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Summary Rabies is an important zoonotic disease in India, Rabies occurs in all parts of Indian subcontinent except Andaman, Nicobar and Lakshwadeep group of islands. Fourteen rabies virus isolates were partially sequenced, phylogenetic analysis were performed in relation to the other Indian isolates and other Asian isolate sequences available in the GenBank with two different regions of the "N" gene. Thus it appears that genetic heterogeneity exists between viruses in India although they could not be geographically specifically delineated. Two genetic groups of rabies viruses appear to co-circulate, one in primarily in Tamil Nadu and the other in the other regions of South India. Further studies using a greater number of isolates from several states in India will be required in order to investigate more precisely the molecular epidemiological status of rabies virus circulating in India. Key words: Rabies virus; Indian isolate, nucleoprotein sequencing, phylogenetic analysis, genetic characterization Introduction Rabies is an important zoonotic disease that still poses a serious problem in many developing countries. In spite of pet animal vaccination and several. rabies control measures initiated by government agencies, eradication of rabies in developing countries has been not very successful. Low vaccination coverage in pet dog vaccination and ineffective management of stray dogs are the most likely reasons for the program's lack of success. Human mortality from endemic canine rabies was estimated to be 55,000 deaths per year with 56% of the deaths estimated to occur in Asia. The majority (84%) of these deaths occur in rural areas (WHO Technical Report Series, 931, 2005). Rabies occurs in all parts of Indian sub-continent except Andaman, Nicobar and Lakshwadeep group of islands. The increasing human population as well as the increase in the stray dog population of the country has resulted in more human-dog contacts. Dogs are responsible for 96% of human rabies cases. The mortality in livestock and pet animals is far higher than the number available due to lack of exact reports (Sehgal, 1997). Rabies virus belongs to the Lyssavirus genus of the Family Rhabdoviridae. This genus is composed of rabies virus (genotype 1) and rabies-related viruses, including Lagos bat virus (genotype 2), Mokola virus (genotype 3), Duvenhage virus (genotype 4), European bat lyssaviruses 1 and 2 (genotypes 5 and 6 respectively), and Australian bat lyssaviruses (genotype 7 (Gould et 01.,1998 ). The nucleoprotein (N) gene of rabies virus is the most popular target for rabies diagnosis using reverse transcription polymerase chain reaction (RT-PCR) because the gene is highly conserved and can be applied to genetic analysis (Arai etal.,1997, Ito etal., 2001, Kamolvarin et 01., 1993, Sacramento et al., 1991). The N gene, although highly conserved, allows viral strains to be accurately differentiated by analyzing genetic differences that are present within the gene (Johnson, et 01., 2002). Molecular epidemiology of rabies has been performed not only at regional levels but also at the global level (Kissi etal., 1995; Smith etal., 1992). However, there is little information available on the molecular epidemiology of rabies virus in Asia (Nishizono et 01., 2002; Susetya et 01., 2003; Ito et 01., 1999; Park, et al., 2005; David et 01., 2000) Regarding India, we previously reported the characterization of nucleoprotein sequence of an Indian isolate of rabies virus (Jayakumar et al., 2004). In the present study, to determine the dynamics of rabies virus in India in more detail, we used two variable regions of the 'N' gene of rabies virus isolates from Southern part of India. Materials and Methods Samples Fourteen field rabies virus isolates were obtained from 12 dogs, one calf and one goat from Chennai. Tamil Nadu, India. These brain specimens were collected during necropsy from suspected rabies animals done at the Madras Veterinary College Hospital. Chennai during the years 2003-2005. Primers Two sets of primers were used, the details of which are shown in Table 1.
Fluorescent antibody test Rabies diagnosis was done by a direct fluorescent antibody test (FAT) using anti-rabies 'N' polyclonal antibody conjugate (Bio-Rad, Australia) on impression smears of hippocampus region of the brain following the procedure of Dean and Abeseth (1973). The FAT positive samples were used for RT-PCR. RNA extraction RNA was extracted from the FAT positive brain samples using a commercial reagent TRlzol® (Invitrogen, USA) following the manufacturer's instructions and the extracted RNA was used immediately in RT-PCR reactions. RT-PCR CDNA was prepared using 1 mg of total RNA using the THERMOSCRIPT kit (Gibco-BRL, USA) and random hexamers according to the manufacturer's instructions. PCR was performed in MJ Research Thermal Cycler (PTC-200) programmed for 5 cycles with denaturation at 94°e for 2 mins; annealing at 45°e for 1 min, and elongation at 72°e for 2 mins. Subsequently, the annealing temperature was increased to 500e for 5 cycles and at 55°C for 20 cycles. The ultimate elongation was at 72°C for 10mins. Nucleotide sequencing and phylogenetic analysis The amplified PCR products were purified using the gel extraction kit (AuPrep, USA) and sequenced using the forward and reverse PCR primers in an ABI Prism 3700 DNA Sequencer (PEApplied Biosystem, USA). The nucleotide sequences thus obtained were aligned to get the partial 'N' gene sequence of isolates and had been deposited in GenBank, the accession numbers of which are presented in Table 2. The other know sequences of rabies viruses from other parts of India and the world, used for comparison and phylogenetic analysis in this study were obtained from the GenBank database and their accession numbers are also presented in the Table 2. AY259846, AY259853, AY259851, AF334790, AY259841, AY259842, AY259843, AY259844, AY259845, AF374721, AY644760, AY644782, AY644780 and AY644761. The sequences used for analysis were downloaded and imported into the Alignment explorer of MEGA 3.1 (Kumar et 01., 2004). Two different regions of nucleoprotein gene (nucleotide positions 5' - 45 to 310 and 5' - 311 to 710) were used for the analysis. Multisequence alignment was done using Clustal W alogorithm and the file was saved in MEGA format and opened in the MEGA window. Phylogenetic tree was created using the neighbor-joining method with Kimura-2 parameter model was used to create a guide tree. Bootstrap analyses with 1000 replications were done to determine the confidence interval of the developed tree (Kumar et al., 2004).
Result and Discussion Fourteen rabies antigen positive samples were obtained from 12 dogs, one calf and one goat in Chennai, Tamil Nadu, India. Phylogenetic analysis of these fourteen isolates sequenced in this study were performed in relation to the other Indian isolates sequenced (Nagaraj an et at., 2005) and other Asian isolate sequences available in the GenBank with two different regions of the 'N' gene. Using sequences from nucleotide 45-310, three different clades could be identified, one comprising of isolates from Kerala, Karnataka, Andhra Pradesh along with others from Pakistan, Korea, Japan and China. Other clade had isolates from China, Thailand and Indonesia. The third clade comprised of isolates from Sri Lanka and Tamil Nadu (Chennai) along with one isolate from Kerala. It is interesting to note that isolates from Southern states of India (Tamil Nadu, Kerala, Karanataka and Andra Pradesh) group differently. All the isolates from Tamil Nadu and Sri Lanka are together and different from those in the adjoining Southern States such as Kerala, Karnataka and Andra Pradesh. This could be due to the closer proximity of Southern Tamil Nadu to Sri Lanka. However Kerala has heterogeneity of isolates, one isolate DQ105964 from a cow grouping itself with those from Tamil Nadu. This could have come from a cow transported from Tamil Nadu to Kerala for slaughter. It is a regular practice that Kerala State getting their cattle from Tamil Nadu for their meat consumption through out the year. Most probably this cow may have been transported to Kerala and died due to rabies. However, this cannot be confirmed. While other isolates of Kerala group themselves with those from Karnataka, Andhra Pradesh (Figure 1).
While sequences from nucleotides 311-700 were not available for isolates from other parts of India, the Tamil Nadu isolates for which they were available again grouped themselves with the Sri Lankan ones, confirming their genetic relationship (Figure 2). The genetic divergence between samples from Sri Lanka and the most closely related Indian samples (Chennai) suggests a similarity distant common ancestor for these two virus populations. Rabies viruses in Sri Lanka and India may be more closely related than described elsewhere (Nanayakkara et 01.,2003, Arai et 01.,2001). However, although considerable genetic diversity was found in the available sequences for rabies virus from the Indian subcontinent only fifteen Indian isolates lineage shared a common ancestry with Sri Lankan samples. It is also interesting to find out that isolates in Iran, a Middle Eastern Country showing lineage with those in Far / South Eastern countries of Japan and Korea. However other South Eastern countries such as Thailand and Indonesia also cluster together. It is interesting that when the 5'- 45-310 region of nucleoprotein was used to analyze the reported sequences the isolates reported from Chennai and one isolate from Kerala (DQ105964) are related to Sri Lankan isolates. The isolates reported from other parts (Karnataka, Andhra Pradesh, Goa (UK citizen bitten while in India-DQ146162) and a reported sequence from an organ transplant from India) were closely related to the Korean isolates. Phylogenetic data suitable for compilation of a large epidemiological study were provided by using a shorter sequence, that of a 200-bp region of the N gene, in a study of 87 isolates collected from areas where dog rabies virus is enzootic in Asia, Africa, Europe and the Americas (Smith et 01., 1992). The analysis supports the earlier report of (Jayakumar et 01., 2004) that an Indian isolate from the state of Tamil Nadu was closely related to Sri Lankan isolates and fall in the Asian cluster. However, the results were analyzed with one isolate from India and the 5' 400 bp region of the N gene was used for analysis. Thus it appears that genetic heterogeneity exists between viruses in India although they could not be geographically specifically delineated. Two different genetic groups of rabies viruses appear to co-circulate, one primarily in Tamil Nadu and the other in the other regions of South India. It would be interesting to relate these viruses to the clinical signs of the viruses noticed. These two lineages also do not appear to be delineated in terms of species of animal affected. Further studies using a greater number of isolates from several states in India will be required in order to investigate more precisely the molecular epidemiological status of rabies viruses circulating in India. Such a genetic database would be useful for tracing the routes of rabies infection and for establishing measures to eliminate the rabies viruses. Acknowledgements The work was supported by the Indian Council of Agricultural Research,Government of India. We thank Drs. K. Thangaraj and Lalji Singh, CCMB, Hyderabad for their immense help rendered in the sequencing and the analysis. References
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