To investigate the hypothesis that neurological sequelae are associated with persistent West Nile virus (WNV) and neuropathology, we developed an electrophysiological motor unit number estimation (MUNE) assay to measure the health of motor neurons temporally in hamsters. The MUNE assay was successful in identifying chronic neuropathology in the spinal cords of infected hamsters. MUNE was suppressed at days 9 to 92 in hamsters injected subcutaneously with WNV, thereby establishing that a long-term neurological sequela does occur in the hamster model. MUNE suppression at day 10 correlated with the loss of neuronal function as indicated by reduced choline acetyltransferase staining (R2 = 0.91). Between days 10 and 26, some α-motor neurons had died, but further neuronal death was not detected beyond day 26. MUNE correlated with disease phenotype, because the lowest MUNE values were detected in paralyzed limbs. Persistent WNV RNA and foci of WNV envelope-positive cells were identified in the central nervous systems of all hamsters tested from 28 to 86 days. WNV-positive staining colocalized with the neuropathology, which suggested that persistent WNV or its products contributed to neuropathogenesis. These results established that persistent WNV product or its proteins cause dysfunction, that WNV is associated with chronic neuropathological lesions, and that this neurological sequela is effectively detected by MUNE. Inasmuch as WNV-infected humans can also experience a poliomyelitis-like disease where motor neurons are damaged, MUNE may also be a sensitive clinical or therapeutic marker for those patients.
An understanding of the different phases of West Nile virus (WNV) disease is important to understand the natural history and the prevention and control of the disease. The initial infection leading to the viremic phase elicits innate (31, 39) and acquired (e.g., WNV-specific cytotoxic T lymphocytes) (41) immune responses. The West Nile fever syndrome may be associated with this phase of infection, which involves fever, headache, malaise, fatigue, and nausea. The virus can infect the central nervous system (CNS) to cause acute neurological clinical syndromes, such as meningitis, encephalitis, and paralysis (13, 34). The innate and acquired immune responses participate to prevent, reduce, or eliminate viral load in the CNS, but they may also contribute to neuroimmunopathology (11). The resulting neurological damage can persist as long-term neurological sequelae, which are widespread among survivors. The pathogenesis and cause of neurological sequelae have not been established, nor is the role that the virus plays in long-term disease known (13, 34).
Some evidence suggests that WNV can establish a persistent infection, which may adversely affect neuronal tissues. WNV was isolated from rhesus monkey brains up to 5½ months after intracerebral inoculations; and the monkeys from which these viruses were isolated possessed subacute inflammatory neurodegeneration of the CNS (27). This property of persistence is shared with some other flaviviruses, (i.e., tick-borne encephalitis virus in primates) (28), Japanese encephalitis virus in pregnant mice (20), Modoc virus in hamsters (8), and St. Louis encephalitis virus in hamsters (40). These preliminary data provided impetus for the hypothesis that neurological sequelae are associated with persistent WNV infection and neuropathology.
Our data (32) and the research of others (38) have established that WNV infects neurons, among which are large α-motor neurons in the spinal cord, to cause cellular damage or death involving apoptotic signaling or necrosis (4). The loss of motor neurons is detected in other neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) (30, 36) and poliomyelitis (25), by using electrophysiological motor unit number estimation (MUNE) (5), in which a motor unit consists of an α-motor neuron and all the muscle fibers it innervates. A presumptive use of MUNE in the human WNV infection was identified in an uncontrolled clinical study (2) in which MUNE appeared to be a marker for degrees of weakness and recovery in patients with neurological sequelae.
In this study, we establish that WNV can persistently infect neurons to cause dysfunction, that WNV is associated with chronic neuropathological lesions in the spinal cord, and that this neurological sequela as detected by MUNE is nearly a universal event in WNV-infected hamsters.