The insular cortex (IC) plays key roles in emotional and regulatory brain functions and is affected across psychiatric diseases. However, the brain-wide connections of the mouse IC have not been comprehensively mapped. Here we traced the whole-brain inputs and outputs of the mouse IC across its rostro-caudal extent. We employed cell-type specific monosynaptic rabies virus tracings to characterize afferent connections onto either excitatory or inhibitory IC neurons, and adeno-associated viral tracings to label excitatory efferent axons. While the connectivity between the IC and other cortical regions was highly bidirectional, the IC connectivity with subcortical structures was often unidirectional, revealing prominent cortical-to-subcortical or subcortical-to-cortical pathways. The posterior and medial IC exhibited resembling connectivity patterns, while the anterior IC connectivity was distinct, suggesting two major functional compartments. Our results provide insights into the anatomical architecture of the mouse IC and thus a structural basis to guide investigations into its complex functions.

1The insular cortex (IC) plays key roles in emotional and regulatory brain functions and is 2 affected across psychiatric diseases. However, the brain-wide connections of the mouse IC have 3 not been comprehensively mapped. Here we traced the whole-brain inputs and outputs of the 4 mouse IC across its rostro-caudal extent. We employed cell-type specific monosynaptic rabies 5 virus tracings to characterize afferent connections onto either excitatory or inhibitory IC neurons, 6 and adeno-associated viral tracings to label excitatory efferent axons. While the connectivity 7 between the IC and other cortical regions was highly reciprocal, the IC connectivity with 8 subcortical structures was often unidirectional, revealing prominent top-down and bottom-up 9pathways. The posterior and medial IC exhibited resembling connectivity patterns, while the 10 anterior IC connectivity was distinct, suggesting two major functional compartments. Our results 11 provide insights into the anatomical architecture of the mouse IC and thus a structural basis to 12 guide investigations into its complex functions. 13 50 subdivisions for the two major neuronal subclasses that is excitatory pyramidal neurons and 51 inhibitory interneurons. 52 Results 53 4 Viral tracing approach to reveal the input-output connectivity of the mouse 54 IC 55To map the connectivity of the entire mouse IC, we injected viral tracers into three evenly spaced 56 locations along the rostro-caudal axis with the aim of comprehensively tracing from its entire 57 extent and to assess possible parcellation of the mouse IC into connectivity-based subdomains. 58The most anterior region, aIC ranged from +2.45 mm to +1.20 mm from Bregma; the medial 59 part, mIC, from +1.20 mm to +0.01 mm from Bregma, and the posterior part, pIC, from +0.01 60 mm to -1.22 mm from Bregma (see also Fig. 1c). 61 In order to trace the monosynaptic inputs to the IC we utilized a modified SAD∆G-eGFP(EnvA) 62 rabies virus (RV), which has been shown to label monosynaptic inputs to selected starter cells 63 with high specificity (Wall, Wickersham, Cetin, De La Parra, & Callaway, 2010; Wickersham, 64 Finke, Conzelmann, & Callaway, 2007). This virus lacks the genes coding for the rabies virus 65 glycoprotein (G) and is pseudotyped with the avian viral envelope EnvA. This restricts its 66 infection to neurons expressing the avian TVA receptor and to monosynaptic retrograde infection 67of afferents ( Fig. 1a). We infected the IC of CamKIIα-Cre and GAD2-Cre expressing mouse 68 lines to specifically target TVA and rabies virus glycoprotein expression to excitatory pyramidal 69 or inhibitory interneurons, respectively (see Fig. 1a and Methods). 70 In order to trace and quantify the axonal projections (outputs) of the IC, we injected Cre-71 dependent adeno-associated virus (AAV2/5-DIO-eYFP) into CamKIIα-Cre and GAD2-Cre 72 transgenic mice (see Fig 1b and Methods). We did not observe long-range projections from IC 73 GAD2-Cre tracings (data not shown). Therefore, we here only present outputs from exc...

Vaccines of outstanding efficiency, safety, and public acceptance are needed to halt the current SARS-CoV-2 pandemic. Concerns include potential side effects caused by the antigen itself and safety of viral DNA and RNA delivery vectors. The large SARS-CoV-2 spike (S) protein is the main target of current COVID-19 vaccine candidates but can induce non-neutralizing antibodies, which might cause vaccination-induced complications or enhancement of COVID-19 disease. Besides, encoding of a functional S in replication-competent virus vector vaccines may result in the emergence of viruses with altered or expanded tropism. Here, we have developed a safe single round rhabdovirus replicon vaccine platform for enhanced presentation of the S receptor-binding domain (RBD). Structure-guided design was employed to build a chimeric minispike comprising the globular RBD linked to a transmembrane stem-anchor sequence derived from rabies virus (RABV) glycoprotein (G). Vesicular stomatitis virus (VSV) and RABV replicons encoding the minispike not only allowed expression of the antigen at the cell surface but also incorporation into the envelope of secreted non-infectious particles, thus combining classic vector-driven antigen expression and particulate virus-like particle (VLP) presentation. A single dose of a prototype replicon vaccine complemented with VSV G, VSVΔG-minispike-eGFP (G), stimulated high titers of SARS-CoV-2 neutralizing antibodies in mice, equivalent to those found in COVID-19 patients, and protected transgenic K18-hACE2 mice from COVID-19-like disease. Homologous boost immunization further enhanced virus neutralizing activity. The results demonstrate that non-spreading rhabdovirus RNA replicons expressing minispike proteins represent effective and safe alternatives to vaccination approaches using replication-competent viruses and/or the entire S antigen.

CD4+ T cells are central mediators of adaptive and innate immune responses and constitute a major reservoir for human immunodeficiency virus (HIV) in vivo. Detailed investigations of resting human CD4+ T cells have been precluded by the absence of efficient approaches for genetic manipulation limiting our understanding of HIV replication and restricting efforts to find a cure. Here we report a method for rapid, efficient, activation-neutral gene editing of resting, polyclonal human CD4+ T cells using optimized cell cultivation and nucleofection conditions of Cas9–guide RNA ribonucleoprotein complexes. Up to six genes, including HIV dependency and restriction factors, were knocked out individually or simultaneously and functionally characterized. Moreover, we demonstrate the knock in of double-stranded DNA donor templates into different endogenous loci, enabling the study of the physiological interplay of cellular and viral components at single-cell resolution. Together, this technique allows improved molecular and functional characterizations of HIV biology and general immune functions in resting CD4+ T cells.

Rhabdoviruses, as single-stranded, negative-sense RNA viruses within the order Mononegavirales, are characterised by bullet-shaped or bacteroid particles that contain a helical ribonucleoprotein complex (RNP). Here, we review the components of the RNP and its higher-order structural assembly.

Hepatitis C virus (HCV) infection is a major problem worldwide. HCV is not limited to liver disease but is frequently complicated by immune-mediated extrahepatic manifestations such as glomerulonephritis or vasculitis. A fatal complication of HCV-associated vascular disease is thrombosis. Polyriboinosinic:polyribocytidylic acid (poly (I:C)), a synthetic analog of viral RNA, induces a Toll-like receptor 3 (TLR3)-dependent arteriolar thrombosis without significant thrombus formation in venules in vivo. These procoagulant effects are caused by increased endothelial synthesis of tissue factor and PAI-1 without platelet activation. In addition to human umbilical endothelial cells (HUVEC), human mesangial cells (HMC) produce procoagulatory factors, cytokines and adhesion molecules after stimulation with poly (I:C) or HCV-containing cryoprecipitates from a patient with a HCV infection as well. Activated protein C (APC) is able to prevent the induction of procoagulatory factors in HUVEC and HMC in vitro and blocks the effects of poly (I:C) and HCV-RNA on the expression of cytokines and adhesion molecules in HMC but not in HUVEC. In vivo, protein C inhibits poly (I:C)-induced arteriolar thrombosis. Thus, endothelial cells are de facto able to actively participate in immune-mediated vascular thrombosis caused by viral infections. Finally, we provide evidence for the ability of protein C to inhibit TLR3-mediated arteriolar thrombosis caused by HCV infection.

An adaptive stress response involves various mediators and circuits orchestrating a complex interplay of physiological, emotional, and behavioral adjustments. We identified a population of corticotropin-releasing hormone (CRH) neurons in the lateral part of the interstitial nucleus of the anterior commissure (IPACL), a subdivision of the extended amygdala, which exclusively innervate the substantia nigra (SN). Specific stimulation of this circuit elicits hyperactivation of the hypothalamic-pituitary-adrenal axis, locomotor activation, and avoidance behavior contingent on CRH receptor type 1 (CRHR1) located at axon terminals in the SN, which originate from external globus pallidus (GPe) neurons. The neuronal activity prompting the observed behavior is shaped by IPACL CRH and GPe CRHR1 neurons coalescing in the SN. These results delineate a previously unidentified tripartite CRH circuit functionally connecting extended amygdala and basal ganglia nuclei to drive locomotor activation and avoidance behavior.

The large SARS-CoV-2 spike (S) protein is the main target of current COVID-19 vaccine candidates but can induce non-neutralizing antibodies, which may cause vaccination-induced complications or enhancement of COVID-19 disease. Besides, encoding of a functional S in replication-competent virus vector vaccines may result in the emergence of viruses with altered or expanded tropism. Here, we have developed a safe single round rhabdovirus replicon vaccine platform for enhanced presentation of the S receptor-binding domain (RBD). Structure-guided design was employed to build a chimeric minispike comprising the globular RBD linked to a transmembrane stem-anchor sequence derived from rabies virus (RABV) glycoprotein (G). Vesicular stomatitis virus (VSV) and RABV replicons encoding the minispike not only allowed expression of the antigen at the cell surface but also incorporation into the envelope of secreted non-infectious particles, thus combining classic vector-driven antigen expression and particulate virus-like particle (VLP) presentation. A single dose of a prototype replicon vaccine, VSVΔG-minispike-eGFP (G), stimulated high titers of SARS-CoV-2 neutralizing antibodies in mice, equivalent to those found in COVID-19 patients. Boost immunization with the identical replicon further enhanced neutralizing activity. These results demonstrate that rhabdovirus minispike replicons represent effective and safe alternatives to vaccination approaches using replication-competent viruses and/or the entire S antigen.