Varicella zoster virus (VZV) is a neurotropic alphaherpesvirus exclusively infecting humans, causing two distinct pathologies: varicella (chickenpox) upon primary infection and herpes zoster (shingles) following reactivation. In susceptible individuals, VZV can give rise to more severe clinical manifestations, including disseminated infection, pneumonitis, encephalitis, and vasculopathy with stroke. Here, we describe a 3-year-old boy in whom varicella followed a complicated course with thrombocytopenia, hemorrhagic and necrotic lesions, pneumonitis, and intermittent encephalopathy. Hemophagocytic lymphohistiocytosis (HLH) was strongly suspected and as the condition deteriorated, HLH therapy was initiated. Although the clinical condition improved, longstanding hemophagocytosis followed despite therapy. We found that the patient carries a rare monoallelic variant in autocrine motility factor receptor ( AMFR ), encoding a ubiquitin ligase involved in innate cytosolic DNA sensing and interferon (IFN) production through the cyclic GMP-AMP synthase–stimulator of IFN genes (cGAS-STING) pathway. Peripheral blood mononuclear cells (PBMCs) from the patient exhibited impaired signaling downstream of STING in response dsDNA and 2'3'-cGAMP, agonists of cGAS and STING, respectively, and fibroblasts from the patient showed impaired type I IFN responses and significantly increased VZV replication. Overexpression of the variant AMFR R594C resulted in decreased K27-linked STING ubiquitination compared to WT AMFR. Moreover, ImageStream technology revealed reduced STING trafficking from ER to Golgi in cells expressing the patient AMFR R594C variant. This was supported by a dose-dependent dominant negative effect of expression of the patient AMFR variant as measured by IFN-β reporter gene assay. Finally, lentiviral transduction with WT AMFR partially reconstituted 2'3'-cGAMP-induced STING-mediated signaling and ISG expression in patient PBMCs. This work links defective AMFR-STING signaling to severe VZV disease and hyperinflammation and suggests a direct role for cGAS-STING in the control of viral infections in humans. In conclusion, we describe a novel genetic etiology of severe VZV disease in childhood, also representing the first inborn error of immunity related to a defect in the cGAS-STING pathway.

Autophagy is a degradational pathway with pivotal roles in cellular homeostasis and survival, including protection of neurons in the central nervous system (CNS). The significance of autophagy as antiviral defense mechanism is recognized and some viruses hijack and modulate this process to their advantage in certain cell types. Here, we present data demonstrating that the human neurotropic herpesvirus varicella zoster virus (VZV) induces autophagy in human SH-SY5Y neuronal cells, in which the pathway exerts antiviral activity. Productively VZV-infected SH-SY5Y cells showed increased LC3-I-LC3-II conversion as well as co-localization of the viral glycoprotein E and the autophagy receptor p62. The activation of autophagy was dependent on a functional viral genome. Interestingly, inducers of autophagy reduced viral transcription, whereas inhibition of autophagy increased viral transcript expression. Finally, the genotype of patients with severe ocular and brain VZV infection were analyzed to identify potential autophagy-associated inborn errors of immunity. Two patients expressing genetic variants in the autophagy genes ULK1 and MAP1LC3B2, respectively, were identified. Notably, cells of both patients showed reduced autophagy, alongside enhanced viral replication and death of VZV-infected cells. In conclusion, these results demonstrate a neuro-protective role for autophagy in the context of VZV infection and suggest that failure to mount an autophagy response is a potential predisposing factor for development of severe VZV disease.

PurposeHerpes simplex virus (HSV) and varicella-zoster virus (VZV) are neurotropic human alphaherpesviruses endemic worldwide. Upon primary infection, both viruses establish lifelong latency in neurons and reactivate intermittently to cause a variety of mild to severe diseases. Acute retinal necrosis (ARN) is a rare, sight-threatening eye disease induced by ocular VZV or HSV infection. The virus and host factors involved in ARN pathogenesis remain incompletely described. We hypothesize an underlying genetic defect in at least part of ARN cases.MethodsWe collected blood from 17 patients with HSV-or VZV-induced ARN, isolated DNA and performed Whole Exome Sequencing by Illumina followed by analysis in Varseq with criteria of CADD score > 15 and frequency in GnomAD < 0.1% combined with biological filters. Gene modifications relative to healthy control genomes were filtered according to high quality and read-depth, low frequency, high deleteriousness predictions and biological relevance.ResultsWe identified a total of 50 potentially disease-causing genetic variants, including missense, frameshift and splice site variants and on in-frame deletion in 16 of the 17 patients. The vast majority of these genes are involved in innate immunity, followed by adaptive immunity, autophagy, and apoptosis; in several instances variants within a given gene or pathway was identified in several patients.DiscussionWe propose that the identified variants may contribute to insufficient viral control and increased necrosis ocular disease presentation in the patients and serve as a knowledge base and starting point for the development of improved diagnostic, prophylactic, and therapeutic applications.

The present study describes a 19-year-old woman with systemic herpes simplex virus (HSV)-1 infection and hemophagocytic lymphohistiocytosis (HLH) postpartum, and a fatal course of neonatal herpesvirus infection. Functional investigation of cells from the mother demonstrated significantly impaired induction of antiviral interferons and cytokines in the context of normal activation of the transcription factors NF-κB and IRF3. Whole-exome sequencing did not reveal any functionally validated genetic variants. We suggest that the functionally impaired antiviral responses, potentially caused by a variant in CASP8 or other variants in noncoding regions of the genome, contributed to the unusually severe disease course observed in two generations.

BackgroundUpon SARS-CoV-2 infection, most individuals develop neutralizing antibodies and T-cell immunity. However, some individuals reportedly remain SARS-CoV-2 PCR positive by pharyngeal swabs weeks after recovery. Whether viral RNA in these persistent carriers is contagious and stimulates SARS-CoV-2-specific immune responses is unknown.MethodsThis cohort study was conducted between April 3rd–July 9th 2020, recruiting COVID-19 recovered individuals that were symptom-free for at least 14 days. We collected serum for SARS-CoV-2-specific total Ig, IgA and IgM detection by ELISA, pharyngeal swabs (two time points) for ddPCR and PBMCs for anti-SARS-CoV-2 CD8 T-cell dextramer analyses.FindingsWe enrolled 203 post-symptomatic participants with a previous RT-PCR-verified SARS-CoV-2 infection. At time point 1, a median of 23 days (range 15–44) after recovery, 26 individuals (12⋅8%) were PCR positive. At time point 2, 90 days (median, range 85–105) after recovery, 5 (5⋅3%) were positive. There was no difference in SARS-CoV-2 antibody levels between the PCR negative and positive group. The persistent PCR positive group however, had SARS-CoV-2-specific CD8 T-cell responses of significantly increased breadth and magnitude. Assisted contact tracing among persistent PCR positive individuals revealed zero new COVID-19 diagnoses among 757 close contacts.InterpretationPersistent pharyngeal SARS-CoV-2 PCR positivity in post-symptomatic individuals is associated with elevated cellular immune responses and thus, the viral RNA may represent replicating virus. However, transmission to close contacts was not observed indicating that persistent PCR positive individuals are not contagious at the post-symptomatic stage of the infection.

BACKGROUND:Infection with varicella zoster virus (VZV) may involve different central nervous system (CNS) manifestations, including meningitis, encephalitis, and vasculitis. In cases in which otherwise healthy individuals are affected, an inborn error of immunity may underlie increased susceptibility or severity of infection.METHODS:We collected a cohort of 17 adults who experienced VZV encephalitis and performed whole exome sequencing. Patient peripheral blood mononuclear cells were infected with VZV, and innate antiviral interferon (IFN) and cytokine responses as well as viral replication were evaluated. Data were analyzed by Mann-Whitney U test.RESULTS:We identified a total of 21 different potentially disease-causing variants in a total of 13 of the 17 patients included. These gene variants were within 2 major functional clusters: (1) innate viral sensors and immune pathways and (2) autophagy pathways. Antiviral IFN and cytokine responses were abnormal in the majority of patients, whereas viral replication was increased in only 2 of 17 patients.CONCLUSIONS:This study identifies a list of variants of pathogenic potential, which may serve as a platform for generating hypotheses for future studies addressing genetic and immunological factors associated with susceptibility to VZV encephalitis. These data, taken together, suggest that disturbances in innate sensing and autophagy pathways may predispose to VZV encephalitis.

Recurrent lymphocytic meningitis, also referred to as Mollaret meningitis, is a rare neurological disease characterized mainly by reactivation of herpes simplex virus 2 (HSV-2) from sensory ganglia. However, the underlying host immune determinants and viral factors rendering some individuals unable to maintain HSV-2 latency are largely unknown. We collected a cohort of 15 patients diagnosed with Mollaret meningitis. By whole-exome sequencing we identified rare host genetic variants predicted to be deleterious in molecules involved in (1) ubiquitin-proteasome pathways, (2) the autophagy machinery, and (3) cell proliferation/apoptosis. Moreover, infection of patient cells with HSV-2 or stimulation by virus-derived double-stranded DNA ligands revealed reduced antiviral interferon responses in most patients. These findings may contribute to a better understanding of disease pathogenesis and protective immunity to HSV in the central nervous system, and may ultimately be of importance for identification of targets for development of improved prophylaxis and treatment of this disease.

Recurrent herpesvirus infections can manifest in different forms of disease, including cold sores, genital herpes, and encephalitis. There is an incomplete understanding of the genetic and immunological factors conferring susceptibility to recurrent herpes simplex virus 2 (HSV2) infection in the central nervous system (CNS). Here, we describe two adult patients with recurrent HSV2 lymphocytic Mollaret’s meningitis that each carry a rare monoallelic variant in the autophagy proteins ATG4A or LC3B2. HSV2-activated autophagy was abrogated in patient primary fibroblasts, which also exhibited significantly increased viral replication and enhanced cell death. HSV2 antigen was captured in autophagosomes of infected cells, and genetic inhibition of autophagy by disruption of autophagy genes, including ATG4A and LC3B2 , led to enhanced viral replication and cell death in primary fibroblasts and a neuroblastoma cell line. Activation of autophagy by HSV2 was sensitive to ultraviolet (UV) irradiation of the virus and inhibited in the presence of acyclovir, but HSV2-induced autophagy was independent of the DNA-activated STING pathway. Reconstitution of wild-type ATG4A and LC3B2 expression using lentiviral gene delivery or electroporation of in vitro transcribed mRNA into patient cells restored virus-induced autophagy and the ability to control HSV2 replication. This study describes a previously unknown link between defective autophagy and an inborn error of immunity that can lead to increased susceptibility to HSV2 infection, suggesting an important role for autophagy in antiviral immunity in the CNS.

Coronavirus disease-19 (COVID-19) describes a set of symptoms that develop following infection by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Whilst COVID-19 disease is most serious in patients with significant co-morbidities, the reason for healthy individuals succumbing to fulminant infection is largely unexplained. In this review, we discuss the most recent findings in terms of clinical features and the host immune response, and suggest candidate immune pathways that may be compromised in otherwise healthy individuals with fulminating COVID-19. On the basis of this early knowledge we reason a potential genetic effect on host immune response pathways leading to increased susceptibility to SARS-CoV-2 infection. Understanding these pathways may help not only in unraveling disease pathogenesis, but also in suggesting targets for therapy and prophylaxis. Importantly such insight should instruct efforts to identify those at increased risk in order to institute preventative measures, such as prophylactic medication and/or vaccination, when such opportunities arise in the later phases of the current pandemic or during future similar pandemics.

Paralytic poliomyelitis is a rare disease manifestation following poliovirus (PV) infection. The disease determinants remain largely unknown. We used whole exome sequencing to uncover possible contributions of host genetics to the development of disease outcome in humans with poliomyelitis. We identified a patient with a variant inATG7, an important regulatory gene in the macroautophagy/autophagy pathway. PV infection did not induce a prominent type I interferon response, but rather activated autophagy in neuronal-like cells, and this was essential for viral control. Importantly, virus-induced autophagy was impaired in patient fibroblasts and associated with increased viral burden and enhanced cell death following infection. Lack of ATG7 prevented control of infection in neuronal-like cells, and reconstitution of patient cells with wild-type ATG7 reestablished autophagy-mediated control of infection. Collectively, these data suggest that ATG7 defect contributes to host susceptibility to PV infection and propose autophagy as an unappreciated antiviral effector in viral infection in humans.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Influenza virus is a common pathogen that mostly causes mild disease but in rare cases may progress to very severe and life-threatening illness. The pathogenesis of severe influenza in otherwise healthy individuals without known predisposing risk factors however, remains largely unknown 1. Influenza virus is predominantly recognized by the immune system through the cytosolic RNA sensor Retinoic acid-inducible gene I (RIG-I) in non-hematopoietic cells such as lung epithelial cells, and by Toll-like receptor (TLR)7 in hematopoietic cells 1. Common for these PRRs is the activation of interferon (IFN) regulatory factor (IRF)3 and IRF7, which are key transcription factors that cooperate in inducing type I (IFN-α/β) and type III (IFN-λ) IFNs in response to virus, which in turn stimulate the production of a whole range of different IFN stimulated genes (ISG)s with antiviral properties 2. The importance of type I and type III IFNs for the control of influenza virus has been highlighted in several murine models, demonstrating significantly increased susceptibility to influenza virus infection and elevated virus titers in mice lacking genes, such as Ifnb, Ifnar1, Stat1 or Ifnlr1 3-5. In addition, several genome-wide association studies have attempted to link host genetics to the susceptibility to severe influenza infection, but these studies have generally been underpowered and without any major findings 6. More recently, autosomal recessive defects in IRF7 and IRF9 were found to cause life-threatening influenza infection in two unrelated children, demonstrating the importance of IRFs in the immune response against influenza virus 7, 8. Here we describe a patient, P1, a 55-year old Caucasian male with severe influenza A virus (IAV) infection, who was heterozygous for a variant in the 3′UTR of the transcription factor IRF3. The variant (c.1576C>T) identified by whole exome sequencing is rare (frequency < 0.001 in gnomAD) and localized within the 3′UTR of IRF3 (Fig. 1A, B and C) in 7 out of 8 splice variants. One of these (IRF3-CL; NM_001197122.1) is placed in the protein sequence and gives rise to an amino acid substitution from proline to serine at position 447. See Supporting Information Table 1 for a complete list of other identified variants. By RT-qPCR we found IRF3 mRNA to be expressed at similar levels in cells from P1 and healthy controls (Fig. 1D). However, Western blotting on PBMC lysates revealed markedly decreased expression of IRF3 protein in P1 compared to controls (Fig. 1E and Supporting Information Fig. 1). These results indicate that the variant in the 3′UTR of IRF3 might affect the translation of IRF3 mRNA into protein, but not IRF3 mRNA stability. Therefore, we performed an in vitro translation analysis relying on HeLa cell lysates which, did however not demonstrate any difference in expression between IRF3 WT or the IRF3 c.1576T variant (Fig. 1F). Moreover, expression of the IRF3 3′UTR variant in HEK293T cells also did not reveal any significant difference in expression of IRF3 protein between the variant and IRF3 WT (data not shown). Next, we examined the induction of IFNs and inflammatory cytokines in peripheral blood mononuclear cells (PBMC)s from P1 and controls by RT-qPCR in response to infection with two different strains of IAV, IAV PR 8 and IAV pdm09, and herpes simplex virus type 1 (HSV-1) for comparison. Strikingly, we found that P1 exhibited impaired and almost abolished induction of IFNA2, IFNB and IFNL1 in response to infection with all three viruses (Fig. 2A–C). The induction of IL6 and TNFA as well as the ISG IFIT1 was selectively impaired in P1 when cells were infected with IAV pdm09, whereas infection of patient PBMCs with IAV PR8 and HSV-1 resulted in normal to increased responses compared to healthy controls (Fig. 2D, Supporting Information Fig. 2A,B)). In addition, PBMCs from P1 and controls were stimulated with different synthetic pathogen-associated molecular patterns (PAMPs). We found that cells from P1 exhibited impaired induction of IFNA2, IFNB, IFNL1, and IFIT1 in response to stimulation with the TLR3 agonist poly(I:C) and the TLR7/8 agonist R848, as well as decreased production of pro-inflammatory cytokines compared to healthy controls (Fig. 2E–H and Supporting Information Fig. 2C,D)). Surprisingly, when transfecting cells with poly(I:C) or dsDNA, agonists that stimulate RIG-I and dsDNA sensors, respectively, cells from P1 exhibited normal to slightly increased expression of IFNs and IFIT1, while production of pro-inflammatory cytokines was normal (Supporting Information Fig. 2E–J). This finding may be due to redundancy between different IRFs in certain cell types. Gene expression analysis of a broad panel of immune genes was performed on patient and healthy control PBMCs left untreated or infected with IAV pdm09 for 6 h using Nanostring technology. Gene expression patterns markedly differed between patient and controls, with the number of significantly differentially expressed genes in patient cells being much lower than those of controls (29 genes in patient samples versus 157 and 142 genes in each control, respectively) (Fig. 2I). Importantly, 89 genes, encoding both innate and adaptive immune mediators, were only significantly up- or down-regulated in controls (Fig. 2I,J). Most notably, expression of both IFNA2 and IFNB was almost completely abolished in patient cells, whereas IFNG expression was decreased to a lesser degree (Fig. 2J). A STRING analysis further revealed a large degree of interconnection and functional diversity of the upregulated genes (Supporting Information Fig. 3B). Finally, IRF7 failed to be upregulated upon IAV infection in P1 compared to controls (Supporting Information Fig. 3C). IRFs are key transcription factors that regulate the induction of IFNs 2. The first and only case of IRF3 deficiency so far was reported by Andersen et. al in a study from 2015, demonstrating a heterozygous missense variant in the regulatory domain of IRF3 causing decreased transcription factor activity and increased susceptibility to herpes simplex encephalitis (HSE) in an adolescent 9. Subsequently, IRF7- and IRF9 deficiencies were identified in two unrelated children with severe disseminated influenza infection, adding to the evidence suggesting an important role of IRFs and IFN in immunity against influenza virus infection 7, 8. More recently, a genetic variant in the cytosolic RNA sensor RIG-I has been identified in an adult patient presenting with severe disseminated influenza infection 10. Here, we report the identification of a variant of the transcription factor IRF3 together with impaired antiviral IFN responses in an adult patient who developed severe life-threatening influenza infection during the H1N1 swine flu pandemic in 2009–2010. We favor a scenario, in which the identified IRF3 3′UTR variant exerts an effect by haploinsufficiency rather than by a dominant negative effect, since the coding region of IRF3 is normal, wherefore any translated protein most likely would function and signal normally. The IRF3-CL transcript variant is believed to function as a negative regulator of the remaining IRF3 transcripts, and a missense variant herein is therefore unlikely to explain the infectious and immunological phenotype. The mechanism, whereby this variant causes less IRF3 protein expression and impaired antiviral responses, thus remains incompletely understood, given that IRF3 mRNA levels were found to be normal in patient PBMCs, and in vitro translation of the IRF3 variant was comparable to WT IRF3. However, the in vitro translation assay may not precisely reflect the physiological situation, since IRF3 protein expression was markedly reduced in cell lysates from patient PBMCs. In summary, the present report is in line with previous studies demonstrating a central role of this family of transcription factors in inducing potent and immediate IFN responses in antiviral immunity to IAV in humans. We would like to thank the patient for participation in the study. THM received funding from Aarhus University Research Foundation (AUFF-E-215-FLS-8-66), the Danish Council for Independent Research-Medical Sciences (# 4004-00047B), and The Lundbeck Foundation (R268-406 2016–3927). RH was funded by the Danish Council for Independent Research-Medical Sciences (7016-00331). The authors declare no financial or commercial conflict of interest. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Influenza infection is common worldwide with many individuals affected each year during epidemics and occasionally pandemics. Previous studies in animal models and a few human cases have established an important role of innate type I and III interferon (IFN) for viral elimination and mounting of antiviral responses. However, genetic and immunological determinants of very severe disseminated influenza virus infection in humans remain incompletely understood. Here, we describe an adult patient with severe influenza virus A (IAV) infection, in whom we identified a rare variant E331V in IFN regulatory factor (IRF)7 by whole-exome sequencing. Examination of patient cells demonstrated a cellular phenotype suggesting functional IRF7 impairment, since priming with IFN was almost abolished and IFN responses to IAV were significantly impaired in patient cells. Moreover, IAV replication was significantly higher in patient cells than in controls. Finally, expression of IRF7 E331V in HEK293 cells demonstrated significantly reduced activation of both IFNA7 and IFNB promoters in a luciferase reporter gene expression assay compared to IRF7 wild type. These findings provide further support for the essential role of IRF7 in amplifying antiviral IFN responses to ensure potent and sustained IFN responses during influenza virus infection in humans.

Deficiency of mitochondrial sulfur dioxygenase (ETHE1) causes the severe metabolic disorder ethylmalonic encephalopathy, which is characterized by early‐onset encephalopathy and defective cytochrome C oxidase because of hydrogen sulfide accumulation. Although the severe systemic consequences of the disorder are becoming clear, the molecular effects are not well defined. Therefore, for further elucidating the effects of ETHE1‐deficiency, we performed a large scale quantitative proteomics study on liver tissue from ETHE1‐deficient mice. Our results demonstrated a clear link between ETHE1‐deficiency and redox active proteins, as reflected by downregulation of several proteins related to oxidation‐reduction, such as different dehydrogenases and cytochrome P450 (CYP450) members. Furthermore, the protein data indicated impact of the ETHE1‐deficiency on metabolic reprogramming through upregulation of glycolytic enzymes and by altering several heterogeneous ribonucleoproteins, indicating novel link between ETHE1 and gene expression regulation. We also found increase in total protein acetylation level, pointing out the link between ETHE1 and acetylation, which is likely controlled by both redox state and cellular metabolites. These findings are relevant for understanding the complexity of the disease and may shed light on important functions influenced by ETHE1 deficiency and by the concomitant increase in the gaseous mediator hydrogen sulfide. All MS data have been deposited in the ProteomeXchange with the dataset identifiers PXD002741 ( http://proteomecentral.proteomexchange.org/dataset/PXD002741 ) and PXD002742 ( http://proteomecentral.proteomexchange.org/dataset/PXD002741 ).