Recent genomic epidemiology of western BTV-4 strains in the Mediterranean basin Alessio Lorusso1 , Soufien Sghaier2 , Corinne Sailleau3 , Damien Vitour3 , Emmanuel Bréard3 , Stéphan Zientara3 , Montserrrat Agüero4 , Rubén Villalba4 , Salah Hammami5 , Francesca Di Giallonardo6 , Ben Hassine Thameur7 , Sara Thabet 5 , María José Ruano4 , Maurilia Marcacci1 , Giovanni Savini1 1) Istituto Zooprofilattico Sperimentale dell’Abruzzo e Molise, Teramo-Italy 2) Department of Virology, Institution of Agricultural Research and Higher Education, Tunisian Institute of Veterinary Research (IRVT) 3) Animal health laboratory, UMR ANSES/INRAE/ENVA, Maisons-Alfort, France 4) Laboratorio Central de Veterinaria-LCV-MAPA, Algete, Madrid, Spain 5) National School of Veterinary Medicine Sidi-Thabet, University of Manouba, Ariana, Tunisia 6) The Kirby Institute, UNSW Sydney, Sydney, NSW 2052, Australia 7) Ministry of Agriculture of Tunisia, General Directorate of Veterinary Services, CRDA Nabeul, Tunisia Corresponding author: a.lorusso@izs.it
Bluetongue (BT) is a non-contagious, viral disease of domestic and wild ruminants caused by bluetongue virus (BTV), an Orbivirus with a double strand RNA segmented genome, mostly transmitted from one animal to another by biting midges (Culicoides spp.). Since 1998, Southern Europe has experienced multiple incursions of different serotypes and topotypes (western (w) or eastern (e)) of BTV. Strains of BTV-1e, BTV-4w, BTV-9e and BTV-16e have all entered the eastern Mediterranean region. In addition, strains of BTV-1w, BTV-2w, BTV-3w and BTV-4w have entered Southern Europe because of wind-driven dissemination of infected midges from Northern African countries. Specifically, the virus had been likely introduced to Europe from Northern Africa via two major gateways: (i) from Morocco to Spain through the Straits of Gibraltar, (ii) from Tunisia or Algeria to Italy and Spain through Sardinia, Sicily, and Balearic Islands, respectively. In recent years (2012-2021), multiple BT outbreaks caused by BTV-4 strains have been notified in several countries facing the Mediterranean basin including Tunisia, Spain (mainland and Balearic Islands), France (mainland and Corsica), and Italy (mainland, Sardinia, and Sicily). In particular, between 2019 and 2021, these outbreaks have caused severe economic losses and a surge in demand for vaccines. However, recent genomic analysis of BTV4 circulating strains was limited and hampered by the COVID-19 pandemic crisis. Therefore, in the present work, a collaborative effort of several Institutions, operating in these countries and active in Orbivirus surveillance, has been put in place to disentangle the recent molecular epidemiology of BTV4. To do so, viral RNA was purified from a total of 60 BTV-4-positive biological samples (whole blood, spleen homogenates) collected throughout the years (2012-2021) in Tunisia, France, Italy, Morocco and Spain and sequenced either by NGS or Sanger sequencing to get the genome constellations of the involved strains. The obtained sequences were compared with those available on GenBank. All strains identified in Northern Africa and Southern Europe throughout the years (2012-2021) belonged to the western topotype. Moreover, results suggest that evolution shaped the recent genomic epidemiology of BTV-4w as strains, characterized by different genome constellations and drift mutations, were identified. In regards to recent years, BTV-4w strains collected in 2021 in Italy (mainland and Sardinia), France (Corsica) and Spain (Balearic island) were remarkably close (>99.56 % of nucleotide identity in all genome segments) to homologous strains collected in Tunisia in 2019, 2020 and 2021. These novel BTV-4w, along with a different genome constellation, were slightly divergent in Seg-2 (97.85% of nt identity) with respect to Balkanic BTV-4w strains isolated from 2014 onward in Europe including also recent French (Corsica, 2020) and Italian (Sicily, 2021) BTV-4w strains. The novel BTV-4w differed also from Spanish BTV-4w strains which have circulated in mainland Spain since 2010 as these latter were related to BTV-4w strains collected in Morocco and Tunisia in 2012 and 2013, respectively. Overall, combined results suggest that the novel BTV-4w strains from 2021 had likely originated in southern Europe as a consequence of a novel wind-driven dissemination of infected midges from Northern
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