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International audience; In Europe, Puumala virus (PUUV) transmitted by the bank vole (Myodes glareolus) is the causative agent of nephropathia epidemica (NE), a mild form of haemorrhagic fever with renal syndrome. In France, very little is known about the spatial and temporal variability of the virus circulating within bank vole populations. The present study involved monitoring of bank vole population dynamics and PUUV microdiversity over a ten-year period (2000–2009) in two forests of the Ardennes region: Elan and Croix-Scaille. Ardennes region is characterised by different environmental conditions associated with different NE epidemiology. Bank vole density and population parameters were estimated using the capture/marking/recapture method, and blood samples were collected to monitor the overall seroprevalence of PUUV in rodent populations. Phylogenetic analyses of fifty-five sequences were performed to illustrate the genetic diversity of PUUV variants between forests. The pattern of the two forests differed clearly. In the Elan forest, the rodent survival was higher, and this limited turn-over resulted in a lower seroprevalence and diversity of PUUV sequences than in the Croix-Scaille forest. Uncovering the links between host dynamics and virus microevolution is improving our understanding of PUUV distribution in rodents and the NE risk.

Pathogen discovery contributes to our knowledge of bat-borne viruses and is linked to the heightened interest globally in bats as recognised reservoirs of zoonotic agents. The transmission of lyssaviruses from bats-to-humans, domestic animals, or other wildlife species is uncommon, but interest in these pathogens remains due to their ability to cause an acute, progressive, invariably fatal encephalitis in humans. Consequently, the detection and characterisation of bat lyssaviruses continues to expand our knowledge of their phylogroup definition, viral diversity, host species association, geographical distribution, evolution, mechanisms for perpetuation, and the potential routes of transmission. Although the opportunity for lyssavirus cross-species transmission seems rare, adaptation in a new host and the possibility of onward transmission to humans requires continued investigation. Considering the limited efficacy of available rabies biologicals it is important to further our understanding of protective immunity to minimize the threat from these pathogens to public health. Hence, in addition to increased surveillance, the development of a niche pan-lyssavirus vaccine or therapeutic biologics for post-exposure prophylaxis for use against genetically divergent lyssaviruses should be an international priority as these emerging lyssaviruses remain a concern for global public health.

Background Hantaviruses are a group of emerging pathogens causing hemorrhagic fever with renal syndrome and Hantavirus cardiopulmonary syndrome in human. This study was conducted to investigate Hantavirus infection among Iranian viral hemorrhagic fever suspected patients. Methods From April 2014 to June 2016, 113 cases from 25 different provinces of Iran were analyzed for Hantavirus infection by IgM/IgG ELISA and pan-Hantavirus RT-PCR tests. Results Although, viral genome was detected in none of the subjects, IgM and IgG antibodies were detected in 19 and 4 cases, respectively. Differentiation of the anti-Hantavirus antibodies according to virus species by EUROLINE Anti-Hantavirus Profile Kit revealed three Puumala virus IgM positive, one Hantaan virus IgM positive, one Hantaan virus IgM borderline, and two Puumala virus IgG borderline cases. Conclusions This study demonstrates the circulation of Hantaviruses in Iran and calls for further investigations of these life-threatening viruses in the country.

Rabies is a deadly viral disease with an extremely high fatality rate in humans. Previously, it was suggested that an enhancement of the blood-brain barrier (BBB) permeability, which allows immune cells and/or antibodies to enter the central nervous system (CNS) tissue, is critical to clear the infection. In this study, we utilised mannitol to increase BBB permeability in mice infected with highly pathogenic silver-haired bat rabies virus (SHBRV). We found that intraperitoneal injection of mannitol causes a slight, transient increase of BBB permeability in the treated mice. SHBRV-infected mice were treated with intraperitoneally administered mannitol daily from day 3 or day 4 post-infection, but no effect of this treatment on the time of disease onset, clinical signs or survival was observed. This data indicates that the increase of BBB permeability by mannitol is not efficient in promoting CNS virus clearance in SHBRV-infected mice.

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.

International audience; ABMA and its analogue DABMA are two molecules of the adamantane family known to perturbate the endosomal pathway and to inhibit cell infection of several RNA and DNA viruses. Their activity against Rabies Virus (RABV) infection has already been demonstrated in vitro. (Wu et al., 2017, 2019). Here, we describe in more details their mechanism of action by comparison to Arbidol (umifenovir) and Ribavirin, two broad spectrum antivirals against emerging viruses such as Lassa, Ebola, influenza and Hantaan viruses. ABMA and DABMA, delivered 2 h pre-infection, inhibit RABV infection in vitro with an EC50 of 7.8 μM and 14 μM, respectively. They act at post-entry, by causing RABV accumulation within the endosomal compartment and DABMA specifically diminishes the expression of the GTPase Rab7a controlling the fusion of early endosomes to late endosomes or lysosomes. This may suggest that ABMA and DABMA act at different stages of the late endosomal pathway as supported by their different profile of synergy/antagonism with the fusion inhibitor Arbidol. This difference is further confirmed by the RABV mutants induced by successive passages under increasing selective pressure showing a particular involvement of the viral G protein in the DABMA inhibition while ABMA inhibition induces less mutations dispersed in the M, G and L viral proteins. These results suggest new therapeutic perspectives against rabies.

Crimean-Congo hemorrhagic fever virus (CCHFV) circulates in many countries of Asia, Africa, and Europe. CCHFV can cause a severe hemorrhagic fever in humans with case-fatality rates of up to 80%. CCHF is considered to be one of the major emerging diseases spreading to and within Europe. Ticks of the genus Hyalomma function as vector as well as natural reservoir of CCHFV. Ticks feed on various domestic animals (e.g. cattle, sheep, goats) and on wildlife (e.g. hares, hedgehogs). Those animal species play an important role in the life cycle of the ticks as well as in amplification of CCHFV. Here we present a competitive ELISA (cELISA) for the species-independent detection of CCHFV-specific antibodies. For this purpose nucleocapsid (N) protein specific monoclonal antibodies (mAbs) were generated against an Escherichia coli (E. coli) expressed CCHFV N-protein. Thirty-three mAbs reacted with homologous and heterologous recombinant CCHFV antigens in ELISA and Western blot test and 20 of those 33 mAbs reacted additionally in an immunofluorescence assay with eukaryotic cells expressing the N-protein. Ten mAbs were further characterized in a prototype of the cELISA and nine of them competed with positive control sera of bovine origin. The cELISA was established by using the mAb with the strongest competition. For the validation, 833 sera from 12 animal species and from humans were used. The diagnostic sensitivity and specificity of the cELISA was determined to be 95% and 99%, respectively, and 2% of the sera gave inconclusive results. This cELISA offers the possibility for future large-scale screening approaches in various animal species to evaluate their susceptibility to CCHFV infection and to identify and monitor the occurrence of CCHFV.

Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne disease in humans caused by the CCHF virus (CCHFV). The detection of anti-CCHFV antibodies in animals is used to reveal infection risk areas. Therefore a simple, quick and reliable multispecies assay for the detection of CCHFV-specific antibodies is needed. This work presents the development and validation of a novel CCHF double-antigen ELISA for the detection of anti-CCHFV nucleoprotein antibodies. The test requires 30 μl of serum, and results are obtained within 90 min. As the ELISA is based on recombinant N-protein of the IbAr10200 virus, it can be run under standard biosafety conditions. For assay validation, sera from 95 cattle and 176 small ruminants from CCHF-endemic regions (origin: Albania, Cameroon, Kosovo, Former Yugoslav Republic of Macedonia, Mauritania, Pakistan, Turkey) served as a positive reference serum panel. The CCHF antibody status of the positive reference samples had been previously confirmed by two serological assays (species-adapted VectorBest ELISA and Euroimmun IFA). CCHFV strains belonging to three different clades are known to circulate in the countries where the positive samples originated. Sera from 402 cattle and 804 small ruminants from Germany and France served as the negative serum panel, as both countries are considered outside of the CCHFV endemic zone. Sera from monkeys, camels, rats, ferrets, raccoon dogs, raccoons, foxes, hares, pigs and humans were also tested, to determine the suitability of this novel ELISA for these species. All negative reference sera were confirmed by the CCHF double-antigen ELISA, indicating a specificity of 100%. 268 of 271 positive reference sera tested positive for CCHFV-specific antibodies, 8sensitivity of 99%9. Further analysis are needed to ensure a recognition of the IbAr10200 nucleoprotein by antibodies directed against all known CCHFV clades. This is planned to be realized with sera from other regions covering the three missing clades.

Hantaviruses are zoonotic agents transmitted from small mammals, mainly rodents, to humans, where they provoke diseases such as Hemorrhagic fever with Renal Syndrome (HFRS) and its mild form, Nephropathia Epidemica (NE), or Hantavirus Cardio-Pulmonary Syndrome (HCPS). Hantaviruses are spread worldwide and monitoring animal reservoirs is of primary importance to control the zoonotic risk. Here, we describe the development of a pan-viral resequencing microarray (PathogenID v3.0) able to explore the genetic diversity of rodent-borne hantaviruses endemic in Europe. Among about 800 sequences tiled on the microarray, 52 correspond to a tight molecular sieve of hantavirus probes covering a large genetic landscape. RNAs from infected animal tissues or from laboratory strains have been reverse transcribed, amplified, then hybridized to the microarray. A classical BLASTN analysis applied to the sequence delivered through the microarray allows to identify the hantavirus species up to the exact geographical variant present in the tested samples. Geographical variants of the most common European hantaviruses from France, Germany, Slovenia and Finland, such as Puumala virus, Dobrava virus and Tula virus, were genetically discriminated. Furthermore, we precisely characterized geographical variants still unknown when the chip was conceived, such as Seoul virus isolates, recently emerged in France and the United Kingdom.

Background Animal control measures in Latin America have decreased the incidence of urban human rabies transmitted by dogs and cats; currently most cases of human rabies are transmitted by bats. In 2004–2005, rabies outbreaks in populations living in rural Brazil prompted widespread vaccination of exposed and at-risk populations. More than 3,500 inhabitants of Augusto Correa (Pará State) received either post-exposure (PEP) or pre-exposure (PrEP) prophylaxis. This study evaluated the persistence of rabies virus-neutralizing antibodies (RVNA) annually for 4 years post-vaccination. The aim was to evaluate the impact of rabies PrEP and PEP in a population at risk living in a rural setting to help improve management of vampire bat exposure and provide additional data on the need for booster vaccination against rabies. Methodology/Principal Findings This prospective study was conducted in 2007 through 2009 in a population previously vaccinated in 2005; study participants were followed-up annually. An RVNA titer >0.5 International Units (IU)/mL was chosen as the threshold of seroconversion. Participants with titers ≤0.5 IU/mL or Equivalent Units (EU)/mL at enrollment or at subsequent annual visits received booster doses of purified Vero cell rabies vaccine (PVRV). Adherence of the participants from this Amazonian community to the study protocol was excellent, with 428 of the 509 (84%) who attended the first interview in 2007 returning for the final visit in 2009. The long-term RVNA persistence was good, with 85–88.0% of the non-boosted participants evaluated at each yearly follow-up visit remaining seroconverted. Similar RVNA persistence profiles were observed in participants originally given PEP or PrEP in 2005, and the GMT of the study population remained >1 IU/mL 4 years after vaccination. At the end of the study, 51 subjects (11.9% of the interviewed population) had received at least one dose of booster since their vaccination in 2005. Conclusions/Significance This study and the events preceding it underscore the need for the health authorities in rabies enzootic countries to decide on the best strategies and timing for the introduction of routine rabies PrEP vaccination in affected areas., Author Summary Animal control measures have decreased the incidence of human rabies in urban regions of Latin America. Currently, most cases of human rabies occur in rural areas and are transmitted by bats. In 2004–2005, rabies outbreaks affecting populations living in remote areas of Brazil prompted the widespread vaccination of exposed and at-risk populations. We evaluated the persistence of the humoral immune response for 4 years after vaccination in a rural population at daily risk of rabies exposure. Our aim was to evaluate the impact of vaccination in a rural setting to help improve management of vampire bat exposure. The participation of this Amazonian community was excellent, with 428 of the 509 (84%) who attended the first interview in 2007 returning for the final visit in 2009. The long-term RVNA persistence was good, with 85–88% of the participants evaluated at each yearly follow-up visit remaining seroconverted. Similar neutralizing antibody persistence levels were observed in participants originally given post-exposure or pre-exposure prophylaxis in 2005. This study and the events preceding it underscore the need for the health authorities in rabies enzootic countries to decide on the best strategies and timing for the introduction of routine rabies PrEP vaccination in affected areas.

In case of a bite by a rabies infected animal, the World Health Organisation recommends a prophylactic treatment including the administration of Human Rabies Immunoglobulins (HRIGs) or highly purified F(ab')2 fragments produced from Equine Rabies Immunoglobulin (F(ab')2 - ERIGs). According to international regulation, quality control of F(ab')2 - ERIGs lots requires potency testing by the in vivo Mouse Neutralisation Test (MNT) prior marketing. However, the strategy of the 3Rs (Reduce, Refine, Replace) for animal testing required by the European Directive encourages the replacement of the in vivo potency test by an in vitro assay. In this context, a competitive ELISA method (c-ELISA) has been developed by the Agence Nationale de Securite du Medicament et des Produits de Sante where F(ab')2 - ERIGs are in competition with a monoclonal antibody recognizing the trimeric native form of the rabies glycoprotein. After a full validation study, the c-ELISA has been applied to commercial batches of F(ab')2 - ERIGs. A correlation study with the MNT demonstrated a similarity between the two methods (r=0.751). Moreover, the c-ELISA method which does not need any species specific reagent has been applied to HRIGs potency testing as an alternative method to Rapid Fluorescent Focus Inhibition Test (RFFIT), thus avoiding the handling of live rabies virus in BSL3 containment. In conclusion, the c-ELISA has shown its potential to replace MNT and possibly RFFIT for the quantification of rabies immunoglobulin. After optimisation it may be used for the quantification of rabies immunoglobulin in any animal species, notably for rabies immunogenicity assay in mice.

The events leading to death in severe cases of Lassa fever (LF) are unknown. Fatality seems to be linked to high viremia and immunosuppression, and cellular immunity, rather than neutralizing antibodies, appears to be essential for survival. We previously compared Lassa virus (LV) with its genetically close but nonpathogenic homolog Mopeia virus (MV), which was used to model nonfatal LF. We showed that strong and early activation of antigen-presenting cells (APC) may play a crucial role in controlling infection. Here we developed an in vitro model of dendritic-cell (DC)-T-cell coculture in order to characterize human T-cell responses induced by MV- or LV-infected DCs. Our results show very different responses to infection with LV and MV. MV strongly and durably stimulated CD8 + and CD4 + T cells, showing early and high activation, a strong proliferative response, and acquisition of effector and memory phenotypes. Furthermore, robust and functional CD4 + and CD8 + cytotoxic T lymphocytes (CTL) were generated. LV, however, induced only weak memory responses. Thus, this study allows an improved understanding of the pathogenesis and immune mechanisms involved in the control of human LV.

SUMMARYIn 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.

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.

Tissue-culture vaccines like purified chick embryo cell vaccine (PCECV) have been shown to provide protection against classical rabies virus (RABV) via pre-exposure or post-exposure prophylaxis. A cross-neutralization study was conducted using a panel of 100 human sera, to determine, to what extent after vaccination with PCECV protection exists against non-classical bat lyssavirus strains like European bat lyssavirus (EBLV) type 1 and 2 and Australian bat lyssavirus (ABLV). Virus neutralizing antibody (VNA) concentrations against the rabies virus variants CVS-11, ABLV, EBLV-1 and EBLV-2 were determined by using a modified rapid fluorescent focus inhibition test. For ABLV and EBLV-2, the comparison to CVS-11 revealed almost identical results (100% adequate VNA concentrations >= 0.5 IU/mL; correlation coefficient r(2) = 0.69 and 0.77, respectively), while for EBLV-1 more scattering was observed (97% adequate VNA concentrations; r(2) = 0.50). In conclusion, vaccination with PCECV produces adequate VNA concentrations against classical RABV as well as non-classical lyssavirus strains ABLV, EBLV-1, and EBLV-2. (C) 2009 Elsevier Ltd. All rights reserved

Although rabies is reputed to be 100% fatal once the symptomatic phase is reached, in recent years an increased interest has arisen for therapeutic approaches. The “Milwaukee” protocol successfully applied to a rabid patient in 2005 has revitalized interest (although highly criticized by some investigators) in the rabies community. Most approaches still remain at a very early and conceptual stage of development. Rabies and its long incubation period is obviously an excellent target to develop an antiviral, even if its status as a neglected disease and its neurotropism are two economical and physiological obstacles, respectively, from this perspective. This review presents the empirical arsenal of substances having shown “some” anti-rabies viral (RABV) activity, then describes two complementary strategies that are currently elaborated for the rationale development of antivirals against RABV and other lyssaviruses: (1) a cognitive approach by destabilization of the known functional interactions between viral components, and (2) a random approach by high-throughput screening of highly diverse libraries of molecules using functional tests.

Puumala virus (PUUV) is the agent of nephropathia epidemica (NE), a mild form of hemorrhagic fever with renal syndrome (HFRS) in Europe. NE incidence presents a high spatial variation throughout France, while the geographical distribution of the wild reservoir of PUUV, the bank vole, is rather continuous. A missing piece of the puzzle is the current distribution and the genetic variation of PUUV in France, which has been overlooked until now and remains poorly understood. During a population survey, from 2008 to 2011, bank voles were trapped in eight different forests of France located in areas known to be endemic for NE or in area from where no NE case has been reported until now. Bank voles were tested for immunoglobulin (Ig)G ELISA serology and two seropositive animals for each of three different areas (Ardennes, Jura and Orleans) were then subjected to laboratory analyses in order to sequence the whole S, M and L segments of PUUV. Phylogenetic analyses revealed that French PUUV isolates globally belong to the central European (CE) lineage although isolates from Ardennes are clearly distinct from those in Jura and Orleans, suggesting a different evolutionary history and origin of PUUV introduction in France. Sequence analyses revealed specific amino acid signatures along the N protein, including in PUUV from the Orleans region from where NE in humans has never been reported. The relevance of these mutations in term of pathophysiology is discussed.

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.

International audience; Background: Crimean-Congo hemorrhagic fever (CCHF) is a zoonotic arboviral infection with hemorrhagic manifestation and often a fatal ending. Human become infected mainly through tick bite or by crushing infected tick, by contact with blood or tissues from viraemic livestock or patient. CCHF virus (CCHFV) has been isolated once in Madagascar but data on the epidemiology of the disease in the country are very scarce. Objectives: To investigate the circulation and the geographic distribution of CCHFV infection among at risk population in Madagascar. Study design: A national cross-sectional serologic survey was performed in 2008–2009 among slaughterhouse workers. Results: A total of 1995 workers were included. A recent CCHFV infection was detected in 1 of the 1995 participants (0.5‰; 95% confidence interval [CI]: 0–0.15%), and a past CCHFV infection was detected in 15 participants (0.75%; 95% CI: 0.37–1.13%). Conclusion: Overall, the percentage of CCHFV infection seen in Madagascar among at-risk professionals is very low compared to endemic countries. An assessment of the prevalence in livestock as a sensitive indicator of CCHFV activity must be considered in order to confirm the lack or the weak endemicity of CCHF in Madagascar.

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.

Antigenic characterisation of over 350 chiropteran rabies viruses of the Americas, especially from species reported rabid in Canada, distinguished 13 viral types. In close accord with this classification, nucleotide sequencing of representative isolates, at both the N and G loci, identified four principal phylogenetic groups (I–IV), sub-groups of which circulated in particular bat species. Amongst the North American bat viruses, there was a notable division between group I specimens associated with colonial, non-migratory bats ( Myotis sp. and Eptesicus fuscus ) and those of group II harbored by solitary, migratory species ( Lasiurus sp. and Lasionycteris noctivagans ). Certain species of Myotis were clearly identified as rabies reservoirs, an observation often obscured previously by their frequent infection by viral variants of other chiroptera. An additional group (III) apparently circulates in E. fuscus, whilst viruses harbored by both insectivorous and haematophagus bats of Latin America clustered to a separate clade (group IV). Comparison of the predicted N and G proteins of these viruses with those of strains of terrestrial mammals indicated a similarity in structural organisation regardless of host species lifestyle. Finally, these sequences permitted examination of the evolutionary relationship of American bat rabies viruses within the Lyssavirus genus.

Bats, which represent approximately 24% of all known mammalian species, frequently act as vectors of lyssaviruses. In particular, insectivorous bats play an important role in the epidemiology of rabies and some rabies-like viruses, while the haematophagous vampire bats are the major wildlife vector for rabies in Latin America. In contrast, the role of fruit bats (flying foxes) in the epidemiology of the recently discovered Australian bat lyssavirus is only just emerging. Information on the pathogenesis of lyssaviruses in bats is scarce. However, in general, mortality in bats infected via a natural route appears to be low, and seroconversion occurs in many of those that survive. While transmission of rabies from an infected bat may be via a bite, other routes are apparently also possible. Methods for the diagnosis of bat lyssavirus infections in bats and terrestrial mammals (including humans) are similar to the classical procedures for rabies. Measures for the prevention and control of these diseases are also similar to those for rabies, although additional innovative methods have been tested, specifically to control vampire bat rabies.

Background The design of efficient rabies control programs within a geographic area requires an appropriate knowledge of the local epidemiological cycles. In Latin America, there is a geographical overlap of the two main epidemiological cycles: (a) the terrestrial cycle, where the dog is the main terrestrial vector and the principal cause of human transmission; and (b) the aerial cycle, in which the vampire bat Desmodus rotundus is representative in Mexico. This bat is the major sylvatic rabies vector transmitting rabies to cattle. The purpose of this study was to distinguish between the epidemiological cycles of rabies virus (aerial and terrestrial) circulating in Mexico, using restriction fragment length polymorphism (RFLP). Methods Thirty positive rabies isolates were obtained from different species (including humans, domestic, and wildlife animals) and geographical regions. The methodology included the extraction of RNA, and synthesis of cDNA, PCR, and RFLP using four restriction endonucleases. To determine the aerial cycle, BsaW I and BsrG I were utilized, and for terrestrial cycle, BamH I and Stu I. Most of the samples belonged to the aerial and terrestrial cycles, except for two skunk isolates from Northwestern Mexico, which were not cut by any of the enzymes. Results Three different migration patterns were detected: (a) the first was observed in six amplicons, which were cut by BsaW I and BsrG I (aerial cycle); (b) 19 amplified samples were digested with BamH I and Stu I enzymes (terrestrial cycle); and (c) two skunk isolates from Northwest Mexico, were not cut by any of the enzymes utilized in the experiments (hypervariable cycle). Conclusions This concludes that RFLP can be used for the classification of rabies field samples in epidemiological studies. Moreover, it has demonstrated its usefulness, not only for differentiating between the main epidemiological rabies cycles present in Mexico, but also to detect new cycles in wildlife species.

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.

Rift Valley fever (RVF) is an emerging zoonosis in Africa which has spread to Egypt, the Arabian Peninsula, Madagascar, and Comoros. RVF virus (RVFV) ( Bunyaviridae family, Phlebovirus genus) causes a wide range of symptoms in humans, from benign fever to fatal hemorrhagic fever. Ruminants are severely affected by the disease, which leads to a high rate of mortality in young animals and to abortions and teratogenesis in pregnant females. Diagnostic tests include virus isolation and genome or antibody detection. During RVFV infection, the nucleoprotein encapsidating the tripartite RNA genome is expressed in large amounts and raises a robust antibody response, while the envelope glycoproteins elicit neutralizing antibodies which play a major role in protection. Much less is known about the antigenicity/immunogenicity of the nonstructural protein NSs, which is a major virulence factor. Here we have developed a competitive enzyme-linked immunosorbent assay (ELISA) enabling detection of low levels of NSs-specific antibodies in naturally infected or vaccinated ruminants. Detection of the NSs antibodies was validated by Western blotting. Altogether, our data showed that the NSs antibodies were detected in only 55% of animals naturally infected by RVFV, indicating that NSs does not induce a consistently high immune response. These results are discussed in light of differentiation between infected and vaccinated animals (DIVA) tests distinguishing naturally infected animals and those vaccinated with NSs-defective vaccines.

Lassa virus (LASV; Arenaviridae) is responsible for severe hemorrhagic fevers in Africa. LASV nucleoprotein (NP) plays important roles in regulating viral transcription and replication and in inhibiting type I interferon (IFN) production. The NP C-terminal domain contains a 3-to-5 exonuclease activity involved in suppressing IFN induction. We have established a murine polymerase (Pol) I reverse genetics system for LASV, showing that residues D389 and G392 of NP were critical for LASV viability, while the D389A/G392A and D389T/392A double mutants were severely altered in the ability to suppress IFN in macrophages and dendritic cells. Assessing their attenuation in vivo may open new perspectives in vaccinology. Lassa fever is a viral hemorrhagic fever caused by an Old World arenavirus, Lassa virus (LASV; family Arenaviridae) (45) transmitted by infected Mastomys natalensis, a peridomestic rodent (35). It is a major public health concern in regions of endemicity in West Africa and a threat for importation and misuse as a bioterrorism agent in industrial countries (9). The severity of the disease varies from asymptomatic infection to fatal hemorrhagic fever (19, 33, 34). Whether infection leads to death seems to depend on host immune responses, although the mechanisms involved remain to be clarified. LASV tropism for antigen-presenting cells (APC), such as dendritic cells (DC) and macrophages (MP), in the early stages of infection probably plays a key role in the defective cellular responses observed for severe cases (5, 28, 29, 51). DC and MP massively release LASV but are not activated and do not produce cytokines, except for a modest type I interferon (IFN) production (5, 7).

SUMMARYRabies 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.

The sequence of the glycoprotein gene of the Mokola virus, the more divergent element of the Lyssavirus genus, has been determined and the predicted protein structure compared to its counterpart in rabies vaccine strains. A global similarity of 54.3% was observed. The divergence affects particularly the rabies antigenic sites involved in the B-cell response. This provides a molecular basis for the absence of cross-protection between Mokola and rabies viruses and argues for the necessity of a specific anti-Mokola vaccine. Toward this goal, a cDNA copy of the glycoprotein gene was cloned into the baculovirus and expressed in Spodoptora frugiperda cells. A recombinant protein was produced in substantial amounts at the surface of the insect cells. Although less strongly glycosylated than the native viral glycoprotein produced in BHK-21 cells, the recombinant protein is antigenically and immunologically similar, it is recognized by specific monoclonal antibodies, and protects mice against an intracerebral challenge with Mokola virus. It therefore constitutes the first experimental genetically engineered vaccine against a rabies-related virus, and fulfills the international standard for protection.

International audience; Safe and efficient vaccination is important for rabies prevention in domestic animals. Replicative vectors expressing the rabies virus glycoprotein, derived from canine adenovirus have been reported to be promising vaccines in various animal models. In this paper we compare the potential of a replicative and a non-replicative vector, both based on canine adenovirus type 2 and expressing the rabies glycoprotein. Upon inoculation in sheep, immune responses against the rabies virus protein elicited by recombinant vectors were monitored. All immunised sheep produced a rapid and potent neutralizing antibody response against rabies virus after a single inoculation of either replicative or non-replicative recombinant canine adenovirus type 2. In addition, the non-replicative vector expressing the rabies glycoprotein stimulated antigen-specific CD4(+) and CD8(+) lymphocyte proliferation as well as IFN-γ production. These results suggest that vectors derived from canine adenovirus 2 could be considered for the development of promising vaccines in the ruminant species.

International audience; 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.

International audience; We infected squirrel monkeys (Saimiri sciureus) with Nipah virus to determine the monkeys' suitability for use as primate models in preclinical testing of preventive and therapeutic treatments. Infection of squirrel monkeys through intravenous injection was followed by high death rates associated with acute neurologic and respiratory illness and viral RNA and antigen production.

Richard Franka was not included in the author byline. He should be listed as the ninth author, and his affiliation is 7: WHO Collaborating Centre for Reference and Research on Rabies, Rabies Section, Division of Viral and Rickettsial Diseases, Viral and Rickettsial Zoonoses Branch, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America. The contributions of this author are as follows: Performed the experiments, analyzed the data, contributed reagents/materials/analysis tools.

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.

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.

We have developed a multivalent vaccine prototype using the DNA technology and chimeric lyssavirus glycoproteins to carry foreign virus epitopes. Lyssaviruses (rabies and rabies-related viruses) induce a fatal encephalomyelitis. They are divided in 7 genotypes (GT) and two principal groups according the cross-reactivity of virus neutralizing antibody (VNAb); group 1: GT 1, 4, 5, 6 and 7; group 2: GT2 and 3. Currently available vaccines belong to GT1. They induce protection against rabies (GT1) and are more or less efficacious against the other members of the group 1. They do not induce protection against group 2 viruses. Lyssavirus glycoprotein (G) is involved in the induction of both VNAb and protection. Rabies G molecule can be divided in two parts separated by a flexible hinge: the NH2 half and the COOH half containing the VNAb-inducing antigenic site II and III respectively. Injection of chimeric plasmid containing the COOH half of Pasteur Virus (PV: GT1) and the NH2 half of GT5 or GT3 G induced VNAb and protection against parental viruses but also enlarged to the other genotypes.

Antigenic differences between rabies virus strains characterized with monoclonal antibodies presently define at least four serotypes within the Lyssavirus genus of the Rhabdoviridae family: classical rabies virus strains (serotype 1), Lagos bat virus (serotype 2), Mokola virus (serotype 3) and Duvenhage virus (serotype 4). The wide distribution of rabies-related virus strains (serotypes 2, 3 and 4) and above all, the weak protection conferred by rabies vaccines against some of them (principally Mokola virus) necessitates the development of new specific vaccines. We first determined the complete nucleotide sequence of a rabies virus strain of serotype 1 (Pasteur virus) and characterized the structure of the viral genes and their regulatory sequences. We then extended this study to the Mokola virus genome. Five non-overlapping open reading frames were found in both viruses and had similar sizes and positions in both. Similarities were also found in the mRNA start and stop sequences and at the genomic extremities. Comparison of both genomes helps to analyze the basis of the particular antigenicity of these two serotypes. The sequence homology in the region coding for the viral glycoprotein was only 58% between the two viruses, compared with 94% between different rabies virus strains within serotype 1. This comparison, extended to other unsegmented negative strand RNA viruses, gives new insight into the understanding of rhabdoviruses and paramyxoviruses. Furthermore, molecular cloning provides a rationale for the genetic engineering of a future vaccine.

International audience; The elimination of rabies from the red fox (Vulpes vulpes) in Western Europe has been achieved by the oral rabies vaccination (ORV) of wildlife with a range of attenuated rabies virus strains. With the exception of the vaccinia rabies glycoprotein recombinant vaccine (VRG), all strains were originally derived from a common ancestor; the Street Alabama Dufferin (SAD) field strain. However, after more than 30 years of ORV it is still not possible to distinguish these vaccine strains and there is little information on the genetic basis for their attenuation. We therefore sequenced and compared the full-length genome of five commercially available SAD vaccine viruses (SAD B19, SAD P5/88, SAG2, SAD VA1 and SAD Bern) and four other SAD strains (the original SAD Bern, SAD VA1, ERA and SAD 1-3670 Wistar). Nucleotide sequencing allowed identifying each vaccine strain unambiguously. Phylogenetic analysis revealed that the majority of the currently used commercial attenuated rabies virus vaccines appear to be derived from SAD B19 rather than from SAD Bern. One commercially available vaccine virus did not contain the SAD strain mentioned in the product information of the producer. Two SAD vaccine strains appeared to consist of mixed genomic sequences. Furthermore, in-del events targeting A-rich sequences (in positive strand) within the 3' non-coding regions of M and G genes were observed in SAD-derivates developed in Europe. Our data also supports the idea of a possible recombination that had occurred during the derivation of the European branch of SAD viruses. If confirmed, this recombination event would be the first one reported among RABV vaccine strains.

Background There are only few assays available for the detection of Crimean-Congo Hemorrhagic Fever Virus (CCHFV)-specific antibodies in animals, and data about diagnostic sensitivity and specificity are incompletely documented for most of these tests. This is unfortunate since CCHFV antibodies in animals can be used as indicator for virus circulation in a geographic area and therewith potential risk of human exposure. This paper therefore reports on a novel ELISA for the detection of CCHFV-specific antibodies in cattle and on its application for testing ruminant sera from the Former Yugoslav Republic of Macedonia. Principal Findings A highly sensitive and specific ELISA was developed to detect CCHFV-specific IgG antibodies in cattle. The assay was validated by using 503 negative serum samples from a country where CCHFV has never been detected until now, and by using 54 positive serum samples. The positive sera were verified by using two commercially available assays (for testing human serum) which we have adapted for use in animals. The sensitivity of the novel ELISA was 98% and its specificity 99%. The presence of Hyalomma ticks was demonstrated in the Former Yugoslav Republic of Macedonia and depending on the region antibody prevalence rates up to 80% were detected in the cattle population. Conclusion This article describes a fully validated, highly sensitive and specific ELISA for the detection of CCHFV-specific IgG antibodies in cattle. Using this assay, CCHFV-specific antibodies were detected for the first time in cattle in the Former Yugoslav Republic of Macedonia, giving evidence for an active circulation of this virus in the country. Supporting this conclusion, the occurrence of the main vector of CCHFV was demonstrated in the present work for the first time in Former Yugoslav Republic of Macedonia., Author Summary Crimean-Congo hemorrhagic fever virus (CCHFV) infections in humans can cause a deadly hemorrhagic fever with high fatality rates. Most humans are infected by ticks or by contact with blood. No vaccine prophylaxis or therapeutic interventions are available. Ticks of the genus Hyalomma function as vector of CCHFV. Domestic ruminants play a crucial role in the transmission and amplification of the virus. Since infected ruminants do not develop clinical signs, CCHFV infections can in most cases only be detected by serology. There are only few assays available for the detection of CCHFV-specific antibodies in animals. In general, the real distribution of CCHFV is fairly unknown. Here we present a novel, highly sensitive and specific ELISA, and the adaptations for two commercially available assays for the detection of CCHFV-specific antibodies in cattle. With these assays, we detected CCHFV-specific antibodies in cattle in the Former Yugoslav Republic of Macedonia. This is the first indication of an active circulation of CCHFV in this country. This knowledge will assist decision makers and public health authorities in deciding on effective countermeasures and can help to prevent outbreaks.

Quality control of human rabies vaccines performed by National Control Laboratories (NCLs) prior to marketing vaccines batches requires in vivo and in vitro potency assays as requested by the relevant European Pharmacopoeia monographs, OMCLs guidelines and WHO technical recommendations. The aim of the present study was to check the suitability of an enzyme-linked immunosorbent assay (ELISA) using a virus neutralizing monoclonal antibody, directed to the rabies virus glycoprotein, to monitor the consistency of the lot to lot rabies vaccines production. Furthermore, this work was implemented to establish in house specifications for the glycoprotein content.

The low-affinity nerve-growth factor receptor p75NTR interacts in vitro with the rabies virus (RV) glycoprotein and serves as a receptor for RV. The Lyssavirus genus comprises seven genotypes (GTs) of rabies and rabies-related viruses. The ability of p75NTR to interact with the glycoprotein of representative lyssaviruses from each GT was investigated. This investigation was based on a specific binding assay between BSR cells infected with a lyssavirus and Spodoptera frugiperda (Sf21) cells expressing p75NTR on the cell surface. A specific interaction was observed with the glycoprotein of GT 1 RV (challenge virus standard or Pasteur virus strains) as well as wild-type RV and the glycoprotein of GT 6 European bat lyssavirus type 2. In contrast, no interaction was detected with the glycoprotein of lyssaviruses of GTs 2–5 and 7. Therefore, p75NTR is only a receptor for some lyssavirus glycoproteins, indicating that the other GTs must use an alternative specific receptor.

Rabies is a fatal encephalomyelitis. Most cases occur in developing countries and are transmitted by dogs. Because of their high cost, cell culture vaccines have not totally replaced the unsafe brain-derived vaccines which are still used in many developing countries. Moreover, there will be a need for vaccines against rabies-related viruses against which classical vaccines are not always effective. DNA vaccines would, therefore, be a valuable alternative for the production of cheaper rabies vaccines against a larger spectrum of viruses. In this review we report published data on DNA-based immunization with sequences encoding rabies and rabies-related virus antigens.

The genetic diversity of representative members of theLyssavirusgenus (rabies and rabies-related viruses) was evaluated using the gene encoding the transmembrane glycoprotein involved in the virus-host interaction, immunogenicity, and pathogenicity. Phylogenetic analysis distinguished seven genotypes, which could be divided into two major phylogroups having the highest bootstrap values. Phylogroup I comprises the worldwide genotype 1 (classicRabies virus), theEuropean bat lyssavirus(EBL) genotypes 5 (EBL1) and 6 (EBL2), the African genotype 4 (Duvenhage virus), and theAustralian bat lyssavirusgenotype 7. Phylogroup II comprises the divergent African genotypes 2 (Lagos bat virus) and 3 (Mokola virus). We studied immunogenic and pathogenic properties to investigate the biological significance of this phylogenetic grouping. Viruses from phylogroup I (Rabies virusand EBL1) were found to be pathogenic for mice when injected by the intracerebral or the intramuscular route, whereas viruses from phylogroup II (Mokola and Lagos bat viruses) were only pathogenic by the intracerebral route. We showed that the glycoprotein R333 residue essential for virulence was naturally replaced by a D333 in the phylogroup II viruses, likely resulting in their attenuated pathogenicity. Moreover, cross-neutralization distinguished the same phylogroups. Within each phylogroup, the amino acid sequence of the glycoprotein ectodomain was at least 74% identical, and antiglycoprotein virus-neutralizing antibodies displayed cross-neutralization. Between phylogroups, the identity was less than 64.5% and the cross-neutralization was absent, explaining why the classical rabies vaccines (phylogroup I) cannot protect against lyssaviruses from phylogroup II. Our tree-axial analysis divided lyssaviruses into two phylogroups that more closely reflect their biological characteristics than previous serotypes and genotypes.

The evolution of rabies viruses of predominantly European origin was studied by comparing nucleotide sequences of the nucleoprotein and glycoprotein genes, and by typing isolates using RFLP. Phylogenetic analysis of the gene sequence data revealed a number of distinct groups, each associated with a particular geographical area. Such a pattern suggests that rabies virus has spread westwards and southwards across Europe during this century, but that physical barriers such as the Vistula river in Poland have enabled localized evolution. During this dispersal process, two species jumps took place – one into red foxes and another into raccoon dogs, although it is unclear whether virus strains are preferentially adapted to particular animal species or whether ecological forces explain the occurrence of the phylogenetic groups.

Rabies is a fatal encephalomyelitis which is transmitted to man, mostly by dogs in developing countries. This zoonosis can be prevented by vaccination of humans before or after exposure. However, a more radical approach is possible, involving the elimination of the principal vector/reservoir by vaccinating dogs. The vaccine must be effective, safe and inexpensive. Mass production of plasmids is possible and DNA-based immunization with a plasmid encoding the antigen responsible for inducing protection seems to be more cost-effective than classical techniques involving cell culture. Beagles were immunized by intramuscular (i.m.) injection with a plasmid encoding the rabies virus (PV strain) glycoprotein. Neutralizing antibodies against both wild-type rabies virus and European Bat Lyssaviruses (EBL1 and EBL2) were detected after a single injection and a boost, but levels of neutralizing antibodies against EBL1 were low. Moreover, all vaccinated dogs were protected against a lethal challenge with a wild-type dog rabies strain. This is one of the first studies to demonstrate that dogs can be protected by DNA vaccines, and opens important perspectives for rabies control.

The PV rabies (genotype 1) G and N proteins were produced by recombinant baculoviruses in insect cells. We tested the ability of recombinant antigens to synergistically induce an immune response and, particularly, to broaden the spectrum of Lyssavirus-neutralizing antibodies produced. Cell-free preparations of recombinant proteins caused an immune response. Recombinant rabies G protein (RRG) from infected cell extract or supernatant induced virus neutralizing antibodies (VNAb) against rabies CVS virus (genotype 1) and in a less extent against European Bat Lyssavirus-1 (EBL-1: genotype 5) Recombinant rabies N protein (RRN) induced antibodies that reacted with the rabies virus ribonucleoprotein (RNP) and primed mice for both the production of VNAb induced by inactivated and purified rabies virus and the protection conferred by RNP. RRN also had an adjuvant effect on VNAb production induced by RRG when the two recombinant proteins were physically associated either encapsulated in liposomes or subjected to ultrasound treatment. However, there was no increase in production of VNAb directed against EBL-1 although classical vaccines (genotype 1) induce partial protection against this virus. Thus, beside its adjuvant effect there is some doubt as to whether including rabies N protein in a rabies subunit vaccine containing the recombinant G protein would be advantageous.

The aetiologic agents of rabies encephalomyelitis are classified into six genotypes within the Lyssavirus genus (1). It is generally accepted that the classical rabies vaccine strains (genotype 1) are inefficient in protecting against distantly rabies-related Lyssaviruses (genotypes 2 to 6). In an attempt to enlarge the protective activity of rabies vaccines, we have expressed the two principal viral antigens as recombinant proteins using a baculovirus expression system: 1° the glycoprotein (G) which constitutes viral spikes and induces virus-neutralizing antibody (VNAb); 2° the nucleoprotein (N) which is the major constituant of the ribonucleoprotein complex (RNP) involved in the T helper cell response. This work was performed with the Pasteur virus strain (PV: genotype 1).

An experimental rabies vaccine was prepared from the BHK-21 cell line adapted to culture in suspension using bioreactors. A new serum-free medium (MDSS2) (Merten et al., Cytotechnology, 1994, 14, 47) developed for the culture of various cell lines and for the production of several biologicals, was used for cell culture and virus production. The PV-Paris/BHK-21 rabies virus strain (adapted to the BHK-21 grown in monolayer) was adapted to BHK-21 cells cultivated in suspension and in the serum-free medium. High titres of rabies virus were obtained with bioreactors equipped with a perfusion system using BHK-21 cells grown in suspension in MDSS2. Experimental vaccines were prepared and had satisfactory protective activity when tested in mice. This new and low cost technology for rabies vaccine production could be suitable for developing countries where rabies is an important health problem.

In the future, numerous biologicals (including vaccine) will be produced by recombinant DNA technology in continuous cell lines (CCLs). Strict criteria exist for the acceptance of these biologicals for human use, notably concerning the residual DNA (rDNA) from cell origin. The use of inactivating agents, which can react with DNA, may lead to revise these criteria. The experimental rabies vaccine prepared from the BHK-21 CCL was used as a model for studying the effects of an inactivating and alkylating agent (beta-propiolactone: BPL) on structural and in vitro biological properties of rDNA. Both rabies infection and at a larger extent BPL treatment induced breaks and nicks in DNA strands. After BPL treatment, the capability of DNA to be used as template for different polymerases decreased dramatically according to BPL concentration. This result indicate that both a quantitative (concentration measurement) and a qualitative (determination of rDNA biological activity) analyses might be taken into account for the acceptance of biologicals.

A mouse model was designed to test in vivo the efficacy of rabies immune globulins and specific neutralizing monoclonal antibodies to prevent European bat lyssavirus I infection. Human or equine rabies immune globulins previously found to contain variable amounts of neutralizing bat lyssavirus crossreactive antibodies were passively transferred to mice receiving intramuscularly a lethal dose of bat lyssavirus type 1. Immune globulins did not protect mice well against bat lyssavirus 1 whereas they reduced the mortality caused by rabies virus. In contrast, mice inoculated with bat lyssavirus 1 or rabies virus survived when passively immunized with bat lyssavirus 1 specific monoclonal antibody (mAb 8-2). This monoclonal antibody, an IgG 2a , recognized an epitope located in the antigenic site IIa of rabies glycoprotein. A mutation replacing the lysine 198 by glutamate in a rabies variant abrogated sensitivity to this neutralizing antibody. Because of its broad neutralizing spectrum against wild virus isolates, including European bat lyssaviruses, this monoclonal antibody should be a good candidate for rabies immune globulin replacement. It could improve efficacy of rabies vaccination, used either alone or in conjunction with human rabies immune globulins or monoclonal antibody cocktail to supplement their lack of crossreactivity to European bat lyssavirus 1.

The predominant role of Eptesicus serotinus in the epizootic of bat rabies in Europe was further outlined by the first isolation of the rabies virus from this species in France. The distribution of the virus was studied in naturally infected E. serotinus bats at the time of death and suggested that the papillae of the tongue and the respiratory mucosa may play a role in virus production and excretion. The analysis of 501 French rabies virus isolates from various animal species by antinucleocapsid monoclonal antibodies indicated that transmission of the disease from bats to terrestrial animals is unlikely. The antigenic profile of two isolates from French bats corresponded to that of European bat lyssavirus type 1 (EBL1). Comparisons of 12 different isolates from bats with antinucleocapsid and antiglycoprotein monoclonal antibodies and by direct sequencing of the polymerase chain reaction amplification product of the N gene indicated that EBL1, EBL2, Duvenhage virus (serotype 4 of lyssavirus), and the European fox rabies virus (serotype 1) are phylogenetically distant. They formed four tight genetic clusters named genotypes. EBL1 was shown to be antigenically and genetically more closely related to Duvenhage virus than to EBL2. We propose that EBL1 and EBL2 constitute two distinct genotypes which further serologic characterization will probably classify as new serotypes. We also report a simple method for the rapid characterization of EBL based on the digestion of the polymerase chain reaction product of the N gene by three restriction endonucleases.