Evidence for an enterovirus as the cause of encephalitis lethargica.

BACKGROUND: The epidemic of encephalitis lethargica (EL), called classical EL, was rampant throughout the world during 1917-1926, affecting half a million persons. The acute phase was lethal for many victims. Post-encephalitic parkinsonism (PEP) affected patients for decades. Our purpose was to investigate the cause of classical EL by studying the few available brain specimens. Cases of PEP and modern EL were also studied. Transmission electron microscopy (TEM) and immunohistochemistry were employed to examine brain from four classical EL cases, two modern EL cases and one PEP case. METHODS: Standard methods for TEM, immunohistochemistry and RTPCR were applied. RESULTS: 27 nm virus-like particles (VLP) were observed in the cytoplasm and nuclei of midbrain neurons in all classical EL cases studied. Large (50 nm) VLP and 27 nm intranuclear VLP were observed in the modern EL cases and the PEP case. Influenza virus particles were not found. VLP were not observed in the control cases. TEM of cell cultures inoculated with coxsackievirus B4 and poliovirus revealed both small and large intranuclear virus particles and small cytoplasmic particles, similar to the VLP in EL neurons. In the EL brains, nascent VLP were embedded in putative virus factories and on endoplasmic reticulum (ER). The VLP in the cases of classical EL survived, whereas ribosomes underwent autolysis due to the lack of refrigeration and slow formaldehyde fixation of whole brain. The VLP were larger than ribosomes from well preserved brain. Immunohistochemistry of classical EL cases using anti-poliovirus and anti-coxsackievirus B polyclonal antibodies showed significant staining of cytoplasm and nuclei of neurons as well as microglia and neuropil. Purkinje cells were strongly stained.A 97-bp RNA fragment of a unique virus was isolated from brain tissue from acute EL case #91558. Sequence analysis revealed up to 95% identity to multiple human Enteroviruses. Additional cases had Enterovirus positive reactions by real time PCR. CONCLUSIONS: The data presented here support the hypothesis that the VLP observed in EL tissue is an Enterovirus.

Supplementary data on virus-like particles in the brainstem of Parkinson's disease patients and controls.

In this study, we present 84 transmission electron microscopy (TEM) images of human brainstem tissue from 11 cases of late onset Parkinson's disease (PD). The tissues were fixed, embedded, sectioned, and stained for TEM application. In addition, we present 14 images from autopsy specimens of 1 case of human poliomyelitis infection as positive controls and 14 images from 8 cases of autopsy specimens of other conditions as negative controls. In the TEM images of the PD cases there were cytoplasmic inclusion bodies consisting of virus-like particles (VLP) 30 nm in diameter that were associated with endoplasmic reticulum membranes.  In the nuclei of the PD neurons there were VLP ranging from 40 nm to 50 nm in diameter. In the poliomyelitis cases, similar particles as were observed in PD which were interpreted to be poliomyelitis virus particles. In the negative controls one case was identified which showed similar VLP (Figure 1, controls).  A Lewy body was found in this "control" case (Figure 10) suggesting that this was an undiagnosed case of PD. Cytoplasmic ribosomes measuring approximately 17 nm were observed in the control neurons.

Virus-like particles and enterovirus antigen found in the brainstem neurons of Parkinson's disease.

Background: In a previous study on encephalitis lethargica, we identified an enterovirus in autopsy brain material. Transmission electron microscopy (TEM), immunohistochemistry (IHC) and molecular analysis were employed.  Our present objective was to investigate, using a similar approach, as to whether virus-like particles (VLP) and enterovirus antigen are present in Parkinson's disease (PD) brainstem neurons. Methods: Fixed tissue from autopsy specimens of late onset PD and control brainstem tissue were received for study. The brain tissue was processed for TEM and IHC according to previous published methods. Results:  We observed VLP in the brainstem neurons of all the cases of PD that were examined.  In the neurons' cytoplasm there were many virus factories consisting of VLP and endoplasmic reticulum membranes. In some neurons, the virus factories contained incomplete VLP. Complete VLP in some neurons' virus factories had an average diameter of 31 nm, larger than control brain ribosomes. In the nuclei, there were VLP with an average diameter of 40 nm. In cases of human poliomyelitis, there were cytoplasmic virus factories and intranuclear virus particles similar to those observed in PD. On preparing PD brain sections for IHC there was positive staining using anti-poliovirus antibody and anti-coxsackie antibody. This result was statistically significant. Conclusions: We present evidence for an enterovirus infection in PD.  For future studies, virus isolation and molecular analysis is suggested.

Immunogold electron microscopy recognizes prion protein-associated particles prepared from scrapie-infected mouse brain.

Previous studies have proposed that the disease isoform of prion protein (PrPSc) is particulate. Our purpose was to search by electron microscopy (EM) for particles in fractions of density gradients prepared from differentially centrifuged homogenates of scrapie-infected, normal, and null mouse brain. Only mild detergents were used during the separation process. The low-density fractions derived from scrapie-infected brain were rich in PrP. Three morphologically distinct types of particle were observed. Type 1 particles, measuring approximately 6.8 nm in mean diameter, were found in abundance in the fractions of scrapie-infected brain at the peak PrP concentrations. They were often clumped and adherent to raft-like structures. Type 2 particles, in low-density fractions from normal brain, were similar to type 1 but were smaller, with the mean diameter measuring approximately 5.3 nm. Type 3 particles from null brain differed morphologically from types 1 and 2 and were not clumped. The low density of the particles indicated a lipid component, which was confirmed by lipid analysis. Immunogold EM using Mab 6H4 labeled a portion of the particles from scrapie mouse brain, but not those from normal or null brain. Dimensions of PrP suggest that the labeled particles carry a PrPSc dimer per particle.