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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: [email protected]
I
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=3
n=7
n=6
The Israeli border isolates
Israel – Jordan – 7 isolates
Israel – Lebanon – 3 isolates Israel – Syria – 31 isolates
Total: 41 isolates
Location of rabies virus
isolates identified on Israeli
borders by animal
species 25 km.
Dead Sea
Syria
Lebanon
Jordan
Egypt
1997
2000
2000
20041997
1997
2005
2002
Legend: 41 cases
Dog- 21
Cattle- 11
Fox- 5
Wolf- 2
Badger- 1
Stone Marten-1
1997
2000
2000
20041997
1997
2005
2002
Legend: 41 cases
Dog- 21
Cattle- 11
Fox- 5
Wolf- 2
Badger- 1
Stone Marten-1
Antigen characterization
MABsMABs//VariantsVariants
V1V1V2V2
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
Antigen typing of
the border isolates
25 km.
Dead S
ea
Syria
Lebanon
Jordan
Egypt
1997
2000
2000
20041997
1997
2005
2002
Legend: 41 rabies cases on the borders
Antigenic variants:
V1 37
V2 4
1997
2000
2000
20041997
1997
2005
2002
Legend: 41 rabies cases on the borders
Antigenic variants:
V1 37
V2 4
MABsMABs//VariantsVariants
EgyEgy..V1V1
TurTur..V1V1
JorJor..V1V1
JorJor..V2V2
C1 63-3-1C1 63-3-1
C2 62-8-1C2 62-8-1
C3 62-11-1C3 62-11-1
C4 62-15-1C4 62-15-1
C5 62-22-3C5 62-22-3
C6 62-23-1C6 62-23-1
C7 62-24-1C7 62-24-1
Polyclonal antibodyPolyclonal antibody
C9 62-52-1C9 62-52-1
C10 62-52-2C10 62-52-2
C11 62-61-1C11 62-61-1
C12 62-62-4C12 62-62-4
103-7103-7
502-2502-2
C15 62-97-3C15 62-97-3
C16 62-97-11C16 62-97-11
C17 62-141-1C17 62-141-1
C18 62-143-2C18 62-143-2
C19 62-146-3C19 62-146-3
C20 64-164-2C20 64-164-2
Antigenic variants of
neighboring countries isolates
Positive Negative
MABsMABs//VariantsVariants
V1V1V2V2 V3V3V4V4V5V5V6V6
C1 63-3-1C1 63-3-1
C2 62-8-1C2 62-8-1
C3 62-11-1C3 62-11-1
C4 62-15-1C4 62-15-1
C5 62-22-3C5 62-22-3
C6 62-23-1C6 62-23-1
C7 62-24-1C7 62-24-1
Polyclonal antibodyPolyclonal antibody
C9 62-52-1C9 62-52-1
C10 62-52-2C10 62-52-2
C11 62-61-1C11 62-61-1
C12 62-62-4C12 62-62-4
103-7103-7
502-2502-2
C15 62-97-3C15 62-97-3
C16 62-97-11C16 62-97-11
C17 62-141-1C17 62-141-1
C18 62-143-2C18 62-143-2
C19 62-146-3C19 62-146-3
C20 64-164-2C20 64-164-2
Antigenic variants of Israeli isolates between
1993-2006
Positive
Negative
Weak positive
Antigen typing of
Israeli and Jordanian
rabies isolates
25 km.
Dead S
ea
Syria
Lebanon
Jordan
Egypt
Atigen Typing:
V1
V2
V3
V4
V5
V6
Reactivity of mAbs for antigenic typing of rabies isolates
Antigenic Variants
Amino acid and their position
Reactivity with mAb panel
101 Others Positive Negative
V1 D or N None Rest C18
V2 N S on 36 Rest C2, C12, C16, C18,103-7
V3 T I on 90S on 133
Rest C2, C7, C11*, C12, C18,103-7
V4 T I on 90A on 134
Rest C2, C7, C12, 103-7, C18
V5 T None Rest C2*, C7, C12, 103-7 , C18
V6 S F on 80 Rest C1, C7, C12, C18
* 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
9 9
N k5630U 2 2 4 8 3
U 2 2 4 8 2
M s 1 9 0 9Ay352475U 22476
U 22707Ay352476
U 43025Ay352464
U 42704U 22839
U 22475U 22840
U 42701U 42706
U 43433U 22474
U 42607T 4
U 22643
U 22637U 22629
U 22630U 22650
U 22651U 22488
U 22645U 22632
U 22638U 22484
PVSad 19
U 2 2 6 2 7
U 22640
U 22644U 22487
A f467949Ay352480
Y 09762A f374721
U 22653
V 5
V 3
V 4
V 4
V 1
V 2
V 1
V 6
V 1
V 1
V 1
V 1
V 5
V 4
V 4
V 1
V 1
V 6
V 1
V 1
Europe
W E
C E
E E
N E E
Turkey
V IIIsrael
IranVIsrael
IIIsrael
IVIsrael
IIsrael
IIIIsrael
Turkey, G eorg ia
A frica 1b
A frica 1a
Vaccine
A frica 4
A frica 3
A frica 2
A sia
A rc tic
M id d leE a st
E u rop e
A rcticA fr ica
A fr icaA sia
A ntigen ic var iants
Saudi A rabiaO m anJordan
C lu ste r s
V I
99
8 9
6 1
47
99
83
9680
8265
9647
44
94
A b041968
U 22639U 22489
U 22485
U 22486U 22634
U 22633
U 22852
T 1Ay352497
9961
59
52
53
9 9
99
9075
3632
4655
99
72
99
99
99
9950
5164
8494
74
4130
57
9985
25
24 99
7 3
C la d e s
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
Dead S
ea
Syria
Lebanon
Jordan
Egypt
12
3
4
25 km.
II
I
III
V
VI
I
VI
VII
I
I
I
V
I
IV
VI
VII
VI
V
V
Legend:
Fox - 41Dog - 29Cattle- 14Wolf- 2Badger- 3Stone Marten - 1 Cat-2Human-3Sheep-1Donkey-2Jackal-1Goat-1Squirrel-1Horse-1
Application of a
molecular clock
10
THI/1983IND/1996
SRI/2001GUI/1986CAM/1987BEN/1986NIG/1990TAC/1992
MAU/1986BUF/1986IVC/1989
R250/1983S4/1979
s1/1999s2/1998s3/1999
GAB/1986GAB2/1986ETH/1987
ALG/1982MAR/1990NIG/1989CAF/1992CAF2/1992TAN/1992
ZAI/1989NAM/1987MOZ/1986
RSP/1981t1/2000t2/2000t4/2000t7/2000
0187/20059613/20057626/20050298/20051211/20045745/2005
HUN/1991R257/1989EST/1991
EST/1985R245/1990
YOU/1972YOU/1986
GER/1991ALL/1991
POL/1985YOU2/1986
FRA/19749147/1991FRA/1992
1353/19971132/19971919/2000j2/1999
IRN/1986IRN/1987OMN/1990
SDR/19875417/2000j3/1998j1/1999j6/19985644/1998145/1997
8437/20053370/19951902/19953205/19975700/19979489/19987411/1998
329/19964230/1997
4828/20049655/1997j4/19980034/2002
6462/19966530/19962323/19968124/19961153/19981064/20029330/2003
1598/1999445/19977230/1999
0699/19965814/1999
2437/20045970/2003
8855/19961956/2000
6967/19979207/1999
3889/19954802/19988389/19980160/19993996/2000
8617/19962180/2001
7106/1998
1550 1600 1650 1700 1750 1800 1850 1900 1950 2000
AsiaAfrica 3
Africa 2
ArcticAfrica 4
Africa 1b
Africa 1a
Turkey
Israel VII
Europe
Israel VI
Iran
Israel V
Israel II
Israel IV
Israel I
Israel III
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 mABs could discriminate the antigen diversity of the Middle
East isolates (at present).
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.
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
Thanks for your attention
