Rabies Virus Strains Research Articles

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.

Reverse genetic approaches allowing the characterization of the rabies virus street strain belonging to the SEA4 subclade

Rabies virus (RABV) is the causative agent of rabies, a lethal neurological disease in mammals. RABV strains can be classified into fixed strains (laboratory strains) and street strains (field/clinical strains), which have different properties including cell tropism and neuroinvasiveness. RABV Toyohashi strain is a street strain isolated in Japan from an imported case which had been bitten by rabid dog in the Philippines. In order to facilitate molecular studies of RABV, we established a reverse genetics (RG) system for the study of the Toyohashi strain. The recombinant virus was obtained from a cDNA clone of Toyohashi strain and exhibited similar growth efficiency as the original virus in cultured cell lines. Both the original and recombinant strains showed similar pathogenicity with high neuroinvasiveness in mice, and the infected mice developed a long and inconsistent incubation period, which is characteristic of street strains. We also generated a recombinant Toyohashi strain expressing viral phosphoprotein (P protein) fused with the fluorescent protein mCherry, and tracked the intracellular dynamics of the viral P protein using live-cell imaging. The presented reverse genetics system for Toyohashi strain will be a useful tool to explore the fundamental molecular mechanisms of the replication of RABV street strains.

Safety and efficacy assessment of an mRNA rabies vaccine in dogs, rodents, and cynomolgus macaques

Rabies is a lethal disease caused by the rabies virus (RABV), which causes acute neurological infections in mammals, including human beings. We previously reported that an mRNA vaccine (LVRNA001) encoding the rabies virus’s glycoprotein induced strong protective immune responses to rabies in mice and dogs. Here, we further evaluate the safety of LVRNA001. First, we performed a confirmative efficacy study in dogs, which showed that LVRNA001 fully protected the animals from the virus, both pre- and post-infection. Moreover, using pre- and post-exposure prophylaxis murine models, we showed that LVRNA001, built from the CTN-1 strain, was able to protect against various representative RABV strains from the China I–VII clades. To evaluate the safety of the vaccine, chronic and reproductive toxicity studies were performed with cynomolgus macaques and rats, respectively. In a repeated-dose chronic toxicity study, vaccinated monkeys displayed no significant alterations in body weight, temperature, or hematological and biochemical markers. Lymphocyte subset measurement and histopathological examination showed that no toxicity was associated with the vaccine. The immunogenicity study in cynomolgus macaques demonstrated that LVRNA001 promoted the generation of neutralizing antibodies and Th1-biased immune response. Evaluation of reproductive toxicity in rats revealed that administration of LVRNA001 had no significant effects on fertility, maternal performance, reproductive processes, and postnatal outcomes. In conclusion, LVRNA001 can provide efficient protection against rabies virus infection in dogs and mice, and toxicity studies showed no significant vaccine-related adverse effects, suggesting that LVRNA001 is a promising and safe vaccine candidate for rabies prophylaxis and therapy.

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

BackgroundRabies 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 resultsThe 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).ConclusionsIn 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.

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

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

Determination of the rabies virus variants circulating in Poland in 2021-2023 and their phylogeny with analysis of the strains in the Mazowieckie and Podkarpackie voivodeships.

Rabies is endemic in Europe and red foxes are the vector and reservoir of the rabies virus (RABV). Based on classification established in the early 1990s, four variants of the rabies virus have been distinguished in Europe. Rabies broke out in January 2021 in the Mazowieckie voivodeship in central north-eastern Poland. The virus spread rapidly, reaching the Świętokrzyskie voivodeship in the central southern part and the Lubelskie voivodeship in the eastern part in the next months. Nine rabies cases were reported in the Podkarpackie voivodeship in south-eastern Poland between 2021 and 2023, mainly in red foxes but also in dogs and wildcat. The aim of the study was the identification of RABV variants in wildlife and domestic animals in Poland between 2021 and 2023. The study involved 157 animal brains tested positive for rabies using a fluorescent antibody test. From 10% w/v brain homogenates, RNA was isolated and full-length RABV genomes were high-throughput sequenced with an RABV-enriched approach. Complete genomes of RABV isolates were phylogenetically analysed and the variants were estimated. Molecular and phylogenetic studies revealed 147 (93.6%) of the RABV strains out of 157 which had rapidly spread in the wildlife of the Mazowieckie, Świętokrzyskie and Lubelskie voivodeships to be Central European strains. Nine RABVs (5.7%) detected in foxes, a wildcat and a dog in the Podkarpackie voivodeship were identified as North-Eastern European. A vaccine-induced rabies case was detected in a red fox in the Lubelskie voivodeship in May 2023. Central European and North-Eastern European RABVs were circulating in Poland between 2021 and 2023.

Construction of Vero cell-adapted rabies vaccine strain by five amino acid substitutions in HEP-Flury strain

Rabies virus (RABV) causes fatal neurological disease. Pre-exposure prophylaxis (PrEP) and post-exposure prophylaxis (PEP) using inactivated-virus vaccines are the most effective measures to prevent rabies. In Japan, HEP-Flury, the viral strain, used as a human rabies vaccine, has historically been propagated in primary fibroblast cells derived from chicken embryos. In the present study, to reduce the cost and labor of vaccine production, we sought to adapt the original HEP-Flury (HEP) to Vero cells. HEP was repeatedly passaged in Vero cells to generate ten- (HEP-10V) and thirty-passaged (HEP-30V) strains. Both HEP-10V and HEP-30V grew significantly better than HEP in Vero cells, with virulence and antigenicity similar to HEP. Comparison of the complete genomes with HEP revealed three non-synonymous mutations in HEP-10V and four additional non-synonymous mutations in HEP-30V. Comparison among 18 recombinant HEP strains constructed by reverse genetics and vesicular stomatitis viruses pseudotyped with RABV glycoproteins indicated that the substitution P(L115H) in the phosphoprotein and G(S15R) in the glycoprotein improved viral propagation in HEP-10V, while in HEP-30V, G(V164E), G(L183P), and G(A286V) in the glycoprotein enhanced entry into Vero cells. The obtained recombinant RABV strain, rHEP-PG4 strain, with these five substitutions, is a strong candidate for production of human rabies vaccine.

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-1β, 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.

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.

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.

Transcriptome analysis of salivary glands of rabies-virus-infected mice.

Rabies is a fatal zoonotic disease that poses a threat to public health. Rabies virus (RABV) is excreted in the saliva of infected animals, and is primarily transmitted by bite. The role of the salivary glands in virus propagation is significant, but has been less studied in the pathogenic mechanisms of RABV. To identify functionally important genes in the salivary glands, we used RNA sequencing (RNA-seq) to establish and analyze mRNA expression profiles in parotid tissue infected with two RABV strains, CVS-11 and PB4. The biological functions of differentially expressed genes (DEGs) were determined by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, which revealed 3,764 DEGs (678 up-regulated and 3,086 down-regulated) in the CVS-11 infected group and 4,557 DEGs (874 up-regulated and 3,683 down-regulated) in the PB4 infected group. Various biological processes are involved, including the salivary secretion pathway and the phosphatidylinositol 3-kinase-Akt (PI3K-Akt) signaling pathway. This study provides the first mapping of the transcriptome changes in response to RABV infection in parotid tissue, offering new insights into the study of RABV-affected salivary gland function and RABV pathogenic mechanisms in parotid tissue. The salivary gland-enriched transcripts may be potential targets of interest for rabies disease control.

Genetic characteristics and geographic distribution of rabies virus in China.

China is one of the largest countries with endemic rabies. In this study, we examined the full-length genome sequences of 87 rabies virus (RABV) strains identified in China from 1931 to 2019. Chinese RABV isolates were divided into two major clades, GI and GII. Clade GI consisted of viruses from the Asian clade, which was further divided into three subclades: Asian1, Asian2, and Asian3. Clade GII consisted of viruses from the Cosmopolitan, Arctic-related, and Indian clades. A phylogeographic network showed that the variation of rabies virus was more closely associated with geographic location than with the host species. Recombination appears to be one of the factors driving the emergence of new viral strains.

Internalization of rabies virus glycoprotein differs between pathogenic and attenuated virus strains.

The zoonotic rabies virus (RABV) is a non-segmented negative-sense RNA virus classified within the family Rhabdoviridae, and is the most common aetiological agent responsible for fatal rabies disease. The RABV glycoprotein (G) forms trimeric spikes that protrude from RABV virions and mediate virus attachment, entry and spread, and is a major determinant of RABV pathogenesis. A range of RABV strains exist that are highly pathogenic in part due to their ability to evade host immune detection. However, some strains are disease-attenuated and can be cleared by host defences. A detailed molecular understanding of how strain variation relates to pathogenesis is currently lacking. Here, we reveal key differences in the trafficking profiles of RABV-G proteins from the challenge virus standard strain (CVS-11) and a highly attenuated vaccine strain SAD-B19 (SAD). We show that CVS-G traffics to the cell surface and undergoes rapid internalization through both clathrin- and cholesterol-dependent endocytic pathways. In contrast, SAD-G remains resident at the plasma membrane and internalizes at a significantly slower rate. Through engineering hybrids of CVS-G and SAD-G, we show that the cytoplasmic tail of CVS-G is the key determinant of these different internalization profiles. Alanine scanning further revealed that mutation of Y497 in CVS-G (H497 in SAD-G) could reduce the rate of internalization to SAD-G levels. Together, these data reveal new phenotypic differences between CVS-G and SAD-G proteins that may contribute to altered in vivo pathogenicity.

Inactivated Recombinant Rabies Virus Displaying the Nipah Virus Envelope Glycoproteins Induces Systemic Immune Responses in Mice.

Nipah virus (NiV) causes severe, lethal encephalitis in humans and pigs. However, there is no licensed vaccine available to prevent NiV infection. In this study, we used the reverse genetic system based on the attenuated rabies virus strain SRV9 to construct two recombinant viruses, rSRV9-NiV-F and rSRV9-NiV-G, which displayed the NiV envelope glycoproteins F and G, respectively. Following three immunizations in BALB/c mice, the inactivated rSRV9-NiV-F and rSRV9-NiV-G alone or in combination, mixed with the adjuvants ISA 201 VG and poly (I:C), were able to induce the antigen-specific cellular and Th1-biased humoral immune responses. The specific antibodies against rSRV9-NiV-F and rSRV9-NiV-G had reactivity with two constructed bacterial-like particles displaying the F and G antigens of NiV. These data demonstrate that rSRV9-NiV-F or rSRV9-NiV-G has the potential to be developed into a promising vaccine candidate against NiV infection.

Bardoxolone methyl inhibits the infection of rabies virus via Nrf2 pathway activation in vitro

BackgroundRabies is a widespread, fatal, infectious disease. Several antivirals against rabies virus (RABV) infection have been reported, but no approved, RABV-specific antiviral drugs that inhibit RABV infection in the clinic after symptom onset are available. Therefore, more effective drugs to reduce rabies fatalities are urgently needed. Bardoxolone methyl (CDDO-Me), an FDA-approved compound that has long been known as an antioxidant inflammatory modulator and one of the most potent nuclear factor erythroid-derived 2-like 2 (Nrf2) activators, protects myelin, axons, and CNS neurons by Nrf2 activation. Therefore, we investigated the potency of its anti-RABV activity in vitro.MethodsThe mouse neuroblastoma cell line Neuro2a (N2a) and three RABV strains of different virulence were used; the cytotoxicity and anti-RABV activity of CDDO-Me in N2a cells were evaluated by CCK-8 assay and direct fluorescent antibody (DFA) assay. Pathway activation in N2a cells infected with the RABV strains SC16, CVS-11 or CTN upon CDDO-Me treatment was evaluated by western blotting (WB) and DFA assay.ResultsCDDO-Me significantly inhibited infection of the three RABV strains of differing virulence (SC16, CVS-11 and CTN) in N2a cells. We also examined whether CDDO-Me activates the Nrf2-associated pathway upon infection with RABV strains of differing virulence. Nrf2, phosphorylated sequestosome (SQSTM1), SQSTM1, hemoglobin oxygenase (HO-1) and NAD(P)H dehydrogenase quinone 1 (NQO1) expression in N2a cells increased to varying degrees with CDDO-Me treatment, accompanied by Kelch-like ECH-associated protein 1 (Keap1) dissociation, upon infection with SC16, CVS-11 or CTN. The activation of SQSTM1 phosphorylation was significantly associated with the degradation of Keap-1 in CDDO-Me-treated N2a cells upon RABV infection. Furthermore, N2a cells pretreated with the Nrf2-specific inhibitor ATRA showed a significant decrease in HO-1 and NQO1 expression and a decrease in the anti-RABV efficacy of CDDO-Me. These inhibitory effects were observed upon infection with three RABV strains of differing virulence.ConclusionCDDO-Me inhibited RABV infection via Nrf2 activation, promoting a cytoprotective defense response in N2a cells. Our study provides a therapeutic strategy for RABV inhibition and neuroprotection during viral infection.

Optimization of Virus Yield as a Strategy to Increase the Efficiency of Rabies Vaccine by BHK-21 Cells in a Bioreactor

Background: Rabies is a usually fatal viral zoonotic and preventable disease. The efficacy and safety of animal rabies vaccination made in permanent BHK-21 cell culture have been proven over a long period of use. By increasing the yield of cells and viruses, the efficacy of the vaccine can be increased. Objectives: The objective of this study was to optimize and maximize the output of a rabies vaccine made on BHK cells in a bioreactor. Methods: This study examined the impacts of independent parameters, such as pH, temperature, cell density, and dissolved oxygen (DO), on rabies virus strain PV-PARIS yield for a central composite design. To achieve high viral production, this study used the central composite approach to optimize cell development. Results: The findings showed that BHK-21 cells were grown under the ideal conditions of pH 7.21, temperature 35.05ºC, 68.75% for DO, and 2.30 × 106 cell/mL of cell density to produce high titers of rabies virus (4.7 × 107 plaque-forming unit [PFU]/mL). High correlation coefficients (0.927) validated that the predicted model was well-fitted with the data, and the statistical analysis of the collected data indicated that the experimental data and predicted model were well-matched. The accuracy of this model’s predictions was correlated with values of adjusted R-squared (R2Adj) and predicted R-squared (R2Pred). Conclusions: These upgrades lead to a more reliable and economical procedure that makes industrialization and commercialization easier.

Large-scale phylogenetic analysis reveals genetic diversity and geographic distribution of rabies virus in South-East and South Asia.

South-East Asia (SEA) and South Asia (SA) are two important geographic regions with the most severe enzootic rabies in the world. In these regions, phylogenetic analysis of rabies virus (RABV) has been conducted only at a country level; the results obtained from different countries are scattered and unequal, with a non-uniform system to name RABV genotypes. Therefore, it is difficult to undertake origin-tracking and compare inter-country RABV evolution and transmission. To avoid the confusion in understanding and to generate a panoramic picture of RABV genetic diversity, distribution, and transmission in SEA and SA, the present study conducted a systematic phylogenetic analysis by combining all sequences representing 2368 RABV strains submitted to GenBank by 14 rabies endemic SEA and SA countries. The results showed that RABVs circulating in two regions were classified into four major clades and many subclades: the Asia clade is circulating only in SEA, the Indian subcontinent, and Arctic-like clades only in SA, while the Cosmopolitan clade has been detected in both regions. The results also showed a wide range of hosts were infected by divergent RABV subclades, with dogs being the major transmission source. However, wildlife rabies was also found to be an important issue with 6 wild carnivore species identified as potential sources of spillover risk for sylvatic rabies to humans, domestic animals, and other wild animals. Current findings indicate that the two regions have separate virus clades circulating thus indicating the absence of cross-transmission between the regions. The study emphasizes the importance of phylogenetic analysis in the regions using uniform genotyping and naming systems for rabies surveillance, to coordinate actions of member countries to eliminate dog-mediated human rabies by 2030.

Hydrogel-based 3D human iPSC-derived neuronal culture for the study of rabies virus infection.

Rabies is a highly fatal infectious disease that poses a significant threat to human health in developing countries. In vitro study-based understanding of pathogenesis and tropism of different strains of rabies virus (RABV) in the central nervous system (CNS) is limited due to the lack of suitable culture models that recapitulate the complex communication pathways among host cells, extracellular matrices, and viruses. Therefore, a three-dimensional (3D) cell culture that mimics cell-matrix interactions, resembling in vivo microenvironment, is necessary to discover relevant underlying mechanisms of RABV infection and host responses. The 3D collagen-Matrigel hydrogel encapsulating hiPSC-derived neurons for RABV infection was developed and characterized based on cell viability, morphology, and gene expression analysis of neuronal markers. The replication kinetics of two different strains of RABV [wild-type Thai (TH) and Challenge Virus Standard (CVS)-11 strains] in both 2D and 3D neuronal cultures were examined. Differential gene expression analysis (DEG) of the neuropathological pathway of RABV-infected 2D and 3D models was also investigated via NanoString analysis. The 3D hiPSC-derived neurons revealed a more physiologically interconnected neuronal network as well as more robust and prolonged maturation and differentiation than the conventional 2D monolayer model. TH and CVS-11 exhibited distinct growth kinetics in 3D neuronal model. Additionally, gene expression analysis of the neuropathological pathway observed during RABV infection demonstrated a vast number of differentially expressed genes (DEGs) in 3D model. Unlike 2D neuronal model, 3D model displayed more pronounced cellular responses upon infection with CVS-11 when compared to the TH-infected group, highlighting the influence of the cell environment on RABV-host interactions. Gene ontology (GO) enrichment of DEGs in the infected 3D neuronal culture showed alterations of genes associated with the inflammatory response, apoptotic signaling pathway, glutamatergic synapse, and trans-synaptic signaling which did not significantly change in 2D culture. We demonstrated the use of a hydrogel-based 3D hiPSC-derived neuronal model, a highly promising technology, to study RABV infection in a more physiological environment, which will broaden our understanding of RABV-host interactions in the CNS.

Whole genome sequencing and phylogenetic characterisation of rabies virus strains from Moldova and north-eastern Romania.

Rabies is the oldest fatal zoonotic disease recognised as a neglected tropical disease and is caused by an RNA virus belonging to the genus Lyssavirus, family Rhabdoviridae. A deep molecular analysis was conducted on full-length nucleoprotein (N) gene and whole genome sequences of rabies virus from 37 animal brain samples collected between 2012 and 2017 to study the circulation of rabies virus (RABV) variants. The overall aim was to better understand their distribution in Moldova and north-eastern Romania. Both Sanger and high throughput sequencing on Ion Torrent and Illumina platforms were performed. Phylogenetic analysis of the RABV sequences from both Moldova and Romania revealed that all the samples (irrespective of the year of isolation and the species) belonged to a single phylogenetic group: north-eastern Europe (NEE), clustering into three assigned lineages: RO#5, RO#6 and RO#7. High throughput sequencing of RABV samples from domestic and wild animals was performed for the first time for both countries, providing new insights into virus evolution and epidemiology in this less studied region, expanding our understanding of the disease.

A simplified method for measuring neutralising antibodies against rabies virus

Rabies virus (RABV) causes a fatal encephalitis that can be prevented through timely vaccination. The levels of virus neutralising antibodies against rabies virus induced by vaccination can be measured using the fluorescent antibody virus neutralisation (FAVN) test. Following incubation of live virus with sera, this method involves the fixation of cell monolayers and staining of rabies virus-specific antigen using fluorescein isothiocyanate (FITC) -conjugated antibody to enable visualisation of rabies virus antigen using a fluorescence microscope. To simplify this procedure, a fluorescent recombinant rabies virus was constructed using reverse genetics by inserting the gene for the mCherry fluorescent protein in front of the ribonucleoprotein gene of the SAD B-19 genome and replacing its glycoprotein with that of the Challenge Virus Standard (CVS)-11 RABV strain to ensure antigenic authenticity with the FAVN. This new recombinant virus (termed mCCCG) expressed the mCherry protein to high levels enabling direct observation of infected cells. In vitro growth kinetics of mCCCG were indistinguishable from that of CVS-11. The stability of the recombinant virus was assessed by sequencing several passages of the rescued virus and only minor changes were detected. Comparative assessment of the virus neutralisation test using mCherry producing virus (NTmCV) against the FAVN demonstrated that test results were equivalent to each other; therefore, mCCCG can be used as an alternative to CVS-11 for measuring antibody titres against the rabies virus. The use of NTmCV removes the need for expensive antibody conjugates and significantly reduces assay time. This would be particularly beneficial for RABV serological assessment in resource limited settings. Moreover, the reading of the plates can be automatically using a cell imaging reader.