Human Rabies Encephalitis Prevention and Treatment: Progress Since Pasteur's Discovery RUNNING TITLE Prevention and Treatment of Human Rabies

Keywords: Antiviral drugs, encephalitis, immunoglobulins, lyssavirus, Milwaukee protocol, postexposure prophylaxis, rabies virus, vaccine

Rabies remains one of the most ancient and deadly of human infectious diseases. This viral zoonosis is transmitted principally by the saliva of infected dogs, inducing a form of encephalomyelitis that is almost invariably fatal. Since the first implementation, by Louis Pasteur in 1885, of an efficient preventive post-exposure treatment, more effective protocols and safer products have been developed, providing almost 100% protection if administered early enough. However, this disease still represents a major, but neglected public health problem, with an estimated 50,000 human deaths due to rabies reported each year, mostly in Africa and Asia. Once the first clinical signs appear, there is no effective treatment. A ray of hope emerged in 2004, with the report of a patient recovering from rabies after aggressive, innovative treatment. However, this case was not clearly reproduced and the identification of targets for antiviral treatment in cases of rabies infection remains a major challenge.

In this context, this review presents the state-of-the art in the prevention and curative treatment of human rabies. We begin by describing the viral etiological agent and the disease it causes, to provide an essential background to rabies. An overview of the post-exposure prophylaxis of rabies in humans is then given, from its initial implementation to possible future developments. Finally, an analysis of the various antiviral compounds tested in rabies in vitro, in animal models or in humans is presented, focusing in particular on potential new strategies.

Introduction

Rabies is an acute, almost invariably fatal form of viral encephalomyelitis in humans. The rabies virus (RABV)one of the 11 species of the genus Lyssavirus in the family Rhabdoviridae-is the main etiological agent. It is acquired from the saliva of infected animals by bites, scratches and mucous membrane exposure and, more rarely, by aerosol exposure and tissue/organ transplantation. Rabies is the infectious disease with the highest case-fatality ratio in humans. An extraordinary leap forward was made with the work of Louis Pasteur at the end of the 19 th Century, with the development of the first efficient postexposure treatment able to prevent rabies in exposed patients. Unfortunately, the original vaccine was derived from brain and nerve tissues and was associated with neurological complications [START_REF] Hemachudha | Myelin basic protein as an encephalitogen in encephalomyelitis and polyneuritis following rabies vaccination[END_REF]. These complications eventually led to the replacement of such vaccines by tissue-cultured vaccines. This disease remains a major but neglected public health concern throughout the world, with an estimated 50,000 cases of rabies still reported in humans each year, mostly in rural area of Africa and Asia, and with a particularly high incidence in young children (under the age of 15 years) [START_REF] Knobel | Re-evaluating the burden of rabies in Africa and Asia[END_REF]. These figures, although high, must be considered an underestimate, as there is undoubtedly a high frequency of misdiagnosis and underreporting [START_REF] Mallewa | Rabies encephalitis in malaria-endemic area, Malawi, Africa[END_REF][START_REF] Ly | Rabies situation in Cambodia[END_REF]. Dogs are the main source of exposure for several billion people worldwide, and remain responsible for almost 99% of all human deaths from rabies [START_REF] Knobel | Re-evaluating the burden of rabies in Africa and Asia[END_REF]. Other mammal species also serve as natural vectors and/or reservoirs of rabies, including various carnivores and several bat species. Even though this zoonotic disease was known and described in the Classical Era, our knowledge of its physiopathological mechanisms remains limited, except for detailed descriptions of the clinical symptoms. Rabies virus, like other lyssaviruses, is a neurotropic agent that is transported via the peripheral nerves from the site of inoculation to the brain, where it replicates massively, subsequently spreading centrifugally to the salivary glands and 4/115 other peripheral innervated tissues [START_REF] Warrell | Rabies and other lyssavirus diseases[END_REF]. The major neurological signs appear at least five to six days after the virus has reached the central nervous system (CNS) [START_REF] Laothamatas | MR imaging in human rabies[END_REF][START_REF] Mitrabhakdi | Difference in neuropathogenetic mechanisms in human furious and paralytic rabies[END_REF]. They include the classic encephalitic (or furious) form with hydrophobia and hyperactivity, but also a paralytic (or dumb) form, occurring in about 30% of cases [START_REF] Rupprecht | Rabies re-examined[END_REF]. The basic molecular mechanisms of this disease and its associated clinical signs remain unknown. However, electrophysiological studies of the nerve and muscle in paralytic rabies patients have shown that different neural structures are involved in encephalitic and paralytic rabies in humans. Peripheral nerve dysfunction (axon-and myelinopathy) underlies the weakness observed in paralytic rabies [START_REF] Mitrabhakdi | Difference in neuropathogenetic mechanisms in human furious and paralytic rabies[END_REF][START_REF] Sheikh | Overlap of pathology in paralytic rabies and axonal Guillain-Barre syndrome[END_REF]. Subclinical anterior horn cell dysfunction is evident only in furious rabies [START_REF] Mitrabhakdi | Difference in neuropathogenetic mechanisms in human furious and paralytic rabies[END_REF]. Rabiesinfected dogs are a relevant model for studies of human rabies [START_REF] Laothamatas | Furious and paralytic rabies of canine origin: neuroimaging with virological and cytokine studies[END_REF]. Little or no apparent histopathological modification is observed in infected brains and neurons. The virus appears to induce neuronal dysfunctions, but these remain to be elucidated. In this context, effective antiviral targets remain to be identified, and there is currently no effective treatment for rabies once the clinical signs have appeared. Nevertheless, various compounds have been studied in vitro and in animal models, and several protocols have also been attempted in individual cases of human rabies.

In this review, we aim to present the state-of-the-art in the preventive and curative treatment of human rabies. We begin by considering general aspects of rabies and then describe the preand post-exposure prophylaxis (PrEP and PEP, respectively) currently available for rabies.

Finally, we present an analysis of the various antiviral compounds tested for the treatment of RABV infection in vitro, in animal models or in humans, focusing in particular on potential new developments.

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1 General considerations relating to rabies and lyssaviruses

Virus presentation

Lyssaviruses are enveloped bullet-shaped particles, rounded on one side and flat on the other, approximately 180 to 200 nm long and 75 nm in diameter. Their genome is composed of a single-strand, negative-sense, non-segmented RNA molecule almost 12,000 nucleotides in length [START_REF] Delmas | Genomic diversity and evolution of the lyssaviruses[END_REF]. The extremities (leader and trailer region at the 3' and 5' ends, respectively) of the genome are conserved and have inverted complementary sequences. The genome contains five monocistronic genes, encoding the five viral proteins, which are, in sequential order from the 3' to the 5' end: the nucleoprotein (N), the phosphoprotein (P), the matrix protein (M), the glycoprotein (G) and the large protein (L) carrying all the enzymatic activities required to fulfill the function of an RNA-dependent RNA polymerase [START_REF] Assenberg | Genomics and structure/function studies of Rhabdoviridae proteins involved in replication and transcription[END_REF]. Each gene is surrounded by initiation and termination sequences. All the encoded proteins are encapsidated in the viral particle. The RNA genome is tightly associated with several copies of N protein in the viral helical nucleocapsid, the structure of which has recently been elucidated [START_REF] Albertini | Crystal structure of the rabies virus nucleoprotein-RNA complex[END_REF][START_REF] Ge | Cryo-EM model of the bullet-shaped vesicular stomatitis virus[END_REF]. The nucleocapsid is associated with smaller amounts of P and L proteins. These two proteins form the polymerase complex and are responsible for the RNA polymerase activity associated with the virions, in association with the N-RNA complex. The M protein condenses the nucleocapsid and provides the virion with its bullet-shaped appearance. The structure of the full-length M protein has been determined, leading to the description of a model for the polymerization of this protein, potentially involving the insertion of the N-terminal part in the membrane and association with the nucleocapsid [START_REF] Graham | Rhabdovirus matrix protein structures reveal a novel mode of self-association[END_REF]. This model was recently confirmed and improved, with the results of cryo-electron microscopy for vesiculovirus, another rhabdovirus [START_REF] Ge | Cryo-EM model of the bullet-shaped vesicular stomatitis virus[END_REF]. The viral envelope consists of a lipid bilayer with spike-like projections composed of trimers of G protein, a transmembrane protein with a very short cytoplasmic domain [START_REF] Roche | Structure of the prefusion form of the vesicular stomatitis virus glycoprotein G[END_REF]. The lipids of the envelope are derived from the host cell membrane, from which 6/115 the virus buds. The G protein is responsible for virus attachment to and penetration into the cell.

Viral replication cycle

Lyssaviruses have a broad cellular tropism in vitro after adaptation to cell culture. These viruses use the exposed region of the glycoprotein to target different surface components, facilitating their entry into host cells. The surface components targeted include specific peripheral membrane receptors, such as the  subunit of the nicotinic acetylcholine receptor (nAChR) on muscle cells, and also neuronal membrane receptors, such as nAChR, the neural cell-adhesion molecule (NCAM or CD56) and the low-affinity nerve growth factor receptor (p75NTR) [START_REF] Lentz | Is the acetylcholine receptor a rabies virus receptor?[END_REF][START_REF] Thoulouze | The neural cell adhesion molecule is a receptor for rabies virus[END_REF][START_REF] Tuffereau | Low-affinity nervegrowth factor receptor (P75NTR) can serve as a receptor for rabies virus[END_REF]. The significance of these non peripheral receptors remains unclear. Other molecules, such as phospholipids and sialylated gangliosides, may also be used, in a non specific manner, for viral entry [START_REF] Superti | Mechanism of rabies virus entry into CER cells[END_REF][START_REF] Superti | Involvement of gangliosides in rabies virus infection[END_REF][START_REF] Superti | Role of phospholipids in rhabdovirus attachment to CER cells. Brief report[END_REF][START_REF] Conti | Effect of concanavalin A on the early events of rabies virus infection of CER cells[END_REF]. Following attachment, RABV enters the cell, mostly via the endocytic pathway, and releases its ribonucleocapsid into the cytoplasm after membrane fusion mediated by a pH-dependent conformational modification of the glycoprotein. The viral genome is transcribed by the polymerase complex, to produce capped and polyadenylated monocistronic messenger RNAs corresponding to each of the five viral genes. Transcription starts at the 3' end (leader region) of the genome and continues to the other end, with a gradient in the amount of each transcript as a function of its order and distance from the 3' end, regulating the relative abundance of the various viral proteins. These mRNAs are translated by the cellular protein synthesis machinery. There is then a switch from transcription to replication, regulated by a subtle mechanism dependent on the quantity of N protein produced. This leads to the generation of intermediate RNAs (antigenome), which act as templates for progeny genomes, and genome molecules then become predominant. Once sufficient quantities of viral proteins and RNA genomes have accumulated in the infected cell, ribonucleoprotein complexes form and virus particles are assembled near 7/115 the plasma membrane, from which mature virions are released after budding.

Taxonomy and epidemiology of lyssaviruses

Taxonomy

Rabies virus is the prototype species of the genus Lyssavirus, which belongs to the family Rhabdoviridae, order Mononegavirales [START_REF]International Committee on Taxonomy of Viruses (ICTV)[END_REF]. Lyssaviruses constitute a distinct monophyletic clade less divergent than other genera in this virus family [START_REF] Dacheux | Application of broadspectrum resequencing microarray for genotyping rhabdoviruses[END_REF][START_REF] Bourhy | Phylogenetic relationships among rhabdoviruses inferred using the L polymerase gene[END_REF][START_REF] Kuzmin | Phylogenetic relationships of seven previously unclassified viruses within the family Rhabdoviridae using partial nucleoprotein gene sequences[END_REF]. The classification of these viruses is based on a combination of knowledge from serological studies and phylogenetic analysis. Serotypes and species (formerly genotypes) were initially used to separate lyssaviruses. Species classification is based on the similarity of the N gene sequence. The cutoff point for species separation was initially set at 80 to 82% nucleotide identity [START_REF] Kissi | Genetic polymorphism in the rabies virus nucleoprotein gene[END_REF].

Based on this cutoff point, 11 different species of lyssaviruses have been recognized by the International Committee on Taxonomy of Viruses (ICTV) (Table 1) [START_REF]International Committee on Taxonomy of Viruses (ICTV)[END_REF] (Figure 1). They include the classical rabies virus (RABV) species (formerly genotype 1) and rabies-related lyssavirus species, including the three African lyssaviruses Lagos bat virus (LBV), Mokola virus (MOKV) and Duvenhage virus (DUVV), the two European bat lyssaviruses type 1 (EBLV-1) and type 2 (EBLV-2), and the Australian bat lyssavirus (ABLV) (formerly genotypes 2 to 7, respectively). Four new lyssavirus species have also been recognized: the Irkut (IRKV), Aravan (ARAV), Khujand (KHUV) and West Caucasian bat (WCBV) viruses, each isolated once from insectivorous bats in Eurasia [START_REF] Botvinkin | Novel lyssaviruses isolated from bats in Russia[END_REF][START_REF] Kuzmin | Bat lyssaviruses (Aravan and Khujand) from Central Asia: phylogenetic relationships according to N, P and G gene sequences[END_REF]. Another two bat lyssaviruses have recently been identified: Ozernoe, which was identified in a fatal case of human rabies, probably contracted after a bat bite nearly one month before the onset of symptoms and in the Far East of Russia during 2007 [START_REF] Leonova | A fatal case of bat lyssavirus infection in Primorye Territory of the Russian Far East[END_REF], and Shimoni bat virus (SHIBV), which was isolated from an insectivorous bat in Kenya in 2009 [START_REF] Kuzmin | Shimoni bat virus, a new representative of the Lyssavirus genus[END_REF].

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Species definition criteria based solely on N gene nucleotide sequence have been shown to be insufficiently powerful to discriminate between closely-related lyssaviruses. Indeed, as the number of new and/or full-length genome sequences available increases, rigorous phylogenetic analysis based on these complete sequences is likely to provide the highest level of discriminatory power for species identification [START_REF] Delmas | Genomic diversity and evolution of the lyssaviruses[END_REF]. For example, the results of such analyses have suggested that viruses currently classified as LBV species (may actually correspond to several different species [START_REF] Delmas | Genomic diversity and evolution of the lyssaviruses[END_REF].

Epidemiology

Rabies is widely distributed throughout the world and is present on all continents, with the exception of certain specific regions, such as Antarctica and some islands (Table 1) [START_REF] Warrell | Rabies and other lyssavirus diseases[END_REF]33].

RABV has the broadest geographic distribution of the 11 species of lyssaviruses identified to date, and the widest spectrum of vectors or reservoirs within the orders Carnivora and Chiroptera. Moreover, RABV has been isolated from almost all the orders of terrestrial mammals. The dog remains the main reservoir and vector of RABV, and is responsible for almost all rabies infections in humans, particularly in Asia and Africa [START_REF] Knobel | Re-evaluating the burden of rabies in Africa and Asia[END_REF]. However, many other species of carnivores are involved in the maintenance and transmission of RABV worldwide. These species include other species from the family Canidae, such as red foxes and raccoon dogs (particularly in Europe), but also arctic foxes in polar regions, gray foxes and coyotes in North America and jackals in Africa. Skunks (family Mephitidae) and raccoons (family Procyonidae) also represent major terrestrial reservoirs in North America and rabies virus circulation has been observed in mongooses (family Herpestidae) in Africa and in the Caribbean. Many other terrestrial mammal species are susceptible to rabies but do not transmit the disease further, acting as epidemiological dead-end hosts (including livestock species, such as cattle and horses). The dog has been identified as the probable main vector involved in inter-species RABV transmission [START_REF] Bourhy | The origin and phylogeography of dog rabies virus[END_REF]. Bats have been identified as an active 9/115 reservoir and vector for RABV only in the Americas, where the chief species involved are insectivorous bat species in North and Latin America and vampire bats in South America [START_REF] Rupprecht | Rabies re-examined[END_REF].

All species of lyssaviruses other than RABV species have been isolated from bats, which are thought to be the sole reservoir of these species, with the exception of MOKV, which has been isolated, in Africa only, from shrews, cats, dogs and a rodent, but for which the reservoir species remains to be identified [START_REF] Nel | Emergence of lyssaviruses in the Old World: the case of Africa[END_REF]. LBV and DUVV have been isolated from frugivorous and insectivorous bats, respectively, in Africa [START_REF] Nel | Emergence of lyssaviruses in the Old World: the case of Africa[END_REF]. Lyssaviruses EBLV-1 and EBLV-2 circulate in specific species of insectivorous chiropterans in Europe [START_REF] Davis | The evolutionary dynamics of canid and mongoose rabies virus in Southern Africa[END_REF] and ABLV has been isolated form insectivorous and frugivorous bats in Australia [START_REF] Warrilow | Australian bat lyssavirus: a recently discovered new rhabdovirus[END_REF]. The various new lyssavirus species identified more recently -ARAV, IRKV, KHUV and WCBV, and the tentative SHIBV and Ozernoe specieswere all isolated from bats, suggesting that there may be more, as yet undiscovered species of lyssavirus in the order Chiroptera (Table 1) [START_REF] Botvinkin | Novel lyssaviruses isolated from bats in Russia[END_REF][START_REF] Kuzmin | Bat lyssaviruses (Aravan and Khujand) from Central Asia: phylogenetic relationships according to N, P and G gene sequences[END_REF][START_REF] Leonova | A fatal case of bat lyssavirus infection in Primorye Territory of the Russian Far East[END_REF][START_REF] Kuzmin | Shimoni bat virus, a new representative of the Lyssavirus genus[END_REF][START_REF] Lumlertdacha | Survey for bat lyssaviruses, Thailand[END_REF].

Moreover, the considerable diversity of the viruses circulating in bats has led to suggestions that RABV may have originated from chiropteran lyssaviruses [START_REF] Badrane | Host switching in Lyssavirus history from the Chiroptera to the Carnivora orders[END_REF]. Spill-over transmissions of bat lyssaviruses (EBLV-1, LBV) to carnivores occur, as demonstrated by a number of reported cases, but without successful adaptation or propagation [START_REF] Nel | Emergence of lyssaviruses in the Old World: the case of Africa[END_REF][START_REF] Dacheux | European bat Lyssavirus transmission among cats, Europe[END_REF].

From a public health standpoint, the complex epidemiological context of rabies poses a continual challenge and threat. Effective mass vaccination campaigns have greatly decreased the incidence of rabies in dogs and have proved efficient for rabies control in developed countries. However, canine rabies remains an increasing problem in Asia and Africa.

Successful rabies eradication within wild reservoirs is also possible, as illustrated by the various oral vaccination campaigns in red fox carried out in Western Europe [START_REF] Bourhy | Rabies in Europe in[END_REF]. However, it may be difficult to transpose these achievements to large geographic regions containing multiple wild reservoirs. Finally, new reservoirs may emerge in rabies-free areas, in Western Europe for example, as already observed for the red fox and raccoon dog, or may be identified 10/115 for the first time, particularly for bats. Furthermore, the reintroduction of rabies through the transport of uncontrolled domestic carnivores (particularly dogs) or through the migration of wild rabid reservoirs across borders (such as the red fox in Italy) poses a real threat [START_REF] Bourhy | Rabies in Europe in[END_REF][START_REF] De Benedictis | Emergence of fox rabies in north-eastern Italy[END_REF].

Pathogenesis

Rabies is transmitted by inoculation with infected material (mostly saliva) through the skin, into muscle and subcutaneous tissues (mostly after bite wounds). Inoculation may also occur through scratches or the licking of mucous membranes or broken skin. The efficacy of virus transmission depends on several factors, including the characteristics of the inoculation process (severity, location and number of bites, for example), the characteristics and dose of the virus, and the susceptibility of the host [START_REF] Warrell | Rabies and other lyssavirus diseases[END_REF]33,[START_REF] Hemachudha | Human rabies: a disease of complex neuropathogenetic mechanisms and diagnostic challenges[END_REF][44]. Other routes of transmission have been described but have a limited impact on rabies epidemiology in humans. They include aerosol contamination [START_REF] Constantine | Rabies transmission by nonbite route[END_REF][START_REF] Winkler | Airborne rabies transmission in a laboratory worker[END_REF][START_REF]CDC Rabies in a laboratory worker--New York[END_REF], contact with and the preparation of infected meat or carcasses [START_REF] Wertheim | Furious rabies after an atypical exposure[END_REF] and human-to-human transmission through the transplantation of various solid organs and tissues, including the cornea and vascular conduits [START_REF] Houff | Human-to-human transmission of rabies virus by corneal transplant[END_REF][START_REF] Maier | Management and outcomes after multiple corneal and solid organ transplantations from a donor infected with rabies virus[END_REF][START_REF] Srinivasan | Transmission of rabies virus from an organ donor to four transplant recipients[END_REF]. The transplacental transmission of rabies seems to be rare, as most of the infants born to rabid mothers are healthy [START_REF] Iehle | Delivery and follow-up of a healthy newborn from a mother with clinical rabies[END_REF]. After inoculation, RABV may reach the peripheral nerves directly, via the nAChR present at the neuromuscular junction. However, an initial period of virus multiplication may be observed, in the muscle, as in the skunk animal model [START_REF] Charlton | The long incubation period in rabies: delayed progression of infection in muscle at the site of exposure[END_REF], or in fibroblasts and epithelial cells in the dermis, as in experimental studies of RABV isolates of bat origin [START_REF] Morimoto | Characterization of a unique variant of bat rabies virus responsible for newly emerging human cases in North America[END_REF]. Direct inoculation of the nerve with the virus is also possible, as in the case of severe bites at the brachial plexus [55]. Having gained access to the peripheral nerves, RABV is transported to the CNS via the fast retrograde axoplasmic flux (experimentally estimated at 50-100 mm/day) within motor axons and, possibly, via the anterograde axoplasmic flux within sensory axons [START_REF] Tsiang | Pathophysiology of rabies virus infection of the nervous system[END_REF]. There is experimental evidence to suggest that viral phosphoprotein or glycoprotein is involved in this axonal transport, through interaction with 11/115 the light chain LC8 protein or after binding to p75NTR, respectively [START_REF] Raux | Interaction of the rabies virus P protein with the LC8 dynein light chain[END_REF][START_REF] Jacob | Cytoplasmic dynein LC8 interacts with lyssavirus phosphoprotein[END_REF][START_REF] Wong | Transduction patterns of pseudotyped lentiviral vectors in the nervous system[END_REF]. During this transport, the virus may replicate again in the dorsal-root ganglia and anterior-horn cells [START_REF] Hemachudha | Human rabies: a disease of complex neuropathogenetic mechanisms and diagnostic challenges[END_REF].

The virus acts as a specific neurotropic agent in vivo, replicating rapidly in infected neuronal cells. The transneuronal migration of the virus along neuroanatomical pathways is not well defined, but trans-synaptic spread dependent on the presence of the viral glycoprotein has been suggested [START_REF] Etessami | Spread and pathogenic characteristics of a G-deficient rabies virus recombinant: an in vitro and in vivo study[END_REF]. Once the virus has gained access to the brain, it disseminates rapidly, particularly in specific anatomical areas such as the brainstem, thalamus, basal ganglia and spinal cord [START_REF] Tirawatnpong | Regional distribution of rabies viral antigen in central nervous system of human encephalitic and paralytic rabies[END_REF]. In the terminal phase of infection, the virus spreads, by axonal anterograde transport, to peripheral sites, including the salivary glands (necessary for the transfer of the virus through saliva and for propagation of the infectious cycle in a natural reservoir) and other innervated tissues [START_REF] Murphy | Comparative pathogenesis of rabies and rabies-like viruses: infection of the central nervous system and centrifugal spread of virus to peripheral tissues[END_REF][START_REF] Jackson | Extraneural organ involvement in human rabies[END_REF].

Despite the severe clinical neurological signs observed in rabies cases, no major histological change can be observed in the brains of rabid patients, and cellular abnormalities are limited in infected neurons. It has been suggested that major dysfunctions in infected and uninfected neurons are the main cause of the neurological symptoms. Several hypotheses have been tested, but no clear or definitive conclusion has been drawn. The hypotheses tested include abnormalities of neurotransmitter functions affecting serotonin, opioids, gamma-aminobutyric acid (GABA), acetylcholine, the involvement of neuronal membrane ion channels, excitatory amino acids, apoptosis, nitric oxide, electrophysiological alteration or cellular RNA and protein synthesis [START_REF] Warrell | Rabies and other lyssavirus diseases[END_REF][START_REF] Hemachudha | Human rabies: a disease of complex neuropathogenetic mechanisms and diagnostic challenges[END_REF][44]. However, peripheral nerve dysfunction has been implicated in the weakness observed in paralytic rabies, whereas anterior horn cell dysfunction may be associated with the furious form of rabies [64].

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Clinical presentation of rabies encephalitis

Human rabies

Rabies is an acute, almost invariably fatal form of viral encephalomyelitis, but is preventable provided that correct prophylaxis is administered soon after exposure (see section 2.2.2). Only a very small number of patients developing symptoms have survived (see section 3.2.1). In addition to RABV, viruses from all the other known lyssavirus species have either been shown or are predicted to cause rabies encephalitis in humans. Classically, the clinical features of the disease can be divided into five stages, including the incubation or asymptomatic period, the prodrome, the acute neurological phase, coma and death [START_REF] Hemachudha | Human rabies: a disease of complex neuropathogenetic mechanisms and diagnostic challenges[END_REF]65].

The mean incubation period is typically 20 to 60 days, but incubation periods of less than seven days or up to several years have been reported [START_REF] Hemachudha | Human rabies: a disease of complex neuropathogenetic mechanisms and diagnostic challenges[END_REF][START_REF] Dacheux | A reliable diagnosis of human rabies based on analysis of skin biopsy specimens[END_REF]. Short incubation periods have been associated with the direct inoculation of nerve tissue, including the brain, in particular.

Post-exposure prophylaxis is effective only during the incubation period, and should be administered as soon as possible after exposure (see section 2.2.2). The non specific prodromal symptoms last for one to 10 days and may include fever, headache, anxiety and irritability [START_REF] Hemachudha | Human rabies: a disease of complex neuropathogenetic mechanisms and diagnostic challenges[END_REF][START_REF] Dacheux | A reliable diagnosis of human rabies based on analysis of skin biopsy specimens[END_REF][START_REF] Warrell | The clinical picture of rabies in man[END_REF]. Pain, paresthesia or pruritus close to the site of viral inoculation is commonly described in rabid patients. It has been suggested that that the prodromal clinical signs are related to the centripetal migration of the virus from the peripheral nerves to the dorsal-root ganglia and, finally, to the CNS [START_REF] Hemachudha | Human rabies: a disease of complex neuropathogenetic mechanisms and diagnostic challenges[END_REF]. Once the virus reaches the CNS, it may cause one of two distinct forms of the disease: classical or encephalitic (furious) rabies or paralytic (dumb) rabies, which affect about 70% and 30% of infected patients, respectively.

The encephalitic form of rabies usually includes fluctuating consciousness with intermittent episodes of confusion, hallucinations, agitation or aggressive behavior [START_REF] Hemachudha | Human rabies: a disease of complex neuropathogenetic mechanisms and diagnostic challenges[END_REF][START_REF] Dacheux | A reliable diagnosis of human rabies based on analysis of skin biopsy specimens[END_REF]. Signs of autonomic dysfunction are also observed, with hypersalivation, excessive sweating, dilated pupils, piloerection or priapism. Inspiratory or phobic spasms occur mostly in the encephalitic 13/115 form, and include hydrophobia (in about 50 to 80% of patients), a characteristic and unique manifestation of rabies, which may occur in association with aerophobia, although these two manifestations may occur independently. Fever and dysphagia are generally associated with encephalitic rabies, and seizures may also be observed. These signs classically persist for one to four days, subsequently progressing to severe flaccid paralysis, coma and multiple organ failure. Patients generally die after paralysis of the cardiorespiratory system, which occurs a mean of five days after onset of symptoms, in the absence of intensive medical care [START_REF] Hemachudha | Human rabies: a disease of complex neuropathogenetic mechanisms and diagnostic challenges[END_REF][START_REF] Dacheux | A reliable diagnosis of human rabies based on analysis of skin biopsy specimens[END_REF].

The paralytic form of rabies remains difficult to diagnose and may be confused with Guillain-Barré syndrome. Flaccid muscle weakness develops early in the course of the disease and may be associated with sphincter involvement, myoedema and phobic spasm, including hydrophobia. Patients presenting paralytic rabies may survive for almost two weeks, with death resulting from respiratory paralysis [START_REF] Hemachudha | Human rabies: a disease of complex neuropathogenetic mechanisms and diagnostic challenges[END_REF]65].

Animal rabies

Natural rabies infection causes an acute neurological illness in almost all mammalian species.

As in humans, the initial signs of rabies are non specific, but both clinical forms of rabies can be observed in infected animals. Indeed, aggressiveness and hyperactivity are frequently observed in carnivores with encephalitic rabies. Paralytic forms have also been described, and both clinical forms may occur alternately in the same infected animal. Dramatic changes in behavior, such as wild animals losing their fear of humans, may be an indication of a rabies infection. Death generally occurs within two weeks of the onset of symptoms, whatever the clinical form observed. However, asymptomatic episodes and survival have been observed or suggested in various species, following the detection of rabies antibodies or viral RNA in apparently healthy animals, including mongooses, skunks, raccoons, dogs, foxes, hyenas, jackals, frugivorous and insectivorous bats [START_REF] Warrell | Rabies and other lyssavirus diseases[END_REF]33].

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Diagnosis

There is currently no test for the diagnosis of rabies infection before the onset of clinical disease. The clinical diagnosis of rabies therefore remains difficult and unreliable. In humans, signs of hydrophobia or aerophobia, when present, may be indicative of rabies infection.

However, rabies diagnosis must be confirmed by laboratory techniques, using approved and validated methods [START_REF]WHO WHO Expert Consultation on Rabies -First report[END_REF]. Post-mortem diagnosis in animals with suspected rabies is based on methods for detecting viral antigens with the gold standard fluorescent antibody test (FAT) [START_REF] Dean | Laboratory Techniques in Rabies[END_REF], ELISA [START_REF] Xu | A simple sandwich ELISA (WELYSSA) for the detection of lyssavirus nucleocapsid in rabies 104/115 suspected specimens using mouse monoclonal antibodies[END_REF][START_REF] Bourhy | Comparative field evaluation of the fluorescent-antibody test, virus isolation from tissue culture, and enzyme immunodiagnosis for rapid laboratory diagnosis of rabies[END_REF], the recently developed direct rapid immunohistochemical test (dRIT) [START_REF] Lembo | Evaluation of a direct, rapid immunohistochemical test for rabies diagnosis[END_REF] and the rapid immunodiagnostic test (RIDT) [START_REF] Kang | Evaluation of a rapid immunodiagnostic test kit for rabies virus[END_REF], or viral isolation techniques using the rapid tissue culture infection test (RTCIT) (which should replace the mouse inoculation test (MIT)) [START_REF]WHO WHO Expert Consultation on Rabies -First report[END_REF][START_REF] Sureau | Rapid technics for the laboratory diagnosis of rabies[END_REF]. Post-mortem diagnosis in humans can be achieved by applying the same techniques to brain biopsy specimens. However, biopsy is not always feasible and the detection of viral RNA in skin biopsy specimens may be an interesting alternative for the diagnosis of rabies encephalitis [START_REF] Dacheux | A reliable diagnosis of human rabies based on analysis of skin biopsy specimens[END_REF]. Several biological samples can also be used for the diagnosis of rabies in living human patients, with lyssavirus RNA detection with molecular diagnostic tools, including RT-PCR, real-time PCR or other techniques, such as nucleic acid sequence-based amplification (NASBA) and loop-mediated isothermal amplification (LAMP) [START_REF] Dacheux | More Accurate Insight into the Incidence of Human Rabies in Developing Countries through Validated Laboratory Techniques[END_REF][76][START_REF] Wacharapluesadee | Ante-and post-mortem diagnosis of rabies using nucleic acid-amplification tests[END_REF]. The samples suitable for such analyses include saliva, cerebral spinal fluid (CSF), skin biopsy specimens containing hair follicles collected at the nape of the neck, extracted hair follicles, tears and urine [START_REF] Dacheux | A reliable diagnosis of human rabies based on analysis of skin biopsy specimens[END_REF][START_REF] Wacharapluesadee | Ante-and post-mortem diagnosis of rabies using nucleic acid-amplification tests[END_REF]. In a recent study, a sensitivity of more than 98% was demonstrated for tests on single skin biopsy samples, regardless of the time at which the sample was collected (i.e., from 1 day after the onset of symptoms to just after death), with a sensitivity of 100% reported when at least three successive saliva samples per patient were analyzed [START_REF] Dacheux | A reliable diagnosis of human rabies based on analysis of skin biopsy specimens[END_REF]. When possible, the positive detection of viral RNA in samples should be confirmed by sequencing, to exclude false-positive results [START_REF] Dacheux | More Accurate Insight into the Incidence of Human Rabies in Developing Countries through Validated Laboratory Techniques[END_REF]. This also makes it possible to 15/115 genotype viral strains, which is useful for epidemiological surveillance. Viral antigens can also be detected in skin biopsy specimens by FAT, and it is possible to isolate the virus from several types of bodily fluid sample, including saliva and CSF. Antibodies against rabies can also be detected in living patients, by the reference techniques, the rapid fluorescent focus inhibition test (RFFIT) [START_REF] Smith | A rapid reproducible test for determining rabies neutralizing antibody[END_REF] and the fluorescent antibody virus neutralization test (FAVN) [START_REF] Cliquet | Development of a fluorescent antibody virus neutralisation test (FAVN test) for the quantitation of rabies-neutralising antibody[END_REF], but also with ELISA [START_REF] Feyssaguet | Multicenter comparative study of a new ELISA, PLATELIA RABIES II, for the detection and titration of anti-rabies glycoprotein antibodies and comparison with the rapid fluorescent focus inhibition test (RFFIT) on human samples from vaccinated and non-vaccinated people[END_REF][START_REF] Welch | An evaluation of two commercially available ELISAs and one in-house reference laboratory ELISA for the determination of human anti-rabies virus antibodies[END_REF][START_REF] Muhamuda | Development and evaluation of a competitive ELISA for estimation of rabies neutralizing antibodies after post-exposure rabies vaccination in humans[END_REF]. However, it is rather difficult to determine rabies antibody titers from serum until eight days of illness have elapsed, and detection does not become possible until even later in the CSF (lower titers than for serum). In cases of infection with dog variant RABV, as in Thailand, CSF antibody remains undetectable regardless of the time after onset [START_REF] Dacheux | More Accurate Insight into the Incidence of Human Rabies in Developing Countries through Validated Laboratory Techniques[END_REF][START_REF] Wacharapluesadee | Ante-and post-mortem diagnosis of rabies using nucleic acid-amplification tests[END_REF]. In bat variant RABV cases, it is possible to detect antibody after eight days.

2 Post-exposure rabies prophylaxis: from history to future development

Historical development of postexposure prophylaxis

The preliminary works of Louis Pasteur

Pasteur's work on a vaccine for rabies occurred in the context of intense research on rabies in France. Pasteur reported the first successful transmission of rabies in rabbits to the Academy of Sciences in 1881. This transmission was achieved by inoculating the CNS tissues and spinal fluid, demonstrating that the causal agent was present not only in the saliva, but also in the tissues of the nervous system. Pasteur also observed that intracerebral inoculation with infected brain material in dogs, an innovation of his associate, Roux, shortened the usual incubation period to 1 to 2 weeks, making this system a useful laboratory model for the disease [START_REF] Pasteur | Sur une maladie nouvelle provoquée par la salive d'un enfant mort de la rage[END_REF][START_REF] Pasteur | Sur la rage[END_REF]. The following year, Pasteur described a small number of cases of animals recovering from rabies after experimental inoculation and that were subsequently immune to the virus [START_REF] Pasteur | Nouveaux faits pour servir à la connaissance de la rage[END_REF].

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By 1885, Pasteur had formulated the fundamental tenets of his vaccination strategy [START_REF] Pasteur | Méthode pour prévenir la rage après morsure[END_REF].

Through a series of passages in rabbits by intracerebral inoculation, Pasteur was able to create a form of rabies with enhanced virulence, decreasing the incubation time and "fixing" it at seven days. The virus populated the entire length of the spinal cord in rabbits, and became progressively more attenuated when infected tissue was suspended in dry air. The tissue lost all virulence after 15 days of desiccation. Dogs repeatedly inoculated with a series of rabbit cord suspensions of increasing virulence, or tissues that had been dried for shorter periods, displayed resistance to rabies, even when challenged via the intracerebral route, presumably due to the development of strong immune responses. Pasteur used 50 dogs for these experiments, a large number even by current standards. The animals were generally challenged with rabies only after the series of vaccinations [START_REF] Vodopija | The Natural History of Rabies[END_REF].

The first human vaccinations against rabies

Pasteur recognized that, given the unpredictable occurrence and clinical biology of rabies, a vaccine would be most beneficial if it could be given after a bite had occurred and if it induced protection over a time period shorter than the clinical incubation period of the disease. In modern terms, he was describing post-exposure vaccine prophylaxis for the prevention of symptomatic disease. Human use of the vaccine was associated with significant risk and controversy, but Pasteur justified such risks based on the invariably fatal outcome of rabies infections.

The first real opportunity for Pasteur to test his novel vaccine "treatment" for human rabies occurred in July 1885 and involved nine-year-old Joseph Meister, from Alsace, who had received numerous, severe bites from a rabid dog but had not yet developed symptoms of rabies. Pasteur, who was not medically qualified, arranged for the boy to be examined by two physicians [START_REF] Debré | [END_REF]. Treatment started about 60 h after bites, an acceptable delay for postexposure prophylaxis by today's standards. The boy was inoculated in the skin of the right upper abdomen, with a suspension of 15-day-old desiccated spinal cord from an infected rabbit, corresponding to the most attenuated viruses in the regimen. He received another 12 inoculations over the next 10 days, each less attenuated than the last and hence containing more virulent rabies virus. The last spinal cord preparation was dried for only one day [START_REF] Pasteur | Méthode pour prévenir la rage après morsure[END_REF].

Meister returned home, feeling well, after three weeks in Paris.

The Meister episode was a major accomplishment, but it did not attract the attention of the world to the subsequent use of rabies vaccines in humans by Pasteur. Later in 1885, a 15year-old shepherd from a small rural village in the Jura, Jean-Baptiste Jupille, was sent to Pasteur after being bitten several times on his left hand by a rabid dog. Again, the risk of rabies was high, but the treatment was successful. This time, the success of the vaccination was reported worldwide. In no time, Pasteur was treating potentially infected people from many different nations. By the end of 1886, more than 2,000 people had received Pasteur's rabies vaccine regimen [START_REF] Geison | The private science of Louis Pasteur[END_REF] and only a few rare failures were reported. In the 1898 annual report of the Institut Pasteur, 96 deaths were reported among 20,166 treated patients, a mortality rate of only 0.5% whereas that for unvaccinated individuals was 16% [START_REF] Pottevin | Les vaccinations antirabiques à l'Institut Pasteur en 1897[END_REF].

Further developments of Pasteur's treatment

Over the next 50 years, the vaccine underwent various modifications. Early on, Pasteur's colleagues began experimenting with variations on the number and timing of inoculations, and of the method for preparing rabbit spinal cords, based on their increasing experience and the individual history of exposure of each case. Many colleagues and students of Louis Early in the 20 th Century, methods involving the inactivation of the virus with phenol were introduced, to minimize the risk of vaccine-induced disease [START_REF] Kammer | Rabies vaccines: from the past to the 21st century[END_REF]. This approach was sporadically associated with incomplete inactivation and subsequent vaccine-induced 18/115 paralysis or serious adverse events related to hypersensitivity to nervous tissues in the vaccine, but nonetheless eventually replaced the method of Pasteur, continuing to be used into the 1950s.

The development of safer products

Vaccines derived from nervous system tissues were known to be associated with allergic encephalomyelitis, triggered by hypersensitivity to residual myelin basic protein in the preparations, causing severe demyelinization in some recipients [START_REF] Javier | Semple rabies vaccine: presence of myelin basic protein and proteolipid protein and its activity in experimental allergic encephalomyelitis[END_REF]. This led to the discontinuation of Pasteur's method of vaccine preparation in 1953. At the end of the 1950s, Fuenzalida and coworkers introduced the first myelin-free inactivated vaccines against RABV, prepared from neonatal mouse brains. These vaccines are still used in some parts of the developing world, due to their low cost, but their production and use are not recommended by the World Health Organization (WHO) [START_REF] Hemachudha | Myelin basic protein as an encephalitogen in encephalomyelitis and polyneuritis following rabies vaccination[END_REF][START_REF] Who Rabies Vaccine | WHO position paper[END_REF]. The recommended approaches are instead based on tissue culture methods developed in the 1950s and 1960s subsequently improved by other technical modifications. The duck embryo vaccine (DEV) was prepared from virus propagated in embryonated duck eggs [START_REF] Culbertson | Duck-embryo rabies vaccine; study of fixed virus vaccine grown in embryonated duck eggs and killed with betapropiolactone (BPL)[END_REF]. The Swiss Serum and Vaccine Institute later improved the DEV by using density gradient ultracentrifugation to generate a purified DEV (PDEV). The PDEV is still produced today and used for human vaccination worldwide [START_REF] Kammer | Rabies vaccines: from the past to the 21st century[END_REF]. In the early 1960s, Hilary Koprowski and his colleagues at the Wistar Institute, Philadelphia, selected the human diploid cell line WI-38, to avoid the problems inherent in the use of primary tissue cultures [START_REF] Hayflick | The establishment of a line (WISH) of human amnion cells in continuous cultivation[END_REF][START_REF] Plotkin | Vaccine production in human diploid cell strains[END_REF][START_REF] Wiktor | Development and clinical trials of the new human rabies vaccine of tissue culture (human diploid cell) origin[END_REF]. They adapted the Pitman-Moore strain of rabies virus to WI-38 cells and the free virus was inactivated with propiolactone and then concentrated by ultracentrifugation [START_REF] Plotkin | Rabies vaccine prepared in human cell cultures: progress and perspectives[END_REF]. This human diploid cell vaccine (HDCV), now prepared from the MRC-5 human diploid cell line, induced much stronger immune responses in animals and humans than any of the other vaccines known at the time. Another vaccine, the purified chick embryo cell vaccine (PCECV), was prepared 19/115 from the RABV strain Flury low-egg passage (LEP), grown in primary cultures of chick embryo fibroblasts [START_REF] Nicholson | Pre-exposure studies with purified chick embryo cell culture rabies vaccine and human diploid cell vaccine: serological and clinical responses in man[END_REF][START_REF] Dreesen | Two-year comparative trial on the immunogenicity and adverse effects of purified chick embryo cell rabies vaccine for pre-exposure immunization[END_REF]. The virus was inactivated with -propiolactone, then purified and concentrated by zonal centrifugation in a sucrose gradient [START_REF] Berlin | Rhesus diploid rabies vaccine (adsorbed), a new rabies vaccine. II. Results of clinical studies simulating prophylactic therapy for rabies exposure[END_REF][START_REF] Burgoyne | Rhesus diploid rabies vaccine (adsorbed): a new rabies vaccine using FRhL-2 cells[END_REF]. The use of this vaccine is essentially restricted to the Americas and Asia. The last vaccine, developed at the end of the 1980s, was the purified Vero cell rabies vaccine (PVRV), based on the Pitman-Moore strain of the RABV produced in the Vero cell line, a continuous African green monkey kidney cell line [START_REF] Montagnon | Polio and rabies vaccines produced in continuous cell lines: a reality for Vero cell line[END_REF]. This vaccine is inactivated with -propiolactone and concentrated and purified by zonal centrifugation and ultrafiltration. PVRV is licensed for use in humans in Europe and in many countries of the developing world. PCECV and PVRV have safety and efficacy records equivalent to those of HDCV and are cheaper [START_REF] Nicholson | Pre-exposure studies with purified chick embryo cell culture rabies vaccine and human diploid cell vaccine: serological and clinical responses in man[END_REF][START_REF] Dreesen | Two-year comparative trial on the immunogenicity and adverse effects of purified chick embryo cell rabies vaccine for pre-exposure immunization[END_REF][START_REF] Ajjan | Comparative study of the safety and protective value, in preexposure use, of rabies vaccine cultivated on human diploid cells (HDCV) and of the new vaccine grown on Vero cells[END_REF]. These purified cell culture and embryonated egg-based rabies vaccines are designed for both PrEP and PEP and have been administered to millions of people worldwide.

Societal and economic problems encountered

About 15 million people receive post-exposure vaccination worldwide each year [START_REF] Bourhy | Is there a need for anti-rabies vaccine and immunoglobulins rationing in Europe?[END_REF]. In industrialized countries, animal vaccination, developed in parallel with that for humans, has almost eliminated rabies as a significant problem. Unfortunately, in developing parts of the world, rabies remains a global public health threat. In these countries, many people who are exposed to rabies do not seek PEP, because they are not even aware of the risk of contracting this deadly disease, or because they live in rural areas, too far away from rabies prevention centers, which are generally located in large cities. Instead, traditional healers are frequently consulted and various types of wound care applied. When subjects at risk of contracting rabies do consult, it is often too late. Furthermore, the cost of rabies prevention or PEP is often too high [START_REF] Dodet | Fighting rabies in Africa: the Africa Rabies Expert Bureau (AfroREB)[END_REF][107]. Vaccines and immunoglobulins remain relatively expensive for much of the target population. The mean cost of the rabies vaccine alone for PEP is $45 in Africa and Asia (where the average salary is $2 person-day). 20/115

State-of-the art for rabies prophylaxis

Pre-exposure prophylaxis

PrEP is recommended for people frequently or continuously at risk of exposure to rabies, such as laboratory workers dealing with rabies, veterinary surgeons and animal handlers in countries in which rabies is enzootic. It is strongly recommended for travelers with extensive outdoor exposure in these countries, when access to appropriate medical care is limited.

Young children visiting or living in areas in which the disease is enzootic should also be vaccinated, as they are more frequently exposed than adults and may not report potential exposure to their parents [START_REF] Who Rabies Vaccine | WHO position paper[END_REF]. Two protocols, by the intramuscular (IM) and intradermal (ID) routes, are recommended by the WHO [START_REF] Who Rabies Vaccine | WHO position paper[END_REF]. IM doses of 0.5 or 1 ml (depending on the type of vaccine) are administered on days 0, 7, 21 or 28, in the deltoid area (adults and children >2 years old) or the anterolateral area of the thigh (children < 2 years old). ID administrations are performed on the same days, but with only 0.1 ml. Booster injections are given only to those at regular risk of exposure [START_REF] Who Rabies Vaccine | WHO position paper[END_REF]. Monitoring of the titer of rabies antibody may be useful for limiting the number of injections. A booster injection is required when titers fall below 0.5 IU/ml.

Post-exposure prophylaxis

Decisions regarding prophylaxis are complex. The indication for PEP depends on the type of contact with the suspected rabid animal, as defined by the WHO [START_REF] Who Rabies Vaccine | WHO position paper[END_REF] (Table 2), but other factors should also be taken into account when deciding whether to initiate PEP. These factors include the epidemiological situation of the country in which exposure occurred, the circumstances in which exposure occurred, the clinical features of the animal, if known, and the availability of the animal for veterinary observation. For cases in countries in which rabies is enzootic, PEP, if required according to WHO criteria, should not be delayed on the basis of epidemiological aspects or the clinical features or vaccination status of the animal. However, PEP may be discontinued if the animal remains healthy after the observation period, starting from the day of the bite (10 days according to the WHO and 15 days according to the legislation in force in some countries). For exposure categories II and III, thorough (~15 minutes) washing of all bite wounds and scratches, which should be flushed with soap/detergent and copious amounts of water, should be carried out immediately, or as soon as possible.

Data concerning the efficacy of prophylaxis are provided by experiments in animals and clinical trials. The PEP schedules recommended by the WHO are safe and highly effective.

PEP consists of three primary elements: wound care, infiltration of rabies immune globulin into the wound and vaccine administration. These three components are reviewed below.

Other measures, such as the use of tetanus toxin or antibiotics, are applied as needed.

Decisions are urgent, because delays may affect the outcome of treatment [START_REF] Wilde | Failures of post-exposure rabies prophylaxis[END_REF]. When completion of PEP with the same cell culture or embryonated egg-based rabies vaccine is not possible, another vaccine of a similar category may be used instead.

IM administration

The schedule of PEP vaccination consists of either four or five doses of vaccine [START_REF] Who Rabies Vaccine | WHO position paper[END_REF] (Figure 2). A dose of 1 or 0.5 ml is administered, depending on the type of vaccine used. Doses are delivered by the IM route, to the deltoid muscle (or the antero-lateral thigh in children < 2 years of age).

ID administration

Many studies have demonstrated the safety and efficacy of the IM delivery method, but modern vaccines are too expensive for many developing countries [START_REF] Dodet | Fighting rabies in Africa: the Africa Rabies Expert Bureau (AfroREB)[END_REF][107][START_REF] Dodet | The fight against rabies in Africa: From recognition to action[END_REF]. ID vaccine regimens, which use smaller amounts of vaccine and are less costly, therefore play an 22/115 important part in strategies to reduce the incidence of rabies. Initial studies demonstrated the safety and dose-sparing benefits of this route. Successful experience in Thailand has encouraged other countries to implement rabies PEP via the ID route and has led vaccine producers to validate the use of these ID regimens with their new vaccines [START_REF] Kamoltham | Rabies intradermal post-exposure vaccination of humans using reconstituted and stored vaccine[END_REF][START_REF] Quiambao | Reducing the cost of post-exposure rabies prophylaxis: efficacy of 0.1 ml PCEC rabies vaccine administered intradermally using the Thai Red Cross post-exposure regimen in patients severely exposed to laboratory-confirmed rabid animals[END_REF]. A twosite regimen, with the injection of 0.1 ml at 2 sites (deltoid and thigh) on days 0, 3, 7 and 28, is currently recommended by the WHO [START_REF] Who Rabies Vaccine | WHO position paper[END_REF]. ID delivery methods for rabies vaccine have been thoroughly investigated in humans and subjected to expert panel review by the WHO, but a number of precautions are required when using the ID technique, including staff training, attention to the conditions and duration of vaccine storage after reconstitution and the use of appropriate 1 ml syringes and short hypodermic needles.

PEP in previously vaccinated individuals

One IM or ID dose of vaccine on days 0 and 3 or four ID doses (equally distributed over the left and right deltoid or prescapular areas) at a single visit should be administered as a booster in patients exposed to rabies that have previously received preventive vaccination or a complete course of PEP with a cell culture or embryonated egg-based rabies vaccine [START_REF] Who Rabies Vaccine | WHO position paper[END_REF].

Passive immunization against rabies

According to WHO guidelines, rabies exposure requires post-exposure vaccination, and rabies immunoglobulins (RIGs) in cases of severe (category III) exposure [START_REF] Who Rabies Vaccine | WHO position paper[END_REF]. The life-saving benefit of adding specific RIG to post-exposure treatments of subjects exposed to rabies has been clearly established, particularly in cases of severe wounds. The underlying rationale is that many studies and observations have confirmed the importance of immediate administration of RIG to inhibit viral spread during the first seven to 14 days, before the immune response to the vaccine becomes sufficiently strong. Unfortunately, in many developing countries, only limited quantities of RIGs may be available via the private sector 23/115 and most of those at high risk of rabies cannot afford them. There are two types of product on the market: human rabies immune globulin (HRIG) and equine rabies immune globulin (ERIG). Crude equine sera have gradually been replaced by ERIGs of various degrees of purity.

Modern commercial preparations of HRIG are much safer than heterologous sera. They may contain antibodies against other agents and may inhibit immune responses to viral vaccines that have not been inactivated. Interference depends on the amount of specific antibody present. The administration of vaccines against other diseases, such as measles and varicella, should be delayed for at least four months after PEP, to allow the degradation of HRIG.

Interest has been growing, in developing countries in which rabies is endemic, in the use of purified ERIG in PEP for rabies, because HRIG is unavailable or unaffordable. Purified ERIG has been reported to generate adverse reactions in about 1% of patients. Life-threatening reactions are very rare. Serum sickness-like reactions to purified ERIG are also rare (less than 0.05%) in patients under the age of 10 years [START_REF] Lang | Evaluation of the safety, immunogenicity, and pharmacokinetic profile of a new, highly purified, heat-treated equine rabies immunoglobulin, administered either alone or in association with a purified, Vero-cell rabies vaccine[END_REF][START_REF] Suwansrinon | Adverse reactions to human rabies immune globulin manufactured by the Thai Red Cross Society[END_REF][START_REF] Wilde | Adverse effects of equine rabies immune gobulin[END_REF][START_REF] Wilde | Purified equine rabies immune globulin: a safe and affordable alternative to human rabies immune globulin[END_REF][START_REF] Wilde | Equine rabies immune globulin: a product with an undeserved poor reputation[END_REF]. In this context, the use of highlypurified ERIG represents a safe and effective alternative. Some of these preparations are digested with pepsin (to remove the Fc part of the immunoglobulin molecules, which might be responsible for some adverse reactions), yielding F(ab′) 2 fragments, corresponding to the antigen-binding part of the molecule. These preparations are effective and safe [START_REF] Lang | Evaluation of the safety, immunogenicity, and pharmacokinetic profile of a new, highly purified, heat-treated equine rabies immunoglobulin, administered either alone or in association with a purified, Vero-cell rabies vaccine[END_REF].

Management of immunocompromised patients

Immunocompromised patients should receive the full course of PEP vaccination by the IM route and the vaccination should be completed by passive immunization for both category II and category III exposures [START_REF] Who Rabies Vaccine | WHO position paper[END_REF]. When feasible, the rabies virus neutralizing antibody titer of the patient should be checked two to four weeks after the last dose of vaccine, to assess the possible need for an additional dose of vaccine.

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Adverse effects and contraindications

The purified cell culture and embryonated egg-based rabies vaccines are safe and well tolerated. However, transient pain, redness and swelling at the site of injection occur in 35 to 45% of vaccinated patients, particularly after ID injections. Occasionally, more severe reactions may occur, such as transient fever, headache, dizziness and gastrointestinal symptoms. There is no contraindication for vaccination, because rabies infection is fatal. Individuals taking chloroquine for malaria treatment may have a weaker response to rabies vaccination, in which case, the IM regimen of PEP is recommended. If a previous severe reaction is documented, the use of a different rabies vaccine is recommended.

Future developments

Several new approaches to vaccine production have been developed: the production of rabies antigens in baculovirus [START_REF] Tordo | Molecular epidemiology of lyssaviruses: focus on the glycoprotein and pseudogenes[END_REF] and the development of edible vaccines through the expression of rabies antigens in plants [START_REF] Fu | Rabies and rabies research: past, present and future[END_REF][START_REF] Mcgarvey | Expression of the rabies virus glycoprotein in transgenic tomatoes[END_REF][START_REF] Modelska | Immunization against rabies with plant-derived antigen[END_REF][START_REF] Yusibov | Antigens produced in plants by infection with chimeric plant viruses immunize against rabies virus and HIV-1[END_REF]. Recombinant poxvirus vaccines are used extensively in animals and have been evaluated for human use. A canarypox vector encoding the rabies G protein has been prepared and tested in humans [START_REF] Cadoz | Immunisation with canarypox virus expressing rabies glycoprotein[END_REF]. Canarypoxvirus, unlike vaccinia virus, cannot replicate in humans and is therefore unlikely to elicit any serious side effects.

Recombinant adenoviruses encoding the G protein have also been tested in mice and dogs [START_REF] Tims | Adult dogs receiving a rabies booster dose with a recombinant adenovirus expressing rabies 107/115 virus glycoprotein develop high titers of neutralizing antibodies[END_REF]. Furthermore, recombinant DNA vaccines are currently being developed for use in humans and in dogs [START_REF] Lodmell | Gene gun particle-mediated vaccination with plasmid DNA confers protective immunity against rabies virus infection[END_REF][START_REF] Lodmell | DNA immunization protects nonhuman primates against rabies virus[END_REF][START_REF] Perrin | Immunization of dogs with a DNA vaccine induces protection against rabies virus[END_REF][START_REF] Xiang | Vaccination with a plasmid vector carrying the rabies virus glycoprotein gene induces protective immunity against rabies virus[END_REF][START_REF] Xiang | Immune responses to nucleic acid vaccines to rabies virus[END_REF]. However, none of these approaches is likely to graduate to use in clinical settings in the near future.

Several technological approaches currently being used to develop human monoclonal antibody mixtures have yielded promising results. Human monoclonal antibodies directed against non overlapping epitopes of the G protein have been shown to mediate the broad neutralization in vitro of a large panel of field isolates of RABV from various animal species, 25/115 and to ensure in vivo protection in a Syrian hamster rabies challenge model; they have also been demonstrated to be effective in phase I trials in humans [START_REF] Bakker | Novel human monoclonal antibody combination effectively neutralizing natural rabies virus variants and individual in vitro escape mutants[END_REF][START_REF] Bakker | First administration to humans of a monoclonal antibody cocktail against rabies virus: safety, tolerability, and neutralizing activity[END_REF][START_REF] Champion | The development of monoclonal human rabies virusneutralizing antibodies as a substitute for pooled human immune globulin in the prophylactic treatment of rabies virus exposure[END_REF][START_REF] Dorfman | Development of human monoclonal antibodies to rabies[END_REF][START_REF] Sloan | Identification and characterization of a human monoclonal antibody that potently neutralizes a broad panel of rabies virus isolates[END_REF]. The successful development of such antibodies would help to ensure the supply of life-saving biological drugs to people exposed to rabies, at a reasonable cost.

3 In search of an effective antiviral treatment for human rabies: where are we now?

During the progress in our understanding of RABV infection, the various steps of the viral cycle have been considered as potential antiviral targets for preventing, limiting or stopping the infection. With this goal in mind, the antiviral activities of various drugs thought to affect early events in the cycle of RABV replication (including virus entry and release into the cytoplasm of infected cells) and transcription/replication steps, have been evaluated in vitro or in animal models. In parallel, well characterized broad-spectrum antiviral compounds, such as interferons and inducers of interferon, and molecules with no suspected antiviral activity have been assessed for use against rabies infection. Some of these compounds were also administered, alone or in combination, in cases of clinical human rabies. However, although some studies have reported interesting preliminary results in vitro, no drug has been shown to be effective, with convincing and reproducible activity against infection in cases in which clinical signs are already present, in animal models or in humans. The successful outcome achieved in 2004, in which a rabid patient treated aggressively subsequently recovered restored some hope. Unfortunately, the efficacy of this protocol was not subsequently confirmed, as all published attempts to reproduce these findings have been unsuccessful. The quest to identify an effective and potent antiviral treatment against human rabies therefore continues.

In this section, we present an overview of the various drug evaluations performed in vitro and in animal models (described in Table 3), followed by the major drug treatments administered in cases of human rabies, including the case of the Milwaukee protocol and subsequent 26/115 unsuccessful attempts at replication (described in Table 4). This section ends with a brief description of potential future advances in the field of antirabies drug discovery.

Overview of drugs evaluated for activity against rabies virus in vitro and in animal models

Inhibitors of viral entry into the cell

The attachment of viruses on target cells and their penetration into those cells are of great interest when considering an antiviral strategy. Indeed, blocking or disturbing these early steps in the cycle of virus replication may decrease or abolish infection. With this aim in mind, viral proteins involved in cell entry (such as viral glycoproteins) and the binding domains of the cellular receptors are considered potential antiviral targets. Several different compounds have been tested in cultured cells infected with rabies. Some of these compounds, including drugs targeting the nicotinic acetylcholine receptor (nAChR) and various lipid molecules and molecules with high affinity for lipids, have been found to have potential antiviral effects.

Antibodies against rabies glycoprotein and rabies vaccines

The antiviral activities of rabies antibodies targeting the viral glycoprotein have been clearly demonstrated in vitro and in animal models. The efficacy of these compounds is now used, in combination with rabies vaccines, in PEP, to prevent rabies infection (see section 2.2).

However, the use of antibodies remains of limited value against clinical rabies infection, for which no efficacy has been demonstrated (see section 3.2.2.2.4).

Live attenuated rabies vaccines have been investigated for the treatment of symptomatic rabies encephalitis in various animal models, including dogs, monkeys and mice. No convincing results have been obtained, despite the potential induction of neutralizing antibodies in the CNS and/or CSF after intrathecal or intracerebral administration [START_REF] Baer | Prolonging morbidity in rabid dogs by intrathecal injection of attenuated rabies vaccine[END_REF][START_REF] Faber | Effective preexposure and postexposure prophylaxis of rabies with a highly attenuated recombinant rabies virus[END_REF][START_REF] Warrell | An attempt to treat rabies encephalitis in monkeys with intrathecal live rabies virus RV 675. Brief report[END_REF].

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Indeed, although promising results were obtained in a preliminary experiment in dogs, in which three symptomatic animals (of 12) treated intrathecally with a live attenuated ERAderived rabies vaccine appeared to recover, a second experiment based on the same treatment resulted only in the probable prolongation of the survival period, with all the symptomatic dogs eventually dying [START_REF] Baer | Prolonging morbidity in rabid dogs by intrathecal injection of attenuated rabies vaccine[END_REF]. In another study conducted in monkeys, a live attenuated Flury LEP-derived rabies vaccine was also administrated intrathecally at the clinical stage, but without success, as all symptomatic animals (n=2) succumbed with no detectable prolongation of the survival period [START_REF] Warrell | An attempt to treat rabies encephalitis in monkeys with intrathecal live rabies virus RV 675. Brief report[END_REF]. A highly attenuated live recombinant RABV (SPBAANGAS-GAS-GAS) derived from the SAD B19 strain and expressing three mutated G genes (mutation R333G), has been evaluated for the prevention of rabies infection (for PrEP or PEP) in the mouse model. Interesting results have been obtained in the preexposure context, but the complete protection of mice was observed only if attenuated recombinant RABV was administered intramuscularly or intracerebrally shortly (4 hours) after infection.

When administered at a time (4 days post-infection) close to the predicted onset of the first symptoms (5-6 days post-infection in this model), survival rates fell to 50% and 30% after intracerebral and intramuscular route of administration of the live attenuated vaccine, respectively. Although potentially promising, effective treatments of human clinical rabies based on such strategies appear to be some way off. Moreover, another major obstacle to the use of live attenuated rabies vaccines in humans is the safety of these vaccines, which must be extremely high, particularly for rabies and taking into account the existence of an effective and safe, albeit complex and costly vaccine [START_REF] Mcgettigan | Experimental rabies vaccines for humans[END_REF].

Receptor-specific inhibitors

Alpha-bungarotoxin (-Btx) is a high-affinity irreversible competitive antagonist of AChR that interacts irreversibly with the ACh binding site on the 40 kDa subunit of this receptor.

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This drug is a snake venom neurotoxin, obtained from the Taiwanese banded krait, Bungarus multicinctus, which inhibits rabies infection in various types of cultured cells, including chick embryo muscle cells [START_REF] Lentz | Is the acetylcholine receptor a rabies virus receptor?[END_REF], cultured primary rat myotubes [START_REF] Tsiang | Infection of cultured rat myotubes and neurons from the spinal cord by rabies virus[END_REF] and IMR-32 human neuroblastoma cells (continuous nerve cell line expressing neuronal nicotinic acetylcholine receptors) [START_REF] Lentz | Rabies virus infection of IMR-32 human neuroblastoma cells and effect of neurochemical and other agents[END_REF]. Competition between -Btx and RABV was observed shortly before infection (from 2 h to 30 min), with a dose-dependent response (from 10 -5 to 10 -7 M in cultured primary rat myotubes) [START_REF] Lentz | Is the acetylcholine receptor a rabies virus receptor?[END_REF][START_REF] Tsiang | Infection of cultured rat myotubes and neurons from the spinal cord by rabies virus[END_REF][START_REF] Lentz | Rabies virus infection of IMR-32 human neuroblastoma cells and effect of neurochemical and other agents[END_REF]. However, the antiviral effect of -Btx was not observed when cells were treated later, 15 minutes before infection, at the time of infection or a few hours later [START_REF] Lentz | Is the acetylcholine receptor a rabies virus receptor?[END_REF][START_REF] Tsiang | Infection of cultured rat myotubes and neurons from the spinal cord by rabies virus[END_REF][START_REF] Reagan | Rabies virus interaction with various cell lines is independent of the acetylcholine receptor[END_REF]. No virucidal effect was identified [START_REF] Lentz | Is the acetylcholine receptor a rabies virus receptor?[END_REF]. In a single study, an inhibitory effect was also demonstrated with d-tubocurarine (dTC), a cholinergic antagonist isolated from the South American plant Chondrodendron tomentosum, when cells were treated before infection [START_REF] Lentz | Is the acetylcholine receptor a rabies virus receptor?[END_REF]. Like -Btx, this toxin interacts with the ACh binding site of the AChR and reversibly and competitively inhibits the binding of -Btx. However, the prevention of rabies infection by this drug was not replicated in other studies [START_REF] Lentz | Rabies virus infection of IMR-32 human neuroblastoma cells and effect of neurochemical and other agents[END_REF][START_REF] Reagan | Rabies virus interaction with various cell lines is independent of the acetylcholine receptor[END_REF].

These toxins probably act by inhibiting the attachment step, as demonstrated for -Btx with A/J (H-2) mouse neuroblastoma cells [START_REF] Briggs | The effect of neurotoxin on rabies virus binding to mouse neuroblastoma cells[END_REF]. These observations, together with experiments aiming to demonstrate the colocalization of rabies virus and AChR, led to the identification of AChR as a rabies virus receptor. Moreover, the amino-acid sequences of the toxic loop of -Btx and the G protein of rabies virus have been shown to be very similar [START_REF] Lentz | Amino acid sequence similarity between rabies virus glycoprotein and snake venom curaremimetic neurotoxins[END_REF] and synthetic peptides of the major neurotoxin determinant on the 1 subunit of AChR inhibit infection [START_REF] Lentz | Rabies virus infection of IMR-32 human neuroblastoma cells and effect of neurochemical and other agents[END_REF]. Both then compete for attachment sites on the AChR in Torpedo sp. [START_REF] Lentz | Amino acid sequence similarity between rabies virus glycoprotein and snake venom curaremimetic neurotoxins[END_REF][START_REF] Lentz | Synthetic peptides in the study of the interaction of rabies virus and the acetylcholine receptor[END_REF][START_REF] Bracci | Antipeptide monoclonal antibodies inhibit the binding of rabies virus glycoprotein and alpha-bungarotoxin to the nicotinic acetylcholine receptor[END_REF].

However, the potential value of this drug as an antiviral agent for treating rabies infection remains limited, because -Btx acts as an inhibitor only at the viral binding step, in preventive action, and has not been shown to be virucidal. Moreover, AChR is not an obligate receptor for susceptibility to rabies infection in different cell types [START_REF] Tsiang | Infection of cultured rat myotubes and neurons from the spinal cord by rabies virus[END_REF][START_REF] Reagan | Rabies virus interaction with various cell lines is independent of the acetylcholine receptor[END_REF].

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Ligands of the NCAM receptor, which has been identified as another possible cellular receptor for RABV, also inhibit rabies infection in vitro. They include heparan sulfate, one of the natural ligands of this receptor, and antibodies targeting NCAM receptors [START_REF] Thoulouze | The neural cell adhesion molecule is a receptor for rabies virus[END_REF]. In this same study, a reduction of virus infectivity was also observed after the incubation of rabies virus with soluble NCAM protein. However, drugs targeting this receptor appear to be of limited value, because NCAM receptor-deficient mice are not resistant to rabies infection [START_REF] Jackson | Experimental rabies virus infection of p75 neurotrophin receptor-deficient mice[END_REF]. Antiviral effects of two neurotrophin molecules (nerve growth factor and neurotrophin-3) have been evaluated on rabies-infected mouse dorsal root ganglia cell cultures [START_REF] Castellanos | Studying neurotrophin antiviral effect on rabies-infected dorsal root ganglia cultures[END_REF]. The inhibition of viral infection has been suggested when these drugs were added at the time of infection or later.

Lipid-based inhibitors

Early in the 1980s, evidence was obtained that lipids, together with gangliosides and phospholipids, might also contribute to the binding of lyssaviruses to cell membranes [START_REF] Superti | Mechanism of rabies virus entry into CER cells[END_REF][START_REF] Superti | Involvement of gangliosides in rabies virus infection[END_REF][START_REF] Superti | Role of phospholipids in rhabdovirus attachment to CER cells. Brief report[END_REF].

A dose-dependent inhibition of viral infection was observed in vitro with chick embryorelated (CER) cells when RABV was incubated with gangliosides before infection. By contrast, the addition of gangliosides before infection did not modify infectivity. This observation suggests that highly sialylated gangliosides are part of the cellular membrane receptor structure for the attachment of infective rabies virus [START_REF] Superti | Mechanism of rabies virus entry into CER cells[END_REF]. These results also suggest that competition occurs between RABV and gangliosides for binding to the cell surface, or possibly to aggregated formations, with the entrapment of virions, thereby preventing the infection of cells. An inhibition of viral attachment was also observed with phospholipids or phospholipase [START_REF] Superti | Mechanism of rabies virus entry into CER cells[END_REF][START_REF] Superti | Role of phospholipids in rhabdovirus attachment to CER cells. Brief report[END_REF]. Indeed, viral inhibition was clearly demonstrated with RABV in the same CER cell line treated with phospholipase A2 before infection, and, to a lesser extent, with various other phospholipases (phospholipase C, D and shingomyelinase). The prior 30/115 incubation of RABV with various phospholipids also exerted an inhibitory effect that was stronger than that observed when these compounds were added during infection (for L-phosphatidylserine and, to a lesser extent, L--phosphatidylethanolamine and L-phosphatidylinositol). However, targeting or using gangliosides and phospholipids for antiviral strategies against rabies infection appears to be of little value, as the inhibitory effects of such treatment are limited to the viral binding step. Indeed, the addition of these molecules before or after infection did not modify infection rates [START_REF] Superti | Mechanism of rabies virus entry into CER cells[END_REF][START_REF] Superti | Involvement of gangliosides in rabies virus infection[END_REF][START_REF] Superti | Role of phospholipids in rhabdovirus attachment to CER cells. Brief report[END_REF]. Moreover, these receptors are not specific and consist of multicomponent structures, the composition of which may differ between cell types.

Lectins and neuraminidases

Some studies have demonstrated an inhibitory activity of lectinssugar-binding proteins that are highly specific for their sugar moietiesin rabies infections of CER cells [START_REF] Conti | Effect of concanavalin A on the early events of rabies virus infection of CER cells[END_REF][START_REF] Marchetti | Inhibition of herpes simplex, rabies and rubella viruses by lectins with different specificities[END_REF].

Concanavalin A, a lectin extracted from the jack-bean Canavalia ensiformis, which specifically binds to -D-mannopyranosyl and -D-glucopyranosyl residues of carbohydratecontaining components on the cell surface, has been shown to have a direct inhibitory effect on virion infectivity [START_REF] Conti | Effect of concanavalin A on the early events of rabies virus infection of CER cells[END_REF]. This inhibitory effect was observed when cells were treated before infection or after the viral binding step, and could be reversed by -D-methylmannopyranoside. In a more recent study, the ability of 11 lectins of various origins to inhibit RABV infection was assessed [START_REF] Marchetti | Inhibition of herpes simplex, rabies and rubella viruses by lectins with different specificities[END_REF]. Five of these lectins seemed to prevent rabies infection in CER cells: the agglutinins of Ulex europaeus (UEA), Limulus polyphemus (LPA), Narcissus pseudonarcissus (NPA), wheat germ (WGA) and Bauhinia purpurea (BPA). A dose-dependent effect was observed for LPA, NPA, WGA and BPA. It was suggested that LPA and NPA prevent virus attachment, whereas WGA and BPA inhibit infection at a later stage. Only two of these four lectins (NPA and BPA) have potentially useful selectivity indices [START_REF] Marchetti | Inhibition of herpes simplex, rabies and rubella viruses by lectins with different specificities[END_REF].

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Neuraminidases, which are also known as sialidases, are glycoside hydrolases that cleave the glycoside bonds of neuraminic acids. The activity of various bacterial neuraminidases, including those of Clostridium perfringens (type V and X) and Vibrio cholerae, was evaluated in vitro in RABV infection [START_REF] Superti | Mechanism of rabies virus entry into CER cells[END_REF][START_REF] Superti | Involvement of gangliosides in rabies virus infection[END_REF]. A dose-dependent inhibition of viral attachment was observed only with target cells that had previously been treated for one hour with the neuraminidase of C. perfringens. The inhibition mediated by neuraminidase treatment persisted for 3 h after removal of the enzyme, the cells gradually recovering their susceptibility to rabies infection thereafter [START_REF] Superti | Mechanism of rabies virus entry into CER cells[END_REF].

Like other drugs targeting the first steps of the viral replication cycle (attachment and penetration into cells), the use of lectins or neuraminidases for the treatment of rabies infection appears to be of little value, as the inhibitory effects of such treatment are not selected, and are transitory and limited to the viral binding step. Moreover, the potential antiviral effects of these molecules have never been evaluated in animal models of rabies infection.

Analog and metabolic inhibitors

It has long been demonstrated that antimetabolic agents affecting DNA synthesis (such as mitomycin C, fluorodeoxyuridine and bromodeoxyuridine) or DNA transcription by RNA polymerase (such as actinomycin D) have no effect on RABV replication in vitro, leading to the classification of RABV as an RNA-containing virus [START_REF] Defendi | Metabolic and autoradiographic studies of rabies virusinfected cells[END_REF][START_REF] Hamparian | Contributions to characterization and classification of animal viruses[END_REF][START_REF] Kissling | Anti-Rabies Vaccine of Tissue Culture Origin[END_REF]. Further therapeutic evaluations of these agents (including mitomycin C, actinomycin D, fluorodeoxyuridine and other antimetabolic drugs and analog inhibitors) provided no evidence of a virucidal effect, inhibition of viral replication or protective effect in mouse models [START_REF] Enright | Experimental rabies chemotherapy in mice[END_REF][START_REF] Flamand | Effect of actinomycin D and cytosine arabinoside on rabies and VSV multiplication[END_REF][START_REF] Maes | The Molecular Biology of Viruses[END_REF].

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However, cytarabin (also known as cytosine arabinoside or ara-C) is a potent inhibitor of DNA synthesis that has surprisingly been shown to be effective in vitro against the replication of laboratory-adapted RABV strains [START_REF] Maes | The Molecular Biology of Viruses[END_REF][START_REF] Campbell | The inhibition of rabies virus by arabinosyl cytosine. Studies on the mechanism and specificity of action[END_REF][START_REF] Harmon | Effects of cytosine arabinoside, adenine arabinoside, and 6azauridine on rabies virus in vitro and in vivo[END_REF]. It has been suggested that this inhibition occurs soon after infection (3 hours post-infection) [START_REF] Maes | The Molecular Biology of Viruses[END_REF][START_REF] Campbell | The inhibition of rabies virus by arabinosyl cytosine. Studies on the mechanism and specificity of action[END_REF] and is specific to this molecule, as other pyrimidine nucleoside analogs or derivatives (such as 2-thio and 5-iodo derivatives of ara-C and uracil arabinoside) have no inhibitory effect [START_REF] Campbell | The inhibition of rabies virus by arabinosyl cytosine. Studies on the mechanism and specificity of action[END_REF]. Inhibition was overcome by adding high concentrations of both deoxycytidine and cytidine, without concomitant reversal of the inhibition of all DNA synthesis [START_REF] Maes | The Molecular Biology of Viruses[END_REF]. It has been suggested that the inhibition of RABV by ara-C requires the induction of a cellular protein, as the addition of antimetabolic agents acting on DNA synthesis partially or completely reverses this inhibition [START_REF] Campbell | The inhibition of rabies virus by arabinosyl cytosine. Studies on the mechanism and specificity of action[END_REF]. This in vitro inhibitory effect of ara-C on virus replication was partly reproduced in another study, in which viral replication was only slightly decreased, even in conditions completely shutting down host DNA synthesis [START_REF] Flamand | Effect of actinomycin D and cytosine arabinoside on rabies and VSV multiplication[END_REF]. Another pyrimidine analog inhibitor, 6-azauridine (6-azu), was shown to have an antiviral effect on laboratory-adapted RABV strain replication in vitro [START_REF] Harmon | Effects of cytosine arabinoside, adenine arabinoside, and 6azauridine on rabies virus in vitro and in vivo[END_REF] that was unlikely to be due to a direct virucidal effect [START_REF] Enright | Experimental rabies chemotherapy in mice[END_REF]. However, neither of these drugs was effective at reducing the mortality rate in mice challenged with non adapted virus strains [START_REF] Harmon | Effects of cytosine arabinoside, adenine arabinoside, and 6azauridine on rabies virus in vitro and in vivo[END_REF]. Only one study suggested beneficial effects of ara-C for rabies prevention in mice, together with iododeoxyuridine, actinomycin D, rifampicin or azathioprine [START_REF] Sodja | Experimental chemotherapy of rabies. Preliminary results[END_REF].

However, this result remains difficult to interpret, due to the complexity and lack of standardization of the various experimental protocols performed in the study concerned.

Purine analogs with antiviral activities against RNA and DNA viruses have also been extensively studied in RABV infection, including adenosine analogs, such as vidarabine (arabinosyl adenosine or ara-A) and dihydroxypropyladenine ((S)-DHPA) [START_REF] De Clercq | Broad-spectrum antiviral activity of adenosine analogues[END_REF]. A dosedependent inhibitory effect of vidarabine on laboratory-adapted RABV strain replication has 33/115 been observed in vitro, but was not further confirmed in the mouse model [START_REF] Harmon | Effects of cytosine arabinoside, adenine arabinoside, and 6azauridine on rabies virus in vitro and in vivo[END_REF][START_REF] Bussereau | Effect of heteropolyanions and nucleoside analogues on rabies virus[END_REF]. This molecule was not virucidal and it was suggested that it acts by inhibiting cell and viral RNA synthesis and viral protein synthesis [START_REF] Bussereau | Effect of heteropolyanions and nucleoside analogues on rabies virus[END_REF]. Four different acyclic derivatives of adenosine (including (S)-DHPA), together with one carbocyclic substance, 3-deazaadenosine (CDZ), have been shown to inhibit RABV production by 90% in single-growth cycle conditions [START_REF] Bussereau | Effect of heteropolyanions and nucleoside analogues on rabies virus[END_REF][START_REF] Bussereau | Search for compounds which have an inhibitory effect on rhabdovirus multiplication in vitro[END_REF]. This inhibition was dose-independent over a large range of concentrations (5 to 500 μg/ml). However, a strong antimitogenic effect was observed with these drugs. No direct virucidal effect was observed, particularly with (S)-DHPA [START_REF] Bussereau | Effect of heteropolyanions and nucleoside analogues on rabies virus[END_REF][START_REF] Bussereau | Search for compounds which have an inhibitory effect on rhabdovirus multiplication in vitro[END_REF][START_REF] Sodja | Effect of 9-(S)-(2,3-dihydroxypropyl)adenine on experimental rabies infection in laboratory mice[END_REF]. It has been suggested that the inhibitory effects of (S)-DHPA on viral multiplication result from the inhibition of viral RNA synthesis [START_REF] Bussereau | Effect of heteropolyanions and nucleoside analogues on rabies virus[END_REF]. The therapeutic effects of this molecule were assessed against rabies infection in mice [START_REF] Bussereau | Treatment of rabies in mice and foxes with antiviral compounds[END_REF]. Despite the use of several different doses, beginning one day after viral challenge and using different routes of administration, no effect was demonstrated, by contrast to reports from a previous study [START_REF] Sodja | Effect of 9-(S)-(2,3-dihydroxypropyl)adenine on experimental rabies infection in laboratory mice[END_REF]. Foxes were treated orally with (S)-DHPA at a dose of 30 mg/kg, on the day of infection. Despite the limited number of animals tested, the time of death was found to be only slightly delayed by treatment [START_REF] Bussereau | Treatment of rabies in mice and foxes with antiviral compounds[END_REF]. Ribavirin is another purine analog that has been shown to have a dose-dependent effect on RABV production, within the 5 to 500 μg/ml range of concentrations, in single-growth cycle conditions, with 90% inhibition of infectious virion release observed at a concentration of 25 μg/ml, and 99.9% inhibition at concentrations above 50 μg/ml [START_REF] Bussereau | Effect of heteropolyanions and nucleoside analogues on rabies virus[END_REF][START_REF] Bussereau | Search for compounds which have an inhibitory effect on rhabdovirus multiplication in vitro[END_REF]. It has been suggested that this drug exerts its antiviral effects by inhibiting cellular and viral RNA synthesis, together with viral protein synthesis [START_REF] Bussereau | Effect of heteropolyanions and nucleoside analogues on rabies virus[END_REF]. No direct virucidal effect was observed, but a strong antimitogenic effect was observed at a concentration of 25 μg/ml. Once again, the antiviral effect was not replicated in mice experimentally infected with rabies virus, and two related molecules (including a lipophilic analog with RTA and a structurally and functionally related drug with selenazofurin) had only a slight effect [START_REF] Bussereau | Treatment of rabies in mice and foxes with antiviral compounds[END_REF]. It has been suggested that 34/115 another purine-related molecule, guanine 7-N-oxide (G-7-Ox), isolated from Streptomyces sp., has broadly inhibitory effects against several RNA viruses in vitro, including RABV [START_REF] Yamamoto | Inhibitory effect of a new antibiotic, guanine 7-N-oxide, on the replication of several RNA viruses[END_REF]. However, these preliminary results were not confirmed in subsequent studies.

No conclusive or strongly demonstrative result was obtained in any of these studies based on the use of analogs and metabolic inhibitors in rabies infection. This suggests that either these drugs are not suitable for rabies treatment or that the right inhibitor has yet to be identified.

Lysosomotropic agents

As described above, RABV is an enveloped virus that makes use of the endocytic pathway as its main route of entry into target cells (see section 1.2). Following the delivery of the virus to an endosomal compartment, membrane fusion occurs, mediated by the viral glycoprotein in a pH-dependent manner and leading to the release of the virus into the cytoplasm. This mechanism has been demonstrated with various lysosomotropic agents (which may selectively enter the lysosomes of certain cell types), including ammonium chloride, chloroquine and quinine, suggesting that it may be possible to target new viral steps in rabies infection. These drugs interfered in vitro with the replication of RABV, by raising the pH of endosomes and lysosomes and then preventing the fusion of the viral envelope with endosomal membranes. An inhibition of infection was observed in IMR-32 human neuroblastoma cells [START_REF] Lentz | Rabies virus infection of IMR-32 human neuroblastoma cells and effect of neurochemical and other agents[END_REF], CER cells [START_REF] Superti | Mechanism of rabies virus entry into CER cells[END_REF] and other cells (including NIE-115 murine neuroblastoma cells, NS20 cell lines, dissociated neuronal cells and cultured rat myotubes) [START_REF] Tsiang | Ammonium chloride and chloroquine inhibit rabies virus infection in neuroblastoma cells. Brief report[END_REF]. Antiviral effects occurred only when drugs were applied shortly (a few hours) after the viral attachment step, with a potentially dose-dependent relationship, at least for chloroquine [START_REF] Superti | Mechanism of rabies virus entry into CER cells[END_REF][START_REF] Lentz | Rabies virus infection of IMR-32 human neuroblastoma cells and effect of neurochemical and other agents[END_REF][START_REF] Tsiang | Ammonium chloride and chloroquine inhibit rabies virus infection in neuroblastoma cells. Brief report[END_REF]. Two other lysosomotropic molecules, methylamine and monensin, displayed dose-dependent virus inhibition in vitro, acting after the virus adsorption step [START_REF] Conti | Effect of inhibitors of cytoplasmic structures and functions on rabies virus infection in vitro[END_REF]. None of these molecules had any effect on viral attachment or virucidal activity. Moreover, the 35/115 antiviral activity of lysosomotropic agents against rabies infection has not yet been evaluated in animal models.

Drugs acting on microfilaments and the microtubule cytoskeleton

In an effort to understand the cellular mechanisms of RABV replication and maturation, several drugs acting on the cellular organization of microfilaments or calmodulin processes have been tested, including cytochalasin B, which acts directly on actin microfilaments [START_REF] Bussereau | Cellular response to rabies virus infection[END_REF][START_REF] Lycke | Rabies virus infection of cultured rat sensory neurons[END_REF][START_REF] Lockhart | Actin-independent maturation of rabies virus in neuronal cultures[END_REF], anti-calmodulin drugs, including trifluoperazine and chlorpromazine [START_REF] Lockhart | Actin-independent maturation of rabies virus in neuronal cultures[END_REF], and inhibitors of Ca 2+ -dependent processes, such as EGTA and nifedipine (reversible L-type Ca 2+ channel antagonist) or the Ca 2+ -specific ionophore A23187 [START_REF] Lockhart | Actin-independent maturation of rabies virus in neuronal cultures[END_REF]. However, no inhibitory effect on viral multiplication was observed, indicating that the actin-based cytoskeleton is not a target for RABV inhibition, as viral maturation was independent of the integrity of microfilament structures and calmodulin-dependent processes in neuronal cells. Only a single study has suggested a potential antiviral effect of cytochalasin B and trifluoperazine in IMR-32 and CER cells, but this finding was not subsequently confirmed [START_REF] Conti | Effect of inhibitors of cytoplasmic structures and functions on rabies virus infection in vitro[END_REF]. An absence of antiviral effects on established rabies infection was also demonstrated in vitro with drugs affecting microtubules, such as vinblastine, colchicine and colcemid [START_REF] Bussereau | Cellular response to rabies virus infection[END_REF][START_REF] Lycke | Rabies virus infection of cultured rat sensory neurons[END_REF]. Moreover, no virucidal effect on RABV was observed with colchicine or vinblastine [START_REF] Tsiang | Evidence for an intraaxonal transport of fixed and street rabies virus[END_REF][START_REF] Bulenga | Post-exposure local treatment of mice infected with rabies with two axonal flow inhibitors, colchicine and vinblastine[END_REF]. By contrast, pharmacological impairment of microtubulin and microfilament function prevented virus transport by axonal flow to differentiated rat embryonic dorsal root ganglion cells and a compartmentalized cell culture system [START_REF] Lycke | Rabies virus infection of cultured rat sensory neurons[END_REF]. Local administration of colchicine or vinblastine to the sciatic nerve of mice was demonstrated to prevent rabies infection, even if given up to 6 to 24 h after viral inoculation [START_REF] Bulenga | Post-exposure local treatment of mice infected with rabies with two axonal flow inhibitors, colchicine and vinblastine[END_REF]. This preventive effect of colchicine was confirmed in an adult rat model in which this drug was administered to the sciatic nerve through a silicone cuff, 4 to 48 hours before viral challenge [START_REF] Tsiang | Evidence for an intraaxonal transport of fixed and street rabies virus[END_REF]. These results suggest that the retrograde transfer of RABV, from the periphery to the neuronal soma, requires the 36/115 integrity of tubulin-and actin-containing structures. However, the use of these drugs in rabies infection appears to be of little value, particularly if antiviral activity is obtained only after local administration.

Interferons and interferon-inducing molecules

Since the discovery of interferon (IFN) and its potent antiviral effect, several experiments have been carried out in vitro and in various animal models, to evaluate the potential use of this molecule for combating rabies infection. These studies were also prompted by failures of PEP with antirabies vaccines developed in the 1970s and 1980s, as a possible way to increase the level of protection provided [START_REF] Anderson | Human rabies after antiserum and vaccine postexposure treatment. Case report and review[END_REF][START_REF] Dean | Potency of commercial rabies vaccine used in man[END_REF].

Treatment with exogenous IFN was rapidly shown to prevent the replication of RABV in cell cultures [START_REF] Barroeta | Inhibiting action of interferon on the development of rabes virion in cell culture[END_REF][START_REF] Depoux | Fixed Rabies Virus and Interferon[END_REF][START_REF] Wiktor | Role of interferon induction in the protective activity of rabies vaccines[END_REF][START_REF] Marcovistz | The effect of interferon treatment in rabies prophylaxis in immunocompetent, immunosuppressed, and immunodeficient mice[END_REF]. This antiviral property was not related to a virucidal effect [START_REF] Postic | Effect of administered interferon on rabies in rabbits[END_REF].

Complete inhibition was obtained when IFN was administered before (12-24 h) or at the time of infection, but only partial inhibition was observed when IFN was administered shortly after infection (12-24 h) [START_REF] Barroeta | Inhibiting action of interferon on the development of rabes virion in cell culture[END_REF][START_REF] Depoux | Fixed Rabies Virus and Interferon[END_REF].

This antiviral effect was rapidly confirmed in animal models. Preliminary studies were performed in rabbits, hamsters and mice infected with IFN-inducing viruses, such as live vaccinia virus [START_REF] Vieuchange | Interference between vacciniavirus and street rabies virus[END_REF][START_REF] Vieuchange | Interference between vaccine virus and fixed rabies virus[END_REF], Newcastle disease virus (NDV) [START_REF] Atanasiu | In vivo inhibition of rabies virus multiplication by an endogenous interferon[END_REF][START_REF] Vieuchange | Interference between Newcastle disease virus and rabies virus in rabbits: utilization of this phenomenon in the prevention of experimental rabies[END_REF] or bovine parainfluenza 3 virus [START_REF] Fayaz | Interference between bovine parainfluenza 3 virus and a street strain of rabies virus in rabbits[END_REF]. Effective protection was observed in all species other than mice. Interferon injection in mice, induced by the intraperitoneal injection of statolon, a fermentation product of a mold, Penicillium stoloniferum, provided no protection [START_REF] Soave | Influence of statolon-induced interferon on rabies virus infection in mice[END_REF].

Treatment with exogenous homologous or heterologous IFN [START_REF] Marcovistz | The effect of interferon treatment in rabies prophylaxis in immunocompetent, immunosuppressed, and immunodeficient mice[END_REF][START_REF] Hilfenhaus | Effect of administered human interferon on experimental rabies in monkeys[END_REF][START_REF] Hilfenhaus | Administration of human interferon to rabies virus-infected monkeys after exposure[END_REF][START_REF] Bahmanyar | Presence of an active substance of the interferon type with respect to street rabies, in extracts of tissues infected with vaccine[END_REF][START_REF] Vieuchange | The action of an interferon regarding experimental rabies of the rabbit[END_REF] or inducers of IFN, such as polyriboinosinic-polyribocytidylic acid (poly I:C) [START_REF] Fenje | Protection of rabbits against experimental rabies of poly I-poly C[END_REF][START_REF] Fenje | Prophylaxis of experimental rabies with the polyriboinosinicpolyribocytidylic acid complex[END_REF][START_REF] Janis | Rabies in rabbits and mice: protective effect of polyriboinosinicpolyribocytidylic acid[END_REF], its derivatives containing kanamycin and CaCl 2 (PICKCa) [START_REF] Lin | A new immunostimulatory complex (PICKCa) in experimental rabies: antiviral and adjuvant effects[END_REF] or other structural molecules [START_REF] Tazulakhova | Russian experience in screening, analysis, and clinical application of novel interferon inducers[END_REF] also completely or partly prevented rabies infection in animal models including mice, rabbits and 37/115 monkeys. Various protocols have been tested, including modifications of the following parameters: area of administration (same or different sites of inoculation), route of administration (intramuscular, intravenous, intralumbar, intraperitoneal, subcutaneous, intracarotid or intracerebral), time of drug administration (before, at the time of viral challenge or after viral challenge), number of doses and amount administered per dose, quantity and strain of virus used, route of inoculation, immunological status of the challenged animals. These experiments showed that efficacy was achieved essentially when large doses of IFN or IFN-inducers were administered before or shortly after viral challenge, close to the site of inoculation. Indeed, treatment administered at later time points after infection offered weaker protection, although the incubation period was prolonged in some treated animals [START_REF] Postic | Effect of administered interferon on rabies in rabbits[END_REF][START_REF] Hilfenhaus | Effect of administered human interferon on experimental rabies in monkeys[END_REF]. Combinations of drugs, including combinations of IFN-inducers, such as poly I:C, and chlorite-oxidized amylose (COAM), a small inducer of interferon [START_REF] Harmon | Post-exposure prophylaxis of murine rabies with polyinosinic-polycytidylic acid and chlorite-oxidized amylose[END_REF], or combinations of rabies vaccines and IFN or IFN inducers [START_REF] Harmon | Therapy of murine rabies after exposure: efficacy of polyriboinosinic-polyribocytidylic acid alone and in combination with three rabies vaccines[END_REF][START_REF] Baer | Successful prophylaxis against rabies in mice and Rhesus monkeys: the interferon system and vaccine[END_REF][START_REF] Wiktor | Role of interferon in prophylaxis of rabies after exposure[END_REF] were also effective only when administered close to the time of viral challenge, and the protective effects obtained were variable. Finally, neither clinical improvement nor extension of the incubation period was observed when IFN-based therapy was initiated in a primate model at the onset of clinical symptoms [START_REF] Hilfenhaus | Effect of administered human interferon on experimental rabies in monkeys[END_REF][START_REF] Weinmann | Intramuscular and/or intralumbar postexposure treatment of rabies virus-infected cynomolgus monkeys with human interferon[END_REF].

Ketamine and dizocilpine (MK-801)

In preliminary studies on experimental rabies infection, brain dysfunctions were described, suggesting that functional alterations might play an important role in rabies pathogenesis [START_REF] Gourmelon | Electrophysiological and sleep alterations in experimental mouse rabies[END_REF][START_REF] Koschel | Rabies virus infection selectively impairs membrane receptor functions in neuronal model cells[END_REF][START_REF] Tsiang | Neuronal function impairment in rabies-infected rat brain[END_REF]. The inhibitory effects of two dissociative anesthetic agents, ketamine and dizocilpine (also known as MK-801) against RABV infection have been evaluated [START_REF] Lockhart | Inhibition of rabies virus transcription in rat cortical neurons with the dissociative anesthetic ketamine[END_REF][START_REF] Lockhart | Ketamine-mediated inhibition of rabies infection in vitro and in rat brain[END_REF][START_REF] Tsiang | Inhibition of rabies virus infection in cultured rat cortical neurons by an N-methyl-D-aspartate noncompetitive antagonist, MK-801[END_REF].

These molecules are non competitive antagonists of the N-methyl-D-aspartate (NMDA) receptor and were initially demonstrated to protect against the neurotoxicity induced by excitatory amino acids, such as L-glutamate and L-aspartate [START_REF] Olney | MK-801 powerfully protects against N-methyl aspartate neurotoxicity[END_REF].

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In a preliminary study, dizocilpine was shown to inhibit rabies infection in rat primary cortical neurons, when present after inoculation, for the duration of the experiment [START_REF] Tsiang | Inhibition of rabies virus infection in cultured rat cortical neurons by an N-methyl-D-aspartate noncompetitive antagonist, MK-801[END_REF]. This inhibitory effect was shown to be dose-dependent only at millimolar concentrations (0-2 mM) and was selective for RABV, because no effect was observed with other viruses, such as herpes simplex virus, vesicular stomatitis virus, poliovirus and human immunodeficiency virus. This molecule was found not to be virucidal and its effect was highly selective for the class of non competitive NMDA receptor antagonists binding to the phencyclidine site, because an inhibitory effect was also observed with ketamine and phencylidine derivatives, but not with AP5, a competitive antagonist. Its effect appeared to be independent of a highaffinity NMDA-binding mechanism [START_REF] Tsiang | Inhibition of rabies virus infection in cultured rat cortical neurons by an N-methyl-D-aspartate noncompetitive antagonist, MK-801[END_REF]. The effects of ketamine on the infectivity of RABV in neuronal culture systems and in vivo have also been investigated [START_REF] Lockhart | Ketamine-mediated inhibition of rabies infection in vitro and in rat brain[END_REF]. This molecule was demonstrated to have a dose-dependent effect (0.2-2 mM) on virus multiplication in infected neuronal cell cultures (for both rat primary cortical neurons and human neuroblastoma cells). However, efficient antiviral effects were achieved only with high concentrations of drug, with a decrease in viral production by a factor of 100 to 1000 observed with 1 to 2 mM ketamine. The antiviral effect of ketamine was confirmed in rats, following intraperitoneal administration (15 mg, twice daily for four days) after the stereotaxic inoculation of the right striatum with RABV [START_REF] Lockhart | Ketamine-mediated inhibition of rabies infection in vitro and in rat brain[END_REF]. A decrease in rabies infection was observed four days after infection in certain regions of the brain, including the thalamus, cortex and hippocampus. In a complementary report, studies of the effects of ketamine and dizocilpine on rat brain and neuronal cell culture (rat cortical neurons) showed that these drugs probably acted specifically on transcription of the RABV genome, inhibiting viral protein synthesis [START_REF] Lockhart | Inhibition of rabies virus transcription in rat cortical neurons with the dissociative anesthetic ketamine[END_REF]. This inhibition was incomplete and displayed time-dependent reversibility, as a recovery of viral transcription and protein synthesis was observed over time.

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Further evidence of the inhibitory activity of ketamine (3 mM) was subsequently obtained in vitro, in McCoy cells, with the PV strain of RABV [START_REF] Chavez | Evaluation of antiviral activity of phenolic compounds and derivatives against rabies virus[END_REF]. However, in a more recent study, neither ketamine nor dizocilpine was observed to protect against rabies infection in the mouse model [START_REF] Weli | Rabies virus infection of primary neuronal cultures and adult mice: failure to demonstrate evidence of excitotoxicity[END_REF]. The absence of a neuroprotective effect was demonstrated in primary cultures of mouse cortical and hippocampus neurons, for 125 μM ketamine and 50 μM dizocilpine. The lack of a therapeutic effect of ketamine was confirmed in vivo, in infected mice (following peripheral or intracerebral inoculation with the CVS virus strain) treated with ketamine at a dose similar to that used in rats (60 mg/kg every 12 h intraperitoneally on days 3 to 6 postinfection) [START_REF] Weli | Rabies virus infection of primary neuronal cultures and adult mice: failure to demonstrate evidence of excitotoxicity[END_REF].

The conflicting results obtained with ketamine and dizocilpine, particularly for in vivo experiments, raise the question of the choice of the most relevant animal model to be used. Indeed, different species (rat and mouse) have been used in different studies and it would be interesting to evaluate these drugs in similar studies using both these species and possibly even other species.

Amantadine

The antiviral effects of amantadine have long been demonstrated, particularly for the prevention of influenza infection. Interesting results have also been obtained with vesicular stomatitis virus, a member of the family Rhabdoviridae, with this drug inhibiting virus uptake into the cell and endocytosis [START_REF] Lenard | Uncoating of enveloped viruses[END_REF][START_REF] Schlegel | Amantadine and dansylcadaverine inhibit vesicular stomatitis virus uptake and receptor-mediated endocytosis of alpha 2-macroglobulin[END_REF][START_REF] Superti | Effect of amantadine on rhabdovirus infection[END_REF]. The effect of this lipophilic amine on RABV replication has been tested in vitro with various laboratory-adapted virus strains; it has been shown to have a dose-dependent antiviral effect [START_REF] Bussereau | Search for compounds which have an inhibitory effect on rhabdovirus multiplication in vitro[END_REF][START_REF] Superti | Effect of amantadine on rhabdovirus infection[END_REF]. In particular, viral replication was shown to be inhibited when amantadine was added shortly after viral inoculation (during the first 2 h), with a minimum incubation of 30 minutes required for the detection of antiviral 40/115 activity [START_REF] Superti | Effect of amantadine on rhabdovirus infection[END_REF]. No virucidal effect was detected in either of these studies. It has been hypothesized that amantadine affects the uncoating of the virus, as no activity is observed during viral attachment or entry into the cell [START_REF] Superti | Effect of amantadine on rhabdovirus infection[END_REF]. Superti and coworkers suggested that the antiviral effect of this lipophilic drug on rhabdovirus infections might be related to its capacity to cross the cell membranes and to increase the natural pH of lysosomes, hindering the uncoating of rhabdoviruses. However, an evaluation of therapeutic effect of this drug against rabies infection in mice demonstrated an absence of beneficial effects, even if treatment was initiated only one day after intramuscular infection and despite the application of several protocols involving different dose schedules (30 or 67 mg/kg/day) or routes of administration [START_REF] Bussereau | Treatment of rabies in mice and foxes with antiviral compounds[END_REF].

Isoprinosine

Isoprinosine, an alkylamino-alcohol complex of inosine, also known as inosine pranobex, has long been recognized as a broad-spectrum antiviral compound. The potential antiviral effect of this drug was evaluated in a large coordinated study of several viruses and animal models, including rabies in mice [START_REF] Glasgow | Isoprinosine: lack of antiviral activity in experimental model infections[END_REF]. The daily intraperitoneal administration of 300 mg/kg isoprinosine, initiated one day before infection and continuing for up to five days, had no protective effect in this model following intracerebral or intramuscular inoculation with RABV. In two other studies conducted in vitro, a slight decrease in the titer of laboratoryadapted virus strains was observed [START_REF] Chavez | Evaluation of antiviral activity of phenolic compounds and derivatives against rabies virus[END_REF][START_REF] Hernandez-Jauregui | In vitro effect of isoprinosine on rabies virus[END_REF].

Corticosteroids

The effect of corticosteroid treatment on RABV infection has been investigated in various rodent models, including rats, hamsters and guinea pigs [START_REF] Enright | Effects of corticosteroids on rabies virus infections in various animal species[END_REF] and mice [START_REF] Enright | The effects of corticosteroids on rabies in mice[END_REF]. Hydrocortisone acetate and prednisolone are the most frequently investigated treatments. In most experiments, the mortality rates of infected animals (with vaccinal, attenuated or virulent 41/115 virus strains) were slightly higher for the treated than for the untreated animals [START_REF] Enright | Effects of corticosteroids on rabies virus infections in various animal species[END_REF][START_REF] Enright | The effects of corticosteroids on rabies in mice[END_REF].

The biological mechanisms underlying this increase in mortality remain unknown. However, these results have not been confirmed by other groups or in other studies.

Heteropolyanions

Heteropolyanion (HPA)-based molecules have been tested as antiviral drugs in RABV infection. In particular, in vitro dose-dependent inhibition has been demonstrated with ammonium-5-tungsto-2-antimoniate heteropolyanion (HPA-23), in tests with the Flury HEP vaccinal strain [START_REF] Tsiang | Inhibition of rabies virus in vitro by the ammonium-5-tungsto-2-antimoniate[END_REF]. Inhibition rates of 50% were obtained with 4.5 g/ml HPA-23, and complete inhibition was obtained with 12.5 μg/ml HPA-23. However, inhibitory effects were observed only when treatment was initiated early, ideally within 18 to 24 hours after inoculation, with no further inhibition observed beyond 48 hours post infection [START_REF] Tsiang | Inhibition of rabies virus in vitro by the ammonium-5-tungsto-2-antimoniate[END_REF]. The antiviral effect of HPA-23 on RABV multiplication was further confirmed in single-growth cycle conditions, with CVS, and similar effects were observed with six other HPAs: HPA-39,-56, -40, -51, -52 and -57 [START_REF] Bussereau | Effect of heteropolyanions and nucleoside analogues on rabies virus[END_REF][START_REF] Bussereau | Search for compounds which have an inhibitory effect on rhabdovirus multiplication in vitro[END_REF]. A dose-dependent effect was observed, with almost complete inhibition at concentrations exceeding 50 μg/ml [START_REF] Bussereau | Effect of heteropolyanions and nucleoside analogues on rabies virus[END_REF][START_REF] Bussereau | Search for compounds which have an inhibitory effect on rhabdovirus multiplication in vitro[END_REF]. No virucidal effect of these drugs was observed in any of these studies, even at high concentrations [START_REF] Bussereau | Effect of heteropolyanions and nucleoside analogues on rabies virus[END_REF][START_REF] Bussereau | Search for compounds which have an inhibitory effect on rhabdovirus multiplication in vitro[END_REF][START_REF] Tsiang | Inhibition of rabies virus in vitro by the ammonium-5-tungsto-2-antimoniate[END_REF].

The antiviral activity of HPA-23 and -39 against RABV was thought to target viral RNA synthesis and cellular protein synthesis [START_REF] Bussereau | Effect of heteropolyanions and nucleoside analogues on rabies virus[END_REF]. These molecules have been evaluated in animal models, including foxes and mice [START_REF] Bussereau | Treatment of rabies in mice and foxes with antiviral compounds[END_REF][START_REF] Pepin | Ammonium-5-tungsto-2-antimoniate (HPA23) can prevent fox rabies[END_REF]. In total, 14 different HPA derivatives have been used at various concentrations and with different protocols of administration in mice, with assessment of their antiviral activity against rabies infection. Only HPA-23 and -39 were found to have a slightly protective effect, essentially when treatment was both initiated soon after virus inoculation (1 day post-infection) and administered intramuscularly at the site of virus inoculation [START_REF] Bussereau | Treatment of rabies in mice and foxes with antiviral compounds[END_REF]. Late treatment with HPA-39 was not protective even when initiated only five days after inoculation. A slight dose-dependent protective effect was also observed with HPA-23 in rabies-infected foxes when this molecule was administered intramuscularly at the time of inoculation; a decrease in the incubation period appeared to occur when the treatment was administered one to two days after viral challenge [START_REF] Bussereau | Treatment of rabies in mice and foxes with antiviral compounds[END_REF][START_REF] Pepin | Ammonium-5-tungsto-2-antimoniate (HPA23) can prevent fox rabies[END_REF]. Moreover, a hypothetical therapeutic effect of HPA-39 has been described when this molecule is administered shortly after the onset of clinical signs [START_REF] Bussereau | Treatment of rabies in mice and foxes with antiviral compounds[END_REF]. However, high mortality rates were observed in both these studies, due to high toxicity of these drugs in foxes, particularly after intraperitoneal, subcutaneaous or intramuscular administration, for doses ranging from 50 to 150 mg/kg. The small numbers of animals studied therefore render interpretation difficult, even if a very small number of animals seem to have survived after treatment (2/7 or 2/15 with HPA-23 and 1/1 with HPA-39) [START_REF] Bussereau | Treatment of rabies in mice and foxes with antiviral compounds[END_REF][START_REF] Pepin | Ammonium-5-tungsto-2-antimoniate (HPA23) can prevent fox rabies[END_REF]. From these results, and given the high toxicity and the low antiviral activity of HPA-based molecules, the use of these compounds in the treatment or even the prevention of animal (including humans) rabies infection remains rather unlikely.

Ascorbic acid (vitamin C)

Surprisingly, a treatment based on the intramuscular injection of ascorbic acid has been proposed as a means of preventing rabies infection in a guinea pig model [START_REF] Banic | Prevention of rabies by vitamin C[END_REF]. Treatment was initiated 6 hours after intramuscular inoculation with RABV and was based on the administration of a dose of 100 mg/kg of body weight twice daily for seven days. Mortality rates were lower in treated animals than in control animals, with 17 of 48 (35.4%) and 35 of 50 (70%) animals, respectively, dying. However, the results were variable between groups of animals and lower doses of vitamin C were less effective. Moreover, this result was not confirmed by further studies conducted in animal models. Only one recent study has suggested that a solution of ascorbic acid (0.5 mg/ml) and copper sulfate (5 g/ml) can completely inactivate RABV in vitro after 72 hours of incubation [START_REF] Madhusudana | In vitro inactivation of the rabies virus by ascorbic acid[END_REF].

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-phenylserine

Partial prophylactic protection of rats against RABV was achieved by daily intraperitoneal administration of -phenylserine, a competitive inhibitor of phenylalanine hydroxylase [START_REF] Pons | The in vivo inhibition by beta-phenylserine of rabies, myxoma, and vaccinia viruses[END_REF].

This protection was obtained when animals were treated for three days before inoculation, and treatment administered 24 hours after infection was also protective. No virucidal effect of phenylserine was demonstrated. The inhibition of rabies infection was abolished if DLphenylalanine or DL-tyrosine was administered in combination with this drug. However, the antiviral effect of -phenylserine was restricted to the rat strain used in this experiment, limiting the potential utility of this molecule [START_REF] Pons | The in vivo inhibition by beta-phenylserine of rabies, myxoma, and vaccinia viruses[END_REF]. A protective effect of this drug was also suggested in rabies-infected mice, but the results could not be uniformly replicated and the differences observed were not statistically significant [START_REF] Enright | Experimental rabies chemotherapy in mice[END_REF].

Minocycline

Minocycline, a derivative of tetracycline with antiapoptotic and anti-inflammatory properties, has been evaluated in experimental models of RABV infection using a cell-adapted rabies virus strain [START_REF] Jackson | Therapy with minocycline aggravates experimental rabies in mice[END_REF]. Only minor effects on neuroprotection or viral multiplication were demonstrated in vitro in mouse cortical and hippocampal primary neurons, even if the drug was added within an hour of inoculation with the virus. Detrimental effects were observed in rabies-infected newborn mice, with higher mortality rates and an earlier onset of neurological symptoms than observed in mock-infected mice, jeopardizing the possible future use of this molecule in the prevention or treatment of rabies infection.

Phosprenyl

It has been claimed that phosprenyl, a molecule obtained by the chemical phosphorylation of polyprenols (natural long-chain isoprenoid alcohols), has broad-spectrum antiviral activity, 44/115 with effects on rabies [START_REF] Danilov | Phosprenyl: a novel drug with antiviral and immunomodulatory activity[END_REF]. Significant results were reported in vivo, but were not confirmed by subsequent studies.

Other drug derivatives from plants or fungi

The inhibitory effects of an antibiotic substance, cinnabarin, extracted from the fungus Pycnoporus sanguineus, against RABV replication in vitro have been evaluated [START_REF] Smania | Toxicity and antiviral activity of cinnabarin obtained from Pycnoporus sanguineus (Fr.) Murr[END_REF]. At non cytotoxic doses (0.155 to 0.31 mg/ml), it has been suggested that cinnabarin has antiviral effects, leading to decreases in viral titer by a factor of two to four (evaluations with the PV strain). These effects were observed after prior incubation of the drug with the viral suspension, and maintenance of the drug in the cell culture medium for 24 h. No tests on animal models or analyses of antiviral activity were carried out. In another study, antiviral activities of exudate fluids from small red beans (Vigna angularis) have been evaluated in vitro against rabies infection with BHK-21 cells [START_REF] Kawai | Small red bean (azuki) sheds biologically active substances as a prerequisite step for germination, one of which displays the antiviral activity against the rabies virus infectivity and infections in culture[END_REF]. Virucidal and dose-dependent virus inhibition effects have been suggested (IC 50% =0.48% dose), especially during the early phase of infection (0-6 h p.i.). Lastly, in another study, four different extracts and fractions from South American plants (Alamanda schottii, Passiflora edulis and Sloanea guianensis) were evaluated to assess their potential in vitro antiviral activity against RABV [START_REF] Muller | Evaluation of antiviral activity of South American plant extracts against herpes simplex virus type 1 and rabies virus[END_REF]. Based on determination of the selectivity index (SI), which is the ratio of the 50% cytotoxic concentration (CC 50% ) to the 50% inhibitory concentration (IC 50% ), only one extract (obtained from Alamanda schottii) had potent antiviral activity (SI > 5). These extracts and fractions were not studied further in animal models of rabies infection, and their molecular composition was not determined.

RNA-interference

RNA interference-based antiviral approaches have been evaluated in vitro for rabies infection.

With short cDNA, short-interfering RNAs (siRNAs) or artificial microRNAs (amiRNAs) 45/115 targeting viral nucleoprotein or phosphoprotein mRNAs, partial inhibition of RABV multiplication was obtained [START_REF] Brandao | Short-interfering RNAs as antivirals against rabies[END_REF][START_REF] Wunner | Selection of genetic inhibitors of rabies virus[END_REF][START_REF] Israsena | Inhibition of rabies virus replication by multiple artificial microRNAs[END_REF]. However, the direct application of such an approach to the treatment of rabies is not yet realistic, and several obstacles remain, including the induction of viral resistance or escape, due to natural genetic variability of the nucleotide target, drug delivery in the CNS, drug efficiency in animal models, etc.

Antiviral peptides

Strategies have been proposed for antiviral peptide discovery in the field of rabies infection, based on genetically encoded combinatorial peptide libraries of intrinsically constrained peptides screened with the viral phosphoprotein [START_REF] Real | Antiviral drug discovery strategy using combinatorial libraries of structurally constrained peptides[END_REF]. This approach led to the identification of peptides with efficient antiviral activities in vitro against RABV. However, these peptides were generated by transfecting host cells with expression vectors before virus infection. No further study based on the direct use of these peptides was carried out. In another study, two peptides covering the first 42 and 60 residues of the N-terminal region of the RABV phosphoprotein were shown to have antiviral activity against rabies infection in vitro [START_REF] Castel | Peptides that mimic the amino-terminal end of the rabies virus phosphoprotein have antiviral activity[END_REF].

The viral infection of neuroblastoma cells was found to be inhibited by the delivery of synthetic peptides shortly after virus inoculation. However, the antiviral activities of these peptides has not been confirmed in animal models of rabies infection.

Multiple-drug screening

Over a number of years, various groups have performed large drug screening studies in vitro or in animal models, to identify compounds effective against RABV infection. For example, the viral inhibitory activity of 21 molecules was tested in vitro (in CER cells) [START_REF] Bussereau | Search for compounds which have an inhibitory effect on rhabdovirus multiplication in vitro[END_REF]. Eightribavirin and seven heteropolyanionsdisplayed dose-dependent inhibition, whereas the othersfour acyclic derivatives of adenosine and carbocyclic 3-deazaadeonisinedisplayed 90% inhibition at all doses used, as previously indicated (see sections 3.1.2 and 46/115 3.1.10). In another study with the IMR-32 human neuroblastoma cell line, Lentz and coworkers screened 25 chemical agents, assessing their ability to inhibit rabies infection in a fluorescent focus assay [START_REF] Lentz | Rabies virus infection of IMR-32 human neuroblastoma cells and effect of neurochemical and other agents[END_REF]. Inhibitory effects were observed for agents with the potential to inhibit the attachment of the virus to the cell surface (with antibodies against the viral glycoprotein, gangliosides, a synthetic peptide of the neurotoxin-binding site of the Torpedo acetylcholine receptor 1 subunit and -bungarotoxin) and lysosomotropic agents, as previously indicated (see sections 3.1.1.2 and 3.1.3). Antiviral activities of 14 natural and semisynthetic polymeric carbohydrates were evaluated in vitro using CER cells and the CVS strain of rabies virus [START_REF] Pietropaolo | Effect of natural and semisynthetic polymers on rabies virus infection in CER cells[END_REF]. Most were able to inhibit infection by interfering with the virus adsorption step, and this was particularly true for the following polysaccharides: scleroglucan, Keltrol  , alginate acid and two sulfated tamarind gums (also known as glyloid sulfates).

Finally, the potential antiviral activity of 24 synthetic phenolic compounds against RABV was recently evaluated in McCoy cells with the PV strain [START_REF] Chavez | Evaluation of antiviral activity of phenolic compounds and derivatives against rabies virus[END_REF]. Only four of these compoundscatechin, quercetin, 3,4,5-trimethoxyacetophenone and 3,4,5trimethoxybenzoic acid ethyl esterdisplayed potent antiviral effects against rabies.

Enright and coworkers used the mouse model to test up to 65 different drugs in PEP [START_REF] Enright | Experimental rabies chemotherapy in mice[END_REF].

These compounds were classified on the basis of their chemical structure or biological activity and included antimetabolites and antibiotics, proteins, nucleic acids and related compounds and miscellaneous substances, including solvents and detergents. Treatments were administered at the site of inoculation (virus entry), and were systematic or blocking (injection of the treatment compound between the site of viral inoculation and the CNS). They followed several different schedules and doses, depending on the compounds tested. The most active compounds were solvents or detergents (probably affecting viral lipids). None of the other protein compounds, enzymes, antimetabolites or antibiotics tested had a significant chemotherapeutic effect. In another study in mice, 17 immunosuppressive, cytostatic or 47/115 antiviral agents were assessed for rabies prevention [START_REF] Sodja | Experimental chemotherapy of rabies. Preliminary results[END_REF]. It was suggested that iododeoxyuridine (IUdR) (intraperitoneal, 20 mg/kg), actinomycin D (intraperitoneal, 0.5-1.0 mg/kg) and ara-C (subcutaneous, 20 mg/kg) inhibited intracerebral infection, and that rifampicin (subcutaneous, 100 mg/kg) and azathioprine (intravenous, 2.5 mg/kg) inhibited intramuscular infection (see section 3.1.2). However, these results remain difficult to interpret, due to a lack of standardization, as studies were carried out in different conditions (drug doses, routes of administration, rabies strains used for inoculation, etc.). In total, 40 different compounds were evaluated for their potential protective activity against rabies in mice and/or in foxes, using different doses, time and schedules of administration [START_REF] Bussereau | Treatment of rabies in mice and foxes with antiviral compounds[END_REF]. Only five HPA molecules (HPA-23, -39, -46, -51 and -56) were identified as effective preventive drugs.

Conclusion

From these numerous studies evaluating the antiviral activities against rabies infection of a large range of molecules (from well characterized broad-spectrum antiviral compounds, such as interferons, to more unexpected compounds, such as ascorbic acid), we can conclude that no drug has been shown to be effective in vivo, particularly in cases in which clinical signs are already present. These results suggest that other rabies virus targets in the viral replication cycle must be identified, and more specific antiviral molecules designed. Moreover, a key issue is the identification of effective drugs able to cross the blood brain barrier, a natural obstacle blocking access to the CNS, the specific target tissue of RABV.

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The history of drug treatment for human rabies

The hope: the existence of rare cases of human recovery after rabies encephalitis

Human rabies is an almost always preventable provided that effective PEP is administered to exposed individuals early enough. However, this acute viral encephalitis is also almost invariably fatal once the first clinical signs have appeared. Nonetheless, a few cases of survival have been reported, raising hopes that it may be possible to cure this infection and to survive rabies. Five well documented cases of prolonged survival or recovery from rabies were specifically associated with the administration of PEP before the onset of symptoms.

However, a presumptive case of abortive rabies was described in 2009, in a patient who recovered from rabies encephalitis despite the absence of PEP before the illness. No other specific antiviral treatment was administered in any of these cases. With the exception of the last case, early, aggressive, intensive support therapy was given to anticipate the problems suffered by many patients, including severe complications. Interestingly, it was not possible to isolate RABV or to detect rabies antigen in any of these patients. Two other less documented cases of human recovery have been also reported in rabid patients given treatment in combination with PEP and aggressive care [START_REF] Gode | Intensive care in rabies therapy. Clinical observations[END_REF] (described in sections 3.2.2.2.2 and 3.2.2.2.3).

Case 1

The first reported case of recovery from rabies was that of a six-year-old boy in November 1970, in Ohio, USA [START_REF] Hattwick | Recovery from rabies. A case report[END_REF]. The child developed rabies 20 days after he was bitten on the left thumb by a big brown bat (Eptesicus fucus) and two days after the completion of a 14-day course of PEP without antirabies serum (multidose therapy with DEV). The bat was confirmed as rabid four days after the bite, leading to vaccination. The patient presented neck pain at the onset of symptoms, extending to the arms, legs and head a few days later.

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Fluctuating consciousness occurred, with neurological abnormalities, and the patient became increasingly lethargic and comatose. Biological analysis of a brain biopsy specimen and a CSF sample confirmed encephalitis. Cardiac and pulmonary complications occurred during the hospitalization of this patient. He later presented focal seizures, which were treated with diphenylhydantoin therapy. Almost 25 days after the onset of symptoms, the patient gradually improved and he was discharged two months later [START_REF] Hattwick | Recovery from rabies. A case report[END_REF]. No detectable psychological or neurological abnormality was observed after a few months of follow-up. A high titer of specific antirabies antibodies was detected in serum and CSF. In particular, antibody levels increased strongly in the patient's serum and CSF during hospitalization, consistent with rabies infection rather than the effects of vaccination. However, RABV was not isolated from any of the samples tested, included a brain biopsy specimen, probably due to the presence of high levels of neutralizing antibodies.

Case 2

In 1972, another case of recovery was described in a 45-year-old female patient from Mendoza, Argentina, who was severely bitten, on the left arm, by a dog with suspected rabies and who developed neurological signs 21 days later [START_REF] Porras | Recovery from rabies in man[END_REF]. As in the first case, this patient received a 14-day course of post-exposure treatment, based on suckling mouse brain vaccine administered 10 days after the bite, with two booster doses, without antirabies antiserum. The encephalitic syndrome persisted for 75 days, and the patient made an almost complete recovery 13 months later. Onset of the symptoms begans at the time of the 12 th dose of vaccine, with paresthesia of the bitten limb, extending to the other arm and accompanied by generalized pain. A severe cerebellar striatal syndrome occurred. Rabies antigens (on corneal smear) were not detected and it was not possible to isolate the virus (from saliva and CSF samples). However, neutralizing antibodies were detected in CSF and serum, the titer increasing with time of infection to a magnitude never observed after vaccination with the 50/115 vaccine used. Chemotherapeutic treatment was based on vitamin B1 and B12, diphenahydramine, diazepam, biperiden, dihphenylhydantoin, betamethasone and ACTH for a period of almost 10 days. After a period of improvement, leading to the discharge of the patient, neurological symptoms reappeared shortly after the first booster dose of vaccine and began to worsen with the second booster dose, with generalized seizures, neurological abnormalities, fluctuating consciousness and tetraparesis, hypertonia, hyperreflexia and Babinski's sign. Clinical improvement then occurred progressively. The patient was discharged a few months later and made an almost complete recovery from rabies 13 months after the onset of symptoms. Rabies diagnosis was based on clinical, epidemiological and laboratory data, supporting the conclusion of a non fatal case of rabies in a human clinical presentation [START_REF] Porras | Recovery from rabies in man[END_REF]. However, the hypotheses of encephalomyelitis or a post-vaccine accident after treatment with suckling mouse brain vaccine, particularly after the administration of rabies vaccine booster doses, could not be formally rejected [65].

Case 3

In 1977, a case of partial recovery from rabies was identified in a 32-year-old male technician working in a rabies laboratory in New York, USA [START_REF]CDC Rabies in a laboratory worker--New York[END_REF][START_REF]CDC Follow-up of rabies--New York[END_REF]. This patient was pre-immunized and was probably exposed to high concentrations of aerosolized modified live RABV strains (derived from the attenuated SAD virus strain) two weeks before the beginning of the illness [START_REF]CDC Rabies in a laboratory worker--New York[END_REF]. The initial symptoms were not specific, with malaise and headache, followed by chills, fever and nausea on the next few days. The patient then became lethargic, with fluctuating consciousness and fell into a deep coma after hospitalization. Two weeks later, he began to show gradual improvement. Three months after the onset of clinical signs, he recovered motor function and was ambulatory but conserved sequelae, with occasional aphasic periods of agitation and spasticity [START_REF]CDC Follow-up of rabies--New York[END_REF]. Rabies was diagnosed purely on the basis of the increase in 51/115 antirabies antibody titer in the serum and CSF, and corneal impression and neck skin biopsy testing by FAT gave negative results.

Case 4

A nine-year old Mexican boy partially recovered from rabies after being bitten on the face by a rabid dog in August, 1992 [START_REF] Alvarez | Partial recovery from rabies in a nine-year-old boy[END_REF]. PEP was administered the day after the bite, according to a modified five-dose regimen of cell-cultured rabies vaccines (PVRV and HDCV), without rabies antiserum or immunoglobulin. Symptoms began 18 days after exposure, with fever and dysphagia, followed by neurological abnormalities and encephalitic signs. Cranial hypertension was observed, leading to coma a few days later. Progressive but limited improvement was then observed, with severe neurological sequelae [START_REF] Alvarez | Partial recovery from rabies in a nine-year-old boy[END_REF]. The patient died less than four years after the onset of symptoms [65]. No rabies antigens were detected on corneal impression and nuchal skin biopsy, and the virus could not be isolated from saliva.

High levels of neutralizing antibodies were observed in the CSF and serum, and the intrathecal production of rabies antibodies was demonstrated.

Case 5

In 2001, in Bangalore, India, a six-year old girl developed rabies 20 days after she had been bitten by a dog on the face and hands [START_REF] Madhusudana | Partial recovery from rabies in a six-year-old girl[END_REF]. Incomplete PEP was given (three doses of PCECV cell vaccine on day 0, 3 and 7 days), without local wound treatment or antirabies immunoglobulin. The symptoms at onset of illness included fever, swallowing difficulties, photophobia, hallucinations and sensory alterations, followed by fluctuating consciousness.

The patient became comatose, with focal motor seizures and excessive salivation and sweating. Methylprednisolone, antibiotics and intravenous fluids were administered, together with an additional dose of rabies vaccine (HDCV). High titers of rabies antibodies were detected in serum and CSF, with a significant increase in titers not associated with rabies 52/115 vaccination, as in the other cases. No rabies antigen was detected on corneal smear and nuchal skin biopsy and it was not possible to isolate the virus from the CSF. Intrathecal synthesis of rabies antibodies was demonstrated. The child remained comatose for three months with the maintenance of supportive care. Steroid doses were tapered and discontinued. The patient's condition gradually improved, but she did not recover completely, retaining severe neurological sequelae [START_REF] Madhusudana | Partial recovery from rabies in a six-year-old girl[END_REF]. She died about two years after the beginning of the disease [65].

Case 6

The first presumptive case of abortive human rabies was described in a 17 year-old female patient in Texas, USA, in 2009 [START_REF]Presumptive abortive human rabies -Texas, 2009[END_REF]. Almost two months after contact with bats, this patient developed symptoms, including headache, followed by photophobia, paresthesia of the upper extremities, neck pain, transient disorientation, fever and severe headache over the next two weeks, requiring hospitalization. Hypercytosis was demonstrated in the CSF. After a short period of recovery, clinical and biological signs of encephalitis recurred and intensified, leading to a second hospitalization with a diagnosis of suspected infectious encephalitis. After a period of weakness and emesis followed by agitation, rabies was suspected. Diagnosis was based purely on the detection of RABV antibodies in CSF and serum samples by indirect fluorescence antibody tests, as neither viral antigen nor viral RNA was detected in saliva or a nuchal skin biopsy specimen. Moreover, neutralizing rabies antibodies were detected in the serum by RFFIT only after the administration of single doses of rabies immune globulin (HRIG, 1500 IU) and rabies vaccine, almost one month after the onset of symptoms. The patient was discharged after the resolution of symptoms, after almost 17 days of hospitalization, but suffered two other episodes of severe headache and potential increase in intracranial pressure. The current clinical status of the patient is unknown [START_REF]Presumptive abortive human rabies -Texas, 2009[END_REF]. In this report, the authors considered this patient to constitute a presumptive case of abortive rabies.

However, the biological confirmation of rabies infection remains limited and debatable.

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Clinical presentation was nonspecific and unusual, with strong alternation between periods of illness and recovery. Additional information is required to provide a full description of this potential case of recovery.

Description of the first trials of treatment for human rabies

The treatment of the rare cases of rabies recovery described to date was specifically designed to maximize the chances of recovery, by preventing all treatable complications of rabies infection through aggressive supportive care [START_REF] Hattwick | Recovery from rabies. A case report[END_REF]. Such rare reports have encouraged physicians to set up rigorous intensive measures to prolong life in patients with rabies, in the hope that the virus may be cleared in due course, with partial or complete recovery. Various antiviral therapies have been used in parallel, following the discovery and evaluation of new antiviral drugs in vitro or in animal models, effective particularly against rabies infection.

Interferon (IFN)

In the 1970s and 1980s, several attempts were made to treat clinical cases of rabies with human leukocyte IFN associated with medical intensive care in France and the Americas, but without success [START_REF] Merigan | Human leukocyte interferon administration to patients with symptomatic and suspected rabies[END_REF]. The first two cases were French children, a 10-year-old girl and a fiveyear-old boy hospitalized in Paris, France, after being bitten by rabid dogs in Africa one and a half months previously [START_REF] Merigan | Human leukocyte interferon administration to patients with symptomatic and suspected rabies[END_REF]. Neither of these patients received PrEP or PEP. IFN treatment was administered just after admission, corresponding to two to three days and five days after the onset of symptoms, respectively. The protocol of human leukocyte IFN-based treatment was as follows: intravenous injection of 3 x 10 6 U on a continuous basis for 12 days and intrathecal administration of 1 x 10 6 U every other day for six days for the girl, and administration of 1 x 10 6 U intramuscularly for 12 days and 0.5 to 2 x 10 6 U intrathecally every other day for six days for the boy. Both died of rabies, 28 and 23 days after the onset of symptoms, respectively.

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A few years later, between 1981 and 1983, three American cases of human rabies were also treated with IFN [START_REF] Merigan | Human leukocyte interferon administration to patients with symptomatic and suspected rabies[END_REF]. All received human leukocyte IFN according to a similar protocol, including the intramuscular injection of 10 x 10 6 U twice daily (12 h apart) and the intraventricular administration of 5 x 10 6 U once daily into a Rickham reservoir, connected by a cannula to a lateral ventricle of the brain. The first case was a 16-year old Brazilian girl who developed rabies after being licked by a rabid dog. She received no PrEP or PEP. Treatment was initiated 14 days after the onset of symptoms and continued until the death of the patient, 10 days later (with a total of 250 x 10 6 U IFN administered) [START_REF] Merigan | Human leukocyte interferon administration to patients with symptomatic and suspected rabies[END_REF]. The other two cases of human rabies treated with IFN occurred in the US. In 1981, a 40-year-old man who had been bitten two months previously by a dog with suspected rabies in Mexico, was treated [START_REF] Merigan | Human leukocyte interferon administration to patients with symptomatic and suspected rabies[END_REF][START_REF] Parham | Rabies in the United States, 1981[END_REF]. This patient received no PEP until his hospitalization for probable rabies, on the sixth day of illness. He was given a single dose of rabies vaccine (HDCV) and of HRIG, and human leukocyte IFN treatment was initiated 10 days after the onset of symptoms. The patient died 14 days later, after 14 days of treatment (total of 350 x 10 6 U IFN given). The other case of human rabies in the USA was treated with human leukocyte IFN, initiated seven days after the onset of symptoms (one day after the biological confirmation of rabies infection) [START_REF] Merigan | Human leukocyte interferon administration to patients with symptomatic and suspected rabies[END_REF]238] and continued for 17 days. This patient, who did not receive PEP, died 28 days after the onset of illness, after receiving a total dose of 425 x 10 6 U of IFN.

An open trial of intensive care and chemotherapy with lymphoblastoid -IFN was later conducted in Thailand, on three patients with confirmed rabies infection (from 1.5 to 7 days of illness before treatment) [START_REF] Warrell | Failure of interferon alfa and tribavirin in rabies encephalitis[END_REF]. High doses of -IFN were administered intravenously and intraventricularly and concentration was monitored. The following protocol was used: loading dose of 50 x 10 6 U/m 2 body surface area over 6 h, repeated 18 h later and followed by a daily continuous intravenous infusion. The first dose of intrathecal IFN was administered by the lumbar route, and subsequent doses were administered to the cerebral ventricle via an 55/115 Ommaya reservoir. A loading dose of 2 x 10 6 U/m 2 body surface area was repeated after 6 h and then daily. Despite the high concentrations of -IFN measured in serum and cerebrospinal fluid, the patients died after 5.5 and 12.5 days of treatment, with no evidence of virostatic or clinical benefits, suggesting that IFN is not effective against rabies encephalitis.

Combined drug therapy including IFN or IFN-inducers and other drugs was also attempted early in the history of rabies treatment. In a retrospective study over a period of 17 years of the management and follow-up of rabid patients in India, 17 of the 54 patients studied received cytarabine, nucleic acid lipoprotein complex or IFN-inducer, in combination with intensive care management [START_REF] Gode | Treatment of 54 clinically diagnosed rabies patients with two survivals[END_REF]. All patients died of rabies despite this treatment. In another study in 1985, a combination of IFN (10 x 10 6 U, administered daily, intramuscularly and intraventricularly), rabies immunoglobulin (1 and 5 ml doses given intramuscularly and intrathecally) and vidarabine (350 mg to 700 mg infused intravenously over 24 h) was given to a 25-year-old Canadian patient who presented clinical signs of rabies, probably contracted after exposure to a bat three months earlier [START_REF] Webster | A case of human rabies in western Canada[END_REF][START_REF] Dolman | Massive necrosis of the brain in rabies[END_REF]. This patient died almost one month after the onset of symptoms (25 days after admission to hospital), and a generalized necrosis of the brain was observed post mortem.

Antimetabolic agents

Ribavirin

In 1984, an experimental treatment with ribavirin was administered to a 12-year-old girl in Houston, Texas, USA, presenting the paralytic form of rabies [START_REF]CDC Human rabies--Texas[END_REF]. This treatment was given after biological confirmation of rabies infection, obtained almost 16 days after the onset of symptoms. Little information is available concerning this case, but the patient is known to have died 27 days after the onset of the first clinical signs. In another study, clinical (furious) rabies was treated with ribavirin in a Thai patient in the early stage of infection (3 days after 56/115 the onset of symptoms), in association with intensive care clinical management [START_REF] Warrell | Failure of interferon alfa and tribavirin in rabies encephalitis[END_REF]. The drug was given intravenously as a loading dose of 2 g (30 mg/kg) over 20 min, followed by 1 g every 6 h (60 mg/kg/day) for 4 days and 0.5 g every 8 h (25 mg/kg/day) daily thereafter. In parallel, intraventricular injections of 100 mg ribavirin (2 mg/kg) were administered daily through an Ommaya reservoir. The patient died of rabies after a nine-day period of treatment, indicating an absence of antiviral or beneficial effects of ribavirin [START_REF] Warrell | Failure of interferon alfa and tribavirin in rabies encephalitis[END_REF]. Later, through a retrospective analysis of 64 potential cases of human rabies admitted to the You-An Infectious Disease Hospital in Beijing, China, over a 15-year period (1974 to 1989), 16

patients were found to have received intravenous doses of 16 to 400 mg ribavirin [START_REF] Kureishi | Rabies in China: recommendations for control[END_REF]. No significant effect on survival was noted.

Other antimetabolic agents

As previously indicated, combined drug treatment including the systemic and intrathecal administration of IFN, rabies immunoglobulin and vidarabine was given to a Canadian rabid patient, without success [START_REF] Webster | A case of human rabies in western Canada[END_REF][START_REF] Dolman | Massive necrosis of the brain in rabies[END_REF].

Intensive treatment for human rabies was attempted in seven Indian patients in the 1970s [START_REF] Gode | Intensive care in rabies therapy. Clinical observations[END_REF]. The measures used included aggressive treatment with cytosine arabinoside (acting as an antiviral agent), in association with Freund's emulsion (used for stimulating antibody formation). In parallel, intensive and critical care was administered, with intermittent positive pressure facilitated by muscle relaxants and sedatives to prevent hypoxia, maintenance of nutrition, correction of electrolyte concentration and blood pH and intensive nursing care.

However, none of the patients survived, although survival was prolonged in some, to up to 17 days after the onset of symptoms [START_REF] Gode | Intensive care in rabies therapy. Clinical observations[END_REF]. The same group subsequently carried out a retrospective study over a period of 17 years including 54 rabid Indian patients treated with the same intensive care regimen but different drugs [START_REF] Gode | Treatment of 54 clinically diagnosed rabies patients with two survivals[END_REF]. Seventeen of these patients 57/115 received cytarabine, nucleic acid lipoprotein complex or IFN-inducer, with no beneficial effect on survival period (see paragraph 3.2.2.1).

Other drugs

In the same retrospective study conducted by Gode and colleagues in 1976 [START_REF] Gode | Intensive care in rabies therapy. Clinical observations[END_REF], 37 of the 54 patients were treated with diphenylhydantoin and vitamin C. It was suggested that these patients survived for longer. However, all but two of these patients died from rabies. The two survivors were still alive and normal after 28 and 13 months, at the time of communication [START_REF] Gode | Treatment of 54 clinically diagnosed rabies patients with two survivals[END_REF]. These two patients received PEP with a course of Semple's vaccine without antiserum, which may have accounted for the favorable outcome independently of the drugs administered. Furthermore, only three of the 54 patients had biologically confirmed rabies, with diagnosis purely on clinical symptoms for the others.

Vaccines and rabies immune globulins

Other therapeutic interventions after the onset of clinical illness, based on intensive supportive care and the administration of rabies immune globulins (given intravenously, subcutaneously, intrathecally or intraventricularly), alone or in combination with antirabies vaccines, have been performed in multiple cases of human rabies. Unfortunately, no beneficial effect was observed in any of these clinical cases [START_REF] Anderson | Human rabies in the United States, 1960 to 1979: epidemiology, diagnosis, and prevention[END_REF][START_REF] Hemachudha | Paralytic complications following intravenous rabies immune globulin treatment in a patient with furious rabies[END_REF][START_REF] Maton | Human rabies encephalomyelitis[END_REF][START_REF] Cohen | A case of rabies in man: some problems in diagnosis and management[END_REF][START_REF] Emmons | A case of human rabies with prolonged survival[END_REF].

Quinine and urea hydrochloride

In 1913, a 73-year-old patient with suspected rabies was given quinine and urea hydrochloride intravenously for two days after admission to hospital in Saint Louis, USA [START_REF] Harris | A clinical report of seven cases of hydrophobia[END_REF]. This treatment was initiated four to five days after the onset of symptoms and the patient recovered four days after hospitalization, eight to nine days after the onset of the illness. However, this case remains inconclusive and dubious as the diagnosis of rabies was based purely on clinical (principally hydrophobia) and epidemiological (dog bite 5-6 weeks before) grounds.

Moreover, no further follow-up of this patient was performed.

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New directions, moving towards combination therapy for human rabies

Expert recommendations for the treatment of human rabies in 2003

Recommendations concerning the management of rabies in humans were presented in 2003 by a group of physicians and researchers with expertise in rabies [START_REF] Jackson | Management of rabies in humans[END_REF]. This group concluded that the routine management of patients with rabies should be palliative, with the anticipation of disease complications, and use of appropriate treatments, such as sedatives, narcotic analgesics, neuromuscular blockers and antiepileptic drugs. However, in some circumstances, an aggressive approach to treatment may be attempted, particularly in previously healthy patients presenting at a very early stage of disease, vaccinated before the onset of symptoms and with access to adequate medical resources and facilities. In these cases, a combination of treatments was suggested, including rabies vaccines (particularly by the multiple-site intradermal route), rabies immune globulin (given intramuscularly, as in PEP, for example), ribavirin (administered intravenously and intraventricularly through an Ommaya reservoir), -IFN (administered in the same way as ribavirin), monoclonal antibodies (in the future and after clinical evaluation) and ketamine (administered intravenously in a continuous infusion).

The administration of corticosteroids for rabies treatment should be avoided [START_REF] Jackson | Management of rabies in humans[END_REF].

In line with these recommendations, a combined therapeutic approach including rabies vaccine, rabies immune globulin, ribavirin and -IFN was administered to a 66-year-old patient on admission to hospital, almost two weeks after the onset of symptoms [START_REF]CDC Human death associated with bat rabies--California, 2003[END_REF].

However, this patient, who had been bitten by a bat five weeks previously, died seven days after admission.

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3.2.3.2

The unexpected successful case of rabies recovery in 2004 and the Milwaukee protocol An unexpected and instructive case of survival was reported in a 15-year-old girl who developed encephalitic rabies after being bitten by a bat one month earlier [START_REF] Willoughby | Survival after treatment of rabies with induction of coma[END_REF]. During the clinical illness, she was treated with an experimental protocol combining antiexcitatory and antiviral drugs, therapeutic coma and supportive intense care. This patient never received rabies vaccine or antirabies immunoglobulins. The rationale of this protocol was based on previous clinical cases suggesting that death from rabies results from a regional imbalance of neurotransmitters or neuromodulators and autonomic failure, and that supportive care could potentially be successful [START_REF] Hattwick | Recovery from rabies. A case report[END_REF][START_REF] Gode | Treatment of 54 clinically diagnosed rabies patients with two survivals[END_REF][START_REF] Bhatt | Human rabies. Diagnosis, complications, and management[END_REF][START_REF] Rubin | A case of human rabies in Kansas: epidemiologic, clinical, and laboratory considerations[END_REF]. Agonists of gamma-aminobutyric acid (GABA)

receptor, such as benzodiazepines (midazolam or diazepam) and barbiturates (phenobarbital)

were used to induce a therapeutic coma, to produce a burst-suppression pattern on electroencephalogram. In parallel, attempts were made to reduce excitotoxicity, brain metabolism and autonomic reactivity by NMDA-receptor antagonism with amantadine and ketamine. Both these drugs also have antiviral activity, reportedly specific against rabies in vitro and in rat models for ketamine (see section 3.1.6). Antiviral therapy was also implemented, with the administration of ribavirin. After 31 days of isolation, the patient was removed from intensive care. She was discharged home after 76 days. The patient survived with mild to moderate neurological sequelae, which seem to have improved with time [START_REF] Willoughby | Survival after treatment of rabies with induction of coma[END_REF][START_REF] Hu | Long-term follow-up after treatment of rabies by induction of coma[END_REF]. However, the efficacy and utility of this protocol remains debatable, as it is unclear whether it played a significant role in recovery, or whether survival resulted from an effective endogenous antirabies immune response developed in this patient, in combination with the quality of critical care implemented during hospitalization [START_REF] Wilde | Viewpoint: Management of human rabies[END_REF][START_REF] Jackson | Recovery from rabies[END_REF]. This patient had antirabies neutralizing antibodies in her CSF and serum shortly after the onset of symptoms, antigen or RNA detection in saliva, CSF and nuchal skin biopsy). This unusual biological presentation was also observed in the other case of survival after rabies that had been vaccinated [START_REF] Hattwick | Recovery from rabies. A case report[END_REF]. It is therefore possible that both these patients managed to mount an unusually effective early immune response to the infection that contributed partially or fully to recovery. However, we also cannot exclude the possibility that survival was due to an unusual variant of RABV that was less pathogenic (a bat-related virus in both cases) or a particular polymorphism in these hosts rendering them less susceptible.

Experimental attempts to reproduce the Milwaukee protocol

The recent case of recovery from human rabies in 2004 and its promotion have led to the repetition of this protocol in a number of individual cases. Various attempts have been made

to use the original or closely related protocols. According to the special rabies registry website dedicated to the promotion of this protocol, it has been repeated at least 25 times

[259]. Regardless of the precise protocol used, most of these trials remained unsuccessful. A brief description of the best described attempts to use this protocol, in chronological order, is provided below.

The first repetition was carried out in multiple cases of rabies transmission via transplanted solid organs from a single infected donor, which occurred in Germany in 2004 [START_REF] Maier | Management and outcomes after multiple corneal and solid organ transplantations from a donor infected with rabies virus[END_REF]. This repetition followed a similar report several months previously in the USA, in which four recipients received solid-organ transplants and vascular grafts from a donor with undetected rabies [START_REF] Srinivasan | Transmission of rabies virus from an organ donor to four transplant recipients[END_REF]. In the German report, six patients received organs (corneas in two cases, kidney, lung, liver, and kidney with pancreas for the others) from the German donor, who was retrospectively diagnosed with rabies 45 days after transplantation. At this time, three of the recipients (who had received lung, liver or kidney with pancreas) developed clinical symptoms and aggressive treatment was initiated, based on the recently described Milwaukee 61/115

protocol. This included deep sedation (with ketamine and midazolam, with or without phenobarbital) and the administration of potentially antiviral drugs (with ribavirin, interferon and amantadine). PEP was also started immediately, including antirabies vaccination (five-dose regimen) and the administration of several HRIG. However, this treatment was unsuccessful, as all these patients developed rabies and died within days of the onset of symptomatic disease. The other three recipients were also given PEP and did not develop clinical signs. Transplanted corneas were removed in two of these cases and tested negative for rabies virus RNA. The third patient, who received a liver transplantation, was treated with ribavirin and -IFN for three weeks, in addition to PEP. This patient did not develop infection, probably due to a previous antirabies vaccination, 20 years previously.

In 2006, two similar unsuccessful therapeutic approaches were attempted. The first was performed in a male Thai patient in 2006 who presented furious rabies [START_REF] Hemachudha | Failure of therapeutic coma and ketamine for therapy of human rabies[END_REF]. An aggressive approach was adopted, with treatment with diazepam and thiopental for a 46 h, in addition to ribavirin (enteral) and ketamine (intravenous), but this patient died after eight days of hospitalization. The second of these cases, which was also not confirmed, was treated with the Milwaukee protocol and concerned a 17-year-old male patient in Texas, USA, possibly contaminated by a bat [261]. However, little information is available concerning this case.

At least four other reports of unsuccessful treatment based on the Milwaukee protocol were assessed in 2007. One of these reports concerned two patients in the US [START_REF]CDC Human rabies--Indiana and California[END_REF]. The first case was a female patient probably contaminated a few months previously by an infected bat at home (Indiana, USA). She received the full protocol (including antiviral agents, antiexcitatory therapy and supplementation) but died after 23 days of treatment. The second case in this report was a male patient probably bitten by a rabid dog two years previously in the 62/115

Philippines. Sedative and antiviral molecules were administered, but the patient died after 27 days of treatment [START_REF]CDC Human rabies--Indiana and California[END_REF]. The second report concerned a German case treated with a modified version of the protocol, including a vaccination strategy based on classical PEP with two additional vaccinations with live attenuated rabies virus vaccine injected intradermally [START_REF] Schmiedel | Case report on fatal human rabies infection in Hamburg, Germany[END_REF].

The aim of this strategy was to induce an early antibody response, in the hope of improving antigen presentation and response in the skin, via the intradermal route. Ribavirin was not given, due to reports of a lack of antirabies activity and the potential of this drug to delay the cellular response [START_REF] Warrell | Failure of interferon alfa and tribavirin in rabies encephalitis[END_REF]. However, this strategy failed. The third report concerned a Dutch patient infected with Duvenhage virus (lyssavirus, species 4), after contact with a suspected rabid bat in Kenya a few weeks earlier [START_REF] Van Thiel | Fatal Human Rabies due to Duvenhage Virus from a Bat in Kenya: Failure of Treatment with Coma-Induction, Ketamine, and Antiviral Drugs[END_REF][START_REF] Van Thiel | Fatal case of human rabies (Duvenhage virus) from a bat in Kenya: The Netherlands[END_REF]. Antiviral and sedative treatment was given, with PEP and aggressive supportive care. The patient died after 20 days of hospitalization.

The last report concerned a patient treated with the Milwaukee protocol in Equatorial Guinea [START_REF] Rubin | Applying the Milwaukee protocol to treat canine rabies in Equatorial Guinea[END_REF]. The authors claimed possible success for this treatment, as some semblance of neurological recovery was observed during the course of hospitalization. The patient nonetheless died and the authors suggested that this was due to complications of malnutrition.

However, this interpretation is highly debatable. Moreover, it should be stressed that the environment for testing this protocol did not appear to be satisfactory and that the patient was not correctly managed.

During 2008 alone, several other treatments were carried out in different part of the world. A modified Milwaukee protocol was tested in a Canadian patient [START_REF] Mcdermid | Human rabies encephalitis following bat exposure: failure of therapeutic coma[END_REF]. This patient presented clinical signs of rabies after being bitten by a bat six months earlier. On day 15 after the onset of symptoms, a therapeutic coma was induced in this patient, with infusions of ketamine, midazolam and propofol. Administration of ribavirin and amantadine was initiated, together with metabolic supplementation (with tetrahydrobiopterin and L-arginine) and intramuscular 63/115 injection of HRIG (1200 IU). The therapeutic coma was stopped after three weeks of treatment but the patient remained comatose for the next four weeks, subsequently undergoing brain death. In Northern Ireland, a modified Milwaukee protocol was used on a female patient 14 days after the onset of symptoms. It included the administration of ketamine and midazolam for coma induction, antiviral treatment with amantadine and ribavirin, and metabolic supplementation with co-enzyme Q, tetrahydrobiopterin and vitamin C [START_REF] Hunter | Immunovirological correlates in human rabies treated with therapeutic coma[END_REF].

Neither rabies vaccine nor rabies immunoglobulin was administered. The patient died after 25 days of treatment. Finally, two other reports of Milwaukee treatment given to patients with clinical rabies have been described in brief. One concerned a nine-or 12-year-old boy from the Goias region of Brazil who contracted rabies from a bat bite. He was treated with antiviral drugs and by coma induction, but died after cardiorespiratory arrest [START_REF]ProMED-mail Rabies, human, bat -Brazil (03[END_REF][270]. The other case was an eight-year-old Colombian girl who was not vaccinated against rabies (not published).

She died one month after the initiation of treatment, about one month after being bitten by a cat. However, very little information is available for either of these two cases.

In the same year, a short description of the potentially successful treatment based on the Milwaukee protocol of a 15-year-old Brazilian boy presenting clinical rabies was published.

This patient developed rabies almost 29 days after being bitten by a hematophagous bat [START_REF]ProMED-mail Rabies, human survival, bat -Brazil: (Pernambuco)[END_REF][272]. According to the few data available, the treatment of this patient, hospitalized in the region of Pernambuco, included the administration of ketamine, midazolan and amantadine, without ribavirin or EEG monitoring. This treatment was initiated seven days after the onset of symptoms and continued for 28 days. Partial PEP was given four days after the bat bite (almost 25 days before the onset of symptoms), including four doses of vaccine without the administration of rabies immunoglobulin. A 5th dose of vaccine was given after the onset of the clinical symptoms. Viral RNA was detected by skin biopsy and an increase in neutralizing 64/115

antibody levels was measured in the CSF and blood. This patient was still alive at least three months after the first published reports [273][START_REF]04): (PE), recovery[END_REF]. He was conscious and speaking (with difficulty), with good cognitive function but with motor limitations (difficulty walking). This case may be interpreted as a failure of PEP, and remains similar to other cases of vaccination failure. Indeed, the survival of children receiving treatment with rabies immune serum or immunoglobulin before the onset of symptoms, associated with intensive, aggressive care is well recognized (see paragraph 3.2.1). More details of this case are required.

Conclusions

All attempts at antiviral treatment for cases of clinical human rabies to date have failed.

Retrospectively, these disappointing results are not particularly surprising, given the absence of convincing and reproducible activity against rabies infection in animal models for most of the compounds tested in human rabies. The role of the Milwaukee protocol in the successful and unexpected recovery from encephalitic rabies in one case in 2004 remains unclear, as all published attempts to reproduce these findings have been unsuccessful. Palliative therapy remains of paramount importance in this fatal disease.

The future development of antiviral treatment for human rabies disease

Despite its long history, human rabies remains an almost invariably fatal disease once the clinical signs have appeared. No curative drug therapy is currently available. Several molecules have been tested in vitro and in animal models, without convincing results.

Antiviral activity against rabies infection has been tested individually for drugs, and the few studies based on large-scale drug screening carried out were too broad and not specific enough. Novel and innovative strategies of antiviral drug identification for use in rabies are clearly required, bearing in mind that such drugs must be actively delivered to the CNS, crossing the blood brain barrier. However, searches for such compounds are limited by the 65/115 lack of knowledge on rabies pathogenesis, particularly at the cellular level. The molecular mechanisms leading to the appearance of clinical signs and the neuronal dysfunction observed in rabies remain unsolved. Integrative research programs, including a basic and comprehensive analysis of the biology of RABV (and lyssaviruses in general), together with the use of advanced technology to identify novel effective antiviral compounds, constitute a promising approach. The VIZIER program, an EU-funded FP6 program, provides an interesting example of such a strategy (www.vizier-europe.org). This program was based on the identification of potential new drug targets in RNA viruses, including rhabdoviruses, through comprehensive structural characterization of the replication machinery [START_REF] Assenberg | Genomics and structure/function studies of Rhabdoviridae proteins involved in replication and transcription[END_REF]. This approach is now being pursued through the SILVER program. Until new effective therapeutic molecules against rabies can be identified, careful evaluation of treatments for human rabies is required. The recent case of recovery from human rabies in 2004, following an experimental protocol combining antiexcitatory and antiviral drugs, therapeutic coma and supportive intense care, has been a source of much hope and has attracted considerable attention. However, multiple repetitions of this protocol have been unsuccessful, suggesting that this protocol probably played only a limited role in the favorable outcome of this patient [START_REF] Jackson | Recovery from rabies[END_REF]. These findings also demonstrate that the application and promotion of a therapeutic protocol that has not been validated with robust and reproducible antiviral studies in vitro and in animal models may lead to unproductive, disappointing results.

Conclusion

With an estimated 55,000 human deaths from rabies occurring each year worldwide, this disease is the infectious disease with the highest mortality case ratio, affecting mostly young children from rural regions of developing countries. Despite the lack of an effective antiviral treatment once the disease is established, tools for decreasing and eliminating human rabies 66/115 viruses are well known, validated and generally available. These tools combine coordinated public health and veterinary programs. In particular, established public health systems and networks of surveillance, education of the population about rabies and widespread access to medical care for the administration of PEP are essential. In parallel, canine rabies elimination is a key step towards decreasing the burden of disease, with mass vaccination campaigns and management of the dog population, associated with a national program of rabies surveillance.

The use of oral vaccination in free-ranging carnivore hosts has demonstrated the feasibility of rabies elimination in wildlife. This approach has proved effective in many countries, including those of Europe in particular.

In parallel, there is clearly an urgent need to increase basic knowledge on rabies virus and the physiopathological mechanisms involved in rabies encephalitis, for the ultimate development of effective antiviral drugs for treating rabies infection.

67/115

  Pasteur exported this methodology to other parts of the world, in which new Pasteur Institutes opened.

  but RABV remained undetectable by classical diagnosis tests (including viral isolation, 60/115

  neutralizing antibodies; N.Av: not available; NCAM: neural cell-adhesion molecule; NDV: Newcastle disease virus; NGF: nerve growth factor; NMDA: N-methyl-D-aspartate; NT-3: neurotrophin-3; o.v.: overnight; PFU : plaque-forming unit; p.i.: post-infection; poly I:C: polyriboinosinicpolyribocytidylic acid; poly ICLC: poly I:C containing poly-L-lysine and carboxymethylcellulose; PM: Pitman-Moore; PV: Pasteur virus; RTA: lipophilic analog of ribavirin; SAD: Street Alabama Dufferin; s.c.: subcutaneous; (S)-DHPA: dihydroxypropyladenine; S.I.: selectivity index (inhibitory concentration/cytotoxicity concentration); siRNA: short interfering RNA; s.s.i.: same site of virus inoculation; (T: time in days (d), weeks (we.), hours (h) or minutes (min) (after virus inoculation, beginning of treatment); U: unit 88/115

Table 1 :

 1 Lyssavirus classification and associated epidemiological characteristics

	Name	Species	Geographic distribution	Reservoirs and/or vectors	Other infected animals	Human cases	Vaccine protection
	Rabies virus	RABV	Worldwide except Antarctica, Australia, Western Europe, United Kingdom, part of Scandinavia and some islands	Dog, wild carnivores, bats (solely for American continent)	Human, wild and domestic carnivores, herbivores	55,000/year (99% due to dogs, <1% due to bats)	Yes
	Lagos bat virus	LBV	Africa: Nigeria, Central African Republic, South Guinea, Zimbabwe Africa, Senegal, Ethiopia,	Frugivorous bats (species Rousettus, Micropteropus) Eidolon, Epomophorus,	Insectivorous bats (species mongoose Nycteris), cats, dogs, aquatic	None	No
	Mokola virus	MOKV	Africa: Nigeria, Central African Republic, Zimbabwe, Cameroon, Ethiopia, South Africa	Undetermined	Human, shrew, cats, dogs, rodent	1 confirmed case suspected (Nigeria, 1969) (Nigeria, 1971), 1	No
	Duvenhage	DUVV	Africa: South Africa, Zimbabwe	Insectivorous bats (species Miniopterus, Nycteris)	Human	3 (South Africa, 1971, Kenya, 2007 2006; Netherlands via	Partial
	European bat lyssavirus type 1, (subtype a or b)	EBLV-1	Europe	Insectivorous bats (mainly species Eptesicus)	Human, others insectivorous bats (?), cats, sheep, stone marten	1 confirmed and 2 suspected (Russia, 1985)	Partial
	European bat lyssavirus type 2	EBLV-2	Europe	Insectivorous bats (mainly species Myotis)	Human	2 (Finland, 1985, Scotland, 2002)	Partial
				Frugivorous bats (species
	Australian bat lyssavirus	ABLV	Australia	Pteropus) and insectivorous bats (mainly species	Human	2 (Australia, 1996, 1998)	Partial
				Saccolaimus)
				Insectivorous bats (species
	Aravan virus	ARAV	Central Asia (Kirghizistan)	Myotis) (isolated once in	?	None	Partial
				1991)
				Insectivorous bats (species
	Khujand virus	KHUV	Central Asia (Tadjikistan)	Myotis) (isolated once in	?	None	Partial
				2001)
				Insectivorous bats (species
	Irkut virus	IRKV	East Siberia	Murina) (isolated once in	?	None	Partial
				2002)
	West Caucasian	WCBV	Caucasian region	Insectivorous bats (species	?	None	No

Table 2 :

 2 Modalities of rabies post-exposure prophylaxis (PEP) according to WHO recommendations[START_REF] Who Rabies Vaccine | WHO position paper[END_REF] For categories II and III, all bite wounds and scratches should be thoroughly washed and flushed (for about 15 minutes if possible) with soap/detergent and copious amounts of water immediately, or as soon as possible.

	Categories of exposure Description of exposure	Prophylaxis
	Category I	touching or feeding animals, licks on intact skin (that is, no	no prophylaxis
		exposure)
	Category II	nibbling of uncovered skin, minor scratches or abrasions	immediate vaccination a
		without bleeding
	Category III	single or multiple transdermal bites or scratches,	immediate vaccination and
		contamination of mucous membrane with saliva from licks,	administration of rabies
		licks on broken skin, exposures to bats	immunoglobulin a
	a

Table 3 :

 3 Overall description of the main drugs tested for antiviral activities against rabies infection in vitro and in animal models

	Molecule	Test a	Treatment	Virus	Evaluation of	Main results	Reference
			(dose, route of	challenge	antiviral	(best results: number of
			administration, starting	(strain, dose,	activities	survivors /total number)
			time, incubation period or	route of
			duration of treatment,	inoculation)
			other)
	Inhibitors of viral entry
	Antibodies against rabies glycoprotein and rabies vaccines
	Live-attenuated ERA-derived	In vivo (dogs, 2	ml in mice); 2 ml (titer = 10 6.5 i.c.LD 50 /0.03	5.92 x 10 6 -Fox strain;	Virus Mortality rate;	effect (recovery of 3/12 dogs), 1 st exp.: suggested therapeutic	[134]
	rabies vaccine	successive	i.t.;	5.92 x 10 4	detection and	but no intra-vitam
	(produced on	experiments)	At the time of clinical signs of	i.c.LD 50	isolation (also	confirmation of rabies
	BHK cells)		rabies	(mice) (1st	intra-vitam for	infection and no use of control
				exp.) or 5.92 x	2 nd exp.);	animal group;
				10 4 i.c.LD 50 (mice) (2 nd	Level of NA (2 nd exp. only)	2 nd exp.: no effect (only 4/7 challenged dogs became
				exp.);		symptomatic and all died),
				i.ma. for both		suggested increase in survival
						period for 2 animals;
						No convincing therapeutic
						activity in clinical rabies
	Live-attenuated	In vivo	1 ml (titer = 10 8.3 i.c.LD 50 /ml	Dog strain	Mortality rate;	No therapeutic effect or	[136]
	Flury HEP	(monkeys)	in mice);	(MR strain);	Virus	increase in survival period
	derived rabies		i.t.;	i.m.	detection and	(only 2/12 challenged
	vaccine (RV		At onset of symptoms		isolation	monkeys were symptomatic
	675 strain,				(intra-vitam	and receive the vaccine, and
	produced on				and post-	both died)
	BHK-21 cells)				mortem);
					Level of NA
	Live-attenuated	In vivo	10 7 FFU for i.c. or 10 8 FFU	Dog strain	Mortality rate	Complete and effective	[135]
	recombinant	(mice)	for i.ma.	(DOG4		protection only when given
	SAD B19-		i.t.;	strain);		i.c. or i.ma. shorly after
	derived rabies vaccine		T4h to T5d p.i.	10 i.m.LD 50 i.m. (10 6 FFU)		infection (4 h p.i.); Number of (healthy) survivors
	(SPBAANGAS-					lower when given >4 h p.i.
	GAS-GAS					(50% or 30% at 4d p.i., after

  Using a compartmentalized cell culture system 6-azu: 6-azauridine; amiRNA: artificial microRNA; AP5: competitive antagonist of NMDA receptor; ara-A: adenosine arabinoside; ara-C: cytosine arabinoside; ara-U: uracyl arabinoside; BHK: baby hamster kidney cells; CDZ : carbocyclic 3-deazaadenosine; CER: chick embryorelated cells; COAM: chlorite-oxidized amylose; CVS: challenge virus strain (laboratory strain); ERA: Evelyn-Rokitnicki-Abelseth; FFU: fluorescent focus unit; G-7-Ox: guanine 7-N-oxide; HPA: heteropolyanion; HEP: high egg passage; i.c.: intracerebral; IC[START_REF] Maier | Management and outcomes after multiple corneal and solid organ transplantations from a donor infected with rabies virus[END_REF] : inhibitory concentration 50%; i.ca.: intracarotidn; i.d.: intradermal; i.l.: intralumbar; i.m.: intramuscular; i.ma.: intramasseter; incub.: incubation; IFN: interferon; i.p.: intraperitoneal; i.t.: intrathecally; IU: international unit; i.v.: intravenous; i.ve.: intraventricular; LBV: Lagos bat virus; LD: lethal dose; LEP: low egg passage; MADU: 5-methylamino-2'-deoxyuridine; MIT: mouse inoculation test; moi: multiplicity of infection; NA:

	IFN-inducers (3 siRNAs	fibroblasts) (BHK-21)	T(-1) h p.i. (with cells), T0 (at Incub. 72 h for both Starting T3 h p.i.; each siRNA; suspension	(PV) moi=10	of infection cycle	No virucidal effect No virucidal effect Dose-dependent inhibition
	Vinblastine, Exogenous Poly I:C or/and Minocycline targeting	In vitro In vitro In vivo	virus attachment step) or T(1-Vinblastine: 1-10 M; 25-6,400 U/ml; poly I:C: 0.1 mg (1 or 2 +/-rabies vaccines Incub. 2-22 h p.i. incub. up to 40 h	Lab. strain Lab. strain Skunk strain; PFU/cell	Counts of Virus titration Mortality rate; (fluorescence conditions)	No virus inhibition; Decrease in virus production Partial protection with poly with 7 HPA, ribavirin and	[164] [183] [192]
	colchicine (human origin) COAM PICKCa nucleoprotein (derivative of mRNA)	(IMR-32 and CER cells) (MCK cells) (mice) In vivo In vitro (mice) (mouse	5) h p.i. (after virus adsorption step) Colchicine: 10-100 M; T(-1) h p.i. (with cells), T0 (at T0 p.i.; Incub. 1 d doses); i.m. s.s.i.; 25-50 g (per dose) (single or In vitro: 5 nM; T1h p.i.; incub. Various doses: 5-500 g/ml COAM: 0.3 mg; i.p.; multiple doses); 1-3 d	(CVS); moi = 5-10 (LEP); i.m. Lab. strain Lab. strain 10 4 mouse (CVS); (derivative of	infected cells Level of IFN Mortality rate, In vitro: virus intensity); and NA Level of IFN titration, cell Virus titration	No virucidal effect I:C alone (30/32) or in Complete to partial protection Slight protective effect in vitro amantadine; combination with COAM (at s.s.i., T ≤6 h p.i., up to with decrease in virus 90% inhibition for 5	[194] [218]
	Incub. 1-24 h Drugs targeting actin microfilamnets or calmodulin processes Drugs acting on the cytoskeleton Cytochalasin B virus attachment step) or T1 h p.i. (after virus adsorption Endogenous (inactivated In vivo (rabbits) Multiple doses; i.c., i.ca., i.d., i.p., i.v., s.c.; T3h p.i. for both poly I:C) T6 h or T3 d p.i., up to 7 s.c. at s.s.i. +/-rabies vaccines cortical and In vivo: 50 mg/kg/d; s.c.; T6 h amiRNAs In vitro Transfection with 1 g of hippocampal p.i., for 17 d (targeting (N2a) DNA; primary nucleoprotein T-12 h p.i. with incub. 72 h; step) In vitro b (rat embryonic DRG cells) 0.1 mM, +/-vinblastine; T0 or T4-24 h p.i.; incub. 72 h Cytochalasin B In vitro (BHK-21 and CER cells) 13-208 M; immediately p.i.; incub. 32 h Cytochalasin B and D, trifluoperazine In vitro (IMR-32 and CER cells) Cytochalasin B and D: 0.128-16 M; Cytochalasin B: 0.128-16 M; Trifluoperazine (1-20 M); T(-1) h p.i. (with cells), T0 (at virus attachment step) or T(1-5) h p.i. (after virus adsorption step) Incub. 1-24 h Cytochalasin B, EGTA, A23187, chloropromazin e, trifluoperazine, nifedipine In vitro (rat primary cortical neurons, IMR-32, BHK-21 and CER cells) Cytochalasin B: 1-20 g/ml; EGTA (0.5-0.37 mM), A23187 (0.05-1.0 M) , chloropromazine (1-30 M), trifluoperazine (1-20 M), nifedipine (1-10 M); T1 h p.i.; Incub. 48 h (up to 4 d with EGTA) Drugs targeting microtubules Vinblastine, colchicine In vitro ** (rat embryonic DRG cells) Vinblastine: 0.1 mM, (+/-cytochalasine); T0 or T4-24 h p.i.; Colchicine: 0.1 mM; T-(1-4) h, T0 or T(1-4) h p.i.; Incub. 24 h Colchicine, colcemid In vitro (BHK-21 and CER cells) Colchicine: 16-250 M, vaccinia virus) T(-24) h or from T24 h p.i.; Poly I:C In vivo (rabbits and Rabbits: 0.5-1 mg (multiple doses); i.m. or i.v.; T(-1) d to neurons) mRNA) 17 compounds In vivo T(-1)d to T3d p.i.; T12 h p.i. with incub. 24 h (immunosuppre (mice) Single or multiple doses; Exogenous In vivo 1.6-6.4 x 10 6 U (total dose); mice) T1 d p.i. In vivo (mice) ssive, cytostatic Various concentrations; colcemid 17-336 M; (rabbit origin) (rabbits) i.v. and/or i.m.; Mice: 0.26 -33 g (i.m.) or Poly ICLC In vivo 0.06 mg equi. poly I:C (per or antiviral i.p., i.v., s.c. or i.m. immediately p.i.; T(0-24) h p.i. 100 g (i.p.) (single or (mice and dose) (mice), 2 mg poly ICLC Antiviral peptides agents) (depending on the drug tested) incub. 32 h Colchicine In vivo (rats) Solution of 2, 5 or 10% colchicine, in the sciatic nerve (delimited with a silicone cuff); T-(4-48) h p.i. Exogenous In vivo rhesus (per dose) (monkeys), Other drugs derived from plants or fungi Peptides In vitro Synthetic peptide: 10 M,; T1 multiple doses); T0-T91 h p.i. 1-10 x 10 6 U (total dose), monkeys) multiple doses; Cinnabarin In vitro 0.155-5 mg/ml; mimicking (BHK-21, h p.i. (rabbit orign) (rabbits) multiple doses, up to 3 we. i.ve. or i.v. and i.m. (in Poly I:C, poly I, In vivo 0.04-1 mg/kg (single dose); T24 h p.i. (both) and T6 h p.i. (monkeys); (from fungus Pycnoporus (NA, C-1300 cells) Added to viral suspension (incub. 30 min), then added to phosphoprotein (first 42 and 60 neuronal cell lines) Plasmid transfection: 2 g; T(-5) h p.i. combination); poly C (rabbits) i.v., i.m. or i.ma.; +/-rabies vaccine sanguineus) cells (incub. 24 h) residues of N-24 synthetic In vitro T0 p.i.; T(0-21) p.i. T0 p.i. Ketamine and dizocilpine (MK-801) 4 extracts from In vitro doses ≤ 50% cytotoxic terminal region) phenolic (McCoy cells) Incub. 96 h; Exogenous In vivo 10 5 U/d, up to 30 d Poly I:C In vivo 1 mg (single dose); Dizocilpine and In vitro 0-2 mM; South American (McCoy cells) concentration; Multiple-drug screening compounds 100 l/dose of Vinblastine, colchicine In vivo (mice) 26-64 g (colchicine), 9-12 g (vinblastine), given locally on sciatic nerves using impregnated blotting papers); T0 or T(2-60) h p.i. (mouse origin) (mice: immuno-i.p.; T1 h p.i. (rabbits) i.v.; T-1 d to T1 d p.i. AP5 (rat primary cortical T1h p.i.; Incub. 4 d plants T0 p.i.; 65 compounds In vivo Administered at the site of concentrations Incub. 96 h (antimetabolites (mice) inoculation, systemically or competent, -neurons) and antibiotics; between the site of inoculation depressed or -deficient) Exogenous (human or In vivo (mice and Poly I:C, NDV In vivo (rhesus Poly I:C: 15 mg/kg. i.v.; NDV: 2.5 x 10 PFU per Exudate fluids In vitro Final concentrations: 0-20%; proteins, nucleic and the CNS; 14 natural and In vitro 0.1-800 g/ml Ketamine and In vitro Ketamine: 1-1.5 mM, from small red (BHK-21 T(-1)h p.i. (with cells or virus) acids and T5 min p.i. (+/-up to 5 d) or semisynthetic (CER cells) T(-1.5) h p.i. (incub. with 0.03 ml (mice), 1 x 10 6 U (monkeys) (multiple doses); monkeys) animal; T(5-8) h p.i. dizocilpine (rat primary cortical dizocilpine: 1 mM; After infection for both; beans cells) or T(0-24)h p.i.; related T1 h p.i.; polymeric virus or with cells) or T0 p.i. Incub. 24h compounds; i.m., i.p., s.c., p.o. carbohydrates (incub. during virus IFN and IFN-inducers mouse origin) rhesus i.m.; +/-rabies vaccines neurons) Incub. 48 h miscellaneous attachment step); IFN Exogenous (mice origin) In vitro (mouse cells) 10-20 x 10 3 U; T(-24) h to T24 h p.i.; incub. up to 96 h Exogenous (dog origin) In vitro (dog cells) Fixed quantity, different batches; T(-(24-1)) h p.i. Exogenous (human or hamster origin) In vitro (BHK cells, primary human Overnight incubation (18 h) with 100 IU/2 ml before infection first clinical signs (CER cells) incubation with virus T(-4) h to T11 d p.i., and after i.m.; ketamine) siRNAs In vitro Transfection with 20 pmols of Lab. strain PFU/cell LD 50 Dog strain; 10 -5.31 (MIT Fox strain; SAD); Lab. strain 1.5-4 x 10 6 , moi=10 (CVS, HEP), 1.5-4 x 10 7 or FFU/cell (in dog strain; Lab. strain (CVS); 5 x 10 6.85 PFU Lab. strain (CVS); moi = 10 PFU/cell Lab. strain (CVS); moi = 5-10 PFU/cell Lab. strain (CVS); 4.6 x 10 6 FFU Lab. strain (CVS); 5 x 10 6.85 PFU Lab. strain (CVS); titration); i.d. Lab. strain (CVS, 1 x vitro), 100 10 or 100 Fox and 10 8 i.c.LD 50 ; FFU i.m. (in TCID 50 rodent strains; Fox strain; 1 rabbit LD 50 ); s.c. vivo) i.m. or i.c. moi = 10 80 rabbit Skunk strain Fox and skunk PFU/cell i.m.LD 50 ; (1 mouse strain; Lab. strain (CVS), fox strain; i.m. for both i.m. LD 75 , 1 rabbit 5-30 mouse Lab strain Fox strain; LD 100 ); peripheral Lab. strain (PV), LBV ≥ 28DL 50 ; i.m. i.m. for both Fox strain; LD 50 (mice); i.m. for both (PV) (species 2); 90% infection, 5-3125 LD 50 ; or moi=5 Lab. strain i.m. or i.ma. Lab. strain PFU/cell (PV); Fox strain; Fox strain; Lab. strain (PV); moi=1 Lab. strain (CVS, 10 7.3 i.c.LD 50 ), fox strain (10 5.3 i.c.LD 50 ); i.m. for both 10 mouseLD 50 ; 25-30 rabbit DL 50 ; (CVS); moi=1 Coyote strain; 10 6 UFF/ml FFU/cell ≥LD 50 i.m. i.ma. NYC strain; Lab. strain Lab. strain 10 5 i.c. mouse Lab. strain (HEP); (CVS); Fox and skunk strain; LD 50 ; i.m. (CVS); moi=0.01 Dilution=50% 10 6 UFF/ml PFU/cell infected cells 5-30 mouse Lab. strain; 650LD 50 Lab. strain (PV); 10 -5 (MIT Lab. strain (ERA); moi=5 i.m. i.d. striatum) (CVS); PFU/cell ey LD 50 ; nearly 1 LD 50 ; (right 21 compounds In vitro T40min p.i. or direct Lab strain monkeys) i.m. and/or i.l.; (mouse)/monk (mice) multiple doses); skunk strains; only with inoculation genes) (human origin) (Cynomolgus (multiple doses and/or days); 10 5.7-6. Poly I:C In vivo 100 g (per dose) (single or bobcat and In vivo (rats, stereotaxic phosphoprotein i.m., i.c. Exogenous In vivo 1-11 x 10 6 U (total dose) NYC strain; (rabbits) i.c.LD 50 , and drugs; titration) +/-rabies vaccine i.m. for both Exogenous (human origin) In vivo (Cynomolgus monkeys) i.m. and/or i.l.; T(1-3) d p.i.; up to 7 d NYC strain; 10 5.7 mouseLD 50 ; i.m. or inactivated) (hamsters, mice, rabbits) hamster); T(-24) h, T0 or T24 h p.i. (CSV, VP10 or 11); i.m. (hamsters and mice), i.c. competitive or non-competitive NMDA receptors cortical neurons, BHK-21 and IMR-32 cells) 4 d In vivo: i.p.: 15 mg, twice daily for 4 d In vitro: 4 x In vivo: nucleoprotein Different concentrations of FFU/ml 10 5 mouse fragments targeting the Incub. 1-3 d moi=0.25 or 2.5 PFU/cell single or multiple doses; 10 2 i.m.LD 50 , binding step 5 x 10 5 -5 x 10 7 (mice, foxes) treatments (up to T13 d p.i.), i.m.; incub. 1-3 d or just during suspension of 10 6 UFF/ml (2 short cDNA (BSR cells) (1-2 x 10 4 cells/well); (CVS); 40 compounds In vivo Early (from T1 d p.i.) or late Fox strains; or with cells), T0 p.i. and Dilutions of 1-10 x 10 5 U/d; NDV (infectious In vivo i.v. (mice), i.p. (rabbit and Lab. strains other (rat primary after virus infection, incub. 1-(CVS) cDNA In vitro Stably transfected BSR cells Lab. strain detergents) (IMR-32 cells) T(-0.5) h p.i. (incub with virus (CVS); monkeys) T24 h p.i. (both) and T(-6) h peripheral Statolon In vivo i.p.; i.m. or i.c. substances with Incub. 1.5 h for all conditions p.i. (monkeys); LD 50 (mice); (mice) T(-(24-18)) h p.i. Ketamine and In vitro In vitro: ketamine: 0.2-2 mM, Lab. strain RNA interference solvents and 26 compounds In vitro Determination of the IC 50 ; Lab. strain	Mortality rate and NA viability Counts of infected cells; In vivo: Quantification Count of infected cells, virus titration Virus titration Detection of infected cells Virus titration; Detection of infected cells Counts of infected cells, virus titration Virus titration Mortality rate, level of IFN Mortality rate, of Mortality rate; histological nucleoprotein Virus titration Mortality rate and NA and mRNA and Mortality rate, immunohistolo virus genome level of IFN gical analysis Mortality rate; level of NA; antigen detection and virus isolation (on sciatic and NA Counts of Mortality rate Virus titration infected cells, Mortality rate, virus titration, minireplicon Level of IFN system IC 50 and S.I. Measurement determination Mortality rate, Mortality rate; Virus titration of cytopathic (inhibition of nerve sections) Mortality rate; Level of NA Titration of NA Level of IFN and NA effect Mortality rate viral cytopathic effect) Mortality rate; Counts of Counts of Level of IFN Count sof infected cells infected cells; Mortality rate, level of IFN and NA infected cells; IC 50 and S.I. Viral titration; determination and NA Radiolabeling; Counts of infected cells (single-growth Counts of infected cells; Virus titration Quantification Slight decrease in viral titer 100% protection with i.d. s.s.i. (5/5); (31/32); No protection of COAM alone (15/33) 100% or 20/20); production Reduction of viral genome, derivatives of adenosine Comparable protective effect nucleoprotein mRNA and including (S)-DHPA; with poly I:C; Not protective in vivo infectious cells No virucidal effect No virus inhibition after established infection (24 h p.i.) No virus inhibition Dose-dependent virus inhibition; Acting after virus adsorption step (T≥1 h p.i.); No virucidal effect No virus inhibition No virus inhibition after established infection (24 h p.i. for vinblastine, 4 h p.i. for colchicine) No virus inhibition Partially protective with i.c. and i.ca. Total to partial protection with poly I:C at highest dose, given Stronger protective effect in (increase in mortality rate) Best protective effect obtained combination with rabies with iododeoxyuridine and Total to partial protection (6/6, around time inoculation (up to vaccines actinomycin D (i.c.) and with i.m., T0 p.i. highest dose); 100% and 94% in rabbits and Partial protection (monkeys= 6-azacytidine and azathioprine No virucidal effect mice, respectively) 8/8, T6 h p.i., + rabies ; Dose-dependent virus vaccine) (mice= 47/50, T0 p.i., Reduction of virus infection Minor or no protective effects Partial protection with i.v. and s.s.i.) Decrease in virus titer (50% with ara-C and vinblastine; inhibition; Protective effect (up to100% i.m. route (2/3) Total to partial protection, with 0.155-0.31 mg/ml) Results remain difficult to interpret or 6/6 with 5% colchicine only with poly I:C (up to 4 compounds with potential solution, T-(24) h p.i.) 100% or 6/6) Virus inhibition for only one antiviral effect (S.I.>3) No virucidal effect Partial protection with nude Total to partial protection (up Specific dose-dependent virus extract (S.I.>5). Protective effect (up to 100% (7/8) and immunocompetent mice (7/14); to 100% until T3 h p.i.) inhibition (range 0.5-2 mM); Most effective drugs: solvents, No effect with AP5; detergent and antirabies or 10/10 at T0 p.i. for both Virucidal effective at high doses; serum; with CSV strain); Protective effect up to 6 h or 24 h with CVS or fox strain, respectively Partial protection with rabies No virucidal effect Dose-dependent virus No therapeutic effect with Dose-dependent virus vaccines (poly I:C = 5/10 and Virus inhibition; proteins, enzymes, inhibition for most of the inhibition (IC 50 =0.48% dose) Partial protection (monkeys= 7/8, T6h p.i., + rabies vaccine) NDV = 2/10); No improvement of protection 100-fold decrease of virus antimetabolites or antibiotics compounds; observed only during early production phase of infection; (including actinomycin D, Acting at the virus binding (mice= 41/50, T0 p.i., s.s.i.) over rabies vaccines alone Virucidal effects (obtained mitomycin D and -step Dose-and time-dependent appearance of clinical signs compounds; Decrease in virus infectivity No effect when given after rabies vaccines (130/150) Virus titration Virus inhibition with 14 (nearly 1 to 3 log) and NA with multiple doses); Level of IFN (83/132) or combined with vivo) nucleosides analogs (80-97-%) and production Level of IFN or 8/10 for i.m.+i.l. at T1d p.i. Mortality rate; Partial protection at s.s.i, alone certain parts of the CNS (in including amantadine and Mortality rate; Partial protection (5/9 for i.m. Potential virus inhibition in gene (coding cDNA) No effect with other molecules Virus titration Decrease in virus production (T(-24) h p.i.) Mortality rate; Level of NA Partial protection (8/10 with i.m. and i.l., T24 h p.i. up to 13 d) rateLevel of IFN Partial protection in hamster (35/51 to 40/57) and rabbits (5/8) (in vivo) No virucidal effect; infected cells targeting the nucleoprotein Level of NA 56); (in vitro); Mortality rate virus production (1-2 mM, in vitro); (ELISA); Counts of with protein of the cDNA isolation; (HPA-23, -39, -46, -51 and -agents Potential direct interactions Virus restricted to 5 HPA molecules step and other lysosomotropic infected cells 100-1000-fold decrease in quantification for both cDNAs); Mortality rate; Effective preventive drugs targeting the virus binding Mortality No protection in mice; counts of inhibition (in vitro); Virus antigen Virus inhibition (up to 100% infected cells compounds, including drugs virus inhibition Mortality rate No protection Northern blot with 3% dose) phenylserine Virus titration, Dose-dependent virus Counts of Virus inhibition with 8	[185-186, 275] [275] [225] [166] [165] [164] [167] [166] [189] [156] [165] [176] [194] [168] [227] [276] [220] [188] [204] [222] [175] [187] [202] [169] [151] [195] [228] [221] [200] [194] [223] [159] [193] [174] [183, 196] [173] [184] [161] [179] [224] [182] [172] [201] [139]
				77/115 78/115 79/115 80/115 85/115 86/115

Legend:

a Administration of drugs: in vitro or in animals 87/115 b

Table 4 :

 4 Description of the principal attempts at the treatment of human rabies Human lymphoblastoid IFN: i.v. = loading dose of 50 x 10 6 U/m 2 body area per 6 h, repeated over the next 18 h, then given daily in continuous infusion, dose halved after the first we.; and i.t. (loading dose of 2 x 10 6 U/m 2 given by the lumbar route, repeated 6 h later then daily through the cerebral ventricle by the Ommaya reservoir, dose halved after the first we.;

	Case	Rabies exposure	Onset of	Drug-based therapeutic treatment	Diagnosis of rabies	Outcome	Reference
	description	(source, time of exposure	symptoms (o.s.)	(time after o.s. a , duration, description		(time after
		before o.s. or treatment,		of treatment)		o.s., exposure
		geographic location,				or
		year, PEP)				hospitalizatio
						n)
	First treatments for cases of human rabies
	Interferon
	Female, 10 y Dog bite=1.5 mo. before	N.Av.	d5 before o.s.;	Detection of rabies virus:	Death (19 d	[236]
		o.s. (shoulder, Africa)		15 d;	+ (saliva, postmortem	after o.s.)
		Paris, France; 1976		Human leukocyte IFN: 3 x 10 6 U i.v. on	brain biopsy)
				a continuous basis for 12 d, 1 x 10 6 U
				i.t. daily for 6 d) (total: 42 x 10 6 U);
				Never received PEP rabies vaccine or
				RIG)
	Male, 5 y	Dog bite=1.5 mo. before	N.Av.	d2-3;	Detection of rabies virus:	Death (28 d	[236]
		o.s. (buttock, Africa);		18 d (?);	+ (saliva, postmortem	after o.s.)
		Paris, France; 1976		Human leukocyte IFN: 1 x 10 6 U i.m.	brain biopsy)
				for 12 d and 0.5-2 x 10 6 U i.t. daily for 6
				d) (total maxi: 24 x 10 6 U);
				Never received PEP rabies vaccine or
				RIG)
	Female, 16 y Dog lick=several weeks	N.Av.	d14;	Viral iso.: + (postmortem	Death (23 d	[236]
		before o.s.;		10 d;	brain biopsy)	after o.s.)
		Sao Paulo, Brazil; 1981-		Human leukocyte IFN: 10 x 10 6 U twice
		1983		daily i.m., 5 x 10 6 U once daily i.ve.
				into a Rickham reservoir (total: 250 x
				10 6 U);
				Never received PEP rabies vaccine or
				RIG)
	Male, 40 y	Probable dog bite (right	Paresthesia and	d10 (for IFN treatment), d8 (?) for PEP,	FAT: + (neck biopsy,	Death (24 d	[236-237]
		hand), 58 d before o.s.;	numbness of the	before o.s.;	several other tissues	after o.s.)
		Arizona, USA (probably	right hand (near	14 d;	collected at post-mortem
		infected in Mexico); 1981	the site of the	1 dose of HDCV and HRIG: d8 (?);	stage, including brain
			bite), then fever	Human leukocyte IFN: d10, i.m. (twice	biopsies);
			malaise and	a d) and i.v. (once a d, using an Omaya	Abs: -(serum and CSF

Virus inhibition: IC 50 less than 3 g/ml [START_REF] Yamamoto | Inhibitory effect of a new antibiotic, guanine 7-N-oxide, on the replication of several RNA viruses[END_REF] Ketamine and dizocilpine

In vitro (mice primary cortical and hippocampus cells)

In vivo (mice, only with ketamine)

In vitro: four doses of vaccine [START_REF] Who Rabies Vaccine | WHO position paper[END_REF]. In cases of category III exposure, rabies immunoglobulin should be administered during the week after the first rabies vaccine injection (ideally at the same time or D0). PEP should be started (D0) immediately after rabies exposure (category II and III), or as soon as possible.

Legend:

D: day; ERIG: equine rabies immunoglobulin; HRIG: human rabies immunoglobulin, RIG: rabies immunoglobulin