In 1885, Louis Pasteur demonstrated that the rabies virus could be attenuated by serial passage in rabbits intracranially. On 6^th July 1885, the first human was treated by giving 13 consecutive and an increasingly virulent dose of a dessicated spinal cord suspension from a rabid animal and the boy Joseph Meister was survived. Pasteur's crude vaccine was later modified by Fermi and Semple. In 1927, the First International Rabies Conference recommended that fixed virus for canine rabies vaccines be completely inactivated or attenuated so that they cause no disease in dogs vaccinated either S/C or I/M (19). For the next several decades, all nervous tissue rabies vaccines were inactivated by phenol described by Semple (42). The nervous tissue vaccines currently in use for mass vaccination campaign in Africa, Latin America and Caribbean are produced from rabies virus infected suckling mouse brains or lamb brains. However, nervous tissue vaccines for dogs and other animals often caused post vaccinal nervous symptoms and death in some vaccinated animals (8).

Embryonated chicken eggs were used by Koprowski and Cox for serial passage of the Flury strain. The virus was initially passaged 136 times in 1 day old chicks followed by passage in embryonated chicken egg. At 40^th to 50^th chicken embryo passage, the rabies virus losses its viscerotropic properties but retained some neurotropic properties and named LEP. It could be safely used in dogs but occasionally caused rabies in young pups, cats and cattle. The LEP was further passaged in embryonated chicken eggs to increase the safety by Koprowski and associates and at 205^th passage level it was found to be safe for I/M use in cats, cattle as well as puppies 3 months of age (43).

The Flury and Kelev strains of rabies virus were used to produce chick embryo origin Modified Live Virus (MLV) vaccines. Street Alabama Dufferin (SAD) strains adapted in hamster kidney cells and Evelyn Rokitnicki Abelseth (ERA) strains grown on porcine kidney cells were used to produce tissue culture based modified live vaccine for parenteral use in animals against rabies. These vaccines are used in carnivores including dogs and cats in Asia, Africa and parts of Europe (4, 44). These non adjuvanted vaccines are cheap and use low amount of virus, but elicited longer duration of immunity and good cell mediated immune response. However, there is chance of regaining the virulence property by the vaccine virus over a long time use. Further, the vaccine virus may have the residual pathogenicity to other non-target species of animals (10).

Modified Live Virus (MLV) vaccines are not recommended for parenteral immunization against rabies in animals as rabies infection can occur as a result of vaccine strain. Several types of MLV oral rabies vaccines have been produced for use in baits for free ranging animals that serve as vectors for the maintenance and transmission of the disease in wildlife (45). SAD B19 and SAD P5/88 vaccines are produced by several cell culture passage of the SAD Berne strain, which is a cell culture adapted derivative of ERA strain, was used extensively in Europe since 1977 and in Canada from 1989 with considerable success (46). Unfortunately the live virus SAD vaccines contained some degree of residual pathogenicity for wild rodents and resulted in partially impaired immune responses in fox cub <8 weeks old born from SAD strain vaccinated vixens and resulting in insufficient protection against rabies (47). The SAD strain used in vaccine has been replaced by SAG-1 and SAG2 (SAD avirulent Gif) strains in the development of vaccines. The SAG (Street Alabama Gif) 2 strain was selected from the SAD Berne strain. The SAG-2, the strain of choice is a double mutants and avirulent following I/C inoculation of immunocompetent mice and protects the mice against challenged with CVS (48). No adverse effects following the oral administration of 10 times the field dose of SAG2 were reported in target species (red fox, dog, raccoon dog and arctic fox) or in non-target species including baboons, different rodent species, two species of cervids, wild boars, badgers, goats, ferrets, hedgehogs and diurnal and nocturnal birds (49). Red foxes, raccoon dogs and dogs were protected from virulent challenge after immunization with single SAG2 bait. No salivary excretion of infective SAG2 virus strain was detected in dogs after vaccination. In bait, SAG2 is either contained in a capsule as a viral suspension or in the bait matrix as freeze dried suspension. The SAG2 strain of rabies virus packaged in chicken head baits has also successfully protected captive African wild dogs against rabies virus challenge (49, 50).

A recombinant vaccinia virus vector in which the G gene of the Evelyn-Rokitniki-Abelseth (ERA) strain of rabies virus was inserted into the thymidine kinase region of the vaccinia virus genome has been developed for immunization against rabies (51). The vaccinia-rabies glycoprotein (V-RG) recombinant virus induced a rapid Virus-Neutralizing Antibody (VNA) response in mice, both by subcutaneous inoculation and by oral administration and the animals were protected against a lethal rabies virus challenge (52). Similarly, other pox viruses including the orthopoxvirus of raccoon and avian pox viruses such the fowl pox and canary pox viruses were investigated as possible alternative vectors for expression of rabies virus antigens (53). Some of these investigations reported the role of rabies virus nucleoprotein (N) for protective cellular immune responses in addition to G protein of rabies virus for the induction of VNA. A recombinant vaccinia virus expressing the rabies virus glycoprotein gene (V-RG) has been developed by inserting the cDNA of the glycoprotein gene of the ERA strain into the thymidine kinase gene of the Copenhagen strain of vaccinia virus (54). When administered orally by direct instillation into the oral cavity or in a bait, a dose of 10⁸ TCID₅₀ of VRG elicits virus neutralizing antibodies and confers protective immune response against rabies virus challenge in a number of carnivorous species (red fox, arctic fox, coyote, raccoon, raccoon dog, domestic dog and golden jackals) (55). In the field, VRG vaccine strain is stable above 56 °C whereas the melting point of bait case is >60 °C. Safety studies conducted in over 50 mammalian and 10 avian species (most of them are rabies vectors) have not revealed any residual pathogenicity. More than 75 million doses of VRG have been used to successfully control or reduce wildlife or canine rabies in a variety of animal species such as red foxes (Belgium, France, Israel, Luxemburg and Ukraine), raccoon dogs (Republic of Korea), coyotes, raccoons and grey foxes (Canada and USA) and domestic dogs (Sri Lanka) (43, 49, 54, 56, 57). The vacciniarabies glycoprotein (V-RG) recombinant virus induced a rapid Virus-Neutralizing Antibody (VNA) response in mice, both by subcutaneous inoculation and by oral administration and the animals were protected against a lethal rabies virus challenge (52). Similarly, other poxviruses, including the orthopoxvirus of raccoon and avian pox viruses, such as the fowl pox and canary pox viruses, were investigated as possible alternative vectors for expression of rabies virus antigens (53). The vaccine fulfilled all the criteria such as readily accepted by target species, rabies virus free, thermostable and provides protection against rabies but no risks of developing rabies if ingested and safe for contact with humans. The vaccine was used in USA and France to immunize raccoons, coyotes, wild dogs, fox, jackals etc with high success and by 1995 the vaccine was conditionally licensed in the US for use in Oral Rabies Vaccination (ORV) programs (43, 49, 58). The vaccine can be given in fish meal or poultry based bait which contains 150 mg of Tetracycline HCl as a bone biomarker and a plastic sachet containing 1.8 ml of vaccine. The concept that a live recombinant virus vector might fulfill the rabies vaccine requirements for the next generation was rapidly gaining credibility (59, 60).

A canary pox rabies glycoprotein recombinant vaccine has been developed and found to be effective as other poxvirus rabies glycolprotein recombinants. Live canarypox virus that expresses the rabies virus glycoprotein has been licensed in the USA as a parenteral monovalent vaccine for cats and as a combination rabies vaccines for cats with feline panleukopenia virus, feline parvovirus, feline calicivirus vaccines included in the product (51). A recombinant adenovirus vectored vaccine expressing rabies virus glycoprotein was shown to be capable of inducing antibody response in greyhound dogs immunized S/C or I/M. This vaccine holds promise as a rabies virus vaccine for dogs (61, 62).

For preparing inactivated vaccines, rabies virus strain (CVS, PM or PV) is grown in chicken embryo fibroblast, BHK-21 or Vero cells (63, 64). Neonatal mice lack the myelin protein that causes allergic reactions occasionally in animals vaccinated earlier with sheep brain tissue origin killed vaccines. On the other hand, tissue culture based rabies vaccines are less allergenic but more immunogenic (57). Although a number of inactivating agents namely phenol, HCHO, UV light, AEI or other amines have been used for inactivation of rabies virus, BPL is most commonly used. After inactivation, adjuvants namely Al(OH)₃, AlPO₄, saponin (cattle vaccines) and rarely oil adjuvant are used to improve the immunogenicity of the vaccine (42). The rabies vaccine also is available along with canine distemper, canine adenovirus, feline parvovirus and feline calicivirus for use in cats or FMD for use in cattle, sheep and goat (10).

Due to higher antigenic mass and use of adjuvants inactivated vaccines may sometimes produce local and systemic reactions. The most common symptoms are soreness, lameness, regional lymphadenopathy in the injected limb, focal vasculitis, granulomas and sarcoma. However, the new generation vectored recombinant vaccines produce less allergic or neoplastic reactions (21, 65).

Inactivated rabies vaccines for animals should have a potency of 1.0 IU/dose as measured by National Institute of Health (NIH) test or other recognized Pharmacopoeia test (66). The NIH rabies vaccine potency test is used internationally for evaluating the efficacy of inactivated rabies vaccine which is performed in vivo. In NIH test, the potency of anti-rabies vaccine is compared with an international reference vaccine while testing in groups of at least ten Swiss albino mice, aged 3–4 weeks inoculated with two doses at one week apart. Different 5 fold dilutions of both the vaccines are inoculated and compared to determine the dilution at which 50% mice are protected against intracerebral challenge 14 days later. The challenge dose should be in the range of 12–50 mouse intra-cranial LD₅₀ (MIC LD₅₀) in 0.03 ml of the suspension. The PD₅₀ and the IU is calculated in the test vaccine by observing and comparing the protection level of the reference vaccine. The duration of immunity should be at least 1 year (66).

The production of rabies vaccine has dramatically changed since Pasteur, who produced and treated the patient with serial injections of increasingly virulent rabies virus infected nerve tissue by drying different time interval. In 1900s Fermi and Semple used phenol to inactivate the rabies virus in nerve tissue and used same suspension for all injections. However, the use of the Fermi vaccines has been discontinued due to presence of residual live fixed rabies virus. The nervous tissue vaccine (Semple) that are presently produced and administered in Africa, Asia and Latin America include vaccines produced from the rabies virus infected brain tissue of sheep, goat and mice (67). Although the production of Semple vaccine was recently stopped in India it is still produced and used in some Asian and African countries even though safer and more efficacious cell culture vaccines are available (7, 9).

Due to post vaccinal reactions associated with Semple rabies vaccine, the need for a safer and more efficacious rabies vaccine was felt to protect humans against rabies. The next development of rabies vaccine was the vaccine derived from suckling mice having less reactogenic property used extensively during 1950s in Latin American countries (19, 68). Suckling mouse brain vaccine is less allergenic because brain tissue is unmyelinated and produced from fixed rabies virus strains originally isolated in Chile (strains 51 and 91 of dog and human origin, respectively) (69). Mice not older than 1 day are injected intracerebrally and brain tissue is harvested approximately 4 days later. The brain tissue infected with rabies virus is diluted to 10% suspension and inactivated with UV light or BPL. The vaccine has a final potency of 1.3 IU/dose. The vaccine is supplied in 1–2 ml vials with a shelf life of 1 year stored at 4 °C (70). The adverse reactions associated with suckling mouse brain vaccines are lower (1:8000) than Semple rabies vaccines (1:142 to 1:7000), but the case fatality rate is higher in affected patients (22% for suckling mouse brain vaccine and 4.8% for Semple vaccines) (21). WHO has recommended that the production of all nervous tissue vaccine should be discontinued and replaced by cell culture rabies vaccine.

The first non-nervous tissue culture rabies vaccine became available in North America was Duck Embryo Vaccine (DEV).

The vaccine was replaced by the first modern cell culture rabies vaccine, the Human Diploid Cell strain Vaccine (HDCV) in 1978 in USA. The HDCV was safe with high immunogenicity and negligible allergic reactions compared to previous vaccines (71). Further, two other cell culture vaccines namely Purified Vero cell culture Rabies Vaccine (PVRV) and Purified Chicken Embryo Cell Vaccine (PCECV) were developed almost simultaneously and became available around the world by international pharmaceutical companies (18).

Presently PVRV and PCECV are the most widely used cell culture vaccines in millions of patients in the world. A more purified version of Vero cell vaccine known as Chromatographically purified Vero cell rabies vaccine was undergone clinical trial in USA and few Asian countries but not marketed in any country (72).

There are many different types of cell substrates viz., primary cell, diploid cell and continuous cell cultures used for the production of cell culture rabies vaccine. In China and former USSR, primary baby hamster kidney cells are used to produce a rabies vaccine (41). Embryonated eggs are a primary cell culture substrate used to produce 3 different types of rabies vaccine in Europe, Japan and India. Rabies vaccines can also be produced in human diploid cell strain (WI-38 and MRC-5). The Vero cell line derived rabies vaccines are available in Asia, Europe and Latin America (73).

The first widely used cell culture rabies vaccine was developed on the human fetal lung diploid fibroblast cell line WI-38 using Pitman Moore strain of rabies virus by Wiktor and his associates at Wistar Institute of Philadelphia. Human diploid cells have a higher but finite life span than primary cells whereas continuous cell lines have a capacity to multiply indefinitely in vitro (71). The cells used for vaccine production from continuous cell lines may have differences in karyotype from the original cell line and may be obtained from healthy or tumor tissues. The FDA (Food and Drug Administration) Center for Biologics Evaluation and Research (CBER) in USA has guidelines for manufacturers using the continuous cell line mentioning that Vero should have less than or equal to 10 ng of cellular DNA per dose for a parenterally administered vaccine (63, 73).

HDCV was the first cell culture rabies vaccine to be developed and this vaccine dramatically changed the perception of human rabies prevention and the concept of rabies vaccination. This vaccine provided a safe and immunogenic means to protect against rabies in both pre and post exposure cases. It is recommended to give booster every 2 years for people with continued risk of exposure to rabies. Although HDCS produces high serologic titers, but the yields of virus is low. Again, due to high production cost, this vaccine is unaffordable by people in most developing countries of the world where over 90% human deaths occur (71, 72).

To overcome the high cost of HDCV and to make rabies vaccine more affordable to common people, other cell culture rabies vaccines were developed that are less expensive to produce and cause fewer adverse reactions. These vaccines are PCECV developed by Barth and his associates using Flury LEP strain of rabies virus propagated in primary chick fibroblast cells. The virus is inactivated by BPL and purified by zonal centrifugation (74). The production of PCECV on chick embryo fibroblasts permits a high yield of virus compared to human diploid cells.

This vaccine technology was transferred from Marburg, Germany to Ankleshwar, India in the late 1980s and cost of production was reduced substantially while maintaining the production standard recommended by WHO. Another transfer was recently occurred from Switzerland to India is purified duck embryo cell vaccine (11). PVRV is a second generation rabies vaccine that is produced on Vero cells (Vervet monkey origin). PVRV is cultivated on microcarriers in large scale biofermentors thus reducing the production cost compared to HDCV which is produced on a monolayer cell culture. PVRV inactivated by BPL and concentrated by ultracentrifugation is widely used throughout the world including Europe and Latin America but it is not licensed to be used in North America (18, 75).

Cell culture and embryonated egg based rabies vaccines are of superior quality compared to nervous tissue vaccines of adult and young animal origin in terms of both safety and efficacy and saved the lives of millions of human victims of animal bites (WHO, 2001). WHO recommends that all cell culture rabies vaccines must contain at least 2.5 IU/dose intramuscularly (I/M) for administration to humans. The vaccines are inactivated with BPL and undergone the safety, sterility, potency and stability testing. The vaccines are highly effective when used both in pre and post exposure cases against rabies (72).

Currently there is no cell culture rabies vaccine that is prepackaged for use intradermally either for pre or post exposure vaccination. The currently available cell culture rabies vaccines intended for I/M use do not have a preservative and the risk of contamination is increased if they are used as a ‘multiuse’ vial as the case for intradermally administration. WHO recommends that when rabies vaccines are administered intradermally (I/D) they should be kept at 2–8 °C and used within 6–8 hrs (17). In North America, intradermal administration of human rabies vaccine is sometimes used as pre-exposure immunization but never practiced for post exposure vaccination. However, I/D administration of rabies vaccines have been extensively used during the last two decades in Asia for post exposure and in Europe for pre-exposure vaccination. At present, WHO only recommends three vaccines (HDCS, PCECV and PVRV) for I/D post exposure prophylaxis (18, 63). Although WHO recommends a potency of 2.5 IU/human dose for the intramuscular administration of rabies vaccines, there is no similar specific potency requirement for an I/D dose of a cell culture rabies vaccines. The same vaccine intended for I/M can be safely given at the rate of 0.1 ml I/D from a 1 ml vial of PCECV that contained 2.5 IU/ml. All the vaccines responded well with acceptable titer by day 14. There is no need to increase the potency requirements for I/D use of cell culture rabies vaccine (64, 76, 77).

The standard (Essen) five doses I/M regimen for PET are unaffordable in developing countries. So, two economical ID PET regimens have been recommended for use by the WHO since 1997: an eight site and a two site regimen. The eight site regimen is used with PCECV and HDCV (1.0 ml/ampoule) and consists of eight doses of about 0.1 ml I/D on day 0 (using a whole ampoule) on the arms, thighs, suprascapular and lower abdomen areas; four I/D doses on day 7 on the arms and thighs and one dose on days 28 and 91 also over the deltoid. It requires four visits to the clinics (Figure 1) (64, 77).

The two site regimen consists of two I/D doses of 0.2 ml each on day 0, 3, 7 and one 0.2 ml I/D dose on day 28 and 91 for PCEV and HDCV whereas in case of PVRV the dose is 0.1 ml in each occasions (Figure 2) (76). It requires 5 visits to the clinics. Ampoules of vaccines are shared on each occasion whereas sharing is needed on three of the four visits with the eight site regimen. A comparison of these I/D regimens shows that the eight site method induce significantly higher levels of antibody than the two site regimen from day 7 to 1 year. The same amount of antigen is used for the two regimens, a total of less than two ampoules (64).

Subunit vaccines consisting of the immunogenic component of a specific virus or merely its immuno-reactive portion were considered, for practical purposes, to be a rather futuristic perception of the ultimate in safe (i.e. genome-free) antiviral vaccines. It has been firmly established that rabies virus G protein forms spike projections on the external surface of the virus membrane and is the major antigen responsible for the induction of VN antibodies and for conferring immunity against lethal infection with rabies virus (78). The rosette structures of G protein (haemagglutinin preparation) which consisted solely of polypeptide chains of G protein was fully protective against a lethal challenge infection with rabies virus in mice. The pre-exposure protective and antigenic values of this haemagglutinin preparation were equal to that of inactivated virus vaccine and at least ten times greater than those for the monomeric form of G protein prepared using Triton X-100 (78).

The G gene coding for the glycoprotein G of rabies virus was inserted into the replication-non-essential E3 locus of the human adenovirus serotype 5 (AdHu5) genome and used as recombinant adenovirus vectored vaccine. The E3 gene locus was deleted to down regulate expression of major histocompatibility complex antigens and protect the adenovirus-infected cells from T-cell-mediated destruction (79). This prototype vaccine tested by parenteral or oral vaccineation in several animal species including mice, dogs, foxes and skunks provided protection against a rabies virus challenge. However, the potential for in vivo replication of this E3-deleted recombinant adenovirus and tendency to cause occasional disease in the vaccinated host posed the question about its safety. Recombinant adenovirus vectored rabies vaccine (recombinant Ad-RG) also capable of inducing high level of anti-rabies virus neutralizing antibodies (VNA) in dogs previously immunized with conventional rabies vaccines. The level of VNA induced following booster immunization with the recombinant Ad-RG was reported to be higher than that of conventional rabies vaccines suggesting that the recombinant Ad-RG vaccine is capable of inducing a strong anamnestic response in dogs (62). Further, activity of recombinant Ad-RG vaccine was not impaired by pre-existing immunity when administered intranasally or orally. With a growing interest at this time to use adenoviruses for gene therapy as well as for vaccination, replication-defective adenovirus vector constructs have been investigated as possible next generation vaccines for rabies (61, 79, 80).

Another area of innovative rabies vaccine development is that of DNA or gene vaccine. DNA-based vaccines offer new approaches and unique strategies for both prophylaxis and post exposure therapy against rabies. DNA-based vaccines were initially developed as a simple and versatile way to induce a broad spectrum of immune responses (both cellmediated and humoral) when injected directly into the host, compared with conventional vaccines. The most appealing and compelling reason to develop DNA-based rabies vaccines is that plasmids (DNA encoding G gene of rabies virus) are easy to construct and can be produced in large quantity inexpensively. DNA plasmids that carry more than one viral gene component, e.g. G and N genes of different rabies virus strains, or multiple plasmids encoding single expressible genes combined in a ‘plasmid vaccine cocktail’, provide a multivalent vaccine approach for simultaneous induction of immunity to more than ‘one viral pathogen’ (81, 82).

Multiple G genes of rabies and rabies related viruses in one vaccine cocktail would protect individuals not only against rabies but also rabies related viruses at the same time. Further, DNA vaccines not only induce VNA and CD₄ ⁺ T cells but also cytotoxic CD₈ ⁺ T cells. The induction of CD₈ ⁺ T cells are not generally induced by recombinant and synthetic peptides. The first DNA vaccine for rabies developed at Wistar Institute induced long lasting immunity following I/M injection but induction of VNA is usually slower than HDCV. Various parameters such as plasmid doses, route, host species, virus challenge and primary and booster mode of inoculation were studied both as pre-exposure and post exposure formats. However, the results have been encouraging in mouse and some large mammals but not successful in totally protecting in non-human primates following pre-exposure and post exposure vaccination. So, further work needs to be carried out to exploit full potential of DNA vaccines in order to provide fully protective and highly desirable vaccines for animals and humans at low cost (81, 83).

Plants have also provided new and promising prospects in the process of developing effective, inexpensive and safe production and delivery systems for the next generation of vaccines for rabies. Plant viruses, such as tomato bushy stunt virus and Tobacco Mosaic Virus(TMV) serving as vectors for expression of foreign antigens in plants, have provided a variety of genetically manufactured vaccines from tomatoes and tobacco leaves, respectively. Among the key advantages of plants and plant crops for protein expression, particularly the tobacco plant as a suitable host for plant virus vectored protein expression, is that they represent a biomass of major proportions for recombinant (foreign) protein production. Mice immunized orally or by feeding on antigen producing spinach leaves or parenterally with plant derived rabies virus specific antigen could be protected from a lethal rabies virus challenge. It offers the advantage of safety, low cost, proper glycosylation of antigen, easy oral administration and devoid of several painful injections. It also can generate local and systematic immune response and protection against rabies virus (84).

It is understood that following vaccination both humoral and cellular immune responses are induced in rabies. A high level of neutralizing antibodies in the sera of vaccinated animals and humans can protect them from lethal infections since antibodies take at least 7–10 days to become effective for virus neutralization. This time is covered by passive immunization with rabies immunoglobulin. The combined serum vaccine therapy is always recommended in exposed persons particularly in severe bite wounds. Purified equine rabies immunoglobulin which occasionally causes adverse reaction is used in most developing countries at 40 IU/kg body weight whereas human rabies immunoglobulin is given at 20 IU/kg body weight (85).

In this method plasmid expressing full length anti-genomic RNA (genome plasmid) and three plasmids expressing N, P and L protein of the virus (helper plasmids) are transfected into a cell. The anti-genomic RNA and the proteins form an anti-genomic ribonucleoprotein (RNP) complex. The anti-genomic RNP complex has the same biological activity as occurs in virus infected cells, genomic RNA is synthesized using this anti-genomic RNP as a template, followed by synthesis of mRNA from genomic RNP and expression of viral protein. The assembly of the genomic RNP and other viral proteins as M and G proteins results in generation of an infectious recombinant rabies virus. Using this manipulation system of rabies virus, an attenuated live virus vaccine can be established quickly than conventional cell culture vaccine with change in biological characters such as improved safety and immunogenicity (84).

A recombinant rabies virus expressing a proapoptotic protein cytochrome c (SPBN-Cyto c(+) strain) strongly induced apoptosis in infected cells, found to be more attenuated than negative control virus carrying inactivated cytochrome c gene (SPBN-Cyto c(-) strain) and induced protective immune response in mice (86).

In rabies virus, the presence of an arginine or lysine residue at position 333 in the G protein is well known for pathogenicity (87, 88). Many recombinant viruses harbouring the G gene from various rabies virus strains with amino acid other than arginine or lysine have been found to be attenuated (55). The deletion of dynein light chain binding site in P protein which is necessary for axonal transport of the virus reduces peripheral infectivity of the recombinant virus in suckling mice (89).

To enhance the immunogenicity of the attenuated vaccine virus, the expression level of G protein in infected cells has been increased by insertion of an additional G gene into the genome (90). Both the G genes contain alteration of amino acid at position 333 position and recombinant rabies virus (SPBNGA-GA strain) produced twice the quantity of G protein in cultured cell compared to virus carrying only a single G gene and that the recombinant virus induced apoptosis more strongly and more efficient protective immunity compared to control one (90).

Recombinant virus lacking the P gene (def-P) has been recovered from the genome plasmid with supplementation of the P protein from helper plasmid. The def-P virus can be produced in cell lines stably expressing P protein. On the other hand, the def-P virus did not effectively grow in normal cells that did not express the P protein. The def-P virus was completely apathogenic for adult and suckling mice inoculated intracerebrally. The def-P virus induced high level VNA and protective immunity sufficient to withstand challenge virus infection in mice. The P protein blocks both type I interferon production and signaling pathway which inhibits host innate immunity. The loss of these functions indef-P may contribute to high immunogenicity (91).

M gene deficient rabies virus (RCHLΔ M) has been developed using gene manipulation system of RC-HL strain used in vaccine production in Japan (92, 93). The M protein is essential for viral assembly and budding (94). The RCHLΔ M strain has been recovered from the genomic plasmid in the presence of helper plasmid coding for M protein. The infectious progeny virus was not detected in a culture supernatant from RCHLΔ M strain infected cells whereas the infectious virus was effectively produced in the supernatant from RC-HL strain infected cells. The BHK21 cell line expressing M protein of RC-HL strain can be used for propagation of RCHLΔ M strain. The RCHLΔ M strain is completely apathogenic for both adult and suckling mice inoculated intracerebrally. The RCHLΔ M induced VNA in adult mice after I/M inoculation or intranasal instillation (95).

The gene manipulation system of rabies virus has opened up the possibility of development of new generation rabies vaccines. However, it is to be carefully investigated to test these vaccines as safety of a recombinant virus with a foreign gene may cause serious side effects in inoculated animals and humans. For example, recombinant virus with a foreign gene or an attenuation related mutation, there is a possibility that the virus will become pathogenic during viral propagation in the body of the inoculated animal due to certain mutation.