e703d8659e472d7d5e08c0684f941e46 pmc_8951896.pdf ad783fe448afef937949e4ba4a4a370556e7ec68 pmc_8951896.pdf 4b5270abf3298c0aa268695e1d0a6a06a87ecb303ca93d49db49af42d288208e pmc_8951896.pdf Title: West Nile Virus Lineage 2 Spreads Westwards in Europe and Overwinters in North-Eastern Spain (2017–2020) Subject: West Nile virus lineage 2 (WNV-L2) emerged in Europe in 2004; since then, it has spread across the continent, causing outbreaks in humans and animals. During 2017 and 2020, WNV-L2 was detected and isolated from four northern goshawks in two provinces of Catalonia (north-eastern Spain). In order to characterise the first Spanish WNV-L2 isolates and elucidate the potential overwintering of the virus in this Mediterranean region, complete genome sequencing, phylogenetic analyses, and a study of phenotypic characterisation were performed. Our results showed that these Spanish isolates belonged to the central-southern WNV-L2 clade. In more detail, they were related to the Lombardy cluster that emerged in Italy in 2013 and has been able to spread westwards, causing outbreaks in France (2018) and Spain (2017 and 2020). Phenotypic characterisation performed in vitro showed that these isolates presented characteristics corresponding to strains of moderate to high virulence. All these findings evidence that these WNV-L2 strains have been able to circulate and overwinter in the region, and are pathogenic, at least in northern goshawks, which seem to be very susceptible to WNV infection and may be good indicators of WNV-L2 circulation. Due to the increasing number of human and animal cases in Europe in the last years, this zoonotic flavivirus should be kept under extensive surveillance, following a One-Health approach. Keywords: West Nile virus; lineage-2; Spain; phylogeny; avian host; northern goshawk; overwintering; Europe Author: Pilar Aguilera-Sepúlveda, Sebastián Napp, Francisco Llorente, Carlos Solano-Manrique, Rafael Molina-López, Elena Obón, Alba Solé, Miguel Ángel Jiménez-Clavero, Jovita Fernández-Pinero and Núria Busquets Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Fri Mar 11 11:58:42 2022 CET ModDate: Fri Mar 11 12:03:31 2022 CET Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 14 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 1482663 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- LRBJIA+VnURWPalladioL Type 1 Custom yes yes no 10 0 LXHCHG+URWPalladioL-Bold Type 1 Custom yes yes no 15 0 ECQVPJ+URWPalladioL-Roma Type 1 Custom yes yes no 20 0 JDWUMT+URWPalladioL-Ital Type 1 Custom yes yes no 24 0 RUGTHW+VnURWPalladioL-Bold Type 1 Custom yes yes no 28 0 MCBCCI+SourceSansRoman-Regular Type 1C Custom yes yes no 36 0 MCBCCM+SourceSansRoman.613wght Type 1C Custom yes yes no 40 0 MCBCCO+SourceSansRoman-Bold Type 1C Custom yes yes no 44 0 HOWKDX+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 56 0 CRXMSX+CMSY10 Type 1 Builtin yes yes no 81 0 OVNEXW+EURM10 Type 1 Builtin yes yes no 85 0 TMMRUN+URWPalladioL-BoldItal Type 1 Custom yes yes no 95 0 OFLBEP+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 102 0 OFLBGB+PalatinoLinotype TrueType WinAnsi yes yes no 105 0 OFLBGD+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 108 0 OFLEJD+Arial CID TrueType Identity-H yes yes yes 119 0 OFLEKE+Tahoma CID TrueType Identity-H yes yes yes 125 0 OFLELF+SimSun CID TrueType Identity-H yes yes yes 131 0 OFLBEP+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 137 0 OFLBFA+PalatinoLinotype CID TrueType Identity-H yes yes yes 140 0 OFLBGB+PalatinoLinotype TrueType WinAnsi yes yes no 146 0 OFLBGD+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 149 0 OFLEJC+Arial,Bold CID TrueType Identity-H yes yes yes 152 0 OFLEJD+Arial CID TrueType Identity-H yes yes yes 164 0 OFLELF+SimSun CID TrueType Identity-H yes yes yes 170 0 OFLFFC+Tahoma,Bold CID TrueType Identity-H yes yes yes 176 0 OFLBEP+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 182 0 OFLBFA+PalatinoLinotype CID TrueType Identity-H yes yes yes 185 0 OFLBGB+PalatinoLinotype TrueType WinAnsi yes yes no 191 0 OFLBGD+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 194 0 OFLEJC+Arial,Bold CID TrueType Identity-H yes yes yes 197 0 OFLBEP+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 212 0 OFLBGB+PalatinoLinotype TrueType WinAnsi yes yes no 215 0 OFLBGD+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 218 0 Jhove (Rel. 1.22.1, 2019-04-17) Date: 2022-04-29 02:25:19 CEST RepresentationInformation: pmc_8951896.pdf ReportingModule: PDF-hul, Rel. 1.12.1 (2019-04-17) LastModified: 2022-04-26 17:59:56 CEST Size: 1482663 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 464 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7b69c6ba Item: Title: Materials and Methods Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@46daef40 Children: Item: Title: Sample Collection Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@12f41634 Item: Title: Initial WNV Molecular Diagnostics Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@13c27452 Item: Title: Virus Isolation Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@262b2c86 Item: Title: Full Genome Sequencing Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@371a67ec Item: Title: Sequence and Phylogenetic Analyses Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@5ed828d Item: Title: Characterisation of the Viral Plaques Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@50d0686 Item: Title: Results Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7a3d45bd Item: Title: Discussion Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1e7c7811 Item: Title: Conclusions Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77ec78b9 Item: Title: References Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1a3869f4 Info: Title: West Nile Virus Lineage 2 Spreads Westwards in Europe and Overwinters in North-Eastern Spain (2017 2020) Author: Pilar Aguilera-Sepúlveda, Sebastián Napp, Francisco Llorente, Carlos Solano-Manrique, Rafael Molina-López, Elena Obón, Alba Solé, Miguel Ángel Jiménez-Clavero, Jovita Fernández-Pinero and Núria Busquets Subject: West Nile virus lineage 2 (WNV-L2) emerged in Europe in 2004; since then, it has spread across the continent, causing outbreaks in humans and animals. During 2017 and 2020, WNV-L2 was detected and isolated from four northern goshawks in two provinces of Catalonia (north-eastern Spain). In order to characterise the first Spanish WNV-L2 isolates and elucidate the potential overwintering of the virus in this Mediterranean region, complete genome sequencing, phylogenetic analyses, and a study of phenotypic characterisation were performed. Our results showed that these Spanish isolates belonged to the central-southern WNV-L2 clade. In more detail, they were related to the Lombardy cluster that emerged in Italy in 2013 and has been able to spread westwards, causing outbreaks in France (2018) and Spain (2017 and 2020). Phenotypic characterisation performed in vitro showed that these isolates presented characteristics corresponding to strains of moderate to high virulence. All these findings evidence that these WNV-L2 strains have been able to circulate and overwinter in the region, and are pathogenic, at least in northern goshawks, which seem to be very susceptible to WNV infection and may be good indicators of WNV-L2 circulation. Due to the increasing number of human and animal cases in Europe in the last years, this zoonotic flavivirus should be kept under extensive surveillance, following a One-Health approach. 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