Recombinant Rabies Virus Research Articles

A novel oral rabies vaccine enhances the immunogenicity through increasing dendritic cells activation and germinal center formation by expressing U-OMP19 in a mouse model

ABSTRACT Rabies remains a public health threat in most parts of the world. Dogs, especially stray dogs, are the main sources of rabies transmission in developing countries, while wild animals are primarily responsible for the spread of rabies in developed countries and play an emerging role in rabies transmission in developing countries. Oral vaccination is the most practical method for rabies control in these animals, and the greatest challenge for oral vaccination is the hostile environment and large quantity of proteases in the gastrointestinal tract. In the present study, a promising adjuvant with potential protease inhibitory activity, unlipidated outer membrane protein 19 (U-OMP19), was inserted into the genome of the recombinant rabies virus (rRABV) strain LBNSE, designated LBNSE-U-OMP19, and the immunogenicity of LBNSE-U-OMP19 was investigated. LBNSE-U-OMP19 could potentially protect viral glycoprotein from digestion by gastrointestinal fluids in vitro. The expression of U-OMP19 attenuated viral pathogenicity by restricting viral replication in the central nervous system (CNS) and repressing the production of inflammatory chemokines and cytokines. After oral vaccination, LBNSE-U-OMP19 recruited dendritic cells (DCs), follicular helper T (TFH) cells and germinal center (GC) B cells, promoted the formation of GCs, and increased the population of plasma cells in immunized mice, resulting in higher levels of RABV-neutralizing antibodies and better protection in mice immunized with LBNSE-U-OMP19 than in those immunized with the parent virus LBNSE. Together, our data suggest that LBNSE-U-OMP19 is a promising candidate for oral rabies vaccines.

Rabies virus matrix protein targets host actin cytoskeleton: a protein-protein interaction analysis.

Multifunctional matrix protein (M) of rabies virus (RABV) plays essential roles in the pathogenesis of rabies infection. Identification of M protein interacting partners in target hosts could help to elucidate the biological pathways and molecular mechanisms involved in the pathogenesis of this virus. In this study, two-dimensional Far-western blotting (2D-Far-WB) technique was applied to find possible matrix protein partners in the rat brainstem. Recombinant RABV M was expressed in Pichia pastoris and was partially purified. Subsequently, 2D-Far-WB-determined six rat brainstem proteins interacted with recombinant M proteins that were identified by mass spectrometry. Functional annotation by gene ontology analysis determined these proteins were involved in the regulation of synaptic transmission processes, metabolic process and cell morphogenesis-cytoskeleton organization. The interaction of viral M protein with selected host proteins in mouse Neuro-2a cells infected with RABV was verified by super-resolution confocal microscopy. Molecular docking simulations also demonstrated the formation of RABV M complexes. However, further confirmation with co-immunoprecipitation was only successful for M-actin cytoplasmic 1 interaction. Our study revealed actin cytoplasmic 1 as a binding partner of M protein, which might have important role(s) in rabies pathogenesis.

Genetically modified rabies virus vector-based bovine ephemeral fever virus vaccine induces protective immune responses against BEFV and RABV in mice.

Bovine ephemeral fever (BEF), caused by the bovine ephemeral fever virus (BEFV), is associated with an acute febrile infection in cattle and widespread in tropical and subtropical areas, leading to great economic losses to cattle and milk industry. However, no efficacious BEF vaccine is currently available in China. Herein, we generated a recombinant rabies virus (RABV) expressing BEFV glycoprotein (LBNSE-BG), utilizing a reverse genetics system based on the recombinant rabies virus strain LBNSE. It was found that mice immunized with LBNSE-BG produced robust neutralizing antibodies against both BEFV and RABV, and developed complete protection from lethal RABV challenge. Further studies showed that LBNSE-BG activated more dendritic cells (DCs), B cells and T cells in immunized mice than the parent virus LBNSE. Collectively, these findings demonstrate that the recombinant LBNSE-BG described here has the potential to be developed as a cost-effective and efficacious bivalent vaccine for cattle use in endemic areas of BEF and rabies.

Purification of rabies virus glycoprotein produced in Drosophila melanogaster S2 cells: An efficient immunoaffinity method.

Most rabies vaccines are based on inactivated virus, which production process demands a high level of biosafety structures. In the past decades, recombinant rabies virus glycoprotein (RVGP) produced in several expression systems has been extensively studied to be used as an alternative vaccine. The immunogenic characteristics of this protein depend on its correct conformation, which is present only after the correct post-translational modifications, typically performed by animal cells. The main challenge of using this protein as a vaccine candidate is to keep its trimeric conformation after the purification process. We describe here a new immunoaffinity chromatography method using a monoclonal antibody for RVGP Site II for purification of recombinant rabies virus glycoprotein expressed on the membrane of Drosophila melanogaster S2 cells. RVGP recovery achieved at least 93%, and characterization analysis showed that the main antigenic proprieties were preserved after purification.

Lyssavirus Vaccine with a Chimeric Glycoprotein Protects across Phylogroups

Rabies is nearly 100% lethal in the absence of treatment, killing an estimated 59,000 people annually. Vaccines and biologics are highly efficacious when administered properly. Sixteen rabies-related viruses (lyssaviruses) are similarly lethal, but some are divergent enough to evade protection from current vaccines and biologics, which are based only on the classical rabies virus (RABV). Here we present the development and characterization of LyssaVax, a vaccine featuring a structurally designed, functional chimeric glycoprotein (G) containing immunologically important domains from both RABV G and the highly divergent Mokola virus (MOKV) G. LyssaVax elicits high titers of antibodies specific to both RABV and MOKV Gs in mice. Immune sera also neutralize a range of wild-type lyssaviruses across the major phylogroups. LyssaVax-immunized mice are protected against challenge with recombinant RABV and MOKV. Altogether, LyssaVax demonstrates the utility of structural modeling in vaccine design and constitutes a broadened lyssavirus vaccine candidate.

SNAP25 regulates the release of the Rabies virus in nerve cells via SNARE complex-mediated membrane fusion

Recent studies have reported that host proteins regulate Rabies virus (RABV) infection via distinct mechanisms. The abnormal neural function caused by RABV infection is related to the abnormal synaptic signal transmission in which the RABV glycoprotein (G) is involved. In the present study, two recombinant Rabies viruses (rRABVs), namely rSAD-SAD-Flag-G and rSAD-CVS-Flag-G, were established and rescued based on rSAD and verified by indirect fluorescence assay (IFA), and western blotting (WB). To investigate how the G protein interacts with synaptosomal-associated protein 25 (SNAP25), primary neuronal cells (PNC) of embryonic mice were cultured and infected with rRABVs. Immunoprecipitation (IP) and LC–MS/MS analysis of glycoprotein-binding proteins, which were flag tagged, were carried out to determine the interaction of G protein and soluble N-ethylmaleimide-sensitive factor attachment protein receptor proteins (SNARE) complex in PNC. G protein and the SNARE member SNAP25 were co-expressed in HEK293 cells or primary neuronal cells to investigate their colocalization. Knockdown of SNAP25 with small interfering RNA (siRNA) was conducted on mNA cells, and rRABV replication was observed by IFA, qRT-PCR, and virus titration. The results indicated that rRABVs were successfully rescued and grew well in PNC. Flag-tag IP and confocal microscopy demonstrated that SNAP25 works together with G protein and colocalizes with G on the cytomembrane of HEK293 cells. The downregulation of SNAP25, using RNA interference, resulted in a significant decrease in the number of viral mRNAs, viral proteins, and virus particles. Furthermore, the regression of SNAP25 did not affect the initial infection of the virus but reduced the infectivity of progeny virions.

Interferon-λ Attenuates Rabies Virus Infection by Inducing Interferon-Stimulated Genes and Alleviating Neurological Inflammation.

Rabies, caused by rabies virus (RABV), is a fatal neurological disease that still causes more than 59,000 human deaths each year. Type III interferon IFN-λs are cytokines with type I IFN-like antiviral activities. Although IFN-λ can restrict the infection for some viruses, especially intestinal viruses, the inhibitory effect against RABV infection remains undefined. In this study, the function of type III IFN against RABV infection was investigated. Initially, we found that IFN-λ2 and IFN-λ3 could inhibit RABV replication in cells. To characterize the role of IFN-λ in RABV infection in a mouse model, recombinant RABVs expressing murine IFN-λ2 or IFN-λ3, termed as rB2c-IFNλ2 or rB2c-IFNλ3, respectively, were constructed and rescued. It was found that expression of IFN-λ could reduce the pathogenicity of RABV and limit viral spread in the brains by different infection routes. Furthermore, expression of IFN-λ could induce the activation of the JAK-STAT pathway, resulting in the production of interferon-stimulated genes (ISGs). It was also found that rRABVs expressing IFN-λ could reduce the production of inflammatory cytokines in primary astrocytes and microgila cells, restrict the opening of the blood-brain barrier (BBB), and prevent excessive infiltration of inflammatory cells into the brain, which could be responsible for the neuronal damage caused by RABV. Consistently, IFN-λ was found to maintain the integrity of tight junction (TJ) protein ZO-1 of BBB to alleviate neuroinflammation in a transwell model. Our study underscores the role of IFN-λ in inhibiting RABV infection, which potentiates IFN-λ as a possible therapeutic agent for the treatment of RABV infection.

Recombinant Rabies Virus Overexpressing OX40-Ligand Enhances Humoral Immune Responses by Increasing T Follicular Helper Cells and Germinal Center B Cells.

Rabies, caused by the rabies virus (RABV), remains a serious threat to public health in most countries. Development of a single-dose and efficacious rabies vaccine is the most important method to restrict rabies virus transmission. Costimulatory factor OX40-ligand (OX40L) plays a crucial role in the T cell-dependent humoral immune responses through T-B cell interaction. In this work, a recombinant RABV overexpressing mouse OX40L (LBNSE-OX40L) was constructed, and its effects on immunogenicity were evaluated in a mouse model. LBNSE-OX40L-immunized mice generated a larger number of T follicular helper (Tfh) cells, germinal center (GC) B cells, and plasma cells (PCs) than the parent virus LBNSE-immunized mice. Furthermore, LBNSE-OX40L induced significantly higher levels of virus-neutralizing antibodies (VNA) as early as seven days post immunization (dpi), which lasted for eight weeks, resulting in better protection for mice than LBNSE (a live-attenuated rabies vaccine strain). Taken together, our data in this study suggest that OX40L can be a novel and potential adjuvant to improve the induction of protective antibody responses post RABV immunization by triggering T cell-dependent humoral immune responses, and that LBNSE-OX40L can be developed as an efficacious and nonpathogenic vaccine for animals.

Immune responses in mice and pigs after oral vaccination with rabies virus vectored Nipah disease vaccines

Nipah virus (NiV) is a re-emerging zoonotic pathogen that causes high mortality in humans and pigs. Oral immunization in free-roaming animals is one of the most practical approaches to prevent NiV pandemics. We previously generated a recombinant rabies viruses (RABV) Evelyn-Rokitnicki-Abelseth (ERA) strain, rERAG333E, which contains a mutation from arginine to glutamic acid at residue 333 of glycoprotein (G333E) and serves as an oral vaccine for dog rabies. In this study, we generated two recombinant RABVs, rERAG333E/NiVG and rERAG333E/NiVF, expressing the NiV Malaysian strain attachment glycoprotein (NiV-G) or fusion glycoprotein (NiV-F) gene based on the rERAG333E vector platform. Both rERAG333E/NiVG and rERAG333E/NiVF displayed growth properties similar to those of rERAG333E and caused marked syncytia formation after co-infection in BSR cell culture. Adult and suckling mice intracerebrally inoculated with the recombinant RABVs showed NiV-G and NiV-F expression did not increase the virulence of rERAG333E. Oral vaccination with rERAG333E/NiVG either singularly or combined with rERAG333E/NiVF induced significant NiV neutralizing antibody against NiV and RABV, and IgG to NiV-G or NiV-F in mice and pigs. rERAG333E/NiVG and rERAG333E/NiVF thus appeared to be suitable candidates for further oral vaccines for potential animal targets in endemic areas of NiV disease and rabies.

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.

Reevaluation of the efficacy of favipiravir against rabies virus using in vivo imaging analysis

Rabies virus (RABV) is a highly neurotropic virus and the causative agent of rabies, an encephalitis with an almost 100% case-fatality rate that remains incurable after the onset of symptoms. Favipiravir (T-705), a broad-spectrum antiviral drug against RNA viruses, has been shown to be effective against RABV in vitro but ineffective in vivo. We hypothesized that favipiravir is effective in infected mice when RABV replicates in the peripheral tissues/nerves but not after virus neuroinvasion. We attempted to clarify this point in this study using in vivo bioluminescence imaging. We generated a recombinant RABV from the field isolate 1088, which expressed red firefly luciferase (1088/RFLuc). This allowed semiquantitative detection and monitoring of primary replication at the inoculation site and viral spread in the central nervous system (CNS) in the same mice. Bioluminescence imaging revealed that favipiravir (300 mg/kg/day) treatment commencing 1 h after intramuscular inoculation of RABV efficiently suppressed viral replication at the inoculation site and the subsequent replication in the CNS. However, virus replication in the CNS was not inhibited when the treatment began 2 days after inoculation. We also found that higher doses (600 or 900 mg/kg/day) of favipiravir could suppress viral replication in the CNS even when administration started 2 days after inoculation. These results support our hypothesis and suggest that a highly effective drug-delivery system into the CNS and/or the enhancement of favipiravir conversion to its active form are required to improve favipiravir treatment of rabies. Furthermore, the bioluminescence imaging system established in this study will facilitate the development of treatment for symptomatic rabies.

Generation of recombinant rabies virus ERA strain applied to virus tracking in cell infection

The mechanism of rabies virus (RABV) infection still needs to be further characterized. RABV particle with self-fluorescent is a powerful viral model to visualize the viral infection process in cells. Herein, based on a reverse genetic system of the Evelyn-Rokitnicki-Abelseth (rERA) strain, we generated a recombinant RABV rERA-N/mCherry strain that stably expresses an additional ERA nucleoprotein that fuses with the red fluorescent protein mCherry (N/mCherry). The rERA-N/mCherry strain retained growth property similar to the parent strain rERA in vitro. The N/mCherry expression showed genetic stability during passage into mouse neuroblastoma (NA) cells and did not change the virulence of the vector. The rERA-N/mCherry strain was then utilized as a visual viral model to study the RABV-cell binding and internalization. We directly observed the red self-fluorescence of rERA-N/mCherry particles binding to the cell surface, and further co-localizing with clathrin in the early stage of infection in NA cells by fluorescence microscopy. Our results showed that the rERA-N/mCherry strain uses clathrin-dependent endocytosis to enter cells, which is consistent with the well-known mechanism of RABV invasion. The recombinant RABV rERA-N/mCherry thus appears to have the potential to be an effective viral model to further explore the fundamental molecular mechanism of rabies neuropathogenesis.

Codon optimization of G protein enhances rabies virus-induced humoral immunity.

Rabies, caused by rabies virus (RABV), is a fatal zoonosis, which still poses a threat to public health in most parts of the world. Glycoprotein of RABV is the only viral surface protein, which is critical for the induction of virus-neutralizing antibodies (VNA). In order to improve the production of VNA, recombinant RABVs containing two copies of G gene and codon-optimized G gene were constructed by using reverse genetics, named LBNSE-dG and LBNSE-dOG, respectively. After being inoculated into the mouse brains, LBNSE-dOG induced more apoptosis and recruited more inflammatory cells than LBNSE-dG and LBNSE, resulting in reduced virulence in vivo. After intramuscular (im) immunization in mice, LBNSE-dOG promoted the formation of germinal centres (GCs), the recruitment of GC B cells and the generation of antibody-secreting cells (ASCs) in the draining lymph nodes (LNs). Consistently, LBNSE-dOG boosted the production of VNA and provided better protection against lethal RABV challenge than LBNSE-dG and LBNSE when it was used as both live and inactivated vaccines. Our results demonstrate that the codon-optimized RABV LBNSE-dOG displays attenuated pathogenicity and enhanced immunogenicity, therefore it could be a potential candidate for the next generation of rabies vaccines.

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.

Tracing of Afferent Connections in the Zebrafish Cerebellum Using Recombinant Rabies Virus.

The cerebellum is involved in some forms of motor coordination and learning, and in cognitive and emotional functions. To elucidate the functions of the cerebellum, it is important to unravel the detailed connections of the cerebellar neurons. Although the cerebellar neural circuit structure is generally conserved among vertebrates, it is not clear whether the cerebellum receives and processes the same or similar information in different vertebrate species. Here, we performed monosynaptic retrograde tracing with recombinant rabies viruses (RV) to identify the afferent connections of the zebrafish cerebellar neurons. We used a G-deleted RV that expressed GFP. The virus was also pseudotyped with EnvA, an envelope protein of avian sarcoma and leucosis virus (ALSV-A). For the specific infection of cerebellar neurons, we expressed the RV glycoprotein (G) gene and the envelope protein TVA, which is the receptor for EnvA, in Purkinje cells (PCs) or granule cells (GCs), using the promoter for aldolase Ca (aldoca) or cerebellin 12 (cbln12), respectively. When the virus infected PCs in the aldoca line, GFP was detected in the PCs’ presynaptic neurons, including GCs and neurons in the inferior olivary nuclei (IOs), which send climbing fibers (CFs). These observations validated the RV tracing method in zebrafish. When the virus infected GCs in the cbln12 line, GFP was again detected in their presynaptic neurons, including neurons in the pretectal nuclei, the nucleus lateralis valvulae (NLV), the central gray (CG), the medial octavolateralis nucleus (MON), and the descending octaval nucleus (DON). GFP was not observed in these neurons when the virus infected PCs in the aldoca line. These precerebellar neurons generally agree with those reported for other teleost species and are at least partly conserved with those in mammals. Our results demonstrate that the RV system can be used for connectome analyses in zebrafish, and provide fundamental information about the cerebellar neural circuits, which will be valuable for elucidating the functions of cerebellar neural circuits in zebrafish.

Rabies-based vaccine induces potent immune responses against Nipah virus

Nipah Virus (NiV) is a re-emerging zoonotic pathogen in the genus Henipavirus of the Paramyxoviridae family of viruses. NiV is endemic to Bangladesh and Malaysia and is highly fatal to both livestock and humans (human case fatality rate = 74.5%). Currently, there is no approved vaccine against NiV on the market. The goal of this study was to use a recombinant RABV vector expressing NiV glycoprotein (NiV G) to develop a bivalent candidate vaccine against NiV disease and rabies virus (RABV) disease, which is also a significant health burden in the regions where NiV is endemic. The rabies vector is a well-established vaccine strain that lacks neurovirulence and can stably expresses foreign antigens that are immunogenic in various animal models. Mice inoculated intranasally with the live recombinant RABV/NiV vaccine (NIPARAB) showed no signs of disease. To test the immunogenicity of the vaccine candidate, groups of C57BL/6 mice were immunized intramuscularly with a single dose of live vaccine particles or two doses of chemically inactivated viral particles. Both vaccination groups showed NiV G-specific seroconversion, and the inactivated (INAC) vaccine group yielded higher titers of NiV G-specific antibodies. Furthermore, cross-reactivity of NiV G-specific immune sera against Hendra virus (HeV), was confirmed by immunofluorescence (IF) and indirect ELISA against soluble recombinant HeV glycoprotein (HeV G). Both live and killed vaccines induced neutralizing antibodies. These results indicate that NIPARAB may be used as a killed virus vaccine to protect humans against NiV and RABV, and possibly as a preventative measure against HeV as well.

A new recombinant rabies virus expressing a green fluorescent protein: A novel and fast approach to quantify virus neutralizing antibodies

The Rapid Fluorescent Focus Inhibition Test (RFFIT) is a standard assay used to detect and assess the titers of rabies virus neutralizing antibodies (RVNA) in blood sera. To simplify the multistep RFFIT procedure by eliminating the immunostaining step, we generated a new recombinant RV expressing a green fluorescent protein (rRV-GFP) and assess its suitability for quantifying RVNA. We rescued the rRV-GFP virus from plasmid DNA carrying a full-length genome of the CVS-N2c strain of RV in which the eGFP gene was inserted between the glycoprotein and RNA-polymerase genes. The recombinant virus was genetically stable and grew efficiently in appropriate cells expressing sufficient GFP fluorescence to detect directly 20 h post infection (hpi). We evaluated the feasibility of using rRV-GFP in RFFIT by comparing RVNA titers in 27 serum samples measured by conventional RFFIT and RFFIT-GFP. A linear regression analysis of the data demonstrated a good agreement between these two methods (r = 0.9776) including results with samples having RVNA titers close to the minimally acceptable vaccine potency threshold (0.5 IU/ml).Study results showed that the rRV-GFP virus could replace the CVS-11 challenge virus currently used in the conventional RFFIT and enabling more rapid, simpler, and less expensive detection and quantitation of RVNA.

A Recombinant Rabies Virus Expressing the Marburg Virus Glycoprotein Is Dependent upon Antibody-Mediated Cellular Cytotoxicity for Protection against Marburg Virus Disease in a Murine Model.

Marburg virus (MARV) is a filovirus related to Ebola virus (EBOV) associated with human hemorrhagic disease. Outbreaks are sporadic and severe, with a reported case mortality rate of upward of 88%. There is currently no antiviral or vaccine available. Given the sporadic nature of outbreaks, vaccines provide the best approach for long-term control of MARV in regions of endemicity. We have developed an inactivated rabies virus-vectored MARV vaccine (FILORAB3) to protect against Marburg virus disease. Immunogenicity studies in our labs have shown that a Th1-biased seroconversion to both rabies virus and MARV glycoproteins (GPs) is beneficial for protection in a preclinical murine model. As such, we adjuvanted FILORAB3 with glucopyranosyl lipid adjuvant (GLA), a Toll-like receptor 4 agonist, in a squalene-in-water emulsion. Across two different BALB/c mouse challenge models, we achieved 92% protection against murine-adapted Marburg virus (ma-MARV). Although our vaccine elicited strong MARV GP antibodies, it did not strongly induce neutralizing antibodies. Through both in vitro and in vivo approaches, we elucidated a critical role for NK cell-dependent antibody-mediated cellular cytotoxicity (ADCC) in vaccine-induced protection. Overall, these findings demonstrate that FILORAB3 is a promising vaccine candidate for Marburg virus disease.IMPORTANCE Marburg virus (MARV) is a virus similar to Ebola virus and also causes a hemorrhagic disease which is highly lethal. In contrast to EBOV, only a few vaccines have been developed against MARV, and researchers do not understand what kind of immune responses are required to protect from MARV. Here we show that antibodies directed against MARV after application of our vaccine protect in an animal system but fail to neutralize the virus in a widely used virus neutralization assay against MARV. This newly discovered activity needs to be considered more when analyzing MARV vaccines or infections.

Deficient Incorporation of Rabies Virus Glycoprotein into Virions Enhances Virus-Induced Immune Evasion and Viral Pathogenicity.

Previous studies have shown that wild-type (wt) rabies virus (RABV) evades the host immune response by restricting expression of glycoprotein (G), which blocks activation of dendritic cells (DCs) and induces production of virus-neutralizing antibodies (VNAs). In the present study, wt RABVs not only restricted G expression but also reduced incorporation of G into mature virions compared with laboratory-adapted viruses. A recombinant RABV expressing triple G was used to further determine whether G expression relates to incorporation. The recombinant virus showed higher expression and incorporation of G and activated more DCs than the virus that expressed a single copy of G. Removal of G from viruses using subtilisin or Dithiothreitol (DTT)/ Nonidet P-40 (NP40) almost completely abolishes DC activation and VNA production. Consequently, these G-depleted viruses cause lethal infection in mice. Thus, wt RABVs can subvert DC-induced antiviral immune response and maintain pathogenicity by decreasing G expression in infected cells and G incorporation into virions.

Higher primate-like direct corticomotoneuronal connections are transiently formed in a juvenile subprimate mammal

The corticospinal (CS) tract emerged and evolved in mammals, and is essentially involved in voluntary movement. Over its phylogenesis, CS innervation gradually invaded to the ventral spinal cord, eventually making direct connections with spinal motoneurons (MNs) in higher primates. Despite its importance, our knowledge of the origin of the direct CS-MN connections is limited; in fact, there is controversy as to whether these connections occur in subprimate mammals, such as rodents. Here we studied the retrograde transsynaptic connection between cortical neurons and MNs in mice by labeling the cells with recombinant rabies virus. On postnatal day 14 (P14), we found that CS neurons make direct connections with cervical MNs innervating the forearm muscles. Direct connections were also detected electrophysiologically in whole cell recordings from identified MNs retrogradely-labeled from their target muscles and optogenetic CS stimulation. In contrast, few, if any, lumbar MNs innervating hindlimbs showed direct connections on P18. Moreover, the direct CS-MN connections observed on P14 were later eliminated. The transient CS-MN cells were distributed predominantly in the M1 and S1 areas. These findings provide insight into the ontogeny and phylogeny of the CS projection and appear to settle the controversy about direct CS-MN connections in subprimate mammals.