Rabies pathogenesis and immunology.

Once clinical disease is manifest, the rabies virus is one of the few pathogens known to science with a near 100% fatality rate and, as such, this zoonotic pathogen has shaped both humanity and the history of science. However, today rabies is still considered to be a neglected tropical disease, despite the fact that it causes more than 59,000 human deaths each year. Although effective vaccines are available to combat the disease, the underlying mechanisms of its pathogenicity and immunology remain poorly defined. In this paper, the existing knowledge of the pathogenesis and immunological response to the rabies virus in infected hosts is described.

Le virus rabique est l'un des rares agents pathogènes connus capables de conduire à une mort quasi certaine lorsque la phase clinique de la maladie est atteinte. En tant que telle, cette zoonose a toujours marqué tant les sociétés humaines que l'histoire de la science. Pourtant, la rage est toujours considérée aujourd'hui comme une maladie tropicale négligée bien qu'elle continue de faire plus de 59 000 victimes humaines chaque année. Si des vaccins efficaces sont disponibles pour prévenir l'infection rabique, les mécanismes sous-jacents de la pathogenèse de la rage ou la réponse immunitaire qu'elle provoque chez l'hôte infecté demeurent peu connus. Les auteurs font le point sur l'état actuel des connaissances dans ce domaine.

Una vez se ha manifestado la enfermedad en su forma clínica, el virus de la rabia es uno de los pocos patógenos conocidos por los científicos que presenta una tasa de letalidad cercana al 100%. De ahí que este patógeno zoonótico haya dejado su impronta tanto en la humanidad como en la historia de la ciencia. Todavía hoy, sin embargo, pese a que causa la muerte de más de 59 000 personas al año, la rabia sigue siendo considerada una enfermedad tropical desatendida. Si bien hay vacunas eficaces para combatir la enfermedad, aún no se conocen bien los mecanismos que subyacen a su patogenicidad e inmunología. Los autores repasan lo que hasta ahora se sabe de la patogénesis y la respuesta inmunitaria al virus de la rabia en los anfitriones infectados.

Crimean-Congo haemorrhagic fever.

Crimean-Congo haemorrhagic fever (CCHF) is one of the most widespread arboviroses in the world. It is present in Africa, south-east Europe, the Middle East and Asia. It is caused by a nairovirus (Bunyaviridae family) transmitted by several species of ticks. The geographical distribution of the disease coincides with the distribution of Hyalomma ticks. While infected livestock do not show signs of illness, humans are severely affected, with a high mortality rate. The most common symptoms are high fever, dizziness, headache, vomiting and haemorrhages. Pathogenesis studies in interferon-receptor-deficient mice indicated that the interferon response is crucial in controlling virus propagation and in protecting against the disease. Detection of the virus in biological material is currently performed by reverse-transcription polymerase chain reaction. Enzyme-linked immunosorbent assay and indirect immunofluorescence are used to detect the presence of CCHF virus-specific antibodies. In the 1970s, a formalin-inactivated vaccine prepared from suckling mouse brain was used in Eastern Europe and the former Soviet Union, but its efficacy remains to be proven. Treatment of patients with ribavirin is recommended by the World Health Organization, but it should be administered as early as possible. Although important progress has been made over the last few decades, many questions about the pathogenesis and epidemiology of the disease are still to be addressed and there is a need to develop efficient vaccines and antivirals.

Fighting rabies in Eastern Europe, the Middle East and Central Asia--experts call for a regional initiative for rabies elimination.

MEEREB is an informal network of rabies experts from the Middle East, Eastern Europe and Central Asia, seeking to eliminate rabies from the region. They met for the second time to review the current rabies situation, both globally and in their respective countries, highlighting current rabies control problems and potential solutions. Success stories in Latin America, in Western Europe, in some Asian countries, as well as in Croatia and Serbia prove that elimination of human rabies is achievable in the MEEREB region. It requires political willingness and cooperation of all stakeholders, including Ministries of Health and of Agriculture; adequate management of animal bites through post-exposure prophylaxis; pre-exposure prophylaxis for populations at high risk of rabies exposure, animal vaccination and humane control of stray dog populations. MEEREB members called for a regional initiative for rabies elimination in Eastern Europe and the Middle East. They are confident that the elimination of human rabies of canine origin can be achieved in the region through adopting a One Health approach, and that campaigns for rabies elimination will have significant benefit for public health, including strengthening the structure for control of other zoonoses.

Experimental infection of Artibeus intermedius bats with serotype-2 dengue virus.

Dengue fever is caused by a flavivirus that primarily infects humans and Aedes sp. mosquitoes. However, viral replication in wild animals other than non-human primates has been scarcely studied. In this report, the susceptibility of Artibeus intermedius frugivorous bat to serotype-2 dengue virus (DENV-2) infection was tested. Twenty-three bats were intraperitoneally inoculated with different viral loads of DENV-2 (New Guinea-C strain). Forty-three percent of the infected bats developed bruises on the chest or on the wings. Histological analyses showed structural alterations in the spleen and bleeding in liver and intestine, but the virus was not detected by RT-PCR in any of the analyzed tissues, and it was found in only one bat (kidney) by semi-nested RT-PCR. In sera, the viral RNA was detected by semi-nested RT-PCR in 39% of bats, but only 8% of bats seroconverted. Overall, these data indicate that DENV-2 replicates poorly in these bats, suggesting they are not suitable hosts to this virus.

Molecular diagnostic and genetic characterization of highly pathogenic viruses: application during Crimean-Congo haemorrhagic fever virus outbreaks in Eastern Europe and the Middle East.

Several haemorrhagic fevers are caused by highly pathogenic viruses that must be handled in Biosafety level 4 (BSL-4) containment. These zoonotic infections have an important impact on public health and the development of a rapid and differential diagnosis in case of outbreak in risk areas represents a critical priority. We have demonstrated the potential of a DNA resequencing microarray (PathogenID v2.0) for this purpose. The microarray was first validated in vitro using supernatants of cells infected with prototype strains from five different families of BSL-4 viruses (e.g. families Arenaviridae, Bunyaviridae, Filoviridae, Flaviviridae and Paramyxoviridae). RNA was amplified based on isothermal amplification by Phi29 polymerase before hybridization. We were able to detect and characterize Nipah virus and Crimean-Congo haemorrhagic fever virus (CCHFV) in the brains of experimentally infected animals. CCHFV was finally used as a paradigm for epidemics because of recent outbreaks in Turkey, Kosovo and Iran. Viral variants present in human sera were characterized by BLASTN analysis. Sensitivity was estimated to be 10(5) -10(6) PFU/mL of hybridized cDNA. Detection specificity was limited to viral sequences having ~13-14% of global divergence with the tiled sequence, or stretches of ~20 identical nucleotides. These results highlight the benefits of using the PathogenID v2.0 resequencing microarray to characterize geographical variants in the follow-up of haemorrhagic fever epidemics; to manage patients and protect communities; and in cases of bioterrorism.

A five-year perspective on the situation of haemorrhagic fever with renal syndrome and status of the hantavirus reservoirs in Europe, 2005-2010.

Hantavirus infections are reported from many countries in Europe and with highly variable annual case numbers. In 2010, more than 2,000 human cases were reported in Germany, and numbers above the baseline have also been registered in other European countries. Depending on the virus type human infections are characterised by mild to severe forms of haemorrhagic fever with renal syndrome. The member laboratories of the European Network for diagnostics of Imported Viral Diseases present here an overview of the progression of human cases in the period from 2005 to 2010. Further we provide an update on the available diagnostic methods and endemic regions in their countries, with an emphasis on occurring virus types and reservoirs.

The occurrence of rabies in pre-Columbian Central America: an historical search.

Rabies is considered one of the oldest infectious diseases known to humans. However, the first written reports on rabies cases in the Americas did not appear until the first decade of the 18th century from Mexico. In an attempt to clarify if the disease was already present in pre-Columbian times, we searched for evidence in the Maya and Aztec cultures. Other sources of information were early manuscripts written by the conquistadors and early explorers. We did not identify any unequivocal direct evidence that the disease rabies was known in pre-Columbian Central America but sufficient circumstantial evidence is available suggesting that (bat) rabies was already present in these early times.

Environmental risk factors for haemorrhagic fever with renal syndrome in a French new epidemic area.

In France, haemorrhagic fever with renal syndrome (HFRS) is endemic along the Belgian border. However, this rodent-borne zoonosis caused by the Puumala virus has recently spread south to the Franche-Comté region. We investigated the space-time distribution of HFRS and evaluated the influence of environmental factors that drive the hantavirus reservoir abundance and/or the disease transmission in this area. A scan test clearly indicated space-time clustering, highlighting a single-year (2005) epidemic in the southern part of the region, preceded by a heat-wave 2 years earlier. A Bayesian regression approach showed an association between a variable reflecting biomass (normalized difference vegetation index) and HFRS incidence. The reasons why HFRS cases recently emerged remain largely unknown, and climate parameters alone do not reliably predict outbreaks. Concerted efforts that combine reservoir monitoring, surveillance, and investigation of human cases are warranted to better understand the epidemiological patterns of HFRS in this area.

Human rabies due to lyssavirus infection of bat origin.

Rabies is a fatal viral encephalitis and results from infection with viruses belonging to the genus Lyssavirus. Infection usually results from a bite from a dog infected with classical rabies virus. However, a small number of cases result from contact with bats. It is within bats that most lyssavirus variants, referred to as genotypes, are found. The lyssaviruses found in bats have a distinct geographical distribution and are often restricted to specific bat species. Most have been associated with rabies in humans and in some cases spill-over to domestic animals. Many diagnostic techniques are unable to differentiate rabies virus from other genotypes so it is possible that some human and animal cases go unreported. Furthermore, current vaccines have limited efficacy against some genotypes.

[Viruses and bats: rabies and Lyssavirus]. / Virus et chauves-souris: Rage et Lyssavirus.

Recent emerging zoonoses (hemorrhagic fevers due to Ebola or Marburg virus, encephalitis due to Nipah virus, severe acute respiratory syndrome due to SRAS virus...) outline the potential of bats as vectors for transmission of infectious disease to humans. Such a potential is already known for rabies encephalitis since seven out of the eight genotypes of Lyssavirus are transmitted by bats. In addition, phylogenetic reconstructions indicate that Lyssavirus have evolved in chiropters before their emergence in carnivores. Nevertheless, carnivores remain the most critical vectors for public health, in particular dogs that are originating 55.000 rabies deaths per year, essentially in developing countries. Rabies control in carnivores by parenteral (dog) or oral (wild carnivores) vaccination is efficacious and campaigns start to be more widely applied. On the other hand, rabies control in bat still remains non realistic, particularly as the pathogenicity of bat Lyssavirus for bats is still under debate, suggesting that a "diplomatic relationship" between partners would have arisen from a long term cohabitation. While comparing the interactions that humans and bats establish with Lyssavirus, scientists try to understand the molecular basis ofpathogenicity in man, a indispensable prerequisite to identify antiviral targets in a perspective of therapy.

Experimental infection of serotine bats (Eptesicus serotinus) with European bat lyssavirus type 1a.

The serotine bat (Eptesicus serotinus) accounts for the vast majority of bat rabies cases in Europe and is considered the main reservoir for European bat lyssavirus type 1 (EBLV-1, genotype 5). However, so far the disease has not been investigated in its native host under experimental conditions. To assess viral virulence, dissemination and probable means of transmission, captive bats were infected experimentally with an EBLV-1a virus isolated from a naturally infected conspecific from Germany. Twenty-nine wild caught bats were divided into five groups and inoculated by intracranial (i.c.), intramuscular (i.m.) or subcutaneous (s.c.) injection or by intranasal (i.n.) inoculation to mimic the various potential routes of infection. One group of bats was maintained as uninfected controls. Mortality was highest in the i.c.-infected animals, followed by the s.c. and i.m. groups. Incubation periods varied from 7 to 26 days depending on the route of infection. Rabies did not develop in the i.n. group or in the negative-control group. None of the infected bats seroconverted. Viral antigen was detected in more than 50% of the taste buds of an i.c.-infected animal. Shedding of viable virus was measured by virus isolation in cell culture for one bat from the s.c. group at 13 and 14 days post-inoculation, i.e. 7 days before death. In conclusion, it is postulated that s.c. inoculation, in nature caused by bites, may be an efficient way of transmitting EBLV-1 among free-living serotine bats.

International network for capacity building for the control of emerging viral vector-borne zoonotic diseases: ARBO-ZOONET.

Arboviruses are arthropod-borne viruses, which include West Nile fever virus (WNFV), a mosquito-borne virus, Rift Valley fever virus (RVFV), a mosquito-borne virus, and Crimean-Congo haemorrhagic fever virus (CCHFV), a tick-borne virus. These arthropod-borne viruses can cause disease in different domestic and wild animals and in humans, posing a threat to public health because of their epidemic and zoonotic potential. In recent decades, the geographical distribution of these diseases has expanded. Outbreaks of WNF have already occurred in Europe, especially in the Mediterranean basin. Moreover, CCHF is endemic in many European countries and serious outbreaks have occurred, particularly in the Balkans, Turkey and Southern Federal Districts of Russia. In 2000, RVF was reported for the first time outside the African continent, with cases being confirmed in Saudi Arabia and Yemen. This spread was probably caused by ruminant trade and highlights that there is a threat of expansion of the virus into other parts of Asia and Europe. In the light of global warming and globalisation of trade and travel, public interest in emerging zoonotic diseases has increased. This is especially evident regarding the geographical spread of vector-borne diseases. A multi-disciplinary approach is now imperative, and groups need to collaborate in an integrated manner that includes vector control, vaccination programmes, improved therapy strategies, diagnostic tools and surveillance, public awareness, capacity building and improvement of infrastructure in endemic regions.

Can rabies be eradicated?

Rabies, an acute progressive encephalitis, is an ancient zoonosis. Its distribution encompasses all continents, except Antarctica. Agents consist of at least 11 species orgenotypes of rhabdoviruses, in the Genus Lyssavirus. Susceptible natural hosts include all mammals. Primary reservoirs reside in the Orders Carnivora and Chiroptera. A plethora of variants, maintained by a diversity of abundant hosts, presents a challenge to a strict concept of true eradication. Globally, the domestic dog remains the most significant species for viral transmission, responsible for millions of suspect human exposures and tens of thousands of fatalities. As such, this single major target provides an ideal opportunity for focused intervention programmes in humane disease prevention and control, driven by laboratory-based surveillance and guided via modern epidemiological insights. Historically, substantial technical progress throughout the 20th century led to the development of safe, affordable and efficacious animal and human vaccines, resulting in declining disease burdens in selected developed and developing countries. Regional and local disease resurgence occurs, due in part to a combination of political and economic instability, environmental perturbations, and shifting government priorities. Society must recall that despite the recent recognition of other important emerging infectious diseases, none exceed the case fatality rate of rabies. Given the clear relevance of rabies in public health, agriculture, and conservation biology, substantive international progress must continue towards enhanced public awareness, human rabies prevention, wildlife rabies control, and canine rabies elimination, with renewed collaborative vigour.

Molecular epidemiology of lyssaviruses in Eurasia.

The Lyssavirus genus, a member of the Rhabdoviridae family, consists of seven established related viruses (genotypes 1-7). Rabies cases in Eurasia are principally attributed to three of these genotypes, namely genotype 1 (RABV, classical rabies) and to a lesser extent genotypes 5 and 6 (European bat lyssaviruses type-1 and -2). In addition, four newly identified divergent lyssaviruses have been isolated from insectivorous bats. The molecular diversity of classical rabies viruses (genotype 1, RABV) has been studied at the global level and reference has been made to the existence of a number of European strains in a range of mammalian species. It is accepted that these viruses cluster within a 'Cosmopolitan Lineage' having ancestral roots in Europe in the 17th century before its widespread dispersal to Asia, Africa and the Americas as a result of European exploration and colonization.

A random grid based molecular epidemiological study on EBLV isolates from Germany.

Germany has reported one of the highest levels of EBLV cases in bats in Europe. So far, all isolates originating from Germany have been identified as EBLV-1, using monoclonal antibodies, and a preliminary epidemiological study has indicated that there is a distinct geographical distribution of EBLV-1 in Germany. To further investigate the spatial and temporal distribution of EBLV-1 variants in Germany and their impact on molecular epidemiology, isolates were selected using a random grid sampling procedure based on GIS. Agrid layer 30 km long over the entire area of Germany was applied to 120 geo-referenced isolates and one isolate of each grid cell containing EBLV isolates was randomly chosen. Once selected, the nucleoprotein (N) plus parts of the phosphoprotein (P) gene of each isolate were sequenced using direct cycle sequencing. Results of the subsequent phylogenetic analysis of the N-gene confirmed previous studies on European EBLVs, showing a high sequence homology between German EBLV-1 isolates. Almost identical sequence homologies within certain geographical regions indicate genomic stability during the transmission cycle of EBLV-1, with little geographic spread or intermixing. Interestingly, a 6 bp insertion as well as a single nucleotide insertion, detected in the N-P intergenic region, has been found in EBLV-1 isolates from Germany.

Canine adenovirus based rabies vaccines.

Adenovirus based vectors are very attractive candidates for vaccination purposes as they induce in mammalian hosts potent humoral, mucosal and cellular immune responses to antigens encoded by the inserted genes. We have generated E1-deleted and replication-competent recombinant canine type-2 adenoviruses expressing the rabies virus glycoprotein (G). The effectiveness of both vectors to express a native G protein has been characterized in vitro in permissive cell lines. We compared the humoral and cellular immune responses induced in mice by intramuscular injection of the recombinant canine adenovirus vectors with those induced by a human (Ad5) E1-deleted virus expressing the same rabies G protein. Humoral responses specific to the adenoviruses or the rabies glycoprotein antigens were studied. The influence of the mouse strain was observed using replication-competent canine adenovirus. A high level of rabies neutralizing antibody was observed upon i.m. inoculation, and 100% of mice survived lethal challenge. These results are very promising in the perspective of oral vaccine for dog rabies control.

Comparative analysis of the full genome sequence of European bat lyssavirus type 1 and type 2 with other lyssaviruses and evidence for a conserved transcription termination and polyadenylation motif in the G-L 3' non-translated region.

We report the first full-length genomic sequences for European bat lyssavirus type-1 (EBLV-1) and type-2 (EBLV-2). The EBLV-1 genomic sequence was derived from a virus isolated from a serotine bat in Hamburg, Germany, in 1968 and the EBLV-2 sequence was derived from a virus isolate from a human case of rabies that occurred in Scotland in 2002. A long-distance PCR strategy was used to amplify the open reading frames (ORFs), followed by standard and modified RACE (rapid amplification of cDNA ends) techniques to amplify the 3' and 5' ends. The lengths of each complete viral genome for EBLV-1 and EBLV-2 were 11 966 and 11 930 base pairs, respectively, and follow the standard rhabdovirus genome organization of five viral proteins. Comparison with other lyssavirus sequences demonstrates variation in degrees of homology, with the genomic termini showing a high degree of complementarity. The nucleoprotein was the most conserved, both intra- and intergenotypically, followed by the polymerase (L), matrix and glyco- proteins, with the phosphoprotein being the most variable. In addition, we have shown that the two EBLVs utilize a conserved transcription termination and polyadenylation (TTP) motif, approximately 50 nt upstream of the L gene start codon. All available lyssavirus sequences to date, with the exception of Pasteur virus (PV) and PV-derived isolates, use the second TTP site. This observation may explain differences in pathogenicity between lyssavirus strains, dependent on the length of the untranslated region, which might affect transcriptional activity and RNA stability.

Rabies: epidemiological tendencies and control tools.

Rabies encephalitis still generates 50,000 human deaths/year. It is due to neuron infection by lyssaviruses. Seven genotypes (GT) are currently distinguished within the Lyssavirus genus which segregate in two phylogroups (PG). This classification is constantly evolving due to isolation of new lyssaviruses within bat populations. Functional differences exist between GTs in term of neurotropism, pathogenesis, induction of apoptosis, immunogenicity, and their molecular basis are starting to be elucidated. Lyssavirus vectors are mammals, preferentially from the Carnivora and Chiroptera orders. Phylogenetic reconstruction strongly supports that lyssaviruses evolved in chiropters long before the emergence of carnivoran rabies which very likely occurred through host-switchings from bats to carnivores. If dog rabies control is possible by vaccination and population control, if oral vaccination demonstrated its potential to eliminate rabies from a terrestrial wildlife reservoir (fox in Western Europe), it is unrealistic today to clear lyssaviruses from bats, while bat rabies is a growing concern for both public and animal health. As bat transmit divergent lyssavirus GTs which are not well prevented by available vaccine strains, there is a need to increase the protection spectrum of vaccines. DNA-based immunization with plasmids expressing chimeric G proteins (fusion of two halves from different GTs) was shown to be effective in inducing a complete immune response and to broaden the spectrum of rabies vaccines toward lyssavirus vaccines. Further, the lyssavirus G protein can carry foreign epitopes/antigens in the perspective of multivalent vaccines against various zoonoses of carnivores.

A simple immuno-capture ELISA to estimate rabies viral glycoprotein antigen in vaccine manufacture.

Rabies is an endemic, fatal zoonotic disease in the developing countries. Prevention and post-exposure therapy require safe and efficacious vaccines. The vaccine potency depends on the amount of immunogenic rabies viral glycoprotein antigen in the vaccine preparation. In order to estimate the rabies viral glycoprotein antigen, a specific monoclonal antibody was developed and used in an immuno-capture ELISA (IC-ELISA). The monoclonal antibody binds a conformational epitope on the natively folded rabies viral glycoprotein as indicated by specific, membrane fluorescence on unfixed, rabies virus infected murine neuroblastoma (MNA) cells and glycoprotein gene encoding plasmid transfected COS cells. In addition, the monoclonal antibody competes with and blocks a glycoprotein antigen site III binding monoclonal antibody (mAb-D1, Institut Pasteur, Paris, France). The monoclonal antibody was used in an IC-ELISA using an in-house standard to quantify the rabies viral glycoprotein antigen in 12 vaccine preparations with potency values ranging from 4 to 18 IU. The results indicated a good correlation with the NIH mouse potency assay (r=0.83). The immuno-capture ELISA described in this study can be used to quantify the immunogenic rabies viral glycoprotein antigen in the inactivated rabies viral antigen preparation in a simple and rapid format, which enables better vaccine formulation.