Development of two competitive ELISAs based on monoclonal antibodies for the serological detection of Bovine ephemeral fever virus.

Bovine ephemeral fever virus (BEFV) is a member of the genus Ephemerovirus in the family Rhabdoviridae. It is an arthropod-borne virus transmitted by many species of midges and mosquitoes. It can cause severe economic consequences due to losses in milk production and the general condition of cattle and water buffalo. BEF occurs in some tropical, subtropical and warm temperate regions of Africa, Australia, the Middle East and Asia with seasonal outbreaks, but its possible spread to other areas (e.g. Europe) cannot be excluded. Therefore, using and developing rapid diagnostic methods with optimal performance is essential for identifying emerging pathogens and their control. In the present study, we developed two competitive serological ELISAs based on monoclonal antibodies (mAbs), designed by using BEFV inactivated antigen and the BEF recombinant nucleoprotein (N), respectively. A panel of 77 BEF-positive and 338 BEF-negative sera was used to evaluate the two tests. With a diagnostic sensitivity of 97.4 % using the inactivated virus and 98.7 % using the recombinant N, and a diagnostic specificity of 100 % using both antigens, our results suggest that these tests are suitable for the serological diagnosis of BEF.

Bovine Ephemeral Fever Viruses in Israel 2014-2023: Genetic Characterization of Local and Emerging Strains.

Bovine ephemeral fever (BEF) is an arthropod-borne viral disease, which frequently causes significant epizootics in susceptible water buffalo and cattle in Africa, Australia, Asia and the Middle East. In the current study, a two-stage protocol for BEFV viral isolation was developed. Data on the clinical signs, geographic distribution and phylogenetic analysis of BEFV strains isolated in Israel in 2015, 2018, 2021 and 2023 were summarized. It was found that during 2015-2021, all BEF outbreaks were caused by local BEFV strains, whereas the epizootic of BEFV in 2023 was caused by a new "Mayotte-like" BEFV strain. A comparison of bluetongue (BT) and BEF outbreaks during 2023 in Israel demonstrated that the incidence of BEFV was 2.21 times higher and its pathogenicity was more serious for the cattle population compared to that caused by BTVs. A phylogenetic analysis of Israeli and global BEFV revealed the emergence of non-local strains in new areas. This finding suggests that BEFV can no longer be classified based only upon geographic distribution. Considering a phylogenetic, genetic and proteomic analysis of all available BEFV strains, we suggest classifying them as a single serotype, which includes four lineages.

The Molecular Epidemiology of Epizootic Hemorrhagic Disease Viruses Identified in Israel between 2015 and 2023.

Epizootic hemorrhagic disease (EHD) is an infectious, non-contagious viral disease seriously affecting cattle and some wild ruminants and has a worldwide distribution. All viruses can be subdivided into "Eastern" and "Western" topotypes according to geographic distribution via the phylogenetic analysis of internal genes. In Israel, during the last decade, three outbreaks were registered: caused by EHDV-6 in 2015, by EHDV-1 in 2016, and by EHDV-7 in 2020. Additionally, RNA of EHDV-8 was found in imported calves from Portugal in 2023. During the same period in other countries of the region, non-Israeli-like EHDV-6 and EHDV-8 were identified. Full genome sequencing, BLAST, and phylogenetic analyses of the locally and globally known EHDV genomes allowed us to presume the probable route and origin of these viruses detected in Israel. Thus, EHDV-6 has probably been circulating in the region for a long period when EHDV-1 and -8 appeared here for the last years, while their route of introduction into the new areas was probably natural; all of them belonged to the "Western" topotype. In contrast, EHDV-7 probably had the "Eastern", anthropogenic origin. Data from the study can facilitate the evaluation of the appearance or reappearance of EHDVs in the Mediterranean area and enhance the planning of prevention measures.

Hefer valley virus: a novel ephemerovirus detected in the blood of a cow with severe clinical signs in Israel in 2022.

A novel ephemerovirus was identified in a Holstein-Friesian cow in the Hefer Valley, Israel, that showed severe and fatal clinical signs resembling an arboviral infection. A sample taken during the acute phase tested negative for important endemic arboviral infectious cattle diseases. However, sequencing from blood revealed the full genome sequence of Hefer Valley virus, which is likely to represent a new species within the genus Ephemerovirus, family Rhabdoviridae. Archived samples from cattle with comparable clinical signs collected in Israel in 2021 and 2022 tested negative for the novel virus, and therefore, the actual distribution of the virus is unknown. As this is a recently identified new viral infection, the viral vector and the prevalence of the virus in the cattle population are still unknown but will be the subject of future investigations.

Comparative Molecular and Epidemiological Analyses of Israeli Bluetongue Viruses Serotype 1 and 9 Causing Outbreaks in 2018-2020.

Israel is endemic to bluetongue virus (BTV). The introduction of novel-for-the-region arboviruses have been recorded annually in recent years. In 2019, previously non-reported in-the-country BTV-1 and BTV-9 were identified. BTV-1 caused a single-season outbreak, probably linked to mild infection in ruminants. BTV-9 was retrospectively detected in the field samples collected from August 2018 until 2020. It was the dominant serotype in 2019, out of the six serotypes recorded during that calendar year. Clinical manifestation of the disease in cases diagnosed with BTV-9 were compared to those in cases determined to have BTV-1. BLAST and phylogenetic analyses of BTV-1 showed that the nucleotide (nt) sequence coding the viral outer protein 1 (VP2) determining the serotype is closely related to BTV-1 isolated in Sudan in 1987, and the coding sequence of the outer protein 2 (VP5) is related to South African BTV-1 from 2017. A probable common ancestor with Libyan BTV-9 strains isolated in 2008 was seen in an analysis of Israeli BTV-9 nt sequences. Notably, the outbreak-caused BTV-9 strains collected in 2019 exhibited a distinct level of genetic reassortment with local Israeli strains compared to BTV-9 strains registered in 2018 and 2020.

Characterization of experimental Shuni virus infection in the mouse.

Shuni virus (SHUV), an orthobunyavirus of the Simbu serogroup, was initially isolated in Nigeria in the 1960s, further detected in other African countries and in the Middle East, and is now endemic in Israel. Transmitted by blood-sucking insects, SHUV infection is associated with neurological disease in cattle and horses, and with abortion, stillbirth, or the birth of malformed offspring in ruminants. Surveillance studies also indicated a zoonotic potential. This study aimed to test the susceptibility of the well-characterized interferon (IFN)-α/ß receptor knock-out mouse model (Ifnar-/-), to identify target cells, and to describe the neuropathological features. Ifnar-/-mice were subcutaneously infected with two different SHUV strains, including a strain isolated from the brain of a heifer showing neurological signs. The second strain represented a natural deletion mutant exhibiting a loss of function of the S-segment-encoded nonstructural protein NSs, which counteracts the host's IFN response. Here it is shown that Ifnar-/-mice are susceptible to both SHUV strains and can develop fatal disease. Histological examination confirmed meningoencephalomyelitis in mice as described in cattle with natural and experimental infections. RNA in situ hybridization was applied using RNA Scope™ for SHUV detection. Target cells identified included neurons and astrocytes, as well as macrophages in the spleen and gut-associated lymphoid tissue. Thus, this mouse model is particularly beneficial for the evaluation of virulence determinants in the pathogenesis of SHUV infection in animals.

Identification of Novel Reassortant Shuni Virus Strain in Clinical Cases of Israeli Ruminants, 2020-2021.

The Shuni virus (SHUV) causes an endemic viral infection in Israel and South Africa. It belongs to the Simbu serogroup within the order Bunyavirales, family Peribunyaviridae, genus Orthobunyavirus. Recently, it has been identified in aborted cases of domestic ruminants, young cattle and horses manifesting neural signs and acute death, symptomatic cows, and in carcasses of wild animals. Moreover, SHUV was isolated and identified in humans. In this study, we describe clinical cases of SHUV infection in Israeli domestic ruminants in 2020-2021, which represented clinical manifestations of simbuviral infection including abortions, a neural lethal case in a fattening calf, and an acute symptomatic case in a beef cow. In all cases, SHUV was confirmed by complete or partial viral genome sequencing. There is a significant difference of M and L segments of the novel strains compared with those of all known SHUV strains, while the S segments have more than 99% nucleotide (nt) identity with Israeli and African "Israeli-like" strains previously circulated in 2014-2019. This indicates a reassortment origin of the strain. At the same time, M and S segment nt sequences showed about 98-99% nt identity with some South African strains collected in 2016-2018. Nevertheless, the viral origin and the geographical place of the reassortment stayed unknown.

Identification and Genetic Characterization of Viral Pathogens in Ruminant Gestation Abnormalities, Israel, 2015-2019.

Infectious agents including viruses are important abortifacients and can cause fetal abnormalities in livestock animals. Here, samples that had been collected in Israel from aborted or malformed ruminant fetuses between 2015 and 2019 were investigated for the presence of the following viruses: the reoviruses bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV), the flaviviruses bovine viral diarrhea virus (BVDV) and border disease virus (BDV), the peribunyaviruses Shuni virus (SHUV) and Akabane virus (AKAV), bovine herpesvirus type 1 (BoHV-1) and bovine ephemeral fever virus (BEFV). Domestic (cattle, sheep, goat) and wild/zoo ruminants were included in the study. The presence of viral nucleic acid or antigen could be confirmed in 21.8 % of abnormal pregnancies (213 out of 976 investigated cases), with peribunyaviruses, reoviruses and pestiviruses being the most prevalent. At least four different BTV serotypes were involved in abnormal courses of pregnancy in Israel. The subtyping of pestiviruses revealed the presence of two BDV and several distinct BVDV type 1 strains. The peribunyaviruses AKAV and SHUV were identified annually throughout the study period, however, variation in the extent of virus circulation could be observed between the years. In 2018, AKAV even represented the most detected pathogen in cases of small domestic ruminant gestation abnormalities. In conclusion, it was shown that various viruses are involved in abnormal courses of pregnancy in ruminants in Israel.

Genomic Analysis Illustrated a Single Introduction and Evolution of Israeli Bluetongue Serotype 8 Virus Population 2008-2019.

Outbreaks of the European Bluetongue virus (BTV) serotype 8 (BTV-8), which are characterized by activity cycles separated by years of inactivity, may be influenced by genetic changes of the virus or by herd immunity. BTV activity in Israel is characterized by similar dynamics, but differs from European countries in its vector population, environmental conditions, and lack of cattle vaccination against this serotype. Comparison of these two geographical systems and characterization of their epidemiological connection is therefore of high interest in-order to better understand the factors influencing BTV-8 evolution. BTV-8, closely related to the European strain, was introduced to Israel in 2008. It was at the center of BT outbreaks in 2010 and 2015-2016 and thereafter was lastly isolated in Israel in 2019. We performed genetic analyses of twelve BTV-8 Israeli strains isolated between 2008 and 2019 and compared them with published sequences of BTV-8 isolated in other countries. The analysis revealed a single introduction of BTV-8 into Israel and thereafter extensive occurrence of genomic drifts and multiple reassortments with local BTV strains. Comparison of the Israeli and Cypriot BTV-8 from 2015 to 2016 suggests transmission of the virus between the two countries and a separate and parallel development from European or other Israeli BTV-8 strains. The parallel development of other BTV-8 strains was demonstrated by the identification of the Israeli BTV-8 ISR-1194/1/19 strain, which exhibited common origin with reassorted Israeli BTV-8 strains from 2010 and additional reassortment of seven segments. In order to reveal the source of BTV-8 introduction into Israel we performed BEAST analysis which showed that a probable common ancestor for both European and Israeli BTV-8 presumably existed in 2003-2004. In 2019, a possible new introduction occurred in Israel, where a novel BTV-8 strain was detected, sharing ~95% identity by segments 2 and 6 with Nigerian BTV-8NIG1982/07 and European-Middle Eastern strains. The results of the study indicate that Israel and neighboring countries consist a separate environmental and evolutionary system, distinct from European ones.

Shuni virus-induced meningoencephalitis after experimental infection of cattle.

Shuni virus (SHUV), an insect-transmitted orthobunyavirus of the Simbu serogroup within the family Peribunyaviridae, may induce severe congenital malformations when naïve ruminants are infected during gestation. Only recently, another clinical presentation in cattle, namely neurological disease after postnatal infection, was reported. To characterize the course of the disease under experimental conditions and to confirm a causal relationship between the virus and the neurological disorders observed in the field, six calves each were experimentally inoculated (subcutaneously) with two different SHUV strains from both clinical presentations, that is encephalitis and congenital malformation, respectively. Subsequently, the animals were monitored clinically, virologically and serologically for three weeks. All animals inoculated with the 'encephalitis strain' SHUV 2162/16 developed viremia for three to four consecutive days, seroconverted, and five out of six animals showed elevated body temperature for up to three days. No further clinical signs such as neurological symptoms were observed in any of these animals. However, four out of six animals developed a non-suppurative meningoencephalitis, characterized by perivascular cuffing and glial nodule formation. Moreover, SHUV genome could be visualized in brain tissues of the infected animals by in situ hybridization. In contrast to the 'encephalitis SHUV strain', in animals subcutaneously inoculated with the strain isolated from a malformed newborn (SHUV 2504/3/14), which expressed a truncated non-structural protein NSs, a major virulence factor, no viremia or seroconversion, was observed, demonstrating an expected severe replication defect of this strain in vivo. The lack of viremia further indicates that virus variants evolving in malformed foetuses may represent attenuated artefacts as has been described for closely related viruses. As the neuropathogenicity of SHUV could be demonstrated under experimental conditions, this virus should be included in differential diagnosis for encephalitis in ruminants, and cattle represent a suitable animal model to study the pathogenesis of SHUV.

Bluetongue Serotype 3 in Israel 2013-2018: Clinical Manifestations of the Disease and Molecular Characterization of Israeli Strains.

In this paper, the results of the diagnostic activities on Bluetongue virus serotype 3 (BTV-3) conducted at Kimron Veterinary Institute (Beit Dagan, Israel) between 2013 and 2018 are reported. Bluetongue virus is the causative agent of bluetongue (BT), a disease of ruminants, mostly transmitted by competent Culicoides species. In Israel, BTV-3 circulation was first detected in 2013 from a sheep showing classical BT clinical signs. It was also evidenced in 2016, and, since then, it has been regularly detected in Israeli livestock. Between 2013 and 2017, BTV-3 outbreaks were limited in sheep flocks located in the southern area only. In 2018, BTV-3 was instead found in the Israeli coastal area being one of the dominant BTV serotypes isolated from symptomatic sheep, cattle and goats. In Israeli sheep, BTV-3 was able to cause BT classical clinical manifestations and fatalities, while in cattle and goats infection ranged from asymptomatic forms to death cases, depending on either general welfare of the herds or on the occurrence of viral and bacterial co-infections. Three different BTV-3 strains were identified in Israel between 2013 and 2018: ISR-2019/13 isolated in 2013, ISR-2153/16 and ISR-2262/2/16 isolated in 2016. Sequencing and phylogenetic analysis of these strains showed more than 99% identity by segment (Seg) 2, 5, 6, 7, and 8 sequences. In contrast, a wide range of diversity among these strains was exhibited in other viral gene segments, implying the occurrence of genome reassortment between these local circulating strains and those originating from Africa. The genome sequences of the BTV-3 isolated in 2017 and 2018 were most closely related to those of the ISR-2153/16 strain suggesting their common ancestor. Comparison of BTV-3 Israeli strains with those recently detected in the Mediterranean region uncovered high percentage identity (98.19-98.28%) only between Seg-2 of all Israeli strains and the BTV-3 Zarzis/TUN2016 strain. A 98.93% identity was also observed between Seg-4 sequences of ISR-2019/13 and the BTV-3 Zarzis/TUN2016 strain. This study demonstrated that BTV-3 has been circulating in the Mediterranean region at least since 2013, but, unlike the other Mediterranean strains, Israeli BTV-3 were able to cause clinical signs also in cattle.

Complete Coding Sequence of a Novel Bluetongue Virus Isolated from a Commercial Sheeppox Vaccine.

The full genome sequences of two isolates of bluetongue virus (BTV) from a commercial sheeppox vaccine were determined. Strain SPvvvv/02 shows low sequence identity to its closest relative, strain BTV-26 KUW2010/02, indicating the probable detection of a novel BTV genotype, whereas strain SPvvvv/03 shows high sequence identity to strain BTV-28/1537/14.

Characterization of bluetongue virus serotype 28.

Bluetongue virus (Reoviridae; Orbivirus, BTV), which is usually transmitted by biting midges, affects wild and domestic ruminants worldwide, thereby causing an economically important disease. Recently, a putative new BTV strain was isolated from contaminated vaccine batches. In this study, we investigated the genomic and clinical characteristics of this isolate, provisionally designated BTV-28. Phylogenetic analysis of BTV-28 segment 2 (Seg-2) showed that it is related to Seg-2 from BTV serotypes 4, 10, 11, 17, 20 and 24, sharing 64%-66% identity in nucleotide sequences (nt) and 59%-62% in amino acid (aa) sequences of BTV VP2. BTV-28 Seg-6 is related to the newly reported XJ1407 BTV isolate, sharing 76.70% nt and 90.87% aa sequence identity. Seg-5 was most closely related to a South African BTV-4 strain, and all other segments showed close similarity to BTV-26. Experimental infection by injection of 6-month-old ewes caused clinical signs in all injected animals, lasting from 2 to 3 days to several weeks post-infection, including high body temperature, conjunctivitis, nasal discharge and rhinitis, facial oedema, oral hyperaemia, coronitis, cough, depression and tongue cyanosis. Naïve control animals, placed together with the infected sheep, displayed clinical signs and were positive for viral RNA, but their acute disease phase was shorter than that of BTV-injected ewes. Control animals that were kept in a separated pen did not display any clinical signs and were negative for viral RNA presence throughout the experiment. Seroconversion was observed in the injected and in one of the two contact-infected animals. These findings demonstrate that BTV-28 infection of sheep can result in clinical manifestation, and the clinical signs detected in the contact animals suggest that it might be directly transmitted between the mammalian hosts.

Emergence of a Novel Reassortant Strain of Bluetongue Serotype 6 in Israel, 2017: Clinical Manifestations of the Disease and Molecular Characterization.

Reassortment contributes to the evolution of RNA viruses with segmented genomes, including Bluetongue virus (BTV). Recently, co-circulation of natural and vaccine BTV variants in Europe, and their ensuing reassortment, were proposed to promote appearance of novel European BTV strains, with potential implications for pathogenicity, spread and vaccination policies. Similarly, the geographical features of the Mediterranean basin, which spans over portions of three continents, may facilitate the appearance of clinically relevant reassortants via co-circulation of BTV strains of African, Asian and European origins. In August-October 2017, BTV serotype 6 (BTV-6) was identified in young animals exhibiting classical clinical signs of Bluetongue (BT) at Israeli sheep and cattle farms. Sequencing and pairwise analysis of this Israeli BTV-6 isolate revealed the closest sequence homology of its serotype-defining Segment 2 was with that of South African reference BTV-6 strain 5011 (93.88% identity). In contrast, the other viral segments showed highest homology (97.0%-99.47% identity) with BTV-3, -4 and -9 of Mediterranean and African origins. Specifically, four viral segments were nearly identical (99.13%-99.47%), with Tunisian and Italian BTV-3 strains (TUN2016 and SAD2018, correspondingly). Together, our data suggest that Mediterranean co-circulation and reassortment of BTV-3 and BTV-6 drove the emergence of a novel and virulent BTV-6 strain.

Detection of Epizootic Hemorrhagic Disease Virus Serotype 1, Israel.

During September 2016-February 2017, we detected epizootic hemorrhagic disease virus (EHDV) in ruminants in Israel. BLAST and phylogenetic analyses of segment 2 in 6 EHDVs isolated from field samples indicated a close relationship to the EHDV serotype 1 strain in Nigeria. Affected cattle had mostly mild or asymptomatic disease.

Shuni virus in Israel: Neurological disease and fatalities in cattle.

The insect-transmitted Shuni virus (SHUV) belongs to the Simbu serogroup of orthobunyaviruses and it is known to induce abortions, stillbirths and severe congenital malformations in ruminants and may cause neurological signs in infected horses. Here, SHUV was detected in brain samples of two Israeli cattle, which suffered from severe neurological signs that led to the deaths of the animals. During histopathological examination of the first case, a 5-month-old calf, small perivascular cuffs, composed mainly of neutrophils with few lymphocytes were observed in the brain stem and cerebrum. Similar infiltrates were also found to a lesser extent in the cerebellar meninges leading to the diagnosis of acute-subacute meningoencephalitis. The histological examination of the brainstem from the second case, a 16-month-old heifer, revealed perivascular infiltration composed of equal numbers of macrophages and neutrophils associated with cerebral and meningeal haemorrhages. In this case encephalitis was diagnosed. Viral RNA was extracted from brain samples of both cattle that suffered from severe neurological signs and was subsequently tested by a polymerase chain reaction PCR assay specific for Simbu serogroup viruses and found positive. The presence of SHUV was subsequently confirmed by the isolation of the virus from one sample and sequence analysis of both brain samples. The comparison of the complete sequences of the coding regions of all three genome segments from both cases revealed a close relationship to Shuni viruses detected in tissue samples of aborted or malformed calves or lambs born during the last years in Israel.

Epizootic hemorrhagic disease virus serotype 6 outbreak in Israeli cattle in 2015.

In September 2015, a large outbreak caused by epizootic hemorrhagic disease virus (EHDV) was identified in Israeli dairy and beef farms. The main clinical signs were reduced milk production, weakness, drooling, lameness and recumbency, fever, slight erythema of nasal and oral mucosae, weight loss, and abortion. Dyspnea, cachexia, and death were observed less frequently. The clinical diagnosis was confirmed by ELISAs and EHDV-specific real-time reverse transcription PCR (RT-rtPCR), followed by conventional RT-PCR of the VP2 gene and sequence analysis. According to the sequence and phylogenetic analysis of theVP2 gene, the 2015 Israeli EHD outbreak was caused by EHDV-6, which was found not only in clinically ill cattle, but also in aborted fetuses.

Bluetongue virus serotype 24 (BTV-24) in Israel: phylogenetic characterization and clinical manifestation of the disease.

Bluetongue (BT), an arthropod-borne viral disease of ruminants, a ects sheep most severely than other domestic animals. Bluetongue virus serotype 24 (BTV-24) is one of 26 known Bluetongue virus (BTV) serotypes. In this article, we present data of phylogenetic analysis of 9 viral genes (Seg1, Seg2, Seg3, Seg4, Seg5, Seg6, Seg8, Seg9, and Seg10) from 8 Israeli BTV-24 isolates and relate the genotype of the BTV-24 isolates to their phenotype with regard to clinical manifestations. The high level of genetic identity (> 99.6%) between Seg2, Seg4 and Seg5 in all 8 BTV-24 isolates indicated that these segments shared the same viral ancestor. Phylogenetic analysis of Seg1, Seg3, Seg5, Seg8, Seg9, and Seg10 revealed that the Israeli BTV-24 strains comprised 4 variants. Five of the viruses revealed high identity among all 9 segments, and represented variant 1. A second variant (BTV24/3027/6/10), isolated in 2010, showed signi cant variation from variant 1 in 3 gene segments (VP-1, VP-3, and NS-3 genes). A third variant (BTV24/3027/1/10) showed signi cant variation from variant 1 in 6 segments (VP-1, VP-3, VP-6 and NS-1, NS-2 and NS-3 genes), while a fourth variant (BTV24/2214/1/10) showed signi cant variation from variant 1 in 4 segments (VP-1, NS-1, NS-2 and NS-3 genes). These marked di erences in sequence identity indicate that a high level of genetic reassortment is occurring between co-circulating BTV strains in Israel.

Unusual clinical manifestations in Israeli ruminant populations infected with Orbiviruses.

Orbiviruses, some of which are virulent in ruminant species, are transmitted by blood- sucking insects. They can cause the smallest blood vessels to leak, leading to oedema, which is presented as Bluetongue (BT) and/or Epizootic haemorrhagic diseases (EHD). Other clinical manifestations include big-muscle necrosis, excessive scialorrea, and coronitis. Pathology and laboratory testing can con rm the involvement of orbivirus. Bluetongue infection in naïve sheep can elicit the 'classical signs' of the disease and, therefore, can warn of Bluetongue virus' (BTV) attacks and of increased vector activity. In 2006, infection of cattle by serotype 7 of the Epizootic haemorrhagic disease virus (EHDV) was detected in Israel, with lesions clinically identical to those of BT disease in sheep. In 2006, serotype 15 of the BTV (BTV-15) was isolated in Israel from sheep with acute BT. In 2008 clinical BT in cattle was reported and con rmed in Israel. To date, additional serotypes (BTV-2, BTV-4, BTV-5, BTV-8, BTV-12, BTV-16, and BTV-24) have been reported, of these BTV-5, BTV-8, BTV-12, and BTV-24 were isolated for the rst time in the region. Some of these serotypes have been detected in animals with simultaneous double/triple infections with di erent BTV serotypes, so that reassortment may also occur during these simultaneous infections. The use of local strains for the development of inactivated or subunit vaccines would however help to ensure antigenic matching. Various changes in orbiviral diseases occurred between 2006 and 2013 in Israel, and similarities and di erences between Israel and Europe have been reported in this study.

Characterization of Shuni viruses detected in Israel.

Shuni virus (SHUV) was recently identified in Israel in several brains of ovine, bovine, and goat fetuses and newborn animals with congenital arthrogryposis-hydranencephaly syndrome. In the present study, the sequences of several Israeli SHUV strains were analyzed in detail; based on the small genome segment which encodes the nucleocapsid protein and the small nonstructural protein (NSs), a very high similarity of 99-100 % among each other was found. In contrast to the highly conserved N protein, several mutations were found within the NSs-coding sequence of SHUVs present in brain samples of malformed fetuses, resulting in a considerably frequent appearance of stop codons. Interferon alpha/beta production was demonstrated in an in-vitro interferon bioassay; hence, the virus isolated from the brain of a malformed sheep fetus acquired mutations, resulting in the loss of its NSs protein function.