Mechanisms underlying Zika virus infection of testicular germ cells, a key cellular reservoir for sexual transmission

"Context: Zika virus (ZIKV) is a teratogenic arbovirus with a high potential for (re)emergence. Its recent epidemic revealed a unique ability to persist in the male genital tract for months after systemic clearance, enabling sexual transmission long after the resolution of acute symptoms. The prolonged sexual transmission of ZIKV not only facilitates its global dissemination but also poses a critical threat to pregnant women and their unborn children, as exposure to infected semen increases the risk of congenital infection. Understanding the reasons for ZIKV prolonged seminal excretion is therefore essential. Results obtained: Using our unique models combined with in vivo analysis, we demonstrated that ZIKV productively infects the human testis without cytopathic effects and persistently replicates in testicular germ cells (TGC), which constitute the main cell reservoir of ZIKV in the semen of infected men (Matusali et al, JCI 2018; Mahé et al, Lancet Infect Dis 2020). We revealed that human TGC fail to mount an antiviral response upon ZIKV infection or stimulation by Poly(I:C), and that a sub-population of TGC lacks type I interferon (IFN) receptor expression (Kuassivi et al., Front Immunol 2022), highlighting the particular innate immune features of these reservoir cells. Objectives: The general objective of our project is to advance our understanding of the early mechanisms leading to ZIKV persistent infection of TGC. Our specific aims are to elucidate the cellular (WP1) and viral factors (WP2) that enable ZIKV to acutely replicate in TGC without triggering a host antiviral responses or cell death, thus favoring prolonged viral replication. Our project rests on the use of primary human TGC and a range of techniques that we or our partner (Team B) fully master. In WP1, we will: Unravel the mechanisms underlying the weak antiviral response of TGC to ZIKV. Our preliminary data show that TGC express low level of MAVS, which is critical to activate the innate antiviral response upon ZIKV sensing. Conversely, TGC express high levels of autophagy actors known to interact with MAVS and ZIKV replication. Here we will determine the role of autophagy on the innate responses and ZIKV replication in TGC. Determine the ability of ZIKV-infected TGC populations to respond to the antiviral activity of exogenous IFN-I and III. Our preliminary data show limited response of TGC to exogenous IFN-I and absence of IFN-I receptors' surface expression in a TGC sub-population. We will investigate IFN-I/III receptors expression and signaling in TGC populations, and determine the effect of IFN-I and III treatment on ZIKV replication in TGC populations, to unveil potential reservoir cells resistant to IFN. In WP2, in collaboration with Team B, we will use ZIKV/DENV chimeras to identify the viral proteins responsible for ZIKV's distinctive tropism for TGC and its non-cytopathic replication. Our preliminary data show that Dengue virus (DENV), which is closely related to ZIKV, does not infect TGC, making it an ideal control. These findings will lay the groundwork for understanding the specific molecular interactions between ZIKV and its reservoir cells, and will provide a predictive model for assessing the potential of other emerging viruses to persist in TGC. Expected outcomes: At the end of this three-year project, we expect to unveil new mechanisms governing antiviral responses and cell survival within ZIKV-infected testicular germ cells. Beyond Zika virus, the findings and models generated in this project will provide a strong foundation for anticipating the ability of other emerging viruses to establish similar infections and persist in semen. Overall, this project will advance our understanding of the early mechanisms driving viral persistence in a key cellular reservoir. This may offer novel perspectives for preventing the prolonged sexual transmission of ZIKV and other emerging viruses."