Host transcriptome profiling for resistance against Lumpy Skin Disease (LSD)

Host transcriptome profiling for resistance against Lumpy Skin Disease (LSD) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Short Report Host transcriptome profiling for resistance against Lumpy Skin Disease (LSD) Mohammad Hossein Banabazi, Steven Borm, Tomas Klingström, Adnan Niazi, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4950902/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 15 Jul, 2025 Read the published version in BMC Research Notes → Version 1 posted 4 You are reading this latest preprint version Abstract Objective Lumpy skin disease (LSD) is an acute or subacute systemic viral disease of cattle that shows variation in the response of cattle to LSD virus infection. To better understand the mechanisms underlying this response diversity in field studies and under carefully controlled artificial infections, we studied the differentially expressed genes (DEGs) between two resilient versus three susceptible Holstein bulls before an infection challenge and three time points after that. Results The host transcriptome profiling revealed that IL1RAP gene expression could be a potential determinant in distinguishing between resilient and susceptible cattle (padj < 0.05). It was significantly shifted from up-regulated prior to infection to down-regulated three days post-infection in the LSD-resilient cattle. Its expression remained up-regulated among the susceptible cattle post-infection compared to pre-infection. The results showed that seven days post-infection may be a critical time point for LSD infection. The Gene Ontology (GO) and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment test showed a few enriched GO terms and pathways relevant to the LSD and the involvement of the IL1RAP gene. This pilot study, with limited statistical power, is the first to investigate bovine gene expression profiling in response to LSDV. Cattle Lumpy Skin Disease (LSD) IL1RAP gene Transcriptome Profiling Host Determinants Differentially Expressed Genes (DEG) INTRODUCTION Lumpy skin disease (LSD), an acute or subacute systemic viral disease of cattle, is a major global health threat to livestock ( 1 ). LSD was first diagnosed in 1929 in Zambia and spread into the Middle East in 2012 and Europe in 2015 ( 2 ). Since 2019, LSD recombinant virus strains are spreading in large parts of Asia ( 3 ). There is host variation in the response of cattle to lumpy skin disease virus (LSDV) infection in field studies. Between 20–50% of the animals have no clinical signs (asymptomatic) when a herd is infected with LSDV ( 4 ). To better understand the mechanisms underlying the response diversity, we studied the differentially expressed genes (DEGs) between symptomatic and asymptomatic cattle at each time point before and after virus challenge as well as among non-recovered animals over time. MAIN TEXT Material and Methods : Five Holstein bulls, that served as the control animals in a large vaccine trial ( 5 ), were sampled for whole blood using Tempus™ Blood RNA Tubes (ThermoFisher Scientific) and experimentally infected five days later at Sciensano (Belgium) with LSDV via injection in the vena jugularis and the neck with a LSDV strain derived from Israel ( 5 ). Three bulls showed LSD symptoms, and two did not. All bulls were also sampled three, seven, and fifteen days post-infection (dpi). Twenty whole RNA samples were isolated using the Tempus™ Spin RNA Isolation Reagent Kit (ThermoFisher Scientific) according to the manufacturer's instructions. All RNA showed RNA Integrity Number equivalent (RINe) scores above 9.0 (High Sensitivity RNA ScreenTape assay, Agilent Technologies). RNA sequencing was performed through a QuantSeq approach ( 6 ) by Illumina NextSeq500 using a 150 HO sequencing kit at the Neuromics Support Facility of VIB University of Antwerp, Center for Molecular Neurology, Belgium. This method results in a single unique fragment per transcript, thus simplifying the quantification of gene expression. After data quality control using FastQC v0.11.8, the 150-bp single reads were trimmed using bbduk.sh script available in BBMAP suite v38.94 ( 7 ). The filtered reads were mapped on the Bos taurus reference genome (ARS-UCD1.2, Ensemble release 105) by HISAT2 aligner ( 8 ). The features included in the Bos taurus annotation (the same release) were counted on the individual assembled transcriptomes by featureCounts v2.0.1( 9 ). Raw read counts were normalized and differential gene expression analysis was performed for the asymptomatic versus symptomatic contrasts in each time point as well as for symptomatic animals in each post-infection time point versus pre-infection time using the DESeq2 v1.32.0 package ( 10 ) under R v. 4.1.3 ( 11 ). The former contrasts reveal the genes involved in the susceptibility to disease, and the last show which genes would be involved when the infection is induced and evolves to symptomatic LSD. After differential expression analysis, the resulting p-values were adjusted for multiple testing using the Benjamini–Hochberg procedure. DEGs with adjusted p-values (p adj ) < 0.05 were considered significant. Gene Set Enrichment Analyses were done for gene ontology terms (GO) and KEGG pathways through R packages: clusterProfiler v4.2.2 ( 12 ) and org.Bt.eg.db V3.14.0 ( 13 ). Results. On average 25.6 million reads per sample were generated by sequencing. The raw reads were minimally trimmed about 0.7% of total numbers. The filtered reads were aligned on the reference genome in an average rate of 95.4% (Table S1 ). One symptomatic sample on three dpi was recognized as an outlier in Principle Component Analysis (PCA) and removed (Figures S1 and S2). This sample had the lowest RNA concentration. The alignment rate for all samples was 95,4% on average. The asymptomatic vs. symptomatic contrasts revealed that 20, 34, 364, and 37 genes were significantly differentially expressed (p adj < 0.05) five days prior to (pre-infection), three, seven, and fifteen days post-infection (dpi), respectively (Table S2 , and Figures S3 -A). Differentially expressed genes (DEGs) in the pre-infection time point reveal good candidate determinants for susceptibility to LSD. They are expressed without the infection being present but may be predictors for disease outcome. The experimental infection may influence in the gene expression and activate GO and pathways that do not necessarily result from the induced infection and propose confounding determinants. The symptomatic vs. asymptomatic contrast on pre-infection revealed a few DEGs with an interesting trend. For example, Interleukin 1 Receptor Accessory Protein (IL1RAP) gene was significantly down-regulated five days pre-infection (p adj < 0.05) in cattle that showed the symptoms after challenge (susceptible) but was up-regulated three dpi in the same animals. This gene is located on chromosome one and includes twelve exons ordered as four transcripts. IL1RAP is an essential regulator of redox homeostasis and a cell-surface protein best known as a co-receptor for interleukin 1 receptor signaling ( 14 ). Also, its expression was significantly up-regulated after challenge over time among symptomatic animals (p adj < 0.05). IL1RAP was thus differentially expressed at each time point post-infection versus pre-infection (Table 1 ). It can be concluded that IL1RAP may have a key role in LSD susceptibility and the control of the disease where high expression of IL1RAP is associated with disease occurrence. Table 1 Gene expression of IL1RAP gene ( ENSBTAG00000013205 ) in different contrasts (p adj < 0.05) Contrast log 2 Fold Change pvalue padj Symp vs. Asymp (BaseMean = 21.432) pre-infection -20.158 6.55E-11 2.28E-07 3 dpi 19.558 1.28E-08 3.81E-05 7 dpi 0.389 0.89 0.99 15 dpi -0.990 0.73 1 Symptomatic over time (Post-infection vs. pre-infection) (BaseMean = 21.528) 3dpi vs. pre-infection 14.998 8.25E-07 0.0022 7dpi vs. pre-infection 17.296 1.19E-08 2.08E-05 15dpi vs. pre-infection 15.721 2.27E-07 0.00034 Day seven post infection showed the highest number of significant DEGs (p adj < 0.05) and the relatively low number of shared ones with other time points (Table S2 , Table S3 , Figure S3 , Figure S4 ). In addition, the enriched GO terms were only seen in symptomatic animals vs. pre-infection ones on 7dpi (Figure S5 ). They were mostly relevant to immune responses. These results indicate that day 7 is critical in LSD infection. Differential expression of IL1RAP show subsequent enrichment for the cytokine-cytokine receptor interaction pathway with five other DEGs (p adj < 0.05) between symptomatic animals on seven dpi compared to pre-infection time (Figure S5 ). The therapeutic potential of targeting interleukin-1 family cytokines has been suggested in chronic human inflammatory skin diseases that show similar symptoms to LSD ( 15 ). It was suggested as promising target for immunotherapy of metastasis ( 16 ) using antibodies in humans ( 17 ). The expression of IL1RAP prior to any LSD outbreak may promise to distinguish the resilient and susceptible cattle for LSD through a high-resolution and low false discovery rate (FDR) diagnostic Real-time PCR test. Some human studies have already shown its involvement in similar diseases. LIMITATIONS The work presented here was a retrospective pilot study to investigate the utility of bio-banked whole blood samples to analyze the transcriptome of cattle at different time points before and after artificial infection with LSDV. While the study clearly showed the merits of routine biobanking for retrospective analyses it also has clear limitations: 1) the number of samples is limited by the experiment that was previously done. Transcriptomics was not the main purpose of the original experiment. 2) the time-points of sampling are ad-hoc and may not reflect the key events in terms of gene expression. Other time points may have given a more informative gene expression signature. The main weakness of the present study is that we only had five biological replicates, with three, respectively two replicates per disease outcome. This means that any statistical inferences from the study are preliminary and require replication in a larger study. Despite the limited sample size, the results make biological sense. Another limitation of the present study was the use of a fragment based sequencing approach as opposed to whole-transcriptome sequencing. This is more cost-effective but prevents the identification of splice variation or transcript length variation between animals. Given the cost and severity of animal experiments involving potentially lethal viruses, transcriptome studies will always have to be planned as a complement to more invasive studies like transmission experiments or vaccine trials. This implies a key role for systematic biobanking to allow the accumulation of sufficient suitable samples across studies. In turn, this requires meticulous data recording and documentation of metadata. Abbreviations DPI Days post infection DEG Differentially Expressed Genes GO Gene Ontology IL1RAP Interleukin 1 Receptor Accessory Protein KEGG Kyoto Encyclopedia of Genes and Genomes LSD Lumpy Skin Disease LSDV Lumpy Skin Disease Virus PCA Principle Component Analysis RINe RNA Integrity Number equivalent Declarations Ethics approval. The present research was based on bio-banked samples stored at Sciensano. The animal experiments were conducted according to the European Union and Belgian regulations on animal welfare in experimentation. The protocol was approved by the joint Ethical Committee of Sciensano, authorization number 20150605-01_EC_Dierproef aanvraag_LSDV_BMG_2015. Consent for publication. Not applicable. No personal data included in this study. Availability of data and materials. Raw RNA Sequencing reads are publicly available in the EBI ArrayExpress repository (https://www.ebi.ac.uk/biostudies/arrayexpress/) with accession number E-MTAB-12547. The blood samples used in this study were finished completely during RNA extractions and are no longer available. Competing Interests. The authors do not declare any competing interests. Funding. This project was funded by the European Union's Horizon 2020 research and innovation program under agreement No 773701. Authors contribution. SVB, KDK, AH and DJK designed the pilot study where the latter was project leader for the pilot study. KDK, AH and LM performed the original infection studies at SCIENSANO. SVB performed RNA extractions and prepared samples for sequencing. MHB, AN and TK performed statistical and bioinformatics analyses. MHB drafted the manuscript with significant input from DJK. All authors provided edits and comments to the manuscript and approved the final manuscript. Acknowledgements Special gratitude to the animal caretakers of the Experimental Centre of Sciensano (Belgium) for collecting the data and samples. RNA sequencing was professionally performed at the VIB Neuromics Support Facility (University of Antwerp, Center for Molecular Neurology, Belgium). The authors thank the SLU Bioinformatics Infrastructure, Uppsala, Sweden for the bioinformatics support. We are very grateful to the Kimron Veterinary Institute (Israel) and the Field Israeli Veterinary Services for providing us with the LSDV strain LSD/OA3-Ts. MORAN. M. seed pass.4.155920/2012.20.1.13. References Yılmaz H. (2017) Lumpy Skin Disease: Global and Turkish Perspectives. Anwar A., Na-Lampang K., Preyavichyapugdee N. & Punyapornwithaya V. (2022) Lumpy Skin Disease Outbreaks in Africa, Europe, and Asia (2005–2022): Multiple Change Point Analysis and Time Series Forecast. Viruses 14, 2203. Vandenbussche F, Mathijs E, Philips W, Saduakassova M, De Leeuw I, Sultanov A, Haegeman A, De Clercq K. (2022) Recombinant LSDV Strains in Asia: Vaccine Spillover or Natural Emergence? Viruses 14, 1429. Calistri EFSA, De Clercq P, Gubbins K, Klement S, Stegeman E, Cortiñas Abrahantes A, Marojevic J, Antoniou D S.-E., Broglia A. (2020) Lumpy skin disease epidemiological report IV: data collection and analysis. EFSA J 18, e06010. Haegeman A, De Leeuw I, Mostin L, Campe WV, Aerts L, Venter E, Tuppurainen E, Saegerman C, De Clercq K. Comparative Evaluation of Lumpy Skin Disease Virus-Based Live Attenuated Vaccines. Vaccines. 2021;9:473. Moll P, Ante M, Seitz A, Reda T. QuantSeq 3′ mRNA sequencing for RNA quantification. Nat Methods. 2014;11:i–iii. Bushnell B. (2014) BBMap: A Fast, Accurate, Splice-Aware Aligner. Technical report, Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Kim D, Paggi JM, Park C, Bennett C, Salzberg SL. Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype. Nat Biotechnol. 2019;37:907–15. Liao Y, Smyth GK, Shi W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics. 2013;30:923–30. Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014;15:550. R Core Team. R: A language and environment for statistical computing. Vienna, Austria: R Foundation for Statistical Computing; 2022. Wu T, Hu E, Xu S, Chen M, Guo P, Dai Z, Feng T, Zhou L, Tang W, Zhan L, Fu X, Liu S, Bo X, Yu G. clusterProfiler 4.0: A universal enrichment tool for interpreting omics data. Innov. 2021;2:100141. Carlson M. & (2021) org.Bt.eg.db: Genome wide annotation for Bovine. R package version 3.14.0. Käll L, Canterbury JD, Weston J, Noble WS, MacCoss MJ. Semi-supervised learning for peptide identification from shotgun proteomics datasets. Nat Methods. 2007;4:923–5. Calabrese L, Fiocco Z, Satoh TK, Peris K, French LE. Therapeutic potential of targeting interleukin-1 family cytokines in chronic inflammatory skin diseases*. Br J Dermatol. 2022;186:925–41. Zhang H-F, Hughes CS, Li W, He J-Z, Surdez D, El-Naggar AM, Cheng H, Prudova A, Delaidelli A, Negri GL, Li X, Ørum-Madsen MS, Lizardo MM, Oo HZ, Colborne S, Shyp T, Scopim-Ribeiro R, Hammond CA, Dhez A-C, Langman S, Lim JKM, Kung SHY, Li A, Steino A, Daugaard M, Parker SJ, Geltink RIK, Orentas RJ, Xu L-Y, Morin GB, Delattre O, Dimitrov DS, Sorensen PH. (2021) Proteomic Screens for Suppressors of Anoikis Identify IL1RAP as a Promising Surface Target in Ewing Sarcoma. Cancer Discovery 11, 2884 – 903. Robbrecht D, Jungels C, Sorensen MM, Spanggaard I, Eskens F, Fretland SØ, Guren TK, Aftimos P, Liberg D, Svedman C, Thorsson L, Steeghs N, Awada A. First-in-human phase 1 dose-escalation study of CAN04, a first-in-class interleukin-1 receptor accessory protein (IL1RAP) antibody in patients with solid tumours. Br J Cancer. 2022;126:1010–7. Additional Declarations No competing interests reported. Supplementary Files FigS1.pdf FigS2.pdf FigS3.pdf FigS4.pdf FigS5.docx TableS1statisticsofsequencereads.xlsx TableS2SDESympvsAsymp.xlsx TableS3SDEOvertimedpivspre.infected.xlsx Cite Share Download PDF Status: Published Journal Publication published 15 Jul, 2025 Read the published version in BMC Research Notes → Version 1 posted Editorial decision: Revision requested 23 Aug, 2024 Editor assigned by journal 22 Aug, 2024 Submission checks completed at journal 22 Aug, 2024 First submitted to journal 21 Aug, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4950902","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":344084598,"identity":"fdf36787-1745-42f8-bd17-7d5ec33fff85","order_by":0,"name":"Mohammad Hossein Banabazi","email":"","orcid":"","institution":"Swedish University of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Mohammad","middleName":"Hossein","lastName":"Banabazi","suffix":""},{"id":344084599,"identity":"a1e5ef0a-ab6e-4220-a62a-94717e9e019c","order_by":1,"name":"Steven 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subacute systemic viral disease of cattle, is a major global health threat to livestock (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). LSD was first diagnosed in 1929 in Zambia and spread into the Middle East in 2012 and Europe in 2015 (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Since 2019, LSD recombinant virus strains are spreading in large parts of Asia (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). There is host variation in the response of cattle to lumpy skin disease virus (LSDV) infection in field studies. Between 20\u0026ndash;50% of the animals have no clinical signs (asymptomatic) when a herd is infected with LSDV (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). To better understand the mechanisms underlying the response diversity, we studied the differentially expressed genes (DEGs) between symptomatic and asymptomatic cattle at each time point before and after virus challenge as well as among non-recovered animals over time.\u003c/p\u003e"},{"header":"MAIN TEXT","content":"\u003cp\u003e \u003cb\u003eMaterial and Methods\u003c/b\u003e: Five Holstein bulls, that served as the control animals in a large vaccine trial (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e), were sampled for whole blood using Tempus\u0026trade; Blood RNA Tubes (ThermoFisher Scientific) and experimentally infected five days later at Sciensano (Belgium) with LSDV via injection in the vena jugularis and the neck with a LSDV strain derived from Israel (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Three bulls showed LSD symptoms, and two did not. All bulls were also sampled three, seven, and fifteen days post-infection (dpi). Twenty whole RNA samples were isolated using the Tempus\u0026trade; Spin RNA Isolation Reagent Kit (ThermoFisher Scientific) according to the manufacturer's instructions. All RNA showed RNA Integrity Number equivalent (RINe) scores above 9.0 (High Sensitivity RNA ScreenTape assay, Agilent Technologies). RNA sequencing was performed through a QuantSeq approach (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e) by Illumina NextSeq500 using a 150 HO sequencing kit at the Neuromics Support Facility of VIB University of Antwerp, Center for Molecular Neurology, Belgium. This method results in a single unique fragment per transcript, thus simplifying the quantification of gene expression. After data quality control using FastQC v0.11.8, the 150-bp single reads were trimmed using bbduk.sh script available in BBMAP suite v38.94 (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). The filtered reads were mapped on the Bos taurus reference genome (ARS-UCD1.2, Ensemble release 105) by HISAT2 aligner (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). The features included in the Bos taurus annotation (the same release) were counted on the individual assembled transcriptomes by featureCounts v2.0.1(\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Raw read counts were normalized and differential gene expression analysis was performed for the asymptomatic versus symptomatic contrasts in each time point as well as for symptomatic animals in each post-infection time point versus pre-infection time using the DESeq2 v1.32.0 package (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e) under R v. 4.1.3 (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). The former contrasts reveal the genes involved in the susceptibility to disease, and the last show which genes would be involved when the infection is induced and evolves to symptomatic LSD. After differential expression analysis, the resulting p-values were adjusted for multiple testing using the Benjamini\u0026ndash;Hochberg procedure. DEGs with adjusted p-values (p\u003csub\u003eadj\u003c/sub\u003e)\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered significant. Gene Set Enrichment Analyses were done for gene ontology terms (GO) and KEGG pathways through R packages: clusterProfiler v4.2.2 (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e) and org.Bt.eg.db V3.14.0 (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eResults.\u003c/b\u003e On average 25.6\u0026nbsp;million reads per sample were generated by sequencing. The raw reads were minimally trimmed about 0.7% of total numbers. The filtered reads were aligned on the reference genome in an average rate of 95.4% (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). One symptomatic sample on three dpi was recognized as an outlier in Principle Component Analysis (PCA) and removed (Figures \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e and S2). This sample had the lowest RNA concentration. The alignment rate for all samples was 95,4% on average. The asymptomatic vs. symptomatic contrasts revealed that 20, 34, 364, and 37 genes were significantly differentially expressed (p\u003csub\u003eadj\u003c/sub\u003e \u0026lt; 0.05) five days prior to (pre-infection), three, seven, and fifteen days post-infection (dpi), respectively (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e, and Figures \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e-A).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDifferentially expressed genes (DEGs) in the pre-infection time point reveal good candidate determinants for susceptibility to LSD. They are expressed without the infection being present but may be predictors for disease outcome. The experimental infection may influence in the gene expression and activate GO and pathways that do not necessarily result from the induced infection and propose confounding determinants. The symptomatic vs. asymptomatic contrast on pre-infection revealed a few DEGs with an interesting trend. For example, Interleukin 1 Receptor Accessory Protein (IL1RAP) gene was significantly down-regulated five days pre-infection (p\u003csub\u003eadj\u003c/sub\u003e \u0026lt; 0.05) in cattle that showed the symptoms after challenge (susceptible) but was up-regulated three dpi in the same animals. This gene is located on chromosome one and includes twelve exons ordered as four transcripts. IL1RAP is an essential regulator of redox homeostasis and a cell-surface protein best known as a co-receptor for interleukin 1 receptor signaling (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAlso, its expression was significantly up-regulated after challenge over time among symptomatic animals (p\u003csub\u003eadj\u003c/sub\u003e \u0026lt; 0.05). IL1RAP was thus differentially expressed at each time point post-infection versus pre-infection (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). It can be concluded that IL1RAP may have a key role in LSD susceptibility and the control of the disease where high expression of IL1RAP is associated with disease occurrence.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eGene expression of \u003cem\u003eIL1RAP\u003c/em\u003e gene (\u003cem\u003eENSBTAG00000013205\u003c/em\u003e) in different contrasts (p\u003csub\u003eadj\u003c/sub\u003e \u0026lt; 0.05)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eContrast\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003elog\u003csub\u003e2\u003c/sub\u003e Fold Change\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003epvalue\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003epadj\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e\u003cb\u003eSymp vs. Asymp\u003c/b\u003e\u003c/p\u003e \u003cp\u003e(BaseMean\u0026thinsp;=\u0026thinsp;21.432)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003epre-infection\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e-20.158\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.55E-11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.28E-07\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e3 dpi\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e19.558\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.28E-08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.81E-05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e7 dpi\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.389\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.99\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e15 dpi\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e-0.990\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cb\u003eSymptomatic over time\u003c/b\u003e\u003c/p\u003e \u003cp\u003e(Post-infection vs. pre-infection)\u003c/p\u003e \u003cp\u003e(BaseMean\u0026thinsp;=\u0026thinsp;21.528)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e3dpi vs. pre-infection\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e14.998\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8.25E-07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.0022\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e7dpi vs. pre-infection\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e17.296\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.19E-08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.08E-05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e15dpi vs. pre-infection\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e15.721\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.27E-07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.00034\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eDay seven post infection showed the highest number of significant DEGs (p\u003csub\u003eadj\u003c/sub\u003e \u0026lt; 0.05) and the relatively low number of shared ones with other time points (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e, Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e, Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e, Figure \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e). In addition, the enriched GO terms were only seen in symptomatic animals vs. pre-infection ones on 7dpi (Figure \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e). They were mostly relevant to immune responses. These results indicate that day 7 is critical in LSD infection.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDifferential expression of \u003cem\u003eIL1RAP\u003c/em\u003e show subsequent enrichment for the cytokine-cytokine receptor interaction pathway with five other DEGs (p\u003csub\u003eadj\u003c/sub\u003e \u0026lt; 0.05) between symptomatic animals on seven dpi compared to pre-infection time (Figure \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e). The therapeutic potential of targeting interleukin-1 family cytokines has been suggested in chronic human inflammatory skin diseases that show similar symptoms to LSD (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). It was suggested as promising target for immunotherapy of metastasis (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e) using antibodies in humans (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe expression of \u003cem\u003eIL1RAP\u003c/em\u003e prior to any LSD outbreak may promise to distinguish the resilient and susceptible cattle for LSD through a high-resolution and low false discovery rate (FDR) diagnostic Real-time PCR test. Some human studies have already shown its involvement in similar diseases.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eLIMITATIONS\u003c/h2\u003e \u003cp\u003eThe work presented here was a retrospective pilot study to investigate the utility of bio-banked whole blood samples to analyze the transcriptome of cattle at different time points before and after artificial infection with LSDV. While the study clearly showed the merits of routine biobanking for retrospective analyses it also has clear limitations: 1) the number of samples is limited by the experiment that was previously done. Transcriptomics was not the main purpose of the original experiment. 2) the time-points of sampling are ad-hoc and may not reflect the key events in terms of gene expression. Other time points may have given a more informative gene expression signature. The main weakness of the present study is that we only had five biological replicates, with three, respectively two replicates per disease outcome. This means that any statistical inferences from the study are preliminary and require replication in a larger study. Despite the limited sample size, the results make biological sense. Another limitation of the present study was the use of a fragment based sequencing approach as opposed to whole-transcriptome sequencing. This is more cost-effective but prevents the identification of splice variation or transcript length variation between animals. Given the cost and severity of animal experiments involving potentially lethal viruses, transcriptome studies will always have to be planned as a complement to more invasive studies like transmission experiments or vaccine trials. This implies a key role for systematic biobanking to allow the accumulation of sufficient suitable samples across studies. In turn, this requires meticulous data recording and documentation of metadata.\u003c/p\u003e \u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eDPI\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDays post infection\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eDEG\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDifferentially Expressed Genes\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eGO\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGene Ontology\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eIL1RAP\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eInterleukin 1 Receptor Accessory Protein\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eKEGG\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eKyoto Encyclopedia of Genes and Genomes\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eLSD\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eLumpy Skin Disease\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eLSDV\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eLumpy Skin Disease Virus\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003ePCA\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePrinciple Component Analysis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eRINe\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eRNA Integrity Number equivalent\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval.\u0026nbsp;\u003c/strong\u003eThe present research was based on bio-banked samples stored at Sciensano. The animal experiments were conducted according to the European Union and Belgian regulations on animal welfare in experimentation. The protocol was approved by the joint Ethical Committee of Sciensano, authorization number 20150605-01_EC_Dierproef aanvraag_LSDV_BMG_2015.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication.\u0026nbsp;\u003c/strong\u003e Not applicable. No personal data included in this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials.\u0026nbsp;\u003c/strong\u003eRaw RNA Sequencing reads are publicly available in the EBI ArrayExpress repository (https://www.ebi.ac.uk/biostudies/arrayexpress/) with accession number E-MTAB-12547. \u0026nbsp;The blood samples used in this study were finished completely during RNA extractions and are no longer available.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests.\u0026nbsp;\u003c/strong\u003eThe authors do not declare any competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding.\u0026nbsp;\u003c/strong\u003eThis project was funded by the European Union\u0026apos;s Horizon 2020 research and innovation program under agreement No 773701.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors contribution.\u0026nbsp;\u003c/strong\u003eSVB, KDK, AH and DJK designed the pilot study where the latter was project leader for the pilot study. KDK, AH and LM performed the original infection studies at SCIENSANO. \u0026nbsp;SVB performed RNA extractions and prepared samples for sequencing. MHB, AN and TK performed statistical and bioinformatics analyses. MHB drafted the manuscript with significant input from DJK. All authors provided edits and comments to the manuscript and approved the final manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSpecial gratitude to the animal caretakers of the Experimental Centre of Sciensano (Belgium) for collecting the data and samples. RNA sequencing was professionally performed at the VIB Neuromics Support Facility (University of Antwerp, Center for Molecular Neurology, Belgium). The authors thank the SLU Bioinformatics Infrastructure, Uppsala, Sweden for the bioinformatics support. We are very grateful to the Kimron Veterinary Institute (Israel) and the Field Israeli Veterinary Services for providing us with the LSDV strain LSD/OA3-Ts. MORAN. M. seed pass.4.155920/2012.20.1.13.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eYılmaz H. (2017) Lumpy Skin Disease: Global and Turkish Perspectives.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAnwar A., Na-Lampang K., Preyavichyapugdee N. \u0026amp; Punyapornwithaya V. (2022) Lumpy Skin Disease Outbreaks in Africa, Europe, and Asia (2005\u0026ndash;2022): Multiple Change Point Analysis and Time Series Forecast. Viruses 14, 2203.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVandenbussche F, Mathijs E, Philips W, Saduakassova M, De Leeuw I, Sultanov A, Haegeman A, De Clercq K. (2022) Recombinant LSDV Strains in Asia: Vaccine Spillover or Natural Emergence? Viruses 14, 1429.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCalistri EFSA, De Clercq P, Gubbins K, Klement S, Stegeman E, Corti\u0026ntilde;as Abrahantes A, Marojevic J, Antoniou D S.-E., Broglia A. (2020) Lumpy skin disease epidemiological report IV: data collection and analysis. EFSA J 18, e06010.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHaegeman A, De Leeuw I, Mostin L, Campe WV, Aerts L, Venter E, Tuppurainen E, Saegerman C, De Clercq K. Comparative Evaluation of Lumpy Skin Disease Virus-Based Live Attenuated Vaccines. Vaccines. 2021;9:473.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoll P, Ante M, Seitz A, Reda T. QuantSeq 3\u0026prime; mRNA sequencing for RNA quantification. Nat Methods. 2014;11:i\u0026ndash;iii.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBushnell B. (2014) BBMap: A Fast, Accurate, Splice-Aware Aligner. Technical report, Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim D, Paggi JM, Park C, Bennett C, Salzberg SL. Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype. Nat Biotechnol. 2019;37:907\u0026ndash;15.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiao Y, Smyth GK, Shi W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics. 2013;30:923\u0026ndash;30.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLove MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014;15:550.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eR Core Team. R: A language and environment for statistical computing. Vienna, Austria: R Foundation for Statistical Computing; 2022.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu T, Hu E, Xu S, Chen M, Guo P, Dai Z, Feng T, Zhou L, Tang W, Zhan L, Fu X, Liu S, Bo X, Yu G. clusterProfiler 4.0: A universal enrichment tool for interpreting omics data. Innov. 2021;2:100141.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCarlson M. \u0026amp; (2021) org.Bt.eg.db: Genome wide annotation for Bovine. R package version 3.14.0.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eK\u0026auml;ll L, Canterbury JD, Weston J, Noble WS, MacCoss MJ. Semi-supervised learning for peptide identification from shotgun proteomics datasets. Nat Methods. 2007;4:923\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCalabrese L, Fiocco Z, Satoh TK, Peris K, French LE. Therapeutic potential of targeting interleukin-1 family cytokines in chronic inflammatory skin diseases*. Br J Dermatol. 2022;186:925\u0026ndash;41.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang H-F, Hughes CS, Li W, He J-Z, Surdez D, El-Naggar AM, Cheng H, Prudova A, Delaidelli A, Negri GL, Li X, \u0026Oslash;rum-Madsen MS, Lizardo MM, Oo HZ, Colborne S, Shyp T, Scopim-Ribeiro R, Hammond CA, Dhez A-C, Langman S, Lim JKM, Kung SHY, Li A, Steino A, Daugaard M, Parker SJ, Geltink RIK, Orentas RJ, Xu L-Y, Morin GB, Delattre O, Dimitrov DS, Sorensen PH. (2021) Proteomic Screens for Suppressors of Anoikis Identify IL1RAP as a Promising Surface Target in Ewing Sarcoma. Cancer Discovery 11, 2884\u0026thinsp;\u0026ndash;\u0026thinsp;903.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRobbrecht D, Jungels C, Sorensen MM, Spanggaard I, Eskens F, Fretland S\u0026Oslash;, Guren TK, Aftimos P, Liberg D, Svedman C, Thorsson L, Steeghs N, Awada A. First-in-human phase 1 dose-escalation study of CAN04, a first-in-class interleukin-1 receptor accessory protein (IL1RAP) antibody in patients with solid tumours. Br J Cancer. 2022;126:1010\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-research-notes","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"resn","sideBox":"Learn more about [BMC Research Notes](http://bmcresnotes.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/resn/default.aspx","title":"BMC Research Notes","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Cattle, Lumpy Skin Disease (LSD), IL1RAP gene, Transcriptome Profiling, Host Determinants, Differentially Expressed Genes (DEG)","lastPublishedDoi":"10.21203/rs.3.rs-4950902/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4950902/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eObjective\u003c/h2\u003e \u003cp\u003eLumpy skin disease (LSD) is an acute or subacute systemic viral disease of cattle that shows variation in the response of cattle to LSD virus infection. To better understand the mechanisms underlying this response diversity in field studies and under carefully controlled artificial infections, we studied the differentially expressed genes (DEGs) between two resilient versus three susceptible Holstein bulls before an infection challenge and three time points after that.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe host transcriptome profiling revealed that IL1RAP gene expression could be a potential determinant in distinguishing between resilient and susceptible cattle (padj\u0026thinsp;\u0026lt;\u0026thinsp;0.05). It was significantly shifted from up-regulated prior to infection to down-regulated three days post-infection in the LSD-resilient cattle. Its expression remained up-regulated among the susceptible cattle post-infection compared to pre-infection. The results showed that seven days post-infection may be a critical time point for LSD infection. The Gene Ontology (GO) and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment test showed a few enriched GO terms and pathways relevant to the LSD and the involvement of the IL1RAP gene. This pilot study, with limited statistical power, is the first to investigate bovine gene expression profiling in response to LSDV.\u003c/p\u003e","manuscriptTitle":"Host transcriptome profiling for resistance against Lumpy Skin Disease (LSD)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-09-23 11:29:36","doi":"10.21203/rs.3.rs-4950902/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-08-23T09:01:59+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-08-23T02:26:28+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-08-23T02:26:04+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Research Notes","date":"2024-08-21T10:32:24+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-research-notes","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"resn","sideBox":"Learn more about [BMC Research Notes](http://bmcresnotes.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/resn/default.aspx","title":"BMC Research Notes","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3d05c780-22a1-4a80-a685-33e4cd87ee11","owner":[],"postedDate":"September 23rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-07-21T16:09:46+00:00","versionOfRecord":{"articleIdentity":"rs-4950902","link":"https://doi.org/10.1186/s13104-025-07388-9","journal":{"identity":"bmc-research-notes","isVorOnly":false,"title":"BMC Research Notes"},"publishedOn":"2025-07-15 16:05:43","publishedOnDateReadable":"July 15th, 2025"},"versionCreatedAt":"2024-09-23 11:29:36","video":"","vorDoi":"10.1186/s13104-025-07388-9","vorDoiUrl":"https://doi.org/10.1186/s13104-025-07388-9","workflowStages":[]},"version":"v1","identity":"rs-4950902","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4950902","identity":"rs-4950902","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}