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International audience; Since the elimination of dog and terrestrial wild animal rabies, bat exposures remain the only source of autochthonous lyssavirus transmission to humans in Western Europe. European bats have already been found infected with several lyssaviruses, and human-bat interactions represent a risk of viral transmission and fatal encephalitis for humans. In this study, we aim to better characterize exposures to bats in metropolitan France from 2003 to 2016 and to identify circumstances associated with exposures to lyssavirus-positive bats. Two complementary sources of data were analysed: 1/ data associated with bats responsible for human exposure received for Lyssavirus testing by the French National Reference Centre for Rabies (NRCR); and 2/ data pertaining to individuals seeking medical care through the French Anti-Rabies Clinics network after contact with a bat. From 2003 to 2016, 425 bats originating from metropolitan France were submitted to the NRCR and 16 (4%) were found positive with a lyssavirus (EBLV-1b was diagnosed in 9 bats, EBLV-1a in 6 and BBLV in one specimen). The two factors associated with bat positivity in our study were the female sex and the bat belonging to the E. serotinus species. During the same study period, 1718 individuals sought care at an Anti-Rabies Clinic after exposure to a bat resulting in an estimated incidence of human-bat interactions of 1.96 per 106 person-years. The two most frequent circumstances of exposure were handling or bites. Interactions mostly involved one adult human being and one live and non-sick-looking bat. Our study provides new insights about circumstances of human-bat interactions and may be helpful to target prevention interventions to improve the awareness of the population of the risk of lyssavirus transmission.

Summary Background Tens of thousands of people die from dog-mediated rabies annually. Deaths can be prevented through post-exposure prophylaxis for people who have been bitten, and the disease eliminated through dog vaccination. Current post-exposure prophylaxis use saves many lives, but availability remains poor in many rabies-endemic countries due to high costs, poor access, and supply. Methods We developed epidemiological and economic models to investigate the effect of an investment in post-exposure prophylaxis by Gavi, the Vaccine Alliance. We modelled post-exposure prophylaxis use according to the status quo, with improved access using WHO-recommended intradermal vaccination, with and without rabies immunoglobulin, and with and without dog vaccination. We took the health provider perspective, including only direct costs. Findings We predict more than 1 million deaths will occur in the 67 rabies-endemic countries considered from 2020 to 2035, under the status quo. Current post-exposure prophylaxis use prevents approximately 56 000 deaths annually. Expanded access to, and free provision of, post-exposure prophylaxis would prevent an additional 489 000 deaths between 2020 and 2035. Under this switch to efficient intradermal post-exposure prophylaxis regimens, total projected vaccine needs remain similar (about 73 million vials) yet 17·4 million more people are vaccinated, making this an extremely cost-effective method, with costs of US$635 per death averted and $33 per disability-adjusted life-years averted. Scaling up dog vaccination programmes could eliminate dog-mediated rabies over this time period; improved post-exposure prophylaxis access remains cost-effective under this scenario, especially in combination with patient risk assessments to reduce unnecessary post-exposure prophylaxis use. Interpretation Investing in post-exposure vaccines would be an extremely cost-effective intervention that could substantially reduce disease burden and catalyse dog vaccination efforts to eliminate dog-mediated rabies. Funding World Health Organization.

International audience; Postexposure prophylaxis (PEP) prevents human rabies and is accessible in Cambodia principally in Phnom Penh, the capital. Timely, affordable access to PEP is a challenge for the mainly rural population. We aimed to identify districts independently associated with PEP noncompletion to position frontline vaccination centers. We analyzed the 2009–2013 database at the Rabies Prevention Center at the Institut Pasteur du Cambodge, Phnom Penh. Logistic regressions identified nongeographic determinants of PEP noncompletion as well as the districts that were independently associated with noncompletion after adjustment for these determinants. The influence of distance by road was estimated using a boosted regression-trees model. We computed a population attributable fraction (rabies index (RI)) for each district and developed a map of this RI distribution. A cartographic analysis based on the statistic developed by Getis and Ord identified clusters of high-RI districts. Factors independently associated with noncompletion were pa-tients' district of residence, male sex, age 15–49 years, initial visit during rice harvest, the dog's status (culled or disappeared), and a prescribed PEP protocol requiring more than 3 PEP sessions (4 or 5). Four clusters of high-RI districts were identified using this analytical strategy, which is applicable to many vaccination or other health services. Positioning frontline PEP centers in these districts could significantly widen access to timely and adequate PEP.

At the crossroad between the NF-κB and the MAPK pathways, the ternary complex composed of p105, ABIN2 and TPL2 is essential for the host cell response to pathogens. The matrix protein (M) of field isolates of rabies virus was previously shown to disturb the signaling induced by RelAp43, a NF-κB protein close to RelA/p65. Here, we investigated how the M protein disturbs the NF-κB pathway in a RelAp43-dependant manner and the potential involvement of the ternary complex in this mechanism. Using a tandem affinity purification coupled with mass spectrometry approach, we show that RelAp43 interacts with the p105-ABIN2-TPL2 complex and we observe a strong perturbation of this complex in presence of M protein. M protein interaction with RelAp43 is associated with a wide disturbance of NF-κB signaling, involving a modulation of IκBα-, IκBβ-, and IκBε-RelAp43 interaction and a favored interaction of RelAp43 with the non-canonical pathway (RelB and p100/p52). Monitoring the interactions between host and viral proteins using protein-fragment complementation assay and bioluminescent resonance energy transfer, we further show that RelAp43 is associated to the p105-ABIN2-TPL2 complex as RelAp43-p105 interaction stabilizes the formation of a complex with ABIN2 and TPL2. Interestingly, the M protein interacts not only with RelAp43 but also with TPL2 and ABIN2. Upon interaction with this complex, M protein promotes the release of ABIN2, which ultimately favors the production of RelAp43-p50 NF-κB dimers. The use of recombinant rabies viruses further indicates that this mechanism leads to the control of IFNβ, TNF and CXCL2 expression during the infection and a high pathogenicity profile in rabies virus infected mice. All together, our results demonstrate the important role of RelAp43 and M protein in the regulation of NF-κB signaling., Author summary Rabies virus is a recurring zoonosis responsible for about 60,000 deaths per year. A key feature of rabies virus is its stealth, allowing it to spread within the host and escape the immune response. To do so, rabies virus developed several mechanisms, including a thorough interference with cell signaling pathways. Here, we focused our attention on the molecular aspects of rabies virus escape to the NF-κB pathway through the interaction between the M protein and the NF-κB protein RelAp43. Monitoring close range interactions, we found that RelAp43 plays an important role in the stabilization of the p105-ABIN2-TPL2 complex, which is essential in the regulation of both NF-κB and MAPK pathways, and we brought a new insight on the dynamics within the host protein complex. These results were confirmed in living cells and in mice. Overall, our data suggest that rabies virus interference with the p105-ABIN2-TPL2 complex is a cornerstone of its stealth strategy to escape the immune response.

The matrix (M) protein of wild isolates of rabies virus such as Tha (M-Tha) was previously shown to be able to interact with RelAp43, a protein of the NF-κB family, and to efficiently suppress NF-κB-dependent reporter gene expression, in contrast with the vaccine strain SAD. Here, we analyze the mechanisms involved in RelAp43-M protein interaction. We demonstrate that the central part of M-Tha, and the specific C-terminal region of RelAp43 are required for this interaction. Four differences in the corresponding amino acid sequences of the M-Tha and M-SAD are shown to be crucial for RelAp43 interaction and subsequent modulation of innate immune response. Furthermore, the capacity of M-Tha to interact with RelAp43 was shown to be crucial for the control of the expression of four genes (IFN, TNF, IL8 and CXCL2) during viral infection. These findings reveal that RelAp43 is a potent regulator of transcription of genes involved in innate immune response during rabies virus infection and that the M protein of wild isolates of rabies virus is a viral immune-modulatory factor playing an important role in this RelAp43-mediated host innate immunity response in contrast to M protein of vaccine strains, which have lost this property.

Background Rabies virus (RABV) causes rabies disease resulting in >55,000 human deaths/year. The multifunctional RABV P-protein has essential roles in genome replication, and forms interactions with cellular STAT proteins that are thought to underlie viral antagonism of interferon-dependent immunity. However, the molecular details of P-protein-STAT interaction, and its importance to disease are unresolved. Methods Studies were performed using sequence/structure analysis, mutagenesis, immunoprecipitation, luciferase and qRT-PCR-based signaling assays, confocal microscopy and reverse genetics/in vivo infection. Results We identified a hydrophobic pocket of the P-protein C-terminal domain as critical to STAT-binding/antagonism. This interface was found to be functionally and spatially independent of the region responsible for N-protein interaction, which is critical to genome replication. Based on these findings, we generated the first mutant RABV lacking STAT-association. Growth of the virus in vitro was unimpaired, but it lacked STAT-antagonist function and was highly sensitive to interferon. Importantly, growth of the virus was strongly attenuated in brains of infected mice, producing no major neurological symptoms, compared with the invariably lethal wild-type virus. Conclusions These data represent direct evidence that P-protein-STAT interaction is critical to rabies, and provide novel insights into the mechanism by which RABV coordinates distinct functions in interferon antagonism and replication.

International audience; In 2015, countries in western Europe were declared free of rabies in nonflying mammals. Surveillance data for 2001-2013 indicate that risk for residual rabies is not 0 because of pet importation from countries with enzootic rabies. However, the risk is so low (7.52 × 10(-10)) that it probably can be considered negligible.

International audience; This first extensive retrospective study of the molecular epidemiology of dog rabies in Cambodia included 149 rabies virus (RABV) entire nucleoprotein sequences obtained from 1998-2011. The sequences were analyzed in conjunction with RABVs from other Asian countries. Phylogenetic reconstruction confirmed the South-East Asian phylogenetic clade comprising viruses from Cambodia, Vietnam, Thailand, Laos and Myanmar. The present study represents the first attempt to classify the phylogenetic lineages inside this clade, resulting in the confirmation that all the Cambodian viruses belonged to the South-East Asian (SEA) clade. Three distinct phylogenetic lineages in the region were established with the majority of viruses from Cambodia closely related to viruses from Thailand, Laos and Vietnam, forming the geographically widespread phylogenetic lineage SEA1. A South-East Asian lineage SEA2 comprised two viruses from Cambodia was identified, which shared a common ancestor with RABVs originating from Laos. Viruses from Myanmar formed separate phylogenetic lineages within the major SEA clade. Bayesian molecular clock analysis suggested that the time to most recent common ancestor (TMRCA) of all Cambodian RABVs dated to around 1950. The TMRCA of the Cambodian SEA1 lineage was around 1964 and that of the SEA2 lineage was around 1953. The results identified three phylogenetically distinct and geographically separated lineages inside the earlier identified major SEA clade, covering at least five countries in the region. A greater understanding of the molecular epidemiology of rabies in South-East Asia is an important step to monitor progress on the efforts to control canine rabies in the region.

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.

High-throughput sequencing furnishes a large number of short sequence reads from uncloned DNA and has rapidly become a major tool for identifying viruses in biological samples, and in particular when the target sequence is undefined. In this study, we assessed the analytical sensitivity of a pipeline for detection of viruses in biological samples based on either the Roche-454 genome sequencer or Illumina genome analyzer platforms. We sequenced biological samples artificially spiked with a wide range of viruses with genomes composed of single or double-stranded DNA or RNA, including linear or circular single-stranded DNA. Viruses were added at a very low concentration most often corresponding to 3 or 0.8 times the validated level of detection of quantitative reverse transcriptase PCRs (RT-PCRs). For the viruses represented, or resembling those represented, in public nucleotide sequence databases, we show that the higher output of Illumina is associated with a much greater sensitivity, approaching that of optimized quantitative (RT-)PCRs. In this blind study, identification of viruses was achieved without incorrect identification. Nevertheless, at these low concentrations, the number of reads generated by the Illumina platform was too small to facilitate assembly of contigs without the use of a reference sequence, thus precluding detection of unknown viruses. When the virus load was sufficiently high, de novo assembly permitted the generation of long contigs corresponding to nearly full-length genomes and thus should facilitate the identification of novel viruses.

The lyssavirus matrix (M) protein induces apoptosis. The regions of the M protein that are essential for triggering cell death pathways are not yet clearly defined. We therefore compared the M proteins from two viruses that have contrasting characteristics in terms of cellular apoptosis: a genotype 3 lyssavirus, Mokola virus (MOK), and a genotype 1 rabies virus isolated from a dog from Thailand (THA). We identified a 20-amino-acid fragment (corresponding to positions 67 to 86) that retained the cell death activities of the full-length M protein from MOK via both the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and inhibition of cytochromecoxidase (CcO) activity. We found that the amino acids at positions 77 and 81 have an essential role in triggering these two cell death pathways. Directed mutagenesis demonstrated that the amino acid at position 77 affects CcO activity, whereas the amino acid at position 81 affects TRAIL-dependent apoptosis. Mutations in the full-length M protein that compromised induction of either of these two pathways resulted in delayed apoptosis compared with the time to apoptosis for the nonmutated control.

Mokola virus (MOKV) is a nonsegmented, negative-sense RNA virus that belongs to the Lyssavirus genus and Rhabdoviridae family. MOKV phosphoprotein P is an essential component of the replication and transcription complex and acts as a cofactor for the viral RNA-dependent RNA polymerase. P recruits the viral polymerase to the nucleoprotein-bound viral RNA (N-RNA) via an interaction between its C-terminal domain and the N-RNA complex. Here we present a structure for this domain of MOKV P, obtained by expression of full-length P in Escherichia coli , which was subsequently truncated during crystallization. The structure has a high degree of homology with P of rabies virus, another member of Lyssavirus genus, and to a lesser degree with P of vesicular stomatitis virus (VSV), a member of the related Vesiculovirus genus. In addition, analysis of the crystal packing of this domain reveals a potential binding site for the nucleoprotein N. Using both site-directed mutagenesis and yeast two-hybrid experiments to measure P-N interaction, we have determined the relative roles of key amino acids involved in this interaction to map the region of P that binds N. This analysis also reveals a structural relationship between the N-RNA binding domain of the P proteins of the Rhabdoviridae and the Paramyxoviridae .

It is difficult to deliver adequate training for people working in rabies control in low and middle-income countries. Popular e-learning systems for low-income settings are not well suited to developing and testing practical skills, including laboratory methods.We customized training in rabies control methods for African professionals and students from different disciplines. Trainees participated in preparatory online sessions, evaluations and exercises before and after a 12-day workshop. Trainees and mentors continued to interact through an online forum up to one year after the workshop.In Africa, 15,000 deaths from rabies occur each year due to a lack of awareness, inaccessibility of post-exposure prophylaxis, inadequate or absent canine rabies-control programmes and lack of governmental financial support.Thirty two trainees - working in health departments, hospitals, veterinary stations and research institutes - were selected to participate; 28 completed the course and passed the final evaluation. Pilot rabies investigation programmes were developed, and two manuscripts submitted for publication. An online forum facilitated further progress for a year after the workshop.A combination of customized online and onsite training is suitable for teaching disease-control personnel in low-income countries. Participation in this course enabled trainees to advocate for the development of national disease-control strategies. Mentoring is needed to develop a strong network of experts in similar settings.Il est difficile de dispenser une formation appropriée aux personnes intervenant dans la lutte contre la rage dans les pays à revenu faible et intermédiaire. Les systèmes populaires d'apprentissage en ligne conçus pour les pays à faible revenu ne sont pas adaptés au développement et au test des compétences pratiques, notamment en ce qui concerne les méthodes analytiques.Nous avons adapté la formation sur les méthodes de lutte contre la rage à l'intention de professionnels et d'étudiants africains issus de différentes disciplines. Les bénéficiaires de la formation ont participé à des séances de préparation en ligne, à des évaluations et à des exercices, avant et après un atelier de 12 jours. Puis, les bénéficiaires de la formation et les tuteurs ont continué à interagir jusqu'à un an après l'atelier via un forum accessible en ligne.En Afrique, 15.000 décès dus à la rage surviennent chaque année. Cette situation vient d'un manque de sensibilisation, de l'inaccessibilité des traitements de prophylaxie post-exposition, de programmes de lutte contre la rage canine inappropriés voire inexistants et d'un manque de soutien financier gouvernemental.Trente-deux bénéficiaires de la formation (qui travaillent dans des services de santé, des hôpitaux, des centres vétérinaires ou des instituts de recherche) ont été sélectionnés pour la formation ; vingt-huit l'ont achevée et ont passé avec succès l'évaluation finale. Des programmes pilotes d'enquête sur la rage ont été élaborés et deux manuscrits ont été soumis pour publication. Un forum en ligne a facilité la poursuite des progrès pendant l'année qui a suivi l'atelier.L'association de modules proposés en ligne et sur place est adaptée à la formation du personnel de lutte contre la maladie dans les pays à faible revenu. La participation à cette formation a permis à ses bénéficiaires de plaider pour l'élaboration de stratégies nationales de lutte contre la maladie. Un tutorat est nécessaire pour constituer un solide réseau d'experts dans des contextes similaires.Es difícil proporcionar una formación adecuada a aquellos que trabajan en el control de la rabia en países de ingresos bajos y medios. Los populares sistemas de aprendizaje en línea en comunidades de ingresos bajos no son adecuados para desarrollar y poner a prueba las habilidades prácticas, incluidos los métodos de laboratorio.Se personalizó la formación en los métodos de control de la rabia para los profesionales y estudiantes africanos de diferentes disciplinas. Los alumnos participaron en sesiones preparatorias, evaluaciones y ejercicios en línea antes y después de un taller de 12 días. Los alumnos y los profesores siguieron interactuando a través de un foro en línea hasta un año después del taller.En África, hay 15.000 muertes de rabia cada año debidas a la falta de concienciación, la inaccesibilidad de la profilaxis postexposición, los programas inadecuados o ausentes de control de la rabia canina y la falta de apoyo financiero del gobierno.Treinta y dos alumnos que trabajan en departamentos de salud, hospitales, clínicas veterinarias e institutos de investigación fueron seleccionados para participar. 28 de ellos completaron el curso y aprobaron la evaluación final. Se desarrollaron programas piloto de investigación de la rabia y se presentaron 2 manuscritos para su publicación. Gracias al foro en línea, se pudo seguir progresando hasta un año después del taller.La combinación de la formación en línea y presencial es adecuada para enseñar al personal de control de enfermedades en países de ingresos bajos. La participación en este curso permitió a los alumnos promocionar el desarrollo de estrategias nacionales de control de enfermedades. La tutoría es necesaria para desarrollar una sólida red de expertos en marcos similares.يصعب تقديم التدريب الكافي للعاملين في مكافحة داء الكَلَب في البلدان منخفضة ومتوسطة الدخل. ولا تُعد نظم التعليم الإلكتروني الشعبية للمواقع منخفضة الدخل مناسبة تمامًا لتطوير واختبار المهارات العملية، بما في ذلك الأساليب المختبرية.لقد قمنا بتخصيص تدريبات في أساليب مكافحة داء الكَلَب للمختصين الأفارقة والطلبة من مختلف الاختصاصات. وشارك المتدربون في الجلسات والتقييمات والتمارين التحضيرية المباشرة على شبكة الإنترنت، قبل وبعد إجراء حلقة عمل لمدةفي أفريقيا، تحدثتم اختيار اثنان وثلاثين متدربًا – يعملون في الأقسام والإدارات الصحية، والمستشفيات، والمحطات البيطرية، ومعاهد الأبحاث – للمشاركة؛ وقد قاممن المناسب الجمع بين التدريبات المباشرة على الإنترنت والتدريبات الميدانية في الموقع لتعليم العاملين المختصين بمكافحة الأمراض في البلدان منخفضة الدخل. كان للمشاركة في هذه الدورة التدريبية أثر في تمكين المتدربين من الدعوة إلى إعداد استراتيجيات وطنية لمكافحة الأمراض. وهناك حاجة إلى التوجيه لإعداد شبكة قوية من الخبراء في بيئات مشابهة.在中低收入国家中难以为从事狂犬病控制的人员提供合适的培训。低收入环境中普及的电子学习系统不太适于开发并测试实践技能，包括化验方法。.我们针对不同学科的非洲专业人员和学生开展了关于狂犬病控制方法的定制化培训。受训人员在为期 12 天的研讨会前后参与了预备在线课程、评估和练习。受训人员和导师在研讨会结束后继续通过在线论坛进行了长达一年的互动交流。.在非洲，每年有 15 000 人死于狂犬病，这是因为缺乏意识、无法在接触后采取预防措施、狂犬病控制计划的制定不充分或没有制定，以及政府财政支持不足。.选出 32 名就职于卫生部门、医院、兽医站和研究所的受训人员参与培训，其中有 28 名完成了课程并通过了最终评估。制定出试行的狂犬病调查计划，并提交了 2 份原稿用于出版。研讨会结束后，在线论坛进一步推动了为期一年的进展。.定制化的在线和现场培训相结合，适合用于教导低收入国家的疾病控制人员。参与此项课程有助于让受训人员拥护国家疾病控制策略的发展。在相似的环境中，需要在指导下开发强大的专家网络。.В странах с низким и средним уровнем доходов населения трудно обеспечить адекватное обучение специалистов по контролю распространения бешенства. Популярные системы электронного обучения для стран с низким уровнем дохода плохо подходят для развития и проверки практических навыков, включая знания лабораторных методов контроля.Обучение методам контроля распространения бешенства было адаптировано для африканских специалистов и студентов различных дисциплин. Обучаемые проходили 12-дневный практический курс. Перед курсом и после него проводились подготовительные занятия, тестирование и практические занятия в режиме онлайн. Обучаемые и кураторы продолжали взаимодействие через онлайн-форум на протяжении одного года после окончания курса.В Африке от бешенства ежегодно умирает 15 000 человек. Причиной тому является слабая информированность населения, недоступность постконтактной профилактики, неэффективность или отсутствие программ контроля распространения бешенства среди собак и отсутствие финансовой поддержки от государства.Для участия в курсе были отобраны 32 сотрудника из департаментов здравоохранения, больниц, ветеринарных станций и исследовательских институтов; 28 человек окончили курс и прошли окончательное тестирование. Были разработаны пилотные программы по исследованию бешенства, и две рукописи были сданы в печать. Онлайн-форум способствовал дальнейшему обучению участников на протяжении одного года после окончания курса.Для обучения специалистов по контролю распространения заболеваний в странах с низким уровнем дохода населения подходит сочетание индивидуализированного обучения онлайн и на местах. Участие в данном курсе позволило обучаемым выступить в поддержку разработки национальных стратегий по контролю распространения заболеваний. Для создания сети высококвалифицированных специалистов в подобных условиях необходимо организовать эффективный процесс наставничества.

International audience; Rabies is a worldwide zoonosis resulting from Lyssavirus infection. In Europe, Eptesicus serotinus is the most frequentlyreported bat species infected with Lyssavirus, and thus considered to be the reservoir of European bat Lyssavirus type 1(EBLV-1). To date, the role of other bat species in EBLV-1 epidemiology and persistence remains unknown. Here, we built anEBLV-12transmission model based on local observations of a three-cave and four-bat species (Myotis capaccinii, Myotismyotis, Miniopterus schreibersii, Rhinolophus ferrumequinum) system in the Balearic Islands, for which a 1995–2011serological dataset indicated the continuous presence of EBLV-1. Eptesicus serotinus was never observed in the systemduring the 16-year follow-up and therefore was not included in the model. We used the model to explore virus persistencemechanisms and to assess the importance of each bat species in the transmission dynamics. We found that EBLV-1 couldnot be sustained if transmission between M. schreibersii and other bat species was eliminated, suggesting that this speciesserves as a regional reservoir. Global sensitivity analysis using Sobol’s method revealed that following the rate ofautumn2winter infectious contacts, M. schreibersii’s incubation- and immune-period durations, but not the infectiousperiod length, were the most relevant factors driving virus persistence.

International audience; Bats have been proposed as major reservoirs for diverse emerging infectious viral diseases, with rabies being the best known in Europe. However, studies exploring the ecological interaction between lyssaviruses and their natural hosts are scarce. This study completes our active surveillance work on Spanish bat colonies that began in 1992. Herein, we analyzed ecological factors that might affect the infection dynamics observed in those colonies. Between 2001 and 2011, we collected and tested 2,393 blood samples and 45 dead bats from 25 localities and 20 bat species. The results for dead confirmed the presence of EBLV-1 RNA in six species analyzed (for the first time in Myotis capaccinii). Samples positive for European bat lyssavirus-1 (EBLV-1)-neutralizing antibodies were detected in 68% of the localities sampled and in 13 bat species, seven of which were found for the first time (even in Myotis daubentonii, a species to date always linked to EBLV-2). EBLV-1 seroprevalence (20.7%) ranged between 11.1 and 40.2% among bat species and seasonal variation was observed, with significantly higher antibody prevalence in summer (July). EBLV-1 seroprevalence was significantly associated with colony size and species richness. Higher seroprevalence percentages were found in large multispecific colonies, suggesting that intra- and interspecific contacts are major risk factors for EBLV-1 transmission in bat colonies. Although bat-roosting behavior strongly determines EBLV-1 variability, we also found some evidence that bat phylogeny might be involved in bat-species seroprevalence. The results of this study highlight the importance of life history and roost ecology in understanding EBLV-1-prevalence patterns in bat colonies and also provide useful information for public health officials.

Background One root cause of the neglect of rabies is the lack of adequate diagnostic tests in the context of low income countries. A rapid, performance friendly and low cost method to detect rabies virus (RABV) in brain samples will contribute positively to surveillance and consequently to accurate data reporting, which is presently missing in the majority of rabies endemic countries. Methodology/Principal findings We evaluated a rapid immunodiagnostic test (RIDT) in comparison with the standard fluorescent antibody test (FAT) and confirmed the detection of the viral RNA by real time reverse transcription polymerase chain reaction (RT-qPCR). Our analysis is a multicentre approach to validate the performance of the RIDT in both a field laboratory (N’Djamena, Chad) and an international reference laboratory (Institut Pasteur, Paris, France). In the field laboratory, 48 samples from dogs were tested and in the reference laboratory setting, a total of 73 samples was tested, representing a wide diversity of RABV in terms of animal species tested (13 different species), geographical origin of isolates with special emphasis on Africa, and different phylogenetic clades. Under reference laboratory conditions, specificity was 93.3% and sensitivity was 95.3% compared to the gold standard FAT test. Under field laboratory conditions, the RIDT yielded a higher reliability than the FAT test particularly on fresh and decomposed samples. Viral RNA was later extracted directly from the test filter paper and further used successfully for sequencing and genotyping. Conclusion/Significance The RIDT shows excellent performance qualities both in regard to user friendliness and reliability of the result. In addition, the test cassettes can be used as a vehicle to ship viral RNA to reference laboratories for further laboratory confirmation of the diagnosis and for epidemiological investigations using nucleotide sequencing. The potential for satisfactory use in remote locations is therefore very high to improve the global knowledge of rabies epidemiology. However, we suggest some changes to the protocol, as well as careful further validation, before promotion and wider use., Author Summary The high fatality and burden of rabies stands in contrast to the very low performance of laboratory-based surveillance in resource-challenged countries. The absence of reliable human and animal rabies incidence data ultimately result in neglect of disease prevention and control and the perpetuation of RABV transmission despite the existence of powerful management tools. Rapid, easy to perform rabies diagnostic tests that do not require expensive equipment or special storage conditions, which can be reliably performed by trained ordinary veterinary professionals, are needed urgently for use in low income countries. Such novel methods will help to accurately assess the global rabies burden and are necessary to monitor rabies control and elimination. The present study evaluates the performance and reliability of a rapid, easy to use rabies diagnostic tool. Overall, the validated test was in high accordance with the standard reference method for the detection of RABV by immunofluorescence microscopy and showed even higher reliability when applied in resource poor laboratory conditions. The obtained results support the high potential for the use of this test in the field but suggest a change of the original technical protocol and a need for wider validation.

Background Until 2008, human rabies had never been reported in French Guiana. On 28 May 2008, the French National Reference Center for Rabies (Institut Pasteur, Paris) confirmed the rabies diagnosis, based on hemi-nested polymerase chain reaction on skin biopsy and saliva specimens from a Guianan, who had never travelled overseas and died in Cayenne after presenting clinically typical meningoencephalitis. Methodology/Principal Findings Molecular typing of the virus identified a Lyssavirus (Rabies virus species), closely related to those circulating in hematophagous bats (mainly Desmodus rotundus) in Latin America. A multidisciplinary Crisis Unit was activated. Its objectives were to implement an epidemiological investigation and a veterinary survey, to provide control measures and establish a communications program. The origin of the contamination was not formally established, but was probably linked to a bat bite based on the virus type isolated. After confirming exposure of 90 persons, they were vaccinated against rabies: 42 from the case's entourage and 48 healthcare workers. To handle that emergence and the local population's increased demand to be vaccinated, a specific communications program was established using several media: television, newspaper, radio. Conclusion/Significance This episode, occurring in the context of a Department far from continental France, strongly affected the local population, healthcare workers and authorities, and the management team faced intense pressure. This observation confirms that the risk of contracting rabies in French Guiana is real, with consequences for population educational program, control measures, medical diagnosis and post-exposure prophylaxis., Author Summary Until 2008, rabies had never been described within the French Guianan human population. Emergence of the first case in May 2008 in this French Overseas Department represented a public health event that markedly affected the local population, healthcare workers and public health authorities. The antirabies clinic of French Guiana, located at Institut Pasteur de la Guyane, had to reorganize its functioning to handle the dramatically increased demand for vaccination. A rigorous epidemiological investigation and a veterinary study were conducted to identify the contamination source, probably linked to a bat bite, and the exposed population. Communication was a key factor to controlling this episode and changing the local perception of this formerly neglected disease. Because similar clinical cases had previously been described, without having been diagnosed, medical practices must be adapted and the rabies virus should be sought more systematically in similarly presenting cases. Sharing this experience could be useful for other countries that might someday have to manage such an emergence.

International audience; Identification of microbial pathogens in clinical specimens is still performed by phenotypic methods that are often slow and cumbersome, despite the availability of more comprehensive genotyping technologies. We present an approach based on whole-genome amplification and resequencing microarrays for unbiased pathogen detection. This 10 h process identifies a broad spectrum of bacterial and viral species and predicts antibiotic resistance and pathogenicity and virulence profiles. We successfully identify a variety of bacteria and viruses, both in isolation and in complex mixtures, and the high specificity of the microarray distinguishes between different pathogens that cause diseases with overlapping symptoms. The resequencing approach also allows identification of organisms whose sequences are not tiled on the array, greatly expanding the repertoire of identifiable organisms and their variants. We identify organisms by hybridization of their DNA in as little as 1-4 h. Using this method, we identified Monkeypox virus and drug-resistant Staphylococcus aureus in a skin lesion taken from a child suspected of an orthopoxvirus infection, despite poor transport conditions of the sample, and a vast excess of human DNA. Our results suggest this technology could be applied in a clinical setting to test for numerous pathogens in a rapid, sensitive and unbiased manner.

International audience; The WHIM syndrome, which features high susceptibility to human papillomavirus (HPV) infection, is a rare immunodeficiency associated with autosomal dominant heterozygous mutations of the CXCR4 che-mokine receptor. CXCL12 and its receptors, CXCR4 and CXCR7, are linked to tumorigenesis, and we reported that abnormal expression of CXCL12 in epidermal keratinocytes correlates with HPV infection. However, the HPV-related pathologies observed in WHIM patients remain mechanistically unexplained. We show that keratinocytes immortalized by oncogenic HPV16 or HPV18 upregulate CXCL12 and its receptors in a manner dependent upon expression of the viral proteins E6 and E7. Autocrine signaling activated by CXCL12-engagement of its receptors controls motility and survival of the infected cells. Strikingly, expression of a WHIM syndrome-related gain-of-function CXCR4 mutant confers transforming capacity to HPV18-immortalized keratinocytes. These results establish a pivotal role for CXCL12 signaling in HPV-mediated transformation and provide a mechanistic basis for understanding HPV pathogenesis in WHIM syndrome.

Rabies is a viral zoonotic infection of the central nervous system caused by a lyssavirus. The disease is fatal without proper post exposure prophylaxis (PEP). In July 1885, Louis Pasteur obtained his first success against rabies by vaccinating Joseph Meister, a 9-year-old boy presenting with multiple deep bite wounds [1]. After more than 700 successful inoculations, Pasteur launched an international subscription and opened the world's first research institute dedicated to the prevention of rabies and other infectious diseases. The Institut Pasteur was born. Due to substantial advances during the 20th century, safe and effective human and animal vaccines based on tissue culture methodologies are available today. Today, 125 years later, Institut Pasteur is at the core of an international network of 30 institutes. Through its numerous institutions established in enzootic areas, the worldwide contribution of the Institut Pasteur International Network to rabies surveillance and control is still of paramount importance. Each year, the International Network is responsible for PEP administration to more than 180,000 exposed patients, mainly in Southeast Asia and Africa. Scientists and health staff are also involved in various national committees that work on the development and successful implementation of rabies control programs.

The authors have declared that no competing interests exist. Major funding for the project in Institut Pasteur was provided by the Institut Pasteur International Network with Actions Concertees InterPasteuriennes 2003/687 and 2006/562. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Understanding the role of humans in the dispersal of predominately animal pathogens is essential for their control. We used newly developed Bayesian phylogeographic methods to unravel the dynamics and determinants of the spread of dog rabies virus (RABV) in North Africa. Each of the countries studied exhibited largely disconnected spatial dynamics with major geo-political boundaries acting as barriers to gene flow. Road distances proved to be better predictors of the movement of dog RABV than accessibility or raw geographical distance, with occasional long distance and rapid spread within each of these countries. Using simulations that bridge phylodynamics and spatial epidemiology, we demonstrate that the contemporary viral distribution extends beyond that expected for RABV transmission in African dog populations. These results are strongly supportive of human-mediated dispersal, and demonstrate how an integrated phylogeographic approach will turn viral genetic data into a powerful asset for characterizing, predicting, and potentially controlling the spatial spread of pathogens., Author Summary At least 15 million doses of anti-rabies post-exposure prophylaxis are administered annually worldwide, and an estimated 55,000 people die of rabies every year. Over 99% of these deaths occur in developing countries, predominantly in Asia and in Africa where rabies is endemic in domestic dogs. Despite the global health burden due to rabies, little is known about the patterns of the spread of dog rabies in these endemic regions. We used recently developed Bayesian analytical methods to unravel the dynamics and determinants of the spatial diffusion of dog rabies viruses in North Africa based on viral genetic data. Our analysis reveals a combination of restricted spread across administrative borders, the occasional long-distance movement of rabies viruses, and a strong fit between spatial spread of the virus and road distances between localities. Together, these data indicate that by transporting dogs, humans have played a key role in the dispersal of a major animal pathogen. Our studies therefore provide essential new information on the transmission dynamics of rabies in Africa, and in doing so will greatly assist in future intervention strategies.

The rapid and accurate identification of pathogens is critical in the control of infectious disease. To this end, we analyzed the capacity for viral detection and identification of a newly described high-density resequencing microarray (RMA), termed PathogenID, which was designed for multiple pathogen detection using database similarity searching. We focused on one of the largest and most diverse viral families described to date, the family Rhabdoviridae . We demonstrate that this approach has the potential to identify both known and related viruses for which precise sequence information is unavailable. In particular, we demonstrate that a strategy based on consensus sequence determination for analysis of RMA output data enabled successful detection of viruses exhibiting up to 26% nucleotide divergence with the closest sequence tiled on the array. Using clinical specimens obtained from rabid patients and animals, this method also shows a high species level concordance with standard reference assays, indicating that it is amenable for the development of diagnostic assays. Finally, 12 animal rhabdoviruses which were currently unclassified, unassigned, or assigned as tentative species within the family Rhabdoviridae were successfully detected. These new data allowed an unprecedented phylogenetic analysis of 106 rhabdoviruses and further suggest that the principles and methodology developed here may be used for the broad-spectrum surveillance and the broader-scale investigation of biodiversity in the viral world.

International audience; 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.; Le Bureau des experts de la rage du continent africain (AfroREB) s’est réuni pour la seconde fois, en mars 2009, pour poursuivre leur évaluation de la situation de la rage en Afrique et définir des plans d’action. Une quarantaine d’éminents spécialistes de la rage venant de 15 pays d’Afrique francophone se sont retrouvés à Dakar avec des représentants de l’Institut Pasteur de Paris, du réseau anglophone SEARG (groupe de spécialistes de la rage d’Afrique australe et orientale) et de l’Alliance mondiale contre la rage. En Afrique, de nombreux cas de rage ne sont ni identifiés ni rapportés. Alors que l’OMS estime à 25 000 le nombre de décès annuels dus à la rage sur ce continent (2 à 3,6 décès pour 100 000 habitants), les membres d’AfroREB ont comptabilisé, en tout et pour tout, en 2008, 146 cas pour leurs 15 pays, soit 0,07 cas pour 100 000 habitants. La priorité est de briser le cercle vicieux de l’indifférence et du manque d’information, afin de pouvoir lutter contre la rage humaine.

International audience; Lyssaviruses are RNA viruses with single-strand, negative-sense genomes responsible for rabies-like diseases in mammals. To date, genomic and evolutionary studies have most often utilized partial genome sequences, particularly of the nucleoprotein and glycoprotein genes, with little consideration of genome-scale evolution. Herein, we report the first genomic and evolutionary analysis using complete genome sequences of all recognised lyssavirus genotypes, including 14 new complete genomes of field isolates from 6 genotypes and one genotype that is completely sequenced for the first time. In doing so we significantly increase the extent of genome sequence data available for these important viruses. Our analysis of these genome sequence data reveals that all lyssaviruses have the same genomic organization. A phylogenetic analysis reveals strong geographical structuring, with the greatest genetic diversity in Africa, and an independent origin for the two known genotypes that infect European bats. We also suggest that multiple genotypes may exist within the diversity of viruses currently classified as 'Lagos Bat'. In sum, we show that rigorous phylogenetic techniques based on full length genome sequence provide the best discriminatory power for genotype classification within the lyssaviruses.

Lyssaviruses are highly neurotropic viruses associated with neuronal apoptosis. Previous observations have indicated that the matrix proteins (M) of some lyssaviruses induce strong neuronal apoptosis. However, the molecular mechanism(s) involved in this phenomenon is still unknown. We show that for Mokola virus (MOK), a lyssavirus of low pathogenicity, the M (M-MOK) targets mitochondria, disrupts the mitochondrial morphology, and induces apoptosis. Our analysis of truncated M-MOK mutants suggests that the information required for efficient mitochondrial targeting and dysfunction, as well as caspase-9 activation and apoptosis, is held between residues 46 and 110 of M-MOK. We used a yeast two-hybrid approach, a coimmunoprecipitation assay, and confocal microscopy to demonstrate that M-MOK physically associates with the subunit I of the cytochrome c (cyt- c ) oxidase (CcO) of the mitochondrial respiratory chain; this is in contrast to the M of the highly pathogenic Thailand lyssavirus (M-THA). M-MOK expression induces a significant decrease in CcO activity, which is not the case with M-THA. M-MOK mutations (K77R and N81E) resulting in a similar sequence to M-THA at positions 77 and 81 annul cyt- c release and apoptosis and restore CcO activity. As expected, the reverse mutations, R77K and E81N, introduced in M-THA induce a phenotype similar to that due to M-MOK. These features indicate a novel mechanism for energy depletion during lyssavirus-induced apoptosis.

Background Phi29 polymerase based amplification methods provides amplified DNA with minimal changes in sequence and relative abundance for many biomedical applications. RNA virus detection using microarrays, however, can present a challenge because phi29 DNA polymerase cannot amplify RNA nor small cDNA fragments ( Results WTA successfully amplified cDNA from a panel of RNA viruses representing the diversity of ribovirus genome sizes. We amplified a range of genome copy numbers from 15 to 4 × 107 using WTA, which yielded quantities of amplified DNA as high as 1.2 μg/μl or 1010 target copies. The amplification factor varied between 109 and 106. We also demonstrated that co-amplification occurred when viral RNA was mixed with bacterial DNA. Conclusion This is the first report in the scientific literature showing that a modified WGA (WTA) approach can be successfully applied to viral genomic RNA of all sizes. Amplifying viral RNA by WTA provides considerably better sensitivity and accuracy of detection compared to random RT-PCR.

NF-κB transcription factors are crucial for many cellular processes. NF-κB is activated by viral infections to induce expression of antiviral cytokines. Here, we identified a novel member of the human NF-κB family, denoted RelAp43, the nucleotide sequence of which contains several exons as well as an intron of the RelA gene. RelAp43 is expressed in all cell lines and tissues tested and exhibits all the properties of a NF-κB protein. Although its sequence does not include a transactivation domain, identifying it as a class I member of the NF-κB family, it is able to potentiate RelA-mediated transactivation and stabilize dimers comprising p50. Furthermore, RelAp43 stimulates the expression of HIAP1, IRF1, and IFN-β - three genes involved in cell immunity against viral infection. It is also targeted by the matrix protein of lyssaviruses, the agents of rabies, resulting in an inhibition of the NF-κB pathway. Taken together, our data provide the description of a novel functional member of the NF-κB family, which plays a key role in the induction of anti-viral innate immune response., Author Summary The homeostasis of living cells is tightly regulated by signaling pathways, most of them being pleiotropic, which makes their understanding crucial in biology. One of them, the NF-κB pathway, includes a family of transcription factors involved in cell survival, proliferation, differentiation, and cell immunity. In this study, we identified a novel human member of the NF-κB family that we named RelAp43. It shares all the main characteristics of the already known NF-κB family members. Moreover, we demonstrated that RelAp43 induced specifically the expression of genes involved in the innate immune response against viruses. Interestingly, we showed that RelAp43 is specifically targeted by the matrix protein of rabies virus, which contributes to the pathogenesis of the virus and its escape from innate immune response. Taken together, our data provide the description of a novel functional member of the NF-κB family, which is involved in the induction of innate immune response against virus infection.