Novel ex vivo models reveal segment-specific infection of the human efferent ducts and epididymis: implications for Zika virus persistence and shedding in semen.

Abstract

Study question: Can human epididymis and efferent ducts (ED) support Zika virus (ZIKV) replication ex vivo and act as a source of prolonged viral shedding in semen of infected men?

Summary answer: Our novel ex vivo models demonstrate that ZIKV exhibits segment-specific tropism, with sustained replication in the ED epithelium and restricted infection of epididymal stromal macrophages, identifying these organs as distinct reservoirs for viral persistence in semen.

What is known already: The 2015-2017 ZIKV outbreak revealed that the virus persists in semen and can be sexually transmitted by men long after the acute phase of infection. While the human testis is an established reservoir, animal studies and longitudinal semen analyses suggest that additional organs within the male genital tract contribute to the prolonged excretion of infectious ZIKV. However, the specific roles of the human EDs and epididymis have remained unexplored due to the lack of physiologically relevant human models.

Study design, size, duration: Organotypic cultures of human EDs and epididymis corpus (EC) were developed and characterized using post-mortem tissues obtained from 11 uninfected organ donors (age range 29-79 years, median age: 58 years). Using this innovative ex vivo platform, we investigated ZIKV replication kinetics, cellular tropism, and the resulting impact on tissue viability and innate immune responses over a 9-day culture period.

Participants/materials, setting, methods: ED and EC tissues were dissected and cultured on PET inserts at the air/medium interface. Tissue integrity was validated via histology, lactate dehydrogenase (LDH) viability assays, and transcriptomic/protein marker expression, assessed by RT-qPCR and immunohistochemistry. Explants were exposed to ZIKV, with replication monitored via viral RNA (vRNA) quantification, as assessed by RT-qPCR, and plaque assays for infectious virion release. Cellular targets were identified using RNAscope combined with immunohistochemistry. To determine the impact of the infection, we analyzed the innate immune response by RT-qPCR and ELISA and cell viability by LDH assays and cleaved caspase 3 expression, as well as the transcriptional and protein expression of a selected panel of cell markers, assessed by RT-qPCR and immunohistochemistry.

Main results and the role of chance: The ex vivo models preserved global tissue architecture, viability, and the expression of epithelial, macrophage and smooth muscle markers throughout the study. ZIKV infected the ED epithelium, with replicative vRNA persisting from 3 days post-infection (dpi) until the end of culture (P = 0.01). Conversely, in the EC, infection was limited to stromal macrophages and smooth muscle cells, peaking at 3 dpi before diminishing (P = 0.002). Both segments exhibited robustly repeatable results across donors. Notably, both ED and EC exhibited limited antiviral responses and weak pro-inflammatory responses to ZIKV infection. ZIKV replication did not induce significant changes in morphology, viability or functional marker expression, suggesting a non-cytopathic infection mechanism.

Limitations, reasons for caution: While these organotypic cultures preserved multi-lineage cell types for 9 days, epithelial morphology and organization were gradually impacted, and the expression of a subset of functional and hormone-responsive markers (e.g. AQP1, ESR1, AR) decreased. Furthermore, the ex vivo setup cannot replicate in vivo viral entry via systemic circulation or luminal flow from the testis, nor can it account for the influence of infiltrating systemic leukocytes and global inflammation.

Wider implications of the findings: These novel human ED and EC culture systems bridge a critical gap in reproductive research. Our findings reveal a dual cellular pathway for ZIKV shedding, epithelial in the ED and macrophage-mediated in the EC, offering a mechanistic explanation for the prolonged presence of ZIKV in human semen. Furthermore, this ex vivo platform provides a new tool for studying male reproductive tract-pathogen interactions, as well as the efficacy of antiviral drugs.

Funding: This study received funding from the European Union's Horizon 2020 research and innovation program under grant agreement no. 733176, ZikaPLAN (grant agreement no. 734584) and ZIKAlliance (grant agreement no. 734548), and was funded in part by Inserm and the University of Rennes.

Disclosures: We declare no conflict of interest.

Trial registration number: N/A.