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We present an unconventional approach to antiviral drug discovery, which is used to identify potent small molecules against rabies virus. First, we conceptualized viral capsid assembly as occurring via a hostcatalyzed biochemical pathway, in contrast to the classical view of capsid formation by self-assembly. This suggested opportunities for antiviral intervention by targeting previously unappreciated catalytic host proteins, which were pursued. Second, we hypothesized these host proteins to be components of heterogeneous, labile, and dynamic multi-subunit assembly machines, not easily isolated by specific target protein-focused methods. This suggested the need to identify active compounds before knowing the precise protein target. A cell-free translation-based small molecule screen was established to recreate the hypothesized interactions involving newly synthesized capsid proteins as host assembly machine substrates. Hits from the screen were validated by efficacy against infectious rabies virus in mammalian cell culture. Used as affinity ligands, advanced analogs were shown to bind a set of proteins that effectively reconstituted drug sensitivity in the cell-free screen and included a small but discrete subfraction of cellular ATP-binding cassette family E1 (ABCE1), a host protein previously found essential for HIV capsid formation. Taken together, these studies advance an alternate view of capsid formation (as a host-catalyzed biochemical pathway), a different paradigm for drug discovery (whole pathway screening without knowledge of the target), and suggest the existence of labile assembly machines that can be rendered accessible as next-generation drug targets by the means described.assembly intermediate | viral-host interaction | whole pathway screen | drug discovery paradigm | protein heterogeneity

Even though rabies is almost uniformly fatal, it is readily preventable with currently available tools. Vaccination is highly efficacious for the pre-exposure prophylaxis (PrEP) of rabies in humans and animals, and prompt postexposure prophylaxis (PEP) with vaccine and rabies immune globulin (RIG) can reliably prevent disease in humans. However, access to these tools and knowledge of their proper use are often limited, especially in impoverished, rabies-enzootic countries with the highest disease burden. In the absence of reliable diagnostic capacity and risk assessments, vaccines and RIG are often administered inappropriately, leading to chronic supply shortages and otherwise preventable deaths. Rather than focusing solely on human prophylaxis, it is more cost-effective over the long term to eliminate canine rabies in its natural terrestrial reservoirs. Because more than 99% of human rabies deaths result from dog bites, prevention efforts should focus on dogs. A versatile "One Health" strategy for canine rabies elimination should aim to create sustainable herd immunity in dogs, using proven vaccination strategies at the local level, coupled with community education and humane population management. Such strategies have succeeded in both developed and developing countries, and can be adapted to any locality. Numerous examples in Africa, Asia, and Latin America have shown that community-based, locally guided vaccination and education programs, based on a shared vision and long-term commitment, can eliminate canine rabies. Such programs should have specific goals and measurable outcomes, and should be conducted under the guidance of supportive governments, in collaboration with international partners and nongovernmental organizations. In addition to currently available tools, rabies prevention can be augmented by new dose-sparing human vaccine schedules, alternative routes of vaccine administration, monoclonal antibodies as an alternative to RIG, sensitive and specific point-of-care diagnostics and the development of canine immunocontraceptive methods. Accurate risk assessments of potential human exposures and support for decentralized laboratory capacity will be essential to ensure the most effective utilization of vaccines and RIG until canine rabies has been eliminated.

Migration and transport of dogs may have caused recent epidemics of human rabies.

Standardized protocols and diagnostic-based surveillance are imperative for detection and elimination.

No abstract

Rabies virus nucleoprotein (N) plays vital roles in regulation of viral RNA transcription and replication by encapsidation of the nascent genomic RNA. Rabies virus N is phosphorylated, and previous studies demonstrated that mutation of the phosphorylated serine at position 389 to alanine resulted in reduction of viral transcription and/or replication of a rabies virus minigenome. In the present study, we mutated the serine (S) at position 389 to alanine (A), glycine (G), aspartic acid (D), asparagine (N), glutamic acid (E), and glutamine (Q) and examined the effects of these mutations on rabies virus transcription and replication in the minigenome. Furthermore, mutations from S to A, S to D, and S to E were also incorporated into the full-length infectious virus. Mutation of the serine to each of the other amino acids resulted in the synthesis of an unphosphorylated N and reduction of viral transcription and replication in the minigenome. Mutations from S to A and S to D also resulted in reduction of both viral transcription and replication in full-length infectious viruses. Growth curve studies indicated that production of the mutant virus with the S-to-A mutation (L16A) was as much as 10,000-fold less than that of the wild-type virus (L16). Northern blot hybridization with rabies virus gene probes revealed that the rates of viral transcription and replication were reduced by as much as 10-fold in the mutant viruses when the N was not phosphorylated. Interpretation of the data from the minigenome system and the full-length infectious virus indicates that phosphorylation of rabies virus N is necessary for replication. Further studies involving cycloheximide treatment of infected cells revealed that viral transcription was also reduced when the N was not phosphorylated. Taken together, these results provide definitive evidence that N phosphorylation plays an important role in the processes of rabies virus transcription and replication.Within the Rhabdoviridae family, rabies virus is the prototype of the Lyssavirus genus and Vesicular stomatitis virus (VSV) is the prototype of the Vesiculovirus genus (23). Rhabdovirus genomic RNA is encapsidated with nucleoprotein (N), and this N-RNA complex, together with the phosphoprotein (P, also termed NS) and RNA-dependent RNA polymerase (L), forms the RNP complex. The N proteins of the rhabdoviruses, like the N proteins from other members of the order Mononegavirales, play vital roles in regulating viral RNA transcription and replication by encapsidating de novo-synthesized viral genomic RNA (for a review, see reference 23). Although rabies virus N and VSV N do not have a high degree of homology in primary nucleotide and protein sequences, they have conserved regions and similar protein characteristics. For example, the N protein of rabies virus has four conserved amino acid stretches homologous with those of VSV (22). In addition, similar helical structures exist in both rabies virus and VSV N proteins. Both N proteins have an ␣-helix continuing from the N terminus through...

A major challenge for global rabies prevention and control is the lack of sufficient and affordable high quality vaccines. Such candidates should be pure, potent, safe, effective and economical to produce, with broad cross-reactivity against viral variants of public health and veterinary importance. The history of licensed human vaccines reviewed herein demonstrates clearly how the field has evolved to the current state of more passive development and postexposure management. Modern cell culture techniques provide adequate viral substrates for production of representative verified virus seeds. In contrast to outdated nervous tissue-based rabies vaccines, once a suitable substrate is identified, production of high titer virus results in a major qualitative and quantitative difference. Given the current scenario of only inactivated vaccines for humans, highly cell-adapted and stable, attenuated rabies viruses are ideal candidates for consideration to meet the need for seed viruses in the future.