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Bluetongue virus (BTV) is a segmented double-stranded RNA virus, existing in multiple serotypes, belonging to the genus Orbivirus of the family Reoviridae. BTV causes Bluetongue (BT), a major OIE-listed disease of ruminants. Identification of BTV serotype is accomplished using multiple typing assays and tends to be executed based on the known epidemiological situation within a given country. Samples containing multiple serotypes, particularly those containing novel introductions, may therefore be missed. The aim of this work was to optimize the nCounter® Analysis System Microarray platform (NanoString technologies), that would simultaneously identify all BTV serotypes and co-infections in analyzed samples. Probes were designed according to all Seg-2 sequences, coding for VP2 proteins which determine serotype specificity, available on line. A specific BTV CodeSet of probes was optimized. Experiments were performed with 30 BTV isolates and with 46 field samples previously shown to be infected with BTV by classical molecular assays. All BTV isolates were correctly identified and the expected BTV serotype was recognized in 35 field samples with C T values between 22.0-33.0. In turn, it was unable to identify 11 samples with C T values between 29.0-38.0. Although specificity of the assay needs to be further investigated against a larger panel of BTVs collected worldwide, RNA loads, which are normally detected in blood samples during the acute phase of infection, are within the range of C T values detectable by the BTV CodeSet. We propose the NanoString RNA microarray as a first-line molecular diagnostic tool for identification and typing of BTV. Once identification of the index cases is performed, diagnosis of the following samples may be performed by specific, more sensitive and cheaper PCR-based tools., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Bluetongue (BT), is one of the OIE-listed major diseases of ruminants. Following the official report of BT virus serotype 3 (BTV-3) in a sheep in Cap Bon (Tunisia), blood and serum samples of ruminants were collected from some areas of Tunisia to further investigate the presence of this virus in the country. A quantitative real time RT-PCR has been first developed for the detection and quantitation of BTV-3 RNA from field specimens. Out of 62 collected blood samples, 23 were shown to be positive for BTV-3 RNA. Isolation on cell cultures was also possible from six samples. Genome sequencing revealed the circulation of two unrelated western strains of BTV-3, one circulating in Cap Bon and neighboring areas, and the other circulating nearby the border with Libya. The presence of a putative novel BTV serotype (BTV-Y TUN2017) in sheep introduced from Libya to Tunisia, genomically related to the BTV strain contaminating a commercially-available sheep pox vaccine and to BTV-26, has been also demonstrated. This finding highlights the pressing need for a prompt production and release of a novel inactivated BTV-3 vaccine to be used in case of emergence or proactively in the areas of Southern Europe at major risk of BTV introduction. The assessment of a novel vaccine will certainly exalt the role and importance of surveillance activities and collaboration with Northern African countries., (Copyright © 2018 Elsevier B.V. All rights reserved.)

In recent years, novel Bluetongue virus (BTV) serotypes have been isolated and/or sequenced by researchers within the field. During Bluetongue surveillance activities, we identified a putative novel BTV serotype in healthy goats from Sardinia, Italy. RNAs purified from blood and serum samples were positive for BTV by a generic real time RT-PCR and c-ELISA, respectively, whereas genotyping and serotyping were unsuccessful. By NGS, the whole genome sequence was obtained from two blood samples (BTV-X ITL2015 strains 34200 and 33531). Overall, Seg 2 of BTV-X ITL2015 shows the highest identity (75.3-75.5% nt/77.4-78.1% aa) with recently isolated BTV-27s from Corsica and with the last discovered BTV XJ1407 from China (75.9% nt /78.2% aa), whereas it is less related with BTV-25 from Switzerland (73.0% nt/75.0% aa) and BTV-26 from Kuwait (62.0% nt/60.5% aa). A specific RT-qPCR targeting Seg 2 of BTV-X ITL2015 was assessed in this study. Considering the Seg 2/VP2 identity of BTV-X ITL2015 with BTV-25, 26, 27s and BTV XJ1407 and that serum of BTV-X ITL2015 infected goats failed to neutralize all tested extant serotypes, we propose the existence of a novel BTV serotype circulating in goats in Sardinia. Isolation was so far unsuccessful thus hampering proper antigenic characterization., (Copyright © 2017 The Author(s). Published by Elsevier B.V. All rights reserved.)

Epizootic hemorrhagic disease virus (EHDV) is a Culicoides -transmitted virus circulating in multiple serotypes. It has become a concern in the European Union as a novel strain of the serotype 8 (EHDV-8) of clear Northern African origin, has been recently discovered in symptomatic cattle in Italy (islands of Sardinia and Sicily), Spain, and Portugal. Current molecular typing methods targeting the S2 nucleotide sequences -coding for the outermost protein of the virion VP2- are not able to detect the novel emerging EHDV-8 strain as they enrolled the S2 sequence of the unique EHDV-8 reference strain isolated in Australia in 1982. Thus, in this study, we developed and validated a novel typing assay for the detection and quantitation of the novel EHDV-8 RNA from field samples, including blood of ruminants and insects. This molecular tool will certainly support EHDV-8 surveillance and control. • Current molecular typing methods are not able to detect the novel Mediterranean EHDV-8 strains. • We developed a Segment 2 typing assay specific for EHDV-8 RNA. • Validation has been performed by using biological specimens of ruminants and Culicoides pool. • The assay's limit of detection was determined to be 10 copies/reaction. [ABSTRACT FROM AUTHOR]