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Infectious hepatitis E virus is associated with the mature sperm head [1]
['Kush K. Yadav', 'Center For Food Animal Health', 'Department Of Animal Sciences', 'The Ohio State University', 'Wooster', 'Ohio', 'United States Of America', 'Department Of Veterinary Preventive Medicine', 'College Of Veterinary Medicine', 'Columbus']
Date: 2024-05
Hepatitis E virus (HEV) is the leading cause of acute viral hepatitis worldwide. HEV associated pregnancy mortality has been reported as up to 30% in humans. Recent findings suggest HEV may elicit effects directly in the reproductive system with HEV protein found in the testis, viral RNA in semen, and viral replication occurring in placental cell types. Using a natural host model for HEV infection, pigs, we demonstrate infectious HEV within the mature spermatozoa and altered sperm viability from HEV infected pigs. HEV isolated from sperm remained infectious suggesting a potential transmission route via sexual partners. Our findings suggest that HEV should be explored as a possible sexually transmittable disease. Our findings propose that infection routes outside of oral and intravenous infection need to be considered for their potential to contribute to higher mortality in HEV infections when pregnancy is involved and in HEV disease in general.
Hepatitis E virus (HEV) is one of the major causes of acute viral hepatitis worldwide. Extrahepatic manifestations have been described for HEV including neurological, pancreatic, and reproductive disorders. Recent findings have suggested that HEV can be found in the testes, is associated with male infertility, and viral RNA can be detected in semen. HEV can replicate in human testicular explants and Sertoli cells, and replicates in the vaginal tissue of rabbits. We demonstrate that infectious HEV is associated with the sperm head. The infectious ability of HEV present in sperm suggests a potentially new transmission route of HEV that necessitates in-depth study. Our study adds to the existing literature highlighting the importance of understanding HEV association with sperm as a potential contributor as a sexually transmittable disease. New studies to evaluate sexual transmission of HEV, its association with pregnancy mortality, and the testes as a potential virus reservoir are necessary.
Funding: Additional salaries and research support were provided by state and federal funds appropriated to the Ohio Agricultural Research and Development Center, The Ohio State University, and from the research funds of National Institute of Allergy and Infectious Diseases (#R21AI151736). Portions of some funds from Ohio State University and the NIAID grant were used to cover salaries of SPK and KKY. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
We used a natural host, the pig, infected with the genotype 3 zoonotic strain of Paslahepevirus balayani HEV (US-2 isolate) as a model to investigate the presence of HEV in sperm cells. We further assessed the infectiousness of virus within sperm cells using a human liver cell culture system to understand the potential transmission ability of HEV via sperm cells.
Recent reports have found the presence of HEV RNA in semen at a higher incidence rate in infertile men [ 6 ]. HEV RNA has also been detected within semen in chronic hepatitis E [ 7 ] patients, but infectious virus could not be cultured in either instance. Approximately 50% of clinical male infertility cases are due to unknown causes [ 8 ]. It is currently unknown whether some of these infertility cases could be linked to asymptomatic HEV infection. Human and animal studies (nonhuman primates, gerbils, mice, and pigs) detected HEV RNA in semen and protein in the testis associated with alteration in sperm motility and morphology [ 6 , 9 – 11 ]. Detection of HEV within the testis could simply be due to local inflammation compromising the blood testis barrier resulting in virus entry and replication at a normally immune privileged site (6). Even though HEV RNA has been detected in human semen and immature spermatogonia, the relationship of the virus with mature spermatozoa is unclear with current evidence lacking for the presence of the virus in the sperm cell itself [ 6 ].
Hepatitis E has been increasingly recognized as an emerging and significant clinical problem in immunocompromised and pregnant humans globally [ 1 ]. HEV infection is the leading cause of acute viral hepatitis worldwide causing significant liver damage [ 2 ], eventually leading to cirrhosis [ 3 ] in some cases, with considerable mortality in humans [ 4 ]. A recent report ranked hepatitis E virus (HEV) 6 th out of 887 wildlife viruses with significant risk of spilling over into humans, behind only Lassa, SARS-CoV-2, Ebola, Hanta, and Nipah viruses, but ahead of viral pathogens such as monkeypox, Marburg, rabies, and other emerging coronaviruses [ 5 ].
(A) Light microscopic observation of 200 live spermatozoa harvested from mock or infected pig epididymis. Sperm cells demonstrated decreased progressive motility when infected by HEV US-2. PR–progressive motility of sperm (moving active, either linearly or in a circle, regardless of speed); NP–non-progressive motility (all other patterns of motility with absent progression. IM–immobility. (B) Light microscopic observation of live sperm cells harvested from mock or infected pig epididymis. Sperm from US-2 HEV infected pigs showed a significant increase in spermatozoa with head abnormalities. No significant changes were seen in tail of the sperm cells. * = p < 0.05, ** = p < 0.01. (C) Histogram plot was used to demonstrate the data from flow cytometry. Sperm cells from mock non-infected pigs and from US-2 HEV infected pigs.
To evaluate whether HEV infection directly impairs sperm quality, as measured by motility and morphological appearance of sperm cells, semen analysis was immediately performed after collection from the epididymis. Approximately, 200 sperm cells were analyzed to characterize the motility and morphological difference between groups. The progressive sperm motility from US-2 HEV infected sperm (PR% = 64% ± 4%) was found to be significantly lower than the sperm from mock infected (PR% = 78% ± 4%) pigs. The non-progressive sperm motility from US-2 HEV infected sperm (PR% = 18% ± 3%) was found to be significantly higher (p < 0.05) than the sperm from mock infected (PR% = 10% ± 3%) pigs. Immobility of sperm was higher in sperm from the US-2 HEV infected pigs (15% ± 3%) when compared to mock infected (7% ± 2%, p < 0.05) ( Fig 3A ). Morphologically, we found a higher percentage of abnormal sperm heads (33% ± 4%, p < 0.01) and tails (11% ± 3%) in sperm from US-2 HEV infected pigs than mock infected pigs ( Fig 3B ). Additionally, flow cytometry analysis of sperm cells quantifies the percentage of sperm cells demonstrating the HEV ORF2 capsid protein ( Fig 3C ).
To study whether the HEV antigen detected in the sperm cells represented infectious virus, we lysed sperm cells and inoculated ~4.8 x 10 5 viral RNA copies onto human liver cells (HepG2/C3A) in-vitro. We assessed events at the single cell level using indirect immunofluorescence employing antibodies directed against ORF2 capsid protein. Immunofluorescence detection of HEV ORF2 in HepG2/C3A cells five days post inoculation with sperm cell lysates demonstrates HEV within the sperm cells remains infectious ( Fig 2C ). In addition, flow cytometry quantification of HepG2/C3A cells demonstrates at least 23% of cells contained HEV antigens ( Fig 2D ). HEV replication ability was further demonstrated by an increase in the viral RNA copies in the HepG2/C3A cellular lysate over time and in the supernatant harvested from inoculated HepG2/C3A cells ( Fig 2E ). Higher HEV RNA copies were detected in sperm cells lysates in comparison to the seminal fluid ( Fig 2F ) but almost equivalent to the bile ( Fig 2E ). Interestingly, sperm cell viral RNA load was higher than seen in the gall bladder, liver, ileum, and epididymis ( Fig 1B )
(A) Immunohistochemical detection of hepatitis E (red) in the head of spermatozoa obtained from the US-2 infected pigs at day 84 post infection; Hepatitis E virus ORF2 is red; DAPI stain is blue (nucleus). (B) Flow cytometry analysis demonstrating the percentage of sperm cells infected with hepatitis E virus (US-2 strain). (C) Immunodetection of hepatitis E demonstrating infectious sperm cells in HepG2/C3A cells. (D) Flow cytometry analysis demonstrating the percentage of HepG2/C3A cells infected with the sperm cell lysates from mock and US-2 HEV infected pigs. (E) Infectious titer of sperm-derived HEV using HepG2/C3A cells. US-2 HEV RNA loads in culture supernatant (S) and cell lysates (CL) of HepG2/C3A cell cultures after inoculation with the lysed sperm cells. Independent biological experiments, mean ± SD of three replicates, are presented. The dotted line represents the cut-off value demonstrating the background referring to the attachments of the virus to the cell surfaces. (F) HEV RNA loads in sperm cells suspension, seminal fluid, and bile from US-2 HEV inoculated pigs. *** = p < 0.001, **** = p < 0.0001.
We report the immunohistochemical detection of HEV in the head of spermatozoa obtained at 84 days post intravenous inoculation of conventional pigs ( Fig 2A ). The use of fluorescence microscopy demonstrated HEV ORF2 antigen within the acrosomal region of the sperm head, while non-infected mock group sperm showed no HEV staining, confirming the specificity of the assay. Using flow cytometry analysis, we were able to demonstrate that at least 19% of the sperm cells contained HEV antigens ( Fig 2B ).
In this study, we utilized a natural HEV host with similar human size and male reproductive physiology, the pig. Viremia, fecal viral shedding, higher RNA titers in the gall bladder, liver, ileum, and epididymis in US-2 HEV inoculated pigs was observed until day 84 and demonstrated active sub-clinical HEV infection ( Fig 1A and 1B ). Although viremia and fecal viral shedding was seen as early as day 7 in the US-2 HEV inoculated group, it could be the remnants of the inoculum. Replication kinetics studies showed a clear increase in the viral RNA in serum and feces over the course of infection.
Discussion
HEV continues to present even more complex clinical manifestations due to newly emerging strains of HEV. These manifestations include an increase in extrahepatic and neurological disorders, pregnancy disorders with adverse fetal outcomes, and a lack of approved treatment options beyond ribavirin [4]. The unexpected detection of viral RNA in the semen of infertile men [6] while presenting no viremia and fecal shedding along with our detection of HEV in the spermatozoa potentially shifts the paradigm of host/hepevirus interaction. Our findings place HEV among those viruses which has the potential to be transmitted via sexual intercourse which is troubling considering the high mortality rate associated with HEV infection of pregnant women.
For epidemiological consideration, how HEV genotypes 1 and 2, which are obligate human infecting viruses, persist in the environment has been an ongoing question within the HEV field. There is currently no data for humans harboring the virus in sperm cells, making them a potential extended viral reservoir. Viral persistence in sperm cells in the absence of viremia or fecal shedding is a major concern and could be related to viral tropism for male sexual cells. Currently, we do not know if there are undefined receptors that are common in liver, intestine, ovary, and the sperm head. However, our data suggest that infectious genotype 3 HEV can reside within the sperm head highlighting the need to further characterize its potential ability to persist and transmit sexually.
Further studies are needed to define the mechanisms of the spermatozoa infection, length of persistence in the testis, length of the virus shedding in the spermatozoa, and whether sexual intercourse can result in systemic HEV infection in partners. Given the presence of HEV in sperm cells, future studies will be directed towards understanding the duration of HEV presence in the semen and its concentration. Data on potential ability of HEV to be sexually transmitted will allow public health authorities to recommend safe practices and list HEV on the list of potential pathogens transmitting sexually. HEV gt1, HEV gt2, and HEV gt4 have been described in several human pregnancy mortality cases [1,12]. Despite multiple studies attempting to delineate the mechanism behind pregnancy mortality, a definitive answer is yet to be found. Recent discoveries demonstrating ability of HEV to replicate in the vagina [13] and human male testicular cells [14] demonstrates a new infection scenario that needs detailed investigation and its association with pregnancy mortality. Our study demonstrating infectious HEV in the sperm head raises concerns on a potentially new route of HEV transmission in humans. Sperm related HEV infection should be further explored for its potential to decrease male fertility which has been reported in a 2018 study where HEV RNA was demonstrated in semen samples (3).
Semen alterations were observed, including a decreased progressive motility in US-2 HEV (human HEV) infected pig specimens. These findings suggest a direct viral infection of the sperm cell potentially hinders sperm cell development, in agreement with reported findings [7]. HEV infection has been suggested to impair sperm quality with reductions in sperm motility and vitality in mice via destruction of the blood testis barrier (BTBs) resulting in germ cell necrosis and apoptosis [11]. In addition, HEV infection in mice was demonstrated to damage Leydig cells resulting in spermatogonia necrosis [11]. Another study in non-human primates illustrates the presence of HEV in the spermatogonia with a reduced number of testicular cell populations, primary spermatocytes and Sertoli cells [6]. Thus, we hypothesize that HEV infection impairs the BTB resulting in inflammation that could be a factor resulting in sperm aberrations. In our study, HEV antigen association with the sperm head was performed in fixed sperm cells while morphology and motility were measured in live sperm. We speculate the sperm aberrations observed in sperm from infected pigs is likely due to HEV infection of spermatogonia, inflammation, BTB breakdown, and reduction in Leydig and germ cells. However, the direct association of HEV antigen in the acrosome necessitates an experimental technique that can demonstrate both HEV presence in the sperm head and has the ability to measure sperm quality in live sperm samples. Furthermore, lysates from infected spermatozoa contained infectious HEV capable of infecting HepG2/C3A liver cells. Our study highlights the need to screen human semen samples in geographical regions where endemic HEV causing pregnancy mortality has been reported.
Interestingly, in 2021, differential genomic sequences of HEV were identified in the semen of a chronically infected patient [7]. The antiviral ribavirin can cross the blood-testis barrier to affect viral replication, however the lack of immune clearance may contribute to generation of quasispecies and serve as a source for reinfection. The findings that HEV persists in the semen after detectable viremia and fecal shedding have subsided, suggests that HEV may persist undetected in the testis. Further research is needed to study whether testicular and spermatogonia HEV infection increases the chances of resistant quasispecies in this immune privileged site.
Studies demonstrating the mode of spermatozoa infection and duration of the virus presence in the sperm head leading to the transmission to the ovum are warranted. In addition, this study forms a basis to evaluate the sexual transmission of HEV to pregnant women possibly contributing to pregnancy mortality. Comparative study of HEV receptors in the liver and the sperm head will allow identification of receptors involved in the attachment and the entry of the virus into the sperm cells. Screening of human sperm bank and male pig semen donors needs to be conducted to identify the risk of HEV transmission via artificial insemination. Longitudinal studies to assess the long-term effects of HEV presence in sperm, duration of presence in the sperm including its impact on fertility and reproductive health over time need to be conducted.
Among the merits of our study in terms of translational research of HEV presence in sperm cells, we used a natural HEV host and reservoir pig animal model. Pigs are anatomically and physiologically closer to humans than other small animal models [15]. Our data suggests that the pig model can be used to study HEV-induced sperm health disorders that have been observed in humans. Our findings suggest that HEV infection routes outside of oral and intravenous infection need to be considered for their potential to contribute to higher mortality disease outcomes present in HEV infections when pregnancy is involved and in HEV disease in general.
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