Zika virus (ZIKV) is an emerging mosquito-borne flavivirus that has recently been found to cause fetal infection and neonatal abnormalities, including microcephaly and neurological dysfunction. ZIKV persists in the semen months after the acute viremic phase in humans. To further understand the consequences of ZIKV persistence in males, we infected Ifnar1 −/− mice via subcutaneous injection of a pathogenic but nonlethal ZIKV strain. ZIKV replication persists within the testes even after clearance from the blood, with interstitial, testosterone-producing Leydig cells supporting virus replication. We found high levels of viral RNA and antigen within the epididymal lumen, where sperm is stored, and within surrounding epithelial cells. Unexpectedly, at 21 days post-infection, the testes of the ZIKV-infected mice were significantly smaller compared to those of mock-infected mice, indicating progressive testicular atrophy. ZIKV infection caused a reduction in serum testosterone, suggesting that male fertility can be affected. Our findings have important implications for nonvector-borne vertical transmission, as well as long-term potential reproductive deficiencies, in ZIKV-infected males.

Keywords

Zika virus (ZIKV), a positive-stranded RNA virus belonging to the Flavivirus family, has recently been associated with several unexpected viral characteristics ( 1 , 2 ). Although the main mode of ZIKV transmission is thought to be mosquito-mediated, as in other flaviviruses ( 3 , 4 ), ZIKV has additionally been found to use both maternal-fetal ( 5 ) and sexual transmission ( 6 ) as a means of virus spread. Upon systemic infection, virus replication can be detected in most organs; it has also been found within several immune-privileged sites, including the brain ( 7 – 18 ), placenta ( 19 , 20 ), eyes ( 21 ), ovaries ( 22 ), and testes ( 23 , 24 ). In humans, persistent shedding of infectious virus has been found in vaginal secretions ( 25 ) and semen ( 26 – 33 ) at times well past the acute viremic and symptomatic stage of virus infection.

RESULTS

To understand the pathology associated with persistence of ZIKV shedding within the testes, we used a murine infection model that leads to high levels of systemic virus replication but does not ultimately result in death (Ifnar1−/− mice challenged with ZIKVMEX). ZIKVMEX-infected Ifnar1−/− mice demonstrated relatively mild body weight loss (Fig. 1A) and developed peak viremia at 5 days post-infection (dpi) (Fig. 1B). We were surprised to find that the ZIKVMEX strain, which has not yet been published in mouse models, did not result in the lethal pathology of previous strains (22, 24). This allowed us to monitor the effects of ZIKV infection of a pandemic strain at later time points of infection. Consistent with these findings, mice sacrificed at 5 dpi were found to have high levels of viral RNA (Fig. 1C) and infectious virus (Fig. 1D) within the brain, testes, and blood. At 9 dpi, ZIKV was essentially cleared from the bloodstream and was undetectable at 21 dpi (Fig. 1B).

Fig. 1 Characterization of ZIKV in mice. Ifnar1−/− mice (n = 4) were infected with 105 plaque-forming units (PFU) of ZIKVMEX via subcutaneous route. (A) Weights were monitored for 15 days after virus infection. The values represent average scores of body weight compared with initial body weight ± SD from four mice. (B) Blood samples were collected at 1, 3, 5, 7, 9, and 21 dpi, and the virus loads were examined by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Three wild-type (WT) and Ifnar1−/− mice were infected subcutaneously with 105 PFU of ZIKVMEX. Multiple organs were collected from mice (n = 3) after challenge on day 5 and subjected to virus loads by qRT-PCR (C) and by plaque assays in Vero cells (D). The results are expressed as the mean titers. The detection limit of plaque assays is 1.8 log 10 PFU/g.

To decipher the cellular tropism within the testes, we performed immunohistochemistry (IHC) using a mouse monoclonal antibody recognizing ZIKV NS1 antigen on testes and epididymis of ZIKVMEX-infected Ifnar1−/− mice at 5 dpi. As shown in Fig. 2A, viral antigens were mainly detected within the interstitial cells of the testes. These regions are mainly composed of Leydig cells, a testicular cell type that supports sperm production through the generation of testosterone (34). We did not detect ZIKV within the seminiferous tubules in ZIKVMEX-infected Ifnar1−/− mice at 5 dpi (Fig. 2A, a and b). Because the epididymis is a highly convoluted tube that stores and carries sperm (35), we also examined epididymal tissue of ZIKVMEX-infected Ifnar1−/− mice for ZIKV antigen and found an overwhelming amount of antigen associated with the sperm mass within the epididymal duct lumen (Fig. 2A, d). In addition, we also observed several epithelial cells lining the lumen that were positive for viral antigen (red arrows in Fig. 2A, d). In further support of ZIKV infection of testes, RNA analysis of several cytokines indicated that an innate immune response was elicited at 5 dpi (Fig. 2B).

Fig. 2 ZIKV infection in testes and epididymis. Ifnar1−/− mice were infected with 105 PFU of ZIKVMEX via subcutaneous route. (A) IHC was performed using an anti-ZIKV NS1 antibody. Left panels (low magnification) show insets shown on the right (higher magnification). Red arrowheads indicate infected epithelial cells. (B and C) At 5 dpi, testes were collected, and total RNA was extracted for qRT-PCR of interferon-β (IFN-β), tumor necrosis factor–α (TNF-α), interleukin-6 (IL-6), IFN-γ, and interferon-stimulated gene 15 (ISG-15) (B). The testicular mRNA expression levels of testosterone-synthetic genes [steroidogenic acute regulatory factor (StAR), cytochrome P-450 side-chain cleavage enzyme (P450scc), and 3β-hydroxysteroid dehydrogenase (3β-HSD)] were examined by qRT-PCR (C). Data are normalized to mouse β-actin (*P < 0.05). (D) Isolated Leydig cells from WT and Ifnar1−/− mice were infected with ZIKV at a multiplicity of infection of 10. At the indicated times after infection, virus titers in the supernatant were determined with Vero cells. The reported values are means ± SD.

Because IHC analysis suggested that Leydig cells, which are responsible for the supply of testosterone in testes, are targets for ZIKV infection, we next determined the expression level of several genes related to testosterone synthesis (36) in ZIKVMEX-infected Ifnar1−/− mice testes and found a consistent reduction among all genes tested as compared in testes of ZIKVMEX-infected Ifnar1−/−mice (Fig. 2C). To confirm the susceptibility of Leydig cells to ZIKV, we infected isolated WT or Ifnar1−/− Leydig cells with ZIKVMEX in vitro. As shown in Fig. 2D, Leydig cells were able to productively support ZIKV replication, indicating that Leydig cells could serve as a target and a reservoir cell type for ZIKV within the testes.

Strikingly and rather unexpectedly, at 21 dpi, the testes of ZIKVMEX-infected Ifnar1−/− mice were significantly reduced in size as compared to those of uninfected mice (Fig. 3A), as determined by both weight (Fig. 3B) and length (Fig. 3C). These findings suggest that persistent ZIKV infection may lead to hypofertility. Because IHC, in vitro infection, and the decreased expression of genes related with testosterone synthesis indicated Leydig cells as putative targets for ZIKV infection within the testes, we next set to determine whether atrophy could occur as the result of decreased testosterone levels in ZIKV-infected mice. We examined levels of testosterone within serum from ZIKVMEX-infected Ifnar1−/− mice at 5 and 21 dpi as compared with mock-infected Ifnar1−/− mice and found a significant decrease in testosterone at 5 dpi with a consistent reduction at 21 dpi (Fig. 3D). Notably, the testosterone levels at 5 dpi (six of seven mice) and at 21 dpi (two of seven mice) were lower than the assay detection limit. These findings support other data documenting ZIKV infection of Leydig cells (Fig. 2, A and D) and suggest a potential mechanism for ZIKV-induced testicular atrophy. Viral RNA was noticeably higher in the epididymis compared to testes (Fig. 3E), consistent with the copious amount of viral antigen detected by IHC within the epididymal lumen (Fig. 2A). It remains conceivable that multiple cell types within the reproductive tract produce virus, and the cell-free virus becomes stored and concentrated in the epididymis before physical expulsion upon sexual activity.