[New aspects of rabies control]. / Nouveaux aspects de la lutte contre la rage.

Rabies still causes about 60,000 human deaths per year, mainly in poor populations in Africa and Asia. However, since Louis Pasteur developed the first vaccine 130 years ago, prophylactic measures have been considerably improved and simplified. They now consist of the vaccine combined with purified rabies immunoglobulins of equine or human origin. In general, however, post-exposure prophylaxis protocols are long and expensive. Furthermore, the immunoglobulins used for associated serotherapy are costly and not widely available in developing countries. Approaches have been developed to deal with these two issues that offer hope for a paradigm shift for the benefit of exposed populations. Finally, mass rabies vaccination in dogs, which are the most cost-effective measure for preventing rabies in humans, are difficult to implement and sometimes have moderate effectiveness. The identification and analysis of the epidemiological drivers conditioning the circulation of the virus in dog populations allow a better understanding of the key control points that need to be associated with these campaigns for a better efficacy.

Active and passive surveillance for bat lyssaviruses in Italy revealed serological evidence for their circulation in three bat species.

The wide geographical distribution and genetic diversity of bat-associated lyssaviruses (LYSVs) across Europe suggest that similar viruses may also be harboured in Italian insectivorous bats. Indeed, bats were first included within the passive national surveillance programme for rabies in wildlife in the 1980s, while active surveillance has been performed since 2008. The active surveillance strategies implemented allowed us to detect neutralizing antibodies directed towards European bat 1 lyssavirus in six out of the nine maternity colonies object of the study across the whole country. Seropositive bats were Myotis myotis, M. blythii and Tadarida teniotis. On the contrary, the virus was neither detected through passive nor active surveillance, suggesting that fatal neurological infection is rare also in seropositive colonies. Although the number of tested samples has steadily increased in recent years, submission turned out to be rather sporadic and did not include carcasses from bat species that account for the majority of LYSVs cases in Europe, such as Eptesicus serotinus, M. daubentonii, M. dasycneme and M. nattereri. A closer collaboration with bat handlers is therefore mandatory to improve passive surveillance and decrypt the significance of serological data obtained up to now.

The European Virus Archive goes global: A growing resource for research.

The European Virus Archive (EVA) was created in 2008 with funding from the FP7-EU Infrastructure Programme, in response to the need for a coordinated and readily accessible collection of viruses that could be made available to academia, public health organisations and industry. Within three years, it developed from a consortium of nine European laboratories to encompass associated partners in Africa, Russia, China, Turkey, Germany and Italy. In 2014, the H2020 Research and Innovation Framework Programme (INFRAS projects) provided support for the transformation of the EVA from a European to a global organization (EVAg). The EVAg now operates as a non-profit consortium, with 26 partners and 20 associated partners from 21 EU and non-EU countries. In this paper, we outline the structure, management and goals of the EVAg, to bring to the attention of researchers the wealth of products it can provide and to illustrate how end-users can gain access to these resources. Organisations or individuals who would like to be considered as contributors are invited to contact the EVAg coordinator, Jean-Louis Romette, at jean-louis.romette@univmed.fr.

Diagnostic tests for human rabies. / Tests diagnostiques de la rage chez l'homme.

The lack of reliable data concerning the number of human deaths from rabies presents one of the principal difficulties in a realistic assessment of the importance of this disease, and this lack of an accurate assessment has led to its underestimation and neglect. Priority should therefore be given to establishing a diagnostic test that can confirm human rabies on the basis of biological results. Indeed, only a laboratory diagnosis can properly identify infection, because clinical diagnosis remains difficult to interpret and is insufficiently specific. Historically, diagnosis has been based solely on post-mortem analysis of a cerebral biopsy using immunofluorescence techniques. Although this remains the standard method, considerable progress has been made with the advent of new molecular techniques and the evaluation of new, less-invasive sampling methods that are more easily accepted by the patient's family. Intra-vitam diagnosis of human rabies is now possible using reliable, robust, validated techniques that can be used everywhere, including in regions with limited resources, using minimally invasive or non-invasive sampling (such as saliva or skin biopsies). In practice, one of the major challenges with the diagnosis of human rabies is still the transfer and accessibility of such validated techniques in centralised reference laboratories located in low-income enzootic countries, in order to achieve the biological confirmation of each suspected case of rabies. At the same time, it is necessary to develop easy, fast and low-cost diagnostic methods that can be used in rural and remote areas in peripheral laboratories, or ideally at the patient's bedside.

L'absence de données fiables concernant le nombre de décès humains dus à la rage représente l'une des limitations majeures à l'évaluation réelle du poids mondial de cette maladie, contribuant ainsi à sa sous-estimation et à son caractère négligé. Devant ce constat, l'établissement d'un diagnostic de confirmation de la rage chez l'homme basé sur des résultats biologiques doit être favorisé. En effet, seul le diagnostic de laboratoire permet de valider l'infection, le diagnostic clinique restant difficile d'interprétation et insuffisamment spécifique. Historiquement, ce diagnostic était réalisé exclusivement au stade post-mortem via l'analyse d'une biopsie cérébrale par technique d'immunofluorescence. Bien qu'il s'agisse encore de la méthode de référence, des progrès considérables ont été faits, avec l'avènement de nouvelles techniques moléculaires et l'évaluation de nouveaux types de prélèvements moins invasifs et facilement acceptés par les proches du patient. Ces progrès autorisent maintenant la mise en oeuvre d'un diagnostic intra-vitam de la rage chez l'homme basé sur des techniques fiables, robustes et validées et pouvant être utilisées à tout niveau y compris dans les zones à ressources limitées à partir de prélèvements peu ou non invasifs (tels la salive ou les biopsies de peau). En effet, l'un des enjeux majeurs du diagnostic de la rage chez l'homme réside aussi dans le transfert et l'accessibilité de ces techniques validées, au niveau des laboratoires de référence situés dans les pays enzootiques à faible revenu, afin de réaliser une confirmation biologique de chaque cas suspect de rage. En parallèle, il est nécessaire de poursuivre les recherches sur le développement de méthodes de diagnostic simplifiées, rapides et de faible coût pouvant être utilisées de façon délocalisée, dans les laboratoires périphériques en zone rurale, voire au lit du patient.

La ausencia de datos fidedignos sobre el número de personas fallecidas a causa de la rabia constituye una de las principales limitaciones a la hora de evaluar con exactitud la carga mundial que impone la enfermedad, lo que contribuye al hecho de que esté subestimada y, por consiguiente, desatendida. De semejante constatación se desprende la necesidad de favorecer la instauración de un diagnóstico de confirmación de la rabia humana basado en resultados biológicos, en la medida en que el diagnóstico de laboratorio es el único modo de validar la presencia de la infección, pues el diagnóstico clínico presenta dificultades de interpretación y no es lo bastante específico. Históricamente este diagnóstico se realizaba únicamente tras la muerte del individuo, mediante el análisis por inmunofluorescencia de una muestra encefálica. Aunque este sigue siendo el método de referencia, el advenimiento de nuevas técnicas moleculares y el estudio de nuevos tipos de muestras, obtenidas por métodos menos invasivos y fácilmente aceptados por los allegados del paciente, han deparado progresos considerables, que permiten hoy realizar un diagnóstico intra-vitam de la rabia humana utilizando técnicas fiables, robustas y validadas que se pueden aplicar en todos los niveles, incluso en zonas con escasos recursos, a partir de muestras obtenidas por procedimientos poco o nada invasivos (muestras de saliva o biopsias de piel). Uno de los principales envites del diagnóstico de la rabia en el ser humano reside, en efecto, en la accesibilidad y la transferencia de estas técnicas validadas a laboratorios de referencia situados en los países enzoóticos de renta baja para poder realizar en ellos una confirmación biológica de todo caso sospechoso de rabia. Paralelamente, es necesario seguir investigando para instituir métodos de diagnóstico simplificados, rápidos y poco costosos que se puedan aplicar de forma descentralizada, esto es, en los laboratorios periféricos de zonas rurales e incluso junto al lecho del paciente.

Update on human rabies in a dog- and fox-rabies-free country.

UNLABELLED: Rabies is responsible for 50,000 deaths per year worldwide. Mainland France has been officially freed from rabies in non-flying animals since 2001. METHOD: We wanted to provide an update on the French situation, using published data, and describe possible options since official guidelines are lacking. RESULTS: Post-exposure prophylaxis (PEP) (early and careful cleaning and dressing of the wound, vaccination, and in case of high-risk exposure, injection of specific anti-rabies immunoglobulins) is known to be efficient except in rare cases. It is recommended after grade II contact (+specific immunoglobulins in immunodepressed patients), or grade III contact (vaccination+immunoglobulins). DISCUSSION: Mainland France being rabies-free, 3 options may be considered in case of bite by a dog or a cat that cannot be monitored in France: (a) consider the risk of rabies as null, so no PEP should be administrated, whatever the severity of bites; (b) consider there is a weak but lethal risk, so the international recommendations should be applied, using immunoglobulins in some cases; (c) consider that the risk is extremely low but cannot be excluded, and that the patient should be vaccinated to be protected, but without adding immunoglobulins (whether in case of grade II or III bites). CONCLUSION: There are no national guidelines for rabies in France, and so the physician managing the patient is the one who will decide to treat or not.

Genomic characterization of Sebokele virus 1 (SEBV1) reveals a new candidate species among the genus Parechovirus.

We determined the genomic features and the taxonomic classification of Sebokele virus 1 (SEBV1), a previously unclassified arbovirus isolated in 1972 from rodents collected in Botambi, Central African Republic. The complete genome sequence was obtained using a deep sequencing approach (Illumina technology) and dedicated bioinformatics workflows for data analysis. Molecular analysis identified SEBV1 as a picornavirus, most closely related to Ljungan viruses of the genus Parechovirus. The genome has a typical Ljungan virus-like organization, including the presence of two unrelated 2A protein motifs. Phylogenetic analysis confirmed that SEBV1 belongs to the parechovirus phylogroup and was most closely related to the Ljungan virus species. However, it appeared clearly distinct from all members of this phylogroup, suggesting that it represents a novel species of the genus Parechovirus.

Conservation of a unique mechanism of immune evasion across the Lyssavirus genus.

The evasion of host innate immunity by Rabies virus, the prototype of the genus Lyssavirus, depends on a unique mechanism of selective targeting of interferon-activated STAT proteins by the viral phosphoprotein (P-protein). However, the immune evasion strategies of other lyssaviruses, including several lethal human pathogens, are unresolved. Here, we show that this mechanism is conserved between the most distantly related members of the genus, providing important insights into the pathogenesis and potential therapeutic targeting of lyssaviruses.

Fatal case of imported human rabies in Amadora, Portugal, August 2011.

We report on a case of imported human rabies in Portugal, in July 2011 in a woman who presented initially complaining of back pain, without relating exposure to animal bites. She had travelled from Portugal to Bissau, Guinea-Bissau, in April where she had been bitten by a dog on 1 May. She was diagnosed with rabies on 26 July and died two weeks later in spite of being treated following the Milwaukee protocol.

Rabid dog illegally imported to France from Morocco, August 2011.

In August 2011, a case of canine rabies was notified to the French veterinary services. The dog was a three-month-old puppy illegally imported from Morocco that presented behavioural changes on 1 August and was admitted to a veterinary clinic on 6 August. It died the following day and the body was shortly sent to the national reference centre where rabies was laboratory-confirmed on 11 August. Contact tracing and post-exposure treatment were initiated immediately.

Genomics and structure/function studies of Rhabdoviridae proteins involved in replication and transcription.

Some mammalian rhabdoviruses may infect humans, and also infect invertebrates, dogs, and bats, which may act as vectors transmitting viruses among different host species. The VIZIER programme, an EU-funded FP6 program, has characterized viruses that belong to the Vesiculovirus, Ephemerovirus and Lyssavirus genera of the Rhabdoviridae family to perform ground-breaking research on the identification of potential new drug targets against these RNA viruses through comprehensive structural characterization of the replicative machinery. The contribution of VIZIER programme was of several orders. First, it contributed substantially to research aimed at understanding the origin, evolution and diversity of rhabdoviruses. This diversity was then used to obtain further structural information on the proteins involved in replication. Two strategies were used to produce recombinant proteins by expression of both full length or domain constructs in either E. coli or insect cells, using the baculovirus system. In both cases, parallel cloning and expression screening at small-scale of multiple constructs based on different viruses including the addition of fusion tags, was key to the rapid generation of expression data. As a result, some progress has been made in the VIZIER programme towards dissecting the multi-functional L protein into components suitable for structural and functional studies. However, the phosphoprotein polymerase co-factor and the structural matrix protein, which play a number of roles during viral replication and drives viral assembly, have both proved much more amenable to structural biology. Applying the multi-construct/multi-virus approach central to protein production processes in VIZIER has yielded new structural information which may ultimately be exploitable in the derivation of novel ways of intervening in viral replication.

[Epidemiologic monitoring of human rabies in an endemic canine rabies area in the Ivory Coast]. / Surveillance épidémiologique de la rage humaine dans un contexte d'endémie de rage canine en Cote d'Ivoire.

AIM: The objective of this study was to analyze the epidemiologic monitoring of human rabies in the rabies medical center of Abidjan. PATIENT: A cross-sectional study was undertaken based on files of patients having consulted in the rabies center after exposure and suspicion of human rabies between January 2001 and June 2009. RESULTS: Twenty-six cases of human rabies were reported in 10,706,136 inhabitants (annual incidence of 0.028 cases per 100,000). The number of detected cases increased considerably after reinforcing the monitoring of the disease. Most cases occurred in an urban environment and concerned both sexes. The most concerned age range was under 31 years. The patients' were mostly school children and housewives. In all cases, the rabid animal was a dog and the type of exposure was a bite. Fifty-six percent of the victims had not received any local treatment and almost all no post-exposure prophylaxis. The mean delay before the first symptoms was 49.5 days after exposure and for death, 4 days after the onset of symptoms. Virological diagnosis was made in four cases. CONCLUSION: In spite of the increase of cases observed since 2006, certainly due to reinforcement of the monitoring network, improvement is needed to assess the real incidence of rabies in the Ivory Coast.

[The fight against rabies in Africa: from recognition to action]. / Lutte contre la rage en Afrique: du constat à l'action.

As a follow-up to the first AfroREB (Africa Rabies Expert Bureau) meeting, held in Grand-Bassam (Côte-d'Ivoire) in March 2008, African rabies experts of the Afro-REB network met a second time to complete the evaluation of the rabies situation in Africa and define specific action plans. About forty French speaking rabies specialists from Northern, Western and Central Africa and Madagascar met in Dakar (Senegal), from March 16th to 19th, 2009. With the participation of delegates from Tunisia, who joined the AfroREB network this year, 15 French speaking African countries were represented. Experts from the Institut Pasteur in Paris, the Alliance for Rabies Control, and the Southern and Eastern African Rabies Group (SEARG, a network of rabies experts from 19 English speaking Southern and Eastern African countries) were in attendance, to participate in the discussion and share their experiences. AfroREB members documented 146 known human rabies cases in all represented countries combined for 2008, for a total population of 209.3 million, or an incidence of 0.07 cases per 100,000 people. Even admitting that the experts do not have access to all reported cases, this is far from the WHO estimation of 2 rabies deaths per 100,000 people in urban areas and 3.6 per 100,000 in rural Africa. It was unanimously agreed that the priority is to break the vicious cycle of indifference and lack of information which is the main barrier to human rabies prevention.

Phylogenetic analysis of rabies viruses from Burkina Faso, 2007.

Genetic characterization of 32 canine rabies viruses circulating in Burkina Faso in 2007 identified two clades both belonging to the Africa 2 lineage. Sequence homology data suggest that transboundary spread is the most likely means of introduction, highlighting an evolving epidemiological situation.

Is there a need for anti-rabies vaccine and immunoglobulins rationing in Europe?

Rabies is a lethal encephalitis caused by a lyssavirus and transmitted from animals to humans via bite wound, scratch wound, or licking of mucous membranes. It is preventable by timely administration of post-exposure prophylaxis (PEP) consisting of four or five doses of rabies vaccine combined, in the most severe cases of exposures, with anti-rabies immunoglobulin (RIG). Although the rabies incidence in humans remains low, rabies is still present in some European countries. Moreover, rabid animals imported from enzootic areas are reported every year in rabies-free areas. These importations threaten the rabies-free status of terrestrial animals in western European countries and challenge the public health surveillance system and the health structures responsible for rabies prophylaxis and control. The importations frequently result in the prescription of a large number of PEP including RIG, especially in western European countries. The situation is inverted in some central and eastern European countries where RIG is underprescribed. Only a limited number of rabies vaccines and particularly of RIG are licensed for use in Europe. Their availability is also limited, a situation that may become worse in the future. It therefore seems important to study the possibility of comparing and unifying national PEP guidelines in Europe, if needed, and to generate effective solutions in the event of a shortage of anti-rabies biological products and RIG in particular, such as rationing these products.

WELYSSA: a simple tool using mouse monoclonal antibodies for the detection of lyssavirus nucleocapsid in rabies suspected specimens.

A Monoclonal antibody (MAb)-based capture enzyme-linked immunosorbent assay (ELISA) named WELYSSA was developed for the diagnosis of rabies suspected specimens using prototype viruses from the different genotypes of lyssavirus and from various geographic origins and phylogenetic lineages. It included a panel of 1,660 specimens received for rabies diagnostic testing, and was found to be highly specific (99.9%) and sensitive (97.0%) when compared to other recommended rabies diagnostic methods.

Active monitoring of EBLV infection in natural colonies of the mouse-eared Bat (Myotis myotis).

Bats are important reservoir hosts of RNA viruses, including lyssaviruses, which can cross the species barrier to infect humans and other domestic and wild non-flying mammals. Six of the seven Lyssavirus genotypes described to date infect bats. In Europe, two genotypes of Lyssavirus, European bat Lyssavirus types 1 and 2 (EBLV-1 and EBLV-2), circulate among several bat species and numerous bats are found infected every year. To provide epidemiologists and public health officials with data to effectively implement public health measures, we have undertaken field studies to identify the temporal dynamics of virus infection in bat colonies by combining multidisciplinary approaches. We have focused our work on a long-term longitudinal survey of different bat colonies in the Balearic Islands. The prevalence of virus RNA and neutralizing antibodies were analysed in captured bats. The bats were banded to allow for individual monitoring of infection and movements between colonies. The results show different lyssavirus infection episodes across the twelve years of study and provide the first evidence that mortality of the mouse-eared bat (Myotis myotis) in their natural environment does not increase significantly after episodes of EBLV-1 infection.

Fighting rabies in Africa: the Africa Rabies Expert Bureau (AfroREB).

Rabies experts from 14 francophone African countries met in Grand Bassam (Côte d'Ivoire), 10-13 March 2008. They presented the situation in their respective countries, acknowledging the lack of rabies awareness among the population, health care workers and health authorities. They recognized that infrastructure for the management of rabies exposure is scarce, modern vaccines are in limited quantity and immunoglobulins are lacking in most of their countries. They defined as a priority the need to have reliable figures on the disease burden, which is necessary for informed decision making and priority setting, and for applying for aid in controlling the disease. This meeting sealed the establishment of the Africa Rabies Expert Bureau (AfroREB).

The VIZIER project: preparedness against pathogenic RNA viruses.

Life-threatening RNA viruses emerge regularly, and often in an unpredictable manner. Yet, the very few drugs available against known RNA viruses have sometimes required decades of research for development. Can we generate preparedness for outbreaks of the, as yet, unknown viruses? The VIZIER (VIral enZymes InvolvEd in Replication) (http://www.vizier-europe.org/) project has been set-up to develop the scientific foundations for countering this challenge to society. VIZIER studies the most conserved viral enzymes (that of the replication machinery, or replicases) that constitute attractive targets for drug-design. The aim of VIZIER is to determine as many replicase crystal structures as possible from a carefully selected list of viruses in order to comprehensively cover the diversity of the RNA virus universe, and generate critical knowledge that could be efficiently utilized to jump-start research on any emerging RNA virus. VIZIER is a multidisciplinary project involving (i) bioinformatics to define functional domains, (ii) viral genomics to increase the number of characterized viral genomes and prepare defined targets, (iii) proteomics to express, purify, and characterize targets, (iv) structural biology to solve their crystal structures, and (v) pre-lead discovery to propose active scaffolds of antiviral molecules.

The evolutionary dynamics of canid and mongoose rabies virus in Southern Africa.

Two variants of rabies virus (RABV) currently circulate in southern Africa: canid RABV, mainly associated with dogs, jackals, and bat-eared foxes, and mongoose RABV. To investigate the evolutionary dynamics of these variants, we performed coalescent-based analyses of the G-L inter-genic region, allowing for rate variation among viral lineages through the use of a relaxed molecular clock. This revealed that mongoose RABV is evolving more slowly than canid RABV, with mean evolutionary rates of 0.826 and 1.676 x 10(-3) nucleotide substitutions per site, per year, respectively. Additionally, mongoose RABV exhibits older genetic diversity than canid RABV, with common ancestors dating to 73 and 30 years, respectively, and while mongoose RABV has experienced exponential population growth over its evolutionary history in Africa, populations of canid RABV have maintained a constant size. Hence, despite circulating in the same geographic region, these two variants of RABV exhibit striking differences in evolutionary dynamics which are likely to reflect differences in their underlying ecology.