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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
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”
1
GRANADA
1987
SI
Eptesicus
serotinus
EBLV1
5
HUELVA
1989
NO
Eptesicus
serotinus
EBLV1
1
GRANADA
1994
SI
Eptesicus
serotinus
EBLV1
3
SEVILLA
1999
1 SI
2 NO
Eptesicus
serotinus
EBLV1
1
MURCIA
1999
SI
Eptesicus
serotinus
EBLV1
4
SEVILLA
2000
NO
Eptesicus
serotinus
EBLV1
1
MURCIA
2002
SI
Eptesicus
serotinus
EBLV1
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
19Ebo SY
Genes: CytB, ND1, RAG2
2900 bp
14Ebo JO
97Ebog IR
98Ebog IR
54Ese NorthSpain
55Ese North Spain
50Ese IT
44Ese CZ
Eptesicus serotinus
64Ese UK
Europe
46Ese DE
(including north Spain)
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
34Eis MO
23Eis LI
28Eis LI
South Spain
North Africa
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: 490-496 )
-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: 372-377)
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):
3678-3683)
- 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 147:349-361)
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 –2002Emerging Infectious Diseases 8 (4): 413-420)
- Detection of EBLV2 specific antibodies on Daubenton´s
Bats from Scotland.(Brookes -2005- Emerging Infectious
Diseases 11(4): 572-578).
Lyssaviruses are not pathogenic for bats (3)
- Survival of Eptesicus fuscus after inoculation of RABV
(Shankar –2004- Journal of Wildlife Diseases 40(3): 403-413)
- Survival of Tadarida brasiliensis after aerosol exposition
to RABV. (Davis –2007- Journal of infectious
Diseases195:1144-1150)
STUDY OF EBLV1 INFECTION ON BAT COLONIES
-19 COLONIES OF Eptesicus isabellinus
-1080 INDIVIDUALS
-1227 CAPTURES (1998-2003)
TUNEL
TUNEL DEL MANZANO
SALOMON
LA ARADILLA
PUENTE TAMUJOSO
TUNEL DEL PICOTE
AZNALCOLLAR (Puente Cañaveroso)
ALCALA DEL RIO
SOTIEL CORONADA
SEVILLA (CATEDRAL)
MOLINO DEL DUENDE
SEVILLA (ALCAZAR)
MOLINO DE NIEBLA
SEVILLA (SECRETARIA)
PUENTE GADEA UMBRETE(Colegio)
MOLINO DEL RINCON
- O-PH SWAB (PCR)
- SERUM (RFFIT)
- IDENTIFIED
- DATA COLLECTED
TRAJANO
ORJIVA (TUNEL GUADALFEO)
ORJIVA (REVOLCADERO)
50 km
GENERAL RESULTS
TOTAL CAPTURAS
NUMERO DE COLONIAS
TOTAL POSITIVOS
Número de capturas
1998
Positivos
Número de capturas
1999
Positivos
Número de capturas
2000
Positivos
Número de capturas
2001
Positivos
Número de capturas
2002
Positivos
Número de capturas
2003
Positivos
RT-PCR
1227
19
34 (2.8%)
164
4 (2.4%)
161
4 (2.5%)
204
16 (7.8.%)
209
0
287
10 (3.4%)
201
0
RFFIT
626
13
51 (8.1%)
151
10 (6.6%)
90
5 (5.6%)
128
12 (9.4%)
96
4 (4.2%)
100
5 (5%)
54
0
- 1 individual PCR positive and 5 antibody positive captured
one year later.
- Only one individual simultaneously carrying RNA and antibodies.
Different temporal pattern of circulation on each colony
Colonia
Det.
N.
1998
1999
2000
Puente
Duende
PCR
Ab
137
107
1 (4.7)
0
0
2 (22.2)
1 (4.5)
1 (4.5)
Puente
Gadea
PCR
Ab
114
89
0
2(6.9)
1 (3.7)
2 (11.11)
0
3 (17.65)
Molino
Rincón
PCR
Ab
67
54
0
1 (12.5)
Tunel
Coronad
a
PCR
Ab
143
85
1 (5.0)
5 (25.0)
Tunel
Salomón
PCR
Ab
20
18
2 (15.7)
1 (9.0)
La
Aradilla
PCR
Ab
7
7
0
1 (14.3)
Tunel del
Picote
PCR
Ab
51
23
Revolcad
.Órgiva
PCR
Ab
71
27
Puente
Cañave.
PCR
Ab.
138
108
2001
2002
2 (8.0)
0
0
3 (23)
1 (4.76)
0
0
1 (8.3)
1 (4.76)
0
1 (4.34)
1 (4.34)
0
1 (3.7)
7 (22.5)
4 (23.5)
2003
Repetitive captures of Eptesicus isabellinus on
three colonies sampled six times
120
79.0
100
80
52.5
40.9
60
26.3
40
18.8
18.2
20
8.8
2.2
25.4
15.3
5.8
6.8
0
1 CAPTURE 2 CAPTURES 3 CAPTURES 1 CAPTURE 2 CAPTURES 3 CAPTURES 4 CAPTURES 5 CAPTURES 1 CAPTURE 2 CAPTURES 3 CAPTURES 4 CAPTURES
Puente Cañaveroso
Puente Molino del Duende
Túnel (tren) Coronada
Individuals captured ALWAYS in the same colony
Different haplotype distribution on each colony
Genetic isolation of colonies
Conclusion
Genetic isolation of Eptesicus isabellinus
natural colonies reflects the lack of interchange of
individuals that causes independent epidemiological
circulation of EBLV1.
BCI
BODY CONDITION INDEX AND PATTERN OF INFECTION
1
0
-1
-2
-3
-4
-5
Male
Female
1: Brain neg., swab neg.
2: Brain pos, swab pos.
3: Brain neg, swab pos.
1
3
2
RT-PCR
Bats with EBLV1 RNA in oral cavity are healthy
Conclusion
Many infections are subclinical with asymptomatic
presence of virus in oro-pharyngeal cavity.
LYSSAVIRUSES ARE NOT THE ONLY BAT RHABDOVIRUSES
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
Protein BLAST of a phragment of 39 aa
Protein BLAST of a phragment of 26 aa
OTHER WELL-KNOWN BAT-ASSOCIATED HUMAN PATHOGENS
EBOLA
SARS CORONAVIRUS
HENIPAVIRUS
OTHER BAT ASSOCIATED VIRUSES (Calisher et al. –2006- Clinical Microbiological Reviews 19(3):531-545)
Familia Orthomyxoviridae: Gripe A (Nyctalus noctula)
Familia Parayxoviridae:género Rubulavirus: Virus Mapuera (Sturnina lilium), Virus Menangle (Pteropus poliocephalus), Virus
(Pteropus hypomelanus), Género no determinado (Rousettus leschenaultia).
Tioman
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).
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
87
EBLV1
subtypes
TWO Eptesicus SPECIES TWO EBLV1 SUBTYPES?
65
Subtype 1a
99
68
9875
64
68
71
70
84 63
94
9380
69
88
87
88
83
87
75
62
Subtype 1b
The Netherlands
Germany
Denmark
France
Poland
Russia
Ucrania
Spain
France
The Netherlands
Germany
Not today, but more work needed on both sides