Enterovirus A71 infection-induced dry eye-like symptoms by damaging the lacrimal glands.

Purpose: To determine the effects of EV-A71 (Enterovirus A71) infection on ocular surface and its mechanism. Methods: AG6 mice aged two to three weeks were randomly divided into control and EV-A71 infected groups. Slit-lamp observation, fluorescein staining, and phenol red thread test were used to assess symptoms of ocular surface at 4 dpi (days post infection). The pathological changes of cornea and lacrimal gland were observed by H&E staining, PAS staining, TUNEL assay, IHC staining and qRT-PCR. Corneas and lacrimal glands from mice were obtained and processed for RNA sequencing analysis. Newly diagnosed HFMD patients caused by EV-A71 were recruited and ensured they met the inclusion criteria. Ocular surface parameters (TMH and NIKBUT) were measured using the OCULUS Keratograph 5M. Tear samples were taken to examine Cxcl1 and IL-6 levels through the ELISA method. Results: Mice studies revealed that EV-A71 infection caused tear film instability, decreased tear secretions, decreased in lacrimal gland size, and distinct goblet cell loss. It also resulted in increased large vacuoles within acinar cells and structural damage in lacrimal gland. Apart from minor damage to the epidermis, there was no obvious inflammatory changes or apoptosis in the cornea. However, there were significant inflammatory injury and apoptosis in the lacrimal gland. RNA-seq analysis showed IL-17 and NF-κB signaling pathways were activated in the lacrimal glands of mice infected with EV-A71. In HFMD patients, the THM was in a low range and NITBUT was significantly shorter than the control group by Oculus Keratograph 5M. ELISA assay showed a higher tear Cxcl1 and IL-6 level in them. Conclusion: EV-A71 infection affected lacrimal gland structure and function and induced dry eye-like symptoms.

Supervised training models with or without manual lesion delineation outperform clinicians in distinguishing pulmonary cryptococcosis from lung adenocarcinoma on chest CT.

BACKGROUND: The role of artificial intelligence (AI) in the discrimination between pulmonary cryptococcosis (PC) and lung adenocarcinoma (LA) warrants further research. OBJECTIVES: To compare the performances of AI models with clinicians in distinguishing PC from LA on chest CT. METHODS: Patients diagnosed with confirmed PC or LA were retrospectively recruited from three tertiary hospitals in Guangzhou. A deep learning framework was employed to develop two models: an undelineated supervised training (UST) model utilising original CT images, and a delineated supervised training (DST) model utilising CT images with manual lesion annotations provided by physicians. A subset of 20 cases was randomly selected from the entire dataset and reviewed by clinicians through a network questionnaire. The sensitivity, specificity and accuracy of the models and the clinicians were calculated. RESULTS: A total of 395 PC cases and 249 LA cases were included in the final analysis. The internal validation results for the UST model showed a sensitivity of 85.3%, specificity of 81.0%, accuracy of 83.6% and an area under the curve (AUC) of 0.93. Similarly, the DST model exhibited a sensitivity of 88.2%, specificity of 88.1%, accuracy of 88.2% and an AUC of 0.94. The external validation of the two models yielded AUC values of 0.74 and 0.77, respectively. The average sensitivity, specificity and accuracy of 102 clinicians were determined to be 63.1%, 53.7% and 59.3%, respectively. CONCLUSIONS: Both models outperformed the clinicians in distinguishing between PC and LA on chest CT, with the UST model exhibiting comparable performance to the DST model.

Folate supports IL-25-induced tuft cell expansion following enteroviral infections.

Intestinal tuft cells, a kind of epithelial immune cells, rapidly expand in response to pathogenic infections, which is associated with infection-induced interleukin 25 (IL-25) upregulation. However, the metabolic mechanism of IL-25-induced tuft cell expansion is largely unknown. Folate metabolism provides essential purine and methyl substrates for cell proliferation and differentiation. Thus, we aim to investigate the roles of folate metabolism playing in IL-25-induced tuft cell expansion by enteroviral infection and recombinant murine IL-25 (rmIL-25) protein-stimulated mouse models. At present, enteroviruses, such as EV71, CVA16, CVB3, and CVB4, upregulated IL-25 expression and induced tuft cell expansion in the intestinal tissues of mice. However, EV71 did not induce intestinal tuft cell expansion in IL-25-/- mice. Interestingly, compared to the mock group, folate was enriched in the intestinal tissues of both the EV71-infected group and the rmIL-25 protein-stimulated group. Moreover, folate metabolism supported IL-25-induced tuft cell expansion since both folate-depletion and anti-folate MTX-treated mice had a disrupted tuft cell expansion in response to rmIL-25 protein stimulation. In summary, our data suggested that folate metabolism supported intestinal tuft cell expansion in response to enterovirus-induced IL-25 expression, which provided a new insight into the mechanisms of tuft cell expansion from the perspective of folate metabolism.

Pralatrexate inhibited the replication of varicella zoster virus and vesicular stomatitis virus: An old dog with new tricks.

Varicella zoster virus (VZV) is associated with herpes zoster (HZ) or herpes zoster ophthalmicus (HZO). All antiviral agents currently licensed for the management of VZV replication via modulating different mechanisms, and the resistance is on the rise. There is a need to develop new antiviral agents with distinct mechanisms of action and adequate safety profiles. Pralatrexate (PDX) is a fourth-generation anti-folate agent with an inhibitory activity on folate (FA) metabolism and has been used as an anti-tumor drug. We observed that PDX possessed potent inhibitory activity against VZV infection. In this study, we reported the antiviral effects and the underlying mechanism of PDX against VZV infection. The results showed that PDX not only inhibited VZV replication in vitro and in mice corneal tissues but also reduced the inflammatory response and apoptosis induced by viral infection. Furthermore, PDX treatment showed a similar anti-VSV inhibitory effect in both in vitro and in vivo models. Mechanistically, PDX inhibited viral replication by interrupting the substrate supply for de novo purine and thymidine synthesis. In conclusion, this study discovered the potent antiviral activity of PDX with a novel mechanism and presented a new strategy for VZV treatment that targets a cellular metabolic mechanism essential for viral replication. The present study provided a new insight into the development of broad-spectrum antiviral agents.

HMGB1 Release Induced by EV71 Infection Exacerbates Blood-Brain Barrier Disruption via VE-cadherin Phosphorylation.

PURPOSE: EV71 (Enterovirus 71) is a major causative agent of the outbreaks of HFMD (hand, foot, and mouth disease), which is associated with neurological damage caused by permeability disruption of BBB (blood-brain barrier). HMGB1 (high-mobility group box 1) is a widely expressed nuclear protein that triggers host inflammatory responses. Our work aimed to explore the function of HMGB1 in EV71 infection and its contributions to EV71-related BBB damage. METHODS: HeLa cells, HT-29 cells and AG6 mice were used to explore the translocation of HMGB1 in EV71 infection in vitro and in vivo. The roles of released HMGB1 on EV71 replication and associated inflammatory cytokines were investigated using recombinant HMGB1 in HeLa cells. The mechanisms of released HMGB1 in EV71-induced BBB injury were explored using recombinant HMGB1 and anti-HMGB1 neutralizing antibodies in monolayer HCMECs (immortalized human brain microvascular endothelial cells) and AG6 mice brain. RESULTS: EV71 induced HMGB1 nucleocytoplasmic translocation and extracellular release in vitro and in vivo. Released HMGB1 acted as an inflammatory mediator in EV71 infection rather than affecting viral replication in vitro. Released HMGB1 disrupted BBB integrity by enhancing VE-cadherin phosphorylation at tyrosine 685 in HCMECs, and reducing total VE-cadherin levels in HCMECs and AG6 mice in EV71 infection. And released HMGB1 induced an increase in activated astrocytes. Neutralization of HMGB1 reversed the increased endothelial hyperpermeability and phosphorylation of VE-cadherin in HCMECs. CONCLUSION: The inflammatory mediator HMGB1 released by EV71 exacerbated BBB disruption by enhancing VE-cadherin phosphorylation, which in turn aggravated EV71-induced neuroinflammation.

Therapeutic Potential of Biochanin A in Herpes Simplex Keratitis.

Herpes simplex keratitis (HSK) is a blinding eye disease that is initiated by the herpes simplex virus type 1 (HSV-1). Resistance to acyclovir (ACV) and the side effects of corticosteroid drugs have become concerning issues, so it is crucial to develop new antivirals for treating HSK. In this study, we report that biochanin A (BCA), a naturally occurring flavonoid compound, provides multifaceted protective effects with anti-viral, anti-inflammatory, anti-oxidative stress and anti-apoptotic activities to alleviate HSK. The results show that BCA significantly inhibited HSV-1 replication in vitro and further proved that BCA principally influenced the early stage of virus infection. We reveal that BCA downregulated the expression of pro-inflammatory factors triggered by HSV-1, including TNF-α, RANTES, IL-1ß and IL-6. Furthermore, BCA treatment alleviated oxidative stress and apoptotic arising from HSV-1 infection. Lastly, we induced HSK in male C57BL/6 mice and treated them with either BCA or phosphate buffer solution (PBS) eye drops. We observed the ocular surface lesions; determined the virus load in the tear fluid, corneas as well as trigeminal ganglions (TGs); and detected the levels of inflammation and apoptosis in the corneas simultaneously. These results show that BCA inhibits HSV-1 and alleviates the corneal lesion degree. Our study illustrates that BCA is a promising therapeutic approach for application in treating HSK.

Sertoli cell-derived extracellular vesicles traverse the blood-testis barrier and deliver miR-24-3p inhibitor into germ cells improving sperm mobility.

Asthenozoospermia, characterized by poor sperm motility, is a common cause of male infertility. Improving energy metabolism and alleviating oxidative stress through drug regimens are potential therapeutic strategies. In this study, we observed upregulated miR-24-3p levels in asthenozoospermia spermatozoa, contributing to energy metabolism disorder and oxidative stress by reducing GSK3ß expression. Thus, reducing miR-24-3p levels using drugs is expected to improve sperm motility. The blood-testis barrier (BTB) protects the testis from xenobiotics and drugs. In this study, we found that Sertoli cell-derived small extracellular vesicles (SC-sEV) can traverse the BTB and enter germ cells. We successfully loaded miR-24-3p inhibitor into SC-sEV, creating the nano-drug SC-sEV@miR-24-3p inhibitor, which effectively delivers miR-24-3p inhibitor into germ cells. In a gossypol-induced mouse asthenozoospermia model, administration of SC-sEV@miR-24-3p inhibitor significantly improved sperm motility, in vitro fertilization success, and blastocyst formation rates. As anticipated, it also improved the litter size of asthenozoospermia mice. These results suggest that SC-sEV@miR-24-3p inhibitor holds promise as a potential clinical treatment for asthenospermia.

METTL3-mediated m6A RNA modification promotes corneal neovascularization by upregulating the canonical Wnt pathway during HSV-1 infection.

BACKGROUND: Corneal neovascularization (CNV) is a symptom of herpes simplex keratitis (HSK), which can result in blindness. The corneal angiogenesis brought on by herpes simplex virus type 1 (HSV-1) is strongly affected by vascular endothelial growth factor A (VEGFA). The N6-methyladenosine (m6A) modification catalyzed by methyltransferase-like 3 (METTL3) is a crucial epigenetic regulatory process for angiogenic properties. However, the roles of METTL3 and m6A in HSK-induced CNV remain unknown. Here, we investigated these roles in vitro and in vivo. METHODS: A PCR array in HSV-1-infected human umbilical vein endothelial cells (HUVECs) was used to screen for METTL3 among the epitranscriptomic genes. Tube formation and scratch assays were conducted to investigate cell migration capacity. The global mRNA m6A abundance was evaluated using a dot blot assay. Gene expression was assessed by RT-qPCR, western blotting, and fluorescence immunostaining. In addition, bioinformatic analysis was conducted to identify the downstream molecules of METTL3 in HUVECs. METTL3 knockdown and STM2457 treatment clarified the specific underlying molecular mechanisms affecting HSV-1-induced angiogenesis in vitro. An acute HSK mouse model was established to examine the effects of METTL3 knockdown or inhibition using STM2457 on pathological angiogenic development in vivo. RESULTS: METTL3 was highly upregulated in HSV-1-infected HUVECs and led to increased m6A levels. METTL3 knockdown or inhibition by STM2457 further reduced m6A levels and VEGFA expression and impaired migration and tube formation capacity in HUVECs after HSV-1 infection. Mechanistically, METTL3 regulated LRP6 expression through post-transcriptional mRNA modification in an m6A-dependent manner, increasing its stability, upregulating VEGFA expression, and promoting angiogenesis in HSV-1-infected HUVECs. Furthermore, METTL3 knockdown or inhibition by STM2457 reduced CNV in vivo. CONCLUSION: Our findings revealed that METTL3 promotes pathological angiogenesis through canonical Wnt and VEGF signaling in vitro and in vivo, providing potential pharmacological targets for preventing the progression of CNV in HSK.

FOLR1-induced folate deficiency reduces viral replication via modulating APOBEC3 family expression.

Folate receptor alpha (FOLR1) is vital for cells ingesting folate (FA). FA plays an indispensable role in cell proliferation and survival. However, it is not clear whether the axis of FOLR1/FA has a similar function in viral replication. In this study, we used vesicular stomatitis virus (VSV) to investigate the relationship between FOLR1-mediated FA deficiency and viral replication, as well as the underlying mechanisms. We discovered that FOLR1 upregulation led to the deficiency of FA in HeLa cells and mice. Meanwhile, VSV replication was notably suppressed by FOLR1 overexpression, and this antiviral activity was related to FA deficiency. Mechanistically, FA deficiency mainly upregulated apolipoprotein B mRNA editing enzyme catalytic subunit 3B (APOBEC3B) expression, which suppressed VSV replication in vitro and in vivo. In addition, methotrexate (MTX), an FA metabolism inhibitor, effectively inhibited VSV replication by enhancing the expression of APOBEC3B in vitro and in vivo. Overall, our present study provided a new perspective for the role of FA metabolism in viral infections and highlights the potential of MTX as a broad-spectrum antiviral agent against RNA viruses.

Targeting hexokinase 1 alleviates NLRP3-mediated inflammation in apical periodontitis: A laboratory investigation.

AIM: The aim of the study was to explore whether hexokinase 1 (HK1) is involved in the inhibition of inflammation mediated by nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) signalling pathway in the development of apical periodontitis (AP). METHODOLOGY: Human AP tissues and normal control tissues were collected in the clinic. First, the levels of glucose, pyruvate, lactate and hexokinase activity were examined in human AP tissues. ECAR and OCR were further measured to detect the level of glycolysis in vitro model of inflammation, which established with lipopolysaccharide (LPS)-stimulated RAW264.7 cell line. Secondly, the expression of HK1, NLRP3, caspase-1 and interleukin (IL)-1ß were measured by Western blot, immunohistochemistry or RT-qPCR. Finally, lentiviral short hairpin RNA (shRNA) silencing technique or the inhibitor 2-deoxy-d-glucose (2-DG) were used to further detect the relationship between HK1-mediated glycolysis and NLRP3-mediated inflammation in the development of AP in vitro. RESULTS: Initially, the level of glycolysis was significantly increased in human AP tissues. Subsequently, the expression of HK1, NLRP3, caspase-1 and IL-1ß were upregulated significantly in human AP tissues. Furthermore, in the model of AP in vitro, a high level of glycolysis and the high expression of HK1, NLRP3, caspase-1 and IL-1ß was observed. Finally, the expression of NLRP3, caspase-1 and IL-1ß mediated by LPS stimulation was significantly reduced via HK1 knockdown or 2-DG treatment in vitro. CONCLUSIONS: Our data support that HK1-mediated glycolysis plays a crucial role in the development of AP via upregulating the NLRP3 signalling pathway. Moreover, targeting HK1 may contribute to prevent the progression of AP, which has a potential clinical translation.

Folate metabolism negatively regulates OAS-mediated antiviral innate immunity via ADAR3/endogenous dsRNA pathway.

BACKGROUND: Folate (FA) is an essential cofactor in the one-carbon (1C) metabolic pathway and participates in amino acid metabolism, purine and thymidylate synthesis, and DNA methylation. FA metabolism has been reported to play an important role in viral replications; however, the roles of FA metabolism in the antiviral innate immune response are unclear. OBJECTIVE: To evaluate the potential regulatory role of FA metabolism in antiviral innate immune response, we establish the model of FA deficiency (FAD) in vitro and in vivo. The molecular and functional effects of FAD on 2'-5'-oligoadenylate synthetases (OAS)-associated antiviral innate immunity pathways were assessed; and the potential relationship between FA metabolism and the axis of adenosine deaminases acting on RNA 3 (ADAR3)/endogenous double-stranded RNA (dsRNA)/OAS was further explored in the present study, as well as the potential translatability of these findings in vivo. METHODS: FA-free RPMI 1640 medium and FA-free feed were used to establish the model of FAD in vitro and in vivo. And FA and homocysteine (Hcy) concentrations in cell culture supernatants and serum were used for FAD model evaluation. Ribonucleoprotein immunoprecipitation assay was used to enrich endogenous dsRNA, and dot-blot was further used for quantitative analysis of endogenous dsRNA. Western-blot assay, RNA isolation and quantitative real-time PCR, immunofluorescence assay, and other molecular biology techniques were used for exploring the potential mechanisms. RESULTS: In this study, we observed that FA metabolism negatively regulated OAS-mediated antiviral innate immune response. Mechanistically, FAD induced ADAR3, which interacted with endogenous dsRNA, to inhibit deaminated adenosine (A) being converted into inosine (I), leading to the cytoplasmic accumulation of dsRNA. Furthermore, endogenous dsRNA accumulated in cytoplasm triggered the host immune activation, thus promoting the expression of OAS2 to suppress the replication of viruses. Additionally, injection of 8-Azaadenosine to experimental animals, an A-to-I editing inhibitor, efficiently enhanced OAS-mediated antiviral innate immune response to reduce the viral burden in vivo. CONCLUSIONS: Taken together, our present study provided a new perspective to illustrate a relationship between FA metabolism and the axis of ADAR3/endogenous dsRNA/OAS, and a new insight for the treatment of RNA viral infectious diseases by targeting the axis of ADAR3/endogenous dsRNA/OAS.

Cigarette smoking induces the activation of RIP2/caspase-12/NF-κB axis in oral squamous cell carcinoma.

Cigarette smoking is one of the major risk factors for the occurrence and progression of oral squamous cell carcinoma (OSCC). Receptor-interacting protein 2 (RIP2) has been involved in mucosal immunity and homeostasis via a positive regulation of nuclear factor κB (NF-κB) transcription factor activity. Caspase-12 can bind to RIP2 and dampen mucosal immunity. However, the roles of RIP2/NF-κB and caspase-12 in OSCC induced by cigarette smoking remain unknown. Herein, we investigated the effects of cigarette smoking on the RIP2/NF-κB and caspase-12 in human OSCC tissues and OSCC cell lines (HSC-3). We first observed that RIP2 mediated NF-κB activation and caspase-12 upregulation in OSCC patients with cigarette smoking and cigarette smoke extract (CSE)-treated HSC-3 cells, respectively. Moreover, we confirmed that the downregulation of RIP2 by siRNA resulted in the reduction of caspase-12 expression and NF-κB activity in the presence of CSE treatment in vitro. In summary, our results indicated that cigarette smoking induced the activation of the RIP2/caspase-12/NF-κB axis and it played an important role in the development of OSCC. The RIP2/caspase-12/NF-κB axis could be a target for OSCC prevention and treatment in the future.

Correlations of Computed Tomography Measurement of Distal Pulmonary Vascular Pruning with Airflow Limitation and Emphysema in COPD Patients.

Background: Pulmonary vascular alteration is an important feature of chronic obstructive pulmonary disease (COPD), which is characterized by distal pulmonary vascular pruning in angiography. We aimed to further investigate the clinical relevance of pulmonary vasculature in COPD patients using non-contrast computed tomography (CT). Methods: Seventy-one control subjects and 216 COPD patients completed the questionnaires, spirometry, and computed tomography (CT) scans within 1 month and were included in the study. Small pulmonary vessels represented by percentage of cross-sectional area of pulmonary vessels smaller than 5 mm2 or 5-10 mm2 to the total lung fields (%CSA<5 or %CSA5-10, respectively) were measured using ImageJ software. Spearman correlation was used to investigate the relationship between %CSA<5 and airflow limitation. A receiver operating characteristic (ROC) curve was built to evaluate the value of %CSA<5 in discriminating COPD patients from healthy control subjects. Segmented regression was used to analyze the relationship between %CSA<5 and %LAA-950 (percentage of low-attenuation areas less than -950 HU). Results: We found a significant correlation between %CSA<5 and forced expiratory volume in one second (FEV1) percentage of predicted value (%pred) (r = 0.564, P < 0.001). The area under the ROC curve for the value of %CSA<5 in distinguishing COPD was 0.816, with a cut-off value of 0.537 (Youden index J, 0.501; sensitivity, 78.24%; specificity, 71.83%). Since the relationship between %CSA<5 and %LAA-950 was not constant, performance of segmented regression was better than ordinary linear regression (adjusted R2, 0.474 vs 0.332, P < 0.001 and P < 0.001, respectively). As %CSA<5 decreased, %LAA-950 slightly increased until an inflection point (%CSA<5 = 0.524) was reached, after which the %LAA-950 increased apparently with a decrease in %CSA<5. Conclusion: %CSA<5 was significantly correlated with both airflow limitation and emphysema, and we identified an inflection point for the relationship between %CSA<5 and %LAA-950.

The potential roles of interleukin-25 in infectious diseases.

Interleukin-25 (IL-25), also known as IL-17E, is a recently identified cytokine of the IL-17 family. Numerous studies illustrated that the expression of IL-25 is regulated by multiple pathogens, including parasitic, viral, and bacterial infections. IL-25 has a dual function in infectious diseases. On the one hand, IL-25 activates type 2 immunity via the relevant cytokines, including IL-4, IL-5, and IL-13, which are associated with the development of pathogenic infection-related allergic diseases. On the other hand, IL-25 involves in the recruitment of group 2 innate lymphoid cells (ILC2) to enhanced T helper 2 (Th2) cell differentiation, which are important to the clearance of pathogens. However, the precise roles of IL-25 in infectious diseases remain largely unknown. Thus, the current review will shed light on the pivotal roles of IL-25 in infectious diseases.

Interleukin-25-Mediated-IL-17RB Upregulation Promotes Cutaneous Wound Healing in Diabetic Mice by Improving Endothelial Cell Functions.

Diabetic foot ulcer (DFU) frequently leads to non-traumatic amputation and finally even death. However, the mechanism of DFU is not fully understood. Interleukin 25 (IL-25), an alarmin cytokine that responds to tissue injury, has been reported to participate in tissue regeneration and maintaining glucose homeostasis. However, the role of IL-25 in diabetic wound healing remains unknown. Here, we showed that interleukin 17 receptor B (IL-17RB), the functional receptor of IL-25, was significantly inhibited in the wound skin of both diabetic patients with DFU and streptozotocin (STZ)-induced diabetic mice. Topical administration of recombinant IL-25 protein improved angiogenesis and collagen deposition in the wound bed and thus ameliorated delayed diabetic wound healing. IL-25 increased endothelial-specific CD31 expression in diabetic wounds and exogenous IL-25 protected endothelial cells from high glucose-impaired cell migration and tube formation in vitro. We further revealed that IL-25-mediated-IL-17RB signaling rescued the downregulation of Wnt/ß-catenin pathway both in vivo in diabetic mice and in vitro in HUVECs and induced the phosphorylation of AKT and ERK 1/2 in HUVECs under high glucose conditions. This study defines a positive regulatory role of IL-25-mediated-IL-17RB signaling in diabetic wound healing and suggests that induction of IL-25-mediated-IL-17RB signaling may be a novel therapeutic strategy for treating poor healing diabetic wounds.

6-Thioguanine Inhibits Herpes Simplex Virus 1 Infection of Eyes.

Herpes simplex virus 1 (HSV-1) infects eye corneal tissues leading to herpetic stromal keratitis (HSK), which is one of the leading causes of blindness. Here in our study, we found that 6-thioguanine (6-TG), a once clinically approved medication for child acute myelogenous leukemia, inhibited multiple strains of HSV-1 infection in vitro and in vivo. 6-TG is more potent than acyclovir (ACV) and ganciclovir (GCV), with the 50% inhibitory concentration (IC50) of 6-TG at 0.104 µM with high stimulation index (SI) (SI = 6,475.48) compared to the IC50 of ACV at 1.253 µM and the IC50 of GCV at 1.257 µM. In addition, 6-TG at 500 µM topically applied to the eyes with HSV-1 infection significantly inhibits HSV-1 replication, alleviates virus-induced HSK pathogenesis, and improves eye conditions. More importantly, 6-TG is effective against ACV-resistant HSV-1 strains, including HSV-1/153 and HSV-1/blue. Knockdown of Rac1 with small interfering RNA (siRNA) negatively affected HSV-1 replication, suggesting that Rac1 facilitated HSV-1 replication. Following HSV-1 infection of human corneal epithelial cells (HCECs), endogenous Rac1 activity was upregulated by glutathione S-transferase (GST) pulldown assay. We further found that Rac1 was highly expressed in the corneal tissue of HSK patients compared to normal individuals. Mechanistic study showed that 6-TG inhibited HSV-1 replication by targeting Rac1 activity in HSV-1 infected cells, and the Rac1 is critical in the pathogenesis of HSK. Our results indicated that 6-TG is a promising therapeutic molecule for the treatment of HSK. IMPORTANCE We reported the discovery of 6-TG inhibition of HSV-1 infection and its inhibitory roles in HSK both in vitro and in vivo. 6-TG was shown to possess at least 10× more potent inhibitory activity against HSV-1 than ACV and GCV and, more importantly, inhibit ACV/GCV-resistant mutant viruses. Animal model studies showed that gel-formulated 6-TG topically applied to eyes locally infected with HSV-1 could significantly inhibit HSV-1 replication, alleviate virus-induced HSK pathogenesis, and improve eye conditions. Further study showed that HSV-1 infection upregulated Rac1 expression, and knockdown of Rac1 using siRNA markedly restricted HSV-1 replication, suggesting that Rac1 is required for HSV-1 replication. In addition, we also documented that Rac1 is highly expressed in corneal tissues from HSK patients, indicating that Rac1 is associated with HSK pathogenesis. In view of the high potency of 6-TG, low cytotoxicity, targeting a distinct therapeutic target, we suggest that 6-TG is a potential candidate for development as a therapeutic agent for HSK therapy.

Corrigendum: DEAD-Box Helicase DDX6 Facilitated RIG-I-Mediated Type-I Interferon Response to EV71 Infection.

[This corrects the article DOI: 10.3389/fcimb.2021.725392.].

DEAD-Box Helicase DDX6 Facilitated RIG-I-Mediated Type-I Interferon Response to EV71 Infection.

Previous studies have shown that DEAD (Glu-Asp-Ala-Glu)-box RNA helicases play important roles in viral infection, either as cytosolic sensors of pathogenic molecules or as essential host factors against viral infection. In the current study, we found that DDX6, an RNA helicase belonging to the DEAD-box family of helicase, exhibited anti-Enterovirus 71 activity through augmenting RIG-I-mediated type-I IFN response. Moreover, DDX6 binds viral RNA to form an RNA-protein complex to positively regulate the RIG-I-mediated interferon response; however, EV71 has evolved a strategy to antagonize the antiviral effect of DDX6 by proteolytic degradation of the molecule through its non-structural protein 2A, a virus-encoded protease.

Harringtonine Inhibits Herpes Simplex Virus Type 1 Infection by Reducing Herpes Virus Entry Mediator Expression.

Herpes simplex virus type 1 (HSV-1) infection induces various clinical disorders, such as herpes simplex encephalitis (HSE), herpes simplex keratitis (HSK), and genital herpes. In clinical intervention, acyclovir (ACV) is the major therapeutic drug used to suppress HSV-1; however, ACV-resistant strains have gradually increased. In the present study, harringtonine (HT) significantly inhibited infection of HSV-1 as well as two ACV-resistant strains, including HSV-1 blue and HSV-1 153. Time-of-drug addition assay further revealed that HT mainly reduced the early stage of HSV-1 infection. We also demonstrated that HT mainly affected herpes virus entry mediator (HVEM) expression as shown by qPCR, Western Blot, and Immunofluorescence. Collectively, HT showed antiviral activity against HSV-1 and ACV-resistant strains by targeting HVEM and could be a promising therapeutic candidate for mitigating HSV-1-induced-pathogenesis.

NSC23766 and Ehop016 Suppress Herpes Simplex Virus-1 Replication by Inhibiting Rac1 Activity.

Herpes simplex virus-1 (HSV-1) infection of the eyes leads to herpes simplex virus keratitis (HSK), the main cause of infectious blindness in the world. As the current therapeutics for HSV-1 infection are rather limited and prolonged use of acyclovir (ACV)/ganciclovir (GCV) and in immunocompromised patients lead to the rise of drug resistant mutants, it underlines the urgent need for new antiviral agents with distinct mechanisms. Our study attempted to establish ras-related C3 botulinum toxin substrate 1 (Rac1) as a new therapeutic target for HSV-1 infection by using Rac1-specific inhibitors to evaluate the in vitro inhibition of virus growth. Our results showed that increased Rac1 activity facilitated HSV-1 replication and inhibition of Rac1 activity by NSC23766 and Ehop016 significantly reduced HSV-1 replication. Thus, we identified NSC23766 and Ehop016 as possessing potent anti-HSV-1 activities by suppressing the Rac1 activity, suggesting that Rac1 is a potential target for treating HSV-1-related diseases.