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J.E. ECHEVARRÍA 1 , S. VÁZQUEZ-MORÓN 1 , C. IBÁÑEZ 2 , J.C. AZNAR 1 , E. RUIZ 3 , J. JUSTE 2

ASYMPTOMATIC RHABDOVIRUS INFECTION IN MERIDIONAL SEROTINE BATS ( Eptesicus isabellinus ) FROM SPAIN. J.E. ECHEVARRÍA 1 , S. VÁZQUEZ-MORÓN 1 , C. IBÁÑEZ 2 , J.C. AZNAR 1 , E. RUIZ 3 , J. JUSTE 2. 1 National Center of Microbiology, Carlos III Institute of Health, Majadahonda, Madrid

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J.E. ECHEVARRÍA 1 , S. VÁZQUEZ-MORÓN 1 , C. IBÁÑEZ 2 , J.C. AZNAR 1 , E. RUIZ 3 , J. JUSTE 2

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  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 1National 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

  3. > 16 % cytb!! 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 Europe (including north Spain) 64Ese UK 46Ese DE Eptesicus serotinus 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 South Spain North Africa 35Eis South Spain 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: 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)

  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): • 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)

  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 fromhealthy colonies in Spain.(Serra-Cobo –2002- EmergingInfectious 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).

  7. Lyssaviruses are not pathogenic for bats (3) • Survival of Eptesicus fuscusafter 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)

  8. TUNEL SALOMON TUNEL DEL MANZANO 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 ORJIVA (TUNEL GUADALFEO) TRAJANO ORJIVA (REVOLCADERO) 50 km STUDY OF EBLV1 INFECTION ON BAT COLONIES • 19 COLONIES OF Eptesicus isabellinus • 1080 INDIVIDUALS • 1227 CAPTURES (1998-2003) - 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

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

  12. 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. 1 0 -1 BCI -2 -3 -4 -5 Male Female 1 3 2 RT-PCR BODY CONDITION INDEX AND PATTERN OF INFECTION 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. –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 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. 87 65 99 68 75 98 64 68 71 70 84 63 94 93 80 69 88 88 87 83 87 75 62 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

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