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Short communication

Phylogenetic analysis of nucleoprotein gene of dog rabies virus isolates fromSouthern India

T. Nagarajan, S.B. Nagendrakumar, B. Mohanasubramanian, S. Rajalakshmi, N.R. Hanumantha,R. Ramya, D. Thiagarajan, V.A. Srinivasan *

Indian Immunologicals Limited, Gachibowli, Hyderabad 500 032, India

1. Introduction

Rabies is considered to be a reemerging zoonosis in many partsof the world, particularly in Asia, Africa and Latin America wherethe disease is enzootic despite the availability of proven preventionand control tools. India has the dubious distinction among therabies enzootic Asian countries in reporting as high as 20,000human rabies deaths annually. Rabies in India occurs mainly in theurban form, in which dogs play an important role as the reservoirand transmitter of the disease to humans and domestic animals.Dog rabies is widespread in India with Lakshadweep, Andamanand Nicobar islands being the exceptions (Sudarshan et al., 2007).India has an estimated stray dog population of 27 million with adog–human ratio of 1:40 (Cliquet et al., 2007). Majority of the dogsare stray, ownerless and unprotected which live in and aroundhuman dwellings. This is why more than 95% of the diagnosedrabies cases have been attributed to dog bites.

Rabies virus (RV; genus Lyssavirus, family Rhabdoviridae)possesses a single-stranded, non-segmented, negative-senseRNA approximately 12 kb in length (Tordo et al., 1986). The viralgenome encodes five structural proteins: nucleoprotein (N),

phosphoprotein (P), matrix protein (M), glycoprotein (G), andRNA-dependent RNA polymerase (L) in the order of 30-N-P-M-G-L-50 (Wunner et al., 1988). RNA viruses are characterized by a highmutation rate during replication due to the lack of proofreadingand post-replication error correction by the RNA polymerase(Domingo and Holland, 1994). The genetic diversity seems toprovide an adaptive potential for RV that can vary according to thenatural history of the virus (Morimoto et al., 1998; Kissi et al.,1999). The N protein is a major component of the virus and themajor protein of the helical nucleoprotein which is abundantlyexpressed in the host after infection by RV (Smith et al., 1992).

Study of canine rabies dynamics in the field would help tounderstand the genetic variants in circulation and their evolu-tionary relationships thereby helping to devise effective controlmeasures. The genetic typing technique, which employs RT-PCR,provides an opportunity to elucidate the epidemiologic andevolutionary relationships between rabies virus (RV) and rabiesrelated viruses (RRVs) (Smith et al., 1992; Nadin-Davis et al., 1994;Kissi et al., 1995). To date, genomic and evolutionary studies havemost often targeted the N and G genes of RV (Sacramento et al.,1991; Kamolvarin et al., 1993; Arai et al., 1997; Nadin-Davis et al.,1999; Badrane et al., 2001; Real et al., 2005; Khawplod et al., 2006;Nagarajan et al., 2006; Sato et al., 2006) and most often utilizedpartial genome sequences (Delmas et al., 2008). The N gene,although highly conserved allows viral strains to be accurately

Infection, Genetics and Evolution 9 (2009) 976–982

A R T I C L E I N F O

Article history:

Received 13 February 2009

Received in revised form 1 April 2009

Accepted 7 April 2009

Available online 15 April 2009

Keywords:

Canines

Epidemiology

India

N gene

Phylogeny

Rabies

A B S T R A C T

India like several other South East Asian and African countries continues to face the public health and

economic problems associated with the disease. Our objective was to perform a limited sequence

analysis of a portion of nucleoprotein gene of 22 rabies virus isolates obtained from domestic animals in

Southern India during 2004–2005. These isolates were compared with rabies virus isolates originating

from Asia, Europe, Africa and North America. The phylogenetic analysis showed that RV isolates in

Southern India belong to genotype 1. They were similar to one another forming a single major genetic

cluster not ordered by geography or species of origin. However, they were dissimilar to RV isolates in

Northern India and in other parts of the world. The data indicated that dog rabies virus variants are the

major circulating viruses and control of dog rabies would result in overall reduction in the burden and

incidence of rabies in India.

� 2009 Elsevier B.V. All rights reserved.

* Corresponding author. Tel.: +91 40 23000894; fax: +91 40 23005958.

E-mail address: srini@indimmune.com (V.A. Srinivasan).

Contents lists available at ScienceDirect

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1567-1348/$ – see front matter � 2009 Elsevier B.V. All rights reserved.

doi:10.1016/j.meegid.2009.04.004

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differentiated by analyzing genetic differences that are presentwithin the gene (Johnson et al., 2002). To date, many molecularepidemiological studies have been performed targeting either to aselected region of the N gene or to the entire N gene at regional(Jayakumar et al., 2004, 2006; Nadin-Davis et al., 2007; Yamagataet al., 2007) and global levels (Smith et al., 1992; Kissi et al., 1995;Arai et al., 1997; Ito et al., 2001; Johnson et al., 2002; Nishizonoet al., 2002; Davis et al., 2005; Kobayashi et al., 2005; Mansfieldet al., 2005).

In our continuing efforts to help the understanding of dog rabiesin India better and to provide much needed and basic informationon rabies locally, phylogenetic analysis of a partial region of the Ngene has been done, for a panel of rabies virus isolates representingdistinct geographical regions of Southern India and has beencompared with the other RVs of Indian and exotic origin.

2. Materials and methods

2.1. Rabies viruses

Twenty-two brain tissue samples from three states in SouthernIndia (Kerala, Karnataka, and Andhra Pradesh) were collectedbetween 2004 and 2005 from dogs (14 isolates), cattle (5 isolates),and goats (3 isolates). A 20% homogenate of the brain tissuesamples was prepared in sterile phosphate-buffered salinecontaining 2% horse serum and was stored in the vapor phase ofliquid nitrogen until further use. Those samples which testedpositive by both the fluorescent-antibody test (Dean et al., 1996)and the mouse inoculation test (Koprowski, 1996) were used fornucleotide sequencing (Table 1).

2.2. RNA extraction and RT-PCR

The total RNA was extracted from the brain tissue homogenatesusing the TRIzol1 reagent (Invitrogen, USA), according to themanufacturer’s instructions. The extracted RNA was immediatelyused for RT-PCR with gene-specific primers as described pre-viously (Johnson et al., 2004). Forward primer JW12 (50-ATG TAACAC CTC TAC AAT G-30), which corresponds to bases 55–74 of thepositive-sense N gene, and reverse primer JW6DPL (50-CAA TTAGCA CAC ATT TTG TG-30), which corresponds to bases 660–641 of

the negative-sense N gene sequence of the Pasteur virus (PV) strain(Tordo et al., 1986) were used for RT-PCR. Briefly, about 1 mg oftotal RNA was used for RT-PCR (One-Step RT-PCR kit; QIAGEN,Germany), according to the manufacturer’s instructions. One cycleof reverse transcription was done at 50 8C for 60 min, followed bydenaturation at 94 8C for 10 min. PCR was followed by 35 cycles ofdenaturation at 94 8C for 30 s, primer annealing at 55 8C for 40 s,and extension at 72 8C for 1 min. Finally a 10-min extension step at72 8C was done to complete the amplification of the target. TheJW12 and JW6DPL primer pair amplified a 605-bp amplicon.

2.3. Nucleotide sequencing and phylogenetic analysis

The amplicons were purified with a QIAquick PCR gel extractionkit (QIAGEN, Germany) and were sequenced with the gene-specificprimers in an automated sequencer (ABI, USA) using BigDyeTerminator cycle sequencing ready reaction kit—v3.1 (ABI, USA).For each RV isolate, the sequences were trimmed to include a 270-nucleotide region of N gene corresponding to bases 238–507 of thePV genome (Tordo et al., 1986), for comparison with the N genesequences available in the GenBank. The nucleotide sequenceswere aligned by using the ClustalW 1.8x program (Thompson et al.,1997), and the neighbor-joining tree (NJ tree) was drawn by usingthe MEGA version 3.1 program (Kumar et al., 2004), withconfidence levels assessed by the use of 1000 bootstrap replica-tions. The RV and RRV sequences available in the GenBankoriginating from different parts of the world like North American(USA, Canada, and Mexico), European (France, Poland, Russia andYugoslavia), Asian (India, China, Thailand, Philippines, Iran, SaudiArabia, Oman, and Nepal), and African (South Africa, Nigeria, Egypt,Tanzania and Ethiopia) continents (Table 2) were included in themultiple-sequence alignment and subsequent comparison usingthe NJ tree. Deduced amino acid sequences were used to comparethe variation in amino acid sequences among the RVs and RRVs.

3. Results

3.1. Analysis of partial N gene

The sequences of the partial N gene of all the 22 RV isolateswere aligned with the sequences of representative RVs and RRVs.

Table 1Epidemiological information for the rabies virus isolates. For each of the 22 RV isolates studied in this paper, the host species, place of origin, year of isolation, as well as the

GenBank accession numbers for the nucleotide sequences of a portion of N gene.

S. no. Virus ID Virus reference in

phylogenetic tree

Host species Place of origin Year of isolation GenBank accession no.

1 R76 INDIA-KETr-DOG1 Dog Thrissur, Kerala 2004 DQ105943

2 R81 INDIA-KETr-DOG2 Dog Thrissur, Kerala 2004 DQ105944

3 R82 INDIA-KETr-GOAT1 Goat Thrissur, Kerala 2004 DQ105945

4 R98 INDIA-KETr-GOAT2 Goat Thrissur, Kerala 2004 DQ105946

5 R105 INDIA-KETr-DOG3 Dog Thrissur, Kerala 2004 DQ105947

6 R107 INDIA-KETr-DOG4 Dog Thrissur, Kerala 2004 DQ105948

7 R109 INDIA-KETr-DOG5 Dog Thrissur, Kerala 2004 DQ105949

8 R110 INDIA-KETr-CATTLE1 Cattle Thrissur, Kerala 2004 DQ105950

9 R114 INDIA-KETr-CATTLE2 Cattle Thrissur, Kerala 2004 DQ105951

10 R116 INDIA-KETr-DOG6 Dog Thrissur, Kerala 2004 DQ105952

11 R117 INDIA-KETr-DOG7 Dog Thrissur, Kerala 2004 DQ105953

12 R121 INDIA-KETr-GOAT3 Goat Thrissur, Kerala 2004 DQ105954

13 R122 INDIA-KETr-CATTLE3 Cattle Thrissur, Kerala 2004 DQ105955

14 R155 INDIA-KEQ-CATTLE Cattle Quilon, Kerala 2005 DQ105964

15 R132 INDIA-KAB-DOG1 Dog Bangalore, Karnataka 2005 DQ105956

16 R134 INDIA-KAB-DOG2 Dog Bangalore, Karnataka 2005 DQ105957

17 R135 INDIA-KAB-CATTLE Cow Bangalore, Karnataka 2005 DQ105958

18 R141 INDIA-APH-DOG Dog Hyderabad, Andhra Pradesh 2005 DQ105959

19 R142 INDIA-KAB-DOG3 Dog Bangalore, Karnataka 2005 DQ105960

20 R143 INDIA-KAB-DOG4 Dog Bangalore, Karnataka 2005 DQ105961

21 R144 INDIA-KAB-DOG5 Dog Bangalore, Karnataka 2005 DQ105962

22 R145 INDIA-KAB-DOG6 Dog Bangalore, Karnataka 2005 DQ105963

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The NJ tree showed that all the South Indian isolates were relatedto RV isolates belonging to genotype 1 (<10% difference) and wereunrelated to RRVs (>15% difference). The South Indian RV isolatesshowed >95% sequence homology even though they werecollected from distinct geographical regions, different host speciesand in different time periods (Fig. 1).

Among the Indian RVs there were three distinct geneticclusters, viz., GC1, GC2 and GC3 (Fig. 1). These three geneticclusters GC1, GC2 and GC3 are very strongly supported bybootstrap values 95.2%, 61.0%, 100%, respectively. RV isolates ofdog, human, cattle and goat origin from three different states, viz.,Kerala, Karnataka and Andhra Pradesh in Southern India, formed asingle major GC1, whereas RV isolates of dog, cattle and cat originfrom Himachal Pradesh, in Northern India formed GC2. Two RVisolates of water buffalo and cat origin from Sri Lanka and one RVisolate of dog origin from Tamil Nadu and one RV isolate of cattleorigin from Kerala formed GC3.

GC1 consisted of 14 RV isolates from dogs, cattle and goats fromthe state of Kerala collected in 2004 and 2005, seven RV isolatesfrom dogs and cattle from the state of Karnataka collected in 2005and one RV isolate of dog origin from the state of Andhra Pradeshcollected in 2005. Apart from these, GC1 also consisted of RVisolates of dogs and humans from the state of Karnataka collected

in 2001–2004 and 2004, respectively and of humans from the stateof Andhra Pradesh collected in 2004. The geographical locations ofthe different genetic clusters of Indian RV isolates are provided inFig. 2.

Analysis of the deduced N terminus amino acid sequences ofnucleoprotein (Fig. 3) revealed that South Indian RV isolatesdiffered from North Indian RV isolates at amino acid 134 (aa134)(I! V). All the South Indian RV isolates exhibited 100% amino acidhomology except two isolates, one from goat and another fromcattle. The goat RV isolate differed at aa168 (R! S) while the cattleRV isolate differed from other South Indian isolates at aa84 (T! S),aa98 (R! Q) and aa135 (P! S). Arctic lineage isolates and theisolates of Europe (ME) and Estonia differed from Indian RV isolatesby a unique non-synonymous substitution at aa115 (D! G).

4. Discussion

Twenty-two brain tissue samples collected from dogs, cattle,and goats over a period of 2 years (2004–2005) were used in thisstudy. They were collected from three states in Southern India. Inthis study, we analyzed a limited sequence of a region on the Ngene from nucleotides 238 to 507. This region corresponds roughlyto a variable region that is present on the N gene from nucleotides

Table 2Epidemiological information for the rabies and rabies related virus isolates available in the GenBank and included for comparison in this study.

S. no. Virus reference Host species Place of

origin

Accession no. S. no. Virus reference Host species Place of

origin

Accession no.

1 INDIA-CATTLE Cattle India AY854599 45 CAN-SH-BAT SH Bat Canada AF351834

2 INDIA-DOG1 Dog India DQ521215 46 USA-SKUNK Skunk USA AF461045

3 INDIA-DOG2 Dog India DQ521216 47 SAF-MONGOOSE Mongoose South Africa AF467949

4 INDIA-DOG3 Dog India DQ521217 48 KRG-BAT Bat Kyrgyzstan AY262023

5 INDIA-DOG4 Dog India DQ521218 49 CANADA-FOX1 Fox Canada L20672

6 INDIA-DOG5 Dog India DQ521219 50 CANADA-ARCTIC FOX Arctic Fox Canada L20673

7 INDIA-DOG6 Dog India DQ521220 51 CANADA-FX2 Fox Canada U03768

8 INDIA-DOG7 Dog India DQ521221 52 CANADA-DOG1 Dog Canada U03769

9 INDIA-DOG8 Dog India DQ521222 53 CANADA-DOG2 Dog Canada U03770

10 INDIA-DOG9 Dog India DQ521223 54 FRANCE-RV Unknown France U22474

11 INDIA-DOG10 Dog India DQ521224 55 RV-9212ALL Unknown ALL U224745

12 INDIA-DOG11 Dog India DQ521225 56 MEXICO-RV Unknown Mexico U22477

13 INDIA-DOG12 Dog India DQ521226 57 FR-GUIANA-DOG Dog French Guiana U22478

14 INDIA-DOG13 Dog India DQ521227 58 86118BRE-RV Unknown BRE U22479

15 INDIA-DOG14 Dog India DQ521228 59 OMAN-REDFOX REDFOX OMA U22480

16 INDIA-DOG15 Dog India DQ521229 60 SAU-REDFOX REDFOX Saudi arabia U22481

17 INDIA-DOG16 Dog India DQ521231 61 IRAN-WOLF1 WOLF Iran U22482

18 INDIA-DOG17 Dog India DQ521234 62 IRAN-WOLF2 WOLF Iran U22483

19 INDIA-DOG18 Dog India DQ521235 63 NIGERIA-RV Unknown Nigeria U22488

20 INDIA-DOG19 Dog India DQ521236 64 EGYPT-RV Unknown Egypt U22627

21 INDIA-DOG20 Dog India DQ521237 65 87012MAR Unknown MAR U22631

22 INDIA-DOG21 Dog India DQ521238 66 ETHIOPIA-RV Unknown Ethiopia U22637

23 INDIA-DOG22 Dog India DQ521239 67 TANZANIA-RV Unknown Tanzania U22645

24 INDIA-DOG23 Dog India DQ521240 68 THAILAND-RV Unknown Thailand U22653

25 INDIA-DOG24 Dog India DQ521241 69 8684GRO Unknown GRO U22654

26 INDIA-DOG25 Dog India AF374721 70 RUSSIA Unknown Russia U22656

27 INDIA-HUMAN1 Human India DQ521242 71 86106YOU Unknown YOU U22839

28 INDIA-HUMAN2 Human India DQ521243 72 POLAND-RV Unknown Poland U22840

29 INDIA-HUMAN3 Human India DQ521244 73 CANADA-RAC2 Raccoon Canada U27221

30 INDIA-HUMAN4 Human India DQ521245 74 EUROPE-ME Unknown Europe (ME) U42707

31 INDIA-HUMAN5 Human India DQ521246 75 9142EST Unknown EST U22476

32 INDIA-HUMAN6 Human India DQ521247 76 Mokola Mokola Ethiopia AY333111

33 INDIA-HUMAN7 Human India DQ521248 77 LBV Lagos Bat Ethiopia AY333110

34 INDIA-HUMAN8 Human India DQ521249 78 WCBV W. Caucasian Bat – AU333113

35 INDIA-HUMAN9 Human India DQ521250 79 IBV Irkut Bat Virus – AY333112

36 INDIA-HUMAN10 Human India DQ521251 80 EBLV European Bat Lyssavirus – AY062089

37 SRILANKA-RV Water Buffalo Sri Lanka AB041969 81 DUV Duvenhage virus South Africa AY062081

38 CHINA-RV1 Unknown China AF155039 82 PV Pasteur virus – NC001542

39 CANADA-SKUNK Skunk Canada AF344304 83 CVS Challenge virus standard – AF406696

40 CANADA-RAC1 Raccoon Canada AF351826 84 SRILANKA-CAT Cat Sri Lanka AB041968

41 RUSSIA-ARCTIC FOX Arctic Fox Russia U22656 85 RUSSIA-RODENT Rodent Russia AY352480

42 RUSSIA-HUMAN1 Human Russia AY352497 86 NEPAL-DOG Dog Nepal U22918

43 RUSSIA-HUMAN2 Human Russia AY322475 87 RUSSIA-FOX Fox Russia AY352464

44 RUSSIA-DOG Dog Russia AY352476 88 PHILIPPINES-RV Unknown Philippines AB070772

89 CHINA-RV2 Unknown China AF387863

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Fig. 1. Neighbor-joining tree showing the genetic relationship of Indian RV isolates as determined by analysis of 270 nucleotides coding for N gene.

T. Nagarajan et al. / Infection, Genetics and Evolution 9 (2009) 976–982 979

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99 to 405 (Kissi et al., 1995) and a region that is capable ofdistinguishing isolates at a sub-continental scale (Johnson et al.,2002). In Southern India, rabies is neither region specific nor hostspecific, as is apparent from the phylogenetic tree. Phylogeneticanalysis of the nucleotide sequence of partial N gene of various RVisolates of Southern and Northern India revealed the existence ofthree different genetic clusters, viz., GC1, GC2, and GC3. The RVisolates of dog, human, cattle and goat origin from three states inSouthern India, irrespective of region and species of animal originformed a single major GC1. The RV isolates of dogs and humanscollected from two states in Southern India such as Karnataka andAndhra Pradesh during different time periods reported elsewhere(Nadin-Davis et al., 2007) grouped with GC1. Limited differencesobtained between viruses from a specific area are thought to be aconsequence of the high level of adaptation required for thoseviruses in order to persist in their host populations; highlyselective pressure apparently operate to limit viral geneticvariation (Nadin-Davis et al., 2006). It is intriguing to note thatRV isolates of dog, cattle and cat origin from Himachal Pradesh, inNorthern India formed GC2. This clearly shows the geneticclustering ordered by geography which is in agreement with ourprevious report which involved phylogenetic analysis of G-CD andPsi-L regions of RV isolates from Southern India (Nagarajan et al.,2006). The genetic isolation of GC1 and GC2 can be attributed to

the distinct geographical features such as mountainous ranges(Vindhya and Sadhpura), valleys and major perennial rivers(Narmada, Godavari and Krishna) which may create physicalbarriers to animal movement and promote localized viralevolution in specialized host niches (Bourhy et al., 1999). Further,the phylogeographical structure of the genetic clusters designatedGC1 and GC2 revealed strong population subdivision, with onlylimited movement of virus among localities (Talbi et al., 2009)including north to south spread.

Besides, two South Indian isolates (INDIA-DOG25 from TamilNadu and INDIA-KEQ-CATTLE from Kerala) along with the two SriLankan isolates of water buffalo and cat origin formed GC3. It wasnot surprising to see the grouping of an isolate INDIA-KEQ-CATTLEfrom the state of Kerala in Southern India with GC3, as similargrouping of a South Indian isolate from Tamil Nadu and a SriLankan isolate has already been reported (Arai et al., 2001;Nanayakkara et al., 2003; Jayakumar et al., 2004, 2006) indicatingthe possibility of RV isolates crossing the Palk Strait between thetwo countries as speculated by Arai et al. (2001). We report here forthe first time the close relatedness of a RV isolate from ageographically distinct state of Kerala located on the west coast ofpeninsular India to Sri Lankan isolates. It is presumable that similarRV isolates may be prevalent among domestic animals in SouthernIndia.

Fig. 2. The map of India showing the different geographical locations of the different clusters of RV isolates of Indian origin.

T. Nagarajan et al. / Infection, Genetics and Evolution 9 (2009) 976–982980

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Analysis of the deduced N terminus amino acid sequences ofnucleoprotein revealed a marker within the genome thatdifferentiated South Indian RV isolates from North Indian RVisolates (aa134; V! I) and the Indian RV isolates from the arcticlineage RV isolates (aa115; D! G).

In India, rabies is enzootic in dogs, which plays a major role inmaintenance and transmission of the disease (Cliquet et al., 2007).They account for more than 95% of the diagnosed human cases.Although RV adapts to different hosts for persistence (Wandeleret al., 1994), dogs continue to remain a single major reservoir inIndia. The data presented here support the fact that dog RV variantsare the single major variants circulating in India (Nagarajan et al.,2006). Millions of dogs live in close contact with humans since theyare well tolerated in the community. When a population density ishigh and/or the animals migrate, the contact rate would increaseand it was postulated that there was a positive correlation betweenthe prevalence of rabies and the population density (Mork andPrestrud, 2004). The relative abundance, natural history, andassociation with humans of dogs influence incidence of dog bites.Several thousands of human rabies deaths reported annually can

be meaningfully reduced provided a nationwide program foreffective dog rabies control is in place. It is clear that changes inpopulation may be particularly important since higher populationdensity, a constant supply of susceptible hosts and the closeproximity of donor species (Holmes et al., 2002), inadequate herdimmunity, and unrestricted animal movement could be thepossible reasons for predominance and persistence of dog RVcirculation in India. Induction of herd immunity through massvaccination practices with either parenteral administration ofrabies vaccines or by use of oral rabies vaccines. However, oralrabies vaccination can be a more practical and a cost-effectivemethod to achieve a progressive reduction in the incidence of dograbies (Cliquet et al., 2007) in developing countries.

In summary, dogs continue to remain the principal reservoir ofRV. The RV isolates of Southern India formed a single major geneticcluster not apparently ordered by region or species of originindicating the existence of a dog variant which got persisted due tohigh level of adaptation to the host population. Continued analysisof more samples from different geographical regions at varyingtime periods would give an opportunity to better understand the

Fig. 3. Alignment of deduced amino acid sequences of the N-terminus nucleoprotein of selected rabies and rabies related virus isolates showing amino acid positions 80–170

of the N gene (based on the PV sequence).

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rabies dynamics to achieve reduction of rabies burden and itsincidence. Studies on other genome targets (like the P gene, fulllength G and N genes) must also be carried out to furthercharacterize the RV isolates of Indian origin.

Acknowledgements

The authors thank Dr. M.R. Saseendranath, Professor and Head,Department of Veterinary Epidemiology and Preventive Medicine,College of Veterinary Sciences, Mannuthy and Dr. S. Raju,Veterinary Surgeon, District Veterinary Center, Quilon for provid-ing with the rabies suspected samples.

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