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1.  Novel approaches and challenges to treatment of CNS viral infections 
Annals of neurology  2013;74(3):412-422.
Existing and emerging viral CNS infections are major sources of human morbidity and mortality. Treatments of proven efficacy are currently limited predominantly to herpesviruses and human immunodeficiency virus. Development of new therapies has been hampered by the lack of appropriate animal model systems for some important viruses and by the difficulty in conducting human clinical trials for diseases that may be rare, or in the case of arboviral infections, often have variable seasonal and geographic incidence. Nonetheless, many novel approaches to antiviral therapy are available including candidate thiazolide and purazinecarboxamide derivatives with potential broad-spectrum antiviral efficacy. New herpesvirus drugs include viral helicase-primase and terminase inhibitors. The use of antisense oligonucleotides and other strategies to interfere with viral RNA translation has shown efficacy in experimental models of CNS viral disease. Identifying specific molecular targets within viral replication cycles has led to many existing antivirals and will undoubtedly continue to be the basis of future drug design. A promising new area of research involves therapies based on enhanced understanding of host antiviral immune responses. Toll-like receptor agonists, and drugs that inhibit specific cytokines as well as interferon preparations have all shown potential therapeutic efficacy. Passive transfer of virus-specific cytotoxic T-lymphocytes have been used in humans and may provide an effective therapies for some herpesvirus infections and potentially for progressive multifocal leukoencephalopathy. Humanized monoclonal antibodies directed against specific viral proteins have been developed and in several cases evaluated in humans in settings including West Nile virus and HIV infection and in pre-exposure prophylaxis for rabies.
doi:10.1002/ana.23988
PMCID: PMC4052367  PMID: 23913580
2.  PML Therapy: “Its Déjà vu All Over Again”* 
Journal of neurovirology  2013;19(4):311-313.
doi:10.1007/s13365-013-0191-9
PMCID: PMC3778683  PMID: 23913113
3.  Death Receptor-Mediated Apoptotic Signaling Is Activated in the Brain following Infection with West Nile Virus in the Absence of a Peripheral Immune Response 
Journal of Virology  2014;88(2):1080-1089.
Apoptosis is an important mechanism of West Nile virus (WNV) pathogenesis within the central nervous system (CNS). The signaling pathways that result in WNV-induced apoptotic neuronal death within the CNS have not been established. In this study, we identified death receptor (DR)-induced apoptosis as a pathway that may be important in WNV pathogenesis, based on the pattern of differential gene expression in WNV-infected, compared to uninfected, brains. Reverse transcription-PCR (RT-PCR) and Western blotting confirmed that genes involved in DR-induced apoptotic signaling are upregulated in the brain following WNV infection. Activity of the DR-associated initiator caspase, caspase 8, was also increased in the brains of WNV-infected mice and occurred in association with cleavage of Bid and activation of caspase 9. These results demonstrate that DR-induced apoptotic signaling is activated in the brain following WNV infection and suggest that the caspase 8-dependent cleavage of Bid promotes intrinsic apoptotic signaling within the brains of infected animals. Utilization of a novel ex vivo brain slice culture (BSC) model of WNV encephalitis revealed that inhibition of caspase 8 decreases virus-induced activation of caspase 3 and tissue injury. The BSC model allows us to examine WNV-induced pathogenesis in the absence of a peripheral immune response. Thus, our results indicate that WNV-induced neuronal injury in the brain is mediated by DR-induced apoptosis signaling and can occur in the absence of infiltrating immune cells. However, astrocytes and microglia were activated in WNV-infected BSC, suggesting that local immune responses influence WNV pathogenesis.
doi:10.1128/JVI.02944-13
PMCID: PMC3911655  PMID: 24198425
4.  Virus-Induced Transcriptional Changes in the Brain Include the Differential Expression of Genes Associated with Interferon, Apoptosis, Interleukin 17 Receptor A, and Glutamate Signaling as Well as Flavivirus-Specific Upregulation of tRNA Synthetases 
mBio  2014;5(2):e00902-14.
ABSTRACT
Flaviviruses, particularly Japanese encephalitis virus (JEV) and West Nile virus (WNV), are important causes of virus-induced central nervous system (CNS) disease in humans. We used microarray analysis to identify cellular genes that are differentially regulated following infection of the brain with JEV (P3) or WNV (New York 99). Gene expression data for these flaviviruses were compared to those obtained following infection of the brain with reovirus (type 3 Dearing), an unrelated neurotropic virus. We found that a large number of genes were up-regulated by all three viruses (using the criteria of a change of >2-fold and a P value of <0.001), including genes associated with interferon signaling, the immune system, inflammation, and cell death/survival signaling. In addition, genes associated with glutamate signaling were down-regulated in infections with all three viruses (criteria, a >2-fold change and a P value of <0.001). These genes may serve as broad-spectrum therapeutic targets for virus-induced CNS disease. A distinct set of genes were up-regulated following flavivirus infection but not following infection with reovirus. These genes were associated with tRNA charging and may serve as therapeutic targets for flavivirus-induced CNS disease.
IMPORTANCE
Viral infections of the central nervous system (CNS) are an important cause of morbidity and mortality. Treatment options for virus-induced CNS disease are limited, and for many clinically important neurotropic viruses, no specific therapy of proven benefit is currently available. We performed microarray analysis to identify genes that are differentially regulated in the brain following virus infection in order to identify pathways that might provide novel therapeutic targets for virus-induced CNS disease. Although several studies have described gene expression changes following virus infection of the brain, this report is the first to directly compare large-scale gene expression data from different viruses. We identified genes that are differentially regulated in infection of the brain with viruses from different families and those which appear to be specific to flavivirus infections.
doi:10.1128/mBio.00902-14
PMCID: PMC3952157  PMID: 24618253
5.  Emerging Viral Infections of the Central Nervous System 
Archives of neurology  2009;66(9):1065-1074.
The first part of this review ended with a discussion of new niches for known viruses as illustrated by viral central nervous system (CNS) disease associated with organ transplant and the syndrome of human herpesvirus 6–associated posttransplant acute limbic encephalitis. In this part, we begin with a continuation of this theme, reviewing the association of JC virus–associated progressive multifocal leukoencephalopathy (PML) with novel immunomodulatory agents. This part then continues with emerging viral infections associated with importation of infected animals (monkeypox virus), then spread of vectors and enhanced vector competence (chikungunya virus [CHIK]), and novel viruses causing CNS infections including Nipah and Hendra viruses and bat lyssaviruses (BLV).
doi:10.1001/archneurol.2009.189
PMCID: PMC2876734  PMID: 19752295
6.  Emerging Viral Infections of the Central Nervous System 
Archives of neurology  2009;66(8):939-948.
In this 2-part review, I will focus on emerging virus infections of the central nervous system (CNS). Part 1 will introduce the basic features of emerging infections, including their definition, epidemiology, and the frequency of CNS involvement. Important mechanisms of emergence will be reviewed, including viruses spreading into new host ranges as exemplified by West Nile virus (WNV), Japanese encephalitis (JE) virus, Toscana virus, and enterovirus 71 (EV71). Emerging infections also result from opportunistic spread of viruses into known niches, often resulting from attenuated host resistance to infection. This process is exemplified by transplant-associated cases of viral CNS infection caused by WNV, rabies virus, lymphocytic choriomeningitis, and lymphocytic choriomeningitis–like viruses and by the syndrome of human herpesvirus 6 (HHV6)–associated posttransplantation acute limbic encephalitis. The second part of this review begins with a discussion of JC virus and the occurrence of progressive multifocal leukoencephalopathy in association with novel immunomodulatory therapies and then continues with an overview of the risk of infection introduced by imported animals (eg, monkeypox virus) and examples of emerging diseases caused by enhanced competence of viruses for vectors and the spread of vectors (eg, chikungunya virus) and then concludes with examples of novel viruses causing CNS infection as exemplified by Nipah and Hendra viruses and bat lyssaviruses.
doi:10.1001/archneurol.2009.153
PMCID: PMC2873855  PMID: 19667214
7.  North American encephalitic arboviruses 
Neurologic clinics  2008;26(3):727-ix.
Synopsis
Arboviruses continue to be a major cause of encephalitis in North America and West Nile virus neuroinvasive disease is now the dominant cause of encephalitis. Transmission to humans of North American arboviruses occurs by infected mosquitoes or ticks. Most infections are asymptomatic or produce a flu-like illness. Elderly, immunosuppressed individuals and infants for some arboviruses have the highest incidence of severe encephalitis. Rapid serum or CSF IgM antibody capture ELISA assays are now available to diagnosis the acute infection for all North American arboviruses. Unfortunately, no antiviral drugs are approved for the treatment of arbovirus infection and current therapy is supportive.
doi:10.1016/j.ncl.2008.03.012
PMCID: PMC2866042  PMID: 18657724
arbovirus; togavirus; flavivirus; bunyavirus; encephalitis; myelitis
8.  West Nile Virus growth is Independent of Autophagy Activation 
Virology  2012;433(1):262-272.
West Nile virus (WNV) is an arthropod-borne virus with a world wide distribution that causes neurologic disease and death. Autophagy is a cellular homeostatic mechanism involved in antiviral responses but can be subverted to support viral growth as well. We show that autophagy is induced by WNV infection in cell culture and in primary neuron cultures. Following WNV infection, lysosomes co-localize with autophagosomes resulting in LC3B-II turnover and autolysosomal acidification. However, activation or inhibition of autophagy has no significant effect on WNV growth but pharmacologic inhibition of PI3 kinases associated with autophagy reduce WNV growth. Basal levels of p62/sequestesome1(SQSTM1) do not significantly change following WNV-induced autophagy activation, but p62 is turned over or degraded by autophagy activation implying that p62 expression is increased following WNV-infection. These data show that WNV-induces autophagy but viral growth is independent of autophagy activation suggesting that WNV-specific interactions with autophagy have diverged from other flaviviruses.
doi:10.1016/j.virol.2012.08.016
PMCID: PMC3444629  PMID: 22939285
Flavivirus; West Nile virus; autophagy; neuron; viral encephalitis; p62
9.  West Nile virus meningoencephalitis 
SUMMARY
Since its first appearance in the US in 1999, West Nile virus (WNV) has emerged as the most common cause of epidemic meningoencephalitis in North America. In the 6 years following the 1999 outbreak, the geographic range and burden of the disease in birds, mosquitoes and humans has greatly expanded to include the 48 contiguous US and 7 Canadian provinces, as well as Mexico, the Caribbean islands and Colombia. WNV has shown an increasing propensity for neuroinvasive disease over the past decade, with varied presentations including meningitis, encephalitis and acute flaccid paralysis. Although neuroinvasive disease occurs in less than 1% of infected individuals, it is associated with high mortality. From 1999–2005, more than 8,000 cases of neuroinvasive WNV disease were reported in the US, resulting in over 780 deaths. In this review, we discuss epidemiology, risk factors, clinical features, diagnosis and prognosis of WNV meningoencephalitis, along with potential treatments.
doi:10.1038/ncpneuro0176
PMCID: PMC3773989  PMID: 16932563
acute flaccid paralysis; encephalitis; meningitis; neuroinvasive disease; West Nile virus
10.  Daxx Upregulation within the Cytoplasm of Reovirus-Infected Cells Is Mediated by Interferon and Contributes to Apoptosis 
Journal of Virology  2013;87(6):3447-3460.
Reovirus infection is a well-characterized experimental system for the study of viral pathogenesis and antiviral immunity within the central nervous system (CNS). We have previously shown that c-Jun N-terminal kinase (JNK) and the Fas death receptor each play a role in neuronal apoptosis occurring in reovirus-infected brains. Death-associated protein 6 (Daxx) is a cellular protein that mechanistically links Fas signaling to JNK signaling in several models of apoptosis. In the present study, we demonstrate that Daxx is upregulated in reovirus-infected brain tissue through a type I interferon-mediated mechanism. Daxx upregulation is limited to brain regions that undergo reovirus-induced apoptosis and occurs in the cytoplasm and nucleus of neurons. Cytoplasmic Daxx is present in Fas-expressing cells during reovirus encephalitis, suggesting a role for Daxx in Fas-mediated apoptosis following reovirus infection. Further, in vitro expression of a dominant negative form of Daxx (DN-Daxx), which binds to Fas but which does not transmit downstream signaling, inhibits apoptosis of reovirus-infected cells. In contrast, in vitro depletion of Daxx results in increased expression of caspase 3 and apoptosis, suggesting that Daxx plays an antiapoptotic role in the nucleus. Overall, these data imply a regulatory role for Daxx in reovirus-induced apoptosis, depending on its location in the nucleus or cytoplasm.
doi:10.1128/JVI.02324-12
PMCID: PMC3592169  PMID: 23302889
11.  JAK-STAT signaling pathways are activated in the brain following reovirus infection 
Journal of neurovirology  2007;13(4):373-383.
Reovirus infection provides a classic experimental model system for studying the pathogenesis of viral infections of the central nervous system (CNS), with apoptosis acting as the major mechanism of cell death. The authors have examined the role of signal transducer and activator of transcription (STAT)1, a component of Janus-activated kinase (JAK)-STAT signaling, a pathway implicated in antiviral responses and pathways regulating apoptosis, following reovirus infection. Infection of primary cortical neuron cultures with reovirus serotype 3 strain Abney (T3A) resulted in phosphorylation of STAT1 at sites critical for transcriptional activity. Activated STAT1 was also detected in the brain of neonatal mice following T3A infection, with a nuclear pattern of expression in areas of virus-induced injury. Activation of STAT proteins is typically mediated by JAKs. The authors observed JAK2 phosphorylation (Tyr 1007/1008) in brain lysates from T3A-infected mice. Inhibition of JAK activity with the inhibitor AG-490 blocked reovirus-induced STAT1 activation in neuronal cultures, indicating reovirus-induced STAT activation is JAK dependent. Pretreatment of neuronal cultures with antibody raised against interferon (IFN)-α/βR2 inhibited T3A-induced STAT1 phosphorylation, whereas neither IFN-γ or IFN-γR2 antibody pretreatment had any effect on T3A-induced STAT1 phosphorylation. Mice lacking the STAT1 gene demonstrated increased susceptibility to reovirus infection, with increased mortality and higher viral titers in the brain compared to wild-type animals. The results demonstrate activation of a type I IFN-mediated, JAK-dependent STAT signaling pathway following reovirus infection and suggest that STAT1 is a key component of host defense mechanisms against reovirus infection in the brain.
doi:10.1080/13550280701344983
PMCID: PMC2367059  PMID: 17849321
apoptosis; Janus-activated kinase; reovirus; signal transducer and activator of transcription; viral encephalitis
12.  Experimental Reovirus-Induced Acute Flaccid Paralysis and Spinal Motor Neuron Cell Death 
Acute flaccid paralysis (AFP) describes the loss of motor function in 1 or more limbs commonly associated with viral infection and destruction of motor neurons in the anterior horns of the spinal cord. Therapy is limited, and the development of effective treatments is hampered by a lack of experimental models. Reovirus infection of neonatal mice provides a model for the study of CNS viral infection pathogenesis. Injection of the Reovirus serot Type 3 strains Abney (T3A) or Dearing (T3D) into the hindlimb of 1-day-old mice resulted in the development of AFP in more than 90% of infected mice. Acute flaccid paralysis began in the ipsilateral hindlimb at 8 to 10 days postinfection and progressed to paraplegia 24 hours later. Paralysis correlated with injury, neuron loss, and spread of viral antigen first to the ipsilateral and then to the contralateral anterior horns. As demonstrated by the activation of caspase 3 and its colocalization with viral antigen in the anterior horn and concomitant cleavage of poly-(adenosine diphosphate-ribose) polymerase, AFP was associated with apoptosis. Calpain activity and inducible nitric oxide synthase expression were both elevated in the spinal cords of paralyzed animals. This study represents the first detailed characterization of a novel and highly efficient experimental model of virus-induced AFP that will facilitate evaluation of therapeutic strategies targeting virus-induced paralysis.
doi:10.1097/NEN.0b013e31816564f0
PMCID: PMC2365907  PMID: 18344914
Acute flaccid paralysis; Apoptosis; Motor neurons; Reovirus; Viral myelitis
13.  A 20-Year-Old Woman With Headache and Transient Numbness 
The Neurohospitalist  2013;3(2):101-110.
doi:10.1177/1941874412473118
PMCID: PMC3726119  PMID: 23983894
meningitis; chronic meningitis; fungal meningitis; Histoplasma capsulatum; histoplasmosis; secondary CNS vasculitis; communicating hydrocephalus
14.  Activation of Innate Immune Responses in the Central Nervous System during Reovirus Myelitis 
Journal of Virology  2012;86(15):8107-8118.
Reovirus infection of the murine spinal cord (SC) was used as a model system to investigate innate immune responses during viral myelitis, including the activation of glia (microglia and astrocytes) and interferon (IFN) signaling and increased expression of inflammatory mediators. Reovirus myelitis was associated with the pronounced activation of SC glia, as evidenced by characteristic changes in cellular morphology and increased expression of astrocyte and microglia-specific proteins. Expression of inflammatory mediators known to be released by activated glia, including interleukin-1β (IL-1β), tumor necrosis factor alpha (TNF-α), chemokine (C-C motif) ligand 5 (CCL 5), chemokine (C-X-C motif) ligand 10 (CXCL10), and gamma interferon (IFN-γ), was also significantly upregulated in the SC of reovirus-infected animals compared to mock-infected controls. Reovirus infection of the mouse SC was also associated with increased expression of genes involved in IFN signaling, including IFN-stimulated genes (ISG). Further, reovirus infection of mice deficient in the expression of the IFN-α/β receptor (IFNAR−/−) resulted in accelerated mortality, demonstrating that IFN signaling is protective during reovirus myelitis. Experiments performed in ex vivo SC slice cultures (SCSC) confirmed that resident SC cells contribute to the production of at least some of these inflammatory mediators and ISG during reovirus infection. Microglia, but not astrocytes, were still activated, and glia-associated inflammatory mediators were still produced in reovirus-infected INFAR−/− mice, demonstrating that IFN signaling is not absolutely required for these neuroinflammatory responses. Our results suggest that activated glia and inflammatory mediators contribute to a local microenvironment that is deleterious to neuronal survival.
doi:10.1128/JVI.00171-12
PMCID: PMC3421655  PMID: 22623770
15.  Neuro-Intensive Care of Patients with Acute CNS Infections 
Neurotherapeutics  2011;9(1):124-138.
Summary
Infections in the central nervous system (CNS) are caused by a wide range of microorganisms resulting in distinct clinical syndromes including meningitis, encephalitis, and pyogenic infections, such as empyema and brain abscess. Bacterial and viral infections in the CNS can be rapidly fatal and can result in severe disability in survivors. Appropriate identification and acute management of these infections often occurs in a critical care setting and is vital to improving outcomes in this group of patients. This review of diagnosis and management of acute bacterial and viral infections in the CNS provides a general approach to patients with a suspected CNS infection and also provides a more detailed review of the diagnosis and management of patients with suspected bacterial meningitis, viral encephalitis, brain abscess, and subdural empyema.
Electronic supplementary material
The online version of this article (doi:10.1007/s13311-011-0086-5) contains supplementary material, which is available to authorized users.
doi:10.1007/s13311-011-0086-5
PMCID: PMC3271160  PMID: 22102180
Nervous system; Infection; Meningitis; Encephalitis; Abscess; Treatment
17.  Rituximab-Associated Progressive Multifocal Leukoencephalopathy in Rheumatoid Arthritis 
Archives of neurology  2011;68(9):1156-1164.
Objective
To describe the development of progressive multifocal leukoencephalopathy (PML) in patients with rheumatoid arthritis (RA) treated with rituximab.
Design
Case study.
Setting
Clinical care for patients with rheumatologic diseases. Most were referred to academic centers for care after diagnosis (Washington University, St Louis, Missouri; Karolinska Insitute, Stockholm, Sweden; and Royal Melbourne Hospital, Melbourne, Australia) while one was cared for in a neurology practice in Dallas, Texas, with consultation by an academic neurovirologist from the University of Colorado in Denver.
Patients
Four patients developing PML in the setting of rituximab therapy for RA.
Intervention
Rituximab therapy.
Main Outcome Measures
Clinical and pathological observations.
Results
Four patients from an estimated population of 129 000 exposed to rituximab therapy for RA are reported in whom PML developed after administration of this drug. All were women older than 50 years, commonly with Sjögren syndrome and a history of treatment for joint disease ranging from 3 to 14 years. One case had no prior biologic and minimal immunosuppressive therapy. Progressive multifocal leukoencephalopathy presented as a progressive neurological disorder, with diagnosis confirmed by detection of JC virus DNA in the cerebrospinal fluid or brain biopsy specimen. Two patients died in less than 1 year from PML diagnosis, while 2 remain alive after treatment withdrawal. Magnetic resonance scans and tissue evaluation confirmed the frequent development of inflammatory PML during the course of the disease.
Conclusion
These cases suggest an increased risk, about 1 case per 25 000 individuals, of PML in patients with RA being treated with rituximab. Inflammatory PML may occur in this setting even while CD20 counts remain low.
doi:10.1001/archneurol.2011.103
PMCID: PMC3428054  PMID: 21555606
18.  Type I interferon signaling limits reoviral tropism within the brain and prevents lethal systemic infection 
Journal of neurovirology  2011;17(4):314-326.
In vivo and ex vivo models of reoviral encephalitis were utilized to delineate the contribution of type I interferon (IFN) to the host’s defense against local central nervous system (CNS) viral infection and systemic viral spread. Following intracranial (i.c.) inoculation with either serotype 3 (T3) or serotype 1 (T1) reovirus, increased expression of IFN-α, IFN-β, and myxovirus-resistance protein (Mx1; a prototypical IFN stimulated gene) was observed in mouse brain tissue. Type I IFN receptor deficient mice (IFNAR−/−) had accelerated lethality, compared to wildtype (B6wt) controls, following i.c. T1 or T3 challenge. Although viral titers in the brain and eyes of reovirus infected IFNAR−/− mice were significantly increased, these mice did not develop neurologic signs or brain injury. In contrast, increased reovirus titers in peripheral tissues (liver, spleen, kidney, heart, and blood) of IFNAR−/− mice were associated with severe intestinal and liver injury. These results suggest that reovirus-infected IFNAR−/− mice succumb to peripheral disease rather than encephalitis per se. To investigate the potential role of type I IFN in brain tissue, brain slice cultures (BSCs) were prepared from IFNAR−/− mice and B6wt controls for ex vivo T3 reovirus infection. Compared to B6wt controls, reoviral replication and virus-induced apoptosis were enhanced in IFNAR−/− BSCs indicating that a type I IFN response, initiated by resident CNS cells, mediates innate viral immunity within the brain. T3 reovirus tropism was extended in IFNAR−/− brains to include dentate neurons, ependymal cells, and meningeal cells indicating that reovirus tropism within the CNS is dependent upon type I interferon signaling.
doi:10.1007/s13365-011-0038-1
PMCID: PMC3163031  PMID: 21671121
Reovirus; Virus; Tropism; Interferon; Encephalitis; Brain
19.  A brain slice culture model of viral encephalitis reveals an innate CNS cytokine response profile and the therapeutic potential of caspase inhibition 
Experimental neurology  2011;228(2):222-231.
Viral encephalitis is a significant cause of human morbidity and mortality in large part due to suboptimal diagnosis and treatment. Murine reovirus infection serves as a classic experimental model of viral encephalitis. Infection of neonatal mice with T3 reoviruses results in lethal encephalitis associated with neuronal infection, apoptosis, and CNS tissue injury. We have developed an ex vivo brain slice culture (BSC) system that recapitulates the basic pathological features and kinetics of viral replication seen in vivo. We utilize the BSC model to identify an innate, brain-tissue specific inflammatory cytokine response to reoviral infection, which is characterized by the release of IL6, CXCL10, RANTES, and murine IL8 analog (KC). Additionally, we demonstrate the potential utility of this system as a pharmaceutical screening platform by inhibiting reovirus-induced apoptosis and CNS tissue injury with the pan-caspase inhibitor, Q-VD-OPh. Cultured brain slices not only serve to model events occurring during viral encephalitis, but can also be utilized to investigate aspects of pathogenesis and therapy that are not experimentally accessible in vivo.
doi:10.1016/j.expneurol.2011.01.006
PMCID: PMC3060973  PMID: 21241693
reovirus; virus; viral; apoptosis; caspase; encephalitis; neuron; ex vivo; organotypic; brain slice; cytokine; MCP-1; IL6; KC; IL8; CXCL10; RANTES; Q-VD-OPh
20.  The Proapoptotic Bcl-2 Protein Bax Plays an Important Role in the Pathogenesis of Reovirus Encephalitis ▿  
Journal of Virology  2011;85(8):3858-3871.
Encephalitis induced by reovirus serotype 3 (T3) strains results from the apoptotic death of infected neurons. Extrinsic apoptotic signaling is activated in reovirus-infected neurons in vitro and in vivo, but the role of intrinsic apoptosis signaling during encephalitis is largely unknown. Bax plays a key role in intrinsic apoptotic signaling in neurons by allowing the release of mitochondrial cytochrome c. We found Bax activation and cytochrome c release in neurons following infection of neonatal mice with T3 reoviruses. Bax−/− mice infected with T3 Abney (T3A) have reduced central nervous system (CNS) tissue injury and decreased apoptosis, despite viral replication that is similar to that in wild-type (WT) Bax+/+ mice. In contrast, in the heart, T3A-infected Bax−/− mice have viral growth, caspase activation, and injury comparable to those in WT mice, indicating that the role of Bax in pathogenesis is organ specific. Nonmyocarditic T3 Dearing (T3D)-infected Bax−/− mice had delayed disease and enhanced survival compared to WT mice. T3D-infected Bax−/− mice had significantly lower viral titers and levels of activated caspase 3 in the brain despite unaffected transneuronal spread of virus. Cytochrome c and Smac release occurred in some reovirus-infected neurons in the absence of Bax; however, this was clearly reduced compared to levels seen in Bax+/+ wild-type mice, indicating that Bax is necessary for efficient activation of proapoptotic mitochondrial signaling in infected neurons. Our studies suggest that Bax is important for reovirus growth and pathogenesis in neurons and that the intrinsic pathway of apoptosis, mediated by Bax, is important for full expression of disease, CNS tissue injury, apoptosis, and viral growth in the CNS of reovirus-infected mice.
doi:10.1128/JVI.01958-10
PMCID: PMC3126143  PMID: 21307199
21.  Impact of rituximab-associated B-cell defects on West Nile virus meningoencephalitis in solid organ transplant recipients 
Clinical transplantation  2009;24(2):223-228.
Evidence suggests that West Nile virus (WNV) neuroinvasive disease occurs more frequently in both solid organ and human stem cell transplant recipients. The effect of concomitant anti-B-cell therapy with rituximab, a CD20+ monoclonal antibody, on WNV infection in this population, however, has not been reported. We describe a case of a patient with alpha-1-antitrypsin deficiency who underwent single lung transplantation in 2005 and was maintained on tacrolimus, cytoxan and prednisone. More recently, she had received two courses of rituximab for recurrent A2–A3 grade rejection with concomitant capillaritis and presented six months later with rapid, fulminant WNV meningoencephalitis. Her diagnosis was made by cerebrospinal fluid (CSF) PCR but serum and CSF WNV IgM and IgG remained negative. She received WNV-specific hyperimmune globulin (Omr-Ig-Am®) through a compassionate protocol. She experienced a rapidly progressive and devastating neurological course despite treatment and died three wk after onset of her symptoms. Autopsy revealed extensive meningoencephalomyelitis.
doi:10.1111/j.1399-0012.2009.01044.x
PMCID: PMC2873850  PMID: 19659514
rituximab; transplant; West Nile virus
22.  Gene expression in the brain during reovirus encephalitis 
Journal of neurovirology  2010;16(1):56-71.
Viral encephalitis remains a significant cause of morbidity and mortality throughout the world. We performed microarray analysis to identify genes and pathways that are differentially regulated during reovirus encephalitis and that may provide novel therapeutic targets for virus-induced diseases of the central nervous system (CNS). An increase in the expression of 130 cellular genes was found in the brains of reovirus-infected mice at early times post infection, compared to mock-infected controls. The up-regulation of these genes was consistent with activation of innate immune responses, particularly interferon signaling. At later times post infection, when significant CNS injury is present and mice exhibit signs of severe neurologic disease, many more (1374) genes were up-regulated, indicating that increased gene expression correlates with disease pathology. Virus-induced gene expression at late times post infection was again consistent with the activation of innate immune responses. However, additional significant pathways included those associated with cytokine signaling and apoptosis, both of which can contribute to CNS injury. This is the first report comparing virus-induced cellular gene and pathway regulation at early and late times following virus infection of the brain. The shift of virus-induced gene expression from innate immune responses at early times post infection to cytokine signaling and apoptosis at later times suggests a potential therapeutic strategy that preserves early protective responses whilst inhibiting later responses that contribute to pathogenesis.
doi:10.3109/13550280903586394
PMCID: PMC2891017  PMID: 20158406
encephalitis; gene expression; reovirus
23.  Caspase-3 activation is required for reovirus-induced encephalitis in vivo 
Journal of neurovirology  2010;16(4):306-317.
Reovirus infection of neonatal mice provides a classic experimental system for understanding the molecular pathogenesis of central nervous system (CNS) viral infection. CNS tissue injury, caused by many human neurotropic viruses, including herpes viruses and West Nile virus, is associated with caspase-dependent apoptotic neuronal cell death. We have previously shown that reovirus-induced CNS tissue injury results from apoptosis and is associated with activation of both death-receptor and mitochondrial apoptotic pathways culminating in the activation of the downstream effector caspase, caspase-3. In order to directly investigate the role of caspase-3 in virus-induced neuronal death and CNS tissue injury during encephalitis, we have compared the pathogenesis of reovirus CNS infection in mice lacking the caspase-3 gene (caspase-3 (–/–)) to syngeneic wild-type mice. Prior studies of antiapoptotic treatments for reovirus-infected mice have indicated that protection from reovirus-induced neuronal injury can occur without altering the viral titer in the brains of infected mice. We now show that reovirus infection of caspase-3(–/–) mice was associated with dramatic reduction in severity of CNS tissue injury, decreased viral antigen and titer in the brain, and enhanced survival of infected mice. Following intracerebral inoculation, the authors also show that virus spread from the brain to the eyes in reovirus-infected caspase-3 (–/–) mice, indicating that viral spread was intact in these mice. Examination of brains of long-term survivors of reovirus infection among caspase-3 (–/–) mice showed that these mice eventually clear their CNS viral infection, and do not manifest residual or delayed CNS tissue injury.
doi:10.3109/13550284.2010.499890
PMCID: PMC3023174  PMID: 20626234
apoptosis; caspase; encephalitis; neuron; reovirus
24.  Disrupted Glutamate Transporter Expression in the Spinal Cord With Acute Flaccid Paralysis Due to West Nile Virus Infection 
Neuroinvasive West Nile virus (WNV) infections may cause acute flaccid paralysis (AFP); in fatal cases anterior horn cell (AHC) loss is presumed to be a due to direct viral infection. In related animal models, however, glutamate excitoxicity mediates bystander injury of uninfected AHCs, suggesting additional pathogenetic mechanisms. We examined expression of the principal excitatory amino acid transporter (EAAT) of astrocytes (i.e. EAAT2 in humans, glutamate transporter-1 in hamsters) in the spinal cord of human WNV AFP patients and in hamsters with WNV AFP by immunohistochemistry. Glial fibrillary acidic protein (GFAP), synaptic and dendritic markers (i.e. synaptophysin, microtubule-associated protein-2 [MAP2]), immune activation (HLA-DR), and viral antigens were also evaluated. Humans and hamsters with WNV-induced AFP had decreased spinal grey matter EAAT expression despite greater numbers of GFAP-positive astrocytes compared to controls. Areas of diminished EAAT expression showed reduced synaptic and dendritic protein expression and prominent local inflammation but few infected neurons. These findings suggest that WNV infection results in local immune activation within the spinal cord that in turn causes a failure of astrocyte glutamate reuptake even as the number of astrocytes increases; rising extracellular glutamate levels may then drive excitotoxic injury of both infected and uninfected AHCs. The pathogenesis of this increasingly common disorder likely involves immune response and excitotoxicity mechanisms that are potential therapeutic targets.
doi:10.1097/NEN.0b013e3181b8ba14
PMCID: PMC2854566  PMID: 19918118
Acute flaccid paralysis; Astrocyte; Excitotoxic injury; Glutamate transporters; West Nile virus
25.  Fas-Mediated Apoptotic Signaling in the Mouse Brain following Reovirus Infection▿  
Journal of Virology  2009;83(12):6161-6170.
Type 3 (T3) reovirus strains induce apoptotic neuronal cell death and lethal encephalitis in infected mice. T3 strain Dearing (T3D)-induced apoptosis in primary neuronal cultures occurs by a Fas-mediated mechanism and requires the activation of caspase 8. We now show that Fas mRNA is upregulated in the brains of mice infected with encephalitic reovirus T3D and T3 strain Abney (T3A) but not following infection with nonencephalitic reovirus type 1 strain Lang. Fas is upregulated in regions of the brain that are injured during infection with T3 reovirus strains and colocalizes with virus antigen in individual neurons. In contrast, levels of FasL mRNA induced by encephalitic and nonencephalitic reovirus strains do not differ significantly. Caspase 8, the initiator caspase associated with Fas-mediated apoptosis, is activated in the cortex and hippocampal regions of both T3D- and T3A-infected mice. Furthermore, Bid cleavage and the activation of caspase 9 in the brains of T3D-infected mice suggest that the caspase 8-dependent activation of mitochondrial apoptotic signaling contributes to virus-induced apoptosis. We have previously shown that the inhibition of c-Jun N-terminal kinase (JNK) signaling blocks T3D-induced apoptosis and improves the outcome of virus-induced encephalitis. We now show that the reovirus-induced upregulation of Fas requires JNK signaling, thereby providing a link between reovirus-induced death receptor signaling and mitogen-activated protein kinase pathways and a potential mechanism for the therapeutic action of JNK inhibition.
doi:10.1128/JVI.02488-08
PMCID: PMC2687381  PMID: 19321603

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