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Viruses Feb 2020Currently, no rabies virus-specific antiviral drugs are available. Ranpirnase has strong antitumor and antiviral properties associated with its ribonuclease activity....
Currently, no rabies virus-specific antiviral drugs are available. Ranpirnase has strong antitumor and antiviral properties associated with its ribonuclease activity. TMR-001, a proprietary bulk drug substance solution of ranpirnase, was evaluated against rabies virus in three cell types: mouse neuroblastoma, BSR (baby hamster kidney cells), and bat primary fibroblast cells. When TMR-001 was added to cell monolayers 24 h preinfection, rabies virus release was inhibited for all cell types at three time points postinfection. TMR-001 treatment simultaneous with infection and 24 h postinfection effectively inhibited rabies virus release in the supernatant and cell-to-cell spread with 50% inhibitory concentrations of 0.2-2 nM and 20-600 nM, respectively. TMR-001 was administered at 0.1 mg/kg via intraperitoneal, intramuscular, or intravenous routes to Syrian hamsters beginning 24 h before a lethal rabies virus challenge and continuing once per day for up to 10 days. TMR-001 at this dose, formulation, and route of delivery did not prevent rabies virus transit from the periphery to the central nervous system in this model ( = 32). Further aspects of local controlled delivery of other active formulations or dose concentrations of TMR-001 or ribonuclease analogues should be investigated for this class of drugs as a rabies antiviral therapeutic.
Topics: Animals; Antiviral Agents; Cell Line; Cells, Cultured; Chiroptera; Cricetinae; Female; Fibroblasts; Mesocricetus; Mice; Rabies; Rabies virus; Ribonucleases; Virus Release; Virus Replication
PubMed: 32033253
DOI: 10.3390/v12020177 -
PLoS Neglected Tropical Diseases Oct 2023Rabies is a zoonotic disease of all warm-blooded animals including humans. There is a paucity of data on the status of rabies in wild animals in Cameroon and the disease...
BACKGROUND
Rabies is a zoonotic disease of all warm-blooded animals including humans. There is a paucity of data on the status of rabies in wild animals in Cameroon and the disease is endemic in the country with dogs being the main source of transmission. Bat habitats are widespread in Cameroon, but there is limited information on the prevalence of rabies in bats, and their role of as potential reservoirs of rabies virus.
METHODS
A cross sectional study was carried out to estimate the prevalence and to assess risk factors of rabies virus in bats in the North Region of Cameroon. A total of 212 bats belonging to three families (Pteropodidae, Vespertilionidae and Molossidae) and 5 species were sampled in 7 localities in the North Region of Cameroon and were tested for rabies virus antigen using direct Immunofluorescence Test (IFA).
RESULTS
Overall, 26.9% (57/212) of the bats collected showed an IFA positive reaction. The prevalence was significantly higher (P<0.05) in adult bats (33.3% (36/108)) compared to young individuals (20.2%; 21/104). The main risk factors identified in the study for human exposure to bats were gender (Male), religion (Christianity), localities (Babla and Lagdo), the practice of bat hunting, bat consumption, unawareness of bat rabies and cohabitation with bats in close proximity.
CONCLUSION
The study revealed the first evidence of Lyssavirus in bats in Cameroon. This finding showed that bat rabies are real and represents a potential public health concern in communities with bat habitats in the North Region of Cameroon. Enhancing the level of public awareness and health education on the potential of bats as reservoirs of Lyssavirus in Cameroon as well as the integration of the "One Health" approach for effective management of animal and human rabies should be emphasized.
Topics: Animals; Humans; Male; Cameroon; Chiroptera; Cross-Sectional Studies; Lyssavirus; Prevalence; Public Health; Rabies; Rabies virus; Female
PubMed: 37871008
DOI: 10.1371/journal.pntd.0010803 -
Journal of Virology Jun 2020Rabies virus (RABV) causes a severe and fatal neurological disease, but morbidity is vaccine preventable and treatable prior to the onset of clinical symptoms. However,...
Rabies virus (RABV) causes a severe and fatal neurological disease, but morbidity is vaccine preventable and treatable prior to the onset of clinical symptoms. However, immunoglobulin (IgG)-based rabies postexposure prophylaxis (PEP) is expensive, restricting access to life-saving treatment, especially for patients in low-income countries where the clinical need is greatest, and does not confer cross-protection against newly emerging phylogroup II lyssaviruses. Toward identifying a cost-effective replacement for the IgG component of rabies PEP, we developed and implemented a high-throughput screening protocol utilizing a single-cycle RABV reporter strain. A large-scale screen and subsequent direct and orthogonal counterscreens identified a first-in-class direct-acting RABV inhibitor, GRP-60367, with a specificity index (SI) of >100,000. Mechanistic characterization through time-of-addition studies, transient cell-to-cell fusion assays, and chimeric vesicular stomatitis virus (VSV) recombinants expressing the RABV glycoprotein (G) demonstrated that GRP-60367 inhibits entry of a subset of RABV strains. Resistance profiling of the chemotype revealed hot spots in conserved hydrophobic positions of the RABV G protein fusion loop that were confirmed in transient cell-to-cell fusion assays. Transfer of RABV G genes with signature resistance mutations into a recombinant VSV backbone resulted in the recovery of replication-competent virions with low susceptibility to the inhibitor. This work outlines a tangible strategy for mechanistic characterization and resistance profiling of RABV drug candidates and identified a novel, well-behaved molecular probe chemotype that specifically targets the RABV G protein and prevents G-mediated viral entry. Rabies PEP depends on anti-RABV IgG, which is expensive and in limited supply in geographical areas with the highest disease burden. Replacing the IgG component with a cost-effective and shelf-stable small-molecule antiviral could address this unmet clinical need by expanding access to life-saving medication. This study has established a robust protocol for high-throughput anti-RABV drug screens and identified a chemically well-behaved, first-in-class hit with nanomolar anti-RABV potency that blocks RABV G protein-mediated viral entry. Resistance mapping revealed a druggable site formed by the G protein fusion loops that has not previously emerged as a target for neutralizing antibodies. Discovery of this RABV entry inhibitor establishes a new molecular probe to advance further mechanistic and structural characterization of RABV G that may aid in the design of a next-generation clinical candidate against RABV.
Topics: Animals; Antibodies, Neutralizing; Antibodies, Viral; Antiviral Agents; Cell Line; Cross Protection; Drug Evaluation, Preclinical; Humans; Peptide Library; Rabies; Rabies Vaccines; Rabies virus; Vesicular stomatitis Indiana virus; Vesiculovirus; Viral Fusion Proteins
PubMed: 32321812
DOI: 10.1128/JVI.00321-20 -
Journal of Visualized Experiments : JoVE Aug 2023Genomic data can be used to track the transmission and geographic spread of infectious diseases. However, the sequencing capacity required for genomic surveillance...
Genomic data can be used to track the transmission and geographic spread of infectious diseases. However, the sequencing capacity required for genomic surveillance remains limited in many low- and middle-income countries (LMICs), where dog-mediated rabies and/or rabies transmitted by wildlife such as vampire bats pose major public health and economic concerns. We present here a rapid and affordable sample-to-sequence-to-interpretation workflow using nanopore technology. Protocols for sample collection and the diagnosis of rabies are briefly described, followed by details of the optimized whole genome sequencing workflow, including primer design and optimization for multiplex polymerase chain reaction (PCR), a modified, low-cost sequencing library preparation, sequencing with live and offline base calling, genetic lineage designation, and phylogenetic analysis. Implementation of the workflow is demonstrated, and critical steps are highlighted for local deployment, such as pipeline validation, primer optimization, inclusion of negative controls, and the use of publicly available data and genomic tools (GLUE, MADDOG) for classification and placement within regional and global phylogenies. The turnaround time for the workflow is 2-3 days, and the cost ranges from $25 per sample for a 96 sample run to $80 per sample for a 12 sample run. We conclude that setting up rabies virus genomic surveillance in LMICs is feasible and can support progress toward the global goal of zero dog-mediated human rabies deaths by 2030, as well as enhanced monitoring of wildlife rabies spread. Moreover, the platform can be adapted for other pathogens, helping to build a versatile genomic capacity that contributes to epidemic and pandemic preparedness.
Topics: Humans; Animals; Dogs; Rabies virus; Rabies; Nanopores; Phylogeny; Animals, Wild; Chiroptera; Technology; Whole Genome Sequencing
PubMed: 37677046
DOI: 10.3791/65414 -
Diagnostic Microbiology and Infectious... Jan 2023Despite the enzootic cycle of rabies in dog populations, laboratory confirmation of human rabies has been hardly reported in Cameroon. This study aimed to determine the...
Despite the enzootic cycle of rabies in dog populations, laboratory confirmation of human rabies has been hardly reported in Cameroon. This study aimed to determine the rate of molecular detection and phylogenetic relatedness of Rabies Virus (RABV) isolates from suspected human rabies cases in Cameroon. From 2014 to 2018, 21 suspected human rabies cases were tested for RABV genomic RNA. Full-length sequence of the nucleoprotein (N) coding gene of RABV isolates detected were generated and subjected to phylogenetic analyses. As results, skin biopsies and/or saliva samples from 10 of the 21 suspected human rabies cases were positive for genomic RABV RNA. Four new N gene sequences were generated from confirmed cases. The studied RABV isolates fell into the Cosmopolitan clades, sub-clades Africa-1a and 1b. This study showed a low rate of molecular detection of RABV in suspected human rabies cases; thus, underscoring the interest of systematic laboratory confirmation.
Topics: Humans; Dogs; Animals; Rabies virus; Rabies; Phylogeny; Cameroon; RNA
PubMed: 36343475
DOI: 10.1016/j.diagmicrobio.2022.115834 -
Archives of Virology Jan 2023Although rabies is endemic in Malawi, there have been no studies in which rabies virus was systematically investigated and characterized in multiple animal hosts in that...
Although rabies is endemic in Malawi, there have been no studies in which rabies virus was systematically investigated and characterized in multiple animal hosts in that country. In order to provide molecular epidemiological data on rabies virus in Malawi, 683 suspected rabies case reports from 2008 to 2021 were examined, and 46 (dog = 40, cow = 5, and cat = 1) viable rabies-positive brain samples archived at the Central Veterinary Laboratory (CVL), Lilongwe, Malawi, were analyzed genetically. The results showed an increase in the submission of brain samples from 2008 to 2010, with the highest number of submissions observed in 2020. Of the 683 case reports analyzed for the period under review, 38.1% (260/683) (CI: 34.44 - 41.84) were confirmed by direct fluorescent antibody test. Among the confirmed cases, 65.4% (170/260) (CI: 59.23 - 71.09) were canine rabies. Further, phylogenetic analysis revealed that sequences from different animal hosts clustered together within the Africa 1b lineage, suggesting that the strains circulating in livestock are similar to those in domestic dogs. This finding supports the hypothesis that canine rabies is spilling over to livestock and emphasizes the need for further studies to provide data for effective control of rabies in Malawi.
Topics: Female; Cattle; Animals; Dogs; Rabies virus; Rabies; Phylogeny; Malawi; Molecular Epidemiology; Dog Diseases; Livestock
PubMed: 36631547
DOI: 10.1007/s00705-022-05635-z -
Viruses Jul 2021The case fatality rate of rabies, nearly 100%, is one of the most unique characteristic of this ancient virus infection. The crucial role rabies virus neutralizing... (Review)
Review
The case fatality rate of rabies, nearly 100%, is one of the most unique characteristic of this ancient virus infection. The crucial role rabies virus neutralizing antibody plays in protection is both well established and explanation of why rabies serology is important. Various laboratory methods can and have been used but serum neutralization methods have long been the gold standard due to the ability to measure function (neutralization), however these methods can be difficult to perform for several reasons. Assays such as enzyme linked absorbance assays (ELISA), indirect fluorescence antibody (IFA) and more recently lateral flow methods are in use. Interpretation of results can be problematic, not only between methods but also due to modifications of the same method that can lead to misinterpretations. A common assumption in review of laboratory test results is that different methods for the same component produce comparable results under all conditions or circumstances. Assumptions and misinterpretations provide the potential for detrimental decisions, ranging from regulatory to clinically related, and most importantly what 'level' is protective. Review of the common challenges in performance and interpretation of rabies serology and specific examples illuminate critical issues to consider when reviewing and applying results of rabies serological testing.
Topics: Antibodies, Neutralizing; Antibodies, Viral; Data Interpretation, Statistical; Enzyme-Linked Immunosorbent Assay; Fluorescent Antibody Technique, Indirect; Hematologic Tests; Humans; Neutralization Tests; Rabies; Rabies virus; Serologic Tests
PubMed: 34452381
DOI: 10.3390/v13081516 -
Viruses Nov 2022Rabies virus (RABV) has a broad host range and infects multiple cell types throughout the infection cycle. Next-generation sequencing (NGS) and minor variant analysis...
Rabies virus (RABV) has a broad host range and infects multiple cell types throughout the infection cycle. Next-generation sequencing (NGS) and minor variant analysis are powerful tools for studying virus populations within specific hosts and tissues, leading to novel insights into the mechanisms of host-switching and key factors for infecting specific cell types. In this study we investigated RABV populations and minor variants in both original (non-passaged) samples and in vitro-passaged isolates of various CNS regions (hippocampus, medulla oblongata and spinal cord) of a fatal human rabies case, and of multiple CNS and non-CNS tissues of experimentally infected mice. No differences in virus populations were detected between the human CNS regions, and only one non-synonymous single nucleotide polymorphism (SNP) was detected in the fifth in vitro passage of virus isolated from the spinal cord. However, the appearance of this SNP shows the importance of sequencing newly passaged virus stocks before further use. Similarly, we did not detect apparent differences in virus populations isolated from different CNS and non-CNS tissues of experimentally infected mice. Sequencing of viruses obtained from pharyngeal swab and salivary gland proved difficult, and we propose methods for improving sampling.
Topics: Humans; Mice; Animals; Rabies virus; Central Nervous System; Rabies; Spinal Cord
PubMed: 36560665
DOI: 10.3390/v14122661 -
Viruses Mar 2021Sylvatic rabies was present in Slovenia between 1973 and 2013, with the red fox as the main reservoir of the rabies virus. The first oral rabies vaccination (ORV)... (Review)
Review
Sylvatic rabies was present in Slovenia between 1973 and 2013, with the red fox as the main reservoir of the rabies virus. The first oral rabies vaccination (ORV) control program in foxes started in 1988, using the manual distribution of baits. Significant improvement of fox vaccination was achieved with the aerial distribution of baits, starting in 1995 and successfully finished with the final, fifty-ninth vaccination campaign in 2019. Between 1979 and 2019, a total of 86,471 samples were tested, and 10,975 (12.69%) rabies-positive animals were identified. Within the ORV, two different vaccines were used, containing modified live virus strain Street Alabama Dufferin (SAD) B19 and SAD Bern, while the last ORV campaigns were completed in 2019, with a vaccine containing a genetically modified strain of SPBN GASGAS. Molecular epidemiological studies of 95 rabies-positive samples, originating from red foxes, badgers, cattle, dogs, martens, cats, and horses, revealed a low genetic diversity of circulating strains and high similarity to strains from neighboring countries. During the elimination program, few vaccine-induced rabies cases were detected: three in red foxes and one case in a marten, with no epidemiological relevance. Slovenia has been officially declared a country free of rabies since 2016.
Topics: Administration, Oral; Animals; Disease Eradication; Foxes; RNA, Viral; Rabies; Rabies Vaccines; Rabies virus; Slovenia; Vaccination
PubMed: 33806582
DOI: 10.3390/v13030405 -
Transboundary and Emerging Diseases Sep 2022Rabies is a lethal zoonosis affecting mammals worldwide. Diagnosis of rabies follows international standard protocols, primarily relying on direct immunofluorescence...
Rabies is a lethal zoonosis affecting mammals worldwide. Diagnosis of rabies follows international standard protocols, primarily relying on direct immunofluorescence (DI) followed by mouse inoculation test (MIT). WHO recommends molecular biology techniques such as RT-qPCR for replacing MIT to diagnose rabies in animal samples. Recently, a real-time PCR protocol that detects all rabies virus variants identified worldwide was validated. This assay is a pan-Lyssavirus TaqMan quantitative RT-PCR called LN34. A modified LN34 assay protocol was tested at the Paraná State Reference Laboratory (Lacen/PR) using animal samples previously tested by DI and MIT, the gold standard (GS). This method has been changed to a RT-qPCR duplex format to better fit the diagnostic routine. The new assay was called duplex LN34 and β-actin RT-qPCR. All the 88 samples evaluated using the GS test, modified pan-Lyssavirus TaqMan RT-qPCR and duplex LN34 and β-actin RT-qPCR showed 100% agreement with each other. This novel duplex RT-qPCR protocol has shown adequate diagnostic performance and may be used in research and surveillance purposes, replacing the standard MIT and ending mice use for rabies diagnosis.
Topics: Actins; Animals; Lyssavirus; Mammals; Mice; Rabies; Rabies virus; Real-Time Polymerase Chain Reaction; Sensitivity and Specificity
PubMed: 35438243
DOI: 10.1111/tbed.14565