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

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

Identification and Characterization of a Small-Molecule Rabies Virus Entry Inhibitor.

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.

The influence of poverty and rabies knowledge on healthcare seeking behaviors and dog ownership, Cameroon.

BACKGROUND: Rabies is a fatal encephalitis caused by lyssaviruses, with most human cases worldwide resulting from rabid dog bites. Although effective animal and human vaccines have been available for over 100 years, control efforts have not been adequately implemented on the global scale and rabies remains one of the greatest global zoonotic threats to human health. We conducted a knowledge, attitudes and practices survey in Northern Cameroon to describe dog ownership characteristics, rates of dog bites, and post-bite healthcare seeking behaviors. METHODS: The survey was performed in four rural Cameroonian communities. A structured community-based questionnaire was conducted over a 20-day period in April 2010, and focused on socio-economic factors correlated with gaps in rabies knowledge. Information pertaining to socio-demographics, as well as attitudes and practices with regard to animal bites and bite treatment practices were recorded. Characteristics of dog ownership such as dog confinement, resources provided to dogs, and dog vaccination status were examined. Human to dog ratios were compared on a linear scale to poverty scores by community. When applicable, 2-tailed Chi-square tests or Fisher's exact tests were calculated to determine relationships between variables. We also used One-way Analysis of Variance (ANOVA) to identify associations between rabies knowledge and wealth with dog ownership, dog vaccination, and human healthcare seeking behaviors. Independent variables were evaluated using multivariate logistic regression analysis. RESULTS: A total of 208 households were enrolled. Respondents were predominantly male (68.3%), with a median age of 43.6 years. Eighty-four households (39.9%) reported owning a total of 141 dogs (human dog ratio 10.4:1). The majority of dogs (61%) were allowed to roam freely. A history of rabies vaccination was reported for 30.8% of owned dogs. Respondents reported 11 bites during the two years preceding the survey (annual bite incidence was 2.6% [95% CI 1.4%- 4.6%]). Only one person (9.1%) received rabies post-exposure prophylaxis (PEP), and none described symptoms of clinical illness consistent with rabies. Respondents who indicated that they would seek medical care and PEP after a dog bite had higher average wealth and rabies knowledge index scores (p = 0.01 and 0.04, respectively). Respondents who indicated that they would seek care from a traditional healer had significantly lower wealth scores, but not significantly different knowledge scores (p < 0.01 and p = 0.49, respectively). CONCLUSIONS: In the communities evaluated, the majority of dogs were allowed to roam freely and had no history of rabies vaccination; factors that favor enzootic transmission of canine rabies virus. We also identified a strong relationship between poverty and dog ownership. Bite events were relatively common among respondents, and very few victims reported utilizing health services to treat wounds. Increased wealth and knowledge were significantly associated with increased likelihood that a respondent would seek medical care and post-exposure prophylaxis. These findings indicate the need for educational outreach to raise awareness of dog rabies and proper prevention measures.

Contrasting landscape epidemiology of two sympatric rabies virus strains.

Viral strain evolution and disease emergence are influenced by anthropogenic change to the environment. We investigated viral characteristics, host ecology, and landscape features in the rabies-striped skunk disease system of the central Great Plains to determine how these factors interact to influence disease emergence. We amplified portions of the N and G genes of rabies viral RNA from 269 samples extracted from striped skunk brains throughout the distribution of two different rabies strains for which striped skunks were the reservoir. Because the distribution of these two strains overlapped on the landscape and were present in the same host population, we could evaluate how viral properties influenced epidemiological patterns in the area of sympatry. We found that South Central Skunk rabies (SCSK) exhibited intense purifying selection and high infectivity, which are both characteristics of an epizootic virus. Conversely, North Central Skunk rabies (NCSK) exhibited relaxed purifying selection and comparatively lower infectivity, suggesting the presence of an enzootic virus. The host population in the area of sympatry was highly admixed, and skunks among allopatric and sympatric areas had similar effective population sizes. Spatial analysis indicated that landscape features had minimal influence on NCSK movement across the landscape, but those same features were partial barriers to the spread of SCSK. We conclude that NCSK and SCSK have different epidemiological properties that interact differently with both host and landscape features to influence rabies spread in the central Great Plains. We suggest a holistic approach for future studies of emerging infectious diseases that includes studies of viral properties, host characteristics, and spatial features.

Small red bean (azuki) sheds biologically active substances as a prerequisite step for germination, one of which displays the antiviral activity against the rabies virus infectivity and infections in culture.

When small red beans (azuki bean; Vigna angularis Ohwi et Ohashi) were soaked and warmed in water or saline, the beans began to absorb water to swell and exuded kinds of substances probably as a prerequisite step for seed germination. Such exudate fluids displayed strong antiviral activity against the rabies virus infections in culture. On the other hand, little anti-rabies activity was detected in the aqueous extracts from the red beans when tested soon after the extraction from powdered beans, while low titers of antiviral activity appeared gradually in the extracts during cold storage. In contrast, no antiviral activity was detected in the exudate fluids from non-colored azuki beans (white azuki), implicating that a certain anthocyanin-related substance is involved in the antiviral activity of red beans. Production of antiviral and cytotoxic activities were affected differently depending on the bean-soaking conditions. In addition, the antiviral activity resisted to 10 min-heating in boiling water, while the cytotoxicity was greatly weakened by the heating, suggesting that different substances are involved in the antiviral and cytotoxic activities. Further studies on the antiviral activity of the exudate fluids demonstrated that anti-rabies activity of the bean exudates affected not only the very early phase of infection cycle, but the viral infectivity was also affected similarly, implicating a possible application of azuki bean exudate fluids to post-exposure treatment of rabid dog-bite injuries in combination with vaccination.

Experimental rabies virus infection in Artibeus jamaicensis bats with CVS-24 variants.

An experimental model of rabies was established in the fruit-eating bat species Artibeus jamaicensis. The infections caused by CVS-N2c and CVS-B2c, which are both stable variants of CVS-24, were compared after inoculation of adult bats in the right masseter muscle. CVS-N2c produced neurologic signs of rabies with paresis, ataxia, and inability to fly, while CVS-B2c did not produce neurologic signs. Bats were sacrificed and the distribution of rabies virus antigen was assessed in tissue sections with immunoperoxidase staining. Both viruses spread to the brain stem and bilaterally to the trigeminal ganglia by days 2 to 3. CVS-N2c had disseminated widely in the central nervous system (CNS) by day 4 and had involved the spinal cord, thalamus, cerebellum, and cerebral cortex. CVS-B2c had infected neurons in the spinal cord on day 5 and in the cerebellum, thalamus, and cerebral cortex on day 6. Infected pyramidal neurons of the hippocampus were observed on day 5 in CVS-N2c infection, but infected neurons were never noted in the hippocampus in CVS-B2c infection. CVS-N2c infected many more neurons and more prominently involved neuronal processes than CVS-B2c. CVS-N2c spread more efficiently in the CNS than CVS-B2c. Morphologic changes of apoptosis or biochemical evidence of DNA fragmentation were not observed in neurons with either virus after this route of inoculation. The different neurovirulent properties of these CVS variants in this model were not related to their in vivo ability to induce apoptosis.

Los anticuerpos monoclonales en la caracterizacion y vigilancia de los virus de la rabia en America Latina y el Caribe / Monoclonal antibodies for rabies virus characterization and epidemiological surveillance in Latin America and the Caribbean

As one of the activities of the Rabies Reference Laboratories Consortium of the Pan AMerican Health Organization, a technical consultation meeting was held in late 1999 where well-known experts from Europe, North America, and South America analyzed the contributions to rabies epidemiological surveillance in Latin America and the Caribbean made by techniques of antigenic typing based on monoclonal antibodies and by techniques of genetic typing based on gene sequencing (AU)

[The efficacy of a cultured rabies vaccine studied in guinea pigs preliminarily infected with a fixed rabies virus]. / Izuchenie éffektivnosti kul'tural'noi antirabicheskoi vaktsiny na morskikh svinkakh v usloviiakh predvaritel'nogo zarazheniia ikh fiksirovannym virusom beshenstva.

In experiments of curative vaccination, carried out with the use of an experimental model similar to the current practice of treatment with antirabies preparations, the advantages of using tissue-culture rabies vaccine with immunogenic potency equal to 1.3 international units (I. U.) were shown. In these experiments the vaccine was introduced into guinea pigs infected with fixed rabies virus, the course of vaccination consisting of 14 daily injections. No correlation between the induction of virus-neutralizing antibodies and the immunogenic potency of tissue-culture rabies vaccine was established: the use of the vaccine with immunogenic potency equal to 0,3 and 1,3 I.U. had no essential influence on the level of antibody formation in the animals.

[The efficacy of a cultured rabies vaccine studied on guinea pigs previously infected with the rabies street virus]. / Izuchenie éffektivnosti kul'tural'noi antirabicheskoi vaktsiny na morskikh svinkakh, predvaritel'no zarazhennykh ulichnym virusom beshenstva.

In this work materials on the development of an experimental model for the study of rabies vaccines are presented. The comparative study of different immunization schedules for vaccines with different protective potency has been carried out. Guinea pigs infected with street rabies virus, strain k, were used as an experimental model. As shown in this investigation the optimum method of infecting the animals with strain k was intramuscular injection causing 50% mortality among the animals, the incubation period lasting 10-24 days. Only those tissue-culture rabies vaccines which had activity equal to 1.0-1.3 I. U. and, when injected into the animals, ensured survival rate ranging from 57% to 76%, depending on the immunization schedule, were shown to possess protective potency. It should be pointed out that survival rate among the animals receiving the preparation according to the reduced schedules recommended by WHO was higher than among those immunized daily for 14 days. In all groups immune response was observed. Still in the animals receiving the preparation according to the reduced schedules a higher level of virus-neutralizing antibodies was registered. Thus, an experimental model capable of being used for the evaluation of the quality of existing and newly developed antirabies preparations was obtained. Besides, we believe it to be expedient to carry out the field trial of rabies vaccines with activity equal to 1.0-1.3 I. U., using the reduced immunization schedules.