University of Toronto T-Space



Figure S1. Phylogenetic tree of ULBP gene family among human, Himalayan marmot, mouse and tree shrew. The branch line thickness indicates the degree of bootstrap-based support for the node with 1000 replications. H. sapiens, human; M. musculus, mouse; M. Himalayana, Himalayan marmot; T. belangeri, Tree shrew.Figure S2. The weighted gene co-expression network analysis dendrogram indicates different gene modules in normal and infected liver and lung Table S5. Hematological data of Y. pestis infected Himalayan marmotsIndexControlInfectionWhite blood cell (10^9/L)6.91±0.777.27±1.35Red blood cell (10^12/L)5.86±0.195.228±0.20Hematoglobin (g/L)151.2±2.48131.4±6.45*Hematocrit (%)44.98±0.8540.66±1.73Mean corpuscular volume (fl)76.98±1.8977.78±1.21Mean corpuscular hemoglobin (Pg)25.84±0.5625.1±0.43Mean corpuscular hemoglobin concentration (g/L)336±2.00322.8±2.85**Erythrocyte hemoglobin distribution width (%)14.14±0.3915.58±0.75Red cell distribution width standard deviation (fL)45.52±0.7244.9±1.96Platelet count (10^9/L)270±68.75330.8±66.51mean platelet volume (fL)7.96±0.359.82±0.36**platelet distribution width15.86±0.0911.8±1.34*Thrombocytocrit (%)0.214±0.050.322±0.07Note: Data are expressed as mean ± SEM. n=5 (3 males and 2 females). *, p < 0.05; **, p < 0.01. Table S6. Transcriptomic data of the liver and lung tissues from Himalayan marmots infected with or without EV76 TissuesSample IDInsert sizeRead lengthRead number (PE)Data (bp)GC (%)Q30 (%)Normal liverN318015024,848,268 7,451,790,417 51.46%91.73%N218015021,148,715 6,342,164,837 50.35%92.82%N518015020,479,628 6,141,078,553 53.39%91.65%N418015026,102,297 7,828,022,886 51.27%92.78%N118015024,114,098 7,230,319,793 52.96%92.53%Infected liverI118015022,455,268 6,734,360,243 50.74%92.38%I518015025,060,501 7,515,885,276 50.92%92.34%I318015019,010,724 5,700,496,869 51.16%90.77%I418015022,210,326 6,659,610,796 52.17%92.47%I218015024,357,406 7,304,353,312 52.08%92.52%Total / Average---229,787,231 68,908,082,982 51.65%92.20%Normal lungN218015016,423,306 4,956,053,872 54.59%85.04%N318015017,782,385 5,362,675,008 55.23%85.10%N418015018,172,449 5,484,967,403 53.94%85.22%Infected lungI118015026,679,630 8,000,441,010 52.78%89.43%I318015023,843,081 7,150,565,628 52.84%91.43%I418015028,300,604 8,487,754,364 51.82%91.90%I218015017,622,671 5,316,395,418 54.81%85.67%Total / Average---148,824,126 44,758,852,703 53.72%87.68% Table S7. Differentially expressed genes in the lungs of Himalayan marmot after EV76 infectionCategoryGene IDGene symbolDescriptionFold ChangeDefense responseMar16137SlipSecretory leukocyte peptidase inhibitor2.57 Mar25600Igk-V26-10aIg kappa chain V-II region 26-101.92 Mar25917Igl-5aImmunoglobulin lambda-like polypeptide 51.92 Mar17026Cst7Cystatin F1.90 Mar12478Rgs1Regulator of G-protein signaling 11.79 Mar07978Mpeg1Macrophage expressed 11.87 Mar10076Fcgr3aFc fragment of IgG, low affinity IIIa, receptor 1.84 Mar03821Emr2EGF-like module-containing mucin-like hormone receptor-like 2-like1.76 Mar13270Clec4eC-type lectin domain family 4, member E 1.72 Mar23142Gbp7Guanylate binding protein 71.65 Mar08060Clec6aC-type lectin domain family 6 member A1.57 Mar17669FybFYN binding protein1.50 Mar23546Gvin1Interferon-induced very large GTPase 1 1.46 Mar05196Gbp6Guanylate-binding protein 61.41 Mar13579Nlrp7NACHT, LRR and PYD domains-containing protein 71.35 Mar15491ItgalIntegrin, alpha L1.33 Mar00079Casp4Caspase-41.25 Mar16447Itga4Integrin, alpha 4 1.24 Mar17506Tra29T-cell receptor alpha chain precursor V and C regions1.19 Mar11694CybbCytochrome b-245 heavy chain1.17 Mar06948CytipCytohesin 1 interacting protein1.13 Antigen processing and presentationMar01950Clec4dC-type lectin domain family 4, member D1.82 Mar07827Clec12aC-type lectin domain family 12, member A1.69 Mar09684MHC-DraMHC class II histocompatibility antigen, DR alpha chain1.22 Mar11137MHC-DPbMHC class II histocompatibility antigen, DP beta 1 chain1.15 Mar07875Psmb8Proteasome subunit, beta type, 80.99 Immune cell proliferation and activationMar10444Ubdubiquitin D2.83 Mar12979Ms4a4aMembrane-spanning 4-domains subfamily A member 4A1.73 Mar00909Clec4aC-type lectin domain family 4 member A1.66 Mar10146Ms4a6bMembrane-spanning 4-domains subfamily A member 6B1.64 Mar12084PtprcProtein tyrosine phosphatase, receptor type, C1.52 Mar23155Cd24Signal transducer CD241.30 Mar26740Slamf1Signaling lymphocytic activation molecule family member 11.25 Mar04837Lcp1Lymphocyte cytosolic protein 11.14 Mar25859Cd53Leukocyte surface antigen CD531.01 Cytokine and receptorMar26682PFpr1Formyl peptide receptor 12.48 Mar16793Ccl8Chemokine ligand 8 1.85 Mar13389Cxcr1Chemokine receptor 11.81 Mar15848Il2rgInterleukin 2 receptor, gamma1.24 Cell growth/ApoptosisMar14485NaipNLR family, apoptosis inhibitory protein 1.72 Mar04804Trpm2Transient receptor potential cation channel, subfamily M, member 21.62 Mar25797Sh3bgriSH3 domain-binding glutamic acid-rich-like protein1.44 Mar06794Birc3Baculoviral IAP repeat containing 31.35 Biosynthetic processMar09122Alas2Aminolevulinate, delta, synthase 2 -2.40 Mar16886HbaHemoglobin subunit theta-1-2.00 Signal transductionMar20020Igfbp2Insulin-like growth factor binding protein 2 -1.40 Mar16984Hmcn2Hemicentin 2 -1.28 Mar13746Znf503Zinc finger protein 503-1.34 Mar12312Ano1Calcium activated chloride channel-1.04 UnknownMar00164MybphMyosin binding protein H1.76 Mar25564UnknownClone VMRC20-433G181.54 Mar26751UnknownClone XX-28672257A7-2.33 Mar14947Fam110dFamily with sequence similarity 110, member D-1.24 Mar12744Klhdc7aKelch domain containing 7A-1.04 Table S8. Differentially expressed genes in Himalayan marmot livers after EV76 infectionCategoryGene IDGene symbolDescriptionFold changeDefense responseMar15378Sftpa1Pulmonary surfactant-associated protein AInfMar04994Scgb3a2Secretoglobin, family 3A, member 2 InfMar06338SftpbSurfactant protein B8.43 Mar14460Bpifb1BPI fold-containing family B member 17.45 Mar10237AgerAdvanced glycosylation end product-specific receptor4.38 Mar13995Klk8Kallikrein-related peptidase 8 3.32 Mar25970IglIg lambda chain V-I region BL22.96 Mar27116Igl-5bImmunoglobulin lambda-like polypeptide 52.54 Mar11760Gprc5aG protein-coupled receptor, family C, group 5, member A2.45 Mar26503Igl-5cImmunoglobulin lambda-like polypeptide 52.35 Mar26172Igk-V26-10bIg kappa chain V-II region 26-102.06 Mar26454Saa3Serum amyloid A-3 protein2.01 Mar08076AoahAcyloxyacyl hydrolase (neutrophil) 1.50 Mar18243Hspa4lHeat shock 70kDa protein 4-like 1.11 Mar01227Col3a1Collagen, type III, alpha 11.07 Mar00079Casp4Caspase-41.00 Mar01822Magee1Melanoma-associated antigen E1-3.14 Mar13263Map1aMicrotubule-associated protein 1A -2.60 Immune cell proliferation and activationMar10444UbdUbiquitin D3.10 Mar00033Il7raInterleukin-7 receptor subunit alpha1.65 Mar10146Ms4a6bMembrane-spanning 4-domains subfamily A member 6B1.25 Mar15582Klf10Kruppel-like factor 100.91 Signal transductionMar03330PtprnProtein tyrosine phosphatase, receptor type, N-InfMar12921Gria2Glutamate receptor, ionotropic, AMPA 2-InfMar10678Pcsk1nProprotein convertase subtilisin/kexin type 1 inhibitor-6.86 Mar02354Ppp2r2cProtein phosphatase 2, regulatory subunit B, gamma-6.38 Mar11154HpcaHippocalcin-6.29 Mar11645Nrsn1Neurensin 1-6.17 Mar02249Bai1Brain-specific angiogenesis inhibitor 1 -5.59 Mar15057Padi2Peptidyl arginine deiminase, type II-3.92 Mar11290Gpr37l1G protein-coupled receptor 37 like 1Mar17617Mapk8ip2Mitogen-activated protein kinase 8 interacting protein 2-3.66 Mar08653Clstn3Calsyntenin 3 -2.99 Mar06434Scg2Secretogranin II-2.62 Mar00844Gnao1G protein, alpha activating activity polypeptide O-2.28 Mar08397Caln1Calneuron 1 -2.17 Mar10762CadpsCa++-dependent secretion activator-2.06 Mar15538-Hip-hop1 splice variant-2.02 Mar04551Chn1Heterocephalus glaber chimerin 1-1.94 Mar12421Disp2Dispatched homolog 2-1.93 Mar25679Pacsin1Protein kinase C and casein kinase substrate in neurons 1-1.81 Mar06689Cnksr1Connector enhancer of kinase suppressor of Ras 1-1.61 Mar13581PsdPleckstrin and Sec7 domain containing-1.53 Mar01614Gng7Guanine nucleotide binding protein gamma 7-1.47 Mar20915Gpc3Glypican 3-1.31 Mar01746Trim72Tripartite motif containing 72-1.26 TranscriptionMar16012Chd5Chromodomain helicase DNA binding protein 5 -3.97Mar18623Olig1Oligodendrocyte transcription factor 1-1.93Mar00483Tbx1T-box 1 -1.35Regulation of protein kinase activityMar16980Elfn2Extracellular leucine-rich repeat and fibronectin type III domain containing 2 -InfMar03910Sez6l2Seizure related 6 homolog (mouse)-like 2-7.52Mar01872Uchl1Ubiquitin carboxyl-terminal esterase L1-5.482Mar09675CamkvCaM kinase-like vesicle-associated-3.47Mar11780Agap2ArfGAP with GTPase domain, ankyrin repeat and PH domain 2 -1.31Glycosylation Mar05672B3gat1beta-1,3-glucuronyltransferase 1-InfMar17750Wbscr17Williams-Beuren syndrome chromosome region 17-3.12Cell growth/ApoptosisMar11255DdnDendrin-InfMar07997Cdk5r2Cyclin-dependent kinase 5, regulatory subunit 2-InfMar11571SncbSynuclein, beta -7.48 Mar01453Faim2Fas apoptotic inhibitory molecule 2-6.01 Mar14955Tubb3Tubulin, beta 3 class III -5.31 Mar04954Aplp1Amyloid beta (A4) precursor-like protein 1 -4.70 Mar13421Camk2aCalcium/calmodulin-dependent protein kinase II alpha -4.65 Mar01891Brsk1BR serine/threonine kinase 1 -3.74 Mar13694Peg3Paternally expressed 3-1.73 Development and differentiationMar02569Gap43Neuromodulin-like-InfMar15773Sez6lSeizure related 6 homolog (mouse)-like -InfMar09609GfapGlial fibrillary acidic protein-InfMar10435Rtn1Reticulon 1-6.97 Mar21001MobpMyelin-associated oligodendrocyte basic protein-6.26 Mar12961BcanBrevican-5.72 Mar13229NrcamNeuronal cell adhesion molecule-3.85 Mar00784Plp1Proteolipid protein 1 -3.62 Mar00862Map2Microtubule-associated protein 2-3.51 Mar05133Cspg5Chondroitin sulfate proteoglycan 5-3.08 Mar05132Stmn2Stathmin-2-2.78 Mar12697Gpm6bGlycoprotein M6B -2.37 Mar09750Gpm6aGlycoprotein M6A -2.33 Mar19893Map1bMicrotubule-associated protein 1B -1.42 Mar16684Prrx1Paired related homeobox 1-1.27 Metabolic processMar19413Pcsk2Proprotein convertase subtilisin/kexin type 2 -InfMar07865PhyhipPhytanoyl-CoA 2-hydroxylase interacting protein-6.90 Mar11998Unc13aUnc-13 homolog A-6.35 Mar02040Scd1Acyl-CoA desaturase 1-5.17 Mar10811Snap25Synaptosomal-associated protein, 25kDa -4.54 Mar15100Eef1a2Eukaryotic translation elongation factor 1 alpha 2 -4.00 Mar02176Nat8lN-acetyltransferase 8-like-3.69 Mar02462LctLactase-2.69 Mar16568Enpp5Ectonucleotide pyrophosphatase/phosphodiesterase 5-2.60 Mar09042Eno2Enolase 2 (gamma, neuronal)-2.37 Mar05410Arg2Arginase 2-1.94 Mar18900AldocAldolase C, fructose-bisphosphate-1.10Mar04464Cyp2c55Cytochrome P450 2C55-like-0.97TransportMar05346Snap91Synaptosomal-associated protein, 91kDa -InfMar01209Cplx1Complexin 1-InfMar09007Syt1Synaptotagmin I-6.72 Mar16265Slc1a3Solute carrier family 1 (glial high affinity glutamate transporter), member 3-5.27 Mar10008Kif1aKinesin family member 1A-4.99 Mar17638NeflNeurofilament, light polypeptide-4.95 Mar03168Cplx2Complexin 2 transcript variant X2-4.91 Mar19114Kcnq2Potassium voltage-gated channel, KQT-like subfamily, member 2-4.37 Mar16565NefmNeurofilament, medium polypeptide (Nefm)-4.24 Mar12050Kif5aKinesin family member 5A-4.13 Mar18092Kif5cKinesin family member 5C-3.86 Mar01501Sv2bSynaptic vesicle glycoprotein 2B-3.29 Mar05787Slc12a5Solute carrier family 12 (potassium/chloride transporter), member 5-3.14 Mar03321Rph3aRabphilin 3A homolog-2.85 Mar04519Slc6a17Solute carrier family 6 (neutral amino acid transporter), member 17-2.56 Mar04054Mlc1Megalencephalic leukoencephalopathy with subcortical cysts 1-2.52 Mar05924Sv2aSynaptic vesicle glycoprotein 2A-2.40 Mar03716Rab6bRAB6B, member RAS oncogene family -2.17 Mar15921Dnm1Dynamin 1-2.05 Mar03938Tmem59lTransmembrane protein 59-like -1.40 Mar09928Tmc3Transmembrane channel-like 3-1.32 Mar15443Flvcr2Feline leukemia virus subgroup C cellular receptor family, member 2 -1.11 MiscellaneousMar08944Aqp5Aquaporin 54.50Mar12729Adam1aDisintegrin and metalloproteinase domain-containing protein 1a2.46Mar00304Slc34a2Solute carrier family 34 (type II sodium/phosphate contransporter), member 22.12 Mar12213Fam43aFamily with sequence similarity 43, member A1.29 Mar07900ChkaCholine kinase alpha0.88 Mar08087Pdzd4PDZ domain containing 4-InfMar20150Cryba4Crystallin, beta A4-5.70 Mar05120Rbfox3RNA binding protein, fox-1 homolog 3-5.63 Mar04450Jph3Junctophilin 3-5.23 Mar09129BaalcBrain and acute leukemia, cytoplasmic -3.92 Mar10266Bcas1Breast carcinoma amplified sequence 1 -3.84 Mar17821Add2Adducin 2-3.31 Mar10818Shank1SH3 and multiple ankyrin repeat domains 1-2.26 Mar07583Prrt1Proline-rich transmembrane protein 1 -2.18 Mar13186Syn1Synapsin I-2.00 Mar14868Nptx1Neuronal pentraxin I -1.54 Mar08052FetubFetuin B-1.40 UnknownMar26449-Uncharacterized LOC101971201 1.94 Mar15606Tmem130Transmembrane protein 130 -3.75 Mar13648Mpped1Metallophosphoesterase domain containing 1-1.62 Mar21967Tmem92Transmembrane protein 92-1.16 #script of detecting DEGs# script of heatmap for DEGslibrary(gplots)library(ggplot2)library(RColorBrewer)library(reshape2)exp <- read.table("heatmap",header = T, stringsAsFactors = F)rownames(exp) <- exp$genedistCor <- function(x) as.dist(1-cor(t(x),method="spearman"))hclustAvg <- function(x) hclust(x, method="average")exp <- as.matrix(exp[,2:24])my_palette <- colorRampPalette(c("blue","blue","white","firebrick1","red"))(n = 100)heatmap.2(exp,col=my_palette,trace="none",dendrogram="col",distfun = distCor, hclustfun = hclustAvg,Rowv=F,Colv=T,scale='row',srtCol=0,key.title=NA,key.xlab="RPKM(Z-score)",keysize=1.5)#script of KEGG & GO enrichment analysis for DEGslibrary(clusterProfiler);go <- read.table("marmot_go_annot.txt",sep="\t",quote="");kegg <- read.table("marmot_kegg_annot.txt",sep="\t",quote="");gene <- read.table("DEG_gene.txt");go_term2gene <- data.frame(go$V1,go$V4);go_term2name <- data.frame(go$V1,go$V2);names(go_term2gene) <- c("go_term","gene");names(go_term2name) <- c("go_term","name");kegg_term2gene <- data.frame(kegg$V2,kegg$V1);kegg_term2name <- data.frame(kegg$V2,kegg$V3);names(kegg_term2gene) <- c("ko_term","gene");names(kegg_term2name) <- c("ko_term","name");gene <- as.vector(gene$V1);go_enrich <- enricher(gene=gene,pvalueCutoff = 0.05,pAdjustMethod = "BH",TERM2GENE = go_term2gene,TERM2NAME = go_term2name);write.csv(as.data.frame(go_enrich),"GO_enrichment.csv",row.names = F);kegg_enrich <- enricher(gene=gene,pvalueCutoff = 0.05,pAdjustMethod = "BH",TERM2GENE = kegg_term2gene,TERM2NAME = kegg_term2name);write.csv(as.data.frame(kegg_enrich),"KEGG_enrichment.csv",row.names = F);#Script for WGCNA analysislibrary(WGCNA);workingDir = ".";setwd(workingDir); options(stringsAsFactors = FALSE);enableWGCNAThreads()Data = read.csv("marmot_exp.csv");dim(Data);names(Data);datExpr0 = as.data.frame(t(Data[, -c(1:11)]));names(datExpr0) = Data$gene;rownames(datExpr0) = names(Data)[-c(1:11)];gsg = goodSamplesGenes(datExpr0, verbose = 3);gsg$allOKif (!gsg$allOK){ # Optionally, print the gene and sample names that were removed: if (sum(!gsg$goodGenes)>0) printFlush(paste("Removing genes:", paste(names(datExpr0)[!gsg$goodGenes], collapse = ", "))); if (sum(!gsg$goodSamples)>0) printFlush(paste("Removing samples:", paste(rownames(datExpr0)[!gsg$goodSamples], collapse = ", "))); # Remove the offending genes and samples from the data: datExpr0 = datExpr0[gsg$goodSamples, gsg$goodGenes]}sampleTree = hclust(dist(datExpr0), method = "average");# Plot the sample tree: Open a graphic output window of size 12 by 9 inches# The user should change the dimensions if the window is too large or too small.sizeGrWindow(12,9)#pdf(file = "Plots/sampleClustering.pdf", width = 12, height = 9);par(cex = 0.6);par(mar = c(0,4,2,0))plot(sampleTree, main = "Sample clustering to detect outliers", sub="", xlab="", cex.lab = 1.5, cex.axis = 1.5, cex.main = 2)# Plot a line to show the cutabline(h = 15, col = "red");# Determine cluster under the lineclust = cutreeStatic(sampleTree, cutHeight = 15, minSize = 10)table(clust)# clust 1 contains the samples we want to keep.keepSamples = (clust==1)datExpr = datExpr0[keepSamples, ]nGenes = ncol(datExpr)nSamples = nrow(datExpr)# Choose a set of soft-thresholding powerspowers = c(c(1:10), seq(from = 12, to=20, by=2))；# Call the network topology analysis functionsft = pickSoftThreshold(datExpr, powerVector = powers, verbose = 5)；# Plot the resultssizeGrWindow(9, 5)；par(mfrow = c(1,2));cex1 = 0.9;# Scale-free topology fit index as a function of the soft-thresholding powerplot(sft$fitIndices[,1], -sign(sft$fitIndices[,3])*sft$fitIndices[,2], xlab="Soft Threshold (power)",ylab="Scale Free Topology Model Fit,signed R^2",type="n", main = paste("Scale independence"));text(sft$fitIndices[,1], -sign(sft$fitIndices[,3])*sft$fitIndices[,2], labels=powers,cex=cex1,col="red");# this line corresponds to using an R^2 cut-off of habline(h=0.90,col="red")；# Mean connectivity as a function of the soft-thresholding powerplot(sft$fitIndices[,1], sft$fitIndices[,5], xlab="Soft Threshold (power)",ylab="Mean Connectivity", type="n", main = paste("Mean connectivity"))；text(sft$fitIndices[,1], sft$fitIndices[,5], labels=powers, cex=cex1,col="red")；net = blockwiseModules(datExpr, power = 6, TOMType = "unsigned", minModuleSize = 30, reassignThreshold = 0, mergeCutHeight = 0.25, numericLabels = TRUE, pamRespectsDendro = FALSE, saveTOMs = TRUE, saveTOMFileBase = "femaleMouseTOM", verbose = 3)；# open a graphics windowsizeGrWindow(12, 9)；# Convert labels to colors for plottingmergedColors = labels2colors(net$colors)；# Plot the dendrogram and the module colors underneathplotDendroAndColors(net$dendrograms[[1]], mergedColors[net$blockGenes[[1]]], "Module colors", dendroLabels = FALSE, hang = 0.03, addGuide = TRUE, guideHang = 0.05)；moduleLabels = net$colors；moduleColors = labels2colors(net$colors)；MEs = net$MEs;geneTree = net$dendrograms[[1]];# Calculate topological dissTOM = 1-TOMsimilarityFromExpr(datExpr, power = 6);# Transform dissTOM with a power to make moderately strong connections more visible in the heatmapplotTOM = dissTOM^7;# Set diagonal to NA for a nicer plotdiag(plotTOM) = NA;# Call the plot functionsizeGrWindow(9,9)；TOMplot(plotTOM, geneTree, moduleColors, main = "Network heatmap plot, all genes")；nSelect = 400；set.seed(10);select = sample(nGenes, size = nSelect);selectTOM = dissTOM[select, select];selectTree = hclust(as.dist(selectTOM), method = "average")；selectColors = moduleColors[select];sizeGrWindow(9,9)；plotDiss = selectTOM^7;diag(plotDiss) = NA;TOMplot(plotDiss, selectTree, selectColors, main = "Network heatmap plot, selected genes")；# Recalculate module eigengenesMEs = moduleEigengenes(datExpr, moduleColors)$eigengenes；# Plot the dendrogramsizeGrWindow(6,6);par(cex = 1.0)；plotEigengeneNetworks(MES, "Eigengene dendrogram", marDendro = c(0,4,2,0), plotHeatmaps = FALSE)；# Plot the heatmap matrixpar(cex = 1.0)；plotEigengeneNetworks(MES, "Eigengene adjacency heatmap", marHeatmap = c(3,4,2,2), plotDendrograms = FALSE, xLabelsAngle = 90)； ................
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