Comparative analysis of rabies pathogenic and vaccine strains detection by RIG-I-like receptors.

Rabies virus (RABV) is a lethal neurotropic virus that causes 60,000 human deaths every year globally. RABV infection is characterized by the suppression of the interferon (IFN)-mediated antiviral response. However, molecular mechanisms leading to RABV sensing by RIG-I-like receptors (RLR) that initiates IFN signaling currently remain elusive. Here, we showed that RABV RNAs are primarily recognized by the RIG-I RLR, resulting in an IFN response in the infected cells, but this response varied according to the type of RABV used. Pathogenic RABV strain RNAs, Tha, were poorly detected in the cytosol by RIG-I and therefore caused a weak antiviral response. However, we revealed a strong IFN activity triggered by the attenuated RABV vaccine strain RNAs, SAD, mediated by RIG-I. We characterized two major 5' copy-back defective interfering (5'cb DI) genomes generated during SAD replication. Furthermore, we identified an interaction between 5'cb DI genomes, and RIG-I correlated with a high stimulation of the type I IFN signaling. This study indicates that wild-type RABV RNAs poorly activate the RIG-I pathway, while the presence of 5'cb DIs in the live-attenuated vaccine strain serves as an intrinsic adjuvant that strengthens its efficiency by enhancing RIG-I detection thus strongly stimulates the IFN response.

Evaluation of the efficacy of vitamin C on the immune response after rabies virus vaccine in BALB/c mice.

OBJECTIVE: Rabies is a lethal zoonotic infection caused by the rabies virus. Interferon- (INF) and interleukins (ILs) are a cytokine that is primarily produced by cells of the immune system. Vitamin C is an essential micronutrient in various biological processes, especially immune responses, and plays an essential part. Vaccination can successfully activate immune responses to virus infection protection. This study aimed to investigate the effect of vitamin C administration on immune responses to an inactivated rabies vaccine. MATERIALS AND METHODS: Thirty male Balb/c mice weighing between 25-30 gm (8 weeks old) were used in the current experimental study and randomly equally divided into three groups. Group I: untreated healthy control group was inoculated with PBS as a negative control. Group II: vaccinated intradermally with rabies vaccine alone using a dose of 4 ml/animal at 0, 7, 21 days. Group III: In addition to the dose of vaccine, mice were injected single intraperitoneally with 10 mg of vitamin C with each dose of vaccine on days 0, 7, 21. At experimental end, serum levels of IFN-γ, IL-4, and IL-5 were measured. RESULTS: The results revealed that vitamin C supplementation significantly elevated IFN-γ, IL-4, and IL-5 levels in vaccinated mice and treated with vitamin C (group III) compared to vaccinated group II and healthy control group I. Similarly, vitamin C supplementation exhibited strong positive correlations between IFN-γ and both IL-4 and IL-5 level in all experimental group. Taken together, these results showed that vitamin C is an important stimulator of interferon, interleukin-4 and -5 during inactivated rabies vaccine vaccination in mice. CONCLUSIONS: Our results supported the hypothesis that indicated the immunological improvement of vitamin C to the effectiveness of the inactivated rabies virus vaccination. High dose of vitamin C increases the levels of interferon and interleukin-4 and interleukin-5.

Roles of traditional medicine and traditional healers for rabies prevention and potential impacts on post-exposure prophylaxis: A literature review.

INTRODUCTION: Globally, traditional medicine is widely used to treat a variety of injuries and illnesses, including dog bites, and exposures that are risky for rabies. However, efficacy of most traditional remedies used for rabies prevention or treatment has not been demonstrated in controlled trials or proven in community-based surveys. METHODS: Six databases were searched including the terms rabies, traditional treatment, traditional remedy, traditional therapy, traditional medicine, and medicinal treatment to review traditional remedies used in the prevention and treatment of rabies. In addition, published literature of rabies transmission dynamics was used to estimate statistical likelihood of dog bite victims developing rabies to provide clarity as to why traditional healers have a high apparent success rate when preventing death from rabies in victims bitten by suspected rabid dogs. RESULTS: Literature review yielded 50 articles, including three controlled experiments, that described use of traditional remedies for rabies prevention and treatment. Traditional remedies for rabies ranged from plant- or animal-based products to spiritual rituals; however, only a few controlled mice trials were conducted, and none of these trials demonstrated efficacy in preventing or treating rabies. Risk of dying from rabies after a bite from a dog with unknown rabies status is low, 1.90% (0.05%-29.60%). Therefore, traditional healers had a 98.10% (70.40%-99.95%) apparent success rate in preventing death from suspected rabid dog bites despite inefficaciousness of herbal remedies. CONCLUSION: There was no universal plant species or route of administration that was consistently used for rabies prevention or treatment across countries. No traditional remedy was efficacious in the prevention or treatment of rabies in randomized controlled experiments. Understanding the cultural context under which traditional remedies are used may facilitate collaboration of traditional healers with the modern medical system to ensure timely and appropriate use of proven therapies for prevention and clinical management of rabies.

Colloidal Manganese Salt Improves the Efficacy of Rabies Vaccines in Mice, Cats, and Dogs.

Rabies, caused by rabies virus (RABV), remains a serious threat to public health in most countries worldwide. At present, the administration of rabies vaccines has been the most effective strategy to control rabies. Herein, we evaluate the effect of colloidal manganese salt (Mn jelly [MnJ]) as an adjuvant of rabies vaccine in mice, cats, and dogs. The results showed that MnJ promoted type I interferon (IFN-I) and cytokine production in vitro and the maturation of dendritic cells (DCs) in vitro and in vivo. Besides, MnJ serving as an adjuvant for rabies vaccines could significantly facilitate the generation of T follicular helper (Tfh) cells, germinal center (GC) B cells, plasma cells (PCs), and RABV-specific antibody-secreting cells (ASCs), consequently improve the immunogenicity of rabies vaccines, and provide better protection against virulent RABV challenge. Similarly, MnJ enhanced the humoral immune response in cats and dogs as well. Collectively, our results suggest that MnJ can facilitate the maturation of DCs during rabies vaccination, which can be a promising adjuvant candidate for rabies vaccines. IMPORTANCE Extending the humoral immune response by using adjuvants is an important strategy for vaccine development. In this study, a novel adjuvant, MnJ, supplemented in rabies vaccines was evaluated in mice, cats, and dogs. Our results in the mouse model revealed that MnJ increased the numbers of mature DCs, Tfh cells, GC B cells, PCs, and RABV-specific ASCs, resulting in enhanced immunogenicity and protection rate of rabies vaccines. We further found that MnJ had the same stimulative effect in cats and dogs. Our study provides the first evidence that MnJ serving as a novel adjuvant of rabies vaccines can boost the immune response in both a mouse and pet model.

Optimization of whole-brain rabies virus tracing technology for small cell populations.

The lateral hypothalamus (LH) is critically involved in the regulation of homeostatic energy balance. Some neurons in the LH express receptors for leptin (LepRb), a hormone known to increase energy expenditure and decrease energy intake. However, the neuroanatomical inputs to LepRb-expressing LH neurons remain unknown. We used rabies virus tracing technology to map these inputs, but encountered non-specific tracing. To optimize this technology for a minor cell population (LepRb is not ubiquitously expressed in LH), we used LepRb-Cre mice and assessed how different titers of the avian tumor virus receptor A (TVA) helper virus affected rabies tracing efficiency and specificity. We found that rabies expression is dependent on TVA receptor expression, and that leakiness of TVA receptors is dependent on the titer of TVA virus used. We concluded that a titer of 1.0-3.0 × 107 genomic copies per µl of the TVA virus is optimal for rabies tracing. Next, we successfully applied modified rabies virus tracing technology to map inputs to LepRb-expressing LH neurons. We discovered that other neurons in the LH itself, the periventricular hypothalamic nucleus (Pe), the posterior hypothalamic nucleus (PH), the bed nucleus of the stria terminalis (BNST), and the paraventricular hypothalamic nucleus (PVN) are the most prominent input areas to LepRb-expressing LH neurons.

Delineating the organization of projection neuron subsets in primary visual cortex with multiple fluorescent rabies virus tracing.

The impressive functions of the brain rely on an extensive connectivity matrix between specific neurons, the architecture of which is frequently characterized by one brain nucleus/region connecting to multiple targets, either via collaterals of the same projection neuron or several, differentially specified neurons. Delineating the fine architecture of projection neuron subsets in a specific brain region could greatly facilitate its circuit, computational, and functional resolution. Here, we developed multiple fluorescent rabies viruses (RV) to delineate the fine organization of corticothalamic projection neuron subsets in the primary visual cortex (V1). By simultaneously retrograde labeling multiple distinct subsets of corticothalamic projection neurons in V1 from their target nuclei in thalamus (dLGN, LP, LD), we observed that V1-dLGN corticothalamic projection neurons were densely concentrated in layer VI, except for several sparsely scattered neurons in layer V, while V1-LP and V1-LD corticothalamic projection neurons were localized to both layers V and VI. Meanwhile, we observed a fraction of V1 corticothalamic projection neurons targeting two thalamic nuclei, which was further confirmed by fMOST whole-brain imaging. The multiple fluorescent RV tracing tools can be extensively applied to resolve the architecture of projection neuron subsets in certain brain regions, with a strong potential to delineate the computational and functional organization of these brain regions.

A Phase 3, Randomized, Open-label, Noninferiority Trial Evaluating Anti-Rabies Monoclonal Antibody Cocktail (TwinrabTM) Against Human Rabies Immunoglobulin (HRIG).

BACKGROUND: Limited supply, cost and potential for severe adverse effects observed with the blood derived rabies immunoglobulin products has led to search for alternative therapies. This issue has been addressed by developing an anti-rabies monoclonal antibody cocktail. METHODS: This is a phase 3, randomized, open-label, noninferiority trial conducted in patients with World Health Organization (WHO) category III exposure with suspected rabid animal. Eligible patients were assigned to either the test arm, TwinrabTM (docaravimab and miromavimab) or the reference arm, human rabies immunoglobulin (HRIG; Imogam® Rabies-HT), in a ratio of 1:1. The primary endpoint was the comparison of responder rates between the 2 arms assessed as percentage of those with rabies virus neutralizing antibodies titers ≥0.5 IU/mL on day 14. RESULTS: A total of 308 patients were equally randomized into the 2 arms. In the per-protocol (PP) population, there were 90.21% responders in the TwinrabTM arm and 94.37% in the HRIG arm. The geometric mean of rapid fluorescent foci inhibition test titers in the PP on day 14 were 4.38 and 4.85 IU/mL, for the TwinrabTM and HRIG arms, respectively. There were no deaths or serious adverse events reported. CONCLUSIONS: This study confirmed that TwinrabTM is noninferior to HRIG in terms of providing an unbroken window of protection up to day 84. This trial in healthy adults with WHO category III exposure from suspected rabid animal also establishes the safety of TwinrabTM in patients with 1 WHO approved vaccine regimen (Essen). CLINICAL TRIALS REGISTRATION: CTRI/2017/07/009038.

Projections from the dorsomedial division of the bed nucleus of the stria terminalis to hypothalamic nuclei in the mouse.

As stressful environment is a potent modulator of feeding, we seek in the present work to decipher the neuroanatomical basis for an interplay between stress and feeding behaviors. For this, we combined anterograde and retrograde tracing with immunohistochemical approaches to investigate the patterns of projections between the dorsomedial division of the bed nucleus of the stria terminalis (BNST), well connected to the amygdala, and hypothalamic structures such as the paraventricular (PVH) and dorsomedial (DMH), the arcuate (ARH) nuclei and the lateral hypothalamic areas (LHA) known to control feeding and motivated behaviors. We particularly focused our study on afferences to proopiomelanocortin (POMC), agouti-related peptide (AgRP), melanin-concentrating-hormone (MCH) and orexin (ORX) neurons characteristics of the ARH and the LHA, respectively. We found light to intense innervation of all these hypothalamic nuclei. We particularly showed an innervation of POMC, AgRP, MCH and ORX neurons by the dorsomedial and dorsolateral divisions of the BNST. Therefore, these results lay the foundation for a better understanding of the neuroanatomical basis of the stress-related feeding behaviors.

Identification and Characterization of a Small-Molecule Rabies Virus Entry Inhibitor.

Rabies virus (RABV) causes a severe and fatal neurological disease, but morbidity is vaccine preventable and treatable prior to the onset of clinical symptoms. However, immunoglobulin (IgG)-based rabies postexposure prophylaxis (PEP) is expensive, restricting access to life-saving treatment, especially for patients in low-income countries where the clinical need is greatest, and does not confer cross-protection against newly emerging phylogroup II lyssaviruses. Toward identifying a cost-effective replacement for the IgG component of rabies PEP, we developed and implemented a high-throughput screening protocol utilizing a single-cycle RABV reporter strain. A large-scale screen and subsequent direct and orthogonal counterscreens identified a first-in-class direct-acting RABV inhibitor, GRP-60367, with a specificity index (SI) of >100,000. Mechanistic characterization through time-of-addition studies, transient cell-to-cell fusion assays, and chimeric vesicular stomatitis virus (VSV) recombinants expressing the RABV glycoprotein (G) demonstrated that GRP-60367 inhibits entry of a subset of RABV strains. Resistance profiling of the chemotype revealed hot spots in conserved hydrophobic positions of the RABV G protein fusion loop that were confirmed in transient cell-to-cell fusion assays. Transfer of RABV G genes with signature resistance mutations into a recombinant VSV backbone resulted in the recovery of replication-competent virions with low susceptibility to the inhibitor. This work outlines a tangible strategy for mechanistic characterization and resistance profiling of RABV drug candidates and identified a novel, well-behaved molecular probe chemotype that specifically targets the RABV G protein and prevents G-mediated viral entry.IMPORTANCE Rabies PEP depends on anti-RABV IgG, which is expensive and in limited supply in geographical areas with the highest disease burden. Replacing the IgG component with a cost-effective and shelf-stable small-molecule antiviral could address this unmet clinical need by expanding access to life-saving medication. This study has established a robust protocol for high-throughput anti-RABV drug screens and identified a chemically well-behaved, first-in-class hit with nanomolar anti-RABV potency that blocks RABV G protein-mediated viral entry. Resistance mapping revealed a druggable site formed by the G protein fusion loops that has not previously emerged as a target for neutralizing antibodies. Discovery of this RABV entry inhibitor establishes a new molecular probe to advance further mechanistic and structural characterization of RABV G that may aid in the design of a next-generation clinical candidate against RABV.

Antibody Response Following Pre-Exposure Immunization Against Rabies in High-Risk Professionals.

Vaccination against rabies and routine antibody testing of subjects participating in programs for the surveillance and control of rabies in animals is strongly recommended. The scope of this study is to describe the antibody level as measured by a commercial enzyme-linked immunosorbent assay (ELISA) after primary and booster intramuscular vaccination with a purified vero-cell rabies vaccine (PVRV) in high-risk professionals and to determine the influence of an array of factors on antibody level, that is, time elapsed since primary immunization series and booster dose, sex, age, pathologic conditions, high-risk occupation, and peak antibody level after initial scheme and booster dose. A primary series of three doses of PVRV was administered and a commercial ELISA was recommended 14 days postimmunization with continuous repetition at 6 months and yearly intervals for the laboratory personnel and the rest of the professionals, respectively. The protective antibody titer was defined as a minimum of 0.5 equivalent units/mL (EU/mL) (seroconvertion) and a booster dose was applied if the titer was determined nonprotective. The seroconversion rate (SCR) after primary vaccination was 100%, with a geometric mean titer (GMT) of 2.90 EU/mL (interquartile range [IQR]: 1.85-3.45). After booster vaccination due to nonprotective titer, the SCR was 100% and the GMT increased by 678% (95% confidence interval [CI]: 514-887) reaching 4.25 EU/mL (IQR: 4.00-4.60), 2.5 times higher than the GMT elicited by the primary vaccine scheme in the respective recipients. The titer dropped by 1.20% per month (95% CI: 0.52-1.89) regardless of booster administration or any other factor. Women had 51% higher titer compared with men (95% CI: 6-116). High-risk professionals should be verified for adequate antibody titers, but routine administration of a single booster dose of PVRV 1 year after the primary series could be considered; more evidence is needed to support the benefit in terms of immunity and logistics.

In Vivo Two-photon Calcium Imaging in Dendrites of Rabies Virus-labeled V1 Corticothalamic Neurons.

Monitoring neuronal activity in vivo is critical to understanding the physiological or pathological functions of the brain. Two-photon Ca2+ imaging in vivo using a cranial window and specific neuronal labeling enables real-time, in situ, and long-term imaging of the living brain. Here, we constructed a recombinant rabies virus containing the Ca2+ indicator GCaMP6s along with the fluorescent protein DsRed2 as a baseline reference to ensure GCaMP6s signal reliability. This functional tracer was applied to retrogradely label specific V1-thalamus circuits and detect spontaneous Ca2+ activity in the dendrites of V1 corticothalamic neurons by in vivo two-photon Ca2+ imaging. Notably, we were able to record single-spine spontaneous Ca2+ activity in specific circuits. Distinct spontaneous Ca2+ dynamics in dendrites of V1 corticothalamic neurons were found for different V1-thalamus circuits. Our method can be applied to monitor Ca2+ dynamics in specific input circuits in vivo, and contribute to functional studies of defined neural circuits and the dissection of functional circuit connections.

A Recombinant Rabies Virus Expressing Fms-like Tyrosine Kinase 3 Ligand (Flt3L) Induces Enhanced Immunogenicity in Mice.

Rabies is a zoonotic disease that still causes 59,000 human deaths each year, and rabies vaccine is the most effective way to control the disease. Our previous studies suggested that the maturation of DC plays an important role in enhancing the immunogenicity of rabies vaccine. Flt3L has been reported to own the ability to accelerate the DC maturation, therefore, in this study, a recombinant rabies virus expressing mouse Flt3L, designated as LBNSE-Flt3L, was constructed, and its immunogenicity was characterized. It was found that LBNSE-Flt3L could enhance the maturation of DC both in vitro and in vivo, and significantly more TFH cells and Germinal Center B (GC B) cells were generated in mice immunized with LBNSE-Flt3L than those immunized with the parent virus LBNSE. Consequently, expressing of Flt3L could elevate the level of virus-neutralizing antibodies (VNA) in immunized mice which provides a better protection from a lethal rabies virus challenge. Taken together, our study extends the potential of Flt3L as a good adjuvant to develop novel rabies vaccine by enhancing the VNA production through activating the DC-TFH-GC B axis in immunized mice.

The influence of poverty and rabies knowledge on healthcare seeking behaviors and dog ownership, Cameroon.

BACKGROUND: Rabies is a fatal encephalitis caused by lyssaviruses, with most human cases worldwide resulting from rabid dog bites. Although effective animal and human vaccines have been available for over 100 years, control efforts have not been adequately implemented on the global scale and rabies remains one of the greatest global zoonotic threats to human health. We conducted a knowledge, attitudes and practices survey in Northern Cameroon to describe dog ownership characteristics, rates of dog bites, and post-bite healthcare seeking behaviors. METHODS: The survey was performed in four rural Cameroonian communities. A structured community-based questionnaire was conducted over a 20-day period in April 2010, and focused on socio-economic factors correlated with gaps in rabies knowledge. Information pertaining to socio-demographics, as well as attitudes and practices with regard to animal bites and bite treatment practices were recorded. Characteristics of dog ownership such as dog confinement, resources provided to dogs, and dog vaccination status were examined. Human to dog ratios were compared on a linear scale to poverty scores by community. When applicable, 2-tailed Chi-square tests or Fisher's exact tests were calculated to determine relationships between variables. We also used One-way Analysis of Variance (ANOVA) to identify associations between rabies knowledge and wealth with dog ownership, dog vaccination, and human healthcare seeking behaviors. Independent variables were evaluated using multivariate logistic regression analysis. RESULTS: A total of 208 households were enrolled. Respondents were predominantly male (68.3%), with a median age of 43.6 years. Eighty-four households (39.9%) reported owning a total of 141 dogs (human dog ratio 10.4:1). The majority of dogs (61%) were allowed to roam freely. A history of rabies vaccination was reported for 30.8% of owned dogs. Respondents reported 11 bites during the two years preceding the survey (annual bite incidence was 2.6% [95% CI 1.4%- 4.6%]). Only one person (9.1%) received rabies post-exposure prophylaxis (PEP), and none described symptoms of clinical illness consistent with rabies. Respondents who indicated that they would seek medical care and PEP after a dog bite had higher average wealth and rabies knowledge index scores (p = 0.01 and 0.04, respectively). Respondents who indicated that they would seek care from a traditional healer had significantly lower wealth scores, but not significantly different knowledge scores (p < 0.01 and p = 0.49, respectively). CONCLUSIONS: In the communities evaluated, the majority of dogs were allowed to roam freely and had no history of rabies vaccination; factors that favor enzootic transmission of canine rabies virus. We also identified a strong relationship between poverty and dog ownership. Bite events were relatively common among respondents, and very few victims reported utilizing health services to treat wounds. Increased wealth and knowledge were significantly associated with increased likelihood that a respondent would seek medical care and post-exposure prophylaxis. These findings indicate the need for educational outreach to raise awareness of dog rabies and proper prevention measures.

Adjuvant activity of ethanol extract of Hippophae rhamnoides leaves with inactivated rabies virus antigen.

CONTEXT: Hippophae rhamnoides L. (Elaeagnaceae), commonly known as seabuckthorn (SBT), is known for its medicinal and nutritional properties. OBJECTIVE: Evaluation of in vivo adjuvant activity of SBT leaf extract (SBTE) with inactivated rabies virus antigen (Rb). MATERIALS AND METHODS: Swiss albino mice were immunized with aqueous-alcoholic SBTE (100 mg/kg body weight) or algel (aluminium hydroxide gel) with or without Rb (5% v/v). After priming, booster was administered on day 14. Rabies virus neutralizing antibody (RVNA) titers were estimated by rapid fluorescent focus inhibition test in sera samples collected on days 7, 14, 21, 28 and 35. Effect of adjuvant administration on cytotoxic T lymphocytes (CTLs), memory T cells, plasma and CD11c+ cells was studied by flow cytometry. In vitro hemolysis was assayed in human RBC. RESULTS: RVNA titers were significantly enhanced (p < 0.05) after booster administration in mice immunized with SBTE + Rb as compared to the controls. In combination, SBTE, algel and Rb, enhanced the RVNA titers. CTLs significantly increased (p < 0.05) in SBTE + Rb immunized mice. Memory T cells and plasma cells were 27.9 and 15.9%, respectively, in SBTE + Rb immunized mice as compared to that of 20.3 and 11.3%, respectively, in Rb immunized group. SBTE + Rb enhanced peritoneal CD11c+ cells (25.8%) as compared to 9.4% cells in Rb immunized mice, showed 3.2-fold increment in LPS induced IL-1ß. No RBC hemolysis was observed with SBTE. CONCLUSIONS: This study demonstrates the potential adjuvant activity of SBTE with Rb by increasing RVNA titers and CTL response.

A novel approach to a rabies vaccine based on a recombinant single-cycle flavivirus vector.

The RepliVax® vaccine (RV) platform is based on flavivirus genomes that are rationally attenuated by deletion. These single-cycle RV vaccine candidates targeting flavivirus pathogens have been demonstrated to be safe, highly immunogenic, and efficacious in animal models, including non-human primates. Here we show utility of the technology for delivery of a non-flavivirus immunogen by engineering several West Nile-based RV vectors to express full-length rabies virus G protein. The rabies virus G protein gene was incorporated in place of different West Nile structural protein gene deletions. The resulting RV-RabG constructs were demonstrated to replicate to high titers (8 log10 infectious particles/ml) in complementing helper cells. Following infection of normal cells, they provided efficient rabies virus G protein expression, but did not spread to surrounding cells. Expression of rabies virus G protein was stable and maintained through multiple rounds of in vitro passaging. A sensitive neurovirulence test in 2-3 day old neonatal mice demonstrated that RV-RabG candidates were completely avirulent indicative of high safety. We evaluated the RV-RabG variants in several animal models (mice, dogs, and pigs) and demonstrated that a single dose elicited high titers of rabies virus-neutralizing antibodies and protected animals from live rabies virus challenge (mice and dogs). Importantly, dogs were protected at both one and two years post-immunization, demonstrating durable protective immunity. The data demonstrates the potential of the RepliVax® technology as a potent vector delivery platform for developing vaccine candidates against non-flavivirus targets.

Large-scale Reconstructions and Independent, Unbiased Clustering Based on Morphological Metrics to Classify Neurons in Selective Populations.

This protocol outlines large-scale reconstructions of neurons combined with the use of independent and unbiased clustering analyses to create a comprehensive survey of the morphological characteristics observed among a selective neuronal population. Combination of these techniques constitutes a novel approach for the collection and analysis of neuroanatomical data. Together, these techniques enable large-scale, and therefore more comprehensive, sampling of selective neuronal populations and establish unbiased quantitative methods for describing morphologically unique neuronal classes within a population. The protocol outlines the use of modified rabies virus to selectively label neurons. G-deleted rabies virus acts like a retrograde tracer following stereotaxic injection into a target brain structure of interest and serves as a vehicle for the delivery and expression of EGFP in neurons. Large numbers of neurons are infected using this technique and express GFP throughout their dendrites, producing "Golgi-like" complete fills of individual neurons. Accordingly, the virus-mediated retrograde tracing method improves upon traditional dye-based retrograde tracing techniques by producing complete intracellular fills. Individual well-isolated neurons spanning all regions of the brain area under study are selected for reconstruction in order to obtain a representative sample of neurons. The protocol outlines procedures to reconstruct cell bodies and complete dendritic arborization patterns of labeled neurons spanning multiple tissue sections. Morphological data, including positions of each neuron within the brain structure, are extracted for further analysis. Standard programming functions were utilized to perform independent cluster analyses and cluster evaluations based on morphological metrics. To verify the utility of these analyses, statistical evaluation of a cluster analysis performed on 160 neurons reconstructed in the thalamic reticular nucleus of the thalamus (TRN) of the macaque monkey was made. Both the original cluster analysis and the statistical evaluations performed here indicate that TRN neurons are separated into three subpopulations, each with unique morphological characteristics.

Pseudo-typed Semliki Forest virus delivers EGFP into neurons.

Semliki Forest virus (SFV), a neurotropic virus, has been used to deliver heterologous genes into cells in vitro and in vivo. In this study, we constructed a reporter SFV4-FL-EGFP and found that it can deliver EGFP into neurons located at the injection site without disseminating throughout the brain. Lacking of the capsid gene of SFV4-FL-EGFP does not block its life cycle, while forming replication-competent virus-like particles (VLPs). These VLPs hold subviral genome by using the packaging sequence (PS) located within the nsP2 gene, and can transfer their genome into cells. In addition, we found that the G protein of vesicular stomatitis virus (VSVG) can package SFV subviral genome, which is consistent with the previous reports. The G protein of rabies virus (RVG) could also package SFV subviral genome. These pseudo-typed SFV can deliver EGFP gene into neurons. Taken together, these findings may be used to construct various SFV-based delivery systems for virological studies, gene therapy, and neural circuit labeling.

Brain-Wide Maps of Synaptic Input to Cortical Interneurons.

Cortical inhibition is mediated by diverse inhibitory neuron types that can each play distinct roles in information processing by virtue of differences in their input sources, intrinsic properties, and innervation targets. Previous studies in brain slices have demonstrated considerable cell-type specificity in laminar sources of local inputs. In contrast, little is known about possible differences in distant inputs to different cortical interneuron types. We used the monosynaptic rabies virus system, in conjunction with mice expressing Cre recombinase in either parvalbumin-positive, somatostatin-positive (SST+), or vasoactive intestinal peptide-positive (VIP+) neurons, to map the brain-wide input to the three major nonoverlapping classes of interneurons in mouse somatosensory cortex. We discovered that all three classes of interneurons received considerable input from known cortical and thalamic input sources, as well as from probable cholinergic cells in the basal nucleus of Meynert. Despite their common input sources, these classes differed in the proportion of long-distance cortical inputs originating from deep versus superficial layers. Similar to their laminar differences in local input, VIP+ neurons received inputs predominantly from deep layers while SST+ neurons received mostly superficial inputs. These classes also differed in the amount of input they received. Cortical and thalamic inputs were greatest onto VIP+ interneurons and smallest onto SST+ neurons. SIGNIFICANCE STATEMENT: These results indicate that all three major interneuron classes in the barrel cortex integrate both feedforward and feedback information from throughout the brain to modulate the activity of the local cortical circuit. However, differences in laminar sources and magnitude of distant cortical input suggest differential contributions from cortical areas. More input to vasoactive intestinal peptide-positive (VIP+) neurons than to somatostatin-positive (SST+) neurons suggests that disinhibition of the cortex via VIP+ cells, which inhibit SST+ cells, might be a general feature of long-distance corticocortical and thalamocortical circuits.

Generation of recombinant rabies viruses encoding NanoLuc luciferase for antiviral activity assays.

Rabies is an invariably fatal disease caused by Rabies virus (RABV), a member of the family Rhabdoviridae, genus Lyssavirus. Once central nervous infection occurs and symptoms develop, the case fatality rate approaches 100% despite availability of post-exposure prophylaxis. Therefore, new antiviral therapies for rabies are urgently required. Antivirals which can inhibit virus replication can be identified through screening of small compounds, however, as RABV infection does not generate easily discernible cytopathic effects in vitro, cell viability assays may not be feasible to observe antiviral activity of small compounds against RABV. In this study, recombinant RABVs (rRABVs) encoding NanoLuc luciferase (NanoLuc) were generated to facilitate the screening of small compound libraries. NanoLuc expression was confirmed in single-step growth cures of virus infection and showed that the rRABVs were capable of viral replication without decrease of luciferase activity through ten serial passages. Furthermore, the rRABVs were able to quantify the antiviral activity of the nucleoside analogue ribavirin against RABV in vitro. These findings confirm the potential of the rRABV encoding NanoLuc system to facilitate screening of small compounds to inhibit RABV infection.

A novel rabies vaccine based-on toll-like receptor 3 (TLR3) agonist PIKA adjuvant exhibiting excellent safety and efficacy in animal studies.

Vaccination alone is not sufficiently effective to protect human from post-exposure rabies virus infection due to delayed generation of rabies virus neutralizing antibodies and weak cellular immunity. Therefore, it is vital to develop safer and more efficacious vaccine against rabies. PIKA, a stabilized chemical analog of double-stranded RNA that interacts with TLR3, was employed as adjuvant of rabies vaccine. The efficacy and safety of PIKA rabies vaccine were evaluated. The results showed that PIKA rabies vaccine enhanced both humoral and cellular immunity. After viral challenge, PIKA rabies vaccine protected 70-80% of animals, while the survival rate of non-adjuvant vaccine group (control) was 20-30%. According to the results of toxicity tests, PIKA and PIKA rabies vaccine are shown to be well tolerated in mice. Thus, this study indicates that PIKA rabies vaccine is an effective and safe vaccine which has the potential to develop next-generation rabies vaccine and encourage the start of clinical studies.