West Nile virus-induced neuroinflammation: glial infection and capsid protein-mediated neurovirulence

West Nile virus (WNV) infection causes neurological disease at all levels of the neural axis, accompanied by neuroinflammation and neuronal loss, although the underlying mechanisms remain uncertain. Given the substantial activation of neuroinflammatory pathways observed in WNV infection, we hypothesized that WNV-mediated neuroinflammation and cell death occurred through WNV infection of both glia and neurons, which was driven in part by WNV capsid protein expression. Analysis of autopsied neural tissues from humans with WNV encephalomyelitis (WNVE) revealed WNV infection of both neurons and glia. Upregulation of proinflammatory genes, CXCL10, interleukin-1beta, and indolamine-2',3'-deoxygenase with concurrent suppression of the protective astrocyte-specific endoplasmic reticulum stress sensor gene, OASIS (for old astrocyte specifically induced substance), was evident in WNVE patients compared to non-WNVE controls. These findings were supported by increased ex vivo expression of these proinflammatory genes in glia infected by WNV-NY99. WNV infection caused endoplasmic reticulum stress gene induction and apoptosis in neurons but did not affect glial viability. WNV-infected astrocytic cells secreted cytotoxic factors, which caused neuronal apoptosis. The expression of the WNV-NY99 capsid protein in neurons and glia by a Sindbis virus-derived vector (SINrep5-WNVc) caused neuronal death and the release of neurotoxic factors by infected astrocytes, coupled with proinflammatory gene induction and suppression of OASIS. Striatal implantation of SINrep5-WNV(C) induced neuroinflammation in rats, together with the induction of CXCL10 and diminished OASIS expression, compared to controls. Moreover, magnetic resonance neuroimaging showed edema and tissue injury in the vicinity of the SINrep5-WNVc implantation site compared to controls, which was complemented by neurobehavioral abnormalities in the SINrep5-WNVc-implanted animals. These studies underscore the important interactions between the WNV capsid protein and neuroinflammation in the pathogenesis of WNV-induced neurological disorders.     

West Nile virus-induced bax-dependent apoptosis.

The mechanism of cell death induced by West Nile virus (WNV), a causative agent of human febrile syndrome and encephalitis, was investigated. WNV-infected K562 and Neuro-2a cells manifested the typical features of apoptosis, including cell shrinkage, chromatin condensation and subdiploid DNA content by flow cytometry. DNA fragmentation into nucleosomal size and changes in outer cell membrane phospholipid composition were also observed in K562 cells. UV-inactivated virus failed to induce the above-mentioned characteristics, suggesting that viral replication may be required for the induction of apoptosis by WNV. Additionally, signals involved in WNV-induced apoptosis are associated with the up-regulation of bax gene expression.     

West Nile virus discriminates between DC-SIGN and DC-SIGNR for cellular attachment and infection

The C-type lectins DC-SIGN and DC-SIGNR bind mannose-rich glycans with high affinity. In vitro, cells expressing these attachment factors efficiently capture, and are infected by, a diverse array of appropriately glycosylated pathogens, including dengue virus. In this study, we investigated whether these lectins could enhance cellular infection by West Nile virus (WNV), a mosquito-borne flavivirus related to dengue virus. We discovered that DC-SIGNR promoted WNV infection much more efficiently than did DC-SIGN, particularly when the virus was grown in human cell types. The presence of a single N-linked glycosylation site on either the prM or E glycoprotein of WNV was sufficient to allow DC-SIGNR-mediated infection, demonstrating that uncleaved prM protein present on a flavivirus virion can influence viral tropism under certain circumstances. Preferential utilization of DC-SIGNR was a specific property conferred by the WNV envelope glycoproteins. Chimeras between DC-SIGN and DC-SIGNR demonstrated that the ability of DC-SIGNR to promote WNV infection maps to its carbohydrate recognition domain. WNV virions and subviral particles bound to DC-SIGNR with much greater affinity than DC-SIGN. We believe this is the first report of a pathogen interacting more efficiently with DC-SIGNR than with DC-SIGN. Our results should lead to the discovery of new mechanisms by which these well-studied lectins discriminate among ligands.     

Chemokine control of West Nile virus infection

West Nile virus (WNV) is a re-emerging pathogen responsible for fatal outbreaks of meningoencephalitis in humans. Recent research using a mouse model of infection has indicated that specific chemokines and chemokine receptors help mediate the host response to WNV acting by at least three mechanisms: control of early neutrophil recruitment to the infection site (Cxcr2), control of monocytosis in blood (Ccr2) and control of leukocyte movement from blood to brain (Cxcr4, Cxcr3, Cxcl10 and possibly Ccr5). CCR5 also appears to be important in human infection, since individuals genetically deficient in this receptor have increased risk of symptomatic disease once infected. These findings provide detailed insight into non-redundant chemokine roles in organ-specific leukocyte recruitment during infection, and emphasize the importance of the balance between pathogen control and immunopathology in determining overall clinical outcome.     

Multi-species interactions in West Nile virus infection

In this paper, we analyse the interaction of different species of birds and mosquitoes on the dynamics of West Nile virus (WNV) infection. We study the different transmission efficiencies of the vectors and birds and the impact on the possible outbreaks. We show that the basic reproductive number is the weighted mean of the basic reproductive number of each species, weighted by the relative abundance of its population in the location. These results suggest a possible explanation of why there are no outbreaks of WNV in Mexico.     

Multi-species interactions in West Nile virus infection

In this paper, we analyse the interaction of different species of birds and mosquitoes on the dynamics of West Nile virus (WNV) infection. We study the different transmission efficiencies of the vectors and birds and the impact on the possible outbreaks. We show that the basic reproductive number is the weighted mean of the basic reproductive number of each species, weighted by the relative abundance of its population in the location. These results suggest a possible explanation of why there are no outbreaks of WNV in Mexico.     

Role of IFN-gamma in an experimental murine model of West Nile virus-induced seizures

Seizures are a major complication of viral encephalitis. However, the mechanisms of seizure-associated neuronal dysfunction remain poorly understood. We report that intranasal inoculation with West Nile virus (WNV) (Sarafend) causes limbic seizures in C57BL/6 mice, but not in interferon (IFN)-gamma-deficient (IFN-gamma-/-) mice. Both strains showed similar levels of virus in the brain, as well as similar concentrations of the cytokines, tumor necrosis factor and interleukin-6, both of which can alter neuronal excitability. Experiments in chimeric IFN-gamma-/- mice reconstituted with IFN-gamma-producing leukocytes showed that IFN-gamma is not required during central nervous system infection for limbic seizure development, suggesting a role for IFN-gamma in the developing brain. This was supported responses to pentylenetetrazole, kainic acid (KA), and N-methyl-d-aspartate (NMDA). Both strains of mice exhibited similar behavior after pentylenetetrazole challenge. However, while NMDA and KA treatment resulted in characteristic seizures in C57BL/6 mice, these responses were diminished (NMDA treatment) or absent (KA treatment) in IFN-gamma-/- mice. Furthermore, NMDA-receptor blockade with MK-801 in WNV-infected C57BL/6 mice abrogated seizures and prolonged survival. Our data show that IFN-gamma plays an important role in the development of the excitatory seizure pathways in the brain and that these cascades become pathogenic in encephalitic WNV infection.     

NS1 protein secretion during the acute phase of West Nile virus infection

The West Nile virus (WNV) nonstructural protein NS1 is a protein of unknown function that is found within, associated with, and secreted from infected cells. We systematically investigated the kinetics of NS1 secretion in vitro and in vivo to determine the potential use of this protein as a diagnostic marker and to analyze NS1 secretion in relation to the infection cycle. A sensitive antigen capture enzyme-linked immunosorbent assay (ELISA) for detection of WNV NS1 (polyclonal-ACE) was developed, as well as a capture ELISA for the specific detection of NS1 multimers (4G4-ACE). The 4G4-ACE detected native NS1 antigens at high sensitivity, whereas the polyclonal-ACE had a higher specificity for recombinant forms of the protein. Applying these assays we found that only a small fraction of intracellular NS1 is secreted and that secretion of NS1 in tissue culture is delayed compared to the release of virus particles. In experimentally infected hamsters, NS1 was detected in the serum between days 3 and 8 postinfection, peaking on day 5, the day prior to the onset of clinical disease; immunoglobulin M (IgM) antibodies were detected at low levels on day 5 postinfection. Although real-time PCR gave the earliest indication of infection (day 1), the diagnostic performance of the 4G4-ACE was comparable to that of real-time PCR during the time period when NS1 was secreted. Moreover, the 4G4-ACE was found to be superior in performance to both the IgM and plaque assays during this time period, suggesting that NS1 is a viable early diagnostic marker of WNV infection.     

Innate immune control of West Nile virus infection

West Nile virus (WNV), from the Flaviviridae family, is a re-emerging zoonotic pathogen of medical importance. In humans, WNV infection may cause life-threatening meningoencephalitis or long-term neurologic sequelae. WNV is transmitted by Culex spp. mosquitoes and both the arthropod vector and the mammalian host are equipped with antiviral innate immune mechanisms sharing a common phylogeny. As far as the current evidence is able to demonstrate, mosquitoes primarily rely on RNA interference, Toll, Imd and JAK-STAT signalling pathways for limiting viral infection, while mammals are provided with these and other more complex antiviral mechanisms involving antiviral effectors, inflammatory mediators, and cellular responses triggered by highly specialized pathogen detection mechanisms that often resemble their invertebrate ancestry. This mini-review summarizes our current understanding of how the innate immune systems of the vector and the mammalian host react to WNV infection and shape its pathogenesis.     

Role of two distinct gammadelta T cell subsets during West Nile virus infection.

gammadelta T cells respond rapidly following West Nile virus (WNV) infection, limiting viremia and invasion of the central nervous system and thereby protecting the host from lethal encephalitis. Here, we investigated the role of two major subpopulations of peripheral gammadelta T cells, Vgamma1(+) and Vgamma4(+) cells, in host immunity against WNV infection. We found initially that aged mice were more susceptible to WNV infection than young mice. Following WNV challenge, Vgamma1(+) cells in young mice expanded significantly whereas Vgamma4(+) cells expanded modestly. In contrast, aged mice exhibited a slower and reduced response of Vgamma1(+) cells but maintained a higher content of Vgamma4(+) cells. Vgamma1(+) cells were the major gammadelta subset producing IFN-gamma during WNV infection. Mice depleted of Vgamma1(+) cells had an enhanced viremia and higher mortality to WNV encephalitis. Vgamma4(+) cells had a higher potential for producing tumor necrosis factor-alpha (TNF-alpha), a cytokine known to be involved in blood-brain barrier compromise and WNV entry into the brain. Depletion of Vgamma4(+) cells reduced TNF-alpha level in the periphery, accompanied by a decreased viral load in the brain and a lower mortality to WN encephalitis. These results suggest that Vgamma1(+) and Vgamma4(+) cells play distinct roles in protection and pathogenesis during WNV infection.     

Identification of novel small-molecule inhibitors of West Nile virus infection

West Nile virus (WNV) has spread throughout the United States and Canada and now annually causes a clinical spectrum of human disease ranging from a self-limiting acute febrile illness to acute flaccid paralysis and lethal encephalitis. No therapy or vaccine is currently approved for use in humans. Using high-throughput screening assays that included a luciferase expressing WNV subgenomic replicon and an NS1 capture enzyme-linked immunosorbent assay, we evaluated a chemical library of over 80,000 compounds for their capacity to inhibit WNV replication. We identified 10 compounds with strong inhibitory activity against genetically diverse WNV and Kunjin virus isolates. Many of the inhibitory compounds belonged to a chemical family of secondary sulfonamides and have not been described previously to inhibit WNV or other related or unrelated viruses. Several of these compounds inhibited WNV infection in the submicromolar range, had selectivity indices of greater than 10, and inhibited replication of other flaviviruses, including dengue and yellow fever viruses. One of the most promising compounds, AP30451, specifically blocked translation of a yellow fever virus replicon but not a Sindbis virus replicon or an internal ribosome entry site containing mRNA. Overall, these compounds comprise a novel class of promising inhibitors for therapy against WNV and other flavivirus infections in humans.     

Cholesterol manipulation by West Nile virus perturbs the cellular immune response

Complex membrane structures induced by West Nile virus (WNV), an enveloped RNA virus, are required for efficient viral replication. How these membranes are induced and how they facilitate the viral life cycle are unknown. We show that WNV modulates host cell cholesterol homeostasis by upregulating cholesterol biosynthesis and redistributing cholesterol to viral replication membranes. Manipulating cholesterol levels and altering concentrations of cellular geranylgeranylated proteins had a deleterious effect on virus replication. Depletion of the key cholesterol-synthesizing enzyme 3-hydroxy-methyglutaryl-CoA reductase drastically hampered virus replication. Significantly, virus-induced redistribution of cellular cholesterol downregulated the interferon-stimulated Jak-STAT antiviral signaling response to infection. This defect could be partially restored by exogenous addition of cholesterol, which increased the ability of infected cells to respond to interferon. We propose that, by manipulating cellular cholesterol, WNV utilizes the cellular response to cholesterol deficiency and dependence of antiviral signaling pathways on cholesterol-rich microdomains to facilitate viral replication and survival.     

Wolbachia infection in West Nile Virus vectors of northwest Iran

Mosquito-borne viral diseases are serious health problems in many countries. Various methods have been used for controlling the vectors of these diseases. Among symbiotic bacteria, the members of the genus Wolbachia are the most ubiquitous symbionts of arthropods and play key roles in their host biological characteristics with various effects on their hosts. The identification of these bacteria in Iranian mosquitoes is limited to a few studies. The current study was carried out to determine (1) the Wolbachia infection of probable arbovirus vectors (Aedes caspius, Culex pipiens, Culex theileri and Culiseta longiareolata), (2) the Wolbachia strain(s) infecting the mosquitoes, and (3) the geographical distribution of the Wolbachia strain(s) in the northwest of Iran. Eight species including Ae. caspius, Anopheles hyrcanus, An. maculipennis, Cx. hortensis, Cx. modestus, Cx. pipiens, Cx. theileri, and Cs. longiareolata were identified, amongst which Ae. caspius with 63.1% and An. hyrcanus with 0.3% were the most and the least abundant species, respectively. The results of semi-nested PCR using Wolbachia surface protein (wsp) fragment assays showed that Wolbachia infection was present in three out of the four above mentioned arboviral vector species (Aedes caspius, Culex pipiens, Culex theileri and Culiseta longiareolata), where the highest infection rate was seen in Cx. pipiens. The infection rates of mosquitoes with Wolbachia in the species of Cx. pipiens, Cs. longiareolata, Cx. theileri, and Ae. caspius were 96.9%, 11.5%, 5.2% and 0%, respectively.