1. ASYMPTOMATIC RHABDOVIRUS INFECTION IN MERIDIONAL
SEROTINE BATS (Eptesicus isabellinus) FROM SPAIN
J.E. ECHEVARRÍA1, S. VÁZQUEZ-MORÓN1, C. IBÁÑEZ2,
J.C. AZNAR1, E. RUIZ3, J. JUSTE2
1 National Center of Microbiology, Carlos III Institute of Health, Majadahonda, Madrid
2 Doñana Biological Station, CSIC, Seville
3 Laboratorio Central Veterinario, Santa Fe, Granada
Spain

2. Lyssavirus-infected bats detected by passive surveillance in Spain
Número de individuos
Localidad
año
Exposición humana
Murciélago
Virus 1
VALENCIA
1987 SI
UNKNOWN
“DUVENHAGE
LIKE”
GRANADA
Eptesicus serotinus
EBLV1 5
HUELVA
1989 NO
1994 3
SEVILLA
1999
1 SI
2 NO
MURCIA 4
2000
2002
Eptesicus
serotinus

3. Eptesicus isabellinus
57Ese SY
E. serotinus or E. Isabellinus?
61Ese SY
60Ese SY
43Ese CY
93Ebtur IR
92Ebtur IR
91Ebtur IR
Filogeny of Eptesicus in the Paleartic
94Ebsp IR
95Ebsp IR
90Ebtur IR
16Ebo SY
Genes: CytB, ND1, RAG2
2900 bp
19Ebo SY
14Ebo JO
97Ebog IR
98Ebog IR
54Ese NorthSpain
55Ese North Spain
50Ese IT
44Ese CZ
Europe
(including north Spain)
64Ese UK
Eptesicus serotinus
46Ese DE
> 16 % cytb!!
65Ese UK
45Ese CZ
66Eni GE
51Ese LA
52Ese LA
42Ese CH
7Ean SY
11Ean TU
8Ean SY
10Ean TU
21Eho SA
22Eho SA
33Eis MO
36Eis South Spain
Eptesicus isabellinus
35Eis South Spain
South Spain
North Africa
34Eis MO
23Eis LI
28Eis LI

4. Lyssaviruses are not pathogenic for bats (1)
Prevalence of VRAB antibodies above 60% in a healthy
colony of tailed bat (Tadarida brasiliensis) in USA
(Steece –1989- Journal of Wildlife Diseases 25: )
Prevalence of EBLV1 antibodies above 20% in several
colonies of serotine bats (Eptesicus isabellinus) in Spain.
One seropositive individual captured healthy one year later
(Pérez Jordá –1994- Journal of Wildlife Diseases 3: )

5. Lyssaviruses are not pathogenic for bats (2)
Presence of EBLV1 ARN on oro-pharyngeal swabs of
healthy serotine bats in Spain, recovery of some positive
individuals one year after.
(Echevarría –2001- Journal of Clinical Microbiology 39(10): )
Presence of EBLV1 in healthy Rousettus aegyptiacus months after an outbreak in a captive colony in The Netherlands. Virus is detected by PCR and IF on brain and other organs.
(Wellenberg –2002- Archives of Virology 147: )

6. Lyssaviruses are not pathogenic for bats (3)
Detection of EBLV1 specific antibodies and RNA on blood
pellets of different bat species other than serotine bats from healthy colonies in Spain. (Serra-Cobo –2002- Emerging Infectious Diseases 8 (4): )
Detection of EBLV2 specific antibodies on Daubenton´s
Bats from Scotland.(Brookes Emerging Infectious
Diseases 11(4): ).

7. Lyssaviruses are not pathogenic for bats (3)
Survival of Eptesicus fuscus after inoculation of RABV
(Shankar –2004- Journal of Wildlife Diseases 40(3): )
Survival of Tadarida brasiliensis after aerosol exposition to RABV. (Davis –2007- Journal of infectious Diseases195: )

8. STUDY OF EBLV1 INFECTION ON BAT COLONIES
19 COLONIES OF Eptesicus isabellinus
1080 INDIVIDUALS
1227 CAPTURES ( )
SEVILLA (SECRETARIA)
SEVILLA (CATEDRAL)
TRAJANO
ALCALA DEL RIO
SOTIEL CORONADA
AZNALCOLLAR (Puente Cañaveroso)
MOLINO DEL DUENDE
MOLINO DE NIEBLA
MOLINO DEL RINCON
TUNEL DEL MANZANO
PUENTE GADEA
LA ARADILLA
PUENTE TAMUJOSO
ORJIVA (TUNEL GUADALFEO)
ORJIVA (REVOLCADERO)
TUNEL DEL PICOTE
TUNEL SALOMON
50 km
UMBRETE(Colegio)
SEVILLA (ALCAZAR)
- O-PH SWAB (PCR)
- SERUM (RFFIT)
- IDENTIFIED
- DATA COLLECTED

9. GENERAL RESULTS
- 1 individual PCR positive and 5 antibody positive captured
one year later.
- Only one individual simultaneously carrying RNA and antibodies.

10. Different temporal pattern of circulation on each colony
Colonia
Det. N.
1998
1999
2000
2001
2002
2003
Puente Duende
PCR Ab
137
107
1 (4.7)
2 (22.2)
1 (4.5)
2 (8.0)
Puente
Gadea
114 89
2(6.9)
1 (3.7)
2 (11.11)
3 (17.65)
3 (23)
1 (8.3)
Molino
Rincón 67 54
1 (12.5)
1 (4.76)
Tunel
Coronada
143 85
1 (5.0)
5 (25.0)
Tunel Salomón 20 18
2 (15.7)
1 (9.0) La
Aradilla 7
1 (14.3)
Tunel del
Picote 51 23
1 (4.34)
Revolcad.Órgiva 71 27
Puente Cañave.
Ab.
138
108
7 (22.5)
4 (23.5)

11. Individuals captured ALWAYS in the same colony
Repetitive captures of Eptesicus isabellinus on
three colonies sampled six times
79.0
18.8
2.2
40.9
26.3
8.8
5.8
18.2
52.5
25.4
15.3
6.8
Individuals captured ALWAYS in the same colony

12. Genetic isolation of colonies
Different haplotype distribution on each colony
Genetic isolation of colonies

13. Conclusion Genetic isolation of Eptesicus isabellinus
natural colonies reflects the lack of interchange of
individuals that causes independent epidemiological
circulation of EBLV1.

14. Bats with EBLV1 RNA in oral cavity are healthy
BODY CONDITION INDEX AND PATTERN OF INFECTION 3 2 1
RT-PCR -1 -2 -3 -4 -5
BCI
Male
Female
1: Brain neg., swab neg.
2: Brain pos, swab pos.
3: Brain neg, swab pos.
Bats with EBLV1 RNA in oral cavity are healthy

15. Conclusion Many infections are subclinical with asymptomatic
presence of virus in oro-pharyngeal cavity.

16. LYSSAVIRUSES ARE NOT THE ONLY BAT RHABDOVIRUSES

17. GENERIC SEARCH OF DHIMARABDOVIRUSES
244 oro-pharyngeal swabs from healthy Eptesicus isabellinus captured in Spain. Two relevant sequences obtained.
50 BP
50 BP
INESPECIFIC AMPLIFICATION
DHIMARHABDO
EBLV1

18. Protein BLAST of a phragment of 39 aa

19. Protein BLAST of a phragment of 26 aa

20. OTHER WELL-KNOWN BAT-ASSOCIATED HUMAN PATHOGENS
EBOLA
SARS CORONAVIRUS
HENIPAVIRUS

21. OTHER BAT ASSOCIATED VIRUSES (Calisher et al
OTHER BAT ASSOCIATED VIRUSES (Calisher et al. –2006- Clinical Microbiological Reviews 19(3): )
Familia Orthomyxoviridae: Gripe A (Nyctalus noctula)
Familia Parayxoviridae:género Rubulavirus: Virus Mapuera (Sturnina lilium), Virus Menangle (Pteropus poliocephalus), Virus Tioman (Pteropus hypomelanus), Género no determinado (Rousettus leschenaultia).
Familia Togaviridae, Género Alphavirus: Virus Chikungunya (Scotophilus sp., Rousettus aegyptiacus, Hipposideros cafer, Chaerephon pumilus), Virus Sindbis (Rinnolophidae, Hipposeridae), Virus de la encefalitis equina venezolana (Desmodus rotundus, Uroderma bilibatum, Artibeus phaeotis).
Familia Flaviviridae, género Flavivirus,: Virus Bukalasa bat (Chaerophon pumilus), Virus Isla Carey (Cynopterus brachiotys), Virus Dakar bat(Cherephon pumilus, Taphozous perforatus, Scotophilus sp, Mops condylurus), Virus Entebbe bat (Chaerophon pumilus, Mops condylurus), Virus de la encefalitis japonesa (Hipposideros armiger, Miniopterus schreibersii, Rinolophus cornutus), Virus Jugra (Cynopterus brachiotis), Virus de la leucoencefalitis del Myotis de Montana (Myotis lucifugus), Virus Phom-Penh bat (Eonycteris spelaea, Cynopterus brachyotis), Virus Rio Bravo (Tadarida brasiliensis), Virus de la encefalitis de St. Louis (Tadarida brasiliensis), virus Saboya (Nycteris gambiensis), virus Sokuluk (Pipistrellus pipistrellus), Virus Tamana Bat (Pteronotus parnelli), virus Uganda (Rousettus sp, Tadarida sp), Virus Yokose (no identificado).
Familia Bunyaviridae, género Bunyavirus: Virus Catu (Molossus obscurus), Virus Guama (no identificado), virus Nepuyo (Artibeus jamaicensis). Género Hantavirus: Virus Hantaan (Eptesicus serotinus, Rhinolophus ferrumequinum). Género Phlebovirus, Virus de la fiebre del valle del Rif (Micropterus pusillus, Hipposideros abae, Hipposideros caffer, Miniopterus schreibersii, Epomops franqueti, Glauconycteris argentata), virus Toscana (Pipistrellus kulhii). Género desconocido, virus Kaeng Khoi (Cheropon plicatus), virus Bangi (Scotophilus sp, Pipstrellus sp, Tadarida sp.).
Familia Reoviridae, género Orbivirus: Virus Ife (Eidolum helvum), virus Japanaut (Syconicteris australis), virus Fomede (Nycteris nana). Género orthoreovirus, virus Nelson Bay (Pterupus poliocephalus), virus Palau (Pteropus hypomelanus), virus Broome (Pteropus alecto).
Familia arenaviridae, virus Tacaribe (Artibeus lituratus).
Familia picornaviridae, género indeterminado: virus Juruaca (no identificado)
No clasificados: virus Issyc-Kul (virus Keterah) (Nyctalus noctula, Eptesicus serotinus, Pipistrellus pipistrellus, Myotis blythii, Rinolophus ferrumequinum, Rinolophus lepidus, Rinolophus horsfeldi, Megaderma spasma, Scotophilus kulhii, Cynopterus branchyotis, Eonycteris spelea, Cherephon plicatus, Hipposideros diadema, Tazophous melanopogon, virus Mojui dos Campos (no identificado), virus Yoge (Rousettus aegyptiacus), virus Kasokero (Rousettus aegyptiacus).

22. 1.- ON THE BAT SIDE
+ Estación Biológica de Doñana, Sevilla
- Javier Juste
- Carlos Ibáñez
2.- ON THE VIRUS SIDE
+ Centro Nacional de Microbiología, Majadahonda, Madrid
- Juan E. Echevarría
- Sonia Vázquez
- Carolina Aznar
- José Miguel Berciano
3.- PATHOLOGIST
+ Laboratorio Central Veterinario. Santa Fe, Granada
- Eduardo Ruiz Villamor

23. Subtype 1a Subtype 1b EBLV1 TWO Eptesicus SPECIES TWO EBLV1 SUBTYPES?87 75 98 62 83 71 69 88 94 63 70 64 80 84 68 93 99 65
EBLV1
subtypes
TWO Eptesicus SPECIES TWO EBLV1 SUBTYPES?
The Netherlands
Germany
Denmark
France
Poland
Russia
Ucrania
Subtype 1a
Spain
France
The Netherlands
Germany
Subtype 1b
Not today, but more work needed on both sides