during 2002 through 2008 , a total of 366 apodemus mice were trapped at 7 different sites in germany ( figure 1 ) . serologic screening of transudates collected from these rodents by using an in - house dobv immunoglobulin ( ig ) g - elisa , with a yeast - expressed nucleocapsid protein of dobv - af as antigen , identified 16 reactive and 5 equivocal samples of 114 a. agrarius trapped at 7 trapping sites in 3 federal states of germany ( figure 1 ; technical appendix ) . additionally , of 237 a. flavicollis mice , 1 equivocal sample and 4 dobv - reactive samples were detected at 4 trapping sites ( figure 1 ; technical appendix ) . in contrast , of 15 wood mice ( a. sylvaticus ) originating from 3 trapping sites , none were found to be dobv - seroreactive . a subsequent focus - reduction neutralization test showed a higher endpoint titer with dobv - aa than dobv - af ( technical appendix ) for 6 of the 8 investigated transudates independently , whether originating from a. agrarius or a. flavicollis . seroprevalence of dobrava - belgrade virus ( dobv ) in apodemus 
 agrarius mice within 3 federal states of germany , central europe . wg , lneburg district , lower saxony ( ls ) ; pe1 and pe3 , gstrow district ; h , nordvorpommern district , k / a1 , demmin district , all mecklenburg - western pomerania ( mwp ) ; to and ka , ostprignitz - ruppin district , brandenburg ( bb ) . for each trapping site , the rate of seroreactive a. agrarius mice is given as a circle ( seroreactive and equivocal samples in black , negative samples in white ) and with the numbers of seroreactive and equivocal samples / negative samples . at sites pe1 , h , k / a1 , and ka , 1 seroreactive a. flavicollis mouse was detected in each location ( marked with stars ) . at site pe1 , 1 equivocal sample was found by the dobv immunoglobulin ( ig ) g elisa . in addition to apodemus mice , 138 rodents of other species , including 116 bank voles ( myodes glareolus ) , were trapped during the same period at sites pe1 , pe3 , k / a1 , ka , and to , but none of the 136 rodents with available transudates reacted in the dobv igg elisa . transudates with an optical density ( od ) value below the lower cutoff ( average of the od values determined for 2 parallel tests of a negative apodemus spp . samples with an od value above the upper cutoff ( 2-fold of the lower cutoff ; average 0.082 ) were regarded as positive . an initial screening by a large ( l ) segment  specific nested reverse transcription  pcr ( rt - pcr ) ( 3 ) of 67 lung samples , representing all seroreactive ( n = 20 ) and equivocal ( n = 6 ) as well as 36 selected seronegative and 5 serologically not - analyzed animals , showed a 390-nt amplification product for 21 samples representing 16 seroreactive , 4 seronegative , and 1 serologically not - investigated animals ( technical appendix ) . to enable a comparison with the only available dobv sequence from germany ( h169 ) , an s segment portion of 559 nt was amplified by rt - pcr from 11 lung tissues ( technical appendix ) . in the phylogenetic analyses , all sequences from germany formed 1 well - supported ( puzzle [ www.tree-puzzle.de ] ) and bootstrap support values the first cluster contained s segment sequences from district gstrow ( trapping sites pe1 and pe3 ) , lneburg ( trapping site wg ) , nordvorpommern ( trapping site h ) , and the previously published dobv sequence from an hfrs patient from northern germany ( h169 ; ; figure 2 , panel a ) . a second cluster was formed by s segment sequences originating from districts ostprignitz - ruppin ( trapping sites ka , to ) and demmin ( trapping site k / a1 ) . notably , the a. flavicollis  derived sequences from sites pe1 , h , ka , and k / a1 clustered together or were completely identical with a. agrarius  derived sequences from the same or neighboring trapping sites , suggesting multiple spillover infections ( figure 2 , panel a , technical appendix table 1 ) . maximum - likelihood ( ml ) phylogenetic trees of dobrava - belgrade virus ( dobv ) based on partial small ( s ) segment nucleotide sequences of 559 nt ( position 377935 ) ( a ) and complete nucleocapsid protein coding nucleotide sequences ( s segment open reading frame ) ( b ) . the ml trees ( tamura - nei evolutionary model ) were calculated using tree - puzzle package ( www.tree-puzzle.de ) . values below the branches are bootstrap values of the corresponding neighbor - joining tree ( tamura - nei evolutionary model ) calculated with the paup * software ( paup.csit.fsu.edu ) from 10,000 bootstrap pseudoreplicates . only values > 70% ( considered significant ) are shown . sangv , sangassou virus ; htnv , hantaan virus ; seov , seoul virus ; saa , saaremaa virus ; aa , apodemus agrarius ; ap , a. ponticus ; af , a. flavicollis . wg , district lneburg , lower saxony ( ls ) ; pe1 and pe3 , district gstrow ; h , district nordvorpommern , k / a1 , district demmin , all mecklenburg - western pomerania ( mwp ) ; to and ka , district ostprignitz - ruppin , brandenburg ( bb ) . before tree construction , automated screening for recombination between the s segment sequences was performed using program rdp3 ( 15 ) , which used 6 recombination detection programs : bootscan , chimeric , geneconv , maxchi , rdp , and siscan with their default parameters . no putative recombinant regions could be conclusively detected by > 3 programs and subsequently verified by phylogenetic trees . the sequences from germany share a common ancestor with the dobv - aa sequences originating from slovakia and russia . together , they form a monophyletic group ( dobv - aa lineage ) that is clearly separated from a. flavicollis  borne ( dobv - af ) and a. ponticus  borne ( dobv - ap ) sequences and from a. agrarius subsequent analysis of nucleotide sequences of the entire nucleocapsid ( n ) protein and glycoprotein precursor ( gpc)encoding regions confirmed these findings ( figure 2 , panel b ; technical appendix ) . a pairwise comparison between nucleotide and amino acid sequences of the complete n and gpc open reading frames from the novel german dobv strains showed divergences of 1.5%8.8% ( 0.3%1.4% ) and 2.1%8.3% ( 0.8%1.8% ) , respectively ( technical appendix table 2 ) . the highest identity values on the nucleotide and amino acid sequence level ( 91.2%91.7% and 99%99.7% ) were found for an s segment sequence from denmark ( lolland/1403 ; genbank accession no . the nucleotide and amino acid sequence divergence to other dobv sequences was much higher , reaching 10.1%14.3% ( 1%3.3% ) and 12.6%20.7% ( 2.9%9.4% ) , respectively . morphologic species determination for all dobv - seroreactive and rt - pcr  positive rodents was confirmed by a mitochondrial cytochrome b gene - specific pcr ( 9,10 ) , sequence determination , and comparison with available genbank sequences from a. agrarius and a. flavicollis ( technical appendix table 1 ) . based on a large panel of the entire n- and gpc - encoding dobv sequences , we report direct molecular evidence that dobv in germany is represented by a genetic lineage associated with a. agrarius ( dobv - aa ) . in contrast , we found no evidence for the occurrence of dobv - af in a. flavicollis or other apodemus species from germany . consistent with the geographic distribution of a. agrarius ( 11 ) and the report of human dobv disease exclusively in northern and northeastern germany , this finding may confirm dobv - aa as the sole causative agent of dobv infections in germany ( 4 ; robert koch - institut , survstat , www.rki.de ) . previously a. agrarius  associated saaremaa virus was experimentally shown to be able to infect a. agrarius and a. flavicollis mice ( 12 ) . we report multiple natural spillover infections of a. flavicollis by a dobv strain originally hosted by a. agrarius . this observation is in contrast to other reports from slovenia and slovakia where , although a. agrarius and a. flavicollis are occurring sympatrically , a. flavicollis is exclusively carrying the dobv - af and a. agrarius the dobv - aa lineage ( 4,13 ) . in contrast to our observations , single dobv - af spillover infections of a. sylvaticus and mus musculus have been reported previously ( 14 ) . the phylogenetic analyses demonstrated 2 well - separated clusters within the dobv - aa lineage . these rodent - derived dobv sequences in germany represent a major contribution to the dobv genomics and phylogenetics . future investigations should help to identify specific features of these dobv - aa strains resulting in its frequent spillover to a. flavicollis and to prove a putative adaptation of dobv - aa on a. flavicollis after spillover , as well as possible reassortment processes . summary of the serologic and reverse transcription - pcr ( rt - pcr ) investigations of all serologically and/or rt - pcr - positive apodemus mice *
we present the molecular identification of apodemus agrarius ( striped field mouse ) as reservoir host of the dobrava - belgrade virus ( dobv ) lineage dobv - aa in 3 federal states of germany . 
 phylogenetic analyses provided evidence for multiple spillover of dobv - aa to a. flavicollis , a crucial prerequisite for host switch and genetic reassortment .