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First Isolation of EBLV-2 in Germany
Freuling Conrad, Ernst Grossmann, Franz J. Conraths, Astrid Schameitat, Jeanette Kliemt, Ernst Auer, Irene Greiser-Wilke, Müller Thomas
To cite this version:
Freuling Conrad, Ernst Grossmann, Franz J. Conraths, Astrid Schameitat, Jeanette Kliemt, et al..
First Isolation of EBLV-2 in Germany. Veterinary Microbiology, Elsevier, 2008, 131 (1-2), pp.26.
�10.1016/j.vetmic.2008.02.028�. �hal-00532404�
Accepted Manuscript
Title: First Isolation of EBLV-2 in Germany
Authors: Freuling Conrad, Ernst Grossmann, Franz J.
Conraths, Astrid Schameitat, Jeanette Kliemt, Ernst Auer, Irene Greiser-Wilke, M¨uller Thomas
PII: S0378-1135(08)00088-6
DOI: doi:10.1016/j.vetmic.2008.02.028
Reference: VETMIC 3970
To appear in: VETMIC Received date: 12-12-2007 Revised date: 14-2-2008 Accepted date: 26-2-2008
Please cite this article as: Conrad, F., Grossmann, E., Conraths, F.J., Schameitat, A., Kliemt, J., Auer, E., Greiser-Wilke, I., Thomas, M., First Isolation of EBLV-2 in Germany,Veterinary Microbiology(2007), doi:10.1016/j.vetmic.2008.02.028
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Accepted Manuscript
First Isolation of EBLV-2 in Germany
1 2
Freuling, Conrad1*; Ernst Grossmann2; Franz J. Conraths1, Astrid Schameitat1, Jeanette 3
Kliemt1, Ernst Auer3, Irene Greiser-Wilke4, Müller, Thomas1 4
1Friedrich-Loeffler-Institute, Federal Research Institute for Animal Health, Institute of 5
Epidemiology, WHO Collaborating Centre for Rabies Surveillance and Research, 16868 6
Wusterhausen, Germany 7
2Staatl.Tierärztl. Untersuchungsamt Aulendorf, 88326 Aulendorf, Germany 8
3Arbeitskreis Fledermäuse, Bodensee-Oberschwaben, Postfach 10 05 38, 78405 Konstanz, 9
Germany 10
4Institute of Virology, Hannover School of Veterinary Medicine, Bünteweg 17, 30559 11
Hannover, Germany 12
13
*Corresponding author. Present address: Friedrich-Loeffler-Institute, Federal Research 14
Institute for Animal Health, Institute of Epidemiology, WHO Collaborating Centre for 15
Rabies Surveillance and Research, 16868 Wusterhausen, Germany, Tel.:+49 33979 80 16
158 17
E-mail address: [email protected] 18
Manuscript
Accepted Manuscript
Abstract 19
20
In Europe, rabies in bats is caused by European Bat Lyssavirus (EBLV) type 1 (EBLV-1) 21
or type 2 (EBLV-2) which form two distinct genotypes (gt 5 and 6) within the genus 22
Lyssavirus of the family ofRhadoviridae. Spill-over infections of EBLV in humans have 23
caused fatal rabies encephalitis and highlighted the relevance of this wildlife disease for 24
public health.
25
The vast majority of the 831 European bat rabies cases reported between 1977 and 2006 26
were identified as EBLV-1. Only few virus isolates originating from Switzerland, The 27
Netherlands and the United Kingdom were characterized as EBLV-2. Here we report the 28
first EBLV-2 case detected in Germany in a Daubenton’s bat (Myotis daubentonii) in 29
August 2007. The bat showed clinical signs of disorders of the central nervous system and 30
subsequently tested positive for rabies. The virus was isolated and characterized as 31
EBLV-2 based on its antigen pattern and by nucleotide sequencing. Phylogenetic analysis 32
indicated an association to EBLV-2 isolates from Switzerland which correlates with the 33
origin of the bat close to the Swiss border.
34 35 36 37
Keywords: European bat lyssavirus, Rabies, bats, EBLV-2, Daubenton’s bat 38
39 40 41 42 43 44 45 46 47 48
Accepted Manuscript
49
Introduction 50
51
Lyssaviruses of the order Mononegavirales are the causative agent of rabies, a fatal 52
encephalitic viral disease. Ten out of eleven (putative) lyssavirus genotypes have been 53
isolated from bats. Only Mokola virus was never detected in this order of animals (Anon., 54
2004). In Europe, bats have been found infected with European Bat Lyssavirus (EBLV) 55
type 1 (EBLV-1) or type 2 (EBLV-2) which form two distinct genotypes (gt 5 and 6) 56
within the genus Lyssavirus of the family of Rhadoviridae (Bourhy et al., 1993;
57
Amengual et al., 1997; Davis et al., 2005). Recently, bats from the Caucasian area and 58
Central Asia tested positive for lyssavirus genotypes described as West Caucasian Bat 59
Lyssavirus (WCBL), Aravan (ARAV) and Khujand (KHUV) which are considered 60
tentative members of the genus Lyssavirus (Botvinkin et al., 2003, Kuzmin et al., 2003).
61
As a consequence, it seems possible that other lyssavirus genotypes circulate in parts of 62
Europe. However, only single isolates of WCBL, ARV and KHUV have been obtained so 63
far.
64
The relevance of bat rabies for public health in Europe is illustrated by the fact that both 65
EBLV-1 and EBLV-2 have caused human cases, although the number is small (Lumio et 66
al., 1986; Selimov et al., 1989; Fooks et al., 2003a). Spill-over infections to other 67
mammals have only been described for EBLV-1 (Müller et al., 2004; Tjørnehøj et al., 68
2006).
69 70
The first case of bat rabies in Europe was found in Hamburg, Germany, in 1954. During 71
subsequent years, bat rabies cases were reported only sporadically from different 72
European countries and were seen as rare incidents. Intensified screening started in the 73
second half of the 1980s, leading to a significant increase in tested bats and positive cases.
74
Accepted Manuscript
From 1977 to 2006, a total of 831 cases of bat rabies, most of them typed as EBLV-1, 75
were detected in Europe and reported to the WHO Collaborating Centre for Rabies 76
Surveillance and Research at the Friedrich-Loeffler-Institute, Germany (Müller et al., 77
2007). The majority of positive bats originated from Denmark, followed by The 78
Netherlands, Germany and Poland. These cases accounted for more than 90 percent of all 79
positive bats recorded for this time period, whereas only sporadic cases were reported 80
from other European countries (Müller et al., 2007). Interestingly, EBLV-2 was 81
discovered only in 15 cases, mostly in Daubenton’s bats (Myotis daubentonii) and pond 82
bats (Myotis dasycneme), in only three countries, The Netherlands, Switzerland and the 83
UK. So far, The Netherlands was the only country where both genotypes had been 84
isolated from rabies-positive bats (Van der Poel et al., 2005).
85 86
Here we describe the first detection of EBLV-2 in Germany after 50 years of rabies 87
surveillance in bats.
88 89 90
Materials and methods 91
92
Case report 93
94
On 18 August 2007 a bat was found grounded and unable to fly at a lake shore near the 95
town of Bad Buchau (WGS84 coordinates: O9°37'13", N48°4'46), Biberach district, 96
Baden-Württemberg, Germany, in a nature conservation area. It was taken to a local bat 97
sanctuary for rehabilitation where it started to show an unusual behaviour on day 2 of the 98
hospitalization. Initially lethargic and inconspicuous, the animal developed agitation and, 99
while awake, was restless and climbing furiously in its cage. It was impossible to feed the 100
animal. During this stage, the bat also showed aggressive behaviour such as biting. The 101
Accepted Manuscript
animal was euthanized by a veterinarian on 21 August 2007 and sent to the regional 102
veterinary laboratory in Aulendorf for rabies testing.
103 104
The animal was identified as a Daubenton’s bat (Myotis daubentonii) on the basis of 105
morphological criteria (Dietz and von Helversen, 2004). This result was confirmed by 106
genetic identification using partial sequencing of the cytochrome B-gene as described 107
elsewhere (Harris et al., 2007a). The bat was tested for rabies with a standard fluorescence 108
antibody test (FAT) (Dean et al., 1996) using a polyclonal FITC-labelled anti-rabies 109
conjugate (SIFIN, Berlin, Germany). Rabies was confirmed by the tissue culture 110
inoculation test (RTCIT) using murine neuroblastoma cells as described (Webster and 111
Casey, 1996). Furthermore, a discriminatory EBLV-1 and EBLV-2 specific RT-PCR was 112
performed using the OneStep RT-PCR Kit (Qiagen, Hilden, Germany) to detect EBLV- 113
specific RNA as described (Müller et al., 2004; Vos et al., 2004). Total RNA was isolated 114
directly from the brain homogenate using the RNeasy Kit (Qiagen, Hilden, Germany) and 115
stored at – 80°C prior to testing. Carryover contaminations were avoided by strictly 116
following the precautions for PCR as described (Kwok and Higuchi, 1989).
117 118
Virus was isolated from the animal and antigenic typing performed in cell culture with a 119
panel of 10 anti-nucleocapsid monoclonal antibodies (mAb) using the method of 120
Schneider (1982).
121 122
Sequencing and phylogenetic analysis 123
124
A modified semi-nested RT-PCR using pan lyssavirus primers for the nucleoprotein (N) 125
gene was used to generate PCR fragments for subsequent sequencing (Heaton et al., 126
1997). PCR-products were run on ethidium bromide-stained polyacrylamide gels and 127
Accepted Manuscript
visualized under UV light as 606 bp long fragments. The amplicon corresponding to the 128
N-terminal region of the nucleoprotein gene was sequenced with a set of IRD-800-labelled 129
primers JW 12 (5’ – ATGTAACACC(C/T)CTACAATTG- 3’) and JW10 DLE2 (5’- 130
GTCATCAAAGTGTG(A/G)TGCTC-3’) using the DYEnamic cycle sequencing kit, (GE 131
Healthcare, Amersham, UK) on a LICOR 4200 sequencing machine (MWG Biotech AG, 132
Ebersberg, Germany). Sequence analysis was performed with the Lasergene 6 package 133
(DNAstar Inc., Madison, USA). Phylogenetic analyses of the first 400 bp of the N-gene 134
were conducted using MEGA version 3.1 (Kumar et al., 2004). Other lyssavirus 135
sequences, including classical rabies virus (RABV), EBLV-1 and EBLV-2 were taken 136
from GenBank for comparison (Table 1).
137 138 139
Results 140
141
Brain smears of the Daubenton’s bat were clearly positive by FAT showing distinct rabies 142
specific fluorescence (Fig.1). The diagnosis was confirmed by virus isolation in murine 143
neuroblastoma cell culture using RTCIT and by the discriminatory RT-PCR for EBLV-1 144
and EBLV-2. Evidence for the presence of EBLV-2 specific genetic material in the brain 145
material was obtained by an RT-PCR in which a 217 bp fragment of the gene encoding 146
the nucleoprotein-phosphoprotein (N-P) junction was amplified (Fig. 2). Samples for all 147
other organ tissues (heart, lung, kidney, bladder, pectoral muscle, spleen, liver, thyroid 148
gland) and the salivary gland tested negative by RT-PCR (data not shown).
149 150
Antigenic typing of the isolate was performed with a panel of 10 anti-nucleocapsid mAbs 151
in cell-culture and clearly identified the virus as EBLV-2 based on positive reactions of 152
mAbs W239.17, MSA6.3, LBV7.36 and S62.1.2.
153
Accepted Manuscript
Further genetic characterization using sequence analysis confirmed the result of the 154
antigenic typing. When aligned with the first 400 bp of the corresponding sequence 155
published for an EBLV-2 isolate from Switzerland, an identity of 97.2 % was found. In 156
contrast, comparison with the sequences of representatives of other genotypes from 157
Europe showed identities of only 73.0 % (SAD B19, vaccine virus strain, gt 1;
158
Conzelmann et al., 1990), 72.7 % (fox variant from Germany; Johnson et al., 2003a) and 159
72.2 % (EBLV-1 isolate from Germany; Marston et al., 2007) (Fig. 3).
160 161
Phylogenetic analysis of the corresponding fragment encompassing the N-terminal coding 162
sequence of the nucleoprotein gene of the Daubenton’s bat isolate with representative 163
EBLV-2 isolates from Europe (Table 1) revealed that the isolate is a member of genotype 164
6. The German EBLV-2 isolate showed the closest relationship to Swiss and UK EBLV-2 165
isolates, whereas the isolate from the fatal human case of a Swiss bat handler in Finland 166
and isolates from The Netherlands were rather distantly related. In this phylogenetic tree, 167
the Dutch isolates formed a more separate cluster (Fig. 4).
168 169
Three other bats (Pipistrellus pipistrellus, Nyctalus noctula, Myotis nattererii) that were 170
kept in the same bat sanctuary in Southern Germany but had no direct contact to the 171
infected Daubenton’s bat were subsequently tested for rabies in the FLI. The animals died 172
or were euthanized in a time period associated with the rabies-positive case (16-24 August 173
2007). These bats tested negative for rabies.
174 175
Discussion 176
So far, EBLV-2 in bats has been reported only from Switzerland, The Netherlands and the 177
United Kingdom. In addition, The Netherlands was the only country where both EBLV-1 178
and EBLV-2 have been found (Van der Poel et al., 2005) This report confirms the 179
Accepted Manuscript
presence of EBLV-2 in Germany for the first time after 50 years of rabies surveillance in 180
bats. During this period, more than 200 cases of rabies in bats were reported and more 181
than 110 EBLV isolates were characterised by the German National Reference Laboratory 182
for rabies as EBLV-1. A previous study reported an EBLV-2 case from Lüneburg, 183
Germany, in 1986 in a Myotis daubentonii bat (Johnson et al., 2003b). However, this 184
finding is not supported by our data, as samples taken from this bat that tested positive in 185
a regional veterinary laboratory could not be confirmed as rabies-positive at the National 186
Reference Laboratory. An EBLV-2 sequence published in GenBank (AY212117) with the 187
additional information that the origin of the sample is Germany is dubious. In fact, virus 188
isolates have been shared among rabies laboratories in the past without proper 189
documentation of the epidemiologically relevant information accompanying the samples, 190
thus causing data with incorrect details concerning the geographic origin of isolates.
191 192
Due to the close vicinity of The Netherlands and Switzerland to Germany, failure to detect 193
EBLV-2 in Germany has been subject of discussion and speculation in the past.
194
Interestingly, the geographic origin of the EBLV-2 positive bat reported here close to the 195
Swiss border, the sequence identity and the findings of the phylogenetic analysis (Fig. 3, 196
4) may suggest an association of the German EBVL-2 case to the occurrence of EBLV-2 197
infections in bats from Switzerland. Seasonal movements of Daubenton’s bats between 198
summer and winter roosts, often within a distance of 100-150 km, have been recorded 199
(Hutterer et al., 2005).
200 201
The differences in the numbers of reported EBLV-1 versus EBLV-2 cases might at least 202
in part be explained by the presumed main reservoir species for these two genotypes.
203
While EBLV-1 has a specific association with the Serotine bat (Eptesicus serotinus), 204
EBLV-2 virus is more commonly associated with the species of Myotis bats (M.
205
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daubentonii and M. dascyneme) (Fooks et al., 2003b). Although Daubenton’s bats are 206
frequently found across Germany and represent one of the most abundant bat species, only 207
few samples have been submitted for rabies diagnosis. Between 1997 and 2007 out of 208
more than 1800 bats tested only 45 animals were Daubenton’s bats and only one was 209
identified as M. dasycneme. None of them tested positive for rabies (Müller et al, 210
unpublished). In the UK, active surveillance data suggest that approximately 2% of the M.
211
daubentonii population is antibody positive and during passive surveillance (n = 113) six 212
Daubenton’s bats tested positive for EBLV-2 (Harris et al., 2007b). In contrast, in The 213
Netherlands, where EBLV-2 was also found in the past, surveillance has not detected any 214
new cases of EBLV-2 since 1997. All bats found EBLV-2 positive in the Netherlands 215
were identified as M. dasycneme, whereas the remaining isolates originate from 216
Daubenton’s bats or humans. Interestingly, the isolates from M. dasycneme group as a 217
separate phylogenetic sublineage. However, based on the limited data available, it remains 218
elusive whether this reflects bat species specific strains in EBLV-2 as described for North- 219
American bat RABV variants (Nadin-Davis et al., 2001) or a correspondence to the 220
geographic origin.
221
In Germany, the bat species was unfortunately not determined for the majority of samples 222
that tested positive for EBLV (Müller et al., 2007) highlighting the urgent need for 223
speciation either by morphological criteria or by genetic testing. Knowledge on the 224
distribution of EBLV-1 and 2 among different bat species will greatly improve our 225
understanding of the epidemiology of the infections. It has also been speculated that the 226
adaptation of EBLV to their main reservoir species might reduce their virulence for the 227
preferred hosts and therefore lead to relatively few lethal cases (Van der Poel et al., 2005;
228
Vos et al., 2007). If the small numbers of tested animals is taken into account, one can 229
speculate that EBLV-2 may have a much wider distribution in Germany and perhaps also 230
in other regions of Europe than the limited number of EBLV-2 cases might suggest.
231
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The fact, that no viral RNA was found in organs other than brain, particularly not even in 232
salivary glands, is elusive. Other case studies on viral distribution of EBLV-2 in 233
daubenton’s bats indicate that virus is distributed in many different organs in infected 234
animals (Johnson et al., 2003b, Johnson et al., 2006, Harris et al., 2007b). In contrast, 235
experimental studies showed that it may not always be possible to detect bat lyssavirus 236
RNA (ABLV) in organs other than brain of infected bats (McColl et al., 2002) or other 237
mammal species infected with EBLVs (Vos et al., 2004, Brookes et al., 2007). Data from 238
The Netherlands also show that the corresponding salivary glands of bats tested EBLV-1 239
positive by RT-PCR during routine diagnosis were only tested positive in 22 out of 30 240
cases (Takumi et al., 2008). We speculate that the observed findings are the result of the 241
time point of testing, comprising a stage during the infection where EBLV-2 virus is 242
propagating within the brain but has not yet started to disseminate into other organs, or 243
that the virus concentration had not reached the detection threshold of the methods used.
244 245
The person who handled the Daubenton’s bat from which EBLV was isolated, had just 246
completed the full pre-exposure vaccination some weeks before the incident. One of the 247
other two people who were in contact to the infected bat but were not bitten received a 248
booster vaccination whereas the other person refused post-exposure prophylaxis. So far, 249
two known human rabies cases have been caused by EBLV-2 (Lumio et al., 1986; Fooks 250
et al., 2003a), stressing the need for education and preventive measures. To estimate the 251
public health risk due to exposure to EBLV-2, further research is needed that focuses on 252
the reservoir species of the virus. Targeted passive surveillance for bat lyssaviruses should 253
be established in all European countries including Germany (Anon., 2006).
254
Accepted Manuscript
Reference List 255
256
Amengual, B., Whitby, J.E., King, A., Cobo, J.S., Bourhy, H., 1997. Evolution of 257
European bat lyssaviruses. J Gen. Virol 78, 2319-2328.
258
Anon., 2004. WHO Expert Consultation on Rabies. World Health Organ Tech. Rep. Ser.
259
931, 1-121.
260
Anon., 2006. Passive and active surveillance of bat lyssavirus infections. WHO Rabies 261
Bulletin Europe 4/05, 5-6.
262
Botvinkin, A.D., Poleschuk, E.M., Kuzmin, I.V., Borisova, T.I., Gazaryan, S.V., Yager, 263
P., Rupprecht, C.E., 2003. Novel lyssaviruses isolated from bats in Russia. Emerging 264
Infectious Diseases 9, 1623-1625.
265
Bourhy, H., Kissi, B., Tordo, N., 1993. Molecular Diversity of the Lyssavirus Genus.
266
Virology 194, 70-81.
267
Brookes, S.M., Klopfleisch, R., Muller, T., Healy, D.M., Teifke, J.P., Lange, E., Kliemt, 268
J., Johnson, N., Johnson, L., Kaden, V., Vos, A., Fooks, A.R., 2007. Susceptibility of 269
sheep to European bat lyssavirus type-1 and -2 infection: a clinical pathogenesis study.
270
Vet. Microbiol. 125, 210-223.
271
Conzelmann, K.-H., Cox, J.H., Schneider, L.G., Thiel, H.-J., 1990. Molecular Cloning and 272
Complete Nucleotide Sequence of the Attenuatend Rabies Virus SAD B19. Virology 175, 273
485-499.
274
Davis, P.L., Holmes, E.C., Larrous, F., Van der Poel, W.H.M., Tjornehoj, K., Alonso, 275
W.J., Bourhy, H., 2005. Phylogeography, Population Dynamics, and Molecular Evolution 276
of European Bat Lyssaviruses. J. Virol. 79, 10487-10497.
277
Accepted Manuscript
Dean, D.J., Abelseth, M.K., Athanasiu, P., 1996. The fluorescence antibody test. In:
278
Meslin, F.-X., Kaplan, M.M., Koprowski, H. (Eds.), Laboratory Techniques in Rabies.
279
World Health Organization, Geneva, Switzerland, pp. 88-93.
280
Dietz, C., von Helversen, O., 2004.
281
Identification key to the bats of Europe - electronical publication, version 1.0. URL:
282
www. uni-tuebingen. de/tierphys/Kontakt/mitarbeiter_seiten/dietz. htm.
283
Fooks, A.R., McElhinney, L.M., Pounder, D.J., Finnegan, C.J., Mansfield, K., Johnson, 284
N., Brookes, S.M., Parsons, G., White, K., McIntyre, P.G., Nathwani, D., 2003a. Case 285
report: Isolation of a European bat lyssavirus type 2a from a fatal human case of rabies 286
encephalitis. Journal of Medical Virology 71, 281-289.
287
Fooks, A.R., Brookes, S.M., Johnson, N., McElhinney, L.M., Hutson, A.M., 2003b.
288
European bat lyssaviruses: an emerging zoonosis. Epidemiology and Infection 131, 1029- 289
1039.
290
Harris, S.L., Johnson, N., Brookes, S.M., Hutson, A.M., Fooks, A.R., Jones, G., 2007a.
291
The Application of Genetic Markers for EBLV Surveillance in European Bat Species.
292
Dev. Biol. (Basel) in press.
293
Harris, S.L., Mansfield, K., Marston, D.A., Johnson, N., Pajamo, K., O'Brien, N., Black, 294
C., McElhinney, L.M., Fooks, A.R., 2007b. Isolation of European bat lyssavirus type 2 295
from a Daubenton's bat (Myotis daubentonii) in Shropshire. Vet Rec.161 (11), 384-386.
296
Heaton, P.R., Johnstone, P., McElhinney, L.M., Cowley, R., O'Sullivan, E., Whitby, J.E., 297
1997. Heminested PCR assay for detection of six genotypes of rabies and rabies-related 298
viruses. J Clin Microbiol 35, 2762-2766.
299
Accepted Manuscript
Hutterer, R., Ivanova, T., Meyer-Cords, C., Rodrigues, L., 2005. Bat migrations in 300
Europe: a review of banding data and literature. Naturschutz und Biologische Vielfalt 28, 301
83-84.
302
Johnson, N., Black, C., Smith, J., Un, H., McElhinney, L.M., Aylan, O., Fooks, A.R., 303
2003a. Rabies emergence among foxes in Turkey. J Wildl Dis 39, 262-270.
304
Johnson, N., Selden, D., Parsons, G., Healy, D., Brookes, S.M., McElhinney, L.M., 305
Hutson, A.M., Fooks, A.R., 2003b. Isolation of a European bat lyssavirus type 2 from a 306
Daubenton's bat in the United Kingdom. Veterinary Record 152, 383-387.
307
Johnson, N., Wakeley P.R., Brookes, S.M., and Fooks A. R.. 2006. European bat 308
lyssavirus type 2 RNA in Myotis daubentonii. Emerg. Infect. Dis. 12,1142-1144.
309
Kumar, S., Tamura, K., Nei, M., 2004. MEGA3: Integrated software for Molecular 310
Evolutionary Genetics Analysis and sequence alignment. Briefings in Bioinformatics 5, 311
150-163.
312
Kuzmin, I.V., Orciari, L.A., Arai, Y.T., Smith, J.S., Hanlon, C.A., Kameoka, Y., 313
Rupprecht, C. E., 2003. Bat lyssaviruses (Aravan and Khujand) from Central Asia:
314
phylogenetic relationships according to N, P and G gene sequences. Virus Res.97, 65-79.
315
Kwok, S., Higuchi, R., 1989. Avoiding false positives with PCR. Nature 339, 237-238.
316
Lumio, J., Hillbom, M., Roine, R., Ketonen, L., Haltia, M., Valle, M., Neuvonen, E., 317
Lähdevirta, J., 1986. Human Rabies of Bat origin in Europe. The Lancet 15, 378.
318
Marston, D.A., McElhinney, L.M., Johnson, N., Muller, T., Conzelmann, K.K., Tordo, N., 319
Fooks, A.R., 2007. Comparative analysis of the full genome sequence of European bat 320
lyssavirus type 1 and type 2 with other lyssaviruses and evidence for a conserved 321
Accepted Manuscript
transcription termination and polyadenylation motif in the G-L 3' non-translated region. J.
322
Gen. Virol. 88, 1302-1314.
323
McColl, K.A., Chamberlain, T., Lunt, R.A., Newberry, K.M., Middleton, D., Westbury, 324
H.A., 2002. Pathogenesis studies with Australian bat lyssavirus in grey-headed flying 325
foxes (Pteropus poliocephalus). Australian Veterinary Journal 80, 636-641.
326
Müller, T., Cox, J., Peter, W., Schafer, R., Johnson, N., McElhinney, L.M., Geue, J.L., 327
Tjornehoj, K., 2004. Spill-over of European bat lyssavirus type 1 into a stone marten 328
(Martes foina) in Germany. Journal of Veterinary Medicine Series B-Infectious Diseases 329
and Veterinary Public Health 51, 49-54.
330
Müller, T., Johnson, N., Freuling, C.M., Fooks, A.R., Selhorst, T., Vos, A., 2007.
331
Epidemiology of bat rabies in Germany. Arch. Virol. 152, 273-288.
332
Nadin-Davis, S.A., Huang, W., Armstrong, J., Casey, G.A., Bahloul, C., Tordo, N.
333
Wandeler, A.I., 2001. Antigenic and genetic divergence of rabies viruses from bat species 334
indigenous to Canada. Virus Research 74, 139-156.
335
Schneider, L.G., 1982. Antigenic Variants of Rabies Virus. Comparative Immunology 336
Microbiology and Infectious Diseases 5, 101-107.
337
Selimov, M.A., Tatarov, A.G., Botvinkin, A.D., Klueva, E.V., Kulikova, L.G., 338
Khismatullina, N.A., 1989. Rabies-related Yuli virus; identification with a panel of 339
monoclonal antibodies. Acta Virol 33, 542-546.
340
Takumi, K., Lina, P.H., Van der Poel, W.H., Kramps, J.A., Van der Giessen, J.W., 2008.
341
Public health risk analysis of European bat lyssavirus infection in The Netherlands.
342
Epidemiol. Infect., 1-7. e-pub ahead of print, doi:10.1017/S0950268807000167 343
Accepted Manuscript
Tjørnehøj, K., Fooks, A.R., Agerholm, J.S., Ronsholt, L., 2006. Natural and experimental 344
infection of sheep with European bat lyssavirus type-1 of Danish bat origin. J Comp 345
Pathol. 134, 190-201.
346
Van der Poel, W.H., Van der, H.R., Verstraten, E.R., Takumi, K., Lina, P.H., Kramps, 347
J.A., 2005. European bat lyssaviruses, The Netherlands. Emerg. Infect. Dis. 11, 1854- 348
1859.
349
Vos, A., Kaipf, I., Denzinger, A., Fooks, A.R., Johnson, N., Müller, T., 2007. European 350
bat lyssaviruses - an ecological enigma. Acta Chiropterologica 9(1), 283-296.
351
Vos, A., Muller, T., Cox, J., Neubert, L., Fooks, A.R., 2004. Susceptibility of ferrets 352
(Mustela putorius furo) to experimentally induced rabies with European Bat Lyssaviruses 353
(EBLV). Journal of Veterinary Medicine Series B-Infectious Diseases and Veterinary 354
Public Health 51, 55-60.
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Figure legend:
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Figure 1: Fluorescence antibody test (FAT) performed on brain smears of the infected bat 358
359
Figure 2: Results of the discriminatory EBLV-1 and EBLV-2 RT-PCR targeting the NP- 360
intergenic region of the EBLV genome. The primers amplify a 367 bp and a 217 bp 361
fragment for EBLV-1 or EBLV-2, respectively. Positive (EBLV-1 PC and EBLV-2 PC) 362
and negative (NC) controls were included.
363 364
Figure 3: Distribution of reported EBLV cases in Germany (1954-2007).
365 366
Figure 4: Phylogenetic tree of EBLV-2 sequences based on a 400 bp fragment of the N- 367
terminal region of the N-gene with EBLV-1 (RV9 GER) as an outgroup using the 368
Neighbour- Joining method as included MEGA 3.0 (Kumar et al, 2004). Bootstrap values 369
are indicated.
370 371
Figure 5: Sequences of the N-terminal 400 bp fragment of the nucleoprotein gene of 372
EBLV-1, EBLV-2 and rabies virus (RABV). The fragment corresponds to positions 71- 373
470 of the EBLV-2 genome (EF157977, Marston et al., 2007).
374
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Table 1: Sequences and isolates used for the phylogenetic analysis
Strain Year Genotype Country City Host species Accession No. Author
16618 GER 2007 EBLV-2 Germany Bad Buchau M. daubentonii this study
02053 SWI 2002 EBLV-2 Switzerland NA ? AY863408 Davis et al, 2005
RV 1333 2002 EBLV-2 United Kingdom M. daubentonii AY247650 Fooks et al, 2003 9337 SWI 1993 EBLV-2 Switzerland Versoix M. daubentonii AY863407 Davis et al, 2005
9375 HOL 1993 EBLV-2 Holland Roden M. dasycneme AY863404 Davis et al, 2005
94112 HOL 1989 EBLV-2 Holland Andijk M. dasycneme AY863405 Davis et al, 2005 9018 HOL 1987 EBLV-2 Holland Wommels M. dasycneme AY863403 Davis et al, 2005
9007 FIN 1986 EBLV-2 Finland Helsinki Human AY863406 Davis et al, 2005
RV 9 1986 EBLV-1 Germany Hamburg ? EF 157976 Marston et al, 2007
Table 1
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Figure 1
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Figure 2
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Figure 3
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Figure 4
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Figure 5
