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.
Faculty of Life Sciences, London Metropolitan University, London, UK. dourmashkin@blueyonder.co.uk
Robert R
RR
Dourmashkin
Glynis
G
Dunn
Victor
V
Castano
Sherman A
SA
McCall
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001921:Brain / Q000821:virology; D018792:Encephalitis, Viral / Q000150:complications / Q000821:virology*; D029822:Enterovirus B, Human / Q000302:isolation & purification*; D004769:Enterovirus Infections / Q000150:complications / Q000821:virology*; D005260:Female; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D046529:Microscopy, Electron, Transmission; D008875:Middle Aged; D010301:Parkinson Disease, Postencephalitic / Q000209:etiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction