TRIM72 restricts lyssavirus infection by inducing K48-linked ubiquitination and proteasome degradation of the matrix protein.

The tripartite motif (TRIM) protein family is the largest subfamily of E3 ubiquitin ligases, playing a crucial role in the antiviral process. In this study, we found that TRIM72, a member of the TRIM protein family, was increased in neuronal cells and mouse brains following rabies lyssavirus (RABV) infection. Over-expression of TRIM72 significantly reduced the viral titer of RABV in neuronal cells and mitigated the pathogenicity of RABV in mice. Furthermore, we found that TRIM72 over-expression effectively prevents the assembly and/or release of RABV. In terms of the mechanism, TRIM72 promotes the K48-linked ubiquitination of RABV Matrix protein (M), leading to the degradation of M through the proteasome pathway. TRIM72 directly interacts with M and the interaction sites were identified and confirmed through TRIM72-M interaction model construction and mutation analysis. Further investigation revealed that the degradation of M induced by TRIM72 was attributed to TRIM72's promotion of ubiquitination at site K195 in M. Importantly, the K195 site was found to be partially conserved among lyssavirus's M proteins, and TRIM72 over-expression induced the degradation of these lyssavirus M proteins. In summary, our study has uncovered a TRIM family protein, TRIM72, that can restrict lyssavirus replication by degrading M, and we have identified a novel ubiquitination site (K195) in lyssavirus M.

Single-cell transcriptomic classification of rabies-infected cortical neurons.

Cortical circuit tracing using modified rabies virus can identify input neurons making direct monosynaptic connections onto neurons of interest. However, challenges remain in our ability to establish the cell type identity of rabies-labeled input neurons. While transcriptomics may offer an avenue to characterize inputs, the extent of rabies-induced transcriptional changes in distinct neuronal cell types remains unclear, and whether these changes preclude characterization of rabies-infected neurons according to established transcriptomic cell types is unknown. We used single-nucleus RNA sequencing to survey the gene expression profiles of rabies-infected neurons and assessed their correspondence with established transcriptomic cell types. We demonstrated that when using transcriptome-wide RNA profiles, rabies-infected cortical neurons can be transcriptomically characterized despite global and cell-type-specific rabies-induced transcriptional changes. Notably, we found differential modulation of neuronal marker gene expression, suggesting that caution should be taken when attempting to characterize rabies-infected cells with single genes or small gene sets.

TRIM21 Promotes Rabies Virus Production by Degrading IRF7 through Ubiquitination.

Rabies, a highly fatal zoonotic disease, is a significant global public health threat. Currently, the pathogenic mechanism of rabies has not been fully elucidated, and no effective treatment for rabies is available. Increasing evidence shows that the tripartite-motif protein (TRIM) family of proteins participates in the host's regulation of viral replication. Studies have demonstrated the upregulated expression of tripartite-motif protein 21 (TRIM21) in the brain tissue of mice infected with the rabies virus. Related studies have shown that TRIM21 knockdown inhibits RABV replication, while overexpression of TRIM21 exerted the opposite effect. Knockdown of interferon-alpha and interferon-beta modulates the inhibition of RABV replication caused by TRIM21 knockdown and promotes the replication of the virus. Furthermore, our previous study revealed that TRIM21 regulates the secretion of type I interferon during RABV infection by targeting interferon regulatory factor 7 (IRF7). IRF7 knockdown reduced the inhibition of RABV replication caused by the knockdown of TRIM21 and promoted viral replication. TRIM21 regulates RABV replication via the IRF7-IFN axis. Our study identified TRIM21 as a novel host factor required by RABV for replication. Thus, TRIM21 is a potential target for rabies treatment or management.

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.

Integrin ß1 Promotes Peripheral Entry by Rabies Virus.

Rabies virus (RABV) is a widespread pathogen that causes fatal disease in humans and animals. It has been suggested that multiple host factors are involved in RABV host entry. Here, we showed that RABV uses integrin ß1 (ITGB1) for cellular entry. RABV infection was drastically decreased after ITGB1 short interfering RNA knockdown and moderately increased after ITGB1 overexpression in cells. ITGB1 directly interacts with RABV glycoprotein. Upon infection, ITGB1 is internalized into cells and transported to late endosomes together with RABV. The infectivity of cell-adapted RABV in cells and street RABV in mice was neutralized by ITGB1 ectodomain soluble protein. The role of ITGB1 in RABV infection depends on interaction with fibronectin in cells and mice. We found that Arg-Gly-Asp (RGD) peptide and antibody to ITGB1 significantly blocked RABV infection in cells in vitro and street RABV infection in mice via intramuscular inoculation but not the intracerebral route. ITGB1 also interacts with nicotinic acetylcholine receptor, which is the proposed receptor for peripheral RABV infection. Our findings suggest that ITGB1 is a key cellular factor for RABV peripheral entry and is a potential therapeutic target for postexposure treatment against rabies.IMPORTANCE Rabies is a severe zoonotic disease caused by rabies virus (RABV). However, the nature of RABV entry remains unclear, which has hindered the development of therapy for rabies. It is suggested that modulations of RABV glycoprotein and multiple host factors are responsible for RABV invasion. Here, we showed that integrin ß1 (ITGB1) directly interacts with RABV glycoprotein, and both proteins are internalized together into host cells. Differential expression of ITGB1 in mature muscle and cerebral cortex of mice led to A-4 (ITGB1-specific antibody), and RGD peptide (competitive inhibitor for interaction between ITGB1 and fibronectin) blocked street RABV infection via intramuscular but not intracerebral inoculation in mice, suggesting that ITGB1 plays a role in RABV peripheral entry. Our study revealed this distinct cellular factor in RABV infection, which may be an attractive target for therapeutic intervention.

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.

Comparison of intra- and inter-host genetic diversity in rabies virus during experimental cross-species transmission.

The development of high-throughput genome sequencing enables accurate measurements of levels of sub-consensus intra-host virus genetic diversity and analysis of the role played by natural selection during cross-species transmission. We analysed the natural and experimental evolution of rabies virus (RABV), an important example of a virus that is able to make multiple host jumps. In particular, we (i) analyzed RABV evolution during experimental host switching with the goal of identifying possible genetic markers of host adaptation, (ii) compared the mutational changes observed during passage with those observed in natura, and (iii) determined whether the colonization of new hosts or tissues requires adaptive evolution in the virus. To address these aims, animal infection models (dog and fox) and primary cell culture models (embryo brain cells of dog and fox) were developed and viral variation was studied in detail through deep genome sequencing. Our analysis revealed a strong unidirectional host evolutionary effect, as dog-adapted rabies virus was able to replicate in fox and fox cells relatively easily, while dogs or neuronal dog cells were not easily susceptible to fox adapted-RABV. This suggests that dog RABV may be able to adapt to some hosts more easily than other host variants, or that when RABV switched from dogs to red foxes it lost its ability to adapt easily to other species. Although no difference in patterns of mutation variation between different host organs was observed, mutations were common following both in vitro and in vivo passage. However, only a small number of these mutations also appeared in natura, suggesting that adaptation during successful cross-species virus transmission is a complex, multifactorial evolutionary process.

Expression Profiling and Bioinformatics Analysis of CircRNA in Mice Brain Infected with Rabies Virus.

Rabies virus (RABV) induces acute, fatal encephalitis in mammals including humans. The circRNAs are important in virus infection process, but whether circRNAs regulated RABV infection remains largely unknown. Here, mice brain with or without the RABV CVS-11 strain were subjected to RNA sequencing and a total of 30,985 circRNAs were obtained. Among these, 9021 candidates were shared in both groups, and 14,610 and 7354 circRNAs were expressed specifically to the control and experimental groups, indicating that certain circRNAs were specifically inhibited or induced on RABV infection. The circRNAs mainly derived from coding exons. In total, 636 circRNAs were differentially expressed in RABV infection, of which 426 significantly upregulated and 210 significantly downregulated (p < 0.05 and fold change ≥2). The expression of randomly selected 6 upregulated and 6 downregulated circRNAs was tested by RT-qPCR, and the expression trend of the 11 out of 12 circRNAs was consistent in RT- qPCR and RNA-seq analysis. Rnase R-resistant assay and Sanger sequencing were conducted to verify the circularity of circRNAs. GO analysis demonstrated that source genes of all differentially regulated circRNAs were mainly related to cell plasticity and synapse function. Both KEGG and GSEA analysis revealed that these source genes were engaged in the cGMP-PKG and MAPK signaling pathway, and HTLV-I infection. Also, pathways related to glucose metabolism and synaptic functions were enriched in KEGG analysis. The circRNA-miRNA-mRNA network was built with 25 of 636 differentially expressed circRNAs, 264 mRNAs involved in RABV infection, and 29 miRNAs. Several miRNAs and many mRNAs in the network were reported to be related to viral infection and the immune response, suggesting that circRNAs could regulate RABV infection via interacting with miRNAs and mRNAs. Taken together, this study first characterized the transcriptomic pattern of circRNAs, and signaling pathways and function that circRNAs are involved in, which may indicate directions for further research to understand mechanisms of RABV pathogenesis.

MALT1 Controls Attenuated Rabies Virus by Inducing Early Inflammation and T Cell Activation in the Brain.

MALT1 is involved in the activation of immune responses, as well as in the proliferation and survival of certain cancer cells. MALT1 acts as a scaffold protein for NF-κB signaling and a cysteine protease that cleaves substrates, further promoting the expression of immunoregulatory genes. Deregulated MALT1 activity has been associated with autoimmunity and cancer, implicating MALT1 as a new therapeutic target. Although MALT1 deficiency has been shown to protect against experimental autoimmune encephalomyelitis, nothing is known about the impact of MALT1 on virus infection in the central nervous system. Here, we studied infection with an attenuated rabies virus, Evelyn-Rotnycki-Abelseth (ERA) virus, and observed increased susceptibility with ERA virus in MALT1-/- mice. Indeed, after intranasal infection with ERA virus, wild-type mice developed mild transient clinical signs with recovery at 35 days postinoculation (dpi). Interestingly, MALT1-/- mice developed severe disease requiring euthanasia at around 17 dpi. A decreased induction of inflammatory gene expression and cell infiltration and activation was observed in MALT1-/- mice at 10 dpi compared to MALT1+/+ infected mice. At 17 dpi, however, the level of inflammatory cell activation was comparable to that observed in MALT1+/+ mice. Moreover, MALT1-/- mice failed to produce virus-neutralizing antibodies. Similar results were obtained with specific inactivation of MALT1 in T cells. Finally, treatment of wild-type mice with mepazine, a MALT1 protease inhibitor, also led to mortality upon ERA virus infection. These data emphasize the importance of early inflammation and activation of T cells through MALT1 for controlling the virulence of an attenuated rabies virus in the brain.IMPORTANCE Rabies virus is a neurotropic virus which can infect any mammal. Annually, 59,000 people die from rabies. Effective therapy is lacking and hampered by gaps in the understanding of virus pathogenicity. MALT1 is an intracellular protein involved in innate and adaptive immunity and is an interesting therapeutic target because MALT1-deregulated activity has been associated with autoimmunity and cancers. The role of MALT1 in viral infection is, however, largely unknown. Here, we study the impact of MALT1 on virus infection in the brain, using the attenuated ERA rabies virus in different models of MALT1-deficient mice. We reveal the importance of MALT1-mediated inflammation and T cell activation to control ERA virus, providing new insights in the biology of MALT1 and rabies virus infection.

Regulation of NF-κB by the p105-ABIN2-TPL2 complex and RelAp43 during rabies virus infection.

At the crossroad between the NF-κB and the MAPK pathways, the ternary complex composed of p105, ABIN2 and TPL2 is essential for the host cell response to pathogens. The matrix protein (M) of field isolates of rabies virus was previously shown to disturb the signaling induced by RelAp43, a NF-κB protein close to RelA/p65. Here, we investigated how the M protein disturbs the NF-κB pathway in a RelAp43-dependant manner and the potential involvement of the ternary complex in this mechanism. Using a tandem affinity purification coupled with mass spectrometry approach, we show that RelAp43 interacts with the p105-ABIN2-TPL2 complex and we observe a strong perturbation of this complex in presence of M protein. M protein interaction with RelAp43 is associated with a wide disturbance of NF-κB signaling, involving a modulation of IκBα-, IκBß-, and IκBε-RelAp43 interaction and a favored interaction of RelAp43 with the non-canonical pathway (RelB and p100/p52). Monitoring the interactions between host and viral proteins using protein-fragment complementation assay and bioluminescent resonance energy transfer, we further show that RelAp43 is associated to the p105-ABIN2-TPL2 complex as RelAp43-p105 interaction stabilizes the formation of a complex with ABIN2 and TPL2. Interestingly, the M protein interacts not only with RelAp43 but also with TPL2 and ABIN2. Upon interaction with this complex, M protein promotes the release of ABIN2, which ultimately favors the production of RelAp43-p50 NF-κB dimers. The use of recombinant rabies viruses further indicates that this mechanism leads to the control of IFNß, TNF and CXCL2 expression during the infection and a high pathogenicity profile in rabies virus infected mice. All together, our results demonstrate the important role of RelAp43 and M protein in the regulation of NF-κB signaling.

Cholesterol 25-hydroxylase suppresses rabies virus infection by inhibiting viral entry.

Cholesterol-25-hydroxylase (CH25H) is a reticulum-associated membrane protein that catalyzes the oxidation of cholesterol to 25-hydroxycholesterol (25HC). Recent studies have revealed that CH25H is an interferon-stimulated gene (ISG) that suppresses infection by several viruses. In the present study, we found that overexpression of both human and murine CH25H inhibited rabies virus (RABV) infection in HEK-293T (293T) cells. In contrast, silencing of CH25H enhanced RABV replication in 293T cells, and a catalytic mutant of CH25H lost its ability to inhibit RABV infection. Treatment with the oxysterol 25-hydroxycholesterol (25HC), the product of CH25H, dramatically decreased RABV replication in 293T, BSR and N2a cells by inhibiting viral membrane penetration. These data provide insights into the antiviral function of CH25H against RABV infection, which can potentially be used as a therapeutic agent for rabies.

Exosome-Mediated Delivery of Inducible miR-423-5p Enhances Resistance of MRC-5 Cells to Rabies Virus Infection.

The human diploid cell line Medical Research Council -5 (MRC-5) is commonly utilized for vaccine development. Although a rabies vaccine developed in cultured MRC-5 cells exists, the poor susceptibility of MRC-5 cells to the rabies virus (RABV) infection limits the potential yield of this vaccine. The underlying mechanism of MRC-5 cell resistance to RABV infection remains unknown. In this study, we demonstrate that viral infection increased exosomal release from MRC-5 cells; conversely, blocking exosome release promoted RABV infection in MRC-5 cells. Additionally, RABV infection up-regulated microRNA (miR)-423-5p expression in exosomes, resulting in feedback inhibition of RABV replication by abrogating the inhibitory effect of suppressor of cytokine signaling 3 (SOCS3) on type I interferon (IFN) signaling. Furthermore, intercellular delivery of miR-423-5p by exosomes inhibited RABV replication in MRC-5 cells. We also show that RABV infection increased IFN-ß production in MRC-5 cells and that blocking the type I IFN receptor promoted RABV infection. In conclusion, MRC-5 cells were protected from RABV infection by the intercellular delivery of exosomal miR-423-5p and the up-regulation of IFN-ß. These findings reveal novel antiviral mechanisms in MRC-5 cells against RABV infection. miR-423-5p, exosomes, and IFN signaling pathways may therefore be potential targets for improving MRC-5 cell-based rabies vaccine production.

Using Viral Gene Sequences to Compare and Explain the Heterogeneous Spatial Dynamics of Virus Epidemics.

Rabies is an important zoonotic disease distributed worldwide. A key question in rabies epidemiology is the identification of factors that impact virus dispersion. Here we apply new analytical methods, based on phylogeographic reconstructions of viral lineage movement, to undertake a comparative evolutionary-epidemiological study of the spatial dynamics of rabies virus (RABV) epidemics in different hosts and habitats. We compiled RABV data sets from skunk, raccoon, bat and domestic dog populations in order to investigate the viral diffusivity of different RABV epidemics, and to detect and compare the environmental factors that impact the velocity of viral spread in continuous spatial landscapes. We build on a recently developed statistical framework that uses spatially- and temporally-referenced phylogenies. We estimate several spatial statistics of virus spread, which reveal a higher diffusivity of RABV in domestic dogs compared with RABV in other mammals. This finding is explained by subsequent analyses of environmental heterogeneity, which indicate that factors relating to human geography play a significant role in RABV dispersion in domestic dogs. More generally, our results suggest that human-related factors are important worldwide in explaining RABV dispersion in terrestrial host species. Our study shows that phylogenetically informed viral movements can be used to elucidate the factors that impact virus dispersal, opening new opportunities for a better understanding of the impact of host species and environmental conditions on the spatial dynamics of rapidly evolving populations.

Pathobiological investigation of naturally infected canine rabies cases from Sri Lanka.

BACKGROUND: The recommended screening of rabies in 'suspect' animal cases involves testing fresh brain tissue. The preservation of fresh tissue however can be difficult under field conditions and formalin fixation provides a simple alternative that may allow a confirmatory diagnosis. The occurrence and location of histopathological changes and immunohistochemical (IHC) labelling for rabies in formalin fixed paraffin embedded (FFPE) canine brain is described in samples from 57 rabies suspect cases from Sri-Lanka. The presence of Negri bodies and immunohistochemical detection of rabies virus antigen were evaluated in the cortex, hippocampus, cerebellum and brainstem. The effect of autolysis and artefactual degeneration of the tissue was also assessed. RESULTS: Rabies was confirmed in 53 of 57 (93%) cases by IHC. IHC labelling was statistically more abundant in the brainstem. Negri bodies were observed in 32 of 53 (60.4%) of the positive cases. Although tissue degradation had no effect on IHC diagnosis, it was associated with an inability to detect Negri bodies. In 13 cases, a confirmatory Polymerase chain reaction (PCR) testing for rabies virus RNA was undertaken by extracting RNA from fresh frozen tissue, and also attempted using FFPE samples. PCR detection using fresh frozen samples was in agreement with the IHC results. The PCR method from FFPE tissues was suitable for control material but unsuccessful in our field cases. CONCLUSIONS: Histopathological examination of the brain is essential to define the differential diagnoses of behaviour modifying conditions in rabies virus negative cases, but it is unreliable as the sole method for rabies diagnosis, particularly where artefactual change has occurred. Formalin fixation and paraffin embedding does not prevent detection of rabies virus via IHC labelling even where artefactual degeneration has occurred. This could represent a pragmatic secondary assay for rabies diagnosis in the field because formalin fixation can prevent sample degeneration. The brain stem was shown to be the site with most viral immunoreactivity; supporting recommended sampling protocols in favour of improved necropsy safety in the field. PCR testing of formalin fixed tissue may be successful in certain circumstances as an alternative test.

High-Throughput Differentiation and Screening of a Library of Mutant Stem Cell Clones Defines New Host-Based Genes Involved in Rabies Virus Infection.

We used a genomic library of mutant murine embryonic stem cells (ESCs) and report the methodology required to simultaneously culture, differentiate, and screen more than 3,200 heterozygous mutant clones to identify host-based genes involved in both sensitivity and resistance to rabies virus infection. Established neuronal differentiation protocols were miniaturized such that many clones could be handled simultaneously, and molecular markers were used to show that the resultant cultures were pan-neuronal. Next, we used a green fluorescent protein (GFP) labeled rabies virus to develop, validate, and implement one of the first host-based, high-content, high-throughput screens for rabies virus. Undifferentiated cell and neuron cultures were infected with GFP-rabies and live imaging was used to evaluate GFP intensity at time points corresponding to initial infection/uptake and early and late replication. Furthermore, supernatants were used to evaluate viral shedding potential. After repeated testing, 63 genes involved in either sensitivity or resistance to rabies infection were identified. To further explore hits, we used a completely independent system (siRNA) to show that reduction in target gene expression leads to the observed phenotype. We validated the immune modulatory gene Unc13d and the dynein adapter gene Bbs4 by treating wild-type ESCs and primary neurons with siRNA; treated cultures were resistant to rabies infection/replication. Overall, the potential of such in vitro functional genomics screens in stem cells adds additional value to other libraries of stem cells. This technique is applicable to any bacterial or virus interactome and any cell or tissue types that can be differentiated from ESCs.

Molecular genetic characterization of rabies virus glycoprotein gene sequences from rabid dogs in Bangkok and neighboring provinces in Thailand, 2013-2014.

Because of its association with dogs, rabies virus (RABV) is still endemic in Thailand, where it is a serious public health problem. The genetic characterization of RABV in Thailand is limited. Therefore, in this study, we investigated the molecular epidemiology and genetic diversity of RABV in the endemic area. Viral RNA from 48 brain specimens from rabid dogs, collected in Bangkok and seven neighboring provinces in 2013-2014, was extracted and sequenced. The complete rabies glycoprotein (G) gene sequences (1575 nt) were aligned, and a phylogenetic analysis was performed using the maximum-likelihood method. All of the Thai rabies virus isolates belonged to lyssavirus genotype 1 and clustered in the same lineage as isolates from South East Asia (SEA) and China. The Thai rabies virus isolates formed two distinct clades, THA-1 and THA-2. Clade THA-1 was the predominant clade and could be divided into two subclades, THA-1A and THA-1B. Clade THA-2 was closely associated with human Thai isolates collected in a previous study. The overall mean rate of evolution based on the G gene was approximately 1.56 × 10(-4) substitutions/site/year. The genetic identities among the isolates from Thailand and other SEA countries were >88.4 % at the nucleotide sequence level and 95 % at the amino acid sequence level. The deduced amino acid sequences of the G proteins of the RABV isolates were compared. A single amino acid change (N194T) in subclade THA-1A distinguished the Thai RABV isolates from other RABV isolates. Our results suggest that these Thai dog RABV isolates share a common ancestor with the RABV isolates circulating in the endemic regions of SEA countries and China. Furthermore, there were strong genetic relationship to RABV from Cambodia, Vietnam and Laos. These data extend our understanding of the relatedness and genetic variation of RABV in Thailand.

Correlation of inducible nitric oxide synthase (iNOS) inhibition with TNF-α, caspase-1, FasL and TLR-3 in pathogenesis of rabies in mouse model.

The role of inflammatory cytokines such as interleukin-1α/ß (IL-1α/ß), IL-6, IL-10, tumour necrosis factor-alpha (TNF-α), interferons, nitric oxide (NO) and inducible nitric oxide synthase (iNOS) in pathogenesis of rabies is being actively pursued. Presently, levels of certain immune molecules in pathogenesis of rabies in mice have been investigated. CVS strain of rabies infection resulted in early increase in iNOS, TNF-α, caspase-1, Fas ligand (FasL) and toll-like receptor-3 (TLR-3) mRNA levels in brain, and nitric oxide levels in serum. The severity of clinical signs and microscopic lesions largely correlated with NO levels. Aminoguanidine (AG; iNOS inhibitor) decreased NO production with delay in development of clinical signs and increase in survival time. Prolonged survival time correlated with reduced viral load evident by real-time PCR, reduced fluorescent signals of rabies antigen in brain and reduced immunohistochemistry signals in neuronal cytoplasm. These parameters suggested that nitric oxide did influence the rabies virus replication. Inhibition of iNOS by AG administration led to decreased expression of TNF-α, caspase-1, FasL and TLR-3 mRNA levels suggesting that increase in NO levels in rabies virus infection possibly contributed to development of disease through inflammation, apoptosis and immune-evasive mechanisms.

Quantitative Proteome Profiling of Street Rabies Virus-Infected Mouse Hippocampal Synaptosomes.

It is well established now that neuronal dysfunction rather than structural damage may be responsible for the development of rabies. In order to explore the underlying mechanisms in rabies virus (RABV) and synaptic dysfunctions, a quantitative proteome profiling was carried out on synaptosome samples from mice hippocampus. Synaptosome samples from mice hippocampus were isolated and confirmed by Western blot and transmission electron microscopy. Synaptosome protein content changes were quantitatively detected by Nano-LC-MS/MS. Protein functions were classified by the Gene Ontology (GO) and KEGG pathway. PSICQUIC was used to create a network. MCODE algorithm was applied to obtain subnetworks. Of these protein changes, 45 were upregulated and 14 were downregulated following RABV infection relative to non-infected (mock) synaptosomes. 28 proteins were unique to mock treatment and 12 were unique to RABV treatment. Proteins related to metabolism and synaptic vesicle showed the most changes in expression levels. Furthermore, protein-protein interaction (PPI) networks revealed that several key biological processes related to synaptic functions potentially were modulated by RABV, including energy metabolism, cytoskeleton organization, and synaptic transmission. These data will be useful for better understanding of neuronal dysfunction of rabies and provide the foundation for future research.

Molecular characterization of nucleoprotein gene of rabies virus from Maharashtra, India.

CONTEXT: Rabies poses a serious public health concern in developing countries such as India. AIMS: The study focuses on molecular diagnosis of street rabies virus (RABV) from human clinical specimens received from Maharashtra, India. MATERIALS AND METHODS: Nucleoprotein gene from eight (of total 20 suspected samples) rabies cases that tested positive for rabies antigen using reverse transcriptase-polymerase chain reaction (RT-PCR) were sequenced. RESULTS: Sequence analysis using basic local alignment search tool (BLAST) and multiple sequence alignment (MSA) and phylogenetic analysis showed similarity to previously reported sequences from India and those of Arctic lineages. CONCLUSIONS: The circulating RABV strains in Maharashtra, India show genetic relatedness to RABV strains reported from Indo-Arctic lineages and India-South and Japan.

[Creation of DNA vaccine vector based on codon-optimized gene of rabies virus glycoprotein (G protein) with consensus amino acid sequence].

An optimized design of the rabies virus glycoprotein (G protein) for use within DNA vaccines has been suggested. The design represents a territorially adapted antigen constructed taking into account glycoprotein amino acid sequences of the rabies viruses registered in the Russian Federation and the vaccine Vnukovo-32 strain. Based on the created consensus amino acid sequence, the nucleotide codon-optimized sequence of this modified glycoprotein was obtained and cloned into the pVAX1 plasmid (a vector of the last generation used in the creation of DNA vaccines). A twofold increase in this gene expression compared to the expression of the Vnukovo-32 strain viral glycoprotein gene in a similar vector was registered in the transfected cell culture. It has been demonstrated that the accumulation of modified G protein exceeds the number of the control protein synthesized using the plasmid with the Vnukovo-32 strain viral glycoprotein gene by 20 times. Thus, the obtained modified rabies virus glycoprotein can be considered to be a promising DNA vaccine antigen.