Supplementary Material for: Bacillus Calmette-Guérin Confers Neuroprotection in a Murine Model of Japanese Encephalitis

Objective: Japanese encephalitis (JE) is a debilitating disease caused by infection with the JE virus (JEV; family: Flaviviridae), which leaves neurological sequelae in survivors but more often leads to mortality. Neurodegeneration caused by inflammation is the primary pathology behind the clinical manifestation of encephalitis caused by JEV. Bacillus Calmette-Guérin (BCG) has been used in immunoprophylaxis for tuberculosis and in the adjuvant therapy of many malignancies, and has exhibited neuroprotective activities in experimental models of Parkinson and Alzheimer disease. This study aimed at assessing the neuroprotective role of BCG in a murine model of JE. Methods: Suckling mice were inoculated with 106 CFU of BCG and at 18 days postinoculation were challenged with 100 LD50 of JEV. PBS-inoculated mice were used as controls. Mice were sacrificed on days 2, 4, 6, and 8. Brain tissue was homogenized for RNA extraction. One-step real-time RT-PCR was performed to assess the relative gene expressions of TNF-α, IL-6, and iNOS. Results: The BCG-inoculated (BCG+JEV) group exhibited a significant delay in the presentation of neuropathological symptoms, longer survival, and a downregulation in the expression of TNF-α, IL-6, and iNOS on days 2, 4, and 6 post-JEV challenge compared to the JEV group. Conclusion: These findings indicate that the administration of BCG offers neuroprotection in the murine model of JE. BCG should therefore be further investigated as an adjuvant in the management of JE. BCG is an accepted vaccine for tuberculosis in many countries that are endemic for JEV. This approach may have a significant impact on the public health burden in these countries.