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Herpes simplex virus type 1 (HSV-1) can establish latency in humans and easily relapse in immunocompromised patients, with significant mortality. Treatment with acyclovir (ACV) can result in the emergence of HSV resistance. A total of 440 frozen HSV-1 isolates collected from 318 patients from January 2014 to July 2019 were obtained from National Cheng Kung University Hospital in southern Taiwan. These 440 isolates were subjected to phenotypic studies for ACV-resistance by initial screening with the plaque reduction assay (PRA) and further validation by the DNA reduction assay (DRA). The ACV-resistant strains were further investigated by Sanger sequencing for the full-length UL23 and UL30 genes, which encode thymidine kinase and DNA polymerase, respectively. Hematological malignancies or hematopoietic stem-cell transplantation patients accounted for 56.9% (124/218) among the immunocompromised patients (218/318) in this study. Repeated sampling for HSV testing was 50% (109/218) in immunocompromised patients. Only 1.38% (3/218) of immunocompromised patients and 0.9% (3/318) of all patients developed ACV-resistant HSV-1 as measured by phenotypic screening assays. It is noteworthy that a novel Y248D mutation in the UL23 gene from an immunocompromised patient was found by both PRA and DRA. In 3D protein predicting analysis, uncharged Y248 was located at an alpha-helix and substituted by negative-charged D248, which may alter the function of viral thymidine kinase. Besides, three unreported mutations related to natural polymorphism were found in virus isolates from two immunocompetent patients, including 683-688 deletion, R227H, and A351D in the UL30 gene. These data show that the prevalence of ACV-resistant HSV-1 among immunocompromised patients in southern Taiwan is low. These results will be helpful for the clinical management and treatment of HSV infections., (© 2023 The Authors. Journal of Medical Virology published by Wiley Periodicals LLC.)

Herpes simplex virus 1 (HSV-1)-induced encephalitis is the most common cause of sporadic, fatal encephalitis in humans. HSV-1 has at least 10 different envelope glycoproteins, which can promote virus infection. The ligands for most of the envelope glycoproteins and the significance of these ligands in virus-induced encephalitis remain elusive. Here, we show that glycoprotein E (gE) binds to the cellular protein, annexin A1 (Anx-A1) to enhance infection. Anx-A1 can be detected on the surface of cells permissive for HSV-1 before infection and on virions. Suppression of Anx-A1 or its receptor, formyl peptide receptor 2 (FPR2), on the cell surface and gE or Anx-A1 on HSV-1 envelopes reduced virus binding to cells. Importantly, Anx-A1 knockout, Anx-A1 knockdown, or treatments with the FPR2 antagonist reduced the mortality and tissue viral loads of infected mice. Our results show that Anx-A1 is a novel enhancing factor of HSV-1 infection. Anx-A1-deficient mice displayed no evident physiology and behavior changes. Hence, targeting Anx-A1 and FPR2 could be a promising prophylaxis or adjuvant therapy to decrease HSV-1 lethality., Competing Interests: The authors have declared that no competing interests exist.

Histone modifications control the lytic gene expression of herpes simplex virus 1 (HSV-1). The heterochromatin mark, trimethylation of histone H3 on lysine (K) 9 (H3K9me3), is detected on HSV-1 genomes at early phases of infection to repress viral gene transcription. However, the components and mechanisms involved in the process are mostly unknown. Integrin-linked kinase (ILK) is activated by PI3K to phosphorylate Akt and promote several RNA virus infections. Akt has been shown to enhance HSV-1 infection, suggesting a pro-viral role of ILK in HSV-1 infection that has not been addressed before. Here, we reveal that ILK enhances HSV-1 replication in an Akt-independent manner. ILK reduces the accumulation of H3K9me3 on viral promoters and replication compartments. Notably, ILK reduces H3K9me3 in a manner independent of ICP0. Instead, we show an increased binding of H3K9 methyltransferase SUV39H1 and corepressor TRIM28 on viral promoters in ILK knockdown cells. Knocking down SUV39H1 or TRIM28 increases HSV-1 lytic gene transcription in ILK knockdown cells. These results show that ILK antagonizes SVU39H1- and TRIM28-mediated repression on lytic gene transcription. We further demonstrate that ILK knockdown reduces TRIM28 phosphorylation on serine 473 and 824 in HSV-1-infected cells, suggesting that ILK facilitates TRIM28 phosphorylation to abrogate its inhibition on lytic gene transcription. OSU-T315, an ILK inhibitor, suppresses HSV-1 replication in cells and mice. In conclusion, we demonstrate that ILK decreases H3K9me3 on HSV-1 DNA by reducing SUV39H1 and TRIM28 binding. Moreover, our results suggest that targeting ILK could be a broad-spectrum antiviral strategy for DNA and RNA virus infections, especially for DNA viruses controlled by histone modifications., 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 © 2022 Tsai, Chen, Chang, Hsiao and Wang.)

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.

Bortezomib suppressing NF-κB activity is an effective therapy for patients with myeloma or lymphoma. However, this drug can cause adverse effects, neutropenia, and recurrent infections of herpes viruses. Among herpes viruses, HSV-1 can reactivate to induce mortality. The important issues regarding how bortezomib diminishes neutrophils, whether bortezomib can induce HSV-1 reactivation, and how bortezomib exacerbates HSV-1 infection, need investigation. Using the murine model, we found that bortezomib induced HSV-1 reactivation. Bortezomib diminished neutrophil numbers in organs of uninfected and HSV-1-infected mice and turned a nonlethal infection to lethal with elevated tissue viral loads. In vitro results showed that bortezomib and HSV-1 collaborated to enhance the death and apoptosis of mouse neutrophils. The leukocyte deficiency induced by chemotherapies is generally believed to be the cause for aggravating virus infections. Here we show the potential of pathogen to exacerbate chemotherapy-induced leukocyte deficiency., (©2019 Society for Leukocyte Biology.)

Herpes simplex virus 1 (HSV-1) establishes latency in neural tissues of immunocompetent mice but persists in both peripheral and neural tissues of lymphocyte-deficient mice. Thymidine kinase (TK) is believed to be essential for HSV-1 to persist in neural tissues of immunocompromised mice, because infectious virus of a mutant with defects in both TK and UL24 is detected only in peripheral tissues, but not in neural tissues, of severe combined immunodeficiency mice (T. Valyi-Nagy, R. M. Gesser, B. Raengsakulrach, S. L. Deshmane, B. P. Randazzo, A. J. Dillner, and N. W. Fraser, Virology 199:484-490, 1994, https://doi.org/10.1006/viro.1994.1150). Here we find infiltration of CD4 and CD8 T cells in peripheral and neural tissues of mice infected with a TK-negative mutant. We therefore investigated the significance of viral TK and host T cells for HSV-1 to persist in neural tissues using three genetically engineered mutants with defects in only TK or in both TK and UL24 and two strains of nude mice. Surprisingly, all three mutants establish persistent infection in up to 100% of brain stems and 93% of trigeminal ganglia of adult nude mice at 28 days postinfection, as measured by the recovery of infectious virus. Thus, in mouse neural tissues, host T cells block persistent HSV-1 infection, and viral TK is dispensable for the virus to establish persistent infection. Furthermore, we found 30- to 200-fold more virus in neural tissues than in the eye and detected glycoprotein C, a true late viral antigen, in brainstem neurons of nude mice persistently infected with the TK-negative mutant, suggesting that adult mouse neurons can support the replication of TK-negative HSV-1., Importance: Acyclovir is used to treat herpes simplex virus 1 (HSV-1)-infected immunocompromised patients, but treatment is hindered by the emergence of drug-resistant viruses, mostly those with mutations in viral thymidine kinase (TK), which activates acyclovir. TK mutants are detected in brains of immunocompromised patients with persistent infection. However, answers to the questions as to whether TK-negative (TK - ) HSV-1 can establish persistent infection in brains of immunocompromised hosts and whether neurons in vivo are permissive for TK - HSV-1 remain elusive. Using three genetically engineered HSV-1 TK - mutants and two strains of nude mice deficient in T cells, we found that all three HSV-1 TK - mutants can efficiently establish persistent infection in the brain stem and trigeminal ganglion and detected glycoprotein C, a true late viral antigen, in brainstem neurons. Our study provides evidence that TK - HSV-1 can persist in neural tissues and replicate in brain neurons of immunocompromised hosts., (Copyright © 2017 American Society for Microbiology.)

Unlabelled: Herpes simplex virus 1 (HSV-1) establishes latency in neurons of the brains and sensory ganglia of humans and experimentally infected mice. The latent virus can reactivate to cause recurrent infection. Both primary and recurrent infections can induce diseases, such as encephalitis. In humans, the majority of encephalitis cases occur as a recurrent infection. However, in the past, numerous mouse studies documented that viral reactivation occurs efficiently in the ganglion, but extremely rarely in the brain, when assessed ex vivo by cultivating minced tissue explants. Here, we compare the brains and the trigeminal ganglia of mice latently infected with HSV-1 (strain 294.1 or McKrae) for levels of viral genomes and in vivo reactivation. The numbers of copies of 294.1 and McKrae genomes in the brain stem were significantly greater than those in the trigeminal ganglion. Most importantly, 294.1 and McKrae reactivation was detected in the brain stems earlier than in the trigeminal ganglia of mice treated with hyperthermia to reactivate latent virus in vivo. In addition, the brain stem yielded reactivated virus at a high frequency compared with the trigeminal ganglion, especially in mice latently infected with 294.1 after hyperthermia treatment. These results provide evidence that recurrent brain infection can be induced by the reactivation of latent virus in the brain in situ., Importance: Herpes simplex virus 1 (HSV-1) establishes latency in neurons of the brains and sensory ganglia of humans and experimentally infected mice. The latent virus can reactivate to cause recurrent infection. In the past, studies of viral reactivation focused on the ganglion, because efficient viral reactivation was detected in the ganglion but not in the brain when assessed ex vivo by cultivating mouse tissue explants. In this study, we report that the brain contains more viral genomes than the trigeminal ganglion in latently infected mice. Notably, the brain yields reactivated virus early and efficiently compared with the trigeminal ganglion after mice are stimulated to reactivate latent virus. Our findings raise the potential importance of HSV-1 latent infection and reactivation in the brain., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Herpes simplex virus 1 (HSV-1) infects the majority of the human population and establishes latency by maintaining viral genomes in neurons of sensory ganglia. Latent virus can undergo reactivation to cause recurrent infection. Both primary and recurrent infections can cause devastating diseases, including encephalitis and corneal blindness. Acyclovir is used to treat patients, but virus resistance to acyclovir is frequently reported. Recent in vitro findings reveal that pretreatment of cells with tranylcypromine (TCP), a drug widely used in the clinic to treat neurological disorders, restrains HSV-1 gene transcription by inhibiting the histone-modifying enzyme lysine-specific demethylase 1. The present study was designed to examine the anti-HSV-1 efficacy of TCP in vivo because of the paucity of reports on this issue. Using the murine model, we found that TCP decreased the severity of wild-type-virus-induced encephalitis and corneal blindness, infection with the acyclovir-resistant (thymidine kinase-negative) HSV-1 mutant, and tissue viral loads. Additionally, TCP blocked in vivo viral reactivation in trigeminal ganglia. These results support the therapeutic potential of TCP for controlling HSV-1 infection.

The majority of encephalitis induced by herpes simplex virus type I (HSV-1) is due to viral reactivation from latency, but few studies have investigated the factors influencing viral reactivation in the brain due to the lack of a sensitive assay. We have established an ex vivo explant assay, which induced efficient viral reactivation in the dissociated mouse brain. Applying this assay, we investigated the infection of four HSV-1 strains with varying degrees of neurovirulence in three mouse strains with different levels of susceptibility to HSV-1 infection. We found that virulent HSV-1 strains and susceptible mouse strains exhibited prolonged viral growth during acute infection, increased latent viral genomes, and efficient explant reactivation in the brain stem. Collectively, both viral neurovirulence and host susceptibility positively correlate with HSV-1 reactivation from the explanted mouse brain., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Acyclovir (ACV)-resistant herpes simplex virus type 1 (HSV-1) causes severe diseases in immunocompromised patients, so identification of new therapies is needed. Interferons (IFNs) are used to treat several other viral infections in the clinic, and IFN-beta and IFN-gamma are known to cooperatively reduce wild-type HSV-1 replication in the corneas of immunocompetent mice. Because IFN-gamma has been shown to exert an antiviral effect mostly through T cells, whether combined IFN treatment can still inhibit ACV-resistant HSV-1 replication, especially in immunocompromised hosts, is unknown. The present study evaluated the efficacy of combined IFN treatment on ACV-resistant HSV-1 mutants. In vitro results showed that IFN-beta acted synergistically with IFN-gamma to inhibit HSV-1 replication in both human and mouse cell lines. Some ACV-resistant mutants were actually hypersensitive to combined IFN treatment. In vivo results showed that topical treatment with a low dose of IFN-beta plus IFN-gamma (200 U each) on mouse corneas efficiently reduced the viral loads by up to 4, 4 and 3 logs, respectively, in the eyes, trigeminal ganglia and brainstems of wild-type and also immunocompromised nude mice infected or co-infected with ACV-resistant HSV-1 in a manner independent of T cells. A highly efficient reduction in HSV acute replication by combined IFN treatment led to a dramatic decrease in subsequent virus reactivation from neural tissues, trigeminal ganglia, brainstems and spinal cords of latently infected mice. Thus, a combination of IFN-beta and IFN-gamma could be a potential treatment for ACV-resistant HSV-1 in immunocompromised patients.

Herpes simplex virus type 1 (HSV-1) infection is the most common cause of sporadic, fatal encephalitis, but current understanding of how the virus interacts with cellular factors to regulate disease progression is limited. Here, we show that HSV-1 infection induced the expression of the cellular transcription factor early growth response 1 (Egr-1) in a human neuronal cell line. Egr-1 increased viral replication by activating promoters of viral productive cycle genes through binding to its corresponding sequences in the viral promoters. Mouse studies confirmed that Egr-1 expression was enhanced in HSV-1-infected brains and that Egr-1 functions to promote viral replication in embryonic fibroblasts. Furthermore, Egr-1 deficiency or knockdown of Egr-1 by a DNA-based enzyme greatly reduced the mortality of HSV-1-infected mice by decreasing viral loads in tissues. This study provides what we believe is the first evidence that Egr-1 increases the mortality of HSV-1 encephalitis by enhancing viral replication. Moreover, blocking this cellular machinery exploited by the virus could prevent host mortality.

Herpes simplex virus type 1 (HSV-1) is an important pathogen related to epilepsy. We have shown previously that corneal inoculation of mice with HSV-1 causes acute spontaneous behavioral and electrophysiological seizures and increases hippocampal excitability and kainite-induced seizure susceptibility. In this study, we aimed to determine whether early-life HSV-1 infection in mice might cause short- and long-term enhanced susceptibility to pentylenetetrazol (PTZ)-induced seizures and to evaluate whether early antiviral drug therapy was effectively ameliorating this deficit. Seizure threshold was calculated by the latency of onset of the myoclonic jerk, generalized clonus, and maximal tonic-clonic convulsion. We demonstrate that the localization of viral antigens was predominantly within the bilateral temporal areas (amygdala, piriform, and entorhinal cortex) of HSV-1-infected mice. We also present evidence that mice of all HSV-1-infected groups had a shorter latency and higher severity to PTZ-induced seizures than in age-matched, mock-infected controls. Treatment of HSV-1-infected mice with valacyclovir, a potent inhibitor of HSV-1 replication, produced a dose-dependent decrease in the signs of neurological deficits, pathological damages, and PTZ-induced seizure severity. Our results are consistent with the hypothesis that early-life HSV-1 infection leads to persistent enhancement of neuronal excitability in limbic circuits, which could result in an overall increased propensity to induce seizures later in life. Additionally, prompt optimal antiviral therapy effectively decreases seizure susceptibility in HSV-1-infected mice by limiting the level of viral replication and inflammatory response induced by virus. The present study provides not only experimental evidence, but also a new therapeutic strategy in HSV-1-associated human epilepsy.

Purpose: Epileptic seizures are frequently seen after viral encephalitis. Herpes simplex virus type 1 (HSV-1) encephalitis is the most common cause of acquired epilepsy in humans. However, little information is available about the neuropathogenesis of HSV-1-associated seizures. We have developed an in vitro HSV-1-infected organotypic hippocampal slice culture to elucidate the underlying mechanisms of HSV-1-associated acute seizure activity., Methods: Hippocampal slice cultures were prepared from postnatal day 10 to 12 rat pups. Wild-type HSV-1 strain RE (1 x 10(5) PFU) was applied to cultures at 14 days in vitro. The excitability of CA3 pyramidal cells and hippocampal network properties were measured with electrophysiological recordings. Hematoxylin-eosin (H&E) and Timm stains were used., Results: HSV-1 infection induces epileptiform activity, neuron loss, and subsequently a dramatic increase of mossy fiber sprouting in the supragranular area. With intracellular recordings, surviving CA3 pyramidal cells exhibited a more depolarizing resting membrane potential concomitant with an increase in membrane input resistance and had a lower threshold to generate synchronized bursts and a decrease in the amplitude of afterhyperpolarization than did controls. When the antiherpes agent acyclovir was applied with a delay of 1 or 24 h after HSV-1 infection, a dramatic inhibition of HSV-1 replication and protection of the neuron loss were observed., Conclusions: These results suggest that a direct change in the excitability of the hippocampal CA3 neuronal network and HSV-1-induced neuron loss resulting in subsequent mossy fiber reorganization may play an important role in the generation of epileptiform activity.

Latent infections by herpes simplex virus are characterized by repression of productive-cycle gene expression. Several hypotheses to explain this repression involve inhibition of expression of the immediate-early gene activator ICP0 during latency. To address these hypotheses, we developed quantitative reverse transcriptase-PCR assays that detected spliced and intron-containing ICP0 transcripts in mouse ganglia latently infected with wild-type virus. In these ganglia, the numbers of spliced ICP0 transcripts correlated better with the numbers of transcripts from the immediate-early gene encoding ICP4 than with those from the early gene encoding thymidine kinase. There were fewer spliced than intron-containing ICP0 transcripts on average, with considerable ganglion-to-ganglion variation. We then investigated whether ICP0 expression in latently infected ganglia is reduced by the latency-associated transcripts (LATs) and whether splicing of ICP0 transcripts is inhibited by the product of open reading frame (ORF) P. A LAT deletion mutation which essentially eliminates expression of the major LATs did not appreciably increase levels of ICP0 transcripts. LAT deletion mutants did, however, appear to express reduced levels of intron-containing ICP0 transcripts. ORF P mutations did not alter levels of ICP0 transcripts in a manner consistent with inhibition of ICP0 splicing by ORF P. Although these results argue against antisense inhibition of ICP0 expression by LATs or inhibition of ICP0 splicing by ORF P, they are consistent with the possibilities of a block between immediate-early and early gene expression and regulation of spliced versus intron-containing ICP0 transcripts in latently infected ganglia.