1. Introduction
Bluetongue (BT) is an arthropode-born disease of domestic and wild ruminants caused by the Bluetongue virus (BTV) [
1,
2]. The agent and its associated diseases present wide global distribution [
2]. Indeed, BT is one of the major infectious diseases of ruminants and is listed as a notifiable disease by the World Organization for Animal Health (OIE). Transmission between mammalian hosts is mainly carried out by competent
Culicoides species [
3,
4,
5,
6]. In addition to
Culicoides-mediated transmission, a limited repertoire of BTV serotypes has recently been characterized as being able to undergo transplacental transmission, oral-based transmission or aerosol-contact based transmission [
7]. Clinical signs of BT are generally most severe in naïve sheep and white-tailed deer, where death is not an uncommon outcome [
1,
2]. Of note, testicular degeneration and azoospermia were also recently identified as clinical features of BTV infection of rams [
8], extending in this manner the range of putative damage which can be inflicted by BTV outbreaks. In contrast, BTV infection in cattle is usually asymptomatic, although it appears that some strains are more pathogenic and are able to induce a clinical disease. These cases are frequently characterized by a sharp reduction in milk production, depression, lethargy, pyrexia, lameness or stiff gait, serous/purulent nasal discharge, excessive salivation, facial edema, cyanosis/petechiae/erosions of the tongue and oral mucosa, and dyspnea [
1,
2,
9,
10,
11].
The genome of BTV (family Reoviridae, genus Orbivirus) comprises ten segments (Seg-1 to Seg-10) of double-stranded linear RNA and codes for seven virus-structural (VP1–VP7) and four non-structural (NS1, NS2, NS3/NS3a and NS4) proteins, while the existence of a fifth non-structural protein has also been suggested [
12]. Based on sequence analysis of outer protein VP2 (the most variable gene), twenty-seven distinct BTV serotypes have been recognized [
7] and several others have been suggested [
13,
14,
15,
16]. Phylogenetic analysis points out the relatedness of several of these serotypes, as in the case of BTV-6, which is closely related to BTV-14 and to BTV-21 [
17], while resembling to a lesser degree BTV-3, BTV-13 and BTV-16.
BTV-6 is widely distributed and has been identified in several continents, including the Americas (South and North America), the Caribbean, Africa and Asia [
18,
19,
20,
21,
22]. Notably, in 2008, a reassortant live vaccine strain of BTV-6 was observed to induce clinical symptoms in cows in the Netherlands [
23]. These observed cases support the notion of the pathogenic potential of BTV-6 in cattle, as well as serving as a cautionary tale of the putative influence of anthropogenic factors in the spread of BTV [
2]. Indeed, a similar event was observed in Israel, where isolation of BTV-6 from clinical samples between 1972 and 1989 was linked to iatrogenic dissemination of the live attenuated vaccine strain [
20].
Genetic recombination of RNA viruses with segmented genomes through reassortment involves the packaging, into a single virion, of genomic segments of different ancestry [
24]. In order for such genome mixing to occur, a given cell needs to be simultaneously infected with more than one virus. Notably, in spite of the very large number of different combinations that can be generated by random reassortment of multiple segments (e.g., the 10 genomic dsRNA segments of BTV), reassortment-related packaging of genome segments is frequently non-random, thus effectively limiting the repertoire of reassorted virions (reviewed in [
24]). Concerning BTV, recent studies support the following notions: (i) All 10 segments can undergo reassortment, which occurs readily upon co-cultivation in vitro and is also evidenced by genetic analysis of circulating BT strains [
25,
26,
27,
28]. (ii) Central characteristics of BTV virulence and/or interactions with the host cell (e.g., molecular preference during cell attachment, replication kinetics, ability to replicate in interferon-competent cells, mode and extent of induction of cell death) can be modulated through reassortment; underscoring the importance of reassortment for BTV evolution [
25,
26,
27]. (iii) Reassortment is non-random in terms of the packaging of viral segments. For example, six of 10 segments (Seg-1, Seg-3, Seg-4, Seg-5, Seg-8, Seg-9) exhibit a bias towards co-packaging, suggestive of evolutionary links (e.g., epistatic interactions stemming from biochemical or physical interactions of the gene products encoded in these segments) [
28].
In 2017, a severe BTV-6 outbreak was recorded throughout Israel. At the end of August 2017, BTV-6 simultaneously appeared in the northern and southern areas of Israel (Galilee and Southern Shfela districts, respectively) and quickly spread throughout the Golan Heights and the central areas of the country. Importantly, this outbreak was characterized by classical clinical manifestations of the disease and death in sheep and cattle.
The present paper describes the clinical outcomes of BTV-6 infection in Israeli sheep and cattle herds, along with the genetic characterization and phylogenetic analysis of this recently identified BTV-6 Israeli strain. Our results demonstrate the introduction of a new and genetically distinct BTV-6 strain in 2017. The novel genetic features of this novel strain, and their correlation with observed clinical symptoms, suggest an ever increasing significance of BTV-6 as a pathogen for domestic ruminants.
2. Materials and Methods
2.1. qRT-PCR Tests for Viruses Other Than BTV
During 2017, a febrile disease was observed in domestic and wild ruminants. A total of 214 cattle samples (212 whole blood EDTA samples, one spleen sample from a dead cow, and one spleen sample from aborted cattle fetus) were tested for bovine ephemeral fever virus (BEFV) by RT-qPCR [
29] (
Table 1). In addition, 226 field cattle samples (217 whole blood EDTA samples from ill cattle, six spleen samples from dead cattle and three aborted cattle fetuses (two spleen, one brain and one placenta samples)) and 8 samples from wild animals (spleen samples from five Nubian ibexes, one giraffe and two mountain gazelles; and one spleen sample from an aborted fetus of the fallow deer) were tested for hemorrhagic disease virus (EHDV) by qRT-PCR (
Table 1). Briefly, RNA (for EHDV tests) was extracted from whole blood samples and tissue using Invisorb Spin Virus RNA Mini Kit (STRATEC Molecular GmbH, Berlin, Germany). EHDV RNA presence was assessed with an Epizootic Hemorrhagic Disease Virus Real-Time PCR Kit (LSI VetMAX, Lissieu, France), as previously described [
30].
2.2. Pan-BTV qRT-PCR
RNA was extracted as previously described for EHDV [
30]. A total of 693 field samples were included in the present study. These originated from 402 cattle whole blood EDTA samples, 35 spleens and 7 aborted cattle fetuses (four spleen, one brain, one placenta and one mixed sample, which included brain, spleen, liver and lung), 173 whole blood sheep EDTA samples, 41 spleens from dead sheep and 10 aborted sheep fetuses (seven spleen, two lung, two brain, and two mixed samples, which included brain, spleen, liver and lung). Whole blood samples from wild animals were obtained from camels (14 samples), elephants (3 samples); spleen samples were attained from 4 mountain gazelles, 5 roe deer, 3 Persian fallow deer, 9 Nubian ibexes, 1 alpaca; 2 aborted Persian fallow deer fetuses, 1 adult giraffe and 1 aborted giraffe fetus (
Table 2). Initial assessment of BTV was performed by using VetMAX™ BTV NS3 All Genotypes Kit (Applied Biosystems™, Thermo Fisher Scientific Inc., France), as described by the manufacturer (referred to hereafter as Pan-BTV qRT-PCR).
2.3. BT Virus Isolation.
RT-qPCR BTV positive samples (total of 166,
Table 2) were inoculated onto 9–11 day old embryonated chicken eggs (ECE) by intravenous delivery, as previously described [
30,
31], with minor modifications. Briefly, tissue samples were prepared by the same method, which was described above, and supernatant from organ samples was filtrated using 0.45 µm filter (Starstedt, Germany) and stored at 4 °C until use. For isolating BTV from whole blood samples, red blood cells (RBC) were washed 3 times with PBS. Then, 100 µL of washed RBC were resuspended in 900 µL of double distillated water to induce hemolysis. A total of 100 µL from prepared samples were inoculated into each ECE (5 eggs per sample) and eggs were observed for 9 days. Dead ECE (between days 2–9), were homogenized. Supernatant from ECE homogenates was used for RNA extraction and was tested by pan-BTV RT-qPCR; positive in pan-BTV RT-qPCR ECE samples were subsequently passaged 3 or 4 times on baby hamster kidney cells (BHK-21) monolayers (until appearance of a cytopathic effect (CPE)).
2.4. BTV Serotype Identification
RNA from ECE homogenates was extracted using Invisorb Spin Virus RNA Mini Kit (STRATEC Molecular GmbH, Berlin, Germany) according to manufacturer’s instructions. ECE homogenates which were found positive by Pan-BTV RT-qPCR were further assessed by RT-PCR for the typing of serotypes 2, 3, 4, 5, 8, 12, 15, 16, 24 and 28 (all with in house developed primer pairs), which are known to be present in Israel and neighboring countries, using One-Step RT-PCR kit (Qiagen, Hilden, Germany). Identification of BTV-6 isolates was performed with the following pair of primers (developed in house): 6VP2-124F 5′- TGTAACCCAAATTCCCACGAA-3′ and 6VP2-1030R 5′-CAGAGGCGGCTATCATA-3′. Amplified fragments were subsequently sequenced.
2.5. Sequencing and Phylogenetic Analysis
Following three passages on BHK-21cells, the BTV strain (ISR-2095/3/17) was sequenced by NGS at Hy Laboratories Ltd., Rehovot, Israel. Sequence gaps were filled following the performance of conventional RT-PCR, employing in house designed primer-pairs. cDNA fragments were purified using MEGAquick-spin™ Total Fragment DNA Purification Kit (iNtRON Biotechnology, Gyeonggi-do, South Korea) and standard Sanger sequencing was performed on ABI 3730xl DNA Analyzer (Hy Laboratories Ltd., Rehovot, Israel).
Nucleotide sequences were assembled and nucleotide (nt) and amino acid (aa) sequences were aligned and pairwise compared using Geneious (version 9.0.5; Biomatters, Auckland, New Zealand). Phylogenetic trees were constructed using Mega 7.1 [
32].
The ten segment codding regions of Israeli BTV-6 ISR-2095/17 isolate were mostly sequenced (Seg-1-positions 1-3934/3944; Seg-2- 17-2892/2922; Seg-3- 21-2738/2772; Seg-4- 17-1967/1981; Seg-5-2-1688/1772; Seg-6-2-1634/1637; Seg-7- 20-1127/1156; Seg-8- 2-1080/1125; Seg-9-13-1035/1067; Seg-10- 1-800/822) and submitted to NCBI GenBank (MH383089-MH383098). Seg-2 of all other BTV-6 isolated in 2017 were sequenced partially (MH791296-MH791313). Several segments (Seg-1, -3, -5, -6, -7 and -10) of some other BTV-6 isolates were also partially sequenced (MH426705-MH426723).
4. Discussion
Israel has a long history of presence and effect of BTV on livestock, beginning with the first BT clinical detections in the late 1940s [
20]. In particular, BTV-6 was identified in Israel between 1972 and 1989 [
20]. Unfortunately, prior to the present study, there was a lack of information about the clinical manifestation of the disease in local Israeli sheep caused by BTV-6. Such a lack of annotated information prevents a systematic comparison of our current findings on the clinical features of BTV-6 in Israeli livestock, with previous documented cases of BTV-6 in Israel. However, a comparison of our data with those obtained from BTV-6 outbreaks in Europe in 2008 reveals considerable differences. There, clinical signs of BT in cows in Netherlands were limited (e.g., to coronitis), a fact which correlated with PCR-based measurements suggestive of very low spread or a very short viremia. This is in sharp contrast to the recent Israeli BTV-6 outbreak, which quickly spread through Israel causing heavy BT clinical manifestations both in sheep and in cattle. We propose that the basis of such a difference stems from the origin and genetic composition of these different viruses, where the European outbreak was most probably derived from a live attenuated South African vaccine strain, which is considered non-virulent, while the Israeli outbreak was caused by the new BTV-6 strain that we describe here [
15,
16]. A comparison of clinical signs caused BTV-6 with other BTV serotypes circulated in Israeli livestock in 2017 revealed that the most prominent clinical signs both in cattle and in sheep were hyperemia and edema, mostly seen in young animals. BTV-2 and BTV-4 usually cause clinical signs in Israeli cattle (adult milk producing cows) and in the susceptible population of sheep. This is in contrast to BTV-6, where clinical signs in cattle were mostly observed in young animals. According to our analysis, a proposed major genetic source of the Israeli BTV-6 is BTV-3. Within this context, multiple recent studies have identified BTV-3 strains in the Mediterranean Basin [
14,
33,
34,
35]. Notably, the reported clinical BT manifestations of BTV-3 were in sheep only, a markedly different pattern than what was observed for the Israeli BTV-6, as our data support the notion that it affects both sheep and cattle. While serving as a basis for detection and characterization of a new BTV-6 strain in Israel, the present study is most certainly an underestimation of the full extent of Israeli distribution of BT in general, and BTV-6 in particular. This is due to the fact that it is based on passive investigation, using only diagnostic samples collected by veterinary doctors from ill animals (usually a small number of samples from affected animals in each farm,
Table 4). Furthermore, the attribution of the clinical phenomena to BTV-6 is also limited by the possibility that the clinical state of the animals may have also reflected other viral and bacterial infections, which could cause pneumonia in fattening calves. Moreover, mixed BTV-3 and -6 infection were registered in at least two out of six BT-affected sheep flocks, raising the possibility that BTV-3 may have also contributed to the observed clinical symptoms.
Genetic analysis of single nucleotide mutation rates and the rate of reassortment (i.e., genetic drift and genetic shift), suggests that the latter is likely to be a major driver of genotypic and phenotypic change in BTV [
28]. In this context, the unique features of Israeli BTV-6, such as the magnitude of its spread and/or its ability to induce clinical signs in both sheep and cattle, may reflect its reassortant origin, where a backbone of segments from currently circulating Mediterranean BTV-3 (Seg-1, 4, 5, 6, 7 and -9) was supplemented by contributions from BTV-6 (Seg-2), BTV-4 (Seg-3), BTV-9 (Seg-8) and an untyped BTV isolate (Seg-10). Notably, Seg-2 and Seg-10, which encode for VP2 and NS3 and have been proposed as determinants of virulence [
25], were not of BTV-3 origin. Concerning the geographical origin of the proposed contributing BTV strains, we propose that co-circulation in Africa played a predominant role in generating Israeli BTV-6. This proposal also includes the segments presenting highest similitude with Italian BTV-3 isolates, as these have also been proposed to be of North African origin [
33]. The notion that BTV distribution in Israel is heavily influenced by strains that circulate in North Africa is further supported by our unpublished results, which show that BTV-3 strains identified in Israel between 2013-2016 (ISR-2019/13, ISR-2153/16 and ISR-2262/2/16) also exhibit high percentages of genetic identity by some of genes with strains originating from Tunisia (
Figure 2b, and
Figures S2, S3 and S5). In summary, following the observation in the field of farm animals presenting BT-like symptoms, we have isolated and genetically characterized a new reassortant BTV-6 strain. This strain exemplifies how novel disease-causing abilities may stem from co-circulation and reassortment of BTV strains.