Sexual transmission of filoviruses was first reported in 1968 after an outbreak of Marburg virus (MARV) disease and recently caused flare-ups of Ebola virus disease in the 2013–2016 outbreak. How filoviruses establish testicular persistence and are shed in semen remain unknown. We discovered that persistent MARV infection of seminiferous tubules, an immune-privileged site that harbors sperm production, is a relatively common event in crab-eating macaques that survived infection after antiviral treatment. Persistence triggers severe testicular damage, including spermatogenic cell depletion and inflammatory cell invasion. MARV mainly persists in Sertoli cells, leading to breakdown of the blood-testis barrier formed by inter-Sertoli cell tight junctions. This disruption is accompanied by local infiltration of immunosuppressive CD4 + Foxp3 + regulatory T cells. Our study elucidates cellular events associated with testicular persistence that may promote sexual transmission of filoviruses and suggests that targeting immunosuppression may be warranted to clear filovirus persistence in damaged immune-privileged sites.

How filovirus persistence is established remains to be understood, and no reliable animal model is available for studying persistence in untreated survivors. We previously reported a relatively high frequency of persistent EBOV infection in the eye and a single case of EBOV persistence in the brain and epididymis in rhesus monkeys (Macaca mulatta) that survived infection in the absence of, or after treatment with, candidate medical countermeasures (MCMs) (). However, we were unable to determine the mechanism underlying persistence in these animals. Here, we report persistent MARV infection in seminiferous tubules, which are the sites of immune privilege and sperm production, in the testes of male crab-eating macaques (Macaca fascicularis) that survived MARV infection after treatment with MCMs. We demonstrate the cellular reservoir of testicular MARV persistence and suggest a possible mechanism leading to such persistence and shedding virus in semen.

Similarly, in one survivor of Ebola virus disease (EVD) with uveitis, EBOV was isolated from the eye (). Another EVD survivor developed meningoencephalitis more than 9 months after convalescence, and EBOV was isolated from the patient's cerebrospinal fluid (). EBOV genomic RNA has been repeatedly detected in semen of EVD survivors () and EBOV has been successfully isolated from a few samples (). Sexual transmission of EBOV has also been implicated in the initiation of alternative EBOV transmission chains (). Filovirus persistence is also hypothesized to cause the plethora of apparent sequelae, including arthralgia, cognitive impairment, headaches, hearing loss, and myalgia, that have been reported in numerous MVD and EVD survivors ().

Resurgence of Ebola virus disease in Guinea linked to a survivor with virus persistence in seminal fluid for more than 500 days.

Persistence and genetic stability of Ebola virus during the outbreak in Kikwit, Democratic Republic of the Congo, 1995.

Persistence of Ebola virus in various body fluids during convalescence: evidence and implications for disease transmission and control.

Persistence and genetic stability of Ebola virus during the outbreak in Kikwit, Democratic Republic of the Congo, 1995.

Resurgence of Ebola virus disease in Guinea linked to a survivor with virus persistence in seminal fluid for more than 500 days.

Marburg virus (MARV) and Ebola virus (EBOV) are the most notorious members of the mononegaviral family Filoviridae. Both viruses cause severe viral hemorrhagic fevers in humans with extraordinarily high case-fatality rates (). Since the discovery of MARV in 1967 (), virus persistence in human survivors has been sporadically reported. For instance, sexual transmission of MARV from a MARV disease (MVD) survivor to his wife occurred in 1967 (). MARV was isolated from the aqueous fluid of the eye of a 1975 MVD survivor with uveitis () and from the semen of an apparently healthy MVD survivor in 1980 (). In addition, MVD recurred in a man who survived an accidental laboratory infection with MARV in 1991 ().

We further analyzed liver tissues for the presence of FOXP3CD4Treg cells in survivor M8 and survivor M10. These survivors had hepatitis ( Figures S1 D and S1G), but the livers were found to be MARV free. Interestingly, only 1% (three of 291) and 0.6% (two of 326) of CD4T cells in the liver sections from survivors 8 and 11, respectively, were FOXP3CD4Treg cells ( Figures 6 I and 6J). These data suggest that abundant FOXP3CD4Treg cells were specifically present in testes with MARV persistence. Furthermore, in testicular sites with MARV persistence, expression of inhibitory receptor CTLA-4 and immunosuppressive cytokine transforming growth factor β (TGF-β) was high, but expression of interleukin-10 (IL-10) was not detectable ( Figures 6 K–6P). These data suggest that immunosuppressive functions of FOXP3CD4Treg cells may operate through inhibitory receptor CTLA-4 and suppressive cytokine TGF-β. Together, the local immunosuppressive environment elicited by Treg cells may sustain MARV infection in the testis.

To detect the mRNA of T-bet, GATA3, RORγt, and FOXP3, we performed ISH. Surprisingly, high levels of FOXP3 expression were only detected in the areas of MARV persistence ( Figure S6 ). Immunofluorescence co-staining using anti-CD4 and anti-FOXP3 antibodies was used to quantify the regulatory CD4FOXP3cell number in the uninfected control testes, testes from macaques with ACDD, and testes from survivors with testicular MARV persistence ( Figures 6 F–6H). Consistently, in the testes of survivor M8 and survivor M10, about 25% (190 of 764) and 26% (423 of 1,608) of CD4T cells were FOXP3CD4Treg cells, respectively ( Figures 6 H and 6J). Conversely, no (0 of 132) CD4T cells expressed FOXP3 in uninfected control testes ( Figures 6 F and 6J), and only 1.3% (two of 156) of CD4T cells were FOXP3CD4Treg cells in testes from macaques with ACDD ( Figures 6 G and 6J).

Both activated CD4 and CD8 T cell responses were detected during the acute phase of EBOV infection in humans (). To further characterize the excessive CD3T cells that infiltrated the testes ( Figure 2 H), we examined CD4 mRNA and CD8 mRNA in testes with MARV persistence and in uninfected control by ISH. Surprisingly, abundant CD4T cells, but a limited number of CD8T cells were detected in the testes with MARV persistence in comparison with that observed in uninfected control testes ( Figures 6 A–6C ). Immunofluorescence staining using anti-CD3, anti-CD8, and anti-CD4 antibodies further verified that only very few CD3T cells were CD8T cells, but abundant CD4T cells were detected in the testes with MARV persistence ( Figures 6 D and 6E). CD4T cells play critical roles in mediating adaptive immunity to various pathogens. At least four distinct CD4 T helper cell subsets (Th1, Th2, Th17, and regulatory T [Treg] cells) were noted, as defined by the function as well as the expression of the key transcription factors T-bet, GATA3, RAR-related orphan receptor (ROR)γt, and FOXP3 ().

(M and N) Detection of interleukin-10 (IL-10) by ISH in control lymph node (M) and testes of survivor (N). Black arrows point to IL-10 + cells in control lymph node.

(F–J) Immunofluorescence staining of CD4 + (green) FOXP3 + (red) Treg cells in control testes (F), testes from macaques with acute course disease death (ACDD) (G), and testes (H) and livers (I) from the same survivors with testicular MARV persistence. Quantification of CD4 + FOXP3 + Treg cells in 10 4 μm 2 testicle and liver tissues (J). Data are represented as mean ± SEM. n = 65, 66, 43, 26, 43, and 28.

(A–C) Detection of CD8 + (A) and CD4 + (B) T cells in control and survivor testes by ISH. (C) Quantification of CD8 + and CD4 + T cells in each 40× objective view of testis in control and survivors. Data are represented as mean ± SEM. n = 21, 23, 21, 21, 23, and 22, respectively.

Immunofluorescence co-staining using anti-MARV GPand anti-GATA4 antibodies further verified MARV infection of Sertoli cells ( Figures 5 K–5M). Furthermore, MARV was detected in DDX4germ cells in the lumen of seminiferous tubules in addition to Sertoli cells by immunofluorescence staining ( Figure 5 N). Interestingly, MARV could also be detected in the sperm in efferent ducts of the testes ( Figure 5 O). In addition, seminiferous tubule walls were degenerated in animals that were euthanized on days 10, 11, and 12 post exposure ( Figures S5 H–S5L and 5 P). We only detected expression of cleaved caspase-3 among infected spermatogenic germ cells but not in Sertoli cells infected with MARV, suggesting cell death only occurred in spermatogenic germ cells ( Figures S5 M and S5N). These data indicate that MARV infection in interstitial tissue during the acute phase of infection progressively disseminates into seminiferous tubules and then into the Sertoli cells, resulting in tubule wall destruction, BTB disruption, and germ cell loss (in yet-to-be-determined order) ( Figures 5 P and 5Q).

We hypothesized that MARV infection of Sertoli cells occurs subsequent to infection of the peritubular myoid cells. To address this hypothesis, we identified archived samples from a study during which crab-eating macaques were exposed IM to 1,000 PFUs of MARV (). On days 8–12 post exposure, seven macaques were euthanized in extremis. Similar to the macaques euthanized on day 8 post exposure in the study depicted in Figures 3 H, 3J–3J′, and 3L, we detected MARV RNA in cells throughout the testicular interstitial tissue. MARV started to infect peritubular myoid cells in all three animals from this study that had an acute course of disease death (ACDD) at day 8 post exposure as indicated by ISH targeting NP ( Figures S5 A–S5C). In the testes of macaques that were euthanized on days 10, 11, or 12 post exposure, we found MARV-infected Sertoli cells in addition to interstitial cells, peritubular myoid cells, and early-stage germ cells ( Figures S5 D–S5G).

To understand the reaction of Sertoli cells to persistent MARV infection, we stained Sertoli cells using antibodies against Sertoli cell-specific markers anti-GATA binding protein 4 (GATA4) and vimentin. In uninfected testes, the GATA4and vimentinSertoli cells were aligned near the basement membrane (a modified form of extracellular matrix) of the seminiferous epithelium of the testes ( Figure 5 A). In contrast, the GATA4and vimentinSertoli cells detached from the basement membrane and moved toward the apical (adluminal) compartment of the seminiferous epithelium in testes with MARV persistence ( Figure 5 B). Therefore, we sought to address whether the blood-testis barrier (BTB) between detached Sertoli cells still remained intact. To evaluate the BTB, we examined the expression of tight junction protein 1 (ZO-1) and 2 (TJP2), the main constituent proteins of the BTB. Interestingly, the expression of both ZO-1 ( Figures 5 C–5E) and TJP2 in testes persistently infected with MARV was significantly diminished compared with uninfected control tissue ( Figures 5 F–5H), indicating BTB disruption. To exclude any artifacts, we further evaluated the expression of ZO-1 and TJP2 in sites with MARV persistence where MARV GPwas detected, and adjacent uninfected tissue where MARV GPwas undetectable, in the same tissue section. Indeed, the expression of ZO-1 and TJP2 was clearly diminished in the sites of MARV persistence compared with that observed in adjacent uninfected areas ( Figures 5 I–5J′).

(P and Q) Schematic illustration of MARV (red dots) infection in Sertoli cells and spermatogenic cells after breakdown of the tubular wall organized by peritubular myoid cells in the late acute phase of infection (P) (see also Figures S5 H–S5L). MARV only persist in seminiferous tubules in survivors, resulting in inflammatory cell infiltration, Sertoli cell detachment, and BTB disruption (Q).

(O) Detection of MARV RNA in sperm at efferent ducts in an animal at day 12 post exposure. Blue, nuclear stain by DAPI. Scale bars, 50 μm in (A)–(N) and 10 μm in (O).

(K–M) GATASertoli cells (green, white arrows) infected with MARV (GP, red) in two animals at day 10 (K and L) and in one animal at day 11 (M) post exposure (see also Figures S5 D–S5F). (N) DDX4spermatogenic cells (green) infected with MARV (GP, red) next to Sertoli cells (white arrow) at day 12 post exposure (see also Figure S5 G).

(J and J′) Detection of tight junction protein 2 (TJP2, green) in sites with MARV persistence (GP 1,2 , red; right of dashed line) and adjacent uninfected area (left of dashed line) in the same testicle section.

(I and I′) Detection of tight junction protein 1 (ZO-1, green) in sites with MARV persistence (GP 1,2 , red; right of dashed line) and adjacent uninfected area (left of dashed line) in the same testicle section.

(H) Quantification of TJP2 immunofluorescence intensity in control and survivors. Data are represented as mean ± SEM; n = 69, 78, and 74 confocal sections.

(E) Quantification of ZO-1 immunofluorescence intensity in control and survivors. Data are represented as mean ± SEM; n = 51, 52, and 72 confocal sections.

(C and D) Detection of tight junction protein 1 (ZO-1, green) in vimentin + (red) Sertoli cells in control (C) and survivors (D).

(B) In survivor testis, GATA4 + (green) and vimentin + (red) Sertoli cells are pushed toward the tubule lumen and detached and disorganized.

(A) Immunofluorescence staining demonstrates that GATA binding protein 4 (GATA4) + (green) and vimentin + (red) Sertoli cells are aligned and attached to the tubular basement membrane in control testis.

In electron micrographs, Sertoli cells are clearly identifiable due to their pyramidal shape and their cytoplasmic processes extending toward the lumen of the seminiferous tubules (). We therefore performed EM analysis of archived FFPE tissue to locate targets of MARV persistence. Although tissue morphology was compromised because of the unconventional EM sample (FFPE tissue), 15 of 17 (88.2%) cells containing obvious MARV particles clearly were Sertoli cells ( Figures 4 E and 4E′). These data indicate that MARV mainly persists in Sertoli cells but also in very few of the remaining germ cells and CD68macrophages/monocytes that infiltrate seminiferous tubules.

We previously reported that EBOV persists mainly in CD68macrophages/monocytes in the eye, epididymis, and brain of rhesus monkeys (). To identify the cellular targets of persistent MARV in testes of crab-eating macaque survivors, we performed immunofluorescence staining using an antibody against cell-specific markers and the other antibody against MARV GP. Surprisingly, 428 of 479 (89.4%) MARV GPcells (n = 30 seminiferous tubule confocal sections) could be labeled by an anti-Sox9 antibody, an established marker for Sertoli cells ( Figures 4 A, 4A′, and 4D). However, only 15 of 456 (3.3%) MARV GPcells (n = 35 sections) expressed DDX4, a germ cell marker ( Figures 4 B, 4B′, and 4D), and only seven of 312 (2.2%) MARV GPcells expressed CD68 (n = 27 sections), a macrophage/monocyte marker ( Figures 4 C and 4D). Atypical punctate GPstaining, possibly representing MARV inclusion bodies, was found in Sertoli cells, germ cells, and macrophages/monocytes ( Figures 4 A–4C′).

(E and E′) Electron microscopic visualization of MARV particles (blue arrow) in Sertoli cells (Sc). (E′) Inset of (E) at high magnification. Scale bars, 1 μm in (E) and 500 nm in (E′).

(D) Quantitative analysis of the cellular targets of MARV persistence. Data are represented as mean ± SEM n = 30, 35, and 27 (confocal sections of seminiferous tubule), respectively.

(C and C′), MARV (GP 1,2 , red) in some CD68 + (green, white arrow) macrophages/monocytes. Blue, nuclear stain by DAPI. Scale bars, 50 μm in (A)–(C) and 10 μm in (A′)–(C′).

These data suggest that early administration of MCMs may prevent testicular MARV persistence. To test this hypothesis, we identified one previous study during which 18 male crab-eating macaques had been divided into four groups, exposed to a lethal dose of MARV subcutaneously, and treated with PMOs at days 0, 1, 2, and 4 post exposure. All the male macaques survived to the end of the study (these animals were also used to analyze the correlation between viremia and viral persistence in Figure S2 A) (). We further analyzed the relationship between testicular MARV persistence and the time of PMO treatment. Interestingly, of the survivors that were treated with PMOs on day 0 or 1 post exposure, none of four (0%) and one of four (25%), respectively, had persistent MARV infection in testes. Of the survivors that received PMO treatment on day 2 or day 4 post exposure, two of five (40%) and four of five (80%) survivors, respectively, had MARV persistence in the testes ( Figure 3 M).

To investigate when and how MARV disseminates into testes, we re-examined archived tissue sections collected during a study during which male crab-eating macaques were exposed intramuscularly (IM) to 1,000 plaque-forming units (PFUs) of MARV and euthanized at days 2 (n = 3), 3 (n = 3), 4 (n = 3), 6 (n = 3), 7 (n = 2), or 8 (n = 2) post exposure (). We detected MARV RNA by ISH targeting nucleoprotein gene (NP) in the lymph nodes, liver, and spleen as early as day 2, 4, and 6 post exposure, respectively ( Table S3 and Figures 3 A–3D ). In contrast, MARV RNA was undetectable in the testes until day 7 post exposure ( Figures 3 E–3H and 3K and Table S3 ) when MARV had just started to emerge into the vascular structure of testicular interstitial tissue ( Figures 3 G and 3K). At that time, the majority of tissues from the main target organs, including liver, lymph nodes, and spleen, were infected by MARV ( Table S3 and Figure 3 C). This finding was further verified by immunofluorescence detection of MARV in blood vessels using anti-MARV GPand anti-CD31 antibodies ( Figure 3 I). MARV further spread and infected all cells of the testicular interstitial tissue, including Leydig cells, at day 8 post exposure ( Figures 3 H, 3J, 3J′, and 3L). Interestingly, MARV had also infected the peritubular myoid cells labeled by anti-alpha-SMA antibody at that time ( Figures 3 J and 3J′), suggesting that MARV may damage the tubule walls before entering seminiferous tubules.

(M) A log-binomial regression was fit to the testicular MARV persistence in survivors treated with PMOs at day 0 (none of four survivors has testicular MARV persistence), day 1 (one of four has testicular MARV persistence), day 2 (two of five survivors had testicular MARV persistence), and day 4 (four of five had testicular MARV persistence) post exposure. A statistically significant association between MARV persistence and day of treatment was observed (Wald chi-squared; p = 0.0467). CI, confidence interval; RP, regression prediction.

(K and L) Schematic illustration of testicular blood vessel-derived MARV (red dots, K) spreading to interstitial Leydig cells and peritubular myoid cells (L). BTB, blood-testis barrier. Scale bars, 50 μm in (A)–(H) and 20 μm in (I) and (J).

(J and J′) On day 8 post exposure, MARV (VP40, green) spread from blood vessels (star) to infect interstitial cells, including Leydig cells (arrowheads) and peritubular myoid cells (arrows) of a seminiferous tubule labeled by anti-alpha-smooth muscle actin (alpha-SMA) antibody (red). Blue, nuclear stain by DAPI.

(E–H) MARV RNA remained undetectable in the vascular structure (black arrow in G) of testes until day 7 post exposure (G) and was distributed throughout the interstitial tissues from the vascular structure at day 8 post exposure (H) (see also Table S3 ). Blue, nuclear stain by hematoxylin.

(A–D) ISH targeting the NP gene indicates hepatic MARV infection at days 4 (A), 6 (B), 7 (C), and 8 (D) after crab-eating macaques were exposed intramuscularly to 1,000 PFU of MARV. MARV RNA was present in livers as early as day 4 post exposure (arrow in A).

Consistent with histological analysis, immunofluorescence staining demonstrated that CD45leukocytes ( Figures 2 E, 2F, S4 C, and S4D), CD68macrophages/monocytes ( Figures 2 G, 2H, S4 E, and S4F), CD3T cells ( Figures 2 G, 2H, and S4 G), and CD20B cells ( Figures 2 I and 2J) infiltrated both interstitial tissues and seminiferous tubules of testicular sites with MARV persistence. Interestingly, many CD45leukocytes and CD68macrophages/monocytes were proliferating as judged by detection of Ki67 expression ( Figures S4 C–S4F). Additionally, abundant IgG antibody was detected in the interstitial tissues and seminiferous tubules ( Figure 2 J), whereas IgG was undetectable in uninfected control testis ( Figure 2 I). Moreover, infiltration of inflammatory cells, including CD3T cells and CD68macrophages/monocytes mainly occurred in sites with MARV persistence where MARV GPwas detected, but not in uninfected areas with undetectable MARV GP Figures S4 A, S4G, and S4H). Together, these data indicate that persistent testicular MARV infection causes focal orchitis, germ cell loss, and abundant IgG antibody accumulation. Furthermore, the damaged tissue architecture and infiltration of multiple types of inflammatory cells, including antibody-secreting B cells, suggest that the seminiferous tubules with persistent MARV infection might have lost or partially lost “immune privilege.”

The basement membrane of seminiferous tubules is surrounded by peritubular myoid cells, which are squamous contractile cells that generate peristaltic waves in the tubules (). Immunofluorescence analysis of peritubular myoid cells labeled by an antibody against alpha-smooth muscle actin (alpha-SMA) revealed degeneration of the seminiferous tubule wall in testes containing MARV compared with uninfected control tissue ( Figures 2 C and 2D). Importantly, prominent DEAD (Asp-Glu-Ala-Asp)-box helicase 4 (DDX4)spermatogenic cell depletion occurred in sites of MARV persistence ( Figures 2 E, 2F, and 2K). DDX4spermatogenic cell depletion only occurred in MARV-infected areas in which MARV GPwas detected but not in uninfected areas in which MARV GPwas undetectable ( Figure S4 B).

Histological analysis of the testes persistently infected with MARV revealed disruption of the normal architecture of the seminiferous tubules concomitantly with presence of necrotic debris and inflammatory cells. The interstitial tissue surrounding the seminiferous tubules of MARV-infected animals was prominently expanded by an infiltrate of mixed inflammatory cells, including lymphocytes, macrophages, and neutrophils in comparison with uninfected control testes and the areas without MARV persistence in the same testis ( Figures 2 A, 2B , and S4 A). Since all survivors with testicular MARV persistence presented a highly consistent infection pattern and highly similar histopathologic changes in their testes ( Table S1 ), we focused our further analyses on tissues from survivors M8 and M10. These two survivors are from two independent studies and both of them have the most abundant archived tissues ( Table S2 ).

(K) Quantification DDX4 + spermatogenic cells in each confocal section of seminiferous tubules in uninfected control and survivors. Data are represented as mean ± SEM; n = 25, 32, and 32 sections of seminiferous tubules.

(I and J) IgG antibody (green) accumulates and CD20 + B cells (red) infiltrate into interstitial tissue and seminiferous tubules in survivors (J) compared with control testis (I). Arrow, IgG + cells in seminiferous tubules; arrowhead, CD20 + IgG + cell in seminiferous tubule. Blue, nuclear stain by DAPI. Scale bars, 50 μm in (A)– (H) and 20 μm in (I) and (J).

(G and H) Infiltration of CD3T cells (red) and CD68macrophage/monocytes (green) both in interstitial tissue and seminiferous tubules in survivor testis (H) in comparison with control testis (G) (see also Figures S4 E–S4H).

(E and F) DDX4spermatogenic cells (green) are markedly diminished, and large numbers of CD45leukocytes (red) are present in both testicular interstitial tissue and seminiferous tubules in survivors (F) in comparison with control testis (E) (see also Figures S4 C and S4D).

(B) Interstitial tissue areas surrounding the seminiferous tubules are expanded by an infiltrate of mixed inflammatory cells, including lymphocytes, macrophages, and neutrophils, in survivors (see also Figure S4 A). Blue, hematoxylin-stained nuclei.

Together, these data suggest that MARV persistence occurs more frequently in testes compared with eyes, a trend opposite to that seen in EBOV persistence. Galidesivir may prevent or clear MARV persistence in testes more efficiently than antisense reagents, and testicular MARV persistence does not seem to be related to viremia. Due to the high number of animals with persistent testicular MARV infection, we decided to focus our further studies on the testes.

Of note, 21 of 62 survivors (33.9%) that had been treated successfully with antisense phosphorodiamidate morpholino oligomers (PMOs) () were persistently infected in the testes. On the other hand, only one of 11 survivors (9.1%) that had been treated successfully with galidesivir (BCX4430) () showed such persistence ( Table S2 ).

In the testes, abundant genomic MARV RNA was observed using ISH multifocally in the seminiferous tubules, the sites of immune privilege and sperm production. In contrast, no MARV genomic RNA was present in the uninfected control ( Figures 1 A, 1B′, and S3 A–S3U′, and Table S1 ). Interestingly, MARV antigenomic RNA was detected by multiplex fluorescence ISH in the seminiferous tubules in addition to MARV genomic RNA in survivors ( Figures 1 C and 1D). Further, MARV glycoprotein (GP) was also detectable in sites of persistence in survivors by immunohistochemistry ( Figures 1 E and 1F, and Table S1 ). Detection of MARV genomic RNA, antigenomic RNA, and GPindicates active MARV replication in seminiferous tubules. Indeed, electron microscopy (EM) analysis of formalin-fixed paraffin-embedded (FFPE) tissues that had tested positive for MARV RNA using ISH identified MARV particles ( Figures 1 G and 1G′) in the cells in seminiferous tubules.

Scale bar, 500 μm in (A) and (B), 100 μm in (A′) and (B′), 50 μm in (C)–(F), 2 μm in (G), and 200 nm in (G′).

(G and G′) Detection of MARV particles using electron microscope analysis of FFPE tissue with MARV ISH signal. (G′) is an inset (MARV particles indicated by arrow) of (G) at high magnification.

(E and F) In comparison with uninfected control testis (E), MARV GP 1,2 is present in the seminiferous tubules of survivors by immunohistochemistry (IHC) (F).

(C and D) Both MARV genome (red) and antigenome/mRNA (green) are present in seminiferous tubules as judged by detection of genomic NP RNA and VP30 antigenomic RNA using multiplex fluorescence in situ hybridization (FISH) (D) but not in tissues of uninfected control (C). Blue, nuclear stain using DAPI.

(A–B′) Genomic MARV RNA (red, NP ISH) is present multifocally in the seminiferous tubules of survivors in hematoxylin-stained (blue) FFPE testis sections but not in uninfected control (A and A′). Single prime figures (A′–B′) are insets (black dashed rectangles) of (A and B) at high magnification (see also Figure S3 ).

Consistently, quantitative reverse transcriptase-PCR failed to detect viremia in tested survivors that were persistently infected (after day 28 and 41 post exposure, respectively) with MARV in testes or both testes and eyes ( Table S1 and Figure S2 A). Interestingly, no statistically significant difference in viremia was detected between survivors without testicular MARV persistence and survivors with testicular MARV persistence on day 8 (peak viremia) and day 10 (when viremia started to drop) during the acute phase of their infection (study 5 in Table S2 and Figures S2 A and S2B). In addition, no significant viremia was detected in survivors with testicular MARV persistence in comparison with survivors without testicular MARV persistence once the acute infection had subsided ( Figure S2 A).

In a previous study, we identified EBOV persistence in the epididymides of only one of 76 examined rhesus monkey survivors, which otherwise were characterized by a relatively high frequency of ocular EBOV persistence (). The mechanism that led to testicular filovirus persistence and filovirus shedding into semen remained elusive. Using in situ hybridization (ISH), we screened a collection of archived tissue samples (brain, eyes, liver, lymph nodes, spleen, and testes) from 97 crab-eating macaques (73 males and 24 females) that had survived experimental MARV infection through day 47 post exposure. Overall, 23 of the 97 survivors (23.7%) had MARV genomic RNA in the testes and/or eye tissues ( Table S1 ). Specifically, only three of 61 tested survivors (4.9%) for which eye tissues were available were persistently infected with MARV in the eyes ( Table S1 ). In these three cases, MARV was detected in the inflammatory cells attached to the ciliary process ( Figures S1 A–S1C″). Of the tested male survivors, 22 of 73 survivors (30.1%) for which testis tissues were available had MARV genomic RNA in the testes ( Tables S1 and S2 ). However, MARV persistence was not detected in the brains of 67 tested survivors. Of the survivors with MARV persistence in the testes and/or eyes, MARV genomic RNA was undetectable in commonly targeted tissues (liver, lymph nodes, and spleen) of acute MARV infection ( Table S1 and Figures S1 D–S1I).

Discussion

Here we reported that persistent MARV infection frequently occurs in the testicular seminiferous tubules of macaques that survived typically lethal infection after treatment with candidate MCMs. This persistence severely damages the testes, leading to the breakdown of the BTB between infected Sertoli cells. We found this process to be accompanied by a local infiltration of immunosuppressive Treg cells. We discovered orchitis to be a sequela in 40 of 73 (54.8%) survivors. However, only a little more than half of these animals (22 of 40, 55%) also had testicular MARV infection ( Table S2 ). This finding suggests that almost half of the survivors had already cleared MARV infection but still had residual testicular tissue damage by day 47 post exposure. Thus, we believe persistent MARV would eventually be cleared from the testes; however, more in-depth studies are needed to understand how frequently clearance occurs.

Zeng et al., 2017 Zeng X.

Blancett C.D.

Koistinen K.A.

Schellhase C.W.

Bearss J.J.

Radoshitzky S.R.

Honnold S.P.

Chance T.B.

Warren T.K.

Froude J.W.

et al. Identification and pathological characterization of persistent asymptomatic Ebola virus infection in rhesus monkeys. Martines et al., 2015 Martines R.B.

Ng D.L.

Greer P.W.

Rollin P.E.

Zaki S.R. Tissue and cellular tropism, pathology and pathogenesis of Ebola and Marburg viruses. Geisbert et al., 2015 Geisbert T.W.

Strong J.E.

Feldmann H. Considerations in the use of nonhuman primate models of Ebola virus and Marburg virus infection. By contrast, in our previous study, we found persistent EBOV infection in epididymides of only one of 76 rhesus monkey (Macaca mulatta) survivors (). EBOV also has been reported to infect seminiferous tubules in a human case (). We speculate that macaques of different species might differ in their susceptibility to persistent filovirus infection as the disease course appears on average slightly faster in crab-eating macaques compared with rhesus macaques (), or the likelihood of persistent MARV and EBOV infection may be different in nonhuman primates because these two viruses are taxonomically distinct and may have different preferred cellular targets in immune-privileged sites. Further studies are needed to test both possibilities.

Zhao et al., 2014 Zhao S.

Zhu W.

Xue S.

Han D. Testicular defense systems: immune privilege and innate immunity. Seminiferous tubules, the site of germination, maturation, and transportation of sperm cells within testes, possess a special immunological environment that protects immunogenic germ cells from systemic immune attack by effectively blocking the invasion of antibodies and lymphoid cells (). In contrast, seminiferous tubules with MARV persistence were not sheltered from the immune system, as shown by degenerate tubule walls, broken tight junctions, abundant IgG antibodies, and infiltrated immune cells including macrophages, T cells, and B cells. In common target organs (liver, lymph nodes, spleen) of acute MARV infection, virus is cleared completely once an animal survives. In contrast, such clearance seemed to be delayed in the damaged testes that have lost their immune privilege. Future genomic characterization of MARV subpopulations in seminiferous tubules of testes in nonhuman primate survivors compared with actively circulating MARV subpopulations in animals suffering acute disease may provide more insight into the molecular basis of Sertoli cell-specific MARV persistence mechanisms.

Virgin et al., 2009 Virgin H.W.

Wherry E.J.

Ahmed R. Redefining chronic viral infection. + T cell subsets that promote pro-inflammatory responses, FOXP3+ Treg cells are a suppressive subset of CD4+ T cells that have potent immunosuppressive properties. Treg cells can prevent potentially damaging autoimmune responses and the number of Treg cells determines the regulatory responses of the immune system. Having too few Treg cells can trigger fatal autoimmune responses, whereas having too many can cause immune suppression ( Liston and Gray, 2014 Liston A.

Gray D.H. Homeostatic control of regulatory T cell diversity. To reduce excessive immunopathology, a complex regulatory network actively inhibits immune responses during chronic viral infections (). In contrast to effector CD4T cell subsets that promote pro-inflammatory responses, FOXP3Treg cells are a suppressive subset of CD4T cells that have potent immunosuppressive properties. Treg cells can prevent potentially damaging autoimmune responses and the number of Treg cells determines the regulatory responses of the immune system. Having too few Treg cells can trigger fatal autoimmune responses, whereas having too many can cause immune suppression ().

+ CD4+Treg cells identified specifically in damaged testicular sites with persistent MARV infection might have inhibited host immunity important for viral clearance and thus may contribute to persistent infection. Indeed, immunosuppressive Treg cells are associated with ineffective immune responses during chronic infections caused by Friend leukemia virus, hepatitis B virus, hepatitis C virus, and HIV-1 ( Virgin et al., 2009 Virgin H.W.

Wherry E.J.

Ahmed R. Redefining chronic viral infection. The FOXP3CD4Treg cells identified specifically in damaged testicular sites with persistent MARV infection might have inhibited host immunity important for viral clearance and thus may contribute to persistent infection. Indeed, immunosuppressive Treg cells are associated with ineffective immune responses during chronic infections caused by Friend leukemia virus, hepatitis B virus, hepatitis C virus, and HIV-1 (). However, in contrast to these infections, we found accumulations of Treg cells only in the testicular sites of MARV persistence. This restricted distribution suggests that the peripheral blood may not always be the most appropriate compartment in which to investigate Treg cell response during human viral persistence. The immunosuppressive Treg cells in testes with MARV persistence may present a potential immunotherapy target to clear persistent filovirus infection in damaged immune-privileged sites. Our study may therefore open a distinct direction to mitigate testicular filovirus persistence and prevent sexual transmission.

Notably, MARV infection in untreated macaques is typically lethal; survivors have thus far been observed predominantly during MCM efficacy evaluation studies. Hence our data are biased toward virus persistence in nonhuman primates being a possible consequence of diverse treatments. With the number of human MVD survivors being very low, it is therefore impossible to assess whether MARV persistence in humans is a common or rare phenomenon and whether nonhuman primate experiments reflect that phenomenon. It will be important to identify and clinically follow survivors of future MVD outbreaks and to develop a nonhumane primate survivor model in the absence of MCM treatment to enable meaningful comparisons.