Herpes Simplex Virus Reactivation Research Articles

Acyclovir for Mechanically Ventilated Patients with Herpes Simplex Virus Oropharyngeal Reactivation

Background: The role of herpes simplex virus (HSV) reactivation on morbidity/mortality in intensive care unit patients requiring mechanical ventilation remains controversial. Methods: In this multicentre study, non-immunosuppressed adults >18 years old, mechanically ventilated for at least 96 hours and expected to remain on mechanical ventilation for ≥48 hours, with HSV oropharyngeal reactivation, were double-blindly randomized to receive intravenous acyclovir (5 mg/kg tid, for 14 days) or a matching placebo (controls). Randomisation (1:1) was stratified by study site, pre-randomisation mechanical ventilation duration and number of organ failure, and performed using a secure web-based system. The primary endpoint was ventilator-free days from randomisation to day 60. Findings: The 16 participating centres randomised 238 patients: 119 to receive acyclovir and 119 the placebo. On day 60, for acyclovir recipients and controls, respectively, the median (IQR) numbers of ventilator-free days were 35 (0-53) and 36 (0-50) (p=0.17 for between-group comparison); 26 (22%) and 39 (33%) patients had died (risk difference, 0.11, 95% CI, - 0.004 to 0.22, p=0.059). The adverse-event frequency was similar for both groups (28% in the acyclovir group and 23% in the placebo group, p= 0.4), particularly with acute renal failure post-randomisation affecting three acyclovir recipients and two controls. Four patients in the acyclovir group (vs. none in the placebo group) stopped the study drug for treatment-related adverse event. Interpretation: In non-immunosuppressed patients mechanically ventilated for ≥96 hours with HSV reactivation in the throat, intravenous acyclovir use (5 mg/kg tid) did not increase the number of ventilator-free days on day 60, compared to a placebo. Trial Registration: The trial is registered on ClinicalTrials.gov, number NCT02152358. Funding Statement: Direction de la Recherche Clinique et du Developpement and the French Ministry of Health (Programme Hospitalier de Recherche Clinique 2011). Declaration of Interests: No potential conflict of interest relevant to this article was reported. The following conflicts of interest are outside the area of the article. Dr Luyt reports receiving lecture fees from ThermoFischer Brahms, Biomerieux, Merck Sharp & Dohme and Aerogen, and consulting fees from Bayer Healthcare, Carmat and Faron. Dr Jaber reports receiving consulting fees from Drager, Fisher-Paykel, Xenios-Fresenius Medical and Medtronic. Dr Rimmele reports receiving consulting or lecture fees from Fresenius Medical Care, Baxter, Medtronic, Biomerieux, BBraun. Dr Lu reports receiving lectures fees from Aerogen. Dr Combes reports receiving grant support and lectures fees from Maquet and Baxter and consulting fees from Hemovent. Dr Wolff reports receiving consulting fees from Merck Sharp & Dohme, Inotrem, Correvio, Menarini and Pfizer. Dr Chastre reports receiving lecture and consulting fees from Bayer Healthcare, Pfizer, Merck Sharp & Dohme, Aridis, the Medicines Company, Astra Zenecca, Tigenix, Accelerate Diagnotics, Inotrem and Glaxo Smith Kline Ethics Approval Statement: An independent Ethics Committee (Comite de Protection des Personnes Sud Mediterranee 5) approved its protocol.

A Case of Inconspicuous Recurrent Herpes Labialis Mimicking Unilateral Angular Cheilitis Unilateral Angular Cheilitis

Recurrent herpes labialis (RHL) is a common manifestation of herpes simplex virus (HSV) reactivation in immunocompetent individuals, whereas angular cheilitis is an inflammatory lesion occurring on one or both lip commissures and is induced by local and/or systemic conditions. We describe a case of RHL eruption on the corner of the mouth, easily mistaken as angular cheilitis. Case Report: A 21-year-old male presented to our dental hospital with a 3 day history of a painful, unilateral lesion on the left corner of his mouth. The lesion featured an erythematous base with a yellowish crust that extended outward. We diagnosed the lesion as RHL. We prescribed chlorhexidine solution and topical acyclovir to be applied onto the lesion. At 2 weeks follow-up, the lesion was resolved. An RHL lesion that erupts on the corner of the mouth may initially resemble angular cheilitis. However, the typical clinical presentation, history of recurrence, and the absence of predisposing factors for other lesions suggested an infection caused by HSV. Conclusion: RHL which occurred at one side of the mouth corner can be similar with unilateral AC. But, detailed history taking and clinical observation led to correct diagnosis and management.

Herpes Simplex Virus 1 Latency and the Kinetics of Reactivation Are Regulated by a Complex Network of Interactions between the Herpesvirus Entry Mediator, Its Ligands (gD, BTLA, LIGHT, and CD160), and the Latency-Associated Transcript.

Recently, we reported that the herpesvirus entry mediator (HVEM; also called TNFRSF14 or CD270) is upregulated by the latency-associated transcript (LAT) of herpes simplex virus 1 (HSV-1) and that the absence of HVEM affects latency reactivation but not primary infection in ocularly infected mice. gD has been shown to bind to HVEM. LIGHT (TNFSF14), CD160, and BTLA (B- and T-lymphocyte attenuator) also interact with HVEM and can interfere with HSV gD binding. It was not known if LIGHT, CD160, or BTLA affected the level of latency reactivation in the trigeminal ganglia (TG) of latently infected mice. To address this issue, we ocularly infected LIGHT-/-, CD160-/-, and BTLA-/- mice with LAT(+) and LAT(-) viruses, using similarly infected wild-type (WT) and HVEM-/- mice as controls. The amount of latency, as determined by the levels of gB DNA in the TG of the LIGHT-/-, CD160-/-, and BTLA-/- mice infected with either LAT(+) or LAT(-) viruses, was lower than that in WT mice infected with LAT(+) virus and was similar in WT mice infected with LAT(-) virus. The levels of LAT RNA in HVEM-/-, LIGHT-/-, CD160-/-, and BTLA-/- mice infected with LAT(+) virus were similar and were lower than the levels of LAT RNA in WT mice. However, LIGHT-/-, CD160-/-, and BTLA-/- mice, independent of the presence of LAT, had levels of reactivation similar to those of WT mice infected with LAT(+) virus. Faster reactivation correlated with the upregulation of HVEM transcript. The LIGHT-/-, CD160-/-, and BTLA-/- mice had higher levels of HVEM expression, and this, along with the absence of BTLA, LIGHT, or CD160, may contribute to faster reactivation, while the absence of each molecule, independent of LAT, may have contributed to lower latency. This study suggests that, in the absence of competition with gD for binding to HVEM, LAT RNA is important for WT levels of latency but not for WT levels of reactivation.IMPORTANCE The effects of BTLA, LIGHT, and CD160 on latency reactivation are not known. We show here that in BTLA, LIGHT, or CD160 null mice, latency is reduced; however, HVEM expression is upregulated compared to that of WT mice, and this upregulation is associated with higher reactivation that is independent of LAT but dependent on gD expression. Thus, one of the mechanisms by which BTLA, LIGHT, and CD160 null mice enhance reactivation appears to be the increased expression of HVEM in the presence of gD. Thus, our results suggest that blockade of HVEM-LIGHT-BTLA-CD160 contributes to reduced HSV-1 latency and reactivation.

Critical Role of Regulatory T Cells in the Latency and Stress-Induced Reactivation of HSV-1

Herpes simplex virus 1 (HSV-1) spreads in populations through a latency entry and reactivation cycle. The role of host immune-suppressive factor regulatory Tcells (Treg cells) in controlling latency establishment and reactivation is not completely understood. Here, using an HSV-1 ocular infection murine model, we observe a positive correlation between the level of Treg cells and viral infectivity and demonstrate the requirement for Treg cells in latency establishment. Furthermore, we show that host stress leads to HSV-1 reactivation via increased Treg cell control of CD8+ Tcells, permitting viral replication under diminished immune surveillance. Together, we propose that Treg cell regulation may serve as a key target for controlling HSV infection.

Promyelocytic leukemia (PML) nuclear bodies (NBs) induce latent/quiescent HSV-1 genomes chromatinization through a PML NB/Histone H3.3/H3.3 Chaperone Axis.

Herpes simplex virus 1 (HSV-1) latency establishment is tightly controlled by promyelocytic leukemia (PML) nuclear bodies (NBs) (or ND10), although their exact contribution is still elusive. A hallmark of HSV-1 latency is the interaction between latent viral genomes and PML NBs, leading to the formation of viral DNA-containing PML NBs (vDCP NBs), and the complete silencing of HSV-1. Using a replication-defective HSV-1-infected human primary fibroblast model reproducing the formation of vDCP NBs, combined with an immuno-FISH approach developed to detect latent/quiescent HSV-1, we show that vDCP NBs contain both histone H3.3 and its chaperone complexes, i.e., DAXX/ATRX and HIRA complex (HIRA, UBN1, CABIN1, and ASF1a). HIRA also co-localizes with vDCP NBs present in trigeminal ganglia (TG) neurons from HSV-1-infected wild type mice. ChIP and Re-ChIP show that vDCP NBs-associated latent/quiescent viral genomes are chromatinized almost exclusively with H3.3 modified on its lysine (K) 9 by trimethylation, consistent with an interaction of the H3.3 chaperones with multiple viral loci and with the transcriptional silencing of HSV-1. Only simultaneous inactivation of both H3.3 chaperone complexes has a significant impact on the deposition of H3.3 on viral genomes, suggesting a compensation mechanism. In contrast, the sole depletion of PML significantly impacts the chromatinization of the latent/quiescent viral genomes with H3.3 without any overall replacement with H3.1. vDCP NBs-associated HSV-1 genomes are not definitively silenced since the destabilization of vDCP NBs by ICP0, which is essential for HSV-1 reactivation in vivo, allows the recovery of a transcriptional lytic program and the replication of viral genomes. Consequently, the present study demonstrates a specific chromatin regulation of vDCP NBs-associated latent/quiescent HSV-1 through an H3.3-dependent HSV-1 chromatinization involving the two H3.3 chaperones DAXX/ATRX and HIRA complexes. Additionally, the study reveals that PML NBs are major actors in latent/quiescent HSV-1 H3.3 chromatinization through a PML NB/histone H3.3/H3.3 chaperone axis.

The effect of levamisole in the treatment of recalcitrant recurrent erythema multiforme major: An observational study

BackgroundErythema multiforme major (EMM) is an immune-mediated mucocutaneous eruption mostly triggered by herpes simplex virus (HSV) infection. A vicious circle of recurrence may be developed due to HSV reactivation and prolonged use of systemic corticosteroids to control EMM. Levamisole is an immunomodulator and has been applied to prevent relapses of recurrent HSV infection. ObjectiveTo evaluate the clinical efficacy and safety of levamisole in patients with recalcitrant recurrent EMM. MethodsWe enrolled 23 patients with recurrent EMM treated with levamisole and 24 controls, and analyzed the demographics, treatments and outcomes. ResultsPatients with recurrent EMM for years (mean 3.99 ± 2.71) showed significantly reduced recurrences after various durations of levamisole treatment (recurrences after and before treatment: 3.98 ± 1.04 vs 6.75 ± 1.45 times per year, p = 1.33x10−8). The recurrences of EMM also significantly reduced after levamisole treatment comparing to that of patients without levamisole treatment (p = 3.77x10−9). No patient was reported to have severe side effects during or after levamisole treatment. ConclusionsLevamisole was effective in reducing recurrences of recalcitrant recurrent EMM and can thus be considered an alternative or add-on therapy for this disorder.

Treating Infantile Spasms with High-Dose Oral Corticosteroids: A Retrospective Review of 87 Children

BackgroundHormonal therapy is the treatment of choice in most patients with infantile spasms, but the optimal way to provide this therapy is unclear. Intramuscular adrenocorticotropic hormone (ACTH) has historically used first-line; however, there are significant logistical and financial issues. Our institution has used high-dose prednisolone as the first-line hormonal treatment of infantile spasms since 2006 and published our early experience with 15 infants in 2009. This study updates our institutional experience over more than 10 years of continuous use. MethodsCharts of infants who presented to the Johns Hopkins Hospital with infantile spasms and were treated with high-dose oral prednisolone (40-60 mg/day) from January, 2006 through December, 2016 were reviewed. Electroclinical response was defined as clinical spasm-freedom and resolution of hypsarrhythmia within two weeks of initiation of therapy. Presence of infantile spasms at three months and adverse effects throughout treatment were evaluated. ResultsOver the 10-year period, 87 infants with new-onset infantile spasms were treated. Electroclinical response occurred in 64% infants within two weeks; 62% were spasm-free at three months. Fifty-two percent had side effects, primarily irritability, weight gain, and gastroesophageal reflux. Five percent had major adverse events, including gastrointestinal bleeding (n = 2), herpes simplex virus reactivation (n = 1), and necrotizing enterocolitis (n = 1). ConclusionsOur results continue to demonstrate that high-dose oral prednisolone is very effective for the treatment of new-onset infantile spasms, with few major adverse effects. Oral prednisolone represents a less expensive, readily available alternative to adrenocorticotropic hormone injections.

Frequency, symptoms, risk factors, and outcomes of autoimmune encephalitis after herpes simplex encephalitis: a prospective observational study and retrospective analysis

Herpes simplex encephalitis can trigger autoimmune encephalitis that leads to neurological worsening. We aimed to assess the frequency, symptoms, risk factors, and outcomes of this complication. We did a prospective observational study and retrospective analysis. In the prospective observational part of this study, we included patients with herpes simplex encephalitis diagnosed by neurologists, paediatricians, or infectious disease specialists in 19 secondary and tertiary Spanish centres (Cohort A). Outpatient follow-up was at 2, 6, and 12 months from onset of herpes simplex encephalitis. We studied another group of patients retrospectively, when they developed autoimmune encephalitis after herpes simplex encephalitis (Cohort B). We compared demographics and clinical features of patients who developed autoimmune encephalitis with those who did not, and in patients who developed autoimmune encephalitis we compared these features by age group (patients ≤4 years compared with patients >4 years). We also used multivariable binary logistic regression models to assess risk factors for autoimmune encephalitis after herpes simplex encephalitis. Between Jan 1, 2014, and Oct 31, 2017, 54 patients with herpes simplex encephalitis were recruited to Cohort A, and 51 were included in the analysis (median age 50 years [IQR 5-68]). At onset of herpes simplex encephalitis, none of the 51 patients had antibodies to neuronal antigens; during follow-up, 14 (27%) patients developed autoimmune encephalitis and all 14 (100%) had neuronal antibodies (nine [64%] had NMDA receptor [NMDAR] antibodies and five [36%] had other antibodies) at or before onset of symptoms. The other 37 patients did not develop autoimmune encephalitis, although 11 (30%) developed antibodies (n=3 to NMDAR, n=8 to unknown antigens; p<0·001). Antibody detection within 3 weeks of herpes simplex encephalitis was a risk factor for autoimmune encephalitis (odds ratio [OR] 11·5, 95% CI 2·7-48·8; p<0·001). Between Oct 7, 2011, and Oct 31, 2017, there were 48 patients in Cohort B with new-onset or worsening neurological symptoms not caused by herpes simplex virus reactivation (median age 8·8 years [IQR 1·1-44·2]; n=27 male); 44 (92%) patients had antibody-confirmed autoimmune encephalitis (34 had NMDAR antibodies and ten had other antibodies). In both cohorts (n=58 patients with antibody-confirmed autoimmune encephalitis), patients older than 4 years frequently presented with psychosis (18 [58%] of 31; younger children not assessable). Compared with patients older than 4 years, patients aged 4 years or younger (n=27) were more likely to have shorter intervals between onset of herpes simplex encephalitis and onset of autoimmune encephalitis (median 26 days [IQR 24-32] vs 43 days [25-54]; p=0·0073), choreoathetosis (27 [100%] of 27 vs 0 of 31; p<0·001), decreased level of consciousness (26 [96%] of 27 vs seven [23%] of 31; p<0·001), NMDAR antibodies (24 [89%] of 27 vs 19 [61%] of 31; p=0·033), and worse outcome at 1 year (median modified Rankin Scale 4 [IQR 4-4] vs 2 [2-3]; p<0·0010; seizures 12 [63%] of 19 vs three [13%] of 23; p=0·001). The results of our prospective study show that autoimmune encephalitis occurred in 27% of patients with herpes simplex encephalitis. It was associated with development of neuronal antibodies and usually presented within 2 months after treatment of herpes simplex encephalitis; the symptoms were age-dependent, and the neurological outcome was worse in young children. Prompt diagnosis is important because patients, primarily those older than 4 years, can respond to immunotherapy. Mutua Madrileña Foundation, Fondation de l'Université de Lausanne et Centre Hospitalier Universitaire Vaudois, Instituto Carlos III, CIBERER, National Institutes of Health, Generalitat de Catalunya, Fundació CELLEX.

Viral Hypothesis and Antiviral Treatment in Alzheimer's Disease.

Viruses, particularly herpes simplex virus (HSV), may be a cause of Alzheimer's disease (AD). The evidence supporting the viral hypothesis suggests that antiviral treatment trials, which have not been conducted, are warranted. HSV1 (oral herpes) and HSV2 (genital herpes) can trigger amyloid aggregation, and their DNA is common in amyloid plaques. HSV1 reactivation is associated with tau hyperphosphorylation and possibly tau propagation. Anti-HSV drugs reduce Aβ and p-tau accumulation in infected mouse brains. Clinically, after the initial oral infection, herpes simplex virus-1 (HSV1) becomes latent in the trigeminal ganglion and recurrent reactivation may produce neuronal damage and AD pathology. Clinical studies show cognitive impairment in HSV seropositive patients, and antiviral drugs show strong efficacy against HSV. An antiviral treatment trial in AD is clearly warranted. A phase II treatment trial with valacyclovir, an anti-HSV drug, recently began with evaluation of clinical and biomarker outcomes.

Delay of alternative antiviral therapy and poor outcomes of acyclovir-resistant herpes simplex virus infections in recipients of allogeneic stem cell transplant - a retrospective study.

Acyclovir is commonly used to prevent and treat herpes simplex virus (HSV) reactivation after hematopoietic cell transplant (HCT), and only few reports have been published on acyclovir-resistant HSV in HCT recipients. We reviewed the medical records of patients with a microbiologic diagnosis of acyclovir-resistant HSV by plaque reduction test who received an HCT from 2002 through 2014. A total of 4 028 HCTs were performed during the study period, and 18 of the recipients met the diagnostic criteria for acyclovir-resistant HSV. All cases had undergone allogeneic HCTs. Most patients were in the pre-engraftment period or on systemic corticosteroid therapy for graft-versus-host disease (GVHD). The median time between diagnosis and susceptibility testing was 15 days, and antiviral therapy was changed at a median of 27 days. Patients required prolonged therapy (~80 days), and many had serious complications including renal failure and hospitalization. In conclusion, acyclovir-resistant HSV infection is more likely during the period of profound deficit in T-cell-mediated immunity and is associated with significant morbidities. Higher doses of acyclovir prophylaxis might be needed for patients with history of HSV during pre-engraftment or GVHD treatment. In patients who do not respond or progress after 1 week of acyclovir therapy, testing for drug-resistant HSV, and early switch to an alternative antiviral should be considered.

Timing Is Everything.

ABSTRACTN. Drayman et al. in their recent article (mBio 8:e01612-17, 2017, https://doi.org/10.1128/mBio.01612-17) have used dynamic proteomics and machine learning to show that the cell cycle state of any individual cell affects the outcome of a productive herpes simplex virus 1 (HSV-1) infection. Cells infected from early G1 through S were most permissive for expression of genes from the HSV-1 genome, whereas cells infected in late G2 to mitosis were much less so. Most of the infected cells that underwent mitosis became permanently nonpermissive for HSV-1 gene expression afterward. The cell cycle stage accounted for 60% of the success of infection, and cell density and motility accounted for most of the rest. To successfully reactivate, HSV-1 must express its genes in neurons and cells of the spinosum and granulosum epidermis strata. These cells are permanently in the cell cycle stages most permissive for HSV-1 gene expression, and none reenters mitosis, thus maximizing the efficiency of a successful HSV-1 reactivation before the adaptive immunity can control it.

The Etiology of Genital Ulcer Disease and Coinfections With Chlamydia trachomatis and Neisseria gonorrhoeae in Zimbabwe: Results From the Zimbabwe STI Etiology Study

In many countries, sexually transmitted infections (STIs) are treated syndromically. Thus, patients diagnosed as having genital ulcer disease (GUD) in Zimbabwe receive a combination of antimicrobials to treat syphilis, chancroid, lymphogranuloma venereum (LGV), and genital herpes. Periodic studies are necessary to assess the current etiology of GUD and assure the appropriateness of current treatment guidelines. We selected 6 geographically diverse clinics in Zimbabwe serving high numbers of STI cases to enroll men and women with STI syndromes, including GUD. Sexually transmitted infection history and risk behavioral data were collected by questionnaire and uploaded to a Web-based database. Ulcer specimens were obtained for testing using a validated multiplex polymerase chain reaction (M-PCR) assay for Treponema pallidum (TP; primary syphilis), Haemophilus ducreyi (chancroid), LGV-associated strains of Chlamydia trachomatis, and herpes simplex virus (HSV) types 1 and 2. Blood samples were collected for testing with HIV, treponemal, and nontreponemal serologic assays. Among 200 GUD patients, 77 (38.5%) were positive for HSV, 32 (16%) were positive for TP, and 2 (1%) were positive for LGV-associated strains of C trachomatis. No H ducreyi infections were detected. No organism was found in 98 (49.5%) of participants. The overall HIV positivity rate was 52.2% for all GUD patients, with higher rates among women compared with men (59.8% vs 45.2%, P < 0.05) and among patients with HSV (68.6% vs 41.8%, P < 0.0001). Among patients with GUD, 54 (27.3%) had gonorrhea and/or chlamydia infection. However, in this latter group, 66.7% of women and 70.0% of men did not have abnormal vaginal or urethral discharge on examination. Herpes simplex virus is the most common cause of GUD in our survey, followed by T. pallidum. No cases of chancroid were detected. The association of HIV infections with HSV suggests high risk for cotransmission; however, some HSV ulcerations may be due to HSV reactivation among immunocompromised patients. The overall prevalence of gonorrhea and chlamydia was high among patients with GUD and most of them did not meet the criteria for concomitant syndromic management covering these infections.

A Therapeutic Antiviral Antibody Inhibits the Anterograde Directed Neuron-to-Cell Spread of Herpes Simplex Virus and Protects against Ocular Disease

Herpes simplex virus (HSV) is a leading cause of blindness and viral encephalitis in the developed world. Upon reactivation from sensory neurons, HSV returns via axonal transport to peripheral tissues where it causes, e.g., severe, potentially blinding ocular diseases. In the present study we investigated whether the HSV-1/2 glycoprotein B-specific antibody mAb 2c or its humanized counterpart mAb hu2c can protect from ocular disease in a mouse model of HSV-1-induced acute retinal necrosis (ARN). In this model the viral spread from the initially infected to the contralateral eye resembles the routes taken in humans upon HSV reactivation. Systemic antibody treatment prior or early after infection effectively protected the mice from the development of ARN. These observations suggest that the antibody potently neutralized the infection and inhibited the viral transmission, since there was almost no virus detectable in the contralateral eyes and trigeminal ganglia of antibody treated mice. Besides of neutralizing free virus or limiting the infection via activating the complement or cellular effector functions, blocking of the anterograde directed neuron-to-cell spread of HSV represents a viable mode of action how mAb 2c protected the mice from ARN. We proved this hypothesis using a microfluidic chamber system. Neurons and epithelial cells were cultured in two separate compartments where the neurons sent axons via connecting microgrooves to the epithelial cells. Neurons were infected with a reporter HSV-1 strain expressing mCherry, and the co-culture was treated with neutralizing antibodies. In contrast to commercial polyclonal human HSV-neutralizing immunoglobulins, mAb 2c effectively blocked the anterograde directed neuron-to-cell transmission of the virus. Our data suggest that the humanized HSV-1/2-gB antibody protects mice from ocular disease by blocking the neuronal spread of HSV. Therefore, mAb hu2c may become a potent novel therapeutic option for severe ocular HSV infections.

Unexpected Reactivation of Herpes Simplex Virus as Keratitis during Chemotherapy

Unexpected Reactivation of Herpes Simplex Virus as Keratitis during Chemotherapy

Inhibitors of the Histone Methyltransferases EZH2/1 Induce a Potent Antiviral State and Suppress Infection by Diverse Viral Pathogens.

ABSTRACTEpigenetic regulation is based on a network of complexes that modulate the chromatin character and structure of the genome to impact gene expression, cell fate, and development. Thus, epigenetic modulators represent novel therapeutic targets used to treat a range of diseases, including malignancies. Infectious pathogens such as herpesviruses are also regulated by cellular epigenetic machinery, and epigenetic therapeutics represent a novel approach used to control infection, persistence, and the resulting recurrent disease. The histone H3K27 methyltransferases EZH2 and EZH1 (EZH2/1) are epigenetic repressors that suppress gene transcription via propagation of repressive H3K27me3-enriched chromatin domains. However, while EZH2/1 are implicated in the repression of herpesviral gene expression, inhibitors of these enzymes suppressed primary herpes simplex virus (HSV) infection in vitro and in vivo. Furthermore, these compounds blocked lytic viral replication following induction of HSV reactivation in latently infected sensory ganglia. Suppression correlated with the induction of multiple inflammatory, stress, and antipathogen pathways, as well as enhanced recruitment of immune cells to in vivo infection sites. Importantly, EZH2/1 inhibitors induced a cellular antiviral state that also suppressed infection with DNA (human cytomegalovirus, adenovirus) and RNA (Zika virus) viruses. Thus, EZH2/1 inhibitors have considerable potential as general antivirals through the activation of cellular antiviral and immune responses.

Role of the JNK Pathway in Varicella-Zoster Virus Lytic Infection and Reactivation.

Mechanisms of neuronal infection by varicella-zoster virus (VZV) have been challenging to study due to the relatively strict human tropism of the virus and the paucity of tractable experimental models. Cellular mitogen-activated protein kinases (MAPKs) have been shown to play a role in VZV infection of nonneuronal cells, with distinct consequences for infectivity in different cell types. Here, we utilize several human neuronal culture systems to investigate the role of one such MAPK, the c-Jun N-terminal kinase (JNK), in VZV lytic infection and reactivation. We find that the JNK pathway is specifically activated following infection of human embryonic stem cell-derived neurons and that this activation of JNK is essential for efficient viral protein expression and replication. Inhibition of the JNK pathway blocked viral replication in a manner distinct from that of acyclovir, and an acyclovir-resistant VZV isolate was as sensitive to the effects of JNK inhibition as an acyclovir-sensitive VZV isolate in neurons. Moreover, in a microfluidic-based human neuronal model of viral latency and reactivation, we found that inhibition of the JNK pathway resulted in a marked reduction in reactivation of VZV. Finally, we utilized a novel technique to efficiently generate cells expressing markers of human sensory neurons from neural crest cells and established a critical role for the JNK pathway in infection of these cells. In summary, the JNK pathway plays an important role in lytic infection and reactivation of VZV in physiologically relevant cell types and may provide an alternative target for antiviral therapy.IMPORTANCE Varicella-zoster virus (VZV) has infected over 90% of people worldwide. While primary infection leads to the typically self-limiting condition of chickenpox, the virus can remain dormant in the nervous system and may reactivate later in life, leading to shingles or inflammatory diseases of the nervous system and eye with potentially severe consequences. Here, we take advantage of newer stem cell-based technologies to study the mechanisms by which VZV infects human neurons. We find that the c-Jun N-terminal kinase (JNK) pathway is activated by VZV infection and that blockade of this pathway limits lytic replication (as occurs during primary infection). In addition, JNK inhibition limits viral reactivation, exhibiting parallels with herpes simplex virus reactivation. The identification of the role of the JNK pathway in VZV infection of neurons reveals potential avenues for the development of alternate antiviral drugs.

Analysis of Herpes Simplex Virus Reactivation in Explant Reveals a Method-Dependent Difference in Measured Timing of Reactivation

ABSTRACTHerpes simplex virus (HSV) infection is widespread in the human population. Following orofacial infection, HSV establishes latency in innervating sensory neurons, primarily located in the trigeminal ganglia. A central feature of HSV pathogenesis is the ability to periodically reactivate in those neurons and be transported back to the body surface. Both transmission and disease, such as keratitis, encephalitis, and neurodegeneration, have been linked to reactivation. Despite invaluable insights obtained from model systems, interactions between viral and host functions that regulate reactivation are still incompletely understood. Various assays are used for measuring reactivation in animal models, but there have been limited comparisons between methods and the accuracy of detecting the timing of reactivation and the corresponding amount of infectious virus produced in the ganglia per reactivation event. Here, we directly compare two approaches for measuring reactivation in latently infected explanted ganglia by sampling media from the explanted cultures or by homogenization of the ganglia and compare the results to viral protein expression in the whole ganglia. We show that infectious virus detection by direct homogenization of explanted ganglia correlates with viral protein expression, but detection of infectious virus in medium samples from explanted cultures does not occur until extensive spread of virus is observed in the ganglia. The medium-sampling method is therefore not reflective of the initial timing of reactivation, and the additional variables influencing spread of virus in the ganglia should be considered when interpreting results obtained using this method.IMPORTANCE The development of treatments to prevent and/or treat HSV infection rely upon understanding viral and host factors that influence reactivation. Progress is dependent on experimental methods that accurately measure the frequency and timing of reactivation in latently infected neurons. In this study, two methods for detecting reactivation using the explant model are compared. We show through direct tissue homogenization that reactivation occurs much earlier than can be detected by the indirect method of sampling media from explanted cultures. Thus, the sampling method does not detect the initial timing of reactivation, and results obtained using this method are subject to additional variables with the potential to obscure reactivation outcomes.

Dok-1 and Dok-2 Are Required To Maintain Herpes Simplex Virus 1-Specific CD8+ T Cells in a Murine Model of Ocular Infection.

Dok-1 and Dok-2 negatively regulate responses downstream of several immune receptors in lymphoid and myeloid cells. Recent evidence showed that Dok proteins are essential in the formation of memory CD8+ T cells to an exogenous epitope expressed by vaccinia virus; however, the importance of Dok-1 and Dok-2 in the control of viral infection is unknown. Here, we investigated the role of Dok proteins in modulating the immune response against herpes simplex virus 1 (HSV-1) in a mouse model of ocular infection. During acute infection, viral titers in the eye were similar in wild-type (WT) and Dok-1 and Dok-2 double-knockout (DKO) mice, and the percentages of infiltrating leukocytes were similar in DKO and WT corneas and trigeminal ganglia (TG). DKO mice exhibited a diminished CD8+ T cell response to the immunodominant HSV-1 glycoprotein B (gB) epitope in the spleen and draining lymph nodes compared to WT mice during acute infection. Remarkably, gB-specific CD8+ T cells almost completely disappeared in the spleens of DKO mice during latency, and the reduction of CD8+ effector memory T (Tem) cells was more severe than that of CD8+ central memory T (Tcm) cells. The percentage of gB-specific CD8+ T cells in TG during latency was also dramatically reduced in DKO mice; however, they were phenotypically similar to those from WT mice. In ex vivo assays, reactivation was detected earlier in TG cultures from infected DKO versus WT mice. Thus, Dok-1 and Dok-2 promote survival of gB-specific CD8+ T cells in TG latently infected with HSV-1.IMPORTANCE HSV-1 establishes lifelong latency in sensory neurons of trigeminal ganglia (TG). In humans, HSV-1 is able to sporadically reactivate from latently infected neurons and establish a lytic infection at a site to which the neurons project. Most herpetic disease in humans is due to reactivation of HSV-1 from latency rather than to primary acute infection. CD8+ T cells are thought to play an important role in controlling recurrent infections. In this study, we examined the involvement of Dok-1 and Dok-2 signaling proteins in the control of HSV-1 infection. We provide evidence that Dok proteins are required to maintain a CD8+ T cell response against HSV-1 during latency-especially CD8+ Tem cells-and that they negatively affect HSV-1 reactivation from latency. Elucidating Dok-mediated mechanisms involved in the control of HSV-1 reactivation from latency might contribute to the development of therapeutic strategies to prevent recurrent HSV-1-induced pathology.

Keratinocytes produce IL-17c to protect peripheral nervous systems during human HSV-2 reactivation.

Despite frequent herpes simplex virus (HSV) reactivation, peripheral nerve destruction and sensory anesthesia are rare. We discovered that skin biopsies obtained during asymptomatic human HSV-2 reactivation exhibit a higher density of nerve fibers relative to biopsies during virological and clinical quiescence. We evaluated the effects of HSV infection on keratinocytes, the initial target of HSV replication, to better understand this observation. Keratinocytes produced IL-17c during HSV-2 reactivation, and IL-17RE, an IL-17c-specific receptor, was expressed on nerve fibers in human skin and sensory neurons in dorsal root ganglia. In ex vivo experiments, exogenous human IL-17c provided directional guidance and promoted neurite growth and branching in microfluidic devices. Exogenous murine IL-17c pretreatment reduced apoptosis in HSV-2-infected primary neurons. These results suggest that IL-17c is a neurotrophic cytokine that protects peripheral nerve systems during HSV reactivation. This mechanism could explain the lack of nerve damage from recurrent HSV infection and may provide insight to understanding and treating sensory peripheral neuropathies.

Herpes Simplex Virus Establishment, Maintenance, and Reactivation: In Vitro Modeling of Latency.

All herpes viruses establish lifelong infections (latency) in their host, and herpes simplex viruses (HSVs) are highly prevalent worldwide. Recurrence of HSV infections contributes to significant disease burden in people and on rare occasion can be fatal. Cell culture models that recapitulate latent infection provide valuable insight on the host processes regulating viral establishment and maintenance of latency. More robust and rapid than infections in live animal studies, advancements in neuronal culture techniques have made the systematic analysis of viral reactivation mechanisms feasible. Only recently have human neuronal cell lines been available, but models in the natural host cell are a critical addition to the currently available models.