Green tea extract reduces viral proliferation and ROS production during Feline Herpesvirus type-1 (FHV-1) infection.

BACKGROUND: Feline Herpesvirus type-1 (FHV-1) is a worldwide spread pathogen responsible for viral rhinotracheitis and conjunctivitis in cats that, in the most severe cases, can lead to death. Despite the availability of a variety of antiviral medications to treat this illness, mainly characterized by virostatic drugs that alter DNA replication, their use is often debated. Phytotherapeutic treatments are a little-explored field for FHV-1 infections and reactivations. In this scenario, natural compounds could provide several advantages, such as reduced side effects, less resistance and low toxicity. The purpose of this study was to explore the potential inhibitory effects of the green tea extract (GTE), consisting of 50% of polyphenols, on FHV-1 infection and reactive oxygen species (ROS) production. RESULTS: Crandell-Reese feline kidney (CRFK) cells were treated with different doses of GTE (10-400 µg/mL) during the viral adsorption and throughout the following 24 h. The MTT and TCID50 assays were performed to determine the cytotoxicity and the EC50 of the extract, determining the amounts of GTE used for the subsequent investigations. The western blot assay showed a drastic reduction in the expression of viral glycoproteins (i.e., gB and gI) after GTE treatment. GTE induced not only a suppression in viral proliferation but also in the phosphorylation of Akt protein, generally involved in viral entry. Moreover, the increase in cell proliferation observed in infected cells upon GTE addition was supported by enhanced expression of Bcl-2 and Bcl-xL anti-apoptotic proteins. Finally, GTE antioxidant activity was evaluated by dichloro-dihydro-fluorescein diacetate (DCFH-DA) and total antioxidant capacity (TAC) assays. The ROS burst observed during FHV-1 infection was mitigated after GTE treatment, leading to a reduction in the oxidative imbalance. CONCLUSIONS: Although further clinical trials are necessary, this study demonstrated that the GTE could potentially serve as natural inhibitor of FHV-1 proliferation, by reducing viral entry. Moreover, it is plausible that the extract could inhibit apoptosis by modulating the intrinsic pathway, thus affecting ROS production.

Cross-Sectional and Longitudinal Associations Between Treatment for Herpes Virus Infection and the Dispensing of Antidementia Medicines: An Analysis of the Australian Pharmaceutical Benefits Scheme Database.

Background: Evidence from previous observational studies suggest that infection by herpes simplex virus (HSV) and varicella zoster virus (VZV) increase the risk of dementia. Objective: To investigate if older adults exposed to HSV treatment have lower risk of dementia than the rest of the population. Methods: We used the 10% Australian Pharmaceutical Benefits Scheme (PBS) database from 2013 to 2022 to ascertain the cross-sectional, time-series and longitudinal association between exposure to HSV treatment and the dispensing of antidementia medicines. Participants were men and women aged 60 years or older. We used Anatomical Therapeutic Chemical (ATC) codes to identify medicines dispensed for the treatment of HSV and dementia. Results: During the year 2022 6,868 (1.2%) of 559,561 of participants aged 60 years or over were dispensed antidementia agent. The odds ratio (OR) of being dispensed an antidementia agent among individuals dispensed treatment for HSV was 0.73 (99% CI = 0.56-0.95). Multilevel logistic regression for the 2013-2022 period for those dispensed HSV treatment was 0.87 (99% CI = 0.75-1.00). Split-time span series from 2013 was associated with hazard ratio of 0.98 (99% CI = 0.89-1.07) for individuals dispensed relative to those not dispensed HSV treatment. All analyses were adjusted for age, sex, and the dispensing of medicines for the treatment of diabetes, hyperlipidemia, hypertension, and ischemic heart disease. Conclusions: The dispensing of antiviral medicines for the treatment of HSV and VZV is consistently, but not conclusively, associated with decreased dispensing of antidementia medicines. This suggests that treatment of HSV and VZV infections may contribute to reduce the risk of dementia.

Rutin prevents EqHV-8 induced infection and oxidative stress via Nrf2/HO-1 signaling pathway.

Introduction: The Nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling pathway has been extensively studied for its role in regulating antioxidant and antiviral responses. The Equid herpesvirus type 8 (EqHV-8) poses a significant threat to the equine industry, primarily manifesting as respiratory disease, abortions, and neurological disorders in horses and donkeys. Oxidative stress is considered a key factor associated with pathogenesis of EqHV-8 infection. Unfortunately, there is currently a dearth of therapeutic interventions available for the effective control of EqHV-8. Rutin has been well documented for its antioxidant and antiviral potential. In current study we focused on the evaluation of Rutin as a potential therapeutic agent against EqHV-8 infection. Methods: For this purpose, we encompassed both in-vitro and in-vivo investigations to assess the effectiveness of Rutin in combatting EqHV-8 infection. Results and Discussion: The results obtained from in vitro experiments demonstrated that Rutin exerted a pronounced inhibitory effect on EqHV-8 at multiple stages of the viral life cycle. Through meticulous experimentation, we elucidated that Rutin's antiviral action against EqHV-8 is intricately linked to the Nrf2/HO-1 signaling pathway-mediated antioxidant response. Activation of this pathway by Rutin was found to significantly impede EqHV-8 replication, thereby diminishing the viral load. This mechanistic insight not only enhances our understanding of the antiviral potential of Rutin but also highlights the significance of antioxidant stress responses in combating EqHV-8 infection. To complement our in vitro findings, we conducted in vivo studies employing a mouse model. These experiments revealed that Rutin administration resulted in a substantial reduction in EqHV-8 infection within the lungs of the mice, underscoring the compound's therapeutic promise in vivo. Conclusion: In summation, our finding showed that Rutin holds promise as a novel and effective therapeutic agent for the prevention and control of EqHV-8 infections.

A Screening Study Identified Decitabine as an Inhibitor of Equid Herpesvirus 4 That Enhances the Innate Antiviral Response.

Equid herpesvirus 4 (EHV-4) is a common respiratory pathogen in horses. It sporadically induces abortion or neonatal death. Although its contribution in neurological disorders is not clearly demonstrated, there is a strong suspicion of its involvement. Despite preventive treatments using vaccines against EHV-1/EHV-4, the resurgence of alpha-EHV infection still constitutes an important threat to the horse industry. Yet very few studies have been conducted on the search for antiviral molecules against EHV-4. A screening of 42 antiviral compounds was performed in vitro on equine fibroblast cells infected with the EHV-4 405/76 reference strain (VR2230). The formation of cytopathic effects was monitored by real-time cell analysis (RTCA), and the viral load was quantified by quantitative PCR. Aciclovir, the most widely used antiviral against alpha-herpesviruses in vivo, does not appear to be effective against EHV-4 in vitro. Potential antiviral activities were confirmed for eight molecules (idoxuridine, vidarabine, pritelivir, cidofovir, valganciclovir, ganciclovir, aphidicolin, and decitabine). Decitabine demonstrates the highest efficacy against EHV-4 in vitro. Transcriptomic analysis revealed the up-regulation of various genes implicated in interferon (IFN) response, suggesting that decitabine triggers the immune antiviral pathway.

Medical management and positive outcome after prolonged recumbency in a case of equine herpesvirus myeloencephalopathy.

A 17-year-old mare presenting with acute fever, weakness and bladder dysfunction was diagnosed with equine herpesvirus myeloencephalopathy (EHM). The mare become transiently recumbent, underwent parenteral fluid therapy, plasma infusion, steroidal/nonsteroidal anti-inflammatory drugs (SAID/NSAIDs) and bladder catheterization. After 10 days the mare was hospitalized. Neurological evaluation revealed ataxia and proprioceptive deficits mainly in the hind limbs. The mare was able to stand but unable to rise from recumbency or walk. Secondary complications included Escherichia coli cystitis, corneal ulcers and pressure sores. A full-body support sling was used for 21 days. Medical treatment included systemic antimicrobials, NSAIDs, gradual discontinuation of SAIDs, parenteral fluid therapy and bladder lavage. The mare tested positive for Varicellovirus equidalpha 1 (EHV-1) DNA in nasal swab and blood samples on day 13 and in urine samples on days 13 and 25 after the onset of fever. Neurological signs improved over a period of 34 days and the mare was discharged with mild hind limb weakness/ataxia. Secondary complications resolved within 2 weeks. At the eight-month follow-up, marked improvement in locomotory function had been achieved.

Pharmacologic interventions for the treatment of equine herpesvirus-1 in domesticated horses: A systematic review.

BACKGROUND: Equine herpes virus type 1 (EHV-1) infection in horses is associated with upper respiratory disease, neurological disease, abortions, and neonatal death. REVIEW QUESTION: Does pharmacological therapy decrease either the incidence or severity of disease or infection caused by EHV-1 in domesticated horses? METHODS: A systematic review was preformed searching AGRICOLA, CAB Abstracts, Cochrane, PubMed, Web of Science, and WHO Global Health Index Medicus Regional Databases to identify articles published before February 15, 2021. Selection criteria were original research reports published in peer reviewed journals, and studies investigating in vivo use of therapeutic agents for prevention or treatment of EHV-1 in horses. Outcomes assessed included measures related to clinical outcomes that reflect symptomatic EHV-1 infection or virus infection. We evaluated risk of bias and performed a GRADE evaluation of the quality of evidence for interventions. RESULTS: A total of 7009 unique studies were identified, of which 9 met the inclusion criteria. Two studies evaluated valacyclovir or small interfering RNAs, and single studies evaluated the use of a Parapoxvirus ovis-based immunomodulator, human alpha interferon, an herbal supplement, a cytosine analog, and heparin. The level of evidence ranged between randomized controlled studies and observational trials. The risk of bias was moderate to high and sample sizes were small. Most studies reported either no benefit or minimal efficacy of the intervention tested. CONCLUSIONS AND CLINICAL IMPORTANCE: Our review indicates minimal or limited benefit either as a prophylactic or post-exposure treatment for any of the studied interventions in the mitigation of EHV-1-associated disease outcome.

Prevention of Oncogenic Gammaherpesvirinae (EBV and HHV8) Associated Disease in Solid Organ Transplant Recipients.

Long-term risk for malignancy is higher among solid organ transplant (SOT) recipients compared to the general population. Four non-hepatitis viruses have been recognized as oncogenic in SOT recipients-EBV, cause of EBV-associated lymphoproliferative diseases; human herpes virus 8 (HHV8), cause of Kaposi sarcoma, primary effusion lymphoma and multicentric Castleman disease; human papilloma virus, cause of squamous cell skin cancers, and Merkel cell polyomavirus, cause of Merkel cell carcinoma. Two of these viruses (EBV and HHV8) belong to the human herpes virus family. In this review, we will discuss key aspects regarding the clinical presentation, diagnosis, treatment, and prevention of diseases in SOT recipients associated with the two herpesviruses.

Clinical evaluation of antiviral combination treatment in cats with feline herpesvirus-1 infection.

Feline herpesvirus-1 (FHV-1) can cause lifelong problems such as rhinotracheitis and ocular disease due to latency and reactivation in affected cats. The particular effects of antiviral drugs have been separately investigated in previous studies for decades and little is known about the combination treatment in active FHV-1 infection. Therefore, we aimed to evaluate the effects of antiviral combination on clinical effectiveness in cats with naturally occurring FHV-1 infection. 28 cats suffering from clinical signs of sneezing, nasal congestion, conjunctivitis, and eye/nose discharge were involved in this study following FHV-1 DNA detection by PCR assay in oculo-oropharyngeal samples. The treatment protocol was as follows: oral famciclovir and L-lysine, ophthalmic acyclovir, and subcutaneous amoxicillin plus clavulanic acid. The symptoms improved each day and total recovery success rate was 80% reduction in clinical scores at the end of the treatment on day 10 (p<0.001). Additionally, PCR was found to be negative for FHV-1 DNA in 82.1% of the samples after the treatment. There were mild decreases in neutrophil and monocyte counts (p>0.05). The arginine to lysine ratio decreased in favour of lysine (p<0.01). As a result, the antiviral combination treatment with famciclovir, L-lysine and ophthalmic acyclovir, and antibacterial drug appears to be clinically effective for the treatment of naturally occurring active FHV-1 infection in cats. In addition, any adverse clinical effect has not been determined associated with the antiviral combination during the study.

Features of Interferon Status and Methods of Its Correction in the Treatment of Patients with Atypical Chronic Active Herpesvirus Infections.

It is known that the imbalance of the IFN system is the cornerstone of the immunopathogenesis of atypical chronic active herpesvirus infections and is often associated not only with congenital, genetically determined defects, but also with acquired system dysregulation at different levels: receptor, molecular, at the level of a nuclear transducer of signal transmission. Based on the studied features of immunopathogenesis and the revealed disturbances in the antiviral immune defense system and the IFN system in patients with atypical chronic herpes viral infections, an integration program for correcting IFN status was developed, its clinical and immunological effectiveness was evaluated. Improved effectiveness of complex rehabilitation of immunocompromised patients with atypical chronic active herpes viral infections was demonstrated.

Current Perspectives on the Management of Herpesvirus Infections in Solid Organ Transplant Recipients.

Solid organ transplant recipients (SOTRs) are at high risk of human herpesvirus (HHV)-related morbidity and mortality due to the use of immunosuppressive therapy. We aim to increase awareness and understanding of HHV disease burden in SOTRs by providing an overview of current prevention and management strategies as described in the literature and guidelines. We discuss challenges in both prevention and treatment as well as future perspectives.

Exogenous Interleukin-37 Alleviates Hepatitis with Reduced Dendritic Cells and Induced Regulatory T Cells in Acute Murine Cytomegalovirus Infection.

Human cytomegalovirus (HCMV) infection is globally distributed, and the liver is one of the major targeting organs. So far, the mechanisms for cell and organ damage have not fully been elucidated and the treatments for the infection are mainly at symptoms. IL-37 has shown a protective role in certain inflammatory diseases. In the present study, potential protective effect of exogenous IL-37 on murine cytomegalovirus- (MCMV-) infected hepatitis was evaluated through analyses of serum transaminases, the liver histopathology and cytokine expression, and functional state of dendritic cells (DCs) and regulatory T cells (Tregs). These analyses showed a significant decrease in serum transaminase levels and a lower Ishak histopathologic score at the early stage of MCMV-infected mice with exogenous IL-37 pretreatment. The frequencies of MHC-Ⅱ, CD40, CD80, and CD86 positive DCs in the liver and spleen were decreased significantly at 7 days postinfection (dpi) in MCMV-infected mice with IL-37 pretreatment when compared with those without the pretreatment, while the total number of DCs in the liver was reduced in IL-37-pretreated mice. The induction of Tregs in the spleen was enhanced at dpi 3 with IL-37 pretreatment in MCMV-infected mice. The mRNA expression levels of cytokines in the liver were decreased significantly (IL-1ß, IL-6, IL-10, IL-4) or to some extent (TGF-ß and TNF-α). The present study suggested that exogenous IL-37 can alleviate MCMV-infected hepatitis, likely through reduced DCs and induced Tregs with a weaker cytokine storm, demonstrating its potential value in clinical management for HCMV-infected hepatitis.

Murine herpesvirus-68-related growth factors treatment correlates with decrease of p53 and HIF-1α protein levels.

Murine herpesvirus 68 (MHV-68) belongs to the subfamily Gammaherpesvirinae of the family Herpesviridae. This exceptional murine herpesvirus is an excellent model for the study of human gammaherpesvirus infections. Cells infected with MHV-68 under nonpermissive conditions for viral replication produce substances designated as MHV-68 growth factors (MHGF-68), that can cause transformation of the cells, or on the other side, turn transformed cells into normal. It was already proposed, that the MHGF-68 fractions cause transformation, disruption of the cytoskeleton and slower growth of the tumors in nude mice. Here, we examined newly extracted fractions of MHGF-68 designated as F5 and F8. Both fractions proved to inhibit the growth of the spheroids and also tumours induced in nude mice. What more, the fractions caused the decrease of the protein levels of wt p53 and HIF-1α. Decreased levels of p53 and HIF-1α activity leads to decreased vascularization, slower tumour growth, and lower adaptation to hypoxic conditions. This would propose MHGF-68 fractions, or their human herpesvirus equivalents, as a potential anticancer drugs in combined chemotherapy.

Evaluation of Histone Demethylase Inhibitor ML324 and Acyclovir against Cyprinid herpesvirus 3 Infection.

Cyprinid herpesvirus 3 (CyHV-3) can cause severe disease in koi and common carp (Cyprinus carpio). Currently, no effective treatment is available against CyHV-3 infection in koi. Both LSD1 and JMJD2 are histone demethylases (HD) and are critical for immediate-early (IE) gene activation essential for lytic herpesvirus replication. OG-L002 and ML324 are newly discovered specific inhibitors of LSD1 and JMJD2, respectively. Here, HD inhibitors were compared with acyclovir (ACV) against CyHV-3 infection in vitro and in vivo. ML324, at 20-50 µM, can completely block ~1 × 103 PFU CyHV-3 replication in vitro, while OG-L002 at 20 µM and 50 µM can produce 96% and 98% inhibition, respectively. Only about 94% inhibition of ~1 × 103 PFU CyHV-3 replication was observed in cells treated with ACV at 50 µM. As expected, CyHV-3 IE gene transcription of ORF139 and ORF155 was blocked within 72 h post-infection (hpi) in the presence of 20 µM ML324. No detectable cytotoxicity was observed in KF-1 or CCB cells treated for 24 h with 1 to 50 µM ML324. A significant reduction of CyHV-3 replication was observed in ~6-month-old infected koi treated with 20 µM ML324 in an immersion bath for 3-4 h at 1-, 3-, and 5-days post-infection compared to the control and ACV treatments. Under heat stress, 50-70% of 3-4-month-old koi survived CyHV-3 infection when they were treated daily with 20 µM ML324 in an immersion bath for 3-4 h within the first 5 d post-infection (dpi), compared to 11-19% and 22-27% of koi in the control and ACV treatments, respectively. Our study demonstrates that ML324 has the potential to be used against CyHV-3 infection in koi.

Evaluation of compounded cidofovir, famciclovir, and ganciclovir for the treatment of feline herpesvirus ocular surface disease in shelter-housed cats.

OBJECTIVE: To assess the efficacy of compounded cidofovir, famciclovir, and ganciclovir for the treatment of feline herpesvirus type 1 (FHV-1) ocular surface disease. ANIMALS STUDIED: 132 shelter-housed cats qPCR positive for FHV-1. PROCEDURES: A masked placebo-controlled study design was utilized. Animals were enrolled in one of four treatment groups: topical ocular placebo + oral placebo (n = 32), compounded cidofovir 0.5% ophthalmic solution + oral placebo (n = 32), compounded famciclovir oral solution (90 mg/kg) + topical ocular placebo (n = 32), and compounded ganciclovir 0.15% ophthalmic solution + oral placebo (n = 36). Cats were treated with each medication twice daily for 7 days and were evaluated on Day 1 and Day 8 using an ocular scoring system, body weight, and qPCR for FHV-1 viral load. RESULTS: Cidofovir significantly decreased viral load from Day 1 to Day 8 compared with placebo (p = .024). Neither famciclovir nor ganciclovir decreased viral load compared with placebo (p = .14, p = .41). There was no significant improvement of ocular scores for any drug group compared with placebo (p = .62). In all groups, 65%-75% of cats improved from Day 1 to Day 8. Juvenile cats had a significant increase in weight gain compared with placebo for cidofovir (p = .025) and ganciclovir (p = .023). All corneal ulcers in placebo animals failed to heal whereas 77% of ulcers in antiviral group animals healed. CONCLUSIONS: Topical ophthalmic cidofovir significantly decreased ocular FHV-1 viral shedding and increased weight gain in juvenile cats. Ganciclovir increased weight gain in juvenile cats. Compounded famciclovir demonstrated limited efficacy for the treatment of FHV-1 ocular surface disease in shelter-housed cats.

Putative targeting by BX795 causes decrease in protein kinase C protein levels and inhibition of HSV1 infection.

Herpes simplex virus type-1 (HSV1) exploits cellular machinery for its own replicative advantage. Current treatment modalities against HSV1 cause toxicity and drug resistance issues. In the search for alternative forms of treatment, we have uncovered a small molecule, BX795, as a candidate drug with strong antiviral potential owing to its multitargeted mode of action. In this study, we show that in addition to a previously known mechanism of action, BX795 can directly interact with the proviral host factor protein kinase C (PKC) in silico. When administered to HSV1 or mock infected human corneal epithelial (HCE) cells, BX795 significantly reduces the protein level and perinuclear localization of proviral PKC-α and PKC-ζ isoforms. This activity closely mimics that of a known PKC inhibitor, Bisindolylmaleimide I (BIM I), which also inhibits viral replication. Taken together our studies demonstrate a previously unknown mechanism by which BX795 exerts its antiviral potential.

Insights into Antiviral Properties and Molecular Mechanisms of Non-Flavonoid Polyphenols against Human Herpesviruses.

Herpesviruses are one of the most contagious DNA viruses that threaten human health, causing severe diseases, including, but not limited to, certain types of cancer and neurological complications. The overuse and misuse of anti-herpesvirus drugs are key factors leading to drug resistance. Therefore, targeting human herpesviruses with natural products is an attractive form of therapy, as it might improve treatment efficacy in therapy-resistant herpesviruses. Plant polyphenols are major players in the health arena as they possess diverse bioactivities. Hence, in this article, we comprehensively summarize the recent advances that have been attained in employing plant non-flavonoid polyphenols, such as phenolic acids, tannins and their derivatives, stilbenes and their derivatives, lignans, neolignans, xanthones, anthraquinones and their derivatives, curcuminoids, coumarins, furanocoumarins, and other polyphenols (phloroglucinol) as promising anti-herpesvirus drugs against various types of herpesvirus such as alpha-herpesviruses (herpes simplex virus type 1 and 2 and varicella-zoster virus), beta-herpesviruses (human cytomegalovirus), and gamma-herpesviruses (Epstein-Barr virus and Kaposi sarcoma-associated herpesvirus). The molecular mechanisms of non-flavonoid polyphenols against the reviewed herpesviruses are also documented.

Efficacy of a multidose acyclovir protocol against cyprinid herpesvirus 3 infection in koi (Cyprinus carpio).

OBJECTIVE: To evaluate the effect of a multidose acyclovir protocol on koi herpesvirus (KHV) viral load and mortality in a cohabitation challenge. ANIMALS: 180 koi fish. PROCEDURES: Forty fish (shedders) were immersed in a 0.5 KHV plaque-forming units/mL static bath for 8 hours. Mock shedders were treated similarly but exposed to cell culture media. KHV shedders were then transferred into 8 tanks (5 shedders per tank) containing 10 naïve fish (cohabitants) each. Fish in the acyclovir group (AT) received a 10 mg/kg acyclovir intracoelomic injection 1, 3, and 6 days after the first confirmed KHV mortality. Positive controls (PC) were treated similarly but received sterile saline injections. Negative controls (NC) were exposed to mock shedders. Morbidity and mortality were evaluated daily for 50 days post-challenge. Quantitative PCR was used to determine viral load in the gill biopsies of shedders and cohabitants collected at days 19 (T1), 22 (T2), 25 (T3), 34 (T4), and 50 (T5) post-challenge. RESULTS: Survival curves analyzed by the Gehan-Breslow-Wilcoxon method revealed a delayed onset of mortalities and a significantly lower KHV load at T2 and T3 detected in AT cohabitant fish (P = .042) compared to PC group. However, there were no significant differences in overall mortality or viral loads at T5. CLINICAL RELEVANCE: The acyclovir protocol used in this study did not control viral infection or mortality at the end of the 50-day trial. Shorter intervals between injections could improve outcomes, but the additional stress inflicted by handling should be considered. Exploring other therapeutic alternatives and doses is warranted.

The Role of Pyrazolopyridine Derivatives on Different Steps of Herpes Simplex Virus Type-1 In Vitro Replicative Cycle.

Herpes simplex virus type-1 (HSV-1) infection causes several disorders, and acyclovir is used as a reference compound. However, resistant strains are commonly observed. Herein, we investigate the effects of N-heterocyclic compounds (pyrazolopyridine derivatives), named ARA-04, ARA-05, and AM-57, on HSV-1 in vitro replication. We show that the 50% effective concentration (EC50) values of the compounds ARA-04, ARA-05, and AM-57 were 1.00 ± 0.10, 1.00 ± 0.05, and 0.70 ± 0.10 µM, respectively. These compounds presented high 50% cytotoxic concentration (CC50) values, which resulted in a selective index (SI) of 1000, 1000, and 857.1 for ARA-04, ARA-05, and AM-57, respectively. To gain insight into which step of the HSV-1 replication cycle these molecules would impair, we performed adsorption and penetration inhibition assays and time-of-addition experiments. Our results indicated that ARA-04 and ARA-05 affected viral adsorption, while AM-57 interfered with the virus replication during its α- and γ-phases and decreased ICP27 content during initial and late events of HSV-1 replication. In addition, we also observed that AM-57 caused a strong decrease in viral gD content, which was reinforced by in silico calculations that suggested AM-57 interacts preferentially with the viral complex between a general transcription factor and virion protein (TFIIBc-VP16). In contrast, ARA-04 and ARA-05 interact preferentially in the proteins responsible for the viral adsorption process (nectin-1 and glycoprotein). Thus, our results suggest that the 1H-pyrazolo[3,4-b]pyridine derivatives inhibit the HSV-1 replicative cycle with a novel mechanism of action, and its scaffold can be used as a template for the synthesis of promising new molecules with antiviral effects, including to reinforce the presented data herein for a limited number of molecules.