c1a8c0ce1ee6c57a918bf9b9c28c9395 JOUV_a2022v96n14.pdf b96ce176cc271509387190d233b0956594c318c8 JOUV_a2022v96n14.pdf 309a64bd4e3a6c2a0a9c7a465bacbd131a2bc516a06694f1bb3b4833d1d01d9f JOUV_a2022v96n14.pdf Title: Removal of the Erns RNase Activity and of the 3′ Untranslated Region Polyuridine Insertion in a Low-Virulence Classical Swine Fever Virus Triggers a Cytokine Storm and Lethal Disease Subject: Vaccines and Antiviral Agents Keywords: classical swine fever virus; virulence factors; innate and adaptive immunity; disease severity; pigs; vaccine designs; cytokine-mediated symptoms; diagnostic tools and vaccine strategies; tonsil; viral replication; viral spread Author: Miaomiao Wang;José Alejandro Bohórquez;Sara Muñoz-González;Markus Gerber;Mònica Alberch;Marta Pérez-Simó;Xavier Abad;Matthias Liniger;Nicolas Ruggli;Llilianne Ganges Creator: PTC Arbortext Advanced Print Publisher 11.1.4012/W Producer: Adobe LiveCycle PDF Generator; modified using iText® 5.5.13.2 ©2000-2020 iText Group NV (AGPL-version) CreationDate: Tue Jul 12 09:28:42 2022 CEST ModDate: Mon Jun 3 11:09:33 2024 CEST Custom Metadata: yes Metadata Stream: yes Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 15 Encrypted: no Page size: 584.957 x 782.986 pts Page rot: 0 File size: 2770242 bytes Optimized: no PDF version: 1.4 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- GFPGKD+AdvP7DA6 Type 1C WinAnsi yes yes no 54 0 GFPGKE+AdvOT46dcae81+fb Type 1C Custom yes yes yes 56 0 GFPGKF+AdvOT07517017+fb Type 1C Custom yes yes yes 570 0 GFPGHN+AdvOTce3d9a73 Type 1C WinAnsi yes yes no 571 0 GFPGHO+AdvOT8817665d Type 1C WinAnsi yes yes no 58 0 GFPGIP+AdvOT07517017 Type 1C WinAnsi yes yes no 572 0 GFPGJA+AdvOT07517017+20 Type 1C WinAnsi yes yes no 573 0 GFPGJB+AdvOT3b30f6db.B Type 1C WinAnsi yes yes no 59 0 GFPGJC+AdvOT46dcae81 Type 1C WinAnsi yes yes no 60 0 Times-Roman Type 1 WinAnsi no no no 2 0 GFPGMF+AdvOT65f8a23b.I Type 1C WinAnsi yes yes no 55 0 GFPGMG+AdvOT46dcae81+20 Type 1C WinAnsi yes yes no 57 0 GFPGNG+AdvMPI4IT Type 1C WinAnsi yes yes no 106 0 GGAHIL+AdvP4C4E74 Type 1C Custom yes yes no 277 0 GGAHIM+AdvP80675 Type 1C WinAnsi yes yes no 278 0 GGAHIK+AdvPSUnvGMP Type 1C WinAnsi yes yes no 279 0 GGAHKM+AdvOT3b30f6db.B+fb Type 1C Custom yes yes yes 310 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2024-06-04 02:36:29 CEST RepresentationInformation: JOUV_a2022v96n14.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2024-06-03 12:06:56 CEST Size: 2770242 Format: PDF Version: 1.4 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 0 FreeObjects: 2 IncrementalUpdates: 0 DocumentCatalog: ViewerPreferences: HideToolbar: false HideMenubar: false HideWindowUI: false FitWindow: false CenterWindow: false DisplayDocTitle: true NonFullScreenPageMode: UseNone Direction: L2R ViewArea: CropBox ViewClip: CropBox PrintArea: CropBox PageClip: CropBox PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: RESULTS Destination: flink1 Children: Item: Title: Inactivation of the Erns RNase and deletion of the poly-U sequence in PdR did not impair virus replication in cell culture compared with that of the parent virus. Destination: slink1 Item: Title: The vPdR-H30K-5U double mutant strongly activated the IFN-α response in pDC. Destination: slink2 Item: Title: The double mutant vPdR-H30K-5U induced acute CSF with high mortality in 5-day-old piglets. Destination: slink3 Item: Title: Infection of 5-day-old piglets with vPdR-H30K-5U resulted in high virus load in tonsils despite low viremia and limited virus secretion. Destination: slink4 Item: Title: Piglets that survived vPdR-H30K-5U infection seroconverted after 3 weeks. Destination: slink5 Item: Title: The vPdR-H30K-5U double mutant induced stronger IFN-α, IL-8, and IL-12 responses than its parent viruses. Destination: slink6 Item: Title: In 3-week-old pigs, the double mutant vPdR-H30K-5U induced a milder disease with lower lethality than in 5-day-old piglets. Destination: slink7 Item: Title: In 3-week-old pigs, the vPdR-H30K-5U mutant resulted in high virus loads in tonsils with limited viremia and secretion as in 5-day-old piglets. Destination: slink8 Item: Title: The 3-week-old pigs that survived vPdR-H30K-5U infection seroconverted from day 11 postinfection. Destination: slink9 Item: Title: In 3-week-old pigs, the vPdR-H30K-5U double mutant induced drastic elevation of IFN-α and IL-12, without IL-8 induction. Destination: slink10 Item: Title: DISCUSSION Destination: flink2 Item: Title: MATERIALS AND METHODS Destination: flink3 Children: Item: Title: Cells and viruses. Destination: slink11 Item: Title: Construction of the infectious clone pPdR-H30K-5U. Destination: slink12 Item: Title: Virus rescue from cDNA. Destination: slink13 Item: Title: Virus replication kinetics in cell culture. Destination: slink14 Item: Title: RNase activity assay. Destination: slink15 Item: Title: Western blot analyses. Destination: slink16 Item: Title: Stimulation of pDC for IFN-α production. Destination: slink17 Item: Title: Experimental infection of 5-day- and 3-week-old pigs. Destination: slink18 Item: Title: IFN-α quantification by ELISA. Destination: slink19 Item: Title: Detection of CSFV in infected pigs. Destination: slink20 Item: Title: Detection of antibody responses by ELISA and virus neutralization test. Destination: slink21 Item: Title: Detection of cytokines by Luminex assay. Destination: slink22 Item: Title: Statistical analyses. Destination: slink23 Item: Title: ACKNOWLEDGMENTS Destination: flink4 Item: Title: REFERENCES Destination: flink5 Filters: FilterPipeline: FlateDecode Images: Image: NisoImageMetadata: FormatName: image/png ImageWidth: 67 ImageHeight: 67 BitsPerSample: 8 BitsPerSampleUnit: integer Filter: FlateDecode Fonts: Type1: Font: BaseFont: GFPGNG+AdvMPI4IT FontSubset: true FirstChar: 97 LastChar: 97 FontDescriptor: FontName: GFPGNG+AdvMPI4IT Flags: Nonsymbolic FontBBox: -104, -229, 989, 770 FontFile3: true Encoding: WinAnsiEncoding Font: BaseFont: GGAHIL+AdvP4C4E74 FontSubset: true FirstChar: 1 LastChar: 1 FontDescriptor: FontName: GGAHIL+AdvP4C4E74 Flags: Symbolic FontBBox: -20, -958, 1114, 770 FontFile3: true EncodingDictionary: Differences: true Font: BaseFont: GFPGKD+AdvP7DA6 FontSubset: true FirstChar: 97 LastChar: 109 FontDescriptor: FontName: GFPGKD+AdvP7DA6 Flags: Nonsymbolic FontBBox: -20, -208, 1000, 770 FontFile3: true Encoding: WinAnsiEncoding Font: BaseFont: GGAHIM+AdvP80675 FontSubset: true FirstChar: 44 LastChar: 50 FontDescriptor: FontName: GGAHIM+AdvP80675 Flags: Nonsymbolic FontBBox: -20, -229, 1010, 812 FontFile3: true Encoding: WinAnsiEncoding Font: BaseFont: GGAHKM+AdvOT3b30f6db.B+fb FontSubset: true FirstChar: 1 LastChar: 1 FontDescriptor: FontName: GGAHKM+AdvOT3b30f6db.B+fb Flags: Symbolic FontBBox: 0, 0, 838, 721 FontFile3: true EncodingDictionary: Differences: true ToUnicode: true Font: BaseFont: GFPGMF+AdvOT65f8a23b.I FontSubset: true FirstChar: 67 LastChar: 121 FontDescriptor: FontName: GFPGMF+AdvOT65f8a23b.I Flags: Nonsymbolic FontBBox: -112, -250, 894, 873 FontFile3: true Encoding: WinAnsiEncoding Font: BaseFont: GGAHIK+AdvPSUnvGMP FontSubset: true FirstChar: 49 LastChar: 49 FontDescriptor: FontName: GGAHIK+AdvPSUnvGMP Flags: Nonsymbolic FontBBox: -20, -229, 1010, 812 FontFile3: true Encoding: WinAnsiEncoding Font: BaseFont: GFPGKE+AdvOT46dcae81+fb FontSubset: true FirstChar: 1 LastChar: 3 FontDescriptor: FontName: GFPGKE+AdvOT46dcae81+fb Flags: Symbolic FontBBox: 0, 0, 742, 721 FontFile3: true EncodingDictionary: Differences: true ToUnicode: true Font: BaseFont: GFPGMG+AdvOT46dcae81+20 FontSubset: true FirstChar: 146 LastChar: 150 FontDescriptor: FontName: GFPGMG+AdvOT46dcae81+20 Flags: Nonsymbolic FontBBox: -9, -205, 1126, 868 FontFile3: true Encoding: WinAnsiEncoding Font: BaseFont: GFPGKF+AdvOT07517017+fb FontSubset: true FirstChar: 1 LastChar: 1 FontDescriptor: FontName: GFPGKF+AdvOT07517017+fb Flags: Symbolic FontBBox: 0, 0, 683, 721 FontFile3: true EncodingDictionary: Differences: true ToUnicode: true Font: BaseFont: GFPGHO+AdvOT8817665d FontSubset: true FirstChar: 35 LastChar: 57 FontDescriptor: FontName: GFPGHO+AdvOT8817665d Flags: Nonsymbolic FontBBox: -30, -210, 1000, 779 FontFile3: true Encoding: WinAnsiEncoding Font: BaseFont: GFPGHN+AdvOTce3d9a73 FontSubset: true FirstChar: 44 LastChar: 243 FontDescriptor: FontName: GFPGHN+AdvOTce3d9a73 Flags: Nonsymbolic FontBBox: -42, -250, 858, 889 FontFile3: true Encoding: WinAnsiEncoding Font: BaseFont: GFPGJB+AdvOT3b30f6db.B FontSubset: true FirstChar: 44 LastChar: 122 FontDescriptor: FontName: GFPGJB+AdvOT3b30f6db.B Flags: Nonsymbolic FontBBox: -39, -250, 875, 897 FontFile3: true Encoding: WinAnsiEncoding Font: BaseFont: GFPGJC+AdvOT46dcae81 FontSubset: true FirstChar: 37 LastChar: 252 FontDescriptor: FontName: GFPGJC+AdvOT46dcae81 Flags: Nonsymbolic FontBBox: -46, -250, 838, 879 FontFile3: true Encoding: WinAnsiEncoding Font: BaseFont: GFPGIP+AdvOT07517017 FontSubset: true FirstChar: 40 LastChar: 243 FontDescriptor: FontName: GFPGIP+AdvOT07517017 Flags: Nonsymbolic FontBBox: -49, -250, 816, 867 FontFile3: true Encoding: WinAnsiEncoding Font: BaseFont: GFPGJA+AdvOT07517017+20 FontSubset: true FirstChar: 146 LastChar: 146 FontDescriptor: FontName: GFPGJA+AdvOT07517017+20 Flags: Nonsymbolic FontBBox: -6, -209, 1046, 868 FontFile3: true Encoding: WinAnsiEncoding XMP: Removal of the Erns RNase Activity and of the 3′ Untranslated Region Polyuridine Insertion in a Low-Virulence Classical Swine Fever Virus Triggers a Cytokine Storm and Lethal Disease 10.1128/jvi.00438-22 Miaomiao Wang José Alejandro Bohórquez Sara Muñoz-González Markus Gerber Mònica Alberch Marta Pérez-Simó Xavier Abad Matthias Liniger Nicolas Ruggli Llilianne Ganges Vaccines and Antiviral Agents Vaccines and Antiviral Agents classical swine fever virus virulence factors innate and adaptive immunity disease severity pigs vaccine designs cytokine-mediated symptoms diagnostic tools and vaccine strategies tonsil viral replication viral spread American Society for Microbiology Wang et al. Copyright © 2022 Wang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. 20220627 2022 ABSTRACT In this study, we assessed the potential synergistic effect of the Erns RNase activity and the poly-U insertion in the 3′ untranslated region (UTR) of the low-virulence classical swine fever virus (CSFV) isolate Pinar de Rio (PdR) in innate and adaptive immunity regulation and its relationship with classical swine fever (CSF) pathogenesis in pigs. We knocked out the Erns RNase activity of PdR and replaced the long polyuridine sequence of the 3′ UTR with 5 uridines found typically at this position, resulting in a double mutant, vPdR-H30K-5U. This mutant induced severe CSF in 5-day-old piglets and 3-week-old pigs, with higher lethality in the newborn (89.5%) than in the older (33.3%) pigs. However, the viremia and viral excretion were surprisingly low, while the virus load was high in the tonsils. Only alpha interferon (IFN-α) and interleukin 12 (IL-12) were highly and consistently elevated in the two groups. Additionally, high IL-8 levels were found in the newborn but not in the older pigs. This points toward a role of these cytokines in the CSF outcome, with age-related differences. The disproportional activation of innate immunity might limit systemic viral spread from the tonsils and increase virus clearance, inducing strong cytokine-mediated symptoms. Infection with vPdR-H30K-5U resulted in poor neutralizing antibody responses compared with results obtained previously with the parent and RNase knockout PdR. This study shows for the first time the synergistic effect of the 3′ UTR and the Erns RNase function in regulating innate immunity against CSFV, favoring virus replication in target tissue and thus contributing to disease severity. IMPORTANCE CSF is one of the most relevant viral epizootic diseases of swine, with high economic and sanitary impact. Systematic stamping out of infected herds with and without vaccination has permitted regional virus eradication. However, the causative agent, CSFV, persists in certain areas of the world, leading to disease reemergence. Nowadays, low- and moderate-virulence strains that could induce unapparent CSF forms are prevalent, posing a challenge for disease eradication. Here, we show for the first time the synergistic role of lacking the Erns RNase activity and the 3′ UTR polyuridine insertion from a low-virulence CSFV isolate in innate immunity disproportional activation. This might limit systemic viral spread to the tonsils and increase virus clearance, inducing strong cytokine-mediated symptoms, thus contributing to disease severity. These results highlight the role played by the Erns RNase activity and the 3′ UTR in CSFV pathogenesis, providing new perspectives for novel diagnostic tools and vaccine strategies. 20220315 20220524 1098-5514 Adobe LiveCycle PDF Generator; modified using iText® 5.5.13.2 ©2000-2020 iText Group NV (AGPL-version)2024-06-03T02:09:33-07:00 Pages: Page: Label: 1 Annotations: Annotation: Subtype: Link Rect: 338, 554, 402, 563 Annotation: Subtype: Link Rect: 410, 124, 506, 131 Annotation: Subtype: Link Rect: 400, 115, 456, 122 Annotation: Subtype: Link Rect: 465, 24, 526, 31 Annotation: Subtype: Link Rect: 508, 683, 544, 719 Page: Label: 2 Annotations: Annotation: Subtype: Link Rect: 150, 636, 155, 645 Destination: 273 Annotation: Subtype: Link Rect: 165, 598, 170, 607 Destination: 273 Annotation: Subtype: Link Rect: 225, 561, 230, 570 Destination: 273 Annotation: Subtype: Link Rect: 235, 561, 239, 570 Destination: 273 Annotation: Subtype: Link Rect: 281, 536, 285, 545 Destination: 273 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