The HSV-1 pUL37 protein promotes cell invasion by regulating the kinesin-1 motor.

Neurotropic alphaherpesviruses, including herpes simplex virus type 1 (HSV-1), recruit microtubule motor proteins to invade cells. The incoming viral particle traffics to nuclei in a two-step process. First, the particle uses the dynein-dynactin motor to sustain transport to the centrosome. In neurons, this step is responsible for long-distance retrograde axonal transport and is an important component of the neuroinvasive property shared by these viruses. Second, a kinesin-dependent mechanism redirects the particle from the centrosome to the nucleus. We have reported that the kinesin motor used during the second step of invasion is assimilated into nascent virions during the previous round of infection. Here, we report that the HSV-1 pUL37 tegument protein suppresses the assimilated kinesin-1 motor during retrograde axonal transport. Region 2 (R2) of pUL37 was required for suppression and functioned independently of the autoinhibitory mechanism native to kinesin-1. Furthermore, the motor domain and proximal coiled coil of kinesin-1 were sufficient for HSV-1 assimilation, pUL37 suppression, and nuclear trafficking. pUL37 localized to the centrosome, the site of assimilated kinesin-1 activation during infection, when expressed in cells in the absence of other viral proteins; however, pUL37 did not suppress kinesin-1 in this context. These results indicate that the pUL37 tegument protein spatially and temporally regulates kinesin-1 via the amino-terminal motor region in the context of the incoming viral particle.

Herpes simplex encephalitis due to a mutation in an E3 ubiquitin ligase.

Encephalitis is a rare and potentially fatal manifestation of herpes simplex type 1 infection. Following genome-wide genetic analyses, we identified a previously uncharacterized and very rare heterozygous variant in the E3 ubiquitin ligase WWP2, in a 14-month-old girl with herpes simplex encephalitis. The p.R841H variant (NM_007014.4:c.2522G > A) impaired TLR3 mediated signaling in inducible pluripotent stem cells-derived neural precursor cells and neurons; cells bearing this mutation were also more susceptible to HSV-1 infection compared to control cells. The p.R841H variant increased TRIF ubiquitination in vitro. Antiviral immunity was rescued following the correction of p.R841H by CRISPR-Cas9 technology. Moreover, the introduction of p.R841H in wild type cells reduced such immunity, suggesting that this mutation is linked to the observed phenotypes.

In Vitro Effect of 9,9'-Norharmane Dimer against Herpes Simplex Viruses.

Herpes simplex virus (HSV) infections are highly widespread among humans, producing symptoms ranging from ulcerative lesions to severe diseases such as blindness and life-threatening encephalitis. At present, there are no vaccines available, and some existing antiviral treatments can be ineffective or lead to adverse effects. As a result, there is a need for new anti-HSV drugs. In this report, the in vitro anti-HSV effect of 9,9'-norharmane dimer (nHo-dimer), which belongs to the ß-carboline (ßC) alkaloid family, was evaluated. The dimer exhibited no virucidal properties and did not impede either the attachment or penetration steps of viral particles. The antiviral effect was only exerted under the constant presence of the dimer in the incubation media, and the mechanism of action was found to involve later events of virus infection. Analysis of fluorescence lifetime imaging data showed that the nHo-dimer internalized well into the cells when present in the extracellular incubation medium, with a preferential accumulation into perinuclear organelles including mitochondria. After washing the host cells with fresh medium free of nHo-dimer, the signal decreased, suggesting the partial release of the compound from the cells. This agrees with the observation that the antiviral effect is solely manifested when the alkaloid is consistently present in the incubation media.

Herpes Simplex Virus ICP27 Protein Inhibits AIM 2-Dependent Inflammasome Influencing Pro-Inflammatory Cytokines Release in Human Pigment Epithelial Cells (hTert-RPE 1).

Although Herpes simplex virus type 1 (HSV-1) has been deeply studied, significant gaps remain in the fundamental understanding of HSV-host interactions: our work focused on studying the Infected Cell Protein 27 (ICP27) as an inhibitor of the Absent-in-melanoma-2 (AIM 2) inflammasome pathway, leading to reduced pro-inflammatory cytokines that influence the activation of a protective innate immune response to infection. To assess the inhibition of the inflammasome by the ICP27, hTert-immortalized Retinal Pigment Epithelial cells (hTert-RPE 1) infected with HSV-1 wild type were compared to HSV-1 lacking functional ICP27 (HSV-1∆ICP27) infected cells. The activation of the inflammasome by HSV-1∆ICP27 was demonstrated by quantifying the gene and protein expression of the inflammasome constituents using real-time PCR and Western blot. The detection of the cleavage of the pro-caspase-1 into the active form was performed by using a bioluminescent assay, while the quantification of interleukins 1ß (IL-1ß) and 18 (IL-18)released in the supernatant was quantified using an ELISA assay. The data showed that the presence of the ICP27 expressed by HSV-1 induces, in contrast to HSV-1∆ICP27 vector, a significant downregulation of AIM 2 inflammasome constituent proteins and, consequently, the release of pro-inflammatory interleukins into the extracellular environment reducing an effective response in counteracting infection.

First parechovirus reported case in Saudi Arabia in hospitalized immunocompromised adult patient.

Human parechovirus, a member of the Picornaviridae family (PeVs), can lead to severe infections, including severe meningitis, meningoencephalitis, and sepsis-like syndrome. We report a case of human parechovirus-related encephalitis in a 52-year-old woman diagnosed with glioblastoma multiforme. She underwent surgical resection in June 2022. Unfortunately, her disease recurred, and she underwent a second resection in August 2022, followed by radiation therapy and Temozolomide therapy. She presented to the hospital with acute confusion followed by seizures, necessitating intubation for airway support. A cerebrospinal fluid (CSF) sample was obtained and processed using the Biofire FilmArray, which reported the detection of HSV-1. Despite being on Acyclovir, the patient did not show signs of improvement. Consequently, a second CSF sample was obtained and sent for next-generation sequencing (NGS), which returned a positive result for Parechovirus. In this presented case, the patient exhibited symptoms of an unknown infectious cause. The utilization of NGS and metagenomic analysis helped identify Parechovirus as the primary pathogen present, in addition to previously identified HSV. This comprehensive approach facilitated a thorough assessment of the underlying infection and guided targeted treatment. In conclusion, the application of NGS techniques and metagenomic analysis proved instrumental in identifying the root cause of the infection.

Anti-respiratory syncytial virus and anti-herpes simplex virus activity of chemically engineered sulfated fucans from Cystoseira indica.

Seaweed polysaccharides, particularly sulfated ones, exhibited potent antiviral activity against a wide variety of enveloped viruses, such as herpes simplex virus and respiratory viruses. Different mechanisms of action were suggested, which may range from preventing infection to intracellular antiviral activity, at different stages of the viral cycle. Herein, we generated two chemically engineered sulfated fucans (C303 and C304) from Cystoseira indica by an amalgamated extraction-sulfation procedure using chlorosulfonic acid-pyridine/N,N-dimethylformamide and sulfur trioxide-pyridine/N,N-dimethylformamide reagents, respectively. These compounds exhibited activity against HSV-1 and RSV with 50 % inhibitory concentration values in the range of 0.75-2.5 µg/mL and low cytotoxicity at concentrations up to 500 µg/mL. The antiviral activities of chemically sulfated fucans (C303 and C304) were higher than the water (C301) and CaCl2 extracted (C302) polysaccharides. Compound C303 had a (1,3)-linked fucan backbone and was branched. Sulfates were present at positions C-2, C-4, and C-2,4 of Fucp, and C-6 of Galp residues of this polymer. Compound C304 had a comparable structure but with more sulfates at C-4 of Fucp residue. Both C303 and C304 were potent antiviral candidates, acting in a dose-dependent manner on the adsorption and other intracellular stages of HSV-1 and RSV replication, in vitro.

Fn-OMV potentiates ZBP1-mediated PANoptosis triggered by oncolytic HSV-1 to fuel antitumor immunity.

Oncolytic viruses (OVs) show promise as a cancer treatment by selectively replicating in tumor cells and promoting antitumor immunity. However, the current immunogenicity induced by OVs for tumor treatment is relatively weak, necessitating a thorough investigation of the mechanisms underlying its induction of antitumor immunity. Here, we show that HSV-1-based OVs (oHSVs) trigger ZBP1-mediated PANoptosis (a unique innate immune inflammatory cell death modality), resulting in augmented antitumor immune effects. Mechanistically, oHSV enhances the expression of interferon-stimulated genes, leading to the accumulation of endogenous Z-RNA and subsequent activation of ZBP1. To further enhance the antitumor potential of oHSV, we conduct a screening and identify Fusobacterium nucleatum outer membrane vesicle (Fn-OMV) that can increase the expression of PANoptosis execution proteins. The combination of Fn-OMV and oHSV demonstrates potent antitumor immunogenicity. Taken together, our study provides a deeper understanding of oHSV-induced antitumor immunity, and demonstrates a promising strategy that combines oHSV with Fn-OMV.

Primary prophylaxis with mTOR inhibitor enhances T cell effector function and prevents heart transplant rejection during talimogene laherparepvec therapy of squamous cell carcinoma.

The application of mammalian target of rapamycin inhibition (mTORi) as primary prophylactic therapy to optimize T cell effector function while preserving allograft tolerance remains challenging. Here, we present a comprehensive two-step therapeutic approach in a male patient with metastatic cutaneous squamous cell carcinoma and heart transplantation followed with concomitant longitudinal analysis of systemic immunologic changes. In the first step, calcineurin inhibitor/ mycophenolic acid is replaced by the mTORi everolimus to achieve an improved effector T cell status with increased cytotoxic activity (perforin, granzyme), enhanced proliferation (Ki67) and upregulated activation markers (CD38, CD69). In the second step, talimogene laherparepvec (T-VEC) injection further enhances effector function by switching CD4 and CD8 cells from central memory to effector memory profiles, enhancing Th1 responses, and boosting cytotoxic and proliferative activities. In addition, cytokine release (IL-6, IL-18, sCD25, CCL-2, CCL-4) is enhanced and the frequency of circulating regulatory T cells is increased. Notably, no histologic signs of allograft rejection are observed in consecutive end-myocardial biopsies. These findings provide valuable insights into the dynamics of T cell activation and differentiation and suggest that timely initiation of mTORi-based primary prophylaxis may provide a dual benefit of revitalizing T cell function while maintaining allograft tolerance.

An oncolytic HSV-1 armed with Visfatin enhances antitumor effects by remodeling tumor microenvironment against murine pancreatic cancer.

Oncolytic viruses (OVs) have shown potential in converting a "cold" tumor into a "hot" one and exhibit effectiveness in various cancer types. However, only a subset of patients respond to oncolytic virotherapy. It is important to understand the resistance mechanisms to OV treatment in pancreatic ductal adenocarcinoma (PDAC) to engineer oncolytic viruses. In this study, we used transcriptome RNA sequencing (RNA-seq) to identify Visfatin, which was highly expressed in the responsive tumors following OV treatment. To explore the antitumor efficacy, we modified OV-mVisfatin, which effectively inhibited tumor growth. For the first time, we revealed that Visfatin promoted the antitumor efficacy of OV by remodeling the tumor microenvironment, which involved enhancing CD8+ T cell and DC cell infiltration and activation, repolarizing macrophages towards the M1-like phenotype, and decreasing Treg cells using single-cell RNA sequencing (scRNA-seq) and flow cytometry. Furthermore, PD-1 blockade significantly enhanced OV-mVisfatin antitumor efficacy, offering a promising new therapeutic strategy for PDAC.

Enterocin DD14 can inhibit the infection of eukaryotic cells with enveloped viruses.

Bacteriocins are ribosomally synthesized bacterial peptides endowed with antibacterial, antiprotozoal, anticancer and antiviral activities. In the present study, we evaluated the antiviral activities of two bacteriocins, enterocin DD14 (EntDD14) and lacticaseicin 30, against herpes simplex virus type 1 (HSV-1), human coronavirus 229E (HCoV-229E) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Vero, Huh7 and Vero E6 cells, respectively. In addition, the interactions of these bacteriocins with the envelope glycoprotein D of HSV-1 and the receptor binding domains of HCoV-229E and SARS-CoV-2 have been computationally evaluated using protein-protein docking and molecular dynamics simulations. HSV-1 replication in Vero cells was inhibited by EntDD14 and, to a lesser extent, by lacticaseicin 30 added to cells after virus inoculation. EntDD14 and lacticaseicin 30 had no apparent antiviral activity against HCoV-229E; however, EntDD14 was able to inhibit SARS-CoV-2 in Vero E6 cells. Further studies are needed to elucidate the antiviral mechanism of these bacteriocins.

Commercial human 3D corneal epithelial equivalents for modeling epithelial infection in herpes keratitis.

Herpes stromal keratitis is the leading cause of infectious blindness in the western world. Infection by HSV1 is most common, but VZV and hCMV also infect the cornea. Multiple models of HSV1 corneal infection exist, but none for VZV and hCMV because of their host specificity. Here, we used commercially available 3D human corneal epithelial equivalents (HCEE) to study infection by these herpesviruses. HCEE was infected by HSV-1 and hCMV without requiring scarification and resulted in spreading infections. Spread of HSV-1 infection was rapid, while that of hCMV was slow. In contrast, infections with VZV required damage to the HCEE and did not spread. Acyclovir dramatically reduced replication of HSV-1 in this model. We conclude that highly quality-controlled, readily available HCEE is a useful model to study human-restricted herpesvirus infection of the human corneal epithelium and for screening of antiviral drugs for treating HSK in an 3D model system.

Clinical features of atypical presentations of mucocutaneous herpes simplex virus infection observed in immunosuppressed individuals. Part II: the knife-cut sign.

The knife-cut sign is a distinctive manifestation of herpes simplex virus (HSV) type 1 or HSV type 2 infection that has been described in at least 10 immunocompromised patients. It appears as an extremely painful linear erosion or fissure in an intertriginous area such as the body folds beneath the breast, or within the abdomen, or in the inguinal region. Also, concurrent HSV infection at other mucocutaneous sites, or viscera, or both have been observed. The patients had medical conditions (at least 9 patients) and/or immunosuppressive drug therapy (6 patients). The diagnosis of HSV infection was confirmed by viral culture (8 patients), biopsy (4 patients), direct fluorescence antibody testing (3 patients), immunohistochemistry staining (2 patients), polymerase chain reaction (2 patients), or Western blot serologic assay (1 patient). Knife-cut sign-associated HSV infection is potentially fatal; three patients died. However, clinical improvement or complete healing occurred in the patients who received oral valacyclovir (1 patient), or intravenous acyclovir (2 patients), or intravenous acyclovir followed by foscarnet (1 patient). In summary, HSV infection associated with a positive the knife-cut sign is a potentially fatal variant of HSV infection that occurs in the intertriginous areas of immunocompromised patients and usually requires intravenous antiviral therapy.

Clinical features of atypical presentations of mucocutaneous herpes simplex virus infection observed in immunosuppressed individuals. Part I: herpetic geometric glossitis.

Herpetic geometric glossitis is a unique morphologic variant of HSV (herpes simplex virus) type 1 infection on the dorsum of the tongue that presents as an extremely painful linear central lingual fissure with a branched pattern. in the center of the tongue; there is a branched pattern of fissures that extend bilaterally from the central linear fissure. Herpetic geometric glossitis has been reported in 11 patients; 8 of these individuals were immunocompromised. Medical conditions and immunosuppressive medication treatment (7 patients) or only medical disorders (3 patients) or neither (1 patient) were present. HSV type 1 infection was diagnosed by viral culture in (7 patients), Tzanck preparation (2 patients) or clinically (2 patients). Mucocutaneous HSV infection at non-lingual locations--including the lips, labial mucosa, face and chest--were observed in 5 patients. All patients' symptoms and lesions responded to treatment with oral antiviral therapy: acyclovir (9 patients), famciclovir (1 patient) or valacyclovir (1 patient). The lingual pain and dorsal tongue fissures completely resolved completely within two to 14 days. In summary, herpetic geometric glossitis is a unique HSV type 1 infection, usually in immunocompromised patients, that occurs on the dorsal tongue and responds completely after treatment with orally administered antiviral therapy.

Practical considerations relevant to treatment with the gene therapy beremagene geperpavec-svdt for dystrophic epidermolysis bullosa.

BACKGROUND/PURPOSE: Dystrophic epidermolysis bullosa (DEB), a rare genetic skin disease caused by loss-of-function mutations in COL7A1, the gene encoding type VII collagen (COL7), is characterized by skin blistering, scarring, and extracutaneous manifestations that markedly reduce patient quality-of-life. Beremagene geperpavec-svdt ('B-VEC') is a gene therapy employing a non-integrating, replication-defective herpes simplex virus type 1 (HSV-1)-based vector encoding two copies of full-length human COL7A1 to restore COL7 protein after topical administration to DEB wounds. B-VEC was approved in the United States in 2023 as the first topical gene therapy and the first approved treatment for DEB. However, few providers have experience with use of this gene therapy. METHODS: Data was obtained through literature review and the experience of providers who participated in the B-VEC clinical study or initiated treatment after B-VEC approval. RESULTS: This review discusses the burden of disease, describes the clinical trial outcomes of B-VEC, and provides physician and patient/caregiver recommendations as a practical guide for the real-world use of B-VEC, which can be administered in-office or at the patient's home. CONCLUSIONS: By continuing to optimize the practical aspects of B-VEC administration, the focus will continue to shift to patient-centric considerations and improved patient outcomes.

Gene editing for latent herpes simplex virus infection reduces viral load and shedding in vivo.

Anti-HSV therapies are only suppressive because they do not eliminate latent HSV present in ganglionic neurons, the source of recurrent disease. We have developed a potentially curative approach against HSV infection, based on gene editing using HSV-specific meganucleases delivered by adeno-associated virus (AAV) vectors. Gene editing performed with two anti-HSV-1 meganucleases delivered by a combination of AAV9, AAV-Dj/8, and AAV-Rh10 can eliminate 90% or more of latent HSV DNA in mouse models of orofacial infection, and up to 97% of latent HSV DNA in mouse models of genital infection. Using a pharmacological approach to reactivate latent HSV-1, we demonstrate that ganglionic viral load reduction leads to a significant decrease of viral shedding in treated female mice. While therapy is well tolerated, in some instances, we observe hepatotoxicity at high doses and subtle histological evidence of neuronal injury without observable neurological signs or deficits. Simplification of the regimen through use of a single serotype (AAV9) delivering single meganuclease targeting a duplicated region of the HSV genome, dose reduction, and use of a neuron-specific promoter each results in improved tolerability while retaining efficacy. These results reinforce the curative potential of gene editing for HSV disease.

Effect of Melatonin on Herpesvirus Type 1 Replication.

Acute HSV-1 infection is associated with mild symptoms, such as fever and lesions of the mouth, face and skin. This phase is followed by a latency period before reactivation, which is associated with symptoms ranging from ulcers to encephalitis. Despite available anti-HSV-1 drugs, the development of new antiviral agents is sought due to the presence of resistant viruses. Melatonin, a molecule secreted by the pineal gland, has been shown to be an antioxidant, inducer of antioxidant enzymes, and regulator of various biological processes. Clinical trials have explored its therapeutic utility in conditions including infections. This study focuses on melatonin's role in HSV-1 replication and the underlying mechanisms. Melatonin was found to decrease the synthesis of HSV-1 proteins in infected Vero cells measured by immunofluorescence, indicating an inhibition of HSV-1 replication. Additionally, it regulates the activities of antioxidant enzymes and affects proteasome activity. Melatonin activates the unfolded protein response (UPR) and autophagy and suppresses apoptosis in HSV-1-infected cells. In summary, melatonin demonstrates an inhibitory role in HSV-1 replication by modulating various cellular responses, suggesting its potential utility in the treatment of viral infections.

Management of Oral Manifestations of Herpes Simplex Virus, Varicella Zoster Virus, and Human Papillomavirus.

Human herpes virus is a family of DNA viruses that includes herpes simplex virus (HSV) and varicella zoster virus (VZV). HSV-1 and HSV-2 are fairly common and result in oral and genital lesions. Recurrent infections of herpes include lesions on the lips resulting in pain and possibly societal stigma, making adequate treatment of these conditions crucial. VZV is the cause of chicken pox and shingles. Acyclovir and other nucleoside analogues have been the gold standard of treatment for HSV and VZV, but newer, more effective treatments are being developed, which is beneficial regarding the issue of resistance to standard antivirals. Human papillomavirus (HPV) is also a DNA virus with different subtypes that result in four common oral benign lesions. The significance and treatments of HSV, VZV, and HPV are discussed, along with certain developing treatments of herpes labialis (HSV).

Human in vivo evidence of associations between herpes simplex virus and cerebral amyloid-beta load in normal aging.

BACKGROUND: Mounting data suggests that herpes simplex virus type 1 (HSV-1) is involved in the pathogenesis of AD, possibly instigating amyloid-beta (Aß) accumulation decades before the onset of clinical symptoms. However, human in vivo evidence linking HSV-1 infection to AD pathology is lacking in normal aging, which may contribute to the elucidation of the role of HSV-1 infection as a potential AD risk factor. METHODS: To shed light into this question, serum anti-HSV IgG levels were correlated with 18F-Florbetaben-PET binding to Aß deposits and blood markers of neurodegeneration (pTau181 and neurofilament light chain) in cognitively normal older adults. Additionally, we investigated whether associations between anti-HSV IgG and AD markers were more evident in APOE4 carriers. RESULTS: We showed that increased anti-HSV IgG levels are associated with higher Aß load in fronto-temporal regions of cognitively normal older adults. Remarkably, these cortical regions exhibited abnormal patterns of resting state-functional connectivity (rs-FC) only in those individuals showing the highest levels of anti-HSV IgG. We further found that positive relationships between anti-HSV IgG levels and Aß load, particularly in the anterior cingulate cortex, are moderated by the APOE4 genotype, the strongest genetic risk factor for AD. Importantly, anti-HSV IgG levels were unrelated to either subclinical cognitive deficits or to blood markers of neurodegeneration. CONCLUSIONS: All together, these results suggest that HSV infection is selectively related to cortical Aß deposition in normal aging, supporting the inclusion of cognitively normal older adults in prospective trials of antimicrobial therapy aimed at decreasing the AD risk in the aging population.

Non-Thermal Plasma Reduces HSV-1 Infection of and Replication in HaCaT Keratinocytes In Vitro.

Herpes simplex virus type 1 (HSV-1) is a lifelong pathogen characterized by asymptomatic latent infection in the trigeminal ganglia (TG), with periodic outbreaks of cold sores caused by virus reactivation in the TG and subsequent replication in the oral mucosa. While antiviral therapies can provide relief from cold sores, they are unable to eliminate HSV-1. We provide experimental results that highlight non-thermal plasma (NTP) as a new alternative therapy for HSV-1 infection that would resolve cold sores faster and reduce the establishment of latent infection in the TG. Additionally, this study is the first to explore the use of NTP as a therapy that can both treat and prevent human viral infections. The antiviral effect of NTP was investigated using an in vitro model of HSV-1 epithelial infection that involved the application of NTP from two separate devices to cell-free HSV-1, HSV-1-infected cells, and uninfected cells. It was found that NTP reduced the infectivity of cell-free HSV-1, reduced viral replication in HSV-1-infected cells, and diminished the susceptibility of uninfected cells to HSV-1 infection. This triad of antiviral mechanisms of action suggests the potential of NTP as a therapeutic agent effective against HSV-1 infection.

Progression of herpesvirus infection remodels mitochondrial organization and metabolism.

Viruses target mitochondria to promote their replication, and infection-induced stress during the progression of infection leads to the regulation of antiviral defenses and mitochondrial metabolism which are opposed by counteracting viral factors. The precise structural and functional changes that underlie how mitochondria react to the infection remain largely unclear. Here we show extensive transcriptional remodeling of protein-encoding host genes involved in the respiratory chain, apoptosis, and structural organization of mitochondria as herpes simplex virus type 1 lytic infection proceeds from early to late stages of infection. High-resolution microscopy and interaction analyses unveiled infection-induced emergence of rough, thin, and elongated mitochondria relocalized to the perinuclear area, a significant increase in the number and clustering of endoplasmic reticulum-mitochondria contact sites, and thickening and shortening of mitochondrial cristae. Finally, metabolic analyses demonstrated that reactivation of ATP production is accompanied by increased mitochondrial Ca2+ content and proton leakage as the infection proceeds. Overall, the significant structural and functional changes in the mitochondria triggered by the viral invasion are tightly connected to the progression of the virus infection.