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 Paris, May 2007

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

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

4. Problems with bat species identification Time (~800 bats per year), quality, knowledge, cryptic species Brandts Whiskered Morphologically highly similar: Dentition - worn Penis shape - age Forearm - overlap Khujand virus identified in Whiskered bat (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. Molecular markers – 2 genes 1.Cytochrome b – mtDNA gene for bat phylogenetics Cryptic species 2. -actin – housekeeping gene for EBLV PCR at VLA 2. -actin – housekeeping gene for EBLV PCR at VLA Rapid identification of species in suspect bat cases So how can we improve rapidity and accuracy of bat species identification?

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

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

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

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

10. Future applications Future applications EBLV Passive Surveillance systems in Europe: 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 Lyssaviruses in other species Cytochrome b gene species ID of LBV-infected Mongoose (Markotter et al., 2006, EID) Disease epidemiology requires accurate information Disease epidemiology requires accurate information known host species > host range > epidemiological patterns > known host species > host range > epidemiological patterns > potential threat to public/animal health

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