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In the present study, optimization of parameters for the preparation of nano formulated whole attenuated rabies vaccine with high immune potency and biocompatibility was synthesised. Response Surface Methodology (RSM) was adapted to study the influence of factors such as the concentration of chitosan (mg/mL), PEG (mg), acetic acid (%), sodium tripolyphosphate (M) and antigen (I.U.) on the structural, functional properties of chitosan PEG nanoparticles loaded rabies whole attenuated viral antigen (CS-PEG-NP-RVAg). Mathematical models were used to study the release kinetics of the nanoformulation adopting various plots such as Zero-order kinetics, First-order kinetics, Huguchi, and Huguchi-Crowell models. The evaluation provides insight into the controlled release of the rabies vaccine. The use of acetic acid (1%), sodium tripolyphosphate (0.1 M), antigen 1.5 (I.U.), PEG (60 µL), chitosan (75 mg) yields the desirable size of nanoformulations, immune potency, notable control release pattern Biocompatibility of the prepared nanovaccine was tested on Vero cell line, peripheral blood cells and probiotic bacterial strains. Nanoformulation, which obtained at the optimum condition was not inducing any toxic sign-on tested model system. Our study provides the basis of developing as effective CS-PEG-OV-RVAg as an effective and biocompatible vaccine formulation.

Background Live attenuated vaccines (LAVs) can mimic natural infection and have advantages to stimulate a robust and sustained immune response as well as to confer long-term protection. However, safety concerns is one of the major obstacles for LAVs development. In an effort to achieve the optimal balance between immunogenicity and safety, researchers currently have taken different strategies for the development of LAVs. Methods We constructed a novel infectious self- p ropagating hybrid r eplicon p article (PRP), VEEV-RABV-G, through replacing the entire structural proteins of the Venezuelan equine encephalitis virus (VEEV) with the glycoprotein of rabies virus (RABV-G) as the single structural protein. We evaluated the potential of VEEV-RABV-G as a safe live attenuated vaccine in mice model. Findings We found that VEEV-RABV-G could self-propagate efficiently in cell culture and induce a robust humoral immunity and provide protection against virulent RABV challenge in immunized mice. Remarkably, VEEV-RABV-G is highly attenuated in both adult and sucking mice, causing much weaker inflammatory and apoptotic effects in the brains of infected adult mice and significantly lower weight loss and morbidity compared with the commonly used RABV-derived LAVs. Interpretation This study reveals the feasibility of developing novel rabies vaccines based on the self-replicating PRPs. Funding This work was supported by the National Key Research and Development Program of China (2016YFD0500400).

In this study the ability of spherical particles (SPs) obtained from the tobacco mosaic virus (TMV) virions to enhance the immunogenic potential of the vaccine was evaluated. TMV SPs were shown to increase the protective properties of the widely used effective Russian adjuvant-free rabies vaccine, composed of killed rabies virions. The results of the NIH potency test showed enhancement of protectivity, that is comparable with the effect of the incomplete Freund׳s adjuvant on the same vaccine. Keywords: Rabies vaccine, Plant virus, Spherical particles, Adjuvant

A simple method was established for the determination of β-propiolactone (BPL) in human inactivated rabies vaccine by gas chromatography-mass spectrometry (GC-MS). The determination was performed on an Agilent HP-INNOWAX (30 m × 0.32 mm i.d., 0.25 µm) capillary column at the temperature of 80 °C. Electrospray ionization (ESI) was used by selective ion detection at m/z 42. The temperature for ESI source and inlet was set at 230 °C and 200 °C, respectively. Helium was used as the carrier gas at a flow rate of 25.1 mL/min. The total run time was 8 min. Acetonitrile and other components in the sample did not interfere with the determination of BPL. The results showed good linearity of BPL in the range of 0.50–10.01 μg/mL, with the limit of detection and the limit of quantification of 0.015 μg/mL and 0.050 μg/mL, respectively. Satisfactory precision was achieved for the current developed method. The method was applied to detect 6 batches of vaccine samples, and the results indicated that the target analyte BPL was present in three batches of unpurified samples, but was not detected in the purified samples, indicating the test samples were qualified. The established method was proved to be simple, versatile and sensitive, which can meet the requirements of quality control of BPL in human inactivated rabies vaccine. Keywords: β-propiolactone, Inactivated human rabies vaccine, GC-MS, Hydrolysis

This chapter discusses serum-free rabies vaccine production. Animal serum is used in cell culture because of its nutritional factors. However, its use is not only potential risky to human health, owing to the possible presence of adventitious agents; it is also the major factor responsible for allergic reactions in dogs during rabies vaccination. Here we present a mini review of other methods for its production and technological development to suppress serum in rabies vaccine for use in veterinary medicine.

Rabies is a viral zoonosis with 61,000 estimated deaths per year. The rabies vaccine is the best treatment before and after exposure and the virus can be produced in animal cells for veterinary use. Lyophilization has permitted the development of vaccines that would otherwise be too unstable for effective use, including a rabies vaccine that has improved stability when freeze-dried. This chapter is divided into two parts: a review of the rabies virus, viral vaccines, and lyophilization; and a case of study of infected rabies cells kinetics and vaccine freeze-drying with different cryoprotectors. The experiment tested three viral inocula in baby hamster kidney-21 cells growing in 1.5-L spinner flasks, comparing kinetics and virus titration to determine the best inoculum. Harvests of the best inoculum were inactivated and 12 formulations of excipients were made and freeze-dried, comparing visual aspects and residual moisture to select the best formulations.

Purified Chick Embryo Cell Vaccine (PCECV) production involves rabies virus (RABV), Flury low egg passage (LEP) strain, cultivation in propagated primary specific pathogen-free (SPF) chick embryo cells, inactivation with beta-propiolactone, and concentration and purification by zonal sucrose-density ultracentrifugation. The final vaccine is formulated with a gelatin stabilizer and lyophilized. This chapter provides details of vaccine production and release tests, including in-process controls, potency testing, and stability testing.

Vero-cell rabies vaccine produced using serum-free medium (SFM) and animal origin-free trypsin-like enzyme reduces the potential risk of inducing hypersensitivity and transmitting contaminants (bacteria, fungi, mycoplasma, bovine and porcine viruses, and others). In this production, Vero cells grown on microcarriers using the SFM in a bioreactor are infected with Pasteur virus (PV) rabies virus (RABV) strain. Virus-infected supernatants of this culture are harvested, filtered, concentrated, purified, and inactivated by β-propiolactone. The inactivated concentrates are mixed and diluted in phosphate buffered saline (PBS) to 5.0 IU/dose and used for filling of the bulk vaccine. The final product may be freeze-dried or stored in liquid form, and in both cases, its potency should be at least 2.5 IU per single human dose. The safety and immunogenicity of this type of rabies vaccine have been demonstrated in clinical trials using this product.

There is a worldwide shortage of human and equine rabies immune globulin (RIG). The equine (ERIG) is less expensive than human (HRIG), and is used for rabies post-exposure prophylaxis (PEP) in developing countries. Rabies is a fatal viral disease and still a major cause of human death in developing countries. The WHO recommends combined administration of rabies vaccine and RIG to patients after exposure.1 One example is produced by the Queen Saovabha Memorial Institute (QSMI), and the Thai Red Cross Society (TRCS) manufactures ERIG (“TRCS ERIG”), which is a F(ab′)2 with high specific activity, purity, and safety. Healthy horses, sero-negative for equine infectious anemia and equine influenza, are immunized with a tissue culture rabies vaccine. Crude plasma is collected by plasmapheresis and immune globulin is converted into F(ab′)2 fragments by pepsin digestion. The F(ab′)2 fragments are purified using caprylic acid precipitation followed by ultrafiltration. At least three consecutive batches of ERIG are validated and tested according to product specification, conforming to the requirements of the WHO and national Pharmacopoeias before each lot release.

Rabies causes one of the most lethal zoonotic diseases, with more than 55,000 deaths reported annually. Prevention is based on pre‑exposure vaccination of individuals at high risk of contracting rabies, and mass vaccination of dog, which are the main vector for transmission to humans. Post-exposure prophylaxis includes vaccination and rabies immunoglobulins treatment. The measurement of neutralizing antibodies in sera of vaccinated individuals is a primary concern to determine the efficacy of immunization schedules or the potency of new vaccines. Antibodies against rabies glycoprotein are considered an ideal indicator. In this work we showed the development of a VERO clone that is able to detect by fluorescence microscopy and flow cytometry, the presence of antibodies against the rabies glycoprotein, specifically in its native conformation anchored in the plasma membrane. These cells could trigger the development of a new rapid method for the detection of rabies virus neutralizing antibodies in vaccinated individuals. Fil: Fontana, Diego Sebastian. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina Fil: Prieto, Claudio. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina Fil: Kratje, Ricardo Bertoldo. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina Fil: Etcheverrigaray, Marina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina

Rabies is an infectious viral disease with a mortality rate close to 100%. Currently, there is a need to generate cheaper and more immunogenic vaccines for the effective prevention of rabies, mostly in developing countries. Virus-like particles have been widely used in viral vaccine production due to their high immunogenicity and safety during the production process. Rabies virus glycoprotein is the major antigen to trigger a protective immune response and the only protein capable of generating virus neutralizing antibodies. In this study we describe the development of a recombinant stable cell line for the production of rabies virus-like particles (RV-VLPs) expressing the rabies virus glycoprotein by lentivirus-based transduction of HEK293 cells. Protein expression was analyzed by flow cytometry, fluorescence microscopy, western blot and ELISA. Particles were purified from culture supernatant and their size and morphology were studied. Furthermore, mice were immunized with RV-VLPs, formulated with adjuvant, and these particles were able to produce a specific antibody response, demonstrating that these virus-like particles present a promising rabies vaccine candidate. Fil: Fontana, Diego Sebastian. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina Fil: Kratje, Ricardo Bertoldo. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina Fil: Etcheverrigaray, Marina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina Fil: Prieto, Claudio. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina

The level of virus-neutralizing antibodies (VNA) against rabies virus (RABV) glycoprotein (G) plays a crucial role in protection against rabies. Functional assays, such as the rapid fluorescent focus inhibition test (RFFIT) and the fluorescent antibody virus neutralization test (FAVN), are suitable for measuring the VNA levels in human and animal sera in the laboratory. However, these assays are time-consuming, expensive, and require live RABV, which means that they can only be performed in restricted reference laboratories. Based on the principle of immunochromatography and the VNA-binding activity of inactivated virus, we developed an immunochromatographic strip test called the “ Rapi d N eutralizing A ntibody” (RAPINA) test for the qualitative or semi-quantitative determination of antibody levels. The theoretical basis of the RAPINA test is that the inactivated CVS-11 RABV binds with antibodies in the serum and is no longer detected by the immunochromatography strip, which detects G, so a band is not produced on the test strip.

Rabies is an acute encephalomyelitis of humans and animals caused by rabies virus, which is usually transmitted by an animal bite. Rabies has a very rich history. Worldwide, dogs are the most important vector, whereas wild animals, especially bats, are the main threat to humans in North America. After a delay at the site of entry, rabies virus spreads through the nervous system by fast axonal transport. Rabies can be effectively prevented after a recognized animal exposure with wound cleansing and administration of rabies vaccine and rabies immune globulin. Rabies typically develops after an incubation period of 20–90 days. Rabies has encephalitic and paralytic forms of disease and rabies is almost universally fatal after clinical onset. Hydrophobia is a characteristic clinical feature of encephalitic rabies. Pathologic changes include the presence of eosinophilic inclusions called Negri bodies in the cytoplasm of neurons and inflammatory changes. Imaging studies may be normal and do not show specific abnormalities. An antemortem laboratory diagnosis can be made with the detection of rabies virus antigen or RNA in tissues (e.g., skin) and/or body fluids (e.g., saliva) and with serologic testing. There is no known effective therapy for rabies, although there have been rare survivors.

Publisher Summary This chapter discusses the possible future developments of rabies and the management approaches related to it. There are various challenges to control and eliminate rabies in animals and to prevent the human disease. A strong commitment from the governments of many countries is essential for this to be accomplished. Cooperation and adequate support of both the human health and veterinary sectors are necessary. An improved understanding of the pathogenesis of rabies can lead to novel approaches to the therapy of the human disease. New antiviral agents and/or immunotherapies may be developed that have efficacy in the established rabies. The rabies diagnosis is difficult when there is no history of an animal exposure. Astute clinical practitioners are essential, even if there are improved diagnostic investigations like—imaging and sensitive assays on body fluids, including saliva and cerebrospinal fluid that allow confirmation of a diagnosis of rabies much earlier than is presently possible. An improved understanding of the role and importance of rabies receptors may be useful in the design of new therapies for rabies.

The control of rabies in wild carnivores has evolved from culling operations targeting vector species, to parenteral and oral vaccination of free-ranging vector populations. In order for a rabies control program to be successful, many variables must be evaluated including the timing of control tactic implementation, the ecology of the rabies vector species, and the density of vaccine-baits required to immunize the target population at a level that will result in the control or eradication of rabies from the target zone. Key to the effective control of rabies in wildlife populations, and prevention of re-initiation of outbreaks following control, will be restrictions on the importation of wildlife species that are at risk of importing disease pathogens such as rabies. It has been proven, both in Europe and in North America, that large-scale oral rabies vaccination programs targeting wild carnivore populations can be cost-effective as well as effective at controlling and eliminating rabies. Key to continued success of wildlife rabies control operations will be the design of effective contingency plans to respond to re-emergence of the disease as well as rabies management plans formulated by multi-jurisdictional collaborations at the international level.

Rabies vaccine is the most valuable tool to prevent rabies in humans and animals. Vaccines provide the best service to public health when used in concert with effective risk management assessment systems that include enhanced surveillance, reliable de-centralized diagnostic laboratories and continued education for health professionals. All modern human rabies vaccines are inactivated, but both inactivated and modified-live or recombinant rabies cell culture rabies vaccines are produced for use in animals.

Although preventative therapy for rabies is highly effective after recognized exposures, there is no established therapy that is effective for patients who develop rabies encephalomyelitis, which remains an important challenge for the future. There have been several survivors from rabies and all except one received rabies vaccine prior to the onset of their clinical illness. Aggressive approaches to therapy of human rabies may be appropriate in certain situations. There is no scientific rationale for the use of therapeutic coma and there are numerous reports of failures using this approach. Therapeutic coma should be abandoned for the therapy of rabies. New approaches such as therapeutic hypothermia should be evaluated in combination with other therapeutic agents. More basic research is needed in order to improve our understanding of the mechanisms involved in rabies pathogenesis, which will hopefully facilitate the development of novel therapeutic approaches in the future.

Rabies has a rich history that goes back to antiquity. The writings of Fracastoro (1546) and Morgagni (1769) showed great early insights into rabies pathogenesis. Zinke (1804) performed the first experimental studies on rabies transmission. Galtier used rabbits in his rabies experiments, which was soon also taken up by Pasteur. Pasteur developed a rabies vaccine and used a series of inoculations of infected spinal cords from rabbits with partial inactivation after periods of desiccation, and he successfully immunized the first patients in 1885. Negri (1903) reported eosinophilic cytoplasmic inclusion bodies in neurons from rabid brains tissues. The earliest electron microscopic studies in rabid animal brains were reported in 1951. In 1958, Goldwasser and Kissling first reported fluorescent antibody staining in rabies, which proved to be an important diagnostic test for rabies and to also be very useful in early pathogenesis studies by Richard Johnson and Frederick Murphy. The first clone of a rabies virus gene was reported in 1983, which lead to the molecular era in rabies diagnosis and also for epidemiological and pathogenesis studies.

Rabies virus is an important causative agent of disease resulting in an acute infection of the nervous system and death. Although curable if treated in a timely manner, rabies remains a serious public health issue in developing countries, and the indigenous threat of rabies continues in developed countries because of wildlife reservoirs. Control of rabies in wildlife is still an important challenge for governmental authorities. There are a number of rabies vaccines commercially available for control of wildlife rabies infection. However, the vaccines currently distributed to wildlife do not effectively immunize all at-risk species, particularly skunks. A replication competent recombinant adenovirus expressing rabies glycoprotein (AdRG1.3) has shown the most promising results in laboratory trials. The adenovirus vectored vaccine is manufactured using HEK 293 cells. This study describes the successful scale-up of AdRG1.3 adenovirus production from 1 to 500 L and the manufacturing of large quantities of bulk material required for field trials to demonstrate efficacy of this new candidate vaccine. The production process was streamlined by eliminating a medium replacement step prior to infection and the culture titer was increased by over 2 fold through optimization of cell culture medium. These improvements produced a more robust and cost-effective process that facilitates industrialization and commercialization. Over 17,000 L of AdRG1.3 adenovirus cultures were manufactured to support extensive field trials. AdRG1.3 adenovirus is formulated and packaged into baits by Artemis Technologies Inc. using proprietary technology. Field trials of AdRG1.3 rabies vaccine baits have been conducted in several Canadian provinces including Ontario, Quebec and New Brunswick. The results from field trials over the period 2006-2009 demonstrated superiority of the new vaccine over other licensed vaccines in immunizing wild animals that were previously difficult to vaccinate.

In recent years, no major paradigm shifts have occurred in the utilization of new products for the prevention and control of rabies. Development of new cost-effective rabies biologics and antiviral drugs is critical in continuing to prevent and reduce disease. Current rabies vaccines are highly effective but have developed largely based on technical improvements in the vaccine industry. In the future, alternative approaches for improved vaccines, including novel avirulent rabies virus (RABV) vectors, should be pursued. Any rabies vaccine that is effective without the need for rabies immune globulin (RIG) will contribute fundamentally to disease prevention by reducing the cost and complexity of postexposure prophylaxis (PEP). The lack of high quality, affordable RIG is a continuing problem. Virus-specific monoclonal antibodies (mAbs) will soon fulfill the PEP requirement for passive immunity, currently met with RIG. Several relevant strategies for mAb production, including use of transgenic mice, humanization of mouse mAbs, and generation of human immune libraries, are underway. As a result of successful PEP and pre-exposure prophylaxis in developed countries, until recently, no significant focused efforts have been devoted to RABV-specific antiviral agents. To date, combination therapy including broad spectrum antiviral agents has been successful in only one case, and reports of antiviral activity are often conflicting. Current antiviral strategies target either the nucleoprotein or phosphoprotein, but drugs targeting the viral polymerase should be considered. Considering the lag from creation of new concepts to experimental development and clinical trials, many years will likely elapse between today's ideas and tomorrow's practices.

Human rabies is essentially a fatal disease once clinical signs develop. Rabies postexposure prophylaxis (PEP) consists of thorough wound care in combination with administration of rabies immunoglobulin and rabies vaccine. This is highly effective in rabies prevention if carried out diligently. Preexposure rabies prophylaxis simplifies PEP in the event of an exposure by eliminating the need for immunoglobulin. Shortened and more convenient and economical PEP regimens are being developed with promising results. They reduce the cost of PEP as well as travel expenses for the often very poor patients. The intradermal PEP regimen can now reduce the vaccine cost by ~60-70%. Although PEP in humans can prevent death, controlling the canine vector by sustained vaccination remains the mainstay of rabies elimination.

We note the comments of Dr. Aaby [1] on 4 March 2009 regarding the use of control vaccinations in our recent studies of RTS,S/AS01E in 5–17-month-old children [2] and RTS,S/AS02D combined with the EPI schedule in young infants [3]. Dr. Aaby and co-workers are concerned that the apparent reduction in severe adverse events (SAEs) in the RTS,S groups may represent an adverse consequence of the control vaccines (rabies and hepatitis B vaccines). To evaluate this hypothesis Dr. Aaaby and co-workers request: “we hope the authors will report the malaria and non-malaria serious adverse events and vaccine efficacy in the two trials separately for each sex” [1]. Many parents, the FDA, local regulatory authorities and IRBs, want children to benefit from a vaccine rather than a placebo where an appropriate choice for a control exists. Young children are at risk of rabies in East Africa, and the disease is fatal if acquired. Pre-exposure prophylaxis is not widespread in Kenya and Tanzania, but is of proven benefit [4]. The licensed human diploid-cell rabies vaccine (rabies vaccine BP, Sanofi-Pasteur) was therefore an appropriate control for RTS,S/AS01E vaccination of 5–17-month-old children. In EPI (extended programme of immunisation) schedules children are routinely vaccinated against hepatitis B, and it would not have been acceptable to withhold this vaccination in the control group [5]. RTS,S/AS02D includes the hepatitis B surface antigen. It raises an antibody response to this antigen in infants [6], and so replaces the hepatitis B vaccine that would otherwise have been part of the EPI schedule. The design of both trials was approved by IRB and regulatory authority review. Dr. Aaby and co-workers have described non-specific deleterious effects of inactivated vaccines in Sub-Saharan Africa [7]. In Dr. Aaby's work non-specific vaccine effects were particularly noted in girls rather than boys [8]. He therefore proposes we re-analyse our safety data separately by gender. In 5–17-month-old children the SAE rates by vaccination allocation appeared consistent by girls and boys (Table 1). In the EPI schedule trial, the difference in SAE rates by vaccination was more pronounced for boys than girls (Table 2). The difference in pneumonia SAEs was also more pronounced for boys. Table 1 Study of 5–17-month-old children [2]. Table 2 Study of young infants [3]. Efficacy against clinical malaria was 53% (95%CI 28–69) in all 5–17-month-old children [2], compared with 63% (95% CI 29–81) in boys and 41% (95% CI −3 to 67) in girls. Efficacy against first infection was 65% (95%CI, 21–85) in the EPI study [3], compared with 52% (95%CI −40 to 84) in boys and 75% (95%CI 6–93) in girls. Our phase II studies were not designed to examine inpatient morbidity in detail. Phase III studies to clarify these findings are underway. There seems little evidence of a non-specific effect by Dr. Aaby's proposed analysis, and control vaccinations will be used in accordance with regulatory authority and IRB advice.

Publisher Summary For the past two decades, investigators are searching for alternative rabies vaccines that have all the advantages of the killed or modified-live (attenuated) rabies virus vaccines for total stimulation of the immune response in animals and humans, without the undesirable side effects sometimes associated with conventional rabies virus vaccines. With the present high-quality cell-culture rabies vaccines for humans that are virtually 100% efficacious when they are used in accordance with the present WHO recommendations for pre- or post-exposure vaccination, one could say that there is little need to develop a more efficacious human rabies vaccine. The principal reason that alternative rabies vaccines are being researched for development is that inexpensive vaccines are desperately needed in developing countries to replace the present nerve tissue origin (NTO) rabies vaccines. Therefore, new rabies vaccines must be developed, which are inexpensive enough, possibly requiring one or two administrations, by oral or parenteral inoculation routes, to vaccinate millions of people at risk following exposure, especially in canine rabies endemic countries. Oral vaccines have certain attractiveness, particularly for humans in developing countries, as they can be administered more rapidly in mass vaccination programs, as was done in the worldwide campaign to eradicate poliovirus.

The control of rabies in wild carnivores has evolved from culling operations targeting vector species, to parenteral and oral vaccination of free-ranging vector populations. In order for a rabies control program to be successful, many variables must be evaluated including the timing of control tactic implementation, the ecology of the rabies vector species, and the density of vaccine-baits required to immunize the target population at a level that will result in the control or eradication of rabies from the target zone. Key to the effective control of rabies in wildlife populations, and prevention of re-initiation of outbreaks following control, will be restrictions on the importation of wildlife species that are at risk of importing disease pathogens such as rabies. It has been proven, both in Europe and in North America, that large-scale oral rabies vaccination programs targeting wild carnivore populations can be cost-effective as well as effective at controlling and eliminating rabies. Key to continued success of wildlife rabies control operations will be the design of effective contingency plans to respond to re-emergence of the disease as well as rabies management plans formulated by multi-jurisdictional collaborations at the international level.

Publisher Summary This chapter discusses the concept of controlling rabies in wildlife. It reviews the diagnosis of suspect wildlife, and the oral rabies vaccine development. The development of the rabies vaccines for oral administration to wildlife is complex. It involves the considerations such as—efficacy testing in the target vector species first by intramuscular, and then by oral routes. The release of any new biologically active agent like—a vaccine, an antibiotic, or a pesticide, has its own associated safety concerns. The release of a pesticide molecule, engineered in a laboratory, is under direct human control when released, and its further release can be limited even though it may persist in the biologic web— like DDT, whereas an organism capable of reproduction, such as a live-virus vaccine, has the potential for amplification and divergence in nature. The safety testing in animals, including humans, that may contact the vaccine bait in the field is therefore a prime consideration.

The impact of recombinant technology in veterinary and human medicine can only be hypothesized at this time. The development of vaccines and other biological products that go beyond the abilities of conventional products demonstrates the benefits of this new technology. Raboral V-RG was developed as an alternative rabies vaccine with the novel attribute of being effective by the oral route. Within 10 years after its first application as an experimental vaccine in European, red foxes it developed into a useful tool and is being used to curtail rabies epizootics in three wildlife species in the United States. The use of this vaccine can be considered as monumental in contributing to the control of rabies in species that were at one time considered to be incapable of vaccination in large-scale campaigns.

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

Publisher Summary The family Rhabdoviridae encompasses more than 100 viruses of vertebrates, invertebrates, and plants, the virions of all having a distinctive bullet-shaped morphology. Important animal pathogens occur in four subgroups of the family: the genus Lyssavirus, the genus Vesiculovirus, and two unnamed subgroups including bovine ephemeral fever virus and the fish rhabdoviruses. Rabies virus is the cause of one of the oldest and most feared diseases of humans and animals—recognized in Egypt before 2300 BC and in ancient Greece, where it was well described by Aristotle. Rabies has the dubious distinction of being the most lethal of all infectious diseases; it also has the distinction of having been the disease that stimulated one of the great early discoveries in biomedical research. In 1885, before the nature of viruses had begun to be comprehended, Louis Pasteur developed, tested, and applied a rabies vaccine and thereby opened the modern era of the prevention of viral diseases by vaccination. Rabies virus can infect all warm-blooded animals and in nearly all instances, the infection ends in death. Dog rabies is still important in many parts of the world; infected dogs cause most of the estimated 75,000 human rabies cases that occur each year worldwide.