Herpes Simplex Virus Research Articles

Seroprevalence of Neonatal Herpes Simplex Virus Infection at A Tertiary Teaching Hospital in Malaysia

INTRODUCTION: Neonatal herpes simplex virus (HSV) infection is generally infrequent, despite being a life-threatening illness. Knowledge of its prevalence is limited in Malaysia since most cases are asymptomatic infections and only limited routine neonatal HSV screening is conducted. This study therefore provides a comprehensive investigation of the seroprevalence of HSV-1 and HSV-2 in neonates. MATERIALS AND METHODS: Serological screening for HSV-1 IgG and HSV-2 IgG antibody tests using the Electrochemiluminescence assay was performed on serum samples of 215 neonates delivered from January until December 2022 at Hospital Universiti Sains Malaysia. RESULTS: Of the neonates, 54.4% were found to be HSV-1 positive, while 4.2% were HSV-2 positive. All the HSV-2 neonates were co-infected with HSV-1. Newborns aged 0–10 days were the most infected group by HSV-1 (92.3%) and HSV-2 (55.6%). The most reported clinical presentation was small gestational age (SGA) (60%). Microcephaly and macrocephaly were observed in one neonate each. The clinical presentations of reactive HSV-1 and HSV-2 cases revealed the presence of fever with rash in both cases. CONCLUSION: The high seroprevalence of HSV-1 is alarming. It is hoped that these data will support the advocacy of screening women for HSV before or during pregnancy as a precautionary approach to reducing the risk of vertical transmission.

Novel Mouse Model of Recurrent Sublethal Herpes Simplex Virus Infection Recapitulates Human Antibody Responses to Primary and Chronic Infection.

Background: Herpes simplex virus (HSV) vaccine development has been impeded by the absence of predictive preclinical models and defined correlates of immune protection. Prior candidates elicited neutralizing responses greater than natural infection but no antibody-dependent cellular cytotoxicity (ADCC) and failed to protect in clinical trials. Primary HSV infection also elicits only neutralizing responses, but ADCC and an expanded antigenic repertoire emerge over time. This evolution may contribute to the decreased frequency and severity of recurrences. To test this notion, we developed a recurrent HSV infection mouse model and evaluated changes in humoral immunity with repeated challenges. Methods: Mice were repeatedly infected intranasally with clinical isolates of HSV-1 or HSV-2 for four months. HSV binding IgG, neutralizing (with or without complement) and ADCC-mediating antibodies were quantified prior to each round of infection. Viral targets were assessed by western blotting. Pooled immune serum (750 μg IgG per mouse) was passively transferred into naïve wild-type or Hvem knockout mice 24 h prior to lethal skin challenge. Results: Repeated exposure to HSV-1 or HSV-2 induced an increase in total HSV-binding IgG but did not boost neutralizing titers. In contrast, ADCC-mediating responses increased significantly from the first to the fourth viral exposure (p < 0.01). The increase was associated with an expanded antigenic repertoire. Passive transfer of fourth round immune serum provided significant protection whereas first round serum failed to protect (p < 0.01). However, protection was lost when serum was transferred into Hvem knockout mice, which are impaired in mediating ADCC killing. Conclusion: This novel model recapitulates clinical responses, highlights the importance of ADCC in protecting against recurrent infection, and provides a strategy for evaluating therapeutic vaccines.

Disseminated Coinfections of Cytomegalovirus and Herpes Simplex Virus in Lupus Nephritis

Disseminated Coinfections of Cytomegalovirus and Herpes Simplex Virus in Lupus Nephritis

Self-reported sexually transmitted infections and associated risk factors among female university students.

The spread of sexually transmitted infections (STIs) is an ongoing public health challenge, and awareness of risk factors is essential for designing effective preventive interventions. This study aimed to assess self-reported STI occurrences and identify risk factors and sexual behaviors associated with STIs among female university students. This is a cross-sectional, online questionnaire study, including 384 female university students seeking contraceptive counseling at a gynecology clinic in Uppsala, Sweden, and reporting having had sex. Associated risk factors and behaviors were assessed by comparing those who reported STIs and those who did not. The mean age of participants was 22.8 years. Seventy-eight (20%) had contracted at least one STI, with seven (9%) experiencing multiple infections. Seventy-three (94%) reported first-date sexual activity without a condom among STI experienced. Chlamydia trachomatis was the most common STI pathogen (68% of all infections), followed by Herpes simplex virus (18%) and Mycoplasma genitalium (13%). Behavioral factors associated with self-reported STIs were first-date sexual activity without a condom, not using condom at first intercourse, younger age at first intercourse, a higher number of sexual partners overall and in the last 12 months, experience of anal sex, dating app usage, and regretting sexual activity after substance use (P < 0.003 for all). Condom use was low among the respondents, and STIs were common regardless of the high level of education in this group. Contraceptive counseling needs to highlight the importance of condom use in addition to contraceptive efficacy. It is also essential to consider the specific risk factors and behaviors prevalent among young adults to reduce the spread of STIs.

S4659 Navigating Diagnostic Challenges: Herpes Simplex Virus as an Uncommon Cause of Acute Liver Failure in a Low-Risk Patient

S4659 Navigating Diagnostic Challenges: Herpes Simplex Virus as an Uncommon Cause of Acute Liver Failure in a Low-Risk Patient

Acute Retinal Necrosis: A Review of Diagnosis and Management

Acute retinal necrosis (ARN) is a profound infection of the retina, marked by acute panuveitis, retinal periarteritis, and widespread necrotizing retinitis. The etiology of ARN involves human herpesviruses, such as herpes simplex virus (HSV) and varicella-zoster virus (VZV), which can lead to severe visual impairment or even blindness. A diagnosis of ARN is based on clinical characteristics and disease progression according to the standard diagnostic criteria established by the American Uveitis Society (AUS) in 1994. The polymerase chain reaction(PCR) of aqueous specimens can enable identification of the type of virus. Early initiation of antiviral medication is essential for treatment efficacy to stop lesion progression, accelerate the healing process, and prevent contralateral eye involvement. Ocular complications of ARN include atrophic retina, multiple retinal breaks, rhegmatogenous retinal detachment (RRD), tractional retinal detachment (TRD), optic atrophy, macular edema, epiretinal membrane (ERM), and retinal and optic disc neovascularization. This review summarizes the clinical features, diagnostic criteria, and recently recommended ARN management.

An improved high-resolution method for quantitative separation of empty and filled AAV8 capsids by strong anion exchange HPLC.

Cell and gene therapy (CGT) is a field of therapeutic medicine that aims to treat, prevent, and cure diseases using engineered cells (stem cells, immune cells, and differentiated adult or fetal cells), vectors [Adeno Associated Virus (AAV), Adeno Virus (AV), Herpes Simplex Virus (HSV), Baculo Virus (BV), Lenti Virus (LV), Retro Virus (RV), etc.], and other carriers [non-viral vectors, virus-like particles (VLP), Lipid Nano-Particles (LNP), etc.]. Among viral CGT vectors, adeno-associated viruses and lentiviruses (AAV and LV) are the most widely applied vector platforms. The presence of non-functional (empty or non-infectious) vectors that carry null or partial genes in the final drug product is classified as an impurity by the FDA. These impurities impair dosage accuracy and induce non-specific immunogenicity and variability in drug efficacy. These non-functional viral vectors in the drug product need to be elucidated following International Conference on Harmonization (ICH) guidelines for clinical manufacturing of the final drug product. This article showcases an ion-exchange chromatography (IEX) high-resolution method supporting ICH guidelines using commercially available AAV8 filled and empty capsids as reference standards. Our method successfully separated empty to full capsids with a resolution of 15 and sustained a linearity greater than 0.98 even under a wide range of empty or full viral particle concentrations (E+9 to E+13 vp/mL), which is an upgrade to other IEX capsid separation methods. The medium-throughput capacity and shorter sample processing time improve testing efficiency and save costs while delivering quality as value. The discussed method is a reliable and reproducible platform to precisely evaluate the presence of non-functional viral particles in AAV8 samples. Aligned with other orthogonal results, the method is a powerful tool to improve the quality of rAAV analytics.

Frequent Evaluation of Herpes Simplex Virus Type 2 in Women with Genital Herpes by Realtime PCR

Background: Herpes simplex type 2 is a common infection worldwide. This disease is common in both developed and developing countries. Early detection of infection is very important to reduce the risk of infection. Real-time reliable PCR is a very sensitive and specific method that can be used as the best marker in determining the therapeutic effect by identifying a viral genome in an individual. The prevalence of herpes simplex virus type 2 in women with genital herpes was evaluated by Real time PCR method. Methods: From January 1999 to March 2010, 45 samples of vaginal swabs and cervix of women with genital herpes were examined for HSV virus DNA detection using Real Time PCR. Results: The mean age of the patients was 35.9 + 5.9. The percentage of positive cases of herpes simplex virus type 2 in the studied women was 22.2% and the history of infection with hpv was 33.3% vs. 12.5%. = 0.094 which was significant. Conclusion: Clinical specimens of vaginal swabs from genital herpes caused by herpes simplex virus 2 can be quantitatively analyzed instead of nucleic acid extraction and amplification by PCR.

The antimicrobial protein RNase 7 directly restricts herpes simplex virus infection of human keratinocytes.

Approximately 22% of moderately to severely affected atopic dermatitis (AD) patients have a history of eczema herpeticum, a disseminated rash primarily caused by herpes simplex virus type 1 (HSV-1). Reduced activity of antimicrobial peptides may contribute to the increased susceptibility of AD patients to HSV-1. We previously demonstrated that the antimicrobial protein RNase 7 limits HSV-1 infection of human keratinocytes by promoting self-DNA sensing. Here, we addressed whether RNase 7 has any effect on HSV-1 infection when infecting keratinocytes without exogenously added costimulatory DNA, and which step(s) of the infection cycle RNase 7 interferes with. We quantified viral gene expression by RT-qPCR and flow cytometry, viral genome replication by qPCR, virucidal effects by plaque titration, and plaque formation and the subcellular localization of incoming HSV-1 particles by microscopy. Recombinant RNase 7 restricted HSV-1 gene expression, genome replication, and plaque formation in human keratinocytes. It decreased HSV-1 immediate-early transcripts independently of the induction of interferon-stimulated genes. Its main effect was on intracellular infection processes and not on extracellular virions or virus binding to cells. RNase 7 reduced the amount of cell-associated capsids and the HSV-1 envelope glycoprotein D at 3 but not at 0.5 h postinfection. Our data show that RNase 7 directly restricts HSV-1 infection of human keratinocytes, possibly by promoting the degradation of incoming HSV-1 particles. This suggests that RNase 7 may limit HSV-1 spread in the skin and that mechanisms that reduce its activity in the lesional skin of AD patients may increase their susceptibility to eczema herpeticum.

An STI rarely comes alone-An overview of sexually transmitted infections

Sexually transmitted infections (STI) or sexually transmitted diseases (STD) can be caused by bacteria, viruses, fungi, protozoa and arthropods. The most frequent bacterial STIs include Chlamydia infections, syphilis and gonorrhea; viral STIs include herpes genitalis (herpes simplex virus 1 and 2), human papillomavirus (HPV) and human immunodeficiency virus (HIV). An empathetic medical history taking in cases of a clinical suspicion can provide important information for adequate diagnostics. When an STI is detected, a complete screening for other STIs should be carried out. The STIs result in awide variety of symptoms and it is often difficult to make a clear diagnosis to a certain pathogen based on the clinical symptoms. To avoid reinfections, it is recommended to also test recent sexual partners. For certain pathogens highly effective postexposure prophylaxis or preventive vaccinations are available.

Mitochondria play a critical role in the replication of Herpes simplex virus of type 1

Mitochondria play a critical role in the replication of Herpes simplex virus of type 1

Single-cell RNA Sequencing Reveals the Diversity of the Immunological Landscape Response to Genital Herpes

Genital herpes (GH) is a common sexually transmitted disease, which is primarily caused by herpes simplex virus type 2 (HSV-2), and continues to be a global health concern. Although our understanding of the alterations in immune cell populations and immunomodulation in GH patients is still limited, it is evident that systemic intrinsic immunity, innate immunity, and adaptive immunity play crucial roles during HSV-2 infection and GH reactivation. To investigate the mechanisms underlying HSV-2 infection and recurrence, single-cell RNA sequencing (scRNA-seq) was performed on immune cells isolated from the peripheral blood of both healthy individuals and patients with recurrent GH. Furthermore, the systemic immune response in patients with recurrent GH showed activation of classical monocytes, CD4+ T cells, natural killer cells (NK cells), and plasmacytoid dendritic cells (pDCs), especially of genes associated with the Toll-like receptor signaling pathway and T cell activation. Circulating immune cells in GH patients show higher expression of genes associated with inflammation and antiviral responses both in the scRNA-Seq data set and in independent quantitative real-time polymerase chain reaction (qRT-PCR) analysis and ELISA experiments. This study demonstrated that localized genital herpes, resulting from HSV reactivation, may influence the functionality of circulating immune cells, suggesting a potential avenue for future research into the role of systemic immunity during HSV infection and recurrence.

Specific clinical and radiological characteristics of anti-NMDA receptor autoimmune encephalitis following herpes encephalitis.

Herpes simplex virus encephalitis (HSE) frequently triggers secondary anti-N-methyl-D-aspartate receptor encephalitis (NMDARE), but markers predicting the occurrence of this entity (HSE-NMDARE) are lacking. We conducted a retrospective description of patients with HSE-NMDARE diagnosed between July 2014 and August 2022 and compared them to both patients with regular forms of HSE and NMDARE. Among the 375 patients with NMDARE, 13 HSE-NMDARE were included. The median age was 19years (0.5-73), 4/13 (31%) were children < 4years old, and 7/13 (54%) were male. The median time between HSE and NMDARE onset was 30days (21-46). During NMDARE, symptoms differed from HSE, including increased behavioral changes (92% vs 23%, p = 0.008), movements disorders (62% vs 0%, p = 0.013), and dysautonomia (54% vs 0%, p = 0.041). Compared to 21 patients with regular HSE, patients with HSE-NMDARE more often achieved severity-associated criteria on initial MRIs, with extensive lesions (11/11, 100% vs 10/21, 48%, p = 0.005) and bilateral diffusion-weighted imaging sequence abnormalities (9/10, 90% vs 6/21, 29%, p = 0.002). Compared to 198 patients with regular NMDARE, patients with HSE-NMDARE were more frequently males (7/13, 54% vs 43/198, 22%; p = 0.015) and children < 4 (4/13, 31% vs 14/198, 7%; p = 0.016), with a worse 12-month mRS (2[1-6] vs 1[0-6], p = 0.023). Herein, patients with HSE-NMDARE have a poorer long-term prognosis than patients with regular NMDARE. We report a greater rate of severity-associated criteria on initial MRIs for HSE-NMDARE compared to regular HSE, which may help identify patients with higher risk of HSE-NMDARE.

Quality of Caregiver-Adolescent Relationship on HIV, Herpes Simplex Virus Type-2, and on Pregnancy Among Young Women in South Africa Enrolled in HIV Prevention Trials Network 068

PurposeAdolescent girls and young women (AGYW) are at an increased risk of acquiring HIV and HSV-2, and unintended pregnancies are high in AGYW. Despite the protective impact of caregiver-adolescent relationships on risk behaviors, less attention has been paid to the association of these relationships and sexually transmitted infections (STIs) and pregnancy. MethodsWe used longitudinal data from HIV Prevention Trial Network 068, which was conducted among 2,533 AGYW (13 – 20 years) over 5 years in Agincourt, South Africa. Kaplan-Meier and Cox models were used to estimate the effect of quality of caregiver-adolescent relationships (caring and closeness) on STIs and pregnancy. Also, we assessed effect measure modification by age (14–19 vs. 20–25 years) for STI risk using stratum-specific estimates and likelihood ratio tests, with a p value <.1 indicative of effect measure modification. ResultsThere were no significant differences in the hazard of HIV by our exposures (caring: hazard ratio (HR): 1.03, 95% CI: 0.75, 1.42; closeness: HR: 0.80, 95% CI: 0.57, 1.11). Among 14–19-year-olds, those who reported caregiver caring were less likely to acquire HSV-2 (HR: 0.69, 95% CI: 0.51, 0.94, likelihood ratio tests= 3.89, p-value = .0487), in contrast, there were no significant differences among 20 – 25-year-olds. AGYW who reported high-quality relationships had a lower hazard of pregnancy incidence (caring: HR: 0.79, 95% CI: 0.68, 0.93; closeness: HR: 0.76; 95% CI: 0.64, 0.91). DiscussionPositive caregiver-adolescent relationships are associated with reduced risk of HSV-2 among younger AGYW and pregnancy incidence.

Joint British Association for Sexual Health and HIV and Royal College of Obstetricians and Gynaecologists national UK guideline for the management of herpes simplex virus (HSV) in pregnancy and the neonate (2024 update).

This updated national UK guideline offers recommendations on the management of genital herpes simplex virus (HSV) in mothers and pregnant people during pregnancy and within 4 weeks following birth. It includes recommendations for first episode and recurrent HSV, HSV in preterm pre-labour rupture of membranes and in co-infection with HSV and HIV. Recommendations around management of the neonate are made, on prevention of postnatal transmission, management of breastfeeding, and the management of clinically discordant couples. This guideline is aimed at healthcare professionals working in sexual health clinics, maternity units, and those working on postnatal wards and neonatal units in the UK. However, the principles of the recommendations should be adopted across all services, including community care.

Monitoring the Seroprevalence of Herpes Simplex Virus-1 Among Egyptian Hemodialysis Patients

Background: Hemodialysis patients are at high risk for infections due to their compromised immune systems. Herpes Simplex Virus (HSV) infections can lead to severe complications in this population. Despite its clinical significance, comprehensive data on the seroprevalence of HSV among hemodialysis patients are limited. Objective: This study aims to evaluate the seroprevalence of HSV-1 among Egyptian hemodialysis patients. Methods: A cross-sectional study was conducted with 218 participants (116 females and 102 males). Among these, 132 (60.6%) were undergoing hemodialysis, while 86 (39.4%) had normal kidney function and were free from HCV, CMV, and HIV infections. Serum samples were analyzed for IgG antibodies against HSV-1. Statistical analyses were performed to determine HSV seroprevalence. Results: The study observed a significantly higher prevalence of HSV-1-IgG antibodies in hemodialysis patients (84.1%) compared to the control group (66.3%, P&lt;0.01). Additionally, HSV-1-IgM positivity was also higher in the hemodialysis group (41.6%) compared to the control group (32.6%, P&lt;0.05). These findings highlight the need for vigilant monitoring and preventive strategies for HSV infections in hemodialysis patients.

Clinicopathological Study of Herpes Simplex Esophagitis in a Tertiary Centre in India

Introduction and objectives: Herpes Simplex Virus (HSV) is a common viral pathogen, known to cause symptomatic disease in the immunocompromised hosts; however, it may occur in healthy individuals as well. Among the varied manifestations of the disease, involving skin, central nervous system and gastrointestinal systems, HSV esophagitis is a less studied entity from the Indian perspective. The aim of the study is to compare clinical, histologic and serologic data of HSV esophagitis patients. Materials and methods: 27 cases, proved to have HSV esophagitis on endoscopic biopsy were included, and the pertinent clinical and serologic characteristics were studied. Results: We found a male preponderance (p&lt;0.01), which has been a well-established risk factor. Other risk factors are retroviral coinfection, Type 2 Diabetes Mellitus, immunosuppression, chemoradiation etc. among others. Eleven patients had retroviral coinfection, with CD4 counts of &gt; 200/μL in all. Anti HSV 1 IgM antibody was most commonly detected on serology. HSV esophagitis affects immunocompromised patients more often than immunocompetent ones. Odynophagia and dysphagia are the most common symptoms. Histological evaluation for the characteristic inclusions helps in early diagnosis. The novelty of this study rests on the clinicopathological and serologic correlation, for a better understanding of the disease process, to prompt future large scale studies on the same. Conclusion: Herpes simplex esophagitis is most seen in immunocompromised patients. Apart from retroviral illness, other risk factors include diabetes mellitus, patients undergoing chemotherapy and other malignancy. CD4 counts in our series was &gt;200/μL, in contrast to the cutoff of &lt;200/μL described in literature.

Unveiling the causal link between herpes virus infection and cutaneous leukocytoclastic angiitis: Insights from Mendelian randomization analysis

AbstractBackgroundCutaneous leukocytoclastic angiitis (CLA) is a clinically relevant condition, with previous studies suggesting an association with herpes virus infections. However, the causality of this association remains unclear. This study aimed to investigate the causal relationship between herpes viruses and CLA.MethodsGenetic variants linked to the herpes virus were retrieved from the Integrative Epidemiology Unit at the University of Bristol open genome‐wide association studies project and FinnGen database. Data on CLA, involving 262 CLA cases and 207,482 healthy controls, were obtained from the FinnGen consortium R7. Mendelian randomization (MR) analysis, including the inverse variance weighted (IVW), MR‐Egger, and weighted median methods, was performed. Sensitivity analyzes were conducted to ensure the accuracy of the results.ResultsOf the 15 herpes viruses investigated, only human herpesvirus 6 (HHV‐6) demonstrated a causal association with CLA (odds ratio: 1.886, 95% confidence interval: 1.053–3.378, p = 0.033), indicating that HHV‐6 infection significantly increases the risk of CLA. Furthermore, IVW and MR‐Egger tests for heterogeneity confirmed homogeneous MR analysis results without evidence of horizontal pleiotropy (p &gt; 0.05). No significant causal relationship was observed for other herpes viruses, such as herpes simplex virus, varicella‐zoster virus, cytomegalovirus, and Epstein‐Barr virus.ConclusionOur MR analyzes strongly support a causal relationship between HHV‐6 and CLA, elucidating the etiology of this condition and highlighting the potential of HHV‐6‐targeted therapeutic interventions in CLA treatment. However, further research is necessary to expound the underlying mechanisms and explore potential therapeutic interventions targeting HHV‐6‐associated CLA.

Prevalence of bacterial vaginosis, sexually transmitted infections and their association with HPV infections in asymptomatic women attending antenatal care in Ethiopia.

Sexually transmitted infections (STIs) and human papillomavirus (HPV) infections are common among women of reproductive age and can lead to infertility, adverse pregnancy outcomes, neonatal infections and cervical cancer. In countries with limited medical coverage, untreated infections contribute to high morbidity. This study aimed to expand the current knowledge on the prevalence of bacterial vaginosis (BV) and STIs in pregnant Ethiopian women and assess the association of these conditions with HPV infections. Socio-demographic data and vaginal lavage samples were collected from 779 asymptomatic women aged 18 to 45 years (median age, 25.9 years) attending antenatal care in seven centres across Ethiopia. Multiplex polymerase chain reaction was used to test for BV, Chlamydia trachomatis, Trichomonas vaginalis, Neisseria gonorrhoeae, herpes simplex virus types 1 and 2 (HSV-1/2), Mycoplasma, Ureaplasma, Candida species and HPV. Overall, 26.8% (95% confidence interval (CI): 23.7-29.9) of women tested positive for BV or one of the following STIs: C. trachomatis, T. vaginalis, N. gonorrhoeae, Mycoplasma genitalium, HSV-1/2 or Ureaplasma urealyticum. Additionally, 22.1% tested positive for at least one high-risk HPV type. Chlamydia trachomatis and HSV-2 were significantly more common among women who were positive for HPV and high-risk HPV. This study reveals a high prevalence of asymptomatic pregnant women who are positive for BV, STIs or HPV, putting them at risk of adverse pregnancy outcomes, secondary infertility or cervical cancer in a country with limited medical coverage. Screening and treating these women could be crucial in reducing morbidity.

Development and characterization of formulations based on combinatorial potential of antivirals against genital herpes.

Herpes simplex virus type 2 (HSV-2) treatment faces challenges due to antiviral resistance and systemic side effects of oral therapies. Local delivery of antiviral agents, such as tenofovir (TDF) and zinc acetate dihydrate (ZAD), may offer improved efficacy and reduced systemic toxicity. This study's objective is to develop and evaluate local unit dose formulations of TDF and ZAD combination for local treatment of HSV-2 infection and exploring their individual and combinatory effects in vitro. The study involved the development of immediate-release film and pessary formulations containing TDF and ZAD. These formulations were characterized for physicochemical properties and in vitro drug release profiles. Cytotoxicity and antiviral activity assays were conducted to evaluate the individual and combinatory effects of TDF and ZAD. Film formulations released over 90% of the drugs within 1h, and pessary formulations within 90min, ensuring effective local drug delivery. ZAD showed moderate antiviral activity while TDF exhibited significant antiviral activity at non-cytotoxic concentrations. The combination of TDF and ZAD demonstrated synergistic effects in co-infection treatments, reducing the concentration required for 50% inhibition of HSV-2. Developed film and pessary formulations offer consistent and predictable local drug delivery, enhancing antiviral efficacy while minimizing systemic side effects. The combination of TDF and ZAD showed potential synergy against HSV-2, particularly in co-infection treatments. Further preclinical studies on pharmacokinetics, safety, and efficacy are necessary to advance these formulations toward clinical application.