Eradication of Rabies with Mass Parental Vaccination, Post-exposure Prophylaxis and Gene Therapy: A Systematic Review

Aims: To review canine rabies, mass parental vaccination, human post-exposure prophylaxis, gene therapy and costs for fighting rabies. Place and Duration of Study: Department of Animal Science – Other, Nelwan Institution for Human Resource Development, Indonesia, between December 2017 and March 2018. Methodology: The author searched the Pubmed Database at NCBI for articles on rabies disease published between 2007 and 2018. All articles were open access and in English. For rabies virus examination, Seller’s test was used. In this article, references written by the author and other relevant publications were included. The author reviewed a rabies dog case kept at Nelwan Institution for Human Resource Development. Results: The dog showed clinical signs such as inappetance, urinary frequency and soaking in a small, juicy drain. Currently, to treat rabies, no drugs are available. For rabies prevention, vaccination is the best way. To eradicate rabies, mass vaccination in dogs, post-exposure prophylaxis, and gene therapy should be used. Fort rabies disease eradication, minimum of 70% of the dog population should receive the vaccination. In addition, humans with category II exposure should receive a rabies vaccine and rabies immunoglobulin. Conclusion: To eradicate rabies, vaccinations are required. In addition, gene therapy can eliminate rabies from the infected neurons by using rAAV-N796. CRISPR/Cas9 system in combination with the MMEJ-based method. Furthermore, mass parental vaccination, postexposure prophylaxis, and gene therapy can reduce costs in controlling rabies disease. Registration Number: PROSPERO (CRD42018084448)


INTRODUCTION
Rabies is one of the oldest diseases on the globe and most feared zoonotic disease known to humankind. The disease is a hazardous, progressive and practically deadly encephalomyelitis [1]. Lyssavirus is the most important rabies virus (RABV) [2]. Rabies can infect both humans and domestic animals. For example, most cases of rabies in animals arise among bats, carnivores, cats, raccoons [3], mongooses, and wolves [4]. In addition, a natural rabies infection in birds has also been reported [5]. Dog (Canis lupus familiaris) [6,7] is the source of more than 99% of rabies cases in humans [8,9]. The animal can transmit RABV from animal to human through bites, or mucous membranes from saliva [2,6] or other potentially infectious material such as neural tissue. In domestic animals, the incubation time is normally around 1-3 months. Moreover, non-bite sources of rabies are salivating, scratches [8,10], and corneal transplantation [10]. Once symptoms of rabies begin, the disease is around 100% fatal [4,6].
In this study, the author reports canine rabies clinical signs that include heat, urinary frequency, and thirst. In addition, this study will provide an overview of the different rabies situations in other countries where the disease has been eliminated. Currently, there is no available drug for rabies [2], but this disease is 100% preventable [9] through vaccination of both dogs and humans who were exposed to the virus. According to the World Organization for Animal Health, mass dog vaccination (MDV) is the most cost-efficient way to eliminate rabies. At least 70% of the dog population in the area needs to be vaccinated to maintain herd immunity. This has been proven to be effective in many countries, including Argentina, Indonesia (Bali) and Mexico. Rabies in humans is avoidable through vaccination. To prevent rabies postexposure prophylaxis (PEP), rabies vaccine administrations and immunoglobin following contamination should be used. There have been few studies regarding possible rabies treatment for animals or humans with λ-CG P32 (carrageenan) and gene therapy. According to Luo et al., carrageenan is an anti-RABV agent that can slow down significantly RABV infection in vitro. It is a sulfated polysaccharide soluble in water obtained from red algae [11]. The idea of gene therapy is to use induced pluripotent stem cells (iPSCs) and CRISPR/Cas9 (clustered regularly palindromic repeats/CRISPRassociated) for rabies treatment. Indeed, rabies is a disease which requires significant financial commitment. MDV, PEP, including mass information drive for pet owners, are seen to be the major components of rabies prevention and control program. Modern trends such as vaccine development and gene therapy studies hold potential for the treatment of rabies.

Systematic Review
The present report follows the guidelines of the PRISMA extension statement for systematic review [12]. These guidelines also correspond to PROSPERO guidelines such as searches and risk of bias assessment [13].

Searches
The author searched articles for rabies in only one database, that is, Pubmed Database at the National Center for Biotechnology Information (NCBI). These included free PMC articles in English published between 2007 and 2018 for open access. Keywords comprised "canine rabies, human rabies, rabies PEP in human, rabies and vaccination, gene therapy, or iPSCs and CRISPR/Cas9 system, and costs for fighting rabies." In addition, the author's articles regarding gene therapy and other relevant publications were included. The study included the report of a canine rabies case kept at the Nelwan Institution for Human Resource Development, Indonesia. In this study, Seller's test was used. Articles used to describe this study included case study reports, research articles, and review articles.

Exclusion Criteria
Criteria for the exclusion of literature included analysis of subgroups or subsets, publications other than English, publications before 2007 and rabies other than human rabies and canine rabies.

RESULTS
The author took 163 articles from the Pubmed Database searches and other relevant publication searches (Fig. 1). After screening titles and abstract, 128 articles were taken for full-text review of these 58 articles met the criteria for data extraction.

A Rabies Case in Indonesia
The dog was a male, and was six years old when it died. It was the only dog in the house and never received a vaccination. The dog had clinical signs that included aggressive behavior, inappetance, urinary frequency, thirst, heat and soaking in a small, juicy drain. Based on Seller's test, the dog was positive for rabies. This case of rabies occurred in Palu, Central Sulawesi, Indonesia.
Forebears of Indonesian RABVs derived from Java. The Java's RABVs offspring transmitted these rabies viruses to Kalimantan, and then to Bali, Flores, and Sumatra. The Flores's offspring transmitted these RABVs viruses to Sulawesi and went back to Kalimantan. In Indonesia, the dog is the only source of infection of other animals [14].
Human deaths in Asia due to rabies exceed 30000 annually [1,40]. For example, India has the highest incidence of rabies, even globally. Human deaths from rabies were 16450 in India and China 7450 in 2010 [40]. Rabies is endemic in Indonesia in 24 of the country's 34 provinces. This disease causes 150 to 300 human deaths annually [45]. In Africa, human deaths due to rabies are about 23000 to 23800 or 24200 annually [1,15,40]. In the Middle East and Central Asia, initial estimation for human's deaths from canine rabies is 350 and 1900, respectively. In Latin America, human rabies derived from dogs decreased from 250 in 1990 to fewer than 10 in 2010 [40].
Human rabies in the United States is rare and is only one to three cases annually. Rabies in this country may derive from bats, dogs, dogmongoose, foxes, and raccoons. Of the 23 cases of rabies in the United States from 2008 through 2017, eleven (47.83%) derived from bats (contact, bite or unknown). Seven (30.43%) were rabies from dog bites. Dog-mongoose, fox, and raccoons were 4.3%, respectively. One (4.3%) was unknown. Eight (34.78%) of 23 cases were from outside of the United States and its territories [46]. Australia is free from carnivore rabies, and many Pacific Island nations have always been free from rabies and related viruses [1,40]. In addition, the United States [2], Canada, Western Europe, Japan [40], Argentina and Chile have successfully controlled canine rabies [47].

Mass Parental Vaccination in Dogs
The World Health Organization (WHO) recommends that to eradicate rabies, at least 70% of the dog population should receive the vaccination [24- 25,35,40,[48][49]. It would avoid the main disease outbreak at least 95.5%, and meets the requirements for eradicating rabies [24]. In the endemic areas of rabies, a minimum of 70% of the dog population in each year during 5-7 years should receive the vaccination [48]. The crucial vaccination coverage ranges from 25-40% [41]. It is essential to interrupt rabies transmission. In addition, mass vaccination under 30% is not beneficial for rabies eradication purposes [28]. For mass vaccination, vaccines such as Rabvac 1 and Inrab 1, used in the United States, can be used for vaccination annually. Route of vaccination is intramuscular or subcutaneous (Rabvac 1) and subcutaneous (Inrab 1) [3]. In a canine rabies-free country, the limit 70% threshold for eradication purposes is irrelevant. Most rabies vaccines are licensed for dogs older than 12 weeks of age [48] and revaccination with a booster is one year later [38,48].
Vaccination approaches include door-to-door campaigns, static point campaigns, and a combination of the two [24,40]. Such posts are usually sufficiently attended only when those posts are at less than 500 m or a 10-minute walk. The option depends on the people on the local level [40]. reporting on the successful implementation of vaccination and reporting on the successful implementation of post-vaccination vaccinations by short message service and paper; implementation of daily, weekly and monthly vaccinations through government coordination; and the implementation of vaccination by trained field staffs [40].
In Africa, KwaZulu-Natal (South Africa) has vaccinated more than 15 million dogs since the commencement of the dog rabies eradication project in 2000. In 2012, more than 630000 dogs were vaccinated. In three years, the incidence of animal rabies had declined. KwaZulu-Natal reported in 2010-2011 a continuous 12-month period without a single human case [40]. Rabies was responsible for 1500 deaths annually in Tanzania. Following the implementation of control activities from 2010 to 2016, human rabies deaths declined to 375 deaths (a 75% decline) [47]. Moreover, vaccination of 66% of domestic dogs in Tanzania resulted in a decrease in dog rabies, human PEP, and the number of positive for rabies wild-type diagnosis [50].
Many countries in Latin America have successfully eradicated rabies (

Post-Exposure Prophylaxis in Humans
There are three categories of PEP, category I, category II, and category III. Category I includes touching or feeding animals and licking undamaged skin with secretions or excretions of a rabid animal or human. It is not an exposure, and does not require PEP. Category II includes skin biting and minor scrapes without bleeding. Finally, category III includes simple transdermal bites, multiple transdermal bites and scratching bites [40].  [40]. Currently, there are available RIGs for clinical use, namely, human rabies immuneglobulin (HRIGs) and equine rabies immunoglobulin (ERIGs) [20,37]. Treatment after the category III exposure is the immediate administration of CCEEVs and RIGs [37]. It requires putting HIRG into the wound or intramuscular for active immune response to vaccine antigen [2]. In addition, new RIG products have been available. Chao et al., introduced SYN023 that is derived from two novel monoclonal antibodies (MAbs) CTB011 and CTB012 [52]. Um et al., developed 16B8-Alexa MAb and evaluated it using RFFIT [39]. SYN023 and 16B8-Alexa could replace the current RIG products. Both of them are safe for PEP.

Treatment with Gene Therapy
Clinical rabies in the mouse model can be treated. Wu et al. created the nucleoprotein (N) gene of RABV (rAAV-N796) to fight rabies virus. In their study, the authors did their study in four groups of mice (Table 4). These groups consist of group A, B, C and D. In the first treatment group, the authors administered of rAAV-N796 or rAAV-Neg intracerebral and administered of10 LD50 of lethal CVS-11 intracerebral 24 hr later (A). In the other groups, they administered of rAAV-N796 or rAAV-Neg intramuscular and administered of 20 LD50 of lethal CVS-11 intramuscular 24 hr later (B). Moreover, the authors administered of rAAV-N796 or rAAV-Neg intracerebral and administered of 20 LD50 of lethal CVS-11 intramuscular 24 hr later (C). In the last group, they administered of 20 LD50 of lethal CVS-11 intramuscular and administered of rAAV-N796 or rAAV-Neg intracerebral 24 hr later. The highest results were observed in the group of mice with an intracerebral administration with rAAV-N796 and administration with 20 LD50 of lethal CVS-11 intramuscular (C). The result was 62% alive on day 21 of infection [54].
The author did not find any reference relating to iPSCs and CRISPR/Cas9 system for treating rabies. However, Nelwan indicated that genedelivery tools, gene-editing tools, NHEJ-based technique, for instance, (M Nelwan, Nelwan Institution for Human Resource Development, INDONESIA, Unpublished results), and iPSCs technique are used to treat monogenic disorders [55][56][57].

Estimated Burden of Rabies in the World
The annual cost for rabies prevention varies from one continent to another. Asia needs as much as US$ 1.5 billion for PEP only. European Union and Pan American spend US$ 6.5 million and US$ 20 million, respectively [40]. The United States needs US$ 300 million annually [1,40]. Latin America needs US$ 129 million for PEP and needs US$ 61 for mass vaccination. It is the most cost-effective approach [26].

DISCUSSION
Humans have been living in fear of rabies outbreaks for thousands of years. Currently, more than half the world's population is still fearful of rabies outbreaks. Indeed, the earliest report of rabies was around 2300 BC [25]. Rabies has existed in Indonesia since 1884 [45].
In addition, this disease is endemic in provinces such as Central Sulawesi, North Sulawesi, and West Java. Rahmadane et al. [45] stated that Indonesian rabies belongs to the Asian lineage; that is, lyssavirus genotype 1. Indonesia regularly controls rabies disease at provincial, district [45], and municipal levels. However, sufficient vaccination coverage has been hard to reach [45]. Indonesia and other ASEAN countries expect to be free of rabies in 2020.
Clinical signs of canine rabies include aggression, abnormal behavior, vocalization changes, paralysis [3,40], ataxia, cranial nerve deficits, dysphagia, inappetance [3], drooling, and convulsions [40]. The dog in this study had clinical signs such as urinary frequency and soaking in a small, juicy drain. Although the dog had an inappetance and aggressive clinical signs, it did not have clinical signs such as ataxia and paralysis. Other clinical signs were heat and thirst. It seems that dog clinical signs are not the same as described in reference 3 and reference 16, except inappetance and aggressive behavior. It seems that heat, urinary frequency, thirst and soaking in a small juicy drain has not been described before.
The Seller's test is a rapid and a simple method for the diagnosis of rabies. The limitation of this test is that it is only suitable for fresh samples. In addition, the Seller's test has a very low sensitivity [1,45].  [15] showed that dogs were the only animal that bit humans in Maputo and Matola cities (Mozambique). This was an expected discovery and confirmed data from other countries. It suggests that rabies eradication efforts should focus on dogs.
Currently, for rabies disease, no effective drugs are available. Medical treatments currently focus on mass vaccination and PEP for instance. However, to treat rabies in the future, gene therapy may be a very useful tool. This technique includes gene delivery vectors such as rAAV [55] and the CRISPR/Cas9 system [55], and iPSCs technique. For disease modeling, drug screening, and stem cell therapy; the iPSCs technique is helpful [56][57][58]. In addition, the iPSCs technique in combination with a CRISPR/Cas9 system or NHJE-based technique for treating rabies may also be developed. To treat rabies in wild-type animals, drugs derived from this combination may be useful. Rabies outbreaks may occur in dogs and other animals such as bats and raccoons. Yang et al., [4] showed that rabies outbreaks in dogs have occurred in Malaysia and Taiwan.
Lavan et al. [50] showed that mass vaccination and human PEP are more cost saving and costeffective than human PEP only. This effective cost estimate comes from the annual cost for six years in the Bhutan government project to fight rabies. This project consisted of three stages and each stage lasted 2 years. Vaccination coverage was 70% in stage 1, 60% in stage 2, and 50% in stage 3.

CONCLUSION
Rabies is a neglected tropical zoonotic disease. The disease is nearly 100% fatal and is 100% avoidable. Clinical signs of rabies disease include urinary frequency, inappetance, and soaking in a small, juicy drain. Vaccines are the only way to fight the rabies virus at present. In the future, to fight rabies, gene therapy, iPSCs technology, and gene-editing tools may become useful. The iPSCs technique in combination with the CRISPR/Cas9 system may be useful to eradicate this disease. Mass vaccination, PEP, and gene therapy can help to eradicate rabies disease worldwide. The cost to fight rabies with mass vaccination and PEP is lower than costs of human PEP only.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.