Venkatraman Siddharthan

Axonal transport mediates West Nile virus entry into the central nervous system and induces acute flaccid paralysis

West Nile virus (WNV) has emerged as a significant cause of epidemic viral encephalitis and flaccid limb paralysis, yet the mechanism by which it enters the CNS remains uncertain. We used compartmentalized neuron cultures to demonstrate that WNV spreads in both retrograde and anterograde directions via axonal transport. Transneuronal spread of WNV required axonal release of viral particles and was blocked by addition of a therapeutic neutralizing antibody. To test the physiologic significance of axonal transport in vivo, we directly inoculated the sciatic nerve of hamsters with WNV. Intrasciatic infection resulted in paralysis of the hind limb ipsilateral but not contralateral to the injection site. Limb paralysis was blocked either by surgical transection of the sciatic nerve or treatment with the therapeutic neutralizing antibody. Collectively, these studies establish that WNV undergoes bidirectional spread in neurons and that axonal transport promotes viral entry into the CNS and acute limb paralysis. Moreover, antibody therapeutics directly inhibit transneuronal spread of WNV infection and prevent the development of paralysis in vivo.

Humanized Monoclonal Antibody against West Nile Virus Envelope Protein Administered after Neuronal Infection Protects against Lethal Encephalitis in Hamsters

Humans infected with West Nile virus (WNV) may clinically present with symptoms that are suggestive of neurological infection. Nearly all treatments of WNV disease have been effective in animal models only if administered before or soon after viral challenge. Here, we evaluated whether a potent neutralizing anti-WNV humanized monoclonal antibody (MAb), hE16, could improve the course of disease in a hamster model when administered after the virus had infected neurons in the brain. Five days after viral injection, WNV was detected in the brains of hamsters by cytopathic assay, quantitative reverse-transcription polymerase chain reaction, and immunohistochemical staining of WNV envelope in neurons. Notably, 80%-90% of the hamsters treated 5 days after viral injection by intraperitoneal injection with hE16 survived the disease, compared with 37% of the placebo-treated hamsters (P< or =.001). The hamsters that received hE16 directly in the brain also exhibited markedly improved survival rates, compared with those in the placebo-treated hamsters. In prospective experiments, hamsters with high levels of infectious WNV in their cerebrospinal fluid were also protected by hE16 when administered 5 days after viral injection. These experiments suggest that humanized MAbs with potent neutralizing activity are a possible treatment for human patients after WNV has infected neurons in the central nervous system.

Efficacy of orally administered T-705 pyrazine analog on lethal West Nile virus infection in rodents

We describe herein that a pyrazine derivative, T-705 (6-fluoro-3-hydroxy-2-pyrazinecarboxamide), is protective for a lethal West Nile virus infection in rodents. Oral T-705 at 200 mg/kg administered twice daily beginning 4h after subcutaneous (s.c.) viral challenge protected mice and hamsters against WNV-induced mortality, and reduced viral protein expression and viral RNA in brains. The minimal effective oral dose was between 20 and 65 mg/kg when administered twice a day beginning 1 day after viral s.c. challenge of mice. Treatment could be delayed out to 2 days after viral challenge to still achieve efficacy in mice. Further development of this compound should be considered for treatment of WNV.

Increased Blood-Brain Barrier Permeability Is Not a Primary Determinant for Lethality of West Nile Virus Infection in Rodents

Blood-brain barrier (BBB) permeability was evaluated in mice and hamsters infected with West Nile virus (WNV, flavivirus) as compared to those infected with Semliki Forest (alphavirus) and Banzi (flavivirus) viruses. BBB permeability was determined by measurement of fluorescence in brain homogenates or cerebrospinal fluid (CSF) after intraperitoneal (i.p.) injection of sodium fluorescein, by macroscopic examination of brains after i.p. injection of Evans blue, or by measurement of total protein in CSF compared to serum. Lethal infection of BALB/c mice with Semliki Forest virus and Banzi virus caused the brain : serum fluorescence ratios to increase from a baseline of 2-4% to as high as 11 and 15%, respectively. Lethal infection of BALB/c mice with WNV did not increase BBB permeability. When C57BL/6 mice were used, BBB permeability was increased in some, but not all, of the WNV-infected animals. A procedure was developed to measure BBB permeability in live WNV-infected hamsters by comparing the fluorescence in the CSF, aspirated from the cisterna magnum, with the fluorescence in the serum. Despite a time-dependent tendency towards increased BBB permeability in some WNV-infected hamsters, the highest BBB permeability values did not correlate with mortality. These data indicated that a measurable increase in BBB permeability was not a primary determinant for lethality of WNV infection in rodents. The lack of a consistent increase in BBB permeability in WNV-infected rodents has implications for the understanding of viral entry, viral pathogenesis and accessibility of the CNS of rodents to drugs or effector molecules.

Defining limits of treatment with humanized neutralizing monoclonal antibody for West Nile virus neurological infection in a hamster model

ABSTRACT A potent anti-West Nile virus (anti-WNV)-neutralizing humanized monoclonal antibody, hE16, was previously shown to improve the survival of WNV-infected hamsters when it was administered intraperitoneally (i.p.), even after the virus had infected neurons in the brain. In this study, we evaluated the therapeutic limit of hE16 for the treatment of WNV infection in hamsters by comparing single-dose peripheral (i.p.) therapy with direct administration into the pons through a convection-enhanced delivery (CED) system. At day 5 after infection, treatments with hE16 by the peripheral and the CED routes were equally effective at reducing morbidity and mortality. In contrast, at day 6 only the treatment by the CED route protected the hamsters from lethal infection. These experiments suggest that hE16 can directly control WNV infection in the central nervous system. In support of this, hE16 administered i.p. was detected in a time-dependent manner in the serum, cerebrospinal fluid (CSF), cerebral cortex, brain stem, and spinal cord in CSF. A linear relationship between the hE16 dose and the concentration in serum was observed, and maximal therapeutic activity occurred at doses of 0.32 mg/kg of body weight or higher, which produced serum hE16 concentrations of 1.3 μg/ml or higher. Overall, these data suggest that in hamsters hE16 can ameliorate neurological disease after significant viral replication has occurred, although there is a time window that limits therapeutic efficacy.

An Inactivated Cell Culture Japanese Encephalitis Vaccine (JE-ADVAX) Formulated with Delta Inulin Adjuvant Provides Robust Heterologous Protection against West Nile Encephalitis via Cross-Protective Memory B Cells and Neutralizing Antibody

ABSTRACT West Nile virus (WNV), currently the cause of a serious U.S. epidemic, is a mosquito-borne flavivirus and member of the Japanese encephalitis (JE) serocomplex. There is currently no approved human WNV vaccine, and treatment options remain limited, resulting in significant mortality and morbidity from human infection. Given the availability of approved human JE vaccines, this study asked whether the JE-ADVAX vaccine, which contains an inactivated cell culture JE virus antigen formulated with Advax delta inulin adjuvant, could provide heterologous protection against WNV infection in wild-type and β2-microglobulin-deficient (β2m−/−) murine models. Mice immunized twice or even once with JE-ADVAX were protected against lethal WNV challenge even when mice had low or absent serum cross-neutralizing WNV titers prior to challenge. Similarly, β2m−/− mice immunized with JE-ADVAX were protected against lethal WNV challenge in the absence of CD8+ T cells and prechallenge WNV antibody titers. Protection against WNV could be adoptively transferred to naive mice by memory B cells from JE-ADVAX-immunized animals. Hence, in addition to increasing serum cross-neutralizing antibody titers, JE-ADVAX induced a memory B-cell population able to provide heterologous protection against WNV challenge. Heterologous protection was reduced when JE vaccine antigen was administered alone without Advax, confirming the importance of the adjuvant to induction of cross-protective immunity. In the absence of an approved human WNV vaccine, JE-ADVAX could provide an alternative approach for control of a major human WNV epidemic.

Persistent West Nile Virus Associated with a Neurological Sequela in Hamsters Identified by Motor Unit Number Estimation

ABSTRACT 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.

West Nile virus–induced acute flaccid paralysis is prevented by monoclonal antibody treatment when administered after infection of spinal cord neurons

Acute flaccid polio-like paralysis occurs during natural West Nile virus (WNV) infection in a subset of cases in animals and humans. To evaluate the pathology and the possibility for therapeutic intervention, the authors developed a model of acute flaccid paralysis by injecting WNV directly into the sciatic nerve or spinal cord of hamsters. By directly injecting selected sites of the nervous system with WNV, the authors mapped the lesions responsible for hind limb paralysis to the lumbar spinal cord. Immunohistochemical analysis of spinal cord sections from paralyzed hamsters revealed that WNV-infected neurons localized primarily to the ventral motor horn of the gray matter, consistent with the polio-like clinical presentation. Neuronal apoptosis and diminished cell function were identified by TUNEL (terminal deoxynucleotidyl transferase—mediated BrdUTP nick end labeling) and choline acetyltransferase staining, respectively. Administration of hE16, a potently neutralizing humanized anti-WNV monoclonal antibody, 2 to 3 days after direct WNV infection of the spinal cord, significantly reduced paralysis and mortality. Additionally, a single injection of hE16 as late as 5 days after WNV inoculation of the sciatic nerve also prevented paralysis. Overall, these experiments establish that WNV-induced acute flaccid paralysis in hamsters is due to neuronal infection and injury in the lumbar spinal cord and that treatment with a therapeutic antibody prevents paralysis when administered after WNV infection of spinal cord neurons.

C3H/HeN mouse model for the evaluation of antiviral agents for the treatment of Venezuelan equine encephalitis virus infection

The TC-83 vaccine strain of Venezuelan equine encephalitis virus (VEEV) causes encephalitis and death in C3H/HeN mice infected by intranasal (i.n.) instillation. Since TC-83 is exempt as a select agent, this mouse model was used in the evaluation of antiviral therapies. Virus titers in the brains of infected mice peaked on 4 dpi and persisted at high levels until death at 9.4+/-0.5 dpi. Mouse brains appeared histologically normal on 2 dpi, but developed meningoencephalitis, neuropil vacuolation, and gliosis by 8 dpi. Results from a protein cytokine array showed significant elevations over time in interleukin (IL)-1alpha, IL-1beta, IL-6, IL-12, MCP-1, IFNgamma, TNFalpha, MIP-1alpha, and RANTES in homogenized brain samples of infected mice. Immunohistochemical staining showed a colocalization of viral antigen with neuron markers. Treatment with interferon-alpha B/D or ampligen significantly improved survival, brain virus titer and cytokine levels, mean day-to-death, and weight change in infected mice. The time-course of infection and disease parameters of mice infected with TC-83 VEEV were similar in many ways to disease parameters in mice infected with other VEEV strains. Thus, infection of C3H/HeN mice with TC-83 VEEV may serve as a suitable model for the evaluation of antiviral compounds for the treatment of this viral disease.

Autonomic nervous dysfunction in hamsters infected with West Nile virus.

Clinical studies and case reports clearly document that West Nile virus (WNV) can cause respiratory and gastrointestinal (GI) complications. Other functions controlled by the autonomic nervous system may also be directly affected by WNV, such as bladder and cardiac functions. To investigate how WNV can cause autonomic dysfunctions, we focused on the cardiac and GI dysfunctions of rodents infected with WNV. Infected hamsters had distension of the stomach and intestines at day 9 after viral challenge. GI motility was detected by a dye retention assay; phenol red dye was retained more in the stomachs of infected hamsters as compared to sham-infected hamsters. The amplitudes of electromygraphs (EMGs) of intestinal muscles were significantly reduced. Myenteric neurons that innervate the intestines, in addition to neurons in the brain stem, were identified to be infected with WNV. These data suggest that infected neurons controlling autonomic function were the cause of GI dysfunction in WNV-infected hamsters. Using radiotelemetry to record electrocardiograms and to measure heart rate variability (HRV), a well-accepted readout for autonomic function, we determined that HRV and autonomic function were suppressed in WNV-infected hamsters. Cardiac histopathology was observed at day 9 only in the right atrium, which was coincident with WNV staining. A subset of WNV infected cells was identified among cells with hyperplarization-activated cyclic nucleotide-gated potassium channel 4 (HCN4) as a marker for cells in the sinoatrial (SA) and atrioventricular (AV) nodes. The unique contribution of this study is the discovery that WNV infection of hamsters can lead to autonomic dysfunction as determined by reduced HRV and reduced EMG amplitudes of the GI tract. These data may model autonomic dysfunction of the human West Nile neurological disease.