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Hepatitis E viral infection causes testicular damage in mice
Journal Pre-proof Hepatitis E viral infection causes testicular damage in mice Jianwen Situ, Wenjing Wang, Feiyan Long, Weimin Yang, Chenchen Yang, Daqiao Wei, Wenhai Yu, Fen Huang PII:
S0042-6822(19)30363-0
DOI:
https://doi.org/10.1016/j.virol.2019.12.009
Reference:
YVIRO 9241
To appear in:
Virology
Received Date: 11 September 2019 Revised Date:
26 December 2019
Accepted Date: 26 December 2019
Please cite this article as: Situ, J., Wang, W., Long, F., Yang, W., Yang, C., Wei, D., Yu, W., Huang, F., Hepatitis E viral infection causes testicular damage in mice, Virology (2020), doi: https://doi.org/10.1016/ j.virol.2019.12.009. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Inc.
Hepatitis E viral infection causes testicular damage in mice Running title: HEV replicates in the male genital tract Jianwen Situ1#, Wenjing Wang1#, Feiyan Long1, Weimin Yang1, Chenchen Yang1, Daqiao Wei1*, Wenhai Yu2*, Fen Huang1*
1. Medical School, Kunming University of Science and Technology, Kunming, China; 2. Institute of Medical Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Kunming, China. # These authors contribute equally in this study. *Correspondence:
Prof Fen Huang, E-mail: [email protected]. Medical School, Kunming
University of Science and Technology, 727 Jing Ming South Road, Kunming, China; Prof Wenhai Yu, Email: [email protected]. Institute of Medical Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, 935 Jiao ling Road, Kunming, China; Prof Daqiao Wei, E-mail: [email protected]. Medical School, Kunming University of Science and Technology, 727 Jing Ming South Road, Kunming, China;
Abstract Hepatitis E virus (HEV) is the main pathogen of hepatitis E infections with multiple extrahepatic replication sites. The presence of HEV RNA in the semen of infertile males suggests HEV replicates in the male genital tract. However, the mechanism is largely remained elusive. A BALB/c-based animal model was used to evaluate the effects of HEV infection on the testicular damage. HEV RNA was detected in feces, blood and livers from 7–28 days post-inoculation (dpi), while was positive in male genital tract from 7–70 dpi. Positive signals of HEV antigens were observed in testes, epididymides and seminal vesicles (SVs). Impaired sperm quality, destroyed the blood–testis barrier (BTB) and drastically decreased spermatogonia suggested that HEV infection causes testicular damage. Antiviral immune response was barely found in the testes. Results demonstrated that HEV replicates in male genital tract, causes testicular damage, and consequently results in flawed fertility. Keywords: HEV infection; male genital tract; sperm quality; testicular damage.
Introduction Hepatitis E virus (HEV) is the leading cause of enterically transmitted viral hepatitis infections worldwide. HEV infection is responsible for 20 million infections and 70,000 deaths each year[1]. Although HEV infections are self-limiting, severe HEV infections cause high mortality rates in pregnant women[2], chronic HEV infections in immunocompromised individuals, such as solid-organ transplant recipients[3] or Human Immunodeficiency Virus (HIV) infected patients, have been reported[4, 5]. HEV mainly replicates in the liver but may also replicate in multiple extrahepatic sites, including the brain[6, 7], kidney[8, 9], muscle[10] and testis[11]. Viruses, such as ZIKA virus (ZIKV)[12, 13], HIV[14], hepatitis B virus (HBV) [15, 16], and hepatitis C virus (HCV)[15, 16], can infect the testes and are considered as high-risk factors of male infertility. Viral infections in the testes may affect sperm quality or even lead to male infertility. ZIKV infection ultimately leads to male infertility by inducing testicular inflammation, epididymitis, testicular atrophy, and pathological lesion formation [12, 13]. HBV destroys the blood–testis barrier (BTB), infects germ cells, and affects sperm quality [16]. HEV infection has been detected in the semen of infertile male with oligospermia, asthenospermia, or necrozoospermia[11]. Furthermore, HEV RNA and antigens have been detected in the testes and epididymides of HEV-infected rhesus macaques[11] or Mongolian gerbils with damaged BTB [17]. The presence and persistence of HEV infection in semen raise several important concerns. First, the cells and tissues of the male genital tract are permissive for HEV replication, thereby the presence of HEV in semen must be clarified. Second, given the high prevalence of HEV in infertile men, how to exclude blood-borne transmission? Third, the duration of HEV infection in semen should be determined. Last but not least, whether the male genital tract can recover from the damages caused by HEV infection must be elucidated. In this study, we investigated the damages of the male reproductive tract caused by HEV infection. We analyzed the reproductive organs, including the testes, epididymides and seminal vesicles (SVs), of HEV-infected male BALB/c mice. HEV RNA and antigens were detected in the testes, epididymides and SVs until 90 dpi, 55 days after viral clearance from HEV-infected mice. HEV infection resulted in the degeneration of germ cells, and subsequently impaired sperm count and quality.
Materials and Methods Animal
SPF BALB/c mice (male, 6 weeks old, 18–22 g, n = 60; female, 6 weeks old, 18–22 g, n = 12) were purchased from Kunming Medical University and maintained in a pathogen-free animal facility. The animal protocols used in this study were approved by the Animal Care and Use Committee of Kunming University of Science and Technology. Mice were negative for anti-HEV antibodies (IgG and IgM) and HEV RNA.
Virus and inoculation Human HEV (Genotype 4, KU-LX strain, GenBank No. MF567574, 5.8×105 copies/mL) was isolated from the stool sample of a patient in Yunnan Province, China. The species of enterovirus A and B were excluded by PCR amplified with universal enteroviruses primers. Fecal suspensions (10%, w/v) were centrifuged at 12,000 ×g at 4 °C for 10 min, filtered through 0.22 µm micro filters, and treated with penicillin and streptomycin for 1 h. Male mice were intravenously inoculated with 200 µL of HEV (n = 30) or phosphate-buffered saline (PBS) (n = 30). Mice inoculated with HEV (n = 3) or PBS (n = 3) were humanely euthanized at 7, 14, 21, 28, 35, 42, 70, and 90 dpi. Three mice per treatment group were euthanized at each time point. At 90 dpi, the remaining mice inoculated with HEV (n = 6) or PBS (n = 6) were mated with uninfected female mice (n = 12). Fluids in the cauda epididymis were collected for HEV RNA detection. The testes were weighed and washed with normal saline for the exclusion of blood contamination. Meanwhile, testes, epididymides, and SVs were fixed in neutral 4% paraformaldehyde for histopathological and immunohistochemical analyses. Gene detection and quantification Total RNA was extracted from stool supernatant, blood, serum, semen, or tissue by using Trizol reagent (Invitrogen, USA) in accordance with the manufacturer’s instructions. Reverse transcription was performed by using a reverse transcriptase kit (AMV, Takara, Japan) with HEV-specific primers or random primer [18]. Negative control was included to exclude PCR contamination. HEV viral titer was quantified by using SYBR green-based quantitative RT-PCR (qRT-PCR) as previously described[19]. The relative expressions of genes, including RIG-1, IFN-β, IFN-λ, TNF-α, IL6 and IL10, were quantified with the specific primers as described in a previous study [20, 21]. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the housekeeping control gene. qRT-PCR was performed by using
an ABI PRISM 7300 Real-Time PCR System. The relative gene expression was determined using the formula 2−(△Ct of gene-△Ct of GAPDH), where Ct is the threshold cycle.
Histopathology, immunohistochemistry, and indirect immunofluorescence analysis Tissue samples were fixed in 10% neutral-buffered formalin and embedded in paraffin. The specimens were cut into 3–4 µm serial sections. Standard hematoxylin and eosin (H&E) staining was performed. Stained specimens were examined under microscopy. In immunohistochemical analyses (IHC) and immunofluorescence assay (IFA), tissues were deparaffinized, hydrated, and heated in a water bath for antigen retrieval and then blocked with 3% hydrogen peroxide for 15 min. Tissue sections were incubated for 2h at 37 °C with primary monoclonal a ntiHEV (MAB8003, Millipore, 1:200 dilution), anti-CD45 (610266, BD, 1:150 dilution), anti-F4/80 (ab6640, Abcam, 1:150 dilution), anti-ETV5 (ab102010, Abcam, 1:200 dilution), anti-TRA98 (ab2527, Abcam, 1:200 dilution), α-SAM(A7248, ABclonal,1:200 dilution), anti-Lin28a (37118, SAB, 1:200 dilution),and 3β-HSD (sc515120, Santa Cruz, 1:100 dilution) antibodies; washed with PBS; and then incubated with HRP-, FITCor TRITC-labeled secondary antibodies as previously described[11]. Terminal deoxynucleotidyl transferase dUTP nick-end labeling assay Apoptotic cells in the testicles of HEV-infected mice were detected by using One Step Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) Apoptosis Assay Kit (C1086, Beyotime). Tissue sections were deparaffinized, hydrated, blocked with 20 µg/mL proteinase K (DNase free) for 15–30 min, and washed with PBS. The sections were then incubated for 60 min at 37 °C with TUNEL liquid and washed with PBS. Nuclei were counterstained with DAPI and viewed under fluorescence microscopy (Nikon Ti-E, Japan). Hormone analysis The levels of testosterone and inhibin B in the serum were determined through enzyme-linked immunosorbent assay (ELISA) kits procured from Shanghai MLBIO Biotechnology Co., Ltd. Statistical analysis
All experiments were performed at least thrice. Data were presented as mean ± standard deviation. Statistical analysis was performed using GraphPad Prism 5 software. Wilcoxon matched-pair test was used to determine the significance of differences between the two groups. In this test, a 0.05 level of probability (P < 0.05) was considered statistically significant. Results
HEV replicates in the testes, epididymis, and SVs To trace the replication of HEV in male genital tract, the BALB/c mice model of HEV infection was established. HEV RNA was detectable in the feces, serum and livers of all mice inoculated with HEV at 7 dpi. The viral titers reached a peak at 7 dpi in feces and 14 dpi in serum and livers, then decreased below the limit of detection at 35 dpi (Fig. 1). Meanwhile, PBS-inoculated uninfected mice were negative for HEV RNA. HEV RNA was first detected in the testes of HEV-infected mice at 7 dpi, similar to ZIKV infection in testes[12]. The copy number of HEV RNA in testes was persistently increasing and reached a peak during 21-28 dpi, which is comparable with course of viral load in blood, then slowly decreased during 35-42 dpi and detectable until 90 dpi. However, HEV RNA in the feces, serum and livers was detected from 7 dpi to 28 dpi, and undetectable at 35 dpi. Interestingly, HEV infection persisted in the testis longer than in the blood. HEV RNA was detected at 14 dpi in epididymal fluid (Epid-fluid), 1 week later than that in the testes. Epid-fluid was persistently positive for HEV RNA until 90 dpi. Meanwhile, the epididymides, the storage site of sperm, were also positive for HEV RNA during 7–70 dpi. The highest viral titer in the epididymis was observed during 21–28 dpi. Similar to epididymides, SVs, another sperm storage site, were positive for HEV RNA from 7 dpi to 42 dpi. Meanwhile, PBS-inoculated uninfected mice were negative for HEV RNA. Viruses were completely cleared from the blood at 35 dpi. Nevertheless, HEV RNA was persistent detected in the semen and epididymides until 90 dpi (Fig. 1), 55 days after viral clearance from the blood, which provides evidence against the blood-borne transmission of HEV. Taken together, these findings suggested that HEV can persistently replicate in the male genital tract.
Time-course analysis of HEV replication in the male genital tract The presence of HEV antigens in the testes, epididymides, and SVs was tested by immunohistochemical analysis (IHC) to further confirm which organs of the male genital tract are
permissive for HEV replication. The livers of HEV-infected mice served as the positive control (Fig. 2a). Positive signals were located in the liver cytoplasm cells (black arrows). The liver of uninfected mice served as the negative control. The liver sections were inoculated with PBS instead of HEV-specific antibody and served as negative control. HEV antigens were observed in the testes and mainly localized in Leydig cells (LCs) and testicular peritubular–myoid cells (TPCs) (Fig. 2b, black arrows). Testes stained positive for HEV antigens at 7 dpi, which indicated that HEV begins to replicate in the testes at the early stage of infection. HEV replication in the testes peaked at 28 dpi, decreased at 70 dpi, and then disappeared at 90 dpi. The testes of uninfected mice that were inoculated with PBS were negative for HEV antigen. The duration of HEV infection in the testes was higher than that of HEV infection in the blood or feces. The intense HEV antigen and RNA signals detected in the testes of HEV-infected mice indicated that HEV replicates in the testes. Spermatozoa that form in the testes enter the caput epididymis, progress to the corpus epididymis, and finally reach the cauda epididymis where they are stored. Given that HEV replicates in the testes and can be detected in Epid-fluid, the role of the epididymis in HEV infection must be identified. Interestingly, at 7 dpi, HEV antigens were observed in the tall-ciliated cells and connective tissues of the epididymides of HEV-infected mice, but not in those of uninfected mice (Fig. 2c, black arrows). HEV antigens persisted at high levels in the epididymis from 7 dpi to 70 dpi. These results suggested that HEV can replicate in the epididymis. SVs mainly secrete fluid that ultimately becomes semen. Approximately 70%–84% of the seminal fluid in humans originates from SVs. From 7 dpi to 42 dpi, scattered HEV antigen signals were observed in the smooth muscle of SVs of HEV-infected mice but were absent from uninfected mice (Fig. 2d, black arrows). The presence of HEV antigens in the testes, epididymides, and SVs of HEV-infected mice indicated that HEV can replicate in the male genital tract even after viral clearance in blood. HEV replicates in TPCs and LCs IFA was performed to identify which cells in the testes are permissive for HEV infection. Intensely fluorescent HEV signals (red) colocalized with TPCs positive for α-smooth muscle actin (α-SMA, green) at 28, 35, and 42 dpi (Fig. 3a). The merged fluorescence signals clearly illustrated that HEV replicates in αSMA-positive TPCs (white arrows, orange or yellow). TPCs from the testes of uninfected mice did not exhibit orange or yellow merged signals. In addition, HEV antigens colocalized with 3β-HSD-positive LCs at
28, 35, and 42 dpi (Fig. 3b). The merged signals from 3β-HSD and HEV ORF2 strongly suggested that HEV targets and replicates in LCs (white arrows, orange or yellow). Meanwhile, no merged signal was observed in LCs from the testes of uninfected mice (Fig. 3c). Surprisingly, Lin28a-positive spermatogonia were negative for the HEV antigen, and no yellow or orange signal was observed in HEV-infected or uninfected mice.
HEV persists in testes with barely activated innate immune responses. To clarify why the longer duration of HEV in the testes than in livers or blood, antiviral immune responses were determined in the blood and testes. The relative gene expression of RIG-I, IFN-β, IFN-λ, TNF-α, IL-6 and IL-10 were determined by qRT-PCR. RIG-I medicated interferon response plays a critical role against HEV infection in hepatoma cells in vitro [20]. Indeed, the gene expression of RIG-I was activated once HEV infection, and was detected in the blood of HEV infected mice. Subsequently, IFN-β was
highly
expressed in the blood to clean the viral infection (Fig. 4a, b). However, the most primary antiviral factors, RIG-I and IFN-β, were barely detected in HEV-infected testes (Fig. 4a, b). The undetectable antiviral factors further confirmed the immune-privilege nature of testes. Interestingly, type III interferon (IFN-III), IFN-λ, was activated in testes once HEV infection occurred (Fig. 4c). It has been confirmed that HEV stimulates a sustained IFN- III response in persistently infected cells[26]. Consistently, HEV persistently replicates in the testes with an activated IFN-λ immune response. Furthermore, pro-inflammation cytokines, such as TNF-α, IL-6 and IL-10 were scarcely detected in the testes (Fig. 4d-f). The inactived innate immune may contribute to the persistent infection in the testes.
HEV infection impairs sperm quality ZIKV infection leads to testicular atrophy and eventually results in infertility[12, 13]. However, the exact damages that HEV infection inflicts on the testes remains unclear. Thus, the morphology and histopathology of the genital tracts of HEV-infected male mice were analyzed in this study. The sizes of the testes, epididymides, or SVs of uninfected and HEV-infected mice at 21 dpi were negligibly differed (Fig. 5a). Body weight and testes weight were also unaffected by HEV infection (Fig. 5b). Given that impairments in sperm quality have been found in HEV-infected infertile males[11], the motility and morphology of sperm cells from HEV-infected mice were evaluated through eosin staining within 30 min after separation. The percentage of dead sperm cells in HEV-infected mice at 21 dpi was significantly higher than that in
uninfected mice (74.63 ± 13.06%, n =460vs.19.84 ± 3.69%, n = 281, P = 0.0148 < 0.05). This result indicated that only approximately 25% of sperm cells from HEV-infected mice were alive (black arrows) (Fig. 5c). In clinical practice, men are considered fertile if their live sperm cell rates exceed 58%. Therefore, HEV-infected males suffered from necrozoospermia. In addition, 11.65 ± 2.46% (n = 717) of sperm cells from HEV-infected mice exhibited nonprogressive motility. The percentage of sperm cells with nonprogressive motility from HEV-infected mice was higher than that from uninfected mice (4.98 ± 1.30%, n = 809, P = 0.0285 < 0.05) (Fig. 5c, red arrows). This result indicated that HEV-infected males are at a high risk of asthenospermia. Moreover, 7.43 ± 1.78% of the sperm cells from HEV-infected mice showed head and body abnormalities (n = 717). The rate of sperm cells with head and body abnormalities of HEV-infected mice, however, was not significantly different from that of uninfected mice (5.5 ± 0.93%, n = 809, P=0.1146) (blue arrows) (Fig. 5c). The drastic reductions in sperm motility and vitality and increased sperm abnormalities in HEV-infected male mice strongly suggested that HEV infection impairs sperm quality. Thus, HEV infection mainly causes infertility among males by reducing sperm quality. HEV infection leads to testicular and epididymal damages
The pathological damages of HEV infection are usually caused by severe inflammation. Histopathological changes and inflammatory responses were analyzed to explore the mechanisms underlying the reductions in sperm quality caused by HEV infection. Testicular tissue sections from uninfected mice were histologically normal, exhibited sperm cells at different stages of spermatogenesis (TRA98-positive), and were surrounded by LCs (Fig.6a, b). Meanwhile, numerous sperm cells were observed in the caput and corpora epididymides of uninfected mice (Fig. 7a-c). SVs from uninfected mice were used as the normal tissue control and did not show any histopathological changes (Fig.7d). The gross images of the male genital tracts of HEV-infected mice revealed the absence of observable morphological changes. Nevertheless, the number of germ cells (TRA98+) in the testes drastically decreased from 7 dpi to 70 dpi (Fig. 6a, b). Meanwhile, the number of sperm cells in the caput epididymitis also decreased (Fig. 7b). The number of sperm cells in the corpora epididymitis of infected mice was not affected by HEV infection at 7 dpi (Fig. 7c), because the HEV-induced damage occurred in the early phase of spermatogenesis.
At 14 days of HEV infection, the number of germ cells (TRA98+) in the testes significantly decreased (black arrows) (Fig. 6a, b). Histological analysis revealed that the architecture of seminiferous tubules was damaged and exhibited basement membrane loss (red arrows) and germ cell degeneration (black arrows) (Fig. 7a). The number of spermatozoa in the lumen of the caput and corpora epididymitis decreased because of the drastic decrease in germ cells (Fig.7b, c). The number of degenerative germ cells in the seminiferous tubules of HEV-infected mice increased until 70 dpi (black arrows) (Fig. 7a). The number of TRA98+ germ cells persistently and dramatically decreased until 90 dpi (black arrows) (Fig. 6a, b). Interesting, the reduced number of germ cells is associated with the increased viral load in testes. Numerous necrotic LCs (yellow arrow) and intensive intratesticular venous congestion (green arrows) were observed the testes of HEV-infected mice (Fig. 7a). The testes were infiltrated by CD45+ leukocytes (white arrows) (Fig. 8a) and F4/80+ macrophages (white arrows) (Fig.8b) particularly at 21–42 dpi when the viral titer in the testes reached the peak. The number of sperm in the epididymal lumen dramatically decreased at 21–70 dpi (black star) (Fig. 7b, c). Meanwhile, ductal epithelial vacuoles (red arrow) and numerous degenerating germ cells (black arrow) were observed in the epididymal lumen (Fig. 7b, c). This result clearly imitated the oligozoospermia in male experiencing HEV infection. Fortunately, the number of germ cells partly recovered at 90 dpi (Fig. 6a, b). The inflammatory responses caused by HEV infection resolved, and CD45+ leukocyte and F4/80+ macrophages were no longer observed in the testes at 90 dpi (Fig. 8a, b). The number of spermatozoa in the epididymal lumen also increased despite the presence of vacuoles and necrotic germ cells in the epididymis (Fig. 7b, c). The SVs of HEV-infected mice were not damaged during the experiment (Fig. 7d).
HEV destroys the BTB and induces apoptosis The BTB protects the testes against pathogen invasion. Inflammation and severe damage eventually occur in the testes once the BTB is damaged[24]. In this study, the ETV5 transcription factor was served as an index of BTB integrity. ETV5 mediates BTB function and testicular immune privilege and is required for continuous spermatogenesis in adult mice [25]. The presence of ETV5+ cells in uninfected mice indicated that the BTB is intact (Fig. 8c). The BTB remained intact in HEV-infected mice at 28 dpi but was destroyed at 35 dpi as indicated by the absence of ETV5+ cells. The breakage of BTB caused by HEV infection may account for the increased infiltration of the seminiferous tubules of HEV-infected mice by CD45+ leukocytes
and F4/80+ macrophages at 35 dpi (Fig. 8a, b). Furthermore, apoptosis was observed at 28, 35, and 42 dpi in testicular cells, and disappeared at 90 dpi, when the integrity of BTB recovered (Fig. 8d). HEV infection leads to hormonal disorders Testicular LCs produce the vast majority of androgens in men. Meanwhile, androgens essential for the lifelong support of spermatogenesis and the development of LCs[27]. However, HEV infection damaged LCs and resulted in spermatogonia necrosis. The levels of androgenic hormones, testosterone and inhibin B, were measured to determine whether HEV infection disturbs hormone secretion. Similar to those in HEV-infected men, testosterone levels in HEV-infected mice decreased at 28 dpi (Fig.9a), which corresponds to the robust replication stage of HEV in the testes. This finding suggested that the infection of LCs by HEV reduced testosterone levels. Inhibin B is secreted by Sertoli cells. It is a more efficient marker of spermatogenesis, which is used as an index of male infertility, than FSH or LH[28, 29]. Inhibin B levels decreased after HEV infection and were declined at 28, 35 and 42 dpi (Fig.9b). The reduction in testosterone and inhibin B levels may be attributed to the inflammation and damage of the testes during HEV infection. The levels of testosterone and inhibin B increased when the number of germ cells recovered to normal levels at 90 dpi upon the clearance of HEV from the testes. HEV-induced infertility can be partly recovered HEV RNA was first detected in infertile men with oligospermia (azoospermia) or dead spermatozoa[11]. Whether the fertility can be fully or partly recovered after the complete clearance of HEV from testes at 90 dpi, when the BTB recovered and the levels of testosterone and inhibin B increased. Thus, the fertilities of HEV-infected male mice were assessed at 90 dpi. HEV-infected male mice (n = 6) were mated with uninfected female mice (n = 6) at 1:1 rate. Uninfected male mice (n = 6) were mated with uninfected female mice (n = 6) as the control. Pregnancy rates among female mice mated once with HEV-infected male mice reached 50%, whereas that among female mice mated with uninfected male mice reached 100% (Fig.9c). The number of viable fetuses fathered by HEV-infected males (14 fetuses) was significantly lower than that of fetuses fathered by uninfected males (50 fetuses) (P = 0.0028 < 0.05), which indicated a statistically significant association between HEV infection and litter size in mice. These results suggested that HEVinduced testicular damage can be partly recovered after the complete clearance of HEV. Discussion
HEV infection usually causes acute and self-limiting diseases in the general population and has an incubation period of 40 days[30]. HEV mainly replicates in the liver but may also replicate in multiple extrahepatic sites, including the brain, kidney, spleen, intestine, uterus, and even muscles[31]. The presence of genotype 4 HEV RNA in the semen of infertile males[11] or boar[32] suggests that HEV can infect the male genital tract and inflict testicular damages. Although no HEV RNA was detected in infertile men in Beijing or Europe (where genotype 3 HEV infection is sporadic)[33, 34], rhesus macaque, Mongolian gerbil and BALB/c mouse models of testicular HEV infection have successfully been established [11, 17]. The persistent infection of HEV in testes beyond the BTB contributes to the detection of HEV in the semen of infertile men. Besides, HEV infection has different epidemiological patterns in the world, even in the same country. The “Third National Viral Hepatitis Prevalence Survey” preformed in China (n=81,775) has demonstrated that “Seven provinces (Sichuan, Guizhou, Yunnan, Xinjiang, Shanghai, Hubei and Jiangsu) had the highest seroprevalence estimates in China”[35], while Beijing is the lowest cities of China, because of the superior economy and sanitary conditions. In most villages of Yunnan province, peoples are still living together with domestic animals, eating raw pork or dinking fresh animals’ blood. Thus, the high prevalence of HEV infection in this region makes it possible to find HEV infection in infertile men.
On the
contrary, it is hardly in those cities where the incidence of HEV is very rare. In addition, chronic HEV infection has been reported in immunocompetent patients (n=55) in this region in 2016 [36], and persistent infection has been established in rhesus macaque models by using a genotype 4 HEV isolated in this region [9]. The special features of HEV prevalent in this region may response for the higher incidence of HEV than other cities. Since the association of infertility and HEV infection remains unclear, more clinical investigations are urgently needed to clarify it in the future. To understand the pathogenesis of HEV in male genital tract, HEV infected animals were used for realtime investigation of HEV-induced testicular damage. In this study, HEV replicates in the male genital tract, and subsequent causes testicular damages from 7 dpi to 90 dpi. HEV RNA was undetectable in feces and serum at 35 dpi, but persistently detectable in the epididymides and epididymal fluid until 90 dpi. Antiviral immune responses were barely detected in the testes, while quickly activated in the serum, which may response to the longer duration of HEV in male genital tract. The immune-privileged nature of the testes may allow viruses, such as ZIKV, HBV and HIV, to persist even after viral clearance [12-16, 37]. HEVpositive signals were still observed in the testes and epididymides 55 days after HEV was cleared from the serum. The spermatogenic cycle of mice (35 days) may account for the longer incubation time of HEV in
the epididymides than that in the testes. Results well expressed the fact that longer duration of HEV in semen than in serum. The histopathological analysis of the genital tracts of HEV-infected male mice revealed that intense HEV-positive signals primarily localized in the testes and epididymides and weak HEV-positive signals mainly localized in SVs. Given that we did not collect prostate samples, we were unable to determine whether HEV can replicate in the prostate. Stem-cell-like TPCs and LCs, tall-ciliated cells, and connective epididymal tissues are particularly vulnerable to HEV infection. TPCs possess a mixed phenotype and express smooth muscle-like characteristics, contribute to the SG stem cell niche, and express pluripotency markers and putative stem LC markers, as well as markers for steroidogenic genes involved in the biosynthesis of sex steroids[38-40]. The replication of HEV in TPCs and testosterone-producing LCs may contribute to the disordered production of androgenic hormones, such as testosterone and inhibin B, in HEV-infected men or male mice. The BTB is the main defense of the male genital tract against pathogen infection. Once the BTB is broken, pathogens will invade the testes and cause severe inflammation and damage[24]. We monitored HEV-induced BTB damage in mice. Interestingly, the BTB was broken (as indicated by the disappearance of ETV5) at 35 dpi after intensive attack by HEV starting from 21 dpi. Severe inflammation (as indicated by the increase in CD45+ leukocyte and F4/80+ macrophage infiltration) was observed in the testes and resulted in germ cell necrosis or apoptosis. The BTB damage caused by infection of viruses, including HIV, HBV and ZIKV, results in testicular damages, reduces sperm quality, and even causes male infertility[15, 16, 41, 42]. Reduced sperm count, increased sperm abnormalities and necrospermia may be the main reasons for infertility among HEV-infected males. Fortunately, the BTB recovered at 90 dpi, 55 days after the clearance of HEV from the feces or blood. The fertility of HEV-infected mice partially recovered. Nevertheless, the sizes of litters fathered by HEV-infected mice were smaller than those of uninfected mice. HEV is mainly transmitted through the oral–fecal route but may also be transmitted through organ transplants, blood transfusions, and intimate contact [43, 44]. Urine from patients with chronic HEV infections is infectious and causes kidney injury [45, 46]. Milk secreted by HEV-infected cows is infectious and leads to acute infection in rhesus macaques [18]. The infectivity of blood, urine and milk from HEVinfected individuals strongly suggests that body fluids are important routes of HEV transmission. However,
whether semen can transmit HEV remains to be investigated through clinical or animal experimentation. Caution should also be practiced to prevent sexual contact transmission of HEV. In conclusion, HEV persistently replicates in the male genital tract and causes testicular damages.
Acknowledgements: We thank Dong Shen for the professional analysis of the histopathologic examination. Funding: This work was supported by National Natural Science Foundation of China (81660338 to F. Huang, and 81960370 to D. Wei), Natural Science Foundation of Yunnan Province (2017FA036 to F. Huang and 2018FB132 to W. Yu), Fundamental Research Funds for the Central Universities (2016ZX310179-3) and PUMC Youth Fund (2017310038) to W. Yu.
Conflict of interest: The authors declare that there is no conflict of interest.
References 1. 2. 3.
4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.
15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.
26. 27.
Debing, Y., et al., Update on hepatitis E virology: Implications for clinical practice. J Hepatol, 2016. 65(1): p. 200-212. Kamar, N., J. Izopet, and L. Rostaing, Hepatitis E virus infection. Curr Opin Gastroenterol, 2013. 29(3): p. 271-8. Wang, Y., et al., Chronic Hepatitis E in a Renal Transplant Recipient: The First Report of Genotype 4 Hepatitis E Virus Caused Chronic Infection in Organ Recipient. Gastroenterology, 2018. 154(4): p. 1199-1201. Zeng, H., et al., Seroprevalence of hepatitis E virus in HIV-infected patients in China. Aids, 2017. 31(14): p. 2019-2021. Ferreira, A.C., et al., Serological and molecular markers of hepatitis E virus infection in HIV-infected patients in Brazil. Arch Virol, 2018. 163(1): p. 43-49. Dalton, H.R., et al., Hepatitis E virus and neurological injury. Nat Rev Neurol, 2016. 12(2): p. 77-85. Scanvion, Q., et al., Neuralgic amyotrophy triggered by hepatitis E virus: a particular phenotype. J Neurol, 2017. 264(4): p. 770-780. Geng, Y., et al., Detection and assessment of infectivity of hepatitis E virus in urine. J Hepatol, 2016. 64(1): p. 37-43. Huang, F., et al., Rhesus macaques persistently infected with hepatitis E shed virus into urine. J Hepatol, 2016. 64(6): p. 1446-7. Mengel, A.M., et al., Hepatitis E-induced severe myositis. Muscle Nerve, 2016. 53(2): p. 317-20. Huang, F., et al., High prevalence of hepatitis E virus in semen of infertile male and causes testis damage. Gut, 2018. 67(6): p. 1199-1201. Govero, J., et al., Zika virus infection damages the testes in mice. Nature, 2016. 540(7633): p. 438-442. Ma, W., et al., Zika Virus Causes Testis Damage and Leads to Male Infertility in Mice. Cell, 2017. 168(3): p. 542. Garrido, N., et al., Semen characteristics in human immunodeficiency virus (HIV)- and hepatitis C (HCV)-seropositive males: predictors of the success of viral removal after sperm washing. Hum Reprod, 2005. 20(4): p. 1028-34. Vicari, E., et al., Sperm output in patients with primary infertility and hepatitis B or C virus; negative influence of HBV infection during concomitant varicocele. Minerva Med, 2006. 97(1): p. 65-77. Garolla, A., et al., Sperm viral infection and male infertility: focus on HBV, HCV, HIV, HPV, HSV, HCMV, and AAV. J Reprod Immunol, 2013. 100(1): p. 20-9. Soomro, M.H., et al., Molecular and structural changes related to hepatitis E virus antigen and its expression in testis inducing apoptosis in Mongolian gerbil model. 2017. 24(8): p. 696-707. Huang, F., et al., Excretion of infectious hepatitis E virus into milk in cows imposes high risks of zoonosis. Hepatology, 2016. 64(2): p. 350-9. Baylis, S.A., et al., World Health Organization International Standard to harmonize assays for detection of hepatitis E virus RNA. Emerg Infect Dis, 2013. 19(5): p. 729-35. Xu, L., et al., RIG-I is a key antiviral interferon-stimulated gene against hepatitis E virus regardless of interferon production. Hepatology, 2017. 65(6): p. 1823-1839. Devhare, P.B., et al., Analysis of antiviral response in human epithelial cells infected with hepatitis E virus. PLoS One, 2013. 8(5): p. e63793. F, H., et al., High prevalence of hepatitis E virus in semen of infertile male and causes testis damage. Gut, 2018. 67(6): p. 1199-1201. Li, W., et al., Seroprevalence and molecular detection of hepatitis E virus in Yunnan Province, China. Archives of Virology, 2011. 156(11): p. 1989-1995. Mruk, D.D. and C.Y. Cheng, The Mammalian Blood-Testis Barrier: Its Biology and Regulation. Endocr Rev, 2015. 36(5): p. 564-91. Morrow, C.M., et al., ETV5 is required for continuous spermatogenesis in adult mice and may mediate blood testes barrier function and testicular immune privilege. Ann N Y Acad Sci, 2007. 1120: p. 144-51. X, Y., et al., Hepatitis E virus persists in the presence of a type III interferon response. PLoS pathogens, 2017. 13(5): p. e1006417. Guan, X., et al., Effects of spermatogenic cycle on Stem Leydig cell proliferation and differentiation. Mol Cell Endocrinol, 2018.
28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38.
39.
40.
41. 42.
43.
44. 45. 46.
Anderson, R.A., et al., Inhibin B in seminal plasma: testicular origin and relationship to spermatogenesis. Hum Reprod, 1998. 13(4): p. 920-6. Mahmoud, A.M., F.H. Comhaire, and C.E. Depuydt, The clinical and biologic significance of serum inhibins in subfertile men. Reprod Toxicol, 1998. 12(6): p. 591-9. Purcell, R.H. and S.U. Emerson, Hepatitis E: an emerging awareness of an old disease. J Hepatol, 2008. 48(3): p. 494-503. Pischke, S., et al., Hepatitis E virus: Infection beyond the liver? J Hepatol, 2017. 66(5): p. 10821095. Li, H., et al., Prevalence of hepatitis E virus (HEV) infection in various pig farms from Shaanxi Province, China: First detection of HEV RNA in pig semen. Transbound Emerg Dis, 2018. Horvatits, T., et al., No link between male infertility and HEV genotype 3 infection. Gut, 2019. Wang, L., et al., Absence of hepatitis E virus RNA in semen samples of infertile male in China. Gut, 2019. Jia, Z., et al., Epidemiology of hepatitis E virus in China: results from the Third National Viral Hepatitis Prevalence Survey, 2005-2006. PLoS One, 2014. 9(10): p. e110837. Liu, R., et al., Epidemiological characteristics and clinical features of sporadic viral hepatitis E (HEV). Pak J Pharm Sci, 2016. 29(6 Spec): p. 2251-2254. The human testis is susceptible to HIV-1. Reprod Biomed Online, 2007. 14(3): p. 299. Flenkenthaler, F., et al., Secretome analysis of testicular peritubular cells: a window into the human testicular microenvironment and the spermatogonial stem cell niche in man. J Proteome Res, 2014. 13(3): p. 1259-69. Spinnler, K., et al., Glial cell line-derived neurotrophic factor is constitutively produced by human testicular peritubular cells and may contribute to the spermatogonial stem cell niche in man. Hum Reprod, 2010. 25(9): p. 2181-7. Albrecht, M., et al., Isolation and cultivation of human testicular peritubular cells: a new model for the investigation of fibrotic processes in the human testis and male infertility. J Clin Endocrinol Metab, 2006. 91(5): p. 1956-60. Smith, D.B., et al., Proposed reference sequences for hepatitis E virus subtypes. J Gen Virol, 2016. 97(3): p. 537-42. Aiman, J., P.F. Brenner, and P.C. MacDonald, Androgen and estrogen production in elderly men with gynecomastia and testicular atrophy after mumps orchitis. J Clin Endocrinol Metab, 1980. 50(2): p. 380-6. Xu, C., et al., An assessment of hepatitis E virus (HEV) in US blood donors and recipients: no
detectable HEV RNA in 1939 donors tested and no evidence for HEV transmission to 362 prospectively followed recipients. Transfusion, 2013. 53(10 Pt 2): p. 2505-11. Aggarwal, R., Clinical presentation of hepatitis E. Virus Res, 2011. 161(1): p. 15-22. Kamar, N., et al., Acute hepatitis and renal function impairment related to infection by hepatitis E virus in a renal allograft recipient. Am J Kidney Dis, 2005. 45(1): p. 193-6. Kamar, N., et al., Hepatitis E virus and the kidney in solid-organ transplant patients. Transplantation, 2012. 93(6): p. 617-23.
Figure legends Fig. 1. HEV genomic RNA copy number of livers, feces, serum, testes, epididymides, seminal vesicles and epididymal fluid from HEV-infected male mice
Fig. 2. HEV antigens detected in the genital tracts of male mice. Immunohistochemical (IHC) analyses of HEV ORF2 antigens in the genital tracts of male mice at indicated times. (a) (Uninfected panel) The liver of an uninfected mouse was used as the negative control. (HEV-infected panel) The liver of an HEV-infected mouse was incubated with PBS or HEV ORF2 antibody to identify the antibody specificity, and used as the positive control. Immunostaining of HEV ORF2 in the testis (b), Epididymis (c) and SV (d) of PBSinoculated uninfected mice and HEV-infected mice during 7–90 dpi.
Fig. 3. HEV replicates in TPCs and LCs in mice testes. IFA of HEV ORF2 in α-SMA-positive TPCs (a), 3β-HSD-positive LCs (b) and Lin28a-positive SGs (c) in the testes of uninfected or HEV-infected mice. Nuclei were stained with DAPI.
Fig. 4. HEV infection barely activate antiviral innate immune response in the testes. The gene expressions of RIG-I (a), IFN-β (b), IFN-λ (c), TNF-α (d), IL-6 (e) and IL-10 (f) in the testes and blood were determined by qRT-PCR.
Fig. 5. HEV infection impairs sperm quality. (a) Morphologies of the male genital tracts of uninfected or HEV-infected mice at 21 dpi. (b) Ratio of testes weight/body weight. (c) Mature sperm cells were collected from the cauda epididymis of uninfected or HEV-infected mice and stained with eosin. Black arrows indicate dead sperm cells. Red arrows indicate sperm cells with asthenospermia. Blue arrows indicate abnormal sperm cells.
Fig. 6. Reduced germ cells in the testes of HEV-infected mice. (a) Immunochemical analysis of germ cells in the testes collected from uninfected or HEV-infected mice. (b) The number of germ cells in HEVinfected mice were compared with uninfected mice. *P < 0.05; ** P < 0.01; ns, not significant.
Fig. 7. Testicular and epididymal damages caused by HEV infection. Histopathological analysis of (a) testis, (b) caput epididymis, (c) corpus epididymis and (d) SV collected from uninfected or HEV-infected mice as revealed stained by H&E.
Fig. 8. HEV infection induces inflammatory responses and apoptosis. Immunofluorescence analysis of CD45 (a), F4/80 (b), ETV5 (c) and apoptosis (d) in the testis of uninfected or HEV-infected mice. Nuclei were stained with DAPI.
Fig. 9. HEV infection leads to hormonal disorders and temporarily testicular damage. Androgen hormones decreased during HEV infection. Testosterone (a) and inhibin B (b) levels in the serum of uninfected or HEV-infected mice. (c) Number of fetuses fathered by male mice with or without HEV infection. **P < 0.01.
1. Hepatitis E virus (HEV) is a major cause of acute hepatitis worldwide. 2. HEV has been detected in the semen of male, but the pathogenesis is unknown. 3. We found HEV replicates in male genital tract with longer duration until 90 dpi. 4. HEV infection impairs sperm quality with reductions in sperm motility and vitality. 5. HEV destroys the BTBs and results in germ cell necrosis and apoptosis.
Hepatitis E viral infection causes testicular damage in mice Running title: HEV replicates in the male genital tract Jianwen Situ1#, Wenjing Wang1#, Feiyan Long1, Weimin Yang1, Chenchen Yang1, Daqiao Wei1*, Wenhai Yu2*, Fen Huang1*
1. Medical School, Kunming University of Science and Technology, Kunming, China; 2. Institute of Medical Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Kunming, China; # These authors contribute equally in this study.
*Correspondence: Prof Huang Fen, E-mail: [email protected]. Medical School, Kunming University of Science and Technology 727 Jing Ming South Road , Kunming, China；Prof Wenhai Yu, E-mail: [email protected]. Institute of Medical Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, 935 Jiao ling Road, Kunming, China; Prof Daqiao Wei, E-mail: [email protected]. Medical School, Kunming University of Science and Technology, 727 Jing Ming South Road, Kunming, China;
Funding: This work was supported by National Natural Science Foundation of China (31360619 and 81660338 to F. Huang), Natural Science Foundation of Yunnan Province (2017FA036 to F. Huang and 2018FB132 to W. Yu), Fundamental Research
Funds for the Central Universities (2016ZX310179-3) and PUMC Youth Fund (2017310038) to W. Yu.
Conflict of interest: The authors declare that there is no conflict of interest to this study.
Author contributions: J. Situ, W. Wang, W. Yang, F. Long and C. Yang performed the experiments, J. Situ and W. Yu analyzed the data and drafted the manuscript, and F. Huang conceived the ideas, designed and discussed experiments, supervised the project and extensively edited the manuscript.
S0042-6822(19)30363-0
DOI:
https://doi.org/10.1016/j.virol.2019.12.009
Reference:
YVIRO 9241
To appear in:
Virology
Received Date: 11 September 2019 Revised Date:
26 December 2019
Accepted Date: 26 December 2019
Please cite this article as: Situ, J., Wang, W., Long, F., Yang, W., Yang, C., Wei, D., Yu, W., Huang, F., Hepatitis E viral infection causes testicular damage in mice, Virology (2020), doi: https://doi.org/10.1016/ j.virol.2019.12.009. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Inc.
Hepatitis E viral infection causes testicular damage in mice Running title: HEV replicates in the male genital tract Jianwen Situ1#, Wenjing Wang1#, Feiyan Long1, Weimin Yang1, Chenchen Yang1, Daqiao Wei1*, Wenhai Yu2*, Fen Huang1*
1. Medical School, Kunming University of Science and Technology, Kunming, China; 2. Institute of Medical Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Kunming, China. # These authors contribute equally in this study. *Correspondence:
Prof Fen Huang, E-mail: [email protected]. Medical School, Kunming
University of Science and Technology, 727 Jing Ming South Road, Kunming, China; Prof Wenhai Yu, Email: [email protected]. Institute of Medical Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, 935 Jiao ling Road, Kunming, China; Prof Daqiao Wei, E-mail: [email protected]. Medical School, Kunming University of Science and Technology, 727 Jing Ming South Road, Kunming, China;
Abstract Hepatitis E virus (HEV) is the main pathogen of hepatitis E infections with multiple extrahepatic replication sites. The presence of HEV RNA in the semen of infertile males suggests HEV replicates in the male genital tract. However, the mechanism is largely remained elusive. A BALB/c-based animal model was used to evaluate the effects of HEV infection on the testicular damage. HEV RNA was detected in feces, blood and livers from 7–28 days post-inoculation (dpi), while was positive in male genital tract from 7–70 dpi. Positive signals of HEV antigens were observed in testes, epididymides and seminal vesicles (SVs). Impaired sperm quality, destroyed the blood–testis barrier (BTB) and drastically decreased spermatogonia suggested that HEV infection causes testicular damage. Antiviral immune response was barely found in the testes. Results demonstrated that HEV replicates in male genital tract, causes testicular damage, and consequently results in flawed fertility. Keywords: HEV infection; male genital tract; sperm quality; testicular damage.
Introduction Hepatitis E virus (HEV) is the leading cause of enterically transmitted viral hepatitis infections worldwide. HEV infection is responsible for 20 million infections and 70,000 deaths each year[1]. Although HEV infections are self-limiting, severe HEV infections cause high mortality rates in pregnant women[2], chronic HEV infections in immunocompromised individuals, such as solid-organ transplant recipients[3] or Human Immunodeficiency Virus (HIV) infected patients, have been reported[4, 5]. HEV mainly replicates in the liver but may also replicate in multiple extrahepatic sites, including the brain[6, 7], kidney[8, 9], muscle[10] and testis[11]. Viruses, such as ZIKA virus (ZIKV)[12, 13], HIV[14], hepatitis B virus (HBV) [15, 16], and hepatitis C virus (HCV)[15, 16], can infect the testes and are considered as high-risk factors of male infertility. Viral infections in the testes may affect sperm quality or even lead to male infertility. ZIKV infection ultimately leads to male infertility by inducing testicular inflammation, epididymitis, testicular atrophy, and pathological lesion formation [12, 13]. HBV destroys the blood–testis barrier (BTB), infects germ cells, and affects sperm quality [16]. HEV infection has been detected in the semen of infertile male with oligospermia, asthenospermia, or necrozoospermia[11]. Furthermore, HEV RNA and antigens have been detected in the testes and epididymides of HEV-infected rhesus macaques[11] or Mongolian gerbils with damaged BTB [17]. The presence and persistence of HEV infection in semen raise several important concerns. First, the cells and tissues of the male genital tract are permissive for HEV replication, thereby the presence of HEV in semen must be clarified. Second, given the high prevalence of HEV in infertile men, how to exclude blood-borne transmission? Third, the duration of HEV infection in semen should be determined. Last but not least, whether the male genital tract can recover from the damages caused by HEV infection must be elucidated. In this study, we investigated the damages of the male reproductive tract caused by HEV infection. We analyzed the reproductive organs, including the testes, epididymides and seminal vesicles (SVs), of HEV-infected male BALB/c mice. HEV RNA and antigens were detected in the testes, epididymides and SVs until 90 dpi, 55 days after viral clearance from HEV-infected mice. HEV infection resulted in the degeneration of germ cells, and subsequently impaired sperm count and quality.
Materials and Methods Animal
SPF BALB/c mice (male, 6 weeks old, 18–22 g, n = 60; female, 6 weeks old, 18–22 g, n = 12) were purchased from Kunming Medical University and maintained in a pathogen-free animal facility. The animal protocols used in this study were approved by the Animal Care and Use Committee of Kunming University of Science and Technology. Mice were negative for anti-HEV antibodies (IgG and IgM) and HEV RNA.
Virus and inoculation Human HEV (Genotype 4, KU-LX strain, GenBank No. MF567574, 5.8×105 copies/mL) was isolated from the stool sample of a patient in Yunnan Province, China. The species of enterovirus A and B were excluded by PCR amplified with universal enteroviruses primers. Fecal suspensions (10%, w/v) were centrifuged at 12,000 ×g at 4 °C for 10 min, filtered through 0.22 µm micro filters, and treated with penicillin and streptomycin for 1 h. Male mice were intravenously inoculated with 200 µL of HEV (n = 30) or phosphate-buffered saline (PBS) (n = 30). Mice inoculated with HEV (n = 3) or PBS (n = 3) were humanely euthanized at 7, 14, 21, 28, 35, 42, 70, and 90 dpi. Three mice per treatment group were euthanized at each time point. At 90 dpi, the remaining mice inoculated with HEV (n = 6) or PBS (n = 6) were mated with uninfected female mice (n = 12). Fluids in the cauda epididymis were collected for HEV RNA detection. The testes were weighed and washed with normal saline for the exclusion of blood contamination. Meanwhile, testes, epididymides, and SVs were fixed in neutral 4% paraformaldehyde for histopathological and immunohistochemical analyses. Gene detection and quantification Total RNA was extracted from stool supernatant, blood, serum, semen, or tissue by using Trizol reagent (Invitrogen, USA) in accordance with the manufacturer’s instructions. Reverse transcription was performed by using a reverse transcriptase kit (AMV, Takara, Japan) with HEV-specific primers or random primer [18]. Negative control was included to exclude PCR contamination. HEV viral titer was quantified by using SYBR green-based quantitative RT-PCR (qRT-PCR) as previously described[19]. The relative expressions of genes, including RIG-1, IFN-β, IFN-λ, TNF-α, IL6 and IL10, were quantified with the specific primers as described in a previous study [20, 21]. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the housekeeping control gene. qRT-PCR was performed by using
an ABI PRISM 7300 Real-Time PCR System. The relative gene expression was determined using the formula 2−(△Ct of gene-△Ct of GAPDH), where Ct is the threshold cycle.
Histopathology, immunohistochemistry, and indirect immunofluorescence analysis Tissue samples were fixed in 10% neutral-buffered formalin and embedded in paraffin. The specimens were cut into 3–4 µm serial sections. Standard hematoxylin and eosin (H&E) staining was performed. Stained specimens were examined under microscopy. In immunohistochemical analyses (IHC) and immunofluorescence assay (IFA), tissues were deparaffinized, hydrated, and heated in a water bath for antigen retrieval and then blocked with 3% hydrogen peroxide for 15 min. Tissue sections were incubated for 2h at 37 °C with primary monoclonal a ntiHEV (MAB8003, Millipore, 1:200 dilution), anti-CD45 (610266, BD, 1:150 dilution), anti-F4/80 (ab6640, Abcam, 1:150 dilution), anti-ETV5 (ab102010, Abcam, 1:200 dilution), anti-TRA98 (ab2527, Abcam, 1:200 dilution), α-SAM(A7248, ABclonal,1:200 dilution), anti-Lin28a (37118, SAB, 1:200 dilution),and 3β-HSD (sc515120, Santa Cruz, 1:100 dilution) antibodies; washed with PBS; and then incubated with HRP-, FITCor TRITC-labeled secondary antibodies as previously described[11]. Terminal deoxynucleotidyl transferase dUTP nick-end labeling assay Apoptotic cells in the testicles of HEV-infected mice were detected by using One Step Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) Apoptosis Assay Kit (C1086, Beyotime). Tissue sections were deparaffinized, hydrated, blocked with 20 µg/mL proteinase K (DNase free) for 15–30 min, and washed with PBS. The sections were then incubated for 60 min at 37 °C with TUNEL liquid and washed with PBS. Nuclei were counterstained with DAPI and viewed under fluorescence microscopy (Nikon Ti-E, Japan). Hormone analysis The levels of testosterone and inhibin B in the serum were determined through enzyme-linked immunosorbent assay (ELISA) kits procured from Shanghai MLBIO Biotechnology Co., Ltd. Statistical analysis
All experiments were performed at least thrice. Data were presented as mean ± standard deviation. Statistical analysis was performed using GraphPad Prism 5 software. Wilcoxon matched-pair test was used to determine the significance of differences between the two groups. In this test, a 0.05 level of probability (P < 0.05) was considered statistically significant. Results
HEV replicates in the testes, epididymis, and SVs To trace the replication of HEV in male genital tract, the BALB/c mice model of HEV infection was established. HEV RNA was detectable in the feces, serum and livers of all mice inoculated with HEV at 7 dpi. The viral titers reached a peak at 7 dpi in feces and 14 dpi in serum and livers, then decreased below the limit of detection at 35 dpi (Fig. 1). Meanwhile, PBS-inoculated uninfected mice were negative for HEV RNA. HEV RNA was first detected in the testes of HEV-infected mice at 7 dpi, similar to ZIKV infection in testes[12]. The copy number of HEV RNA in testes was persistently increasing and reached a peak during 21-28 dpi, which is comparable with course of viral load in blood, then slowly decreased during 35-42 dpi and detectable until 90 dpi. However, HEV RNA in the feces, serum and livers was detected from 7 dpi to 28 dpi, and undetectable at 35 dpi. Interestingly, HEV infection persisted in the testis longer than in the blood. HEV RNA was detected at 14 dpi in epididymal fluid (Epid-fluid), 1 week later than that in the testes. Epid-fluid was persistently positive for HEV RNA until 90 dpi. Meanwhile, the epididymides, the storage site of sperm, were also positive for HEV RNA during 7–70 dpi. The highest viral titer in the epididymis was observed during 21–28 dpi. Similar to epididymides, SVs, another sperm storage site, were positive for HEV RNA from 7 dpi to 42 dpi. Meanwhile, PBS-inoculated uninfected mice were negative for HEV RNA. Viruses were completely cleared from the blood at 35 dpi. Nevertheless, HEV RNA was persistent detected in the semen and epididymides until 90 dpi (Fig. 1), 55 days after viral clearance from the blood, which provides evidence against the blood-borne transmission of HEV. Taken together, these findings suggested that HEV can persistently replicate in the male genital tract.
Time-course analysis of HEV replication in the male genital tract The presence of HEV antigens in the testes, epididymides, and SVs was tested by immunohistochemical analysis (IHC) to further confirm which organs of the male genital tract are
permissive for HEV replication. The livers of HEV-infected mice served as the positive control (Fig. 2a). Positive signals were located in the liver cytoplasm cells (black arrows). The liver of uninfected mice served as the negative control. The liver sections were inoculated with PBS instead of HEV-specific antibody and served as negative control. HEV antigens were observed in the testes and mainly localized in Leydig cells (LCs) and testicular peritubular–myoid cells (TPCs) (Fig. 2b, black arrows). Testes stained positive for HEV antigens at 7 dpi, which indicated that HEV begins to replicate in the testes at the early stage of infection. HEV replication in the testes peaked at 28 dpi, decreased at 70 dpi, and then disappeared at 90 dpi. The testes of uninfected mice that were inoculated with PBS were negative for HEV antigen. The duration of HEV infection in the testes was higher than that of HEV infection in the blood or feces. The intense HEV antigen and RNA signals detected in the testes of HEV-infected mice indicated that HEV replicates in the testes. Spermatozoa that form in the testes enter the caput epididymis, progress to the corpus epididymis, and finally reach the cauda epididymis where they are stored. Given that HEV replicates in the testes and can be detected in Epid-fluid, the role of the epididymis in HEV infection must be identified. Interestingly, at 7 dpi, HEV antigens were observed in the tall-ciliated cells and connective tissues of the epididymides of HEV-infected mice, but not in those of uninfected mice (Fig. 2c, black arrows). HEV antigens persisted at high levels in the epididymis from 7 dpi to 70 dpi. These results suggested that HEV can replicate in the epididymis. SVs mainly secrete fluid that ultimately becomes semen. Approximately 70%–84% of the seminal fluid in humans originates from SVs. From 7 dpi to 42 dpi, scattered HEV antigen signals were observed in the smooth muscle of SVs of HEV-infected mice but were absent from uninfected mice (Fig. 2d, black arrows). The presence of HEV antigens in the testes, epididymides, and SVs of HEV-infected mice indicated that HEV can replicate in the male genital tract even after viral clearance in blood. HEV replicates in TPCs and LCs IFA was performed to identify which cells in the testes are permissive for HEV infection. Intensely fluorescent HEV signals (red) colocalized with TPCs positive for α-smooth muscle actin (α-SMA, green) at 28, 35, and 42 dpi (Fig. 3a). The merged fluorescence signals clearly illustrated that HEV replicates in αSMA-positive TPCs (white arrows, orange or yellow). TPCs from the testes of uninfected mice did not exhibit orange or yellow merged signals. In addition, HEV antigens colocalized with 3β-HSD-positive LCs at
28, 35, and 42 dpi (Fig. 3b). The merged signals from 3β-HSD and HEV ORF2 strongly suggested that HEV targets and replicates in LCs (white arrows, orange or yellow). Meanwhile, no merged signal was observed in LCs from the testes of uninfected mice (Fig. 3c). Surprisingly, Lin28a-positive spermatogonia were negative for the HEV antigen, and no yellow or orange signal was observed in HEV-infected or uninfected mice.
HEV persists in testes with barely activated innate immune responses. To clarify why the longer duration of HEV in the testes than in livers or blood, antiviral immune responses were determined in the blood and testes. The relative gene expression of RIG-I, IFN-β, IFN-λ, TNF-α, IL-6 and IL-10 were determined by qRT-PCR. RIG-I medicated interferon response plays a critical role against HEV infection in hepatoma cells in vitro [20]. Indeed, the gene expression of RIG-I was activated once HEV infection, and was detected in the blood of HEV infected mice. Subsequently, IFN-β was
highly
expressed in the blood to clean the viral infection (Fig. 4a, b). However, the most primary antiviral factors, RIG-I and IFN-β, were barely detected in HEV-infected testes (Fig. 4a, b). The undetectable antiviral factors further confirmed the immune-privilege nature of testes. Interestingly, type III interferon (IFN-III), IFN-λ, was activated in testes once HEV infection occurred (Fig. 4c). It has been confirmed that HEV stimulates a sustained IFN- III response in persistently infected cells[26]. Consistently, HEV persistently replicates in the testes with an activated IFN-λ immune response. Furthermore, pro-inflammation cytokines, such as TNF-α, IL-6 and IL-10 were scarcely detected in the testes (Fig. 4d-f). The inactived innate immune may contribute to the persistent infection in the testes.
HEV infection impairs sperm quality ZIKV infection leads to testicular atrophy and eventually results in infertility[12, 13]. However, the exact damages that HEV infection inflicts on the testes remains unclear. Thus, the morphology and histopathology of the genital tracts of HEV-infected male mice were analyzed in this study. The sizes of the testes, epididymides, or SVs of uninfected and HEV-infected mice at 21 dpi were negligibly differed (Fig. 5a). Body weight and testes weight were also unaffected by HEV infection (Fig. 5b). Given that impairments in sperm quality have been found in HEV-infected infertile males[11], the motility and morphology of sperm cells from HEV-infected mice were evaluated through eosin staining within 30 min after separation. The percentage of dead sperm cells in HEV-infected mice at 21 dpi was significantly higher than that in
uninfected mice (74.63 ± 13.06%, n =460vs.19.84 ± 3.69%, n = 281, P = 0.0148 < 0.05). This result indicated that only approximately 25% of sperm cells from HEV-infected mice were alive (black arrows) (Fig. 5c). In clinical practice, men are considered fertile if their live sperm cell rates exceed 58%. Therefore, HEV-infected males suffered from necrozoospermia. In addition, 11.65 ± 2.46% (n = 717) of sperm cells from HEV-infected mice exhibited nonprogressive motility. The percentage of sperm cells with nonprogressive motility from HEV-infected mice was higher than that from uninfected mice (4.98 ± 1.30%, n = 809, P = 0.0285 < 0.05) (Fig. 5c, red arrows). This result indicated that HEV-infected males are at a high risk of asthenospermia. Moreover, 7.43 ± 1.78% of the sperm cells from HEV-infected mice showed head and body abnormalities (n = 717). The rate of sperm cells with head and body abnormalities of HEV-infected mice, however, was not significantly different from that of uninfected mice (5.5 ± 0.93%, n = 809, P=0.1146) (blue arrows) (Fig. 5c). The drastic reductions in sperm motility and vitality and increased sperm abnormalities in HEV-infected male mice strongly suggested that HEV infection impairs sperm quality. Thus, HEV infection mainly causes infertility among males by reducing sperm quality. HEV infection leads to testicular and epididymal damages
The pathological damages of HEV infection are usually caused by severe inflammation. Histopathological changes and inflammatory responses were analyzed to explore the mechanisms underlying the reductions in sperm quality caused by HEV infection. Testicular tissue sections from uninfected mice were histologically normal, exhibited sperm cells at different stages of spermatogenesis (TRA98-positive), and were surrounded by LCs (Fig.6a, b). Meanwhile, numerous sperm cells were observed in the caput and corpora epididymides of uninfected mice (Fig. 7a-c). SVs from uninfected mice were used as the normal tissue control and did not show any histopathological changes (Fig.7d). The gross images of the male genital tracts of HEV-infected mice revealed the absence of observable morphological changes. Nevertheless, the number of germ cells (TRA98+) in the testes drastically decreased from 7 dpi to 70 dpi (Fig. 6a, b). Meanwhile, the number of sperm cells in the caput epididymitis also decreased (Fig. 7b). The number of sperm cells in the corpora epididymitis of infected mice was not affected by HEV infection at 7 dpi (Fig. 7c), because the HEV-induced damage occurred in the early phase of spermatogenesis.
At 14 days of HEV infection, the number of germ cells (TRA98+) in the testes significantly decreased (black arrows) (Fig. 6a, b). Histological analysis revealed that the architecture of seminiferous tubules was damaged and exhibited basement membrane loss (red arrows) and germ cell degeneration (black arrows) (Fig. 7a). The number of spermatozoa in the lumen of the caput and corpora epididymitis decreased because of the drastic decrease in germ cells (Fig.7b, c). The number of degenerative germ cells in the seminiferous tubules of HEV-infected mice increased until 70 dpi (black arrows) (Fig. 7a). The number of TRA98+ germ cells persistently and dramatically decreased until 90 dpi (black arrows) (Fig. 6a, b). Interesting, the reduced number of germ cells is associated with the increased viral load in testes. Numerous necrotic LCs (yellow arrow) and intensive intratesticular venous congestion (green arrows) were observed the testes of HEV-infected mice (Fig. 7a). The testes were infiltrated by CD45+ leukocytes (white arrows) (Fig. 8a) and F4/80+ macrophages (white arrows) (Fig.8b) particularly at 21–42 dpi when the viral titer in the testes reached the peak. The number of sperm in the epididymal lumen dramatically decreased at 21–70 dpi (black star) (Fig. 7b, c). Meanwhile, ductal epithelial vacuoles (red arrow) and numerous degenerating germ cells (black arrow) were observed in the epididymal lumen (Fig. 7b, c). This result clearly imitated the oligozoospermia in male experiencing HEV infection. Fortunately, the number of germ cells partly recovered at 90 dpi (Fig. 6a, b). The inflammatory responses caused by HEV infection resolved, and CD45+ leukocyte and F4/80+ macrophages were no longer observed in the testes at 90 dpi (Fig. 8a, b). The number of spermatozoa in the epididymal lumen also increased despite the presence of vacuoles and necrotic germ cells in the epididymis (Fig. 7b, c). The SVs of HEV-infected mice were not damaged during the experiment (Fig. 7d).
HEV destroys the BTB and induces apoptosis The BTB protects the testes against pathogen invasion. Inflammation and severe damage eventually occur in the testes once the BTB is damaged[24]. In this study, the ETV5 transcription factor was served as an index of BTB integrity. ETV5 mediates BTB function and testicular immune privilege and is required for continuous spermatogenesis in adult mice [25]. The presence of ETV5+ cells in uninfected mice indicated that the BTB is intact (Fig. 8c). The BTB remained intact in HEV-infected mice at 28 dpi but was destroyed at 35 dpi as indicated by the absence of ETV5+ cells. The breakage of BTB caused by HEV infection may account for the increased infiltration of the seminiferous tubules of HEV-infected mice by CD45+ leukocytes
and F4/80+ macrophages at 35 dpi (Fig. 8a, b). Furthermore, apoptosis was observed at 28, 35, and 42 dpi in testicular cells, and disappeared at 90 dpi, when the integrity of BTB recovered (Fig. 8d). HEV infection leads to hormonal disorders Testicular LCs produce the vast majority of androgens in men. Meanwhile, androgens essential for the lifelong support of spermatogenesis and the development of LCs[27]. However, HEV infection damaged LCs and resulted in spermatogonia necrosis. The levels of androgenic hormones, testosterone and inhibin B, were measured to determine whether HEV infection disturbs hormone secretion. Similar to those in HEV-infected men, testosterone levels in HEV-infected mice decreased at 28 dpi (Fig.9a), which corresponds to the robust replication stage of HEV in the testes. This finding suggested that the infection of LCs by HEV reduced testosterone levels. Inhibin B is secreted by Sertoli cells. It is a more efficient marker of spermatogenesis, which is used as an index of male infertility, than FSH or LH[28, 29]. Inhibin B levels decreased after HEV infection and were declined at 28, 35 and 42 dpi (Fig.9b). The reduction in testosterone and inhibin B levels may be attributed to the inflammation and damage of the testes during HEV infection. The levels of testosterone and inhibin B increased when the number of germ cells recovered to normal levels at 90 dpi upon the clearance of HEV from the testes. HEV-induced infertility can be partly recovered HEV RNA was first detected in infertile men with oligospermia (azoospermia) or dead spermatozoa[11]. Whether the fertility can be fully or partly recovered after the complete clearance of HEV from testes at 90 dpi, when the BTB recovered and the levels of testosterone and inhibin B increased. Thus, the fertilities of HEV-infected male mice were assessed at 90 dpi. HEV-infected male mice (n = 6) were mated with uninfected female mice (n = 6) at 1:1 rate. Uninfected male mice (n = 6) were mated with uninfected female mice (n = 6) as the control. Pregnancy rates among female mice mated once with HEV-infected male mice reached 50%, whereas that among female mice mated with uninfected male mice reached 100% (Fig.9c). The number of viable fetuses fathered by HEV-infected males (14 fetuses) was significantly lower than that of fetuses fathered by uninfected males (50 fetuses) (P = 0.0028 < 0.05), which indicated a statistically significant association between HEV infection and litter size in mice. These results suggested that HEVinduced testicular damage can be partly recovered after the complete clearance of HEV. Discussion
HEV infection usually causes acute and self-limiting diseases in the general population and has an incubation period of 40 days[30]. HEV mainly replicates in the liver but may also replicate in multiple extrahepatic sites, including the brain, kidney, spleen, intestine, uterus, and even muscles[31]. The presence of genotype 4 HEV RNA in the semen of infertile males[11] or boar[32] suggests that HEV can infect the male genital tract and inflict testicular damages. Although no HEV RNA was detected in infertile men in Beijing or Europe (where genotype 3 HEV infection is sporadic)[33, 34], rhesus macaque, Mongolian gerbil and BALB/c mouse models of testicular HEV infection have successfully been established [11, 17]. The persistent infection of HEV in testes beyond the BTB contributes to the detection of HEV in the semen of infertile men. Besides, HEV infection has different epidemiological patterns in the world, even in the same country. The “Third National Viral Hepatitis Prevalence Survey” preformed in China (n=81,775) has demonstrated that “Seven provinces (Sichuan, Guizhou, Yunnan, Xinjiang, Shanghai, Hubei and Jiangsu) had the highest seroprevalence estimates in China”[35], while Beijing is the lowest cities of China, because of the superior economy and sanitary conditions. In most villages of Yunnan province, peoples are still living together with domestic animals, eating raw pork or dinking fresh animals’ blood. Thus, the high prevalence of HEV infection in this region makes it possible to find HEV infection in infertile men.
On the
contrary, it is hardly in those cities where the incidence of HEV is very rare. In addition, chronic HEV infection has been reported in immunocompetent patients (n=55) in this region in 2016 [36], and persistent infection has been established in rhesus macaque models by using a genotype 4 HEV isolated in this region [9]. The special features of HEV prevalent in this region may response for the higher incidence of HEV than other cities. Since the association of infertility and HEV infection remains unclear, more clinical investigations are urgently needed to clarify it in the future. To understand the pathogenesis of HEV in male genital tract, HEV infected animals were used for realtime investigation of HEV-induced testicular damage. In this study, HEV replicates in the male genital tract, and subsequent causes testicular damages from 7 dpi to 90 dpi. HEV RNA was undetectable in feces and serum at 35 dpi, but persistently detectable in the epididymides and epididymal fluid until 90 dpi. Antiviral immune responses were barely detected in the testes, while quickly activated in the serum, which may response to the longer duration of HEV in male genital tract. The immune-privileged nature of the testes may allow viruses, such as ZIKV, HBV and HIV, to persist even after viral clearance [12-16, 37]. HEVpositive signals were still observed in the testes and epididymides 55 days after HEV was cleared from the serum. The spermatogenic cycle of mice (35 days) may account for the longer incubation time of HEV in
the epididymides than that in the testes. Results well expressed the fact that longer duration of HEV in semen than in serum. The histopathological analysis of the genital tracts of HEV-infected male mice revealed that intense HEV-positive signals primarily localized in the testes and epididymides and weak HEV-positive signals mainly localized in SVs. Given that we did not collect prostate samples, we were unable to determine whether HEV can replicate in the prostate. Stem-cell-like TPCs and LCs, tall-ciliated cells, and connective epididymal tissues are particularly vulnerable to HEV infection. TPCs possess a mixed phenotype and express smooth muscle-like characteristics, contribute to the SG stem cell niche, and express pluripotency markers and putative stem LC markers, as well as markers for steroidogenic genes involved in the biosynthesis of sex steroids[38-40]. The replication of HEV in TPCs and testosterone-producing LCs may contribute to the disordered production of androgenic hormones, such as testosterone and inhibin B, in HEV-infected men or male mice. The BTB is the main defense of the male genital tract against pathogen infection. Once the BTB is broken, pathogens will invade the testes and cause severe inflammation and damage[24]. We monitored HEV-induced BTB damage in mice. Interestingly, the BTB was broken (as indicated by the disappearance of ETV5) at 35 dpi after intensive attack by HEV starting from 21 dpi. Severe inflammation (as indicated by the increase in CD45+ leukocyte and F4/80+ macrophage infiltration) was observed in the testes and resulted in germ cell necrosis or apoptosis. The BTB damage caused by infection of viruses, including HIV, HBV and ZIKV, results in testicular damages, reduces sperm quality, and even causes male infertility[15, 16, 41, 42]. Reduced sperm count, increased sperm abnormalities and necrospermia may be the main reasons for infertility among HEV-infected males. Fortunately, the BTB recovered at 90 dpi, 55 days after the clearance of HEV from the feces or blood. The fertility of HEV-infected mice partially recovered. Nevertheless, the sizes of litters fathered by HEV-infected mice were smaller than those of uninfected mice. HEV is mainly transmitted through the oral–fecal route but may also be transmitted through organ transplants, blood transfusions, and intimate contact [43, 44]. Urine from patients with chronic HEV infections is infectious and causes kidney injury [45, 46]. Milk secreted by HEV-infected cows is infectious and leads to acute infection in rhesus macaques [18]. The infectivity of blood, urine and milk from HEVinfected individuals strongly suggests that body fluids are important routes of HEV transmission. However,
whether semen can transmit HEV remains to be investigated through clinical or animal experimentation. Caution should also be practiced to prevent sexual contact transmission of HEV. In conclusion, HEV persistently replicates in the male genital tract and causes testicular damages.
Acknowledgements: We thank Dong Shen for the professional analysis of the histopathologic examination. Funding: This work was supported by National Natural Science Foundation of China (81660338 to F. Huang, and 81960370 to D. Wei), Natural Science Foundation of Yunnan Province (2017FA036 to F. Huang and 2018FB132 to W. Yu), Fundamental Research Funds for the Central Universities (2016ZX310179-3) and PUMC Youth Fund (2017310038) to W. Yu.
Conflict of interest: The authors declare that there is no conflict of interest.
References 1. 2. 3.
4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.
15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.
26. 27.
Debing, Y., et al., Update on hepatitis E virology: Implications for clinical practice. J Hepatol, 2016. 65(1): p. 200-212. Kamar, N., J. Izopet, and L. Rostaing, Hepatitis E virus infection. Curr Opin Gastroenterol, 2013. 29(3): p. 271-8. Wang, Y., et al., Chronic Hepatitis E in a Renal Transplant Recipient: The First Report of Genotype 4 Hepatitis E Virus Caused Chronic Infection in Organ Recipient. Gastroenterology, 2018. 154(4): p. 1199-1201. Zeng, H., et al., Seroprevalence of hepatitis E virus in HIV-infected patients in China. Aids, 2017. 31(14): p. 2019-2021. Ferreira, A.C., et al., Serological and molecular markers of hepatitis E virus infection in HIV-infected patients in Brazil. Arch Virol, 2018. 163(1): p. 43-49. Dalton, H.R., et al., Hepatitis E virus and neurological injury. Nat Rev Neurol, 2016. 12(2): p. 77-85. Scanvion, Q., et al., Neuralgic amyotrophy triggered by hepatitis E virus: a particular phenotype. J Neurol, 2017. 264(4): p. 770-780. Geng, Y., et al., Detection and assessment of infectivity of hepatitis E virus in urine. J Hepatol, 2016. 64(1): p. 37-43. Huang, F., et al., Rhesus macaques persistently infected with hepatitis E shed virus into urine. J Hepatol, 2016. 64(6): p. 1446-7. Mengel, A.M., et al., Hepatitis E-induced severe myositis. Muscle Nerve, 2016. 53(2): p. 317-20. Huang, F., et al., High prevalence of hepatitis E virus in semen of infertile male and causes testis damage. Gut, 2018. 67(6): p. 1199-1201. Govero, J., et al., Zika virus infection damages the testes in mice. Nature, 2016. 540(7633): p. 438-442. Ma, W., et al., Zika Virus Causes Testis Damage and Leads to Male Infertility in Mice. Cell, 2017. 168(3): p. 542. Garrido, N., et al., Semen characteristics in human immunodeficiency virus (HIV)- and hepatitis C (HCV)-seropositive males: predictors of the success of viral removal after sperm washing. Hum Reprod, 2005. 20(4): p. 1028-34. Vicari, E., et al., Sperm output in patients with primary infertility and hepatitis B or C virus; negative influence of HBV infection during concomitant varicocele. Minerva Med, 2006. 97(1): p. 65-77. Garolla, A., et al., Sperm viral infection and male infertility: focus on HBV, HCV, HIV, HPV, HSV, HCMV, and AAV. J Reprod Immunol, 2013. 100(1): p. 20-9. Soomro, M.H., et al., Molecular and structural changes related to hepatitis E virus antigen and its expression in testis inducing apoptosis in Mongolian gerbil model. 2017. 24(8): p. 696-707. Huang, F., et al., Excretion of infectious hepatitis E virus into milk in cows imposes high risks of zoonosis. Hepatology, 2016. 64(2): p. 350-9. Baylis, S.A., et al., World Health Organization International Standard to harmonize assays for detection of hepatitis E virus RNA. Emerg Infect Dis, 2013. 19(5): p. 729-35. Xu, L., et al., RIG-I is a key antiviral interferon-stimulated gene against hepatitis E virus regardless of interferon production. Hepatology, 2017. 65(6): p. 1823-1839. Devhare, P.B., et al., Analysis of antiviral response in human epithelial cells infected with hepatitis E virus. PLoS One, 2013. 8(5): p. e63793. F, H., et al., High prevalence of hepatitis E virus in semen of infertile male and causes testis damage. Gut, 2018. 67(6): p. 1199-1201. Li, W., et al., Seroprevalence and molecular detection of hepatitis E virus in Yunnan Province, China. Archives of Virology, 2011. 156(11): p. 1989-1995. Mruk, D.D. and C.Y. Cheng, The Mammalian Blood-Testis Barrier: Its Biology and Regulation. Endocr Rev, 2015. 36(5): p. 564-91. Morrow, C.M., et al., ETV5 is required for continuous spermatogenesis in adult mice and may mediate blood testes barrier function and testicular immune privilege. Ann N Y Acad Sci, 2007. 1120: p. 144-51. X, Y., et al., Hepatitis E virus persists in the presence of a type III interferon response. PLoS pathogens, 2017. 13(5): p. e1006417. Guan, X., et al., Effects of spermatogenic cycle on Stem Leydig cell proliferation and differentiation. Mol Cell Endocrinol, 2018.
28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38.
39.
40.
41. 42.
43.
44. 45. 46.
Anderson, R.A., et al., Inhibin B in seminal plasma: testicular origin and relationship to spermatogenesis. Hum Reprod, 1998. 13(4): p. 920-6. Mahmoud, A.M., F.H. Comhaire, and C.E. Depuydt, The clinical and biologic significance of serum inhibins in subfertile men. Reprod Toxicol, 1998. 12(6): p. 591-9. Purcell, R.H. and S.U. Emerson, Hepatitis E: an emerging awareness of an old disease. J Hepatol, 2008. 48(3): p. 494-503. Pischke, S., et al., Hepatitis E virus: Infection beyond the liver? J Hepatol, 2017. 66(5): p. 10821095. Li, H., et al., Prevalence of hepatitis E virus (HEV) infection in various pig farms from Shaanxi Province, China: First detection of HEV RNA in pig semen. Transbound Emerg Dis, 2018. Horvatits, T., et al., No link between male infertility and HEV genotype 3 infection. Gut, 2019. Wang, L., et al., Absence of hepatitis E virus RNA in semen samples of infertile male in China. Gut, 2019. Jia, Z., et al., Epidemiology of hepatitis E virus in China: results from the Third National Viral Hepatitis Prevalence Survey, 2005-2006. PLoS One, 2014. 9(10): p. e110837. Liu, R., et al., Epidemiological characteristics and clinical features of sporadic viral hepatitis E (HEV). Pak J Pharm Sci, 2016. 29(6 Spec): p. 2251-2254. The human testis is susceptible to HIV-1. Reprod Biomed Online, 2007. 14(3): p. 299. Flenkenthaler, F., et al., Secretome analysis of testicular peritubular cells: a window into the human testicular microenvironment and the spermatogonial stem cell niche in man. J Proteome Res, 2014. 13(3): p. 1259-69. Spinnler, K., et al., Glial cell line-derived neurotrophic factor is constitutively produced by human testicular peritubular cells and may contribute to the spermatogonial stem cell niche in man. Hum Reprod, 2010. 25(9): p. 2181-7. Albrecht, M., et al., Isolation and cultivation of human testicular peritubular cells: a new model for the investigation of fibrotic processes in the human testis and male infertility. J Clin Endocrinol Metab, 2006. 91(5): p. 1956-60. Smith, D.B., et al., Proposed reference sequences for hepatitis E virus subtypes. J Gen Virol, 2016. 97(3): p. 537-42. Aiman, J., P.F. Brenner, and P.C. MacDonald, Androgen and estrogen production in elderly men with gynecomastia and testicular atrophy after mumps orchitis. J Clin Endocrinol Metab, 1980. 50(2): p. 380-6. Xu, C., et al., An assessment of hepatitis E virus (HEV) in US blood donors and recipients: no
detectable HEV RNA in 1939 donors tested and no evidence for HEV transmission to 362 prospectively followed recipients. Transfusion, 2013. 53(10 Pt 2): p. 2505-11. Aggarwal, R., Clinical presentation of hepatitis E. Virus Res, 2011. 161(1): p. 15-22. Kamar, N., et al., Acute hepatitis and renal function impairment related to infection by hepatitis E virus in a renal allograft recipient. Am J Kidney Dis, 2005. 45(1): p. 193-6. Kamar, N., et al., Hepatitis E virus and the kidney in solid-organ transplant patients. Transplantation, 2012. 93(6): p. 617-23.
Figure legends Fig. 1. HEV genomic RNA copy number of livers, feces, serum, testes, epididymides, seminal vesicles and epididymal fluid from HEV-infected male mice
Fig. 2. HEV antigens detected in the genital tracts of male mice. Immunohistochemical (IHC) analyses of HEV ORF2 antigens in the genital tracts of male mice at indicated times. (a) (Uninfected panel) The liver of an uninfected mouse was used as the negative control. (HEV-infected panel) The liver of an HEV-infected mouse was incubated with PBS or HEV ORF2 antibody to identify the antibody specificity, and used as the positive control. Immunostaining of HEV ORF2 in the testis (b), Epididymis (c) and SV (d) of PBSinoculated uninfected mice and HEV-infected mice during 7–90 dpi.
Fig. 3. HEV replicates in TPCs and LCs in mice testes. IFA of HEV ORF2 in α-SMA-positive TPCs (a), 3β-HSD-positive LCs (b) and Lin28a-positive SGs (c) in the testes of uninfected or HEV-infected mice. Nuclei were stained with DAPI.
Fig. 4. HEV infection barely activate antiviral innate immune response in the testes. The gene expressions of RIG-I (a), IFN-β (b), IFN-λ (c), TNF-α (d), IL-6 (e) and IL-10 (f) in the testes and blood were determined by qRT-PCR.
Fig. 5. HEV infection impairs sperm quality. (a) Morphologies of the male genital tracts of uninfected or HEV-infected mice at 21 dpi. (b) Ratio of testes weight/body weight. (c) Mature sperm cells were collected from the cauda epididymis of uninfected or HEV-infected mice and stained with eosin. Black arrows indicate dead sperm cells. Red arrows indicate sperm cells with asthenospermia. Blue arrows indicate abnormal sperm cells.
Fig. 6. Reduced germ cells in the testes of HEV-infected mice. (a) Immunochemical analysis of germ cells in the testes collected from uninfected or HEV-infected mice. (b) The number of germ cells in HEVinfected mice were compared with uninfected mice. *P < 0.05; ** P < 0.01; ns, not significant.
Fig. 7. Testicular and epididymal damages caused by HEV infection. Histopathological analysis of (a) testis, (b) caput epididymis, (c) corpus epididymis and (d) SV collected from uninfected or HEV-infected mice as revealed stained by H&E.
Fig. 8. HEV infection induces inflammatory responses and apoptosis. Immunofluorescence analysis of CD45 (a), F4/80 (b), ETV5 (c) and apoptosis (d) in the testis of uninfected or HEV-infected mice. Nuclei were stained with DAPI.
Fig. 9. HEV infection leads to hormonal disorders and temporarily testicular damage. Androgen hormones decreased during HEV infection. Testosterone (a) and inhibin B (b) levels in the serum of uninfected or HEV-infected mice. (c) Number of fetuses fathered by male mice with or without HEV infection. **P < 0.01.
1. Hepatitis E virus (HEV) is a major cause of acute hepatitis worldwide. 2. HEV has been detected in the semen of male, but the pathogenesis is unknown. 3. We found HEV replicates in male genital tract with longer duration until 90 dpi. 4. HEV infection impairs sperm quality with reductions in sperm motility and vitality. 5. HEV destroys the BTBs and results in germ cell necrosis and apoptosis.
Hepatitis E viral infection causes testicular damage in mice Running title: HEV replicates in the male genital tract Jianwen Situ1#, Wenjing Wang1#, Feiyan Long1, Weimin Yang1, Chenchen Yang1, Daqiao Wei1*, Wenhai Yu2*, Fen Huang1*
1. Medical School, Kunming University of Science and Technology, Kunming, China; 2. Institute of Medical Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Kunming, China; # These authors contribute equally in this study.
*Correspondence: Prof Huang Fen, E-mail: [email protected]. Medical School, Kunming University of Science and Technology 727 Jing Ming South Road , Kunming, China；Prof Wenhai Yu, E-mail: [email protected]. Institute of Medical Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, 935 Jiao ling Road, Kunming, China; Prof Daqiao Wei, E-mail: [email protected]. Medical School, Kunming University of Science and Technology, 727 Jing Ming South Road, Kunming, China;
Funding: This work was supported by National Natural Science Foundation of China (31360619 and 81660338 to F. Huang), Natural Science Foundation of Yunnan Province (2017FA036 to F. Huang and 2018FB132 to W. Yu), Fundamental Research
Funds for the Central Universities (2016ZX310179-3) and PUMC Youth Fund (2017310038) to W. Yu.
Conflict of interest: The authors declare that there is no conflict of interest to this study.
Author contributions: J. Situ, W. Wang, W. Yang, F. Long and C. Yang performed the experiments, J. Situ and W. Yu analyzed the data and drafted the manuscript, and F. Huang conceived the ideas, designed and discussed experiments, supervised the project and extensively edited the manuscript.