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author:("Bourhy, hervs")
1.  A Preliminary Study of Viral Metagenomics of French Bat Species in Contact with Humans: Identification of New Mammalian Viruses 
PLoS ONE  2014;9(1):e87194.
The prediction of viral zoonosis epidemics has become a major public health issue. A profound understanding of the viral population in key animal species acting as reservoirs represents an important step towards this goal. Bats harbor diverse viruses, some of which are of particular interest because they cause severe human diseases. However, little is known about the diversity of the global population of viruses found in bats (virome). We determined the viral diversity of five different French insectivorous bat species (nine specimens in total) in close contact with humans. Sequence-independent amplification, high-throughput sequencing with Illumina technology and a dedicated bioinformatics analysis pipeline were used on pooled tissues (brain, liver and lungs). Comparisons of the sequences of contigs and unassembled reads provided a global taxonomic distribution of virus-related sequences for each sample, highlighting differences both within and between bat species. Many viral families were present in these viromes, including viruses known to infect bacteria, plants/fungi, insects or vertebrates, the most relevant being those infecting mammals (Retroviridae, Herpesviridae, Bunyaviridae, Poxviridae, Flaviviridae, Reoviridae, Bornaviridae, Picobirnaviridae). In particular, we detected several new mammalian viruses, including rotaviruses, gammaretroviruses, bornaviruses and bunyaviruses with the identification of the first bat nairovirus. These observations demonstrate that bats naturally harbor viruses from many different families, most of which infect mammals. They may therefore constitute a major reservoir of viral diversity that should be analyzed carefully, to determine the role played by bats in the spread of zoonotic viral infections.
doi:10.1371/journal.pone.0087194
PMCID: PMC3906132  PMID: 24489870
2.  Recent Emergence and Spread of an Arctic-Related Phylogenetic Lineage of Rabies Virus in Nepal 
Rabies is a zoonotic disease that is endemic in many parts of the developing world, especially in Africa and Asia. However its epidemiology remains largely unappreciated in much of these regions, such as in Nepal, where limited information is available about the spatiotemporal dynamics of the main etiological agent, the rabies virus (RABV). In this study, we describe for the first time the phylogenetic diversity and evolution of RABV circulating in Nepal, as well as their geographical relationships within the broader region. A total of 24 new isolates obtained from Nepal and collected from 2003 to 2011 were full-length sequenced for both the nucleoprotein and the glycoprotein genes, and analysed using neighbour-joining and maximum-likelihood phylogenetic methods with representative viruses from all over the world, including new related RABV strains from neighbouring or more distant countries (Afghanistan, Greenland, Iran, Russia and USA). Despite Nepal's limited land surface and its particular geographical position within the Indian subcontinent, our study revealed the presence of a surprising wide genetic diversity of RABV, with the co-existence of three different phylogenetic groups: an Indian subcontinent clade and two different Arctic-like sub-clades within the Arctic-related clade. This observation suggests at least two independent episodes of rabies introduction from neighbouring countries. In addition, specific phylogenetic and temporal evolution analysis of viruses within the Arctic-related clade has identified a new recently emerged RABV lineage we named as the Arctic-like 3 (AL-3) sub-clade that is already widely spread in Nepal.
Author Summary
Rabies is endemic in most Asian countries and represents a serious public health issue, with an estimated 31,000 people dying each year of this disease. The majority of human cases are transmitted by domestic dogs, which act as the principal reservoir host and vector. However, molecular epidemiology and evolutionary dynamics of the main etiological agent, the rabies virus (RABV), remains largely unappreciated in some regions such as in Nepal. Based on a subset of 24 new Nepalese isolates collected from 2003 to 2011 and representative RABV strains at a global scale, phylogenetic analysis based on the complete nucleoprotein and glycoprotein genes sequences revealed the presence of a surprising wide genetic diversity of RABV circulating in this country. The presence of three different co-existing phylogenetic groups was identified: an Indian subcontinent clade and two different Arctic-like sub-clades within the Arctic-related clade, namely Arctic-like (AL)-1, lineage a (AL-1a), and AL-3. Among these clusters, the AL-3 sub-clade appears as the major Nepalese phylogroup which emerged relatively recently in this country, within the last 30 years. These data has raised some concerns about the exchange of RABV between different countries, and provided key elements for implementation of effective control measures of rabies in Nepal.
doi:10.1371/journal.pntd.0002560
PMCID: PMC3836727  PMID: 24278494
3.  Rabies Risk: Difficulties Encountered during Management of Grouped Cases of Bat Bites in 2 Isolated Villages in French Guiana 
In French Guiana, from 1984 to 2011, 14 animal rabies cases and 1 human rabies case (2008) were diagnosed. In January 2011, vampire-bat attacks occurred in 2 isolated villages. In mid-January, a medical team from the Cayenne Centre for Anti-Rabies Treatment visited the sites to manage individuals potentially exposed to rabies and, in April, an anti-rabies vaccination campaign for dogs was conducted. Twenty individuals were bitten by bats in 1 month, most frequently on the feet. The median time to start management was 15 days. The complete Zagreb vaccination protocol (2 doses on day 0 and 1 dose on days 7 and 21) was administered to 16 patients, 12 also received specific immunoglobulins. The antibody titration was obtained for 12 patients (different from those who received immunoglobulins). The antibody titers were ≥0.5 EU/mL for all of them. The serology has not been implemented for the 12 patients who received immunoglobulins. Accidental destruction of a vampire-bat colony could be responsible for the attacks. The isolation and absence of sensitization of the populations were the main explanations for the management difficulties encountered. Sensitization programs should be conducted regularly.
Author Summary
Rabies is a disease almost invariably fatal in humans once the first clinical signs appear. In French Guiana bats represent the virus reservoir, especially vampire bats. From 1984 to 2011, 14 animal rabies cases and 1 human rabies case (2008) were diagnosed. In case of bat bite, anti-rabies immunoglobulins (RIG) and rabies vaccine must be rapidly administrated. The specific rabies management is exclusively performed by Centre for Anti-Rabies Treatment (CART), located at the Institut Pasteur in Cayenne, the prefecture of French Guiana, and 6 Anti-Rabies Treatment Outposts distributed along the coastal edge and along the two main rivers. Only a CART physician can administer RIG. In January 2011, vampire-bat attacks occurred in 2 isolated villages. In mid-January, a medical team from the CART visited the sites to manage individuals potentially exposed to rabies and, in April, an anti-rabies vaccination campaign for dogs was conducted. The most relevant contribution of this study is to underline difficulties to provide rabies post-exposure prophylaxis to remote populations exposed to bat rabies in the Amazonian region and to show the lack of awareness of these rural populations concerning rabies and the risk associated to vampire bats.
doi:10.1371/journal.pntd.0002258
PMCID: PMC3694830  PMID: 23826400
4.  Ecological Factors Associated with European Bat Lyssavirus Seroprevalence in Spanish Bats 
PLoS ONE  2013;8(5):e64467.
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.
doi:10.1371/journal.pone.0064467
PMCID: PMC3659107  PMID: 23700480
5.  Surveillance and control of rabies in La Reunion, Mayotte, and Madagascar 
Veterinary Research  2013;44(1):77.
Mayotte and La Reunion islands are currently free of animal rabies and surveillance is performed by the French Human and Veterinary Public Health Services. However, dog rabies is still enzootic in Madagascar with 4 to 10 confirmed human cases each year. The number of antirabies medical centres in Madagascar is still scarce to provide easy access to the local population for post-exposure rabies prophylaxis. Furthermore, stray dog populations are considerable and attempts to control rabies by mass campaigns of dog vaccination have not received sufficient attention from the national health authorities. To address these challenges, an expanded program to control rabies needs to be initiated by the Malagasy authorities.
doi:10.1186/1297-9716-44-77
PMCID: PMC3848982  PMID: 24016204
6.  RelAp43, a Member of the NF-κB Family Involved in Innate Immune Response against Lyssavirus Infection 
PLoS Pathogens  2012;8(12):e1003060.
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.
doi:10.1371/journal.ppat.1003060
PMCID: PMC3521698  PMID: 23271966
7.  The origin and phylogeography of dog rabies virus 
The Journal of General Virology  2008;89(Pt 11):2673-2681.
Rabies is a progressively fatal and incurable viral encephalitis caused by a lyssavirus infection. Almost all of the 55 000 annual rabies deaths in humans result from infection with dog rabies viruses (RABV). Despite the importance of rabies for human health, little is known about the spread of RABV in dog populations, and patterns of biodiversity have only been studied in limited geographical space. To address these questions on a global scale, we sequenced 62 new isolates and performed an extensive comparative analysis of RABV gene sequence data, representing 192 isolates sampled from 55 countries. From this, we identified six clades of RABV in non-flying mammals, each of which has a distinct geographical distribution, most likely reflecting major physical barriers to gene flow. Indeed, a detailed analysis of phylogeographic structure revealed only limited viral movement among geographical localities. Using Bayesian coalescent methods we also reveal that the sampled lineages of canid RABV derive from a common ancestor that originated within the past 1500 years. Additionally, we found no evidence for either positive selection or widespread population bottlenecks during the global expansion of canid RABV. Overall, our study reveals that the stochastic processes of genetic drift and population subdivision are the most important factors shaping the global phylogeography of canid RABV.
doi:10.1099/vir.0.2008/003913-0
PMCID: PMC3326349  PMID: 18931062
8.  Evaluation of High-Throughput Sequencing for Identifying Known and Unknown Viruses in Biological Samples▿† 
Journal of Clinical Microbiology  2011;49(9):3268-3275.
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.
doi:10.1128/JCM.00850-11
PMCID: PMC3165575  PMID: 21715589
9.  First Human Rabies Case in French Guiana, 2008: Epidemiological Investigation and Control 
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.
doi:10.1371/journal.pntd.0001537
PMCID: PMC3283561  PMID: 22363830
10.  Risk for Rabies Importation from North Africa 
Emerging Infectious Diseases  2011;17(12):2187-2193.
doi:10.3201/eid1712.110300
PMCID: PMC3311213  PMID: 22185767
Rabies; North Africa; Europe; viruses; importation; travelers; zoonoses
11.  Recent Advances in Prevention and Control of Rabies 
doi:10.4061/2011/956428
PMCID: PMC3180785  PMID: 21991447
12.  Laboratory Surveillance of Rabies in Humans, Domestic Animals, and Bats in Madagascar from 2005 to 2010 
Background. Rabies virus (RABV) has circulated in Madagascar at least since the 19th century. Objectives. To assess the circulation of lyssavirus in the island from 2005 to 2010. Materials and Methods. Animal (including bats) and human samples were tested for RABV and other lyssavirus using antigen, ribonucleic acid (RNA), and antibodies detection and virus isolation. Results. Half of the 437 domestic or tame wild terrestrial mammal brains tested were found RABV antigen positive, including 54% of the 341 dogs tested. This percentage ranged from 26% to 75% across the period. Nine of the 10 suspected human cases tested were laboratory confirmed. RABV circulation was confirmed in 34 of the 38 districts sampled. No lyssavirus RNA was detected in 1983 bats specimens. Nevertheless, antibodies against Lagos bat virus were detected in the sera of 12 among 50 Eidolon dupreanum specimens sampled. Conclusion. More than a century after the introduction of the vaccine, rabies still remains endemic in Madagascar.
doi:10.4061/2011/727821
PMCID: PMC3170745  PMID: 21991442
13.  Renewed Global Partnerships and Redesigned Roadmaps for Rabies Prevention and Control 
Canine rabies, responsible for most human rabies deaths, is a serious global public health concern. This zoonosis is entirely preventable, but by focusing solely upon rabies prevention in humans, this “incurable wound” persists at high costs. Although preventing human deaths through canine rabies elimination is feasible, dog rabies control is often neglected, because dogs are not considered typical economic commodities by the animal health sector. Here, we demonstrate that the responsibility of managing rabies falls upon multiple sectors, that a truly integrated approach is the key to rabies elimination, and that considerable progress has been made to this effect. Achievements include the construction of global rabies networks and organizational partnerships; development of road maps, operational toolkits, and a blueprint for rabies prevention and control; and opportunities for scaling up and replication of successful programs. Progress must continue towards overcoming the remaining challenges preventing the ultimate goal of rabies elimination.
doi:10.4061/2011/923149
PMCID: PMC3135331  PMID: 21776359
14.  Lyssavirus Detection and Typing Using Pyrosequencing▿#‖ 
Journal of Clinical Microbiology  2011;49(5):1932-1938.
Rabies is a fatal zoonosis caused by a nonsegmented negative-strand RNA virus, namely, rabies virus (RABV). Apart from RABV, at least 10 additional species are known as rabies-related lyssaviruses (RRVs), and some of them are responsible for occasional spillovers into humans. More lyssaviruses have also been detected recently in different bat ecosystems, thanks to the application of molecular diagnostic methods. Due to the variety of the members of the genus Lyssavirus, there is the necessity to develop a reliable molecular assay for rabies diagnosis able to detect and differentiate among the existing rabies and rabies-related viruses. In the present study, a pyrosequencing protocol targeting the 3′ terminus of the nucleoprotein (N) gene was applied for the rapid characterization of lyssaviruses. Correct identification of species was achieved for each sample tested. Results from the pyrosequencing assay were also confirmed by those obtained using the Sanger sequencing method. A pan-lyssavirus one-step reverse transcription (RT)-PCR was developed within the framework of the pyrosequencing procedure. The sensitivity (Se) of the one-step RT-PCR assay was determined by using in vitro-transcribed RNA and serial dilutions of titrated viruses. The assay demonstrated high analytical and relative specificity (Sp) (98.94%) and sensitivity (99.71%). To date, this is the first case in which pyrosequencing has been applied for lyssavirus identification using a cheaper diagnostic approach than the one for all the other protocols for rapid typing that we are acquainted with. Results from this study indicate that this procedure is suitable for lyssavirus detection in samples of both human and animal origin.
doi:10.1128/JCM.02015-10
PMCID: PMC3122702  PMID: 21389152
15.  Two Overlapping Domains of a Lyssavirus Matrix Protein That Acts on Different Cell Death Pathways ▿  
Journal of Virology  2010;84(19):9897-9906.
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 cytochrome c oxidase (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.
doi:10.1128/JVI.00761-10
PMCID: PMC2937802  PMID: 20631119
16.  Application of Broad-Spectrum Resequencing Microarray for Genotyping Rhabdoviruses▿  
Journal of Virology  2010;84(18):9557-9574.
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.
doi:10.1128/JVI.00771-10
PMCID: PMC2937603  PMID: 20610710
17.  Correction: Phylodynamics and Human-Mediated Dispersal of a Zoonotic Virus 
PLoS Pathogens  2011;7(1):10.1371/annotation/66c3b26f-7b78-4412-96d0-c960b8d74b50.
doi:10.1371/annotation/66c3b26f-7b78-4412-96d0-c960b8d74b50
PMCID: PMC3029409
18.  Rabies, Still Neglected after 125 Years of Vaccination 
doi:10.1371/journal.pntd.0000839
PMCID: PMC2994912  PMID: 21152052
20.  Phylodynamics and Human-Mediated Dispersal of a Zoonotic Virus 
PLoS Pathogens  2010;6(10):e1001166.
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.
doi:10.1371/journal.ppat.1001166
PMCID: PMC2965766  PMID: 21060816
21.  Structure of the Nucleoprotein Binding Domain of Mokola Virus Phosphoprotein▿  
Journal of Virology  2009;84(2):1089-1096.
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.
doi:10.1128/JVI.01520-09
PMCID: PMC2798355  PMID: 19906936
22.  Imported Episodic Rabies Increases Patient Demand for and Physician Delivery of Antirabies Prophylaxis 
Background
Imported cases threaten rabies reemergence in rabies-free areas. During 2000–2005, five dog and one human rabies cases were imported into France, a rabies-free country since 2001. The Summer 2004 event led to unprecedented media warnings by the French Public Health Director. We investigated medical practice evolution following the official elimination of rabies in 2001; impact of subsequent episodic rabies importations and national newspaper coverage on demand for and delivery of antirabies prophylaxis; regular transmission of epidemiological developments within the French Antirabies Medical Center (ARMC) network; and ARMC discussions on indications of rabies post-exposure prophylaxis (RPEP).
Methodology/Principal Findings
Annual data collected by the National Reference Center for Rabies NRCR (1989–2006) and the exhaustive database (2000–2005) of 56 ARMC were analyzed. Weekly numbers of patients consulting at ARMC and their RPEP- and antirabies-immunoglobulin (ARIG) prescription rates were determined. Autoregressive integrated moving-average modeling and regression with autocorrelated errors were applied to examine how 2000–2005 episodic rabies events and their related national newspaper coverage affected demand for and delivery of RPEP. A slight, continuous decline of rabies-dedicated public health facility attendance was observed from 2000 to 2004. Then, during the Summer 2004 event, patient consultations and RPEP and ARIG prescriptions increased by 84%, 19.7% and 43.4%, respectively. Moreover, elevated medical resource use persisted in 2005, despite communication efforts, without any secondary human or animal case.
Conclusions
Our findings demonstrated appropriate responsiveness to reemerging rabies cases and effective newspaper reporting, as no secondary case occurred. However, the ensuing demand on medical resources had immediate and long-lasting effects on rabies-related public health resources and expenses. Henceforth, when facing such an event, decision-makers must anticipate the broad impact of their media communications to counter the emerging risk on maintaining an optimal public health organization and implement a post-crisis communication strategy.
Author Summary
Rabies has been eliminated from a large part of the European Union and, thus, any newly imported cases threaten its reemergence. The 2000–2005 data derived from the exhaustive surveillance system implemented in France was analyzed to evaluate the impact on demand for and delivery of antirabies prophylaxis following introduction of five rabies-infected dogs and one infected human into this rabies-free area. Using these events, we were able to illustrate the difficulties encountered in reducing the demand for and prescription of post-exposure rabies prophylaxis in this context of episodic importation. Moreover, we highlighted the need for public health decision-makers to anticipate the broad spectrum of consequences of their media communications and to prepare appropriate responses (in terms of health resources) to maintain an optimally effective public health organization after importation of an exotic infectious agent or its emergence. These responses are particularly relevant in the context of limited availability of rabies post-exposure prophylaxis, especially antirabies immunoglobulin.
doi:10.1371/journal.pntd.0000723
PMCID: PMC2889823  PMID: 20582307
23.  Expression, purification and crystallization of a lyssavirus matrix (M) protein 
The expression, purification and crystallization of the full-length matrix protein from three lyssaviruses is described.
The matrix (M) proteins of lyssaviruses (family Rhabdoviridae) are crucial to viral morphogenesis as well as in modulating replication and transcription of the viral genome. To date, no high-resolution structural information has been obtained for full-length rhabdovirus M. Here, the cloning, expression and purification of the matrix proteins from three lyssaviruses, Lagos bat virus (LAG), Mokola virus and Thailand dog virus, are described. Crystals have been obtained for the full-length M protein from Lagos bat virus (LAG M). Successful crystallization depended on a number of factors, in particular the addition of an N-terminal SUMO fusion tag to increase protein solubility. Diffraction data have been recorded from crystals of native and selenomethionine-labelled LAG M to 2.75 and 3.0 Å resolution, respectively. Preliminary analysis indicates that these crystals belong to space group P6122 or P6522, with unit-cell parameters a = b = 56.9–57.2, c = 187.9–188.6 Å, consistent with the presence of one molecule per asymmetric unit, and structure determination is currently in progress.
doi:10.1107/S1744309108004557
PMCID: PMC2374255  PMID: 18391421
matrix proteins; Rhabdoviridae; lyssaviruses; Lagos bat virus; SUMO tag
24.  Rabies Situation in Cambodia 
Background
Rabies, a fatal but preventable zoonosis, is a major public health problem in developing countries. In Cambodia the disease burden is largely underestimated because patients with encephalitis following dog bites are rarely hospitalized and die at home. Since 1998 Institut Pasteur in Cambodia (IPC), Phnom Penh has been the only source of free post-exposure prophylaxis (PEP) and post-mortem diagnosis.
Methods
The 1998–2007 data compiled by IPC was analyzed to describe all treated patients for PEP, results of human testing and confirmed rabies cases, and results of animal testing. From dog bites' characteristics, we defined a suspected rabid dog bite injury (SRDBI) in humans as a bite that was unprovoked, from a dog that died spontaneously, or from a dog that was reported sick. We applied a deterministic probability model to estimate 2007 rabies human mortality nationwide from the estimated incidence of rabid dog bites, the body distribution of bite wounds, and the probability of PEP access.
Results
During 1998–2007, 124,749 patients received PEP at IPC (average 12,470; range 8,907–14,475), and 63 fatal human cases presenting with encephalitis following a dog bite were reported, in which 73% were confirmed positive for rabies by direct immunofluorescence assay or by reverse-transcriptase polymerase chain reaction. During 1998–2007, IPC tested 1,255 animal brain samples; 1,214 (97%) were from dogs including 610 (49%) positive samples. In 2007, 14,475 patients received PEP (100 PEP/100,000 people in Cambodia) including 95% who resided in Phnom Penh (615 PEP/100,000) or five neighboring provinces. The predictive model estimated 810 human rabies deaths would occur in 2007 (95%confidence interval [CI] 394–1,607), an incidence of 5.8/100,000 (95% CI 2.8–11.5).
Conclusions
Access to PEP is only sufficient for Phnom Penh residents. In 2007, the estimated rabies related mortality exceeded that of malaria and that of dengue. A national rabies control program is needed to improve surveillance and access to PEP, and to initiate vaccination campaigns in dogs.
Author Summary
In Cambodia, rabies still elicits fear in the communities. Since 1998 the Institut Pasteur in Cambodia (IPC), Phnom Penh has been the only source of free post-exposure prophylaxis (PEP) and post mortem diagnosis. During 1998–2007, on average ∼12,400 patients received PEP annually at IPC (range 8,907–14,475) and 63 fatal human cases presenting with encephalitis following a dog bite were reported including 73% who tested positive by fluorescent-antibody test on brain samples or/and by reverse-transcriptase polymerase chain reaction on skin, cerebrospinal fluid, or urine. In 2007, 14,475 patients received PEP (100 PEP/100,000 people in Cambodia) including 95% who resided in Phnom Penh city (615 PEP/100,000) or five neighboring provinces. Using a step-by-step probability model, we estimated that 810 human rabies deaths would occur in 2007 (95% confidence interval [CI] 394–1,607); an incidence of 5.8/100,000 (95%CI 2.8–11.5). As a result, despite high attendance at the IPC's PEP center most Cambodians living in peripheral provinces in Cambodia may not have adequate access to PEP. Finally, the model generated one of the highest incidences of rabies worldwide. A national rabies control program is needed to improve surveillance and access to PEP, and to initiate vaccination campaigns in dogs.
doi:10.1371/journal.pntd.0000511
PMCID: PMC2731168  PMID: 19907631
25.  European Bat Lyssavirus Transmission among Cats, Europe 
Emerging Infectious Diseases  2009;15(2):280-284.
We identified 2 cases of European bat lyssavirus subtype 1 transmission to domestic carnivores (cats) in France. Bat-to-cat transmission is suspected. Low amounts of virus antigen in cat brain made diagnosis difficult.
doi:10.3201/eid1502.080637
PMCID: PMC2657616  PMID: 19193273
Rabies; lyssavirus; chiroptera; cats; diagnosis; spillover; transmission; Europe; dispatch

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