in november 2009 , a bat was found on the ground in bokeloh , lower saxony , germany ( 522513.99n ; 92331.56e ) .
it was given mealworms and water ad libitum supplemented with minerals and vitamins . in february 2010 , the bat began to act aggressively , directly approaching any moving object , vigorously trying to bite , and screaming ferociously .
this agitated stage lasted for 7 days and was followed by general weakness , lethargy , and paralysis . after the first 3 days of the clinical course
the bat was submitted for testing , and rabies diagnosis was performed by using immunohistochemical analysis .
lyssavirus antigen was detected in numerous neurons of the cerebral cortex , cerebellum , and especially the nucleus funiculi lateralis and the nucleus olivaris of the medulla ( figure 1 ) .
organs other than the central nervous system , e.g. , the salivary glands , did not contain lyssavirus antigen . after the first cell passage in rabies tissue culture infection test ( 9 ) , virus was isolated from brain tissue .
antigenic typing performed with a panel of 10 antinucleocapsid monoclonal antibodies ( 10 ) clearly differentiated the isolated virus from all other tested lyssavirus species ( table ) .
immunohistochemical analysis of brain of natterer s bat for lyssavirus antigen by using the avidin biotin complex method .
cytoplasmic granular - to - diffuse staining for rabies antigen is visible in the perikarya and neuronal processus .
b ) medulla and neurons of the nucleus funiculi lateralis showing strong cytoplasmic staining for rabies antigen .
bblv , bokeloh bat lyssavirus ; rabv , rabies virus ; lbv , lagos bat virus ; mokv , mokola virus ; duvv , duvenhage virus ; eblv , european bat lyssavirus ; ablv , australian bat lyssavirus ; + + + , strongly positive ;  , negative .
results of discriminatory reverse transcription pcr results for eblv-1 and eblv-2 ( 11,12 ) were negative , and only a generic reverse transcription pcr ( 13 ) yielded a 605-bp amplification product similar to that of the positive control .
the nucleotide sequence was determined by using standard methods ( primers and protocols are available upon request ) .
sequence analysis of the nucleoprotein gene performed with mega version 4.0 software ( www.megasoftware.net/mega4/mega.html ) showed that bblv differed from all other published lyssavirus sequences with the highest nucleotide identity to khuv ( 80% ) , followed by arav ( 79% ) , eblv-2 ( 79% ) , australia bat lyssavirus ( 77% ) , eblv-1 ( 77% ) , irkut virus ( 76% ) , shimoni virus ( 76% ) , rabv ( 73 - 75% ) and duvenhage virus ( 75% ) .
lagos bat virus ( 72 - 74% ) , mokola virus ( 72% ) , and west caucasian bat virus cbv ( 72% ) showed the highest divergence to bblv .
also , phylogenetic analysis based on concatenated n - p - m - g - l nucleotide sequences showed that bblv is most closely related to khuv , followed by eblv-2 ( figure 2 ) .
phylogenetic tree inferred from concatenated n - p - m - g - l sequences of bat lyssaviruses .
the neighbor - joining method ( kimura 2-parameter ) was used as implemented in mega4 software ( www.megasoftware.net ) .
rabv , rabies virus ; ablv , australian bat lyssavirus ; arav , aravan virus ; khuv , khujand virus ; bblv , bokeloh bat lyssavirus ; european bat lyssavirus ; irkv , irkut virus ; duvv , duvenhage virus ; shibv , shimoni bat virus ; lbv , lagos bat virus ; mokv , mokola virus ; wcbv , west caucasian bat virus .
we report the discovery of a lyssavirus ( designated as bblv ) from a natterer s bat that died with rabies - like clinical signs . initially , a distinctive pattern in the reaction with a panel of antinucleocapsid monoclonal antibodies indicated the presence of an antigenically atypical isolate .
the differentiation from other lyssavirus species was confirmed by phylogenetic analysis ( figure 2 ) .
bblv is pathogenic because it caused a fatal disease in the natterer s bat that was similar to the clinical picture of rabies seen in other bats .
viral antigen was present in many locations of the brain ( figure  1 ) but surprisingly not in the salivary glands .
since the exact date of infection is unknown , the incubation period can only be estimated as > 4 months . whether the natterer s bat is the natural reservoir species of bblv or whether it was
bats ( eblv-2 from m. daubentonii , m. dasycneme , khuv from m. mystacinus , and arav from m. blythii ) indicating that myotis spp .
if one considers the history of bat rabies in europe , it seems unlikely that bblv had spread from a distant origin into central europe or that the bat itself was translocated over long distances .
the fact that bblv has been identified only in 1 bat is puzzling , considering the relatively high level of surveillance in germany . also , of 63 natterer s bats tested during 19992010 in a retrospective study , none tested positive for rabies ( t. mller et al .
germany is the only country where several bat species other than serotine bats , i.e. , pipistrellus nathusii , pipistrellus pipistrellus , and plecotus auritus , have been found infected with eblv-1 during this study ( t. mller et al .
data ) , and this is the second discovery in recent years of a new lyssavirus species through routine passive bat rabies surveillance .
also , in 2007 a daubenton s bat found on the ground was taken to a rehabilitation center , where it died and subsequently tested positive for eblv-2 ( 7 ) . in both cases ,
the person who took care of the animal had completed the full preexposure vaccination , as a required risk - mitigating measure .
an encounter with a bblv - infected natterer s bat could lead to a fatal outcome because bat lyssaviruses have caused several human cases of infection ( 4 ) . in europe , species conservation and research require the handling of bats by bat workers . during 20002010 ,
a total of 37,140 handlings were recorded for the natterer s bat ( bat marking centre , saxon state office for environment and geology , dresden , germany ) , underlining the need for adequate prophylaxis for bat handlers . if one considers the close phylogenetic relationship between bblv and eblv-2 humans who receive rabies prophylaxis
however , recent studies of the antigenic relationships of lyssaviruses have shown the difficulty of interpreting antigenic differences by using sequences alone ( 14 ) .
thus , in vitro and in vivo cross - neutralization and protection studies with current anti - rabv vaccines are urgently required for assessing the public health risk posed by this new lyssavirus .
 a virus isolated from a natterer s bat ( myotis nattererii ) in germany was differentiated from other lyssaviruses on the basis of the reaction pattern of a panel of monoclonal antibodies . 
 phylogenetic analysis supported the assumption that the isolated virus , bokeloh bat lyssavirus , may represent a new member of the genus lyssavirus . 
The Study
Conclusions