Periventricular demyelination and axonal pathology is associated with subependymal virus spread in a murine model for multiple sclerosis.

OBJECTIVES: Theiler's murine encephalomyelitis virus (TMEV) infection of mice is a widely used animal model for demyelinating disorders, such as multiple sclerosis (MS). The aim of the present study was to identify topographical differences of TMEV spread and demyelination in the brain of experimentally infected susceptible SJL/J mice and resistant C57BL/6 mice. METHODS: Demyelination was confirmed by Luxol fast blue and cresyl violet staining and axonal damage by neurofilament-specific and ß-amyloid precursor protein-specific immunohistochemistry. Viral dissemination within the central nervous system (CNS) was quantified by immunohistochemistry and in situ hybridization. Further, the phenotype of infected cells was determined by confocal laser scanning microscopy. RESULTS: An early transient infection of periventricular cells followed by demyelination and axonopathies around the fourth ventricle in SJL/J mice was noticed. Periventricular and brain stem demyelination was associated with a predominant infection of microglia/macrophages and oligodendrocytes. CONCLUSIONS: Summarized, the demonstration of ependymal infection and subjacent spread into the brain parenchyma as well as regional virus clearance despite ongoing demyelination and axonal damage in other CNS compartments allows new insights into TME pathogenesis. This novel aspect of TMEV CNS interaction will enhance the understanding of region-specific susceptibilities to injury and regenerative capacities of the brain in this MS model.

Intraventricular injection of myxoma virus results in transient expression of viral protein in mouse brain ependymal and subventricular cells.

Oncolytic viruses that selectively infect and lyse cancer cells have potential as therapeutic agents. Myxoma virus, a poxvirus that is known to be pathogenic only in rabbits, has not been reported to infect normal tissues in humans or mice. We observed that when recombinant virus was injected directly into the lateral ventricle of the mouse brain, virally encoded red fluorescent protein was expressed in ependymal and subventricular cells. Cells were positive for nestin, a marker of neural stem cells. Rapamycin increased the number of cells expressing the virally encoded protein. However, protein expression was transient. Cells expressing the virally encoded protein did not undergo apoptosis and the ependymal lining remained intact. Myxoma virus appears to be safe when injected into the brain despite the transient expression of virally derived protein in a small population of periventricular cells.

Human Hendra virus infection causes acute and relapsing encephalitis.

AIM: To study the pathology of two cases of human Hendra virus infection, one with no clinical encephalitis and one with relapsing encephalitis. METHODS: Autopsy tissues were investigated by light microscopy, immunohistochemistry and in situ hybridization. RESULTS: In the patient with acute pulmonary syndrome but not clinical acute encephalitis, vasculitis was found in the brain, lung, heart and kidney. Occasionally, viral antigens were demonstrated in vascular walls but multinucleated endothelial syncytia were absent. In the lung, there was severe inflammation, necrosis and viral antigens in type II pneumocytes and macrophages. The rare kidney glomerulus showed inflammation and viral antigens in capillary walls and podocytes. Discrete necrotic/vacuolar plaques in the brain parenchyma were associated with antigens and viral RNA. Brain inflammation was mild although CD68(+) microglia/macrophages were significantly increased. Cytoplasmic viral inclusions and antigens and viral RNA in neurones and ependyma suggested viral replication. In the case of relapsing encephalitis, there was severe widespread meningoencephalitis characterized by neuronal loss, macrophages and other inflammatory cells, reactive blood vessels and perivascular cuffing. Antigens and viral RNA were mainly found in neurones. Vasculitis was absent in all the tissues examined. CONCLUSIONS: The case of acute Hendra virus infection demonstrated evidence of systemic infection and acute encephalitis. The case of relapsing Hendra virus encephalitis showed no signs of extraneural infection but in the brain, extensive inflammation and infected neurones were observed. Hendra virus can cause acute and relapsing encephalitis and the findings suggest that the pathology and pathogenesis are similar to Nipah virus infection.

A TAT-modified fusion protein efficiently penetrates mouse hypoglossal nuclei from transduced ependyma.

Future gene therapy for brainstem variant amyotrophic lateral sclerosis may be technically difficult if gene therapy vectors are injected near vital cardiorespiratory centers or if large portions of the tongue and pharyngeal muscles must be peripherally injected for retrograde transport of vectors to motor neurons. In this study we show that it is possible to deliver recombinant proteins to the hypoglossal nuclei without brainstem or muscle injections, by taking advantage of enhanced uptake of fusion proteins containing the protein transduction domain from the human immunodeficiency virus TAT protein. Adenoviral vectors expressing either TAT-modified or native beta-glucuronidase (beta-gluc) were injected into the lateral cerebral ventricles of mice, transducing ventricular epithelium down to the level of the obex in the brainstem. There was significant uptake into the hypoglossal nuclei of TAT-modified but not native beta-glucuronidase. The TAT-modified beta-gluc appeared to encompass half or more of the hypoglossal nuclei as visualized by retrograde labeling with cholera toxin subunit b in adjacent sections. TAT-modification of gene products may allow a relatively non-invasive approach to brainstem gene therapy via cerebroventricular injection.

Adeno-associated virus type 4 (AAV4) targets ependyma and astrocytes in the subventricular zone and RMS.

The subventricular zone (SVZ) is one of the neurogenic niches in the adult mammalian brain. The SVZ is of interest for studies on neurogenesis and stem cell therapy. Here, we report specific transduction of ependyma and/or astrocytes by recombinant adeno-associated virus type 4 (AAV4) viral vectors. AAV4 vectors encoding beta-galactosidase or eGFP were injected into the lateral ventricles of neonatal and adult C57BL/6 mouse brains. In addition, SVZ injections were conducted on adult mice. AAV4 vectors show a characteristic transduction of the ependyma independent of delivery route. However, AAV4 virus injected into the SVZ targeted GFAP positive astrocytes forming the glial tube in the SVZ and rostral migratory stream (RMS). Our results introduce AAV4 as a new tool by which to manipulate glial cells in the RMS.

Transduction of the choroid plexus and ependyma in neonatal mouse brain by vesicular stomatitis virus glycoprotein-pseudotyped lentivirus and adeno-associated virus type 5 vectors.

Evaluation of gene transfer into the developing mouse brain has shown that when adeno-associated virus serotype 1 (AAV1) or AAV2 vectors are injected into the cerebral lateral ventricles at birth, widespread parenchymal transduction occurs. Lentiviral vectors have not been tested by this route. In this study, we found that injection of lentiviral vectors pseudotyped with vesicular stomatitis virus glycoprotein (VSV-G) resulted in targeted transduction of the ependymal cells lining the ventricular system and the choroid plexus along the entire rostrocaudal axis of the brain, whereas a Mokola pseudotype transduced only a few cells after injection into the neonatal ventricle. In contrast, when lentiviral vectors pseudotyped with either VSV-G or Mokola glycoprotein are injected into the adult mouse brain, they transduce similar patterns of cells. An Ebola-Zaire-pseudotyped vector did not transduce any neonatal CNS cells, as was also the case for adult parenchymal injections. Long-term gene expression (12 months) occurred with a constitutively active mammalian promoter and a self-inactivating long terminal repeat (LTR), whereas the cytomegalovirus promoter in a vector with an intact LTR was expressed only in short-term experiments. We found that an AAV5 vector also targeted the ependymal and choroid plexus cells throughout the ventricular system. This vector exhibited limited penetration from the ventricle to other structures, which was significantly different from the previously reported patterns of transduction after intraventricular injection of AAV1 and AAV2 vectors.

Recombinant AAV serotype 1 transduction efficiency and tropism in the murine brain.

Recombinant adeno-associated virus serotype 2 (rAAV2) vectors have shown promise as therapeutic agents for neurologic disorders. However, intracerebral administration of this vector leads to preferential transduction of neurons and a restricted region of transgene expression. The recently developed rAAV vectors based upon nonserotype 2 viruses have the potential to overcome these limitations. Therefore, we directly compared a rAAV type 1 to a type 2 vector in the murine brain. The vectors were engineered to carry identical genomes (AAV2 terminal repeat elements flanking an enhanced green fluorescent protein expression cassette) and were administered by stereotaxic-guided intracerebral injection. We found that the rAAV1 vector (rAAV1-GFP) had a 13- to 35-fold greater transduction efficiency than that of the rAAV2 vector (rAAV2-GFP). Also, rAAV1-transduced cells were observed at a greater distance from the injection site than rAAV2-transduced cells. Neurons were the predominant cell type transduced by both vector types. However, in contrast to rAAV2-GFP, rAAV1-GFP was capable of transducing glial and ependymal cells. Thus, rAAV1-based vectors have biologic properties within the brain distinct from that of rAAV2. These differences might be capitalized upon to develop novel gene transfer strategies for neurologic disorders.

VZV fulminant necrotizing encephalitis with concomitant EBV-related lymphoma and CMV ventriculitis: report of an AIDS case.

A case of AIDS with varicella zoster virus fulminant necrotizing encephalitis associated with cytomegalovirus ependymitis-subependymitis and a periventricular Epstein-Barr virus-related lymphoma is described. The patient had no herpes zoster cutaneous eruptions and died three days after the onset of symptoms. Varicella zoster virus and cytomegalovirus antigens were found by immunohistochemistry in the same area around a necrotic periventricular lesion; a periventricular lymphoma, large B cell type, was also observed. In situ hybridization with Epstein-Barr virus-encoded- RNAs probe was positive in about 40% of the neoplastic cells. The association of herpes-related lesions in the same cerebral region should be consistent in AIDS cases with acute neurological symptoms.

A neuroattenuated ICP34.5-deficient herpes simplex virus type 1 replicates in ependymal cells of the murine central nervous system.

Herpes simplex virus type 1 (HSV-1) variant 1716 is deleted in the gene encoding ICP34.5 and is neuroattenuated after intracranial inoculation of mice. Although the mechanism of attenuation is unclear, this property has been exploited to eliminate experimental brain tumours. Previously, it was shown that infectious 1716 was recoverable for up to 3 days after intracranial inoculation suggesting that there may be limited replication in the central nervous system (CNS). Here it is demonstrated that 1716 replicates in specific cell types (predominantly CNS ependymal cells) of BALB/c mice, using immunohistochemical, immunofluorescence, in situ hybridization and virus titration studies. While 1716-infected mice exhibited no overt signs of encephalitis, histological analysis showed a persistent loss of the ependymal lining. Thus, although ICP34.5-deficient viruses are neuroattenuated, they do retain the ability to replicate in and destroy the ependyma of the murine CNS. A detailed understanding of the mechanism(s) of neuroattenuation and limited replication could lead to the rational design of safe HSV vectors for cancer and gene therapy in the CNS.

CMV-infected subependymoma in the fourth ventricle of an HIV-1 infected patient.

A case of an AIDS patient with a CMV-infected subependymoma of the fourth ventricle is presented. The tumor was incidentally found at autopsy and was not suspected during the clinical course. This is the first report of a subependymoma in HIV-1 infection. Moreover, infection of a tumor with CMV is an extremely rare condition, which has until now been described only once in an anaplastic astrocytoma.