Rabies Virus Regulates Inflammatory Response in BV-2 Cells through Activation of Myd88 and NF-κB Signaling Pathways via TLR7.

Rabies is a fatal neurological infectious disease caused by rabies virus (RABV), which invades the central nervous system (CNS). RABV with varying virulence regulates chemokine expression, and the mechanisms of signaling pathway activation remains to be elucidated. The relationship between Toll-like receptors (TLRs) and immune response induced by RABV has not been fully clarified. Here, we investigated the role of TLR7 in the immune response induced by RABV, and one-way analysis of variance (ANOVA) was employed to evaluate the data. We found that different RABV strains (SC16, HN10, CVS-11) significantly increased CCL2, CXCL10 and IL-6 production. Blocking assays indicated that the TLR7 inhibitor reduced the expression of CCL2, CXCL10 and IL-6 (p < 0.01). The activation of the Myd88 pathway in BV-2 cells stimulated by RABV was TLR7-dependent, whereas the inhibition of Myd88 activity reduced the expression of CCL2, CXCL10 and IL-6 (p < 0.01). Meanwhile, the RABV stimulation of BV-2 cells resulted in TRL7-mediated activation of NF-κB and induced the nuclear translocation of NF-κB p65. CCL2, CXCL10 and IL-6 release was attenuated by the specific NF-κB inhibitor used (p < 0.01). The findings above demonstrate that RABV-induced expression of CCL2, CXCL10 and IL-6 involves Myd88 and NF-κB pathways via the TLR7 signal.

ß-d-N4-hydroxycytidine, a metabolite of molnupiravir, exhibits in vitro antiviral activity against rabies virus.

Rabies is a fatal neurological disorder caused by rabies virus (RABV) infection. Approximately 60,000 patients die from rabies annually, and there are no effective treatments for this disease. Nucleoside analogs are employed as antiviral drugs based on their broad antiviral spectrum, and certain nucleoside analogs have been reported to exhibit anti-RABV activity. The nucleoside analog ß-d-N4-hydroxycytidine (NHC) has antiviral effects against a range of RNA viruses. Molnupiravir (MPV), a prodrug of NHC, is clinically used as an oral antiviral drug for coronavirus infections. Despite its broad-spectrum activity, the antiviral activity of NHC against RABV remains unclear. In this study, we reveal that NHC exhibits comparable in vitro anti-RABV activity as ribavirin and favipiravir (also known as T-705) with a 90% effective concentration of 6 µM in mouse neuroblastoma cells. NHC reduced viral loads in neuronal and nonneuronal cells in a dose-dependent manner. Both laboratory and field RABVs (fixed and street strains, respectively) were susceptible to NHC. However, no increase in survival or reduction in viral titers in the brain was observed in RABV-infected mice treated prophylactically with MPV. These findings highlight the potential and challenges of NHC in the treatment of RABV infection.

Application prospects of the 2BS cell-adapted China fixed rabies virus vaccine strain 2aG4-B40.

BACKGROUND: Rabies is a fatal zoonotic disease whose pathogenesis has not been fully elucidated, and vaccination is the only effective method for protecting against rabies virus infection. Most inactivated vaccines are produced using Vero cells, which are African green monkey kidney cells, to achieve large-scale production. However, there is a potential carcinogenic risk due to nonhuman DNA contamination. Thus, replacing Vero cells with human diploid cells may be a safer strategy. In this study, we developed a novel 2BS cell-adapted rabies virus strain and analysed its sequence, virulence and immunogenicity to determine its application potential as a human diploid cell inactivated vaccine. METHODS AND RESULTS: The 2BS cell-adapted rabies virus strain 2aG4-B40 was established by passage for 40 generations and selection of plaques in 2BS cells. RNA sequence analysis revealed that mutations in 2BS cell-adapted strains were not located at key sites that regulate the production of neutralizing antibodies or virulence in the aG strain (GQ412744.1). The gradual increase in virulence (remaining above 7.0 logLD50/ml from the 40th to 55th generation) and antigen further indicated that these mutations may increase the affinity of the adapted strains for human diploid cells. Identification tests revealed that the 2BS cell-adapted virus strain was neutralized by anti-rabies serum, with a neutralization index of 19,952. PrEP and PEP vaccination and the NIH test further indicated that the vaccine prepared with the 2aG4-B40 strain had high neutralizing antibody levels (2.24 to 46.67 IU/ml), immunogenicity (protection index 270) and potency (average 11.6 IU/ml). CONCLUSIONS: In this study, a 2BS cell-adapted strain of the 2aG4 rabies virus was obtained by passage for 40 generations. The results of sequencing analysis and titre determination of the adapted strain showed that the mutations in the adaptive process are not located at key sequence regions of the virus, and these mutations may enhance the affinity of the adapted strain for human diploid cells. Moreover, vaccines made from the adapted strain 2aG4-B40 had high potency and immunogenicity and could be an ideal candidate rabies virus strain for inactivated vaccine preparation.

Rabies Virus Infection Causes Pyroptosis of Neuronal Cells.

Rabies virus (RABV) is a neurotropic virus that causes fatal neurological disease, raising serious public health issues and attracting extensive attention in society. To elucidate the molecular mechanism of RABV-induced neuronal damage, we used hematoxylin-eosin staining, transmission electron microscopy, transcriptomics analysis, and immune response factor testing to investigate RABV-infected neurons. We successfully isolated the neurons from murine brains. The specificity of the isolated neurons was identified by a monoclonal antibody, and the viability of the neurons was 83.53-95.0%. We confirmed that RABV infection induced serious damage to the neurons according to histochemistry and transmission electron microscope (TEM) scanning. In addition, the transcriptomics analysis suggested that multiple genes related to the pyroptosis pathway were significantly upregulated, including gasdermin D (Gsdmd), Nlrp3, caspase-1, and IL-1ß, as well as the chemokine genes Ccl2, Ccl3, Ccl4, Ccl5, Ccl7, Ccl12, and Cxcl10. We next verified this finding in the brains of mice infected with the rRC-HL, GX074, and challenge virus standard strain-24 (CVS-24) strains of RABV. Importantly, we found that the expression level of the Gsdmd protein was significantly upregulated in the neurons infected with different RABV strains and ranged from 691.1 to 5764.96 pg/mL, while the basal level of mock-infected neurons was less than 100 pg/mL. Taken together, our findings suggest that Gsdmd-induced pyroptosis is involved in the neuron damage caused by RABV infection.

The human alpha7 nicotinic acetylcholine receptor is a host target for the rabies virus glycoprotein.

The rabies virus enters the nervous system by interacting with several molecular targets on host cells to modify behavior and trigger receptor-mediated endocytosis of the virion by poorly understood mechanisms. The rabies virus glycoprotein (RVG) interacts with the muscle acetylcholine receptor and the neuronal α4ß2 subtype of the nicotinic acetylcholine receptor (nAChR) family by the putative neurotoxin-like motif. Given that the neurotoxin-like motif is highly homologous to the α7 nAChR subtype selective snake toxin α-bungarotoxin (αBTX), other nAChR subtypes are likely involved. The purpose of this study is to determine the activity of the RVG neurotoxin-like motif on nAChR subtypes that are expressed in brain regions involved in rabid animal behavior. nAChRs were expressed in Xenopus laevis oocytes, and two-electrode voltage clamp electrophysiology was used to collect concentration-response data to measure the functional effects. The RVG peptide preferentially and completely inhibits α7 nAChR ACh-induced currents by a competitive antagonist mechanism. Tested heteromeric nAChRs are also inhibited, but to a lesser extent than the α7 subtype. Residues of the RVG peptide with high sequence homology to αBTX and other neurotoxins were substituted with alanine. Altered RVG neurotoxin-like peptides showed that residues phenylalanine 192, arginine 196, and arginine 199 are important determinants of RVG peptide apparent potency on α7 nAChRs, while serine 195 is not. The evaluation of the rabies ectodomain reaffirmed the observations made with the RVG peptide, illustrating a significant inhibitory impact on α7 nAChR with potency in the nanomolar range. In a mammalian cell culture model of neurons, we confirm that the RVG peptide binds preferentially to cells expressing the α7 nAChR. Defining the activity of the RVG peptide on nAChRs expands our understanding of basic mechanisms in host-pathogen interactions that result in neurological disorders.

TRIM44 Promotes Rabies Virus Replication by Autophagy-Dependent Mechanism.

Tripartite motif (TRIM) proteins are a multifunctional E3 ubiquitin ligase family that participates in various cellular processes. Recent studies have shown that TRIM proteins play important roles in regulating host-virus interactions through specific pathways, but their involvement in response to rabies virus (RABV) infection remains poorly understood. Here, we identified that several TRIM proteins are upregulated in mouse neuroblastoma cells (NA) after infection with the rabies virus using RNA-seq sequencing. Among them, TRIM44 was found to regulate RABV replication. This is supported by the observations that downregulation of TRIM44 inhibits RABV replication, while overexpression of TRIM44 promotes RABV replication. Mechanistically, TRIM44-induced RABV replication is brought about by activating autophagy, as inhibition of autophagy with 3-MA attenuates TRIM44-induced RABV replication. Additionally, we found that inhibition of autophagy with rapamycin reverses the TRIM44-knockdown-induced decrease in LC3B expression and autophagosome formation as well as RABV replication. The results suggest that TRIM44 promotes RABV replication by an autophagy-dependent mechanism. Our work identifies TRIM44 as a key host factor for RABV replication, and targeting TRIM44 expression may represent an effective therapeutic strategy.

Single Amino Acid Substitution in the Matrix Protein of Rabies Virus Is Associated with Neurovirulence in Mice.

Rabies is a fatal encephalitic infectious disease caused by the rabies virus (RABV). RABV is highly neurotropic and replicates in neuronal cell lines in vitro. The RABV fixed strain, HEP-Flury, was produced via passaging in primary chicken embryonic fibroblast cells. HEP-Flury showed rapid adaptation when propagated in mouse neuroblastoma (MNA) cells. In this study, we compared the growth of our previously constructed recombinant HEP (rHEP) strain-based on the sequence of the HEP (HEP-Flury) strain-with that of the original HEP strain. The original HEP strain exhibited higher titer than rHEP and a single substitution at position 80 in the matrix (M) protein M(D80N) after incubation in MNA cells, which was absent in rHEP. In vivo, intracerebral inoculation of the rHEP-M(D80N) strain with this substitution resulted in enhanced viral growth in the mouse brain and a significant loss of body weight in the adult mice. The number of viral antigen-positive cells in the brains of adult mice inoculated with the rHEP-M(D80N) strain was significantly higher than that with the rHEP strain at 5 days post-inoculation. Our findings demonstrate that a single amino acid substitution in the M protein M(D80N) is associated with neurovirulence in mice owing to adaptation to mouse neuronal cells.

RABV induces biphasic actin cytoskeletal rearrangement through Rac1 activity modulation.

Rabies virus (RABV) is highly lethal and triggers severe neurological symptoms. The neuropathogenic mechanism remains poorly understood. Ras-related C3 botulinum toxin substrate 1 (Rac1) is a Rho-GTPase that is involved in actin remodeling and has been reported to be closely associated with neuronal dysfunction. In this study, by means of a combination of pharmacological inhibitors, small interfering RNA, and specific dominant-negatives, we characterize the crucial roles of dynamic actin and the regulatory function of Rac1 in RABV infection, dominantly in the viral entry phase. The data show that the RABV phosphoprotein interacts with Rac1. RABV phosphoprotein suppress Rac1 activity and impedes downstream Pak1-Limk1-Cofilin1 signaling, leading to the disruption of F-actin-based structure formation. In early viral infection, the EGFR-Rac1-signaling pathway undergoes a biphasic change, which is first upregulated and subsequently downregulated, corresponding to the RABV entry-induced remodeling pattern of F-actin. Taken together, our findings demonstrate for the first time the role played by the Rac1 signaling pathway in RABV infection and may provide a clue for an explanation for the etiology of rabies neurological pathogenesis.IMPORTANCEThough neuronal dysfunction is predominant in fatal rabies, the detailed mechanism by which rabies virus (RABV) infection causes neurological symptoms remains in question. The actin cytoskeleton is involved in numerous viruses infection and plays a crucial role in maintaining neurological function. The cytoskeletal disruption is closely associated with abnormal nervous symptoms and induces neurogenic diseases. In this study, we show that RABV infection led to the rearrangement of the cytoskeleton as well as the biphasic kinetics of the Rac1 signal transduction. These results help elucidate the mechanism that causes the aberrant neuronal processes by RABV infection and may shed light on therapeutic development aimed at ameliorating neurological disorders.

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.

Virología molecular de la rabia: un enfoque clínico / Rabies molecular virology: a clinical approach

Introducción: La rabia es una enfermedad zoonótica asociada al virus RABV, el cual tiene características neurotrópicas. El virus se transmite por el contacto con saliva de animales infectados; la mordedura de un perro es la causa más común. Es un virus que causa la muerte de miles de personas cada año. Objetivo: Describir a profundidad los principios moleculares de la infección por rabia, así como su patogenia, diagnóstico y tratamiento. Métodos: Se realizó una búsqueda de bibliografía en PubMed, SciELO, Scopus, Researchgate; se consultaron 163 referencias y se seleccionaron 51 fuentes que contenían la información más relevante para cumplir con el objetivo del trabajo. Conclusión: Actualmente es posible entender de mejor manera los mecanismos de transmisión y propagación del virus en el organismo; existe nuevo conocimiento sobre los receptores involucrados, así como la función de estos en la replicación viral. Sin embargo, el objetivo de la erradicación de la rabia a corto plazo es complejo. La invasión de territorios selváticos vuelve a la rabia un posible patógeno reemergente; la vacunación de especies transmisoras es el medio ideal para conseguir el control de la enfermedad.

Introduction: Rabies is a zoonotic disease associated with the RABV virus, which has neurotropic characteristics. The virus is transmitted by contact with saliva from infected animals; a dog's bite is the most common cause. This virus causes the death of thousands of people every year. Objective: To describe in depth the molecular principles of rabies infection, as well as its pathogenesis, diagnosis and treatment. Methods: A literature search was conducted in PubMed, SciELO, Scopus, and Researchgate. A total of 163 references were consulted, and 51 sources containing the most relevant information were selected to fulfill the objective of the work. Conclusions: It is currently possible to better understand the mechanisms of transmission and spread of the virus in the organism; there is new knowledge about the receptors involved, as well as their function in viral replication. However, the goal of eradicating rabies in the short term is complex. The invasion of wild territories makes rabies a possible re-emerging pathogen; vaccination of transmitting species is the ideal means to achieve disease control.

Lipid Droplets Are Beneficial for Rabies Virus Replication by Facilitating Viral Budding.

Rabies is an old zoonotic disease caused by rabies virus (RABV), but the pathogenic mechanism of RABV is still not completely understood. Lipid droplets (LDs) have been reported to play a role in pathogenesis of several viruses. However, their role in RABV infection remains unclear. Here, we initially found that RABV infection upregulated LD production in multiple cells and mouse brains. After treatment with atorvastatin, a specific inhibitor of LDs, RABV replication in N2a cells decreased. Then we found that RABV infection could upregulate N-myc downstream regulated gene-1 (NDRG1), which in turn enhanced the expression of diacylglycerol acyltransferase 1/2 (DGAT1/2). DGAT1/2 could elevate cellular triglyceride synthesis and ultimately promote intracellular LD formation. Furthermore, we found that RABV-M and RABV-G, which were mainly involved in the viral budding process, could colocalize with LDs, indicating that RABV might utilize LDs as a carrier to facilitate viral budding and eventually increase virus production. Taken together, our study reveals that lipid droplets are beneficial for RABV replication, and their biogenesis is regulated via the NDRG1-DGAT1/2 pathway, which provides novel potential targets for developing anti-RABV drugs. IMPORTANCE Lipid droplets have been proven to play an important role in viral infections, but their role in RABV infection has not yet been elaborated. Here, we find that RABV infection upregulates the generation of LDs by enhancing the expression of N-myc downstream regulated gene-1 (NDRG1). Then NDRG1 elevated cellular triglycerides synthesis by increasing the activity of diacylglycerol acyltransferase 1/2 (DGAT1/2), which promotes the biogenesis of LDs. RABV-M and RABV-G, which are the major proteins involved in viral budding, could utilize LDs as a carrier for transport to cell membrane, resulting in enhanced virus budding. Our findings will extend the knowledge of lipid metabolism in RABV infection and help to explore potential therapeutic targets for RABV.

Research Advances on the Interactions between Rabies Virus Structural Proteins and Host Target Cells: Accrued Knowledge from the Application of Reverse Genetics Systems.

Rabies is a lethal zoonotic disease caused by lyssaviruses, such as rabies virus (RABV), that results in nearly 100% mortality once clinical symptoms appear. There are no curable drugs available yet. RABV contains five structural proteins that play an important role in viral replication, transcription, infection, and immune escape mechanisms. In the past decade, progress has been made in research on the pathogenicity of RABV, which plays an important role in the creation of new recombinant RABV vaccines by reverse genetic manipulation. Here, we review the latest advances on the interaction between RABV proteins in the infected host and the applied development of rabies vaccines by using a fully operational RABV reverse genetics system. This article provides a background for more in-depth research on the pathogenic mechanism of RABV and the development of therapeutic drugs and new biologics.

Point Mutations in the Glycoprotein Ectodomain of Field Rabies Viruses Mediate Cell Culture Adaptation through Improved Virus Release in a Host Cell Dependent and Independent Manner.

Molecular details of field rabies virus (RABV) adaptation to cell culture replication are insufficiently understood. A better understanding of adaptation may not only reveal requirements for efficient RABV replication in cell lines, but may also provide novel insights into RABV biology and adaptation-related loss of virulence and pathogenicity. Using two recombinant field rabies virus clones (rRABV Dog and rRABV Fox), we performed virus passages in three different cell lines to identify cell culture adaptive mutations. Ten passages were sufficient for the acquisition of adaptive mutations in the glycoprotein G and in the C-terminus of phosphoprotein P. Apart from the insertion of a glycosylation sequon via the mutation D247N in either virus, both acquired additional and cell line-specific mutations after passages on BHK (K425N) and MDCK-II (R346S or R350G) cells. As determined by virus replication kinetics, complementation, and immunofluorescence analysis, the major bottleneck in cell culture replication was the intracellular accumulation of field virus G protein, which was overcome after the acquisition of the adaptive mutations. Our data indicate that limited release of extracellular infectious virus at the plasma membrane is a defined characteristic of highly virulent field rabies viruses and we hypothesize that the observed suboptimal release of infectious virions is due to the inverse correlation of virus release and virulence in vivo.

Dynein- and kinesin- mediated intracellular transport on microtubules facilitates RABV infection.

Rabies, caused by rabies virus (RABV), is one of the most important neurotropic zoonoses and poses a severe threat to human and animal health. Exploration of its mechanism of neural transmission is meaningful but still insufficient. Here, we described the effects of microtubule-depolymerizing drugs and inhibitors of microtubule motor proteins on RABV infection. Colchicine, a microtubule-depolymerizing drug, significantly impeded RABV production in N2a cells. Overexpression of CC1 or p50 attenuated viral infection through the functional disruption of cytoplasmic dynein, which was consistent with the inhibitory effect of Na3VO4, a dynein activity inhibitor. Moreover, transfection with Flag-KHCct impaired RABV infection, as cytoplasmic kinesin-based motility was blocked. These results demonstrated that RABV can infect N2a cells in a manner that depends on microtubule integrity as well as dynein and kinesin function.

Definition of the immune evasion-replication interface of rabies virus P protein.

Rabies virus phosphoprotein (P protein) is a multifunctional protein that plays key roles in replication as the polymerase cofactor that binds to the complex of viral genomic RNA and the nucleoprotein (N protein), and in evading the innate immune response by binding to STAT transcription factors. These interactions are mediated by the C-terminal domain of P (PCTD). The colocation of these binding sites in the small globular PCTD raises the question of how these interactions underlying replication and immune evasion, central to viral infection, are coordinated and, potentially, coregulated. While direct data on the binding interface of the PCTD for STAT1 is available, the lack of direct structural data on the sites that bind N protein limits our understanding of this interaction hub. The PCTD was proposed to bind via two sites to a flexible loop of N protein (Npep) that is not visible in crystal structures, but no direct analysis of this interaction has been reported. Here we use Nuclear Magnetic Resonance, and molecular modelling to show N protein residues, Leu381, Asp383, Asp384 and phosphor-Ser389, are likely to bind to a 'positive patch' of the PCTD formed by Lys211, Lys214 and Arg260. Furthermore, in contrast to previous predictions we identify a single site of interaction on the PCTD by this Npep. Intriguingly, this site is proximal to the defined STAT1 binding site that includes Ile201 to Phe209. However, cell-based assays indicate that STAT1 and N protein do not compete for P protein. Thus, it appears that interactions critical to replication and immune evasion can occur simultaneously with the same molecules of P protein so that the binding of P protein to activated STAT1 can potentially occur without interrupting interactions involved in replication. These data suggest that replication complexes might be directly involved in STAT1 antagonism.

An inactivated recombinant rabies virus displaying the Zika virus prM-E induces protective immunity against both pathogens.

The global spread of Zika virus (ZIKV), which caused a pandemic associated with Congenital Zika Syndrome and neuropathology in newborns and adults, prompted the pursuit of a safe and effective vaccine. Here, three kinds of recombinant rabies virus (RABV) encoding the prM-E protein of ZIKV were constructed: ZI-D (prM-E), ZI-E (transmembrane domain (TM) of prM-E replaced with RABV G) and ZI-F (signal peptide and TM domain of prM-E replaced with the region of RABV G). When the TM of prM-E was replaced with the region of RABV G (termed ZI-E), it promoted ZIKV E protein localization on the cell membrane and assembly on recombinant viruses. In addition, the change in the signal peptide with RABV G (termed ZI-F) was not conducive to foreign protein expression. The immunogenicity of recombinant viruses mixed with a complex adjuvant of ISA 201 VG and poly(I:C) was tested in BALB/c mice. After immunization with ZI-E, the anti-ZIKV IgG antibody lasted for at least 10 weeks. The titers of neutralizing antibodies (NAbs) against ZIKV and RABV at week 6 were all greater than the protective titers. Moreover, ZI-E stimulated the proliferation of splenic lymphocytes and promoted the secretion of cytokines. It also promoted the production of central memory T cells (TCMs) among CD4+/CD8+ T cells and stimulated B cell activation and maturation. These results indicate that ZI-E could induce ZIKV-specific humoral and cellular immune responses, which have the potential to be developed into a promising vaccine for protection against both ZIKV and RABV infections.

Analyses of cell death mechanisms related to amino acid substitution at position 95 in the rabies virus matrix protein.

We previously reported that the avirulent fixed rabies virus strain Ni-CE induces a clear cytopathic effect in mouse neuroblastoma cells, whereas its virulent progenitor, the Nishigahara strain, does not. Infection with Nishigahara and Ni-CE mutants containing a single amino acid substitution in the matrix protein (M) demonstrated that the amino acid at position 95 of M (M95) is a cytopathic determinant. The characteristics of cell death induced by Ni-CE infection resemble those of apoptosis (rounded and shrunken cells, DNA fragmentation), but the intracellular signalling pathway for this process has not been fully investigated. In this study, we aimed to elucidate the mechanism by which M95 affects cell death induced by human neuroblastoma cell infection with the Nishigahara, Ni-CE and M95-mutated strains. We demonstrated that the Ni-CE strain induced DNA fragmentation, cell membrane disruption, exposure of phosphatidylserine (PS), activation of caspase-3/7 and anti-poly (ADP-ribose) polymerase 1 (PARP-1) cleavage, an early apoptosis indicator, whereas the Nishigahara strain did not induce DNA fragmentation, caspase-3/7 activation, cell membrane disruption, or PARP-1 cleavage, but did induce PS exposure. We also demonstrated that these characteristics were associated with M95 using M95-mutated strains. However, we found that Ni-CE induced cell death despite the presence of a caspase inhibitor, Z-VAD-FMK. In conclusion, our data suggest that M95 mutation-related cell death is caused by both the caspase-dependent and -independent pathways.

Development of Monoclonal Antibodies for Detection of Conserved and Variable Epitopes of Large Protein of Rabies Virus.

Rabies virus (RABV) causes fatal neurological encephalitis and results in approximately 6000 human death cases worldwide every year. The large (L) protein of RABV, possessing conserved domains, is considered as the target for detection. In this study, three monoclonal antibodies (mAbs), designated as 3F3, 3A6 and L-C, against L protein were generated by using the recombinant truncated L protein (aa 1431-1754) and the epitopes were also identified using a series of overlapping truncated polypeptides for testing the reactivity of mAbs with different RABV strains. The 1479EIFSIP1484, 1659RALSK1663 and 1724VFNSL1728 were identified as the minimal linear epitopes recognized by mAbs 3F3, 3A6 and L-C, respectively. Amino acid alignment showed epitope 1724VFNSL1728 recognized by mAb L-C is completely conserved among RABV strains, indicating that mAb L-C could be used to detect all of the RABV strains. Epitope 1479EIFSIP1484 is highly conserved among RABV strains except for a P1484S substitution in a China I sub-lineage strain of Asian lineage, which eliminated the reactivity of the epitope with mAb 3F3. However, the epitope 1659RALSK1663 was only completely conserved in the Africa-2 and Indian lineages, and a single A1660T substitution, mainly appeared in strains of the China I belonging to Asian lineage and a Cosmopolitan lineage strain, still retained the reactivity of the epitope with mAb 3A6. While both A1660T and K1663R substitutions in a China I lineage strain, single K1663R/Q substitution in some China II strains of Asian lineage and some Arctic-like lineage strains and R1659Q mutation in a strain of Africa-3 lineage eliminated the reactivity of the epitope with mAb 3A6, suggesting mAb 3A6 could be used for differentiation of variable epitopes of some strains in different lineages. Thus, variability and conservation of the three epitopes of L protein showed the reactive difference of mAbs among RABV strains of different lineages. These results may facilitate future studies in development of detection methods for RABV infection, the structure and function of RABV L protein.

Development of competitive inhibition ELISA as an effective potency test to analyze human rabies vaccines and assessment of the antigenic epitope of rabies glycoprotein.

The potency of all modern tissue culture human rabies vaccines is measured based on the National Institute of Health (NIH) potency test that is laborious, time-consuming, involves large test variations and requires sacrifice of large number of animals. To circumvent these limitations, several researchers and WHO expert working groups have discussed development of alternative in vitro methods to replace the NIH potency test. Although several immunochemical methods have been proposed to quantify rabies glycoprotein (G-protein) using multiple murine monoclonal antibodies, we report an In vitro competitive inhibition ELISA (CIA) method based on the use of a neutralizing rabies glycoprotein site III directed novel therapeutic human rabies monoclonal antibody (RAB1) that shows equivalence to the mice NIH potency test in recognition of neutralization site of the glycoprotein. In vitro potency testing of WHO 7th International Standard for rabies vaccine (IS) by CIA using RAB1 and In-house reference standard (IHRS) as a standard to assess its suitability for the assessment of validation parameters showed accurate and precise values with <15% coefficient variance. The method was validated using 5PL standard curve with linearity r2 > 0.98 and LLOQ of 0.125 IU/mL indicating sensitivity of the method. The method was found to be precise, robust and accurate to quantitate intact rabies glycoprotein in final vaccine and showed a strong correlation (Pearson's r = 0.81) with the NIH potency values of licensed Vero cell rabies vaccine. The CIA test using RAB1 was able to accurately quantitate degradation of rabies vaccine and assess loss in antigenicity of lyophilized and reconstituted liquid rabies vaccine under thermal stress conditions. The method was able to differentiate between potent and reduced potency vaccine samples. The new in vitro competitive inhibition ELISA method using RAB1 thus can be a valid alternative to the NIH test.

Interferon-Inducible GTPase 1 Impedes the Dimerization of Rabies Virus Phosphoprotein and Restricts Viral Replication.

Rabies, caused by rabies virus (RABV), is an ancient zoonosis and still a major public health problem for humans, especially in developing countries. RABV can be recognized by specific innate recognition receptors, resulting in the production of hundreds of interferon-stimulated genes (ISGs), which can inhibit viral replication at different stages. Interferon-inducible GTPase 1 (IIGP1) is a mouse-specific ISG and belongs to the immunity-related GTPases (IRGs) family. IIGP is reported to constrain intracellular parasite infection by disrupting the parasitophorous vacuole membrane. However, the role of IIGP1 in restricting viral replication has not been reported. In this present study, we found that IIGP1 was upregulated in cells and mouse brains upon RABV infection. Overexpression of IIGP1 limited RABV replication in cell lines and reduced viral pathogenicity in a mouse model. Consistently, deficiency of IIGP1 enhanced RABV replication in different parts of mouse brains. Furthermore, we found that IIGP1 could interact with RABV phosphoprotein (P protein). Mutation and immunoprecipitation analyses revealed that the Y128 site of P protein is critical for its interaction with IIGP1. Further study demonstrated that this interaction impeded the dimerization of P protein and thus suppressed RABV replication. Collectively, our findings for the first reveal a novel role of IIGP1 in restricting a typical neurotropic virus, RABV, which will provide fresh insight into the function of this mouse-specific ISG.IMPORTANCE Interferon and its downstream products, ISGs, are essential in defending against pathogen invasion. One of the ISGs, IIGP1, has been found to constrain intracellular parasite infection by disrupting their vacuole membranes. However, the role of IIGP1 in limiting viral infection is unclear. In this study, we show that infection with a typical neurotropic virus, RABV, can induce upregulation of IIGP1, which, in turn, suppresses RABV by interacting with its phosphoprotein (P protein) and thus blocking the dimerization of P protein. Our study provides the first evidence that IIGP1 functions in limiting viral infection and provides a basis for comprehensive understanding of this important ISG.