Encephalitis lethargica and Influenza: a case of mistaken identity with a twist

Following the Influenza epidemic in 1918, physicians observed an increase in cases of Encephalitis lethargica  (EL), the acute (often lethal) phase followed by post encephalitic parkinsonism (PEP), the latter affecting patients for several decades.

Based on the observation that these patients might have been infected with the 1918 Influenza virus, the conclusion was reached that a long-term consequence of Influenza might be the development of a neurological disease. This notion was supported by experimental studies in mice inoculated with neurovirulent variants of Influenza virus strains A/NWS/33 and A/WSN/33  - both strains which are highly lab adapted, but still used in Influenza virus research - as well as circumstantial evidence suggested that patients in 1918 died not only of pulmonary edema but also of neurogenic congestive heart failure. Furthermore one of the variants of the 1918 Influenza virus contains single change in the amino acid sequence that changes its binding properties to receptors, thus making the virus -potentially- neurovirulent

Other evidence dating back to the 1918/1919 epidemic suggested that the neuropathology of EL during and after the epidemic was unique. Arguments against a link between the Influenza virus epidemic and the occurrence of EL were already presented in the 1920s, mainly pointing out that the increase in EL preceded the epidemic by at least two years. This assumes however that the epidemic truly started in 1918 - as I pointed out earlier there is some evidence that local outbreaks of Influenza occurred in 1915 in both Great Britain and France.

What is the relation between Influenza virus and neuropathological abnormalities, namely death of neuronal cells? As I mentioned, mice were infected with neurovirulent strains of A/NWS/33 and A/WSN/33 and the brain tissue was subsequently analyzed for abnormalities. Following nasal inoculation of susceptible mice with the WSN virus, cytotoxic T- Lymphocytes are activated as well as Microglia and neuronal cells of the olfactory bulb are infected. As part of the antiviral response, cytotoxic CD8+ T- Lymphocytes produce cytokines and release cytolytic proteins such Perforin, Granzyme B, and FasL. FasL -or Fas ligand- can bind to the Fas receptor and induces the external/Caspase-3 dependent pathway of apoptosis, in other words leading to cell death, whilst

Perforin/Granzyme B inserts into the cell membrane and forms pores - similar to a bag filled with water which slowly leaks if a tiny hole is present. 

Infection of neuronal cells (as well of T- Lymphocytes) with Influenza A induces the expression of both the Fas ligand and the Fas receptor, thus inducing the clearance of infected cells in an autocrine manner. As mentioned in a different post, the 1918 virus also expressed a PB1-F2 protein with a high capacity for apoptosis. Taken together this explains the why the experimental neurovirulent strain of WSN induces cell death of (mouse) neurons. The infection of Microglia  - which are antigen presenting cells and thus part of the immune system- with neurovirulent WSN leads not only to the activation of macrophages, but also to the secretion of neurotrophin (NT), Annexin V and basal fibroblast growth factor (bFGF) into the surrounding tissue, thus protecting non infected neuronal cells. Activated macrophages on the other hand, secrete tumor necrosis factor (TNF)-α, reactive oxygen Intermediates (ROI) and Nitric Oxyde (NO), all of them leading to the death of infected cells and -potentially- to inflammation. 

The importance of the neuroprotective effect was also highlighted by studies in mice using the neurovirulent strain R404BP virus which is a experimental variant of the neurovirulent A/H1N1/WSN expres-sing the matrix and neuraminidase genes of the neurovirulent WSN strain (H1N1) and other genes of the non-neurovirulent A/Aichi/2/68 strain (H3N2). This strain is only fatal if injected directly into the CNS of mice but not upon nasal infection of the olfactory neuroepithelium, suggesting that the viral infection is cleared after the initial infection.

If Influenza would have been the causative agent of Encephalitis lethargica traces of the Influenza genome or viral particles should be detectable in brain specimens from individuals who died from Encephalitis lethargica. In order to detect remains of viral particles or whole viral particles, brain specimens from patients who died either of modern EL or PEP were analyzed by both Transmission electron microscopy and immunohistochemistry.

Indeed 27nm virus like particles (VLP) were detected in the cytoplasm and in nuclei of neurons in samples of confirmed classic Encephalitis lethargica patients and both larger (50 nm) and smaller particles (27nm) intranuclear particles in modern EL cases as well as in a PEP case. Immunohistochemistry analysis however revealed that these particles derived from two Enteroviruses, Poliovirus and Coxsackievirus B4. Both viruses are known to cause viral meningitis. These results were confirmed by real time PCR and by comparing cell cultures infected with Poliovirus and Coxsackievirus B4. The VLP observed in the patient samples therefore most likely represent viral factories.

Does it mean that the 1918 Influenza virus and modern Influenza viruses do not infect the neuronal system? No it doesn’t. But as I pointed out the infection of neuronal cells does not only have apoptotic effects but also induces neuroprotection in mice. Other Influenza viruses that show a high degree of neuropathogenesis are those that are classified as highly pathogenic avian influenza viruses, including A/H7N1, A/H5N1, and A/Whistling Swan/Shimane/499/83 (H5N3). As in the case of the most recent human pandemic Influenza virus, A/H1N1/2009, these exhibit both α2,3- and α2,6-linked sialic acid receptor binding properties (i.e. they bind both to human and avian cells as well to cells located in both the upper and lower human respiratory tract) and exhibit neurological symptoms. Experimental infection of the human neuroblastoma cell lines SK-N-SH and SH-SY5Y the human GBM847 glioblastoma patient isolate with A/CA/7/2009 (an isolate of pandemic A/H1N1/2009) showed that those are permissive for A/CA/7/2009. Hypothetically it is possible that neurovirulent Influenza viruses might infect neurons and then -over time- use the neuronal network to to migrate to the brain, thus leading to neurodegenerative diseases, such as Parkinson. So far however no link has been proven.

What about Encephalitis lethargica then? After all it might be an autoimmune disease and not caused (directly) by pathogens.

However, as the controversy surrounding Encephalitis lethargica illustrates is that seemingly benign viruses such as Enteroviruses harbor serious side effects and emphasizes the importance for a careful analysis of epidemiological data. 

Further reading:

Maurizi CP (2010). Influenza caused epidemic encephalitis (encephalitis lethargica): the circumstantial evidence and a challenge to the nonbelievers. Medical hypotheses, 74 (5), 798-801 PMID: 20060230

Foley PB (2009). Encephalitis lethargica and the influenza virus. II. The influenza pandemic of 1918/19 and encephalitis lethargica: epidemiology and symptoms. Journal of neural transmission (Vienna, Austria : 1996), 116 (10), 1295-308 PMID: 19707848

Fujimoto I, Takizawa T, Ohba Y, & Nakanishi Y (1998). Co-expression of Fas and Fas-ligand on the surface of influenza virus-infected cells. Cell death and differentiation, 5 (5), 426-31 PMID: 10200492 

Ward AC (1996). Neurovirulence of influenza A virus. Journal of neurovirology, 2 (3), 139-51 PMID: 8799206

Nichols JE, Niles JA, & Roberts NJ Jr (2001). Human lymphocyte apoptosis after exposure to influenza A virus. Journal of virology, 75 (13), 5921-9 PMID: 11390593

Gomes, C., Ferreira, R., George, J., Sanches, R., Rodrigues, D., Gonçalves, N., & Cunha, R. (2013). Activation of microglial cells triggers a release of brain-derived neurotrophic factor (BDNF) inducing their proliferation in an adenosine A2A receptor-dependent manner: A2A receptor blockade prevents BDNF release and proliferation of microglia Journal of Neuroinflammation, 10 (1) DOI: 10.1186/1742-2094-10-16

Chaves AJ, Busquets N, Valle R, Rivas R, Vergara-Alert J, Dolz R, Ramis A, Darji A, & Majó N (2011). Neuropathogenesis of a highly pathogenic avian influenza virus (H7N1) in experimentally infected chickens. Veterinary research, 42 PMID: 21982125

Mori I, Goshima F, Imai Y, Kohsaka S, Sugiyama T, Yoshida T, Yokochi T, Nishiyama Y, & Kimura Y (2002). Olfactory receptor neurons prevent dissemination of neurovirulent influenza A virus into the brain by undergoing virus-induced apoptosis. The Journal of general virology, 83 (Pt 9), 2109-16 PMID: 12185263

Lo KC, Geddes JF, Daniels RS, & Oxford JS (2003). Lack of detection of influenza genes in archived formalin-fixed, paraffin wax-embedded brain samples of encephalitis lethargica patients from 1916 to 1920. Virchows Archiv : an international journal of pathology, 442 (6), 591-6 PMID: 12695912

McCall S, Henry JM, Reid AH, & Taubenberger JK (2001). Influenza RNA not detected in archival brain tissues from acute encephalitis lethargica cases or in postencephalitic Parkinson cases. Journal of neuropathology and experimental neurology, 60 (7), 696-704 PMID: 11444798

Dale RC, Church AJ, Surtees RA, Lees AJ, Adcock JE, Harding B, Neville BG, & Giovannoni G (2004). Encephalitis lethargica syndrome: 20 new cases and evidence of basal ganglia autoimmunity. Brain : a journal of neurology, 127 (Pt 1), 21-33 PMID: 14570817