Abstract Zika virus (ZIKV) is unique among mosquito-borne flaviviruses in that it is also vertically and sexually transmitted by humans. The male reproductive tract is thought to be a ZIKV reservoir; however, the reported magnitude and duration of viral persistence in male genital tissues varies widely in humans and non-human primate models. ZIKV tissue and cellular tropism and potential effects on male fertility also remain unclear. The objective of this study was to resolve these questions by analyzing archived genital tissues from 51 ZIKV-inoculated male macaques and correlating data on plasma viral kinetics, tissue tropism, and ZIKV-induced pathological changes in the reproductive tract. We hypothesized that ZIKV would persist in the male macaque genital tract for longer than there was detectable viremia, where it would localize to germ and epithelial cells and associate with lesions. We detected ZIKV RNA and infectious virus in testis, epididymis, seminal vesicle, and prostate gland. In contrast to prepubertal males, sexually mature macaques were significantly more likely to harbor persistent ZIKV RNA or infectious virus somewhere in the genital tract, with detection as late as 60 days post-inoculation. ZIKV RNA localized primarily to testicular stem cells/sperm precursors and epithelial cells, including Sertoli cells, epididymal duct epithelium, and glandular epithelia of the seminal vesicle and prostate gland. ZIKV infection was associated with microscopic evidence of inflammation in the epididymis and prostate gland of sexually mature males, which could have significant effects on male fertility. The findings from this study increase our understanding of persistent ZIKV infection which can inform risk of sexual transmission during assisted reproductive therapies as well as potential impacts on male fertility. Author Summary Zika virus (ZIKV) spread since 2015 led to establishment of urban epidemic cycles involving humans and Aedes mosquitoes. ZIKV is also sexually and vertically transmitted and causes congenital Zika syndrome. Together, these features show that ZIKV poses significant global public health risks. By virtue of similar reproductive anatomy and physiology to humans, macaques serve as a useful model for ZIKV infection. However, macaque studies to date have been limited by small sample size, typically 1 to 5 animals. Although mounting evidence identifies the male reproductive tract as a significant ZIKV reservoir, data regarding the duration of ZIKV persistence, potential for sexual transmission, and male genitourinary sequelae remain sparse. Here, we analyzed archived genital tissues from more than 50 ZIKV-inoculated male macaques. Our results show that ZIKV can persist in the male macaque reproductive tract after the resolution of viremia, with virus localization to sperm precursors and epithelial cells, and microscopic evidence of inflammation in the epididymis and prostate gland. Additionally, we show that freezing is not a viable method of destroying infectious ZIKV. Our findings help explain cases of sexual transmission of ZIKV in humans, which also carries a risk for transmission via assisted fertility procedures, even after resolution of detectable viremia.

Zika virus persistence in the male macaque reproductive tract