Rabies is one of the diseases in the spotlight in Indonesia. Based on data from the Ministry of Health of the Republic of Indonesia during the period 2011-2017, there were more than 500,000 cases of Animal Bite Transmission (GHPR) occurred in Indonesia. Of these 836 cases tested positive for Rabies (depkes.go.id, accessed August 1, 2019).
Rabies, which is a zoonotic disease has not been able to be overcome until now because the virus circulating in Indonesia is different from Rabies vaccine seed that has been used so far (Susetya et al., 2011). This condition causes the formation of antibodies that can neutralize viruses that infect perfectly.
Rabies virus has five structural proteins, namely Nucleoprotein (N), Matrix protein (M), Phospoprotein (P), Polymerase protein (L), and Glycoprotein (G) envelope protein (Fenner, 2011). Nucleoprotein is one of the structural proteins of the Rabies Virus that plays a role in virus replication and antibody induction.
The nucleoprotein is known to have four antigenic sites. Antigenic sites I and IV are arranged by linear epitope, whereas antigenic sites II and III are arranged by conformation-dependent epitopes (Hideo et al., 2000; Suwarno, 2005). This antigenic site plays a role in the induction of antibodies. Molecular analysis of Nucleoprotein Rabies Virus isolates in Indonesia needs to be done as one way to determine the right strategy for overcoming Rabies in Indonesia.
Samples in the form of brains collected from dogs confirmed to have been infected with Rabies Virus from Sumatra (Regional Research and Investigation Institute II Bukittinggi-Sumatra), Kalimantan (Regional Research and Investigation Center for Veterinary Regional V Banjarbaru-Kalimantan), Sulawesi (Balai Besar Veterinary Maros-Sulawesi), and Bali (Denpasar Veterinary Center). A total of 12 samples were isolated from the four islands. Each sample is made a suspension with a concentration of 10%.
The suspension that has been made is processed for RNA extraction. Extracted RNA was amplified through Reverse-Transcriptase Polymerase Chain Reaction (RT-PCR) (Hideo et al., 2000). The primer used amplified the antigenic site of the nucleoprotein coding gene weighing 1047 bp (nucleotides 71-1118) (Yang et al., 2011).
The results of the amplification are then processed in 1% gel agarose electrophoresis to ensure the primer used amplifies the targeted region. Electrophoresis results were observed under ultraviolet light with a wavelength of 302 nm (Suwarno, 2005; Sambrook and Russel, 2001). The RT-PCR product was purified then sequenced.
The sequencing results are processed for homology, phylogenetic, and mutation analysis (in the antigenic site area). Homological and phylogenetic analyses were performed using the Basic Local Alignment Search Tool of NCBI (https: //www.ncbi. Nlm.nih.gov) and MEGA 5.05 (N-J branched-chain method) to determine the characteristics of the isolated rabies virus. Homological and phylogenetic analyses were performed by comparing samples with the Rabies Virus in other Asian countries such as Indonesia, China, Thailand, India, Korea, and the vaccine seed virus (Pasteur). Analysis of the antigenic site part of the sample was carried out to determine the possibility of mutations.
Molecular detection results showed that all samples isolated were Rabies Virus. The results of homology analysis between samples and the Rabies Virus in Indonesia are 98-99%. It shows that the isolated virus did not experience much change compared to the previously isolated Rabies Virus.
The homology score between samples with Rabies Virus from China is 92-93%, while the results of homology scores between samples with Rabies Virus from Thailand, India, Korea, and Pasteur Virus are 88-89%, 86-88%, 85-87%, and 84-85%. Phylogenetic analysis shows that the Rabies Virus isolated in Indonesia with the Rabies Virus isolated in China share a common ancestor.
It causes the homology score between Rabies Virus isolates in Indonesia and Rabies Virus in China to be high. Meanwhile, between Indonesian Rabies Virus isolate, and Pasteur Virus did not share the same ancestor, so the homology score was quite low. The difference in homology score is thought to be due to rapid mutation and lack of proofreading in RNA virus replication.
Besides, environmental conditions also trigger mutations (Knipe and Howley, 2013). It is not yet known why Indonesian Rabies Virus shares the same ancestor with the Rabies Virus from China. Allegedly due to human migration from China to Indonesia and this condition also occurs in the spread of Rabies Virus in Indonesia.
The movement of humans and animals carrying rabies (HPR) between islands is thought to be a means of spreading rabies in Indonesia. An analysis of antigenic sites from nucleoproteins showed that there was only one mutation in the fourth antigenic site between one of the isolates from Bali and Virus Pasture.
It shows that the antigenic site of the nucleoprotein is conserved. Studies that have been done explains that nucleoprotein of Rabies Virus is quite stable, but the possibility of mutation and evolution still exists (Nagaraja et al., 2018). From this study, it can be concluded that one way to prevent the spread of Rabies is by using local isolates as vaccine seeds because the antibodies produced will be able to neutralize the infected Rabies Virus perfectly. (*)
