To determine the differentiation status of the effector CD4+ T cells induced by ZIKV infection, we stimulated total splenocytes from ZIKV-infected mice with phorbol myristate acetate (PMA) and ionomycin in the presence of Brefeldin A for 3 h and analyzed cytokine production with intracellular cytokine staining via flow cytometry (Fig 5). Here, CD4 is linked to Zika virus infectious disease.