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Full Text: 1 Introduction Rabies is a fatal disease posing serious public health concern, particularly in Asia and Africa (Singh et al., 2017). The word rabies has its origin from the Latin word ‘rabere’ which means ‘to be mad’. History and clinical recognition of rabies dates to twenty third century BC, where the first reference to animal rabies was recorded in the laws of Eshunna in Mesopotamia. Demonstration of neurotropism of the virus and the development of a rabies vaccine followed by its successful administration by Louis Pasteur during 1980’s was a great triumph in the history of rabies. With the advent of science and modern understanding of the virus and its immune protection, highly effective vaccines for the prevention and control of rabies have been developed. Despite the availability of safe and effective vaccines and understanding of the disease, the dog-mediated rabies causes approximately 61,000 annual deaths worldwide in humans, of which about 16,450 cases occur in India alone (WHO, 2013). The reasons being lack of awareness about the bite wound cleaning, vaccination, use of anti-rabies immunoglobulins in bite cases, non availability of the vaccines in remote places, etc. Due to under-reporting, rabies falls among the eighteen Neglected Tropical Diseases (Hampson et al., 2015). The true burden remains underestimated as the diagnostic facilities and disease surveillance are restricted in both animals as well as humans. Regardless of the concerted efforts made on  global scale, with enhanced focus on implementation of control schemes and awareness programmes, the disease remains endemic in Asia and Africa, contributing 95% of reported cases worldwide  (WHO, 2013). Rabies is completely preventable through exhaustive mass immunization of dogs and post-exposure prophylaxis (PEP) of humans as well as animals. It is imperative to create awareness among the vulnerable population to avoid exposure (Balaram et al., 2016). Rabies virus (RABV) induces mild to severe neurological signs described as furious and dumb forms, followed by paralysis, coma and death. The rabies virus  circulates in two interrelated cycles, the first one involving domestic mammals (predominantly dogs and cats) and the second one involving wild mammals (mongoose, fox, raccoon, wolf, jackal, badger, bat, etc.) (Kuzmin et al., 2012). Stray dogs and probably jackals in India are the main vectors or reservoirs that sustain the disease in humans and other animals through bite of the infected ones (Singh et al., 2017). Bat mediated rabies, which is most prevalent in Latin American countries, has not been reported in India. With the aim to eradicate the dog-borne rabies in humans by 2030, Global Alliance for Rabies Control (GARC), a non-profit organization in collaboration with international rabies stakeholders (WHO/OIE/FAO) and various other communities is engaged in rabies control activities. They have been working over a decade, hand-in-hand, at local, national and global levels raising awareness as well as financial support. Apart from such collective approaches, several intersectoral projects were launched in rabies endemic countries within Asia and Africa for the elimination of dog-mediated rabies that involves enhanced educational awareness and mass immunization of dogs at risk (WHO, 2015). The present review emphasizes the current status, epidemiology, prevention and control strategies of rabies especially in Indian context. 2 Rabies virus Rabies virus is a, negative sense, single-stranded, enveloped, bullet shaped RNA virus of the genus Lyssavirus under the family Rhabdoviridae. Presently, 7 distinct genotypes of RABV have been found to circulate in nature. Classical rabies virus genotype 1 (street and laboratory strains) is the globally prevalent and causing disease in >99% cases in humans as well as animals (King et al., 2011). The other 6 genotypes named as rabies- related viruses (RRVs) such like Lagos bat virus (genotype-2), Mokola bat virus (genotype-3), Duvenhage virus (genotype-4), European bat Lyssaviruses (genotypes-5 and 6), and Australian bat Lyssavirus (genotype-7), are prevalent in certain areas of Africa, Western and Eastern Europe and Australia (Gould et al., 1998; Heaton et al., 1999). The fatal classical rabies - like disease in humans as well as animals is known to be caused by all the RRVs, except Lagos bat virus. So far, the role of RRVs has been established in the death of 5 human patients (Smith, 1996). The genome of RABV contains 5 highly conserved genes in the following order from 3' leader sequence, N, NS, M, G and L (Yousaf et al., 2012). The N gene encodes a highly conserved nucleoprotein with ‘group specific’ antigenic determinants shared by all rabies viruses and is exploited for diagnosis and virus identification. The G gene encodes trans-membrane glycoprotein, a ‘serotype specific’ antigen responsible for pathogenesis and induces neutralizing antibodies in the host (Singh et al., 2017). 3 Epidemiology of rabies Rabies affects all warm blooded animals and incidences have been reported from all continents except Australia and Antartica (Singh et al., 2017). Asia remains the hot spot in terms of both incidences and reservoirs. In Asia, over 3 billion human beings are at the risk of canine mediated rabies and it witnesses 30000 deaths annually (1 death every 15 minutes). The mortality rate in children below 15 years of age is more. The South Asian region records a maximum incidence of rabies outbreak, with India and Bangladesh in the lead. Other nations in the region and its vicinity with a considerable prevalence of rabies are Nepal, Bhutan, Myanmar, Thailand and Indonesia. The prevalence across domesticated species of animals in the region is in the range of 20-50%. In India, rabies is widely prevalent except Andaman and Nicobar islands (Sehgal & Bhatia, 1985). Increasing trend in stray dog population, urbanization and the lack of environmental hygiene among the densely populated rural communities are considered as critical risk factors in the predominance of rabies in Asian as well as African countries. On the contrary, European nations zero down rabies in humans by compulsory vaccination in animals, especially in dogs (Singh et al., 2017).  
4 TransmissionThe susceptibility of animals to RABV depends on several factors and there is difference in the reservoirs of disease among different geographical regions. Various studies have documented prevalence of rabies as 61.4% in cattle and buffaloes, 48.7% in goats, 48% in dogs, 45% in horses and 21.9% in cats (WHO, 2005; Sudarshan et al., 2006; Singh et al., 2017). Rabies outbreaks in domestic and wild animals in India are reported round the year with all such cases traced back to the bite of rabid dogs (Singh et al., 1990; Singh et al., 1995). According to the National Health Profile-2015, Ministry of Health and Family Welfare (MoHFW), Government of India (GOI), 104 cases has been reported from January to December, 2014 and the number showed a decline from the 132 cases of human rabies during 2013. West Bengal and Uttar Pradesh recorded maximum number of rabies outbreaks during 2014. In > 99% cases of human rabies worldwide, rabid dog has been found as a main source of infection (WHO, 2013). Eradication efforts in endemic countries have suffered heavily from systemic deficiencies, starting with under-reporting that has hampered synergized global response by hiding its real magnitude (Singh et al., 2017). A study conducted from 11 states/union territories of India during January 2012 to December 2014 showed about 52.3% of rabies cases were from Karnataka, 18% from Maharashtra, 7.8% from Tamil Nadu, 6.3% from Kerala and 3.1% from Andhra Pradesh states. West Bengal was the worst affected state, with 47 rabies deaths in 2016, followed by Karnataka with 19 deaths (Mani et al., 2016).     Rabies in human beings develops from the bites of infected animals, predominantly dogs (91.5%) that have contracted the virus from feral or fellow infected creatures (Menezes, 2008). The transmission of virus occurs only when the saliva of the infected animal comes into contact with a broken skin or mucous membrane (Figure 1). The risk associated with the infection is reported to be high through the bite (5%-80%) than scratches or licks (0.1% -1%) from the affected animal. Fatality depends on the site of bite and the quantum of virus in the saliva of bitten rabid animal (Hemachudha et al., 2013). However, disease transmission through non- bite exposures such as cornea and organ transplants and contact with the infected materials such as brain tissues have also been reported (Winkler et al., 1973; Gibbons, 2002; Hellenbrand et al. 2005; Takayama, 2005; Milton et al., 2015). Bat colonies are known wildlife reservoirs for RABV variants in northern hemispheres. Invasive activities like skinning of infected animals accompanied with improper operating procedures enhances the risk of contraction of disease. Transmission through gastro-intestinal tract has also been documented (CDC, 1999). Exposure to live attenuated vaccines during the production stage may have potential risk. The persons who are involved in capturing the animals for birth control programmes are also at potential risk because of the threat of bite and scratch. Even though birds have all along been considered to be resistant to rabies, an unusual case of natural rabies virus infection following the bite of rabid dog has been reported recently in Kerala, India (Baby et al., 2015). 5 Pathogenesis Detailed pathogenesis of rabies virus has been recently reviewed by Singh et al. (2017). The sequence of events in the disease process involves: 1) replication of the virus in the peripheral tissues, 2) spread along the peripheral nerves and the spinal cord to the brain, 3) dissemination within the central nervous system (CNS) and its 4) centrifugal spread along nerves to various tissues and organs. These sequential steps have been elucidated from a number of experiments in mouse models using laboratory fixed strains (Mifune et al., 1980). Although these events may not mimic closely the natural disease process either in human or in rabies vector, yet understanding of these events is necessary for treatment and control of the disease.                 5.1 Entry of virus                   The RABV binds neurons or muscle cells through receptors on the cell surface. A critical step in the disease process of street virus which differs from fixed virus is the involvement of muscle cell and occasional fibroblast infection (Johnson, 1965; Coulon et al., 1989). The virus remains undetected at the site when it enters an eclipse phase and replicates in muscles sequestered at or near the ingress site prior to nerve entry (Murphy et al., 1973). After the variable incubation period, the uptake of virus is completely restricted to motor end plates and axons in the peripheral nervous system (PNS) through receptor mediated endocytosis without affecting the sensory and autonomic nerve endings (Ugolini, 2008). However, there is a possible chance of transmission of bat RABV variants via sensory or sympathetic skin innervation (Ugolini, 2011). Velandia-Romero et al. (2013) reported that large sensory neurons preferentially capture and passage RABV to the dorsal root ganglion after inoculation in plantar foot-pad of adult mice. Nicotinic acetylcholine receptor (nAchR) is localized in the post synaptic membrane of motor neurons, neuromuscular junction (NMJ) on myotubes and in neurons of the CNS. Binding to nAchR concentrates the virus at the NMJ and aids in the uptake by nerve terminals by binding of RABV-G to micro-domains formed by another protein receptor known as neural cell adhesion molecule (NCAM)/ CD56 through gangliosides (Lafon et al., 2008). NCAM is present in cell bodies of pre-synaptic membranes of neurons of CNS as well as non-myelinated axons of the PNS. Low affinity neurotrophin receptor /p75NTR is the third protein receptor expressed at synapses of neurons (Tuffereau et al.
, 1998). Direct penetrating injury facilitates direct entry of virus to nerve terminals without replication in muscle (Coulon et al., 1989; Shankar et al., 1991). 5.2 Replication of virus Upon getting endocytosed in the peripheral neurons, transmission of virus to the CNS occurs by fast retrograde axoplasmic flow, but drugs and neurectomy prevent spread, whereas binding with p75NTR facilitates transcytosis (Gluska et al., 2014). The virus then reaches the neurons of the CNS in dorsal root ganglia and spinal cord where it multiplies and further spread to brain. Rapid multiplication of the virus is facilitated in almost all areas of the brain, however, with minimal pathological findings. Affection of neurons in limbic areas of brain results in behavioural changes, which leads to transmission through bite of infected animals. Neuronal dysfunction rather than neuronal damage is believed to be the mechanism by which RABV causes disease (Tsiang, 1982). The possible mechanisms of neuronal dysfunction were extensively studied and described as alteration of neurotransmission (Dumrongphol et al., 1996), disturbance of ion channels (Iwata et al., 1999), degeneration of neuronal processes and synaptic structure disruption (Li et al., 2005), down regulation of protein synthesis (Dhingra et al., 2007) and increased production of nitric oxide (Nakamichi et al., 2004). The possible strategy employed by the virus to favour its advancement through the nervous system is inhibition of apoptosis as there is an inverse relation between induction of apoptosis and the capacity of a RABV strain to invade brain (Thoulouze et al., 2003). By slow centrifugal anterograde axoplasmic flow, the virus moves from the CNS to muscle spindles, skin, hair follicles, salivary glands, lungs, heart muscle and abdominal visceral organs (Hemachudha et al., 2002). 5.3 Evasion strategies of virus The evasion strategy used by RABV is by inhibiting both innate and adaptive immune responses. Toll - like receptor 3 (TLR3) is an intracellular innate immune receptor in neurons (Jackson et al., 2006). In normal neurons, TLR3 is present within the endosomes in the neuronal cytoplasm; however, RABV sequesters TLR3 in Negri bodies (Menager et al., 2009). As there is lack of antigen presenting cells and lymphatic drainage, the CNS cannot induce an adaptive immune response against RABV. However, activated T cells especially CD8+ T cells and monocytes cross the intact blood-brain barrier but undergo death (Lafon, 2008). B cells needs disruption of the barrier in order to reach brain (Hooper et al., 2009). Antibody response is elicited during the later phase of infection with a low titre in the CSF, the source of which is not clear whether; it is from the virus in the periphery or from CNS (Johnson et al., 2010). RABV adopts stealth to keep away from stimulating the neutralizing host immunity by regulating the rabies virus glycoprotein (RABV-G) expression at the infection site as well as in neural tissues during its movement towards the CNS and there is an inverse association between pathogenicity and RABV-G expression (Yan et al., 2001; Zhang et al., 2013). On contrary, Wirblich & Schnell (2011) surmised that the RABV-G expression level plays a role in pathogenicity, but it will not attenuate a pathogenic RABV and is not a decisive dominant factor. 6 Diagnosis of rabies Prompt and precise laboratory diagnosis of rabies in humans and animals is imperative for astute administration of post-exposure preventive measures (Figure 1). The clinical signs of rabies are misdiagnosed with other neurological disorders, therefore cannot be relied upon. The isolation of RABV in albino mouse, cell lines like Neuro2a/CCL 131, BHK- 21/C13, Vero and McCoy are the most dependable and consistent methods in diagnosis of rabies. The CCL 131 cell line without any adaptation is most susceptible to street RABV. The results are obtained in 18 h post inoculation and hence routinely preferred for isolation (Shankar, 2009). An alternative highly sensitive method is mouse inoculation test (MIT), which is also used principally in developing countries (Singh et al., 2017). Many laboratories are targeting Negri bodies in brain impressions and sections using Seller’s and Mann’s staining till date. Due to poor sensitivity of these tests, direct fluorescent antibody test (dFAT) is the most comprehensively recommended test for diagnosis and interpretation of rabies in fresh brain samples in animals (WHO, 2013). An indirect rapid immuno-histochemistry test (IRIT) has been established and evaluated to detect as well as differentiate RABV variants; however, it needs further evaluation by traditional microscopy. So, a direct rapid immunohistochemical test (dRIT) for detection of rabies in humans as well as animals has also been developed (Madhusudana et al., 2012). Lateral flow technique employing immunochromatographic principle having 100% specificity and more than 88% sensitivity enabled the early diagnosis of diverse rabies virus strains (RABV species 1, 5, 6 and 7) when compared to earlier mentioned tests (Servat et al., 2012). RT-PCR–ELISA has recently been identified for the detection of RABV (AravindhBabu et al., 2014). The dFAT, having 100% sensitivity to detect virus from fresh brain tissues is used regularly by all the laboratories envisaged to diagnose rabies and confirmative diagnosis can be made within 2 h. Moreover, the dFAT has                   also been found appropriate to detect rabies positive cases (100%) even in formalin-fixed or paraffin-embedded materials                     (Singh et al., 2017). Serological tests are rarely used to diagnose rabies as the infected animal or human do not survive the disease long enough to release sufficient antibodies to detect its presence in serum. Serological tests are usually employed for detection of rabies antibodies by targeting G and N proteins and are chiefly used to assess potency of various rabies vaccines (Wasniewski & Cliquet, 2012). Serum neutralizing tests are employed for virus serotyping that requires mouse neuroblastoma cells and G-protein reactive monoclonal antibodies (mAb-Gs). The approaches currently recommended by WHO to estimate RABV neutralizing antibodies are rapid fluorescent focus inhibition test (RFFIT) and fluorescent antibody virus neutralization test (FAVN) (Cliquet et al., 1998). A minimum measurable protective antibody titre of 0.5IU/ml represents the level of immunity in humans and animals to rabies infection (OIE, 2016). For the transport of pet dogs from countries considered infected with rabies, an international veterinary certificate must be produced indicating that the animal has not shown any signs of rabies on the day or prior to the travel and were either vaccinated or revaccinated against rabies following the standard protocol or should have undergone an antibody titration test not less than 3 months or not more than 12 months prior to the day of travel with a  protective antibody titre of not less than 0.5IU/ml. RFFIT is widely used to estimate the sero-conversion following preventive measures like prophylactic vaccination, and to help clinical detection of rabies in suspected cases. Diagnosis of RABV nucleic acid in the clinical samples like cerebrospinal fluid (CSF), saliva, skin biopsy and corneal impression smear by polymerase chain reaction (PCR) is documented as reliable for the ante-mortem diagnosis of rabies (Madhusudana & Sukumaran, 2008). Other tests which are established, which are in use in WHO or World Organization for Animal Health (OIE) reference laboratories to diagnose rabies includes tests based on the detection of nucleic acid like in situ hybridization, genome sequencing, etc (Singh et al., 2017). These methods are able to detect extremely specific viral RNA molecular subunits in brain material of experimental or routine specimens. PCR based tests are established for studying viral pathogenesis and epidemiological analysis apart from diagnosis. Though FAT can detect RABV within 2 h of receiving sample, it is not sensitive with RRVs and fails in the decomposed tissues (Heaton et al., 1999). In contrast, PCR being independent on such conditions detect even few numbers of viral particles in such samples (Singh et al., 2017). There are no diagnostic tests available to diagnose rabies in humans during pre-clinical phase, and till the rabies-pathognomonic signs like hydrophobia or aerophobia are developed, the clinical diagnosis might be difficult (Chacko et al., 2016). Several tests are required for diagnosis of rabies ante-mortem in humans due to the variability of the virus in different samples, the timing of collection of sample, and the antibody response of the host. Clinical samples like saliva, serum, spinal fluid, and skin biopsies of hair follicles at the neck region can be included for diagnostic tests (Warrell & Warrell, 2004). Conventional RT-PCR, Real-time PCR, Direct dot-blot enzyme immunoassay (EIA) on saliva and Polymerase chain reaction (RT-hnPCR) on skin biopsy (non-neural) are available with variable sensitivity and 100% specificity (Madhusudana et al., 2004; Nagaraj et al., 2006; Dacheux et al., 2008; Chacko et al., 2016). Saliva is a sample of choice for virus isolation as well as reverse transcription polymerase chain reaction (RT-PCR). Moreover, serum and spinal fluid can be tested for antibodies against rabies virus. Skin biopsy materials are examined for rabies antigen in the cutaneous nerve endings at the base of hair follicles (Madhusudana & Sukumaran, 2008; Dacheux et al., 2008). 7 Vaccination approaches in control of rabies: Pioneering anti-rabies immunization was performed on a boy (Joseph Meister) by Louis Pasteur in late phase of 19th century. After that plenty of effective and safe, second and third generation vaccines have been developed for use in animals and humans. Currently, a number of vaccines such as recombinant rabies virus strains or rabies antigen-glycoprotein (G protein), either as a component of non-pathogenic viruses, or in plants / form of DNA vaccines are being developed (Ohara et al., 2013). To stimulate the host immunity against rabies infection, normally different forms (live intact, inactivated, attenuated) or purified components of pathogens (outer coat proteins of rabies virus) with high immunogenicity are used. This generates immune response within 2 weeks. These vaccines are administered by IM or ID routes. If dog population is sufficiently (>70%) covered by rabies vaccination, it will reduce the disease burden in humans. Currently, the rabies vaccines are accessible to prevent and control rabies in animal population (dogs, cats, wild carnivores and bats). The vaccines administered within first few days of post-exposure are reported to decrease the disease by attenuating the virus considerably. Later on the 4 dose series (day 0, 3, 7 and 14) would take care. Treatment of the category III wounds (single or multiple transdermal bites or scratches, licks on broken skin; contamination of mucous membrane with saliva from licks) with rabies immunoglobulin (RIG) should be commenced as soon as possible after exposure to prevent disease development in human. In the United States alone, 11 different categories of rabies vaccines are licensed for dogs, 12 for cats, 5 for sheep, 4 for cattle, 3 for horses, and 1 for ferrets (Briggs et al., 2007). The most challenged obstacle to develop vaccines for rabies is the need to work with the active virus, for which appropriate bio safety levels are essential. Virus like particles (VLPs) seems to be promising in this context for development of rabies vaccine. The efficacy of glycoprotein sequences of Pasteur virus (PV), Challenge Virus Standard (CVS), Evelyn- Rokitnicki-Abelseth (ERA), or street virus isolates as DNA vaccines have been evaluated. Also, the advancement in science in the recent years has made momentous progress in the design of vectors suited for gene delivery of the virus components. However, drawbacks related to poor immunogenicity and requirement of larger doses of DNA in animals remain as a fathomless issue in rabies DNA vaccination (Yang et al., 2013). Glycoprotein expressed on the surface of the vaccinia virus, canary pox virus (Yang et al., 2013), canine adenovirus (Zhang et al. 2008), chimeric lyssavirus glycoprotein with segments from Rabies virus and Mokola virus (Badrane et al., 2001) are the recombinant vaccines which provide immunization against more than one lyssavirus. DNA vaccination with glycoprotein cloned into a plasmid vector has also been developed as vaccines against rabies. Vaccination in wild animals is an essential part in control of rabies so as to prevent its spread to domestic animals and human. Oral Rabies Vaccine (ORV) laden baits have been successfully established for red, Arctic, and gray foxes; coyotes, raccoon dogs, raccoons, skunks, and domestic dogs (Yang et al., 2013). ORV was explicated to vaccinate free-ranging wild animals in geographically large enzootic areas, to subjugate the development and extent of a rabies epizootic, or to establish a rabies-free buffer zone (Muller et al., 2001). Vaccination for rabies can be either pre- or post-exposure. Pre-exposure vaccination is performed to safeguard those who are at high risk of rabies exposure. Post-exposure vaccination is performed after the bite of rabies suspected animal to prevent the development of disease. The vaccines used for pre-exposure and post-exposure prophylaxis remains the same, but the schedule varies. Inactivated animal nerve tissue vaccines (NTV) from rabbit brain and later sheep and goat brain were used previously, but later discontinued due to the high levels of myelin that caused sensitization in some vaccine recipients and, in extreme cases, fatal encephalitis (Fishbein et al., 1993; Hicks et al., 2012). In India, NTV was used for post-exposure treatment, but the production was stopped in December, 2004 due to the adverse reactions. NTV was later replaced by cell culture adopted or embryonated egg passaged vaccines which were found safer and more efficient than older vaccines. 7.1 Pre-exposure vaccination   Table 1 Type of contact, exposure and recommended post-exposure prophylaxis  
Category Type of contact Recommended post-exposure prophylaxis I Touching or feeding of animals Licks on intact skin Contact of intact skin with secretions and excretions of rabid animal and human case None, if reliable case history is available II Nibbling of uncovered skin Minor scratches or abrasions without bleeding Wound management Anti-rabies vaccine III Single or multiple transdermal bites or  
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