Persistence of Zika virus RNA in the epididymis of the male reproductive tract

Zika virus (ZIKV) can infect developing fetuses in utero and cause severe congenital defects. This in utero transmission can occurs following ZIKV infection during pregnancy via sexual transmission or mosquito bite. Infected men may shed ZIKV RNA in semen for over six months post symptom onset, indicating that ZIKV may persistently infect the male reproductive tract (MRT). However, the site of persistent infection in the MRT and whether ZIKV can recrudesce in the MRT is unknown. We hypothesized that if ZIKV establishes a persistent infection in the MRT, then immunosuppressant treatment should stimulate ZIKV replication. We tested this hypothesis in a wild-type mouse model of ZIKV sexual transmission. Male mice were infected with ZIKV and immunosuppressed when they no longer shed infectious virus in their ejaculates. After immunosuppression, ejaculates and MRT tissues were monitored for infectious virus and ZIKV RNA. Our results show that ZIKV recrudescence did not occur following immunosuppression, as we did not detect significant levels of infectious virus in ejaculates or MRT tissues following immunosuppression. We did detect ZIKV RNA in the epididymides of mice treated with the immunosuppressant cyclophosphamide. Further analysis revealed that this ZIKV RNA was contained within the lumen of the epididymis. Our findings suggest that ZIKV persistently infects the epididymis within the male reproductive tract. This study provides insight into the mechanisms behind ZIKV sexual transmission, which may inform public health decisions regarding ZIKV risks. Importance Zika virus (ZIKV) is an emerging mosquito-transmitted virus that typically causes mild and self-limiting febrile illness in humans; however, during the recent epidemic of ZIKV in the Americas, severe birth defects, such as microcephaly and club foot, were reported in infants born to ZIKV infected mothers. Additionally, sexual transmission has been identified as a secondary method of ZIKV transmission. Since ZIKV can be isolated from semen of infected men long after initial infection, it is imperative to understand the mechanism(s) of ZIKV infection of the male reproductive tract to prevent sexual transmission and ZIKV-associated birth defects. The significance of our research is in identifying a site of persistent ZIKV infection in the male reproductive tract and in assessing the likelihood that a persistently infected individual will begin shedding infectious virus in semen again. This information will enhance our understanding of ZIKV sexual transmission and inform health decisions regarding ZIKV risks. ### Competing Interest Statement The authors have declared no competing interest.