Investigation of Rabies virus in wild mammals of the atlantic forest in Rio de Janeiro, Brazil.

With the successful control of rabies transmitted by dogs in Brazil, wild animals have played a relevant epidemiological role in the transmission of rabies virus (RABV). Bats, non-human primates and wild canines are the main wild animals that transmit RABV in the country. It is worth highlighting the possibility of synanthropic action of these species, when they become adapted to urban areas, causing infections in domestic animals and eventually in humans. This work aimed to evaluate the circulation of RABV in the Pedra Branca Forest, an Atlantic Forest area, located in the state of Rio de Janeiro, Southeast Brazil. Saliva and blood samples were obtained from 60 individuals of eight species of bats, captured with mist nets, and 13 individuals of callitrichid primates, captured with tomahawk traps. Saliva samples were subjected to Reverse Transcription Polymerase Chain Reaction (RT-PCR), targeting the RABV N gene, with all samples being negative. Blood samples of all animals were submitted to the Rapid Fluorescent Focus Inhibition Test (RFFIT) to detect neutralizing antibodies (Ab) for RABV. Six bat samples (8%) were seropositive for RABV with antibody titers greater than or equal to 0.1 IU/mL. The detection of Ab but not viral RNA indicates exposure rather than current RABV transmission in the analyzed populations. The results presented here reinforce the importance of serological studies in wildlife to access RABV circulation in a region.

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.

Introduction: 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 Swietokrzyskie 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. Material and Methods: 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. Results: 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, Swietokrzyskie 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. Conclusion: Central European and North-Eastern European RABVs were circulating in Poland between 2021 and 2023.

Rabies Virus Infection Causes Pyroptosis of Neuronal Cells.

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

Human cat borne rabies as the new epidemiology of the disease in the Andes mountains.

BACKGROUND: Rabies virus (RABV) is the etiologic agent of rabies, a fatal brain disease in mammals. Rabies circulation has historically involved the dog has the main source of human rabies worldwide. Nevertheless, in Colombia, cats (Felis catus) have become a relevant species in the epidemiology of rabies. AIMS: To characterize rabies cases in humans in Colombia in the last three decades in the context of the epidemiology of the aggressor animal. MATERIALS AND METHODS: We conducted a retrospective longitudinal epidemiological study of human rabies caused by cats' aggression, collecting primary and secondary information. Variables considered included the demography of the patient, symptoms, information about the aggressor animal as the source of infection and the viral variant identified. RESULTS: We found that the distribution of rabies incidence over the years has been constant in Colombia. Nevertheless, between 2003 and 2012 a peak of cases occurred in rural Colombia where cats were the most frequent aggressor animal reported. Most cats involved in aggression were unvaccinated against rabies. Cat's clinical signs at the time of the report of the human cases included hypersalivation and changes in behaviour. Human patients were mostly children and female and the exposure primarily corresponded to bite and puncture lacerations in hands. The RABV lineage detected in most cases corresponded to variant 3, linked to the common vampire bat (Desmodus rotundus). The geographical presentation of cat borne RABV in humans occurred along the Andes mountains, epidemiologically known as the rabies red Andean corridor. DISCUSSION: By finding cats as the primary source of rabies spillover transmission in Colombia, this report highlights the importance of revising national rabies control and prevention protocol in countries in the Andes region. CONCLUSION: Our results demonstrate that rabies vaccination for outdoor cats needs to prioritize to reduce the number of rabies-related human deaths.

The matrix protein of lyssavirus hijacks autophagosome for efficient egress by recruiting NEDD4 through its PPxY motif.

Lyssaviruses are well-known worldwide and often cause fatal encephalitis. Previous studies have shown that autophagy is beneficial for the replication of rabies virus (RABV), the representative lyssavirus, but the detailed mechanism remains obscure. In this study, we showed that the rabies virus matrix protein (RABV-M) used its PPxY motif to interact with the E3 ubiquitin-protein ligase NEDD4. NEDD4 then recruited MAP1LC3/LC3 via its LC3-interacting region (LIR). Interestingly, after binding to the ubiquitinated RABV-M, NEDD4 could bind more LC3 and enhance autophagosome accumulation, while NEDD4 knockdown significantly reduced M-induced autophagosome accumulation. Further study revealed that RABV-M prevented autophagosome-lysosome fusion and facilitated viral budding. Inhibition of RABV-M-induced autophagosome accumulation reduced the production of extracellular virus-like particles. We also found that M proteins of most lyssaviruses share the same mechanism to accumulate autophagosome by hijacking NEDD4. Collectively, this study revealed a novel strategy for lyssaviruses to achieve efficient viral replication by exploiting the host autophagy system.Abbreviations: ABLV: Australian bat lyssavirus; ATG5: autophagy related 5; Baf A1:bafilomycin A1;co-IP: co-immunoprecipitation; CQ: chloroquine; DAPI:4',6-diamidino-2'-phenylindole; DMSO: dimethyl sulfoxide; EBLV:European bat lyssavirus; GFP: green fluorescent protein; GST:glutathione S-transferase; hpi: hours post-infection; hpt: hourspost-transfection; LIR: LC3-interactingregion;MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; mCherry:red fluorescent protein; MOI: multiplicity of infection; NC: negativecontrol; MVB: multivesicular body; NEDD4: neural precursorcell-expressed developmentally down-regulated 4; RABV: rabies virus;SQSTM1/p62: sequestosome 1; VLP: virus-like particle; VPS4B: vacuolarprotein sorting 4B; TEM: transmission electron microscopy; WB:western blotting; WT: wild-type; µm: micrometer; µM: micromole.

Comparative analysis of rabies pathogenic and vaccine strains detection by RIG-I-like receptors.

Rabies virus (RABV) is a lethal neurotropic virus that causes 60,000 human deaths every year globally. RABV infection is characterized by the suppression of the interferon (IFN)-mediated antiviral response. However, molecular mechanisms leading to RABV sensing by RIG-I-like receptors (RLR) that initiates IFN signaling currently remain elusive. Here, we showed that RABV RNAs are primarily recognized by the RIG-I RLR, resulting in an IFN response in the infected cells, but this response varied according to the type of RABV used. Pathogenic RABV strain RNAs, Tha, were poorly detected in the cytosol by RIG-I and therefore caused a weak antiviral response. However, we revealed a strong IFN activity triggered by the attenuated RABV vaccine strain RNAs, SAD, mediated by RIG-I. We characterized two major 5' copy-back defective interfering (5'cb DI) genomes generated during SAD replication. Furthermore, we identified an interaction between 5'cb DI genomes, and RIG-I correlated with a high stimulation of the type I IFN signaling. This study indicates that wild-type RABV RNAs poorly activate the RIG-I pathway, while the presence of 5'cb DIs in the live-attenuated vaccine strain serves as an intrinsic adjuvant that strengthens its efficiency by enhancing RIG-I detection thus strongly stimulates the IFN response.

A rabies mRNA vaccine with H270P mutation in its glycoprotein induces strong cellular and humoral immunity.

Rabies is a lethal zoonotic disease that kills approximately 60,000 people each year. As the sole virion-surface protein, the rabies virus glycoprotein (RABV-G) mediates its host-cell entry. RABV-G's pre-fusion conformation displays major known neutralizing antibody epitopes, which can be used as immunogen for prophylaxis. H270P targeted mutation can stabilize RABV-G in the pre-fusion conformation. Herein, we report the development of a highly promising rabies mRNA vaccine composed of H270P targeted mutation packaged in lipid nanoparticle (LNP), named LNP-mRNA-G-H270P. Humoral and cellular immunity of this vaccine were assessed in mice comparing to the unmodified LNP-mRNA-G and a commercially available inactivated vaccine using one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparisons test. The results show the titer of RABV-G-specific IgG and virus-neutralization antibody titers (VNTs) in LNP-mRNA-G-H270P group were significant higher than those in LNP-mRNA-G and inactivated vaccine groups. Likewise, IFN-γ-secreting splenocytes, level of IL-2 in the supernatant of spleen cells, as well as IFN-γ-producing CD4+ T cells in LNP-mRNA-G-H270P group were significant higher than those in the other two vaccine groups. Hence, these results demonstrated that targeting the H270P mutation in RABV-G through an mRNA-LNP vaccine platform represents a promising strategy for developing a more efficacious rabies vaccine.

Effect of bioreactor scale-up culture of micro-carrier Vero cells after extra-tank trypsinization on virus-producing ability of rabies virus CTN-1Ⅴ strain / 中国生物制品学杂志

@#Objective To investigate the effect of amplification culture of micro-carrier Vero cells from 30 L bioreactor to 300 L bioreactor after extra-tank trypsinization on the virus-producing ability of rabies virus(RABV)CTN-1Ⅴ strain.Methods The 140-passage of Vero cells were cultured at 37 ℃ for 72-120 h,then amplified by passaging at a cell density ratio of 1∶4 into the 10 × cell factory. After incubation at 37 ℃ for 72-120 h,the monolayer cells were detached and inoculated into the 30 L bioreactor with micro-carriers 7-10 g/L,culture temperature 37 ℃,pH 7. 0-7. 4,dissolved oxygen 30%-80%,stirring speed 10-50 r/min,and continuous perfusion culture 72-120 h. Total three batches of microcarrier Vero cells were cultured,which were amplified to the 300 L bioreactor after extra-tank trypsinization,with microcarrier5-8 g/L,culture temperature 37 ℃,pH 7. 0-7. 4,dissolved oxygen 30%-80%,stirring speed 30-80 r/min,and perfusion culture 72-120 h. RABV CTN-1Ⅴ was inoculated at the MOI of 0. 05,and the virus solution was harvested every 24 h and detected for the virus titer and antigen content. Results The cell density was about 1 × 10~7 cells/mL after culture for 96 h in the 30 L bioreactor,and was about 7. 4 × 10~6 cells/mL after culture for 96 h in the 300 L bioreactor. At 96 h after virus inoculation,the virus harvest solution reached the peak potency,with the average virus titer of 6. 8 lgLD_(50)/mL and the average antigen content of 2. 58 IU/mL. Conclusion The scale-up culture process of micro-carrier Vero cells after extra-tank trypsinization from 30 L bioreactor to 300 L bioreactor is stable and feasible,with no significant effect on the virus-producing ability of RABV CTN-1Ⅴ strain,which provides a reference for the large-scale production of inactivated RABV vaccine

Development of large-scale amplification process of rabies virus in 150 L bioreactor / 中国生物制品学杂志

@#Objective To develop a large-scale culture process for rabies virus(RABV)in 150 L bioreactor,and lay a foundation for the further development of a larger-scale and high-density microcarrier reactor process.Methods Vero cells and RABV strain CTN-1V were cultured in 30 L(model:C30-2)and 150 L(model:VESSEL FERMENTER 300L)bioreactors by perfused culture with 20 g/L Cytodex-1 microcarrier and DO 20%-60%,at culture temperature 36-38 ℃ and pH 7.0-7.4.During the culture process,the cell density and virus titer were measured.The virus culture media was harvested for consecutive 13 d and detected for the sterility,mycoplasma,and the residues of antigen,host cell protein(HCP),bovine serum albumin(BSA)and DNA.Results The density of cultured cells in 30 L and 150 L bioreactors all reached above 1.2 ×10~7cell/mL.There was no significant difference in cell density at different time points during the culture(t = 0.225-2.173,P = 0.096-0.833).The highest virus titer(8.5 lgLD_(50)/mL)was found in the both bioreactors 6 d after infection with no significant difference(t = 1.000,P = 0.374).The residues of antigen,HCP,BSA and DNA in the virus suspension from the two bioreactors were basically the same.Conclusion 150 L bioreactor can be used for the large-scale culture of RABV,and the harvested virus conformed to the relevant standards in Chinese Pharmacopoeia(Volume Ⅲ,2020 edition).

TMP269, a small molecule inhibitor of class IIa HDAC, suppresses RABV replication in vitro.

TMP269, a small molecular inhibitor of IIa histone deacetylase, plays a vital role in cancer therapeutic. However, the effect of TMP269 on the regulation of viral replication has not been studied. In the present study, we found that TMP269 treatment significantly inhibited RABV replication at concentrations without significant cytotoxicity in a dose-dependent manner. In addition, TMP269 can reduce the viral titers and protein levels of RABV at an early stage in the viral life cycle. RNA sequencing data revealed that immune-related pathways and autophagy-related genes were significantly downregulated after RABV infection treated with TMP269. Further exploration shows that autophagy enhances RABV replication in HEK-293T cells, while TMP269 can inhibit autophagy to decrease RABV replication. Together, these results provide a novel treatment strategy for rabies.

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.

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.

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.

Genomic Surveillance of Rabies Virus in Georgian Canines.

Rabies is a fatal zoonosis that is considered a re-emerging infectious disease. Although rabies remains endemic in canines throughout much of the world, vaccination programs have essentially eliminated dog rabies in the Americas and much of Europe. However, despite the goal of eliminating dog rabies in the European Union by 2020, sporadic cases of dog rabies still occur in Eastern Europe, including Georgia. To assess the genetic diversity of the strains recently circulating in Georgia, we sequenced seventy-eight RABV-positive samples from the brain tissues of rabid dogs and jackals using Illumina short-read sequencing of total RNA shotgun libraries. Seventy-seven RABV genomes were successfully assembled and annotated, with seventy-four of them reaching the coding-complete status. Phylogenetic analyses of the nucleoprotein (N) and attachment glycoprotein (G) genes placed all the assembled genomes into the Cosmopolitan clade, consistent with the Georgian origin of the samples. An amino acid alignment of the G glycoprotein ectodomain identified twelve different sequences for this domain among the samples. Only one of the ectodomain groups contained a residue change in an antigenic site, an R264H change in the G5 antigenic site. Three isolates were cultured, and these were found to be efficiently neutralized by the human monoclonal antibody A6. Overall, our data show that recently circulating RABV isolates from Georgian canines are predominantly closely related phylogroup I viruses of the Cosmopolitan clade. Current human rabies vaccines should offer protection against infection by Georgian canine RABVs. The genomes have been deposited in GenBank (accessions: OQ603609-OQ603685).

Uncovering the endemic circulation of rabies in Cambodia.

In epidemiology, endemicity characterizes sustained pathogen circulation in a geographical area, which involves a circulation that is not being maintained by external introductions. Because it could potentially shape the design of public health interventions, there is an interest in fully uncovering the endemic pattern of a disease. Here, we use a phylogeographic approach to investigate the endemic signature of rabies virus (RABV) circulation in Cambodia. Cambodia is located in one of the most affected regions by rabies in the world, but RABV circulation between and within Southeast Asian countries remains understudied. Our analyses are based on a new comprehensive data set of 199 RABV genomes collected between 2014 and 2017 as well as previously published Southeast Asian RABV sequences. We show that most Cambodian sequences belong to a distinct clade that has been circulating almost exclusively in Cambodia. Our results thus point towards rabies circulation in Cambodia that does not rely on external introductions. We further characterize within-Cambodia RABV circulation by estimating lineage dispersal metrics that appear to be similar to other settings, and by performing landscape phylogeographic analyses to investigate environmental factors impacting the dispersal dynamic of viral lineages. The latter analyses do not lead to the identification of environmental variables that would be associated with the heterogeneity of viral lineage dispersal velocities, which calls for a better understanding of local dog ecology and further investigations of the potential drivers of RABV spread in the region. Overall, our study illustrates how phylogeographic investigations can be performed to assess and characterize viral endemicity in a context of relatively limited data.

IFITM3 restricts RABV infection through inhibiting viral entry and mTORC1- dependent autophagy.

Rabies, which caused by rabies virus (RABV), is a zoonotic and life-threatening disease with 100% mortality, and there is no effective treatment thus far due to the unclear pathogenesis and less of treatment targets. Interferon-induced transmembrane protein 3 (IFITM3) has recently been identified as an important anti-viral host effector induced by type I interferon. However, the role of IFITM3 in RABV infection has not been elucidated. In this study, we demonstrated that IFITM3 is a crucial restriction factor for RABV, the viral-induced IFITM3 significantly inhibited RABV replication, while knockdown of IFITM3 had the opposite effect. We then identified that IFNß induces the upregulation of IFITM3 in the absence or presence of RABV infection, meanwhile, IFITM3 positively regulates RABV-triggered production of IFNß in a feedback manner. In-depth research we found that IFITM3 not only inhibits the virus absorb and entry, but also inhibits viral replication through mTORC1-dependent autophagy. All these findings broaden our understanding of IFITM3 function and uncover a novel mechanism against RABV infection.

Bif-1c Attenuates Viral Proliferation by Regulating Autophagic Flux Blockade Induced by the Rabies Virus CVS-11 Strain in N2a Cells.

Bax-interacting factor-1 (Bif-1) is a multifunctional protein involved in apoptosis, autophagy, and mitochondrial morphology. However, the associations between Bif-1 and viruses are poorly understood. As discrete Bif-1 isoforms are selectively expressed and exert corresponding effects, we evaluated the effects of neuron-specific/ubiquitous Bif-1 isoforms on rabies virus (RABV) proliferation. First, infection with the RABV CVS-11 strain significantly altered Bif-1 expression in mouse neuroblastoma (N2a) cells, and Bif-1 knockdown in turn promoted RABV replication. Overexpression of neuron-specific Bif-1 isoforms (Bif-1b/c/e) suppressed RABV replication. Moreover, our study showed that Bif-1c colocalized with LC3 and partially alleviated the incomplete autophagic flux induced by RABV. Taken together, our data reveal that neuron-specific Bif-1 isoforms impair the RABV replication process by abolishing autophagosome accumulation and blocking autophagic flux induced by the RABV CVS-11 strain in N2a cells. IMPORTANCE Autophagy can be triggered by viral infection and replication. Autophagosomes are generated and affect RABV replication, which differs by viral strain and infected cell type. Bax-interacting factor-1 (Bif-1) mainly has a proapoptotic function but is also involved in autophagosome formation. However, the association between Bif-1-involved autophagy and RABV infection remains unclear. In this study, our data reveal that a neuron-specific Bif-1 isoform, Bif-1c, impaired viral replication by unchoking autophagosome accumulation induced by RABV in N2a cells to a certain extent. Our study reveals for the first time that Bif-1 is involved in modulating autophagic flux and plays a crucial role in RABV replication, establishing Bif-1 as a potential therapeutic target for rabies.

Pseudotyped Viruses for Lyssavirus.

Lyssaviruses, which belong to the family Rhabdoviridae, are enveloped and bullet-shaped ssRNA viruses with genetic diversity. All members of Lyssavirus genus are known to infect warm-blooded animals and cause the fatal disease rabies. The rabies virus (RABV) in lyssavirus is the major pathogen to cause fatal rabies. The pseudotyped RABV is constructed to study the biological functions of G protein and evaluation of anti-RABV products including vaccine-induced antisera, rabies immunoglobulins (RIG), neutralizing mAbs, and other antiviral inhibitors. In this chapter, we focus on RABV as a representative and describe the construction of RABV G protein bearing pseudotyped virus and its applications. Other non-RABV lyssaviruses are also included.

Rabies virus uniquely reprograms the transcriptome of human monocyte-derived macrophages.

Macrophages are amongst the first immune cells that encounter rabies virus (RABV) at virus entry sites. Activation of macrophages is essential for the onset of a potent immune response, but insights into the effects of RABV on macrophage activation are scarce. In this study we performed high-throughput sequencing on RNA extracted from macrophages that were exposed to RABV for 48 hours, and compared their transcriptional profiles to that of non-polarized macrophages (M0), and macrophages polarized towards the canonical M1, M2a and M2c phenotypes. Our analysis revealed that RABV-stimulated macrophages show high expression of several M1, M2a and M2c signature genes. Apart from their partial resemblance to these phenotypes, unbiased clustering analysis revealed that RABV induces a unique and distinct polarization program. Closer examination revealed that RABV induced multiple pathways related to the interferon- and antiviral response, which were not induced under other classical polarization strategies. Surprisingly, our data show that RABV induces an activated rather than a fully suppressed macrophage phenotype, triggering virus-induced activation and polarization. This includes multiple genes with known antiviral (e.g. APOBEC3A, IFIT/OAS/TRIM genes), which may play a role in anti-RABV immunity.

An inactivated recombinant rabies virus chimerically expressed RBD induces humoral and cellular immunity against SARS-CoV-2 and RABV.

Many studies suggest that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can infect various animals and transmit among animals, and even to humans, posing a threat to humans and animals. There is an urgent need to develop inexpensive and efficient animal vaccines to prevent and control coronavirus disease 2019 (COVID-19) in animals. Rabies virus (RABV) is another important zoonotic pathogen that infects almost all warm-blooded animals and poses a great public health threat. The present study constructed two recombinant chimeric viruses expressing the S1 and RBD proteins of the SARS-CoV-2 Wuhan01 strain based on a reverse genetic system of the RABV SRV9 strain and evaluated their immunogenicity in mice, cats and dogs. The results showed that both inactivated recombinant viruses induced durable neutralizing antibodies against SARS-CoV-2 and RABV and a strong cellular immune response in mice. Notably, inactivated SRV-nCoV-RBD induced earlier antibody production than SRV-nCoV-S1, which was maintained at high levels for longer periods. Inactivated SRV-nCoV-RBD induced neutralizing antibodies against both SARS-CoV-2 and RABV in cats and dogs, with a relatively broad-spectrum cross-neutralization capability against the SARS-CoV-2 pseudoviruses including Alpha, Beta, Gamma, Delta, and Omicron, showing potential to be used as a safe bivalent vaccine candidate against COVID-19 and rabies in animals.