A novel borna disease virus vector system that stably expresses foreign proteins from an intercistronic noncoding region.

Borna disease virus (BDV), a nonsegmented, negative-strand RNA virus, infects a wide variety of mammalian species and readily establishes a long-lasting, persistent infection in brain cells. Therefore, this virus could be a promising candidate as a novel RNA virus vector enabling stable gene expression in the central nervous system (CNS). Previous studies demonstrated that the 5' untranslated region of the genome is the only site for insertion and expression of a foreign gene. In this study, we established a novel BDV vector in which an additional transcription cassette has been inserted into an intercistronic noncoding region between the viral phosphoprotein (P) and matrix (M) genes. The recombinant BDV (rBDV) carrying green fluorescent protein (GFP) between the P and M genes, rBDV P/M-GFP, expressed GFP efficiently in cultured cells and rodent brains for a long period of time without attenuation. Furthermore, we generated a nonpropagating rBDV, ΔGLLP/M, which lacks the envelope glycoprotein (G) and a splicing intron within the polymerase gene (L), by the transcomplementation system with either transient or stable expression of the G gene. Interestingly, rBDV ΔGLLP/M established a persistent infection in cultured cells with stable expression of GFP in the absence of the expression of G. Using persistently infected rBDV ΔGLLP/M-infected cells, we determined the amino acid region in the cytoplasmic tail (CT) of BDV G important for the release of infectious rBDV particles and also demonstrated that the CT region may be critical for the generation of pseudotyped rBDV having vesicular stomatitis virus G protein. Our results revealed that the newly established BDV vector constitutes an alternative tool not only for stable expression of foreign genes in the CNS but also for understanding the mechanism of the release of enveloped virions.

Prevalence of JC polyomavirus genomic sequences from the large T-antigen and non-coding control regions among Bulgarian patients with primary brain tumors.

A total of 111 fresh brain biopsies from patients with primary brain tumors were examined for JC polyomavirus sequences from the Large T antigen encoding region (LT) and the viral non-coding control region (NCCR). SYBR Green and TaqMan real-time polymerase chain reaction assays were used. In the glioblastoma group of 39 patients 48.7% were positive for LT sequences. Among the astrocytoma group (19 patients) and the oligodendroglioma group (12 patients) 31.6% and 33.3% were also positive. The prevalence of LT genomic sequences among the other groups was as follows: in 2 out of 3 oligoastrocytomas; in 3 out 5 gangliogliomas; in 2 out of 5 meduloblastomas; in 1 out 3 pineocytomas; and in none of the tested 5 ependimomas. All positive samples had a late threshold cycle that varied from 36 to 49, indicative of very low starting viral number. Only 21 of all the 111 samples were positive for NCCR. Low copy number in range of 10-1,000 was present. Notably, only 8 of all NCCR positive specimens were also LT positive. It might be suggested that the disproportion between the results for LT and NCCR is either due to clonally integrated LT fragments, with loss of genetic material, or changes in the NCCR. The latter would alter the productive course of the infection and may establish a premise for continuous interaction of viral regulatory proteins with cell molecules that are responsible for the control of the cell cycle. This may lead subsequently to malignant transformation.

Detection of JC polyomavirus DNA sequences and cellular localization of T-antigen and agnoprotein in oligodendrogliomas.

PURPOSE: Productive infection of the human neurotropic polyomavirus JCPyV in oligodendrocytes leads to the development of progressive multifocal leukoencephalopathy, a fatal demyelinating disorder of the central nervous system. In addition to its role in viral infection, JCPyV T-antigen can transform cells in vitro and induce tumors in experimental animals in the absence of viral DNA replication and late gene expression. The goal of this study is to examine the presence of JCPyV DNA sequences and viral antigens in a series of human oligodendrogliomas. EXPERIMENTAL DESIGN: A total of 20 well-characterized oligodendrogliomas were examined for detection of the JCPyV genome by PCR and immunohistochemistry for expression of viral proteins. RESULTS: Gene amplification has revealed the presence of JCPyV DNA sequences corresponding to the NH2-terminal of T-antigen in 15 of 20 samples. DNA sequences corresponding to late regions, which are responsible for production of the capsid protein, VP1, were detected in 14 of 20 samples. Sequencing of the viral control region determined the presence of JCPyV Mad-4 or JCPyV(CY) in these tumors. By immunohistochemistry, T-antigen expression was detected in the nuclei of tumor cells from 10 samples that also contained corresponding DNA sequences by PCR. Eleven of 20 tumors exhibited immunoreactivity for the late auxiliary gene product, agnoprotein. None of the samples showed immunoreactivity for the capsid proteins, ruling out productive infection of neoplastic cells by JCPyV. CONCLUSIONS: Collectively, these observations provide new evidence in support of the association of the oncogenic human neurotropic JCPyV and oligodendrogliomas.

Adeno-associated virus (AAV) as a vector for gene transfer into glial cells of the human central nervous system.

To examine the potential of AAV as a vector for gene transfer in glial cells, an established astrocytoma cell line and short-term cultures derived from human oligodendroglioma have been coinfected with AAV and helper adenovirus. The level of AAV replication in glioma cells was high indicating that they express receptors for AAV.

A case of diffuse leptomeningeal oligodendrogliomatosis associated with HHV-6 variant A.

We describe a rare case of diffuse leptomeningeal oligodendrogliomatosis associated with the human herpes virus 6 variant A (HHV-6 A). A 2-year-old boy presented with progressive neurological symptoms and hydrocephalus. The patient had a VP shunt placement but did not fully recover. HHV-6 A was detected in both CSF and serum by nested PCR. His symptoms improved repeatedly, but temporarily, on antiviral treatment. An open brain biopsy, ten months after presentation, revealed leptomeningeal tumour as well as the presence of viral DNA in the tumour tissue. The role of HHV-6 A could be that of a reactivated opportunist. However, this case also raises the question whether this neurotropic virus, with malignant transforming properties in vitro, may have a role in pathogenesis in some cases of brain malignancy.

Progressive multifocal leukoencephalopathy and oligodendroglioma in a monkey co-infected by simian immunodeficiency virus and simian virus 40.

A rhesus monkey experimentally inoculated with simian immunodeficiency virus (SIV) mac251 was killed 42 months later because of poor general condition. CD4 lymphocyte count which was 3,430/mm3 before inoculation, had decreased to 638/mm3 2 months before death. Neuropathological examination revealed changes characteristic of progressive multifocal leukoencephalopathy (PML) in the white matter of the cerebral hemispheres and brain stem. In situ hybridization was negative for JC virus but markedly positive for simian virus 40 (SV40) in the nuclei of many oligodendrocytes. Many oligodendrocytes also expressed p53. Within an area involved by PML, there was a densely cellular tumor with honeycomb appearance and elongated vessels characteristic of oligodendrogliomas. Within the tumor in situ hybridization for SV40 and immunocytochemistry for p53 were negative. Opportunistic infection by SV40 has been occasionally reported in experimentally SIV-infected monkeys resulting in PML or malignant astrocytoma. Association of JC virus-induced PML and astrocytomas has been reported in three human cases without AIDS. In those cases, as in our monkey, polyomaviruses (SV40 or JC virus) were expressed in the areas with PML but not in the glial tumor. Association of PML and oligodendroglioma has not been reported previously to our knowledge. The relationship between oligodendrocyte proliferation and polyomavirus infection of oligodendrocytes is unclear. Our findings suggest that binding of the viral protein to p53 may result in inactivation of the pro-apoptotic protein favoring the proliferation of a randomly occurring tumoral clone of oligodendrocytes.

Detection of JC virus DNA sequence and expression of the viral oncoprotein, tumor antigen, in brain of immunocompetent patient with oligoastrocytoma.

We describe molecular and clinical findings in an immunocompetent patient with an oligoastrocytoma and the concomitant presence of the human papovavirus, JC virus (JCV), which is the etiologic agent of the subacute, debilitating demyelinating disease, progressive multifocal leukoencephalopathy. Histologic review revealed a glial neoplasm consisting primarily of a moderately cellular oligodendroglioma with distinct areas of a fibrillary astrocytoma. Immunohistochemical analysis revealed nuclear staining of tumor cells with antibodies against the viral oncoprotein [tumor antigen (T antigen)], the proliferation marker (Ki67), and the cellular proliferation regulator (p53). Using primers specific to the JCV control region, PCR yielded amplified DNA that was identical to the control region of the Mad-4 strain of the virus. PCR analysis demonstrated the presence of the genome for the viral oncoprotein, T antigen, and results from primer extension studies revealed synthesis of the viral early RNA for T antigen in the tumor tissues. The presence of viral T antigen in the tumor tissue was further demonstrated by immunoblot assay. To our knowledge, this is the first report of the presence of JCV DNA, RNA, and T antigen in tissue in which viral T antigen is localized to tumor cell nuclei and suggests the possible association of JCV with some glial neoplasms.