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Members of the family Rhabdoviridae have unsegmented, negative-strand RNA genomes. Rod- or bullet-shaped virions are enveloped with helical nucleocapsids. There are well over 100 named rhabdoviruses isolated from hosts as diverse as insects, vertebrate animals, and plants. The overall replication strategy of the rhabdoviruses is similar to that of other families in the order Mononegavirales. Rabies virus (RABV), a member of the genus Lyssavirus, is an ancient and much feared pathogen. The name rabies comes from the Latin rabidus meaning raving, furious, or mad, a name that accurately describes many animals suffering from this invariably fatal neurologic infection. Suspected RABV exposures (i.e., bites) call for the administration of antirabies immune globulin and postexposure vaccination. Postexposure vaccination is effective due to the long incubation period as RABV travel up axons to the CNS.

1. Understanding the role of different species in the transmission of multi-host pathogens, such as rabies virus, is vital for effective control strategies. Across most of sub-Saharan Africa domestic dogs Canis familiaris are considered the reservoir for rabies, but the role of wildlife has been long debated. Here we explore the multi-host transmission dynamics of rabies across south-east Tanzania. 2. Between January 2011 and July 2019, data on probable rabies cases were collected in the regions of Lindi and Mtwara. Hospital records of animal-bite patients presenting to healthcare facilities were used as sentinels for animal contact tracing. The timing, location and species of probable rabid animals were used to reconstruct transmission trees to infer who infected whom and the relative frequencies of within- and between-species transmission. 3. During the study, 688 probable human rabies exposures were identified, resulting in 47 deaths. Of these exposures, 389 were from domestic dogs (56.5%) and 262 from jackals (38.1%). Over the same period, 549 probable animal rabies cases were traced: 303 in domestic dogs (55.2%) and 221 in jackals (40.3%), with the remainder in domestic cats and other wildlife species. 4. Although dog-to-dog transmission was most commonly inferred (40.5% of transmission events), a third of inferred events involved wildlife-to-wildlife transmission (32.6%), and evidence suggested some sustained transmission chains within jackal populations. 5. A steady decline in probable rabies cases in both humans and animals coincided with the implementation of widespread domestic dog vaccination during the first 6 years of the study. Following the lapse of this program, dog rabies cases began to increase in one of the northernmost districts. 6. Synthesis and applications. In south-east Tanzania, despite a relatively high incidence of rabies in wildlife and evidence of wildlife-to-wildlife transmission, domestic dogs remain essential to the reservoir of infection. Continued dog vaccination alongside improved surveillance would allow a fuller understanding of the role of wildlife in maintaining transmission in this area. Nonetheless, dog vaccination clearly suppressed rabies in both domestic dog and wildlife populations, reducing both public health and conservation risks and, if sustained, has potential to eliminate rabies from this region.

Viruses are obligatory intracellular parasites that use cell proteins to take the control of the cell functions in order to accomplish their life cycle. Studying the viral-host interactions would increase our knowledge of the viral biology and mechanisms of pathogenesis. Studies on pathogenesis mechanisms of lyssaviruses, which are the causative agents of rabies, have revealed some important host protein partners for viral proteins, especially for most studied species, i.e. Rabies virus. In this review article, the key physical lyssavirus-host protein interactions, their contributions to rabies infection, and their exploitation are discussed to improve the knowledge about rabies pathogenesis.

Background Inaccurate diagnosis of encephalitis is a major issue as immunosuppressive treatments can be deleterious in case of viral infection. The European bat lyssavirus type 1 (EBLV-1), a virus related to rabies virus, is endemic in European bats. No human case has yet been reported in Western Europe. A 59-year-old patient without specific past medical history died from encephalitis. A colony of bats lived in an outbuilding of his house. No diagnosis was made using standard procedures. Methods We used a next generation sequencing (NGS) based transcriptomic protocol to search for pathogens in autopsy samples (meninges and brain frontal lobe). Results were confirmed by polymerase chain reaction (PCR) and by antibody testing in serum. Immunochemistry was used to characterize inflammatory cells and viral antigens in brain lesions. Cells and mice were inoculated with brain extracts for virus isolation. Results The patient’s brain lesions were severe and diffuse in white and gray matter. Perivascular inflammatory infiltrates were abundant and rich in plasma cells. NGS identified European bat lyssavirus type 1a in brain, which was confirmed by PCR. A high titer of neutralizing antibodies was found in serum. No viral antigen was detected, and the virus could not be isolated by cell culture or by mouse inoculation. Conclusions The patient died from European bat lyssavirus type 1a infection. NGS was key to identifying this unexpected viral etiology in an epidemiological context that did not suggest rabies. People exposed to bats should be strongly advised to be vaccinated with rabies vaccines, which are effective against EBLV-1.

he purpose of this study was to analyze the genomic information of the glycoprotein gene of rabies virus (RABV) in several countries of Asia. The genomics information of vaccine virus strain and some local isolates were retrieved from GeneBank® (National Center for Biotechnology Information, USA). Ten genomics informations are consist of ERA vaccine virus, two viruses from Indonesia, a virus from Myanmar, India, Nepal, South Korea, Japan, Sri Lanka, and Iran. This bioinformatics process the ten genomics data in order to find pattern in 1,575 bp of glycoprotein gene of RABV. In conclusion, this study have revealed the data of homology, B-cell epitope prediction, genetic distance, and molecular phylogenic analysis of RABV in various countries of Asia. We indicated that the glycoprotein, the immunogenic surface protein of RABV, has important part for vaccine design Keywords: Bioinformatics Lyssavirus glycoprotein rabies vaccine

Background Palearctic bats host a diversity of lyssaviruses, though not the classical rabies virus (RABV). As surveillance for bat rabies over the Palearctic area covering Central and Eastern Europe and Siberian regions of Russia has been irregular, we lack data on geographic and seasonal patterns of the infection. Results To address this, we undertook serological testing, using non-lethally sampled blood, on 1027 bats of 25 species in Bulgaria, the Czech Republic, Poland, Russia and Slovenia between 2014 and 2018. The indirect enzyme-linked immunosorbent assay (ELISA) detected rabies virus anti-glycoprotein antibodies in 33 bats, giving an overall seroprevalence of 3.2%. Bat species exceeding the seroconversion threshold included Myotis blythii, Myotis gracilis, Myotis petax, Myotis myotis, Murina hilgendorfi, Rhinolophus ferrumequinum and Vespertilio murinus. While Myotis species (84.8%) and adult females (48.5%) dominated in seropositive bats, juveniles of both sexes showed no difference in seroprevalence. Higher numbers tested positive when sampled during the active season (10.5%), as compared with the hibernation period (0.9%). Bat rabies seroprevalence was significantly higher in natural habitats (4.0%) compared with synanthropic roosts (1.2%). Importantly, in 2018, we recorded 73.1% seroprevalence in a cave containing a M. blythii maternity colony in the Altai Krai of Russia. Conclusions Identification of such “hotspots” of non-RABV lyssavirus circulation not only provides important information for public health protection, it can also guide research activities aimed at more in-depth bat rabies studies.

Serology is a core component of the surveillance and management of viral zoonoses. Virus neutralization tests are a gold standard serological diagnostic, but requirements for large volumes of serum and high biosafety containment can limit widespread use. Here, focusing on Rabies lyssavirus, a globally important zoonosis, we developed a pseudotype micro‐neutralization rapid fluorescent focus inhibition test (pmRFFIT) that overcomes these limitations. Specifically, we adapted an existing micro‐neutralization test to use a green fluorescent protein‐tagged murine leukaemia virus pseudotype in lieu of pathogenic rabies virus, reducing the need for specialized reagents for antigen detection and enabling use in low‐containment laboratories. We further used statistical models to generate rapid, quantitative predictions of the probability and titre of rabies virus‐neutralizing antibodies from microscopic imaging of neutralization outcomes. Using 47 serum samples from domestic dogs with neutralizing antibody titres estimated using the fluorescent antibody virus neutralization test (FAVN), pmRFFIT showed moderate sensitivity (78.79%) and high specificity (84.62%). Despite small conflicts, titre predictions were correlated across tests repeated on different dates both for dog samples (r = 0.93) and in a second data set of sera from wild common vampire bats (r = 0.72, N = 41), indicating repeatability. Our test uses a starting volume of 3.5 µl of serum, estimates titres from a single dilution of serum rather than requiring multiple dilutions and end point titration, and may be adapted to target neutralizing antibodies against alternative lyssavirus species. The pmRFFIT enables high‐throughput detection of rabies virus‐neutralizing antibodies in low‐biocontainment settings and is suited to studies in wild or captive animals where large serum volumes cannot be obtained.

Lyssavirus P protein is a multifunctional protein that interacts with numerous host-cell proteins. The C-terminal domain (CTD) of P is important for inhibition of JAK-STAT signaling enabling the virus to evade host immunity. Several regions on the surface of rabies virus P are reported to interact with host factors. Among them, an extended, discrete hydrophobic patch of P CTD is notable. Although structures of P CTD of two strains of rabies virus, and of mokola virus have been solved, the structure of P CTD for Duvenhage virus, which is functionally divergent from these species for immune evasion function, is not known. Here, we analyze the structures of P CTD of Duvenhage and of a distinct rabies virus strain to gain further insight on the nature and potential function of the hydrophobic surface. Molecular contacts in crystals suggest that the hydrophobic patch is important to intermolecular interactions with other proteins, which differ between the lyssavirus species.

Although rabies is enzootic in the Democratic Republic of the Congo, there is very little molecular epidemiological information about the viruses circulating in animals. In this study, a fragment of the rabies virus (RABV) nucleoprotein gene was amplified and sequenced from 21 animal brain samples collected in two western provinces of the country between 2008 and 2017. The samples tested were from cat (n = 1), dog (n = 17), goat (n = 2), and sheep (n = 1). Phylogenetic analysis revealed that the sequences generated were highly similar to each other and belonged to lineage Africa 1b clustering with a single sample identified in a canine in the Republic of Congo in 2014. This is the first molecular epidemiological study of RABV in the DRC and the data generated will assist authorities in the development of effective control strategies for rabies in the country.

Rabies is an acute viral disease caused by a virus of the Rhabdoviridae family of the Lyssavirus genus, which affects the central nervous system and is characterised by absolute mortality. Vaccination is the only way to prevent the disease in humans. One of the products used for vaccination is a cultural concentrated purified inactivated dry rabies vaccine produced by the Federal State Budgetary Institution of Science “Chumakov Federal Scientific Center for Research and Development of Immune-and-Biological Products of Russian Academy of Sciences” (hereinafter—Chumakov Center).The aim of the study was to examine the structure of the working virus seed of Vnukovo-32 strain used by the Chumakov Center for rabies vaccine production, to assess its genetic stability during production, to explore the possibility of using molecular genetic methods for identification of the production strain in the finished dosage form, and to study the nucleotide sequence of the CVS strain.Materials and methods: Vnukovo-32 rabies virus production strain, working virus seeds, finished batches of the rabies vaccine, CVS fixed rabies virus strain used in the assessment of specific immunity. The molecular genetic study was performed using RT-PCR followed by restriction and sequencing.Results: the paper presents the results of nucleotide sequence analysis of the G gene fragment obtained from the Vnukovo-32 production strain, batches of the working virus seed, and finished batches of the rabies vaccine produced in 2012, 2018, and 2019, and the CVS fixed rabies virus strain used in the assessment of the vaccine’s specific immunity. The study demonstrated that restriction analysis could be used for Vnukovo-32 strain identification at all production stages, including the finished dosage form.Conclusion: Vnukovo-32 and CVS strains used by the Chumakov Center are rabies viruses. Analysis of the nucleotide sequence of the G gene fragment showed that the Vnukovo-32 strain remains stable throughout different production stages. The obtained nucleotide sequence of gene G of the Vnukovo-32 strain was deposited in GenBank (accession number MN116503). The study demonstrated that restriction analysis could be used for Vnukovo-32 strain identification at all production stages, including the finished dosage form.

Introduction . Rabies caused by the neurotropic virus of the genus Lyssavirus , Rhabdoviridae family, which infects all warm-blooded vertebrates including human beings. The homology level of the amino acid sequences for Lyssaviruses nucleoprotein reaches 78–93%. Aim – study the genetic diversity and molecular epidemiology of Lyssaviruses circulated in the Russian Federation in 1985–2016. Material and methods . 54 isolates of rabies virus isolated from animals, and 2 isolates from humans, 4 vaccine strains of rabies virus: RV-97, ERA, Shchelkovo 51, ERAG333 used in phylogenetic study. Phylogenetic analysis was performed using Genbank data on genome fragments of 73 rabies virus isolates and 9 EBLV-1 isolates. DNASTAR V.3.12, Bio Edit 7.0.4.1 and MEGA v.10.0.5, Primer Premier 5 programs have been used. Results . Comparative molecular genetic analysis of genomes fragments of 130 Lissaviruses, isolated on the territory of the RF, Ukraine in 1985-2016, vaccine strains of rabies virus, showed their distribution by geographical feature. Comparison of the nucleoprotein fragments of the rabies virus isolates with vaccine strains revealed 4 marker mutations: V56I (Eurasian group), L/V95W (Central group), D101N/S/T, and N/G106D. Phylogenetic analysis of the isolate «Juli», isolated from a human bitten by a bat proved his belonging to the European Bat lyssavirus-1a. Discussion . Study of the molecular epidemiology of rabies within the Russian Federation allows for the genotyping of the viruses and helps to study the hidden mechanisms of rabies infection in animal and human populations, and to characterize vaccine strains, including during oral vaccination. Conclusion . Further study of the molecular epidemiology of rabies within the Russian Federation and the countries bordering it is important.

Bats have been identified as a natural reservoir of several potentially zoonotic viruses, including Lyssavirus, Ebola virus, Marburg virus, Hendra virus, Nipah virus, as well as severe acute respiratory syndrome and Middle East respiratory syndrome coronavirus (CoV). Here, we performed a molecular epidemiological investigation of South Korean bat viruses. Genetic comparative analysis was performed on the spike glycoprotein gene of the detected MERS-related CoVs. Among 1640 samples (348 oral swabs, 1199 faecal samples, 83 urine samples and 10 bat carcass) collected across 24 South Korean provinces during 2017-2019, CoV was detected in 82 samples (75 faeces and seven oral swab samples) from 11 provinces. Surveillance over the 3 years during which samples were collected revealed significantly higher CoV detection rates between spring and autumn, and a high detection rate in Vespertillionidae and Rhinolophidae bats. Our phylogenetic analysis shows that Korean bat CoVs are genetically diverse regardless of their spatiotemporal distribution and their host species, and that the discovered bat CoVs belong to various subgenera within the Alpha- and Betacoronavirus genera. Twenty detected MERS-related CoVs belonging to the genus Betacoronavirus were similar to the Ia io bat CoV NL140422 and NL13845 strains. A comprehensive genetic analysis of two Korean bat MERS-related CoV spike receptor binding domain (RBDs) (176 and 267 strains) showed that the 18 critical residues that are involved in interactions with the human DPP4 receptor are most similar to the NL13845 strain, which is known to not bind with hDPP4. A deeper analysis of the interfacing residues in the Korean bat MERS-related CoVs RBD-hDPP4 complexes showed that the Korean bat CoVs has fewer polar contacts than the NL13845 strain. Although further study will be needed, these results suggest that Korean bat MERS-related CoVs are unlikely to bind with hDPP4. Nevertheless, these findings highlight the need for continuous monitoring to identifying the origin of new infectious diseases, specifically mutant CoV.

A plethora of bat-associated lyssaviruses potentially capable of causing the fatal disease rabies are known today. Transmitted via infectious saliva, occasionally-reported spillover infections from bats to other mammals demonstrate the permeability of the species-barrier and highlight the zoonotic potential of bat-related lyssaviruses. However, it is still unknown whether and, if so, to what extent, viruses from different lyssavirus species vary in their pathogenic potential. In order to characterize and systematically compare a broader group of lyssavirus isolates for their viral replication kinetics, pathogenicity, and virus release through saliva-associated virus shedding, we used a mouse infection model comprising a low (102 TCID50) and a high (105 TCID50) inoculation dose as well as three different inoculation routes (intramuscular, intranasal, intracranial). Clinical signs, incubation periods, and survival were investigated. Based on the latter two parameters, a novel pathogenicity matrix was introduced to classify lyssavirus isolates. Using a total of 13 isolates from ten different virus species, this pathogenicity index varied within and between virus species. Interestingly, Irkut virus (IRKV) and Bokeloh bat lyssavirus (BBLV) obtained higher pathogenicity scores (1.14 for IRKV and 1.06 for BBLV) compared to rabies virus (RABV) isolates ranging between 0.19 and 0.85. Also, clinical signs differed significantly between RABV and other bat lyssaviruses. Altogether, our findings suggest a high diversity among lyssavirus isolates concerning survival, incubation period, and clinical signs. Virus shedding significantly differed between RABVs and other lyssaviruses. Our results demonstrated that active shedding of infectious virus was exclusively associated with two RABV isolates (92% for RABV-DogA and 67% for RABV-Insectbat), thus providing a potential explanation as to why sustained spillovers are solely attributed to RABVs. Interestingly, 3D imaging of a selected panel of brain samples from bat-associated lyssaviruses demonstrated a significantly increased percentage of infected astrocytes in mice inoculated with IRKV (10.03%; SD±7.39) compared to RABV-Vampbat (2.23%; SD±2.4), and BBLV (0.78%; SD±1.51), while only individual infected cells were identified in mice infected with Duvenhage virus (DUVV). These results corroborate previous studies on RABV that suggest a role of astrocyte infection in the pathogenicity of lyssaviruses., Author summary Globally, there are at present 17 different officially recognized lyssavirus species posing a potential threat for human and animal health. Bats have been identified as carriers for the vast majority of those zoonotic viruses, which cause the fatal disease rabies and are transmitted through infectious saliva. The occurrence of sporadic spillover events where lyssaviruses are spread from bats to other mammalian species highlights the importance of studying pathogenicity and virus shedding in regard to a potentially sustained onward cross-species transmission. Therefore, as part of this study, we compared 13 different isolates from ten lyssavirus species in a standardized mouse infection model, focusing on clinical signs, incubation periods, and survival. Based on the latter two, a novel pathogenicity index to classify different lyssavirus species was established. This pathogenicity index varied within and between different lyssavirus species and revealed a higher ranking of other bat-related lyssaviruses in comparison to the tested Rabies virus (RABV) isolates. Altogether, our results demonstrate a high diversity among the investigated isolates concerning pathogenicity and clinical picture. Furthermore, we comparatively analyzed virus shedding via saliva and while there was no indication towards a reduced pathogenicity of bat-associated lyssaviruses as opposed to RABV, shedding was increased in RABV isolates. Additionally, we investigated neuronal cell tropism and revealed that bat lyssaviruses are not only capable of infecting neurons but also astrocytes.

Lyssaviruses are the causative agents for rabies, a zoonotic and fatal disease. Bats are the ancestral reservoir host for lyssaviruses, and at least three different lyssaviruses have been found in bats from Germany. Across Europe, novel lyssaviruses were identified in bats recently and occasional spillover infections in other mammals and human cases highlight their public health relevance. Here, we report the results from an enhanced passive bat rabies surveillance that encompasses samples without human contact that would not be tested under routine conditions. To this end, 1236 bat brain samples obtained between 2018 and 2020 were screened for lyssaviruses via several RT-qPCR assays. European bat lyssavirus type 1 (EBLV-1) was dominant, with 15 positives exclusively found in serotine bats (Eptesicus serotinus) from northern Germany. Additionally, when an archived set of bat samples that had tested negative for rabies by the FAT were screened in the process of assay validation, four samples tested EBLV-1 positive, including two detected in Pipistrellus pipistrellus. Subsequent phylogenetic analysis of 17 full genomes assigned all except one of these viruses to the A1 cluster of the EBLV-1a sub-lineage. Furthermore, we report here another Bokeloh bat lyssavirus (BBLV) infection in a Natterer’s bat (Myotis nattereri) found in Lower Saxony, the tenth reported case of this novel bat lyssavirus.

Mongooses, a nonnative species, are a known reservoir of rabies virus in the Caribbean region. A cross-sectional study of mongooses at 41 field sites on the US Virgin Islands of St. Croix, St. John, and St. Thomas captured 312 mongooses (32% capture rate). We determined the absence of rabies virus by antigen testing and rabies virus exposure by antibody testing in mongoose populations on all three islands. USVI is the first Caribbean state to determine freedom-from-rabies for its mongoose populations with a scientifically-led robust cross-sectional study. Ongoing surveillance activities will determine if other domestic and wildlife populations in USVI are rabies-free., Author summary Mongooses in the Caribbean region are known to carry rabies and infect and kill humans with the deadly virus. While many countries in the Caribbean region assume they are rabies-free, there is a lack of scientifically based surveillance. We prospectively designed a statistically valid study to determine that mongoose populations in the US Virgin Islands are rabies-free and pose no risk of transmission to human or animal populations. We are pursuing further surveillance efforts to establish rabies freedom in all wildlife and domestic animal population in the US Virgin Islands.

Rabies remains a public health threat in China, and most transmissions are dog-mediated. In this study, we studied 31 clinically diagnosed human rabies patients that had been scratched or bitten by dogs. Real-time reverse transcription polymerase chain reaction (RT-qPCR) and nested RT-PCR were performed on saliva samples or cerebrospinal fluid, and samples from 28 patients tested positive for rabies virus. A total of one near-complete genome sequence, 15 complete glycoprotein (G) gene sequences, and five partial G gene sequences were determined. Phylogenetic analysis was performed, based on complete G gene sequences, using the maximum-likelihood method. The results indicated that the isolates belonged to the lyssavirus genotype I lineage and China I lineage. The Chinese rabies virus can be divided into six major lineages. The China I lineage was the dominant clade and could be divided into four subclades. Isolates 17HN19, 17HN75, and 18HN162 fell within clade IC subgroup, and the other isolates were assigned to the clade IA subgroup. This study provides epidemiological and genetic information on rabies incidence in humans.

Rabies virus (RABV), an enveloped virus with a single-stranded and negative-sense RNA genome, is the type species of the Lyssavirus Genus within the Rhabdoviridae family. As the causative agent of rabies with a nearly 100% fatality, the neurotropic RABV pose a serious threat to the global public health. Though a great effort has been made toward understanding the molecular mechanism underlying virus infection cycle, there are still many aspects need to be elucidated, especially on the early events during virus replication cycle. With the application of the multiple advanced technologies, much progress has been made on these aspects. To date, multiple receptors, such as nAChR, NCAM, p75NTR, mGluR2, carbohydrates, and gangliosides, have been identified. Following initial attachment, RABV internalization occurs through clathrin-mediated endocytosis (CME) with the help of actin. After viral entry, intracellular trafficking occurs. Two retrograde trafficking models, stating that either whole virions are parceled into vesicles or only the viral capsids are transported, have been proposed. Moreover, complete enveloped virions or G-containing vesicle-associated ribonucleoproteins (RNPs) may be formed during anterograde transport, which remains poorly characterized but is important for viral budding. Combining the data elucidating the molecular mechanisms of RABV attachment, entry, and intracellular trafficking, this review provides an integrated view of the early events in the viral life cycle.

Rabies virus (RABV) constitutes a major social and economic burden associated with 60 000 deaths annually worldwide. Although pre-exposure and post-exposure treatment options are available, they are efficacious only when initiated before the onset of clinical symptoms. Aggravating the problem, the current RABV vaccine does not cross-protect against the emerging zoonotic phylogroup II lyssaviruses. A requirement for an uninterrupted cold chain and high cost of the immunoglobulin component of rabies prophylaxis generate an unmet need for the development of RABV-specific antivirals. We discuss desirable anti-RABV drug profiles, past efforts to address the problem and inhibitor candidates identified, and examine how the rapidly expanding structural insight into RABV protein organization has illuminated novel druggable target candidates and paved the way to structure-aided drug optimization. Special emphasis is given to the viral RNA-dependent RNA polymerase complex as a promising target for direct-acting broad-spectrum RABV inhibitors.

Aim: This study was conducted to know the genetic variability of rabies viruses (RVs) from wild animals in India. Materials and Methods: A total of 20 rabies suspected brain samples of wild animals from different states of India were included in the study. The samples were subjected for direct fluorescent antibody test (dFAT), reverse transcription polymerase chain reaction (RT-PCR), and quantitative reverse transcriptase real-time PCR (RT-qPCR). The phylogenetic analysis of partial nucleoprotein gene sequences was performed. Results: Of 20 samples, 11, 10, and 12 cases were found positive by dFAT, RT-PCR, and RT-qPCR, respectively. Phylogenetic analysis showed that all Indian wild RVs isolates belonged to classical genotype 1 of Lyssavirus and were closely related to Arctic/Arctic-like single cluster indicating the possibility of a spillover of rabies among different species. Conclusion: The results indicated the circulation of similar RVs in sylvatic and urban cycles in India. However, understanding the role of wild animals as reservoir host needs to be studied in India.

International audience; The ecology of Ebola virus (EBV) remains largely unknown, but the previous detec‐tion of viral RNA and anti‐EBV antibodies in African bats suggests that they might play a role in the EBV reservoir. Moreover, African bats also carry other potentially zoonotic agents such as Henipah‐like viruses, coronaviruses and lyssaviruses. Today only little information is available on interactions between humans and bats. The objective of our exploratory study was to describe the extent and modes of contacts between humans and bats in southern Cameroon, considered as an area at risk for future EBV outbreaks. The survey was conducted in 11 villages of four distinct rural areas in southern Cameroon. A total of 135 respondents were interviewed using semi‐structured questionnaires, between February and May 2017. The study showed that direct contacts between bats and humans are relatively common. Bat bushmeat appeared to be an occasional meat resource; 40% of respondents consume bats with a median annual consumption of three, and 28% of respondents hunt them. About 22% of the respondents reported children catching bats. Indirect contact also ap‐peared to be common; 55% of hunters use caves as shelters and 67% of interviewees eat fruits previously chewed by bats. Bat consumption varied significantly between regions (from 0% to 87%) and between pygmies and bantus in the extreme south‐east of Cameroon. The study revealed considerable diversity in practices among in‐terviewees, most of them being subsistence cultivators and relying on self‐hunted bushmeat. Geographical diversity of contacts and perceptions regarding bats in Cameroon emphasizes the need to adjust zoonotic pathogen surveillance and educa‐tion campaigns to the specificities of the communities and their context of interac‐tion with wildlife.

Objectives Domestic dogs are the main reservoir of rabies virus (RABV) infection in Nigeria, thus surveillance of rabies in dog populations is crucial in order to understand the patterns of spread of infection and ultimately devise an appropriate rabies control strategy. This study determined the presence of lyssavirus antigen in brain tissues and anti-rabies antibodies in sera of apparently healthy and suspected-rabid dogs slaughtered for human consumption at local markets in South-Eastern Nigeria. Results Our findings demonstrated that 8.3% (n = 23) of brain tissues were lyssavirus positive and 2.5% (n = 25) of sera had rabies antibody levels as percentage blocking of 70% and above correlating with a cut-off value ≥ 0.5 IU/mL in the fluorescent antibody neutralization test. There was an inverse correlation between lyssavirus positivity and rabies antibody levels confirming that infected individuals most often do not develop virus neutralizing antibodies to the disease. The low percentage of rabies antibodies in this dog population suggests a susceptible population at high risk to RABV infection. These findings highlight a huge challenge to national rabies programs and subsequent elimination of the disease from Nigeria, considering that majority of dogs are confined to rural communal areas, where parenteral dog vaccination is not routinely undertaken. Electronic supplementary material The online version of this article (10.1186/s13104-018-4024-z) contains supplementary material, which is available to authorized users.

The genus Lyssavirus includes rabies virus as well as multiple diverse and recently described novel species. Using next-generation sequencing technologies, we have obtained the whole-genome sequence of Matlo bat lyssavirus, which was isolated from a Natal long-fingered bat (Miniopterus natalensis) in South Africa.

Rabies is a fatal encephalitis caused by an important group of viruses within the Lyssavirus genus. The prototype virus, rabies virus, is still the most commonly reported lyssavirus and causes approximately 59,000 human fatalities annually. The human and animal burden of the other lyssavirus species is undefined. The original reports for the novel lyssavirus, Kotalahti bat lyssavirus (KBLV), were based on the detection of viral RNA alone. In this report we describe the successful generation of a live recombinant virus, cSN-KBLV, where the full-length genome clone of RABV vaccine strain, SAD-B19, was constructed with the glycoprotein of KBLV. Subsequent in vitro characterisation of cSN-KBLV is described here. In addition, the ability of a human rabies vaccine to confer protective immunity in vivo following challenge with this recombinant virus was assessed. Naïve or vaccinated mice were infected intracerebrally with a dose of 100 focus-forming units/30 µL of cSN-KBLV, all naïve mice and 8% (n = 1/12) of the vaccinated mice succumbed to the challenge, whilst 92% (n = 11/12) of the vaccinated mice survived to the end of the experiment. This report provides strong evidence for cross-neutralisation and cross-protection of cSN-KBLV using purified Vero cell rabies vaccine.

Human fatalities caused by rabies are rarely reported in Jordan; however, domestic animals are more likely to fall victim to rabies compared to wild animals, at least this is the case in Jordan due to the presence of canine rabies. In this study, twelve brain samples from domestic and wild animals suspected of being infected with rabies virus from different regions of Jordan were collected during 2019. Seven of them tested positive using the fluorescent antibody test and real-time SYBR RT-PCR assay. Five specimens were from stray dogs and two from foxes. The whole genome sequences were obtained from the positive samples. Sequence analysis showed that one dog virus from Al Quwaysimah city located in Amman governorate, was closely related to an Israeli strain belonging to a Cosmopolitan ME1a clade. The genomes of the remaining six viruses (four from dogs and two from foxes) collected from different areas of Jordan were genetically-related to each other and clustered together with sequences from Iran and Turkey; all belong to Cosmopolitan ME2 clade. These sequences were analyzed with six other Jordanian rabies virus nucleoprotein (N) gene sequences available in the public database, five of them belong to ME1a clade and one belongs to ME1b clade. Rabies virus whole genome data is scarce across the Middle East. This study provides a better understanding of the molecular epidemiology of rabies virus in the region., Author summary In this study, we performed whole genome sequencing (WGS) for rabies virus (RABV) isolates from seven samples, five of which were of stray dogs, and the other two were from foxes. Specimens were collected from animals across Jordan, including Balqa, Amman, Irbid, Tafilah, and Madaba governorates. Six out of the seven isolates were belonging to the Cosmopolitan ME2 clade, which related to the Iranian and Turkish sequences. This is not the case previously, where the majority of the Jordanian isolates belong to Cosmopolitan ME1a clade and closely related to the sequences from Israel. This shift might be due to the applied regulations across borders between Jordan and Israel. Besides the growth in travel and trade movement between Jordan and Turkey, where the latter is a border country with Iran. These collected data, where such studies are not common in the Middle East countries, will enhance our understanding of the RABV evolution and epidemiology in the region for rapid and effective response for rabies virus outbreaks.

Post-exposure prophylaxis (PEP) is highly effective in preventing disease progression of rabies when used in timely and appropriate manner. The key treatment for PEP is infiltration of rabies immune globulin (RIG) into lesion site after bite exposure, besides wound care and vaccination. Unfortunately, however, RIG is expensive and its supply is limited. Currently, several anti-rabies virus monoclonal antibody (mAb) products are under development as alternatives to RIG, and two recently received regulatory approval in India. In this study, fully human mAbs that recognize different rabies virus glycoprotein conformational antigenic site (II and III) were created from peripheral blood mononuclear cells of heathy vaccinated subjects. These mAbs neutralized a diverse range of lyssavirus types. As at least two anti-rabies virus mAbs are recommended for use in human PEP to ensure broad coverage against diverse lyssaviruses and to minimize possible escape variants, two most potent mAbs, NP-19-9 and 11B6, were selected to be used as cocktail treatment. These two mAbs were broadly reactive to different types of lyssaviruses isolates, and were shown to have no interference with each other. These results suggest that NP-19-9 and 11B6 are potent candidates to be used for PEP, suggesting further studies involving clinical studies in human.

Big brown bats (Eptesicus fuscus) are the bat species in North America most frequently found to be rabid because of their high rate of human contact and thus submissions for rabies testing, of which, 4-5% are positive. The social behavior of big brown bats during the summer months may drive space use and potential viral exposure to conspecifics and mesocarnivores. We collected 88 unique genetic samples via buccal swabs from big brown bats captured at four maternity roosts surrounding a golf course during the summer of 2013. We used seven microsatellite loci to estimate genetic relatedness among individuals and genetic structure within and among colonies to infer whether females selected roosts based on kinship and used genetics and radio telemetry to determine the frequency of roost switching. We found roost switching through genetics and telemetry, and no evidence of elevated genetic relatedness within colonies or genetic structure among colonies. Social cohesion based on relatedness may not act to constrain the pathogen to a particular roost area, and thus, geographic mobility may increase viral exposure of bats in neighboring areas.

Bats are reservoirs of many pathogenic viruses, including the lyssaviruses rabies virus (RABV) and Australian bat lyssavirus (ABLV). Lyssavirus strains are closely associated with particular host reservoir species, with evidence of specific adaptation. Associated phenotypic changes remain poorly understood but are likely to involve phosphoprotein (P protein), a key mediator of the intracellular virus–host interface. Here, we examine the phenotype of P protein of ABLV, which circulates as two defined lineages associated with frugivorous and insectivorous bats, providing the opportunity to compare proteins of viruses adapted to divergent bat species. We report that key functions of P protein in the antagonism of interferon/signal transducers and activators of transcription 1 (STAT1) signaling and the capacity of P protein to undergo nuclear trafficking differ between lineages. Molecular mapping indicates that these differences are functionally distinct and appear to involve modulatory effects on regulatory regions or structural impact rather than changes to defined interaction sequences. This results in partial but significant phenotypic divergence, consistent with “fine-tuning” to host biology, and with potentially distinct properties in the virus–host interface between bat families that represent key zoonotic reservoirs.

During 2017 – 2019, the Pennsylvania Department of Health Bureau of Laboratories (PABOL) tested 6,855 animal samples for rabies using both the gold standard direct fluorescent antibody (DFA) test and LN34 pan-lyssavirus reverse transcriptase quantitative PCR (RT-qPCR). Two samples (0.03 %) were identified as LN34 RT-qPCR positive after failure to detect rabies virus antigen during initial DFA testing: an adult raccoon collected in 2017 and a juvenile raccoon collected in 2019. After the positive PCR result, additional tissues were collected and re-tested by DFA, where very sparse, disperse antigen was observed. Tissues from both animals were submitted to the Centers for Disease Control and Prevention (CDC) for confirmatory testing, and were confirmed positive. At both PABOL and CDC, rabies virus antigen and RNA levels were much lower than for a typical rabies case. In addition, rabies virus antigen and RNA levels were higher in brain stem and rostral spinal cord than cerebellum, hippocampus and cortex. Cross-contamination was ruled out in the case of the 2019 juvenile raccoon by sequencing, as nucleoprotein and glycoprotein gene sequences displayed >1% nucleotide differences to sequences from all positive samples processed at PABOL within two weeks of the juvenile raccoon. Taken together, the low level of rabies virus in the central nervous system combined with presence in more caudal brain structures suggest the possibility of an early infection in both cases. These two cases highlight the increased sensitivity and ease of interpretation of LN34 RT-qPCR in rabies diagnostics for the identification of low positive cases.

Australian bat lyssavirus (ABLV) is a rhabdovirus that circulates in four species of pteropid bats (ABLVp) and the yellow-bellied sheath-tailed bat (ABLVs) in mainland Australia. In the three confirmed human cases of ABLV, rabies illness preceded fatality. As with rabies virus (RABV), post-exposure prophylaxis (PEP) for potential ABLV infections consists of wound cleansing, ad-ministration of the rabies vaccine and injection of rabies immunoglobulin (RIG) proximal to the wound. Despite the efficacy of PEP, the inaccessibility of human RIG (HRIG) in the developing world and the high immunogenicity of equine RIG (ERIG) has led to consideration of human monoclonal antibodies (hmAbs) as a passive immunization option that offers enhanced safety and specificity. Using a recombinant vesicular stomatitis virus (rVSV) expressing the glycoprotein (G) protein of ABLVs and phage display, we identified two hmAbs, A6 and F11, which completely neutralize ABLVs/ABLVp, and RABV at concentrations ranging from 0.19-3.12 µg/mL and 0.39-6.25 µg/mL respectively. A6 and F11 recognize overlapping epitopes in the lyssavirus G protein, ef-fectively neutralizing phylogroup 1 lyssaviruses, while having little effect on phylogroup 2 and non-grouped diverse lyssaviruses. These results suggest A6 and F11 could be effective therapeutic and diagnostic tools for phylogroup 1 lyssavirus infections.

If the goal of eliminating dog-mediated human rabies by 2030 is to be achieved, effective mass dog vaccination needs to be complemented by effective prophylaxis for individuals exposed to rabies. Aptamers and short-interfering RNAs (siRNAs) have been successful in therapeutics, but few studies have investigated their potential as rabies therapeutics. In this study, siRNAs and aptamers—using a novel selection method—were developed and tested against rabies virus (RABV) in a post-infection (p.i.) scenario. Multiple means of delivery were tested for siRNAs, including the use of Lipofectamine and conjugation with the developed aptamers. One siRNA (N53) resulted in an 80.13% reduction in viral RNA, while aptamer UPRET 2.03 demonstrated a 61.3% reduction when used alone at 2 h p.i. At 24 h p.i., chimera UPRET 2.03-N8 (aptamer-siRNA) resulted in a 36.5% inhibition of viral replication. To our knowledge, this is the first study using siRNAs or aptamers that (1) demonstrated significant inhibition of RABV using an aptamer, (2) tested Lipofectamine RNAi-Max as a means for delivery, and (3) produced significant RABV inhibition at 24 h p.i. This study serves as a proof-of-concept to potentially use aptamers and siRNAs as rabies immunoglobulin (RIG) replacements or therapeutic options for RABV and provides strong evidence towards their further investigation.

Rabies excitant is a neurophilic virus (Rabiesvirus), regarding to Mononegavirales order, Rhabdoviridae family, Lyssavirus genus. The situation with rabies morbidity in Russia is characterized as extremely unpleasant. Saliva, tear liquid, patient’s cornea prints were used in the work as well as, after their death, various segments of brain and salivary glands were used. Additionally, antibody concentration in blood serum of humans passing antirabies therapy after animal bites were investigated. Research Research of eye cornea prints from patient A, suffered from wolf bite, with antibody fluorescent method showed positive result 6 days before the death. The obtained results were confirmed postmortem while the investigation of brain various segments and salivary glands with the use of antibody fluorescent spectroscopy and enzyme-linked analysis method, ELISA, and in biosamples. Intravital research of various biosamples picked from the patient B, suffered from homeless dog bite, showed the precedence of Virus 9 days before the death. Rabies virus antigen was discovered on the basis of research results of eye cornea prints with confocal spectroscopy, saliva samples - with ELISA method, as well as rabies virus genome in saliva and tear liquid was demonstrated with Nested Enzyme-Linked Analysis with Reverse Transcription (Nested RT-PCR). Intravital diagnostics results of hydrophobia patient were confirmed afterwards postmortem while the investigation of brain various segments with confocal fluorescence spectroscopy, ELISA, method and biopsy. Hydrophobia of injured was going on in clinically well-expressed manner. The results of intravital and postmortem laboratory diagnostics of hydrophobia clinical cases in Russian Federation were considered. These cases were the result of wolf and homeless dog bites. Both hydrophobia cases, caused by wild predator and dog bite, were laboratory confirmed yet during patient’s life. Introduction of intravital laboratory rabies diagnostics into medical practice is an important element of differential diagnostics.

Bats are a host and reservoir for a large number of viruses, many of which are zoonotic. In North America, the big brown bat (Eptesicus fuscus) is widely distributed and common. Big brown bats are a known reservoir for rabies virus, which, combined with their propensity to roost in human structures, necessitates testing for rabies virus following human exposure. The current pandemic caused by severe acute respiratory syndrome coronavirus 2, likely of bat origin, illustrates the need for continued surveillance of wildlife and bats for potentially emerging zoonotic viruses. Viral metagenomic sequencing was performed on 39 big brown bats and one hoary bat submitted for rabies testing due to human exposure in South Dakota. A new genotype of American bat vesiculovirus was identified in seven of 17 (41%) heart and lung homogenates at high levels in addition to two of 23 viscera pools. A second rhabdovirus, Sodak rhabdovirus 1 (SDRV1), was identified in four of 23 (17%) viscera pools. Phylogenetic analysis placed SDRV1 in the genus Alphanemrhavirus, which includes two recognized species that were identified in nematodes. Finally, a highly divergent rhabdovirus, Sodak rhabdovirus 2 (SDRV2), was identified in two of 23 (8.7%) big brown bats. Phylogenetic analysis placed SDRV2 as ancestral to the dimarhabdovirus supergroup and Lyssavirus. Intracranial inoculation of mouse pups with rhabdovirus-positive tissue homogenates failed to elicit clinical disease. Further research is needed to determine the zoonotic potential of these non-rabies rhabdoviruses.

Many infectious pathogens can be transmitted by highly mobile species, like bats that can act as reservoir hosts for viruses such as henipaviruses, lyssaviruses and coronaviruses. In this study, we investigated the seroepidemiology of protein antigens to Severe acute respiratory syndrome virus (SARS-CoV-1) and Middle eastern respiratory syndrome virus (MERS-CoV) in Grey-headed flying foxes (Pteropus poliocephalus) in Adelaide, Australia sampled between September 2015 and February 2018. A total of 301 serum samples were collected and evaluated using a multiplex Luminex binding assay, and median fluorescence intensity thresholds were determined using finite-mixture modelling. We found evidence of antibodies reactive to SARS-CoV-1 or a related antigen with 42.5% (CI: 34.3%-51.2%) seroprevalence but insufficient evidence of reactivity to MERS-CoV antigen. This study provides evidence that the Grey-headed flying foxes sampled in Adelaide have been exposed to a SARS-like coronavirus.

As countries with endemic canine rabies progress towards elimination by 2030, it will become necessary to employ techniques to help plan, monitor, and confirm canine rabies elimination. Sequencing can provide critical information to inform control and vaccination strategies by identifying genetically distinct virus variants that may have different host reservoir species or geographic distributions. However, many rabies testing laboratories lack the resources or expertise for sequencing, especially in remote or rural areas where human rabies deaths are highest. We developed a low-cost, high throughput rabies virus sequencing method using the Oxford Nanopore MinION portable sequencer. A total of 259 sequences were generated from diverse rabies virus isolates in public health laboratories lacking rabies virus sequencing capacity in Guatemala, India, Kenya, and Vietnam. Phylogenetic analysis provided valuable insight into rabies virus diversity and distribution in these countries and identified a new rabies virus lineage in Kenya, the first published canine rabies virus sequence from Guatemala, evidence of rabies spread across an international border in Vietnam, and importation of a rabid dog into a state working to become rabies-free in India. Taken together, our evaluation highlights the MinION&rsquo, s potential for low-cost, high volume sequencing of pathogens in locations with limited resources.

Implementation of lateral flow devices (LFDs) for rabies antigen detection is expected to improve surveillance through the efficient detection of rabid animals in resource-limited settings; however, the use of LFDs for diagnosis remains controversial because some commercially available kits show low sensitivity. Therefore, we compared the diagnostic efficacy of three LFDs (ADTEC, Bionote, and Elabscience kits) paralleled with the direct fluorescent antibody test (dFAT) using fresh samples and investigated the diagnostic accuracies. To do so, we evaluated rabies-suspected samples submitted to the Regional Animal Disease Diagnostic Laboratory III, Philippines. Furthermore, we conducted real-time RT-PCR and sequencing to measure the accuracy of field laboratory diagnosis. The total number of animals submitted during this study period was 184 cases, including negative control samples. Of these, 53.9% (84 cases) were positive in the dFAT. Dogs were the most common rabies-suspected animal (n = 135). The sensitivities of the ADTEC and Bionote kits were 0.88 (74 cases) and 0.95 (80 cases), respectively. The specificity of both kits was 1.00 (100 cases). Furthermore, the sensitivity and specificity of the ADTEC kit after directly homogenizing the samples in assay buffer without dilution in phosphate-buffered saline (ADTEC kit DM) were 0.94 (79 cases) and 1.00 (100 cases), respectively. By contrast, there were no positive results using the Elabscience kit among all dFAT-positive samples. The sensitivity and specificity of LFDs make these tests highly feasible if properly used. Therefore, LFD tests can be used to strengthen the surveillance of rabies-infected animals in endemic and resource-limited settings., Author summary Implementation of lateral flow devices (LFDs) for rabies antigen detection is expected to improve surveillance through the efficient detection of rabid animals in resource-limited settings because they are not only accurate but rapid, user-friendly, and low-cost tools. Although LFDs may positively contribute to accurate data reporting, the use of LFDs for definitive diagnosis remains controversial because some LFDs showed inadequate ability. In this study, we evaluated the diagnostic accuracy of three LFDs (ADTEC, Bionote, and Elabscience kits) paralleled with the direct fluorescent antibody test (dFAT) and real-time RT-PCR using fresh samples and investigated discrepancies among these tests. Of these, half of the submitted samples were positive in the dFAT. The sensitivity and specificity of the simplified ADTEC method (ADTEC kit DM) and Bionote kits were comparable with those of dFAT, but no positive reactions were observed using the Elabscience kit. Despite controversial discussions regarding the use of LFDs, we demonstrated that the diagnostic accuracy of ADTEC and Bionote kits was satisfactory under regional diagnostic laboratory conditions. Our results support the potential of LFD tests for in-field diagnosis of rabies in endemic countries and the feasibility for practical use when they are properly used.

Rabies is enzootic in over one hundred countries worldwide. In the European Union/European Economic Area (EU/EEA), the vast majority of human rabies cases are travellers bitten by dogs in rabies-enzootic countries, mostly in Asia and Africa. Thus, EU/EEA travellers visiting rabies enzootic countries should be aware of the risk of being infected with the rabies virus when having physical contact with mammals. They should consider pre-exposure vaccination following criteria recommended by the World Health Organization and if unvaccinated, immediately seek medical attention in case of bites or scratches from mammals. As the majority of the EU/EEA countries are free from rabies in mammals, elimination of the disease (no enzootic circulation of the virus and low number of imported cases) has been achieved by 2020. However, illegal import of potentially infected animals, mainly dogs, poses a risk to public health and might threaten the elimination goal. Additionally, newly recognised bat lyssaviruses represent a potential emerging threat as the rabies vaccine may not confer protective immunity. To support preparedness activities in EU/EEA countries, guidance for the assessment and the management of the public health risk related to rabies but also other lyssaviruses, should be developed.

Rabies is a neglected disease caused by a neurotropic Lyssavirus, transmitted to humans predominantly by the bite of infected dogs. Rabies is preventable with vaccines or proper post‐exposure prophylaxis (PEP), but it still causes about 60,000 deaths every year. No cure exists after the onset of clinical signs, and the case‐fatality rate approaches 100% even with advanced supportive care. Here, we report that a combination of two potent neutralizing human monoclonal antibodies directed against the viral envelope glycoprotein cures symptomatic rabid mice. Treatment efficacy requires the concomitant administration of antibodies in the periphery and in the central nervous system through intracerebroventricular infusion. After such treatment, recovered mice presented good clinical condition, viral loads were undetectable, and the brain inflammatory profile was almost normal. Our findings provide the unprecedented proof of concept of an antibody‐based therapeutic approach for symptomatic rabies., Rabies is an invariably fatal disease after the rabies virus has invaded the central nervous system and clinical signs have been manifested. This study brings the proof‐of‐concept that a cocktail of human monoclonal antibodies (mAbs) can cure symptomatic rabies.

Several Lyssaviruses are known to be a causative agent of rabies and rabies like syndrome. There are no proven effective treatment strategies for symptomatic rabies patient. Risk of infection from dog variant of rabies virus is highest with deep bite reaching muscular layer and much higher when compared to scratch. Failure of viral eradication at the central nervous system (CNS) is partly due to inadequate immune response. Favipiravir selectively inhibit viral RNA polymerase and has been shown to reduce rabies replication in neuronal cell and mouse model system. Endocannabinoid system has emerged as an important regulator for CNS integrity, cell fate and may serve as an important novel neuroprotective agent. Cannabinoid may be able to regulate the impaired homeostasis induced by rabies virus by promoting infected cell survival and promote complete autophagy in infected cell.

Rhabdoviruses, as single-stranded, negative-sense RNA viruses within the order Mononegavirales, are characterised by bullet-shaped or bacteroid particles that contain a helical ribonucleoprotein complex (RNP). Here, we review the components of the RNP and its higher-order structural assembly.

Rabies virus infections normally cause universally lethal encephalitis across mammals. However, ‘abortive infections’ which are resolved prior to the onset of lethal disease have been described in bats and a variety of non-reservoir species. Here, we surveyed rabies virus neutralizing antibody titers in 332 unvaccinated livestock of 5 species from a vampire bat rabies endemic region of southern Peru where livestock are the main food source for bats. We detected rabies virus neutralizing antibody titers in 11, 5 and 3.6% of cows, goats and sheep respectively and seropositive animals did not die from rabies within two years after sampling. Seroprevalence was correlated with the number of local livestock rabies mortalities reported one year prior but also one year after sample collection. This suggests that serological status of livestock can indicate the past and future levels of rabies risk to non-reservoir hosts. To our knowledge, this is the first report of anti-rabies antibodies among goats and sheep, suggesting widespread abortive infections among livestock in vampire bat rabies endemic areas. Future research should resolve the within-host biology underlying clearance of rabies infections. Cost-effectiveness analyses are also needed to evaluate whether serological monitoring of livestock can be a viable complement to current monitoring of vampire bat rabies risk based on animal mortalities alone., Author summary Rabies virus is the deadliest virus affecting mammals. In Latin America, rabies transmitted by vampire bats is one of the most recognized zoonoses affecting humans and livestock. For unknown reasons, species such as bats and cattle can produce antibodies against rabies that clear viral infection prior to the development of deadly clinical signs. However, the extend of this phenomenon in naturally exposed populations remains unknows. In this study, we detected anti-rabies antibodies among cattle and, for the first time, goats and sheep bitten by vampire bats across as large geographic area endemic of vampire bat rabies in South Peru. We also showed that the presence of antibodies in livestock can indicate the past and future levels of cattle mortality due to rabies in the area. Future research should resolve the within-host biology underlying clearance of rabies infections and evaluate if antibody detection of healthy animals can be used for monitoring vampire bat rabies risk.

Currently, Slovakia is a rabies-free country, but the epizootiological situation of rabies was not always favorable. The main reservoir species of rabies virus in the first half of the last century was the domestic dog. Since 1906, hundreds of cases were reported, of which approximately 90% were infected dogs. The disease had a typical urban character. Since 1929, the number of rabid domestic animals decreased due to the implementation of dog vaccination campaigns in particular parts of Slovakia. From the second half of 1950s, red foxes (Vulpes vulpes) have become an important reservoir of the RABV. In this time period urban rabies in Slovakia changed into sylvatic form. One effective method of prevention and control of wildlife rabies is an oral rabies vaccination of red foxes. It is carried out in Slovakia since 1993. A detailed development of the rabies epizootiological situation on the territory of the Slovak Republic until the application of oral antirabies immunisation of foxes and the current situation after its performance is the main object of this review. Keywords: rabies; Lyssavirus; red fox; incidence; oral vaccination.

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. IMPORTANCE 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.

Rabies is a generally fatal encephalitis caused by a negative-sense single-stranded RNA lyssavirus transmitted to humans mainly from dog bite. Despite the recommendation by WHO and OIE to use the direct immunofluorescence test as standard method, molecular diagnostic assays like reverse transcription quantitative polymerase chain reaction (RT-qPCR) are increasing as a confirmatory method. However, both technologies are inaccessible in resource-limited settings. Moreover, the available point-of-need molecular assay is of poor detection limit for African strains. Herein, we developed a reverse transcription recombinase polymerase amplification (RT-RPA) assay as potential point-of-need diagnostic tool for rapid detection of various strains of rabies virus including locally isolated African strains. The sensitivity and specificity of the method was evaluated using a molecular RNA standard and different Rabies-related viruses belonging to the Rhabdoviridea family, respectively. The RABV-RPA performances were evaluated on isolates representative of the existing diversity and viral dilutions spiked in non-neural clinical specimen. The results were compared with RT-qPCR as a gold standard. The RABV-RPA detected down to 4 RNA molecules per reaction in 95% of the cases in less than 10 min. The RABV-RPA assay is highly specific as various RABV isolates were identified, but no amplification was observed for other member of the Rhabdoviridea family. The sample background did not affect the performance of the RABV-RPA as down to 11 RNA molecules were identified, which is similar to the RT-qPCR results. Our developed assay is suitable for use in low-resource settings as a promising alternative tool for ante-mortem rabies diagnosis in humans for facilitating timely control decisions.

SummarySerology is a core component of the surveillance and management of viral zoonoses. Virus neutralization tests are a gold standard serological diagnostic, but requirements for large volumes of serum and high biosafety containment can limit widespread use. Here, focusing onRabies lyssavirus,a globally important zoonosis, we developed a pseudotype micro-neutralization rapid fluorescent focus inhibition test (pmRFFIT) that overcomes these limitations. Specifically, we adapted an existing micro-neutralization test to use a green fluorescent protein–tagged murine leukemia virus pseudotype in lieu of pathogenic rabies virus, reducing the need for specialized reagents for antigen detection and enabling use in low-containment laboratories. We further used statistical analysis to generate rapid, quantitative predictions of the probability and titer of rabies virus neutralizing antibodies from microscopic imaging of neutralization outcomes. Using 47 serum samples from domestic dogs with neutralizing antibody titers estimated using the fluorescent antibody virus neutralization test (FAVN), pmRFFIT showed moderate sensitivity (78.79%) and high specificity (84.62%). Despite small conflicts, titer predictions were correlated across tests repeated on different dates both for dog samples (r = 0.93), and for a second dataset of sera from wild common vampire bats (r = 0.72, N = 41), indicating repeatability. Our test uses a starting volume of 3.5 μL of serum, estimates titers from a single dilution of serum rather than requiring multiple dilutions and end point titration, and may be adapted to target neutralizing antibodies against alternative lyssavirus species. The pmRFFIT enables high-throughput detection of rabies virus neutralizing antibodies in low-biocontainment settings and is suited to studies in wild or captive animals where large serum volumes cannot be obtained.

Despite proactive measures to prevent raccoon rabies entering Canada from the United States, several incursions of this disease have occurred. The largest outbreak, first reported in December 2015 in the city of Hamilton, Ontario, has resulted in the reporting of 449 animal cases as of December 31, 2018. Initial phylogenetic studies on the index case suggested that this outbreak was not due to local cross-border spread from the Niagara region of the United States where raccoon rabies has persisted for several years. Phylogenetic analysis of whole genome sequences of a viral collection from the Hamilton area and several US states indicates that a long-distance translocation of a diseased animal from southeastern New York State was responsible for this incursion. The role of the skunk as a potential secondary host supporting persistence and / or spread of the virus is also examined., Author summary Distinct variants of rabies virus are harbored by several wildlife species across North America. The variant associated with the raccoon host is especially problematic given its rapid spread throughout the eastern seaboard of the United States over the second half of the 20th century and the high population density of this species in urban habitats that raises the potential for disease spread to both companion animals and their owners. Accordingly, extensive efforts to control raccoon rabies have been undertaken at many jurisdictional levels. This study uses current methods of genome analysis to explore the origins of a raccoon rabies outbreak in and around the city of Hamilton, Ontario, Canada, located close to the border with western New York state. In contrast to previous Canadian outbreaks of raccoon rabies, which were due to cross-border spread of the virus, this study demonstrates that the incursion into the Hamilton area was the result of a long-distance animal translocation from south-eastern New York. The implications of this event for strategies to control raccoon rabies are discussed.

Background Rabies is a deadly preventable viral disease that affects all warm-blooded animals and widespread in many regions including Africa. The disease remains of major public health importance in Uganda. The purpose of this study was to establish Knowledge, Attitude, Practice (KAP) of Rabies in Moyo and Ntoroko districts and to characterize Rabies virus (RABV) strains from seven districts of Uganda with consistent prevalence of rabies. Methods KAP survey data were collected based on animal biting history by interviewing the head of the veterinary departments, the medical centers and selected households from the study sites. Data were obtained from 84 households in Ntoroko and Moyo districts. Thirty-five (35) brain samples were collected from bovine, dogs, goats, foxes, jackals ad sheep between 2011 and 2013. Samples were tested using fluorescent antibody test (FAT), One step RT-PCR (following RNA extraction) and partial RABV N gene was sequenced by Sanger method before phylogenetic and phylogeographic analyses of sequences. Results Scarcity of post-exposure prophylaxis services in the health centers was noted. Poor attitude of wound washing and deficiency of knowledge on how to handle wounds related to dog bites and the significance among household participants lacked. There is a high risk of rabies infection due to a limited dog’s vaccination. Dog biting episodes in humans were of 75.00 and 62.50% in Moyo and Ntoroko districts respectively. Twenty-seven (27) samples tested positive for rabies by FAT and PCR. Ugandan sequences were closely related (97% nucleotide id) to the rabies virus sequences from Tanzania, Rwanda, Burundi, Nigeria, Central African Republic and Sudan with both the “Africa 1A” and “Africa 1B” RABV clades represented. A putative new clade 1D was also detected. Conclusions Rabies remains a public health hazard in Uganda. There is urgent need to establish advocacy programs in both schools and communities to curtail the spread of rabies. Increasing the knowledge regarding wound washing, post-exposure prophylaxis and dogs vaccination would enhance prevention of rabies. A strong collaboration between medical and veterinary sectors under a one health platform is required to ensure sufficient preventative services to the communities.

Rabies is a lethal viral disease and dogs are the major disease reservoir in the Philippines. Spatio-temporal variations in environmental factors are known to affect disease dynamics. Some rabies-affected countries considered investigating the role of weather components in driving rabies cases and it has helped them to strategize their control efforts. In this study, cointegration analysis was conducted between the monthly reported rabies cases and the weather components, such as temperature and precipitation, to verify the effect of weather components on rabies incidence in Davao City, Philippines. With the Engle-Granger cointegration tests, we found that rabies cases are cointegrated into each of the weather components. It was further validated, using the Granger causality test, that each weather component predicts the rabies cases and not vice versa. Moreover, we performed the Johansen cointegration test to show that the weather components simultaneously affect the number of rabies cases, which allowed us to estimate a vector-error correction model for rabies incidence as a function of temperature and precipitation. Our analyses showed that canine rabies in Davao City was weather-sensitive, which implies that rabies incidence could be projected using established long-run relationship among reported rabies cases, temperature, and precipitation. This study also provides empirical evidence that can guide local health officials in formulating preventive strategies for rabies control and eradication based on weather patterns.

A canine rabies epidemic started in early 2015 in Arequipa, Peru and the rabies virus continues to circulate in the dog population. Some city residents who suffer dog bites do not seek care or do not complete indicated post-exposure prophylaxis (PEP) regimens, increasing the risk of human rabies. The objectives of our study are to qualitatively assess knowledge about rabies, and preventive practices, such as rabies vaccine administration, following a dog bite. We conduct eight focus group discussions in peri-urban and urban communities with 70 total participants. In our results, we observe low awareness of rabies severity and fatality, and different practices following a dog bite, depending on the community type: for example, whereas participants in the urban communities report cleaning the wound with hydrogen peroxide rather than soap and water, participants in peri-urban areas cover the wound with herbs and hair from the dog that bit them. Misconceptions about rabies vaccines and mistreatment at health centers also commonly prevent initiating or completing PEP. We identify important behavioral and structural barriers and knowledge gaps that limit evidence-based preventive strategies against rabies and may threaten successful prevention of dog-mediated human rabies in this setting., Author summary The city of Arequipa, Peru has been experiencing an outbreak of dog rabies since 2015. The Peruvian Ministry of Health has implemented measures to prevent human infection, including no-cost rabies vaccinations for people bitten by dogs, but health posts report that many people do not utilize the service or complete treatment. We conduct focus groups in urban and peri-urban areas of Arequipa to examine community perceptions and attitudes towards PEP, and organizational and behavioral barriers to initiate and complete the PEP regimen. We observe low awareness of rabies severity, and different practices following a dog bite depending on the community type. Misconceptions about rabies vaccines and mistreatment at health centers also commonly prevent participants from initiating or completing PEP. We identify important behavioral and structural barriers and knowledge gaps that limit preventive strategies against rabies and may threaten successful prevention of dog-mediated human rabies in this setting. Finally, disparate landscapes and topography seem to have different effects on urban and peri-urban participants’ use of healthcare resources. Thus, strategies should target the specific needs of each population.