South African bovine ephemeral fever virus glycoprotein sequences are phylogenetically distinct from those from the rest of the world.

Bovine ephemeral fever virus (BEFV) is an economically important arbovirus affecting cattle and water buffalo. Currently, isolates can be separated into three phylogenetic groups, differentiated by the place of isolation, namely, East Asia, Australia, and the Middle East. BEFV surface glycoprotein (G) genes from 14 South African field strains collected between 1968 and 1999 were sequenced and compared to 154 published sequences. The BEFV isolates from South Africa were found to be phylogenetically distinct from those from other parts of the world.

Antigenic variation of bovine ephemeral fever viruses isolated in Iran, 2012-2013.

Bovine ephemeral fever virus (BEFV) is an economic arthropod-borne virus distributed in Africa, Asia, and Australia. Based on the sequence of the gene encoding the surface glycoprotein G, the viral antigenic determinant, BEFV has been phylogenetically classified into three clusters, including Australia, East Asia, and the Middle East. Here, we provide evidence for antigenic variations among the BEFV isolates in Iran during the period of 2012 to 2013 and also the exotic YHL strain, which are all classified into the East Asian cluster of the virus. For this propose, the entire length of the G gene of the viruses were sequenced and phylogenetically compared. The corresponding antigenic sites (G1-G4) were analyzed and antigenic relatedness among these viruses was measured. The two Iranian viruses, which displayed substitutions at residues E503K in the site G1 and E461K in the predicted site G4, were partially neutralized by each other's antisera (R value = 63.23%); however, these two viruses exhibited much lower cross-neutralization that measured by R value as 28.28% and 22.82%, respectively. The crucial substitution at amino acid R218K in the site G3a is believed to be the foremost cause of these declines. The data emphasize the frequent evolution of BEFV in different time periods and geographic regions, in which the new variants can emerge and likely escape from the pre-existing immunities. Thus, continuous monitoring of the circulating viruses is necessary for understanding the viral evolution and evaluation of protective immunity induced by the heterologous viruses.

Development of a quick dot blot assay for the titering of bovine ephemeral fever virus.

BACKGROUND: Bovine ephemeral fever virus (BEFV) causes fever and muscle stiffness in cattle, resulting in negative economic impact for cattle and dairy farms. During the manufacturing process of inactivated vaccine for virus control, it is important to determine the virus titer, but traditional methods such as plaque assay and TCID50 assay require days of waiting time. We sought to develop a quick dot blot assay for BEFV titering. RESULTS: Three different kinds of BEFV antigens were prepared to raise primary antibodies for BEFV detection in dot blot assays: 1) purified BEFV particles, 2) Escherichia coli (E. coli)-expressed BEFV G1 region, and 3) E. coli-expressed BEFV N protein. Results showed that antibodies raised against purified BEFV particles can detect BEFV particles, but it also showed a high background level from the proteins of BHK-21 cells. Antibodies raised against E.coli-expressed BEFV G1 region could not detect BEFV particles in dot blot assays. Finally, antibodies raised against E.coli-expressed BEFV N protein detected BEFV particles with a high signal-to-noise ratio in dot blot assays. CONCLUSIONS: E.coli-expressed N protein is a suitable antigen for the production of antiserum that can detect BEFV particles with a high signal-to-noise ratio. A dot blot assay kit using this antiserum can be developed as a quick and economical way for BEFV titering.

Development of a recombinase polymerase amplification combined with lateral-flow dipstick assay for detection of bovine ephemeral fever virus.

Bovine ephemeral fever virus (BEFV), identified as the causative pathogen of bovine ephemeral fever (BEF), is responsible for increasing numbers of epidemics/outbreaks and has a significant harmful effect on the livestock industry. Therefore, a rapid detection assay is imperative for BEFV diagnosis. In this study, we described the development of lateral-flow dipstick isothermal recombinase polymerase amplification (LFD-RPA) assays for detection of BEFV. RPA primers and LF probes were designed by targeting the specific G gene, and the amplification product can be visualized on a simple lateral flow dipstick with the naked eyes. The amplification reaction was performed at 38 °C for 20 min and LFD incubation time within 5 min. The detection limit of this assay was 8 copies per reaction, and there was no cross-reactivity with other bovine infectious viruses such as bovine viral diarrhea virus, infectious bovine rhinotracheitis virus, bovine respiratory syncytial virus, bovine coronavirus, bovine parainfluenza virus type 3, bovine vesicular stomatitis virus. In addition, the assay was performed with total 128 clinical specimens and the diagnostic results were compared with conventional RT-PCR, real-time quantative(q) PCR. The result showed that the coincidence rate of BEFV LFD-RPA and real-time qPCR was 96.09% (123/128), which was higher than conventional RT-PCR. The RPA combined with LFD assay probably provides a rapid and sensitive alternative for diagnosis of BEFV infections outbreak.

Risk analysis and seroprevalence of bovine ephemeral fever virus in cattle in the Kingdom of Saudi Arabia.

Bovine ephemeral fever virus (BEFV) is an arthropod-borne rhabdovirus that causes disabling clinical signs and major economic losses in cattle and water buffalo. The disease is well documented in Asia, Africa, and the Middle East; however, the seroprevalence of BEFV in different regions and bovine breeds in the Kingdom of Saudi Arabia (KSA) is unknown. The aim of this study was to analyze risk factors which affect the prevalence of antibodies against BEFV in small herds of cattle in four geographical regions of KSA. A total of 1480 serum samples from non-BEFV vaccinated small herds of cattle were collected from the Eastern, Jizan, Qasim, and Riyadh regions (370 samples per region) during the summer of 2010. Serum neutralization test was used to detect antibodies against BEFV. There was a significant effect of region, breed, sex, and age on the seroprevalence of BEFV. Seropositive ratios were 18, 18, 26, and 12 % for the Eastern, Jizan, Qasim, and Riyadh regions, respectively (P = 0.00002); 23.2 % for dairy and 13.7 % for non-dairy breeds (P = 0.00004); 24.4 % for males and 14.6 % for females (P = 0.00004); and 15.4, 29.1, and 11.4 % for animals <1 year, 1-3 years, and >3 years, respectively (P < 0.001). Risk analysis showed a significant effect of different regions of KSA on the seroprevalence of BEFV. Host risk factors (age, sex, and breed) showed also a significant effect on the seroprevalence of BEFV. This indicates active circulation of this virus in small herds of cattle. Insect control strategies and BEFV vaccination programs during the spring are recommended to reduce the spread of BEFV and minimize subsequent economic losses as this is adopted in many enzootic countries.

Evolution of bovine ephemeral fever virus in the Australian episystem.

Bovine ephemeral fever virus (BEFV) is an arthropod-borne rhabdovirus that causes a debilitating disease of cattle in Africa, Asia, and Australia; however, its global geodynamics are poorly understood. An evolutionary analysis of G gene (envelope glycoprotein) ectodomain sequences of 97 BEFV isolates collected from Australia during 1956 to 2012 revealed that all have a single common ancestor and are phylogenetically distinct from BEFV sampled in other geographical regions. The age of the Australian clade is estimated to be between 56 and 65 years, suggesting that BEFV has entered the continent on few occasions since it was first reported in 1936 and that the 1955-1956 epizootic was the source of all currently circulating viruses. Notably, the Australian clade has evolved as a single genetic lineage across the continent and at a high evolutionary rate of ∼10(-3) nucleotide substitutions/site/year. Screening of 66 isolates using monoclonal antibodies indicated that neutralizing antigenic sites G1, G2, and G4 have been relatively stable, although variations in site G3a/b defined four antigenic subtypes. A shift in an epitope at site G3a, which occurred in the mid-1970s, was strongly associated with a K218R substitution. Similarly, a shift at site G3b was associated primarily with substitutions at residues 215, 220, and 223, which map to the tip of the spike on the prefusion form of the G protein. Finally, we propose that positive selection on residue 215 was due to cross-reacting neutralizing antibody to Kimberley virus (KIMV). This is the first study of the evolution of BEFV in Australia, showing that the virus has entered the continent only once during the past 50 to 60 years, it is evolving at a relatively constant rate as a single genetic lineage, and although the virus is relatively stable antigenically, mutations have resulted in four antigenic subtypes. Furthermore, the study shows that the evolution of BEFV in Australia appears to be driven, at least in part, by cross-reactive antibodies to KIMV which has a similar distribution and ecology but has not been associated with disease. As BEFV and KIMV are each known to be present in Africa and Asia, this interaction may occur on a broader geographic scale.

Phylogenetic relationships of the glycoprotein gene of bovine ephemeral fever virus isolated from mainland China, Taiwan, Japan, Turkey, Israel and Australia.

BACKGROUND: The glycoprotein (G) gene sequences of bovine ephemeral fever virus (BEFV) strains derived from mainland China have not been compared with those of the isolates from other countries or areas. Therefore, the G genes of four BEFV isolates obtained from mainland China were amplified and sequenced. A phylogenetic tree was constructed in order to compare and analyze the genetic relationships of the BEFV isolates derived from mainland China and different countries and areas. RESULTS: The complete BEFV G gene was successfully amplified and sequenced from four isolates that originated from mainland China. A total of fifty-one BEFV strains were analyzed based on the G gene sequence and were found to be highly conserved. A phylogenetic tree showed that the isolates were grouped into three distinct lineages depending on their source of origin. The antigenic sites of G1, G2 and G3 are conserved among the isolates, except for several substitutions in a few strains. CONCLUSIONS: The phylogenetic relationships of the BEFV isolates that originated from mainland China, Taiwan, Japan, Turkey, Israel and Australia were closely related to their source of origin, while the antigenic sites G1, G2 and G3 are conserved among the BEFV isolates used in this work.

A SYBR green I-based quantitative RT-PCR assay for bovine ephemeral fever virus and its utility for evaluating viral kinetics in cattle.

We developed a SYBR green I-based reverse-transcription quantitative PCR (RT-qPCR) assay for bovine ephemeral fever virus (BEFV). Analytical sensitivity of the assay was ~ 100 times higher than conventional RT-PCR. The precision of the RT-qPCR established for RNA standards was high, with intra-assay and inter-assay coefficients of variation of 0.23-0.89% and 0.23-1.02%, respectively. The test was highly specific for BEFV strains, with no cross-reactivity with other viruses of veterinary significance. The assay detected BEFV RNA as early as 1 d post-infection (dpi) and up to 7-8 dpi in the blood samples of experimentally infected cattle. The most stable reference gene, peptidylprolyl isomerase A (PPIA), was selected for the quantification of BEFV. Viral RNA loads reached peak level at 3-5 dpi and then decreased rapidly through 7-8 dpi. Our assay provides a reliable approach for the detection of BEFV in the early infection stage and for use in the profiling of BEFV kinetics in vivo.

Molecular characterization of bovine ephemeral fever virus in Thailand between 2013 and 2017.

Bovine ephemeral fever (BEF) is an arthropod-borne disease caused by bovine ephemeral fever virus (BEFV), a negative sense, single-stranded RNA virus. BEFV is endemic in tropical and sub-tropical regions including Thailand, a country in mainland Southeast Asia. However, there are few studies on BEFV and no available information regarding molecular characteristics of BEFV in Thailand. Therefore, the aims of this study were to genetically characterize Thai BEFVs and reveal their evolutions by phylogenetic analysis of G gene ectodomain sequences. From 2013 to 2017, blood samples were collected from bovine that matched with BEF case definition from three regions of Thailand. Thai BEFV G genes and a whole genome of an isolate, East Asia/TH/LRI0045/2016, were sequenced and characterized. Additionally, their phylogenies were constructed. This is the first report on genetics of BEFV in Southeast Asia. G ectodomain encoding region of Thai BEFV found during 2013-2017 are closely related to the second and third sub-clades of East Asia lineage. In addition, we observed mutation in the putative P' ORF of all Thai BEFVs which generated a premature stop codon. Thai G gene sequences are closely related to those of mainland Chinese and Taiwanese isolates. The whole genomic sequences of Thai BEFV and East Asia/China/JT02 L/2002 possess common characteristics, suggesting shared evolutionary relationship between East and Southeast Asian strains. Further studies on relationship between animal translocation, circulation of BEFV in Greater Mekong subregion and acquisition of more G gene sequences may improve understanding of BEFV epidemiology in mainland Southeast Asia.

Post-viraemic detection of bovine ephemeral fever virus by use of autogenous lymphoid tissue-derived bovine primary cell cultures.

BACKGROUND: The potential tissue replication sites and specific cell types that support in vivo virus survival beyond the acute phase of bovine ephemeral fever virus (BEFV) infection have not been fully defined in cattle. To clarify the knowledge gap, tissue specimens were tested after collection from an adult steer necropsied 1 week after acute BEF. CASE REPORT: Significant necropsy findings included fibrinoproliferative synovitis in the stifle joints and fibrin clot-laden fluid in serous body cavities. Moderate numbers of infiltrating neutrophils were demonstrated in sections of the prefemoral lymph nodes and haemal node, and lymphoid hyperplasia in the spleen, haemal node and prefemoral lymph nodes. Viral RNA was detected by qRT-PCR in fresh spleen, haemal node, prefemoral lymph node, synovial fluid and in several spleen-derived cell cultures. BEFV was isolated from autogenously derived splenic primary cell cultures 6 days after cessation of viraemia, and characteristic bullet-shaped virions were confirmed by electron microscopy of an ultrathin haemal node section. In sections of the spleen, haemal node and other tissues, immunohistochemistry demonstrated BEFV antigens that were intracellularly associated with probable histiocytic cells. CONCLUSION: BEFV has preferential tropism for bovine lymphoid tissues and the spleen and haemal node may be potential sites for post-viraemic virus replication.

High-resolution melting (HRM) for genotyping bovine ephemeral fever virus (BEFV).

In recent years there have been several major outbreaks of bovine ephemeral disease in the Middle East, including Israel. Such occurrences raise the need for quick identification of the viruses responsible for the outbreaks, in order to rapidly identify the entry of viruses that do not belong to the Middle-East BEFV lineage. This challenge was met by the development of a high-resolution melt (HRM) assay. The assay is based on the viral G gene sequence and generation of an algorithm that calculates and evaluates the GC content of various fragments. The algorithm was designed to scan 50- to 200-base-long segments in a sliding-window manner, compare and rank them using an Order of Technique of Preference by Similarity to Ideal Solution (TOPSIS) the technique for order preference by similarity to ideal solution technique, according to the differences in GC content of homologous fragments. Two fragments were selected, based on a match to the analysis criteria, in terms of size and GC content. These fragments were successfully used in the analysis to differentiate between different virus lineages, thus facilitating assignment of the viruses' geographical origins. Moreover, the assay could be used for differentiating infected from vaccinated animales (DIVA). The new algorithm may therefore be useful for development of improved genotyping studies for other viruses and possibly other microorganisms.

Resurgence of bovine ephemeral fever in mainland Japan in 2015 after a 23-year absence.

In September and October 2015, suspected cases of bovine ephemeral fever (BEF) were reported in the mainland region of Kagoshima Prefecture and on Tanegashima Island. The genome of the BEF virus (BEFV) was detected in the diseased cows and the cows that had recovered. The serum obtained from the affected cows contained high titers of BEFV-neutralizing antibody. In total, 18 affected cows were demonstrated to be infected with BEFV during the outbreak. Our findings showed evidence that BEF occurred in mainland Japan after a 23-year absence. Phylogenetic analysis based on the surface glycoprotein (G) gene revealed that BEFVs detected in the affected cows were genetically distinct from previous Japanese BEFVs, but were close to BEFVs circulating in Taiwan and mainland China in recent years. Amino acid substitution in the neutralizing epitope domains of the G protein was limited between the detected viruses and the vaccine strain (YHL isolate), and high titers of the neutralizing antibody against the YHL isolate were induced in the infected cattle during the disease occurrences. Therefore, current BEF vaccines probably elicit protective immunity against the BEFVs detected in 2015, although their effectiveness should be assessed. Since the BEFV vaccination rates are estimated to be low, a BEF outbreak should be considered a possibility in mainland Japan.

Bovine ephemeral fever in Australia and the world.

Bovine ephemeral fever (BEF) is a disabling viral disease of cattle and water buffaloes. It can cause significant economic impact through reduced milk production in dairy herds, loss of condition in beef cattle and loss of draught animals at the time of harvest. Available evidence indicates clinical signs of BEF, which include bi-phasic fever, anorexia, muscle stiffness, ocular and nasal discharge, ruminal stasis and recumbency, are due primarily to a vascular inflammatory response. In Australia, between 1936 and 1976, BEF occurred in sweeping epizootics that commenced in the tropical far north and spread over vast cattle grazing areas of the continent. In the late 1970s, following several epizootics in rapid succession, the disease became enzootic in most of northern and eastern Australia. In Africa, the Middle East and Asia, BEF occurs as also epizootics which originate in enzootic tropical areas and sweep north or south to sub-tropical and temperate zones. The causative virus is transmitted by haematophagous insects that appear to be borne on the wind, allowing rapid spread of the disease. Bovine ephemeral fever virus (BEFV) has been classified as the type species of the genus Ephemerovirus in the Rhabdoviridae. It has a complex genome organization which includes two glycoprotein genes that appear to have arisen by gene duplication. The virion surface glycoprotein (G protein) contains four major antigenic sites that are targets for neutralizing antibody. An analysis of a large number of BEFV isolates collected in Australia between 1956 and 1992 has indicated remarkable stability in most neutralization sites. However, epitope shifts have occurred in the major conformational site G3 and these have been traced to specific mutations in the amino acid sequence. BEFV isolates from mainland China and Taiwan are closely related to Australian isolates, but some variations have been detected. Natural BEFV infection induces a strong neutralizing antibody response and infection usually induces durable immunity. Several forms of live-attenuated, inactivated and recombinant vaccines have been reported but with variable efficacy and durability of protection. The BEFV G protein is a highly effective vaccine antigen, either as a purified subunit or expressed from recombinant viral vectors.

Invasion of exotic bovine ephemeral fever virus into Taiwan in 2013-2014.

Bovine ephemeral fever virus is a member of the family Rhabdoviridae and bovine ephemeral fever has frequently affected cattle population in Taiwan since 1967. During the outbreaks in 2013 and 2014, exotic bovine ephemeral fever viruses were detected by reverse transcription polymerase chain reaction and nucleotide sequencing. Sequence comparison showed that the exotic viruses shared 99.0-99.4% nucleotide identities (99.4-100.0% amino acid identities) with Chinese viruses and, on the contrary, 96.2-97.2% nucleotide identities (97.8-98.6% amino acid identities) with indigenous Taiwanese viruses. Additionally, our phylogenetic analysis also supported that the newly invaded bovine ephemeral fever viruses were closely related to the Chinese strains. These exotic 2013-2014 viruses have become prevalent and displaced indigenous virus strains since their appearance.

Viral neurotropism, peripheral neuropathy and other morphological abnormalities in bovine ephemeral fever virus-infected downer cattle.

OBJECTIVE: This study assessed the neurotropism of bovine ephemeral fever (BEF) virus (BEFV) and described histomorphological abnormalities of the brain, spinal cord and peripheral nerves that may causally contribute to paresis or paralysis in BEF. METHODS: Four paralysed and six asymptomatic but virus-infected cattle were monitored, and blood and serum samples screened by qRT-PCR, virus isolation and neutralisation tests. Fresh brain, spinal cord, peripheral nerve and other tissues were qRT-PCR-tested for viral RNA, while formalin-fixed specimens were processed routinely and immunohistochemically evaluated for histomorphological abnormalities and viral antigen distribution, respectively. RESULTS: The neurotropism of BEFV was immunohistochemically confirmed in the brain and peripheral nerves and peripheral neuropathy was demonstrated in three paralysed but not the six aneurological but virus-infected animals. Wallerian degeneration (WD) was present in the ventral funicular white matter of the lumbar spinal cord of a paralysed steer and in cervical and thoracic spinal cord segments of three paralysed animals. Although no spinal cord lesions were seen in the steer euthanased within 7 days of illness, peripheral neuropathy was present and more severe in nerves of the brachial plexuses than in the gluteal or fibular nerves. The only steer with WD in the lumbar spinal cord also showed intrahistiocytic cell viral antigen that was spatially distributed within areas of moderate brain stem encephalitis. CONCLUSION: The data confirmed neurotropism of BEFV in cattle and documented histomorphological abnormalities in peripheral nerves and brain which, together with spinal cord lesions, may contribute to chronic paralysis in BEFV-infected downer cattle.

Development of a reliable assay protocol for identification of diseases (RAPID)-bioactive amplification with probing (BAP) for detection of bovine ephemeral fever virus.

A rapid, sensitive, and specific assay, RAPID-BAP assay, was developed to detect and quantify the G protein-encoding gene of bovine ephemeral fever virus (BEFV). This new technique uses a nested PCR and magnetic bead-based DNA probing assay. The optimal conditions for the assay were examined. By applying a nested PCR, a minimum of 1 copy/mul of the BEFV plasmid DNA could be detected by the assay. The optimal hybridization conditions at 50 degrees C in 5x SSC and 0.5% SDS with a 20-min incubation allowed clear discrimination between negative and positive controls. The assay was also highly specific as all negative controls failed to show any positive detection. The diagnostic sensitivity of the RAPID-BAP assay, real-time RT-PCR, and conventional RT-PCR in the detection of 34 clinical blood samples suspected to have BEFV infections were 72.73, 36.36, and 18.18%, respectively. The results indicated that the RAPID-BAP assay developed in this study was more sensitive than the conventional RT-PCR and real-time RT-PCR assays for the detection of BEFV. The novel RAPID-BAP assay is an excellent diagnostic tool with high sensitivity, specificity, and fast turnaround time.

A real-time RT-quantative(q)PCR for the detection of bovine ephemeral fever virus.

A quantitative reverse-transcriptase real-time PCR assay, using TaqMan chemistry, for detecting bovine ephemeral virus (BEFV) is described. Available G gene sequences of viral RNA were aligned, and primers and probes were designed to recognize the virus. To quantitate the viruses, cDNA containing the real-time amplicon was prepared with a forward primer carrying the T7 promoter sequences. Run-off transcription from the T7 promoter amplicon template was used to prepare cRNA. Ten-fold dilutions of the run-off viral transcript were used as templates for the reaction in which they served as standards to quantitate unknown viral samples. By using this system it was shown that as few as 10-100 copies of a viral genome could be detected.

Large-scale serological survey of bovine ephemeral fever in China.

Bovine ephemeral fever (BEF) is caused by the arthropod-borne bovine ephemeral fever virus (BEFV), which is classified in the family Rhabdoviridae and the genus Ephemerovirus. A debilitating and sometimes fatal viral disease, BEF affects cattle and water buffalo. The epizootiology of BEF among cattle in China has not been fully determined. We examined the seroprevalence of the BEFV among cattle in China between January 2012 and June 2014. Among the 2822 serum samples collected from various cattle breeds in 26 provinces in China, the seropositive rate for the BEFV ranged from 0% to 81% between regions and species. Our findings show that BEFV was prevalent in the all of the regions tested in our study and provide the first reliable reference regarding BEF surveillance in China.

Occurrence of bovine ephemeral fever in Okinawa Prefecture, Japan, in 2012 and development of a reverse-transcription polymerase chain reaction assay to detect bovine ephemeral fever virus gene.

In September 2012, several cows and a calf showed decreased activity, anorexia and fever on Ishigaki Island, Okinawa Prefecture, Japan, and the cases were diagnosed as bovine ephemeral fever (BEF). We isolated BEF virus (BEFV) from one of the affected cows and then determined the complete genome sequence of the G gene, which encodes a class I transmembrane glycoprotein of BEFV. The BEFV isolate in this case, ON-3/E/12, was sorted into the same cluster as other BEFV isolates in Japan, Taiwan and China obtained in 1996-2004 and was most closely related to a 2002 Chinese isolate, JT02L, according to the phylogenetic analysis of the complete G gene. Since inactivated vaccines for BEF available in Japan are considered effective against the ON-3/E/12 isolate as well as other isolates in East Asia from 1996-2004, annual vaccination should be conducted to prevent BEF in Okinawa. Additionally, in this study, we developed an RT-PCR assay to detect the BEFV gene in Japan and neighboring countries. Our assay was able to amplify target sequences in all of the tested BEFV isolates, including 18 isolates in Japan and another isolate in Australia. The assay was found to be useful also for testing RNA samples extracted from bovine peripheral blood mononuclear cells, and the detection limit of the assay was 10 copies per tube. We believe that our assay would be an important tool for the screening of BEFV infection and the diagnosis of BEF.

Seroprevalence of bovine ephemeral fever virus in domesticated and wildlife species during epidemic and inter-epidemic periods (2000-2009) in Israel.

Bovine ephemeral fever (BEF) is an economically important vector-borne viral disease of cattle and buffalo. It has been reported from most of the world's tropical and subtropical regions. In the last few decades, outbreaks of BEF have occurred in Israel almost every other year. Several serological studies have demonstrated a wide range of wild animal species that are positive for BEF virus (BEFV) antibodies. However, the question of whether wild animals and domesticated species other than cattle also play an important role in the maintenance and transmission of BEFV in Israel remains. Here, we examined the prevalence of anti-BEFV antibodies in 942 samples collected from various wild, semi-captive and domesticated animal species during the years 2000-2009 using the serum neutralization (SN) method. SN test revealed the presence of BEFV-neutralizing antibodies in nine samples (0.96%), from three species: Bubalus bubalis (4/29, 13.79%), Gazella g. gazella (3/68, 4.44%) and Dama d. mesopotamica (2/296, 0.68%). All positive samples were collected in areas of earlier outbreaks. The low prevalence of positive animals and the solid correlation with prior outbreaks indicate that the tested species probably do not serve as virus reservoirs and may play only a minor role in the maintenance of BEFV in the Middle East.