Viral infections and implications for male reproductive health.

Viral infections have haunted humankind since times immemorial. Overpopulation, globalization, and extensive deforestation have created an ideal environment for a viral spread with unknown and multiple shedding routes. Many viruses can infect the male reproductive tract, with potential adverse consequences to male reproductive health, including infertility and cancer. Moreover, some genital tract viral infections can be sexually transmitted, potentially impacting the resulting offspring's health. We have summarized the evidence concerning the presence and adverse effects of the relevant viruses on the reproductive tract (mumps virus, human immunodeficiency virus, herpes virus, human papillomavirus, hepatitis B and C viruses, Ebola virus, Zika virus, influenza virus, and coronaviruses), their routes of infection, target organs and cells, prevalence and pattern of virus shedding in semen, as well as diagnosis/testing and treatment strategies. The pathophysiological understanding in the male genital tract is essential to assess its clinical impact on male reproductive health and guide future research.

The Chinese herbal prescription JZ-1 induces autophagy to protect against herpes simplex Virus-2 in human vaginal epithelial cells by inhibiting the PI3K/Akt/mTOR pathway.

ETHNOPHAMACOLOGICAL RELEVANCE: The Chinese herbal prescription JieZe-1 (JZ-1) is based on the modification of Yihuang Tang, which was first described in Fu Qingzhu Nvke by the famous Qing Dynasty doctor Shan Fu as a treatment for leukorrheal diseases. As an in-hospital preparation, JZ-1 has been used in Tongji Hospital for many years to treat various infectious diseases of the lower female genital tract, including cervicitis, vaginitis, genital herpes and condyloma acuminatum. Our previous studies have shown that JZ-1 has curative effects on Candida albicans, Trichomonas vaginalis and Ureaplasma urealyticum infections. AIM OF THE STUDY: Genital herpes is among the most common sexually transmitted diseases (STDs) worldwide and is mainly caused by herpes simplex virus type-2 (HSV-2). Current therapies can relieve symptoms in patients but do not cure or prevent the spread of the virus. This study was designed to investigate the effect of JZ-1 on HSV-2 infection and its mechanism, which is based on autophagy induction, to provide new ideas and a basis for the study of antiviral drugs. MATERIALS AND METHODS: Evaluation of the antiviral activity of JZ-1 was conducted by MTT assay and western blotting. Then, Western blot and immunofluorescence analyses, observations through transmission electron microscopy and experiments with the recombinant lentivirus vector mRFP-GFP-LC3B were used to monitor autophagic flux in VK2/E6E7 cells. To explore the mechanism by which JZ-1 regulates autophagy, western blotting and real-time quantitative PCR (qRT-PCR) were used to determine the expression of phosphoinositide 3-kinase (PI3K)/Akt/mTOR pathway proteins and to detect changes in critical molecules in the pathway after the application of a PI3K inhibitor. Additionally, the mRNA expression levels of inflammatory cytokines, namely, IL-6, IFN-α, IFN-ß and TNF-α, were measured with qRT-PCR. RESULTS: HSV-2 infection inhibited autophagy in the VK2/E6E7 cells. Further study revealed that the activation of the PI3K/Akt/mTOR pathway induced by HSV-2 infection may result in the blocked autophagic flux and inhibited autophagosome and autolysosome formation. JZ-1 exhibited significant antiviral activity in the VK2/E6E7 cells, which showed increased cell vitality and reduced viral protein expression, namely, earliest virus-specific infected cell polypeptides 5 (ICP5) and glycoprotein D (gD). We found that JZ-1 treatment inhibited the upregulation of the PI3K/Akt/mTOR pathway proteins and promoted autophagy to combat HSV-2 infection, while PI3K inhibitor pretreatment prevented the enhanced autophagy induced by JZ-1. Moreover, JZ-1 attenuated the increase in inflammatory cytokines that had been induced HSV-2 infection. CONCLUSION: Our results showed that JZ-1 protects against HSV-2 infection, and this beneficial effect may be mediated by inducing autophagy via inhibition of the PI3K/Akt/mTOR signaling axis.

Cervical HSV-2 infection causes cervical remodeling and increases risk for ascending infection and preterm birth.

Preterm birth (PTB), or birth before 37 weeks gestation, is the leading cause of neonatal mortality worldwide. Cervical viral infections have been established as risk factors for PTB in women, although the mechanism leading to increased risk is unknown. Using a mouse model of pregnancy, we determined that intra-vaginal HSV2 infection caused increased rates of preterm birth following an intra-vaginal bacterial infection. HSV2 infection resulted in histological changes in the cervix mimicking cervical ripening, including significant collagen remodeling and increased hyaluronic acid synthesis. Viral infection also caused aberrant expression of estrogen and progesterone receptor in the cervical epithelium. Further analysis using human ectocervical cells demonstrated a role for Src kinase in virus-mediated changes in estrogen receptor and hyaluronic acid expression. In conclusion, HSV2 affects proteins involved in tissue hormone responsiveness, causes significant changes reminiscent of premature cervical ripening, and increases risk of preterm birth. Studies such as this improve our chances of identifying clinical interventions in the future.

Keratinocytes produce IL-17c to protect peripheral nervous systems during human HSV-2 reactivation.

Despite frequent herpes simplex virus (HSV) reactivation, peripheral nerve destruction and sensory anesthesia are rare. We discovered that skin biopsies obtained during asymptomatic human HSV-2 reactivation exhibit a higher density of nerve fibers relative to biopsies during virological and clinical quiescence. We evaluated the effects of HSV infection on keratinocytes, the initial target of HSV replication, to better understand this observation. Keratinocytes produced IL-17c during HSV-2 reactivation, and IL-17RE, an IL-17c-specific receptor, was expressed on nerve fibers in human skin and sensory neurons in dorsal root ganglia. In ex vivo experiments, exogenous human IL-17c provided directional guidance and promoted neurite growth and branching in microfluidic devices. Exogenous murine IL-17c pretreatment reduced apoptosis in HSV-2-infected primary neurons. These results suggest that IL-17c is a neurotrophic cytokine that protects peripheral nerve systems during HSV reactivation. This mechanism could explain the lack of nerve damage from recurrent HSV infection and may provide insight to understanding and treating sensory peripheral neuropathies.

In Situ Detection of Regulatory T Cells in Human Genital Herpes Simplex Virus Type 2 (HSV-2) Reactivation and Their Influence on Spontaneous HSV-2 Reactivation.

BACKGROUND: Herpes simplex virus type 2 (HSV-2) reactivation is accompanied by a sustained influx of CD4(+) and CD8(+) T cells that persist in genital tissue for extended periods. While CD4(+) T cells have long been recognized as being present in herpetic ulcerations, their role in subclinical reactivation and persistence is less well known, especially the role of CD4(+) regulatory T cells (Tregs). METHODS: We characterized the Treg (CD4(+)Foxp3(+)) population during human HSV-2 reactivation in situ in sequential genital skin biopsy specimens obtained from HSV-2-seropositive subjects at the time of lesion onset up to 8 weeks after healing. RESULTS: High numbers of Tregs infiltrated to the site of viral reactivation and persisted in proximity to conventional CD4(+) T cells (Tconvs) and CD8(+) T cells. Treg density peaked during the lesion stage of the reactivation. The number of Tregs from all time points (lesion, healed, 2 weeks after healing, 4 weeks after healing, and 8 weeks after healing) was significantly higher than in control biopsy specimens from unaffected skin. There was a direct correlation between HSV-2 titer and Treg density. CONCLUSIONS: The association of a high Treg to Tconv ratio with high viral shedding suggests that the balance between regulatory and effector T cells influences human HSV-2 disease.

Herpes Simplex Virus Cervicitis Mimicking Preterm Premature Rupture of Membranes.

BACKGROUND: The diagnosis of preterm premature rupture of membranes (PROM) is based on pooling, ferning, and Nitrazine tests; definitive diagnosis is made with a blue dye test. CASE: A 21-year-old woman, gravida 1 para 0, at 25 5/7 weeks of gestation was admitted for preterm PROM with positive findings of pooling, Nitrazine, and ferning. Her cervix was bluish with white plaques. Amniotic fluid volume was normal. On hospital day 8, her discharge ceased; examination was negative for pooling, Nitrazine, and ferning. A blue dye tampon test was negative. A Pap test result from her hospitalization returned consistent with herpes infection. CONCLUSION: The diagnosis of preterm PROM should be constantly reevaluated in the setting of a normal amniotic fluid volume.

HSV-2-driven increase in the expression of α4ß7 correlates with increased susceptibility to vaginal SHIV(SF162P3) infection.

The availability of highly susceptible HIV target cells that can rapidly reach the mucosal lymphoid tissues may increase the chances of an otherwise rare transmission event to occur. Expression of α4ß7 is required for trafficking of immune cells to gut inductive sites where HIV can expand and it is expressed at high level on cells particularly susceptible to HIV infection. We hypothesized that HSV-2 modulates the expression of α4ß7 and other homing receptors in the vaginal tissue and that this correlates with the increased risk of HIV acquisition in HSV-2 positive individuals. To test this hypothesis we used an in vivo rhesus macaque (RM) model of HSV-2 vaginal infection and a new ex vivo model of macaque vaginal explants. In vivo we found that HSV-2 latently infected RMs appeared to be more susceptible to vaginal SHIVSF162P3 infection, had higher frequency of α4ß7high CD4+ T cells in the vaginal tissue and higher expression of α4ß7 and CD11c on vaginal DCs. Similarly, ex vivo HSV-2 infection increased the susceptibility of the vaginal tissue to SHIVSF162P3. HSV-2 infection increased the frequencies of α4ß7high CD4+ T cells and this directly correlated with HSV-2 replication. A higher amount of inflammatory cytokines in vaginal fluids of the HSV-2 infected animals was similar to those found in the supernatants of the infected explants. Remarkably, the HSV-2-driven increase in the frequency of α4ß7high CD4+ T cells directly correlated with SHIV replication in the HSV-2 infected tissues. Our results suggest that the HSV-2-driven increase in availability of CD4+ T cells and DCs that express high levels of α4ß7 is associated with the increase in susceptibility to SHIV due to HSV-2. This may persists in absence of HSV-2 shedding. Hence, higher availability of α4ß7 positive HIV target cells in the vaginal tissue may constitute a risk factor for HIV transmission.

Role of Fas/FasL in regulation of inflammation in vaginal tissue during HSV-2 infection.

To assess the role of Fas in lesion development during genital HSV-2 infection, we used a well-established HSV-2 murine model applied to MRL-Fas(lpr)/J (Fas-/-) and C3-Fasl(gld)/J (FasL-/-) C57BL6 mice. In vitro infection of murine keratinocytes and epithelial cells was used to clarify molecular details of HSV-2 infection. Despite upregulation of Fas and FasL, HSV-2-infected keratinocytes and epithelial cells showed a moderate level of apoptosis due to upregulated expression of the anti-apoptotic factors Bcl-2, Akt kinase and NF-κB. Inflammatory lesions within the HSV-2-infected epithelium of C57BL6 mice consisted of infected cells upregulating Fas, FasL and Bcl-2, uninfected cells upregulating Fas and neutrophils expressing both Fas and FasL. Apoptosis was detected in HSV-2-infected cells and to even higher extent in non-infected cells surrounding HSV-2 infection sites. HSV-2 infection of Fas- and FasL-deficient mice led to increased apoptosis and stronger recruitment of neutrophils within the infection sites. We conclude that the Fas pathway participates in regulation of inflammatory response in the vaginal epithelium at the initial stage of HSV-2 infection.

Loss of the type I interferon pathway increases vulnerability of mice to genital herpes simplex virus 2 infection.

The mouse model of genital herpes relies on medoxyprogesterone treatment of female mice to render the vaginal lumen susceptible to inoculation with herpes simplex virus 2 (HSV-2). In the present study, we report that mice deficient in the A1 chain of the type I interferon receptor (CD118(-/-)) are susceptible to HSV-2 in the absence of medroxyprogesterone preconditioning. In the absence of hormone pretreatment, 2,000 PFU of a clinical isolate of HSV-2 was sufficient to establish a productive infection in the vagina of 75% ± 17% and in the spinal cord of 71% ± 14% of CD118(-/-) mice, whereas the same dose of HSV-2 replicated to detectable levels in only 13% ± 13% of vaginal samples and 0% of spinal cord samples from wild-type mice, as determined at day 5 postinfection. The susceptibility to HSV-2 infection in the CD118(-/-) mice was associated with a significant reduction in the infiltration of HSV-specific cytotoxic T lymphocytes into the vaginal tissue, the local production of gamma interferon (IFN-γ), and the expression of T cell-recruiting chemokines CCL5, CXCL9, and CXCL10. Collectively, the results underscore the significant contribution of type I IFNs in resistance to genital HSV-2 infection.

Comparative efficacy and immunogenicity of replication-defective, recombinant glycoprotein, and DNA vaccines for herpes simplex virus 2 infections in mice and guinea pigs.

Many candidate vaccines are effective in animal models of genital herpes simplex virus type 2 (HSV-2) infection. Among them, clinical trials showed moderate protection from genital disease with recombinant HSV-2 glycoprotein D (gD2) in alum-monophosphoryl lipid A adjuvant only in HSV women seronegative for both HSV-1 and HSV-2, encouraging development of additional vaccine options. Therefore, we undertook direct comparative studies of the prophylactic and therapeutic efficacies and immunogenicities of three different classes of candidate vaccines given in four regimens to two species of animals: recombinant gD2, a plasmid expressing gD2, and dl5-29, a replication-defective strain of HSV-2 with the essential genes UL5 and UL29 deleted. Both dl5-29 and gD2 were highly effective in attenuating acute and recurrent disease and reducing latent viral load, and both were superior to the plasmid vaccine alone or the plasmid vaccine followed by one dose of dl5-29. dl5-29 was also effective in treating established infections. Moreover, latent dl5-29 virus could not be detected by PCR in sacral ganglia from guinea pigs vaccinated intravaginally. Finally, dl5-29 was superior to gD2 in inducing higher neutralizing antibody titers and the more rapid accumulation of HSV-2-specific CD8+ T cells in trigeminal ganglia after challenge with wild-type virus. Given its efficacy, its defectiveness for latency, and its ability to induce rapid, virus-specific CD8(+)-T-cell responses, the dl5-29 vaccine may be a good candidate for early-phase human trials.

Isopentenyl pyrophosphate-reactive Vgamma9Vdelta 2 T helper 1-like cells are the major gammadelta T cell subset recovered from lesions of patients with genital herpes.

Herpes simplex virus (HSV)-specific T cells are essential to control and resolve genital herpes (GH). To investigate the potential involvement of gamma delta T cells in GH, T cells were recovered and expanded, by mitogenic stimulation, to T cell lines from the genital lesions of 17 patients with GH and 5 control subjects who had other diseases. Relatively high numbers of gamma delta T cells--predominantly, V gamma 9V delta 2 T cells--were detected only in the T cell lines of the patients with GH. Intralesional V gamma 9V delta 2 T cell clones did not recognize HSV-infected cells, but they showed reactivity to isopentenyl pyrophosphate and Daudi cells. The T cell clones secreted interferon- gamma, tumor necrosis factor- alpha, interleukin (IL)-8, macrophage inflammatory protein-1 alpha, and RANTES (regulated on activation, normally T cell expressed or secreted), but they secreted no or limited IL-4. The results of the present study suggest the infiltration and putative involvement of isopentenyl pyrophosphate-reactive V gamma 9V delta 2 T helper 1-like cells in individuals with GH.

Critical involvement of CD40 in protection against herpes simplex virus infection in a murine model of genital herpes.

We studied the requirement for CD40+ cells in the resolution of vaginal infection with avirulent herpes simplex virus type I (HSV-1) in vivo using CD40-deficient mice, which were susceptible to infection with avirulent HSV-1. Compared with wild-type mice, CD40-deficient mice could not eliminate HSV-1 virus effectively from the vaginal mucosa and produced lower amounts of interleukin-12 and interferon-gamma. These results show that the induction and activation of CD40+ cells are important for HSV prevention, facilitating the activation of T cells to induce an efficient HSV clearance from the vaginal mucosa and to prevent lethal illness due to HSV infection.

Does supplemental creatine prevent herpes recurrences?

While functioning as a general practitioner at the Camp Pendleton Marine Base, the first author treated numerous patients with recurrent genital herpes. Beginning in 1998, a number of these patients failed to return for periodic acyclovir therapy. Inquiries revealed that these patients had all commenced supplemental creatine after their last outbreak, and had experienced no further outbreaks. A literature search uncovered a report that cyclocreatine, a synthetic compound structurally and functionally homologous to creatine, inhibits the replication of cytomegalovirus, varicella-zoster, and herpes simplex types 1 and 2, in low millimolar concentrations; furthermore, dietary cyclocreatine reduces morbidity and mortality in mice infected with HSV-2. The fact that both creatine and cyclocreatine exert neuroprotective and cancer-retardant effects in rodents, encourages the speculation that creatine shares the anti-viral activity of cyclocreatine. Pilot studies to assess the impact of creatine loading on recurrence of oral and genital herpes appear warranted; the impact of creatine on shingles occurrence in high-risk patients could also be explored. Although initially conceived as an aid to athletic performance, creatine loading may prove to have broad preventive and therapeutic applications.

Herpes simplex virus infection of the uterine cervix--relationship with a cervical factor?

OBJECTIVE: To determine the prevalence of genital herpes simplex virus (HSV) in women of reproductive age and to evaluate a potential relation of asymptomatic HSV shedding with a cervical factor. DESIGN: Prospective study. SETTING: Outpatient infertility clinic of a university hospital. PATIENT(S): Randomly chosen asymptomatic women (n = 1,262) with a median age of 30 years. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Prevalence of cervical HSV, cervical index parameters, and other variables of CM quality, including CM penetrability in vivo and in vitro. RESULT(S): The prevalence of HSV infection of the uterine cervix was 5.2% (identified with cell culture). There was a tendency toward increased viscosity of the CM in HSV-positive women, but no significant relation with the other variables of CM quality (amount, spinnbarkeit, ferning, cervical appearance, and cellularity of the CM), or with the summarized Insler score or the cervical index according to World Health Organization guidelines. Postcoital testing and the in vitro penetration test, using either partners' or donors' semen, showed that the penetrability of the CM did not differ significantly between women with and without cervical HSV shedding. Asymptomatic cervical HSV infection was not significantly associated with bacterial colonization of the lower genital tract, with leukocyte counts in cervical secretions, with the pH of the CM or the vaginal fluid, or with antisperm antibodies in the CM. CONCLUSION(S): The results suggest that in asymptomatic women under controlled endocrine conditions, cervical HSV infection is not a significant cause of impaired quality and penetrability of the CM.

Herpes. Atypical clinical manifestations.

Herpes simplex viruses (HSV) 1 and 2 are responsible for genital herpes which is usually recognized as vesicles that ulcerate and eventually heal but recur periodically. Atypical genital herpes is often described in immunocompromised patients and can present as large, chronic, hyperkeratotic ulcers. Acyclovir-resistant HSV is occasionally isolated from such ulcers. Most cases of HSV infection reproduce subtle signs and symptoms, or more commonly, asymptomatic viral shedding. Such subclinical presentations may be responsible for most of the 30% increase in the prevalence of genital herpes in the United States during the past two decades.

Severe genital herpes infections in HIV-infected individuals with impaired herpes simplex virus-specific CD8+ cytotoxic T lymphocyte responses.

The specific mechanisms underlying the varied susceptibility of HIV-infected (HIV+) individuals to opportunistic infections (OI) are still incompletely understood. One hypothesis is that quantitative differences in specific T cell responses to a colonizing organism determine the development of an AIDS-defining OI. We evaluated this hypothesis for herpes simplex virus (HSV) infection, a common OI in HIV+ patients. Using limiting dilution analyses, the frequency of HSV-specific CD8+ cytotoxic T lymphocyte precursors (pCTL) and proliferative precursors were quantitated in peripheral blood mononuclear cells from 20 patients coinfected with HIV and HSV-2. The frequency of HSV-specific CD8+ pCTL in HSV+HIV+ individuals was significantly lower than in HSV+HIV- individuals (1 in 77,000 vs. 1 in 6,000, P = .0005) and was not different than in HSV-HIV- individuals (1 in 100,000, P = .24). HIV+ patients who suffered more severe genital herpes recurrences had significantly lower HSV-specific CD8+ pCTL frequencies than those patients with mild recurrences (1 in 170,000 vs. 1 in 26,000, P = .03). In contrast, no significant difference was seen in proliferative precursor frequencies between those patients with mild vs. severe genital herpes (1 in 3,800 vs. 1 in 6,600, P > .5). Quantitative differences in pCTL frequency to HSV appear to be the most important host factor influencing the frequency and severity of HSV reactivation in HIV+ patients. Studies to reconstitute such immunity, especially in people with acyclovir-resistant HSV, appear warranted.

Herpes genital na gravidez / Genital herpes in pregnancy

Os autores fazem uma atualizaçÝoda infecçÝo das gestantes pelo herpes vírus simples tipo 2, mostrando a sua fisiopatologia, epidemiologia, classificaçÝo, diagnóstico, conduta obstétrica e uso de aciclovir na gravidez, identificando e discutindo soluçSes para questSes ainda nÝo resolvidas. Face ao aumento progressivo desta afecçÝo, é necessária uma permanente atualizaçÝo, buscando uma diminuiçÝo da morbidade nestas gestantes e, consequentemente, morbidade e mortalidade fetais

Asymptomatic vaginal herpes simplex virus infections in mice: virology and pathohistology.

One of the causes of genital tract infections in humans are herpes simplex virus types 1 and 2 (HSV-1, HSV-2). Although primary and recurrent infections can be clinically apparent and in part very serious, many infections are asymptomatic and result only in temporary genital shedding of virus (recurrences). During our investigations of vaginitis, strain IES of HSV-1 produced an asymptomatic infection. Replication in the murine vaginal (vag.) epithelium as well as antibody formation after vag. infection was comparable to those of survivors after infection with highly virulent strains. Titration of liver, spleen, ovaries, adrenal glands spinal cord, or brain after vag. IES infection revealed no virus, whereas after i.p. infection virus could be demonstrated in many organs examined. Histological examination with a DNA probe (in situ hybridisation), HSV antibodies (immunohistochemistry), and haematoxylin and eosin (HE) staining showed only small focal HSV lesions of the vaginal epithelium in early stages of the infection, never exceeding to the subepithelial tissue. Severe infiltrations and ulcerations after infection with highly virulent strains (17syn +, ER-) could never be demonstrated after IES vag. infection. Identical replication rates of both groups of HSV despite much greater areas of epithelial necrosis with the virulent strains may be explained by the large number of virus inactivating granulocytes induced by the virulent strains, thus inactivating the hypothetical higher virus load.