Identification of novel canine rabies virus clades in the ... - CiteSeerX

Journal of General Virology (2007), 88, 967–980

DOI 10.1099/vir.0.82352-0

Identification of novel canine rabies virus clades in the Middle East and North Africa Dan David,1 Gareth J. Hughes,2 Boris A. Yakobson,1 Irit Davidson,3 Hikmat Un,4 Orhan Aylan,4 Ivan V. Kuzmin5 and Charles E. Rupprecht5 1

Rabies Laboratory, Pathology Division, Kimron Veterinary Institute, Bet Dagan 50250, Israel

Correspondence Dan David

2

Laboratory for Clinical and Molecular Virology, The University of Edinburgh, Edinburgh EH9 1QH, UK

[email protected]

3

Division of Avian Diseases, Kimron Veterinary Institute, Bet Dagan 50250, Israel

4

Etlik Central Veterinary Control and Research Institute, Etlik, Ankara, Turkey

5

Rabies Unit, Viral and Rickettsial Zoonoses Branch, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA

Received 4 July 2006 Accepted 21 November 2006

Four novel phylogenetic clades of canine rabies virus (RABV) variants have been identified in the Middle East and North Africa. The three novel Middle Eastern clades comprise RABV isolates from the borders between Israel and neighbouring countries. The North African clade (Africa 4) comprises four RABV isolates from Egypt and one from Israel. We characterized various RABV lineages antigenically by using a panel of monoclonal antibodies to the nucleoprotein (N) and phylogenetically by analysis of entire N gene sequences. The estimated mean substitution rate for the N gene alignment (2.7610”4 substitutions per site per year) is comparable with previous estimates for RABV. The application of a molecular clock indicates the emergence of current canine RABV diversity to have occurred at about the same time (approx. 1870) in the Middle East and Europe, following divergence from established lineages in Africa and Asia.

INTRODUCTION Rabies is a fatal disease of humans and all other warmblooded animals. The virus belongs to the genus Lyssavirus, family Rhabdoviridae. Rabies virus (RABV) has a negativesense RNA genome of approximately 12 kb and consists of five genes encoding the nucleoprotein (N), phosphoprotein (P), matrix protein, glycoprotein and polymerase (Tordo et al., 1986). The original method for typing RABV was a panel of monoclonal antibodies (mAbs) to the viral nucleoprotein (N) (Smith, 1989; Wiktor & Koprowski, 1978). However that method is quite antiquated and was replaced by phylogenetic analysis based on genetic variability (Sacramento et al., 1991; Smith et al., 1992). There are seven recognized genotypes of lyssavirus defined on the basis of their genetic similarity: rabies virus (genotype 1), Lagos bat virus (genotype 2), Mokola (genotype 3), Duvenhage virus (genotype 4), European bat lyssavirus type 1 (genotype 5), European bat lyssavirus type 2 (genotype 6) and Australian bat lyssavirus (genotype 7). Additionally four novel genotypes: Aravan virus, Khujand virus, Irkut virus and West Caucasian (Kuzmin et al., 2003; Botvinkin et al., 2003) were recently recovered from bats in Eurasia. Genotype 1 is responsible for classical rabies in terrestrial mammals throughout the world. 0008-2352 G 2007 SGM

Printed in Great Britain

Rabies is endemic in Asia and Africa, where the primary reservoir and vector of the RABV is the domestic dog. Worldwide human mortality from enzootic canine rabies is estimated to be in excess of 55 000 deaths per year, of which approximately 56 % occur in Asia and 44 % in Africa (WHO, 2005). Rabies has been known in the Middle East since biblical times, but the modern Hebrew name ‘Kalevet’ was coined only at the beginning of the 20th century by a physician named Beham, who established the Jerusalem Pasteur Institute in 1913 (Yakobson et al., 2004). Between 1930 and 1960, the domestic dog was considered to be the primary reservoir of RABV in Israel (Goor, 1949). Komarov & Hornstein (1953) developed a rabies vaccine based on the Kelev RABV strain isolated in 1950 from a naturally infected dog in Israel. The original street virus virulence was modified by 100 passages in chick embryos (Komarov & Hornstein, 1953), the virus was attenuated and then used for cattle vaccination in Israel until the early 1960s (Kalmar & Tadmor, 1968). The Kelev vaccine has been used for cat and dog vaccination in Turkey since 1968 (Orhan et al., 1998). Today, RABV circulates in Israel among wild canids, with occasional transmission to humans and domestic animals. 967

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Since 1979, red foxes (Vulpes vulpes) have been the most important reservoir of RABV in Israel (Shimshony, 1997). Molecular epidemiological studies of rabies in Israel between 1993 and 1998 revealed five phylogenetic lineages, distributed among four geographical regions (David et al., 2000). An oral vaccination (ORV) programme directed at wild animals has been implemented since 1998 in the northern regions of Israel. In 2004 the programme was extended and it currently covers all of the area controlled by Israel and the Palestinian Authority (Yakobson et al., 2006). Israel is the only country in the Middle East that implements ORV, and to ensure the success of the programme extensive rabies surveillance along Israel’s borders is required. Rabies is enzootic throughout the Middle East. Turkey still reports dog-mediated rabies and several cases of rabies in wildlife have been confirmed there (Johnson et al., 2003). Rabies is also a serious enzootic disease in Jordan, Syria, Lebanon and Iran, where stray dogs maintain RABV circulation and there is frequent spillover into wildlife such as jackals, squirrels, stone martens, foxes, monkeys and wolves (Al-Qudah et al., 1997; Bizri et al., 2000; Nadin-Davis et al., 2003; Yakobson et al., 2004). Stray dogs are the reservoir of most of the RABV circulation in Egypt. The number of human deaths caused by rabies in Egypt since 1990, as reported to the WHO, has been approximately 30–40 cases per annum (Matter et al., 2004). Kissi et al. (1995) studied the molecular epidemiology of rabies in Africa and described three phylogenetic lineages of RABV: Africa 1, 2 and 3. The divergence was believed to reflect descent from differing progenitor viruses. The Africa 1 and 2 lineages were isolated from dogs or humans bitten by rabid dogs, whereas the Africa 3 lineage was found associated with mongoose species, principally the yellow mongoose (Cynictis penicillata) from the Republic of South Africa. The Africa 1 lineage was subdivided into two subgroups: 1a, restricted to North and West Africa; and 1b, limited to South East Africa. In general, the Africa 1 lineage was the most similar to current Eurasian RABV lineages, suggesting its recent introduction to Africa (Kissi et al., 1995; Swanepoel et al., 1993). The Africa 2 lineage includes wild-type strains that originated from several central and eastern African countries, and is phylogenetically ancestral to the cluster that includes the Eurasian and Africa 1 RABVs. The Africa 3 lineage, of mongoose origin, is distant from all dog RABV variants. The single Egyptian RABV isolate that Kissi et al. (1995) included in their study was a human isolate (GenBank accession no. U22627), and it was unique within the study as the authors were unable to group it with any of the three African RABV lineages. In the present paper we describe the antigenic and phylogenetic analysis of the Middle Eastern and Egyptian RABV isolates, as well as the estimation of their age based on a molecular clock. In the light of this analysis, we have identified novel canine RABV lineages in the Middle East, 968

designated V, VI and VII and one novel lineage associated with North Africa, designated Africa 4.

METHODS Viruses. RABV-containing samples were collected in Israel (n=90) and neighbouring countries (n=16), with particular attention to the border areas (n=28) (Table 1). Seven isolates were collected on the border between Israel and Jordan, three on the border between Israel and Lebanon and 18 on the border between Israel and Syria (Fig. 1). In addition we analysed three isolates from Egypt, six from Jordan and seven from Turkey (Table 1). The dead, suspected animals were collected and sent to the rabies laboratory at the Kimron Veterinary Institute for post-mortem examination. The brains were desiccated and touch impressions from three regions, hippocampus, cerebellum and medulla, were taken immediately for direct fluorescent antibody and for monoclonal antibody (mAb) typing.

The brains were frozen at 280 uC until processing for virus isolation. All the viruses were isolated by intracerebral mouse inoculation test using suckling mice (David et al., 2002). The attenuated Kelev RABV vaccine strain was grown in a mouse neuroblastoma cell culture according to David et al. (2002). Direct fluorescent antibody test. From each brain sample, three

anatomical regions were subjected to the direct fluorescent antibody test with FITC-conjugated anti-RABV monoclonal (Centocor, Fujirebio Diagnostic) and polyclonal (Chemicon International) antibodies, according to the manufacturers’ recommendations. mAb typing. Indirect immunofluorescent antibody staining was

used to detect N antigen in acetone-fixed touch brain impressions and cell monolayers infected with RABV isolates on Teflon-coated microscope slides (Erie Scientific) (David et al., 1999a; Smith et al., 1990). A panel of 19 mAbs developed at the Centers for Disease Control and Prevention and mAbs 103-7 and 502-2 (Wistar Institute) used in the present study (Table 2) have been characterized previously (Wiktor & Koprowski, 1978). All the mAbs were prepared as mouse ascitic fluid and reacted against the N epitopes of RABV as previously described (Smith et al., 1984). The slides were examined at 6200 magnification with a BX-40 fluorescence microscope (Olympus Optical Co.). RT-PCR and direct sequencing. Total RNA from brains was

extracted with TRI Reagent LS (Molecular Research Center) according to the manufacturer’s instructions. For reverse transcription, 1 ml RNA was heated to 95 uC for 1 min, chilled on ice and added to 20 ml reverse transcription reaction mixture containing reaction buffer (25 mM Tris/HCl, pH 8.3 at 42 uC, 25 mM KCl, 5 mM MgCl2, 5 mM DTT, 0.25 mM spermidine), 250 mM each of four dNTPs, 100 pmol forward primer 10g (59-CTACAATGGATGCCGAC-39; specific to the RABV N gene), 25 U RNAsin (Promega) and 10 U AMV reverse transcriptase (Promega). After incubation at 42 uC for 90 min, 2 ml cDNA product was added to 50 ml PCR mixture, using Ex-Taq polymerase (Takara Bio) according to the manufacturer’s instructions. The primers 10g and 304 (59GAGTCACTCGAATATGTC-39) were used for PCR (Smith, 2002). The following thermocycling program was used: 95 uC for 2 min, 37 uC for 1 min, 68 uC for 1.5 min, 98 uC for 20 s. This was repeated once, followed by a further 16 repetitions under identical conditions, except that an extra 20 s was added to each elongation step. A final 10 min elongation step followed. The 1461 bp PCR product comprised the 1350 bp of the entire N gene, the non-coding region between the N and P genes, and the initial 20 bp of the P gene. PCR products were visualized on 1.5 % agarose gels, purified with the Wizard PCR Preps Journal of General Virology 88

Novel Middle East and N. Africa canine rabies clades

Table 1. Rabies isolates analysed in the present study Sample

Country

SL9655 Kz4230 Sn7411 Sn8460 Am4828 Sm0034 329 J4 J5 Am4168 Ks6616 Kz5918 Ms3520 Kt2969 Vs3370 Kt1902 Mz5644 Tm3205 Ah8437 145 Bm9877 Mi0338 Ns3056 Ho6890 Th6967 Yt3700 Tl8852 Dm2897 Bq7184 Ak0904 Af8045 Bs0448 445 Qd8617 So0047 Ya6530 Hr1510 Ar3889 Ma3752 Bi7106 Eg0699 Hb1153 Jr6462 Nh8389 Rz4879 Bn8124 Br2323 Bs7230 Bs9207 Dt5814 Hm0160 Om1598 Rt6224

South Lebanon Israel Israel Israel Israel Israel Israel Jordan Jordan Israel Israel Israel Israel Israel Israel Israel Israel Israel Israel Israel Israel Israel Israel Israel Israel Israel Israel Israel Israel Israel Israel Israel Israel Israel Israel Israel Israel Israel Israel Israel Israel Israel Israel Israel Israel Israel Israel Israel Israel Israel Israel Israel Israel

http://vir.sgmjournals.org

Place

Date

1/9/1997 Kazerin 21/2/1997 Senir 1/4/1998 Senir 14/4/1998 Aramsha 27/5/2004 Shadmot Mehola 19/2/2002 Golan Heights 12/1996 Irbid 5/10/1998 Ajloun 4/11/1998 Aramsha 23/5/2004 Keshet 7/11/1996 Kidmat Zvi 18/7/1997 Magdel Shams 19/10/1997 Kiryat Tivon 1/10/1999 Salama 3/9/1995 Kiryat Tivon 17/8/1995 Mizpa 8/3/1998 Timrat 10/2/1997 Ahihud 10/7/2005 Gedieda 12/1997 Beer El Maxsur 23/8/1999 Misgav 4/1/1998 Neot Smadar 2/2/2004 Homesch 2/12/1997 Teqoa 4/12/1997 Yatir 17/5/2004 Tel Aviv 29/3/2004 Dimona 8/2/1999 Beqaot 27/7/1998 Abu Korinat 13/1/2000 Afula 1/8/1996 Beer Sheva 7/1/1996 Yeruham 5/2003 Qedar 7/8/1996 Soham 5/5/1996 Yafit 14/3/1996 Herut 18/1/2004 Ariel 8/9/1995 Masua 11/2/1996 Bat Ain 27/7/1998 Ein Gedi 29/8/1996 Hebron 14/9/1998 Jerusalem 14/3/1996 Nahashonim 13/4/1998 Ramat Raziel 25/2/1998 Beit Nehemia 8/4/1996 Bareqet 2/6/1996 Beer Sheva 15/11/1999 Beer Sheva 8/12/1999 Aderet 11/3/1999 Hamera 25/8/1999 Omer 10/9/1999 Rahat 17/3/1999

Mammal species Stone marten Fox Cow Dog Cow Fox Human Cow Squirrel Cow Cow Fox Fox Fox Fox Fox Dog Fox Cow Human Sheep Fox Fox Fox Dog Fox Jackal Fox Fox Fox Cat Badger Human Fox Dog Fox Fox Fox Fox Fox Fox Dog Dog Cat Fox Dog Fox Fox Fox Dog Fox Fox Fox

Source/reference This This This This This This This This This This This This This This This This This This This This This This This This This This This This This This This This This This This This This This This This This This This This This This This This This This This This This

study study study study study study study study study study study study study study study study study study study study study study study study study study study study study study study study study study study study study study study study study study study study study study study study study study study study study

GenBank Antigenic accession no. variant DQ837465 DQ837438 DQ837470 DQ837471 DQ837390 DQ837466 DQ837383 DQ837425 DQ837426 DQ837389 DQ837433 DQ837439 DQ837444 DQ837435 DQ837484 DQ837434 DQ837448 DQ837483 DQ837387 DQ837382 DQ837397 DQ837442 DQ837452 DQ837417 DQ837481 DQ837487 DQ837482 DQ837404 DQ837399 DQ837388 DQ837386 DQ837401 DQ837384 DQ837458 DQ837472 DQ837485 DQ837418 DQ837391 DQ837440 DQ837396 DQ837406 DQ837415 DQ837429 DQ837449 DQ837460 DQ837398 DQ837400 DQ837402 DQ837403 DQ837405 DQ837416 DQ837456 DQ837459

V1 V1 V1 V1 V1 V1 V1 V1 V1 V1 V1 V1 V1 V1 V1 V1 V1 V1 V1 V1 V1 V1 V1 V3 V5 V1 V5 V1 V1 V1 V1 V1 V1 V4 V2 V1 V5 V4 V1 V4 V5 V1 V1 V3 V4 V2 V2 V5 V5 V5 V3 V5 V5

Genetic variant I I I I I I I I I I I I I II II II II II II II II II III III III III III III III III III III III III III III III III III III III III III III III III III III III III III III III

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Table 1. cont. Sample

Country

Ga5970 Nr8863 Ab2437 Eg8855 Mu3996 Om1956 Ih9330 Ft2180 Hb1064 Ih5700 Sp1662 Gr9489 OMN SDR J1 J3 J6 Ha9119 Ha5417 Nk5630 Io3216 Ms1909 Ke1132 Mg1353 J2 Od6220 Ar8606 Nt1211 Kz4209 Az5745 Az7134 Kz2844 Yo6867 Od9613 Ek0187 Ks1772 Si2236 Mv7626 Az0298 Sm0313 Sn4414 Sm3563 T1 T2 T3 T4 T5 T6 T7 IRN IRN POL ALL

Israel Israel Israel Israel Israel Israel Israel Israel Israel Israel Israel Israel Oman Saudi Arabia Jordan Jordan Jordan Israel Israel Israel Israel Israel Israel Israel Jordan Israel Israel Israel Israel Israel Israel Israel Israel Israel Israel Israel Israel Israel Israel Israel Israel Israel Turkey Turkey Turkey Turkey Turkey Turkey Turkey Iran Iran Poland Germany

970

Place Givat Ada Naran Avihail Ein Gedi Mashua Omer Iahel Efrat Hebron Iahel Sapir Grofit

Amman Irbid Mafrak Hazeva Hazeva Neot Hakikar Ir Ovot Mizpe Shalem Qetura Merom Golan Karak Odem Elrom Natur Kimat Zvi Ein Zivan Ein Zivan Kazerin Yonatan Odem Ein Kinya Keshet Sar Yeshuv Merhavia Ein Zivan Shamir Senir Shamir Sanlurfa Istanbul Istanbul Istanbul Istanbul Istanbul Istanbul

Date

Mammal species

29/12/2003 7/2/2002 26/1/2004 3/12/1996 18/2/2000 25/1/2000 27/10/2003 14/10/2001 28/2/2002 17/11/1997 23/9/1997 28/4/1998 1990 1987 3/1/1999 19/9/1998 6/12/1998 2/8/2000 26/6/2000 15/10/2000 13/9/2000 21/12/2000 21/5/1997 18/9/1997 6/3/1999 20/6/2005 12/10/2005 27/10/2004 31/8/2005 9/3/2005 23/3/2005 14/2/2005 21/3/2005 18/4/2005 21/4/2005 10/5/2005 16/5/2005 1/10/2005 23/4/2005 26/4/2005 8/12/2005 28/2/2006 2000 2000 2000 2000 2000 2000 2000

Dog Fox Fox Fox Fox Fox Fox Dog Donkey Fox Wolf Fox Fox Goat Cow Badger Fox Fox Fox Fox Dog Dog Cow Donkey Wolf Badger Cow Dog Dog Dog Dog Dog Dog Dog Dog Dog Cow Dog Cow Cow Cow Cow Dog Dog Dog Dog Dog Dog

Source/reference This study This study This study This study This study This study This study This study This study This study This study This study Kissi et al. Kissi et al. This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study Kissi et al. Kissi et al. Kissi et al. Kissi et al.

(1995) (1995)

(1995) (1995) (1995) (1995)

GenBank Antigenic accession no. variant DQ837410 DQ837451 DQ837385 DQ837407 DQ837446 DQ837457 DQ837420 DQ837409 DQ837414 DQ837419 DQ837473 DQ837411 U22480 U22481 DQ837422 DQ837424 DQ837427 DQ837413 DQ837412 DQ837450 DQ837421 DQ837443 DQ837430 DQ837441 DQ837423 DQ837454 DQ837392 DQ837453 DQ837437 DQ837394 DQ837395 DQ837436 DQ837486 DQ837455 DQ837408 DQ837432 DQ837464 DQ837447 DQ837393 DQ837467 DQ837469 DQ837468 DQ837474 DQ837475 DQ837476 DQ837477 DQ837478 DQ837479 DQ837480 U22482 U22483 U22840 U42701

V5 V5 V5 V5 V3 V5 V1 V4 V1 V1 V1 V1

V6 V6 V6 V6 V6 V6 V6 V1 V1 V1 V1 V1 V1 V1 V1 V1 V1 V1 V1 V1 V1 V1 V1 V1 V1 V1 V1 V1 V1 V1 V1 V1 V1 V1 V1

Genetic variant III III III III III III III III III IV IV IV V V V V V V V V V VI VI VI VI VII VII VII VII VII VII VII VII VII VII VII VII VII VII VII VII VII Turkey Turkey Turkey Turkey Turkey Turkey Turkey Middle East Middle East Europe Europe

Journal of General Virology 88


Table 1. cont. Sample ALL YOU YOU YOU FRA FRA EST EST HUN RUS RUS RUS RUS GEO ALG MAR ETH GAB GAB CAF CAF NIG TAN ZAI MOZ RSP GUI TAC BUF CAM BEN NIG MAU IVC RSP Kelev S1 S2 S3 S4 THI IND SARL

Country Germany Yugoslavia Yugoslavia Yugoslavia France France Estonia Estonia Hungary Russia Russia Russia Russia Georgia Algeria Morocco Ethiopia Gabon Gabon CAR* CAR Nigeria Tanzania Zaire Mozambique RSAD Guinea Chad Burkina Faso Cameroon Benin Niger Mauritania Ivory Coast RSA Israel Egypt Egypt Egypt Egypt Thailand India Sri Lanka

Place

Date

Mammal species

Fox Human Dog Rodent Human

Cairo Faium Cairo

16/11/1950 3/9/1998 25/5/1998 3/10/1999 1979

Dog Dog Dog Dog Human

Source/reference Kissi et al. (1995) Kissi et al. (1995) Kissi et al. (1995) Bourhy et al. (1999) Bourhy et al. (1999) Bourhy et al. (1999) Kissi et al. (1995) Bourhy et al. (1999) Bourhy et al. (1999) Kuzmin et al. (2004) Kuzmin et al. (2004) Kuzmin et al. (2004) Kuzmin et al. (2004) Kuzmin et al. (2004) Kissi et al. (1995) Kissi et al. (1995) Kissi et al. (1995) Kissi et al. (1995) Kissi et al. (1995) Kissi et al. (1995) Kissi et al. (1995) Kissi et al. (1995) Kissi et al. (1995) Kissi et al. (1995) Kissi et al. (1995) Kissi et al. (1995) Kissi et al. (1995) Kissi et al. (1995) Kissi et al. (1995) Kissi et al. (1995) Kissi et al. (1995) Kissi et al. (1995) Kissi et al. (1995) Kissi et al. (1995) GenBank This study This study This study This study Kissi et al. (1995) Kissi et al. (1995) Jayakumar et al. (2004) Arai et al. (2001)

GenBank Antigenic accession no. variant U22475 U42706 U22839 U42704 U42607 U43433 U22476 U42707 U43025 AY352464 AY352475 AY352476 AY352480 AY352497 U22643 U22852 U22637 U22629 U22630 U22650 U22651 U22488 U22645 U22638 U22484 U22633 U22487 U22644 U22486 U22634 U22485 U22640 U22489 U22639 AF467949 DQ837431 DQ837461 DQ837462 DQ837463 U22627 U22653 AF374721 AB041968

V1 V1 V1 V1

Genetic variant Europe Europe Europe Europe Europe Europe Europe Europe Europe West Siberia Europe Europe West Siberia Europe Africa 1b Africa 1b Africa 1b Africa 1b Africa 1b Africa 1a Africa 1a Africa 1a Africa 1a Africa 1a Africa 1a Africa 1a Africa 2 Africa 2 Africa 2 Africa 2 Africa 2 Africa 2 Africa 2 Africa 2 Africa 3 Africa 4 Africa 4 Africa 4 Africa 4 Africa 4 Asia Asia Asia

*CAR, Central African Republic DRSA, Republic of South Africa

DNA purification system (Promega) and sequenced with an automatic sequencer 3700 DNA Analyzer (Applied Biosystems), according to the manufacturer’s instructions. Phylogenetic analysis. The nucleotide sequences were aligned

with the

PILEUP

sequence analysis package (Genetics Computer


Group). A phylogenetic tree of 1350 bp of the N gene was constructed by the neighbour-joining method, with the distance calculated using the Kimura two-parameter with the computer program MEGA, version 3.1 (Kumar et al., 2004). The reliability of the phylogenetic groupings was evaluated using bootstrapping with 1000 replicates. 971

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Table 2. Antigenic profiles of the Israeli, Egyptian and South Lebanon RABV isolates

Jordanian,

Black, no reaction; white, positive reaction; grey, diminished reaction with ten times less diluted antibody. S L, South Lebanon; Tur, Turkey; Egy, Egypt.

mAbs

Israel Israel

JordanEgy EgyTur TurS SLL Jordan

V1 V3 V4 V4 V5 V5V6V6V1V1V6V6V1V1 V1 V1 V1 V1 V1 V2 V3 C1/63-3-1 C1/63-3-1 C2/62-8-2 C2/62-8-2 C3/62-11-1 C3/62-11-1 C4/62-15-2 C4/62-15-2 C5/62-22-3 C5/62-22-3 C6/62-23-4 C6/62-23-4 C7/62-24-1 C7/62-24-1 Polyclonal Polyclonal C9/62-52-1 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 C20/62-164-2 Fig. 1. Map of Israel and its borders on which all RABV isolates are marked by their place of isolation except isolates originating from Turkey and Egypt. The map was subdivided into four geographical regions and four clades, based on molecular classification. The regions were: 1, Golan heights and upper Galilee; 2, Galilee; 3, central and southern area and 4, Arava valley. The legend details the rabid mammals from which RABV was isolated. Six RABV isolates were obtained from Jordan, and they were located at the site of detection and classified molecularly. Symbol colour: light grey, 28 sampling sites of RABV isolates from the borders collected between 1997 and 2006 and one isolate from the Galilee. Black, RABV isolates detected inside Israel during 1995–2005.

substitution was selected with the MODELTEST software (Posada & Crandall, 1998) and the model selected was used as the basis for BEAST analysis. Full details of the models used are available from the authors upon request. Jeffrey’s priors for substitution rate and population size were used for each BEAST analysis (Drummond et al., 2002). Two population dynamics models were used (constant population size and exponential population growth) and their likelihoods compared (Davis et al., 2005). The MCMC analysis was optimized according to the criteria suggested in the program documentation. These included an operator acceptance probability of approximately 25 % and an effective sample size >100. The BEAST output was evaluated with the Tracer program (available from http://www.evolve.zoo.ox.ac.uk/beast/).

(substitutions per site per year), of the complete N gene alignment were obtained by using the BEAST program (available from http:// www.evolve.zoo.ox.ac.uk/beast/). This program uses a Bayesian Markov Chain Monte Carlo (MCMC) method, which requires no assumptions to be made regarding tree topology (Drummond et al., 2002). This method was previously applied to analysis of RABV datasets (Davis et al., 2006; Hughes et al., 2004, 2005).

Divergence times for individual clades were estimated with the TipDate software (Rambaut, 2000). An ML tree generated by PAUP* (Swofford, 2000) by means of the ML nucleotide substitution model selected by MODELTEST was used as input for TipDate. The PAUP* ML trees were constrained to contain no polytomy. An ML tree was generated in TipDate under the assumption of a single rate of nucleotide substitution, by using single rate dated tips (SRDT) model. Trees were generated by using the nucleotide substitution parameters estimated by MODELTEST. For the SRDT tree, the mean rate of substitution estimated in the BEAST analysis and the upper and lower 95 % highest probability density (HPD) values were used to scale trees and the ML root estimated by TipDate.

For each alignment, an input file for BEAST was generated by using the BEAUti program (available from http://www.evolve.zoo.ox.ac.uk/beast/ ) with sequences dated according to the year of isolation. Identical sequences with the same year of isolation were removed from the analysis, as were laboratory-generated and vaccine-derived sequences. For each dataset, the maximum likelihood (ML) model of nucleotide

To assess the degree of substitution saturation, graphical plots of the accumulation of transitions (TS) and transversions (TV) against evolutionary distance were generated with the DAMBE software (Xia & Xie, 2001) for the complete N gene alignment. The TN93 model was used to calculate evolutionary distance. When multiple transition substitutions occur at the same site, the phylogenetic signal for the

Estimation of the evolutionary rate and application of a molecular clock. Estimates of the rate of molecular evolution, m

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Novel Middle East and N. Africa canine rabies clades earlier changes are essentially lost; saturation of this nature is then visible because transversions accumulate more rapidly than transitions, i.e. the TS line flattens. These methods have been used previously to assess substitution saturation during the evolution of RNA viruses (Hanada et al., 2004; Moury et al., 2002; Van Dooren et al., 2001).

RESULTS Antigenic analysis The mAb typing of the Israeli RABV isolates revealed six antigenic variants: V1–V6 (Table 2). All variants were identified in a large spectrum of mammalian species, including humans (Table 1). The mAb 62-15-2 (C4), which does not react with any lyssavirus other than RABV (Nanayakkara et al., 2003), reacted with all isolates, suggesting that no other lyssavirus was present among these samples. The V1 antigenic variant was the one most frequently found in Israel. It was identified in the northern and southern parts of Israel (Table 1), and characterized all isolates that belonged to clades I, II, IV, V and VII and to some of the clade III isolates. The antigenic variants V2–V5 circulate in the central and southern areas of Israel and characterized some of the isolates that belonged to clade III (Fig. 2). The V1 antigenic variant included three human isolates, two of which (nos 329 and 145) originated from the northern region of Israel (David et al., 1999a, b). The third (no. 445) was obtained in 2003 from the southern area. The RABV isolates, originating from three dogs in Egypt and from six dogs and one cow in Turkey, were also typed by mAbs as members of the V1 antigenic variant. The Jordanian RABV isolates were typed as members of two antigenic variants, V1 and V6; variant V6, represented by four fox RABVs, was first identified in Israel during 2000, along the border between Israel and Jordan, in the Arava Valley. The results summarized in Table 2 showed that Middle Eastern RABV isolates that belong to antigenic variants V2–V5 were negative to mAb 103-7. Variable reactivity with mAb 103-7 has also been reported for French RABV isolates (Sureau et al., 1983). To investigate the genetic basis for the Middle Eastern RABV antigenic variation, representative Middle Eastern isolates belonging to antigenic variants V1–V6 were selected for N gene sequencing. Fig. 2 illustrates the comparison between the Middle Eastern and the Israeli antigenic variants, including the predicted amino acid differences and the reference RABV isolate KT1902. Several amino acids are crucial for the determination the antigenic profiles of the Middle Eastern RABV isolates (summarized in Fig. 2 and Table 3). For antigenic variant V1, the amino acid 101 can either be D or N and mAb C18 is unreactive. The change of one amino acid at position 101 to T prevents the binding of C18 and four additional mAbs to the RABV isolates belonging to antigenic variant V5 (Table 3). Therefore, the criteria for antigenic profile classification imply a change in antigenic variants. Change in two amino acids at position 90 and 101 to I and T respectively prevents the binding of four mAbs to the isolates belonging to antigenic variant V4. For http://vir.sgmjournals.org

antigenic variant V2, two amino acids S and N at positions 36 and 101, respectively, prevent the reaction of four mAbs. For antigenic variant V3, three amino acids, I, T and S or A at positions 90, 101 and 133 or 134, respectively, prevent the reaction of five mAbs. Change of three amino acids, S at position 101, F at position 80 and E at position 371, prevents the reaction of three mAbs with the isolates belonging to V6 antigenic variant. In summary, the amino acid at position 101 of all isolates that belong to antigenic variant V1 is either N or D, and one change in that amino acid only, from N or D to T, is sufficient to prevent recognition of that isolate by mAbs (Table 3). The amino acid L at position 80 of the N protein characterized the Lebanese isolates, Jordanian isolates belong to clade I, Israeli isolates belong to clades I, II, III and IV and Egyptian RABV isolates belong to Africa 4 clade. All the RABV isolates that belonged to the clades V (four isolates), VI (three isolates) and VII (18 isolates), which were identified on the borders, have F at this position (Fig. 2). Phylogenetic characterization To facilitate a comprehensive comparison of isolates originating from the Middle East and Egypt, we incorporated into the dataset representatives of all the RABV lineages from Africa and the middle latitudes of Eurasia that had been previously described (Fig. 3). Analysis of the entire N gene sequences revealed novel canine RABV clades in the Middle East and Africa (Fig. 3). Seven clades (I–VII) were identified in Israel and one (Africa 4) in Egypt. The Israeli lineages I–IV have been previously described and are distributed among four geographical regions: (I) Golan Heights and Upper Galilee, (II) Galilee, (III) the central southern Israel area, including the Palestinian Authority in the West Bank, and (IV) the Arava Valley (David et al., 2000). The three novel clades (V, VI and VII) were identified on the borders between Israel and neighbouring countries. All these lineages are included in the cluster of RABV variants circulating in the middle latitudes of Eurasia and the Middle East. Clade V was represented by four RABV isolates that were found on the border between Israel and Jordan, and also by isolates that occurred in the Gulf countries, Oman (GenBank accession no. U22480) and Saudi Arabia (accession no. U22481). The Israeli RABV isolates of clade V were closely related to isolates originating from the latter countries (bootstrap support of 99 %) (Fig. 3). The viruses included in the clade VI were isolated from rabid animals on the borders between Israel, Jordan and Syria (bootstrap support of 99 %). The clade VI was represented by four isolates: the first (KE1132) was found in an Israeli dog in 1997 in the Arava Valley on the border with Jordan. The second isolate (MG1353) was obtained from a rabid Israeli cow found in 1997 on the border with Syria. The third isolate (J2) was identified in a Jordanian donkey in 1999, which was found to be identical to the Israeli dog isolate 973

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Fig. 2. Comparison of the predicted N sequences of representative antigenic groups that circulate in the Middle East. The amino acid alignment was performed on the entire nucleoprotein of 450 aa; however, the figure shows only aa 1–150 and 300–450, as aa 151–300 were identical.


Table 3. Summary of amino acid requirements and reactivity with mAbs for antigenic typing of rabies virus isolates Antigenic variant

Amino acids and their position 101

V1 V2 V3

D, N N T

V4 V5 V6

T T S

Others None S on 36 I on 90 S on 133 or A on 134 I on 90 None F on 80 E on 371

Reactivity with the mAb panel Negative C18 C2, C12, C16, C18, 103-7 C2, C7, C11*, C12, C18, 103-7 C2, C7, C12, C18, 103-7 C2*, C7, C12, C18, 103-7 C1, C7, C12, C18

*Weak reaction.

KE1132. The fourth isolate (MS9119) was from an Israeli dog located near the Dead Sea in 2001. The Israel cluster VII comprised 18 RABV isolates obtained on the borders between Israel and Syria during 2004–2006 (bootstrap support of 99 %) and one isolate (MV7626) from a cow in the Galilee region (Fig. 1). These isolates were more closely related to Turkish isolates than the clade I isolate that circulated in Israel before the ORV programme was implemented. The bovine isolate MV7626 isolated in the Galilee region may have been transmitted by an infected dog from the Golan Heights. Molecular analysis of the Jordanian sequences J1–J6, all of which were found on the border between Israel and Jordan, revealed the presence of three clades: Israel I, V and VI. Israel shares clade I with Jordan, and the two sequences of the Jordanian isolates J4 and J5 were found to be similar to the Israeli isolates that circulated until 1998 on the Golan Heights. The isolate SM0034, of vulpine origin, was identified as a member of clade I; it was obtained from the Jordan Valley on the border with Jordan in 2002. Three sequences of the Jordanian isolates J1, J3 and J6 were identified as members of Israel V; these sequences were closely related to Israeli RABV sequences obtained along the border between Israel and Jordan during 2000.

value 99 %, indicating that this clade is part of the cosmopolitan canine RABV lineage that is believed to have originated in Europe and widely disseminated as a consequence of colonial activity during the 16th to 19th centuries (Smith et al., 1992). The lineages that were placed more ancestrally (Arctic, Africa 2 and 3 and Asia) were not consistently joined to the cosmopolitan cluster. The Kelev RABV vaccine strain and the Egyptian human RABV isolate S4 share 97.8 % nucleotide identity, and show 97.3–97.6 % identity with the new Egyptian isolates S1, S2 and S3. These samples also shared 98.2–99.3 % amino acid identity. All other Israeli RABV isolates available for comparison (n=89) belonged to the Middle Eastern clade that is maintained primarily by red foxes. This clade was further incorporated into the cluster of RABVs circulating within foxes and raccoon dogs in the middle latitudes of Eurasia. Estimation of the evolutionary rate of the N gene and application of a molecular clock

The bovine isolates AM4828 and AM4816 were obtained on the border between Israel and Lebanon during 2004. They belong to the same genetic variant that circulated in the Golan Heights and Upper Galilee until 1998. These isolates were closely related to the stone marten isolate SL9655 that was obtained in 1997 in South Lebanon.

For estimation of the substitution rate of the N gene, a total of 106 taxa were included in the analysis. The constant population size model was significantly favoured over the exponential growth model. BEAST analysis gave an estimated rate of 2.761024 substitutions per site per year (95 % HPD: 1.861024 to 3.761024) with a corresponding root height of 470 years (95 % HPD: 306 to 651). Application of the BEAST-calculated rates to TipDate resulted in SRDT trees with very similar root heights (448 years, 330–689). The SRDT generated by using the mean substitution rate is shown in Fig. 4.

The phylogenetic tree showed that the three Egyptian dog RABV isolates, together with the Kelev vaccine strain, clustered with the previously described Egyptian human isolate S4 (Kissi et al., 1995). This novel clade, Africa 4, was supported by a high bootstrap value (99 %) and was placed ancestral to the cluster of RABV variants that were associated with the Middle East and the middle latitudes of Eurasia, and to Africa 1. The ancestral position of the Africa 4 clade was supported by the very high bootstrap

From the SRDT trees, the first major division leads to RABV variants in Asia and Africa (1557, 1316–1675). Although only a small number of Asian taxa were used, the time of most recent common ancestor (TMRCA) for these sequences is close to the root, indicating that, of all the canine RABVs analysed, the Asian variants share the oldest common ancestor. Current RABV diversity in Europe and the Middle East has a TMRCA of 1867 (1799–1901) (the apparently anomalous Turkish isolate T1 was excluded from


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these calculations). Following this division, distinct lineages in Europe appear to have established rapidly, whereas the current clades in the Middle East have much more recent TMRCAs. Other than Israel V (TMRCA 1964, 1949–1971), all the current RABV diversity associated with Israel is estimated to have emerged within the last approximately 20 years. Although the TMRCA of current Israel clade VII diversity is similar to those of other extant Israeli variants, it does appear that the progenitor virus for this lineage (which includes isolates from Turkey) diverged earlier than those that subsequently established the other existing Israeli variants.

DISCUSSION Molecular and antigenic analyses have revealed four novel RABV clades, three from the Middle East and one from North Africa. Three sources were hypothesized to be the origins of the three novel genetic clades V, VI and VII that were identified on the Israeli borders. Clade V appears to originate from Oman and Saudi Arabia, clade VI from Iran and clade VII from Turkey. The RABV isolates belonging to clade V, which circulated in the Gulf countries during 1990–1991 (Ata et al., 1993; Novelli & Malankar, 1991) and in Jordan during 1998, were detected on the Israeli border during 2000. The RABV isolates belonging to clade VI circulate in Syria and Jordan and are related to RABV isolates from Iran. At least three clades (I, V and VI) circulate in Jordan; viruses representing all these variants were identified on the border between Israel and Jordan. RABV that circulates in Syria appears to belong to the clades VI and VII, and the Israeli RABV isolates that belong to clade VI are closely related to isolates from Jordan and Syria. The RABV isolates identified on the Israeli border with Syria belong to clade VII and were found to be closely related to the Turkish isolates (Fig. 3). The clade I isolates appear to be circulating in Lebanon as well as Israel and Jordan. The last rabies cases caused by the clade I isolates in Israel occurred in 1998 in the Golan Heights region, whereas the isolates of this variant were identified on the border with Jordan in 2000 and on the border with Lebanon during 2004. The isolates identified as belonging to the three Middle Eastern clades V, VI and VII, which were identified on the Israeli borders, are likely to have penetrated from neighbouring countries, and isolates of variant I from the Israeli border probably represent penetrations by variants circulating in Jordan and Lebanon. The present antigenic typing is an attempt to provide a comprehensive classification. Reports published until now

have described antigenic typing of isolates from Iran only (Nadin-Davis et al., 2003). These variants indicated the limited geographical variability of RABV, within a large cluster of cosmopolitan RABVs. Antigenic characterization of representative Israeli and other Middle Eastern isolates identified eight mAbs that are useful for group discrimination: 63-3-1 (C1), 62-8-2 (C2), 62-24-1 (C7), 62-61-1 (C11), 62-62-4 (C12), 62-97-11 (C16), 62-143-2 (C18) and 103-7. The present study exemplifies that concerted substitutions of a limited number of N amino acids are responsible for the antigenic classification of the Middle Eastern RABV isolates. This finding is consistent with the results of antigenic characterization of viruses from Iran (Nadin-Davis et al., 2003). The Iranian specimens of NV703 (Nadin-Davis et al., 2003), which belong to the Iranian antigen variant 1, exhibit the same antigenic profiles as the J2 isolate belonging to Jordanian antigenic variant V1 and to Israeli isolate KE1132 antigenic variant V1. Apart from confirmation of the presence of the novel antigenic variant V6 on the border between Israel and Jordan, the antigen typing method could not distinguish between the RABV isolates belonging to other border-associated clades. Furthermore, it is unknown whether the antigenic epitopes for the mAbs are linear or conformational. It is possible that the amino acid substitutions described above did not preclude antibody binding to a linear site, but changed the secondary structure and conformational epitope, with consequent alteration of mAb binding. One example of this phenomenon is the substitution of a single amino acid in an Arctic RABV N, a substitution that prevented reactions with seven mAbs (Kuzmin et al., 2004). Since 1979, the rabies reservoir in Israel has been the red fox, whereas in Turkey and Egypt the main host has been dogs. Bourhy et al. (1999) suggested that the specificity to the fox could depend on the substitution of D with N or A at position 101 of the RABV N. However, this was not true of some fox isolates originating from eastern Europe and Asia, which contained S, T or unsubstituted D at this position (Kuzmin et al., 2004). Similarly, the Israeli fox isolates belonging to the genetic variants I–IV contained N or T, and those belonging to variant V contained S at position 101. Thus, it is still impossible to make a valid conclusion regarding specific amino acids at position 101 of the RABV N and an association with alteration in species specificity. In contrast, all the dog isolates in this study (Africa 4 clade, Israeli genetic variants VI and VII and the Turkish isolates) had D in that position. For this reason, the alternative

Fig. 3. Neighbour-joining phylogenetic tree of rabies viruses isolated from Africa and Eurasia, based on the entire N gene sequence. Isolate names are given according to Table 1 and GenBank. Mokola virus (GenBank accession no. Y09762) is used as the outgroup. Bootstrap values are shown for key nodes and branch lengths are drawn to scale. The lineage names are given according to Bourhy et al. (1999) and Kuzmin et al. (2004). NEE, North-eastern Europe; EE, eastern Europe; CE, central Europe and WE, western Europe. http://vir.sgmjournals.org

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Fig. 4. ML SRDT tree for complete N gene alignment. The tree is scaled according to time using the mean substitution rate estimated from BEAST analysis. Sequence names have been altered to include the year of isolation.

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proposal that D101 is characteristic of dog RABV variants may be more plausible. The Africa 4 clade includes RABV isolates from Egypt and Israel, whereas previous studies identified only one Egyptian human isolate that could not be classified within any of the previously identified African lineages (Kissi et al., 1995). Because of the limited dataset, we cannot conclude whether other RABV lineages, which are phylogenetically related to the other African or Eurasian groups, circulate within the territory of Egypt. We have shown for the first time that the Kelev RABV vaccine strain belongs to the Africa 4 lineage, whereas the other Israeli RABV isolates discussed here are similar to those described previously (David et al., 2000). We consider, therefore, that the Kelev RABV is probably an ‘imported’ Egyptian isolate that was most likely brought to Israel by the transfer of dogs across the Suez Canal, during the armed conflict that occurred at that time. Alternatively, the Africa 4 lineage might have circulated previously within a broader geographical area, but was subsequently replaced in the Middle East by other variants. The origin of the existing canine RABV variants is currently unclear. Although canine RABV variants have been hypothesized to evolve from bat RABV variants (Badrane & Tordo, 2001), the undoubted involvement of extinct variants in this evolutionary pathway ensures that clarification of this process remains impossible. Indeed, carnivore rabies was described 4000 years ago (Theodoridies, 1986), although those infections were doubtless caused by now extinct viruses that may have shared only a distant genetic relationship with current RABV variants. Previous research has dated the emergence of canine rabies between 888 and 1459 years ago, with the TMRCA of cosmopolitan canine RABV variants dated to 284–504 years ago (Badrane & Tordo, 2001). Another study estimated that current global RABV diversity arose within the last 500 years (Holmes et al., 2002). These values are close to our present estimate of the root height of the N gene alignment at about 450 years. Our estimate of the substitution rate of the N gene (2.761024 substitutions per site per year) is close to the value of 2.361024 estimated for bat RABV variants in the Americas (Hughes et al., 2005) and 3.961024 estimated for terrestrial mammals (Davis et al., 2006). The recent isolation in Eurasia of the most divergent lyssavirus to date, the West Caucasian bat virus (Kuzmin et al., 2005), coupled with the present observation that Asian canine RABV variants appear to share the closest common ancestor to the progenitor, is indicative that now extinct Eurasian lineages may have served as a crucial genetic component in the evolution of current global RABV diversity. The divergence and eventual establishment of the newly described Africa 4 clade appear to have occurred at a branching point earlier than the divergence of the lineage that has evolved to generate the vast majority of the current global diversity of RABV. In turn, lineages associated with areas of Europe, middle latitudes of Asia and the Middle East http://vir.sgmjournals.org

appear to have been established later. The current diversity in the Middle East is suggested to have recent origins, which suggests that the establishment of new lineages of RABV must occur fairly rapidly in this region.

ACKNOWLEDGEMENTS We are indebted to the Rabies Laboratories of Jordan and Egypt for providing us with rabies samples.

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