we sequenced 12 bat brains positive for lyssavirus antigen detected by immunofluorescence and reverse transcription the 400-bp 5 variable extreme of the nucleoprotein gene of these eblv-1 strains was amplified by specific eblv-1 nested rt - pcr and sequenced by using the following primers : seqvar1f 5-1acgcttaacaaccagatcaaag22 - 3 , seqvar2f 5-51aaaaatgtaacacyyctaca70 - 3 , eblvseqvar1r 5-596cagtctcaaagatctgttccat575 - 3 , and eblvseqvar2r 5-552tagttcccagtattctgtcc533 - 3. all rabies - positive serotine bats came from southern spain ( huelva , seville , murcia , and badajoz ) and were molecularly identified as e. isabellinus ( 8) . an alignment was performed by using clustalx ( www.clustal.org ) to combine the obtained sequences and other available eblv-1 sequences from genbank , including a duvenhage virus used as the outgroup ( table a1 ) . before conducting further analyses , we used jmodeltest ( http://darwin.uvigo.es/software/jmodeltest.html ) to select the best fitting substitution model for our sequences according to the corrected akaike information criterion . maximum - likelihood phylogenies were reconstructed by using phyml ( http://atgc.lirmm.fr/phyml ) software and by using a generalized time - reversible model and the  parameter estimated in the analyses . maximum - parsimony analyses were conducted by using paup * 4.0b10 ( http://paup.csit.fsu.edu/ ) weighting transversions 15 according to the transitions / transversion ratio estimated in the jmodeltest analyses . confidence in the topologies for the maximum - likelihood and the maximum - parsimony analyses was established with 1,000 bootstrap replicates . a bayesian phylogenetic inference was obtained by using mrbayes version 3.1 ( http://mrbayes.csit/fsu.edu/ ) with random starting trees without constraints . two simultaneous runs of 10 generations were conducted , each with 4 markov chains , and the trees were sampled every 100 generations . net p - distances between groups were calculated by using mega4 ( www.megasoftware.net ) ( figure 1 ) . european bat lyssavirus 1 ( eblv-1 ) phylogenetic reconstruction based on the first 400 bp of the nucleoprotein gene . the tree was obtained by bayesian inference run for 10 generations ; trees were sampled every 100 generations . bootstrap supports after 1,000 replicates for each node are also shown for maximum - parsimony ( green numbers ) and maximum - likelihood ( blue numbers ) analyses . net p - distance values ( as percentages ) between groups are indicated by arrows . a parsimony - based network is presented for each major lineage ; sizes of yellow circles are proportional to the number of individuals sharing a given haplotype , and reconstructed haplotypes ( median vectors ) are shown in red . duvv , duvenhage virus . the genetic structure and relationships between haplotypes were examined within the main lineages through a parsimony - based network built with a median - joining algorithm implemented in the network 4.5.1 program ( 11 ) . to evaluate and compare genetic variability and polymorphism among lineages , we estimated the number of haplotypes , mutations , and segregating sites as well as haplotype diversity and nucleotide diversity by using dnasp version 4.5 ( 12 ) for the major clades ( table ) . finally , to investigate population dynamics across lineages , the fu fs and tajima d statistics were calculated ( table ) . these 2 statistics are considered to be the most powerful tests for detecting expansion events ( 13 ) . * eblv , european bat lyssavirus ; n , no . sequences ; s , no . segregating sites ; eta , no . mutations ; hap , no . haplotypes ; hd , haplotype diversity ; varhd , haplotype variance ; pi , nucleotide diversity ; thetanuc , estimated population mutation rate per site ; k , average no . all phylogenetic analyses , regardless of the reconstruction criterion used , formed a monophyletic cluster of the eblv-1 strains from spain ( only the bayesian inference reconstruction is shown ) . the bayesian inference , maximum - likelihood , and maximum - parsimony analyses identified the cluster from spain and eblv-1a and eblv-1b as being monophyletic ( figure 1 ) , although only maximum - likelihood and maximum - parsimony analyses suggested a closer relationship between eblv-1a and the cluster from spain . the genetic differentiation of the eblv-1 strains from the iberian peninsula matches their association with another bat species ( figure 2 ) , which suggests that the host bat s evolutionary history plays a major role in eblv-1 molecular epidemiology , as has been proposed for rabies virus in bats in north america ( 14 ) . geographic distribution of eptesicus serotinus bats ( red ) , e. isabellinus bats ( blue ) , and cases of rabies in bats ( green dots ) , europe , 19902009 . obtained from rabies bulletin europe ( www.who-rabies-bulletin.org/ ) . the low genetic diversity and the fu fs and tajima d statistics ( table ) all suggest rapid population expansion of eblv-1a , which is consistent with the star - like structure of the network for this lineage ( figure 1 ) . conversely , haplotype and nucleotide diversity descriptors ( table ) have the highest values for eblv-1b and a complex network structure with differentiated subnetworks . the lineage from spain also has low diversity and a star - shaped network , but neutral evolution can not be rejected on the basis of the fs and d statistics . net distances are similar within and between lineages , except for eblv-1a , which is slightly more differentiated ( figure 1 ) . consequently , the suggested eblv-1 expansion from spain into europe ( 15 ) is not supported by our results , which record the highest variability and most complex phylogenetic structure for france and the netherlands ( figure 1 ) . this complex structure suggests either a longer evolutionary history in these areas or a recent contact of distinct bat lineages in this zone . the results of this study show that the strains from spain do not belong to subtype 1b because of their association with a different reservoir ( e. isabellinus bats ) . moreover , what is currently considered to be eblv-1b seems to include at least 4 lineages that are more genetically diverse and have a complex history . eblv-1a , however , has low genetic diversity despite its extensive geographic distribution , suggesting a relatively recent and successful expansion of this lineage . these results call into question the current classification of eblv-1 into 2 single subtypes . to provide a better understanding of eblv-1 molecular epidemiology in europe , additional studies that consider different genes should be conducted and the current classification should be revised accordingly .
to better understand the epidemiology of european bat lyssavirus 1 ( eblv-1 ) in europe , we phylogenetically characterized lyssavirus from eptesicus isabellinus bats in spain . an independent cluster of eblv-1 possibly resulted from geographic isolation and association with a different reservoir from other european strains . 
 eblv-1 phylogeny is complex and probably associated with host evolutionary history .