HSV-1 Replication Research Articles

The type I interferon receptor is not required for protection in the Chlamydia muridarum and HSV-2 murine super-infection model.

Chlamydia trachomatis/HSV-2 vaginal co-infections are seen clinically, suggesting that these sexually transmitted pathogens may interact. We previously established an intravaginal Chlamydia muridarum/HSV-2 super-infection model and observed that chlamydial pre-infection protects mice from a subsequent lethal HSV-2 challenge. However, the mechanism of protection remains unknown. The type I interferon, IFN-β, binds to the type I interferon receptor (IFNR), elicits a host cellular antiviral response and inhibits HSV replication in vitro and in vivo. Previous studies have demonstrated that C. muridarum infection stimulates genital tract (GT) IFN-β production; therefore, we hypothesized that chlamydial pre-infection protects mice from HSV-2 challenge via the IFN-β/IFNR-induced antiviral response. To test this prediction, we quantified IFN-β levels in vaginal swab samples. Detection of IFN-β in C. muridarum singly infected, but not in mock-infected animals, prompted the use of the super-infection model in IFNR knockout (IFNR−/−) mice. We observed that C. muridarum pre-infection reduces HSV-2-induced mortality by 40% in wild-type mice and by 60% IFNR−/− mice. Severity of HSV-2 disease symptoms and viral shedding was also similarly reduced by C. muridarum pre-infection. These data indicate that, while chlamydial infection induces GT production of IFN-β, type I IFN-induced antiviral responses are likely not required for the observed protective effect.

Bowman‒Birk Inhibitor Suppresses Herpes Simplex Virus Type 2 Infection of Human Cervical Epithelial Cells

The Bowman‒Birk inhibitor (BBI), a protease inhibitor derived from soybeans, has been extensively studied in anti-tumor and anti-inflammation research. We recently reported that BBI has an anti-HIV-1 property in primary human macrophages. Because HSV-2 infection plays a role in facilitating HIV-1 sexual transmission, we thus examined whether BBI has the ability to inhibit HSV-2 infection. We demonstrated that BBI could potently inhibit HSV-2 replication in human cervical epithelial cells (End1/E6E7). This BBI-mediated HSV-2 inhibition was partially through blocking HSV-2-mediated activation of NF-κB and p38 MAPK pathways. In addition, BBI could activate the JAK/STAT pathway and enhance the expression of several antiviral interferon-stimulated genes (ISGs). Furthermore, BBI treatment of End1/E6E7 cells upregulated the expression of tight junction proteins and reduced HSV-2-mediated cellular ubiquitinated proteins’ degradation through suppressing the ubiquitin‒proteasome system. These observations indicate that BBI may have therapeutic potential for the prevention and treatment of HSV-2 infections.

Experimental Dissection of the Lytic Replication Cycles of Herpes Simplex Viruses in vitro.

Herpes simplex viruses type 1 and type 2 (HSV-1 and HSV-2) produce lifelong infections and are highly prevalent in the human population. Both viruses elicit numerous clinical manifestations and produce mild-to-severe diseases that affect the skin, eyes, and brain, among others. Despite the existence of numerous antivirals against HSV, such as acyclovir and acyclovir-related analogs, virus variants that are resistant to these compounds can be isolated from immunosuppressed individuals. For such isolates, second-line drugs can be used, yet they frequently produce adverse side effects. Furthermore, topical antivirals for treating cutaneous HSV infections usually display poor to moderate efficacy. Hence, better or novel anti-HSV antivirals are needed and details on their mechanisms of action would be insightful for improving their efficacy and identifying specific molecular targets. Here, we review and dissect the lytic replication cycles of herpes simplex viruses, discussing key steps involved in cell infection and the processes that yield new virions. Additionally, we review and discuss rapid, easy-to-perform and simple experimental approaches for studying key steps involved in HSV replication to facilitate the identification of the mechanisms of action of anti-HSV compounds.

Herpes Simplex Virus 1 γ134.5 Protein Inhibits STING Activation That Restricts Viral Replication.

The γ134.5 gene of herpes simplex virus 1 (HSV-1) encodes a virulence factor that promotes viral pathogenesis. Although it perturbs TANK-binding kinase 1 (TBK1) in the complex network of innate immune pathways, the underlying mechanism is obscure. Here we report that HSV-1 γ134.5 targets stimulator of interferon genes (STING) in the intracellular DNA recognition pathway that regulates TBK1 activation. In virus-infected cells the γ134.5 protein associates with and inactivates STING, which leads to downregulation of interferon regulatory factor 3 (IRF3) and IFN responses. Importantly, HSV-1 γ134.5 disrupts translocation of STING from the endoplasmic reticulum to Golgi apparatus, a process necessary to prime cellular immunity. Deletion of γ134.5 or its amino-terminal domain from HSV-1 abolishes the observed inhibitory activities. Consistently, an HSV mutant that lacks functional γ134.5 replicated less efficiently in STING+/+ than in STING-/- mouse embryonic fibroblasts. Moreover, reconstituted expression of human STING in the STING-/- cells activated IRF3 and reduced viral growth. These results suggest that control of the DNA sensing pathway by γ134.5 is advantageous to HSV infection.IMPORTANCE Viral inhibition of innate immunity contributes to herpes simplex virus pathogenesis. Although this complex process involves multiple factors, the underlying events remain unclear. We demonstrate that an HSV virulence factor γ134.5 precludes the activation of STING, a central adaptor in the intracellular DNA sensing pathway. Upon HSV infection, this viral protein engages with and inactivates STING. Consequently, it compromises host immunity and facilitates HSV replication. These observations uncover an HSV mechanism that is likely to mediate viral virulence.

HSV-2 glycoprotein J promotes viral protein expression and virus spread

HSV-2 spread is predominantly dependent on cell-to-cell contact. However, the underlying mechanisms remain to be determined. Here we demonstrate that HSV-2 gJ, which was previously assigned no specific function, promotes HSV-2 cell-to-cell spread and syncytia formation. In the context of viral infection, knockout or knockdown of gJ impairs HSV-2 cell-to-cell spread among epithelial cells or from epithelial cells to neuronal cells, which leads to decreased virus production, whereas ectopic expression of gJ enhances virus production. Mechanistically, gJ increases the expression levels of HSV-2 proteins, and also enhances viral protein expression and replication of heterologous viruses like HIV-1 and JEV, suggesting that HSV-2 gJ likely functions as a regulator of viral protein expression and virus production. Findings in this study provide a basis for further understanding the role of gJ in HSV-2 replication.

In Vitro Study of Multi-Therapeutic Properties of Thymus bovei Benth. Essential Oil and Its Main Component for Promoting Their Use in Clinical Practice

Thymus bovei Benth. (TB) is an important plant in the traditional medicine of the Mediterranean region. This study investigates the health-promoting properties of TB essential oil (TB-EO) for its possible use in clinical practice with regards to its cytotoxic, anti-herpes simplex virus type 2 (HSV-2), and antihypertensive (through inhibition of human angiotensin-converting enzyme; ACE) properties. The phytochemical profile of EO (99.9%) was analyzed by Gas Chromatography with Flame-Ionization Detection (GC-FID) and Gas Chromatography-Mass Spectrometry (GC-MS). In this study, all biological methods were performed at the level of in vitro studies. The results showed that TB-EO exerted remarked cytotoxic properties against human cervical carcinoma cells, colon cancer cells, and lung adenocarcinoma cells with the half-maximal inhibitory concentration (IC50) values of 7.22, 9.30, and 8.62 µg/mL, respectively, in comparison with that of standard anticancer drug cisplatin with IC50 values of 4.24, 5.21, and 5.43 µg/mL, respectively. Fascinatingly, TB-EO showed very weak cytotoxicity on the healthy human fetal lung fibroblast cells with an IC50 value of 118.34 µg/mL compared with that of cisplatin (IC50 = 10.08 µg/mL). TB-EO, its main component geraniol, TB-EO combined with acyclovir (ACV) along with standard ACV, have displayed pronounced inhibitory properties against the replication of HSV-2 with the half-maximal effective concentration (EC50) values of 2.13, 1.92, 0.81 and 1.94 µg/mL, respectively, with corresponding selectivity indices (SI) 98.59, 109.38, 259.26 and 108.25, respectively. TB-EO and geraniol at a concentration of 15 µg/mL showed prominent inhibitory activities against ACE with % of inhibition 95.4% and 92.2%, respectively, compared with that of standard inhibitor captopril (99.8%; 15 µg/mL). Molecular docking studies were performed to unveil the mechanism of action of geraniol as well as structural parameters necessary for anti-HSV-2 activity (through the inhibition of HSV-2 protease) and ACE inhibition. This is the first report on the chemical composition of Egyptian TB-EO along with the above-mentioned biological activities. Our results may be considered as novel findings in the course of a search for new and active anticancer, anti-HSV-2 and antihypertensive agents, and expand the medicinal value of this plant and its phytochemicals in clinical practice.

Virucidal, antiviral and immunomodulatory activities of β-escin and Aesculus hippocastanum extract.

β-Escin, one of the constituents of Aesculus hippocastanum L. (Hippocastanaceae) seed extract (AH), inhibits NF-κB activation, which plays an important role in HSV-1 replication. The aim was to examine the antiherpetic activity of β-escin and AH, as well as their effect on the activation of NF-κB and AP-1 and cytokine secretion in epithelial cells and macrophages. Cell viability was evaluated using MTT assay, and antiviral and virucidal activity was determined by plaque assay. The effect on NF-κB and AP-1 signalling pathways activation was determined by a luciferase reporter assay, and cytokine production was measured by ELISA. β-Escin and AH had virucidal and anti-HSV-1 activities, and the antiviral activity was discovered for other enveloped viruses (VSV and Dengue). Moreover, β-escin and AH significantly reduced NF-κB and AP-1 activation and cytokine production in macrophages stimulated with HSV-1 and TLRs ligands. However, an enhanced activation of these pathways and an increase in the levels of pro-inflammatory cytokines in β-escin and AH-treated HSV-1-infected epithelial cells were found. This study demonstrates virucidal and broad-spectrum antiviral activities for β escin and AH. Besides, β-escin and AH modulate cytokine production depending on the stimuli (viral or non-viral) and the cell type under study.

Extracellular Vesicles Released by Herpes Simplex Virus 1-Infected Cells Block Virus Replication in Recipient Cells in a STING-Dependent Manner.

Herpes simplex virus 1 (HSV-1)-infected cells release extracellular vesicles (EVs) that deliver to uninfected cells viral factors and host components, such as the stimulator of interferon genes (STING), which activates type I interferon upon foreign DNA sensing. The functions of EVs released by HSV-1-infected cells have remained unknown. Here, we describe a procedure to separate the EVs from HSV-1 virions that is based on an iodixanol/sucrose gradient. STING, along with the EV markers CD63 and CD9, was found in light-density fractions, while HSV components accumulated in heavy-density fractions. HSV-1 infection stimulated the release of EVs from the cells. The EVs derived from infected cells, but not from uninfected cells, activated innate immunity in recipient cells and suppressed viral gene expression and virus replication. Moreover, only the EVs derived from infected cells stimulated the expression of a subset of M1-type markers in recipient macrophages. Conversely, EVs derived from STING-knockdown cells failed to stimulate the expression of these M1-type markers, they activated innate immune responses to a lesser extent in recipient cells, and they did not sustain the inhibition of virus replication. These data suggest that STING from the EV donor cells contributes to the antiviral responses in cells receiving EVs from HSV-1-infected cells. Perturbations in the biogenesis of EVs by silencing CD63 or blocking the activity of the neutral spingomyelinase-2 (nSMase-2) increased the HSV-1 yields. Overall, our data suggest that the EVs released from HSV-1-infected cells negatively impact the infection and could control the dissemination of the virus.IMPORTANCE Extracellular vesicles (EVs) are released by all types of cells as they constitute major mechanism of intercellular communication and have the capacity to alter the functions of recipient cells despite their limited capacity for cargo. How the EVs released by HSV-infected cells could alter the surrounding microenvironment and influence the infection currently remains unknown. The cargo of EVs reflects the physiological state of the cells in which they were produced, so the content of EVs originating from infected cells is expected to be substantially different from that of healthy cells. Our studies indicate that the EVs released by HSV-1-infected cells carry innate immune components such as STING and other host and viral factors; they can activate innate immune responses in recipient cells and inhibit HSV-1 replication. The implication of these data is that the EVs released by HSV-1-infected cells could control HSV-1 dissemination promoting its persistence in the host.

The aryl hydrocarbon receptor and interferon gamma generate antiviral states via transcriptional repression.

The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor whose activation induces the expression of numerous genes, with many effects on cells. However, AhR activation is not known to affect the replication of viruses. We show that AhR activation in macrophages causes a block to HIV-1 and HSV-1 replication. We find that AhR activation transcriptionally represses cyclin-dependent kinase (CDK)1/2 and their associated cyclins, thereby reducing SAMHD1 phosphorylation, cellular dNTP levels and both HIV-1 and HSV-1 replication. Remarkably, a different antiviral stimulus, interferon gamma (IFN-γ), that induces a largely non-overlapping set of genes, also transcriptionally represses CDK1, CDK2 and their associated cyclins, resulting in similar dNTP depletion and antiviral effects. Concordantly, the SIV Vpx protein provides complete and partial resistance to the antiviral effects of AhR and IFN-γ, respectively. Thus, distinct antiviral signaling pathways converge on CDK/cyclin repression, causing inhibition of viral DNA synthesis and replication.

Anti-Herpes Simplex Virus Type 1 Activity of Specially Selected Groups of Tannins.

Anti-herpes simplex virus (HSV-1) activity of 9 ellagitannins, including 6 natural compounds (castalin, vescalin, acutissimin A, epiacutissimins A and B, mongolicain) and 3 vescalagin synthetic derivatives (VgSBuSH, VgSOctSH, VgOMe), and 13 gallotannin-type compounds [Gal-01A, Gal-01B, Gal-02A, Gal-02B, Gal-03M, Gal-04A, Gal-04B, Gal-05M, Gal-07, Gal-08, Gal-09, Gal-11M (tannic acid), as well as Gal-12 (gallic acid), Gal-13 and Gal-14 (ellagic acid)] were examined in MDBK monolayer cell culture. Their antiviral activity was determined by the cytopathic effect (CPE) inhibition test and their cytotoxicity was evaluated through the neutral red uptake assay. In general, the series of ellagitannins showed a significantly stronger activity against HSV-1 replication than that of the gallotannins. Six of the tested ellagitannins manifested a well-pronounced activity: epiacutissimin B (selectivity index, SI>60.6), epiacutissimin A (SI>55.5), acutissimin A (SI>34.8), mongolicain (SI>32.5), VgSBuSH (SI>24.6) and VgOMe (SI>22.0). Four gallotannin-type compounds inhibited the replication of HSV-1 at a lower but still significant extent: Gal-04B (SI>35.7), Gal-04A (SI>28.5), Gal-11M (tannic acid) (SI>25) and Gal-05M (SI=15.6).

A Polyphenol Rich Extract from Solanum melongena L. DR2 Peel Exhibits Antioxidant Properties and Anti-Herpes Simplex Virus Type 1 Activity In Vitro.

DR2B and DR2C extracts, obtained by ethanolic maceration of peel from commercially and physiologically ripe aubergine berries, were studied for the antioxidative cytoprotective properties and anti-HSV-1 activity, in line with the evidence that several antioxidants can impair viral replication by maintaining reducing conditions in host cells. The antioxidative cytoprotective effects against tBOOH-induced damage were assessed in Caco2 cells, while antiviral activity was studied in Vero cells; polyphenolic fingerprints were characterized by integrated phytochemical methods. Results highlighted different compositions of the extracts, with chlorogenic acid and delphinidin-3-rutinoside as the major constituents; other peculiar phytochemicals were also identified. Both samples reduced reactive oxygen species (ROS) production and exhibited scavenging and chelating properties. DR2C partly counteracted the tBOOH-induced cytotoxicity, with a remarkable lowering of lactate metabolism under both normoxia and hypoxia; interestingly, it increased intracellular GSH levels. Furthermore, DR2C inhibited the HSV-1 replication when added for 24 h after viral adsorption, as also confirmed by the reduction of many viral proteins’ expression. Since DR2C was able to reduce NOX4 expression during HSV-1 infection, its antiviral activity may be correlated to its antioxidant properties. Although further studies are needed to better characterize DR2C activity, the results suggest this extract as a promising new anti-HSV-1 agent.

Regulation of Herpes Simplex Virus 2 Protein Kinase UL13 by Phosphorylation and Its Role in Viral Pathogenesis.

UL13 proteins are serine/threonine protein kinases encoded by herpes simplex virus 1 (HSV-1) and HSV-2. Although the downstream effects of the HSV protein kinases, mostly those of HSV-1 UL13, have been reported, there is a lack of information on how these viral protein kinases are regulated in HSV-infected cells. In this study, we used a large-scale phosphoproteomic analysis of HSV-2-infected cells to identify a physiological phosphorylation site in HSV-2 UL13 (i.e., Ser-18) and investigated the significance of phosphorylation of this site in HSV-2-infected cell cultures and mice. Our results were as follows. (i) An alanine substitution at UL13 Ser-18 (S18A) significantly reduced HSV-2 replication and cell-to-cell spread in U2OS cells to a level similar to those of the UL13-null and kinase-dead mutations. (ii) The UL13 S18A mutation significantly impaired phosphorylation of a cellular substrate of this viral protein kinase in HSV-2-infected U2OS cells. (iii) Following vaginal infection of mice, the UL13 S18A mutation significantly reduced mortality, HSV-2 replication in the vagina, and development of vaginal disease to levels similar to those of the UL13-null and the kinase-dead mutations. (iv) A phosphomimetic substitution at UL13 Ser-18 significantly restored the phenotype observed with the UL13 S18A mutation in U2OS cells and mice. Collectively, our results suggested that phosphorylation of UL13 Ser-18 regulated UL13 function in HSV-2-infected cells and that this regulation was critical for the functional activity of HSV-2 UL13 in vitro and in vivo and also for HSV-2 replication and pathogenesis.IMPORTANCE Based on studies on cellular protein kinases, it is obvious that the regulatory mechanisms of protein kinases are as crucial as their functional consequences. Herpesviruses each encode at least one protein kinase, but the mechanism by which these kinases are regulated in infected cells remains to be elucidated, with a few exceptions, although information on their functional effects has been accumulating. In this study, we have shown that phosphorylation of the HSV-2 UL13 protein kinase at Ser-18 regulated its function in infected cells, and this regulation was critical for HSV-2 replication and pathogenesis in vivo UL13 is conserved in all members of the family Herpesviridae, and this is the first report clarifying the regulatory mechanism of a conserved herpesvirus protein kinase that is involved in viral replication and pathogenesis in vivo Our study may provide insight into the regulatory mechanisms of the other conserved herpesvirus protein kinases.

Genome-Wide Association Study in Vestibular Neuritis: Involvement of the Host Factor for HSV-1 Replication.

Objective: In order to identify genetic variants associated with vestibular neuritis, a common cause of peripheral vertigo with a potential causative link to the reactivation of herpes simplex type 1 (HSV-1), we conducted a genome-wide association study.Methods: Association was assessed using approximately 8 million variants. 131 patients with vestibular neuritis and 2,609 controls of European ancestry were included.Results: Genome-wide associations with vestibular neuritis were detected in 4 regions containing protein coding genes assignable to two functional groups: virus hypothesis and insulin metabolism. Genes of set 1 are related to viral processes: nuclear receptor subfamily 3 group C member 2 (NR3C2) is a receptor for mineralocorticoids and glucocorticoids and was shown to be a host factor for HSV-1 replication. Ankyrin repeat domain 30A (ANKRD30A) encodes a host factor for human immunodeficiency virus-1 (HIV-1) infection. It shows rapid evolution and is induced by interferon stimulation. Mediator complex 30 (MED30), an important member of the mediator complex, has been shown to be involved in replication of HIV-1, a knockdown leading to impaired viral replication. The second set of genes LIM homeobox transcription factor 1 alpha (LMX1A), solute carrier family 30 member 8 (SLC30A8) is associated with insulin metabolism and resistance, a feature of some patients in whom type 2 diabetes is an accompanying comorbidity of vestibular neuritis.Conclusions: Using a GWAS approach to evaluate the etiology of vestibular neuritis these findings provide another piece of evidence that it may be caused by a viral inflammation.

Anti-Infectivity against Herpes Simplex Virus and Selected Microbes and Anti-Inflammatory Activities of Compounds Isolated from Eucalyptus globulus Labill.

Herpes simplex virus (HSV) causes numerous mild-to-serious human diseases, including mucocutaneous herpes infections and life-threatening herpes encephalitis. Moreover, herpes viral lesions can be complicated by inflammation and secondary bacterial infections. The development of resistance to antiviral drugs along with the undesirable side effects of these drugs are relevant argue for the development of new anti-HSV drugs with diverse mechanisms of action. Eucalyptus extracts have been used for decades to combat various infectious diseases. We isolated and studied 12 pure compounds and one mixture of two constitutional isomers from the leaves and twigs of E. globulus. The structures were identified by spectroscopic methods (NMR, HR-MS, IR) and all of them were tested for antiherpetic activity against the replication of antigen types HSV-1 and HSV-2. Tereticornate A (12) (IC50: 0.96 μg/mL; selectivity index CC50/IC50: 218.8) showed the strongest activity in the anti-HSV-1 assay, even greater than acyclovir (IC50: 1.92 μg/mL; selectivity index CC50/IC50: 109.4), a standard antiviral drug. Cypellocarpin C (5) (EC50: 0.73 μg/mL; selectivity index CC50/EC50: 287.7) showed the most potent anti-HSV-2 activity, also more intensive than acyclovir (EC50: 1.75 μg/mL; selectivity index CC50/EC50: 120.0). The antimicrobial activity of the isolated compounds was also evaluated against the bacteria Staphylococcus aureus, Bacillus cereus, Escherichia coli, and Pseudomonas aeruginosa and the yeast Candida albicans. The anti-inflammatory potential was examined using LPS-stimulated THP-1-XBlue™-MD2-CD14 and THP-1 macrophages and focusing on the influences of the NF-κB/AP-1 activity and the secretion of pro-inflammatory cytokines IL-1β and TNF-α.

Herpes Simplex Virus 1 Inhibits TANK-Binding Kinase 1 through Formation of the Us11-Hsp90 Complex.

The Us11 protein of herpes simplex virus 1 (HSV-1) is an accessory factor with multiple functions. In virus-infected cells, it inhibits double-stranded RNA-dependent protein kinase (PKR), 2',5'-oligoadenylate synthetase, RIG-I, and MDA-5. However, its precise role is incompletely defined. By screening a human cDNA library, we showed that the Us11 protein targets heat shock protein 90 (Hsp90), which inactivates TANK binding kinase 1 (TBK1) and antiviral immunity. When ectopically expressed, HSV-1 Us11 precludes TBK1 from access to Hsp90 and interferon (IFN) promoter activation. Consistently, the Us11 protein, upon HSV infection, suppresses the expression of beta interferon (IFN-β), RANTES, and interferon-stimulated genes. This is mirrored by a blockade in the phosphorylation of interferon regulatory factor 3. Mechanistically, the Us11 protein associates with endogenous Hsp90 to disrupt the Hsp90-TBK1 complex. Furthermore, Us11 induces destabilization of TBK1 through a proteasome-dependent pathway. Accordingly, Us11 expression facilitates HSV growth. In contrast, TBK1 expression restricts viral replication. These results suggest that control of TBK1 by Us11 promotes HSV-1 infection.IMPORTANCE TANK binding kinase 1 plays a key role in antiviral immunity. Although multiple factors are thought to participate in this process, the picture is obscure in herpes simplex virus infection. We demonstrated that the Us11 protein of HSV-1 forms a complex with heat shock protein 90, which inactivates TANK binding kinase 1 and IFN induction. As a result, expression of the Us11 protein promotes HSV replication. These experimental data provide a new insight into the molecular network of virus-host interactions.

Antifungal drug ciclopirox olamine reduces HSV-1 replication and disease in mice

Herpes simplex virus (HSV)-1 and HSV-2 cause painful blisters and shallow ulcers in exposed skin and mucosae during primary or recurrent infection. In addition, recurrent and potentially blinding HSV-1 infections of the eye afflict nearly half a million persons in the U.S. Current clinical therapies rely on nucleoside analog drugs such as acyclovir (ACV) or ganciclovir to ameliorate primary infections and reduce the frequency and duration of reactivations. However, these treatments do not fully suppress viral shedding and drug-resistant mutants develop in the eye and in vulnerable, immunosuppressed patients. Herpesvirus DNA replication requires several enzymes in the nucleotidyl transferase superfamily (NTS) that have recombinase and nuclease activities. We previously found that compounds which block NTS enzymes efficiently inhibit replication of HSV-1 and HSV-2 by up to 1 million-fold in Vero and human foreskin fibroblasts. Among the compounds with potent suppressive effects in culture is the anti-fungal drug ciclopirox. Here we report that topical application of ciclopirox olamine to the eyes of mice infected with HSV-1 reduced virus shed from the corneal epithelium compared with saline control, and reduced development of blepharitis to the level of mice treated with ACV. Results were dose-dependent. In addition, treatment with ciclopirox olamine significantly reduced acute and latent HSV-1 infection of the peripheral nervous system. These results support further development of ciclopirox olamine as a repurposed topical agent for HSV infections.

PEDF Reduces the Severity of Herpetic Simplex Keratitis in Mice.

The purpose of this study was to explore the effects of pigment epithelium derived factor (PEDF) and PEDF-derived peptides Mer44 and Mer34 on the severity of herpetic simplex keratitis (HSK) in mice. Adult C57BL/6 mice were infected ocularly with the herpes simplex virus type 1 (HSV-1, McKrae strain) and injected subconjunctivally with PEDF, Mer44, or Mer34. Corneal nerve degeneration, neovascularization, sensitivity, neutrophils, macrophages and CD4+ T-cell infiltration, virus contents, and expressions of VEGF, PEDF, and proinflammatory factors were evaluated during acute period. The direct inhibitory effect of PEDF on HSV-1 replication was further evaluated in cultured monkey Vero cells. Following HSV-1 infection, corneal PEDF expression decreased at 3 and 7 days postinfection (dpi) but increased at 15 dpi, and returned to the similar level of normal mice at 45 dpi, which was accompanied with the progress of corneal nerve degeneration and neovascularization. Exogenous PEDF application attenuated corneal nerve degeneration and neovascularization and improved the impaired corneal sensitivity. Moreover, PEDF attenuated the neutrophils, but not macrophage or CD4+ T-cell infiltration, with the reduced expressions of IL-1β, IL-6, TNF-α, and VEGF. In addition, PEDF inhibited the replication of HSV-1 both in vitro and in mice. Mer44 attenuated corneal nerve degeneration more significantly than Mer34, whereas Mer34 inhibited corneal neovascularization. PEDF and its derived peptides reduce the severity of herpetic simplex keratitis in mice, representing the potential therapeutic approach to control HSK lesions.

Correction: IFI16 Restricts HSV-1 Replication by Accumulating on the HSV-1 Genome, Repressing HSV-1 Gene Expression, and Directly or Indirectly Modulating Histone Modifications.

[This corrects the article DOI: 10.1371/journal.ppat.1004503.].

Antiviral Effects of Saffron and its Major Ingredients.

The lack of an effective vaccine against viral infections, toxicity of the synthetic anti-viral drugs and the generation of resistant viral strains led to discover novel inhibitors. Recently, saffron and its compounds were used to treat different pathological conditions. In this study, we tested the anti-HSV-1 and anti-HIV-1 activities of Iranian saffron extract and its major ingredients including crocin and picrocrocin as well as cytotoxicity in vitro. The data showed that the aqueous saffron extract was not active against HIV-1 and HSV-1 virions at certain doses (i.e., a mild activity), but crocin and picrocrocin indicated significant anti-HSV-1 and also anti-HIV-1 activities. Crocin inhibited the HSV replication at before and after entry of virions into Vero cells. Indeed, crocin carotenoid suppressed HSV penetration in the target cells as well as disturbed virus replication after entry into the cells. Picrocrocin was also effective for inhibiting virus entry and also its replication. This monoterpen aldehyde showed higher anti-HSV effects after virus penetrating in the cells. Generally, these sugar-containing compounds extracted from saffron showed to be effective antiherpetic drug candidates. The recent study is the first report suggesting antiviral activities for saffron extract and its major ingredients. Crocin and picrocrocin could be a promising anti-HSV and anti-HIV agent for herbal therapy against viral infections.

CD8+ T cells control HSV-1 replication in the nervous system but not the cornea in HSV-1 0ΔNLS-vaccinated mice lacking virus-neutralizing antibody

Abstract HSV-1 is a successful human pathogen that can elicit benign pathology including cold sores to more severe to extreme life-threatening manifestations like blinding necrotizing stromal keratitis and encephalitis. To date, there is no commercially available vaccine for HSV-1 despite many failed experimental prototypes. We have evaluated the use of a live-attenuated HSV-1 strain (HSV-1 0ΔNLS) as a vaccine in a mouse model of ocular infection. This vaccine has been shown to prevent virus replication, spread, and establishment of latency while functionally preserving the visual axis and corneal integrity. The correlate of protection elicited by the vaccine is antibody, and optimal efficacy requires expression of the neonatal Fc receptor in the corneal epithelium. The present study was undertaken to determine if T cell receptor transgenic mice expressing CD8 T cells specific for glycoprotein B of HSV-1 (gBT-I.1) benefit from vaccination. gBT-I.1 mice were vaccinated with HSV-1 0ΔNLS or vehicle and subsequently ocularly challenged with HSV-1. The results showed HSV-1 0ΔNLS vaccinated gBT-I.1 mice had no measureable neutralizing antibody titer but did show enhanced survival compared to vehicle-vaccinated gBT-I.1 mice. The efficacy correlated with less infectious virus in the nervous system of HSV-1 0ΔNLS-vaccinated mice during acute infection (p < 0.05). However, viral titers and pathology were similar in the corneas of both groups. Collectively, the results suggest in the absence of antibody, vaccine-primed HSV-specific CD8+ T cells enhance control of HSV-1 replication and spread in the nervous system but are not effective in the cornea.