Assay for Assessing Protective Effect of Z2 Against ZIKV Infection in Testicular Tissue of Male A129 Mice

To determine the protection of Z2 against ZIKV-induced testis damage, male A129 mice (7–8 weeks old) were randomly divided into three groups (n = 5): Z2-treated group, vehicle-treated group, and mock-infected group. Mice in the Z2- or vehicle-treated group were intraperitoneally injected (i.p.) with 200 PFU ZIKV with Z2 (15 mg/kg) or vehicle on day 0, followed by i.p. administration of Z2 (15 mg/kg) or vehicle once daily for six consecutive days, respectively. Mice in the mock-infected group only received PBS as normal control. The body weight was monitored daily for 14 days, and blood was collected at 1, 3, 7, 11, and 16 days post-infection (d.p.i.) for detection of ZIKV copies in sera. At 16 d.p.i., mice were euthanized by CO₂ inhalation, and the testes and epididymides were removed. The weight and size of testes were measured as previously described (de La Vega et al., 2018). After imaging, the left testes and epididymides were immersed in Bouin’s for hematoxylin-eosin (H&E) staining. The right testes and epididymides were soaked in RNAiso Plus reagent at −80°C for further detection of ZIKV copies.