Sahar Ahmed

Mohamed Abd

El-Fatah Mahmoud

Cyril Viarouge

Corinne Sailleau

Stephan Zientara

Emmanuel Breard
email: emmanuel.breard@anses.fr

PRESENCE OF BLUETONGUE AND EPIZOOTIC HEMORRHAGIC DISEASE VIRUSES IN EGYPT IN 2016 AND 2017

Keywords: Bluetongue virus, EHDV: Epizootic hemorrhagic disease virus, EHDV-1: EHDV serotype 1, BTV BTV, EHDV, Egypt, ELISA, genome detection

BTV and EHDV are closely-related orbiviruses that are transmitted between domestic and wild ruminants via the bites of hematophagous midges. Previous studies have reported seropositivity against BTV antibodies in sheep and goats in two Egyptian governorates (Beni Suef and Menoufia).

However, no recent data are available on the BTV serotype(s) circulating in Egypt and the likely presence of EHDV has never been explored. This study investigated the presence of BTV and EHDV among cattle which had been found BTV-seropositive by ELISA method. These cattle living in proximity to sheep and goats previously found BTV-seropositive. These cattle displayed no clinical signs of BT but reproductive problems had been reported in herds. A total of 227 cattle blood samples were therefore collected in 2016 and 2017. Ninety-four of the 227 animals tested by a BTV ELISA were positive for BTV antibodies (41.4%). Of these 94 ELISA-positive cattle, only 83 EDTA-blood samples were available and therefore tested for BTV and EHDV genome detection by RT-PCR and sequencing.

Of the cattle sampled in 2016, results revealed that two were RT-PCR-positive for BTV and seven for EHDV. Sequencing showed the presence of EHDV-1 and BTV-3 genome sequences. EHDV-1 S2 shared 99.5% homology with an EHDV-1 S2 from a strain isolated in 2016 in Israel. BTV-3 S2 and S8 sequences shared more than 99.8% nucleotide similarity with the BTV-3 Zarzis S2 and S8 sequences (Tunisian BTV, also detected in 2016). Of the 66 blood samples tested following their collection in 2017, they were all EHDV-negative by RT-qPCR while five were BTV-positive by RT-qPCR.

However, attempts to identify the BTV serotype of these five samples were unsuccessful. Only part of BTV S8 was sequenced and it showed 79% nucleotide similarity with S8 of atypical BTV serotypes (particularly with BTV-26 and another BTV serotype strain isolated from a sheep pox vaccine).

Introduction

Bluetongue (BT) and Epizootic hemorrhagic disease (EHD) are vector-borne viral diseases affecting domestic and wild ruminants, notifiable under OIE rules. BT and EHD viruses are species of the genus Orbivirus, with structural, antigenic and molecular similarities. Their genomes are composed of ten segments (S1 to S10) of double-stranded RNA which encode seven structural proteins (VP) and five nonstructural proteins (NS) [START_REF] Ratinier | Identification and characterization of a novel non-structural protein of bluetongue virus[END_REF]. VP2 forms the outer capsid, and is therefore a target of neutralizing antibodies, as well as determining the virus serotype.

Both viruses induce variable clinical signs that mainly depend on the strain and the affected ruminant species (or breed). Both diseases have been reported to involve seasonal circulation and transmission by hematophagous midges (Culicoides) [START_REF] Anthony | Genetic and phylogenetic analysis of the outer-coat proteins VP2 and VP5 of epizootic haemorrhagic disease virus (EHDV): comparison of genetic and serological data to characterise the EHDV serogroup[END_REF][START_REF] Maclachlan | The pathology and pathogenesis of bluetongue[END_REF].

Twenty-four traditional BTV serotypes in addition to novel and atypical serotypes have been identified to date [START_REF] Bumbarov | Detection and isolation of Bluetongue virus from commercial vaccine batches[END_REF][START_REF] Chaignat | Toggenburg Orbivirus, a new bluetongue virus: initial detection, first observations in field and experimental infection of goats and sheep[END_REF][START_REF] Lorusso | Analysis of bluetongue serotype 3 spread in Tunisia and discovery of a novel strain related to the bluetongue virus isolated from a commercial sheep pox vaccine[END_REF][START_REF] Maan | Novel bluetongue virus serotype from Kuwait[END_REF][START_REF] Marcacci | One after the other: A novel Bluetongue virus strain related to Toggenburg virus detected in the Piedmont region (North-western Italy), extends the panel of novel atypical BTV strains[END_REF][START_REF] Savini | Novel putative Bluetongue virus in healthy goats from Sardinia, Italy[END_REF][START_REF] Sun | Emergence of a Novel Bluetongue Virus Serotype, China 2014[END_REF][START_REF] Zientara | Novel bluetongue virus in goats, Corsica, France[END_REF]. Since 2009, there has been a consensus recognizing seven EHDV serotypes (1, 2, and 4 to 8) [START_REF] Anthony | Genetic and phylogenetic analysis of the outer-coat proteins VP2 and VP5 of epizootic haemorrhagic disease virus (EHDV): comparison of genetic and serological data to characterise the EHDV serogroup[END_REF].

The global distribution of BT and EHD has recently expanded, possibly due to a variety of factors including increased global commerce and climate change [START_REF] Guis | Modelling the effects of past and future climate on the risk of bluetongue emergence in Europe[END_REF][START_REF] Maclachlan | The pathology and pathogenesis of bluetongue[END_REF][START_REF] Wilson | Bluetongue in Europe: past, present and future[END_REF]. In the last 20 years, various BTV serotypes have been reported in Europe and other countries bordering the Mediterranean basin, whereas EHDV has sporadically been detected in countries in the south or east of the Mediterranean basin (the Maghreb, Israel, Turkey, and Jordan) [START_REF] Ben Dhaou | Molecular characterisation of epizootic haemorrhagic disease virus associated with a Tunisian outbreak among cattle in 2006[END_REF][START_REF] Temizel | Epizootic hemorrhagic disease in cattle, Western Turkey[END_REF].

From 1998 to 2005, at least seven BTV incursions (involving five BTV serotypes (serotypes 1, 2, 4, 9 and 16)) spread across the Mediterranean basin (the Maghreb, Mediterranean islands and Southern Europe), inducing clinical signs in sheep and goats. These BTV strains entered the Mediterranean Basin and Europe through two main corridors: eastern via Turkey and Greece and western via North Africa [START_REF] Lorusso | Molecular epidemiology of bluetongue virus serotype 1 circulating in Italy and its connection with northern Africa[END_REF]. In this last case, BTV first spread through North Africa, then across multiple Mediterranean islands and finally, across southern European countries (Spain, Italy and/or France).

In 2006, a BTV-8 strain emerged in Northern Europe and rapidly infected naive ruminants across northern and eastern European countries. It was not only virulent in sheep but also, unexpectedly, in cattle and its origin is still unknown. Experimental and field studies conducted on a large number of abortions reported transplacental transmission of BTV-8 in cattle [START_REF] Zanella | Bluetongue virus serotype 8: abortion and transplacental transmission in cattle in the Burgundy region, France, 2008-2009[END_REF]. After compulsory nationwide vaccination campaigns, Europe became free of BTV-8 in 2012. However, the same BTV-8 strain re-emerged in 2015 in France [START_REF] Breard | Complete Genome Sequence of Bluetongue Virus Serotype 8, Which Reemerged in France in August 2015[END_REF], has since spread and is now (in 2019) present in mainland France, Switzerland and eastern Germany. A BTV-4 strain from Eastern Europe (detected in Greece in 2014) moved westwards (through Balkan countries) and has been found in Italy and mainland France in 2019 [START_REF] Sailleau | Emergence of bluetongue virus serotype 4 in mainland France in November 2017[END_REF].

In 2018, an exotic serotype (BTV-3) was detected in sheep in Sardinia. This strain was shown to be almost identical (>99% nucleotide identity) across all segments to the BTV-3 strain isolated in 2016 in Tunisia [START_REF] Cappai | Western Bluetongue serotype 3 in Sardinia, diagnosis and characterization[END_REF]. This new BTV-serotype incursion in the Mediterranean Basin, spreading northwards, bears resemblance with the distinctive corridor where BTV-1, -2 and -4 strains had previously been observed spreading from Africa into Europe [START_REF] Lorusso | Bluetongue virus serotypes 1 and 4 in Sardinia during autumn 2012: new incursions or re-infection with old strains?[END_REF]. Indeed, serotype 3 constitutes a possible future threat for Southern Europe.

Common to each Orbivirus is the variability of clinical outcomes after infection. In many cases, BTV and EHDV induce mild or unapparent clinical infections, whereas in others they induce fever, depression, respiratory distress and anorexia [START_REF] Maclachlan | Experimental reproduction of severe bluetongue in sheep[END_REF][START_REF] Maclachlan | The pathology and pathogenesis of bluetongue[END_REF][START_REF] Savini | Novel putative Bluetongue virus in healthy goats from Sardinia, Italy[END_REF]. In the case of BTV, sheep are the most sensitive species. Although goats and cattle are fully susceptible to BTV infection, they do not generally show signs of disease; it remains mild or subclinical. Cattle show longer periods of BTV viremia than sheep and are considered reservoirs of the virus. Experimental and field studies have reported clinical signs of BT in cattle and goats, particularly when outbreaks have occurred in non-endemic areas [START_REF] Maclachlan | Bluetongue: pathogenesis and duration of viraemia[END_REF]. Historically, EHDV has been associated with disease in wild cervids [START_REF] Jessup | Epidemiology of two orbiviruses in California's native wild ruminants: preliminary report[END_REF][START_REF] Odiawa | Bluetongue and epizootic hemorrhagic disease in ruminants in Georgia: survey by serotest and virologic isolation[END_REF]. Severe clinical signs induced by EHDV have also been observed in infected cattle [START_REF] Maclachlan | Epizootic haemorrhagic disease[END_REF][START_REF] Omori | Ibaraki virus, an agent of epizootic disease of cattle resembling bluetongue. I. Epidemiologic, clinical and pathologic observations and experimental transmission to calves[END_REF]. EHDV-6 or -7 has sporadically spread in countries surrounding the Mediterranean Basin and has induced clinical forms in cattle [START_REF] Ben Dhaou | Molecular characterisation of epizootic haemorrhagic disease virus associated with a Tunisian outbreak among cattle in 2006[END_REF][START_REF] Temizel | Epizootic hemorrhagic disease in cattle, Western Turkey[END_REF][START_REF] Yadin | Epizootic haemorrhagic disease virus type 7 infection in cattle in Israel[END_REF]. More precisely, in 2006, outbreaks of EHDV-6 were reported in cattle from Turkey, Morocco, Algeria, Tunisia and Jordan when an EHDV-7 strain was involved in outbreaks in Israel. The more recent outbreaks of EHD in the Mediterranean Basin were recognized clinically in 2015 In Israel involving an EHDV-6 strain [START_REF] Golender | Epizootic hemorrhagic disease virus serotype 6 outbreak in Israeli cattle in 2015[END_REF]. The introduction origins of these EHDV-6 and 7 strains detected in Mediterranean countries since 2006 are still unknown, however, analyses of their genome segments indicate a common "African/Arabian Peninsula and Indian Ocean Asia" origin [START_REF] Ben Dhaou | Molecular characterisation of epizootic haemorrhagic disease virus associated with a Tunisian outbreak among cattle in 2006[END_REF][START_REF] Golender | Epizootic hemorrhagic disease virus serotype 6 outbreak in Israeli cattle in 2015[END_REF].

Studies concerning the presence of orbiviruses have been conducted in various countries neighboring Egypt and have confirmed the endemic nature of BT and EHD in these areas [START_REF] Mejri | Epizootic haemorrhagic disease virus circulation in Tunisia[END_REF][START_REF] Sghaier | A novel Bluetongue virus serotype 3 strain in Tunisia, November 2016[END_REF]. In Egypt, several serological studies reported BTV seroprevalence [START_REF] Mahmoud | seroprevalence of bluetongue in sheep and goats in Egypt[END_REF][START_REF] Mahmoud | Serological investigation of some important RNA viruses affecting sheep and goats in Giza and Beni-Suef governorates in Egypt[END_REF]. In 1977, a BTV-4 strain from Egypt was isolated by the Pirbright Institute (ReoID). In 1987, BTV serotypes 1, 4, 10, 12 and 16 were identified through seroneutralization assays in retrospective studies from Egyptian sera [START_REF] Ismail | Bluetongue neutralization test with different virus under variable conditions[END_REF].

Recent investigations carried out on sheep and goats have revealed the presence of antibodies against BTV in these domestic species in two Egyptian governorates (Beni Suef and Menoufia) [START_REF] Mahmoud | Serological investigation of some important RNA viruses affecting sheep and goats in Giza and Beni-Suef governorates in Egypt[END_REF]. Egyptian veterinary authorities wanted to determine whether BTV could have been involved in the reproductive problems reported in 2016 and 2017 in several herds located in these two Egyptian governorates. This study reports the results of the molecular detection of BTV and EHDV that was carried out on cattle samples collected in this context.

Material and methods

Animals and samples collected

A total of 227 cattle blood samples were collected from two herds in Beni Suef (72 animals sampled in 2016) and three herds in Menoufia (155 animals sampled in 2017). The animals were apparently healthy, but reproductive problems had been reported in these herds. Serum and EDTAblood samples were stored in the National Research Centre (NRC) (Egypt) at -20°C until use.

ELISA

The ID Screen Bluetongue Competition ELISA kit (IDVet, Grabels, France) was used as per the manufacturer's instructions to initially detect BTV VP7 antibodies in all 227 serum samples. Eightythree EDTA-blood samples from ELISA-positive cattle were then used for the molecular diagnosis of BTV and EHDV.

RNA extraction

A first batch of 17 RNA extracts came from 17 EDTA-blood samples collected in 2016 in Beni Suef.

This RNA was purified in Egypt by the National Research Centre (NRC) using the TRIzol method. Two hundred and fifty µl of EDTA-blood was extracted using TRIzol LS Reagent as per the manufacturer's instructions. Total RNA was eluted into 30 µl of RNase-free water. The extraction products were then sent to ANSES's Laboratory for Animal Health (Maisons-Alfort, France) for RT-PCR assays and sequencing.

The second batch consisted of 66 EDTA-blood samples (from ELISA-tested cattle found positive for BTV) sent to ANSES's Laboratory for Animal Health in order for RNA extraction to be carried out using the MagVet Universal Isolation kit (Thermo Fisher Scientific, Lissieu, France). Total RNA was then extracted from 100 μl of EDTA-blood using an automated method (KingFisher Flex automated extraction platform (ThermoFisher Scientific) and the MagVet Universal nucleic acid extraction kit (Thermo Fisher)). Total RNA was eluted into 80 μl of RNase-free water.

RT-PCRs

Five µl of denatured RNA (heated at 95°C for 3 minutes) was then tested using each RT-PCR method (real-time RT-PCR (RT-qPCR) or conventional RT-PCR). Commercial RT-qPCR kits were used for pan-BTV detection (Adiavet BTV kit (Bio-X Diagnostics, Saint Brieuc, France)) and for detecting BTV-1, -2, -4, -8, -9 or -16 (serotypes currently found in Europe) (VetMAX European BTV Typing Kit), as per the manufacturer's instructions. An in-house BTV-3 RT-qPCR [START_REF] Lorusso | Analysis of bluetongue serotype 3 spread in Tunisia and discovery of a novel strain related to the bluetongue virus isolated from a commercial sheep pox vaccine[END_REF] was also used.

In-house RT-qPCRs were used to specifically detect the EHDV genome; these RT-qPCRs were EHDVgroup or -serotype specific [START_REF] Viarouge | Duplex Real-Time RT-PCR Assays for the Detection and Typing of Epizootic Haemorrhagic Disease Virus[END_REF].

For each of the in-house RT-qPCRs targeting EHDV or BTV segments, different primers and probe sets were designed in order to amplify part of the targeted gene using the AgPath One-Step RT-qPCR Kit (Life Technologies). Amplifications were carried out using the same cycling parameters for all RT-qPCR assays: 45°C for 10 min, 95°C for 10 min, followed by 40 cycles of 95°C for 15 s then 60°C for 1 min. These same cycling parameters were also applied when using commercial RT-qPCR kits: in-house RT-qPCR methods have been developed to be used together, in a single amplification run that could incorporate various commercial RT-qPCR mixes. Conventional RT-PCRs for BTV S2, S8, S9 and S10 sequence amplifications [START_REF] Breard | Molecular epidemiology of bluetongue virus serotype 4 isolated in the Mediterranean Basin between 1979 and 2004[END_REF][START_REF] Sailleau | Co-circulation of bluetongue and epizootic haemorrhagic disease viruses in cattle in Reunion Island[END_REF][START_REF] Viarouge | Identification of bluetongue virus and epizootic hemorrhagic disease virus serotypes in French Guiana in 2011 and 2012[END_REF] were also performed using the Qiagen OneStep RT-PCR kit (Qiagen, France). The amplification products were directly sequenced, in both directions, according to the Sanger method by Eurofins.

Phylogenetic analyses

Sequences were assembled and analyzed using EditSeq, SeqMan and Megalign Software (DNA Star Inc.) and aligned using ClustalW (DNAstar programs, Lasergene). The EditSeq program was used to split the sequence into individual files, SeqMan was used to assemble the reads of each sequence into contigs, and the Megalign program was then used to calculate sequence relationships, compare phylogenetic associations among BTV strains and construct phylogenetic trees based on the nucleotide sequences. The Megalign program uses the Neighbor-Joining method based on a matrix of "distances" between all sequences. The phylogenetic trees generated by Megalign are rooted trees, regardless of the method used for alignment.

Isolation assays

Isolation assays were carried out (at NRC and ANSES) on embryonated eggs (Clavijo et al., 2000) and KC cells (Culicoides sonorensis cell line) [START_REF] Wechsler | Cell lines from Culicoides variipennis (Diptera: Ceratopogonidae) support replication of bluetongue virus[END_REF]) using blood samples found positive for BTV or EHDV by RT-qPCR. The blood samples were inoculated onto KC cells. Briefly, a confluent monolayer of KC cells was inoculated with EDTA-blood samples diluted 10 -1 in sterile PBS. The inoculum was removed 24 hours after inoculation and the cells were incubated with an appropriate cell culture medium (Schneider's Drosophila Media (ThermoFisher Scientific) and 10% of foetal calf serum). After 7 days of incubation at 28°C, KC cell culture supernatants were tested for the presence of BTV or EHDV RNA by RT-qPCR.

In parallel, groups of three embryonated chicken eggs were each inoculated intravenously with 0.1-0.2 ml of a 10 -1 dilution of lysed blood samples. The eggs were incubated for 7 days at 35°C and examined daily using a cold candling lamp. The embryos that died within 24 h were discarded. After 1 week of incubation, the embryos were weighed and homogenized in Minimum Essential Medium (weight/volume: 1 / 10). Total RNA was then extracted from 100 µl of embryo lysate and tested by EHDV or BTV RT-qPCR.

Results and discussion

Serological analysis by ELISA revealed that 41.4% (94/227) of blood samples tested using the VP7 competitive ELISA were positive for BTV antibodies (Table 1). In each governorate, the percentage of cattle found positive for BTV by ELISA was similar (Table 1).

The BTV genome was looked for only in these ELISA-positive cattle. This limit was imposed due to findings from previous studies: when serum and EDTA-blood from BTV-infected animals (in the field) were tested, the animals that tested positive for BTV by RT-qPCR were mainly also found positive for BTV by the ELISA method [START_REF] Breard | Bluetongue virus in the French Island of Reunion[END_REF][START_REF] Verdezoto | Novel serotype of bluetongue virus in South America and first report of epizootic haemorrhagic disease virus in Ecuador[END_REF][START_REF] Viarouge | Identification of bluetongue virus and epizootic hemorrhagic disease virus serotypes in French Guiana in 2011 and 2012[END_REF]). An ELISA (commercial kit) is easy to perform in any laboratory and in our studies enables noninfected herds to be set aside in order not to undergo molecular analyses. Furthermore, when samples come from potentially endemic areas of BTV (Ecuador, Galapagos Islands and French Guiana in South America, Reunion Island in the Indian Ocean, and Tunisia in Africa), we systematically look for the EHDV genome or its antibodies [START_REF] Ben Dhaou | Molecular characterisation of epizootic haemorrhagic disease virus associated with a Tunisian outbreak among cattle in 2006[END_REF][START_REF] Breard | Outbreak of epizootic haemorrhagic disease on the island of Reunion[END_REF][START_REF] Breard | Bluetongue virus in the French Island of Reunion[END_REF][START_REF] Sailleau | Co-circulation of bluetongue and epizootic haemorrhagic disease viruses in cattle in Reunion Island[END_REF][START_REF] Verdezoto | Novel serotype of bluetongue virus in South America and first report of epizootic haemorrhagic disease virus in Ecuador[END_REF][START_REF] Vinueza | Bluetongue virus and epizootic hemorrhagic disease virus survey in cattle of the Galapagos Islands[END_REF]. In all these studies, EHDV was always present in areas where BTV was also found. It is also common to find animals co-infected by both viruses.

Eighty-three EDTA-blood samples from these 94 ELISA-positive cattle were available and tested for BTV and EHDV genome detection by RT-qPCR. Of the first 17 RNA extracts sampled in 2016 and sent to ANSES, two head of cattle (C19 and C44) were positive by BTV group-specific RT-qPCR (Table 2). Both of these samples were also found to be positive by RT-qPCR specific to BTV-3 and negative by the RT-qPCR that amplified BTV-1, -2, -4, -8, -9, and -16. S2 (VP2) and S8 (NS2) sequences (754 bp (GenBank Accession no. MH706764) and 942 bp (GenBank Accession no. MH706766) respectively) were obtained by conventional RT-PCR from C44 RNA, showing 99.86% and 99.89% homology with the Tunisian BTV-3 strain (Zarzis) isolated in 2016 [START_REF] Sghaier | A novel Bluetongue virus serotype 3 strain in Tunisia, November 2016[END_REF]. A phylogenetic tree (Figure 1) illustrates the homology between Egyptian BTV-3 S2 and the S2 from BTV-3 strains isolated across the world. These results demonstrate that the BTV-3 variant detected in Tunisia in 2016 was also present in Egypt that same year and supports the hypothesis that BTV-3 Zarzis had moved westwards after originating from North-East Africa.

A particular surprise was to detect a specific EHDV genome in seven cattle blood samples collected in 2016. EHDV-1-specific RT-qPCR [START_REF] Viarouge | Duplex Real-Time RT-PCR Assays for the Detection and Typing of Epizootic Haemorrhagic Disease Virus[END_REF] allowed the EHDV serotype to be determined for six of the seven pan-EHDV-positive cattle (Table 2). Only the sample with a Ct of 34.6

was not positive by EHDV-1 RT-qPCR, no doubt due to its low viral load. The tree is based on a single sequence sample (longer sequences were obtained with RNA from C17). The serotype was confirmed using conventional RT-PCR in three out of the seven EHDV-positive cattle and sequencing results demonstrated that the amplification products (1839 bp, GenBank Accession no. MH706767) shared 99.5% homology with S2 of an EHDV-1 strain isolated in 2016 in Israel (GenBank Accession no. MG808409), 95.86% homology with an EHDV-1 S2 isolated in Nigeria in 1967 and less than 87% with other known EHDV-1 S2 sequences available in GenBank (Figure 2). One S2 sequence was obtained from C20 and C21 (about 800 bp) with 100% homology with sequence S2 from C17. To our knowledge, this study is the first report of EHDV-1 in North Africa. EHDV-6 and 7 have previously been found in the Maghreb, Turkey and Israel [START_REF] Ben Dhaou | Molecular characterisation of epizootic haemorrhagic disease virus associated with a Tunisian outbreak among cattle in 2006[END_REF][START_REF] Temizel | Epizootic hemorrhagic disease in cattle, Western Turkey[END_REF][START_REF] Yadin | Epizootic haemorrhagic disease virus type 7 infection in cattle in Israel[END_REF] whereas EHDV has never yet been reported on any Mediterranean islands or in Europe.

Out of the 66 blood samples collected in 2017 in Menoufia and extracted in ANSES's laboratory, five were found positive for BTV by RT-qPCR and all were negative for EHDV by RT-qPCR (Tables 1 and2). No amplification product was obtained when either classical or serotype-specific RT-qPCR targeted BTV S2. In this study, the authors tried to determine the BTV serotype(s) by RT-qPCR or conventional RT-PCR. Finding no success for some samples, we subsequently tried to amplify and sequence BTV segments other than S2 (easier to amplify because they have more conserved nucleotide sequences than S2) [START_REF] Breard | Bluetongue virus in the French Island of Reunion[END_REF][START_REF] Sailleau | Co-circulation of bluetongue and epizootic haemorrhagic disease viruses in cattle in Reunion Island[END_REF]. The objective was then to confirm, using a BTV genome sequence, the positive results obtained for the group by pan-BTV RT-qPCR (targeting S10). Indeed, we tried to amplify S8, S9, and S10 , but only segment 8 amplification worked for one out of the five BTV-positive animals sampled in 2017 (914 bp, GenBank Accession no. MH706765). The S8 sequence showed 79.86% homology with S8 of a BTV strain isolated from a sheep pox vaccine [START_REF] Bumbarov | Detection and isolation of Bluetongue virus from commercial vaccine batches[END_REF] and 78.99% with BTV-26 [START_REF] Maan | Novel bluetongue virus serotype from Kuwait[END_REF] (data not shown). These five RT-qPCR-positive blood samples obtained in 2017 were also negative with BTV-3 RT-qPCR. Considered as a whole, the BTV RT-PCR results suggest that other BTV serotypes were able to circulate at least in the Menoufia governorate in 2017, and that one of these BTV serotypes was atypical, like many other similar BTV serotypes recently discovered in the Mediterranean Basin and Europe [START_REF] Chaignat | Toggenburg Orbivirus, a new bluetongue virus: initial detection, first observations in field and experimental infection of goats and sheep[END_REF][START_REF] Lorusso | Analysis of bluetongue serotype 3 spread in Tunisia and discovery of a novel strain related to the bluetongue virus isolated from a commercial sheep pox vaccine[END_REF][START_REF] Schulz | Bluetongue virus serotype 27: detection and characterization of two novel variants in Corsica, France[END_REF][START_REF] Sghaier | A novel Bluetongue virus serotype 3 strain in Tunisia, November 2016[END_REF].

Unfortunately, no virus was isolated from EHDV-or BTV-positive blood samples, most likely due to their pre-analysis storage at -20°C or the fact that animals were sampled after the viremia period, when the virus had been neutralized but viral RNA was still present in the blood and detectable by RT-PCR. Isolating EHDV or BTV strains in most cases allowed us to obtain full EHDV or BTV genomes by the NGS method [START_REF] Breard | Complete Genome Sequence of Bluetongue Virus Serotype 8, Which Reemerged in France in August 2015[END_REF][START_REF] Schulz | Bluetongue virus serotype 27: detection and characterization of two novel variants in Corsica, France[END_REF][START_REF] Verdezoto | Novel serotype of bluetongue virus in South America and first report of epizootic haemorrhagic disease virus in Ecuador[END_REF].

Obtaining the full genome for every strain would have allowed us to verify that the Egyptian BTV-3, which has the same S2 and 8 as the Tunisian Zarzis strain, was not a reassortant virus for the other eight RNA segments.

Conclusions

This study showed that BTV-3 (Zarzis strain) and EHDV-1 were present in Egypt in 2016 in the same period during which BTV-3 was detected in Tunisia and EHDV-1 in Israel. These data confirm that Egypt constitutes an area of particular interest for investigating BTV and EHDV epidemiology in the Mediterranean Basin. Additional studies are necessary in order to estimate BTV and EHDV seroprevalence in this part of Africa, and to isolate BTV and EHDV strains so as to characterize the full genome of these orbiviruses present in the Mediterranean basin. Further collaboration must be

Figure 1 :

 1 Figure 1: Phylogenetic tree of full-length segment 2, showing the relationships between EHDV-1 2016

Figure 2 :

 2 Figure 2: Phylogenetic tree of full-length segment 2, showing the relationships between BTV-3 Egypt

  Figure 1

Table 1 :

 1 : only NS2 sequence obtained. ELISA and PCR results from 277 Egyptian cattle sampled in 2016 and 2017 in two Egyptian governorates.

	Table 1
					BTV ELISA				PCR
	year of sampling	governorate	number of cattle tested	positive negative	% of positive cattle	number of ELISA-positive cattle tested	BTV-positive	EHDV-positive
	2016	Beni Suef	72	28	44	38.9	17		2	4
	2017	Menoufia	155	66	89	42.6	66		5	0
		Total	227	94	133	41.4	83		7	4
	Table 2
			Date of	Animal	Ct value	Ct value
	Location	sampling	n°	with pan-	with pan-		Serotype
						BTV PCR	EHDV PCR
			13/10/2016	C44	19.1	-		BTV-3 a b
					C13	-		30		EHDV-1 a
					C17	-		26.7		EHDV-1 a b
					C19	31.6	-		BTV-3 a
	Beni Suef			C20	-		26.8		EHDV-1 a b
			6/11/2016	C21	-		26		EHDV-1 a b
					C7	-		34.6	undetermined
					C1	-		29.7		EHDV-1 a
					C9	-		27		EHDV-1 a b
					C60	31.7	-	undetermined
					C61	36.9	-	undetermined
	Menoufia	27/07/2017	C69	29.4	-	undetermined c
					C86	31.2	-	undetermined
					C87	30.9	-	undetermined
	a : determined with RT-qPCR.

b

: determined with sequencing. c

Table 2 :

 2 RT-PCR and sequencing results of the 14 head of cattle found positive for BTV or EHDV by RT-qPCR sampled in 2016 and 2017 in Beni Suef and Menoufia governorates.

Funding: This work was supported by the EMHOTEP Program-Campus France (No. 35156PK) and by the H2020 PALE BLU project (Grant Agreement no. 727393).

established between European and Mediterranean countries in order to control the spread of BTV more easily and effectively.

Conflict of Interest Statement:

Declarations of interest: none.