Herpes Simplex Virus ICP27 Protein Inhibits AIM 2-Dependent Inflammasome Influencing Pro-Inflammatory Cytokines Release in Human Pigment Epithelial Cells (hTert-RPE 1).

Although Herpes simplex virus type 1 (HSV-1) has been deeply studied, significant gaps remain in the fundamental understanding of HSV-host interactions: our work focused on studying the Infected Cell Protein 27 (ICP27) as an inhibitor of the Absent-in-melanoma-2 (AIM 2) inflammasome pathway, leading to reduced pro-inflammatory cytokines that influence the activation of a protective innate immune response to infection. To assess the inhibition of the inflammasome by the ICP27, hTert-immortalized Retinal Pigment Epithelial cells (hTert-RPE 1) infected with HSV-1 wild type were compared to HSV-1 lacking functional ICP27 (HSV-1∆ICP27) infected cells. The activation of the inflammasome by HSV-1∆ICP27 was demonstrated by quantifying the gene and protein expression of the inflammasome constituents using real-time PCR and Western blot. The detection of the cleavage of the pro-caspase-1 into the active form was performed by using a bioluminescent assay, while the quantification of interleukins 1ß (IL-1ß) and 18 (IL-18)released in the supernatant was quantified using an ELISA assay. The data showed that the presence of the ICP27 expressed by HSV-1 induces, in contrast to HSV-1∆ICP27 vector, a significant downregulation of AIM 2 inflammasome constituent proteins and, consequently, the release of pro-inflammatory interleukins into the extracellular environment reducing an effective response in counteracting infection.

Ancestral allele of DNA polymerase gamma modifies antiviral tolerance.

Mitochondria are critical modulators of antiviral tolerance through the release of mitochondrial RNA and DNA (mtDNA and mtRNA) fragments into the cytoplasm after infection, activating virus sensors and type-I interferon (IFN-I) response1-4. The relevance of these mechanisms for mitochondrial diseases remains understudied. Here we investigated mitochondrial recessive ataxia syndrome (MIRAS), which is caused by a common European founder mutation in DNA polymerase gamma (POLG1)5. Patients homozygous for the MIRAS variant p.W748S show exceptionally variable ages of onset and symptoms5, indicating that unknown modifying factors contribute to disease manifestation. We report that the mtDNA replicase POLG1 has a role in antiviral defence mechanisms to double-stranded DNA and positive-strand RNA virus infections (HSV-1, TBEV and SARS-CoV-2), and its p.W748S variant dampens innate immune responses. Our patient and knock-in mouse data show that p.W748S compromises mtDNA replisome stability, causing mtDNA depletion, aggravated by virus infection. Low mtDNA and mtRNA release into the cytoplasm and a slow IFN response in MIRAS offer viruses an early replicative advantage, leading to an augmented pro-inflammatory response, a subacute loss of GABAergic neurons and liver inflammation and necrosis. A population databank of around 300,000 Finnish individuals6 demonstrates enrichment of immunodeficient traits in carriers of the POLG1 p.W748S mutation. Our evidence suggests that POLG1 defects compromise antiviral tolerance, triggering epilepsy and liver disease. The finding has important implications for the mitochondrial disease spectrum, including epilepsy, ataxia and parkinsonism.

Reanalysis of single-cell RNA sequencing data does not support herpes simplex virus 1 latency in non-neuronal ganglionic cells in mice.

Most individuals are latently infected with herpes simplex virus type 1 (HSV-1), and it is well-established that HSV-1 establishes latency in sensory neurons of peripheral ganglia. However, it was recently proposed that latent HSV-1 is also present in immune cells recovered from the ganglia of experimentally infected mice. Here, we reanalyzed the single-cell RNA sequencing (scRNA-Seq) data that formed the basis for that conclusion. Unexpectedly, off-target priming in 3' scRNA-Seq experiments enabled the detection of non-polyadenylated HSV-1 latency-associated transcript (LAT) intronic RNAs. However, LAT reads were near-exclusively detected in mixed populations of cells undergoing cell death. Specific loss of HSV-1 LAT and neuronal transcripts during quality control filtering indicated widespread destruction of neurons, supporting the presence of contaminating cell-free RNA in other cells following tissue processing. In conclusion, the reported detection of latent HSV-1 in non-neuronal cells is best explained using compromised scRNA-Seq datasets.IMPORTANCEMost people are infected with herpes simplex virus type 1 (HSV-1) during their life. Once infected, the virus generally remains in a latent (silent) state, hiding within the neurons of peripheral ganglia. Periodic reactivation (reawakening) of the virus may cause fresh diseases such as cold sores. A recent study using single-cell RNA sequencing (scRNA-Seq) proposed that HSV-1 can also establish latency in the immune cells of mice, challenging existing dogma. We reanalyzed the data from that study and identified several flaws in the methodologies and analyses performed that invalidate the published conclusions. Specifically, we showed that the methodologies used resulted in widespread destruction of neurons which resulted in the presence of contaminants that confound the data analysis. We thus conclude that there remains little to no evidence for HSV-1 latency in immune cells.

Mice with type I interferon signaling deficiency are prone to epilepsy upon HSV-1 infection.

Viral encephalitis continues to be a significant public health concern. In our previous study, we discovered a lower expression of antiviral factors, such as IFN-ß, STING and IFI16, in the brain tissues of patients with Rasmussen's encephalitis (RE), a rare chronic neurological disorder often occurred in children, characterized by unihemispheric brain atrophy. Furthermore, a higher cumulative viral score of human herpes viruses (HHVs) was also found to have a significant positive correlation with the unihemispheric atrophy in RE. Type I IFNs (IFN-I) signaling is essential for innate anti-infection response by binding to IFN-α/ß receptor (IFNAR). In this study, we infected WT mice and IFNAR-deficient A6 mice with herpes simplex virus 1 (HSV-1) via periocular injection to investigate the relationship between IFN-I signaling and HHVs-induced brain lesions. While all mice exhibited typical viral encephalitis lesions in their brains, HSV-induced epilepsy was only observed in A6 mice. The gene expression matrix, functional enrichment analysis and protein-protein interaction network revealed four gene models that were positively related with HSV-induced epilepsy. Additionally, ten key genes with the highest scores were identified. Taken together, these findings indicate that intact IFN-I signaling can effectively limit HHVs induced neural symptoms and brain lesions, thereby confirming the positive correlation between IFN-I signaling repression and brain atrophy in RE and other HHVs encephalitis.

TREM2 is down-regulated by HSV1 in microglia and involved in antiviral defense in the brain.

Immunological control of viral infections in the brain exerts immediate protection and also long-term maintenance of brain integrity. Microglia are important for antiviral defense in the brain. Here, we report that herpes simplex virus type 1 (HSV1) infection of human induced pluripotent stem cell (hiPSC)-derived microglia down-regulates expression of genes in the TREM2 pathway. TREM2 was found to be important for virus-induced IFNB induction through the DNA-sensing cGAS-STING pathway in microglia and for phagocytosis of HSV1-infected neurons. Consequently, TREM2 depletion increased susceptibility to HSV1 infection in human microglia-neuron cocultures and in the mouse brain. TREM2 augmented STING signaling and activation of downstream targets TBK1 and IRF3. Thus, TREM2 is important for the antiviral immune response in microglia. Since TREM2 loss-of-function mutations and HSV1 serological status are both linked to Alzheimer's disease, this work poses the question whether genetic or virus-induced alterations of TREM2 activity predispose to post-infection neurological pathologies.

IFI16 phase separation via multi-phosphorylation drives innate immune signaling.

The interferon inducible protein 16 (IFI16) is a prominent sensor of nuclear pathogenic DNA, initiating innate immune signaling and suppressing viral transcription. However, little is known about mechanisms that initiate IFI16 antiviral functions or its regulation within the host DNA-filled nucleus. Here, we provide in vitro and in vivo evidence to establish that IFI16 undergoes liquid-liquid phase separation (LLPS) nucleated by DNA. IFI16 binding to viral DNA initiates LLPS and induction of cytokines during herpes simplex virus type 1 (HSV-1) infection. Multiple phosphorylation sites within an intrinsically disordered region (IDR) function combinatorially to activate IFI16 LLPS, facilitating filamentation. Regulated by CDK2 and GSK3ß, IDR phosphorylation provides a toggle between active and inactive IFI16 and the decoupling of IFI16-mediated cytokine expression from repression of viral transcription. These findings show how IFI16 switch-like phase transitions are achieved with temporal resolution for immune signaling and, more broadly, the multi-layered regulation of nuclear DNA sensors.

The E3 ubiquitin ligase ARIH1 promotes antiviral immunity and autoimmunity by inducing mono-ISGylation and oligomerization of cGAS.

The cytosolic DNA sensor cyclic GMP-AMP synthase (cGAS) plays a critical role in antiviral immunity and autoimmunity. The activity and stability of cGAS are fine-tuned by post-translational modifications. Here, we show that ariadne RBR E3 ubiquitin protein ligase 1 (ARIH1) catalyzes the mono-ISGylation and induces the oligomerization of cGAS, thereby promoting antiviral immunity and autoimmunity. Knockdown or knockout of ARIH1 significantly inhibits herpes simplex virus 1 (HSV-1)- or cytoplasmic DNA-induced expression of type I interferons (IFNs) and proinflammatory cytokines. Consistently, tamoxifen-treated ER-Cre;Arih1fl/fl mice and Lyz2-Cre; Arih1fl/fl mice are hypersensitive to HSV-1 infection compared with the controls. In addition, deletion of ARIH1 in myeloid cells alleviates the autoimmune phenotypes and completely rescues the autoimmune lethality caused by TREX1 deficiency. Mechanistically, HSV-1- or cytosolic DNA-induced oligomerization and activation of cGAS are potentiated by ISGylation at its K187 residue, which is catalyzed by ARIH1. Our findings thus reveal an important role of ARIH1 in innate antiviral and autoimmune responses and provide insight into the post-translational regulation of cGAS.

Controlling Herpes Simplex Virus-Induced Immunoinflammatory Lesions Using Metabolic Therapy: a Comparison of 2-Deoxy-d-Glucose with Metformin.

Herpes simplex virus (HSV) infection of the eye can result in a blinding immunoinflammatory lesion in the cornea called herpetic stromal keratitis (HSK). This lesion is orchestrated by T cells and can be reduced in magnitude by anti-inflammatory drugs and procedures that change the balance of cellular participants in lesions. This report evaluates the effect of drugs that cause metabolic reprogramming on lesion expression using two drugs that affect glucose metabolism: 2-deoxy-d-glucose (2DG) and metformin. Both drugs could limit HSK severity, but 2DG therapy could result in herpes encephalitis if used when replicating virus was still present. The reason metformin was a safer therapy was its lack of marked inhibitory effects on inflammatory cells particularly interferon-γ (IFN-γ)-producing Th1 and CD8 T cells in the trigeminal ganglion (TG), in which HSV latency is established and sustained. Additionally, whereas 2DG in TG cultures with established latency accelerated the termination of latency, this did not occur in the presence of metformin, likely because the inflammatory cells remained functional. Our results support the value of metabolic reprogramming to control viral immunoinflammatory lesions, but the approach used should be chosen with caution. IMPORTANCE Herpes simplex virus (HSV) infection of the eye is an example where damaging lesions are in part the consequence of a host response to the infection. Moreover, it was shown that changing the representation of cellular participants in the inflammatory reaction can minimize lesion severity. This report explores the value of metabolic reprogramming using two drugs that affect glucose metabolism to achieve cellular rebalancing. It showed that two drugs, 2-deoxy-d-glucose (2DG) and metformin, effectively diminished ocular lesion expression, but only metformin avoided the complication of HSV spreading to the central nervous system (CNS) and causing herpetic encephalitis. The report provides some mechanistic explanations for the findings.

MARCH8 attenuates cGAS-mediated innate immune responses through ubiquitylation.

Cyclic GMP-AMP synthase (cGAS) binds to microbial and self-DNA in the cytosol and synthesizes cyclic GMP-AMP (cGAMP), which activates stimulator of interferon genes (STING) and downstream mediators to elicit an innate immune response. Regulation of cGAS activity is essential for immune homeostasis. Here, we identified the E3 ubiquitin ligase MARCH8 (also known as MARCHF8, c-MIR, and RNF178) as a negative regulator of cGAS-mediated signaling. The immune response to double-stranded DNA was attenuated by overexpression of MARCH8 and enhanced by knockdown or knockout of MARCH8. MARCH8 interacted with the enzymatically active core of cGAS through its conserved RING-CH domain and catalyzed the lysine-63 (K63)-linked polyubiquitylation of cGAS at Lys411. This polyubiquitylation event inhibited the DNA binding ability of cGAS, impaired cGAMP production, and attenuated the downstream innate immune response. Furthermore, March8-deficient mice were less susceptible than their wild-type counterparts to herpes simplex virus 1 (HSV-1) infection. Together, our findings reveal a mechanism underlying the functional regulation of cGAS and the fine-tuning of the innate immune response.

Small Noncoding RNA (sncRNA1) within the Latency-Associated Transcript Modulates Herpes Simplex Virus 1 Virulence and the Host Immune Response during Acute but Not Latent Infection.

The HSV-1 latency-associated transcript (LAT) locus contains two small noncoding RNA (sncRNA) sequences (sncRNA1 and sncRNA2) that are not microRNAs (miRNAs). We recently reported that sncRNA1 is more important for in vitro activation of the herpesvirus entry mediator than sncRNA2, but its in vivo function is not known. To determine the role, if any, of sncRNA1 during herpes simplex virus 1 (HSV-1) infection in vivo, we deleted the 62-bp sncRNA1 sequence in HSV-1 strain McKrae using dLAT2903 (LAT-minus) virus, creating ΔsncRNA1 recombinant virus. Deletion of the sncRNA1 in ΔsncRNA1 virus was confirmed by complete sequencing of ΔsncRNA1 virus and its parental virus (i.e., McKrae). Replication of ΔsncRNA1 virus in tissue culture or in the eyes of infected mice was similar to that of HSV-1 strain McKrae and dLAT2903 viruses. However, the absence of sncRNA1 significantly reduced the levels of ICP0, ICP4, and gB but not LAT transcripts in infected rabbit skin cells in vitro. In contrast, the absence of sncRNA1 did reduce LAT expression in trigeminal ganglia (TG), but not in corneas, by day 5 postinfection (p.i.) in infected mice. Levels of eye disease in mice infected with ΔsncRNA1 or McKrae virus were similar, and despite reduced LAT levels in TG during acute ΔsncRNA1 infection, McKrae and ΔsncRNA1 viruses did not affect latency or reactivation on day 28 p.i. However, mice infected with ΔsncRNA1 virus were more susceptible to ocular infection than their wild-type (WT) counterparts. Expression of host immune response genes in corneas and TG of infected mice during primary infection showed reduced expression of beta interferon (IFNß) and IFNγ and altered activation of key innate immune pathways, such as the JAK-STAT pathway in ΔsncRNA1 virus compared with parental WT virus. Our results reveal novel functions for sncRNA1 in upregulating the host immune response and suggest that sncRNA1 has a protective role during primary ocular HSV-1 infection. IMPORTANCE HSV-1 latency-associated transcript (LAT) plays a major role in establishing latency and reactivation; however, the mechanism by which LAT controls these processes is largely unknown. In this study, we sought to establish the role of the small noncoding RNA1 (sncRNA1) encoded within LAT during HSV-1 ocular infection. Our results suggest that sncRNA1 has a protective role during acute ocular infection by modulating the innate immune response to infection.

Sensory Nerve Retraction and Sympathetic Nerve Innervation Contribute to Immunopathology of Murine Recurrent Herpes Stromal Keratitis.

Purpose: Herpes stromal keratitis (HSK) represents a spectrum of pathologies which is caused by herpes simplex virus type 1 (HSV-1) infection and is considered a leading cause of infectious blindness. HSV-1 infects corneal sensory nerves and establishes latency in the trigeminal ganglion (TG). Recently, retraction of sensory nerves and replacement with "unsensing" sympathetic nerves was identified as a critical contributor of HSK in a mouse model where corneal pathology is caused by primary infection. This resulted in the loss of blink reflex, corneal desiccation, and exacerbation of inflammation leading to corneal opacity. Despite this, it was unclear whether inflammation associated with viral reactivation was sufficient to initiate this cascade of events. Methods: We examined viral reactivation and corneal pathology in a mouse model with recurrent HSK by infecting the cornea with HSV-1 (McKrae) and transferring (intravenous [IV]) human sera to establish primary infection without discernible disease and then exposed the cornea to UV-B light to induce viral reactivation. Results: UV-B light induced viral reactivation from latency in 100% of mice as measured by HSV-1 antigen deposition in the cornea. Further, unlike conventional HSK models, viral reactivation resulted in focal retraction of sensory nerves and corneal opacity. Dependent on CD4+ T cells, inflammation foci were innervated by sympathetic nerves. Conclusions: Collectively, our data reveal that sectoral corneal sensory nerve retraction and replacement of sympathetic nerves were involved in the progressive pathology that is dependent on CD4+ T cells after viral reactivation from HSV-1 latency in the UV-B induced recurrent HSK mouse model.

Hygiene Hypothesis Indicators and Prevalence of Antinuclear Antibodies in US Adolescents.

Autoimmunity prevalence, as measured by antinuclear antibodies (ANA), is increasing in U.S. adolescents. Improved hygiene and cleaner environments in childhood may reduce exposure to infections and other immune challenges, resulting in improper immune responses to later-life exposures. We examined associations of hygiene hypothesis indicators, including asthma, allergies, and antibodies to infectious agents, with ANA prevalence, measured by HEp-2 immunofluorescence, in adolescents (aged 12-19 years) over a 25-year time span in the National Health and Nutrition Examination Survey (NHANES) (N=2,709), adjusting for age, sex, race/ethnicity, body mass index, education and survey cycle, overall and within individual time periods, using logistic regression. Prevalence of ANA in adolescents increased from 5.0% in 1988-1991 to 12.8% in 2011-2012. ANA were positively associated with diagnosis of asthma in early childhood (OR: 2.07, CI: 1.09-3.99) and the effect estimate for current hay fever was elevated but not statistically significant (OR: 1.55, CI: 0.85-2.84). Fewer than 2% of those with ANA in 1988-1991 had been diagnosed with asthma, compared with 18% in 1999-2000, and 27% in 2003-2004 and 2011-2012. ANA trended negatively with Helicobacter pylori antibodies (OR: 0.49, CI: 0.24-0.99). ANA may be useful as an additional indicator of inadequate immune education in adolescence, a critical period of growth and development.

Skin Injury Activates a Rapid TRPV1-Dependent Antiviral Protein Response.

The skin serves as the interface between the body and the environment and plays a fundamental role in innate antimicrobial host immunity. Antiviral proteins (AVPs) are part of the innate host defense system and provide protection against viral pathogens. How breach of the skin barrier influences innate AVP production remains largely unknown. In this study, we characterized the induction and regulation of AVPs after skin injury and identified a key role of TRPV1 in this process. Transcriptional and phenotypic profiling of cutaneous wounds revealed that skin injury induces high levels of AVPs in both mice and humans. Remarkably, pharmacologic and genetic ablation of TRPV1-mediated nociception abrogated the induction of AVPs, including Oas2, Oasl2, and Isg15 after skin injury in mice. Conversely, stimulation of TRPV1 nociceptors was sufficient to induce AVP production involving the CD301b+ cells‒IL-27‒mediated signaling pathway. Using IL-27 receptor‒knockout mice, we show that IL-27 signaling is required in the induction of AVPs after skin injury. Finally, loss of TRPV1 signaling leads to increased viral infectivity of herpes simplex virus. Together, our data indicate that TRPV1 signaling ensures skin antiviral competence on wounding.

Noncognate Signals Drive Enhanced Effector CD8+ T Cell Responses through an IFNAR1-Dependent Pathway after Infection with the Prototypic Vaccine, 0ΔNLS, against Herpes Simplex Virus 1.

Previous studies by our group identified a highly efficacious vaccine 0ΔNLS (deficient in the nuclear localization signal of infected cell protein 0) against herpes simplex virus 1 (HSV-1) in an experimental ocular mouse model. However, details regarding fundamental differences in the initial innate and adaptive host immune response were not explored. Here, we present a side-by-side analysis of the primary infection characterizing differences of the host immune response in mice infected with 0ΔNLS versus the parental, GFP105. The results show that local viral infection and replication are controlled more efficiently in mice exposed to 0ΔNLS versus GFP105 but that the clearance of infectious virus is equivalent when the two groups are compared. Moreover, the 0ΔNLS-infected mice displayed enhanced effector CD8+ but not CD4+ T cell responses from the draining lymph nodes at day 7 postinfection measured by gamma interferon (IFN-γ) and tumor necrosis factor alpha production along with changes in cell metabolism. The increased effector function of CD8+ T cells from 0ΔNLS-infected mice was not driven by changes in antigen presentation but lost in the absence of a functional type I IFN pathway. These results are further supported by enhanced local expression of type I IFN and IFN-inducible genes along with increased IL-12 production by CD8α+ dendritic cells in the draining lymph nodes of 0ΔNLS-infected mice compared to the GFP105-infected animals. It was also noted the recall to HSV-1 antigen by CD8+ T cells was elevated in mice infected with HSV-1 0ΔNLS compared to GFP105. Collectively, the results underscore the favorable qualities of HSV-1 0ΔNLS as a candidate vaccine against HSV-1 infection. IMPORTANCE Cytotoxic T lymphocytes (CTLs) play a critical role in the clearance for many viral pathogens including herpes simplex virus 1 (HSV-1). Here, we compared the cellular innate and adaptive immune response in mice infected with an attenuated HSV-1 (0ΔNLS) found to be a highly successful experimental prophylactic vaccine to parental HSV-1 virus. We found that CD8+ T cell effector function is elevated in 0ΔNLS-infected mice through noncognate signals, including interleukin-12 and type I interferon pathways along with changes in CD8+ T cell metabolism, whereas other factors, including cell proliferation, costimulatory molecule expression, and antigen presentation, were dispensable. Thus, an increase in CTL activity established by exposure to HSV-1 0ΔNLS in comparison to parental HSV-1 likely contributes to the efficacy of the vaccine and underscores the nature of the attenuated virus as a vaccine candidate for HSV-1 infection.

The oncolytic virus VT09X optimizes immune checkpoint therapy in low immunogenic melanoma.

Tumors with a low level of pre-existing immune cell infiltration respond poorly to immune checkpoint therapies. Oncolytic viruses optimize immunotherapies by modulating the tumor microenvironment and affecting multiple steps in the cancer-immunity cycle, making them an attractive agent for combination strategies. We engineered an HSV-1-based oncolytic virus and investigated its antitumor effects in combination with the marketed PD-1 antibody Keytruda (pembrolizumab) in hPD-1 knock-in mice bearing non-immunogenic B16-F10 melanoma. Our results showed enhanced CD8+ and CD4+ T cell infiltration, IFN-γ secretion and PD-L1 expression in tumors, subsequently leading to the prolonged overall survival of mice. Systemic changes in lymphocyte cell proportions were also observed in the peripheral blood. In summary, these findings provide evidence that oncolytic viruses can be engineered as a potential platform for combination therapies, especially to treat tumors that are poorly responsive to immune checkpoint therapy.

Innate Immune Signaling and Role of Glial Cells in Herpes Simplex Virus- and Rabies Virus-Induced Encephalitis.

The environment of the central nervous system (CNS) represents a double-edged sword in the context of viral infections. On the one hand, the infectious route for viral pathogens is restricted via neuroprotective barriers; on the other hand, viruses benefit from the immunologically quiescent neural environment after CNS entry. Both the herpes simplex virus (HSV) and the rabies virus (RABV) bypass the neuroprotective blood-brain barrier (BBB) and successfully enter the CNS parenchyma via nerve endings. Despite the differences in the molecular nature of both viruses, each virus uses retrograde transport along peripheral nerves to reach the human CNS. Once inside the CNS parenchyma, HSV infection results in severe acute inflammation, necrosis, and hemorrhaging, while RABV preserves the intact neuronal network by inhibiting apoptosis and limiting inflammation. During RABV neuroinvasion, surveilling glial cells fail to generate a sufficient type I interferon (IFN) response, enabling RABV to replicate undetected, ultimately leading to its fatal outcome. To date, we do not fully understand the molecular mechanisms underlying the activation or suppression of the host inflammatory responses of surveilling glial cells, which present important pathways shaping viral pathogenesis and clinical outcome in viral encephalitis. Here, we compare the innate immune responses of glial cells in RABV- and HSV-infected CNS, highlighting different viral strategies of neuroprotection or Neuroinflamm. in the context of viral encephalitis.

Seroprevalence of Toxoplasma gondii, Rubella, Group A Streptococcus, CMV and HSV-1 in COVID-19 Patients with Vitamin D Deficiency.

<b>Background and Objective:</b> In recent years, respiratory tract viral infections have caused many pandemics that impact the whole world. To investigate the seropositivity of <i>Toxoplasma gondii</i>, rubella, CMV, HSV-1 and group A <i>Streptococcus</i> in recovered COVID-19 patients and correlate these findings with vitamin D levels. <b>Materials and Methods:</b> A total of 417 COVID-19 patients with diarrhoea were enrolled in this study. Vitamin D and seroprevalence for <i>Toxoplasma gondii</i>, rubella, CMV, HSV-1 and group A <i>Streptococcus</i> were evaluated and correlated. <b>Results:</b> It was found that recent infection in COVID-19 patients with HSV-1, rubella, <i>Toxoplasma</i> and CMV, respectively. IgG was detected indicating the development of adaptive immunity with all microbes. <b>Conclusion:</b> Current study detected a correlation between vitamin D levels and HSV-1 and no correlation between this infection and vitamin D deficiency with the other microbes.

Alphaherpesvirus-induced activation of plasmacytoid dendritic cells depends on the viral glycoprotein gD and is inhibited by non-infectious light particles.

Plasmacytoid dendritic cells (pDC) are important innate immune cells during the onset of viral infections as they are specialized in the production of massive amounts of antiviral type I interferon (IFN). Alphaherpesviruses such as herpes simplex virus (HSV) or pseudorabies virus (PRV) are double stranded DNA viruses and potent stimulators of pDC. Detailed information on how PRV activates porcine pDC is lacking. Using PRV and porcine primary pDC, we report here that PRV virions, so-called heavy (H-)particles, trigger IFNα production by pDC, whereas light (L-) particles that lack viral DNA and capsid do not. Activation of pDC requires endosomal acidification and, importantly, depends on the PRV gD envelope glycoprotein and O-glycosylations. Intriguingly, both for PRV and HSV-1, we found that L-particles suppress H-particle-mediated activation of pDC, a process which again depends on viral gD. This is the first report describing that gD plays a critical role in alphaherpesvirus-induced pDC activation and that L-particles directly interfere with alphaherpesvirus-induced IFNα production by pDC.

Stress Induced Transcription Factors Transactivate the Herpes Simplex Virus 1 Infected Cell Protein 27 (ICP27) Transcriptional Enhancer.

Following acute infection, herpes simplex virus 1 (HSV-1) establishes lifelong latency in neurons, including sensory neurons within trigeminal ganglia. During latency, lytic cycle viral gene expression is silenced. However, stressful stimuli can trigger reactivation from latency. The viral tegument protein, VP-16, transactivates all immediate early (IE) promoters during productive infection. Conversely, cellular factors are expected to trigger viral gene expression during early stages of reactivation from latency and in non-neuronal cells that do not support high levels of productive infection. The glucocorticoid receptor (GR), synthetic corticosteroid dexamethasone, and certain stress-induced transcription factors cooperatively transactivate infected cell protein 0 (ICP0) and ICP4 promoters. Since ICP27 protein expression is required for productive infection, we hypothesized that the ICP27 promoter is transactivated by stress-induced transcription factors. New studies have demonstrated that ICP27 enhancer sequences were transactivated by GR and Krüppel-like factor 15 (KLF15). Mutation of a consensus Sp1 binding site within ICP27 enhancer sequences impaired transactivation by GR and KLF15. Chromatin immunoprecipitation studies have demonstrated that GR and KLF15 occupy ICP27 promoter sequences during productive infection. Cells transfected with an ICP27 enhancer fragment revealed the GR and KLF15 occupancy of ICP27 enhancer sequences required the intact Sp1 binding site. Notably, GR and KLF15 form a feed-forward transcription loop in response to stress, suggesting these cellular factors promote viral replication following stressful stimuli.

Tupaia GBP1 Interacts with STING to Initiate Autophagy and Restrict Herpes Simplex Virus Type 1 Infection.

Stimulator of IFN genes (STING) is a key molecule that binds to cyclic dinucleotides produced by the cyclic GMP-AMP synthase to activate IFN expression and autophagy in the fight against microbial infection. The regulation of STING in the activation of IFN expression has been extensively reported, whereas the regulation of STING in the initiation of autophagy is still insufficiently determined. IFN-inducible guanylate-binding proteins (GBPs) are central to the cell-autonomous immunity in defending a host against viral, bacterial, and protozoan infections. In this study using the Chinese tree shrew (Tupaia belangeri chinensis), which is genetically close to primates, we found that Tupaia GBP1 (tGBP1) combines with Tupaia STING (tSTING), promotes autophagy, and moderately inhibits HSV type 1 (HSV-1) infection. The antiviral effects of tGBP1 are IFN independent. Mechanistically, tGBP1 interacted with tSTING, Tupaia sequestosome 1, and Tupaia microtubule associated protein 1 L chain 3, forming a complex which promotes autophagy in response to HSV-1 infection. This function of tGBP1 against HSV-1 infection was lost in tSTING knockout cells. Overexpression of either tSTING or its mutant tSTING-ΔCTT that can only activate autophagy rescued the anti-HSV-1 activity of tGBP1 in tSTING knockout cells. Our study not only elucidated the underlying mechanism of tGBP1 antiviral activity against HSV-1 infection, but also uncovered the regulation of tSTING in the initiation of autophagy in response to HSV-1 infection.