Fig 2: Loss of CLEC5A reduces testicular inflammation and damage. a Neutrophil infiltration in the testes and epididymis (caput, corpus, and cauda) collected from ZIKV-infected stat1−/− mice (n ≥ 8) and stat1−/−clec5a−/− mice (n ≥ 8) at 7 dpi and from uninfected C57BL/6 control mice (n ≥ 6) was examined and quantified via immunohistochemical staining with anti-Ly6G antibody. Levels of b chemokine MCP-1 and c proinflammatory cytokine TNF-α in serum and testes from infected mutant mice or uninfected WT mice. The serum was collected at 2 dpi, and the testicular tissues were collected at 7 dpi. The concentrations of MCP-1 and TNF-α in the serum were determined via a standard ELISA assay, and the RNA levels of MCP-1 and TNF-α expressed in the testicular tissues were assessed by quantitative real-time PCR assay. d Apoptosis assay using tissue sections from the testes and epididymis (caput, corpus, and cauda) of ZIKV-infected stat1−/− mice (n ≥ 4) and stat1−/−clec5a−/− mice (n ≥ 4), with uninfected C57BL/6 mice (n = 3) used as a negative control. DNase I-treated samples served as a positive control. The graphs show the ratio of TUNEL-staining intensity in the ZIKV-infected and uninfected testicular tissue to that in the DNase I-treated samples. Neutrophil invasion and apoptosis in the testicular tissues were evaluated at 7 dpi. Quantitative data are presented as mean ± SD. *p < 0.05; **p < 0.01; N.S., not significant. Scale bars: (a) 100 μm; (d) 250 μm

Fig 3: CLEC5A function is related to malfunctioning spermatozoa following ZIKV infection. a A cytomorphological study of mature spermatozoa harvested from the cauda of the epididymis was conducted using Papanicolaou stain. Aberrant morphology in the head or tail of spermatozoa (red arrows) was observed in ZIKV-infected stat1−/− mice and stat1−/−clec5a−/− mice compared to that in uninfected C57BL/6 mice. The proportions of normal and abnormal sperm were quantified for statistical analysis. b Sperm motility assay. At least three randomly selected fields of view were analyzed for each group (a). c Immunofluorescence staining of sperm allowed the visualization of ZIKV (green, anti-NS1-eGFP), cytoplasm (red, CellTracker), and nuclei (blue, DAPI). ZIKV-positive sperm were counted from more than 150 sperm cells in three random fields of view per mouse and are presented as percentages of all cells. Samples were taken from ZIKV-infected stat1−/−(n = 12) and stat1−/−clec5a−/− mice (n = 8) (7 dpi) and uninfected C57BL/6 mice (n = 3), which served as negative controls. Quantitative data are presented as mean ± SD. *p < 0.05; ***p < 0.001; N.D., not detected. Scale bars: (a) 50 μm; (c) 25 μm

Fig 4: Clec5a knockout attenuates ZIKV infectivity in the testicular tissues of mice. Bodyweight (recorded daily) (a) and survival (monitored every 2 days) (b) in male stat1−/− and stat1−/−clec5a−/− mice infected with ZIKV via mosquitoes. c ZIKV titer in serum samples (n ≥ 3) collected at 2 days post-infection (dpi) and in various tissue samples (n ≥ 4) collected at 7 dpi. Plaque assay was used to quantify infectious ZIKV and the limit of detection was estimated by counting numbers of plaque formation in original lysates. No plaque formation was observed in the livers, kidneys and spleens of some ZIKV-infected mice. d Immunohistochemical staining of tissues from the testes and epididymis (caput, corpus, and cauda) collected from ZIKV-infected mice (n ≥ 4) at 7 dpi and from uninfected C57BL/6 mice (n = 3), which were used as controls. Quantitative data are presented as mean ± standard deviation (SD). *p < 0.05. Scale bars: 100 µm

Fig 5: Loss of CLEC5A attenuates oligospermia following ZIKV infection. Hematoxylin-and-eosin staining histological analysis of tissue sections of the seminiferous tubules (a) in the testis and (b) the mesonephric tubules in the epididymis. c Sperm count for epididymis tissues from mice. d Spermatid density and distribution in the epididymis of ZIKV-infected stat1−/− mice (n ≥ 4) and stat1−/−clec5a−/− mice (n ≥ 5) (7 dpi), with uninfected C57BL/6 mice (n = 6) as negative controls, quantified using Panoramic Viewer software. For a statistical analysis of the distribution of sperm, more than two randomly selected fields of view of the section of the epididymis tissue were assessed for each mouse, and more than three mice were analyzed for each group. Quantitative data are presented as mean ± SD. *p < 0.05; **p < 0.01. Scale bars: 200 μm