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. 2022 Apr 23;53(3):1279–1287. doi: 10.1007/s42770-022-00761-x

Early infection of Zika virus in the male reproductive system of AG129 mice: molecular and immunohistochemical evaluation

Maria Letícia Duarte Lima 1, Ágata Silva Cabral 1, Cintia Bittar 1,✉, Luiz Roberto Falleiros Junior 1, Luiz Henrique Alves Guerra 1, Bruno Moreira Carneiro 1,2, Luis Carlos de Souza Ferreira 3, Maurício Lacerda Nogueira 4, Sebastião Roberto Taboga 1, Marilia Freitas Calmon 1,#, Paula Rahal 1,#
PMCID: PMC9433484  PMID: 35460509

Abstract

Sexual transmission of Zika virus (ZIKV), an important arbovirus, and the virus persistence in semen raise several questions about how and where it circulates in the male reproductive system (MRS). Several studies reported detection of the virus in testes, epididymis, and prostate at 5 days post-infection (dpi) or more in animal models. In the present study, we investigated the interactions of ZIKV with mouse MRS using the AG129 strain, a ZIKV permissive immunodeficient mouse strain, at two dpi. Viral RNA was detected in blood, testes, epididymis, and prostatic complexes (prostate and seminal vesicles). Immunohistochemical (IHC) analyses, based on the envelope protein, showed an early infection in organs of MRS since ZIKV positive antigens were detected in cells within or surrounding blood vessels, Sertoli, and germ cells in testes and epithelial cells in epididymis and prostate. Positive antigens for NS5 protein, the virus RNA-dependent RNA polymerase, were also detected by IHC in these organs and circulating leukocytes, suggesting that the virus replicates in these sites as early as 2 days post-infection. Analysis of the early stages of ZIKV infection in MRS may improve the current knowledge about this issue and contribute to the development of therapies directed to the infection at this site.

Supplementary Information

The online version contains supplementary material available at 10.1007/s42770-022-00761-x.

Keywords: ZIKV, Zika virus, Male reproductive system, Mice, AG129

Introduction

Zika virus (ZIKV), an arbovirus member of the Flaviviridae family, genus Flavivirus was first identified in 1947 from the blood of sentinel Rhesus monkeys in the Zika forest of Uganda [1]. Until 2007, few cases of human infections by ZIKV were reported; however, in that year, an epidemic occurred in Yap Island, Federated States of Micronesia. [2]. The first autochthonous case in Brazil was reported in 2015 [3], and recent outbreaks in Central and South America demonstrated that the infection is associated with severe health conditions, such as congenital Zika syndrome in the fetus [4] and Guillain-Barré syndrome in adults [5].

ZIKV is mainly transmitted by mosquitoes from the genus Aedes [6]. Other forms of transmission that have been reported are blood transfusion [7], vertical (maternal–fetal) [8–10], and sexual transmission [11–13].

The first suspected case of ZIKV sexual transmission was reported in 2011 [12]. An American man traveled to Senegal and contracted ZIKV. Upon returning home, his wife, who had not traveled to places where there were vectors of Zika virus, also had Zika virus infection, and the results indicated that the man could have transmitted ZIKV to his wife through sexual transmission [12]. Other similar cases were described, and they occurred from man-to-woman [14, 15], man-to-man [11], and woman-to-man [16]. Additionally, this virus is capable of causing a long-term persistence in semen [17, 18], and ZIKV RNA was already detected in this fluid 370 days after the symptom onset [19].

The male reproductive system (MRS) has been shown to be a site of persistence of the Zika virus and animal models have been used to investigate the infection. In mice, most studies that detected ZIKV in testes and epididymis also observed inflammation in these organs [20, 21]. The virus was detected in the prostate and seminal vesicle as well, but in a lower quantity [22]. Prostatitis cases also were reported in mice and macaques [23]. In other studies, ZIKV also was found in testes, prostate, and seminal vesicles of macaques [24], besides testes and epididymis of olive baboons [25]. Here, we analyzed the infection of ZIKV in different organs of the AG129 mouse MRS, an immunodeficient mouse strain lacking interferon types I and II receptors, permissive to virus replication, at 2 days post-infection (dpi) and reported the early events associated with ZIKV infection in MRS of AG129 mouse.

Methods

Cell culture

Cell culture of Vero E6 cells (monkey African green kidney cell line) (ATCC CRL-1586) was done in Dulbecco’s Modified Eagle Medium (DMEM) (Cultilab, Brazil), with supplementation of 10% fetal bovine serum (FBS) (Cultilab, Brazil), 1000 U/mL penicillin–streptomycin (P/S) (Thermo Scientific, USA). They were maintained in an incubator with humidification, 5% CO2, and at 37 °C. The mosquito C6/36 cell line (Aedes albopictus, Banco de células do Rio de Janeiro, Brazil) was cultured in Leibovitz L-15 medium (Cultilab, Brazil) with supplementation of 10% fetal bovine serum (FBS) and 1000 U/mL penicillin–streptomycin (P/S) at 28 °C.

Virus preparation

Zika virus used was a Brazilian strain (ZIKVBR) that was obtained from a patient from northeastern Brazil [26], kindly provided by Dr. Pedro Vasconcelos, Evandro Chagas Institute, Brazil. The viral stock was prepared by infection of C6/36 cells with a sample of this virus and incubation for 96–144 h until cytopathic effects could be observed. Cells were frozen, thawed, and the supernatant was filtered and stored at − 80 °C. Viral stock was tittered by plaque assay in Vero E6 cells, and viral titer was obtained in plaque-forming units per microliter (PFU/mL).

Animals

Matrices (three females and one male) of the eleven male mice were obtained at the Bioterium of the University of São Paulo (USP)—São Paulo campus; they were courtesy of Dr. Luis Carlos de Souza Ferreira, University of São Paulo—Brazil, and maintained in pathogen-free conditions.

Nine 7-week-old male AG129 mice were intradermally injected into the footpad with 50µL of ZIKVBR at 1 × 105 PFU/mL (5 × 103 PFU). Animals were euthanized in 2 dpi, peak of viremia according to Aliota et al. [27]. Besides them, two uninfected mice were euthanized as the negative control. The euthanasia occurred by an overdose of the anesthetic Dopalen (Ketamine Hydrochloride, Agribrands Brasil Ltda, Brazil) -500 mg/kg, injected intraperitoneally.

The brain, testes, epididymis, prostatic complex (prostate and seminal vesicle), and blood were collected. For real-time PCR (qPCR), five animals were used, four infected and one control. Six animals were used for immunohistochemistry (IHC), five infected, and one control.

RNA extraction, cDNA synthesis, and real-time PCR

Total RNA extraction of the organs and serum was performed by macerating the organs or mixed serum in TRIzol (Invitrogen, USA) and proceeding with RNA extraction following the manufacturer’s instructions. High Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, USA) was used to prepare the complementary DNA (cDNA), following the guidelines of the manufacturer, using 10 µL of extracted RNA.

Absolute quantification of viral RNA levels was determined by RT-PCR based on a standard curve using ZIKV 1086, ZIKV 1162c, and 1107-FAM primers [28] and TaqMan Universal PCR Master Mix, No AmpErase® UNG (Thermo Fisher Scientific, USA) QuantStudio™ 12 K Flex Real-Time PCR System (Applied Biosystems, USA). Viral load was expressed on a log10 scale as ZIKV RNA copies/μL.

Immunohistochemistry

Sections of the organs paraformaldehyde-fixed, paraffin-embedded (5 μm) were submitted to immunohistochemistry staining using the Immunohistochemistry Application Solutions Kit (Rabbit) (#13,079, Cell Signaling, USA). After deparaffinization and rehydration of tissues, the antigen retrieval was done with citrate buffer (pH 6.0) at 98 °C for 25 min, followed by the endogenous peroxidase blocking for 10 min with Peroxide Blocking Reagent (Previously Covance catalog # SIG-31128, BioLegend®, USA). The blockage of nonspecific proteins was done for 2 h with a Goat Serum 10% blocking solution (Invitrogen, USA) at room temperature. The slides were then incubated with primary antibodies, NS5 antibody (NBP2-42,900, rabbit, polyclonal, 1:500, Novus Biologicals®, USA), and Zika virus envelope protein (GeneTex Cat # GTX133314, RRID: AB_2747413, rabbit, polyclonal, 1:500, GeneTex, USA) for 2 h at 25 °C. The tissues were submitted to SignalStain® Boost IHC Detection Reagent (HRP, Rabbit) (kit) for 30 min. Diaminobenzidine (DAB) was used to detect the reaction and counterstained with hematoxylin (Merck, Germany). The slides were analyzed in Olympus BX60 (Olympus, Japan) light microscope using the DP‐BSW V3.1 software (Olympus, Japan).

Data analysis

Data were first submitted to descriptive analysis for determination of normality by the D’Agostino-Pearson test. Once the normal distribution was confirmed, analysis of variance (one-way ANOVA) was performed to compare the organs and serum, followed by the Bonferroni test. For statistical significance, values of p < 0.05 were considered. The GraphPad Prism 5 software (GraphPad Software, Inc., USA) was used to perform the statistical analysis. Microsoft Excel (Microsoft, USA) was also used to aid in data processing. The data were demonstrated as a mean, and ± represents standard error.

Results

ZIKV RNA was detected in male reproductive system organs at 2 dpi

First, we verified the titers of ZIKV RNA copies/μL in the organs and serum by real-time PCR analysis. The virus was detected in all analyzed samples. The highest titer was observed in serum (1.61 × 108 ZIKV RNA copies/μL), followed by testes (8.85 × 106 ZIKV RNA copies/μL) and epididymis (3.28 × 105 ZIKV RNA copies/μL), and the lowest titer was detected in the prostatic complex (2.13 × 104 ZIKV RNA copies/μL). Figure 1 presents the mean viral titer, measured by RNA copies/μL, of each organ of MRS, brain, and serum considering four infected mice. Statistical differences were observed by comparison only between serum and the tested MRS organs (p value < 0.0001). Comparisons between organs were not statistically significant. The brain was used as a positive control due to the previously described neurotropism of ZIKV [29–31]. In the brain, viral titer (1,98 × 105 ZIKV RNA copies/μL) was lower than in testes and epididymis but higher than in prostatic complex (Fig. 1).

Fig. 1.

Fig. 1

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Zika virus RNA levels in organs of MRS, brain, and serum. Mean of the ZIKV RNA copies/μL (log) in brain, testes, epididymis, prostatic complex, and serum of four 7-week-old AG129 mice infected with 1 × 105 PFU/mL of ZIKVBR, measured in RNA copies by two-step qPCR, at 2 dpi. Statistical analysis was performed with one-way ANOVA followed by the Bonferroni test, ***p < 0.0001, n = 4. Data presented as a mean and ± standard error (SEM). Viral loads in organs and serum were not detected in uninfected mouse

MRS organs display an initial infection at 2 dpi by ZIKV

Sections of the brain, testes, epididymis, and prostatic complex of infected mice (Supplementary S1c to S1d and S2c to S2d; Figs. 2d to 2g and 3d to 3h Figs) and an uninfected mouse (Supplementary S1a to S1b and S2a to S2b Figs; Figs. 2a to 2c and  3a to 3c) were analyzed after staining by immunohistochemistry to determine the location of ZIKV in these organs at 2 dpi. For this purpose, we used a marker for the viral envelope protein (ENV) and another for the NS5 nonstructural protein (NS5), which is the viral RNA polymerase. The two antigens for ZIKV were detected in all organs analyzed as early as 2 dpi.

Fig. 2.

Fig. 2

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Photomicrograph of ZIKV envelope protein antigen (ENV) in MRS organs of 7-week-old AG129 mice, 2 days post-infection. a–c Represent organs of the uninfected mouse, and d–g show the immunoreactivity for ENV in organs of infected mice. d, d′, g Testes with Sertoli cells (yellow arrows) and germ cells (red arrows) positive to ZIKV ENV antigen. e, e′ Immunoreactivity to ZIKV ENV in some cells in the epithelium of epididymis (black arrow). f, f′ Positive cells in the glandular epithelium of the prostate (black arrow). a′ Bar = 50.0 μM, polyclonal anti-Zika virus envelope protein; counterstained hematoxylin; b′, c′, d′, e′, f′, g bar = 20.0 μM, polyclonal anti-Zika virus envelope protein; counterstained hematoxylin; a, b, c, d, e, f bar = 10.0 μM; polyclonal anti-Zika virus envelope protein; counterstained hematoxylin

Fig. 3.

Fig. 3

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Photomicrograph of flavivirus NS5 protein antigen (NS5) in MRS organs of 7-week-old AG129 mice, 2 days post-infection. a–c Represents organs of the uninfected mouse, and d–h show the immunoreactivity for NS5 in organs of infected mice. d, d′ Seminiferous tubules with the Sertoli cells (yellow arrows) and the germ cells (red arrow) immunoreactive to NS5 antigen. e, e′, g Positive cells in the epithelium of the epididymis (black arrows). f, f′ Positive cells in the glandular epithelium of the prostate (black arrows). h Positive leukocytes within blood vessels (blue arrow) and transmigrating to tissue in the prostate (orange arrow). a′, b′, c′, e′, f′ Bar = 20.0 μM, polyclonal anti-NS5 protein (NS5); counterstained hematoxylin; d′ bar = 50.0 μM, polyclonal anti-NS5 protein (NS5); counterstained hematoxylin; a, b, c, d, e, f, g, h bar = 10.0 μM; polyclonal anti-NS5 protein (NS5); counterstained hematoxylin

More specifically, in the testes, this virus was found in Sertoli cells (Figs. 2d and 3d—yellow arrows) and germ cells (Figs. 2d, 2g, and 3d—red arrows) in basal regions of seminiferous tubules for both ENV and NS5 antigens. Regarding the epididymis, the immunoreactivity occurred in cells of epididymal epithelium (Figs. 2e, 3e, and 3g—black arrows), more evidently in NS5 immunoreactivity (Figs. 3e and 3g—black arrows). In the prostate, the ZIKV antigens were detected in cells of the glandular epithelium (Figs. 2f and 3f—black arrows).

The positive ZIKV antigens were often found inside or nearby blood vessels (Fig. 3h). Inside the blood vessels, immunoreactivity for NS5 antigen was seen many times in leukocytes in all organs analyzed (Fig. 3h—blue arrow, Supplementary S2c Fig—black arrows). In Fig. 3h, obtained from the prostate of an infected mouse, the orange arrow demonstrates a leukocyte positive to NS5 protein of ZIKV transmigrating from the blood vessels into the connective tissue, overcoming the endothelial barrier.

In the brain, the antigen of envelope protein was detected in some neurons (Supplementary S1c Fig—black arrows) and cells of the granular layer (Supplementary S1d Fig—black arrow). NS5 antigen was also positive in this last site (Supplementary S2d Fig—black arrow) and in leukocytes within blood vessels (Supplementary S2c Fig—black arrows).

ZIKV replicates in murine MRS organs

In the present study, we used an NS5-specific antibody for immunohistochemistry analysis. NS5 protein of flavivirus, like Zika virus, has in the C-terminal domain the RNA-dependent RNA polymerase (RdRp), which is responsible for the polymerization of RNA negative strand (intermediate RNA template) and generation of RNA positive strands [32, 33]. Thus, the immunoreactivity to this antibody infers replication of this virus in the cell or tissue.

As previously described, all organs analyzed showed immunoreactivity for this antigen at 2 dpi (Fig. 3). Although many positive leukocytes for this antibody were found within blood vessels in the organs (Fig. 3h—blue arrow), we can also see positive cells in the tissue (Figs. 3d to 3h), mainly in the testes and epididymis. These findings suggest that ZIKV is replicating in these organs early, at 2 dpi.

Discussion

Due to reports of ZIKV sexual transmission [11–13, 34], data that showed the presence of ZIKV in semen [17–19] and persistence of viral RNA in this fluid for 370 days [19], it is necessary to study the infection of this virus in the MRS. Several authors analyzed, under experimental conditions, the infection by this virus of murine MRS 5 or more dpi [20–23, 35]. In this study, we evaluated an early ZKV infection of the mouse MRS in 7-week-old immunodeficient AG129 mice. For this, we infected the animals, and the euthanasia occurred at 2 dpi, peak of viremia according to Aliota et al. [27], and the infection in MRS was analyzed by qPCR and immunohistochemistry. Our results indicate that, even at this early infection stage, ZIKV infects different MRS cells at levels higher than those detected in the brain and supports the sexual transmission features of the infection.

The AG129 mice were characterized as a murine model to develop ZIKV studies by Rossi and collaborators. They detected that ZIKV viremia peaked on day 2 post-infection when the mice were infected by intraperitoneal inoculation on day 3 for intradermal infection. All infected mice died on day 6, and some of their organs were analyzed, showing the highest viral loads were in the brain and testes [36]. Another study tested different ages of AG129 mice, inoculum doses, and challenge routes and verified that in adult mice, 8-week-old, infected in the footpads, the peak of viremia was 2 dpi [27].

Several studies found the Zika virus in organs of the MRS, mainly in testes and epididymis [20, 22, 37]. Concerning the prostate, some researchers reported ZIKV infection in this site in mice and prostatic cells culture [22, 23, 38]. However, other studies that rated the infection in MRS of mice did not detect ZIKV in the prostate or did not evaluate the infection in this site [20, 21]. Most of these researches evaluated the infection in later infection periods (5 dpi or more).

In testes, our findings are consistent with some reports that also detected ZIKV in germ cells [20, 35] and Sertoli cells [35, 39, 40] in vivo at 5 dpi or more. Regarding the epididymis, we found the virus in some cells in the epithelium, which was also reported by Clancy et al. at 5 dpi [22]. In the prostate, the virus was detected in some regions of granular epithelium, as also seen in the literature [22].

A study evaluated the infectious ZIKV in seminal fluids of AG129 male mice infected and observed that the infectivity started at 7 dpi and persisted for 2 weeks. The researchers also verified that the male-infected mice transmitted Zika virus sexually to females during the infectivity period in half of the mattings. Furthermore, even after the end of the infectious period, viral RNA was detected for weeks in the semen of the mice [41].

The male reproductive system has some sites with immune privilege. In the testes, the sperm development is essentially protected by a physical barrier located between adjacent Sertoli cells (SCs) and formed by tight junctions, the blood-testis-barrier (BTB). The BTB avoids the autoimmune attack, thus enabling the complete meiosis of germ cells [42]. Besides that, the BTB is known to collaborate in defense against pathogens [40]. A previous study showed that the Zika virus might infect and replicate in a lineage of human Sertoli cells, and when these cells were exposed to inflammatory mediators originating from macrophages, a tight junction protein, the ZO-1 protein, that may compose the BTB, was degraded. This increased the permeability of BTB in an in vitro model and may be one of the important points in the access of ZIKV to seminiferous tubules [40]. Our findings detected structural and non-structural components of ZIKV in SCs, the envelope protein, and NS5 protein, respectively, showing that this virus replicates in SC in the early stages of infection in AG129 mice.

Epididymis has its physical barrier, the blood-epididymis-barrier (BEB), which, like in testes, protects sperm maturation from the attack of immune cells and pathogens. It is formed by attached epithelial cells with tight junctions between them [43]. However, when compared with BTB, the BEB seems to be less effective because immune cells may be observed more frequently in the epididymal epithelium along with leukocytes infiltration in the lumen of the epididymis, than in the testes [44]. McDonald et al. verified that epithelial cells of epididymis were the main origin of replication intermediaries of ZIKV in MRS of AG129 mice, after 8 and 10 dpi, in comparison with testis. The authors also demonstrated the presence of ZIKV in leukocytes and the replication in these cells together with epithelial cells of the epididymis correlated with the peak of potential sexual transmission. They suggested that these cells may be the main origin of ZIKV in semen [45]. These may be related to the major susceptibility of barrier-epididymis-blood, as previously described [44]. In the present study, we detected the presence of NS5 antigen in the epithelium of epididymis and circulating leukocytes presented in all analyzed organs. Immunoreactivity to the NS5 protein of flavivirus indicates replication in these sites.

Many studies suggest that the testes and epididymis are the main organs in which the ZIKV replicates in MRS, but cases of vasectomized men in whom ZIKV was detected in the semen [14, 46, 47] demonstrate that Zika virus can be replicated in other sites. When a man undergoes a vasectomy, the ductus deferens are cut, so the communication between epididymis and testis with the rest of MRS is ceased; therefore, viral replication in these organs would not be detected in semen [38].

Duggal et al. studied vasectomized AG129 mice and observed that the vasectomy reduced significantly the levels of the infectious ZIKV in semen, but sexual transmission to uninfected females still occurred. These data suggested that sexual transmission may occur even with the low levels of infectious ZIKV in semen and that the testes may not be the only viral source of sexual transmission [41]. They also found that ZIKV RNA was detected in seminal fluids of these mice, and this can indicate that other organs contribute to the shed of viral RNA in these fluids [41].

In that way, accessory glands such as the prostate may be a potential replication site. Although some studies did not detect ZIKV in the prostate [20], others have described it as an important site of ZIKV persistence in MRS. Studies have shown that the Zika virus can infect and replicate in lineages of prostate cells. [38]. Also, Halabi et al. analyzed the infection in the prostate of mice and macaques, and they observed that ZIKV may cause acute and chronic inflammation in this site in both animal models [23]. We also detected the ZIKV antigens, envelope, and NS5 proteins, in the glandular epithelium of the prostate in our IHC analysis at 2 dpi which indicates both infection and replication, respectively, in this site. Therefore, the prostate may be an important site of ZIKV infection in MRS, besides the testes and epididymis.

When comparing MRS and brain infection, ZIKV seems to start the infection faster in the former. The mean of titers detected in the brain of mice was 1.98 × 105 ZIKV RNA copies/μL, and IHC analysis showed that the infection in this tissue seems to be modest, being seen in a few neurons and cells of the granular layer at 2 dpi. Siemann et al. compared two in vitro models of blood barrier: the BTB and the blood–brain-barrier (BBB). They observed that the Zika virus may cross the model of BTB and be released in the lumen with greater efficiency than in BBB [40]. Such observations support our findings at an earlier infection stage by ZIKV in MRS and demonstrate that, under the tested experimental conditions, infection of MRS is more considerable than in the brain in this AG129 mouse model. Altogether, comprehension of the infection in the early stage might aid the development of drugs and vaccines directed to treat Zika virus infection in MRS.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (PDF 320 KB) (319.9KB, pdf)

Acknowledgements

We would like to thank Gustavo Matheus Amaro and Nayara Fernanda da Costa Castro for helping with mice euthanasia.

Author contribution

Cintia Bittar, Bruno Moreira Carneiro, Luis Carlos de Souza Ferreira, Maurício Lacerda Nogueira, Sebastião Roberto Taboga, Marilia Freitas Calmon, and Paula Rahal conceived and designed the experiments; Maria Letícia Duarte Lima, Ágata Silva Cabral, Luiz Roberto Falleiros-Junior, Luiz Henrique Alves Guerra, and Marilia Freitas Calmon performed the experiments; Maria Letícia Duarte Lima, Ágata Silva Cabral, Cintia Bittar, Sebastião Roberto Taboga, and Marilia Freitas Calmon analyzed the data; Maria Letícia Duarte Lima, Cintia Bittar, Sebastião Roberto Taboga, Marilia Freitas Calmon, and Paula Rahal wrote and edited the paper with contributions of all authors.

Funding

This study was financed by the Fundação de Amparo à Pesquisa do Estado de São Paulo – Brasil (FAPESP), Brazil Grant number 2014/22198–0; Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) – Finance Code 001; 2046/2016 and by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brazil grant number 440723/2016–7.

Declarations

Ethics approval

This study was submitted, approved, and performed in accordance with instructions of the Ethics Committee on Animal Use (CEUA) of the Institute of Biosciences, Humanities, and Exact Sciences, São Paulo State University (IBILCE / UNESP) in São José do Rio Preto, SP, with approval no. 163/2017—CEUA.

Consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher's note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Marilia Freitas Calmon and Paula Rahal contributed equally to this work.

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