1. Identification of novel canine rabies clades in the Middle East and North Africa Dan David Rabies Laboratory, Pathology Division, Kimron Veterinary Institute, Bet Dagan 50250, P.O.B. 12, Israel E-mail: davidd@int.gov.il I

2. Rabies in the Middle East Rabies is enzootic throughout the Middle East The main reservoirs and transmitters of the virus are dogs and foxes

3. Aim of the study Israel is the only country in the Middle East that has implemented an oral vaccination program. To ensure the success of the program, rabies surveillance is performed along Israeli borders to detect disease ingress

4. Material and Methods 1) Brain tissue samples were collected from animals in Israel (n=54) and from the border areas (n=41) during the years 1993-2006. 2) Samples were considered as positive for rabies by direct IF assay. 3) Antigen typing made using CDC panel of 19 monoclonal antibodies to detect the nucleoprotein. 4) Direct sequencing of 1350 bp of the nucleoprotein gene. 5) Molecular analysis by the MEGA computer program.

5. Kelev strain rabies virus isolate Isolated in Israel in 1950 from a naturally infected dog. The original street virus virulence was modified by 100 passages in chick embryos (Komarov & Hornstein, 1953). The Kelev vaccine has been used for cat and dog vaccination in Turkey since 1968 (Orhan et al., 1998).

6. Middle Eastern isolates used in this study n=3 n=7 n=6

7. The Israeli border isolates Israel – Jordan – 7 isolates Israel – Lebanon – 3 isolates Israel – Syria – 31 isolates Total: 41 isolates

8. Location of rabies virus isolates identified on Israeli borders by animal species

9. Antigen characterization

10. V2V1 MABs / Variants C1 63-3-1 C2 62-8-1 C3 62-11-1 C4 62-15-1 C5 62-22-3 C6 62-23-1 C7 62-24-1 Polyclonal antibody C9 62-52-1 C10 62-52-2 C11 62-61-1 C12 62-62-4 103-7 502-2 C15 62-97-3 C16 62-97-11 C17 62-141-1 C18 62-143-2 C19 62-146-3 C20 64-164-2 Antigenic variants of the Israeli border isolates Positive Negative

11. Antigen typing of the border isolates

12. Jor.V2Jor.V1Tur.V1Egy.V1 MABs / Variants C1 63-3-1 C2 62-8-1 C3 62-11-1 C4 62-15-1 C5 62-22-3 C6 62-23-1 C7 62-24-1 Polyclonal antibody C9 62-52-1 C10 62-52-2 C11 62-61-1 C11 62-61-1 C12 62-62-4 103-7 502-2 C15 62-97-3 C16 62-97-11 C17 62-141-1 C18 62-143-2 C18 62-143-2 C19 62-146-3 C20 64-164-2 Antigenic variants of neighboring countries isolates Positive Negative

13. V6V5V4 V3 V3V2V1 MABs / Variants C1 63-3-1 C2 62-8-1 C3 62-11-1 C4 62-15-1 C5 62-22-3 C6 62-23-1 C7 62-24-1 Polyclonal antibody C9 62-52-1 C10 62-52-2 C11 62-61-1 C11 62-61-1 C12 62-62-4 103-7 502-2 C15 62-97-3 C16 62-97-11 C17 62-141-1 C18 62-143-2 C18 62-143-2 C19 62-146-3 C20 64-164-2 Antigenic variants of Israeli isolates between 1993-2006 Positive Negative Weak positive

14. Antigen typing of Israeli and Jordanian rabies isolates

15. Reactivity of mAbs for antigenic typing of rabies isolates Antigenic Variants Amino acid and their position Reactivity with mAb panel 101Others Positive Negative V1D or NNoneRest C18 V2NS on 36Rest C2, C12, C16, C18,103-7 V3TI on 90 S on 133 Rest C2, C7, C11*, C12, C18, 103-7 V4TI on 90 A on 134 Rest C2, C7, C12, 103-7, C18 V5TNoneRest C2*, C7, C12, 103-7, C18 V6SF on 80Rest C1, C7, C12, C18 * Weak reaction

16. Molecular characterization

17. Phylogenetic analysis of rabies isolates based on the nucleoprotein gene sequence David D. et al., J. Gen. Virol. 2007, 88: 967-980

18. Distribution of the rabies Border isolates and Jordanian isolates based on genetic analysis David D. et al., J. Gen. Virol. 2007, 88: 967-980 Israeli rabies isolates belongs to clades I to IV between 1995-2004 Rabies isolates on the border belongs to clade VII Rabies isolates on the border belongs to clade VI Legend: Rabies isolates on the border belongs to clade V Rabies isolates on the borders belongs to clade I

19. Application of a molecular clock David D. et al., J. Gen. Virol. 2007, 88: 967-980

20. Conclusions -1 1) The isolates that created the new clades (V, VI and VII) penetrated into Israel from neighbouring countries. 2) A panel of 8 mABs could discriminate the antigen diversity of the Middle East isolates (at present).

21. 4) The Middle East and European appeared in the same period of time, around 1870. Conclusions - 2 3) The Kelev strain, which was classified as a member of Africa 4 clade, was probably translocated during the 1950 Israel –Egypt conflict.

22. Thanks for contributions from: Gareth J. Hughes-University of Edinburgh, UK Ivan V. Kuzmin, Charles E. Rupprecht- Rabies Lab. CDC, Atlanta, GA, USA Hikmet Hun, Orhan Aylan - Etlik, Ankara, Turkey Boris Yakobson, Neli Dvers, Irit Davidson – Kimron Veterinary Institute

23. Thanks for your attention