Murdoch University Research Repository

Welcome to the Murdoch University Research Repository

The Murdoch University Research Repository is an open access digital collection of research
created by Murdoch University staff, researchers and postgraduate students.

Learn more

The growth and characterisation of a morbilli-like virus isolated from multiple sclerosis brain tissue

White, Anthony Robert (1995) The growth and characterisation of a morbilli-like virus isolated from multiple sclerosis brain tissue. PhD thesis, Murdoch University.

[img]
PDF - Whole Thesis
Available Upon Request

Abstract

Antibodies to closely related multiple sclerosis (MSV) and feline (MV631 and CCA147) viral isolates were adsorbed to an affinity chromatography column containing one of several activated supports and through this was passed homogenised brain tissue from MS or non-MS patients. Morbillivirus-like particles were identified in 10 separate elutions from 6 MS brains but not in eluates obtained from 3 control brains. The virus-like particles were pleomorphic, 150-350 nm in diameter, often revealed a limiting membrane and contained 16-18 nm diameter tubules morphologically similar to morbillivirus nucleocapsids. The tubules occasionally revealed periodic cross-striations indicative of helical coiling but the limiting membranes did not possess glycoprotein spikes common to morbillivirus particles. Elution profile analysis of MS and control brain eluates revealed significantly higher protein levels in the MS eluates.

Primary rat and mouse glial cell cultures were prepared and maintained for 4-5 weeks post seeding. The presence of oligodendrocytes was established by phase contrast microscopy and confirmed using light and electron microscopic immunocytochemistry with horse radish peroxidase (HRP) labelled antisera to 2', 3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) and galactocerebroside (GalC). Cultures were inoculated at 4 to 7 days in vitro with affinity chromatography purified viral isolates from 5 different cases of MS or with identically prepared control eluates.

A cytopathic effect (CPE), in the form of multinucleated giant cells (MNGCs) and inclusion body formation, was seen 5-14 days post infection in some of the cultures inoculated with each MS viral isolate but not in cultures inoculated with non-MS eluates or in uninoculated cultures. The MNGCs were CNPase and GalC positive and the presence of viral antigen in these cells was confirmed using HRP-labelled antisera to MS and antigenically related feline viral isolates. Viral antigen was also detected in non-cytopathic oligodendrocytes in virally-infected cultures and the presence of nucleic acid in viral inclusions was suggested through acridine orange staining. Replication of the agent in oligodendrocytes did not result in apparent cellular necrosis or immune-related activation (as assessed by staining for macrophage and microglial cell markers). Possible morbillivirus intranuclear inclusions were identified in many MNGCs but not in noncytopathic cells. In addition, oligodendrocytes in infected cultures cross-reacted with antisera to measles (MV) and canine distemper (CDV) viruses.

Oligodendrocyte-like cells continued to express viral protein up to 40 days post-infection. Viral infection of astrocytes was not observed but astrocytic growth was inhibited adjacent to multinucleated oligodendroglia. Viral-antigen expression but not CPE could be passaged to fresh cultures via concentrated media from cytopathogenic cultures.

Ultrastructural examination of infected oligodendroglia showed the presence of cytoplasmic inclusions which contained 16-18 nm diameter nucleocapsid-like tubules. The surface of some of the infected cells showed viral budding with a morphology typical of that seen in paramyxovirus replication. The cytoplasmic inclusions and viral budding were stained with the HRP-labelled antisera to the viral isolates. Electron microscopic examination of negatively stained media from infected cultures revealed the presence of virions morphologically similar to those isolated from MS brain tissue by affinity chromatography.

ELISA analysis of MS brain eluates revealed significantly higher absorbance readings with antisera to MS and feline isolates than with antisera to unrelated viruses. The same antisera (anti-MSV and anti- MV631) also produced high absorbance readings on lysates of MS virus cultures. Antisera to MV and CDV similarly produced higher absorbance readings than unrelated viral antisera on MS eluates and cultures.

Immunoblot analysis was performed on MS viral eluates, infected glial cell culture lysates, infected cultures of CVIs and a CRFK cell-line infected with a similar feline virus (MV631) using antisera to the viral isolates. A protein with an approximate molecular weight (Mr) of 66,000 (66K) was detected in all isolates and infected cell-lines but not in control material using antisera to MS and feline viral isolates. An additional protein with a molecular weight of 45,000 (45K) was also detected in some infected cell lysates but not MS isolates. The 66K protein was shown to cross-react with antisera to measles and canine distemper viruses and may represent a morbillivirus nucleocapsid protein. Preliminary analysis of MS sera involving ELISA and immunocytochemical techniques suggested that MS patients may express specific antibodies to the viral proteins.

Polymerase chain reaction (PCR) studies using primer pairs against conserved morbillivirus and paramyxovirus sequences have, however, proved negative on RNA extracted from MS and feline isolates and infected tissue cultures. Successful amplification of Z?-actin mRNA from infected and control cultures and consistent detection of positive control viral and plasmid RNA suggested that RNA integrity was conserved. Highly sensitive nested PCR also failed to detect any known viral sequences in the infected cultures or isolates using conserved primers. Further attempts to determine the sequence of the putative viral nucleic acid are in progress.

The results of the study described here indicate that a cytopathic agent repeatedly isolated from MS brain tissue has an affinity for oligodendrocytes in vitro. The identity of the agent is yet to be confirmed but reveals several features in common with the Paramyxoviridae family of viruses. Although the infection of oligodendroglia could be associated with the demyelinating process seen in MS and cats, the pathological mechanism by which this may occur is not known. The infection of oligodendroglia by the MS-derived agents did not result in necrosis or other morphological changes in this cell-type (syncytial formation did not involve a large proportion of the infected oligodendrocytes), however, the possibility that the virus contains peptide sequences cross-reactive with myelin components should be investigated, as a virus-triggered autoimmune attack may be responsible for initiating demyelination. Alternatively, the agent may be sequestered at the site of inflammation and is not involved in the aetiology of lesion formation.

Item Type: Thesis (PhD)
Murdoch Affiliation: School of Biological and Environmental Sciences
Notes: Note to the author: If you would like to make your thesis openly available on Murdoch University Library's Research Repository, please contact: repository@murdoch.edu.au. Thank you.
Supervisor(s): Cook, Robert
URI: http://researchrepository.murdoch.edu.au/id/eprint/52305
Item Control Page Item Control Page