Background:  In the HAART era, progressive multifocal leukoencephalopathy (PML), remains an important cause of morbidity and mortality among AIDS patients. The pathogenesis of PML, specifically how JC virus (JCV), the etiological agent of PML, crosses the blood-brain barrier (BBB) remains unclear. We hypothesized that cell-free JCV during viremia may infect the human brain microvascular endothelial cells (HBMVEC), a critical component of the BBB and cross the BBB to further infect other brain cells, specifically, oligodendrocytes.

Methods:  Primary HBMVEC were infected with JCV (Mad1) and were analyzed for the expression of early viral protein, T antigen (TAg), by reverse transcriptase real-time PCR. An in vitro BBB model was developed to study the transmigration of cell-free JCV across the BBB by seeding primary human brain cortical astrocytes and primary HBMVEC, on the lower and upper surfaces, respectively, of fibronectin-coated, 3-μm pore, 24-well format cell culture inserts. Moreover, the effect of several serotonin inhibitors including chlorpromazine, kentaserin, and ritanserin in blocking the JCV infection of HBMVEC or its transmigration across the BBB model was tested.

Results:  Expression of TAg in JCV-infected HBMVEC increased 2- to 4-fold from day 5 to day 10 post-infection, and serotonin blockers were unable to inhibit JCV infection. About 1% of the JCV inoculated in the upper chamber was recovered from the lower chamber supernatant by day 7 post-inoculation, whereas only 0.2% of the heat-inactivated JCV crossed the BBB by day 7. Moreover, 90% of the heat-inactivated JCV recovered from the lower chamber crossed the BBB within the first 6 hour; in contrast 85% of JCV crossed the BBB after the first 6 hours, with a gradual increase till 148 hours. The serotonin inhibitors did not block JCV transmigration across the in vitro BBB model.

Conclusions:  Our data indicates that cell-free JCV can infect the HBMVEC, JCV can migrate across the BBB, and based on the infection of primary human fetal glial cells, the migrated JCV is infectious. The transmigration of JCV was slow in early hours post-infection but continued to increase slowly and gradually over the next 7 days. Inability of the heat-inactivated JCV to continue to cross the BBB after 6 hours indicates that infectious JCV is essential to efficiently cross the BBB. Moreover, JCV does not employ serotonin receptors to infect HBMVEC, nor to cross the BBB, and serotonin blockers may not prevent the spread of JCV to brain cells.