Intrinsic Innate Immunity Fails To Control Herpes Simplex Virus and Vesicular Stomatitis Virus Replication in Sensory Neurons and Fibroblasts

Herpes simplex virus 1 (HSV-1) establishes lifelong latent infections in the sensory neurons of the trigeminal ganglia (TG), wherein it retains the capacity to reactivate. The interferon (IFN)-driven antiviral response is critical for the control of HSV-1 acute replication. We therefore sought to further investigate this response in TG neurons cultured from adult mice deficient in a variety of IFN signaling components. Parallel experiments were also performed in fibroblasts isolated concurrently. We showed that HSV-1 replication was comparable in wild-type (WT) and IFN signaling-deficient neurons and fibroblasts. Unexpectedly, a similar pattern was observed for the IFN-sensitive vesicular stomatitis virus (VSV). Despite these findings, TG neurons responded to IFN-β pretreatment with STAT1 nuclear localization and restricted replication of both VSV and an HSV-1 strain deficient in γ34.5, while wild-type HSV-1 replication was unaffected. This was in contrast to fibroblasts in which all viruses were restricted by the addition of IFN-β. Taken together, these data show that adult TG neurons can mount an effective antiviral response only if provided with an exogenous source of IFN-β, and HSV-1 combats this response through γ34.5. These results further our understanding of the antiviral response of neurons and highlight the importance of paracrine IFN-β signaling in establishing an antiviral state

Viral adaptation to immune selection pressure by HLA class I–restricted CTL responses targeting epitopes in HIV frameshift sequences

CD8+ cytotoxic T lymphocyte (CTL)–mediated immune responses to HIV contribute to viral control in vivo. Epitopes encoded by alternative reading frame (ARF) peptides may be targeted by CTLs as well, but their frequency and in vivo relevance are unknown. Using host genetic (human leukocyte antigen [HLA]) and plasma viral sequence information from 765 HIV-infected subjects, we identified 64 statistically significant (q < 0.2) associations between specific HLA alleles and sequence polymorphisms in alternate reading frames of gag, pol, and nef that did not affect the regular frame protein sequence. Peptides spanning the top 20 HLA-associated imprints were used to test for ex vivo immune responses in 85 HIV-infected subjects and showed responses to 10 of these ARF peptides. The most frequent response recognized an HLA-A*03–restricted +2 frame–encoded epitope containing a unique A*03-associated polymorphism at position 6. Epitope-specific CTLs efficiently inhibited viral replication in vitro when viruses containing the wild-type sequence but not the observed polymorphism were tested. Mutating alternative internal start codons abrogated the CTL-mediated inhibition of viral replication. These data indicate that responses to ARF-encoded HIV epitopes are induced during natural infection, can contribute to viral control in vivo, and drive viral evolution on a population level

Overview of progress in European medium sized tokamaks towards an integrated plasma-edge/wall solution

Integrating the plasma core performance with an edge and scrape-off layer (SOL) that leads to tolerable heat and particle loads on the wall is a major challenge. The new European medium size tokamak task force (EU-MST) coordinates research on ASDEX Upgrade (AUG), MAST and TCV. This multi-machine approach within EU-MST, covering a wide parameter range, is instrumental to progress in the field, as ITER and DEMO core/pedestal and SOL parameters are not achievable simultaneously in present day devices. A two prong approach is adopted. On the one hand, scenarios with tolerable transient heat and particle loads, including active edge localised mode (ELM) control are developed. On the other hand, divertor solutions including advanced magnetic configurations are studied. Considerable progress has been made on both approaches, in particular in the fields of: ELM control with resonant magnetic perturbations (RMP), small ELM regimes, detachment onset and control, as well as filamentary scrape-off-layer transport. For example full ELM suppression has now been achieved on AUG at low collisionality with n  =  2 RMP maintaining good confinement ${{H}_{\text{H}\left(98,\text{y}2\right)}}\approx 0.95$ . Advances have been made with respect to detachment onset and control. Studies in advanced divertor configurations (Snowflake, Super-X and X-point target divertor) shed new light on SOL physics. Cross field filamentary transport has been characterised in a wide parameter regime on AUG, MAST and TCV progressing the theoretical and experimental understanding crucial for predicting first wall loads in ITER and DEMO. Conditions in the SOL also play a crucial role for ELM stability and access to small ELM regimes

Real-time plasma state monitoring and supervisory control on TCV

In ITER and DEMO, various control objectives related to plasma control must be simultaneously achieved by the plasma control system (PCS), in both normal operation as well as off-normal conditions. The PCS must act on off-normal events and deviations from the target scenario, since certain sequences (chains) of events can precede disruptions. It is important that these decisions are made while maintaining a coherent prioritization between the real-time control tasks to ensure high-performance operation. In this paper, a generic architecture for task-based integrated plasma control is proposed. The architecture is characterized by the separation of state estimation, event detection, decisions and task execution among different algorithms, with standardized signal interfaces. Central to the architecture are a plasma state monitor and supervisory controller. In the plasma state monitor, discrete events in the continuous-valued plasma state are modeled using finite state machines. This provides a high-level representation of the plasma state. The supervisory controller coordinates the execution of multiple plasma control tasks by assigning task priorities, based on the finite states of the plasma and the pulse schedule. These algorithms were implemented on the TCV digital control system and integrated with actuator resource management and existing state estimation algorithms and controllers. The plasma state monitor on TCV can track a multitude of plasma events, related to plasma current, rotating and locked neoclassical tearing modes, and position displacements. In TCV experiments on simultaneous control of plasma pressure, safety factor profile and NTMs using electron cyclotron heating (ECH) and current drive (ECCD), the supervisory controller assigns priorities to the relevant control tasks. The tasks are then executed by feedback controllers and actuator allocation management. This work forms a significant step forward in the ongoing integration of control capabilities in experiments on TCV, in support of tokamak reactor operation

Neuronal Interferon Signaling Is Required for Protection against Herpes Simplex Virus Replication and Pathogenesis

<div><p>Interferon (IFN) responses are critical for controlling herpes simplex virus 1 (HSV-1). The importance of neuronal IFN signaling in controlling acute and latent HSV-1 infection remains unclear. Compartmentalized neuron cultures revealed that mature sensory neurons respond to IFNβ at both the axon and cell body through distinct mechanisms, resulting in control of HSV-1. Mice specifically lacking neural IFN signaling succumbed rapidly to HSV-1 corneal infection, demonstrating that IFN responses of the immune system and non-neuronal tissues are insufficient to confer survival following virus challenge. Furthermore, neurovirulence was restored to an HSV strain lacking the IFN-modulating gene, γ34.5, despite its expected attenuation in peripheral tissues. These studies define a crucial role for neuronal IFN signaling for protection against HSV-1 pathogenesis and replication, and they provide a novel framework to enhance our understanding of the interface between host innate immunity and neurotropic pathogens.</p></div

Neural STAT1 expression is required for host survival.

<p>Survival of Stat1^N-/- or Stat1^fl/fl mice infected via the cornea with 2 x 10⁶ PFU/eye strain 17 (st17) or Δγ34.5 virus. Mice were monitored over time and were euthanized upon reaching end-point criteria, here referred to as survival. Data represent two independent experiments where Stat1^fl/fl WT n = 14 mice, Stat1^N-/- WT n = 12, Stat1^fl/fl Δγ34.5 n = 13 and Stat1^N-/- Δγ34.5 n = 12 mice. Survival was plotted on a Kaplan-Meier curve and statistics performed with Log-rank test where ** p<0.01, ***p<0.001.</p

Neural STAT1 expression is critical for controlling HSV-1 replication <i>in vivo</i>.

<p>Viral titers in the TG, brain stem and brain of Stat1^N-/- or Stat1^fl/fl mice infected via corneal scarification with 2 x 10⁶ PFU/eye WT (strain 17) or Δγ34.5 virus. Viral titers were measured at 3 dpi (A) and 5 dpi (B). Dashed lines represent the limit of detection. Error bars represent SEM of a minimum of 13 mice total over 2 experiments. Two-way ANOVA was performed where ** p<0.01, ***p<0.001.</p

Neural deletion of STAT1 affects viral tropism in the trigeminal ganglia.

<p>A) Immunofluorescence of TG sections from Stat1^N-/- or Stat1^fl/fl mice 5 days post infection with 2 x 10⁶ PFU/eye WT (strain 17) or Δγ34.5 virus via the cornea. Tissue sections show immunostaining for HSV antigen (red), the neuronal marker NeuN (green), and nuclei (DAPI, blue). The ophthalmic branch of the TG is indicated and white dotted lines mark the trigeminal root entry zone (TREZ) delineating the boundary between PNS and CNS. Scale bar = 500 μm. B) Quantification of the percentage of total cells that are HSV antigen-positive. C) Quantification of the percent of total neurons that are HSV antigen-positive. D) Quantification of the percent of HSV antigen-positive cells that are neurons. Quantification was done using ImageJ/Fuji. Individual data points represent the average of >5 tissue slices per TG with a minimum of 7 TGs over 3 experiments. Student’s t-test was performed where *p<0.05, ** p<0.01, ***p<0.001.</p

Axonal IFNβ signaling restricts HSV-1 titers through mechanisms independent of establishment of an antiviral state at the soma.

<p>A) Titers of WT (strain 17) or Δγ34.5 viruses in the soma compartment of 129SVEV neuron cultures 72 hours post infection via the soma. Cultures were untreated (white bars) or treated with 12.5U/mL IFNβ in the soma (grey bars) or axon compartment (black bars) 18 hours prior to infection. B) Titers of VSV in the soma compartment 24 hours post infection via the soma of 129SVEV neuron cultures. Cultures were treated with IFNβ as in (A). C) Representative image of immunofluorescence staining for STAT1 (red) in chambers treated with IFNβ at the soma for 3 hours (left) or axon for 3, 5, 7 or 16 hours (right). Cells were counterstained for nuclei (DAPI, blue). DiO (green) was added to the axon compartment, thereby labeling neurons that extended axons through the central barrier. Scale bar = 50μm. D) Expression of IFIT1 and ISG15 transcripts in the soma compartment of chambers treated with IFNβ for 6 or 12 hours at the soma or axon. Error bars represent SEM of a minimum of 4 samples over 2 experiments. E) HSV genome copy number of WT (strain 17) or Δγ34.5 virus measured from the soma compartment of axonally infected 129SVEV neuron cultures at 1hpi and 3hpi. Cultures were treated with 100μM ACV, and with 12.5 U/mL IFNβ in the axon compartment for 18 hours, or 10μM capsaicin (red bar) in both compartments for 0.5 hour prior to infection with 10⁸ PFU. Error bars represent SEM of a minimum of 3 repeats with >3 chambers each. Two-way ANOVA was performed where *p<0.05, ** p<0.01, ***p<0.001.</p

Paracrine IFNβ signaling at the cell body and distal axon reduces HSV-1 titers upon axonal infection.

<p>A) Diagram of Campenot chambers that were modified by removing one of two central barriers. Trigeminal ganglia (TG) neurons from adult mice were isolated and seeded in soma compartment. Micrograph shows βIII-tubulin staining of neurons extending axons through a single barrier guided by grooves to the axonal compartment. Scale bar = 250μm. B) Diagram depicting the compartment where virus was added (indicated by a red virion), and where sampling occurred (blue background). Titers of WT (strain 17) or Δγ34.5 viruses in the soma compartment 72 hours post-axonal infection with 10⁸ PFU in 129SVEV and STAT1^-/- neurons. Cultures were untreated or treated with 12.5U/mL IFNβ in the soma or axon compartment. Dashed line represents the limit of detection. Error bars represent SEM of a minimum of 3 experiments with >2 chambers each. Significance was evaluated by two-way ANOVA where ** p<0.01, ***p<0.001.</p