Fig 1.
ZIKV replication in the mouse epididymis and simian primary cells.
Eight-week-old male AG6 mice were inoculated with ZIKV at 105 plaque-forming units (PFU) in bilateral footpads. (A) Viral loads in the testis and epididymis at 2, 5, and 8 days post infection (dpi) were detected by RT-qPCR. n = 5. Each data point represents an individual mouse. (B) Distribution of ZIKV antigens in various epididymal segments in AG6 mice at 2, 5, and 8 dpi was determined by immunofluorescent assay (IFA). IT, intertubular space; Lu, lumen. Primary Seroli cells (SC), epididymal epithelial cells (EEC) and Leydig cells (LC) were isolated from a two-year-old male African green monkey. Simian primary cells were infected with ZIKV at multiplicity of infection (MOI) = 1. Supernatant was harvested every 24 hours until 120 hours. (C) Viral proliferation kinetics in simian SC, EEC and LC were determined by RT-qPCR. Three independent experiments were repeated. (D) Distribution of ZIKV antigens in simian SC, EEC and LC at 48 and 72 hours post infection (hpi) was detected by IFA.
Fig 2.
Histopathological changes in the epididymis after ZIKV infection.
Eight-week-old male AG6 mice were inoculated with 105 plaque-forming units (PFU) of CAS-ZK01 strain of ZIKV in bilateral footpads. Epididymis was collected at 2, 5, and 8 days post infection (dpi). Uninfected mice were used as a control. n = 5. (A) Histology of the epididymal segments was analyzed by HE staining. The atrophied epithelium was indicated by black asterisks. Blockage of cell debris in lumen was indicated by white arrows. The changes in epithelial thickness (B) and luminal diameter (C) of the segments at indicated time points were measured. Average epithelial thickness and luminal diameter in bilateral epididymis of 5 mice in each group were assessed. n = 10. *, P < 0.05 and **, P < 0.01, according to t test. (D) Ultrastructure of the caput (a-f) and cauda (g-j) with or without ZIKV infection was observed using a transmission electron microscope (TEM). Unilateral epididymis from 3 mice in each group was used. After ZIKV infection, epithelial thickness is considerably decreased, microvilli become sparse, and vacuoles (indicated by black arrows) are detected between the cells (a, b, d, e). The foam macrophages (indicated by black stars) are accumulated, and the density of the sperms in the lumen is decreased (c, f). In the cauda, epithelial cell layer is disrupted after the infection (g, h, i). Sperm directly contacts the basement membrane of the tubules (indicated by rectangle, j). IT, intertubular space; BM, basement membrane; Mv, microvilli; Lu, lumen; EC, epithelial cells; V, vesicles.
Fig 3.
Inflammation in the epididymis after ZIKV infection.
Eight-week-old male AG6 mice were inoculated with 105 plaque-forming units (PFU) of the CAS-ZK01 strain of ZIKV in bilateral footpads. IL-6 (A) and IL-28 (B) in the epididymis and testis at 2, 5, and 8 dpi were detected by protein microarray. Each data point represents the results obtained in an individual mouse. n = 5. **, P < 0.01, according to the t test. (C) The types of immunocytes were determined by immunohistochemistry staining (IHC). CD45, CD11c, and F4/80 staining correspond to pan-leukocytes, myeloid dendritic cells, and macrophages marker, respectively. Changes in the expression and distribution were detected in the epididymis at 8 dpi after ZIKV infection (CD45 indicated by black arrows, CD11c indicated by white arrows, F4/80 indicated by black stars).
Fig 4.
Expression and distribution of tight junction proteins in the epididymis after ZIKV infection.
Eight-week-old male AG6 mice were inoculated with 105 plaque-forming units (PFU) of ZIKV in bilateral footpads. n = 5. Epididymis was harvested at 8 days post infection (dpi), paraffin sections were prepared. ZO-1 (A) and Cldn-1 (B) in each segment were detected by immunofluorescent staining. Uninfected mice were used as a control. The disruptions were indicated by white arrows.
Fig 5.
Changes in lipocalins (Lcns) and aquaporins (AQPs) in the epididymis after ZIKV infection.
Eight-week-old male AG6 mice were inoculated with 105 plaque-forming units (PFU) of ZIKV in bilateral footpads. Epididymis at 5 days post infection (dpi) was harvested and subjected to transcriptome analysis. Uninfected mice were used as the control. Transcription levels of selected genes associated with Lcns (A) and AQPs (B) were compared in ZIKV-infected and uninfected mice. FPKM referred to fragments per kilobase million. The results were expressed as Log10(FPKM). The results were pooled data from 3 mice. The significant differences were displayed according to q value, FDR-adjusted p-value of the test statistic. *, Q < 0.05, **, Q < 0.01. The distribution of Lcn8 (C) and AQP1 (D) in the epididymis at 8 dpi was analyzed by immunohistochemistry staining. The changes were indicated by asterisks.