Inhibitors of One or More Cellular Aurora Kinases Impair the Replication of Herpes Simplex Virus 1 and Other DNA and RNA Viruses with Diverse Genomes and Life Cycles.

We utilized a high-throughput cell-based assay to screen several chemical libraries for inhibitors of herpes simplex virus 1 (HSV-1) gene expression. From this screen, four aurora kinase inhibitors were identified that potently reduced gene expression during HSV-1 lytic infection. HSV-1 is known to interact with cellular kinases to regulate gene expression by modulating the phosphorylation and/or activities of viral and cellular proteins. To date, the role of aurora kinases in HSV-1 lytic infection has not been reported. We demonstrated that three aurora kinase inhibitors strongly reduced the transcript levels of immediate-early (IE) genes ICP0, ICP4, and ICP27 and impaired HSV-1 protein expression from all classes of HSV-1, including ICP0, ICP4, ICP8, and gC. These restrictions caused by the aurora kinase inhibitors led to potent reductions in HSV-1 viral replication. The compounds TAK 901, JNJ 7706621, and PF 03814735 decreased HSV-1 titers by 4,500-, 13,200-, and 8,400-fold, respectively, when present in a low micromolar range. The antiviral activity of these compounds correlated with an apparent decrease in histone H3 phosphorylation at serine 10 (H3S10ph) during viral infection, suggesting that the phosphorylation status of H3 influences HSV-1 gene expression. Furthermore, we demonstrated that the aurora kinase inhibitors also impaired the replication of other RNA and DNA viruses. These inhibitors significantly reduced yields of vaccinia virus (a poxvirus, double-stranded DNA, cytoplasmic replication) and mouse hepatitis virus (a coronavirus, positive-sense single-strand RNA [ssRNA]), whereas vesicular stomatitis virus (rhabdovirus, negative-sense ssRNA) yields were unaffected. These results indicated that the activities of aurora kinases play pivotal roles in the life cycles of diverse viruses. IMPORTANCE We have demonstrated that aurora kinases play a role during HSV-1 lytic infection. Three aurora kinase inhibitors significantly impaired HSV-1 immediate-early gene expression. This led to a potent reduction in HSV-1 protein expression and viral replication. Together, our results illustrate a novel role for aurora kinases in the HSV-1 lytic cycle and demonstrate that aurora kinase inhibitors can restrict HSV-1 replication. Furthermore, these aurora kinase inhibitors also reduced the replication of murine coronavirus and vaccinia virus, suggesting that multiple viral families utilize the aurora kinases for their own replication.

Blue light irradiation exerts anti-viral and anti-inflammatory properties against herpes simplex virus type 1 infection.

The aim of this study was to investigate the antiviral and anti-inflammatory functions of blue light (BL) in cutaneous viral infections. Previously, we examined the photo-biogoverning role of 450 nm BL in SARS-CoV-2-infected cells, which showed that photo-energy could inhibit viral activation depending on the number of photons. However, the communication network between photo-energy irradiation and immune cells involved in viral infections has not been clarified. We verified viral activation, inflammatory responses, and relevant downstream cascades caused by human simplex virus type I (HSV-1) after BL irradiation. To examine the antiviral effect of BL, we further tested whether BL could disturb viral absorption or entry into host cells. The results showed that BL irradiation, but not green light (GL) exposure, specifically decreased plaque-forming activity and viral copy numbers in HSV-1-infected cells. Accumulated BL irradiation inhibited the localization of viral proteins and the RNA expression of characteristic viral genes such as UL19, UL27, and US6, thus exerting to an anti-viral effect. The results also showed that BL exposure during viral absorption interfered with viral entry or destroyed the virus, as assessed by plaque formation and quantitative PCR assays. The levels of the pro-inflammatory mediators interleukin (IL)-18 and IL-1ß in M1-polarized macrophages were increased by HSV-1 infection. However, these increases were attenuated by BL irradiation. Importantly, BL irradiation decreased cGAS and STING expression, as well as downstream NF-κB p65, in M1-polarized HSV-1-infected macrophages, demonstrating anti-viral and anti-inflammatory properties. These findings suggest that BL could serve as an anti-viral and anti-inflammatory therapeutic candidate to treat HSV-1 infections.

Complete and Prolonged Inhibition of Herpes Simplex Virus Type 1 Infection In Vitro by CRISPR/Cas9 and CRISPR/CasX Systems.

Almost all people become infected with herpes viruses, including herpes simplex virus type 1 (HSV-1), during their lifetime. Typically, these viruses persist in a latent form that is resistant to all available antiviral medications. Under certain conditions, such as immunosuppression, the latent forms reactivate and cause disease. Moreover, strains of herpesviruses that are drug-resistant have rapidly emerged. Therefore, it is important to develop alternative methods capable of eradicating herpesvirus infections. One promising direction is the development of CRISPR/Cas systems for the therapy of herpesvirus infections. We aimed to design a CRISPR/Cas system for relatively effective long-term and safe control of HSV-1 infection. Here, we show that plasmids encoding the CRISPR/Cas9 system from Streptococcus pyogenes with a single sgRNA targeting the UL30 gene can completely suppress HSV-1 infection of the Vero cell line within 6 days and provide substantial protection within 9 days. For the first time, we show that CRISPR/CasX from Deltaproteobacteria with a single guide RNA against UL30 almost completely suppresses HSV-1 infection of the Vero cell line for 3 days and provides substantial protection for 6 days. We also found that the Cas9 protein without sgRNAs attenuates HSV-1 infection. Our results show that the developed CRISPR/Cas systems are promising therapeutic approaches to control HSV-1 infections.

Broadly Applicable, Virus-Free Dual Reporter Assay to Identify Compounds Interfering with Membrane Fusion: Performance for HSV-1 and SARS-CoV-2.

Membrane fusion constitutes an essential step in the replication cycle of numerous viral pathogens, hence it represents an important druggable target. In the present study, we established a virus-free, stable reporter fusion inhibition assay (SRFIA) specifically designed to identify compounds interfering with virus-induced membrane fusion. The dual reporter assay is based on two stable Vero cell lines harboring the third-generation tetracycline (Tet3G) transactivator and a bicistronic reporter gene cassette under the control of the tetracycline responsive element (TRE3G), respectively. Cell-cell fusion by the transient transfection of viral fusogens in the presence of doxycycline results in the expression of the reporter enzyme secreted alkaline phosphatase (SEAP) and the fluorescent nuclear localization marker EYFPNuc. A constitutively expressed, secreted form of nanoluciferase (secNLuc) functioned as the internal control. The performance of the SRFIA was tested for the quantification of SARS-CoV-2- and HSV-1-induced cell-cell fusion, respectively, showing high sensitivity and specificity, as well as the reliable identification of known fusion inhibitors. Parallel quantification of secNLuc enabled the detection of cytotoxic compounds or insufficient transfection efficacy. In conclusion, the SRFIA reported here is well suited for high-throughput screening for new antiviral agents and essentially will be applicable to all viral fusogens causing cell-cell fusion in Vero cells.

Genome-wide CRISPR screen for HSV-1 host factors reveals PAPSS1 contributes to heparan sulfate synthesis.

Herpes simplex virus type 1 (HSV-1) is a ubiquitous pathogen that causes various diseases in humans, ranging from common mucocutaneous lesions to severe life-threatening encephalitis. However, our understanding of the interaction between HSV-1 and human host factors remains incomplete. Here, to identify the host factors for HSV-1 infection, we performed a human genome-wide CRISPR screen using near-haploid HAP1 cells, in which gene knockout (KO) could be efficiently achieved. Along with several already known host factors, we identified 3'-phosphoadenosine 5'-phosphosulfate synthase 1 (PAPSS1) as a host factor for HSV-1 infection. The KO of PAPSS1 in HAP1 cells reduced heparan sulfate (HepS) expression, consequently diminishing the binding of HSV-1 and several other HepS-dependent viruses (such as HSV-2, hepatitis B virus, and a human seasonal coronavirus). Hence, our findings provide further insights into the host factor requirements for HSV-1 infection and HepS biosynthesis.

RespiCoV: Simultaneous identification of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and 46 respiratory tract viruses and bacteria by amplicon-based Oxford-Nanopore MinION sequencing.

Since December 2019 the world has been facing the outbreak of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Identification of infected patients and discrimination from other respiratory infections have so far been accomplished by using highly specific real-time PCRs. Here we present a rapid multiplex approach (RespiCoV), combining highly multiplexed PCRs and MinION sequencing suitable for the simultaneous screening for 41 viral and five bacterial agents related to respiratory tract infections, including the human coronaviruses NL63, HKU1, OC43, 229E, Middle East respiratory syndrome coronavirus, SARS-CoV, and SARS-CoV-2. RespiCoV was applied to 150 patient samples with suspected SARS-CoV-2 infection and compared with specific real-time PCR. Additionally, several respiratory tract pathogens were identified in samples tested positive or negative for SARS-CoV-2. Finally, RespiCoV was experimentally compared to the commercial RespiFinder 2SMART multiplex screening assay (PathoFinder, The Netherlands).

Development of a multiplex droplet digital PCR assay for detection of enterovirus, parechovirus, herpes simplex virus 1 and 2 simultaneously for diagnosis of viral CNS infections.

BACKGROUND: Enterovirus (EV), parechovirus (HPeV), herpes simplex virus 1 and 2 (HSV1/2) are common viruses leading to viral central nervous system (CNS) infections which are increasingly predominant but exhibit deficiency in definite pathogen diagnosis with gold-standard quantitative PCR method. Previous studies have shown that droplet digital PCR (ddPCR) has great potential in pathogen detection and quantification, especially in low concentration samples. METHODS: Targeting four common viruses of EV, HPeV, HSV1, and HSV2 in cerebrospinal fluid (CSF), we developed a multiplex ddPCR assay using probe ratio-based multiplexing strategy, analyzed the performance, and evaluated it in 97 CSF samples collected from patients with suspected viral CNS infections on a two-channel ddPCR detection system. RESULTS: The four viruses were clearly distinguished by their corresponding fluorescence amplitude. The limits of detection for EV, HPeV, HSV1, and HSV2 were 5, 10, 5, and 10 copies per reaction, respectively. The dynamic range was at least four orders of magnitude spanning from 2000 to 2 copies per reaction. The results of 97 tested clinical CSF specimens were identical to those deduced from qPCR/qRT-PCR assays using commercial kits. CONCLUSION: The multiplex ddPCR assay was demonstrated to be an accurate and robust method which could detect EV, HPeV, HSV1, and HSV2 simultaneously. It provides a useful tool for clinical diagnosis and disease monitoring of viral CNS infections.

Herpes Simplex Virus 1 Anterior Uveitis following Coronavirus Disease 2019 (COVID-19) Vaccination in an Asian Indian Female.

PURPOSE: To describe a case of herpes simplex virus 1 (HSV 1) infection following coronavirus disease 2019 (COVID-19) vaccination in an Asian Indian female. METHODS: Retrospective case report. RESULT: A 40-year-old female presented with decreased vision, pain, and photophobia of 2 weeks duration. She reported receiving the second dose of COVISHIELDTM (ChAdOx1-S [recombinant]) 1 week prior to the onset of ocular symptoms. Left eye examination revealed granulomatous anterior uveitis. Aqueous sample from the left eye tested positive for HSV1 by polymerase chain reaction(PCR) method. She was managed with oral antiviral therapy, topical steroids, and cycloplegic agent and showed significant improvement of inflammation within 1 week and resolved within 3 weeks. CONCLUSION: This report demonstrates a potential association of HSV 1 anterior uveitis with COVID-19 vaccination. A high index of suspicion of viral etiology is warranted when uveitis presents with reduced corneal sensations and pigmented keratic precipitates, following a recent history of COVID-19 vaccination.

Identification and characterisation of SARS-CoV-2 and Human alphaherpesvirus 1 from a productive coinfection in a fatal COVID-19 case.

BACKGROUND: During routine Coronavirus disease 2019 (COVID-19) diagnosis, an unusually high viral load was detected by reverse transcription real-time polymerase chain reaction (RT-qPCR) in a nasopharyngeal swab sample collected from a patient with respiratory and neurological symptoms who rapidly succumbed to the disease. Therefore we sought to characterise the infection. OBJECTIVES: We aimed to determine and characterise the etiological agent responsible for the poor outcome. METHODS: Classical virological methods, such as plaque assay and plaque reduction neutralisation test combined with amplicon-based sequencing, as well as a viral metagenomic approach, were performed to characterise the etiological agents of the infection. FINDINGS: Plaque assay revealed two distinct plaque phenotypes, suggesting either the presence of two severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strains or a productive coinfection of two different species of virus. Amplicon-based sequencing did not support the presence of any SARS-CoV-2 genetic variants that would explain the high viral load and suggested the presence of a single SARS-CoV-2 strain. Nonetheless, the viral metagenomic analysis revealed that Coronaviridae and Herpesviridae were the predominant virus families within the sample. This finding was confirmed by a plaque reduction neutralisation test and PCR. MAIN CONCLUSIONS: We characterised a productive coinfection of SARS-CoV-2 and Herpes simplex virus 1 (HSV-1) in a patient with severe symptoms that succumbed to the disease. Although we cannot establish the causal relationship between the coinfection and the severity of the clinical case, this work serves as a warning for future studies focused on the interplay between SARS-CoV-2 and HSV-1 coinfection and COVID-19 severity.

Herpes Simplex Virus Keratitis Reactivation after SARS-CoV-2 BNT162b2 mRNA Vaccination: A Report of Two Cases.

PURPOSE: To report two cases of herpes simplex virus keratitis reactivation following Pfizer-BioNTech COVID-19 (BNT162b2) mRNA vaccination. METHODS: Two patients (one male, age 42 years, and one female, age 29 years) who are known to have herpetic keratitis presented to our emergency room in a time frame between 4 days and 4 weeks of receiving the vaccine. One patient presented with necrotizing stromal keratitis; the other presented with endotheliitis and epithelial keratitis. PCR for herpes simplex virus (HSV) was obtained from the two patients, and all cases received systemic acyclovir. RESULTS: PCR for HSV came positive in both cases. Patients responded well to the provided treatment. CONCLUSION: Ocular herpetic infection may be activated by COVID-19 (BNT162b2) mRNA vaccine. Treating physician should be alert to such associations, and patients should be followed closely. No direct causality has been proven, but further reporting and investigating similar conditions is recommended.

Expression of SARS coronavirus 1 spike protein from a herpesviral vector induces innate immune signaling and neutralizing antibody responses.

SARS coronavirus 1 (SARS-CoV-1) causes a respiratory infection that can lead to acute respiratory distress characterized by inflammation and high levels of cytokines in the lung tissue. In this study we constructed a herpes simplex virus 1 replication-defective mutant vector expressing SARS-CoV-1 spike protein as a potential vaccine vector and to probe the effects of spike protein on host cells. The spike protein expressed from this vector is functional in that it localizes to the surface of infected cells and induces fusion of ACE2-expressing cells. In immunized mice, the recombinant vector induced antibodies that bind to spike protein in an ELISA assay and that show neutralizing activity. The spike protein expressed from this vector can induce the expression of cytokines in an ACE2-independent, MyD88-dependent process. These results argue that the SARS-CoV-1 spike protein intrinsically activates signaling pathways that induce cytokines and contribute directly to the inflammatory process of SARS.

Hypopharyngeal Ulcers in COVID-19: Histopathological and Virological Analyses - A Case Report.

In coronavirus disease 2019 (COVID-19), ulcerative lesions have been episodically reported in various segments of the gastrointestinal (GI) tract, including the oral cavity, oropharynx, esophagus, stomach and bowel. In this report, we describe an autopsy case of a COVID-19 patient who showed two undiagnosed ulcers at the level of the anterior and posterior walls of the hypopharynx. Molecular testing of viruses involved in pharyngeal ulcers demonstrated the presence of severe acute respiratory syndrome - coronavirus type 2 (SARS-CoV-2) RNA, together with herpes simplex virus 1 DNA. Histopathologic analysis demonstrated full-thickness lympho-monocytic infiltration (mainly composed of CD68-positive cells), with hemorrhagic foci and necrosis of both the mucosal layer and deep skeletal muscle fibers. Fibrin and platelet microthrombi were also found. Cytological signs of HSV-1 induced damage were not found. Cells expressing SARS-CoV-2 spike subunit 1 were immunohistochemically identified in the inflammatory infiltrations. Immunohistochemistry for HSV1 showed general negativity for inflammatory infiltration, although in the presence of some positive cells. Thus, histopathological, immunohistochemical and molecular findings supported a direct role by SARS-CoV-2 in producing local ulcerative damage, although a possible contributory role by HSV-1 reactivation cannot be excluded. From a clinical perspective, this autopsy report of two undiagnosed lesions put the question if ulcers along the GI tract could be more common (but frequently neglected) in COVID-19 patients.

Exploring novel and potent cell penetrating peptides in the proteome of SARS-COV-2 using bioinformatics approaches.

Among various delivery systems for vaccine and drug delivery, cell-penetrating peptides (CPPs) have been known as a potent delivery system because of their capability to penetrate cell membranes and deliver some types of cargoes into cells. Several CPPs were found in the proteome of viruses such as Tat originated from human immunodeficiency virus-1 (HIV-1), and VP22 derived from herpes simplex virus-1 (HSV-1). In the current study, a wide-range of CPPs was identified in the proteome of SARS-CoV-2, a new member of coronaviruses family, using in silico analyses. These CPPs may play a main role for high penetration of virus into cells and infection of host. At first, we submitted the proteome of SARS-CoV-2 to CellPPD web server that resulted in a huge number of CPPs with ten residues in length. Afterward, we submitted the predicted CPPs to C2Pred web server for evaluation of the probability of each peptide. Then, the uptake efficiency of each peptide was investigated using CPPred-RF and MLCPP web servers. Next, the physicochemical properties of the predicted CPPs including net charge, theoretical isoelectric point (pI), amphipathicity, molecular weight, and water solubility were calculated using protparam and pepcalc tools. In addition, the probability of membrane binding potential and cellular localization of each CPP were estimated by Boman index using APD3 web server, D factor, and TMHMM web server. On the other hand, the immunogenicity, toxicity, allergenicity, hemolytic potency, and half-life of CPPs were predicted using various web servers. Finally, the tertiary structure and the helical wheel projection of some CPPs were predicted by PEP-FOLD3 and Heliquest web servers, respectively. These CPPs were divided into: a) CPP containing tumor homing motif (RGD) and/or tumor penetrating motif (RXXR); b) CPP with the highest Boman index; c) CPP with high half-life (~100 hour) in mammalian cells, and d) CPP with +5.00 net charge. Based on the results, we found a large number of novel CPPs with various features. Some of these CPPs possess tumor-specific motifs which can be evaluated in cancer therapy. Furthermore, the novel and potent CPPs derived from SARS-CoV-2 may be used alone or conjugated to some sequences such as nuclear localization sequence (NLS) for vaccine and drug delivery.

Positive detection of SARS-CoV-2 combined HSV1 and HHV6B virus nucleic acid in tear and conjunctival secretions of a non-conjunctivitis COVID-19 patient with obstruction of common lacrimal duct.

BACKGROUND: The current outbreak of COVID-19 has spread rapidly all over the world. Respiratory droplets and contaction with infected patients are the two major transmission routes. However, the value of tear virus nucleic acid is still not clear. We dynamic detected the SARS-CoV-2 in eye sample of one COVID-19 patient with obstruction of common lacrimal ducts. METHODS: Besides the routine examination, nasopharyngeal and eye swab were continuously measured by polymerase chain reaction assay and next-generation sequencing (NGS). Gene detection was performed for drug use guidance, and flow cytometry was performed to analyse the lymphocyte subsets. RESULTS: Nasopharyngeal swabs were positive for 22 days, but eye swabs were still continuously positive for 2 weeks after nasopharyngeal swabs turned negative. The low level of lymphocyte and the high level IL-6 lasted for almost 4 weeks, then became near normal. Next-generation sequencing (NGS) confirmed the existing of SARS-CoV-2, HSV1 and HHV6B virus nucleic acid. The gene detection for drug use guidance showed the genetic locus ABCB1 (3435T>C) rs1045642 belonged to type CC and it mean the efficiency of lopinavir-ritonavir would be significantly decreased. The flow cytometry of lymphocyte subsets showed PD-1+  CD95+ cells was accounting for 94.8% in CD3+  CD8+ T subset and for 94.8% in CD3+  TCRγδ+ T subset. CONCLUSIONS: As obstruction of common lacrimal duct, positively detection in one eye for 2 weeks more after nasopharyngeal swab became negative. More eye swabs should be collected from COVID-19 patients, especially from those immunocompromised, those with eye symptoms and those had a history of ocular diseases.