West Nile virus-induced cell adhesion molecules on human brain microvascular endothelial cells regulate leukocyte adhesion and modulate permeability of the in vitro blood-brain barrier model.

Characterizing the mechanisms by which West Nile virus (WNV) causes blood-brain barrier (BBB) disruption, leukocyte infiltration into the brain and neuroinflammation is important to understand the pathogenesis of WNV encephalitis. Here, we examined the role of endothelial cell adhesion molecules (CAMs) in mediating the adhesion and transendothelial migration of leukocytes across human brain microvascular endothelial cells (HBMVE). Infection with WNV (NY99 strain) significantly induced ICAM-1, VCAM-1, and E-selectin in human endothelial cells and infected mice brain, although the levels of their ligands on leukocytes (VLA-4, LFA-1and MAC-1) did not alter. The permeability of the in vitro BBB model increased dramatically following the transmigration of monocytes and lymphocytes across the models infected with WNV, which was reversed in the presence of a cocktail of blocking antibodies against ICAM-1, VCAM-1, and E-selectin. Further, WNV infection of HBMVE significantly increased leukocyte adhesion to the HBMVE monolayer and transmigration across the infected BBB model. The blockade of these CAMs reduced the adhesion and transmigration of leukocytes across the infected BBB model. Further, comparison of infection with highly neuroinvasive NY99 and non-lethal (Eg101) strain of WNV demonstrated similar level of virus replication and fold-increase of CAMs in HBMVE cells suggesting that the non-neuropathogenic response of Eg101 is not because of its inability to infect HBMVE cells. Collectively, these results suggest that increased expression of specific CAMs is a pathological event associated with WNV infection and may contribute to leukocyte infiltration and BBB disruption in vivo. Our data further implicate that strategies to block CAMs to reduce BBB disruption may limit neuroinflammation and virus-CNS entry via 'Trojan horse' route, and improve WNV disease outcome

Neutralizing antibodies against cell adhesion molecules partially reverse the disruption of the <i>in vitro</i> BBB model during leukocyte transmigration.

<p>The integrity of the <i>in vitro</i> BBB model was determined by measuring the TEER before and after four hrs of incubation with monocytes and lymphocytes in the presence or absence of the cocktail of neutralizing antibodies against CAMs. The decrease in the TEER values was represented as percentage change as compared to mock-infected controls. The data is mean ± SD of two independent experiments. *p<0.05 compared to mock.</p

WNV-Eg101 efficiently infects HBMVE cells and induces CAMs.

<p>Primary human HBMVE cells were infected with WNV-NY99 or Eg101 (MOI5). (<b>A</b>) Culture supernatants recovered at 48 and 72 hrs after infection were used to determine titers using plaque assay on Vero cells. Data represent the mean ± SD PFU per mL of supernatant from two independent experiments. (<b>B</b>) qRT-PCR analysis of CAMs in Eg101 infected HBMVE cells at 48 and 72 hrs after infection. The data are normalized to the values of GAPDH and are expressed as relative fold increase compared to uninfected controls.</p

WNV induces expression of specific CAMs in the HBMVE cells and mouse brain.

<p>(<b>A</b>) The changes in the mRNA of multiple CAMs was measured in HBMVE cells at 72 hours after infection. (<b>B</b>) qRT-PCR was conducted on the RNA extracted from WNV-infected human monocytes to determine fold-change in the VLA-4, MAC-1 and L-selectin gene expression. (<b>C</b>) WNV titers and fold-change of ICAM-1, VCAM-1 and E-selectin in WNV-infected mouse brain as determined by qRT-PCR. The data are normalized to the values of GAPDH and are expressed as relative fold increase compared to uninfected controls. (<b>D</b>) Validation of the increase in the protein expression of ICAM-1 and E-selectin in HBMVE cells at 72 hrs after infection by immunostaining. The data are expressed as means ± SD from two independent experiments in duplicate. *p<0.05 compared to mock.</p

WNV infection of HMBVE cells, but not of leukocytes, mediates the disruption of the <i>in vitro</i> BBB model.

<p>The effect of leukocyte migration on the permeability of the <i>in vitro</i> BBB model was determined by TEER measurements. (<b>A)</b> WNV infected monocytes (2.5 x 10⁵) at 24 hrs after infection were added in the upper compartment of the uninfected BBB model and permeability was measured at 2 and 4 hrs after incubation. (<b>B</b>) At 72 hrs after WNV infection of BBB model, transmigration of uninfected monocytes significantly decreased the TEER as compared to control. (<b>C and D</b>) A chemotactic gradient was established by the addition of 100 ng/mL recombinant human CCL2 to the lower compartment and uninfected monocytes and lymphocytes were allowed to migrate across infected BBB. Results are the mean of data from two independent experiments in duplicate. *p<0.05 compared to mock. C, inserts with control HBMVE cells without leukocytes.</p

Primer sequences used for qRT-PCR.

<p>Primer sequences used for qRT-PCR.</p

Combined treatment of adenosine nucleoside inhibitor NITD008 and histone deacetylase inhibitor vorinostat represents an immunotherapy strategy to ameliorate West Nile virus infection

Abstract West Nile virus (WNV), a member of the Flaviviridae family, is the leading cause of viral encephalitis in the United States. Despite efforts to control the spread of WNV, there has been an increase in the number of outbreaks and clinical cases with neurological problems. There are no antiviral compounds currently in trials for WNV. NITD008 is an adenosine analogue inhibitor that interrupts the RNA-dependent RNA polymerase of flaviviruses. Previous studies demonstrated NITD008 as a potent antiviral for dengue virus, however this drug was associated with preclinical toxicity. The ability of NITD008 to block WNV replication is only shown in Vero cells. Neuroinflammation is also a major cause of the WNV-associated pathology, therefore we evaluated the effect of NITD008 and a newly characterized anti-inflammatory drug vorinostat (SAHA), a histone deacetylase inhibitor, on WNV replication and disease progression in a mouse model. When administered at 10 and 25 mg/kg at days 1-6 after WNV infection in C57BL/6 mice, NITD008 conferred complete protection from clinical symptoms and death, which correlated with reduced viral load in the serum and restriction of virus-CNS entry. Delay of NITD008 treatment to days 3-6 and days 5-9 after infection, when WNV replication was high in the periphery and brain, resulted in the gradual loss of protection against WNV infection. However, co-treatment with SAHA and NITD008 during the CNS phase of disease improved disease outcome significantly by reducing inflammation and neuronal death. Our results support potential synergistic effect of combination therapy of NITD008 with SAHA for the treatment of WNV encephalitis

Impaired virus clearance, compromised immune response and increased mortality in type 2 diabetic mice infected with West Nile virus.

Clinicoepidemiological data suggest that type 2 diabetes is associated with increased risk of West Nile virus encephalitis (WNVE). However, no experimental studies have elucidated the role of diabetes in WNV neuropathogenesis. Herein, we employed the db/db mouse model to understand WNV immunopathogenesis in diabetics. Nine-week old C57BL/6 WT and db/db mice were inoculated with WNV and mortality, virus burden in the periphery and brain, and antiviral defense responses were analyzed. db/db mice were highly susceptible to WNV disease, exhibited increased tissue tropism and mortality than the wild-type mice, and were unable to clear the infection. Increased and sustained WNV replication was observed in the serum, peripheral tissues and brain of db/db mice, and heightened virus replication in the periphery was correlated with enhanced neuroinvasion and replication of WNV in the brain. WNV infection in db/db mice was associated with enhanced inflammatory response and compromised antiviral immune response characterized by delayed induction of IFN-α, and significantly reduced concentrations of WNV-specific IgM and IgG antibodies. The compromised immune response in db/db mice correlated with increased viremia. These data suggest that delayed immune response coupled with failure to clear the virus leads to increased mortality in db/db mice. In conclusion, this study provides unique mechanistic insight into the immunopathogenesis of WNVE observed in diabetics and can be used to develop therapeutics for the management of WNVE among diabetic patients