Search Results (308)

Background: Madariaga virus (MADV), formerly known as the South American variant of Eastern Equine Encephalitis virus (EEEV), is an alphavirus that belongs to the Togaviridae family and has been periodically infecting equids in Venezuela since its first identification in 1975. This study reports the isolation and molecular characterization of MADV isolated from a horse in December 2024 in the context of MADV cases reported in Venezuela. Methods: Antibodies to the rabies virus were detected by indirect immunofluorescence, and to the Equine Infectious Anemia virus (EIAV) by passive immunodiffusion. MADV RNA was detected by qRT-PCR. The sequence of the complete viral genome was obtained by next-generation sequencing. Results: The sequence of this virus was highly similar to that of the only human case of MADV reported in the country in 2016, as well as to a sequence of a virus isolated from a horse in Colombia in 2002. The horse was found to be co-infected with EIAV. Conclusions: The continuous circulation of MADV in Venezuela warrants reinforcing the preventive measures against these alphaviruses, which ignore borders, and may cause important animal and human health concerns. Full article

Background: Rabies is among the oldest known zoonotic viral diseases and is caused by members of the Lyssavirus genus. The prototype species, Lyssavirus rabies, effectively evades the host immune response, allowing the infection to progress unnoticed until the onset of clinical signs. At this stage, the disease is irreversible and invariably fatal, with definitive diagnosis possible only post-mortem. Given the advances in modern proteomics, this study aimed to identify potential protein biomarkers for antemortem diagnosis of rabies in dogs, which are the principal reservoir hosts of the rabies virus. Methods: Two hundred and thirty-one samples (brain tissues (BT), cerebrospinal fluids (CSF), and serum (SR) samples) were collected from apparently healthy dogs brought for slaughter for human consumption in South-East and North-Central Nigeria. All the BT were subjected to a direct fluorescent antibody test to confirm the presence of lyssavirus antigen, and 8.7% (n = 20) were positive. Protein extraction, quantification, reduction, and alkylation were followed by on-bead (HILIC) cleanup and tryptic digestion. The resulting peptides from each sample were injected into the Evosep One LC system, coupled to the timsTOF HT MS, using the standard dia-PASEF short gradient data acquisition method. Data was processed using Spectronaut^TM (v19). An unpaired t-test was performed to compare identified protein groups (proteins and their isoforms) between the rabies-infected and uninfected BT, CSF, and SR samples. Results: The study yielded 54 significantly differentially abundant proteins for the BT group, 299 for the CSF group, and 280 for the SR group. Forty-five overlapping differentially abundant proteins were identified between CSF and SR, one between BT and CSF, and two between BT and SR; none were found that overlapped all three groups. Within the BT group, 33 proteins showed increased abundance, while 21 showed decreased abundance in the rabies-positive samples. In the CSF group, 159 proteins had increased abundance and 140 had decreased abundance in the rabies-positive samples. For the SR group, 215 proteins showed increased abundance, and 65 showed decreased abundance in the rabies-positive samples. Functional enrichment analysis revealed that pathways associated with CSF, spinocerebellar ataxia, and neurodegeneration were among the significant findings. Conclusion: This study identified canonical proteins in CSF and SR that serve as candidate biomarkers for rabies infection, offering insights into neuronal dysfunction and potential tools for early diagnosis. Full article

Bats are vital parts of ecosystems but also act as important reservoirs of viruses that can infect humans. Studying bat-borne viruses is essential for preventing spillover events, and passive surveillance offers a promising, bat-friendly approach. Among these viruses, lyssaviruses are a major public health concern because infection in humans is almost always fatal. In Europe, the European bat lyssavirus type 1 (EBLV-1) is the most widespread. We aimed to expand the Hungarian rabies surveillance system into a One Health-based passive surveillance framework for multiple bat viruses, using lyssaviruses and filoviruses as examples. Dead-found or injured bats that died despite care were collected for examination. In total, 208 bats from 15 species were tested for lyssaviruses and Lloviu virus. Three serotine bats (Cnephaeus serotinus, formerly Eptesicus) tested positive for EBLV-1, and complete viral genomes were obtained. All sequences belonged to the EBLV-1a lineage, closely related to previously described Hungarian strains. All bats tested negative for Lloviu virus. These results confirm the feasibility of passive surveillance for detecting bat-borne viruses and highlight the importance of collaborative monitoring systems for future zoonotic outbreak preparedness. Full article

Protein–protein interactions (PPIs) are fundamental to viral replication, regulating transcription, assembly, and genome packaging. Despite their biological importance, few FDA-approved therapeutics directly target these complexes. The split luciferase complementation assay (SLCA) is a quantitative bioluminescence system to measure protein–protein interactions in vitro after the proteins in question have been fused in-frame to N and C luciferase fragments. The SLCA can be performed both in vitro using purified protein components and in live cells, as the luciferase substrate luciferin is cell-permeable, allowing detection of protein interactions in intact cells. Assay performance, however, depends on the expression level and stability of the fusion proteins used. SLCA has been successfully applied to target Rev–Rev interactions in human immunodeficiency virus type 1 (HIV-1) for high-throughput small-molecule screening, establishing a proof-of-concept to target other parts of the viral life cycle. The system can be extended to other pathogens that currently do not have specific antiviral therapies such as HIV-1 Tat–cyclin T1, Capsid dimerization in Dengue virus, capsid interactions in equine encephalitis viruses, capsid assembly in Epstein–Barr virus, and nucleoprotein oligomerization in rabies virus. These applications demonstrate how the assay’s ability to quantify multimeric structural interactions is essential to viral replication, providing an avenue to identify small-molecule inhibitors that prevent viral replication and spread. Although there are challenges to protein stability and assay optimization, the sensitivity and adaptability of the SLCA has broader implications in virology to accelerate antiviral drug development. Full article

Background: Rabies virus (RABV) causes approximately 59,000 human deaths annually. Current pre- and post-exposure vaccination relies on inactivated vaccines (INVs) with limited yield and immunogenicity. We engineered a dual-cationic LNP-based nucleocapsid-like nanostructure (NLS) that co-encapsulates RABV G-mRNA and recombinant RABV-N to engage MHC-I/II pathways and enhance protection. Methods: A pVAX-RABV-G plasmid containing 5′/3′UTRs, Kozak, and poly(A) was transcribed in vitro. RABV-N with an N-terminal 6× His tag was expressed in E. coli BL21(DE3) and purified by Ni-Sepharose affinity chromatography. Dual-cationic LNPs (DHA, DOTAP Cl, mPEG-DTA2K, DOPC) were formulated by microfluidics at a 4:1 (G-mRNA:RABV-N) mass ratio. Vaccine quality was assessed by encapsulation efficiency, DLS, PDI, zeta potential, and TEM. Mice received empty LNPs, INV, G-mRNA, or NLS under varied schedules and doses. ELISA measured RABV-G/N-IgG; RFFIT determined neutralizing antibody (nAb) titers; ELISPOT quantified CTL response; qPCR assessed T-cell activation genes. On day 35 after the first immunization of vaccines, mice were challenged intramuscularly with 25 LD₅₀ of CVS-24. Results: G-mRNA purity was >95% and drove strong RABV-G expression in 293T cells. Purified RABV-N was approximately 52 kDa, >90% pure, and reactive to anti-His and anti-N antibodies. NLS achieved >95% encapsulation, a diameter of 136.9 nm, PDI 0.09, and a +18.7 mV zeta potential. A single dose yielded approximately 10 IU mL⁻¹ nAb by day 7; two doses peaked at approximately 1000 IU mL⁻¹. Mice showed 100% survival and no viral rebound in brain, spinal cord, and sciatic nerve. NLS induced stronger MHC-I/II-linked cellular immunity and higher RABV G/N-specific IFN-γ spot frequencies than G-mRNA or INV. Conclusions: The dual-antigen NLS vaccine co-delivering G-mRNA and RABV-N via dual-cationic LNPs robustly activates MHC-I/II, rapidly generates high-titer nAb (≥10 IU mL⁻¹ within 1 week), and sustains potent CD8⁺ CTL and CD4⁺ Th responses. A two-dose regimen (days 0 and 21) conferred complete protection, supporting the NLS platform as a next-generation rabies vaccine candidate. Full article

Background/Objectives: To this day, rabies remains a significant global threat. This threat remains even with the availability of vaccines for humans, wildlife, and domestic animals, which are used as part of a series of interventions to attempt to control the infection and disease. The number of annual human deaths from rabies globally remains significant, with infections being mainly caused by domestic dogs. Although a number of vaccines exist for domestic animals, most contain inactivated rabies virus with adjuvants. Methods: To investigate alternatives to conventional rabies vaccines for dogs and cats, we developed a novel, non-adjuvanted, low-volume (0.5 mL) vaccine, based on the Venezuelan equine encephalitis virus (VEEV) TC-83-derived RNA particle (RP) expressing the rabies glycoprotein (G). This novel vaccine combines the safety profile of a non-adjuvanted vaccine while inducing consistently high efficacy and an extended duration of immunity similar to that shown by adjuvanted vaccines. Results: In multiple studies, we demonstrated that young kittens and puppies can be safely vaccinated without serious adverse effects. In graded dose experiments with cats and dogs, the RNA particle vaccine induced neutralizing levels of antibodies. Additionally, in vaccination/challenge studies, 100% protection from virulent rabies was demonstrated in excess of three years post-vaccination from a single dose at 12 weeks of age in both dogs and cats. The safety of the RP-Rabies vaccine in dogs and cats as young as twelve weeks of age was demonstrated in field safety studies using two vaccine serials formulated at a field dose. Conclusions: Data from these studies suggest that the RP-Rabies vaccine offers an excellent alternative to current vaccines combining the safety of a non-adjuvanted vaccine in a low-volume, single dose with the induction of an extended duration of immunity of at least three years in both dogs and cats. Full article

Background/Objectives: Japan is a rabies-free country; therefore, pre-exposure prophylaxis (PrEP) is primarily recommended for travelers to rabies-endemic regions. However, no prior studies in Japan have assessed long-term immunogenicity after PrEP vaccination. Methods: This descriptive study evaluated the long-term persistence of rabies virus-neutralizing antibodies among Japanese adult travelers who had received PrEP. Neutralizing antibody levels were measured using the rabies rapid fluorescent focus inhibition test more than two years post-vaccination. Results: Among 97 participants, 86.6% remained seropositive, with a median interval of 8.5 years since vaccination. Individuals who received three or more doses had significantly higher geometric mean titers than those who received only two doses. A notable proportion of those vaccinated with PCECV-KMB, an older subcutaneous formulation, were seronegative after a long interval. Conclusion: Antibody levels were strongly influenced by the number of vaccine doses, with reduced persistence in those who received only two. If testing confirms sufficient titers, PrEP booster doses may not be needed. However, for individuals with only two doses, older vaccinations, or those given PCECV-KMB, a risk-based assessment is recommended—especially for travelers to rabies-endemic areas. Full article

Activation of cGAS, a receptor recognizing cytosolic DNA, in macrophages might be associated with rabies (an RNA virus) through mitochondrial damage. A similar mortality rate was observed between cGAS-deficient (cGAS-/-) and wild-type (WT) mice post-CVS-11 strain injection. However, 2 out of 12 cGAS-/- mice (but not WT) survived for 15 days post-injection. At 7 days post-infection, less severe brain inflammation in cGAS-/- mice was demonstrated by the viral abundance in the hippocampus, the expression of proinflammatory genes (TNF-α and IL-1β), and the Evans blue dye assay (blood–brain barrier defect) with the presence of higher anti-inflammatory genes (TGF-β and arginase-1). Fecal Proteobacteria was more prominent in the infected WT mice, while serum cytokines (TNF-α and IL-1β) were similar in both mouse strains. There were less prominent responses against the rabies virus in cGAS-/- macrophages than in WT cells, as indicated by supernatant IL-6 and the gene expression of TLR-3, RIG-1, MDA-5, and iNOS. On the other hand, mitochondrial injury and cGAS activation were more prominent in WT macrophages over cGAS-/- cells, as indicated by cGAS expression, supernatant cGAMP (a secondary messenger of cGAS), and mitochondrial oxidative stress (MitoSox) together with a decrease in mitochondrial DNA and maximal respiration (extracellular flux analysis). In conclusion, (i) rabies-damaged mitochondria led to cGAS activation that was less severe in cGAS-/- than in WT, (ii) rabies-induced dysbiosis was demonstrated, and (iii) cGAS manipulation and gut–brain axis-associated inflammation warrants further investigation. Full article

Background/Objectives: Rabies is a fatal zoonotic disease caused by the rabies virus (RABV), and effective therapeutic treatments are currently lacking. Vaccination remains the primary strategy for rabies control. The Semliki Forest virus-rabies virus glycoprotein (SFV-RVG), a virus-like vesicle rabies vaccine combining Semliki Forest virus replicase and rabies glycoprotein, has shown potential as a promising vaccine candidate. This study aimed to optimize the production of SFV-RVG and evaluate adjuvant formulations to improve its immunogenicity in both mice and dogs. Methods: SFV-RVG production was optimized by determining the optimal multiplicity of infection (MOI) at 0.03 and cell density at 1 × 10⁶–1.3 × 10⁶ cells/mL, followed by scaling up the process in bioreactors. Eleven adjuvant formulations were tested in mice and dogs to assess their effects on immunogenicity. Cytokine analysis and antibody responses were measured, including IFN-γ, IL-4, IgG2a/IgG1 ratios, and neutralizing antibody titers. Results: The optimized SFV-RVG production was successfully scaled up, and M103 adjuvant induced rapid early antibody titers in mice. In dogs, GEL02 led to the highest neutralizing antibody levels, exceeding 40 IU/mL by 28 days post-immunization. Cytokine analysis indicated that both M103 and GEL02 significantly enhanced IFN-γ and IL-4 expression, balancing the Th1/Th2 immune response. SFV-RVG with GEL02 demonstrated stronger immunogenicity than a commercial vaccine, and challenge studies confirmed robust protection against lethal RABV in mice. Conclusions: This study establishes GEL02 as a superior adjuvant for rabies vaccines and provides a scalable SFV-RVG production process. These findings highlight SFV-RVG with GEL02 as a promising rabies vaccine candidate for dogs, offering significant potential for rabies control. Full article

The rabies virus remains a significant public health threat, particularly in regions with limited access to vaccination. This study shows that the BelloStage™-3000 bioreactor, operating on the “Tide Motion” principle, in combination with BioNOC^® II macrocarriers, ensures highly efficient rabies virus cultivation in BSR and Vero cells grown in serum-free OptiPRO™ SFM medium. This system supports effective cell attachment, formation of a dense and metabolically active cell layer, and reduces microbial contamination risks associated with serum-containing media. For comparison, rabies virus cultivation was also performed on Cytodex 1 and Cytodex 3 microcarriers in spinner flasks. The use of the BelloStage™-3000 bioreactor system with BelloCell™ 500A disposable vials and BioNOC II^® macrocarriers resulted in significantly higher virus titers compared to traditional Cytodex 1 and Cytodex 3 microcarrier culture systems. Thus, in the BSR cell culture, the maximum virus titer reached 5.6 × 10⁸ FFU/mL by day 4 of cultivation, which exceeded the titers obtained on Cytodex 1 and Cytodex 3 microcarriers by about 19.3-fold and 15.3-fold, respectively. A similar trend was observed for the Vero cell line: the peak titer was 2.0 × 10⁸ FFU/mL by day 5 of culturing, which was higher than the values obtained on Cytodex 1 and Cytodex 3 by about 14.0-fold and 9.6-fold, respectively. These findings demonstrate that the integrated use of BioNOC^® II macrocarriers, the BelloStage™-3000 bioreactor, and a serum-free medium provides a scalable, reproducible, and biosafe platform for rabies virus production, offering substantial advantages over traditional microcarrier-based systems. Full article

Stray cats (Felis vaga) are key hosts for feline and zoonotic pathogens. From June to August 2024, we conducted a cross-sectional study across six districts in Shenzhen, China, involving 126 cats sampled from three types of sites. Multiple specimens were tested via quantitative real-time PCR (qPCR) for feline coronavirus type I (FCoV-I), feline calicivirus (FCV), feline herpesvirus type I (FHV-I), feline panleukopenia virus (FPV), and rabies virus (RABV); serum was analyzed for RABV-neutralizing antibodies by the fluorescent antibody virus neutralization (FAVN) assay. The overall pathogen positivity was 89.68%. FPV was most prevalent (61.90%), followed by FCV (57.14%), FCoV-I (46.83%), and FHV-I (23.02%). No RABV nucleic acid was detected. The co-infection rate reached 62.70%, primarily dual infections (33.33%). Geographical variation was observed, with significantly higher FCoV-I in Longgang than Futian (p < 0.05). RABV seropositivity was only 6.00%. FCV and FPV co-occurred most frequently (Jaccard = 0.456). All pathogen pairs had relative risk (RR) > 1, suggesting non-random co-infections, though not significant after correction. In summary, major feline pathogens are widespread with frequent co-infections among Shenzhen stray cats, while low rabies immunity indicates potential public health risk. Targeted control measures are warranted. Full article

Rabies, a zoonotic infectious disease causing central nervous system inflammation, remains a threat to public health in regions with limited medical resources. Vaccination effectively reduces rabies incidence and mortality, underscoring the need for vaccines that are cost-effective, immunogenic, protective, and safe. This study constructed a recombinant rabies virus (rRABV)-overexpressing glucocorticoid-induced tumor necrosis factor receptor ligand (GitrL), named rLBNSE-GitrL, using a reverse genetic operating system. rLBNSE-GitrL exhibited similar in vitro phenotypic characteristics and immune safety as the parent RABV (rLBNSE). This recombinant virus stimulated the production of a greater number of activated dendritic cells (DCs) compared to rLBNSE. The enhanced innate immune response induced by rLBNSE-GitrL may be mediated through the activation of innate immune-related signaling pathways, such as the tumor necrosis factor (TNF), and chemokine signaling pathways, and the upregulation of a series of innate immune-related genes, including MMP2, IL-6, CXCL9, TIMP1, IL-17d, and TNF-α. Consequently, rLBNSE-GitrL elicited significantly higher levels of RABV vaccine-induced virus-neutralizing antibodies (VNA), IgG, and IgM compared to rLBNSE as early as 3 days post-immunization (dpi), thereby improving the protective effect in mice. Collectively, the overexpression of GitrL facilitated the induction of early and potent antibody responses following RABV immunization. Full article

Background: Rabies remains a fatal zoonotic disease, necessitating effective and affordable vaccines. While current vaccines are effective, they require multiple doses and may not induce long-lasting immunity in all settings. The rabies virus glycoprotein (RABV-G) is the principal antigen responsible for eliciting virus-neutralizing antibodies, but its recombinant monomeric forms often suffer from poor immunogenicity due to misfolding and aggregation. Methods: A recombinant trimeric RABV-G ectodomain (rRABV-G-XVIII) was engineered by fusing it to a human collagen XVIII-derived trimerization domain. The protein was expressed in E. coli, purified under denaturing conditions, and refolded. Trimer formation was verified using size-exclusion chromatography. Mice were immunized with rRABV-G-XVIII, with or without adjuvant, and compared to a monomeric form (rRABV-GE). Antigen-specific antibody responses were measured by ELISA, neutralizing activity was assessed, and protective efficacy was evaluated via intracerebral challenge with the CVS-27 rabies strain. Results: rRABV-G-XVIII formed stable trimers and induced strong humoral immune responses, with high ELISA titers and virus-neutralizing activity comparable to an inactivated rabies vaccine. Mice immunized with rRABV-GE showed lower antibody responses and partial protection, which improved with adjuvant. All rRABV-G-XVIII-immunized mice were fully protected against rabies challenge, independent of adjuvant use. Conclusions: Stabilization of RABV-G in its native trimeric conformation markedly improves immunogenicity and protective efficacy. This approach offers a promising strategy for the development of rabies subunit vac-cines with simplified formulations and potential for cost-effective production in bacterial systems. Full article

Studies spanning decades provide important information about the epidemiology and occurrence of a broad range of diseases that affect the central nervous system (CNS) of dogs. This study analyzed records and formalin-fixed paraffin-embedded (FFPE) CNS tissue samples from necropsied dogs with neuropathologic changes between 1962 and 2022. A total of 134,854 animals, including 20,117 dogs, were submitted for necropsy during this time span. Of these dogs, 2646 displayed alterations of the CNS. Degenerative and non-suppurative inflammatory lesions were the most common changes, accounting for 35.6% and 28.6%, respectively. Vascular diseases, neoplasms, congenital malformations, and suppurative inflammation represented 13.8%, 8.6%, 7.2%, and 5.4% of cases, respectively. Morbillivirus canis, the agent of canine distemper, was the most commonly diagnosed. The second most commonly detected virus, varicellovirus suidalpha1, the agent of pseudorabies, occurred almost exclusively between the mid-1970s and 1990s. Other pathogens, including Lyssavirus rabies, canine herpes virus, tick-borne encephalitis virus, apicomplexan parasites, such as Neospora caninum and Toxoplasma gondii, as well as fungal and other parasitic infections, were less frequently diagnosed. Interestingly, 47.6% of cases with non-suppurative inflammation remained etiologically undetermined. This study provides insights into the epidemiology of canine neurotropic infections and shows the value of FFPE material for investigations of past disease outbreaks. Full article

Mexico has not only successfully eliminated dog-mediated human rabies in recent years, but also the last rabies case in a dog infected with the canine variant of the rabies virus was reported in 2016. Mass dog vaccination campaigns were the cornerstone of these achievements. Unfortunately, the rabies virus still circulates in wildlife and, thus, spill-over infections in humans, livestock, and pets, including dogs, still occurs. Especially dogs that cohabit at interfaces shared with wildlife, like shepherd dogs, are at risk. These dogs are often free-roaming and difficult to restrain for vaccination purposes. Oral rabies vaccination (ORV) as an alternative vaccination strategy was tested in several rural villages in Querétaro State, Mexico. Bait acceptance and immunogenicity studies were conducted to test a licensed vaccine bait in terms of attractiveness and if the oral rabies vaccine strain, SPBN GASGAS, was able to induce an adequate immune response in local dogs, respectively. Although the egg(-flavored) bait was less well accepted (68.4%) by the dogs than the two other bait types included in the study, a bait made from boiled intestine segments (71.2%) and a bait with fish meal as an attractant (72.3%), dogs offered the egg bait were more often considered successfully vaccinated. 83.3% of the dogs offered an egg bait seroconverted during the immunogenicity study. Hence, ORV can be a suitable alternative by increasing the overall vaccination coverage of dogs that cannot be easily restrained and handled for vaccination. Full article