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.

Immunogenicity evaluation of a bivalent vaccine based on a recombinant rabies virus expressing gB protein of FHV-1 in mice and cats.

Feline viral rhinotracheitis (FVR) is caused by the feline herpesvirus-1 (FHV-1), which commonly results in upper respiratory symptoms, and can result in death in the kittens and weak cats. Rabies is an infectious disease with zoonotic characteristics highly relevant to public health and also poses a serious threat to cats. Vaccines are the most effective method to control the spread of both FHV-1 and RABV and have the advantage that they produce long-term specific immune responses. In this study, we constructed a bivalent vaccine against FHV-1 and rabies virus (RABV) simultaneously. The vaccine was constructed by cloning FHV-1 gB into a RABV based vector, and the recombinant RABV (SRV9-FHV-gB) expressing the FHV-1 gB protein was rescued. The growth characteristics of SRV9-FHV-gB were analyzed on NA and BSR cells. To assess the immunogenicity of the vaccine, mice and cats were immunized with SRV9-FHV-gB supplemented with Gel02 adjuvant. The SRV9-FHV-gB exhibited the same growth characteristics as the parent virus SRV9 in both BSR cells and NA cells. The safety of SRV9-FHV-gB was evaluated using 5-day-old and 14-day-old suckling mice. The results showed that mice infected with the SRV9-FHV-gB survived for longer than those in the SRV9 group. Mice immunized with inactivated SRV9-FHV-gB produced high titers of specific antibodies against FHV-1 and neutralizing antibodies against RABV. Cats that received three immunizations with SRV9-FHV-gB also produced neutralizing antibodies against both FHV-1 and RABV. This study represents the first time that a bivalent vaccine targeting FHV-1 and RABV has been constructed, laying the foundations and providing inspiration for the development of other multivalent vaccines.

Rabies virus-based barcoded neuroanatomy resolved by single-cell RNA and in situ sequencing.

Mapping the connectivity of diverse neuronal types provides the foundation for understanding the structure and function of neural circuits. High-throughput and low-cost neuroanatomical techniques based on RNA barcode sequencing have the potential to map circuits at cellular resolution and a brain-wide scale, but existing Sindbis virus-based techniques can only map long-range projections using anterograde tracing approaches. Rabies virus can complement anterograde tracing approaches by enabling either retrograde labeling of projection neurons or monosynaptic tracing of direct inputs to genetically targeted postsynaptic neurons. However, barcoded rabies virus has so far been only used to map non-neuronal cellular interactions in vivo and synaptic connectivity of cultured neurons. Here we combine barcoded rabies virus with single-cell and in situ sequencing to perform retrograde labeling and transsynaptic labeling in the mouse brain. We sequenced 96 retrogradely labeled cells and 295 transsynaptically labeled cells using single-cell RNA-seq, and 4130 retrogradely labeled cells and 2914 transsynaptically labeled cells in situ. We found that the transcriptomic identities of rabies virus-infected cells can be robustly identified using both single-cell RNA-seq and in situ sequencing. By associating gene expression with connectivity inferred from barcode sequencing, we distinguished long-range projecting cortical cell types from multiple cortical areas and identified cell types with converging or diverging synaptic connectivity. Combining in situ sequencing with barcoded rabies virus complements existing sequencing-based neuroanatomical techniques and provides a potential path for mapping synaptic connectivity of neuronal types at scale.

In the brain, messages are relayed from one cell to the next through intricate networks of axons and dendrites that physically interact at junctions known as synapses. Mapping out this synaptic connectivity ­ that is, exactly which neurons are connected via synapses ­ remains a major challenge. Monosynaptic tracing is a powerful approach that allows neuroscientists to explore neural networks by harnessing viruses which spread between neurons via synapses, in particular the rabies virus. This pathogen travels exclusively from 'postsynaptic' to 'presynaptic' neurons ­ from the cell that receives a message at a synapse, back to the one that sends it. A modified variant of the rabies virus can therefore be used to reveal the presynaptic cells connecting to a population of neurons in which it has been originally introduced. However, this method does not allow scientists to identify the exact postsynaptic neuron that each presynaptic cell is connected to. One way to bypass this issue is to combine monosynaptic tracing with RNA barcoding to create distinct versions of the modified rabies virus, which are then introduced into separate populations of neurons. Tracking the spread of each version allows neuroscientists to spot exactly which presynaptic cells signal to each postsynaptic neuron. So far, this approach has been used to examine synaptic connectivity in neurons grown in the laboratory, but it remains difficult to apply it to neurons in the brain. In response, Zhang, Jin et al. aimed to demonstrate how monosynaptic tracing that relies on barcoded rabies viruses could be used to dissect neural networks in the mouse brain. First, they confirmed that it was possible to accurately detect which version of the virus had spread to presynaptic neurons using both in situ and single-cell RNA sequencing. Next, they described how this information could be analysed to build models of potential neural networks, and what type of additional experiments are required for this work. Finally, they used the approach to identify neurons that tend to connect to the same postsynaptic cells and then investigated what these have in common, showing how the technique enables a finer understanding of neural circuits. Overall, the work by Zhang, Jin et al. provides a comprehensive review of the requirements and limitations associated with monosynaptic tracing experiments based on barcoded rabies viruses, as well as how the approach could be optimized in the future. This information will be of broad interest to scientists interested in mapping neural networks in the brain.

A flowchart for adequate controls in virus-based monosynaptic tracing experiments identified Cre-independent leakage of the TVA receptor in RΦGT mice.

BACKGROUND: A pseudotyped modified rabies virus lacking the rabies glycoprotein (G-protein), which is crucial for transsynaptic spread, can be used for monosynaptic retrograde tracing. By coupling the pseudotyped virus with transgene expression of the G-protein and the avian leukosis and sarcoma virus subgroup A receptor (TVA), which is necessary for cell entry of the virus, researchers can investigate specific neuronal populations. Responder mouse lines, like the RΦGT mouse line, carry the genes encoding the G-protein and TVA under Cre-dependent expression. These mouse lines are valuable tools because they reduce the number of viral injections needed compared to when using helper viruses. Since RΦGT mice do not express Cre themselves, introducing the pseudotyped rabies virus into their brain should not result in viral cell entry or spread. RESULTS: We present a straightforward flowchart for adequate controls in tracing experiments, which we employed to demonstrate Cre-independent expression of TVA in RΦGT mice. CONCLUSIONS: Our observations revealed TVA leakage, indicating that RΦGT mice should be used with caution for transgene expression of TVA. Inaccurate tracing outcomes may occur if TVA is expressed in the absence of Cre since background leakage leads to nonspecific cell entry. Moreover, conducting appropriate control experiments can identify the source of potential caveats in virus-based neuronal tracing experiments.

Lyssavirus M protein degrades neuronal microtubules by reprogramming mitochondrial metabolism.

Infection with neurotropic viruses may result in changes in host behavior, which are closely associated with degenerative changes in neurons. The lyssavirus genus comprises highly neurotropic viruses, including the rabies virus (RABV), which has been shown to induce degenerative changes in neurons, marked by the self-destruction of axons. The underlying mechanism by which the RABV degrades neuronal cytoskeletal proteins remains incomplete. In this study, we show that infection with RABV or overexpression of its M protein can disrupt mitochondrial metabolism by binding to Slc25a4. This leads to a reduction in NAD+ production and a subsequent influx of Ca2+ from the endoplasmic reticulum and mitochondria into the cytoplasm of neuronal cell lines, activating Ca2+-dependent proteinase calpains that degrade α-tubulin. We further screened the M proteins of different lyssaviruses and discovered that the M protein of the dog-derived RABV strain (DRV) does not degrade α-tubulin. Sequence analysis of the DRV M protein and that of the lab-attenuated RABV strain CVS revealed that the 57th amino acid is vital for M-induced microtubule degradation. We generated a recombinant RABV with a mutation at the 57th amino acid position in its M protein and showed that this mutation reduces α-tubulin degradation in vitro and axonal degeneration in vivo. This study elucidates the mechanism by which lyssavirus induces neuron degeneration.IMPORTANCEPrevious studies have suggested that RABV (rabies virus, the representative of lyssavirus) infection induces structural abnormalities in neurons. But there are few articles on the mechanism of lyssavirus' effect on neurons, and the mechanism of how RABV infection induces neurological dysfunction remains incomplete. The M protein of lyssavirus can downregulate cellular ATP levels by interacting with Slc25a4, and this decrease in ATP leads to a decrease in the level of NAD+ in the cytosol, which results in the release of Ca2+ from the intracellular calcium pool, the endoplasmic reticulum, and mitochondria. The presence of large amounts of Ca2+ in the cytoplasm activates Ca2+-dependent proteases and degrades microtubule proteins. The amino acid 57 of M protein is the key site determining its disruption of mitochondrial metabolism and subsequent neuron degeneration.

A modified recombinant adenovirus vector containing dual rabies virus G expression cassettes confers robust and long-lasting humoral immunity in mice, cats, and dogs.

During the COVID-19 epidemic, the incidence of rabies has increased in several countries, especially in remote and disadvantaged areas, due to inadequate surveillance and declining immunization coverage. Multiple vaccinations with inactivated rabies virus vaccines for pre- or post-exposure prophylaxis are considered inefficient, expensive and impractical in developing countries. Herein, three modified human recombinant adenoviruses type 5 designated Adv-RVG, Adv-E1-RVG, and Adv-RVDG, carrying rabies virus G (RVG) expression cassettes in various combinations within E1 or E3 genomic regions, were constructed to serve as rabies vaccine candidates. Adv-RVDG mediated greater RVG expression both in vitro and in vivo and induced a more robust and durable humoral immune response than the rabies vaccine strain SAD-L16, Adv-RVG, and Adv-E1-RVG by more effectively activating the dendritic cells (DCs) - follicular helper T (Tfh) cells - germinal centre (GC) / memory B cells (MBCs) - long-lived plasma cells (LLPCs) axis with 100% survival after a lethal RABV challenge in mice during the 24-week study period. Similarly, dogs and cats immunized with Adv-RVDG showed stronger and longer-lasting antibody responses than those vaccinated with a commercial inactivated rabies vaccine and showed good tolerance to Adv-RVDG. In conclusion, our study demonstrated that simultaneous insertion of protective antigens into the E1 and E3 genomic regions of adenovirus vector can significantly enhance the immunogenicity of adenoviral-vectored vaccines, providing a theoretical and practical basis for the subsequent development of multivalent and multi-conjugated vaccines using recombinant adenovirus platform. Meanwhile, our data suggest Adv-RVDG is a safe, efficient, and economical vaccine for mass-coverage immunization.

Long-term labeling and imaging of synaptically connected neuronal networks in vivo using double-deletion-mutant rabies viruses.

Rabies-virus-based monosynaptic tracing is a widely used technique for mapping neural circuitry, but its cytotoxicity has confined it primarily to anatomical applications. Here we present a second-generation system for labeling direct inputs to targeted neuronal populations with minimal toxicity, using double-deletion-mutant rabies viruses. Viral spread requires expression of both deleted viral genes in trans in postsynaptic source cells. Suppressing this expression with doxycycline following an initial period of viral replication reduces toxicity to postsynaptic cells. Longitudinal two-photon imaging in vivo indicated that over 90% of both presynaptic and source cells survived for the full 12-week course of imaging. Ex vivo whole-cell recordings at 5 weeks postinfection showed that the second-generation system perturbs input and source cells much less than the first-generation system. Finally, two-photon calcium imaging of labeled networks of visual cortex neurons showed that their visual response properties appeared normal for 10 weeks, the longest we followed them.

Sf9 Cell Metabolism Throughout the Recombinant Baculovirus and Rabies Virus-Like Particles Production in Two Culture Systems.

This work aimed to assess the Sf9 cell metabolism during growth, and infection steps with recombinant baculovirus bearing rabies virus proteins, to finally obtain rabies VLP in two culture systems: Schott flask (SF) and stirred tank reactor (STR). Eight assays were performed in SF and STR (four assays in each system) using serum-free SF900 III culture medium. Two non-infection growth kinetics assays and six recombinant baculovirus infection assays. The infection runs were carried out at 0.1 pfu/cell multiplicity of infection (MOI) for single baculovirus bearing rabies glycoprotein (BVG) and matrix protein (BVM) and a coinfection with both baculoviruses at MOI of 3 and 2 pfu/cell for BVG and BVM, respectively. The SF assays were done in triplicate. The glucose, glutamine, glutamate, lactate, and ammonium uptake or release specific rates were quantified over the exponential growth phase and infection stage. The highest uptake specific rate was observed for glucose (42.5 × 10-12 mmol cell/h) in SF and for glutamine (30.8 × 10-12 mmol/cell/h) in STR, in the exponential growth phases. A wave pattern was observed for assessed analytes throughout the infection phase and the glucose had the highest wave amplitude within the 10-10 mmol cell/h order. This alternative uptake and release behavior is in harmony with the lytic cycle of baculovirus in insect cells. The virus propagation and VLP generation were not limited by glucose, glutamine, and glutamate, neither by the toxicity of lactate nor ammonium under the conditions appraised in this work. The findings from this work can be useful to set baculovirus infection processes at high cell density to improve rabies VLP yield, purity, and productivity.

Development of a novel multi-epitope oral DNA vaccine for rabies based on a food-borne microbial vector.

Rabies has been with humans for a long time, and its special transmission route and almost 100 % lethality rate made it once a nightmare for humans. In this study, by predicting the rabies virus glycoprotein outer membrane region and nucleoprotein B-cell antigenic epitopes, the coding sequence of the predicted highly antigenic polypeptide region obtained was assembled using the eukaryotic expression vector pcDNA3.1(-), and then E. coli was used as the delivery vector. The immunogenicity and protective properties of the vaccine were verified by in vivo and in vitro experiments, which demonstrated that the vaccine could produce antibodies in mice and prolong the survival time of mice exposed to the strong virus without any side effects. This study demonstrated that the preparation of an oral rabies DNA vaccine using food-borne microorganisms as a transport vehicle is feasible and could be a new strategy to eradicate rabies starting with wild animals.

High risk of bat bites in an indigenous village in Brazil: Warning of the re-emergence of rabies among the Maxakali People.

Bat-mediated human rabies is a viral zoonotic disease that poses a serious threat to the public health of traditional peoples, especially indigenous populations that maintain primitive cultural and social habits, such as the Maxakali ethnic group, located in the southeastern region of Brazil. The sociocultural habit of this population led to the emergence between April and May 2022 of the viral spillover of rabies transmitted by bats, which decimated the lives of four children from this population who maintained contact with this animal as a recreational practice. Because the vampire bats Desmodus rotundus have exceptional ecology and social characteristics that can have important effects on the dynamics of viral dispersion in this indigenous population, I present the dynamics of contact between native children and the bat and the meaning of this relationship, which involves ritualistic and recreational significance. As important as knowing the reasons for this practice is discussing some intrinsic and extrinsic factors that imply risks that intensify the vulnerability of this population to the transmission of the rabies virus at any time. In view of this, I warn of the need to adopt efficient strategies to mitigate the risks of a new emergency in this region. Although emergency containment measures were carried out during the critical period of the outbreak, such animal and environmental control actions must become routine programmatic and structuring interventions. Essential for rabies surveillance in this population is to develop culturally adapted interethnic health education campaigns to guarantee the accessibility of the Maxakali indigenous people to the content taught, so that any attempt at domestication, captivity and recreational practices with bats of any species is discouraged, thus avoiding a possible re-emergence of this anthropozoonosis that has impacted not only the epidemiological scenario in this region, but throughout Brazil, and also throughout Latin America.

Phylogenetic relationship between rabies virus (Lyssavirus rabies) variants from two different species / Relações filogenéticas do vírus da raiva (Lyssavirus rabies) em duas diferentes espécies / Relaciones filogenéticas del virus de la rabia (Lyssavirus rabies) en dos especies diferentes

Rabies is a fatal zoonotic disease that affects several mammals. Hematophagous bats are recognized hosts of the rabies virus, and their main food source is the blood of other mammals, particularly cattle. During feeding, bats transmit the virus to cattle, which are victims of the disease, contributing to economic losses and increasing the risk of infection for humans. Based on this affinity in the rabies cycle between bats and cattle, the objective of this study was to analyze the phylogenetic relationships of rabies virus samples in cattle and bats. The G gene of the rabies virus was chosen for this study because it is directly related to the infection process. Nucleotide sequences of the viral G gene were selected from GenBank for samples obtained from infected cattle and bats. Maximum parsimony analyses were conducted using the Molecular Evolutionary Genetics Analysis software. The Maxima Parsimony tree indicated a phylogenetic relationship between the G genes of both hosts, indicating that the virus evolved from bats to cattle. Analysis of parsimoniously informative sites revealed that the viral G gene carried specific mutations in each host. Knowledge of the evolutionary relationships between the rabies virus and its hosts is critical for identifying potential new hosts and the possible routes of infection for humans.

A Raiva é uma zoonose fatal que infecta várias espécies de mamíferos. Os morcegos hematófagos são reconhecidos como hospedeiros do vírus da Raiva e sua principal fonte de alimento é o sangue de outros mamíferos, especialmente os bovinos. Quando se alimentam, os morcegos transmitem o vírus para o bovino os quais são vítimas da doença, contribuindo para perdas econômicas e riscos de infecção para humanos. Baseado nesta afinidade do ciclo da Raiva entre morcegos e bovinos, o objetivo deste estudo foi analisar as relações filogenéticas de amostras do vírus da Raiva em ambos os hospedeiros, bovinos e morcegos. O gene G do vírus da Raiva foi escolhido para esta pesquisa porque ele está diretamente relacionado ao processo de infecção. Sequências de nucleotídeos do gene G viral foram selecionadas no GenBank a partir de amostras obtidas de bovinos e morcegos infectados. Análises de Máxima Parcimônia foram conduzidas utilizando o software Molecular Evolutionary Genetics Analysis. A árvore de Máxima Parcimônia indicou uma relação filogenética entre o gene G de ambos os hospedeiros, indicando que o vírus evoluiu dos morcegos para os bovinos. A análise dos sítios parcimoniosamente informativos revelou que o gene G viral apresentou mutações específicas em cada hospedeiro. O conhecimento sobre as relações evolutivas do vírus da Raiva e seus hospedeiros é crucial para identificar nos hospedeiros potenciais e novas rotas possíveis de infecção para humanos.

La rabia es una zoonosis fatal que infecta a varias especies de mamíferos. Los murciélagos hematófagos son reconocidos como huéspedes del virus de la rabia y su principal fuente de alimentación es la sangre de otros mamíferos, especialmente del ganado. Al alimentarse, los murciélagos transmiten el virus al ganado que es víctima de la enfermedad, contribuyendo a pérdidas económicas y riesgos de infección para los humanos. Basado en esta afinidad del ciclo de la rabia entre murciélagos y ganado, el objetivo de este estudio fue analizar las relaciones filogenéticas de las muestras de virus de la rabia tanto en huéspedes, ganado y murciélagos. El gen G del virus de la rabia fue elegido para esta investigación porque está directamente relacionado con el proceso de infección. Las secuencias de nucleótidos del gen G viral se seleccionaron en GenBank a partir de muestras obtenidas de bovinos y murciélagos infectados. Los análisis de parsimonia máxima se realizaron utilizando el software Molecular Evolutionary Genetics Analysis. El árbol de Máxima Parsimônia indicó una relación filogenética entre el gen G de ambos huéspedes, indicando que el virus evolucionó de murciélagos a bovinos. El análisis de los sitios parsimoniosamente informativos reveló que el gen G viral presentaba mutaciones específicas en cada huésped. El conocimiento sobre las relaciones evolutivas del virus de la rabia y sus huéspedes es crucial para identificar huéspedes potenciales y nuevas posibles rutas de infección para humanos.

An mRNA vaccine against rabies provides strong and durable protection in mice.

Introduction: Rabies is a serious public health problem worldwide for which an effective treatment method is lacking but can be prevented by vaccines. Current vaccines are produced in cell or egg cultures, which are both costly and time consuming. Methods: Here, a non-replicating mRNA vaccine (RV021) encoding the rabies virus glycoprotein was developed in vitro, and its immunogenicity and protective efficacy against live virus was evaluated in mice. Results: A two-dose vaccination with 1 µg of RV021 at 7-day intervals induced a protective level of neutralizing antibody that was maintained for at least 260 days. RV021 induced a robust cellular immune response that was significantly superior to that of an inactivated vaccine. Two doses of 1 µg RV021 provided full protection against challenge with CVS of 30~60-fold lethal dose, 50%. Vaccine potency testing (according to the National Institutes of Health) in vivo revealed that the potency of RV021 at 15 µg/dose was 7.5 IU/dose, which is substantially higher than the standard for lot release of rabies vaccines for current human use. Conclusion: The mRNA vaccine RV021 induces a strong protective immune response in mice, providing a new and promising strategy for human rabies prevention and control.

Third-generation rabies viral vectors have low toxicity and improved efficiency as retrograde labeling tools.

In this issue of Cell Reports Methods, Jin et al. report the generation and validation of a rabies variant, RVΔL, for projection-based neuronal labeling. RVΔL shows little toxicity in vivo and has an improved growth advantage over another variant, RVΔGL, making it a useful tool for a wide variety of systems neuroscience-based studies.

Third-generation rabies viral vectors allow nontoxic retrograde targeting of projection neurons with greatly increased efficiency.

Rabies viral vectors have become important components of the systems neuroscience toolkit, allowing both direct retrograde targeting of projection neurons and monosynaptic tracing of inputs to defined postsynaptic populations, but the rapid cytotoxicity of first-generation (ΔG) vectors limits their use to short-term experiments. We recently introduced second-generation, double-deletion-mutant (ΔGL) rabies viral vectors, showing that they efficiently retrogradely infect projection neurons and express recombinases effectively but with little to no detectable toxicity; more recently, we have shown that ΔGL viruses can be used for monosynaptic tracing with far lower cytotoxicity than the first-generation system. Here, we introduce third-generation (ΔL) rabies viral vectors, which appear to be as nontoxic as second-generation ones but have the major advantage of growing to much higher titers, resulting in significantly increased numbers of retrogradely labeled neurons in vivo.

Genomic stability of self-inactivating rabies.

Transsynaptic viral vectors provide means to gain genetic access to neurons based on synaptic connectivity and are essential tools for the dissection of neural circuit function. Among them, the retrograde monosynaptic ΔG-Rabies has been widely used in neuroscience research. A recently developed engineered version of the ΔG-Rabies, the non-toxic self-inactivating (SiR) virus, allows the long term genetic manipulation of neural circuits. However, the high mutational rate of the rabies virus poses a risk that mutations targeting the key genetic regulatory element in the SiR genome could emerge and revert it to a canonical ΔG-Rabies. Such revertant mutations have recently been identified in a SiR batch. To address the origin, incidence and relevance of these mutations, we investigated the genomic stability of SiR in vitro and in vivo. We found that "revertant" mutations are rare and accumulate only when SiR is extensively amplified in vitro, particularly in suboptimal production cell lines that have insufficient levels of TEV protease activity. Moreover, we confirmed that SiR-CRE, unlike canonical ΔG-Rab-CRE or revertant-SiR-CRE, is non-toxic and that revertant mutations do not emerge in vivo during long-term experiments.

A novel mRNA rabies vaccine as a promising candidate for rabies post-exposure prophylaxis protects animals from different rabies viruses.

Rabies, caused by the rabies virus (RABV), is the most fatal zoonotic disease. It is a neglected tropical disease which remains a major public health problem, causing approximately 59,000 deaths worldwide annually. Despite the existence of effective vaccines, the high incidence of human rabies is mainly linked to tedious vaccine immunisation procedures and the overall high cost of post-exposure prophylaxis. Therefore, it is necessary to develop an effective vaccine that has a simple procedure and is affordable to prevent rabies infection in humans. RABV belongs to the genus Lyssavirus and family Rhabdoviridae. Previous phylogenetic analyses have identified seven major clades of RABV in China (China I-VII), confirmed by analysing nucleotide sequences from both the G and N proteins. This study evaluated the immunogenicity and protective capacity of SYS6008, an mRNA rabies vaccine expressing rabies virus glycoprotein, in mice and cynomolgus macaques. We demonstrated that SYS6008 induced sufficient levels of rabies neutralising antibody (RVNA) in mice. In addition, SYS6008 elicited strong and durable RVNA responses in vaccinated cynomolgus macaques. In the pre-exposure prophylaxis murine model, one or two injections of SYS6008 at 1/10 or 1/30 of dosage provided protection against a challenge with a 30-fold LD50 of rabies virus (China I and II clades). We also demonstrated that in the post-exposure prophylaxis murine model, which was exposed to lethal rabies virus (China I-VII clades) before vaccination, one or two injections of SYS6008 at both 1/10 and 1/30 dosages provided better protection against rabies virus challenge than the immunization by five injections of commercial vaccines at the same dosage. In addition, we proved that SYS6008-induced RVNAs could neutralise RABV from the China I-VII clades. Finally, 1/10 of the dosage of SYS6008 was able to stimulate significant RABV-G specificity in the T cell response. Furthermore, we found that SYS6008 induced high cellular immunity, including RABV-G-specific T cell responses and memory B cells. Our results imply that the SYS6008 rabies vaccine, with a much simpler vaccination procedure, better immunogenicity, and enhanced protective capacity, could be a candidate vaccine for post-exposure prophylaxis of rabies infections.

Genetic analyses of rabies virus glycoprotein and nucleoprotein gene sequences reveal the emergence of multiple lineages in animals in Arkhangai province, a central region of Mongolia.

Genetic characterizations of rabies viruses circulating in carnivore and non-carnivore animals were investigated for the first time in Arkhangai province, a central region of Mongolia. Also, glycoprotein gene of the rabies virus was sequenced for the first time in Mongolia. The nucleotide sequences of the glycoprotein and nucleoprotein genes were analysed, revealing the presence of multiple lineages in this area. Of particular concern are the lineages identified in carnivores, which might emerge to spread throughout Mongolia, further facilitating transboundary transmission to neighbouring countries, including China and Russia.

Immunogenicity and Antigenicity of the Ectodomain of Rabies Virus Glycoprotein Stably Expressed in HEK293T Cells.

Rabies continues to be a huge threat to public health. The rabies virus envelope glycoprotein (RABV G) is a major rabies virus antigen and contains neutralizing epitopes, which are primary candidates for subunit vaccines and diagnostic antigens. However, the production and purification of rRABV G while retaining its antigenic and immunogenic remains to be a challenge. Here, we aimed to establish a platform for rRABV G production and purification, and determine the immunogenicity and antigenicity of rRABV G. The cDNA fragment encoding the soluble form of RABV G was synthesized and cloned into a lentiviral expressing vector. Recombinant lentiviral vector LV-CMV-RABV G-eGFP was packaged, titered, and then transduced into HEK 293T cells. The cell culture supernatant was purified using nickel affinity chromatography and subsequently confirmed through Western Blot analysis and indirect enzyme-linked immunosorbent assay (ELISA). The ELISA utilized human sera obtained from individuals who had been vaccinated with the human commercial Purified Vero Cells Rabies Vaccine (PVRV). Notably, we observed a neutralizing antibody response in immunized pigs rather than in mice. This discrepancy could potentially be attributed to factors such as the instability of the rRABV G protein, variations in host responses, and variances in the adjuvant used. Taking all these findings into account, the rRABV G protein generated in this study exhibits promise as a potential vaccine candidate for the prevention of rabies.

Evaluation of Taxonomic Characteristics of Matlo and Phala Bat Rabies-Related Lyssaviruses Identified in South Africa.

We report the genetic characterization of two potentially novel rabies-related lyssaviruses identified from bats in Limpopo province, South Africa. Matlo bat lyssavirus (MBLV) was identified in two Miniopterus natalensis (Natal long-fingered) bats in 2015 and 2016, and Phala bat lyssavirus (PBLV) was identified in a Nycticeinops schlieffeni (Schlieffen's) bat in 2021. The distribution of both of these bat species is largely confined to parts of Africa, with limited reports from the Arabian Peninsula. MBLV and PBLV were demonstrated to group with the unassigned and phylogroup I lyssaviruses, respectively. MBLV was most closely related to Lyssavirus caucasicus (WCBV), whereas PBLV was most closely related to Lyssavirus formosa (TWBLV-1) and Taiwan bat lyssavirus 2 (TWBLV-2), based on analysis of the N and G genes, the concatenated N + P + M + G + L coding sequence, and the complete genome sequence. Based on our analysis, MBLV and WCBV appeared to constitute a phylogroup separate from Lyssavirus lleida (LLEBV) and Lyssavirus ikoma (IKOV). Analysis of the antigenic sites suggests that PBLV will likely be serologically distinguishable from established lyssaviruses in virus-neutralization tests, whereas MBLV appeared to be antigenically highly similar to WCBV. Taken together, the findings suggested that, while PBLV is likely a new lyssavirus species, MBLV is likely related to WCBV.

A single immunization with core-shell structured lipopolyplex mRNA vaccine against rabies induces potent humoral immunity in mice and dogs.

The persistence and clinical consequences of rabies virus (RABV) infection have prompted global efforts to develop a safe and effective vaccines against rabies. mRNA vaccines represent a promising option against emerging and re-emerging infectious diseases, gaining particular interest since the outbreak of COVID-19. Herein, we report the development of a highly efficacious rabies mRNA vaccine composed of sequence-modified mRNA encoding RABV glycoprotein (RABV-G) packaged in core-shell structured lipopolyplex (LPP) nanoparticles, named LPP-mRNA-G. The bilayer structure of LPP improves protection and delivery of RABV-G mRNA and allows gradual release of mRNA molecules as the polymer degrades. The unique core-shell structured nanoparticle of LPP-mRNA-G facilitates vaccine uptake and demonstrates a desirable biodistribution pattern with low liver targeting upon intramuscular immunization. Single administration of low-dose LPP-mRNA-G in mice elicited potent humoral immune response and provided complete protection against intracerebral challenge with lethal RABV. Similarly, single immunization of low-dose LPP-mRNA-G induced high levels of virus-neutralizing antibody titers in dogs. Collectively, our data demonstrate the potential of LPP-mRNA-G as a promising next-generation rabies vaccine used in human and companion animals.