Guido van Marle

West Nile Virus-Induced Neuroinflammation: Glial Infection and Capsid Protein-Mediated Neurovirulence

ABSTRACT 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-1β, 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-WNVC 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.

Compartmentalization of the gut viral reservoir in HIV-1 infected patients

BackgroundRecently there has been an increasing interest and appreciation for the gut as both a viral reservoir as well as an important host-pathogen interface in human immunodefiency virus type 1 (HIV-1) infection. The gut associated lymphoid tissue (GALT) is the largest lymphoid organ infected by HIV-1. In this study we examined if different HIV-1 quasispecies are found in different parts of the gut of HIV-1 infected individuals.ResultsGut biopsies (esophagus, stomach, duodenum and colorectum) were obtained from eight HIV-1 infected preHAART (highly active antiretroviral therapy) patients. HIV-1 Nef and Reverse transcriptase (RT) encoding sequences were obtained through nested PCR amplification from DNA isolated from the gut biopsy tissues. The PCR fragments were cloned and sequenced. The resulting sequences were subjected to various phylogenetic analyses. Expression of the nef gene and viral RNA in the different gut tissues was determined using real-time RT-PCR. Phylogenetic analysis of the Nef protein-encoding region revealed compartmentalization of viral replication in the gut within patients. Viral diversity in both the Nef and RT encoding region varied in different parts of the gut. Moreover, increased nef gene expression (p < 0.05) and higher levels of viral genome were observed in the colorectum (p < 0.05). These differences could reflect an adaptation of HIV-1 to the various tissues.ConclusionOur results indicated that different HIV-1 quasispecies populate different parts of the gut, and that viral replication in the gut is compartmentalized. These observations underscore the importance of the gut as a host-pathogen interface in HIV-1 infection.

Peripheral neuropathy in lentivirus infection: evidence of inflammation and axonal injury.

Objective: As distal sensory polyneuropathy (DSP) is a major neurological complication of HIV-1 infection, we investigated the extent of peripheral nervous system disease in animals infected with the lentivirus, feline immunodeficiency virus (FIV), because it causes neurological disease and immunosuppression in cats similar to HIV-1 in humans. Methods: After infection with a neurovirulent FIV molecular clone, neurobehavioral testing, nerve morphology, viral detection and load measurements were performed. Results: Neurobehavioral studies showed delayed withdrawal in response to a noxious stimulus among FIV-infected animals compared with sham-infected controls (P < 0.05). Dorsal root ganglia and sciatic nerves from FIV-infected ammals showed activated macrophages that were increased in number and size compared with controls. In addition, TNF-α messenger RNA was detectable in most nerves and spinal cords from the FIV-infected group, but was infrequently detected in controls. Viral RNA copy numbers in plasma and sciatic nerves were detectable in all FIV-infected animals at high levels. Studies of sural nerves identified myelinated fiber atrophy in 12-week FIV-infected animals compared with age-matched control animals, which was accompanied by reduced myelin sheath thickness (P < 0.05). The footpads of FIV-infected animals displayed reduced intraepidermal fiber density compared with control animals (P < 0.01). Conclusion: FIV infection results in the rapid onset of peripheral neuropathy, defined by axonal injury and macrophage activation, together with abundant virus within the nerve, indicating that it may serve as a model of HIV-related DSP.

Hepatitis B virus quasispecies in hepatic and extrahepatic viral reservoirs in liver transplant recipients on prophylactic therapy

The characterization of hepatitis B virus (HBV) quasispecies in different compartments in liver transplant (LT) recipients may be helpful in optimizing prophylaxis regimens. The aims of this study were to evaluate liver, peripheral blood mononuclear cells (PBMC), and plasma samples for HBV and to compare the quasispecies in hepatic and extrahepatic sites in LT recipients on long‐term prophylaxis. For 12 patients followed for up to 15 years post‐LT, liver, plasma, and PBMC samples [all HBV DNA–negative according to conventional polymerase chain reaction (PCR) assays] were evaluated for HBV DNA by a sensitive nested PCR method [covalently closed circular DNA (cccDNA) for liver and PBMC samples] and by the sequencing and phylogenetic analysis of polymerase quasispecies. For the 10 patients on prophylaxis with no clinical recurrence (median time post‐LT = 15.5 months, range = 12‐96 months), liver samples were HBV DNA–reactive in 9 of 10 cases, plasma samples were HBV DNA–reactive in 3 of 10 cases, and PBMC samples were HBV DNA–reactive in 2 of 7 cases (including 1 case with HBV cccDNA in PBMCs). The sequence analysis showed that all HBV clones had a wild‐type (WT) sequence in the liver and PBMCs. In 2 patients with early HBV recurrence post‐LT who were treated with nucleosides only, HBV DNA was detected in serum, PBMC, and liver samples, and HBV cccDNA was found in liver samples. An HBV lamivudine‐resistant variant with an M204I mutation was identified in liver (70% and 18% of the clones) and plasma samples (100% of the clones), but a WT sequence was found in 70% and 100% of the PBMC clones. In conclusion, despite prophylaxis and the absence of HBV DNA in serum according to conventional assays, HBV is detectable in the serum, liver, and PBMCs of almost all patients, and this supports the use of continued anti‐HBV therapy in this group. Antiviral drug–resistant variants are more frequent in the liver versus PBMCs, but both compartments are potential sources of reinfection. Liver Transpl 17:955–962, 2011.

Human immunodeficiency virus type 1 genetic diversity in the nervous system: Evolutionary epiphenomenon or disease determinant?

Over the past decade there has been a revolution in the understanding and care of human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS)-associated disease. Much of this progress stems from a broader recognition of the importance of differences in viral types, including receptor preference(s), replication properties, and reservoirs, as contributing factors to immunosuppresion and disease progression. In contrast, there is limited conceptualizatin of viral diversity and turnover in the brain and circulation in relation to neurocognitive impairments. Herein, the authors review current concepts regarding viral molecular diversity and phenotypes together with features of HIV-1 neuroinvasion, neurotropism, neurovirulence, and neurosusceptiblity. Viral genetic and antigenic diversity is reduced within the brain compared to blood or other systemic organs within individuals. Conversely, viral molecular heterogeneity is greater in patients with HIV-associated dementia compared to nondemented patients, depending on the viral gene examined. Individual viral proteins exert multiple neuropathogenic effects, although the neurological consequences of different viral polymorphisms remain uncertain. Nonetheless, host genetic polymorphisms clearly influence neurological disease outcomes and likely dictate both acquired and innate immune responses, which in turn shape viral evolution within the host. Emerging issues include widespread antiretroviral therapy resistance and increasing awareness of viral superinfections together with viral recombination, all of which are likely to impact on both HIV genetic variation and neuropathogenesis. With the increasing prevalence of HIV-induced neurocognitive disabilities, despite marked improvements in managing immunosuppression, it remains imperative to fully define and understand the mechanisms by which viral dynamics and diversity contribute to neurological disease, permitting the development of new therapeutic strategies.

Hiv dementia patients exhibit reduced viral neutralization and increased envelope sequence diversity in blood and brain

Objectives: To examine the relationship between the humoral immune response and viral envelope diversity among HIV/AIDS patients with or without HIV-associated dementia (HAD). Methods: Whole blood and sera were collected from age- and disease-progression matched AIDS-defined patients with and without neuro-cognitive impairment at two centers. Peripheral blood mononuclear cells were isolated from whole blood and separated into monocyte/macrophage and peripheral blood lymphocyte (PBL) preparations. Genomic DNA, isolated from the PBL population, was used as template to amplify HIV-1 C2V3 envelope sequences in a nested PCR protocol. The resulting fragments were sequenced and subjected to a phylogenetic analysis. Results: Sera from non-demented (ND; n = 21) patients neutralized infection of CCR5-dependent, but not CXCR4-dependent viruses, more efficiently than sera from HAD patients (n = 15) (P < 0.05). A recombinant virus containing a brain derived C2V3 sequence was also neutralized less efficiently by sera from HAD patients (P < 0.05). C2V3 envelope sequences amplified from PBL revealed significantly greater diversity within the V3 region from HAD compared with ND patients (P < 0.001). The number of non-synonymous substitutions was positively correlated with the severity of neuro-cognitive impairment of patients (P < 0.005). Similarly, brain derived V3 sequences exhibited significantly increased diversity among HAD patients (P < 0.001). Conclusion: Our findings imply that HAD patients exhibited impaired serological responses that may lead to the emergence of viral mutants that potentially could infect the brain and mediate neurodegeneration.

Inflammation and epithelial cell injury in AIDS enteropathy: involvement of endoplasmic reticulum stress

Immunosuppressive lentivirus infections, including human, simian, and feline immunodeficiency viruses (HIV, SIV, and FIV, respectively), cause the acquired immunodeficiency syndrome (AIDS), frequently associated with AIDS enteropathy. Herein, we investigated the extent to which lentivirus infections affected mucosal integrity and intestinal permeability in conjunction with immune responses and activation of endoplasmic reticulum (ER) stress pathways. Duodenal biopsies from individuals with HIV/AIDS exhibited induction of IL‐1β, CD3ε, HLA‐DRA, spliced XBP‐1(Xbp‐1s), and CHOP expression compared to uninfected persons (P<0.05). Gut epithelial cells exposed to HIV‐1 Vpr demonstrated elevated TNF‐α., IL‐1β, spliced Xbp‐1s, and CHOP expression (P<0.05) together with calcium activation and disruption of epithelial cell monolayer permeability. In addition to reduced blood CD4+ T lymphocyte levels, viral loads in the gut and plasma were high in FIV‐infected animals (P<0.05). FIV‐infected animals also exhibited a failure to gain weight and increased lactulose/mannitol ratios compared with uninfected animals (P<0.05). Proinflammatory and ER stress gene expression were activated in the ileum of FIV‐infected animals (P<0.05), accompanied by intestinal epithelial damage with loss of epithelial cells and leukocyte infiltration of the lamina propria. Lentivirus infections cause gut inflammation and ensuing damage to intestinal epithelial cells, likely through induction of ER stress pathways, resulting in disruption of gut functional integrity.—Maingat, F., Halloran, B., Acharjee, S., van Marle, G., Church, D., Gill, M. J., Uwiera, R. R. E., Cohen, E. A., Meddings, J., Madsen, K., Power, C. Inflammation and epithelial cell injury in AIDS enteropathy: involvement of endoplasmic reticulum stress. FASEB J. 25, 2211‐2220 (2011). www.fasebj.org

In Vivo Impairment of Neutrophil Recruitment during Lentivirus Infection

Evidence indicates that the lentivirus, HIV, infection affects neutrophil response to bacteria and bacterial products in vitro. We used a novel model of rapid onset immunosuppression following infection with a similar lentivirus, feline immunodeficiency virus (FIV), in cats to examine neutrophil function within the microvasculature in vivo and to determine the steps that are impaired in the neutrophil recruitment cascade. In uninfected cats and cats infected neonatally with FIV, the mesentery was exteriorized, but remained autoperfused during intravital microscopy for 4 h. When the tissue was superfused with 10 μg/ml of LPS for 4 h, intravital microscopy displayed a profound increase in neutrophil rolling at both 8 and 12 wk of age in uninfected cats. At 12 wk of age, FIV-infected animals showed a profound decrease in the number of rolling neutrophils. In vitro studies revealed that neutrophils from infected and uninfected animals rolled equally well on surrogate selectin substrata. In addition, in vivo neutrophil adhesion and emigration out of the vasculature were severely reduced, and in vitro neutrophil chemotaxis from FIV-infected animals was significantly impaired in response to fMLP or IL-8. However, FIV infection of neutrophils could not be detected. In summary, in vivo lentivirus infection with immunosuppression leads to a severe impairment in neutrophil rolling, adhesion, and emigration in response to bacterial stimulants potentially involving both endothelial and neutrophil dysfunction. These in vivo studies also indicate that neutrophil dysfunction should be taken into account when treating infections and tissue injury.

West Nile Virus Infection Causes Endocytosis of a Specific Subset of Tight Junction Membrane Proteins

West Nile virus (WNV) is a blood-borne pathogen that causes systemic infections and serious neurological disease in human and animals. The most common route of infection is mosquito bites and therefore, the virus must cross a number of polarized cell layers to gain access to organ tissue and the central nervous system. Resistance to trans-cellular movement of macromolecules between epithelial and endothelial cells is mediated by tight junction complexes. While a number of recent studies have documented that WNV infection negatively impacts the barrier function of tight junctions, the intracellular mechanism by which this occurs is poorly understood. In the present study, we report that endocytosis of a subset of tight junction membrane proteins including claudin-1 and JAM-1 occurs in WNV infected epithelial and endothelial cells. This process, which ultimately results in lysosomal degradation of the proteins, is dependent on the GTPase dynamin and microtubule-based transport. Finally, infection of polarized cells with the related flavivirus, Dengue virus-2, did not result in significant loss of tight junction membrane proteins. These results suggest that neurotropic flaviviruses such as WNV modulate the host cell environment differently than hemorrhagic flaviviruses and thus may have implications for understanding the molecular basis for neuroinvasion.

Aberrant cortical neurogenesis in a pediatric neuroAIDS model: neurotrophic effects of growth hormone.

Objective:To study the effects of HIV-1 and feline immunodeficiency virus (FIV) on neural stem cell viability, together with the neurotrophic properties of growth hormone (GH) in models of pediatric neuroAIDS. Design and methods:Mouse neural stem cells were infected in vitro with a Sindbis virus vector (SIN-HIVenv) expressing the envelope protein from the brain-derived HIV-1 strain JR-FL using a vector expressing enhanced green fluorescent protein (SIN-EGFP) as control. Cell survival and alterations in expression of neural stem cell markers upon GH treatment was assessed. Neonatal cats were infected with a neurovirulent FIV strain and 6 weeks after infection treated with GH for 6 weeks. Twelve weeks post-infection, neural progenitor cell marker expression, neuronal loss and neuroinflammation in brain were examined using real time reverse transcription–PCR and immunohistochemical analyses. Results:HIV-1 envelope expression in neural stem cells reduced nestin expression (P < 0.05) and induced cell death (P < 0.001), which was blocked by GH. In the frontal cortex of FIV-infected cats neuroinflammation, loss of differentiated neurons (P < 0.01) and aberrant neuronal progenitor cell gene expression (P < 0.05) were observed. FIV envelope expression was detected in neural progenitor and monocytoid cells. GH treatment of FIV-infected animals induced insulin-like growth factor-1 expression in neurons (P < 0.01), enhanced neuronal survival (P < 0.01) and increased nestin expression (P < 0.05). Moreover, improved neurobehavioral performance (P < 0.01) and immunological status (P < 0.001) were observed, among GH-treated animals infected with FIV. Conclusion:GH protects neural stem cells that are susceptible to lentivirus-mediated injury. Thus, GH may be a potential treatment for pediatric neuroAIDS because of its neurotrophic actions.