An organoid-derived bronchioalveolar model for SARS-CoV-2 infection of human alveolar type II-like cells.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19), which may result in acute respiratory distress syndrome (ARDS), multiorgan failure, and death. The alveolar epithelium is a major target of the virus, but representative models to study virus host interactions in more detail are currently lacking. Here, we describe a human 2D air-liquid interface culture system which was characterized by confocal and electron microscopy and single-cell mRNA expression analysis. In this model, alveolar cells, but also basal cells and rare neuroendocrine cells, are grown from 3D self-renewing fetal lung bud tip organoids. These cultures were readily infected by SARS-CoV-2 with mainly surfactant protein C-positive alveolar type II-like cells being targeted. Consequently, significant viral titers were detected and mRNA expression analysis revealed induction of type I/III interferon response program. Treatment of these cultures with a low dose of interferon lambda 1 reduced viral replication. Hence, these cultures represent an experimental model for SARS-CoV-2 infection and can be applied for drug screens.

Herpes simplex virus infection induces necroptosis of neurons and astrocytes in human fetal organotypic brain slice cultures.

BACKGROUND: Herpes simplex virus (HSV) encephalitis (HSE) is a serious and potentially life-threatening disease, affecting both adults and newborns. Progress in understanding the virus and host factors involved in neonatal HSE has been hampered by the limitations of current brain models that do not fully recapitulate the tissue structure and cell composition of the developing human brain in health and disease. Here, we developed a human fetal organotypic brain slice culture (hfOBSC) model and determined its value in mimicking the HSE neuropathology in vitro. METHODS: Cell viability and tissues integrity were determined by lactate dehydrogenase release in supernatant and immunohistological (IHC) analyses. Brain slices were infected with green fluorescent protein (GFP-) expressing HSV-1 and HSV-2. Virus replication and spread were determined by confocal microscopy, PCR and virus culture. Expression of pro-inflammatory cytokines and chemokines were detected by PCR. Cell tropism and HSV-induced neuropathology were determined by IHC analysis. Finally, the in situ data of HSV-infected hfOBSC were compared to the neuropathology detected in human HSE brain sections. RESULTS: Slicing and serum-free culture conditions were optimized to maintain the viability and tissue architecture of ex vivo human fetal brain slices for at least 14 days at 37 °C in a CO2 incubator. The hfOBSC supported productive HSV-1 and HSV-2 infection, involving predominantly infection of neurons and astrocytes, leading to expression of pro-inflammatory cytokines and chemokines. Both viruses induced programmed cell death-especially necroptosis-in infected brain slices at later time points after infection. The virus spread, cell tropism and role of programmed cell death in HSV-induced cell death resembled the neuropathology of HSE. CONCLUSIONS: We developed a novel human brain culture model in which the viability of the major brain-resident cells-including neurons, microglia, astrocytes and oligodendrocytes-and the tissue architecture is maintained for at least 2 weeks in vitro under serum-free culture conditions. The close resemblance of cell tropism, spread and neurovirulence of HSV-1 and HSV-2 in the hfOBSC model with the neuropathological features of human HSE cases underscores its potential to detail the pathophysiology of other neurotropic viruses and as preclinical model to test novel therapeutic interventions.

Varicella zoster virus-induced autophagy in human neuronal and hematopoietic cells exerts antiviral activity.

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.

Varicella-zoster virus proteome-wide T-cell screening demonstrates low prevalence of virus-specific CD8 T-cells in latently infected human trigeminal ganglia.

BACKGROUND: Trigeminal ganglia (TG) neurons are an important site of lifelong latent varicella-zoster virus (VZV) infection. Although VZV-specific T-cells are considered pivotal to control virus reactivation, their protective role at the site of latency remains uncharacterized. METHODS: Paired blood and TG specimens were obtained from ten latent VZV-infected adults, of which nine were co-infected with herpes simplex virus type 1 (HSV-1). Short-term TG-derived T-cell lines (TG-TCL), generated by mitogenic stimulation of TG-derived T-cells, were probed for HSV-1- and VZV-specific T-cells using flow cytometry. We also performed VZV proteome-wide screening of TG-TCL to determine the fine antigenic specificity of VZV reactive T-cells. Finally, the relationship between T-cells and latent HSV-1 and VZV infections in TG was analyzed by reverse transcription quantitative PCR (RT-qPCR) and in situ analysis for T-cell proteins and latent viral transcripts. RESULTS: VZV proteome-wide analysis of ten TG-TCL identified two VZV antigens recognized by CD8 T-cells in two separate subjects. The first was an HSV-1/VZV cross-reactive CD8 T-cell epitope, whereas the second TG harbored CD8 T-cells reactive with VZV specifically and not the homologous peptide in HSV-1. In silico analysis showed that HSV-1/VZV cross reactivity of TG-derived CD8 T-cells reactive with ten previously identified HSV-1 epitopes was unlikely, suggesting that HSV-1/VZV cross-reactive T-cells are not a common feature in dually infected TG. Finally, no association was detected between T-cell infiltration and VZV latency transcript abundance in TG by RT-qPCR or in situ analyses. CONCLUSIONS: The low presence of VZV- compared to HSV-1-specific CD8 T-cells in human TG suggests that VZV reactive CD8 T-cells play a limited role in maintaining VZV latency.

Microglia Activate Early Antiviral Responses upon Herpes Simplex Virus 1 Entry into the Brain to Counteract Development of Encephalitis-Like Disease in Mice.

Spread of herpes simplex virus 1 (HSV1) from the periphery to the central nervous system (CNS) can lead to extensive infection and pathological inflammation in the brain, causing herpes simplex encephalitis (HSE). It has been shown that microglia, the CNS-resident macrophages, are involved in early sensing of HSV1 and induction of antiviral responses. In addition, infiltration of peripheral immune cells may contribute to the control of viral infection. In this study, we tested the effect of microglia depletion in a mouse model of HSE. Increased viral titers and increased disease severity were observed in microglia-depleted mice. The effect of microglia depletion was more pronounced in wild-type than in cGas-/- mice, revealing that this immune sensor contributes to the antiviral activity of microglia. Importantly, microglia depletion led to reduced production of type I interferon (IFN), proinflammatory cytokines, and chemokines at early time points after viral entry into the CNS. In line with this, in vitro experiments on murine primary CNS cells demonstrated microglial presence to be essential for IFN RNA induction, and control of HSV1 replication. However, the effect of microglia depletion on the expression of IFNs, and inflammatory cytokines was restricted to the early time point of HSV1 entry into the CNS. There was no major alteration of infiltration of CD45-positive cells in microglia-depleted mice. Collectively, our data demonstrate a key role for microglia in controlling HSV1 replication early after viral entry into the CNS and highlight the importance of a prompt antiviral innate response to reduce the risk of HSE development. IMPORTANCE One of the most devastating and acute neurological conditions is encephalitis, i.e., inflammation of brain tissue. Herpes simplex virus 1 (HSV1) is a highly prevalent pathogen in humans, and the most frequent cause of viral sporadic encephalitis called herpes simplex encephalitis (HSE). HSV1 can infect peripheral neurons and reach the central nervous system (CNS) of humans, where it can be detected by brain resident cells and infiltrating immune cells, leading to protective and damaging immune responses. In this study, we investigated the effects of microglia depletion, the main brain-resident immune cell type. For this purpose, we used a mouse model of HSE. We found that viral levels increased, and disease symptoms worsened in microglia-depleted mice. In addition, mice lacking a major sensor of viral DNA, cGAS, manifested a more pronounced disease than wild-type mice, highlighting the importance of this immune sensor in the activity of microglia. Microglia depletion led to reduced production of many known antiviral factors, most notably type I interferon (IFN). The importance of microglia in the early control of HSV1 spread and the generation of antiviral responses is further demonstrated by experiments on murine mixed glial cell cultures. Interestingly, mice with microglia depletion exhibited an unaltered activation of antiviral responses and recruitment of immune cells from the periphery at later time points of infection, but this did not prevent the development of the disease. Overall, the data highlight the importance of rapid activation of the host defense, with microglia playing a critical role in controlling HSV1 infection, which eventually prevents damage to neurons and brain tissue.

The architecture of the simian varicella virus transcriptome.

Primary infection with varicella-zoster virus (VZV) causes varicella and the establishment of lifelong latency in sensory ganglion neurons. In one-third of infected individuals VZV reactivates from latency to cause herpes zoster, often complicated by difficult-to-treat chronic pain. Experimental infection of non-human primates with simian varicella virus (SVV) recapitulates most features of human VZV disease, thereby providing the opportunity to study the pathogenesis of varicella and herpes zoster in vivo. However, compared to VZV, the transcriptome and the full coding potential of SVV remains incompletely understood. Here, we performed nanopore direct RNA sequencing to annotate the SVV transcriptome in lytically SVV-infected African green monkey (AGM) and rhesus macaque (RM) kidney epithelial cells. We refined structures of canonical SVV transcripts and uncovered numerous RNA isoforms, splicing events, fusion transcripts and non-coding RNAs, mostly unique to SVV. We verified the expression of canonical and newly identified SVV transcripts in vivo, using lung samples from acutely SVV-infected cynomolgus macaques. Expression of selected transcript isoforms, including those located in the unique left-end of the SVV genome, was confirmed by reverse transcription PCR. Finally, we performed detailed characterization of the SVV homologue of the VZV latency-associated transcript (VLT), located antisense to ORF61. Analogous to VZV VLT, SVV VLT is multiply spliced and numerous isoforms are generated using alternative transcription start sites and extensive splicing. Conversely, low level expression of a single spliced SVV VLT isoform defines in vivo latency. Notably, the genomic location of VLT core exons is highly conserved between SVV and VZV. This work thus highlights the complexity of lytic SVV gene expression and provides new insights into the molecular biology underlying lytic and latent SVV infection. The identification of the SVV VLT homolog further underlines the value of the SVV non-human primate model to develop new strategies for prevention of herpes zoster.

T-cells in human trigeminal ganglia express canonical tissue-resident memory T-cell markers.

BACKGROUND: Trigeminal ganglia (TG) neurons are the main site of lifelong latent herpes simplex virus type 1 (HSV-1) infection. T-cells in ganglia contribute to long-term control of latent HSV-1 infection, but it is unclear whether these cells are bona fide tissue-resident memory T-cells (TRM). We optimized the processing of human post-mortem nervous tissue to accurately phenotype T-cells in human TG ex vivo and in situ. METHODS: Peripheral blood mononuclear cells (PBMC; 5 blood donors) were incubated with several commercial tissue digestion enzyme preparations to determine off-target effect on simultaneous detection of 15 specific T-cell subset markers by flow cytometry. Next, optimized enzymatic digestion was applied to ex vivo phenotype T-cells in paired PBMC, normal appearing white matter (NAWM) and TG of 8 deceased brain donors obtained < 9 h post-mortem by flow cytometry. Finally, the phenotypic and functional markers, and spatial orientation of T-cells in relation to neuronal somata, were determined in TG tissue sections of five HSV-1-latently infected individuals by multiparametric in situ analysis. RESULTS: Collagenase IV digestion of human nervous tissue was most optimal to obtain high numbers of viable T-cells without disrupting marker surface expression. Compared to blood, majority T-cells in paired NAWM and TG were effector memory T-cells expressing the canonical TRM markers CD69, CXCR6 and the immune checkpoint marker PD1, and about half co-expressed CD103. A trend of relatively higher TRM frequencies were detected in TG of latently HSV-1-infected compared to HSV-1 naïve individuals. Subsequent in situ analysis of latently HSV-1-infected TG showed the presence of cytotoxic T-cells (TIA-1+), which occasionally showed features of proliferation (KI-67+) and activation (CD137+), but without signs of degranulation (CD107a+) nor damage (TUNEL+) of TG cells. Whereas majority T-cells expressed PD-1, traits of T-cell senescence (p16INK4a+) were not detected. CONCLUSIONS: The human TG represents an immunocompetent environment in which both CD4 and CD8 TRM are established and retained. Based on our study insights, we advocate for TRM-targeted vaccine strategies to bolster local HSV-1-specific T-cell immunity, not only at the site of recurrent infection but also at the site of HSV-1 latency.

No Evidence of Varicella-Zoster Virus Infection in Temporal Artery Biopsies of Anterior Ischemic Optic Neuropathy Patients With and Without Giant Cell Arteritis.

BACKGROUND: To test the hypothesis that varicella-zoster virus (VZV) infection contributes to temporal arteritis pathogenesis, comprehensive in situ analysis was performed on temporal artery biopsies of 38 anterior ischemic optic neuropathy (AION) patients, including 14 (37%) with giant cell arteritis. METHODS: Biopsies were completely sectioned, and, on average, 146 serial sections per patient were stained for VZV glycoprotein E. RESULTS: Four of 38 AION patients showed VZV glycoprotein E staining, but VZV infection was not confirmed by staining for VZV IE63 protein and VZV-specific polymerase chain reaction on adjacent sections. CONCLUSIONS: This study refutes the premise that VZV is casually related to AION with and without giant cell arteritis.

High Levels of Neutrophil Extracellular Traps Persist in the Lower Respiratory Tract of Critically Ill Patients With Coronavirus Disease 2019.

Lower respiratory tract (LRT) disease induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can deteriorate to acute respiratory distress syndrome (ARDS). Because the release of neutrophil extracellular traps (NETs) is implicated in ARDS pathogenesis, we investigated the presence of NETs and correlates of pathogenesis in blood and LRT samples of critically ill patients with COVID-19. Plasma NET levels peaked early after intensive care unit admission and were correlated with the SARS-CoV-2 RNA load in sputum and levels of neutrophil-recruiting chemokines and inflammatory markers in plasma samples. The baseline plasma NET quantity was correlated with disease severity but was not associated with soluble markers of thrombosis or with development of thrombosis. High NET levels were present in LRT samples and persisted during the course of COVID-19, consistent with the detection of NETs in bronchi and alveolar spaces in lung tissue from deceased patient with COVID-19. Thus, NETs are produced and retained in the LRT of critically ill patients with COVID-19 and could contribute to SARS-CoV-2-induced ARDS disease.

Ileocolic Intussusception as the Presenting Symptom of Primary Enteric Varicella-Zoster Virus Infection in a 7-Month-Old Infant.

Ileocolic intussusception is the invagination of ileum into the colon. In a subset of patients, the disease is caused by mesenteric lymphadenopathy in response to (viral) infection. We present a case of an ileocolic intussusception necessitating surgery in a 7-month-old immunocompetent infant with concurrent primary wild-type varicella-zoster virus (VZV) infection, in whom chickenpox rash developed 2 days after surgery. Detailed in situ analyses of resected intestine for specific cell type markers and VZV RNA demonstrated VZV-infected lymphocytes and neurons in the gut wall and in ganglion cells of the myenteric plexus.

Large, Stable, Contemporary Interspecies Recombination Events in Circulating Human Herpes Simplex Viruses.

BACKGROUND: The ubiquitous human pathogens, herpes simplex virus (HSV)-1 and HSV-2, are distinct viral species that diverged approximately 6 million years ago. At least 4 small, ancient HSV-1 × HSV-2 interspecies recombination events have affected the HSV-2 genome, with recombinants and nonrecombinants at each locus circulating today. However, it is unknown whether interspecies recombination can affect other loci and whether new recombinants continue to be generated. METHODS: Using 255 newly sequenced and 230 existing HSV genome sequences, we comprehensively assessed interspecies recombination in HSV. RESULTS: Our findings show that the sizes and locations of interspecies recombination events in HSV-2 are significantly more variable than previously appreciated and that they can impact species-specific T-cell recognition of HSV. CONCLUSIONS: We describe 2 large (>5 kb) recombination events, one of which arose in its current host, demonstrating that interspecies recombination continues to occur today. These results raise concerns about the use of live-attenuated HSV-2 vaccines in high HSV-1 prevalence areas.

Attenuation of Simian Varicella Virus Infection by Enhanced Green Fluorescent Protein in Rhesus Macaques.

Simian varicella virus (SVV), the primate counterpart of varicella-zoster virus, causes varicella (chickenpox), establishes latency in ganglia, and reactivates to produce zoster. We previously demonstrated that a recombinant SVV expressing enhanced green fluorescent protein (rSVV.eGFP) is slightly attenuated both in culture and in infected monkeys. Here, we generated two additional recombinant SVVs to visualize infected cells in vitro and in vivo One harbors eGFP fused to the N terminus of open reading frame 9 (ORF9) (rSVV.eGFP-2a-ORF9), and another harbors eGFP fused to the C terminus of ORF66 (rSVV.eGFP-ORF66). Both recombinant viruses efficiently expressed eGFP in cultured cells. Both recombinant SVV infections in culture were comparable to that of wild-type SVV (SVV.wt). Unlike SVV.wt, eGFP-tagged SVV did not replicate in rhesus cells in culture. Intratracheal (i.t.) or i.t. plus intravenous (i.v.) inoculation of rhesus macaques with these new eGFP-tagged viruses resulted in low viremia without varicella rash, although SVV DNA was abundant in bronchoalveolar lavage (BAL) fluid at 10 days postinoculation (dpi). SVV DNA was also found in trigeminal ganglia of one monkey inoculated with rSVV.eGFP-ORF66. Intriguingly, a humoral response to both SVV and eGFP was observed. In addition, monkeys inoculated with the eGFP-expressing viruses were protected from superinfection with SVV.wt, suggesting that the monkeys had mounted an efficient immune response. Together, our results show that eGFP expression could be responsible for their reduced pathogenesis.IMPORTANCE SVV infection in nonhuman primates has served as an extremely useful animal model to study varicella-zoster virus (VZV) pathogenesis. eGFP-tagged viruses are a great tool to investigate their pathogenesis. We constructed and tested two new recombinant SVVs with eGFP inserted into two different locations in the SVV genome. Both recombinant SVVs showed robust replication in culture but reduced viremia compared to that with SVV.wt during primary infection in rhesus macaques. Our results indicate that conclusions on eGFP-tagged viruses based on in vitro results should be handled with care, since eGFP expression could result in attenuation of the virus.

Varicella zoster virus glycoprotein C increases chemokine-mediated leukocyte migration.

Varicella zoster virus (VZV) is a highly prevalent human pathogen that establishes latency in neurons of the peripheral nervous system. Primary infection causes varicella whereas reactivation results in zoster, which is often followed by chronic pain in adults. Following infection of epithelial cells in the respiratory tract, VZV spreads within the host by hijacking leukocytes, including T cells, in the tonsils and other regional lymph nodes, and modifying their activity. In spite of its importance in pathogenesis, the mechanism of dissemination remains poorly understood. Here we addressed the influence of VZV on leukocyte migration and found that the purified recombinant soluble ectodomain of VZV glycoprotein C (rSgC) binds chemokines with high affinity. Functional experiments show that VZV rSgC potentiates chemokine activity, enhancing the migration of monocyte and T cell lines and, most importantly, human tonsillar leukocytes at low chemokine concentrations. Binding and potentiation of chemokine activity occurs through the C-terminal part of gC ectodomain, containing predicted immunoglobulin-like domains. The mechanism of action of VZV rSgC requires interaction with the chemokine and signalling through the chemokine receptor. Finally, we show that VZV viral particles enhance chemokine-dependent T cell migration and that gC is partially required for this activity. We propose that VZV gC activity facilitates the recruitment and subsequent infection of leukocytes and thereby enhances VZV systemic dissemination in humans.

T helper 17.1 cells associate with multiple sclerosis disease activity: perspectives for early intervention.

Interleukin-17-expressing CD4+ T helper 17 (Th17) cells are considered as critical regulators of multiple sclerosis disease activity. However, depending on the species and pro-inflammatory milieu, Th17 cells are functionally heterogeneous, consisting of subpopulations that differentially produce interleukin-17, interferon-gamma and granulocyte macrophage colony-stimulating factor. In the current study, we studied distinct effector phenotypes of human Th17 cells and their correlation with disease activity in multiple sclerosis patients. T helper memory populations single- and double-positive for C-C chemokine receptor 6 (CCR6) and CXC chemokine receptor 3 (CXCR3) were functionally assessed in blood and/or cerebrospinal fluid from a total of 59 patients with clinically isolated syndrome, 35 untreated patients and 24 natalizumab-treated patients with relapsing-remitting multiple sclerosis, and nine patients with end-stage multiple sclerosis. Within the clinically isolated syndrome group, 23 patients had a second attack within 1 year and 26 patients did not experience subsequent attacks during a follow-up of >5 years. Low frequencies of T helper 1 (Th1)-like Th17 (CCR6+CXCR3+), and not Th17 (CCR6+CXCR3-) effector memory populations in blood strongly associated with a rapid diagnosis of clinically definite multiple sclerosis. In cerebrospinal fluid of clinically isolated syndrome and relapsing-remitting multiple sclerosis patients, Th1-like Th17 effector memory cells were abundant and showed increased production of interferon-gamma and granulocyte macrophage colony-stimulating factor compared to paired CCR6+ and CCR6-CD8+ T cell populations and their blood equivalents after short-term culturing. Their local enrichment was confirmed ex vivo using cerebrospinal fluid and brain single-cell suspensions. Across all pro-inflammatory T helper cells analysed in relapsing-remitting multiple sclerosis blood, Th1-like Th17 subpopulation T helper 17.1 (Th17.1; CCR6+CXCR3+CCR4-) expressed the highest very late antigen-4 levels and selectively accumulated in natalizumab-treated patients who remained free of clinical relapses. This was not found in patients who experienced relapses during natalizumab treatment. The enhanced potential of Th17.1 cells to infiltrate the central nervous system was supported by their predominance in cerebrospinal fluid of early multiple sclerosis patients and their preferential transmigration across human brain endothelial layers. These findings reveal a dominant contribution of Th1-like Th17 subpopulations, in particular Th17.1 cells, to clinical disease activity and provide a strong rationale for more specific and earlier use of T cell-targeted therapy in multiple sclerosis.

Immunobiology of Varicella-Zoster Virus Infection.

Varicella-zoster virus (VZV) causes clinically significant illness during acute and recurrent infection accompanied by robust innate and acquired immune responses. Innate immune cells in skin and ganglion secrete type I interferon (IFN-I) and proinflammatory cytokines to control VZV. Varicella-zoster virus subverts pattern recognition receptor sensing to modulate antigen presentation and IFN-I production. During primary infection, VZV hijacks T cells to disseminate to the skin and establishes latency in ganglia. Durable T- and B-cell memory formed within a few weeks of infection is boosted by reactivation or re-exposure. Antigen-specific T cells are recruited and potentially retained in VZV-infected skin to counteract reactivation. In latently VZV-infected ganglia, however, virus-specific T cells have not been recovered, suggesting that local innate immune responses control VZV latency. Antibodies prevent primary VZV infection, whereas T cells are fundamental to resolving disease, limiting severity, and preventing reactivation. In this study, we review current knowledge on the interactions between VZV and the human immune system.

Satellite glial cells in human trigeminal ganglia have a broad expression of functional Toll-like receptors.

Toll-like receptors (TLRs) orchestrate immune responses to a wide variety of danger- and pathogen-associated molecular patterns. Compared to the central nervous system (CNS), expression profile and function of TLRs in the human peripheral nervous system (PNS) are ill-defined. We analyzed TLR expression of satellite glial cells (SGCs) and microglia, glial cells predominantly involved in local immune responses in ganglia of the human PNS and normal-appearing white matter (NAWM) of the CNS, respectively. Ex vivo flow cytometry analysis of cell suspensions obtained from human cadaveric trigeminal ganglia (TG) and NAWM showed that both SGCs and microglia expressed TLR1-5, TLR7, and TLR9, although expression levels varied between these cell types. Immunohistochemistry confirmed expression of TLR1-TLR4 and TLR9 by SGCs in situ. Stimulation of TG- and NAWM-derived cell suspensions with ligands of TLR1-TLR6, but not TLR7 and TLR9, induced interleukin 6 (IL-6) secretion. We identified CD45LOW CD14POS SGCs and microglia, but not CD45HIGH leukocytes and CD45NEG cells as the main source of IL-6 and TNF-α upon stimulation with TLR3 and TLR5 ligands. In conclusion, human TG-resident SGCs express a broad panel of functional TLRs, suggesting their role in initiating and orchestrating inflammation to pathogens in human sensory ganglia.

Whole-Genome Next-Generation Sequencing to Study Within-Host Evolution of Norovirus (NoV) Among Immunocompromised Patients With Chronic NoV Infection.

Background: The genus Norovirus comprises large genetic diversity, and new GII.4 variants emerge every 2-3 years. It is unknown in which host these new variants originate. Here we study whether prolonged shedders within the immunocompromised population could be a reservoir for newly emerging strains. Methods: Sixty-five fecal samples from 16 immunocompromised patients were retrospectively selected. Isolated viral RNA was enriched by hybridization with a custom norovirus whole-genome RNA bait set and deep sequenced on the Illumina MiSeq platform. Results: Patients shed virus for average 352 days (range, 76-716 days). Phylogenetic analysis showed distinct GII.4 variants in 3 of 13 patients (23%). The viral mutation rates were variable between patients but did not differ between various immune status groups. All within-host GII.4 viral populations showed amino acid changes at blocking epitopes over time, and the majority of VP1 amino acid mutations were located at the capsid surface. Conclusions: This study found viruses in immunocompromised hosts that are genetically distinct from viruses circulating in the general population, and these patients therefore may contain a reservoir for newly emerging strains. Future studies need to determine whether these new strains are of risk to other immunocompromised patients and the general population.

Chlamydia trachomatis Biovar L2 Infection in Women in South Africa.

We detected Chlamydia trachomatis biovar L2 in vaginal swab specimens of 7 women with vaginal discharge in South Africa. Whole-genome sequencing directly from clinical specimens identified a closely related cluster of strains. The clinical role of this infection in the context of syndromic management should be clarified.

Norovirus Infection in Harbor Porpoises.

A norovirus was detected in harbor porpoises, a previously unknown host for norovirus. This norovirus had low similarity to any known norovirus. Viral RNA was detected primarily in intestinal tissue, and specific serum antibodies were detected in 8 (24%) of 34 harbor porpoises from the North Sea.