reported. Nonetheless, its close relationship to another paramyxovirus, the Menangle virus which had caused diseases in humans and pigs [Philbey, A.W., Kirkland, P.D., Ross, A.D., Davis, R.J., Gleeson, A.B., Love, R.J., Daniels, P.W., Gould, A.R., Hyatt, A.D., 1998. An apparently new virus (family Paramyxoviridae) infectious for pigs, humans, and fruit bats. Emerg. Infect. Dis. 4, 269-271], raises the possibility that it may be potentially pathogenic. In this study, mice were experimentally infected with Tioman virus by intraperitoneal and intracerebral routes, and the cellular targets and topographical distribution of viral genome and antigens were examined using in situ hybridization and immunohistochemistry, respectively. The possible association between viral infection and apoptosis was also investigated using the TUNEL assay and immunohistochemistry to FasL, Caspase-3, Caspase-8, Caspase-9 and bcl-2. The results showed that Tioman virus inoculated intracerebrally was neurotropic causing plaque-like necrotic areas, and appeared to preferentially replicate in the neocortex and limbic system. Viral infection of inflammatory cells was also demonstrated. TUNEL and Caspase-3 positivity was found in inflammatory cells but not in neurons, while FasL, Caspase-8 and Caspase-9 were consistently negative. This suggests that neuronal infection was associated with necrosis rather than apoptosis. Moreover, the data suggest that there may be an association between viral infection and apoptosis in inflammatory cells, and that it could, at least in part, involve Caspase-independent pathways. Bcl-2 was expressed in some neurons and inflammatory cells indicating its possible role in anti-apoptosis. There was no evidence of central nervous system infection via the intraperitoneal route.

PKR and RNase L contribute to protection against lethal West Nile Virus infection by controlling early viral spread in the periphery and replication in neurons.

West Nile virus (WNV) is a neurotropic, mosquito-borne flavivirus that can cause lethal meningoencephalitis. Type I interferon (IFN) plays a critical role in controlling WNV replication, spread, and tropism. In this study, we begin to examine the effector mechanisms by which type I IFN inhibits WNV infection. Mice lacking both the interferon-induced, double-stranded-RNA-activated protein kinase (PKR) and the endoribonuclease of the 2',5'-oligoadenylate synthetase-RNase L system (PKR(-/-) x RL(-/-)) were highly susceptible to subcutaneous WNV infection, with a 90% mortality rate compared to the 30% mortality rate observed in congenic wild-type mice. PKR(-/-) x RL(-/-) mice had increased viral loads in their draining lymph nodes, sera, and spleens, which led to early viral entry into the central nervous system (CNS) and higher viral burden in neuronal tissues. Although mice lacking RNase L showed a higher CNS viral burden and an increased mortality, they were less susceptible than the PKR(-/-) x RL(-/-) mice; thus, we also infer an antiviral role for PKR in the control of WNV infection. Notably, a deficiency in both PKR and RNase L resulted in a decreased ability of type I IFN to inhibit WNV in primary macrophages and cortical neurons. In contrast, the peripheral neurons of the superior cervical ganglia of PKR(-/-) x RL(-/-) mice showed no deficiency in the IFN-mediated inhibition of WNV. Our data suggest that PKR and RNase L contribute to IFN-mediated protection in a cell-restricted manner and control WNV infection in peripheral tissues and some neuronal subtypes.

Cell specificity and efficiency of the Semliki forest virus vector- and adenovirus vector-mediated gene expression in mouse cerebellum.

Establishing efficient gene transfer and expression in post-mitotic neurons is important in understanding the genetic basis of neural circuits with cellular complexity. This study evaluates the properties of exogenous green fluorescent protein (GFP) expression mediated by the Semliki forest virus (SFV) and adenovirus (Ad) vectors in dissociated and slice cultures of the mouse cerebellum. Infection with SFV-GFP resulted in early-onset and high-level GFP expression in about 90% of Purkinje cells and in about 40% of granule cells in dissociated cultures at 1 day after infection. Two days after infection, GFP-positive cells showed signs of SFV-derived cytotoxicity. Ad-GFP infected almost all astrocytes and granule cells in dissociated cultures, and showed a steady increase in GFP fluorescence with a plateau at around 2 days post-infection. Ad vector-mediated GFP expression lasted for several weeks with no significant cell damage. In the slice cultures, both viral vectors mainly infected astroglial cells, but also showed a similar cell preference as that in dissociated cultures. These data indicate that the use of different viral vectors and infection conditions offers a powerful means of expressing exogenous genes in cerebellar cultures with different cell-type specificity and timing and duration of expression.

Retrograde and anterograde labeling of cerebellar afferent projection by the injection of recombinant adenoviral vectors into the mouse cerebellar cortex.

Adenoviral vectors have recently been recognized as highly efficient systems for gene delivery into various tissues. We show that a reporter gene introduced into nerve terminals via an adenovirus can be used to label cell bodies retrogradely and then label the axons and nerve terminals of the infected neurons anterogradely in vivo. We injected a replication-defective recombinant adenovirus carrying the E. coli beta-galactosidase gene (lacZ) into the cerebellar cortex of the adult mouse. The first evidence of retrograde labeling was obtained at 2 days after the infection when neurons in the pontine nuclei and the reticulotegmental nucleus of the pons weakly expressed beta-galactosidase, and at 3 days post-infection when neurons in all precerebellar nuclei, known to project to the cerebellar cortex, were strongly stained with X-gal in a Golgi-like manner. Anterograde transport of lacZ gene products was recognized at 3 days post-infection; beta-galactosidase-positive axons arose from somata or dendrites of retrogradely labeled neurons, passed through the middle or inferior cerebellar peduncles, and entered the cerebellum. Anterogradely labeled mossy terminals were recognized on the injection side at 8 days post-infection, and on the contralateral side at 14 days post-infection. Beta-galactosidase expression persisted for up to two months, with a decrease in the total number of labeled cells over time. We could not find any signs of anterograde or retrograde transsynaptic labeling in the nuclei synaptically linked to the cerebellar cortex at any time point after injection up to 58 days post-infection.