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The application of genetic markers for European Bat Lyssavirus (EBLV) surveillance in bats

The application of genetic markers for European Bat Lyssavirus (EBLV) surveillance in bats. Sarah Harris Rabies and Wildlife Zoonoses Group (VLA – Weybridge, UK) [WHO Collaborating Centre, Med-Vet-Net] Paris, May 2007. The application of genetic markers for EBLV surveillance in bats.

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The application of genetic markers for European Bat Lyssavirus (EBLV) surveillance in bats

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  1. The application of genetic markers for European Bat Lyssavirus (EBLV) surveillance in bats Sarah Harris Rabies and Wildlife Zoonoses Group (VLA – Weybridge, UK) [WHO Collaborating Centre, Med-Vet-Net] Paris, May 2007

  2. The application of genetic markers for EBLV surveillance in bats • EBLV passive surveillance in UK bats • Current methods of bat species ID and problems • Application of genetic markers: Cytochrome b gene: cryptic species b-actin gene: EBLV-2 virus +ve bat case • Future applications

  3. EBLV UK passive surveillance (1987-2007) • Bats submitted > 6,700, 16 UK sp, numbers vary (Harris et al., 2006) • 12 bats of 7 non-UK species: e.g. Pond bat (EBLV-2) • UK cases: 5 EBLV-2 virus +ve Daubenton’s • Accurate species identification essential Morphological characters:body size, nose-leaf, forearm

  4. Time (~800 bats per year), quality, knowledge, cryptic species Problems with bat species identification Morphologically highly similar: Dentition - worn Penis shape - age Forearm - overlap Brandt’s Whiskered Khujand virus identified in Whiskeredbat (Tajikstan, Kuzmin et al., 2003) Older cases in unusual species: P. Pipistrellus/P. nathusii: EBLV-1 Germany (1985-1992) N. noctula: EBLV-1 (1991) EBLV-2 (1985) Ukraine Canine isolate (1955) Former Yugoslavia – species not present

  5. So how can we improve rapidity and accuracy of bat species identification? Molecular markers – 2 genes 1. Cytochrome b –mtDNA genefor bat phylogenetics Cryptic species 2. b-actin – housekeeping gene for EBLV PCR at VLA Rapid identification of species in suspect bat cases

  6. Development of Cytochrome b markers Method • Cytochrome b (1,200 bp) • Primers designed (~800 bp), PCR, sequencing • Phylogenetic analysis (PAUP: ML analysis, 1000 BS) Results • Identified markers for 13 UK species + haplotypes • Correctly identified 2 cryptic species Morphological ID was incorrect in some cases

  7. Natterer’s bat Greater Mouse-eared bat Daubenton’s bat (2 haplotypes) Bechstein bat Whiskered (n = 16) Brandt’s (n = 12) + 4 ‘Whiskered’ Cytochrome b phylogenetic analysis: Myotis genus 20% of morphological ID Whiskered bat’s were genetically ID as Brandt’s bats Current UK pop estimates: 40,000 and 30,000 – accurate for conservation?

  8. Development of b-actin markers Method • RNA extracted from brain sample of bats • PCR (primers ~313 bp) b-act1 / b-act2 (Murray et al., 1990), sequencing • Phylip phylogenetic analysis (ML analysis, 100 BS) Results • b-actin markers for 11 UK species • Species ID of EBLV-2 +ve bat

  9. Application of b-actin in virus positive cases • September 2004, suspect bat submitted (Surrey, UK) • Standard tests – positive for EBLV-2 • Amplified cDNA generated from RNA (brain), b-actin PCR • b-actin sequence aligned with 32 UK bat sequences • 99.85% similarity with Daubenton’s bat 348 bpb-Actin:M. daubentonii EBLV-2 +ve bat M + - 603/04 M: marker + : ‘+ve’ mouse brain - : ‘-ve’ control 603/04: M. daubentonii 603 bps b-Actin (348 bps) 310 bps

  10. Future applications • EBLV Passive Surveillance systems in Europe: Essential for ID of -ve bat cases as well as +ve Up until 2004: >200 EBLV+ve bat cases with species unknown Markers will also enable ID of incomplete/damaged carcasses • Lyssaviruses in other species Cytochrome b gene species ID of LBV-infected Mongoose (Markotter et al., 2006, EID) • Disease epidemiology requires accurate information known host species > host range > epidemiological patterns > potential threat to public/animal health

  11. Acknowledgements • University of Bristol (Prof. Gareth Jones) • VLA (Dr. Nick Johnson, Dr. Sharon Brookes, Prof. Tony Fooks, and other RWZG members) • Joint Nature Conservation Committee (JNCC) • Defra • Tony Hutson

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