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
Rabies in the Middle East Rabies is enzootic throughout the Middle East The main reservoirs and transmitters of the virus are dogs and foxes
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
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.
“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).
Middle Eastern isolates used in this study n=7 n=6 n=3
The Israeli border isolates Israel – Jordan – 7 isolates Israel – Lebanon – Israel – Syria 3 isolates – 31 isolates Total: 41 isolates
Location of rabies virus isolates identified on Israeli borders by animal species
Antigen characterization
MABs/ Variants Antigenic variants of the Israeli border isolates C 1 63 -3 -1 C 2 62 -8 -1 C 3 62 -11 -1 C 4 62 -15 -1 C 5 62 -22 -3 C 6 62 -23 -1 C 7 62 -24 -1 Polyclonal antibody C 9 62 -52 -1 C 10 62 -52 -2 C 11 62 -61 -1 C 12 62 -62 -4 103 -7 502 -2 C 15 Positive Negative 62 -97 -3 C 16 62 -97 -11 C 17 62 -141 -1 C 18 62 -143 -2 C 19 62 -146 -3 C 20 64 -164 -2 V 1 V 2
Antigen typing of the border isolates
MABs/ Variants Antigenic variants of neighboring countries isolates C 1 63 -3 -1 C 2 62 -8 -1 C 3 62 -11 -1 C 4 62 -15 -1 C 5 62 -22 -3 C 6 62 -23 -1 C 7 62 -24 -1 Polyclonal antibody C 9 62 -52 -1 C 10 62 -52 -2 C 11 62 -61 -1 C 12 62 -62 -4 103 -7 502 -2 C 15 C 16 Positive Negative 62 -97 -3 62 -97 -11 C 17 62 -141 -1 C 18 62 -143 -2 C 19 62 -146 -3 C 20 64 -164 -2 Egy. V 1 Tur. V 1 Jor. V 2
Antigenic variants of Israeli isolates between 1993 -2006 MABs/ Variants C 1 63 -3 -1 C 2 62 -8 -1 C 3 62 -11 -1 C 4 62 -15 -1 C 5 62 -22 -3 C 6 62 -23 -1 C 7 62 -24 -1 Polyclonal antibody C 9 62 -52 -1 C 10 62 -52 -2 C 11 62 -61 -1 C 12 62 -62 -4 103 -7 502 -2 C 15 Positive Negative Weak positive 62 -97 -3 C 16 62 -97 -11 C 17 62 -141 -1 C 18 62 -143 -2 C 19 62 -146 -3 C 20 64 -164 -2 V 1 V 2 V 3 V 4 V 5 V 6
Antigen typing of Israeli and Jordanian rabies isolates
Reactivity of m. Abs for antigenic typing of rabies isolates Antigenic Variants Amino acid and their position Reactivity with m. Ab panel 101 Others Positive Negative V 1 D or N None Rest C 18 V 2 N S on 36 Rest C 2, C 16, C 18, 103 -7 V 3 T I on 90 S on 133 Rest C 2, C 7, C 11*, C 12, C 18, 103 -7 V 4 T I on 90 A on 134 Rest C 2, C 7, C 12, 103 -7, C 18 V 5 T None Rest C 2*, C 7, C 12, 103 -7 , C 18 V 6 S F on 80 Rest C 1, C 7, C 12, C 18 * Weak reaction
Molecular characterization
Phylogenetic analysis of rabies isolates based on the nucleoprotein gene sequence David D. et al. , J. Gen. Virol. 2007, 88: 967 -980
Distribution of the rabies Border isolates and Jordanian isolates based on genetic analysis Legend: 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 V Rabies isolates on the borders belongs to clade I David D. et al. , J. Gen. Virol. 2007, 88: 967 -980
Applicatio n of a molecular clock David D. et al. , J. Gen. Virol. 2007, 88: 967 -980
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 m. ABs could discriminate the antigen diversity of the Middle East isolates (at present).
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. 4) The Middle East and European appeared in the same period of time, around 1870.
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
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