DNase SISPA-Next Generation Sequencing Confirms Schmallenberg Virus in Belgian Field Samples and Identifies Genetic Variation in Europe 英文参考文献.docVIP

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DNase SISPA-Next Generation Sequencing Confirms Schmallenberg Virus in Belgian Field Samples and Identifies Genetic Variation in Europe 英文参考文献.doc

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DNase SISPA-Next Generation Sequencing Confirms Schmallenberg Virus in Belgian Field Samples and Identifies Genetic Variation in Europe 英文参考文献

DNaseSISPA-NextGenerationSequencingConfirms SchmallenbergVirusinBelgianFieldSamplesand IdentifiesGeneticVariationinEurope ToonRosseel1,MatthiasScheuch2,DirkHo¨per2,NickDeRegge3,AnnBrigitteCaij3, FrankVandenbussche1,StevenVanBorm1* 1Virological Platform Unit (VIRPLAT), Operational Direction Viral Diseases, Veterinary and Agrochemical Research Center, Brussels, Belgium, 2Institute of Diagnostic Virology,Friedrich-Loeffler-Institut,Greifswald-InselRiems,Germany,3UnitofEnzooticand(Re)emergingViralDiseases(ENZOREM),OperationalDirectionViralDiseases, VeterinaryandAgrochemicalResearchCenter,Brussels,Belgium Abstract In2011,anovelOrthobunyaviruswasidentifiedincattleandsheepinGermanyandtheNetherlands.Thisviruswasnamed Schmallenberg virus (SBV). Later, presence of the virus was confirmed using real time RT-PCR in cases of congenital malformations of bovines and ovines in several European countries, including Belgium. In the absence of specific sequencingprotocolsforthisnovelvirusweconfirmeditspresenceinRT-qPCRpositivefieldsamplesusingDNaseSISPA- next generation sequencing (NGS), a virus discovery method based on random amplification and next generation sequencing.AninvitrotranscribedRNAwasusedtoconstructastandardcurveallowingthequantificationofviralRNAin thefieldsamples.Twofieldsamplesofabortedlambscontaining7.66and7.64log10RNAcopiespermLtotalRNAallowed unambiguousidentificationofSBV.Onesampleyielded192SBVreadscoveringabout81%oftheLsegment,56%oftheM segmentand13%oftheSsegment.Theothersampleresultedin8readsdistributedovertheLandMsegments.Three weakpositivefieldsamples(onefromanabortedcalf,twofromabortedlambs)containingvirusquantitiesequivalentto 4.27–4.89log10RNAcopiespermLdidnotallowidentificationusingDNaseSISPA-NGS.Thispartialsequenceinformation was compared to the whole genome sequence of SBV isolated from bovines in Germany, identifying several sequence differences. The applied viral discovery method allowed the confirmation of SBV in RT-qPCR positive brain samples. Howeve