Management of Refractory/Resistant Herpes Simplex Virus Infections in Haematopoietic Stem Cell Transplantation Recipients: A Literature Review.

Herpes simplex virus (HSV) infections in allogeneic haematopoietic stem cell transplantation (HSCT) recipients pose significant challenges, with higher incidence, severity, and risk of emergence of resistance to antivirals due to impaired T-cell mediated immunity. This literature review focuses on acyclovir-refractory/resistant HSV infections in HSCT recipients. The review addresses the efficacy of antiviral prophylaxis, the incidence of acyclovir-refractory/resistant HSV infections, and the identification of risk factors and potential prognostic impact associated with those infections. Additionally, alternative therapeutic options are discussed. While acyclovir prophylaxis demonstrates a significant benefit in reducing HSV infections in HSCT recipients and, in some cases, overall mortality, concerns arise about the emergence of drug-resistant HSV strains. Our systematic review reports a median incidence of acyclovir-resistant HSV infections of 16.1%, with an increasing trend in recent years. Despite limitations in available studies, potential risk factors of emergence of HSV resistance to acyclovir include human leucocyte antigen (HLA) mismatches, myeloid neoplasms and acute leukaemias, and graft-versus-host disease (GVHD). Limited evidences suggest a potentially poorer prognosis for allogeneic HSCT recipients with acyclovir-refractory/resistant HSV infection. Alternative therapeutic approaches, such as foscarnet, cidofovir, topical cidofovir, optimised acyclovir dosing, and helicase-primase inhibitors offer promising options but require further investigations. Overall, larger studies are needed to refine preventive and therapeutic strategies for acyclovir-refractory/resistant HSV infections in allogeneic HSCT recipients and to identify those at higher risk.

Role of Neurotropic Viruses in Brain Metastasis of Breast Cancer: Mechanisms and Therapeutic Implications.

Neurotropic viruses have been implicated in altering the central nervous system microenvironment and promoting brain metastasis of breast cancer through complex interactions involving viral entry mechanisms, modulation of the blood-brain barrier, immune evasion, and alteration of the tumour microenvironment. This narrative review explores the molecular mechanisms by which neurotropic viruses such as Herpes Simplex Virus, Human Immunodeficiency Virus, Japanese Encephalitis Virus, and Rabies Virus facilitate brain metastasis, focusing on their ability to disrupt blood-brain barrier integrity, modulate immune responses, and create a permissive environment for metastatic cell survival and growth within the central nervous system. Current therapeutic implications and challenges in targeting neurotropic viruses to prevent or treat brain metastasis are discussed, highlighting the need for innovative strategies and multidisciplinary approaches in virology, oncology, and immunology.

Spatiotemporally Distinct Interactions with Dendritic Cell Subsets Facilitates CD4+ and CD8+ T Cell Activation to Localized Viral Infection.

The dynamics of when and where CD4(+) T cells provide help for CD8(+) T cell priming and which dendritic cells (DCs) activate CD4(+) T cells in vivo after localized infection are poorly understood. By using a cutaneous herpes simplex virus infection model combined with intravital 2-photon imaging of the draining lymph node (LN) to concurrently visualize pathogen-specific CD4(+) and CD8(+) T cells, we found that early priming of CD4(+) T cells involved clustering with migratory skin DCs. CD8(+) T cells did not interact with migratory DCs and their activation was delayed, requiring later clustering interactions with LN-resident XCR1(+) DCs. CD4(+) T cells interacted with these late CD8(+) T cell clusters on resident XCR1(+) DCs. Together, these data reveal asynchronous T cell activation by distinct DC subsets and highlight the key role of XCR1(+) DCs as the central platform for cytotoxic T lymphocyte activation and the delivery of CD4(+) T cell help.

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.

Inhibiting Glucose Metabolism Results in Herpes Simplex Encephalitis.

This report evaluates how HSV enters the brain to cause herpes simplex encephalitis following infection at a peripheral site. We demonstrate that encephalitis regularly occurred when BALB/c mice were infected with HSV and treated daily with 2-deoxy-d-glucose (2DG), which inhibits glucose use via the glycolysis pathway. The outcome of infection in the trigeminal ganglion (TG), the site to which the virus spreads, replicates, and establishes latency, showed marked differences in viral and cellular events between treated and untreated animals. In control-untreated mice, the replicating virus was present only during early time points, whereas in 2DG recipients, replicating virus remained for the 9-d observation period. This outcome correlated with significantly reduced numbers of innate inflammatory cells as well as T cells in 2DG-treated animals. Moreover, T cells in the TG of treated animals were less activated and contained a smaller fraction of expressed IFN-γ production compared with untreated controls. The breakdown of latency was accelerated when cultures of TG cells taken from mice with established HSV latency were cultured in the presence of 2DG. Taken together, the results of both in vivo and in vitro investigations demonstrate that the overall effects of 2DG therapy impaired the protective effects of one or more inflammatory cell types in the TG that normally function to control productive infection and prevent spread of virus to the brain.

Oncolytic herpes simplex virus HF10 (canerpaturev) promotes accumulation of CD8+ PD-1- tumor-infiltrating T cells in PD-L1-enriched tumor microenvironment.

Oncolytic viruses (OVs) remodel the tumor microenvironment by switching a "cold" tumor into a "hot" tumor with high CD8+ T-cell infiltration. CD8+ T-cell activity plays an essential role in the antitumor efficacy of OVs. However, the activity of T cells is impaired by the programmed cell death protein-1/programmed cell death-ligand 1 (PD-1/PD-L1) interaction. To date, it remains unclear why OVs alone have a significant antitumor activity even when PD-L1 expression persists on tumor or immune cells. In this study, we found that canerpaturev (C-REV) treatment significantly suppressed tumor growth, even though it induced a significant increase in PD-L1 expression in tumors in vivo as well as persistence of high PD-L1 expression on antigen-presenting cells (macrophage and dendritic cells [DCs]). Surprisingly, we observed that C-REV treatment increased the abundance of activated CD8+ PD-1- tumor-infiltrating lymphocytes (TILs) in the tumor on both the injected and contralateral sides, although infiltration of CD8+ PD-1high TILs into the tumor was observed in the control group. Moreover, the difference in PD-1 expression was observed only in tumors after treatment with C-REV, whereas most CD8+ T cells in the spleen, tumor-draining lymph nodes and blood were PD-1-negative, and this did not change after C-REV treatment. In addition, changes in expression of T-cell immunoglobulin and mucin-domain containing-3 and T-cell immune-receptor with Ig and ITIM domains were not observed on CD8+ TILs after C-REV treatment. Taken together, our findings may reveal mechanisms that allow OVs to trigger an antitumor immune response, irrespective of a PD-L1-enriched tumor microenvironment, by recruitment of CD8+ PD-1- TILs.

Bacterial Pigment Prodigiosin Demonstrates a Unique Antiherpesvirus Activity That Is Mediated through Inhibition of Prosurvival Signal Transducers.

Herpes simplex virus (HSV) is among the most prevalent viral infections worldwide and remains incurable. While nucleoside analogs are used to relieve symptoms of infection, they suffer from having serious adverse effects and are unable to abolish the virus from the host. Here, we demonstrate a unique antiviral effect of prodigiosin (PG), a natural secondary metabolite produced by Serratia marcescens, on HSV infection. We show that PG naturally exerts antiviral activity against HSV-1 and HSV-2 infections. PG treatment resulted in robust inhibition of viral replication in vitro and ex vivo in cultured porcine corneas. Additionally, PG protected against HSV-1 infection and disease progression in a murine model of ocular infection. In our quest to determine the molecular mechanisms of its antiviral activity, we show that PG specifically inhibits NF-κB and Akt signaling pathways and promotes accelerated cell death in HSV-infected cells. Our findings reveal novel antiviral properties of PG, suggesting its high potential as an alternative treatment for herpetic diseases. They also provide new information on antiviral effects of HSV-bacterial metabolite interactions.IMPORTANCE In this article, we provide a new role for a commonly found bacterial pigment in controlling herpes simplex virus infection, for which diverse and multimodal antiviral agents are needed to prevent drug resistance. Serratia marcescens is a red pigment (prodigiosin)-producing Gram-negative bacillus that is naturally found in soil and water. It is associated with many kinds of human infections, including wound and eye infections, and meningitis. Taking cues from previous studies on prodigiosin, including possible proapoptotic anticancer properties, we investigated how it might affect HSV infection. Interestingly, we found that it is a potent virucidal compound that disrupts host signaling pathways needed for HSV growth and survival. The mode of antiviral action suggests potentially broad activity against enveloped viruses. Our results also indicate that interactions with commensal bacteria may inhibit HSV infection, underscoring the importance of studying these microbial metabolites and their implications for viral pathogenesis and treatment.

COVID-19 pandemic as a risk factor for the reactivation of herpes viruses.

The appearance on the skin of herpes virus lesions, concomitantly with the coronavirus disease 2019 (COVID-19) pandemic, leads us to suspect an underlying infection with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Diagnostic reverse transcriptase polymerase chain reaction tests and immunoglobulin M (IgM) and IgG seroconversion studies have therefore been carried out. We present three cases of herpes virus infections in immunocompetent patients: one of the infections was herpes simplex 1 in a 40-year-old woman, and the other two were herpes varicella-zoster infections in a 62-year-old man and a 25-year-old woman. The patients were in the care of the southern health district of Seville of the SAS (Andalusian Health Service) during the Spanish state of alarm over the COVID-19 pandemic. The SARS-CoV-2 infection was confirmed in only one of the three cases. In this study, we briefly review the etiopathogenic role of the COVID-19 pandemic situation, whereby immunodeficiencies are generated that favour the appearance of other viral infections, such as herpes virus infections.

Herpes simplex viruses activate phospholipid scramblase to redistribute phosphatidylserines and Akt to the outer leaflet of the plasma membrane and promote viral entry.

Herpes simplex virus (HSV) entry is associated with Akt translocation to the outer leaflet of the plasma membrane to promote a complex signaling cascade. We hypothesized that phospholipid scramblase-1 (PLSCR1), a calcium responsive enzyme that flips phosphatidylserines between membrane leaflets, might redistribute Akt to the outside during entry. Confocal imaging, biotinylation of membrane proteins and flow cytometric analysis demonstrated that HSV activates PLSCR1 and flips phosphatidylserines and Akt to the outside shortly following HSV-1 or HSV-2 exposure. Translocation was blocked by addition of a cell permeable calcium chelator, pharmacological scramblase antagonist, or transfection with small interfering RNA targeting PLSCR1. Co-immunoprecipitation and proximity ligation studies demonstrated that PLSCR1 associated with glycoprotein L at the outer leaflet and studies with gL deletion viruses indicate that this interaction facilitates subsequent restoration of the plasma membrane architecture. Ionomycin, a calcium ionophore, also induced PLSCR1 activation resulting in Akt externalization, suggesting a previously unrecognized biological phenomenon.

DNA Damage Kills Bacterial Spores and Cells Exposed to 222-Nanometer UV Radiation.

This study examined the microbicidal activity of 222-nm UV radiation (UV222), which is potentially a safer alternative to the 254-nm UV radiation (UV254) that is often used for surface decontamination. Spores and/or growing and stationary-phase cells of Bacillus cereus, Bacillus subtilis, Bacillus thuringiensis, Staphylococcus aureus, and Clostridioides difficile and a herpesvirus were all killed or inactivated by UV222 and at lower fluences than with UV254B. subtilis spores and cells lacking the major DNA repair protein RecA were more sensitive to UV222, as were spores lacking their DNA-protective proteins, the α/ß-type small, acid-soluble spore proteins. The spore cores' large amount of Ca2+-dipicolinic acid (∼25% of the core dry weight) also protected B. subtilis and C. difficile spores against UV222, while spores' proteinaceous coat may have given some slight protection against UV222 Survivors among B. subtilis spores treated with UV222 acquired a large number of mutations, and this radiation generated known mutagenic photoproducts in spore and cell DNA, primarily cyclobutane-type pyrimidine dimers in growing cells and an α-thyminyl-thymine adduct termed the spore photoproduct (SP) in spores. Notably, the loss of a key SP repair protein markedly decreased spore UV222 resistance. UV222-treated B. subtilis spores germinated relatively normally, and the generation of colonies from these germinated spores was not salt sensitive. The latter two findings suggest that UV222 does not kill spores by general protein damage, and thus, the new results are consistent with the notion that DNA damage is responsible for the killing of spores and cells by UV222IMPORTANCE Spores of a variety of bacteria are resistant to common decontamination agents, and many of them are major causes of food spoilage and some serious human diseases, including anthrax caused by spores of Bacillus anthracis Consequently, there is an ongoing need for efficient methods for spore eradication, in particular methods that have minimal deleterious effects on people or the environment. UV radiation at 254 nm (UV254) is sporicidal and commonly used for surface decontamination but can cause deleterious effects in humans. Recent work, however, suggests that 222-nm UV (UV222) may be less harmful to people than UV254 yet may still kill bacteria and at lower fluences than UV254 The present work has identified the damage by UV222 that leads to the killing of growing cells and spores of some bacteria, many of which are human pathogens, and UV222 also inactivates a herpesvirus.

Phenotypic and Functional Signatures of Herpes Simplex Virus-Specific Effector Memory CD73+CD45RAhighCCR7lowCD8+ TEMRA and CD73+CD45RAlowCCR7lowCD8+ TEM Cells Are Associated with Asymptomatic Ocular Herpes.

HSV type 1 (HSV-1)-specific CD8+ T cells protect from herpes infection and disease. However, the nature of protective CD8+ T cells in HSV-1 seropositive healthy asymptomatic (ASYMP) individuals (with no history of clinical herpes disease) remains to be determined. In this study, we compared the phenotype and function of HSV-specific CD8+ T cells from HLA-A*02:01-positive ASYMP and symptomatic (SYMP) individuals (with a documented history of numerous episodes of recurrent ocular herpetic disease). We report that although SYMP and ASYMP individuals have similar frequencies of HSV-specific CD8+ T cells, the "naturally" protected ASYMP individuals have a significantly higher proportion of multifunctional HSV-specific effector memory CD8+ T cells (CD73+CD45RAhighCCR7lowCD8+ effector memory RA (TEMRA) and CD73+CD45RAlowCCR7lowCD8+ effector memory (TEM) as compared with SYMP individuals. Similar to humans, HSV-1-infected ASYMP B6 mice had frequent multifunctional HSV-specific CD73+CD8+ T cells in the cornea, as compared with SYMP mice. Moreover, in contrast to wild type B6, CD73-/- deficient mice infected ocularly with HSV-1 developed more recurrent corneal herpetic infection and disease. This was associated with less functional CD8+ T cells in the cornea and trigeminal ganglia, the sites of acute and latent infection. The phenotypic and functional characteristics of HSV-specific circulating and in situ CD73+CD8+ T cells, demonstrated in both ASYMP humans and mice, suggest a positive role for effector memory CD8+ T cells expressing the CD73 costimulatory molecule in the protection against ocular herpes infection and disease. These findings are important for the development of safe and effective T cell-based herpes immunotherapy.

Effect of antiviral therapy on the outcomes of mechanically ventilated patients with herpes simplex virus detected in the respiratory tract: a systematic review and meta-analysis.

BACKGROUND: Herpes simplex virus (HSV) is frequently detected in the respiratory tract of mechanically ventilated patients. The aim of this study was to assess current evidence to determine whether antiviral therapy is associated with better outcomes in these patients. METHODS: MEDLINE, ISI Web of Science, Cochrane Database and ClinicalTrials.gov were searched from inception to 25 May 2020. All clinical studies investigating the effects of antiviral therapy on the outcome of mechanically ventilated ICU patients in whom HSV was detected in the respiratory tract were eligible for inclusion, regardless of study design, publication status or language. Titles and abstracts were reviewed independently by two authors. If the articles seemed eligible, full-text articles were reviewed and data extracted. We performed a random-effects meta-analysis to estimate relative risks (RRs) with corresponding 95% confidence intervals (CIs). The primary endpoint was hospital all-cause mortality. RESULTS: Nine studies were included in the meta-analysis (one randomized controlled trial, eight cohort studies). Antiviral treatment was associated with lower hospital mortality (with antiviral treatment, 40.6% (189 out of 465 patients); without, 52.7% (193 out of 366 patients); RR 0.74 [0.64, 0.85]; eight studies, low quality of evidence). Furthermore, antiviral treatment was associated with lower 30-day mortality (RR 0.75 [0.59, 0.94]; three studies, very low quality of evidence). We did not observe evidence for differences in ICU mortality (RR 0.73 [0.51, 1.05]; three studies, very low quality of evidence). CONCLUSIONS: This meta-analysis of the available data shows that antiviral therapy might result in lower hospital and 30-day all-cause mortality in mechanically ventilated ICU patients who are positive for HSV in the respiratory tract. However, this result must be interpreted with great caution due to the high risk of bias and limited number of patients. Large, well-designed randomized controlled clinical trials are urgently needed. TRIAL REGISTRATION: The study was registered in advance on International Prospective Register of Systematic Reviews (CRD42020180053) .

Versatile cell ablation tools and their applications to study loss of cell functions.

Targeted cell ablation is a powerful approach for studying the role of specific cell populations in a variety of organotypic functions, including cell differentiation, and organ generation and regeneration. Emerging tools for permanently or conditionally ablating targeted cell populations and transiently inhibiting neuronal activities exhibit a diversity of application and utility. Each tool has distinct features, and none can be universally applied to study different cell types in various tissue compartments. Although these tools have been developed for over 30 years, they require additional improvement. Currently, there is no consensus on how to select the tools to answer the specific scientific questions of interest. Selecting the appropriate cell ablation technique to study the function of a targeted cell population is less straightforward than selecting the method to study a gene's functions. In this review, we discuss the features of the various tools for targeted cell ablation and provide recommendations for optimal application of specific approaches.

The SP100 component of ND10 enhances accumulation of PML and suppresses replication and the assembly of HSV replication compartments.

Nuclear domain 10 (ND10) bodies are small (0.1-1 µM) nuclear structures containing both constant [e.g., promyelocytic leukemia protein (PML), SP100, death domain-associated protein (Daxx)] and variable proteins, depending on the function of the cells or the stress to which they are exposed. In herpes simplex virus (HSV)-infected cells, ND10 bodies assemble at the sites of DNA entering the nucleus after infection. In sequence, the ND10 bodies become viral replication compartments, and ICP0, a viral E3 ligase, degrades both PML and SP100. The amounts of PML and SP100 and the number of ND10 structures increase in cells exposed to IFN-ß. Earlier studies have shown that PML has three key functions. Thus, (i) the interaction of PML with viral components facilitates the initiation of replication compartments, (ii) viral replication is significantly less affected by IFN-ß in PML-/- cells than in parental PML+/+ cells, and (iii) viral yields are significantly lower in PML-/- cells exposed to low ratios of virus per cell compared with parental PML+/+ cells. This report focuses on the function of SP100. In contrast to PML-/- cells, SP100-/- cells retain the sensitivity of parental SP100+/+ cells to IFN-ß and support replication of the ΔICP0 virus. At low multiplicities of infection, wild-type virus yields are higher in SP100-/- cells than in parental HEp-2 cells. In addition, the number of viral replication compartments is significantly higher in SP100-/- cells than in parental SP100+/+ cells or in PML-/- cells.

Bioactive Natural Antivirals: An Updated Review of the Available Plants and Isolated Molecules.

Viral infections and associated diseases are responsible for a substantial number of mortality and public health problems around the world. Each year, infectious diseases kill 3.5 million people worldwide. The current pandemic caused by COVID-19 has become the greatest health hazard to people in their lifetime. There are many antiviral drugs and vaccines available against viruses, but they have many disadvantages, too. There are numerous side effects for conventional drugs, and active mutation also creates drug resistance against various viruses. This has led scientists to search herbs as a source for the discovery of more efficient new antivirals. According to the World Health Organization (WHO), 65% of the world population is in the practice of using plants and herbs as part of treatment modality. Additionally, plants have an advantage in drug discovery based on their long-term use by humans, and a reduced toxicity and abundance of bioactive compounds can be expected as a result. In this review, we have highlighted the important viruses, their drug targets, and their replication cycle. We provide in-depth and insightful information about the most favorable plant extracts and their derived phytochemicals against viral targets. Our major conclusion is that plant extracts and their isolated pure compounds are essential sources for the current viral infections and useful for future challenges.

Kinesin-1 Proteins KIF5A, -5B, and -5C Promote Anterograde Transport of Herpes Simplex Virus Enveloped Virions in Axons.

Herpes simplex virus (HSV) and other alphaherpesviruses must spread from sites of viral latency in sensory ganglia to peripheral tissues, where the viruses can replicate to higher titers before spreading to other hosts. These viruses move in neuronal axons from ganglia to the periphery propelled by kinesin motors moving along microtubules. Two forms of HSV particles undergo this anterograde transport in axons: (i) unenveloped capsids that become enveloped after reaching axon tips and (ii) enveloped virions that are transported within membrane vesicles in axons. Fundamental to understanding this axonal transport is the question of which of many different axonal kinesins convey HSV particles. Knowing which kinesins promote axonal transport would provide clues to the identity of HSV proteins that tether onto kinesins. Prominent among axonal kinesins are the kinesin-1 (KIF5A, -5B, and -5C) and kinesin-3 (e.g., KIF1A and -1B) families. We characterized fluorescent forms of cellular cargo molecules to determine if enveloped HSV particles were present in the vesicles containing these cargos. Kinesin-1 cargo proteins were present in vesicles containing HSV particles, but not kinesin-3 cargos. Fluorescent kinesin-1 protein KIF5C extensively colocalized with HSV particles, while fluorescent kinesin-1 KIF1A did not. Silencing of kinesin-1 proteins KIF5A, -5B, and -5C or light chains KLC1 and KLC2 inhibited the majority of HSV anterograde transport, while silencing of KIF1A had little effect on HSV transport in axons. We concluded that kinesin-1 proteins are important in the anterograde transport of the majority of HSV enveloped virions in neuronal axons and kinesin-3 proteins are less important.IMPORTANCE Herpes simplex virus (HSV) and other alphaherpesviruses, such as varicella-zoster virus, depend upon the capacity to navigate in neuronal axons. To do this, virus particles tether onto dyneins and kinesins that motor along microtubules from axon tips to neuronal cell bodies (retrograde) or from cell bodies to axon tips (anterograde). Following reactivation from latency, alphaherpesviruses absolutely depend upon anterograde transport of virus particles in axons in order to reinfect peripheral tissues and spread to other hosts. Which of the many axonal kinesins transport HSV in axons is not clear. We characterized fluorescent cellular cargo molecules and kinesins to provide evidence that HSV enveloped particles are ferried by kinesin-1 proteins KIF5A, -5B, and -5C and their light chains, KLC1 and KLC2, in axons. Moreover, we obtained evidence that kinesin-1 proteins are functionally important in anterograde transport of HSV virions by silencing these proteins.

Using Antibodies and Mutants To Localize the Presumptive gH/gL Binding Site on Herpes Simplex Virus gD.

HSV virus-cell and cell-cell fusion requires multiple interactions between four essential virion envelope glycoproteins, gD, gB, gH, and gL, and between gD and a cellular receptor, nectin-1 or herpesvirus entry mediator (HVEM). Current models suggest that binding of gD to receptors induces a conformational change that leads to activation of gH/gL and consequent triggering of the prefusion form of gB to promote membrane fusion. Since protein-protein interactions guide each step of fusion, identifying the sites of interaction may lead to the identification of potential therapeutic targets that block this process. We have previously identified two "faces" on gD: one for receptor binding and the other for its presumed interaction with gH/gL. We previously separated the gD monoclonal antibodies (MAbs) into five competition communities. MAbs from two communities (MC2 and MC5) neutralize virus infection and block cell-cell fusion but do not block receptor binding, suggesting that they block binding of gD to gH/gL. Using a combination of classical epitope mapping of gD mutants with fusion and entry assays, we identified two residues (R67 and P54) on the presumed gH/gL interaction face of gD that allowed for fusion and viral entry but were no longer sensitive to inhibition by MC2 or MC5, yet both were blocked by other MAbs. As neutralizing antibodies interfere with essential steps in the fusion pathway, our studies strongly suggest that these key residues block the interaction of gD with gH/gL.IMPORTANCE Virus entry and cell-cell fusion mediated by HSV require gD, gH/gL, gB, and a gD receptor. Neutralizing antibodies directed against any of these proteins bind to residues within key functional sites and interfere with an essential step in the fusion pathway. Thus, the epitopes of these MAbs identify critical, functional sites on their target proteins. Unlike many anti-gD MAbs, which block binding of gD to a cellular receptor, two, MC2 and MC5, block a separate, downstream step in the fusion pathway which is presumed to be the activation of the modulator of fusion, gH/gL. By combining epitope mapping of a panel of gD mutants with fusion and virus entry assays, we have identified residues that are critical in the binding and function of these two MAbs. This new information helps to define the site of the presumptive interaction of gD with gH/gL, of which we have limited knowledge.

Restriction of Replication of Oncolytic Herpes Simplex Virus with a Deletion of γ34.5 in Glioblastoma Stem-Like Cells.

Oncolytic viruses, including herpes simplex viruses (HSVs), are a new class of cancer therapeutic engineered to infect and kill cancer cells while sparing normal tissue. To ensure that oncolytic HSV (oHSV) is safe in the brain, all oHSVs in clinical trial for glioma lack the γ34.5 genes responsible for neurovirulence. However, loss of γ34.5 attenuates growth in cancer cells. Glioblastoma (GBM) is a lethal brain tumor that is heterogeneous and contains a subpopulation of cancer stem cells, termed GBM stem-like cells (GSCs), that likely promote tumor progression and recurrence. GSCs and matched serum-cultured GBM cells (ScGCs), representative of bulk or differentiated tumor cells, were isolated from the same patient tumor specimens. ScGCs are permissive to replication and cell killing by oHSV with deletion of the γ34.5 genes (γ34.5- oHSV), while patient-matched GSCs were not, implying an underlying biological difference between stem and bulk cancer cells. GSCs specifically restrict the synthesis of HSV-1 true late (TL) proteins, without affecting viral DNA replication or transcription of TL genes. A global shutoff of cellular protein synthesis also occurs late after γ34.5- oHSV infection of GSCs but does not affect the synthesis of early and leaky late viral proteins. Levels of phosphorylated eIF2α and eIF4E do not correlate with cell permissivity. Expression of Us11 in GSCs rescues replication of γ34.5- oHSV. The difference in degrees of permissivity between GSCs and ScGCs to γ34.5- oHSV illustrates a selective translational regulatory pathway in GSCs that may be operative in other stem-like cells and has implications for creating oHSVs.IMPORTANCE Herpes simplex virus (HSV) can be genetically engineered to endow cancer-selective replication and oncolytic activity. γ34.5, a key neurovirulence gene, has been deleted in all oncolytic HSVs in clinical trial for glioma. Glioblastoma stem-like cells (GSCs) are a subpopulation of tumor cells thought to drive tumor heterogeneity and therapeutic resistance. GSCs are nonpermissive for γ34.5- HSV, while non-stem-like cancer cells from the same patient tumors are permissive. GSCs restrict true late protein synthesis, despite normal viral DNA replication and transcription of all kinetic classes. This is specific for true late translation as early and leaky late transcripts are translated late in infection, notwithstanding shutoff of cellular protein synthesis. Expression of Us11 in GSCs rescues the replication of γ34.5- HSV. We have identified a cell type-specific innate response to HSV-1 that limits oncolytic activity in glioblastoma.

Recombination promoted by DNA viruses: phage λ to herpes simplex virus.

The purpose of this review is to explore recombination strategies in DNA viruses. Homologous recombination is a universal genetic process that plays multiple roles in the biology of all organisms, including viruses. Recombination and DNA replication are interconnected, with recombination being essential for repairing DNA damage and supporting replication of the viral genome. Recombination also creates genetic diversity, and viral recombination mechanisms have important implications for understanding viral origins as well as the dynamic nature of viral-host interactions. Both bacteriophage λ and herpes simplex virus (HSV) display high rates of recombination, both utilizing their own proteins and commandeering cellular proteins to promote recombination reactions. We focus primarily on λ and HSV, as they have proven amenable to both genetic and biochemical analysis and have recently been shown to exhibit some surprising similarities that will guide future studies.

Ocular manifestations of herpes simplex virus.

PURPOSE OF REVIEW: To review ocular manifestations and complications of herpes simplex virus (HSV) and discuss recent advancements in diagnostic and treatment strategy. RECENT FINDINGS: In-vivo confocal microscopy has expanded our understanding of corneal nerve degeneration, corneal dendritic cell activity, and changes in biomechanical properties in HSV keratitis. Although currently available only as a research tool, metagenomic deep sequencing has the potential to improve diagnostic accuracy beyond the well established PCR technology, especially in atypical cases. Development of an HSV vaccine has shown some encouraging results in a murine model. New treatment options for neurotrophic cornea offer promise, specifically cenegermin nerve growth factor. SUMMARY: Ocular herpes simplex infection and its complications continue to cause significant visual burden and decreased quality of life. Familiarity with its clinical features, wider adoption of viral PCR diagnostic technology, and recognition of the need for long-term maintenance medications for recurrent or chronic cases form the basis for effective management. Metagenomic deep sequencing, the development of a herpes vaccine, and cenegermin nerve growth factor offer promise as diagnostic, preventive, and therapeutic options, respectively.