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Herpes Simplex Virus Type 1 (HSV-1) is a widespread human pathogen known for causing a spectrum of clinical manifestations, ranging from mild cold sores to severe complications like encephalitis. Understanding the strain-specific variations of HSV-1 is crucial for elucidating its pathogenesis and developing targeted therapeutic interventions. In this multifaceted study, we investigated the strain-specific characteristics of HSV-1 using an in vivo rat model. Firstly, a pilot study was conducted to assess the capacity of three HSV-1 strains (Fisher (F), KOS (K), and MacIntyre (M)) to induce cold sores in rats. Remarkably, the F strain exhibited pronounced pathogenicity, inducing erythema, swelling, and disrupted epidermis with ulceration, distinguishing it from the K and M strains. Subsequently, the treatment capability of intravenous acyclovir injection in HSV-1 F strain-infected rats was evaluated. Acyclovir treatment resulted in a significant reduction in HSV-1 viral copy numbers in serum and dissected neuronal tissues, particularly in the spinal cord, brain, and lower lip. Lastly, whole genome sequencing data revealed that high-impact mutations occurred in the K and M strains within the UL49, US2, and US3 genes. These mutations may play a pivotal role in influencing viral replication, dissemination, pathogenesis, and infectivity. In contrast, the moderate missense variant mutations detected in the US12, US8, UL3, UL30, UL31, and UL36 genes appeared to have no effect on viral pathogenesis and infectivity, based on RT-PCR data for spinal cord, trigeminal nerve, brain, and the lower lip. These strain-specific mutations underscore the dynamic nature of HSV-1 evolution. Collectively, our findings contribute to a deeper understanding of HSV-1 strain diversity and pave the way for the development of targeted therapeutic strategies against this medically significant virus., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The periodontal ligament (PDL) is a complex connective tissue that connects the tooth root to the dental alveolar bone and plays crucial mechanical roles. PDL also exhibits regenerative roles and regulatory functions to maintain periodontium integrity and homeostasis. While PDL exposure to oral microbial pathogens is common, virtually nothing is known regarding viral infections of PDL. In particular, human herpes simplex virus type 1 (HSV-1) persistently infects the oral cavity through infections of the oral epithelium, connective tissue and neurons. While the oral spread of HSV-1 is generally asymptomatic, this virus has also been implicated in various oral pathologies. In this study, using a primary cell model derived from PDL (PDL cells), and whole surgical fragments of PDL, we provide evidence supporting the efficient infection of PDL by HSV-1 and the promotion of cytopathic effects. Infection of PDL by HSV-1 was also associated with an acute innate inflammatory response, as illustrated by the production of antiviral interferons and pro-inflammatory cytokines. Furthermore, this inflammatory response to HSV-1 was exacerbated in the presence of bacterial-derived products, such as peptidoglycans. This work therefore highlights the ability of HSV-1 to infect mesenchymal cells from PDL, suggesting that PDL may serve as a viral reservoir for the periodontal spread of HSV-1. Moreover, this raises questions about HSV-1 oral pathogenesis, as HSV-1-associated cytopathic and inflammatory effects may contribute to profound alterations of PDL integrity and functioning.

Most cases of herpes simplex virus 1 (HSV-1) encephalitis (HSE) remain unexplained 1,2 . Here, we report on two unrelated people who had HSE as children and are homozygous for rare deleterious variants of TMEFF1, which encodes a cell membrane protein that is preferentially expressed by brain cortical neurons. TMEFF1 interacts with the cell-surface HSV-1 receptor NECTIN-1, impairing HSV-1 glycoprotein D- and NECTIN-1-mediated fusion of the virus and the cell membrane, blocking viral entry. Genetic TMEFF1 deficiency allows HSV-1 to rapidly enter cortical neurons that are either patient specific or derived from CRISPR-Cas9-engineered human pluripotent stem cells, thereby enhancing HSV-1 translocation to the nucleus and subsequent replication. This cellular phenotype can be rescued by pretreatment with type I interferon (IFN) or the expression of exogenous wild-type TMEFF1. Moreover, ectopic expression of full-length TMEFF1 or its amino-terminal extracellular domain, but not its carboxy-terminal intracellular domain, impairs HSV-1 entry into NECTIN-1-expressing cells other than neurons, increasing their resistance to HSV-1 infection. Human TMEFF1 is therefore a host restriction factor for HSV-1 entry into cortical neurons. Its constitutively high abundance in cortical neurons protects these cells from HSV-1 infection, whereas inherited TMEFF1 deficiency renders them susceptible to this virus and can therefore underlie HSE., (© 2024. The Author(s).)

Although the herpes simplex virus type 1 (HSV-1) genome was thought to contain approximately 80 different protein coding sequences (CDSs), recent multi-omics analyses reported HSV-1 encodes more than 200 potential CDSs. However, few of the newly identified CDSs were confirmed to be expressed at the peptide or protein level in HSV-1-infected cells. Furthermore, the impact of the proteins they encode on HSV-1 infection is largely unknown. This study focused on a newly identified CDS, UL31.6. Re-analyzation of our previous chemical proteomics data verified that UL31.6 was expressed at the peptide level in HSV-1-infected cells. Antisera raised against a viral protein encoded by UL31.6 (pUL31.6) reacted with a protein with an approximate molecular mass of 37  kDa in lysates of Vero cells infected with each of three HSV-1 strains. pUL31.6 was efficiently dissociated from virions in high-salt solution. A UL31.6-null mutation had a minimal effect on HSV-1 gene expression, replication, cell-to-cell spread, and morphogenesis in Vero cells; in contrast, it significantly reduced HSV-1 cell-to-cell spread in three neural cells but not in four non-neural cells including Vero cells. The UL31.6-null mutation also significantly reduced the mortality and viral replication in the brains of mice after intracranial infection, but had minimal effects on pathogenic manifestations in and around the eyes, and viral replication detected in the tear films of mice after ocular infection. These results indicated that pUL31.6 was a tegument protein and specifically acted as a neurovirulence factor by potentially promoting viral transmission between neuronal cells in the central nervous system.IMPORTANCERecent multi-omics analyses reported the herpes simplex virus type 1 (HSV-1) genome encodes an additional number of potential coding sequences (CDSs). However, the expressions of these CDSs at the peptide or protein levels and the biological effects of these CDSs on HSV-1 infection remain largely unknown. This study annotated a cryptic orphan CDS, termed UL31.6, an HSV-1 gene that encodes a tegument protein with an approximate molecular mass of 37  kDa, which specifically acts as a neurovirulence factor. Our study indicates that HSV-1 proteins important for viral pathogenesis remain to be identified and a comprehensive understanding of the pathogenesis of HSV-1 will require not only the identification of cryptic orphan CDSs using emerging technologies but also step-by-step and in-depth analyses of each of the cryptic orphan CDSs., Competing Interests: The authors declare no conflict of interest.

Experimental autoimmune encephalomyelitis (EAE) is a demyelinating disease affecting the central nervous system (CNS) in animals that parallels several clinical and molecular traits of multiple sclerosis in humans. Herpes simplex virus type 1 (HSV-1) infection mainly causes cold sores and eye diseases, yet eventually, it can also reach the CNS, leading to acute encephalitis. Notably, a significant proportion of healthy individuals are likely to have asymptomatic HSV-1 brain infection with chronic brain inflammation due to persistent latent infection in neurons. Because cellular senescence is suggested as a potential factor contributing to the development of various neurodegenerative disorders, including multiple sclerosis, and viral infections may induce a premature senescence state in the CNS, potentially increasing susceptibility to such disorders, here we examine the presence of senescence-related markers in the brains and spinal cords of mice with asymptomatic HSV-1 brain infection, EAE, and both conditions. Across all scenarios, we find a significant increases of senescence biomarkers in the CNS with some differences depending on the analyzed group. Notably, some senescence biomarkers are exclusively observed in mice with the combined conditions. These results indicate that asymptomatic HSV-1 brain infection and EAE associate with a significant expression of senescence biomarkers in the CNS., (© 2024. The Author(s).)

Background: A. baumannii is an important and common clinical pathogen, especially in the intensive care unit (ICU). This study aimed to characterize one hypervirulent A. baumannii strain in a patient with community-acquired pneumonia and herpes simplex type 1 virus infection., Methods: Minimum inhibitory concentrations (MICs) were determined using the Kirby-Bauer (K-B) and broth microdilution methods. Galleria mellonella infection model experiment was conducted. Whole-genome sequencing (WGS) was performed using the Illumina and Nanopore platforms. The resistance and virulence determinants were identified using the ABRicate program with ResFinder and the VFDB database. The capsular polysaccharide locus (K locus) and lipooligosaccharide outer core locus (OC locus) were identified using Kleborate with Kaptive. Phylogenetic analyses were conducted using the BacWGSTdb server., Results: A. baumannii XH2146 strain belongs to ST10 Pas and ST447 Oxf . The strain was resistant to cefazolin, ciprofloxacin, and trimethoprim/sulfamethoxazole (TMP-SMX). Bautype and Kaptive analyses showed that XH2146 contains OCL2 and KL49. WGS analysis revealed that the strain harbored bla ADC-76 , bla OXA-68 , ant(3'')-IIa, tet(B), and sul2. Notably, tet(B) and sul2, both were located within a 114,700-bp plasmid (designated pXH2146-1). Virulence assay revealed A. baumannii XH2146 possessed higher virulence than A. baumannii AB5075 at 12 h. Comparative genomic analysis showed that A. baumannii ST447 strains were mainly isolated from the USA and exhibited a relatively close genetic relationship. Importantly, 11 strains were observed to carry bla OXA-58 ; bla OXA-23 was identified in 11 isolates and three ST447 A. baumannii strains harbored bla NDM-1 ., Conclusions: Early detection of community-acquired hypervirulent Acinetobacter baumannii strains is recommended to prevent their extensive spread in hospitals., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Herpes simplex virus type 1 (HSV-1) and herpes simplex virus type 2 (HSV-2) are two of the most prevalent human viruses worldwide. They are known to cause a variety of diseases including genital herpes, meningitis, encephalitis, cold sores and herpes stromal keratitis. The seropositive rate for HSV-1 is around 90%, whereas for HSV-2 it remains around 20-25% for the general adult population. The infections caused by these viruses remain difficult to study because a large proportion of infected individuals are asymptomatic. Furthermore, given the neurotropic characteristics of the virus, studies aimed at understanding the complex pathogenesis in humans is difficult. As a result, animal models have been developed to understand several characteristics of HSV biology, pathogenesis, disease and host responses to infection. These models are also commonly used as the first evaluation of new drugs and vaccines. There are several well-established animal models to study infection with HSV, including mice, guinea pigs and rabbits. Variables within the animal models depend on the species of animal, route of infection, viral strain, dosage, etc. This review aims at summarizing the most commonly used animal models to study HSV pathogenesis and therapies.

Neurodegenerative diseases (NDs) constitute a group of disorders characterized by the progressive deterioration of nervous system functionality. Currently, the precise etiological factors responsible for NDs remain incompletely elucidated, although it is probable that a combination of aging, genetic predisposition, and environmental stressors participate in this process. Accumulating evidence indicates that viral infections, especially neurotropic viruses, can contribute to the onset and progression of NDs. In this review, emerging evidence supporting the association between viral infection and NDs is summarized, and how the autophagy pathway mediated by viral infection can cause pathological aggregation of cellular proteins associated with various NDs is discussed. Furthermore, autophagy-related genes (ARGs) involved in Herpes simplex virus (HSV-1) infection and NDs are analyzed, and whether these genes could link HSV-1 infection to NDs is discussed. Elucidating the mechanisms underlying NDs is critical for developing targeted therapeutic approaches that prevent the onset and slow the progression of NDs., (© 2023 The Authors. CNS Neuroscience & Therapeutics published by John Wiley & Sons Ltd.)

Microbial infection as a type of environmental risk factors is considered to be associated with long-term increased risk of dementia, including Alzheimer's disease (AD). AD is characterized by two neuropathologically molecular hallmarks of hyperphosphorylated tau and amyloid-β (Aβ), the latter generated by several biochemically reactive enzymes, including γ-secretase. However, how infectious risk factors contribute to pathological development of the AD core molecules remains to be addressed. In this work, we utilized a modified herpes simplex virus type 1 (mHSV-1) and found that its hippocampal infection locally promotes Aβ pathology in 5 × FAD mice, the commonly used amyloid model. Mechanistically, we identified HSV-1 membrane glycoprotein US7 (Envelope gI) that interacts with and modulates γ-secretase and consequently facilitates Aβ production. Furthermore, we presented evidence that adenovirus-associated virus-mediated locally hippocampal overexpression of the US7 aggravates Aβ pathology in 5 × FAD mice. Collectively, these findings identify a herpesviral factor regulating γ-secretase in the development and progression of AD and represent a causal molecular link between infectious pathogens and neurodegeneration., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Viral encephalitis continues to be a significant public health concern. In our previous study, we discovered a lower expression of antiviral factors, such as IFN-β, STING and IFI16, in the brain tissues of patients with Rasmussen's encephalitis (RE), a rare chronic neurological disorder often occurred in children, characterized by unihemispheric brain atrophy. Furthermore, a higher cumulative viral score of human herpes viruses (HHVs) was also found to have a significant positive correlation with the unihemispheric atrophy in RE. Type I IFNs (IFN-I) signaling is essential for innate anti-infection response by binding to IFN-α/β receptor (IFNAR). In this study, we infected WT mice and IFNAR-deficient A6 mice with herpes simplex virus 1 (HSV-1) via periocular injection to investigate the relationship between IFN-I signaling and HHVs-induced brain lesions. While all mice exhibited typical viral encephalitis lesions in their brains, HSV-induced epilepsy was only observed in A6 mice. The gene expression matrix, functional enrichment analysis and protein-protein interaction network revealed four gene models that were positively related with HSV-induced epilepsy. Additionally, ten key genes with the highest scores were identified. Taken together, these findings indicate that intact IFN-I signaling can effectively limit HHVs induced neural symptoms and brain lesions, thereby confirming the positive correlation between IFN-I signaling repression and brain atrophy in RE and other HHVs encephalitis., Competing Interests: Conflict of interest Prof. Jing An is an editorial board member for Virologica Sinica and was not involved in the editorial review or the decision to publish this article. The authors declare that they have no conflict of interest., (Copyright © 2024 The Authors. Publishing services by Elsevier B.V. All rights reserved.)

Importance: Herpesviruses present a major global disease burden. Understanding the host cell mechanisms that block viral infections, as well as how viruses can evolve to counteract these host defenses, is critically important for understanding viral disease pathogenesis. This study reveals that the major human variant of the antiviral protein myxovirus resistance protein B (MxB) inhibits the human pathogen herpes simplex virus (HSV-1), whereas a minor human variant and orthologous MxB genes from even closely related primates do not. Thus, in contrast to the many antagonistic virus-host interactions in which the virus is successful in thwarting the host's defense systems, here the human gene appears to be at least temporarily winning at this interface of the primate-herpesvirus evolutionary arms race. Our findings further show that a polymorphism at amino acid 83 in a small fraction of the human population is sufficient to abrogate MxB's ability to inhibit HSV-1, which could have important implications for human susceptibility to HSV-1 pathogenesis., Competing Interests: The authors declare no conflict of interest.

Piwi-interacting RNAs (piRNAs) are a class of non-coding RNAs that play a key role in spermatogenesis. However, little is known about their expression characterization and role in somatic cells infected with herpes simplex virus type 1 (HSV-1). In this study, we systematically investigated the cellular piRNA expression profiles of HSV-1-infected human lung fibroblasts. Compared with the control group, 69 differentially expressed piRNAs were identified in the infection group, among which 52 were up-regulated and 17 were down-regulated. The changes in the expression of 8 piRNAs were further verified by RT-qPCR with a similar expression trend. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that the target genes of piRNAs were mainly involved in antiviral immunity and various human disease-related signaling pathways. Furthermore, we tested the effects of four up-regulated piRNAs on viral replication by transfecting piRNA mimics. The results showed that the virus titers of the group transfected with piRNA-hsa-28,382 (alias piR-36,233) mimic decreased significantly, and that of the group transfected piRNA-hsa-28,190 (alias piR-36,041) mimic significantly increased. Overall, our results revealed the expression characteristics of piRNAs in HSV-1-infected cells. We also screened two piRNAs that potentially regulate HSV-1 replication. These results may promote a better understanding of the regulatory mechanism of pathophysiological changes induced by HSV-1 infection., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Following acute infection, herpes simplex virus 1 (HSV-1) establishes lifelong latency in neurons. The latency associated transcript (LAT) is the only viral gene abundantly expressed during latency. Wild-type (WT) HSV-1 reactivates more efficiently than LAT mutants because LAT promotes establishment and maintenance of latency. While sensory neurons in trigeminal ganglia (TG) are important sites for latency, brainstem is also a site for latency and reactivation from latency. The principal sensory nucleus of the spinal trigeminal tract (Pr5) likely harbors latent HSV-1 because it receives afferent inputs from TG. The locus coeruleus (LC), an adjacent brainstem region, sends axonal projections to cortical structures and is indirectly linked to Pr5. Senescent cells accumulate in the nervous system during aging and accelerate neurodegenerative processes. Generally senescent cells undergo irreversible cell cycle arrest and produce inflammatory cytokines and chemokines. Based on these observations, we hypothesized HSV-1 influences senescence and inflammation in Pr5 and LC of latently infected mice. This hypothesis was tested using a mouse model of infection. Strikingly, female but not age-matched male mice latently infected with a LAT null mutant (dLAT2903) exhibited significantly higher levels of senescence markers and inflammation in LC, including cell cycle inhibitor p16, NLRP3 (NOD-, LRR- and pyrin domain-containing protein 3), IL-1α, and IL-β. Conversely, Pr5 in female but not male mice latently infected with WT HSV-1 or dLAT2903 exhibited enhanced expression of important inflammatory markers. The predilection of HSV-1 to induce senescence and inflammation in key brainstem regions of female mice infers that enhanced neurodegeneration occurs. IMPORTANCE HSV-1 (herpes simplex virus 1), an important human pathogen, establishes lifelong latency in neurons in trigeminal ganglia and the central nervous system. In contrast to productive infection, the only viral transcript abundantly expressed in latently infected neurons is the latency associated transcript (LAT). The brainstem, including principal sensory nucleus of the spinal trigeminal tract (Pr5) and locus coeruleus (LC), may expedite HSV-1 spread from trigeminal ganglia to the brain. Enhanced senescence and expression of key inflammatory markers were detected in LC of female mice latently infected with a LAT null mutant (dLAT2903) relative to age-matched male or female mice latently infected with wild-type HSV-1. Conversely, wild-type HSV-1 and dLAT2903 induced higher levels of senescence and inflammatory markers in Pr5 of latently infected female mice. In summary, enhanced inflammation and senescence in LC and Pr5 of female mice latently infected with HSV-1 are predicted to accelerate neurodegeneration.

Herpes simplex virus (HSV) infection of the eye can result in a blinding immunoinflammatory lesion in the cornea called herpetic stromal keratitis (HSK). This lesion is orchestrated by T cells and can be reduced in magnitude by anti-inflammatory drugs and procedures that change the balance of cellular participants in lesions. This report evaluates the effect of drugs that cause metabolic reprogramming on lesion expression using two drugs that affect glucose metabolism: 2-deoxy-d-glucose (2DG) and metformin. Both drugs could limit HSK severity, but 2DG therapy could result in herpes encephalitis if used when replicating virus was still present. The reason metformin was a safer therapy was its lack of marked inhibitory effects on inflammatory cells particularly interferon-γ (IFN-γ)-producing Th1 and CD8 T cells in the trigeminal ganglion (TG), in which HSV latency is established and sustained. Additionally, whereas 2DG in TG cultures with established latency accelerated the termination of latency, this did not occur in the presence of metformin, likely because the inflammatory cells remained functional. Our results support the value of metabolic reprogramming to control viral immunoinflammatory lesions, but the approach used should be chosen with caution. IMPORTANCE Herpes simplex virus (HSV) infection of the eye is an example where damaging lesions are in part the consequence of a host response to the infection. Moreover, it was shown that changing the representation of cellular participants in the inflammatory reaction can minimize lesion severity. This report explores the value of metabolic reprogramming using two drugs that affect glucose metabolism to achieve cellular rebalancing. It showed that two drugs, 2-deoxy-d-glucose (2DG) and metformin, effectively diminished ocular lesion expression, but only metformin avoided the complication of HSV spreading to the central nervous system (CNS) and causing herpetic encephalitis. The report provides some mechanistic explanations for the findings.

Herpes simplex virus 1 (HSV-1)-TK (8UAG) expresses a truncated thymidine kinase (TK) translated from the second initiation codon due to a stop codon (UAG) at the 8th position (counted from the first initiation codon). Here, we showed that the sensitivity of HSV-1-TK (8UAG) to acyclovir (ACV) is similar to that of the control HSV-1 wild-type (WT), which expresses an intact TK protein. However, HSV-1-TK (44UAG), which expresses a truncated TK due to a UAG codon at position 44, showed lower sensitivity to ACV. A mouse infection model was used to compare the virulence of HSV-1-TK (8UAG) and HSV-1-TK (44UAG) to that of HSV-1 WT. The 50% lethal dose (LD 50 ) for HSV-1-TK (44UAG) was 7.8-fold higher than that for HSV-1-TK (8UAG), whereas the LD 50 for HSV-1-TK (8UAG) was the same as that for the parental HSV-1 WT. There were no statistically significant differences among HSV-1-TK (44UAG), HSV-1-TK (8UAG), and HSV-1 WT with respect to replication capacity and viral TK mRNA expression in the mouse brain. Thus, the virulence of HSV-1 expressing the truncated viral TK translated from the second initiation codon might depend on the position of the UAG stop codon.

The HSV-1 latency-associated transcript (LAT) locus contains two small noncoding RNA (sncRNA) sequences (sncRNA1 and sncRNA2) that are not microRNAs (miRNAs). We recently reported that sncRNA1 is more important for in vitro activation of the herpesvirus entry mediator than sncRNA2, but its in vivo function is not known. To determine the role, if any, of sncRNA1 during herpes simplex virus 1 (HSV-1) infection in vivo , we deleted the 62-bp sncRNA1 sequence in HSV-1 strain McKrae using dLAT2903 (LAT-minus) virus, creating ΔsncRNA1 recombinant virus. Deletion of the sncRNA1 in ΔsncRNA1 virus was confirmed by complete sequencing of ΔsncRNA1 virus and its parental virus (i.e., McKrae). Replication of ΔsncRNA1 virus in tissue culture or in the eyes of infected mice was similar to that of HSV-1 strain McKrae and dLAT2903 viruses. However, the absence of sncRNA1 significantly reduced the levels of ICP0, ICP4, and gB but not LAT transcripts in infected rabbit skin cells in vitro . In contrast, the absence of sncRNA1 did reduce LAT expression in trigeminal ganglia (TG), but not in corneas, by day 5 postinfection (p.i.) in infected mice. Levels of eye disease in mice infected with ΔsncRNA1 or McKrae virus were similar, and despite reduced LAT levels in TG during acute ΔsncRNA1 infection, McKrae and ΔsncRNA1 viruses did not affect latency or reactivation on day 28 p.i. However, mice infected with ΔsncRNA1 virus were more susceptible to ocular infection than their wild-type (WT) counterparts. Expression of host immune response genes in corneas and TG of infected mice during primary infection showed reduced expression of beta interferon (IFNβ) and IFNγ and altered activation of key innate immune pathways, such as the JAK-STAT pathway in ΔsncRNA1 virus compared with parental WT virus. Our results reveal novel functions for sncRNA1 in upregulating the host immune response and suggest that sncRNA1 has a protective role during primary ocular HSV-1 infection. IMPORTANCE HSV-1 latency-associated transcript (LAT) plays a major role in establishing latency and reactivation; however, the mechanism by which LAT controls these processes is largely unknown. In this study, we sought to establish the role of the small noncoding RNA1 (sncRNA1) encoded within LAT during HSV-1 ocular infection. Our results suggest that sncRNA1 has a protective role during acute ocular infection by modulating the innate immune response to infection.

The objective of this critical review is to provide an overview of how emerging bioanalytical techniques are expanding our understanding of the complex physicochemical nature of virus interactions with host cell surfaces. Herein, selected model viruses representing both non-enveloped (simian virus 40 and human norovirus) and enveloped (influenza A virus, human herpes simplex virus, and human immunodeficiency virus type 1) viruses are highlighted. The technologies covered utilize a wide range of cell membrane mimics, from supported lipid bilayers (SLBs) containing a single purified host membrane component to SLBs derived from the plasma membrane of a target cell, which can be compared with live-cell experiments to better understand the role of individual interaction pairs in virus attachment and entry. These platforms are used to quantify binding strengths, residence times, diffusion characteristics, and binding kinetics down to the single virus particle and single receptor, and even to provide assessments of multivalent interactions. The technologies covered herein are surface plasmon resonance (SPR), quartz crystal microbalance with dissipation (QCM-D), dynamic force spectroscopy (DFS), total internal reflection fluorescence (TIRF) microscopy combined with equilibrium fluctuation analysis (EFA) and single particle tracking (SPT), and finally confocal microscopy using multi-labeling techniques to visualize entry of individual virus particles in live cells. Considering the growing scientific and societal needs for untangling, and interfering with, the complex mechanisms of virus binding and entry, we hope that this review will stimulate the community to implement these emerging tools and strategies in conjunction with more traditional methods. The gained knowledge will not only contribute to a better understanding of the virus biology, but may also facilitate the design of effective inhibitors to block virus entry., (© 2021. The Author(s).)

Pathogens use virulence factors to inhibit the immune system 1 . The guard hypothesis 2,3 postulates that hosts monitor (or 'guard') critical innate immune pathways such that their disruption by virulence factors provokes a secondary immune response 1 . Here we describe a 'self-guarded' immune pathway in human monocytes, in which guarding and guarded functions are combined in one protein. We find that this pathway is triggered by ICP0, a key virulence factor of herpes simplex virus type 1, resulting in robust induction of anti-viral type I interferon (IFN). Notably, induction of IFN by ICP0 is independent of canonical immune pathways and the IRF3 and IRF7 transcription factors. A CRISPR screen identified the ICP0 target MORC3 4 as an essential negative regulator of IFN. Loss of MORC3 recapitulates the IRF3- and IRF7-independent IFN response induced by ICP0. Mechanistically, ICP0 degrades MORC3, which leads to de-repression of a MORC3-regulated DNA element (MRE) adjacent to the IFNB1 locus. The MRE is required in cis for IFNB1 induction by the MORC3 pathway, but is not required for canonical IFN-inducing pathways. As well as repressing the MRE to regulate IFNB1, MORC3 is also a direct restriction factor of HSV-1 5 . Our results thus suggest a model in which the primary anti-viral function of MORC3 is self-guarded by its secondary IFN-repressing function-thus, a virus that degrades MORC3 to avoid its primary anti-viral function will unleash the secondary anti-viral IFN response., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Herpes simplex virus 1 (HSV-1) infects the majority of the human population and can induce encephalitis, which is the most common cause of sporadic, fatal encephalitis. An increase of microglia is detected in the brains of encephalitis patients. The issues regarding whether and how microglia protect the host and neurons from HSV-1 infection remain elusive. Using a murine infection model, we showed that HSV-1 infection on corneas increased the number of microglia to outnumber those of infiltrating leukocytes (macrophages, neutrophils, and T cells) and enhanced microglia activation in brains. HSV-1 antigens were detected in brain neurons, which were surrounded by microglia. Microglia depletion increased HSV-1 lethality of mice with elevated brain levels of viral loads, infected neurons, neuron loss, CD4 T cells, CD8 T cells, neutrophils, interferon (IFN)-β, and IFN-γ. In vitro studies demonstrated that microglia from infected mice reduced virus infectivity. Moreover, microglia induced IFN-β and the signaling pathway of signal transducer and activator of transcription (STAT) 1 to inhibit viral replication and damage of neurons. Our study reveals how microglia protect the host and neurons from HSV-1 infection.

Herpes simplex virus type-1 (HSV-1) ocular infection is one of the leading causes of infectious blindness in developed countries. The resultant herpetic keratitis (HK) is caused by an exacerbated reaction of the adaptive immune response that persists beyond virus clearance causing substantial damage to the cornea. Intramuscular immunization of mice with the HSV-1(VC2) live-attenuated vaccine strain has been shown to protect mice against lethal ocular challenge. Herein, we show that following ocular challenge, VC2 vaccinated animals control ocular immunopathogenesis in the absence of neutralizing antibodies on ocular surfaces. Ocular protection is associated with enhanced intracorneal infiltration of γδ T cells compared to mock-vaccinated animals. The observed γδ T cellular infiltration was inversely proportional to the infiltration of neutrophils, the latter associated with exacerbated tissue damage. Inhibition of T cell migration into ocular tissues by the S1P receptors agonist FTY720 produced significant ocular disease in vaccinated mice and marked increase in neutrophil infiltration. These results indicate that ocular challenge of mice immunized with the VC2 vaccine induce a unique ocular mucosal response that leads into the infiltration of γδ T cells resulting in the amelioration of infection-associated immunopathogenesis., Competing Interests: Louisiana State University has licensed the VC2 vaccine for genital herpes to Rational Vaccines. KGK serves as a consultant for Rational Vaccines, Inc., (Copyright © 2021 Nabi, Lewin, Collantes, Chouljenko and Kousoulas.)

Increasing studies demonstrated that oncolytic activities of oHSV-1 are limited to the capacity of virus replicating in tumors. In order to potentiate the oHSV-1 oncolytic activity and expand the application of oHSV-1 treatment in multiple types of tumors, it is critical to explore the potential factors or mechanisms mediating tumor resistance to oHSV-1 infection. Here we evaluated the levels of oHSV-1 multiplication in various tumor cell lines and showed that glioblastoma cell line A172 had the lowest virus yields but intrinsically accumulated the highest levels of Mx2 protein. Subsequently we demonstrated that genetic depletion of Mx2 specifically enhanced oHSV-1 productive replication in A172 cells through promoting the nuclear translocation of uncoated viral genomic DNA and down-regulating innate antiviral response. In the further investigation, we found that Mx2 knockdown could alter the intrinsic mRNA accumulation of diverse sets innate immune genes in A172 cells, in particular DHX36 and MyD88. Mx2 depletion led to a decrease in mRNA levels of MyD88 and DHX36 in A172 cells and MyD88/DHX36 knockdown increased virus yield in A172 cells and decreased the production of IFNα, activation of IRF3 activity and NF-κB signaling in A172 cells. This shed new lights on understanding the roles of some intrinsic antiviral genes in oHSV-1 resistance, facilitating to offer potential targets to improve oHSV-1 oncolytic efficacy and develop candidates of biomarkers to predict the efficiency of oHSV-1 multiplication in tumors., (© 2021. The Author(s).)

Herpes viruses are known for infecting epithelial cells and manifesting as vesicles. However, herpes viruses can also infect stromal cells. While established in the ocular setting, cutaneous stromal herpes (deep herpes) is previously unreported and may evade clinical and microscopic detection. We searched for skin biopsies with herpes stromal disease. Clinical information was retrieved via electronic medical records and pathology records system. Hematoxylin and eosin slides, immunohistochemical staining, and polymerase chain reaction detection of viral DNA was performed. We identified 12 specimens from 10 patients with cutaneous stromal herpes simplex virus 1/2 (n=7) or varicella-zoster virus infection (n=5). The most common site involved was the buttocks/perianal region (n=6). Ulceration was a frequent dermatologic finding (n=8). Pyoderma gangrenosum was clinically suspected in 6 specimens (50%). Eight patients (80%) were immunosuppressed. Biopsies frequently demonstrated a dense dermal mixed inflammatory infiltrate with subcutaneous extension and enlarged cells with viral cytopathic changes confirmed by herpes simplex virus 1/2 or varicella-zoster virus immunohistochemistry (n=10) or polymerase chain reaction (n=2). Most specimens (67%) lacked evidence of characteristic epidermal keratinocyte infection. This study presents the first known report of the ability of herpes virus to infect deep stromal cells of the dermis. We raise awareness of cutaneous stromal herpes in patients presenting with atypical clinical lesions, particularly while immunocompromised. Establishing the correct diagnosis is critical for initiating therapy., Competing Interests: Conflicts of Interest and Source of Funding: The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Herpes simplex virus 1 (HSV1) infects the stratified epithelia of the epidermis, oral or genital mucosa, where the main cell type is the keratinocyte. Here we have used nTERT human keratinocytes to generate a CRISPR-Cas9 knockout (KO) of the primary candidate HSV1 receptor, nectin1, resulting in a cell line that is refractory to HSV1 entry. Nonetheless, a small population of KO cells was able to support infection which was not blocked by a nectin1 antibody and hence was not a consequence of residual nectin1 expression. Strikingly at later times, the population of cells originally resistant to HSV1 infection had also become infected. Appearance of this later population was blocked by inhibition of virus genome replication, or infection with a ΔUL34 virus defective in capsid export to the cytoplasm. Moreover, newly formed GFP-tagged capsids were detected in cells surrounding the initial infected cell, suggesting that virus was spreading following replication in the original susceptible cells. Additional siRNA depletion of the second major HSV1 receptor HVEM, or PTP1B, a cellular factor shown elsewhere to be involved in cell-to-cell transmission, had no effect on virus spread in the absence of nectin1. Neutralizing human serum also failed to block virus transmission in nectin1 KO cells, which was dependent on the receptor binding protein glycoprotein D and the cell-to-cell spread glycoproteins gI and gE, indicating that virus was spreading by direct cell-to-cell transmission. In line with these results, both HSV1 and HSV2 formed plaques on nectin1 KO cells, albeit at a reduced titre, confirming that once the original cell population was infected, the virus could spread into all other cells in the monolayer. We conclude that although nectin1 is required for extracellular entry in to the majority of human keratinocytes, it is dispensable for direct cell-to-cell transmission., Competing Interests: The authors have declared no competing interests exist.

Infection by herpes simplex virus 1 (HSV-1) impacts nearly all steps of host cell gene expression. The regulatory mechanisms by which this occurs, and the interplay between host and viral factors, have yet to be fully elucidated. We investigated how the occupancy of RNA polymerase II (Pol II) on the host genome changes during HSV-1 infection and is impacted by the viral immediate early protein ICP4. Pol II ChIP-seq experiments revealed ICP4-dependent decreases and increases in Pol II levels across the bodies of hundreds of genes. Our data suggest ICP4 represses host transcription by inhibiting recruitment of Pol II and activates host genes by promoting release of Pol II from promoter proximal pausing into productive elongation. Consistent with this, ICP4 was required for the decrease in levels of the pausing factor NELF-A on several HSV-1-activated genes after infection. In the absence of infection, exogenous expression of ICP4 activated, but did not repress, transcription of some genes in a chromatin-dependent context. Our data support the model that ICP4 decreases promoter proximal pausing on host genes activated by infection and that ICP4 is necessary, but not sufficient, to repress transcription of host genes during viral infection.

Microglia are the primary cellular source of type I interferons (I-IFNs) in the brain upon neurotropic virus infection. Although the I-IFN-based antiviral innate immune response is crucial for eliminating viruses, overproduction led to immune disorders. Therefore, the relatively long-lasting I-IFNs must be precisely controlled, but the regulatory mechanism for the innate antiviral response in microglia remains largely unknown. Long non-coding RNAs (lncRNAs) are being recognized as crucial factors in numerous diseases, but their regulatory roles in the innate antiviral response in microglia are undefined. Methods: The high-throughput RNA sequencing was performed to obtain differentially expressed lncRNAs (DELs) in primary microglia infected with or without the neurotropic herpes simplex virus type 1 (HSV-1). We selected four DELs ranked in the top 15 in basic level and their fold change induced by HSV-1, i.e., FPKM HSV-1 /FPKM Cells .We subsequently found a key lncRNA affecting the innate antiviral response of microglia significantly. We next used dual-luciferase reporter assays, bioinformatical tools, and truncation mutants of both lncRNA and targeted proteins to elucidate the downstream and upstream mechanism of action of lncRNA. Further, we established microglia-specific knock-in (KI) mice to investigate the role of lncRNA in vivo . Results: We identified a long intergenic non-coding RNA, linc-AhRA, involved in regulating the innate antiviral response in murine microglia. linc-AhRA is activated by aryl hydrocarbon receptor (AhR) and restricts I-IFN production in microglia upon neurotropic herpesvirus infection and innate immune stimulation. Mechanistically, linc-AhRA binds to both tripartite motif-containing 27 (TRIM27) and TANK-binding kinase 1 (TBK1) through its conserved 117nt fragment as a molecular scaffold to enhance TRIM27-TBK1 interaction. This interaction facilitates the TRIM27-mediated ubiquitination of TBK1 and results in ubiquitin-proteasome-dependent degradation of TBK1. Consequently, linc-AhRA suppresses I-IFN production through facilitating TBK1 degradation and limits the microglial innate immune response against neurotropic herpesvirus infection. Microglia-specific KI of linc-AhRA mice shows a weakened antiviral immune response upon neurotropic herpesvirus challenge due to a reduction of TBK1 in microglia. Conclusion: Our findings indicate that linc-AhRA is a negative regulator of I-IFN production in microglia to avoid excessive autoimmune responses. These findings uncover a previously unappreciated role for lncRNA conserved fragments in the innate antiviral response, providing a strong foundation for developing nucleotide drugs based on conserved functional fragments within lncRNAs., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

During lytic infection, herpes simplex virus (HSV) 1 induces a rapid shutoff of host RNA synthesis while redirecting transcriptional machinery to viral genes. In addition to being a major human pathogen, there is burgeoning clinical interest in HSV as a vector in gene delivery and oncolytic therapies, necessitating research into transcriptional control. This review summarizes the array of impacts that HSV has on RNA Polymerase (Pol) II, which transcribes all mRNA in infected cells. We discuss alterations in Pol II holoenzymes, post-translational modifications, and how viral proteins regulate specific activities such as promoter-proximal pausing, splicing, histone repositioning, and termination with respect to host genes. Recent technological innovations that have reshaped our understanding of previous observations are summarized in detail, along with specific research directions and technical considerations for future studies.

We report on the efficacy of the non-attenuated HER2-retargeted oHSV named R-337 against the immunologically hot CT26-HER2 tumor, and an insight into the basis of the immune protection. Preliminarily, we conducted an RNA immune profiling and immune cell content characterization of CT26-HER2 tumor in comparison to the immunologically cold LLC1-HER2 tumor. CT26-HER2 tumor was implanted into HER2-transgenic BALB/c mice. Hallmarks of R-337 effects were the protection from primary tumor, long-term adaptive vaccination directed to both HER2 and CT26-wt cell neoantigens. The latter effect differentiated R-337 from OncoVEXGM-CSF. As to the basis of the immune protection, R-337 orchestrated several changes to the tumor immune profile, which cumulatively reversed the immunosuppression typical of this tumor (graphical abstract). Thus, Ido1 (inhibitor of T cell anticancer immunity) levels and T regulatory cell infiltration were decreased; Cd40 and Cd27 co-immunostimulatory markers were increased; the IFNγ cascade was activated. Of note was the dampening of IFN-I response, which we attribute to the fact that R-337 is fully equipped with genes that contrast the host innate response. The IFN-I shut-down likely favored viral replication and the expression of the mIL-12 payload, which, in turn, boosted the antitumor response. The results call for a characterization of tumor immune markers to employ oncolytic herpesviruses more precisely.

The purpose of this study was to determine whether administration of the microorganism Eubacterium rectale ( E. rectale ) could regulate dendritic cell (DC) activation and systemic inflammation in herpes simplex virus type 1-induced Behçet's disease (BD). E. rectale , butyrate-producing bacteria, was administered to BD mice. Peripheral blood leukocytes (PBL) and lymph node cells were isolated and analyzed by flow cytometry. 16S rRNA metagenomic analysis was performed in the feces of mice to determine the differences in the composition of the microbial population between normal and BD mice. Serum cytokine levels were measured by enzyme-linked immunosorbent assay. The frequency of DC activation marker CD83 positive cells was significantly increased in PBL of BD mice. Frequencies of CD83+ cells were also significantly increased in patients with active BD. 16S rRNA metagenomic analysis revealed different gut microbiota composition between normal and BD mice. The administration of E. rectale to BD mice reduced the frequency of CD83+ cells and significantly increased the frequency of NK1.1+ cells with the improvement of symptoms. The co-administration of colchicine and E. rectale also significantly reduced the frequency of CD83+ cells. Differences in gut microbiota were observed between normal mice and BD mice, and the administration of E. rectale downregulated the frequency of CD83, which was associated with BD deterioration. These data indicate that E. rectale could be a new therapeutic adjuvant for BD management., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Islam, Ryu, Sayeed, Byun, Jung, Kim, Suh and Sohn.)

Current therapy against herpes simplex viruses (HSV) relies on the use of a few nucleoside antivirals such as acyclovir, famciclovir and valacyclovir. However, the current drugs are ineffective against latent and drug-resistant HSV infections. A series of amidinourea compounds, designed as analogues of the antiviral drug moroxydine, has been synthesized and evaluated as potential non-nucleoside anti-HSV agents. Three compounds showed micromolar activity against HSV-1 and low cytotoxicity, turning to be promising candidates for future optimization. Preliminary mode of action studies revealed that the new compounds act in an early stage of the HSV replication cycle, just after the viral attachment and the entry phase of the infection.

The inflammatory chemokines, monocyte chemoattractant protein (MCP)-1 and IL-8, are produced by normal trabecular meshwork cells (TM) and elevated in the aqueous humor of primary open angle glaucoma (POAG) and hypertensive anterior uveitis associated with viral infection. However, their role in TM cells and aqueous humor outflow remains unclear. Here, we explored the possible involvement of MCP-1 and IL-8 in the physiology of TM cells in the context of aqueous outflow, and the viral anterior uveitis. We found that the stimulation of human TM cells with MCP-1 and IL-8 induced significant increase in the formation of actin stress fibers and focal adhesions, myosin light chain phosphorylation, and the contraction of TM cells. MCP-1 and IL-8 also demonstrated elevation of extracellular matrix proteins, and the migration of TM cells. When TM cells were infected with HSV-1 and CMV virus, there was a significant increase in cytoskeletal contraction and Rho-GTPase activation. Viral infection of TM cells revealed significantly increased expression of MCP-1 and IL-8. Taken together, these results indicate that MCP-1 and IL-8 induce TM cell contractibility, fibrogenic activity, and plasticity, which are presumed to increase resistance to aqueous outflow in viral anterior uveitis and POAG., (© 2021. The Author(s).)

Alzheimer's disease (AD) is a devastating fatal neurodegenerative disease. An alternative to the amyloid cascade hypothesis is that a viral infection is key to the etiology of late-onset AD, with β-amyloid (Aβ) peptides playing a protective role. In the current study, young 5XFAD mice that overexpress mutant human amyloid precursor protein with the Swedish, Florida, and London familial AD mutations were infected with one of two strains of herpes simplex virus 1 (HSV-1), 17syn+ and McKrae, at three different doses. Contrary to previous work, 5XFAD genotype failed to protect mice against HSV-1 infection. The region- and cell-specific tropisms of HSV-1 were not affected by the 5XFAD genotype, indicating that host-pathogen interactions were not altered. Seven- to ten-month-old 5XFAD animals in which extracellular Aβ aggregates were abundant showed slightly better survival rate relative to their wild-type (WT) littermates, although the difference was not statistically significant. In these 5XFAD mice, HSV-1 replication centers were partially excluded from the brain areas with high densities of Aβ aggregates. Aβ aggregates were free of HSV-1 viral particles, and the limited viral invasion to areas with a high density of Aβ aggregates was attributed to phagocytic activity of reactive microglia. In the oldest mice (12-15 months old), the survival rate did not differ between 5XFAD and WT littermates. While the current study questions the antiviral role of Aβ, it neither supports nor refutes the viral etiology hypothesis of late-onset AD., Competing Interests: Conflicts of interest The authors declare that they have no conflict of interests with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Herpes simplex virus encephalitis (HSE) is the most common sporadic fatal encephalitis. Although timely administered acyclovir treatment decreases mortality, neuropsychiatric sequelae is still common among survivors. Magnetic resonance imaging is frequently utilized for the diagnosis of HSE, which typically involves temporal lobe(s) and can be mixed with brain tumors involving the same area. Here, we report a case of HSE, who received acyclovir with a delay of 90 days because of presumptive tumor diagnosis and survived with minimal sequelae.

The frequency of central nervous system infections due to herpesvirus have been studied in various populations; however, studies in Mexican mestizo patients are scant. This paper documents the frequency of herpesvirus encephalitis in Mexican mestizo patients from the National Institute of Neurology and Neurosurgery (NINN) of Mexico. To study the frequency of herpetic viral encephalitis at the NINN in the period from 2004 to 2009. We reviewed clinical records from patients with clinically suspected encephalitis; polymerase chain reaction assays were done for detection of herpesviruses in cerebrospinal fluid (CSF) samples. The total number of patients studied was 502; in 59 (12%), the diagnosis of herpetic encephalitis was confirmed by PCR-based testing of CSF. Of them, 21 (36%) were positive for herpes simplex virus type 1, 15 (25%) for Epstein-Barr virus, 10 (17%) for varicella zoster virus, 8 (14%) for cytomegalovirus, 3 (5%) for human herpesvirus 6, and 2 (3%) for herpes simplex virus 2. Our results show a varied frequency of viral encephalitis in mestizo patients due to herpesviruses in a tertiary neurological center and point out the importance of modern molecular technology to reach the etiological diagnosis in cases of encephalitis.

Neutrophil responses against pathogens must be balanced between protection and immunopathology. Factors that determine these outcomes are not well-understood. In a mouse model of genital herpes simplex virus-2 (HSV-2) infection, which results in severe genital inflammation, antibody-mediated neutrophil depletion reduced disease. Comparative single-cell RNA-sequencing analysis of vaginal cells against a model of genital HSV-1 infection, which results in mild inflammation, demonstrated sustained expression of interferon-stimulated genes (ISGs) only after HSV-2 infection primarily within the neutrophil population. Both therapeutic blockade of IFNα/β receptor 1 (IFNAR1) and genetic deletion of IFNAR1 in neutrophils concomitantly decreased HSV-2 genital disease severity and vaginal IL-18 levels. Therapeutic neutralization of IL-18 also diminished genital inflammation, indicating an important role for this cytokine in promoting neutrophil-dependent immunopathology. Our study reveals that sustained type I interferon (IFN) signaling is a driver of pathogenic neutrophil responses and identifies IL-18 as a novel component of disease during genital HSV-2 infection., Competing Interests: TL, YL, AC, PA, XJ, JS, MF, AO, CP, RC, MA, MD, HS No competing interests declared, (© 2021, Lebratti et al.)

Corneal transparency is an essential characteristic necessary for normal vision. In response to microbial infection, the integrity of the cornea can become compromised as a result of the inflammatory response and the ensuing tissue pathology including neovascularization (NV) and collagen lamellae destruction. We have previously found complement activation contributes to cornea pathology-specifically, denervation in response to HSV-1 infection. Therefore, we investigated whether the complement system also played a role in HSV-1-mediated neovascularization. Using wild type (WT) and complement component 3 deficient (C3 KO) mice infected with HSV-1, we found corneal NV was accelerated associated with an increase in inflammatory monocytes (CD11b + CCR2 + CD115 +/- Ly6G - Ly6C high ), macrophages (CD11b + CCR2 + CD115 + Ly6G - Ly6C high ) and a subpopulation of granulocytes/neutrophils (CD11b + CCR2 - CD115 + Ly6G + Ly6C low ). There were also increases in select pro-inflammatory and pro-angiogenic factors including IL-1α, matrix metalloproteinases (MMP)-2, MMP-3, MMP-8, CXCL1, CCL2, and VEGF-A that coincided with increased inflammation, neovascularization, and corneal opacity in the C3 KO mice. The difference in inflammation between WT and C3 KO mice was not driven by changes in virus titer. However, viral antigen clearance was hindered in C3 KO mouse corneas suggesting the complement system has a dynamic regulatory role within the cornea once an inflammatory cascade is initiated by HSV-1.

Herpes simplex virus type 1 (HSV-1) is a leading cause of infectious blindness. Current treatments for HSV-1 do not eliminate the virus from the site of infection or latent reservoirs in the trigeminal ganglia. Here, we target HSV-1 genomes directly using mRNA-carrying lentiviral particles that simultaneously deliver SpCas9 mRNA and viral-gene-targeting guide RNAs (designated HSV-1-erasing lentiviral particles, termed HELP). We show that HELP efficiently blocks HSV-1 replication and the occurrence of herpetic stromal keratitis (HSK) in three different infection models. HELP was capable of eliminating the viral reservoir via retrograde transport from corneas to trigeminal ganglia. Additionally, HELP inhibited viral replication in human-derived corneas without causing off-target effects, as determined by whole-genome sequencing. These results support the potential clinical utility of HELP for treating refractory HSK.

Herpes-Simplex Virus 1 (HSV-1) infects most humans when they are young, sometimes with fatal consequences. Gene expression occurs in a temporal order upon lytic HSV-1 infection: immediate early (IE) genes are expressed, then early (E) genes, followed by late (L) genes. During this infection cycle, the HSV-1 genome has the potential for exposure to APOBEC3 (A3) proteins, a family of cytidine deaminases that cause C>U mutations on single-stranded DNA (ssDNA), often resulting in a C>T transition. We developed a computational model for the mutational pressure of A3 on the lytic cycle of HSV-1 to determine which viral kinetic gene class is most vulnerable to A3 mutations. Using in silico stochastic methods, we simulated the infectious cycle under varying intensities of A3 mutational pressure. We found that the IE and E genes are more vulnerable to A3 than L genes. We validated this model by analyzing the A3 evolutionary footprints in 25 HSV-1 isolates. We find that IE and E genes have evolved to underrepresent A3 hotspot motifs more so than L genes, consistent with greater selection pressure on IE and E genes. We extend this model to two-step infections, such as those of polyomavirus, and find that the same pattern holds for over 25 human Polyomavirus (HPyVs) genomes. Genes expressed earlier during infection are more vulnerable to mutations than those expressed later., Competing Interests: The authors have declared that no competing interests exist.

The oral cavity is often the first site where viruses interact with the human body. The oral epithelium is a major site of viral entry, replication and spread to other cell types, where chronic infection can be established. In addition, saliva has been shown as a primary route of person-to-person transmission for many viruses. From a clinical perspective, viral infection can lead to several oral manifestations, ranging from common intraoral lesions to tumors. Despite the clinical and biological relevance of initial oral infection, little is known about the mechanism of regulation of the viral life cycle in the oral cavity. Several viruses utilize host epigenetic machinery to promote their own life cycle. Importantly, viral hijacking of host chromatin-modifying enzymes can also lead to the dysregulation of host factors and in the case of oncogenic viruses may ultimately play a role in promoting tumorigenesis. Given the known roles of epigenetic regulation of viral infection, epigenetic-targeted antiviral therapy has been recently explored as a therapeutic option for chronic viral infection. In this review, we highlight three herpesviruses with known roles in oral infection, including herpes simplex virus type 1, Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus. We focus on the respective oral clinical manifestations of these viruses and their epigenetic regulation, with a specific emphasis on the viral life cycle in the oral epithelium.

The drive to withstand environmental stresses and defend against invasion is a universal trait extant in all forms of life. While numerous canonical signaling cascades have been characterized in detail, it remains unclear how these pathways interface to generate coordinated responses to diverse stimuli. To dissect these connections, we followed heparanase (HPSE), a protein best known for its endoglycosidic activity at the extracellular matrix but recently recognized to drive various forms of late-stage disease through unknown mechanisms. Using herpes simplex virus-1 (HSV-1) infection as a model cellular perturbation, we demonstrate that HPSE acts beyond its established enzymatic role to restrict multiple forms of cell-intrinsic defense and facilitate host cell reprogramming by the invading pathogen. We reveal that cells devoid of HPSE are innately resistant to infection and counteract viral takeover through multiple amplified defense mechanisms. With a unique grasp of the fundamental processes of transcriptional regulation and cell death, HPSE represents a potent cellular intersection with broad therapeutic potential.

Herpes simplex virus 1 causes recurrent diseases by reactivating from latency, which requires the viral thymidine kinase (TK) gene. An acyclovir-resistant mutation in TK, V204G, was previously repeatedly identified in a patient with recurrent herpetic keratitis. We found that compared with its parental strain KOS, a laboratory-derived V204G mutant virus was impaired in replication in cultured neurons despite little defect in non-neuronal cells. After corneal inoculation of mice, V204G exhibited defects in ocular replication that were modest over the first three days but severe afterward. Acute replication of V204G in trigeminal ganglia was significantly impaired. However, V204G established latency with viral loads as high as KOS and reactivated with high frequency albeit reduced kinetics. Acyclovir treatment that drastically decreased ocular and ganglionic replication of KOS had little effect on V204G. Thus, despite reduced neuronal replication due to impaired TK activity, this clinically relevant drug-resistant mutant can efficiently establish reactivatable latency., (Copyright © 2021 Elsevier Inc. All rights reserved.)

RIG-I and MDA5 are cytoplasmic RNA sensors that mediate cell intrinsic immunity against viral pathogens. While it has been well-established that RIG-I and MDA5 recognize RNA viruses, their interactive network with DNA viruses, including herpes simplex virus 1 (HSV-1), remains less clear. Using a combination of RNA-deep sequencing and genetic studies, we show that the γ134.5 gene product, a virus-encoded virulence factor, enables HSV growth by neutralization of RIG-I dependent restriction. When expressed in mammalian cells, HSV-1 γ134.5 targets RIG-I, which cripples cytosolic RNA sensing and subsequently suppresses antiviral gene expression. Rather than inhibition of RIG-I K63-linked ubiquitination, the γ134.5 protein precludes the assembly of RIG-I and cellular chaperone 14-3-3ε into an active complex for mitochondrial translocation. The γ134.5-mediated inhibition of RIG-I-14-3-3ε binding abrogates the access of RIG-I to mitochondrial antiviral-signaling protein (MAVS) and activation of interferon regulatory factor 3. As such, unlike wild type virus HSV-1, a recombinant HSV-1 in which γ134.5 is deleted elicits efficient cytokine induction and replicates poorly, while genetic ablation of RIG-I expression, but not of MDA5 expression, rescues viral growth. Collectively, these findings suggest that viral suppression of cytosolic RNA sensing is a key determinant in the evolutionary arms race of a large DNA virus and its host., Competing Interests: The authors have declared that no competing interests exist.

Background: Talimogene laherparepvec (T-VEC), an oncolytic virus, was designed to selectively replicate in and lyse tumor cells, releasing tumor-derived antigen to stimulate a tumor-specific immune response., Methods: In this phase II study in patients with unresectable stage IIIB-IV melanoma, we evaluated non-injected lesions to establish whether baseline or change in intratumoral CD8 + T-cell density (determined using immunohistochemistry) correlated with T-VEC clinical response., Results: Of 112 enrolled patients, 111 received ≥1 dose of T-VEC. After a median follow-up of 108.0 weeks, objective/complete response rates were 28%/14% in the overall population and 32%/18% in patients with stage IIIB-IVM1a disease. No unexpected toxicity occurred. Baseline and week 6 change from baseline CD8 + T-cell density results were available for 91 and 65 patients, respectively. Neither baseline nor change in CD8 + T-cell density correlated with objective response rate, changes in tumor burden, duration of response or durable response rate. However, a 2.4-fold median increase in CD8 + T-cell density in non-injected lesions from baseline to week 6 was observed. In exploratory analyses, multiparameter immunofluorescence showed that after treatment there was an increase in the proportion of infiltrating CD8 + T-cells expressing granzyme B and checkpoint markers (programmed death-1, programmed death-ligand 1 (PD-L1) and cytotoxic T-lymphocyte antigen-4) in non-injected lesions, together with an increase in helper T-cells. Consistent with T-cell infiltrate, we observed an increase in the adaptive resistance marker PD-L1 in non-injected lesions., Conclusions: This study indicates that T-VEC induces systemic immune activity and alters the tumor microenvironment in a way that will likely enhance the effects of other immunotherapy agents in combination therapy., Trial Registration Number: NCT02366195., Competing Interests: Competing interests: JM has acted as a consultant/advisor for and has received honoraria from Almirall, Amgen, Bristol-Myers Squibb, GlaxoSmithKline, Merck Sharp & Dohme, Leo Pharma, Novartis, Pierre Fabre and Roche. IS has acted as a consultant and served on a speaker’s bureau for Biocad, Bristol-Myers Squibb, Merck Sharp & Dohme, Novartis and Roche. DS has received research support from Bristol-Myers Squibb and Novartis; has acted as a consultant for and received royalties from Amgen, Array, AstraZeneca, Bristol-Myers Squibb, Merck Sharp & Dohme, Novartis, Pierre Fabre, and Roche; and has acted as a consultant for Immunocore, Incyte, LeoPharma, Merck Serono, Pfizer, Philogen, and Regeneron. RG has received research support from Johnson & Johnson, Novartis, Merck Serono, Amgen and Pfizer; has received honoraria for advice and/or lectures from Almirall Hermal, Amgen, Bayer, Bristol-Myers Squibb, Merck Serono, Merck Sharp & Dohme, Novartis, Pierre Fabre, Roche, Sanofi/Regeneron, SUN Pharma and 4SC. J-JG has acted as a consultant/in an advisory role for and has received honoraria from Bristol-Myers Squibb, GlaxoSmithKline, Merck Sharp & Dohme, Merck-Pfizer, Pierre Fabre, and Roche. JJS has received research support from AstraZeneca, Bristol-Myers Squibb and Immunocore; has acted as a consultant/in an advisory role for Immunocore, Delcath, Merck Sharp & Dohme, Amgen and Pierre Fabre; and served on a speaker’s bureau for Bristol-Myers Squibb. KSG and AA are employees and stock holders of Amgen. KL is an employee of Amgen. CAP was previously an employee of Amgen. HG has acted as a consultant/in an advisory role for Bristol-Myers Squibb, Merck Sharp & Dohme, Novartis, Amgen, Roche and Pierre Fabre., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

To investigate the acute clinical, immunological, and corneal nerve changes following corneal HSV-1 KOS-63 strain inoculation. Corneas of C57BL/6 mice were inoculated with either low dose (Ld) or high dose (Hd) HSV-1 KOS-63 or culture medium. Clinical evaluation was conducted up to 7 days post inoculation (dpi). Viral titers were assessed by standard plaque assay. Excised corneas were stained for CD45 and beta-III tubulin. Corneal flow cytometry was performed to assess changes in leukocyte subpopulations. Corneal sensation was measured using a Cochet-Bonnet esthesiometer. Naïve, sham-infected (post scarification), and McKrae-infected C57BL/6 corneas served as two negative and positive controls, respectively. Compared to Ld infected mice, Hd HSV-1 KOS-63 demonstrated higher incidence of corneal opacity (1.5 ×) and neovascularization (2.6 × ; p < 0.05). At 7 dpi Hd infected mice showed more severe corneal opacity (2.23 vs. 0.87; p = 0.0003), neovascularization (6.00 vs. 0.75; p < 0.0001), and blepharitis (3.11 vs. 2.06; p = 0.001) compared to the Ld group. At 3 dpi epitheliopathy was significantly larger in the Hd group (23.59% vs. 3.44%; p = 0.001). Similarly, corneal opacity was significantly higher in Hd McKrae-infected corneas as compared with Ld McKrae-infected corneas at 3 and 5 dpi. No significant corneal opacity, neovascularization, blepharitis, and epitheliopathy were observed in naïve or sham-infected mice. Higher viral titers were detected in corneas (1 and 3 dpi) and trigeminal ganglia (TG) (3 and 5 dpi) in Hd versus Ld KOS-63 groups (p < 0.05). Leukocyte density showed a gradual increase over time from 1 to 7 dpi in both KOS-63 and McKrae-infected corneas. Corneal flow cytometric analysis (3 dpi) demonstrated a higher percentage of Gr-1 + (71.6 vs. 26.3) and CD11b + (90.6 vs. 41.1) cells in Hd versus Ld KOS-63 groups. Corneal nerve density significantly decreased in both Hd KOS-63 and Hd McKrae infected corneas in comparison with naïve and sham-infected corneas. At 3 dpi corneal nerve density was lower in the Hd versus Ld KOS-63 groups (16.79 vs. 57.41 mm/mm2; p = 0.004). Corneal sensation decreased accordingly at 5 and 7 dpi in both Ld and Hd KOS-63-infected mice. Corneal inoculation with HSV-1 KOS-63 strain shows acute keratitis and nerve degeneration in a dose-dependent fashion, demonstrating virulence of this strain.

Multiple sclerosis (MS) is an increasingly prevalent progressive autoimmune and debilitating chronic disease that involves the detrimental recognition of central nervous system (CNS) antigens by the immune system. Although significant progress has been made in the last decades on the biology of MS and the identification of novel therapies to treat its symptoms, the etiology of this disease remains unknown. However, recent studies have suggested that viral infections may contribute to disease onset. Interestingly, a potential association between herpes simplex virus type 1 (HSV-1) infection and MS has been reported, yet a direct relationship among both has not been conclusively demonstrated. Experimental autoimmune encephalomyelitis (EAE) recapitulates several aspects of MS in humans and is widely used to study this disease. Here, we evaluated the effect of asymptomatic brain infection by HSV-1 on the onset and severity of EAE in C57BL/6 mice. We also evaluated the effect of infection with an HSV-1-mutant that is attenuated in neurovirulence and does not cause encephalitis. Importantly, we observed more severe EAE in mice previously infected either, with the wild-type (WT) or the mutant HSV-1, as compared to uninfected control mice. Also, earlier EAE onset was seen after WT virus inoculation. These findings support the notion that a previous exposure to HSV-1 can accelerate and enhance EAE, which suggests a potential contribution of asymptomatic HSV-1 to the onset and severity of MS., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Duarte, Altamirano-Lagos, Tabares-Guevara, Opazo, Díaz, Navarrete, Muza, Vallejos, Riedel, Bueno, Kalergis and González.)

Background: Talimogene laherparepvec (T-VEC) is a licensed therapy for use in melanoma patients of stage IIIB-IVM1a with injectable, unresectable metastatic lesions in Europe. Approval was based on the Oncovex Pivotal Trial in Melanoma study, which also included patients with distant metastases and demonstrated an overall response rate (ORR) of 40.5% and a complete response (CR) rate of 16.6%., Objectives: The aim of this study was to assess the outcome of melanoma patients treated with T-VEC in a real-life clinical setting., Methods: Based on data from 10 melanoma centers in Austria, Switzerland and southern Germany, we conducted a retrospective chart review, which included 88 patients (44 male, 44 female) with a median age of 72 years (range 36-95 years) treated with T-VEC during the period from May 2016 to January 2020., Results: 88 patients fulfilled the inclusion criteria for analysis. The ORR was 63.7%. 38 patients (43.2%) showed a CR, 18 (20.5%) had a partial response, 8 (9.1%) had stable disease and 24 (27.3%) patients had a progressive disease. The median treatment period was 19 weeks (range: 1-65), an average of 11 doses (range: 1-36) were applied. 39 (45.3%) patients developed adverse events, mostly mild, grade I (64.1%)., Conclusion: This real-life cohort treatment with T-VEC showed a high ORR and a large number of durable CRs., Competing Interests: Competing interests: JMR received project funding by Amgen, Speakers bureau of Amgen and Bristol Myers Squibb and travel support from Bristol Myers Squibb, Pierre Fabre outside of the submitted work. JMR has intermittent project focused consultant or advisory relationships with Merck/Pfizer, Merck Sharp & Dohme, Amgen, Novartis, Bristol Myers and Squibb and Pierre Fabre and has received travel support from Ultrasun, L’ oreal, Merck Sharp & Dohme, Bristol Myers and Squibb and Pierre Fabre outside of the submitted work. PK has received honoraria for travel/congress support and consulting/advisory roles for Roche, Bristol Myers Squibb (BMS), Merck Sharp and Dome (MSD), Novartis, Amgen, Pierre Fabre and Sanofi Aventis unrelated to the submitted work. ER Honoraria, consulting or advisory role: Amgen, Bayer, Bristol Myers Squibb, MSD, Merck, Novartis, Pierre Fabre, Roche, SanofiSpeakers'bureau: Amgen, Bristol Myers Squibb, MSD, Merck, Novartis, Pierre Fabre, SanofiResearch funding site PI: Amgen, Bristol Myers Squibb, MSD, Novartis, Pierre Fabre, Roche Research funding steering committee: Novartistravel, accommodations, expenses: Amgen, Bristol Myers Squibb, MSD, Merck, Novartis, Pierre Fabre, Roche, Sanofi. CH is associated with consulting or advisory role for Bristol-Myers Squibb, Amgen, Merck Sharp and Dohme, Novartis, Pierre Fabre and Speaker’s bureau of Bristol-Myers Squibb, Amgen, Merck Sharp & Dohme, Pierre Fabre and received travel/accommodations/expenses from Amgen, Bristol-Myers Squibb, Merck Sharp and Dohme, Pierre Fabre.C.HO. is associated with advisory role for Advisory Boards: Amgen, Astra Zeneca, BMS, Inzyte, MSD, Novartis, Pierre Fabre, Roche and Speakers bureau of Amgen, BMS, MSD, Novartis, Roche., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

This review presents the data on the spreading of all known human herpesviruses (НHVs) in female urogenital tract. According to the WHO almost 500 million people worldwide suffer from genital infection caused by НHVs. НHVs were detected in various inflammatory diseases of female upper and lower genital tract (vaginitis and cervicitis), in extrauterine pregnancy (in fallopian tubes), in infertility (cervical channel, endometrium and ovaries). Herpes simplex virus 1 (HSV‑1) was identified for the first time in oocytes after failed in vitro fertilization (IVF). НHVs produce negative effect on the entire reproductive process from conception to childbirth. It was established that HSV, cytomegalovirus (CMV) and human herpesvirus 6 (HHV-6) markedly increase the risk of spontaneous abortion, preterm birth and stillbirth. Intrauterine НHV infection is a major cause of congenital malformations. Data on humoral and cell immunity in genital herpesvirus infections (НHVI) are also reviewed. Intravaginal HSV‑2 infection changes cell composition of vaginal mucosa, i.e., together with cells mobilized from the blood, protective role is performed by resident memory T‑cells (TRM), natural killer cells (NK‑cells) and regulatory T‑cells (Treg) whose function consists in maintaining the balance of the activities of lymphocytes. Constant НHVI spreading is largely explained by transition of primary infection to potentially reactivating latent form, since latent virus is unavailable to immune recognition and medicines. The genome editing system CRISPR/Cas9 can recognize and modify not only active but also latent viruses. The promising pilot results with the use of this system offer the possibility of developing innovative technologies for НHV elimination and НHVI eradication.

Introduction: Herpes simplex viruses type 1 (HSV-1) are extremely widespread throughout the world and, similar to other herpesviruses, establish lifelong persistent infection in the host. Reactivating sporadically, HSV-1 elicits recurrences in both immunocompetent and immunocompromised individuals and can cause serious diseases (blindness, encephalitis, generalized infections). The currently available antiherpetic drugs that aimed mainly at suppressing replication of viral DNA are not always effective enough, for example, due to the development of drug resistance. As we showed earlier the newly discovered compound LAS-131 exhibits the strong and highly selective inhibitory activity against HSV‑1, including strain resistant to acyclovir (selective index, SI = 63). The presence of LAS-131 at a concentration of 20 μg/ml leads to a decrease in the titer of HSV-1 (strain L2) by 4 lg in a one round of HSV-1 replication., Material and Methods: To establish the step(s) of the virus life cycle that is sensitive to the action of LAS-131, we have applied a widely used approach, that made it possible to determine how long the addition of a compound can be postponed before it loses its antiviral activity (time-of-addition assay), and to compare this indicator with the crucial time of application of inhibitors with a well-known mechanism of action (in cell culture)., Results: It has been shown for the first time that LAS-131 retains a pronounced antiviral effect when introduced into the experimental system no later than 9 hours post-infection (p.i.). However, LAS-131 does not affect the release of HSV-1 from the cell., Discussion: Together with published data on the termination of the synthesis of viral DNA 9-12 h after the adsorption in a cell culture infected with HSV with a high multiplicity (≥1 PFU/cell), our results suggest that LAS-131 interferes the life cycle of HSV-1 during synthesis of viral DNA. Further studies of the mechanism of action are necessary to establish definitely the biological target for this compound,.

Herpesvirus genomes enter the eukaryotic nucleus as large linear double stranded DNA molecules that are free of any proteins (naked DNA). Once inside the nucleus, the HSV-1 genomes immediately associate with proteins that will be instrumental in the organization and regulation of these genomes. These initial interactions are thought to determine the fate of the infecting genomes. In general, the host cell has evolved several mechanisms to suppress viral genomes and induce latent or abortive infections. On the other hand, the virus has evolved to use viral and cellular factors to promote lytic infection. Recent findings suggest that not all viral genomes in the infected nucleus will develop progeny and that not all genetically identical cells will support successful virus propagation. Thus, the decision between different fates of infection is determined at both single-cell and single-genome levels. Here we summarize current knowledge on the conditions and interactions that lead to each outcome and discuss the unknown determinants.