**Background:** Zika virus (ZIKV) gained global attention during the 2015 outbreak in Central and South America, when it was linked to congenital Zika virus syndrome and microcephaly. Subsequent research demonstrated that ZIKV can infect the male reproductive tract, persist in semen, and be sexually transmitted. While studies in mice and nonhuman primates have confirmed testicular tropism, most research has focused on adults, leaving the effects on immature or pubertal individuals poorly understood. This study aimed to characterize the clinical, hormonal, and histopathological effects of ZIKV infection on the testes of pubertal male squirrel monkeys (Saimiri collinsi), a neotropical primate species recognized as a suitable model for ZIKV research.
**Methods:** Five pubertal male S. collinsi (1 year 7 months to 2 years 7 months) from the National Primate Center (CENP) in Brazil were selected. Two animals served as controls (G1) and were inoculated with a 0.5 mL suspension of VERO cells. Three animals (G2) were experimentally infected intradermally with 0.5 mL of Asian genotype ZIKV (strain BE H815744, 1.0 × 10^5 PFU/mL). Animals were monitored from day -5 to day 21 post-infection (dpi). Fecal samples were collected daily for testosterone measurement via chemiluminescence assay. Blood was collected on alternate days for RT-qPCR (viral RNA detection) and IgM ELISA. Testicular biometry and ultrasonographic examinations (B-mode, 8-18 MHz) were performed weekly. At 21 dpi, animals were euthanized, and testes were collected for histopathology (hematoxylin-eosin staining), histomorphometry (tubular area, seminiferous epithelium area, diameter, epithelium height), Johnsen's score assessment, and immunohistochemistry (IHC) for ZIKV antigen detection using an anti-ZIKV polyclonal antibody. Statistical analyses included two-way ANOVA, Student's t-test, and Pearson's correlation.
**Key Results:** Infected animals showed fever (average 39.5°C) between 3 and 10 dpi, with peak temperatures of 40.5°C at 7 dpi. Viremia was detected from 3 dpi, peaking at 5 dpi (mean 5.73 × 10^6 copies/µL RNA copies). IgM seroconversion occurred from 14 dpi and persisted to 21 dpi. Infected animals exhibited significant testicular volume loss and lower gonadosomatic index (GSI) compared to controls during both acute (p=0.016 and p=0.035, respectively) and convalescent phases (p=0.013 and p=0.011, respectively). Fecal testosterone levels showed no significant difference between groups overall, but intragroup analysis revealed a significant reduction in G2 between pre-inoculation and acute phases (p=0.006) and between acute and convalescent phases (p=0.006). Testicular echogenicity loss was significant in G2 during the convalescent phase compared to the acute phase (p=0.010). Postmortem, a very strong inverse correlation was observed between GSI and testicular viral RNA load (R= -0.9986, p=0.033). Histopathological analysis at 21 dpi revealed degeneration and necrosis of seminiferous tubule cells, perivascular mononuclear inflammatory infiltrate (neutrophils, lymphocytes), tubular retraction, and interstitial edema in all infected animals. No spermatozoa were observed in the tubular lumen of infected animals, while controls had spermatozoa present. Histomorphometry showed significant differences between G1 and G2 for all parameters: tubular area (21,292 ± 7,334 vs. 11,475 ± 2,373 µm², p=0.000), seminiferous epithelium area (15,983 ± 6,568 vs. 7,961 ± 1,741 µm², p=0.000), larger diameter (179.6 ± 39.8 vs. 131.2 ± 20.7 µm, p=0.000), smaller diameter (148.1 ± 27.3 vs. 109.5 ± 10.2 µm, p=0.000), epithelium height (45.7 ± 13.5 vs. 30.6 ± 4.8 µm, p=0.000), and Johnsen's score (4.9 ± 1.05 vs. 3.25 ± 0.47, p=0.000). IHC confirmed ZIKV antigen in germ cells, Sertoli cells, and Leydig cells of all infected animals, with an average of 2.83 × 10^2 cells/mm².
**Clinical Implications:** This study demonstrates that ZIKV infection in pubertal S. collinsi causes significant testicular damage, including germ cell degeneration, reduced spermatogenesis, and testicular atrophy, with viral persistence in testicular tissue at 21 dpi. The findings suggest that ZIKV can overcome the blood-testis barrier and impair male reproductive function even in immature individuals. The inverse correlation between GSI and testicular viral load indicates that greater testicular damage is associated with higher viral burden. The study validates noninvasive methods (fecal testosterone, ultrasonography) for monitoring testicular effects. These results raise concerns about ZIKV's potential to cause fertility issues in both humans and free-living neotropical primate populations, particularly given the species' potential role as a viral reservoir. The authors recommend longer-term studies to assess whether testicular lesions are reversible or lead to permanent infertility.