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, Madrid2 Doñana Biological Station, CSIC, Seville3 Laboratorio Central Veterinario, Santa Fe, Granada

Spain

ASYMPTOMATIC RHABDOVIRUS INFECTION IN MERIDIONALSEROTINE BATS (Eptesicus isabellinus) FROM SPAIN

Número de individuos

Localidad añoExposición humana

Murciélago Virus

1 VALENCIA 1987 SI UNKNOWN“DUVENHAGE

LIKE”

1 GRANADA 1987 SIEptesicus serotinus

EBLV1

5 HUELVA 1989 NOEptesicus serotinus

EBLV1

1 GRANADA 1994 SIEptesicus serotinus

EBLV1

3 SEVILLA 19991 SI

2 NOEptesicus serotinus

EBLV1

1 MURCIA 1999 SIEptesicus serotinus

EBLV1

4 SEVILLA 2000 NOEptesicus serotinus

EBLV1

1 MURCIA 2002 SIEptesicus

serotinusEBLV1

Lyssavirus-infected bats detected by passive surveillance in Spain

Genes: CytB, ND1, RAG22900 bp

57Ese SY

90Ebtur IR

93Ebtur IR

92Ebtur IR

91Ebtur IR

94Ebsp IR

95Ebsp IR

61Ese SY

60Ese SY

43Ese CY

16Ebo SY

19Ebo SY

14Ebo JO

97Ebog IR

98Ebog IR

54Ese NorthSpain

55Ese North Spain

50Ese IT

44Ese CZ

64Ese UK

46Ese DE

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

35Eis South Spain

34Eis MO

23Eis LI

28Eis LI

Eptesicus Eptesicus serotinusserotinus

Eptesicus Eptesicus isabellinusisabellinus

> 1

6 %

> 1

6 %

cytb

!!

cytb

!!

Filogeny of Eptesicus in the Paleartic

Europe (including north Spain)

South SpainNorth Africa

E. serotinus or E. Isabellinus?

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)

- 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)

- 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)

Lyssaviruses are not pathogenic for bats (2)

- Detection of EBLV2 specific antibodies on Daubenton´sBats from Scotland.(Brookes -2005- Emerging Infectious Diseases 11(4): 572-578).

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): 413-420)

- Survival of Tadarida brasiliensis after aerosol exposition to RABV. (Davis –2007- Journal of infectious Diseases195:1144-1150)

- Survival of Eptesicus fuscus after inoculation of RABV(Shankar –2004- Journal of Wildlife Diseases 40(3): 403-413)

Lyssaviruses are not pathogenic for bats (3)

SEVILLA (SECRETARIA)

SEVILLA (CATEDRAL)

TRAJANO

ALCALA DEL RIOSOTIEL CORONADAAZNALCOLLAR (Puente Cañaveroso)

MOLINO DEL DUENDEMOLINO 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)

-19 COLONIES OF Eptesicus isabellinus-1080 INDIVIDUALS-1227 CAPTURES (1998-2003)

STUDY OF EBLV1 INFECTION ON BAT COLONIES

- O-PH SWAB (PCR) - SERUM (RFFIT) - IDENTIFIED - DATA COLLECTED

GENERAL RESULTS

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

RTRT--PCR PCR RFFITRFFIT12271227 6266261919 1313

34 (2.8%) 51 (8.1%)NNúúmero de capturasmero de capturas 164164 151151PositivosPositivos 4 (2.4%)4 (2.4%) 10 (6.6%)10 (6.6%)NNúúmero de capturasmero de capturas 161161 9090PositivosPositivos 4 (2.5%)4 (2.5%) 5 (5.6%)5 (5.6%)NNúúmero de capturasmero de capturas 204204 128128PositivosPositivos 16 (7.8.%)16 (7.8.%) 12 (9.4%)12 (9.4%)NNúúmero de capturasmero de capturas 209209 9696PositivosPositivos 00 4 (4.2%)4 (4.2%)NNúúmero de capturasmero de capturas 287287 100100PositivosPositivos 10 (3.4%)10 (3.4%) 5 (5%)5 (5%)NNúúmero de capturasmero de capturas 201201 5454PositivosPositivos 00 00

NUMERO DE COLONIASNUMERO DE COLONIASTOTAL CAPTURASTOTAL CAPTURAS

20002000

20012001

20022002

20032003

19981998

19991999

TOTAL POSITIVOSTOTAL POSITIVOS

RTRT--PCR PCR RFFITRFFIT12271227 6266261919 1313

34 (2.8%) 51 (8.1%)NNúúmero de capturasmero de capturas 164164 151151PositivosPositivos 4 (2.4%)4 (2.4%) 10 (6.6%)10 (6.6%)NNúúmero de capturasmero de capturas 161161 9090PositivosPositivos 4 (2.5%)4 (2.5%) 5 (5.6%)5 (5.6%)NNúúmero de capturasmero de capturas 204204 128128PositivosPositivos 16 (7.8.%)16 (7.8.%) 12 (9.4%)12 (9.4%)NNúúmero de capturasmero de capturas 209209 9696PositivosPositivos 00 4 (4.2%)4 (4.2%)NNúúmero de capturasmero de capturas 287287 100100PositivosPositivos 10 (3.4%)10 (3.4%) 5 (5%)5 (5%)NNúúmero de capturasmero de capturas 201201 5454PositivosPositivos 00 00

NUMERO DE COLONIASNUMERO DE COLONIASTOTAL CAPTURASTOTAL CAPTURAS

20002000

20012001

20022002

20032003

19981998

19991999

TOTAL POSITIVOSTOTAL POSITIVOS

Different temporal pattern of circulation on each colony

Colonia Det. N. 1998 1999 2000 2001 2002 2003Puente Duende

PCR

Ab

137

107

1 (4.7)

0

0

2 (22.2)

1 (4.5)

1 (4.5)

2 (8.0)

0

Puente

Gadea

PCR

Ab

114

89

0

2(6.9)

1 (3.7)

2 (11.11)

0

3 (17.65)

0

3 (23)

0

1 (8.3)

Molino

Rincón

PCR

Ab

67

54

0

1 (12.5)

1 (4.76)

0

1 (4.76)

0

Tunel

Coronada

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

1 (4.34)

1 (4.34)

Revolcad.Órgiva

PCR

Ab

71

27

0

1 (3.7)

Puente Cañave.

PCR

Ab.

138

108

7 (22.5)

4 (23.5)

Repetitive captures of Eptesicus isabellinus onthree colonies sampled six times

0

20

40

60

80

100

120

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

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

Genetic isolation of colonies

Different haplotype distribution on each colony

Conclusion

Genetic isolation of Eptesicus isabellinus

natural colonies reflects the lack of interchange of

individuals that causes independent epidemiological

circulation of EBLV1.

321RT-PCR

10-1-2-3-4-5

BC

I

1: Brain neg., swab neg.

2: Brain pos, swab pos.

3: Brain neg, swab pos.

Male

Female

BODY CONDITION INDEX AND PATTERN OF INFECTION

Bats with EBLV1 RNA in oral cavity are healthy

Many infections are subclinical with asymptomatic

presence of virus in oro-pharyngeal cavity.

Conclusion

LYSSAVIRUSES ARE NOT THE ONLY BAT RHABDOVIRUSES

244 oro-pharyngeal swabs from healthy Eptesicus isabellinus captured in Spain. Two relevant sequences obtained.

GENERIC SEARCH OF DHIMARABDOVIRUSES

EBLV1 DHIMARHABDOINESPECIFIC AMPLIFICATION

50 BP 50 BP

Protein BLAST of a phragment of 39 aa

Protein BLAST of a phragment of 26 aa

SARS CORONAVIRUS EBOLA

HENIPAVIRUS

OTHER WELL-KNOWN BAT-ASSOCIATED HUMAN PATHOGENS

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).

OTHER BAT ASSOCIATED VIRUSES (Calisher et al. –2006- Clinical Microbiological Reviews 19(3):531-545)

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

TWO Eptesicus SPECIES TWO EBLV1 SUBTYPES?

87

7598

6275

8387

71

6988

88

946370

64

87

80

84

68

93

6899

65

Subtype 1a

Subtype 1b

The NetherlandsGermanyDenmarkFrancePolandRussiaUcrania

Spain FranceThe NetherlandsGermany

Not today, but more work needed on both sides

EBLV1subtypes