Wild-type Rabies Virus Research Articles

Structural Heterogeneity of the Rabies Virus Virion.

Rabies virus (RABV) is among the first recognized viruses of public health concern and has historically contributed to the development of viral vaccines. Despite these significances, the three-dimensional structure of the RABV virion remains unknown due to the challenges in isolating structurally homogenous virion samples in sufficient quantities needed for structural investigation. Here, by combining the capabilities of cryogenic electron tomography (cryoET) and microscopy (cryoEM), we determined the three-dimensional structure of the wild-type RABV virion. Tomograms of RABV virions reveal a high level of structural heterogeneity among the bullet-shaped virion particles encompassing the glycoprotein (G) trimer-decorated envelope and the nucleocapsid composed of RNA, nucleoprotein (N), and matrix protein (M). The structure of the trunk region of the virion was determined by cryoEM helical reconstruction, revealing a one-start N-RNA helix bound by a single layer of M proteins at an N:M ratio of 1. The N-M interaction differs from that in fellow rhabdovirus vesicular stomatitis virus (VSV), which features two layers of M stabilizing the N-RNA helix at an M:N ratio of 2. These differences in both M-N stoichiometry and binding allow RABV to flex its N-RNA helix more freely and point to different mechanisms of viral assembly between these two bullet-shaped rhabdoviruses.

The NF-κB pathway negatively regulates the replication of rabies virus by triggering inflammatory responses

Rabies, which is caused by the rabies virus (RABV), is a neurological disease of mammals, including humans. The disease is untreatable, as drugs and antibodies are prevented from entering brain tissue by the blood-brain barrier (BBB). However, it has been reported that BBB permeability can be enhanced in mice infected with a laboratory-attenuated strain of RABV, which is not the case in those infected with wild-type RABV. Moreover, it is not the RABV infection directly that enhances BBB, but rather the inflammatory molecules that are induced by the virus. Understanding the mechanism underlying the RABV-induced production of chemokines and cytokines is crucial for the prevention and management of rabies. In this study, we found that mice infected with the RABV challenge strain CVS11 showed significant inflammatory infiltration in the later stages of infection. At the cellular level, we confirmed that the NF-κB signaling pathway was activated following RABV infection, leading to significant upregulation of the expression of downstream chemokines and inflammatory factors (CCL2, CCL5, CXCL10, TNF-α, and IL-6). Surprisingly, the replication of RABV was upregulated when the NF-κB pathway was inhibited, and the levels of its downstream chemokines and inflammatory factors were reduced following treatment with the inhibitor JSH-23. This suggests that RABV infection activates the NF-κB pathway, which is then able to negatively regulate the replication of RABV. This pathway is therefore a potential target for drugs and other therapies in the treatment of rabies.

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.

A disynaptic basal ganglia connection to the inferior olive: potential for basal ganglia influence on cerebellar learning.

Recent studies have shown that the cerebellum and the basal ganglia are interconnected at subcortical levels. However, a subcortical basal ganglia connection to the inferior olive (IO), being the source of the olivocerebellar climbing fiber system, is not known. We have used classical tracing with CTb, retrograde transneuronal infection with wildtype rabies virus, conditional tracing with genetically modified rabies virus, and examination of material made available by the Allen Brain Institute, to study potential basal ganglia connections to the inferior olive in rats and mice. We show in both species that parvalbumin-positive, and therefore GABAergic, neurons in the entopeduncular nucleus, representing the rodent equivalent of the internal part of the globus pallidus, innervate a group of cells that surrounds the fasciculus retroflexus and that are collectively known as the area parafascicularis prerubralis. As these neurons supply a direct excitatory input to large parts of the inferior olivary complex, we propose that the entopeduncular nucleus, as a main output station of the basal ganglia, provides an inhibitory influence on olivary excitability. As such, this connection may influence olivary involvement in cerebellar learning and/or could be involved in transmission of reward properties that have recently been established for olivocerebellar signaling.

Gene Expression Profile Induced by Two Different Variants of Street Rabies Virus in Mice.

Pathogenicity and pathology of rabies virus (RABV) varies according to the variant, but the mechanisms are not completely known. In this study, gene expression profile in brains of mice experimentally infected with RABV isolated from a human case of dog rabies (V2) or vampire bat-acquired rabies (V3) were analyzed. In total, 138 array probes associated with 120 genes were expressed differentially between mice inoculated with V2 and sham-inoculated control mice at day 10 post-inoculation. A single probe corresponding to an unannotated gene was identified in V3 versus control mice. Gene ontology (GO) analysis revealed that all of the genes upregulated in mice inoculated with V2 RABV were involved in the biological process of immune defense against pathogens. Although both variants are considered pathogenic, inoculation by the same conditions generated different gene expression results, which is likely due to differences in pathogenesis between the dog and bat RABV variants. This study demonstrated the global gene expression in experimental infection due to V3 wild-type RABV, from the vampire bat Desmodus rotundus, an important source of infection for humans, domestic animals and wildlife in Latin America.

Early Transcriptional Changes in Rabies Virus-Infected Neurons and Their Impact on Neuronal Functions.

Rabies is a zoonotic disease caused by rabies virus (RABV). As rabies advances, patients develop a variety of severe neurological symptoms that inevitably lead to coma and death. Unlike other neurotropic viruses that can induce symptoms of a similar range, RABV-infected post-mortem brains do not show significant signs of inflammation nor the structural damages on neurons. This suggests that the observed neurological symptoms possibly originate from dysfunctions of neurons. However, many aspects of neuronal dysfunctions in the context of RABV infection are only partially understood, and therefore require further investigation. In this study, we used differentiated neurons to characterize the RABV-induced transcriptomic changes at the early time-points of infection. We found that the genes modulated in response to the infection are particularly involved in cell cycle, gene expression, immune response, and neuronal function-associated processes. Comparing a wild-type RABV to a mutant virus harboring altered matrix proteins, we found that the RABV matrix protein plays an important role in the early down-regulation of host genes, of which a significant number is involved in neuronal functions. The kinetics of differentially expressed genes (DEGs) are also different between the wild type and mutant virus datasets. The number of modulated genes remained constant upon wild-type RABV infection up to 24 h post-infection, but dramatically increased in the mutant condition. This result suggests that the intact viral matrix protein is important to control the size of host gene modulation. We then examined the signaling pathways previously studied in relation to the innate immune responses against RABV, and found that these pathways contribute to the changes in neuronal function-associated processes. We further examined a set of regulated genes that could impact neuronal functions collectively, and demonstrated in calcium imaging that indeed the spontaneous activity of neurons is influenced by RABV infection. Overall, our findings suggest that neuronal function-associated genes are modulated by RABV early on, potentially through the viral matrix protein-interacting signaling molecules and their downstream pathways.

Change in the Single Amino Acid Site 83 in Rabies Virus Glycoprotein Enhances the BBB Permeability and Reduces Viral Pathogenicity.

Lab-attenuated rabies virus (RABV) is a highly cellular adaptation and less pathogenic than wild-type RABV. However, the molecular mechanisms that regulate the cellular adaptation and pathogenicity remain obscure. In this work, we isolated a wild-type RABV (CNIM1701) from a rabid bovine in northern China. The original CNIM1701 was lethal in adult mice and restricted replication in cell cultures. After 20 serial passages in the brains of suckling mice, the virus was renamed CNIM1701-P20, which was safe in adult mice and replicated well in cell cultures. In addition, sequence comparison analysis of the original CNIM1701 and CNIM1701-P20 identified 2 amino acid substitutions on G protein (Lys83 → Arg83 and Pro367 → Ser 367) related to pathogenesis and cellular adaptation. Using site-directed mutagenesis to exchange Lys83 with Arg83 and Pro367 with Ser 367 in the G protein of the RABV SAD strain, the pathogenicity of rSAD-K83R was significantly decreased. Our data indicate that the decreased pathogenicity of rSAD-K83R is due to increasing the expression of RABV-G, which also induced a higher level of apoptosis in infected cells. Furthermore, the K83 mutation induced high expression of MMP-2 and MMP-9 on DCs and promoted blood–brain barrier (BBB) permeability. These results demonstrate that the pathogenesis of RABV is partially dependent on G expression and BBB permeability, which may help in the design and development of highly safe, live-RABV vaccines.

Comparison of lncRNA and mRNA expression in mouse brains infected by a wild-type and a lab-attenuated Rabies lyssavirus

Rabies is a lethal disease caused by Rabies lyssavirus, commonly known as rabies virus (RABV), and results in nearly 100 % death once clinical symptoms occur in human and animals. Long non-coding RNAs (lncRNAs) have been reported to be associated with viral infection. But the role of lncRNAs involved in RABV infection is still elusive. In this study, we performed global transcriptome analysis of both of lncRNA and mRNA expression profiles in wild-type (WT) and lab-attenuated RABV-infected mouse brains by using next-generation sequencing. The differentially expressed lncRNAs and mRNAs were analysed by using the edgeR package. We identified 1422 differentially expressed lncRNAs and 4475 differentially expressed mRNAs by comparing WT and lab-attenuated RABV-infected brains. Then we predicted the enriched biological pathways by the Gene Ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) database based on the differentially expressed lncRNAs and mRNAs. Our analysis revealed the relationships between lncRNAs and RABV-infection-associated immune response and ion transport-related pathways, which provide a fresh insight into the potential role of lncRNA in immune evasion and neuron injury induced by WT RABV.

Brain tissue-resident Immune Memory Cells are Required for long-term Protection Against CNS Infection With Rabies Virus

Immune memory cells residing in previously infected, nonlymphoid tissues play a role in immune surveillance. In the event that circulating antibodies fail to prevent virus spread to the tissues in a secondary infection, these memory cells provide an essential defense against tissue reinfection. CNS tissues are isolated from circulating immune cells and antibodies by the blood-brain barrier, making the presence of tissue-resident immune memory cells particularly needed to combat recurrent infection by neurotropic viruses. Wild-type and laboratory-engineered rabies virusesare neurotropic, differ in pathogenicity, and have varying effects on BBB functions. These viruses have proven invaluable tools in demonstrating the importance of tissue-resident immune memory cells in the reinfection of CNS tissues. Only Type 1 immune memory is effective at therapeutically clearing a secondary infection with wild-type rabies viruses from the CNS and does so despite the maintenance of blood-brain barrierintegrity.

Phosphoprotein Gene of Wild-Type Rabies Virus Plays a Role in Limiting Viral Pathogenicity and Lowering the Enhancement of BBB Permeability.

Enhancement of blood–brain barrier (BBB) permeability is necessary for clearing virus in the central nervous system (CNS). It has been reported that only laboratory-attenuated rabies virus (RABV) induces inflammatory response to lead BBB transient breakdown rather than wild-type (wt) strains. As a component of ribonucleoprotein (RNP), phosphoprotein (P) of RABV plays a key role in viral replication and pathogenicity. To our knowledge, the function of RABV P gene during RABV invasion was unclear so far. In order to determine the role of RABV P gene during RABV infection, we evaluated the BBB permeability in vivo after infection with wt RABV strain (GD-SH-01), a lab-attenuated RABV strain (HEP-Flury), and a chimeric RABV strain (rHEP-SH-P) whose P gene cloned from GD-SH-01 was expressed in the genomic backbone of HEP-Flury. We found that rHEP-SH-P caused less enhancement of BBB permeability and was less pathogenic to adult mice than GD-SH-01 and HEP-Flury. In an effort to investigate the mechanism, we found that the replication of rHEP-SH-P has been limited due to the suppressed P protein expression and induced less response to maintain BBB integrity. Our data indicated that the P gene of wt RABV was a potential determinant in hampering viral replication in vivo, which kept BBB integrity. These findings provided an important foundation for understanding the viral invasion and development of novel vaccine.

Generation of Monoclonal Antibodies against Variable Epitopes of the M Protein of Rabies Virus.

Rabies virus (RABV), the causative agent of rabies, is highly neurovirulent for warm-blooded animals with a mortality rate of up to 100%. The RABV matrix protein (M) is required for virus particle assembly and budding. However, little is known about antigenic differences in the M protein. In this study, five monoclonal antibodies (mAbs), designated 3B9, 4A1, 2B11, 2C1, and 4B11, against the RABV M protein were generated using a recombinant M protein. All five mAbs reacted with the CVS-11 strain but showed no reactivity against the HEP-Flury strain in indirect immunofluorescence and western blotting. The epitope targeted by these mAbs was further identified by peptide scanning using GST-fused peptides. The 25PPYDDD30 peptide was defined as the minimal linear epitope. Alignment of amino acid sequences and phylogenetic analysis of different RABV strains indicated that the variable epitope 25PPDGDD30 is only present in the HEP-Flury and variant Flury strains of clade III, while the other strains resembling ERA and SRVA9 within the clade had another variable epitope, 25PLDDDD30. A Y27D mutation within the epitope was found among the rest of the RABV strains distributed in different clades. However, a single D28G mutation eliminated the reactivity of these five mAbs. In addition, the mAbs were able to recognize wildtype RABV strain in indirect immunofluorescence and western blotting and detect RABV-infected brain tissue using immunohistochemistry. The newly established mAbs and identified epitope may facilitate future investigations in the structure and function of the M protein and the development of diagnostic methods for the detection of different RABV strains worldwide. Most importantly, the epitope recognized by the mAbs against M protein might serve as a novel target for the development of a vaccine targeting RABV virulent strains.

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.

Molecular characterization of a rabies virus isolated from trade dogs in Plateau State, Nigeria

To have a better understanding of the prevalence of rabies virus (RABV) in the dog trading markets in Plateau State of Nigeria, a total of 532 brain samples collected between 2010 and 2011 were subjected to rabies virus antigen detection and molecular characterization. RABV antigen was detected in 92 out of 532 (17.3%) brain samples from two major commercial dog markets in Plateau State of Nigeria by direct fluorescence antibody assay, indicating the possibility of a high prevalence of RABV in the dog trading markets. The complete genomic sequence was obtained from one of these RABV positive samples. The overall organization of this virus (DRV-NG11) was typical of all the other wild-type RABV. Phylogenetic analysis using either the complete or partial genomic sequence of DRV-NG11 demonstrates that this isolate is most closely related to viruses previously shown to circulate in Nigeria, belonging to the Africa 2 Clade. To our knowledge, this is the first report on the molecular characterization of the complete genome of RABV from trade dogs, which provides a better understanding of the molecular epidemiology, pathogenesis and rabies control in this country.Keywords: Genome, Nigeria, Rabies virus, Street strain, Trade dogs

Immunization with recombinant rabies virus expressing Interleukin-18 exhibits enhanced immunogenicity and protection in mice.

Several studies have shown that interleukin-18 (IL-18) plays an important role in both innate and adaptive immune responses. In this study, we investigated the pathogenicity and immunogenicity of recombinant rabies virus expressing IL-18 (rHEP-IL18). Experimental results showed that Institute of Cancer Research (ICR) mice that received a single intramuscular immunization with rHEP-IL18 elicited the highest titers of serum neutralizing antibodies and the strongest cell-mediated immune responses to prevent the development of rabies disease, compared with immunization with the parent virus HEP-Flury. Mice inoculated with rHEP-IL18 developed significantly higher IFN-γ responses, increased percentages of CD4+ and CD8+ T-lymphocytes compared to HEP-Flury. Flow cytometry results show that rHEP-IL18 recruited more activated T- and B-cells in lymph nodes or peripheral blood, which is beneficial for virus clearance in the early stages of infection. A higher percentage of mice immunized with rHEP-IL18 survived wild-type rabies virus (RABV) challenge, compared to HEP-Flury mice. Our results show that rHEP-IL18 is promising as a novel vaccine for RABV prevention and control.

Phosphoprotein Gene Contributes to the Enhanced Apoptosis Induced by Wild-Type Rabies Virus GD-SH-01 In Vitro.

Previous research demonstrated that the matrix protein (M) and glycoprotein (G) of attenuated rabies virus (RABV) strains are involved in the induction of host cell apoptosis. In this work, we show that wild-type (wt) RABV GD-SH-01 induces significantly greater apoptosis than the attenuated strain HEP-Flury. In order to identify the gene(s) accounting for this phenotype, five recombinant RABVs (rRABVs) were constructed by replacing each single gene of HEP-Flury with the corresponding gene of GD-SH-01. By using these rRABVs, we found that not only M and G, but also the phosphoprotein (P) plays an important role in inducing apoptosis. In order to figure out the different role of P gene in inducing apoptosis from the highly divergent background, another rRABV rGDSH-P, which carries the P gene of HEP-Flury in the background of the GD-SH-01 was generated. It was found that infection of NA cells with GD-SH-01 or the recombinant strain rHEP-shP, which carries P gene of GD-SH-01, induced significantly greater apoptosis than HEP-Flury or rGDSH-P in a caspase-dependent pathway that ultimately leads to the activation of the intrinsic apoptotic pathway, which is well characterized with the downregulation of bcl-2, the decrease of mitochondrial membrane potential, the release of mitochondrial cytochrome c, the activation of caspase-9 and caspase-3, and finally the cleavage of poly (ADP-ribose) polymerase. Our results imply that wt P from GD-SH-01 mediates this effect may partly by facilitating viral RNA synthesis but not by viral replication. In sum, we demonstrate a wt RABV strain GD-SH-01 to induce stronger apoptosis than an attenuated RABV HEP-Flury and propose that wt P from GD-SH-01 is involved in this process.

Protection Against CNS-Targeted Rabies Virus Infection is Dependent upon Type-1 Immune Mechanisms Induced by Live-Attenuated Rabies Vaccines.

Rabies remains a major public health issue worldwide, especially in developing countries where access to medical care can represent a real challenge. While there is still no cure for rabies, it is a vaccine-preventable disease with pre- and post-exposure prophylaxis regimens approved by the World Health Organization (WHO). However, many rabies-exposed individuals have limited access to vaccines and virus-neutralizing antibodies approved for post-exposure prophylaxis. Unfortunately, any delay in the administration of these reagents can have lethal consequences. This highlights the need to develop cost-effective immunological reagents with a greater window of efficacy. Live-attenuated vaccine strains of rabies virus presents a potential treatment in filling this gap. We show here that immunization with live-attenuated vaccines provide long-lasting rabies immunity, superior to the protection induced by inactivated vaccines. In the absence of an immunostimulatory adjuvant, vaccination with multiple doses of inactivated rabies virus induces a type-2 immune response. This type of immunity is highly effective at inducing neutralizing antibody but has limited efficacy in clearing the virus from central nervous system (CNS) tissues. In contrast, a single infection with live-attenuated rabies vaccine safely drives a type-1 immune response, associated with both the production of a neutralizing antibody and the clearance of wild-type rabies virus from CNS tissues. These results indicate that live-attenuated rabies strains have the potential to be more effective in post-exposure prophylaxis than conventional inactivated vaccines.

Rescue of a wild-type rabies virus from cloned cDNA and assessment of the proliferative capacity of recombinant viruses.

Reverse genetic systems (RGS) have been widely used for fixed rabies virus (RABV) strains. However, RGS, for wild-type (wt) strains, have been seldom reported despite the value of this approach in defining the biological characteristics of these strains. In this work, we developed a wt RGS using a swine-origin RABV strain (GD-SH-01) for the first time. In order to have a better understanding of the contribution and function of individual gene on viral proliferation for wt RABV isolates, we constructed a full-length cDNA clone of GD-SH-01 and exchanged the single genes encoding RABV protein of a highly attenuated RABV strain HEP-Flury with those of the virulent strain. Analysis of the viral growth kinetics, cell-to-cell spread, and genomic RNA (gRNA) synthesis of the both the rescued and parental virus strains revealed that replacement of the HEP-Flury N or L genes with those from GD-SH-01 resulted in higher proliferative capacity of both chimeric rHEP-shN and rHEP-shL while the former seemed to have a better viral gRNA synthesis ability, the latter spread faster. Replacement of HEP-Flury P gene with GD-SH-01 P gene resulted in reduction of the virus titer in cell culture supernatants with a poor replicative and spreading ability. However, replacement of HEP-Flury M or G genes with those from GD-SH-01 seemed to impact less on viral proliferation. Taken together, we show that we have successfully rescued a wt RABV strain, and assessed the impact of each gene on viral proliferative capacity using a series of single-gene-substituted viruses.

The ectodomain of rabies virus glycoprotein determines dendritic cell activation

The immune evasion of wild-type (wt) rabies virus (RABV) has been attributed to its glycoprotein (G), particularly to their inefficiency to bind/enter into dendritic cells (DCs). However, the domain responsible for G-mediated DC activation is not clear. In the present study, attempts were made to map the domain(s) on the G involved in differential DC activation using laboratory-adapted and wt viruses. Recombinant RABVs with exchange in each of the structural domains such as signal peptide (sp), ectodomain (et), transmembrane domain (tm), cytoplasmic tail (ct) of the G between wt and laboratory-adapted strains were constructed. Characterizations of these recombinant RABVs show that the viruses containing the sp, tm and ct from the wt G are capable of growing in high titer by efficient cell-to-cell spread, similar to laboratory-adapted virus. On the other hand, recombinant virus containing the et domain from wt G was inefficient in cell-to-cell spread and grew in lower levels, similar to the wt RABV. Analysis of DC activation shows that viruses containing sp and tm from wt G are efficient in binding to and activating DCs. However, viruses containing the et domain from wt G are incompetent in binding to and activating DCs. Analysis of the G expression in the infected cells suggests that the level of G expression is regulated solely by the ct domain, indicating the level of G expression and DC activation are governed by different domains. Together, our results demonstrate that G-mediated DC activation is regulated by the et domain while the level of G expression by the ct domain.

Ineffectiveness of rabies vaccination alone for post-exposure protection against rabies infection in animal models

Most reported vaccination failures among rabies-exposed patients were due to fail to timely co-administer rabies immunoglobulin (RIG). Considering that such protection failure might be caused by low antigen titers in the vaccine, scientists improved antigen titers to 4.0 IU or even higher, yet the failure remained. Therefore, it becomes vital to develop more efficacious vaccine against rabies. In our evaluation of a novel PIKA rabies vaccine, we used multiple animal models (beagles, golden hamsters and Kunming mice) to mimic post-exposure scenarios. All animals were challenged with wild-type rabies virus, followed by vaccination with either rabies vaccines commercially available or PIKA rabies vaccines. As 100% of animals survived after administration of traditional rabies vaccines and rabies immunoglobulin, 80% of animals survived with rabies immunoglobulin alone. Strikingly, animals receiving traditional rabies vaccines alone showed extremely low survival rates, indicating insignificant benefit for exposed animals (p > 0.05, compared to unvaccinated control groups). To the contrary, 40–80% of animals receiving the experimental PIKA rabies vaccines were protected (p < 0.05, compared to unvaccinated control groups). If the above results are fully confirmed, we may conclude that currently as high as 99% of post-exposure patients who are seeking protection against rabies, but only receiving rabies vaccination, could be meaningless.

Wild-type rabies virus induces autophagy in human and mouse neuroblastoma cell lines

ABSTRACTDifferent rabies virus (RABV) strains have their own biological characteristics, but little is known about their respective impact on autophagy. Therefore, we evaluated whether attenuated RABV HEP-Flury and wild-type RABV GD-SH-01 strains triggered autophagy. We found that GD-SH-01 infection significantly increased the number of autophagy-like vesicles, the accumulation of enhanced green fluorescent protein (EGFP)-LC3 fluorescence puncta and the conversion of LC3-I to LC3-II, while HEP-Flury was not able to induce this phenomenon. When evaluating autophagic flux, we found that GD-SH-01 infection triggers a complete autophagic response in the human neuroblastoma cell line (SK), while autophagosome fusion with lysosomes was inhibited in a mouse neuroblastoma cell line (NA). In these cells, GD-SH-01 led to apoptosis and mitochondrial dysfunction while triggering autophagy, and apoptosis could be decreased by enhancing autophagy. To further identify the virus constituent causing autophagy, 5 chimeric recombinant viruses carrying single genes of HEP-Flury instead of those of GD-SH-01 were rescued. While the HEP-Flury virus carrying the wild-type matrix protein (M) gene of RABV triggered LC3-I to LC3-II conversion in SK and NA cells, replacement of genes of nucleoprotein (N), phosphoprotein (P) and glycoprotein (G) produced only minor autophagy. But no one single structural protein of GD-SH-01 induced autophagy. Moreover, the AMPK signaling pathway was activated by GD-SH-01 in SK. Therefore, our data provide strong evidence that autophagy is induced by GD-SH-01 and can decrease apoptosis in vitro. Furthermore, the M gene of GD-SH-01 may cooperatively induce autophagy.