Bioluminescent Imaging and Histopathologic Characterization of WEEV Neuroinvasion in Outbred CD-1 Mice

Western equine encephalitis virus (WEEV; Alphavirus) is a mosquito-borne virus that can cause severe encephalitis in humans and equids. Previous studies have shown that intranasal infection of outbred CD-1 mice with the WEEV McMillan (McM) strain result in high mortality within 4 days of infection. Here in vivo and ex vivo bioluminescence (BLM) imaging was applied on mice intranasally infected with a recombinant McM virus expressing firefly luciferase (FLUC) to track viral neuroinvasion by FLUC detection and determine any correlation between BLM and viral titer. Immunological markers of disease (MCP-1 and IP-10) were measured and compared to wild type virus infection. Histopathology was guided by corresponding BLM images, and showed that neuroinvasion occurred primarily through cranial nerves, mainly in the olfactory tract. Olfactory bulb neurons were initially infected with subsequent spread of the infection into different regions of the brain. WEEV distribution was confirmed by immunohistochemistry as having marked neuronal infection but very few infected glial cells. Axons displayed infection patterns consistent with viral dissemination along the neuronal axis. The trigeminal nerve served as an additional route of neuroinvasion showing significant FLUC expression within the brainstem. The recombinant virus WEEV.McM.FLUC had attenuated replication kinetics and induced a weaker immunological response than WEEV.McM but produced comparable pathologies. Immunohistochemistry staining for FLUC and WEEV antigen showed that transgene expression was present in all areas of the CNS where virus was observed. BLM provides a quantifiable measure of alphaviral neural disease progression and a method for evaluating antiviral strategies.     

The role of the blood-brain barrier during Venezuelan equine encephalitis virus infection

Venezuelan equine encephalitis (VEE) virus is a mosquito-borne alphavirus associated with sporadic outbreaks in human and equid populations in the Western Hemisphere. After the bite of an infected mosquito, the virus initiates a biphasic disease: a peripheral phase with viral replication in lymphoid and myeloid tissues, followed by a neurotropic phase with infection of central nervous system (CNS) neurons, causing neuropathology and in some cases fatal encephalitis. The mechanisms allowing VEE virus to enter the CNS are currently poorly understood. Previous data have shown that the virus gains access to the CNS by infecting olfactory sensory neurons in the nasal mucosa of mice. However, at day 5 after inoculation, the infection of the brain is multifocal, indicating that virus particles are able to cross the blood-brain barrier (BBB). To better understand the role of the BBB during VEE virus infection, we used a well-characterized mouse model system. Using VEE virus replicon particles (VRP), we modeled the early events of neuroinvasion, showing that the replication of VRP in the nasal mucosa induced the opening of the BBB, allowing peripherally administered VRP to invade the brain. Peripheral VEE virus infection was characterized by a biphasic opening of the BBB. Further, inhibition of BBB opening resulted in a delayed viral neuroinvasion and pathogenesis. Overall, these results suggest that VEE virus initially enters the CNS through the olfactory pathways and initiates viral replication in the brain, which induces the opening of the BBB, allowing a second wave of invading virus from the periphery to enter the brain.     

Lesion of the Olfactory Epithelium Accelerates Prion Neuroinvasion and Disease Onset when Prion Replication Is Restricted to Neurons

Natural prion diseases of ruminants are moderately contagious and while the gastrointestinal tract is the primary site of prion agent entry, other mucosae may be entry sites in a subset of infections. In the current study we examined prion neuroinvasion and disease induction following disruption of the olfactory epithelium in the nasal mucosa since this site contains environmentally exposed olfactory sensory neurons that project directly into the central nervous system. Here we provide evidence for accelerated prion neuroinvasion and clinical onset from the olfactory mucosa after disruption and regeneration of the olfactory epithelium and when prion replication is restricted to neurons. In transgenic mice with neuron restricted replication of prions, there was a reduction in survival when the olfactory epithelium was disrupted prior to intranasal inoculation and there was >25% decrease in the prion incubation period. In a second model, the neurotropic DY strain of transmissible mink encephalopathy was not pathogenic in hamsters by the nasal route, but 50% of animals exhibited brain infection and/or disease when the olfactory epithelium was disrupted prior to intranasal inoculation. A time course analysis of prion deposition in the brain following loss of the olfactory epithelium in models of neuron-restricted prion replication suggests that neuroinvasion from the olfactory mucosa is via the olfactory nerve or brain stem associated cranial nerves. We propose that induction of neurogenesis after damage to the olfactory epithelium can lead to prion infection of immature olfactory sensory neurons and accelerate prion spread to the brain.     

N-Acetyl-cysteine inhibition of encephalomyelitis Theiler's virus-induced nitric oxide and tumour necrosis factor-alpha production by murine astrocyte cultures

The pathological mechanisms that cause central nervous system (CNS) dysfunction in most neurological diseases are not well established. Theiler's murine encephalomyelitis virus (TMEV) is known to interact with cells of the CNS and its intracerebral inoculation to susceptible mice strains causes neurological disorders resembling multiple sclerosis (MS). In this study, we reported that primary astrocyte cultures from SJL/J susceptible mice when infected with TMEV released important amounts of nitrites (NO2-) to the culture medium, as measured in the supernatants 24 hours after infection. In addition, we observed an increment in the production of tumour necrosis factor alpha (TNF-alpha) by susceptible SJL/J strain derived astrocytes infected with TMEV. The treatment with the thiolic antioxidant N-acetyl-cysteine partially suppressed the virus-stimulated production of nitric oxide and TNF-alpha, in a dose response fashion. These results indicate that during viral infection astrocytes are an important cellular source of nitric oxide and TNF-alpha, substances which play important roles during CNS inflammatory events. The effects of the antioxidant N-acetyl-cysteine, modulating the production of the above compounds by TMEV-infected astrocytes may be a significant factor in preventing CNS demyelination.     

Crucial role for prion protein membrane anchoring in the neuroinvasion and neural spread of prion infection

In nature prion diseases are usually transmitted by extracerebral prion infection, but clinical disease results only after invasion of the central nervous system (CNS). Prion protein (PrP), a host-encoded glycosylphosphatidylinositol (GPI)-anchored membrane glycoprotein, is necessary for prion infection and disease. Here, we investigated the role of the anchoring of PrP on prion neuroinvasion by studying various inoculation routes in mice expressing either anchored or anchorless PrP. In control mice with anchored PrP, intracerebral or sciatic nerve inoculation resulted in rapid CNS neuroinvasion and clinical disease (154 to 156 days), and after tongue, ocular, intravenous, or intraperitoneal inoculation, CNS neuroinvasion was only slightly slower (193 to 231 days). In contrast, in anchorless PrP mice, these routes resulted in slow and infrequent CNS neuroinvasion. Only intracerebral inoculation caused brain PrPres, a protease-resistant isoform of PrP, and disease in both types of mice. Thus, anchored PrP was an essential component for the rapid neural spread and CNS neuroinvasion of prion infection.     

Immune-mediated protection from measles virus-induced central nervous system disease is noncytolytic and gamma interferon dependent

Neurons of the mammalian central nervous system (CNS) are an essential and largely nonrenewable cell population. Thus, virus infections that result in neuronal depletion, either by virus-mediated cell death or by induction of the cytolytic immune response, could cause permanent neurological impairment of the host. In a transgenic mouse model of measles virus (MV) infection of neurons, we have previously shown that the host T-cell response was required for resolution of infection in susceptible adult mice. In this report, we show that this protective response did not result in neuronal death, even during the peak of T-cell infiltration into the brain parenchyma. When susceptible mice were intercrossed with specific immune knockout mice, a critical role for gamma interferon (IFN-gamma) was identified in protection against MV infection and CNS disease. Moreover, the addition of previously activated splenocytes or recombinant murine IFN-gamma to MV-infected primary neurons resulted in the inhibition of viral replication in the absence of neuronal death. Together, these data support the hypothesis that the host immune response can promote viral clearance without concomitant neuronal loss, a process that appears to be mediated by cytokines.     

Enterovirus‐A71 exploits peripherin and Rac1 to invade the central nervous system

Enterovirus‐A71 (EV‐A71) has been associated with severe neurological forms of hand, foot, and mouth disease (HFMD). EV‐A71 infects motor neurons at neuromuscular junctions (NMJs) to invade the central nervous system (CNS). Here, we investigate the role of peripherin (PRPH) during EV‐A71 infection, a type III intermediate neurofilament involved in neurodegenerative conditions. In mice infected with EV‐A71, PRPH co‐localizes with viral particles in the muscles at NMJs and in the spinal cord. In motor neuron‐like and neuroblastoma cell lines, surface‐expressed PRPH facilitates viral entry, while intracellular PRPH influences viral genome replication through interactions with structural and non‐structural viral components. Importantly, PRPH does not play a role during infection with coxsackievirus A16, another causative agent of HFMD rarely associated with neurological complications, suggesting that EV‐A71 ability to exploit PRPH represents a unique attribute for successful CNS invasion. Finally, we show that EV‐A71 also exploits some of the many PRPH‐interacting partners. Of these, small GTP‐binding protein Rac1 represents a potential druggable host target to limit neuroinvasion of EV‐A71.     

A new model for Hendra virus encephalitis in the mouse

Hendra virus (HeV) infection in humans is characterized by an influenza like illness, which may progress to pneumonia or encephalitis and lead to death. The pathogenesis of HeV infection is poorly understood, and the lack of a mouse model has limited the opportunities for pathogenetic research. In this project we reassessed the role of mice as an animal model for HeV infection and found that mice are susceptible to HeV infection after intranasal exposure, with aged mice reliably developing encephalitic disease. We propose an anterograde route of neuroinvasion to the brain, possibly along olfactory nerves. This is supported by evidence for the development of encephalitis in the absence of viremia and the sequential distribution of viral antigen along pathways of olfaction in the brain of intranasally challenged animals. In our studies mice developed transient lower respiratory tract infection without progressing to viremia and systemic vasculitis that is common to other animal models. These studies report a new animal model of HeV encephalitis that will allow more detailed studies of the neuropathogenesis of HeV infection, particularly the mode of viral spread and possible sequestration within the central nervous system; investigation of mechanisms that moderate the development of viremia and systemic disease; and inform the development of improved treatment options for human patients.     

Neuropeptide Y Has a Protective Role during Murine Retrovirus-Induced Neurological Disease

Viral infections in the central nervous system (CNS) can lead to neurological disease either directly by infection of neurons or indirectly through activation of glial cells and production of neurotoxic molecules. Understanding the effects of virus-mediated insults on neuronal responses and neurotrophic support is important in elucidating the underlying mechanisms of viral diseases of the CNS. In the current study, we examined the expression of neurotrophin- and neurotransmitter-related genes during infection of mice with neurovirulent polytropic retrovirus. In this model, virus-induced neuropathogenesis is indirect, as the virus predominantly infects macrophages and microglia and does not productively infect neurons or astrocytes. Virus infection is associated with glial cell activation and the production of proinflammatory cytokines in the CNS. In the current study, we identified increased expression of neuropeptide Y (NPY), a pleiotropic growth factor which can regulate both immune cells and neuronal cells, as a correlate with neurovirulent virus infection. Increased levels of Npy mRNA were consistently associated with neurological disease in multiple strains of mice and were induced only by neurovirulent, not avirulent, virus infection. NPY protein expression was primarily detected in neurons near areas of virus-infected cells. Interestingly, mice deficient in NPY developed neurological disease at a faster rate than wild-type mice, indicating a protective role for NPY. Analysis of NPY-deficient mice indicated that NPY may have multiple mechanisms by which it influences virus-induced neurological disease, including regulating the entry of virus-infected cells into the CNS.The early innate immune response to virus infection in the central nervous system (CNS) plays an important regulatory role in controlling both viral infection and pathogenesis. The neuroinflammatory response can limit virus replication through production of type I interferons and recruitment of virus-specific T cells to the CNS (5, 9, 12, 15, 19). However, the neuroinflammatory response can also lead to chronic gliosis, the production of cytokines that are toxic to neurons, and the recruitment of virus-infected cells to the CNS (6, 8, 18, 35). Understanding the relationship between the innate immune response and viral disease is essential in order to manipulate this response to control virus infection in the CNS.To better understand the role of the innate immune responses in viral pathogenesis in the CNS, we have utilized a mouse model of polytropic retrovirus infection. In this model, neuropathogenesis is indirect, since the polytropic retroviruses do not productively infect neurons. Instead, the viruses predominantly infect macrophages and microglia in the CNS (32). Despite severe neurological disease development following polytropic retrovirus infection, the only histologic changes observed in the brain are the activations of microglia and astrocytes (31). In addition, we have found high levels of proinflammatory cytokines and chemokines in brain tissue from infected mice, including tumor necrosis factor (TNF); interleukin 1 alpha (IL-1α), IL-1β, and IL-6; and the chemokines chemokine ligand 2 (CCL2/MCP-1), CCL3 (MIP-1α), CCL4 (MIP-1β), CCL5 (RANTES), and chemokine (C-X-C motif) ligand 10 (CXCL10/IP-10) (28). Studies with different chemokine receptors and cytokine-deficient mice demonstrated that at least two of these proinflammatory cytokines, CCL2 and TNF, can contribute to retrovirus-induced neurological disease (26, 27). However, neither of these molecules was necessary for neurological disease for all of the neurovirulent polytropic retroviruses studied, suggesting that other host factors contribute to retroviral pathogenesis.Analysis of the envelope protein of the neurovirulent polytropic retrovirus identified key residues in the envelope protein that influence the ability of the virus to induce neurological disease (28). In this study, we utilized neurovirulent and nonneurovirulent chimeric viruses that differ by only a few amino acid residues in these envelope regions to identify host response factors whose expression correlated with neurovirulence. We also utilized two different mouse strains, Inbred Rocky Mountain White (IRW) and 129S6, to confirm that expression of these host response factors is consistently induced or suppressed during neurovirulent virus infection. We determined that, although a number of host response genes are induced by polytropic retrovirus infection of the CNS, the expression of several of these factors correlated only with neuroinvasion and was not strongly correlative of neurovirulence. However, we have identified a neurotrophin, neuropeptide Y (NPY), whose expression strongly correlates with neurovirulence. We found that NPY had a protective influence on retroviral neuropathogenesis and examined the mechanisms by which NPY influences retrovirus infection of the CNS.     

Mouse erythrocyte tropomodulin in the brain reported by lacZ knocked-in downstream from the E1 promoter

Erythrocyte tropomodulin (E-Tmod, Tmod1) is a tropomyosin-binding protein that caps the slow-growing end of actin filaments. In erythrocytes, it may favor the formation of short actin protofilaments needed for elastic cell deformation. Previously we created a knockout mouse model in which lacZ was knocked-in downstream of the E1 promoter to report the expression of full length E-Tmod. Here we utilize E-Tmod^+/lacZ mice to study E-Tmod expression patterns in the CNS. X-gal staining and in situ hybridization of adults revealed its restricted expression in the olfactory bulb, hippocampus, cerebral cortex, basal ganglia, nuclei of brain stem and cerebellum. In neonates, signals in the cortex and caudate putamen increased from days 15 to 40. Immunohistochemistry also revealed that signals for β-galactosidase coincided with that of NeuN, a post-mitotic nuclear marker for neurons, but not that for GFAP+ astrocytes or APC+ oligodendrocytes, suggesting E-Tmod/lacZ-positive cells in the CNS were neurons. Large neurons, e.g., mitral cells in olfactory bulb and mossy cells in hilus of the dentate gyrus are among those that expressed very high levels of E-Tmod in the CNS.     

CNS recruitment of CD8+ T lymphocytes specific for a peripheral virus infection triggers neuropathogenesis during polymicrobial challenge

Although viruses have been implicated in central nervous system (CNS) diseases of unknown etiology, including multiple sclerosis and amyotrophic lateral sclerosis, the reproducible identification of viral triggers in such diseases has been largely unsuccessful. Here, we explore the hypothesis that viruses need not replicate in the tissue in which they cause disease; specifically, that a peripheral infection might trigger CNS pathology. To test this idea, we utilized a transgenic mouse model in which we found that immune cells responding to a peripheral infection are recruited to the CNS, where they trigger neurological damage. In this model, mice are infected with both CNS-restricted measles virus (MV) and peripherally restricted lymphocytic choriomeningitis virus (LCMV). While infection with either virus alone resulted in no illness, infection with both viruses caused disease in all mice, with ～50% dying following seizures. Co-infection resulted in a 12-fold increase in the number of CD8+ T cells in the brain as compared to MV infection alone. Tetramer analysis revealed that a substantial proportion (>35%) of these infiltrating CD8+ lymphocytes were LCMV-specific, despite no detectable LCMV in CNS tissues. Mechanistically, CNS disease was due to edema, induced in a CD8-dependent but perforin-independent manner, and brain herniation, similar to that observed in mice challenged intracerebrally with LCMV. These results indicate that T cell trafficking can be influenced by other ongoing immune challenges, and that CD8+ T cell recruitment to the brain can trigger CNS disease in the apparent absence of cognate antigen. By extrapolation, human CNS diseases of unknown etiology need not be associated with infection with any particular agent; rather, a condition that compromises and activates the blood-brain barrier and adjacent brain parenchyma can render the CNS susceptible to pathogen-independent immune attack.     

Selective tropism of a neurotropic coronavirus for ependymal cells, neurons, and meningeal cells.

The ability of a neurotropic virus, mouse hepatitis virus type 3 (MHV3), to invade the central nervous system (CNS) and to recognize cells selectively within the brain was investigated in vivo and in vitro. In vivo, MHV3 induced in C3H mice a genetically controlled infection of meningeal cells, ependymal cells, and neurons. In vitro, purified MHV3 bound to the surface of isolated ependymal cells and cultured cortical neurons but not to oligodendrocytes or cultured astrocytes. MHV3 replicated within cultured cortical neurons and neuroblastoma cells (NIE 115); infected cultured neurons nonetheless survived and matured normally for a 7-day period postinfection. On the other hand, MHV3 had a low affinity for cortical glial cells or glioma cells (C6 line), both of which appear to be morphologically unaltered by viral infection. Finally, MHV3 infected and disrupted cultured meningeal cells. This suggests that differences in the affinity of cells for MHV3 are determinants of the selective vulnerability of cellular subpopulations within the CNS. In vivo, a higher titer of virus was needed for CNS penetration in the genetically resistant (A/Jx) mice than in the susceptible (C57/BL6) mouse strain. However, in spite of viral invasion, no neuropathological lesions developed. In vitro viral binding to adult ependymal cells of susceptible and resistant strains of mice was identical. Genetic resistance to MHV3-CNS infection appeared to be mediated both by a peripheral mechanism limiting viral penetration into the CNS and by intra-CNS mechanisms, presumably at a stage after viral attachment to target cells.     

Long-term persistence of infectious Zika virus: Inflammation and behavioral sequela in mice

The neurodevelopmental defects associated with ZIKV infections early in pregnancy are well documented, however the potential defects and long-term consequences associated with milder infections in late pregnancy and perinatal period are less well understood. To model these, we challenged 1 day old (P1) immunocompetent C57BL/6 mice with ZIKV. The animals developed a transient neurological syndrome including unsteady gait, kinetic tremors, severe ataxia and seizures 10–15 days post-infection (dpi) but symptoms subsided after a week, and most animals survived. Despite apparent recovery, MRI of convalescent mice show reduced cerebellar volume that correlates with altered coordination and motor function as well as hyperactivity and impulsivity. Persistent mRNA levels of pro-inflammatory genes including Cd80, Il-1α, and Ifn-γ together with Cd3, Cd8 and perforin (PrfA), suggested persistence of low-grade inflammation. Surprisingly, the brain parenchyma of convalescent mice harbor multiple small discrete foci with viral antigen, active apoptotic processes in neurons, and cellular infiltrates, surrounded by activated astrocytes and microglia as late as 1-year post-infection. Detection of negative-sense strand viral RNA and isolation of infectious virus derived from these convalescent mice by blinded passage in Vero cells confirmed long-term persistence of replicating ZIKV in CNS of convalescent mice. Although the infection appears to persist in defined reservoirs within CNS, the resulting inflammation could increase the risk of neurodegenerative disorders. This raises concern regarding possible long-term effects in asymptomatic children exposed to the virus and suggests that long-term neurological and behavioral monitoring as well as anti-viral treatment to clear virus from the CNS may be useful in patients exposed to ZIKV at an early age.     

A non-mouse-adapted enterovirus 71 (EV71) strain exhibits neurotropism, causing neurological manifestations in a novel mouse model of EV71 infection

Enterovirus 71 (EV71) is a neurotropic pathogen that has been consistently associated with the severe neurological forms of hand, foot, and mouth disease. The lack of a relevant animal model has hampered our understanding of EV71 pathogenesis, in particular the route and mode of viral dissemination. It has also hindered the development of effective prophylactic and therapeutic approaches, making EV71 one of the most pressing public health concerns in Southeast Asia. Here we report a novel mouse model of EV71 infection. We demonstrate that 2-week-old and younger immunodeficient AG129 mice, which lack type I and II interferon receptors, are susceptible to infection with a non-mouse-adapted EV71 strain via both the intraperitoneal (i.p.) and oral routes of inoculation. The infected mice displayed progressive limb paralysis prior to death. The dissemination of the virus was dependent on the route of inoculation but eventually resulted in virus accumulation in the central nervous systems of both animal groups, indicating a clear neurotropism of the virus. Histopathological examination revealed massive damage in the limb muscles, brainstem, and anterior horn areas. However, the minute amount of infectious viral particles in the limbs from orally infected animals argues against a direct viral cytopathic effect in this tissue and suggests that limb paralysis is a consequence of EV71 neuroinvasion. Together, our observations support that young AG129 mice display polio-like neuropathogenesis upon infection with a non-mouse-adapted EV71 strain, making this mouse model relevant for EV71 pathogenesis studies and an attractive platform for EV71 vaccine and drug testing.     

Intranasal inoculation with the olfactory bulb line variant of mouse hepatitis virus causes extensive destruction of the olfactory bulb and accelerated turnover of neurons in the olfactory epithelium of mice

Viral upper respiratory infections are the most common cause of clinical olfactory dysfunction, but the pathogenesis of dysosmia after viral infection is poorly understood. Biopsies of the olfactory mucosa in patients that complain of dysosmia after viral infection fall into two categories: one in which no olfactory epithelium is seen and another in which the epithelium is disordered and populated mainly by immature neurons. We have used intranasal inoculation with an olfactory bulb line variant of MHV to study the consequences of viral infection on peripheral olfactory structures. MHV OBLV has little direct effect on the olfactory epithelium, but causes extensive spongiotic degeneration and destruction of mitral cells and interneurons in the olfactory bulb such that the axonal projection from the bulb via the lateral olfactory tract is markedly reduced. Moreover, surviving mitral cells apparently remain disconnected from the sensory neuron input to the glomerular layer, judging from retrograde labeling studies using Dil. The damage to the bulb indirectly causes a persistent, long-term increase in the turnover of sensory neurons in the epithelium, i.e. the relative proportion of immature to mature sensory neurons and the rate of basal cell proliferation both increase. The changes that develop after inoculation with MHV OBLV closely resemble the disordering of the olfactory epithelium in some patient biopsies. Thus, damage to the olfactory nerve or bulb may contribute to a form of post-viral olfactory dysfunction and MHV OBLV is a useful model for studying the pathogenesis of this form of dysosmia.     

Herpes simplex virus US3 protein kinase regulates virus-induced apoptosis in olfactory and vomeronasal chemosensory neurons in vivo

A role for the US3 protein kinase of herpes simplex virus (HSV) in regulating virus-induced neuronal apoptosis was investigated in an experimental mouse system, in which wild-type HSV invades the central nervous system (CNS) via the olfactory and vomeronasal systems upon intranasal infection. Wild-type HSV-2 strain 186 infected a fraction of olfactory and vomeronasal chemosensory neurons without inducing apoptosis and was transmitted to the CNS, precipitating lethal encephalitis. In sharp contrast, an US3-disrupted mutant, L1BR1, induced neuronal apoptosis in these peripheral conduits upon infection, blocking viral transmission to the CNS and causing no signs of disease. An US3-repaired mutant, L1B(-)11, behaved similarly to the wild-type virus. Only 5 p.f.u. of L1BR1 was sufficient to compromise mice when the mutant virus was introduced directly into the olfactory bulb, a viral entry site of the CNS. These results suggest that the US3 protein kinase of HSV regulates virus-induced neuronal apoptosis in peripheral conduits and determines the neuroinvasive phenotype of HSV. Furthermore, virus-induced neuronal apoptosis of peripheral nervous system cells may be a protective host response that blocks viral transmission to the CNS.     

Early and late pathogenic events of newborn mice encephalitis experimentally induced by itacaiunas and curionópolis bracorhabdoviruses infection

In previous reports we proposed a new genus for Rhabdoviridae and described neurotropic preference and gross neuropathology in newborn albino Swiss mice after Curionopolis and Itacaiunas infections. In the present report a time-course study of experimental encephalitis induced by Itacaiunas and Curionopolis virus was conducted both in vivo and in vitro to investigate cellular targets and the sequence of neuroinvasion. We also investigate, after intranasal inoculation, clinical signs, histopathology and apoptosis in correlation with viral immunolabeling at different time points. Curionopolis and Itacaiunas viral antigens were first detected in the parenchyma of olfactory pathways at 2 and 3 days post-inoculation (dpi) and the first clinical signs were observed at 4 and 8 dpi, respectively. After Curionopolis infection, the mortality rate was 100% between 5 and 6 dpi, and 35% between 8 and 15 dpi after Itacaiunas infection. We identified CNS mice cell types both in vivo and in vitro and the temporal sequence of neuroanatomical olfactory areas infected by Itacaiunas and Curionopolis virus. Distinct virulences were reflected in the neuropathological changes including TUNEL immunolabeling and cytopathic effects, more intense and precocious after intracerebral or in vitro inoculations of Curionopolis than after Itacaiunas virus. In vitro studies revealed neuronal but not astrocyte or microglial cytopathic effects at 2 dpi, with monolayer destruction occurring at 5 and 7 dpi with Curionopolis and Itacaiunas virus, respectively. Ultrastructural changes included virus budding associated with interstitial and perivascular edema, endothelial hypertrophy, a reduced and/or collapsed small vessel luminal area, thickening of the capillary basement membrane, and presence of phagocytosed apoptotic bodies. Glial cells with viral budding similar to oligodendrocytes were infected with Itacaiunas virus but not with Curionopolis virus. Thus, Curionopolis and Itacaiunas viruses share many pathological and clinical features present in other rhabdoviruses but distinct virulence and glial targets in newborn albino Swiss mice brain.     

Limited Trafficking of a Neurotropic Virus Through Inefficient Retrograde Axonal Transport and the Type I Interferon Response

Poliovirus is an enteric virus that rarely invades the human central nervous system (CNS). To identify barriers limiting poliovirus spread from the periphery to CNS, we monitored trafficking of 10 marked viruses. After oral inoculation of susceptible mice, poliovirus was present in peripheral neurons, including vagus and sciatic nerves. To model viral trafficking in peripheral neurons, we intramuscularly injected mice with poliovirus, which follows a muscle–sciatic nerve–spinal cord–brain route. Only 20% of the poliovirus population successfully moved from muscle to brain, and three barriers limiting viral trafficking were identified. First, using light-sensitive viruses, we found limited viral replication in peripheral neurons. Second, retrograde axonal transport of poliovirus in peripheral neurons was inefficient; however, the efficiency was increased upon muscle damage, which also increased the transport efficiency of a non-viral neural tracer, wheat germ agglutinin. Third, using susceptible interferon (IFN) α/β receptor knockout mice, we demonstrated that the IFN response limited viral movement from the periphery to the brain. Surprisingly, the retrograde axonal transport barrier was equivalent in strength to the IFN barrier. Illustrating the importance of barriers created by the IFN response and inefficient axonal transport, IFN α/β receptor knockout mice with muscle damage permitted 80% of the viral population to access the brain, and succumbed to disease three times faster than mice with intact barriers. These results suggest that multiple separate barriers limit poliovirus trafficking from peripheral neurons to the CNS, possibly explaining the rare incidence of paralytic poliomyelitis. This study identifies inefficient axonal transport as a substantial barrier to poliovirus trafficking in peripheral neurons, which may limit CNS access for other viruses.     

CXCR3 mediates region-specific antiviral T cell trafficking within the central nervous system during West Nile virus encephalitis

Regional differences in inflammation during viral infections of the CNS suggest viruses differentially induce patterns of chemoattractant expression, depending on their cellular targets. Previous studies have shown that expression of the chemokine CXCL10 by West Nile virus (WNV)-infected neurons is essential for the recruitment of CD8 T cells for the purpose of viral clearance within the CNS. In the current study we used mice deficient for the CXCL10 receptor, CXCR3, to evaluate its role in leukocyte-mediated viral clearance of WNV infection within various CNS compartments. WNV-infected CXCR3-deficient mice exhibited significantly enhanced mortality compared with wild-type controls. Immunologic and virologic analyses revealed that CXCR3 was dispensable for control of viral infection in the periphery and in most CNS compartments but, surprisingly, was required for CD8 T cell-mediated antiviral responses specifically within the cerebellum. WNV-specific, CXCR3-expressing T cells preferentially migrated into the cerebellum, and WNV-infected cerebellar granule cell neurons expressed higher levels of CXCL10 compared with similarly infected cortical neurons. These results indicate that WNV differentially induces CXCL10 within neuronal populations and suggest a novel model for nonredundancy in chemokine-mediated inflammation among CNS compartments.     

Pattern Recognition Receptor MDA5 Modulates CD8+ T Cell-Dependent Clearance of West Nile Virus from the Central Nervous System

Many viruses induce type I interferon responses by activating cytoplasmic RNA sensors, including the RIG-I-like receptors (RLRs). Although two members of the RLR family, RIG-I and MDA5, have been implicated in host control of virus infection, the relative role of each RLR in restricting pathogenesis in vivo remains unclear. Recent studies have demonstrated that MAVS, the adaptor central to RLR signaling, is required to trigger innate immune defenses and program adaptive immune responses, which together restrict West Nile virus (WNV) infection in vivo. In this study, we examined the specific contribution of MDA5 in controlling WNV in animals. MDA5^−/− mice exhibited enhanced susceptibility, as characterized by reduced survival and elevated viral burden in the central nervous system (CNS) at late times after infection, even though small effects on systemic type I interferon response or viral replication were observed in peripheral tissues. Intracranial inoculation studies and infection experiments with primary neurons ex vivo revealed that an absence of MDA5 did not impact viral infection in neurons directly. Rather, subtle defects were observed in CNS-specific CD8⁺ T cells in MDA5^−/− mice. Adoptive transfer into recipient MDA5^+/+ mice established that a non-cell-autonomous deficiency of MDA5 was associated with functional defects in CD8⁺ T cells, which resulted in a failure to clear WNV efficiently from CNS tissues. Our studies suggest that MDA5 in the immune priming environment shapes optimal CD8⁺ T cell activation and subsequent clearance of WNV from the CNS.