The 2015–2016 Zika virus (ZIKV) epidemic, resulting in> 1.5 million cases in the Americas, emerged as a global threat to public health due to the unique findings of sexual and in utero transmission, which have not been reported for other flaviviruses 1. In ZIKV endemic regions, the role of sexual transmission in disease spread may be difficult to predict; however, a recent study demonstrated that 56% of ZIKV serum-positive males were also semenpositive for virus for up to 108 days after infection 2. This suggests a higher contribution in disease spread and a longer infectious phase as compared to other mosquitoborne flaviviruses. Further, Joguet et al. 3 reported significantly higher seminal viral loads than in other bodily fluids of ZIKV infected men. This was accompanied by testosterone decline and significant decrease in sperm count early after symptoms onset, indicating ZIKV infection may also affect fertility 3. The mammalian testis is divided into two compartments: seminiferous tubules composed of Sertoli cells (SC), peritubular cells, and various generations of germ cells (spermatogonial stem cells (SSC), meiotic spermatocytes, and spermatozoa); and interstitial space (between seminiferous tubules) consisting of blood vessels, tissue-resident immune cells, and testosterone-producing Leydig cells (LC) 4. The testicular immune environment is tightly governed by communication between resident cell types, notably SC and LC, through production of transforming growth factor beta (TGF-β), testosterone, activin, and inhibin complexes, as well as pro-inflammatory cytokines and type I Interferons 5.

Little is known of the cell types that support ZIKV persistence within the human testes. We recently demonstrated that primary human SC are highly susceptible to ZIKV infection compared to dengue virus, and are capable of robust antiviral and inflammatory responses 6. Using an in vitro Sertoli cell barrier (SCB) model we further demonstrated that ZIKV could cross the barrier more efficiently compared to dengue virus without altering barrier permeability 6. However, the limitation of monolayer two-dimensional (2D) culture systems is that they do not allow assessment of multiple testicular cell interactions and the effect of ZIKV on the fate of germ cells. Thus, novel tools that mimic functionality of native tissue to study complex consequences of ZIKV infection are greatly needed. Previous studies have used threedimensional (3D) mouse testicular tissue cultured on softagar, but a human 3D culture model is considered optimal to mimic human testes function 7. A restricted number of research groups, including ours have reported and characterized testicular organoids, as organ-like structures that relatively model testicular histology and physiology by way of reorganization of dissociated testicular cells in vitro 8–10. Our recently developed human testicular organoid (HTO) culture system is composed of SSC, SC, LC, and peritubular cells that maintain> 85% viability throughout long-term culture of 23 days 10. These HTO also produce testosterone continuously and support limited germ cell differentiation, and thus partially recapitulate the composition, diversity, and function of human testes 10.