S. L. Cosby

An Immunohistochemical Study of the Distribution of the Measles Virus Receptors, CD46 and SLAM, in Normal Human Tissues and Subacute Sclerosing Panencephalitis

We have compared the expression of the known measles virus (MV) receptors, membrane cofactor protein (CD46) and the signaling lymphocyte-activation molecule (SLAM), using immunohistochemistry, in a range of normal peripheral tissues (known to be infected by MV) as well as in normal and subacute sclerosing panencephalitis (SSPE) brain. To increase our understanding of how these receptors could be utilized by wild-type or vaccine strains in vivo, the results have been considered with regard to the known route of infection and systemic spread of MV. Strong staining for CD46 was observed in endothelial cells lining blood vessels and in epithelial cells and tissue macrophages in a wide range of peripheral tissues, as well as in Langerhans’ and squamous cells in the skin. In lymphoid tissues and blood, subsets of cells were positive for SLAM, in comparison to CD46, which stained all nucleated cell types. Strong CD46 staining was observed on cerebral endothelium throughout the brain and also on ependymal cells lining the ventricles and choroid plexus. Comparatively weaker CD46 staining was observed on subsets of neurons and oligodendrocytes. In SSPE brain sections, the areas distant from lesion sites and negative for MV by immunocytochemistry showed the same distribution for CD46 as in normal brain. However, cells in lesions, positive for MV, were negative for CD46. Normal brain showed no staining for SLAM, and in SSPE brain only subsets of leukocytes in inflammatory infiltrates were positive. None of the cell types most commonly infected by MV show detectable expression of SLAM, whereas CD46 is much more widely expressed and could fulfill a receptor function for some wild-type strains. In the case of wild-type stains, which are unable to use CD46, a further as yet unknown receptor(s) would be necessary to fully explain the pathology of MV infection.

Inhibition of In Vitro Leukocyte Proliferation by Morbilliviruses

ABSTRACT Immune suppression associated with morbillivirus infections may influence the mortality rate by allowing secondary bacterial infections that are lethal to the host to flourish. Using an in vitro proliferation assay, we have shown that all members of the genus Morbillivirus inhibit the proliferation of a human B-lymphoblast cell line (BJAB). Proliferation of freshly isolated, stimulated bovine and caprine peripheral blood lymphocytes is also inhibited by UV-inactivated rinderpest (RPV) and peste-des-petits ruminants viruses. As for measles virus, coexpression of both the fusion and the hemagglutinin proteins of RPV is necessary and sufficient to induce immune suppression in vitro.

Cerebral endothelial cell infection by measles virus in subacute sclerosing panencephalitis : ultrastructural and in situ hybridization evidence

Infection of vascular endothelium plays a central role in the pathogenesis of acute measles virus infection outside the central nervous system (CNS) but has not been described in the human CNS. An ultrastructural survey was made of blood vessels in five cases of subacute sclerosing panencephalitis (SSPE) to determine whether or not infection of cerebral vascular endothelium occurred in this persistent fatal CNS disease caused by measles virus. Morbillivirus nucleo‐capsids were found in a few endothelial cells in three necropsy cases but not in the limited tissue available from two biopsies. In a severe parenchymal lesion in one necropsied case, endothelial cells hybridized in situ with a biotinylated probe specific for the N genomic RNA of measles virus. It is concluded that human cerebral endothelium is susceptible to measles virus infection.

Examination of eight cases of multiple sclerosis and 56 neurological and non-neurological controls for genomic sequences of measles virus, canine distemper virus, simian virus 5 and rubella virus.

In situ hybridization studies have been carried out on brain samples from eight cases of multiple sclerosis (MS) and 56 non-neurological and neurological controls, using single-stranded 35S-labelled RNA probes prepared against genomic RNA sequences of measles virus, canine distemper virus, rubella virus and simian virus 5. Foci of hybridization were found using probes against the measles virus nucleocapsid protein (N), phosphoprotein and fusion protein gene sequences in two of the MS cases, and also in one control, a case of disseminated cytomegalovirus infection with spinal cord necrosis. This result was confirmed using biotinylated probes prepared against the measles virus N genomic sequence. No hybridization was found in any of the MS or control cases using any of the other viral genome-specific probes.

Apoptosis in measles virus-infected human central nervous system tissues

The extent of apoptotic cell death was examined in central nervous system (CNS) tissues from three cases of subacute sclerosing panencephalitis (SSPE). Apoptosis was demonstrated by in situ end‐labelling of DNA in formalin‐fixed, paraffin‐embedded tissue sections. Measles virus and cell types were labelled by immunohistochemistry and/or in situ hybridization. Furthermore, bcl‐2 expression in SSPE was examined by immunohistochemistry. All three cases exhibited varying degrees of apoptosis in all CNS areas studied. Brain tissue from a non‐neurological control case did not show any significant apoptosis. Characterization of cell types demonstrated neurons, oligodendrocytes, lymphocytes and microglia undergoing apoptosis. A linear relationship could not be established between virus burden and the extent of apoptosis in any particular area. Virus‐negative cells were observed which were undergoing apoptosis. Bcl‐2 immunoreactivity in SSPE was confined to the infiltrating cell population. These results suggest that apoptosis of various cell types may contribute to the neuropathogenesis of measles virus infection in the human CNS, either as a direct effect of viral infection or by cytokine‐mediated responses.

Rhinovirus upregulates transient receptor potential channels in a human neuronal cell line: implications for respiratory virus-induced cough reflex sensitivity

Background The mechanism underlying respiratory virus-induced cough hypersensitivity is unknown. Upregulation of airway neuronal receptors responsible for sensing physical and chemical stimuli is one possibility, and the transient receptor potential (TRP) channel family are potential candidates. We have used an in vitro model of sensory neurons and human rhinovirus (HRV-16) to study the effect of virus infection on TRP expression. Methods IMR-32 neuroblastoma cells were differentiated in culture to express three TRP channels: TRPV1, TRPA1 and TRPM8. Flow cytometry and qRT-PCR were used to measure TRP channel protein and mRNA levels following inoculation with live virus, inactivated virus, virus-induced soluble factors or pelleted virus particles. Multiplex bioassay was used to determine nerve growth factor (NGF), interleukin (IL)-1β, IL-6 and IL-8 levels in response to infection. Results Early upregulation of TRPA1 and TRPV1 expression occurred 2–4 h post infection. This was independent of replicating virus as virus-induced soluble factors alone were sufficient to increase channel expression 50-fold and 15-fold, respectively. NGF, IL-6 and IL-8 levels, increased in infected cell supernatants, represent possible candidates. In contrast, TRPM8 expression was maximal at 48 h (9.6-fold) and required virus replication rather than soluble factors. Conclusions We show for the first time that rhinovirus can infect neuronal cells. Furthermore, infection causes upregulation of TRP channels by channel-specific mechanisms. The increase in TRPA1 and TRPV1 levels can be mediated by soluble factors induced by infection whereas TRPM8 requires replicating virus. TRP channels may be novel therapeutic targets for controlling virus-induced cough.

Development and clinical validation of a loop-mediated isothermal amplification method for the rapid detection of Neisseria meningitidis

Loop-mediated isothermal amplification (LAMP) is an innovative technique that allows the rapid detection of target nucleic acid sequences under isothermal conditions without the need for complex instrumentation. The development, optimization, and clinical validation of a LAMP assay targeting the ctrA gene for the rapid detection of capsular Neisseria meningitidis were described. Highly specific detection of capsular N. meningitidis type strains and clinical isolates was demonstrated, with no cross-reactivity with other Neisseria spp. or with a comprehensive panel of other common human pathogens. The lower limit of detection was 6 ctrA gene copies detectable in 48 min, with positive reactions readily identifiable visually via a simple color change. Higher copy numbers could be detected in as little as 16 min. When applied to a total of 394 clinical specimens, the LAMP assay in comparison to a conventional TaqMan® based real-time polymerase chain reaction system demonstrated a sensitivity of 100% and a specificity of 98.9% with a κ coefficient of 0.942. The LAMP method represents a rapid, sensitive, and highly specific technique for the detection of N. meningitidis and has the potential to be used as a point-of-care molecular test and in resource-poor settings.

ADHESION MOLECULE EXPRESSION AND LYMPHOCYTE ADHESION TO CEREBRAL ENDOTHELIUM : EFFECTS OF MEASLES VIRUS AND HERPES SIMPLEX 1 VIRUS

Expression of endothelial cell (EC) adhesion molecules is increased in inflammatory neurological disorders and this may regulate lymphocyte homing to the central nervous system (CNS). Viral encephalitis is characterised by lymphocytic infiltration of the CNS and one mechanism of this response may be EC adhesion molecule induction with consequent inflammatory cell/EC binding. This report characterises the effects of herpes simplex 1 (HSV1) or measles virus (MV) infection of BALB/c brain microvascular EC in vitro on adhesion of naive syngenic splenocytes and levels of ICAM-1. Adhesion was enhanced by 42% for MV-infected cells and by 73% for HSV-1-infected EC. At the multiplicities of infection employed, levels of ICAM-1 were upregulated on HSV-1-infected EC, but not on MV-infected EC. It is concluded that ICAM-1/ligand interactions do not play a role in mediation of MV enhancement of adherence, but represent one mechanism responsible for increased lymphocyte adherence to HSV-1-infected cerebral EC.

Approaches in the understanding of morbillivirus neurovirulence.

Certain members of the morbillivirus genus, canine distemper virus, phocine distemper virus, and the cetacean viruses of dolphins and porpoises exhibit high levels of central nervous system (CNS) infection in their natural hosts. CNS complications are rare for measles virus (MV) and are not associated with rinderpest virus (RPV) and peste des petits ruminants virus (PPRV) infection. However, both RPV and PPRV are neurovirulent in permissive murine strains. Human postmortem tissue, neural cell cultures, and animal models have been used to answer major questions concerning morbillivirus neurovirulence. Studies of the MV CNS complication subacute sclerosing panencephalitis (SSPE) indicate that virus could enter the CNS either by direct infection of endothelial cells or in infected leucocytes, followed by infection of predominately neurones and oligodendrocytes. It has been established that MV neurovirulence in mice is partially determined by the virus-receptor specificity. The two known MV receptors, CD46 and SLAM, have been examined in normal and SSPE brain tissue and the findings suggest that further receptors may be necessary to explain infection of the CNS with wild-type strains of MV. In both humans and mice (and in vitro), once infection of neurones has been established, virus spreads transneuronally. It is possible that all morbilliviruses transiently infect the CNS in their natural hosts, but development of disease is dependent on the efficiency of the immune response. Alternatively, for RPV and PPRV, virus entry may be restricted due either to absence of viral receptors or failure of virus to replicate or spread in the CNS.

Advantages of using recombinant measles viruses expressing a fluorescent reporter gene with vibratome slice technology in experimental measles neuropathogenesis

Aims: In this study of experimental measles neuropathogenesis, the utility of enhanced green fluorescent protein (EGFP) as a sensitive indicator of measles virus (MV) cell‐to‐cell spread in the central nervous system (CNS) has been assessed in vibratome‐cut brain slices to demonstrate the degree and mechanism of viral spread in the rodent CNS. Methods: Recombinant MVs expressing EGFP were visualized at different levels in 200‐μm vibratome‐cut brain sections from infected animals by confocal scanning laser microscopy (CSLM). Comparison was made with 7‐μm microtome sections, stained for the N protein of measles by immunocytochemistry (ICC). Results: The recombinant viruses were readily visualized in infected brain tissue, with no loss of neuropathogenicity. No difference was found in the sites of infection when MV infection was detected through EGFP fluorescence or by ICC. MV‐infected cells were detected in the cerebral cortex, olfactory bulb and tract, hippocampus, thalamus, hypothalamus, ependyma and subventricular zone. However, the 200‐μm vibratome‐cut sections and confocal microscopy proved excellent for demonstrating virus distribution in neurites and for in‐depth analysis of the extent of tract infection in the white matter of the cerebral hemispheres such as selective infection of the internal capsule and anterior commissure. Conclusions: The use of self‐tracing recombinant MVs, viewed in thick vibratome‐cut sections by CSLM, demonstrated that in experimental MV neuropathogenesis the infection is selective and spreads predominately by neurites using defined anatomical pathways.