Isolation and phylogenetic grouping of Equine encephalosis virus in Israel.
Equine encephalomyelitis (Diagnosis)
Equine encephalomyelitis (Research)
Polymerase chain reaction (Usage)
Maan, Narender Singh
|Publication:||Name: Emerging Infectious Diseases Publisher: U.S. National Center for Infectious Diseases Audience: Academic; Professional Format: Magazine/Journal Subject: Health Copyright: COPYRIGHT 2011 U.S. National Center for Infectious Diseases ISSN: 1080-6040|
|Issue:||Date: Oct, 2011 Source Volume: 17 Source Issue: 10|
|Topic:||Event Code: 310 Science & research|
|Product:||Product Code: 0272010 Horses; 0272000 Horses & Other Equines NAICS Code: 11292 Horse and Other Equine Production SIC Code: 0752 Animal specialty services|
|Geographic:||Geographic Scope: Israel Geographic Code: 7ISRA Israel|
Equine encephalosis is an arthropod-borne, noncontagious, febrile
disease of horses. It was first described >100 years ago by A.
Theiler (1) under the name equine ephemeral fever. The disease is caused
by Equine encephalosis virus (EEV; genus Orbivirus: subfamily
Sedoreovirinae: family Reoviridae) (2,3), which is transmitted by
Culicoides spp. biting midges (4). Before 2008, EEV had been isolated
only in South Africa, where 7 antigenically distinct serotypes, EEV-1-7,
have been identified and characterized (3).
Orbiviruses encode at least 7 structural and 4 nonstructural (NS) proteins from 10 linear dsRNA genome segments (5). The smallest genome segment, segment 10 (Seg-10), encodes NS3, which mediates the release of virus particles from infected cells, and NS3A. The second largest of the EEV genome segments, Seg-2, encodes virus protein (VP) 2, the larger of the 2 outer-capsid proteins. By analogy with bluetongue virus (BTV), the Orbivirus type species, the virus serotype is determined by the specificity of interactions between VP2 and neutralizing antibodies generated during infection of the mammalian host. Consequently, VP2 and Seg-2 show sequence variations that correlate with serotype and, thus, can be used to determine the virus serotype (6).
From October 2008 through January 2009, a febrile horse disease that was diagnosed as equine encephalosis was observed in dozens of stables across Israel (7). The recent emergence of novel orbivirus strains (including BTV and epizootic hemorrhagic disease virus) in Europe, North America, Asia, and Australia (8) is of major concern to the worldwide livestock industry. Furthermore, the similarity of EEV to African horse sickness virus, one of the most devastating pathogens of equids, warranted further investigation of the outbreaks and molecular characterization of the virus. The molecular and sequence analyses reported here confirm the existence of EEV in Israel and identify the virus and its serotype, as well as its phylogenetic roots.
During October-November 2009, samples of whole blood from 8 febrile horses (H1-8; temperatures 39.5[degrees]C-42[degrees]C) in Israel were collected into EDTA tubes and analyzed at the Koret School of Veterinary Medicine (Hebrew University, Rehovot, Israel). Vero cell (American Type Culture Collection, Manassas, VA, USA) culture results of blood from H3, H5, and H8 were positive for EEV (Table 1; Figure 1).
Total RNA was extracted from the fifth and sixth passages of all 3 samples by using the QIAamp Viral RNA Mini Kit (QIAGEN, Valencia, CA, USA) to obtain sufficient viral load for the subsequent analyses and replications. RNA was reverse transcribed into cDNA by using the Verso cDNA Kit (Thermo Fisher Scientific, Epsom, UK). PCR amplification of the gene encoding NS3 (Seg-10) was performed on the 3 isolates by using GoTaq Green Master Mix (Promega, Madison, WI, USA) with the following primers: 5'-[sup.1]GTT AAG TTT CTG CGC CAT GT[sup.23]-3', 5'-[sup.741]GTA ACA CGT TTC CGC CAC G[sup.760]-3'. Thermal cycling conditions for the PCR were as previously described (9); the primer annealing temperature was modified to 53.5[degrees]C. PCR products were purified by using a cDNA purification kit (ExoSAP-IT; USB, Cleveland, OH, USA), and sequencing was conducted by BigDye terminator cycle sequencing chemistry (Applied Biosystems, Foster City, CA, USA) in an ABI 3700 DNA Analyzer (Applied Biosystems) by using ABI data collection and sequence analysis software. Further analysis of the NS3 sequence was performed with Sequencer software, version 4.8 (Gene Codes Corp., Ann Arbor, MI, USA). Sequences were deposited in GenBank under accession nos. HQ441245 for H5, HQ441246 for H3, and HQ441247 for H8. The NS3 genes (Seg-10) were compared with those of different EEVs (9) and other related orbiviruses (Table 2). Phylogenic trees were generated by using the neighbor-joining and maximum-likelihood methods (Phylip Inference Package version 3.68, Seqboot Program; J. Felsenstein, University of Washington, Seattle, WA, USA) to create 100 datasets (bootstrapping) and the DNA Maximum Likelihood Program version 3.5 (http:// cmgm.stanford.edu/phylip/dnaml.html) to construct the trees. Finally, the Consense program version 3.5c (http:// cmgm.stanford.edu/phylip/consense.html) was used to create a final consensus tree for our dataset. Broadhaven virus, a tick-borne orbivirus, was used as the outgroup in the phylogram for the gene encoding NS3.
[FIGURE 1 OMITTED]
The phylogenetic analyses of EEV Seg-10 grouped the Israeli isolates with other EEV isolates but as a distinct group with no close relation to African horse sickness virus, BTV, or epizootic hemorrhagic disease virus. Within the EEV group, 3 discrete clusters (A, B, C) were recognized; the Israeli isolates formed one of these clusters (C; Figure 2). The Israeli isolates have 85%-86% nt identity to cluster A and 75%-76% nt identity to cluster B.
In addition, full-length cDNA copies of individual EEV (from H3 and H8) genome segments were synthesized and amplified by reverse transcription PCR by using the anchor spacer-ligation method as described (10,11). Partial sequences (for the upstream 450 bp) of Seg-2 from the different Israeli isolates were identical, showing 92.3% nt and 95.7% aa sequence identity with Seg-2 and VP2 of the Kaalplaas isolate, the reference isolate of EEV-3 (GenBank accession numbers are listed in Table 2). Previous phylogenetic comparisons of Seg-2/VP2 from different BTV types showed a maximum of 71% nt and 78% aa acid identity between serotypes (6), indicating that the isolates from Israel also belong to EEV type 3.
Equine encephalosis virus has long been enzootic to southern Africa, but it has not been isolated in other parts of the world. We report the characterization of an EEV strain isolated outside of Africa. Phylogenetic analysis of Seg-2 showed 92% sequence identity to EEV-3 (Kaalplaas).
Analysis of Seg-10 (the gene encoding NS3) of different orbiviruses showed 2 clusters of South African EEV strains (A and B), in agreement with previously published studies (9). These 2 clusters appear to correlate with the geographic origins of the viruses in South Africa, independent of their isolation date. It has been suggested that the 2 EEV Seg-10 clusters in South Africa are related to the distribution of their Culicoides spp. midge vectors, C. imicola (senso stricto) and C. bolitinos. The former is the most abundant Culicoides spp. midge in Israel (12). However, the EEV isolates from Israel group as a distinct cluster (C) with similar distances to the 2 South African clusters, raising questions concerning the geographic origin of this virus. A similar finding has been observed in African horse sickness virus Seg-10, which also forms into 3 distinct groups (13).
The question of how and when the virus was initially introduced to Israel remains unanswered. Because the clinical manifestations of equine encephalosis are usually mild, it is often overlooked and underdiagnosed. EEV could have been introduced to Israel before the virus was first isolated in 2009. Alternatively, the virus might have been introduced into neighboring countries and transmitted into Israel by infected vectors carried by winds, as described for other orbiviruses (14,15). The fact that the Israeli strain of EEV-3 grouped in a different cluster than the 2 South African strains, supports the idea that it has evolved in this region for a sufficient time to accumulate these changes and most likely was not recently introduced into Israel from South Africa.
[FIGURE 2 OMITTED]
We thank Irit Orr for helping with the phylogenetic analysis.
Test development and analyses at Institute for Animal Health Pirbright were supported by Department for Environment, Food, and Rural Affairs, Biotechnology and Biological Sciences Research Council, and by European Union contracts OrbiVac-245266, WildTech-222633-2, and OrbiNet-K1303206.
Dr Aharonson-Raz is a veterinarian and a PhD candidate at the Koret School of Veterinary Medicine, Israel. Her primary research interest is epidemiology of arboviruses and infectious diseases of horses.
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Affiliations: The Hebrew University, Rehovot, Israel (K. AharonsonRaz, A. Steinman, Y. Gottlieb, E. Klement); Kimron Veterinary Institute, Bet Dagan, Israel (V. Bumbarov); Institute for Animal Health, Pirbright, UK (S. Maan, N.S. Maan, K. Nomikou, C. Batten, P. Mertens); and Deltamune (Pty) Ltd, Lyttelton, South Africa (C. Potgieter)
Address for correspondence: Eyal Klement, Koret School of Veterinary Medicine, Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University, PO Box 12, Rehovot 76100, Israel; email: eyal. email@example.com
Table 1. Clinical signs for horses whose blood was tested to determine the cause of a febrile disease, Israel, October-November 2009 Horse no. Clinical signs 1 Temperature 39.5[degrees]C, lack of appetite 2 Temperature 39.5[degrees]C, lack of appetite 3 Temperature 40[degrees]C, lack of appetite 4 Temperature 39.5[degrees]C, lack of appetite 5 Fever, colic, lethargy, congested mucous membranes, rapid pulse, lack of appetite 6 Temperature 39.5[degrees]C, lack of appetite 7 Temperature 42[degrees]C, apathy 8 Temperature 39.7[degrees]C, lack of appetite, colic Date of first Duration of Date of Horse clinical clinical blood Virus no. sign signs, d collection isolated * 1 Oct 25 5 Nov 2 No 2 Oct 29 3 Nov 2 No 3 Oct 30 3 Nov 3 Yes 4 Nov 8 Unknown Nov 8 No 5 Nov 5 5 Nov 9 Yes 6 Nov 17 4 Nov 22 No 7 Nov 25 2 Nov 26 No 8 Nov 26 3 Nov 27 Yes * Positive cases were confirmed by reverse transcription PCR of dsRNA genome segment 10. Table 2. GenBank accession numbers for orbiviruses used for phylogenic analyses of strain isolated from horses in Israel, 2009 * Accession no. Virus Strain Seg-2 Seg-10 EEV-1 (Cascara) FLD1 AY115878 FLD2 AY115876 FLD3 AY115875 FLD4 AY115877 Ref AY115865 Ref ([dagger]) AY115864 EEV-2 (Gamil) Ref AY115871 EEV-3 (Kaalplaas) FLD1 AY115874 Ref HQ630933 AY115867 EEV-4 (Bryanston) Ref AY115868 EEV-5 (Kyalami) Ref AY115869 EEV-6 Ref AY115866 (Potchefstroom) FLD1 AY115872 FLD2 AY115873 EEV-7 (N Rand) Ref AY115870 AHSV-2 AF276700 AHSV-4 AJ007305 AHSV-7 AJ007306 BRDV M83197 BTV- 2 AF135224 BTV-12 AF135227 PALV (Chuzan) AB018091 EHDV-1 NC_013405.1 EHDV-2 AM745086.1 Israel EEV H5 Animal H5 HQ441245 Israel EEV H3 Animal H3 JF495411 HQ441246 (ISR2009/20) ([double dagger]) Israel EEV H8 Animal H8 JF495412 HQ441247 (ISR2009/21) ([double dagger]) * Seg, dsRNA genome segment; EEV, equine encephalosis virus; FLD, field strain; ref, reference strain; N Rand, North Rand; AHSV, African horse sickness virus; BRDV, Broadhaven virus; BTV, bluetongue virus; PALV, Palyam virus; EHDV, epizootic hemorrhagic disease virus; H5, H3, H8, horses 5, 3, 8. ([dagger]) Unknown origin. ([double dagger]) Isolate numbers for the Orbivirus reference collection (www.reoviridae.org/dsRNA_virus_proteins/ReoID/ EEV-isolates.htm).
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