on a november morning in 2009 , a  wild cat  appeared in the garden of a basic health clinic in moneragala district , uva province , sri lanka . the animal , which showed aggressive behavior , was suspected to be rabid and was thus killed to prevent transmission of rabies virus to humans . the animal s head was packed in ice to avoid decomposition and sent to the medical research institute ( colombo , sri lanka ) for testing . we detected rabies virus in the animal s brain by using the fluorescent antibody test and extracted viral rna and dna by using trizol ( invitrogen , carlsbad , ca , usa ) . the whole genome of the virus was sequenced directly from the sample as described ( 5 ) . to confirm the species of the rabid animal , we determined the nucleotide sequence of the mitochondrial cytochrome b ( cytb ) gene and performed a blast search ( www.ncbi.nlm.nih.gov/blast/blast.cgi ) for similarity with other sequences . by aligning nucleotide sequences of the cytb gene of mitochondrial dna of domestic cat , jungle cat , fishing cat , asiatic golden cat , marbled pole cat , european pole cat , lynx , puma , leopard , african lion , tiger , jaguar , civet , and palm civet with clustalw2 ( www.ebi.ac.uk/clustalw ) , we designed primer felis cytb - f , 5-atgaccaacattcgaaaatcacacc-3 ( nt 125 ) , and primer felis cytb - r , 5-caataatgcctgagatgggtattag-3 ( nt 10931,117 ) . using these primers , we performed pcr as follows : initial denaturation at 94c for 2 min , followed by 94c for 30 s , 55c for 30 s , and 72c for 1 min for 35 cycles , followed by a final extension at 72c for 5 min . analysis showed that the sequence has 100% identity with the partial ( 224-nt ) sequence of the cytb gene of p. zeylonensis ( genbank accession no . the sequence also has 95% identity with p. jerdoni and 90%92% identity with p. hermaphroditus . we inferred a maximum clade credibility phylogenetic tree by using the bayesian markov chain monte carlo method available in beast version 1.6.1 ( 6 ) . the analysis used a relaxed ( uncorrelated lognormal ) molecular clock and gtr +  + i model of nucleotide substitution . we selected the model on the basis of akaike information criterion by using jmodeltest software ( 7 ) . this procedure resulted in an effective sample size of > 2,000 for all estimated parameters . the posterior densities were calculated with 10% burn - in and checked for convergence by using tracer version 1.5 ( http://beast.bio.ed.ac.uk/main_page ) . the mean rate of nucleotide substitution estimated for the n gene was 2.2  10 substitutions / site / year ( 95% highest posterior density [ hpd ] values 1.33.2  10 substitutions / site / year ) . approximately 155.5 years ago ( 95% hpd 91.3249.5 years)that is , circa 1854 ( 95% hpd range 17601918)rabies viruses from sri lanka and southern india diverged from their most recent common ancestor . approximately 76.4 years ago ( 95% hpd 46.4123.0 years)that is , in 1933 ( 95% hpd range 18861963)strain h-1413 - 09 diverged from canine rabies virus in sri lanka . compared with the genome sequence of rabies virus strain h-08 - 1320 from sri lanka , the genome sequence of strain h-1413 - 09 had a nucleotide deletion at residue 2,417 and an addition at nt 11,807 . as a result , the start and stop signals of the mrna and the start and stop positions of coding sequences of different genes were altered ( table 1 ) . these altered start and the p - m region and terminal sequence of strain h-08 - 1320 were 1 nt shorter ( 91 nt ) and longer ( 136 nt ) , respectively . the nucleotide and amino acid identities between the coding regions of strains h-1413 - 09 and h-08 - 1320 are shown in table 1 . the substitutions detected in the deduced amino acid sequences of h-1413 - 09 were compared with the genomic sequence of h-08 - 1320 ( table 2 ) . by using the mega5 tamura - nei model ( www.megasoftware.net ) , we determined the genetic distance of the n gene to determine whether strain h-1413 - 09 is more diverse than other rabies viruses in sri lanka ( figure ) . the rate of variation among sites was modeled with a gamma distribution ( shape parameter  =  0.5 ) . the genetic distances between strain h-1413 - 09 and other rabies viruses from sri lanka ( 0.0270.036 ) were greater than the genetic distances among other rabies viruses ( 0.0010.011 ) . these results support our finding that strain h-1413 - 09 differs from other rabies viruses circulating in sri lanka . * h-08 - 1320 is a human strain typical of canine rabies virus circulating in sri lanka ; h-1413 - 09 is a novel sylvatic rabies virus variant from a golden palm civet in sri lanka . * h-08 - 1320 is a human strain typical of canine rabies virus circulating in sri lanka ; h-1413 - 09 is a novel sylvatic rabies virus variant from a golden palm civet in sri lanka . blank spaces indicate no site / domain / region has been identified in that portion of the protein . bayesian maximum - credibility tree representing the genealogy of rabies virus as obtained by analyzing nucleotide sequences of full n gene sequences ( 1,350 nt ) . nodes correspond to mean age at which lineages are separated from the most recent common ancestor ; blue horizontal bars at nodes represent 95% highest posterior density of the most recent common ancestor . red arrow indicates strain h-1413 - 09 ; blue arrow indicates strain h-08 - 1320 . nucleotide sequence data for strains from sri lanka appear in nucleotide sequence databases of dna databank of japan , european molecular biology laboratory , and genbank with accession no . ab635373 ( rabies virus strain h-1413 - 09 ) , ab638767 ( strain h-219 - 08 ) , ab638768 ( strain h-1218 - 08 ) , ab638769 ( strain h-1281 - 08 ) , ab638770 ( strain h-15 - 09 ) , ab638771 ( strain h-15609 ) , ab638772 ( strain h-1366 - 09 ) , and ab636165 ( golden palm civet [ paradoxurus zeylonensis ] strain h-1413 - 09 ) . rabies virus probably survives favorably in the wild because it can infect a large spectrum of animals , thereby maximizing replication and dispersal opportunities ( 9 ) . most viruses replicate poorly when transferred to new hosts , but greater genetic variation assists in such species adaptation ( 10 ) . increased mutation in an rna virus like rabies virus can give rise to variants with altered levels of fitness to persist and spread . a large number of substitutions were found in strain h-1413 - 09 compared with strain h-08 - 1320 ; these substitutions might represent changes that resulted from species adaptation . phylogenetic analysis and comparative sequence data indicated that strain h-1413 - 09 is a variant rabies virus . palm civets are facing extinction in sri lanka because the species is losing its habitat , being hunted for its meat , and dying of parasitic diseases ( www.sundaytimes.lk/090118/plus/sundaytimesplus_01.html ) . our study indicates that rabies might be another risk factor for extinction of these animals . identification of a variant rabies virus in wildlife has serious implications for rabies control in sri lanka . identification of such a virus would help provide epidemiologic data about the spread of rabies and its incursion into new geographic regions and would justify allocation of increased resources to help control rabies ( 11,12 ) . several rabies virus variants associated with wildlife are known in the americas and africa ( 1,1315 ) , and this report identified classical sylvatic rabies in asia . whether p. zeylonensis is a reservoir of rabies virus or represents spillover from another animal deserves extensive investigation . the detection of rabies in wildlife indicates that much remains to be discovered in the tropical ecosystem of sri lanka . the circulation of a sylvatic variant rabies virus may be another hurdle in the rabies - control effort in sri lanka .
information is scarce about sylvatic rabies virus in asia and about rabies in palm civets . 
 we report a novel sylvatic rabies virus variant detected in a golden palm civet in sri lanka . 
 evolutionary analysis suggests the virus diverged from canine rabies viruses in sri lanka in 1933 ( range 18861963 ) .