**3. Flavivirus and ZIKV Features**

The *Flavivirus* genus is composed by viruses of small single-stranded RNA. The flaviviruses can cause mild symptoms, such as fever, pain, and cutaneous rash but also covers severe disturbances, such as encephalitis, neurological complications, and hemorrhagic fever [44]. Flaviviruses are arthropod-borne pathogens typically transmitted by mosquitoes or tick vectors and are related to significant mortality and morbidity worldwide [45]. Members with clinical relevance of this genus include Dengue virus (DENV), Yellow Fever virus (YFV), Japanese Encephalitis virus (JEV), West Nile virus (WNV) and ZIKV. The geographic distribution of flaviviruses and the diversity of arthropod

vectors make them of grea<sup>t</sup> interest for epidemiological surveillance. Moreover, the easy entry and adaptation of these viruses in new environments make this genus relevant to extensive research and experimental studies [44].

ZIKV is a vector-borne flavivirus belonging to the *Flaviviridae* family, with two main lineages: the African and the Asian lineage [46]. It is an enveloped virus measuring about 50 nm in diameter with a non-segmented, positive single-stranded ribonucleic acid (RNA) genome (Figure 2). The genome is made up around of 11 kb with a single open reading frame that codes structural proteins: Capsid (C), Envelope (E), precursor membrane (prM); and non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) [47] (Figure 2).

The first ZIKV isolate was identified in primates in 1947 in Uganda Protectorate in a program for surveillance of yellow fever in primates [48]. The first human infection was reported in 1954 in Nigeria; for decades, ZIKV cases were restricted to Africa and Asia [49]. Since 1954, several outbreaks with increasing number cases have been reported worldwide [50,51]. The last outbreak was documented in 2015 in America, which was the largest epidemic ever described of ZIKV a ffecting more than 20 countries [52,53]. In 2016, WHO considered ZIKV a public health emergency of international concern [20].

**Figure 2.** Zika virus (ZIKV) structure and features. ZIKV is an enveloped positive-sense single-stranded RNA virus composed by envelope, capsid, membrane protein, and single-stranded positive-sense RNA. The lower part represents the polyprotein which is cleaved by viral and cellular proteases four structural proteins: capsid (C), envelope (E), precursor membrane (prM), and membrane (M) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). During infection, the ZIKV E proteins bind to host cell receptors and the viral particle is endocytosed. The E proteins enable the fusion of the virus with the endosomal membrane, leading the release of the genomic RNA into the host cell cytoplasm. The translation of the RNA genome occurs in the endoplasmic reticulum. The RNA is translated as a single polypeptide chain encompassing all the viral proteins: C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5.

ZIKV has di fferent pathways of transmission. The ZIKV transmission in humans was firstly reported through bites of infected *Aedes aegypti* or *Aedes albopictus* mosquito [54]. However, the virus was identified and isolated from seventeen di fferent *Aedes mosquitos* species, *Culex quinquefasciatus*, *Culex perfuscus*, *Mansonia uniformis*, *Anopheles coustani,* and *Anopheles gambiae* mosquitoes [55–59]. Another important fact about ZIKV transmission became apparent during the 2015 outbreak, when several cases of ZIKV vertical transmission were identified from an infected mother through the placenta to the fetus and sexual transmission (male-to-female; female-to-male; male-to-male) [60]. This novel mode of ZIKV transmission in humans had never been reported before in flavivirus infection [60–62]. ZIKV was the first arbovirus detected in human semen [63]. While needing more consistent evidence about the ZIKV transmission, these findings sugges<sup>t</sup> the complexity of ZIKV dynamics transmission [64,65].

## **4. ZIKV on Male Reproductive Tract**

The male reproductive system includes the penis, scrotum, testicles, epididymis, vas deferens, prostate and seminal vesicles (Figure 3). Recent studies have demonstrated the presence of ZIKV RNA in semen, as well as in male and female reproductive tracts, indicating the occurrence of the sexual transmission [66]. The first sexual transmission became evident in 2011, and many cases have supported the idea of one potential transmission pathway [62]. Moreover, ZIKV could be detected in semen six months after infection in negative ZIKV serum from a patient [67]. Similarly, ZIKV RNA was detected in semen in symptomatic and asymptomatic-infected patients [68–70]. A case report showed ZIKV RNA presence in total semen and also in the sperm fraction used in assisted reproductive technology up to 112 days after infection [71]. Taken together, all these data indicate that infected men can be a potential reservoir for sexual transmission, even a long time after the infection [72].

In a mouse model, ZIKV sexual transmission was recently characterized, showing that epididymal epithelial cells and leukocytes should be the main source of ZIKV RNA shedding [73]. ZIKV can persist and replicate in MRT [74]. In cases of ZIKV infection, is it known that SCs can support a high level of ZIKV replication [75,76]. In the early stages of infection, ZIKV suppresses cell growth, cell proliferation, and dysregulation of germ cell–SC junction signaling [77]. ZIKV downregulated the secretion of inhibin B, a hormone mostly produced by SCs [78]. Strange and colleagues demonstrated a unique cross-talk between ZIKV infection and SC immune response, which in the course of infection, the viral persistence was associated with activation of canonical pro-inflammatory pathways. That includes the upregulation of genes of the human leukocyte antigen (HLA) class I, pro-inflammatory genes such as interleukin-23 subunit alpha (IL23A) and lymphotoxin beta (LTB), NF-kappa-B-epsilon (NFKBIE), IL6, STAT1, STAT2, and IFN [77].

The IFN response is a strong key in the innate immune response against virus dissemination in testicles. Two animal models of*Mus musculus* species, susceptible to ZIKV infection, are important for understanding the pathogenesis of this virus. These models are A129 and AG129 mice, both immunocompromised mice. A129 mice do not have the receptor for interferon type I (IFN α/β). AG129 mice do not have the receptor for interferon type I and II (IFN α/β/γ) [79]. IFNAR−/− mice are one of the best mice models for ZIKV susceptibility studies [80]. Siemann and colleagues have shown that in the first hours of infection, ZIKV does not induce IFN-α in SC, but it presents a modest induction after 48 and 72 h of infection. However, high levels of pro-inflammatory cytokines such as interleukin-1α (IL-1α), IL-1β, IL-6, IL-8, and TNF-α were found in the supernatant of infected SC, and in the chemokines such as RANTES (CCL5), fractalkine (CX3CL1), and IP-10 (CXCL10). These levels increased significantly 72 h after infection. Although SCs generate a strong immune response against ZIKV, the virus can persist in the male reproductive tract for a long time [81].

The TAM receptor, AXL, promotes the ZIKV entrance in SCs and contributes negatively to the antiviral states of SCs [82]. SCs are one type of cell that expresses high levels of TAM receptors, TGF-β expression, and activin-A to maintain the immune regulation in the seminiferous tubules. SCs play an important role in testicular physiology, creating a BTB and contributing to the nourishment of the spermatozoa. This cellular physiology and ZIKV modulation can develop an important factor that may lead to the establishment of viruses in this organ. Other cell types in the testicle can support the ZIKV infection, such as testicular fibroblast, germ cells, and spermatocyte [43,83].

**Figure 3.** ZIKV reservoir in the male reproductive tract. ZIKV has been found in several portions of the male reproductive tract, including the prostate gland, testicle, epididymis, and seminiferous tubules. ZIKV-infected men have presented prostatitis, hematospermia, and microhematospermia. ZIKV RNA has been detected in semen from ZIKV-infected men and sexual transmission is an important route of contagious ZIKV. Some testicular cells are susceptible to ZIKV infection, such as spermatogonia, primary spermatocytes, Sertoli cells, and spermatozoa. Moreover, ZIKV can infect and replicate in mature sperm, leading to male infertility.

LCs and testicular macrophages are part of the first line of defense in the seminiferous tubules [84]. LCs are not highly susceptible to ZIKV infection in mice models, but more studies in humans are necessary. However, LCs are the main source of testosterone in testis, and during ZIKV infection, the levels of testosterone are significantly modulated [78]. Testicular macrophages are infected by ZIKV [33], and the infection promotes an increase of mRNA transcript levels of the IFN-α and IFIT1 genes, inducing the secretion of pro-inflammatory cytokine TNF-<sup>α</sup>, IL-1<sup>α</sup>, and IL-8 and chemokines, such as GRO, IP-10, and monocyte chemoattractant protein 1 (MCP-1). These inflammatory mediators are correlated with the possibility that ZIKV infection can compromise SC barrier integrity [81]. ZIKV does not modulate the expression of tight junction proteins (TJPs). The virus can cross BTB efficiently and persist in abluminal side seminiferous tubules by the induction of adhesion molecules expression such as VCAM-1, which facilitates the adhesion of immune cells, compromising BTB permeability [81].

In spermatogonia, the infection can promote cell death, leading to the destruction of seminiferous tubules and triggering male infertility by damaging the male reproductive system [75]. Low sperm counts are observed in patients infected with ZIKV [69,85]. Several studies have shown the effect of the ZIKV infection promoting genital damage, modulation of testicular immunity leading to orchitic and viral replication, promoting a long infection establishment. ZIKV does not affect only the testes. In mice and monkey models, ZIKV infection causes acute and chronic prostatitis [86]. Male rats infected with the Mexican ZIKV strain presented a significant decrease in testicle size compared to uninfected rats. Testicle atrophy may have occurred due to decreased testosterone levels in cells infected with this virus [87].

Several studies have shown alterations in mature sperm infected by ZIKV [85,88]. Such findings may also be an additional indication that ZIKV reduces male fertility. Furthermore, it is important to evaluate sperm banks regarding the presence of ZIKV-infection in donors due to the implications for assisted reproduction.

Therefore, ZIKV is capable of entering the testicular microenvironment, disrupting cellular metabolism, altering testicular physiology, and activating an intense immune response, which can result in severe testicular damage and infertility. A better understanding of how ZIKV a ffects the regulation of cell survival pathways and the testicle physiology can help evaluate pathogenesis and may be used for vaccine studies to identify intervention strategies (Figure 4).

**Figure 4.** Testicular cells infection by ZIKV. ZIKV infection can cause serious physiological, immunological, and endocrine damage in the testes, impairing spermatogenesis. ZIKV can infect several cells in the male reproductive tract. Leydig cells are less susceptible to the infection when compared to other cells in the male reproductive tract. Testosterone, the main hormone produced by Leydig cells, is modulated by ZIKV, impairing the endocrinological function. Testicular macrophage is infected by ZIKV, triggering upregulation of IFN-<sup>α</sup>, IFIT1, TNF-<sup>α</sup>, IL-1a and IL-8, GRO, IP-10, and MCP-1. Inside the seminiferous tubule, Sertoli cells have high expression levels of AXL receptors,

which is used by ZIKV to invade cells. Sertoli cells support high levels of ZIKV replication, and the infection promotes the upregulation of genes related to antigen presentation (HLA-1), proinflammatory cytokines (lymphotoxin-beta LTB, IL-6, IL-23a) and transcription factor related to inflammation (NF-kb, STAT1, and STAT2). The release of proinflammatory cytokines such as IL-1<sup>α</sup>, IL-1β, IL-6, IL-8, TNF-<sup>α</sup>, and chemokines such as RANTES, CXC3CL1, and CXCL10 in SCs is also promoted by the infection. These molecules can promote the chemoattraction of more immunological cells and lead to an inflammatory profile, impairing e fficient spermatogenesis. Inhibin-B, produced predominantly by SCs, can control follicle-stimulating hormone (FSH) secretion and is downregulated by ZIKV infection. ZIKV increases the expression of VCAM-1 in SCs which can facilitate the immune cells adhesion. Inside the seminiferous tubules, ZIKV can infect spermatogonia, primary spermatocytes, and mature spermatozoa.

#### **5. The Immune System of Testis during Viral Infection**

MRT requires a homeostatic microenvironment for viable germ cell production and nutrition. The crosstalk between SCs and LCs is fundamental to spermatozoa development [89,90].

In the testicular surroundings, an important immunological component maintains a proper environment for spermatogenesis, turning the testis into an immune-privileged organ [91]. Once MRT homeostasis is broken, spermatogenesis key steps are impaired and inflammation can be trigged. Many pathogens have shown to infect and persist in the MRT [3,26,84]. Testicular abnormalities, infertility, or sexual transmission are some of the major consequences of pathogen persistence in the MRT. Considering the important findings regarding ZIKV RNA detection in the semen, the scientific community has turned their attention to the possibility that other flaviviruses promote similar effects [92]. Once their detection becomes proven, the possibility of sexual transmission or impaired spermatogenesis is another important factor to be explored. Preliminary studies about this have provided us with information on a possible threat derived from di fferent flaviviruses in the MRT. Nevertheless, this question is far from clear and molecular mechanisms still under investigation.

Some studies have been reported flavivirus infection in the MRT [19]. The viral load could be found for some of them, and the presence of leukocytes in the semen suggests an inflammatory process caused by the infection. Salam and colleagues found viruses from several families in the semen, including *Adenoviridae*, *Filoviridae*, *Flaviviridae*, *Herpesviridae*, and *Retroviridae* [19].

DENV is a considerably more common flavivirus than ZIKV, and the knowledge about DENV effects in the testis is scarce. The first case report linking DENV infection to MRT modulation was published in 2011 [93]. In this report, scrotal and penile edema was a rare complication associated with DENV infection. However, the mechanism by which this edema was formed was not evaluated; neither could DENV be detected in penile fluids. Currently, there is no data reporting if testicular abnormalities could be trigged by DENV-associated inflammation in MRT. In 2018, two controversial publications raised questions about the possible impact of DENV in the MRT. The first one demonstrated that DENV RNA was not detected in the semen of five confirmed patients during the acute infection [94]. The second one is a case report released a few days later, demonstrating that DENV was detected in the semen of an infected man 37 days after the related symptoms. The report demonstrated DENV RNA in the cellular fraction, suggesting the possibility of sexual transmission [95]. New evidence of DENV sexual transmission was published in 2019, where a case report from Spain detected the viral RNA in the semen of two men who were partners [96]. Only one of the men had contact with a DENV endemic area and his partner presented the symptoms a few days after the first one. This is the first evidence of DENV sexual transmission. Nevertheless, clinical trials aiming to concisely respond to this question are underway and may be published soon (Clinical Trial Identifier: NCT03612609).

In 2018, a case report was published regarding YFV RNA detection in the semen and urine of a Brazilian man in the convalescent phase of the disease [97]. The integrity and infectivity of the viral particles were accessed and confirmed in the report. This strongly suggests that this virus can be sexually transmitted once it is capable of maintaining infective parameters, although no data are

available confirming the capability of YFV persistence and impact in the MRT, or sexual transmission associated with the infection.

Zheng and colleagues showed that the JEV infection induces inflammation of pig testicles by activating RIG-I/NF-kB pathway signaling [98]. This also leads to orchitis, which is a type of chronic inflammation in the testes caused by viral or bacterial infections, associated with pain, swelling, along with blood and swelling in prostate ejaculate [81,99]. Testes infection with JEV showed a di fferential production of pro-inflammatory cytokines, such as IL-1β, IL-6, IL-8, chemokine RANTES, and TNFα, as well as an increased presence of NS5 (non-structural protein of the virus), RIG-I, TLR3 and -7 [98]. Smith and colleagues showed that a 43-year-old patient presented signs of encephalitis and orchitis caused by WNV [100]. In this report, histological sections showed lymphocyte, SCs, and interstitial multinucleated cells infiltrate, as well as marked thickening of the basement tubular membranes and absence of spermatogenesis, an indication of atrophy. Numerous foci of dense chronic interstitial inflammatory infiltrate and necrotic cell death was observed in the seminiferous tubules [100].

DENV, YFV, and JEV are classified both as arbovirus and flavivirus and present major clinical relevance within these groups. Nevertheless, another important virus that compounds arbovirus group but is a member of a distinct family, presents important findings regarding MRT infection. For this reason, an analysis of the available data for this arbovirus is relevant and will also be explored in this section.

The Chikungunya virus (CHIKV) is a small, enveloped, single-stranded positive-sense RNA virus that belongs to *Alphavirus* genus and *Togaviridae* family. Chikungunya is a vector-borne disease, also transmitted by the bites of mosquitoes from the *Aedes* genus, mainly *Ae. aegypti* and *Ae. albopictus*, causing arthritis or arthralgia, which is accompanied by fever and rash [101]. CHIKV RNA has been detected in semen and urine, as reported in a case published in 2016. This study showed a patient presenting CHIKV and DENV (type 3) dual infection, in which only CHIKV was detected [102] in both the acute and convalescence phases of the disease, within 30 days after symptoms. Thereby, it is important to emphasize that CHIKV presents tropism and cytotoxic e ffects on monocyte-derived macrophages [103], which can be later recruited to testicular microenvironment [32]. In this context, macrophages are being identified as a possible source of CHIKV RNA in the testis, acting as a testicular trojan horse. However, more studies are necessary to verify this hypothesis [102].

Numerous questions related to viruses infection in MRT remain to be answered. The long-term effects of persistent infection for several flaviviruses in male reproductive function, as well as production and fertility of spermatozoa need to be investigated. Importantly, in the case of ZIKV, cryptorchidism, hypospadias and micropenis have been reported in newborn infants of infected mothers [104], although its prevalence is unknown. An e ffect of arboviruses infection in male fertility will only be fully understood in long-term epidemiological studies and suitable animal model experiment design.

#### **6. ZIKV Vaccines and Treatment to Improve the Host Response in the MRT**

Sexual transmission of ZIKV and the viral persistence in the MRT are the strongest challenges for outbreaks control, vaccines, and antiviral drug development [105–107]. The impact of ZIKV infection in the population leads to a significant global e fforts to develop vaccines. Spectacular progress has been made in ZIKV vaccine development, and several strategies have been proposed to increase vaccine protection in immune-privileged organs [105,108–112].

Antibody usage has shown a promising strategy to protect ZIKV in the testicle. Some subclasses of immunoglobulin (IgG) can cross the BTB [113]. The administration of human antibodies to DENV E-dimer epitope (EDE1-B10) 3 days after infection was able to reduce the viral load in testis, reducing the inflammation and preserving sperm count. The protection is not e ffective for the long duration [114]. Further studies in this area have explored the pathogenesis pathways and the host cellular response, suggesting potential targets to develop vaccines, including DNA-based vaccines. DNA-based vaccines, and live attenuated ZIKV have shown testicular protection against infection, avoiding atrophy, damage, and male infertility [74,115].

The combined strategies of DNA-based vaccines and live attenuated ZIKV vaccines demonstrated efficacy when used in a single-dose in A129 mice. This vaccination promotes the complete prevention of testicle infection, injury, and oligospermia [116]. Another live-attenuated ZIKV vaccine, which presents one deletion in the 3 untranslated region of the ZIKV genome (ZIKV-3UTR-LAV), presented protection after a single vaccination in mice and non-human primates. This protection was evaluated for preventing mother-child vertical transmission and the prevention of testicle damages [117].

DNA-based vaccination of recombinant chimpanzee adenovirus type 7 (AdC7) expressing ZIKV M/E glycoproteins presents high e fficacy in a single vaccination. AdC7-M/E induced a potent neutralizing antibody in immunocompetent and immunodeficient mice and full protection against ZIKV-induced testicular damage [118]. Another DNA-based vaccine, encoding ZIKV pre-membrane and envelope (prME) in pVAX vector, protected mice completely against ZIKV, promoting protection in testes and sperm and decreasing viral persistence in MRT [115]. Moreover, this vaccine was also effective in reversing mouse infertility [119].

A few drugs against ZIKV have also been proposed and may have an impact on testicles [106,120,121]. Recently, Z2 an amphipathic peptide derived from the stem region of ZIKV envelope protein was reported to inhibit vertical ZIKV transmission in a mouse model and reduce viral load in the testicle and epididymis. This was also reported to reduce pathological damage while improving sperm quality [122]. Simanjuntak and colleagues demonstrated that ZIKV-infected testicles presented progressive damage with a significant oxidative microenvironment, with high levels of reactive oxygen species, nitric oxide, glutathione peroxidase 4 and pro-inflammatory cytokines as IL-1β, IL-6, and G-CSF. They proposed the use of the antioxidant ebselen (EBS) to prevent the sexual transmission of the virus and to improve host testicular immune response [123].
