Open Access RESEARCH ARTICLE. Genetics Selection Evolution

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1 DOI /s Genetis Seletion Evolution RESEARCH ARTICLE Open Aess Comprison of host geneti ftors influening pig response to infetion with two North Amerin isoltes of porine reprodutive nd respirtory syndrome virus Andrew S. Hess 1, Zeenth Islm 2, Melnie K. Hess 1, Rymond R. R. Rowlnd 3, Jon K. Lunney 4, Andre Doeshl Wilson 2, Grhm S. Plstow 5 nd Jk C. M. Dekkers 1* Abstrt Bkground: Porine reprodutive nd respirtory syndrome (PRRS) is one of the most importnt swine diseses in the world nd geneti seletion of pigs for inresed resistne to PRRS is n ttrtive method to improve the helth sttus of the swine herd. This study ompred phenotypi nd geneti responses to infetion with one of two genetilly distint type 2 PRRS virus (PRRSV) isoltes: NVSL (NVSL) nd KS (KS06), nd evluted whether the single nuleotide polymorphism (SNP) WUR (WUR) on hromosome 4 tht ws ssoited with virl lod nd weight gin under infetion with NVSL lso hs n effet on response to infetion ross North Amerin PRRSV isoltes. Wood s lttion urve ws fitted to repeted viremi mesurements to derive five urve hrteristis tht were evluted. Results: Infetion with NVSL ws hrterized by rehing 14 ± 2 % higher pek viremi (PV) 2.5 ± 0.6 dys erlier (time to pek; TP) thn KS06, followed by 36 ± 1 % fster virus lerne, whih ourred 3.9 ± 0.7 dys sooner. Weight gin from 0 to 42 dys post-infetion (WG) tended to be higher under infetion with KS06 thn NVSL (3.7 ± 1.5 kg). Estimtes of heritbility were moderte for both PRRSV isoltes for virl lod from 0 to 21 dys postinfetion (VL) (NVSL: 0.31 ± 0.06; KS06: 0.51 ± 0.09) nd WG (NVSL: 0.33 ± 0.06; KS06: 0.31 ± 0.09). Strong negtive geneti orreltions were observed between VL nd WG for both NVSL ( 0.74 ± 0.10) nd KS06 ( 0.52 ± 0.17) infeted pigs. Pigs with genotype AB t the WUR SNP hd more desirble phenotype thn AA pigs for ll trits under infetion with NVSL, but only for VL nd PV with KS06; effets on other trits were smller nd not signifintly different from zero (P > 0.05). Geneti orreltions of host response between isoltes were strong for VL, WG nd PV. Aounting for WUR genotype hd little impt on these orreltions, suggesting tht response to PRRSV infetion hs substntil polygeni omponent tht is ommon between these two isoltes. Conlusions: These results suggest tht the KS06 PRRSV isolte is less virulent thn NVSL but tht geneti seletion for inresed resistne to either of these genetilly distint isoltes is expeted to inrese resistne to the other isolte. Bkground Porine reprodutive nd respirtory syndrome (PRRS) osts the US swine industry $664 million per yer [1]. Pst efforts to ontin PRRS hve hd limited suess, *Correspondene: jdekkers@istte.edu 1 Deprtment of Animl Siene, Iow Stte University, Ames, IA, USA Full list of uthor informtion is vilble t the end of the rtile in lrge prt due to the high muttion rte nd ntigeni vribility of PRRS virus (PRRSV), whih hve enumbered efforts to produe vines tht re ross-protetive to heterogeneous PRRSV isoltes [2]. PRRSV isoltes re lssified into two types: type 1 or Europen isoltes nd type 2 or North Amerin isoltes [3]. These two 2016 The Author(s). This rtile is distributed under the terms of the Cretive Commons Attribution 4.0 Interntionl Liense ( whih permits unrestrited use, distribution, nd reprodution in ny medium, provided you give pproprite redit to the originl uthor(s) nd the soure, provide link to the Cretive Commons liense, nd indite if hnges were mde. The Cretive Commons Publi Domin Dedition wiver ( publidomin/zero/1.0/) pplies to the dt mde vilble in this rtile, unless otherwise stted.

2 Pge 2 of 20 types re distint both genetilly [4] nd pthogenilly [5, 6]. Geneti seletion of pigs tht re more resistnt to PRRS n be n ttrtive method to improve the helth sttus of the swine herd [7]. The gol of the PRRS Host Genetis Consortium (PHGC) is to identify host genes or genomi regions tht re ssoited with inresed resistne of pigs to PRRSV infetion [8]. Previous studies using multiple ontemporry North Amerin rossbred wener pigs tht were experimentlly infeted with North Amerin isolte of PRRSV, NVSL (NVSL), identified heritble geneti omponents to virl lod nd weight gin following infetion, nd identified quntittive trit lous (QTL) on hromosome 4 whereby the single nuleotide polymorphism (SNP) WUR (WUR) hd strong ssoition with these two host response trits [9 11]. A puttive ustive muttion tht is in high linkge disequilibrium with the WUR SNP, in the gunylte binding protein 5 (GBP5) gene, ws identified by Koltes et l. [12]. The protein produed by the GBP5 gene plys ruil role in NLRP3-medited formtion of the inflmmsome, whih is involved with inflmmtory response [13]. It is urrently not known whether seletion for improved host resistne to single PRRSV isolte will improve resistne to other PRSSV isoltes. Thus, the objetives of this study were to: (1) ompre host responses to infetion with NVSL nd the genetilly distint North Amerin PRRSV isolte KS (KS06); (2) estimte the geneti prmeters of response to infetion when pigs re infeted with either NVSL or KS06; nd (3) estimte the ssoitions of the WUR SNP with response following infetion with NVSL or KS06. It ws hypothesized tht the host s geneti mke-up tht is involved in the response to infetion is highly orrelted between these two virus isoltes nd tht ssoitions of the WUR SNP with host response to infetion re similr for these two isoltes. The Wood s lttion funtion ws previously shown to ppropritely model PRRS serum viremi following experimentl infetion [14]. Thus, urve hrteristis of the fitted viremi profiles tht re derived from the Wood s urve prmeters were used to quntify different spets of the dynmis of host response to PRRSV infetion with these two isoltes. Methods Study design A detiled desription of the design, dt olletion nd moleulr tehniques used in the PHGC trils is in Lunney et l. [8]. The Knss Stte University Institutionl Animl Cre nd Use Committee pproved ll experimentl protools for these trils. Pigs used for this study were from 14 PHGC trils of ~200 wener pigs (Tble 1). Pigs were provided from ommeril breeding progrms in the United Sttes nd Cnd. Within eh tril, pigs were from single high helth frm nd geneti bkground, exept for trils 5, 8 nd 12, whih eh inluded pigs from one geneti bkground but from two frms. All soure frms were free of PRRS, Myoplsm hyopneumonie, nd swine influenz. Four breeding ompnies supplied pigs of the sme breed ross for more thn one tril, with pigs in one tril infeted with KS06 nd in one or more trils with NVSL (Tble 1). Pigs from the sme breeding ompny nd the sme breed ross were from the sme geneti bkground. Tble 1 Animl omposition of the PHGC trils PRRS virus isolte Tril # Number of nimls Number of sires Number of dms Breed ross Geneti bkground NVSL LW LR A Duro LW/LR B Duro LR/LW C LR LR D Pietrin LW/LR E Duro LW/LR F Pietrin LW G KS Pietrin LW G LW LR A LR LW H 13 b Duro LW/LR F Duro LR/LW C LW lrge white, LR lndre b Geneti bkground is defined s pigs from the sme breeding ompny nd the sme breed ross Tril 13 ws exluded from nlyses due to unusul viremi profiles s seen in Additionl file 1: Figure S1

3 Pge 3 of 20 For eh tril, nimls were trnsported to Knss Stte University t wening (verge ge of 21 dys) nd rndomly pled into pens of 10 to 15 pigs. After 7-dy limtion period, pigs were experimentlly infeted, both intrmusulrly nd intrnslly, with 10 5 TCID50 of NVSL , highly virulent type 2 PRRSV isolte [15], for trils 1 to 8 nd 15 nd 10 5 TCID50 of KS , more ontemporry type 2 PRRSV isolte, for trils 10 to 14. NVSL nd KS06 were isolted from different geogrphi regions nerly ten yers prt, shre 89 % similrity t both the glyoprotein 5 (GP5) nuleotide nd mino id sequene levels [16, 17] nd re loted in two distint moleulr phylogeneti brnhes of PRRSV [16]. The GP5 gene is often used to ssess geneti differenes between PRRSV isoltes nd is suggestive of differenes in virulene between isoltes [18, 19]. Forsberg et l. [20] found tht, on verge, PRRSV isoltes hve substitution rte of per nuleotide in GP5, with the mximum substitution rte between two isoltes being 0.153; NVSL nd KS06 hve substitution rte of 0.11 per nuleotide in the GP5 gene [17]. Blood smples were olleted t 6, 0, 4, 7, 11, 14, 21, 28, 35 nd 42 dys post-infetion (dpi). Body weight ws mesured t 0, 7, 14, 21, 28, 35 nd 42 dpi. Pigs were euthnized t 42 dpi. Trils 7 nd 8 were stopped t 35 dpi due to unvilbility of the fility. Serum viremi ws mesured using semi-quntittive TqMn polymerse hin retion ssy for PRRSV ribonulei id (RNA), s desribed in Boddiker et l. [9 11] nd Ldinig et l. [16]. Assy results were reported s the log 10 of PRRSV RNA opies per ml of serum. A time ourse of viremi levels for eh niml within tril ws plotted in order to provide n initil ssessment of response to infetion nd to onfirm tht ll nimls were infeted (see Additionl file 1: Figure S1). Tril 13 ws exluded from further nlyses due to unusul viremi profiles tht were not observed in ny other PHGC tril; some tril 13 nimls showed delyed presene of serum viremi nd ll pigs hd low nd highly vrible viremi levels over time ompred to the other trils, whih suggested tht the virus ws ttenuted or the pigs were not nïve. The ltter ould be due to the presene of mternl ntibodies resulting from previous infetion or vintion in the soure herd [21]. Aross ll nine trils infeted with NVSL, 12 % of pigs died or were euthnized for humne resons before 42 dpi. Mortlity rte ws similr in the five KS06 trils, with 9 % pigs dying or euthnized before 42 dpi. Ded pigs were neropsied nd subsequent gross nd mirosopi pthology by bord-ertified pthologist identified PRRS ssoited disese s the mjor soure of mortlity, exept for tril 6. Mortlity rte ws high in tril 6 (46 % by dy 42), due to seondry bteril infetions, s identified by pthology. Seondry bteril infetions inluded Esherihi oli, Streptoous suis, Stphyloous ureus, nd Myoplsm hyopneumonie [7], whih were subsequently tred to the pig supplier. The impt of this o-infetion on pig performne ws further investigted by Boddiker et l. [22]. The nimls from ll other trils remined negtive of seondry infetions. Genotyping nd pedigree Er tissue ws olleted from ll pigs for DNA isoltion. DNA smples from trils 1 to 10 were genotyped with Illumin s Porine SNP60 Bedhip [23] v1 (Sn Diego, Cliforni) t GeneSeek In. (Linoln, Nebrsk) nd smples from trils 11 to 15 were genotyped with Illumin s Porine SNP60 Bedhip v2 (Sn Diego, Cliforni) t Delt Genomis (Edmonton, Albert). Only SNPs tht were on both versions of the Illumin s Porine SNP60 Bedhip were used in this study. SNPs were removed if they were fixed within tril or if they were unmpped or mpped to sex hromosome in build 10.2 of the swine genome (GenBnk Aession: GCA_ ); this left 48,164 SNPs. No dditionl filters were pplied to the nimls or genotypes. The niml with the smllest number of lled SNPs hd ll rte of 0.82 (the 99th perentile ws 0.98), while the SNP with the lowest ll rte mong nimls hd ll rte of 0.62 (99th perentile 0.97). Missing genotypes were ssigned the verge genotype (on 0, 1, 2 sle) for nimls in tht tril for tht SNP. This set of SNPs will be referred to s 60k SNPs. Pedigree informtion ws vilble for ll pigs in ll trils. Trils 1, 2 nd 3, whih onsisted of nimls from the sme breeding ompny in onseutive prities, hd the most extensive pedigree informtion, with reords up to two genertions bk, while only sire nd dm informtion ws vilble for the other trils. As suh, there were no reltionships between nimls in different trils, exept for trils 1, 2, nd 3. Pedigree ws orreted using prentl genotypes for trils 1 through 8, s desribed by Boddiker et l. [11]. The 1250 highest qulity 60k SNPs, bsed on GC sore nd ll rte, were used in Cervus 3.0 [15] to verify pedigree informtion for trils 11 nd 15, nd ssign sires for trils 12 nd 13, whih used pooled semen [24]. Prentl genotypes were not vilble for trils 10 nd 14 nd the pedigree provided ws ssumed to be orret. Viremi urve hrteristis In previous studies, Boddiker et l. [9 11] used virl lod, defined s re under the urve of log 10 virl opies/ml of serum from 0 to 21 dpi, s mesurement of response to PRRSV infetion. Are under the urve is summry phenotype of the virl burden but it does not expliitly pture the dynmis of n individul niml s

4 Pge 4 of 20 urve tht n influene this virl burden; two nimls tht hve different viremi urves n hve the sme virl lod. Anlysis of different spets of the viremi urve my id in understnding differenes in virulene of the two virus isoltes, s well s provide insight into the role tht the host s genetis plys in response to infetion [14]. The geneti mehnisms for one urve hrteristi my be onserved ross isoltes, while nother urve hrteristi my be vrible ross isoltes. The Wood s urve, n inomplete gmm funtion often used to model lttion yield in diry ttle [25 27], ws shown to ppropritely model viremi profiles in PHGC trils 1 8 [14]: V(t) = 1 t b 1 e 1t, where V(t) is serum viremi on the log 10 sle t t dpi, 1 is prmeter tht impts the mgnitude of ll points on the urve, b 1 is n inditor of the initil rte of inrese to pek viremi, nd 1 is n inditor of the rte of deline fter the pek nd domintes the viremi profile t lter stges of infetion. These three funtion prmeters were estimted for eh individul tht hd mesurements for t lest five time points, using Byesin inferene with likelihood frmework, implemented by Mrkov hin Monte Crlo method, s desribed in Islm et l. [14]. The rw viremi profiles of some pigs ppered bimodl, so n extended Wood s urve ws lso fitted for eh piglet using the sme methodology [14]: ( ) V(t) = 1 t b 1 e 1t + mx 0, 2 (t t 0 ) b 2 e 2(t t 0 ), where t 0 denotes the time of onset of the seond phse of the profile, whih is ssumed to follow the sme Wood s shpe s the primry phse nd is thus defined by seond set of Wood s model prmeters. A piglet ws lssified s experiening viremi rebound bsed on the Akike s Informtion Criterion (AIC) if AIC WOOD S -AIC EXTENDED- WOOD S ws greter thn 1.488, whih orresponds to the 95 % signifine level of the likelihood rtio test between these models [14]. For summry of estimtes of the urve prmeters (â 1, b 1, ĉ 1, â 2, b 2, ĉ 2 ) nd the proportion of nimls lssified s hving lered, rebound, or persistent serum viremi profile, see Additionl file 2: Tble S1. A more detiled desription of the fitting of the Wood s nd Extended Wood s urve, inluding visuliztion of the urves, is provided in Islm et l. [14]. Using the estimtes of the urve prmeters (â 1, b 1, ĉ 1 ) of the single or the extended Wood s urve for eh pig, five urve hrteristis were derived to desribe the viremi profile of eh pig. For pigs with extended Wood s urves, only estimtes of prmeters of the primry phse were used in this study beuse this phse ws previously shown to hve heritble geneti omponent, while heritbility for rebound ws previously estimted to be low (0.03), suggesting tht rebound is lrgely governed either by virl espe or other environmentl ftors [14]. The first hrteristi evluted, re under the Wood s urve, herefter referred to s virl lod (VL), ws given by the definite integrl for eh individul (i): VL i = 21 0 â 1i t b 1i e ĉ 1it dt. VL is mesure of both the level of viremi nd the extent to whih viremi is mintined. The rnge 0 21 dpi ws hosen to pture the uni-modl phse of infetion ommon to ll pigs. Previous nlyses of viremi from trils 1 to 8 fitted LOESS urve through viremi nd integrted to obtin re under the urve from 0 to 21 dpi [9 11]. This mesure is denoted by VL B. Sine it ws not known how similr VL B nd VL were, whih my impt interprettion nd omprisons with previous studies, bivrite model using pedigree informtion ws fitted to the VL nd VL B dt, seprtely for the KS06 nd NVSL trils, using ASReml 3.0 [28]. Bsed on similr heritbilities nd high geneti nd phenotypi orreltions between VL nd VL B for both isoltes, it ws onluded tht VL bsed on the Wood s urve desribes the sme biologil trit s VL B (Tble 2). Therefore, VL derived from the Wood s urve ws used for ll remining nlyses. The seond urve hrteristi evluted ws time (in dpi) to pek viremi (TP), derived by setting the first derivtive of the Wood s eqution to zero nd solving for t, resulting in: TP i = b 1i ĉ 1i. The third urve hrteristi ws pek viremi (PV), whih ws lulted by setting t = TP in the expression for the Wood s urve: ( ) b1i b1i PV i = â 1i e b 1i. ĉ 1i Tble 2 Comprison of virl lod of Boddiker (VL B ) nd virl lod bsed on the Wood s urve (VL) for the two virus isoltes (NVSL nd KS06) VL B versus VL Heritbility Geneti orreltion Dt from NVSL nd KS06 infetion trils were nlyzed seprtely All trils exept tril 13 were used in the nlysis Phenotypi orreltion NVSL VL B 0.23 (0.10) 0.98 (0.03) 0.90 (0.01) NVSL VL 0.22 (0.10) KS06 VL B 0.35 (0.09) 0.98 (0.02) 0.90 (0.01) KS06 VL 0.35 (0.09)

5 Pge 5 of 20 TP nd PV re relted to the host s bility to respond during the replition-dominnt phse of erly PRRSV infetion [29]. PV is rehed when the rte of virus lerne from serum is equl to the number of virus prtiles relesed into the blood strem. TP is the time it tkes to reh PV, with the nimls tht n mount response erly in infetion expeted to hve shorter TP. Curve hrteristis tht relte to the host s response during the post-pek, lerne-dominnt phse of PRRSV infetion were lso evluted. The mximl dey rte (Vmx) is rehed when the rte of virl lerne from serum is highest ompred to the rte of virl replition. Time to mximl dey (Tmx) ws derived by setting the seond derivtive of the Wood s eqution to zero nd solving for t: b 1i + b 1i Tmx i =. ĉ 1i Substituting this vlue for t in the first derivtive nd tking the bsolute vlue gives Vmx: Vmx i = â 1i b 1i b b 1i 1 ) 1i + b 1i ( b1i e + b1i ĉ. 1i Vmx ws defined s the bsolute vlue, suh tht for n niml with lrger Vmx viremi ws lered more quikly from the serum. Body weights Body weights were olleted weekly nd used to interpolte dily weights. To obtin seprte weight gin urves for eh pig, rndom regression model ws fitted to the weight dt of ll nimls, seprtely for the NVSL nd KS06 trils, using seond order Legendre polynomils in the following model tht ws implemented in ASReml 3.0 [28]: W ijklmop (t) = 2 L ni (t) + P j + A k + S l + n=0 + 2 L ni (t) R m n=0 2 L ni (t) An i + Tr o + Pen(Tr) po + ε ijklmop, n=0 where L ni (t) denotes the nth order Legendre polynomil t t dpi for individul i. L n (t), P, A, S nd L ni (t) R were fitted s fixed effets. L ni (t) ws fitted s ovrite, with t rnging from 0 to 42 dpi, P is the prity of dm, lssified s first, seond, or lter prities, A is the ge of the individul t inoultion, S is the sex of the individul, nd L ni (t) R is the intertion between the nth order Legendre polynomil t t dpi (L ni (t)) nd rebound sttus (R). L ni (t) An, Tr, nd Pen(Tr) were inluded s rndom effets nd denote the intertion between the nth order Legendre polynomil t t dpi nd niml, tril, nd the nested effet of pen within tril, respetively. The term L n (t) An models n individul s weight t eh time point nd ptures both geneti nd permnent environmentl effets, with n unstrutured vrine ovrine struture for polynomil prmeters of given niml nd independene of prmeters between nimls. Residul vrines were modeled seprtely for eh dpi, in order to llow for n inrese in vrine over time. Tril nd Tril * Pen were inluded to pture systemti environmentl effets. This model ws then used to obtin fitted vlues of eh pig s weight for eh dpi (0 to 42) ((Ŵ(t)), using ll oeffiients estimted from the bove model. Genomi reltionship mtries Due to the limited pedigree informtion nd vilbility of genotypes on ll nimls with phenotypes, genomi reltionship mtrix (G) ws onstruted from the 60k SNP genotype dt, using the method of VnRden [30]. The G-mtrix inluded reltionships mong ll nimls ross trils. In some ses, fitting reltionships between breeds n bsorb between-breed differenes tht ould be due to seletion, whih n overestimte the geneti vrine beuse the bse popultion is the popultion from whih the breeds subsequently diverged [31]. Thus, blok digonl G-mtrix ws lso onstruted (G B ) tht only onsidered reltionships between nimls from the sme geneti bkground, with zero reltionships between nimls from different ompnies. Results from nlyses with G B re expeted to be similr to wht would be found with pedigree-bsed nlysis of these dt if the pedigree ws more extensive. A third G-mtrix ws onstruted tht ws the sme s G B but only inluded nimls from trils tht were pired ross isoltes (G P ), to ssess whether the estimtes of orreltions of trits between NVSL nd KS06 infeted pigs ould be bised due to different breed rosses being evluted for eh isolte. In order to ssess the impt of the WUR SNP on these geneti orreltions, mtries G, G B nd G P were lso onstruted fter exluding the 118 SNPs in the 5 Mb region surrounding the WUR SNP. These new mtries were designted s G W, G B W nd G P W, respetively. Sttistil models for phenotypi nd geneti omprisons of NVSL nd KS06 All nlyses used to evlute responses to NVSL nd KS06 infetions were onduted using n niml model in ASReml 3.0 [28]. The univrite model ws: Y ijklmnopq = µ + P i + A j + W k + S l + R m + An n + Li o + Tr p + Pen(Tr) qp + ε ijklmnopq,

6 Pge 6 of 20 where Y is the dependent vrible of dily fitted viremi vlues, fitted weights, VL, TP, PV, Tmx, Vmx, or weight gin from 0 to 42 dpi (WG). Prity of the dm (P), lssified s first, seond, or lter prity, nd sex of the piglet (S) were fitted s fixed lss effet. To ount for potentil model differenes in urve fittings between rebound nd non-rebound pigs, the fixed lss effet of rebound (R) ws inluded in the model. Age (A) nd weight (W) of the piglet t infetion (0 dpi) were inluded s liner ovrites. Rndom effets inluded niml geneti effets (An; using the full G-mtrix), litter (Li), tril (Tr), Pen nested within tril (Pen(Tr)), nd ε s the residul. The rndom effet of niml, An, ws ssumed to hve vrine ovrine struture proportionl to the genomi reltionship mtrix generted from SNP genotypes. Norml distributions of the error nd rndom effet of niml were ssumed N(0, Iσ ε ) nd N ( 0, Gσ g ), respetively. The phenotypi vrine ws obtined by summing the niml, litter, nd pen withintril vrine omponents. Heritbility ws obtined by dividing the niml vrine omponent by the phenotypi vrine. Comprison of host response to NVSL nd KS06 infetion Dt from pired trils (Tble 1) nd the full G-mtrix were used to estimte the effet of isolte on dily fitted viremi vlues, weekly weights, VL, TP, PV, Tmx, Vmx or WG by inluding isolte s fixed lss effet into the bove model. Phenotypi differenes between virus isoltes were ssessed using the t-sttisti reported by ASReml 3.0 [28], with signifine utoff of α = Geneti prmeters by isolte In order to quntify reltionships between the response trits, heritbilities nd phenotypi nd geneti orreltions were estimted seprtely for eh isolte for VL, WG, TP, PV, Tmx, nd Vmx, using the full G-mtrix. Heritbilities nd litter effets were estimted using univrite model. A multivrite model using ll trits ws ttempted for geneti nd phenotypi orreltions between trits but this model did not hieve onvergene, so bivrite models were used insted. Estimtes of orreltions were onsidered sttistilly signifint t α = 0.05 bsed on t-test with 1496 degrees of freedom for NVSL nd 670 degrees of freedom for KS06. The geneti orreltion between viremi nd weight gin ws expeted to hnge during the ourse of infetion. Thus, the geneti orreltion between V(t) nd 3-dy weight gin t dy t (ŴG(t)) were estimted for every other dpi (i.e. 1, 3, 5,, 41), seprtely for eh isolte, using bivrite model. Three-dy weight gin ws derived from the fitted dily weights nd inluded weight 3 dys before s ovrite insted of weight t dy zero. Weight gin t 1 dpi ws djusted for weight t infetion. These bivrite nlyses resulted in two mtries of geneti orreltions between viremi nd 3-dy weight gin, whih were visulized in het mps. Assoition of the WUR genotype with response to NVSL nd KS06 infetion Assoitions of the genotype t the WUR SNP with VL, WG, TP, PV, Tmx, nd Vmx, were estimted seprtely for infetion with NVSL nd KS06 by inluding the intertion of isolte with WUR genotype into the bove model, with the full G-mtrix representing the reltionships between nimls. This model ws lso fitted to dily fitted viremi vlues nd fitted weights to generte viremi nd weight urves for eh isolte nd WUR genotype. For these nlyses, lleles t the WUR SNP were reported using the Illumin A/B genotype referene system, s ws used in the originl studies tht reported the ssoition of this SNP with host response following PRRSV infetion [9 11]. Sttistil differenes between eh isolte by WUR genotype ombintion were ssessed using the t-sttisti reported in ASReml [28] nd the residul degrees of freedom from the model, with signifine utoff of α = Geneti orreltions of response between isoltes The different G-mtries desribed bove were used to estimte geneti orreltions of VL, WG, TP, PV, Tmx, nd Vmx between the two virus isoltes using bivrite model. Geneti orreltions were evluted for sttistil signifine bsed on t-test with 2168 degrees of freedom when using G, G W, G B, nd G B W, nd with 1378 degrees of freedom when using only pired trils (G P nd G P W ). Results Comprison of host response to infetion with NVSL nd KS06 Rw viremi profiles suggested differenes in pig response to infetion with the NVSL versus the KS06 PRRSV isolte (see Additionl file 1: Figure S1). To sttistilly quntify these differenes, seletion of urve hrteristis were derived from the Wood s funtion prmeters nd ompred between isoltes using dt from trils tht were pired by geneti bkground to remove onfounding between isolte nd geneti bkground (Tble 1). Pigs infeted with NVSL hd 16 ± 2 % higher VL thn pigs infeted with KS06 (Tble 3; Fig. 1). Pigs infeted with NVSL hd 14 ± 2 % higher PV nd rehed PV 2.5 ± 0.6 dys erlier (TP) thn pigs infeted with KS06 (Tble 3; Fig. 1). Compred to pigs infeted with KS06, NVSL-infeted nimls rehed mximl PRRSV lerne 3.9 ± 0.7 dys erlier (Tmx) nd

7 Pge 7 of 20 Tble 3 Lest squre mens, heritbilities, litter effets, nd phenotypi stndrd devitions (SD) of responses to infetion with the NVSL nd KS06 isoltes Trit LS mens,b P vlue Heritbility b,d Litter b,d Phenotypi sd b,d NVSL (se) KS06 (se) NVSL (se) KS06 (se) NVSL (se) KS06 (se) NVSL KS06 WG 15.8 (1.1) 19.5 (1.4) (0.06) 0.31 (0.09) 0.07 (0.03) 0.03 (0.04) VL (1.4) 95.0 (1.6) < (0.06) 0.51 (0.09) 0.24 (0.03) 0.01 (0.04) TP 7.0 (0.4) 9.5 (0.4) (0.05) 0.20 (0.09) 0.16 (0.03) 0.10 (0.05) PV 6.6 (0.1) 5.8 (0.1) < (0.05) 0.45 (0.08) 0.27 (0.04) 0.00 (0.00) Tmx 15.4 (0.5) 19.3 (0.6) (0.05) 0.16 (0.09) 0.15 (0.03) 0.14 (0.05) Vmx 0.30 (0.02) 0.22 (0.02) (0.05) 0.26 (0.09) 0.08 (0.03) 0.01 (0.04) WG weight gin (kg), VL virl lod (re under the Wood s urve of log 10 serum viremi from 0 to 21 dys post-infetion; viremi * dys), TP time to pek viremi (dys), PV pek viremi (log 10 serum viremi), Tmx time to mximl rte of viremi dey (dys), Vmx mximl rte of viremi dey (log 10 serum viremi/dy), se stndrd error Full G-mtrix used b Estimtes were obtined by fitting isolte in the model nd only inluded trils in whih pigs from the sme geneti bkground were infeted with both NVSL nd KS06 P vlue for the differene in the estimted lest squre (LS) mens between NVSL nd KS06 d Estimtes were obtined by using the full G-mtrix; NVSL nd KS06 estimtes were estimted seprtely, nd inluded ll nimls infeted with tht isolte, exept tril 13 Pek Viremi Time to Pek Log 10 (Viremi) Mximl Dey Rte Time to Mximl Dey Rte Viremi Weight Dys Post Infetion Fig. 1 Comprison of response to infetion when pigs re infeted with NVSL (red) or KS06 (blue) PRRSV isoltes. For omprison between viruses, trils were mthed bsed on geneti bkground. Lest squre mens of dily viremi, predited using the monophsi Wood s urve prmeters, nd weights were estimted using ASReml [28] with the full G-mtrix. Viremi, Weight, Time to Pek, Pek Viremi, Time to Mximl Dey, nd Mximl Dey were ompred when pigs were infeted with either the NVSL or KS06 PRRSV isolte Weight (kg) lered t 36 ± 14 % fster mximl rte (Vmx) thn their KS06-infeted ounterprts (Tble 3; Fig. 1). When ompring the impt tht infetion hd on weight gin, pigs infeted with the NVSL isolte hd tendeny to grow slower thn their KS06-infeted ounterprts (Tble 3; Fig. 1). This omprison of viremi hrteristis nd WG between isoltes (Tble 3; Fig. 1) indites tht NVSL is more virulent thn KS06 beuse it rehed higher PV more rpidly nd resulted in higher VL nd slower growth of the pigs. KS06 ppers to be more persistent thn NVSL, s shown by longer time to mximl dey rte, lower mximl dey, nd lrger perentge of pigs lssified s persistently infeted, defined s non-rebound pig with fitted log 10 serum viremi vlue greter thn 1 t 42 dpi (56 % for KS06 vs. 40 % for NVSL; Additionl file 2: Tble S1). Heritbility estimtes for viremi urve hrteristis nd weight gin All evluted trits were estimted to be modertely to highly heritble, exept for Vmx under infetion with NVSL (Tble 3). The trits with the highest estimted heritbility under infetion with NVSL were VL nd WG nd these trits lso hd high heritbility estimtes under infetion with KS06. Vmx hd the lowest estimted heritbility under infetion with NVSL ompred to the other PRRSV urve hrteristis, but moderte heritbility under infetion with KS06 (Tble 3). The

8 Pge 8 of 20 estimted geneti vrine for Vmx ws similr under infetion with NVSL nd KS06 ( vs ), so the differene in heritbility ws primrily driven by lrger environmentl vrine under infetion with NVSL ompred to KS06 ( vs ). Heritbility estimtes were similr between NVSL nd KS06 for WG, TP, nd Tmx (Tble 3). These trits lso hd similr estimtes of the litter omponent for NVSL nd KS06 infeted pigs. Trits VL, PV, nd Vmx hd lower heritbilities nd lrger litter omponents for NVSL ompred to KS06. Summing the heritbility nd litter omponents gve similr results for the two isolte for VL nd PV, lthough heritbilities were quite different between isoltes (Tble 3). The lrger number of nimls infeted with the NVSL isolte ompred to KS06 my result in more urte seprtion of geneti nd litter omponents for NVSL. Geneti prmeter estimtes using different G mtries Estimtes of the geneti orreltion between isoltes for PV were very similr when using the full G mtrix (G) or the G mtrix with only geneti reltionships within geneti bkground (G B ; Tble 4), inditing tht there is little vrition in host geneti ftors influening PV between geneti bkgrounds. The geneti orreltion between isoltes for WG ws slightly higher when using G B ompred to G (Tble 4), suggesting tht the impt of PRRSV infetion on WG of pigs within the sme geneti bkground ws more similr thn between pigs of different geneti bkgrounds. The estimte of the geneti orreltion of VL between pigs infeted with NVSL nd KS06 dropped substntilly when G B ws used, ompred to using G (Tble 4). The estimte of geneti orreltion between isoltes for TP inresed when using G B rther thn G (Tble 4), suh tht the estimte bsed on G B ws no longer signifintly different from 1 but lso not signifintly different from 0. Estimtes of the geneti orreltion between isoltes for Tmx nd Vmx were not signifintly different from 0 or 1 when using either G B or G (Tble 4). In generl, estimtes obtined when inluding only reltionships within geneti bkground were similr, whether ll nimls were used (G B ) or only those from trils tht were pired ross isoltes (G P ). Geneti orreltions mong virl lod nd viremi urve hrteristis VL, defined s re under the Wood s urve from 0 to 21 dpi, ws lrgely driven by PV, s shown by the high geneti nd phenotypi orreltions between these two trits for both isoltes (Tbles 5, 6). PV hd the highest geneti orreltion between PRRSV isoltes nd ws not signifintly different from 1 (Tble 4). No other urve hrteristi hd between virus isolte geneti orreltion estimte tht ws signifintly different from 0. This suggests tht the observed geneti orreltion between isoltes for VL is primrily due to the high geneti orreltion observed between isoltes for PV. Tmx nd Vmx hd strong negtive geneti orreltions with eh other for both isoltes but they were only highly orrelted with VL for NVSL (Tbles 5, 6). Time to mximl dey rte (Tmx) ws 19.3 dys for KS06 but 15.4 dys for NVSL (Tble 3). Thus, Vmx ws expeted to ply lrger role in VL for NVSL thn for KS06, beuse VL ws lulted from 0 to 21 dpi. No onlusions n be drwn bout the geneti orreltions between isolte for Tmx or Vmx beuse the estimtes were not signifintly different from 0 or 1 due to lrge stndrd errors (Tble 4). Tble 4 Estimtes of geneti orreltions [genet or. (stndrd errors)] between response to infetion with the NVSL versus KS06 isoltes nd using different reltionship mtries Trit Full (G) Blok digonl (G B ) Pired blok digonl (G p ) Heritbility Genet or. Heritbility Genet or. Heritbility Genet or. NVSL KS06 NVSL KS06 NVSL KS06 VL 0.32 (0.06) 0.53 (0.07) 0.86 (0.19) 0.40 (0.06) 0.53 (0.08) 0.51 (0.24) 0.51 (0.08) 0.54 (0.09) 0.57 (0.22) WG 0.33 (0.05) 0.30 (0.09) 0.86 (0.27) 0.37 (0.06) 0.32 (0.10) 0.96 (0.34) 0.41 (0.08) 0.38 (0.11) 0.90 (0.31) TP 0.22 (0.05) 0.21 (0.09) 0.25 (0.33) 0.28 (0.06) 0.28 (0.10) 0.40 (0.36) 0.32 (0.08) 0.30 (0.12) 0.43 (0.36) PV 0.17 (0.05) 0.46 (0.07) 0.94 (0.28) 0.23 (0.06) 0.43 (0.08) 0.94 (0.33) 0.29 (0.07) 0.42 (0.09) 0.91 (0.30) Tmx 0.21 (0.05) 0.14 (0.09) 0.82 (0.53) 0.26 (0.06) 0.16 (0.10) 0.86 (0.59) NE NE NE Vmx 0.10 (0.05) 0.25 (0.09) 0.63 (0.51) 0.13 (0.05) 0.22 (0.10) 0.32 (0.67) 0.06 (0.06) 0.23 (0.12) 0.41 (0.92) G = full G-mtrix with ll reltionships inluded; G B = blok digonl G-mtrix, with the reltionships between nimls from different geneti bkgrounds set to zero; G p = pired blok digonl G-mtrix, with the reltionships between nimls from different geneti bkgrounds set to zero, nd only inluded trils in whih pigs from the sme geneti bkground were infeted with both NVSL nd KS06 WG weight gin (kg), VL virl lod (re under the Wood s urve of log 10 serum viremi from 0 to 21 dys post infetion; viremi * dys), TP time to pek viremi (dys), PV pek viremi (log 10 serum viremi), Tmx time to mximl rte of viremi dey (dys), Vmx mximl rte of viremi dey (log 10 serum viremi/dy) NE: were not estimted beuse the model did not hieve onvergene in ASReml

9 Pge 9 of 20 Tble 5 Estimtes of orreltions (stndrd error) of response to infetion with PRRSV isolte NVSL Trit VL WG TP PV Tmx Vmx VL 0.33 (0.03) 0.10 (0.03) 0.66 (0.02) 0.36 (0.03) 0.27 (0.03) WG 0.74 (0.10) 0.02 (0.03) 0.22 (0.03) 0.16 (0.03) 0.12 (0.03) TP 0.31 (0.15) 0.27 (0.16) 0.09 (0.03) 0.72 (0.01) 0.12 (0.03) PV 0.85 (0.07) 0.73 (0.13) 0.05 (0.19) 0.23 (0.03) 0.40 (0.03) Tmx 0.81 (0.10) 0.11 (0.16) 0.83 (0.07) 0.50 (0.21) 0.51 (0.02) Vmx 0.72 (0.21) 0.45 (0.22) 0.11 (0.26) 0.27 (0.33) 0.57 (0.19) WG weight gin (kg), VL virl lod (re under the Wood s urve of log 10 serum viremi from 0 to 21 dys post infetion; viremi * dys), TP time to pek viremi (dys), PV pek viremi (log 10 serum viremi), Tmx time to mximl rte of viremi dey (dys), Vmx mximl rte of viremi dey (log 10 serum viremi/dy) Phenotypi orreltions (bove digonl) nd geneti orreltions (below digonl) were estimted using n niml model in ASReml nd the full G-mtrix Tble 6 Correltions of response to infetion with PRRSV isolte KS06 using the full G-mtrix Trit VL WG TP PV Tmx Vmx VL 0.23 (0.05) 0.06 (0.05) 0.76 (0.02) 0.13 (0.05) 0.16 (0.05) WG 0.52 (0.17) 0.05 (0.05) 0.13 (0.05) 0.06 (0.06) 0.13 (0.05) TP 0.08 (0.22) 0.10 (0.24) 0.02 (0.05) 0.80 (0.02) 0.02 (0.05) PV 0.91 (0.05) 0.30 (0.18) 0.08 (0.25) 0.19 (0.05) 0.52 (0.04) Tmx 0.19 (0.23) 0.42 (0.23) 0.69 (0.19) 0.24 (0.28) 0.52 (0.04) Vmx 0.01 (0.20) 0.42 (0.21) 0.12 (0.28) 0.51 (0.13) 0.75 (0.18) WG weight gin (kg), VL virl lod (re under the Wood s urve of log 10 serum viremi from 0 to 21 dys post infetion; viremi * dys), TP time to pek viremi (dys), PV pek viremi (log 10 serum viremi), Tmx time to mximl rte of viremi dey (dys), Vmx mximl rte of viremi dey (log 10 serum viremi/dy) Phenotypi orreltions (bove digonl) nd geneti orreltions (below digonl) were estimted using n niml model in ASReml nd the full G-mtrix The two time-relted trits TP nd Tmx hd strong positive geneti nd phenotypi orreltions with ( ) eh other for both isoltes (Tbles 5, 6) beuse TP b ( is ) omponent of Tmx Tmx = TP + TP. The geneti orreltion between isoltes ws signifintly different from 1 for TP (0.25 ± 0.33, Tble 4), inditing tht host geneti ontrol of the time until mximl virus dey rte my differ between virus isoltes. Geneti orreltions of weight gin with viremi urve hrteristis PV hd moderte negtive geneti orreltion with WG for NVSL-infeted pigs (Tble 5) but this geneti orreltion ws not signifintly different from 0 for KS06- infeted pigs (Tble 6), due to lrger stndrd error nd less negtive estimte. Vmx lso hd signifint geneti orreltion with WG. These results suggest tht the redution in growth is used by n overll high viremi level over prolonged period of time, whih is further supported by the finding tht WG hd the highest estimted geneti nd phenotypi orreltions with VL for both isoltes (Tbles 5, 6). Geneti orreltions of viremi with weight gin The geneti nd phenotypi orreltions between VL nd WG were negtive nd of similr mgnitude for the two isoltes (Tbles 5, 6). Between isoltes, geneti orreltion estimtes for VL nd WG were high nd not signifintly different from 1 (Tble 4), inditing tht host geneti ontrol of VL nd WG ws very similr under infetion with either the NVSL or KS06 isolte. A more thorough explortion of the reltionship between PRRS viremi nd weight gin ws obtined by estimting geneti orreltions between fitted viremi nd 3-dy weight gin ross the infetion period (Fig. 2). Geneti orreltions generlly showed similr pttern between NVSL nd KS06, but orreltions for NVSL were more extreme (rnge = 1 to 0.43) thn KS06 (rnge = 0.75 to 0). A more detiled explntion of these results n be found in the Disussion setion Geneti orreltion of viremi with weight gin ross time. Assoitions of the WUR genotype with response to infetion with NVSL nd KS06 Lest squre mens of dily viremi nd weight were estimted by fitting the intertion between isolte nd WUR genotype for ll trils simultneously nd re in Fig. 3. Very few pigs hd the BB genotype t the WUR SNP, so estimtes of lest squre mens for the BB genotype hd high stndrd errors. For VL, both AB nd BB nimls were signifintly different from AA, while BB

10 Pge 10 of 20 Fig. 2 Het Mp of geneti orreltions between viremi nd weight gin during the ourse of infetion with the NVSL or b KS06 PRRSV isolte. Geneti orreltions from fitting bivrite niml model in ASReml [28] using the full G-mtrix. NVSL nd KS06 were nlyzed seprtely. All trils, exept tril 13, were used in the nlysis. Eh squre in the het mp represents the geneti orreltion between viremi t given time point t (X xis) nd the 3-dy weight gin t time point t* (Y xis) nimls were not signifintly different from AB, suggesting omplete dominne, s previously reported by Boddiker et l. [9 11]. Results for BB nimls re not disussed further. Pigs with the AA WUR genotype hd 4.5 ± 0.4 % higher VL (P < 0.001; Fig. 4A) nd grew 2.0 ± 0.2 kg less thn pigs with the AB genotype fter infetion with NVSL (P < 0.001; Fig. 4B). These estimtes re onsistent

11 Pge 11 of 20 Log 10 (Viremi) Viremi NVSL AA Viremi NVSL AB Weight NVSL AA Weight NVSL AB Weight (kg) Dys Post Infetion 0 b Log 10 (Viremi) Viremi KS06 AA Weight KS06 AA Viremi KS06 AB Weight KS06 AB Dys Post Infetion Fig. 3 Lest squre mens of the effet of the WUR genotype for predited viremi nd weight in pigs infeted with either the NVSL or KS06 PRRSV isolte. Lest squre mens of the WUR genotype for predited viremi nd weight for NVSL () nd KS06 (b) when fitting the Isolte * WUR intertion into the niml model in ASReml [28] using the full G-mtrix. All trils, exept tril 13, were used for the nlysis Weight (kg) with previous estimtes of the ssoition of WUR under infetion with NVSL [11]. Genotype t the WUR SNP ws lso found to be ssoited with VL under infetion with KS06, for whih VL ws 4.2 ± 0.9 % higher in AA nimls thn in AB nimls (P < 0.001) (Fig. 4A). However, in ontrst to infetion with NVSL, the WUR genotype did not hve signifint ssoition with WG (P = 0.32), lthough the diretion of the effet ws onsistent, with AA pigs growing 0.4 ± 0.4 kg less thn pigs with the AB genotype (Fig. 4B). Genotype t the WUR SNP ws ssoited with ll viremi urve hrteristis in pigs infeted with NVSL (Figs. 3, 4A). Compred to AA nimls, AB nimls hd 2.8 ± 0.4 % lower PV (P < 0.001; Fig. 4D), whih ws rehed 0.20 ± 0.09 dys erlier (P < 0.02; Fig. 4C). AB nimls lso hd 3.8 ± 1.5 % fster mximl dey rte (P < 0.02; Fig. 4F), whih ws rehed 0.68 ± 0.16 dys sooner (P < 0.001; Fig. 4E). In KS06 trils, genotype t the WUR SNP ws ssoited with 3.4 ± 0.7 % higher PV in AA nimls ompred to AB nimls (P < 0.001; Fig. 4D) but no ssoition ws found with Vmx (P = 0.36; Fig. 4F) nd the diretion of the effet for Vmx ws opposite to tht of NVSL-infeted nimls, with AB hving 3.1 ± 3.4 % slower mximl dey rte thn AA nimls. Compred to AA nimls, AB nimls tended to reh pek viremi 0.30 ± 0.16 (P = 0.052) dys sooner (Fig. 4C) nd the mximl dey rte 0.47 ± 0.29 (P = 0.078) dys lter (Fig. 4E). The effet of WUR genotype ws signifintly different between the NVSL nd KS06 isoltes for either WG (P = 0.001) nd Vmx (P = 0.041; Fig. 4). Plotting verge weight urves nd viremi using the Wood s urve prmeters from the primry phse of infetion (i.e. not inluding rebound) for pigs with the AA nd AB genotypes t the WUR SNP by isolte (Fig. 3), provides visuliztion of the overll differenes in the shpe of the viremi nd weight urves. For KS06, the effet of the WUR genotype on VL ws minly driven by differenes in PV but the differene in viremi level between AA nd AB ws not mintined due to slightly lower rte of lerne in AB ompred to AA nimls, resulting in similr viremi levels t 42 dpi. Conversely,

12 Pge 12 of 20 A Woods Virl Lod (Log 10 (Viremi)*Dys) b b d d n=1079 n=374 n=44 n=573 n=97 n=6 AA AB BB AA AB BB NVSL KS06 B Weight Gin (kg) b b n=1065 n=373 n=44 AA AB BB NVSL n=555 n=92 n=6 AA AB BB KS06 C Time to Pek (Dys) E Time to Mximl Dey (Dys) b b n=1079 n=374 NVSL n=44 n=573 n=97 KS06 n=6 D Pek Viremi (Log 10 (Viremi)) AA AB BB AA AB BB AA AB BB AA AB BB n=1079 b n=374 b n=44 n=573 n=97 n=6 n=1079 b n=374 NVSL NVSL KS06 NVSL KS06 Fig. 4 Lest squre mens of the effet of the WUR genotype for weight gin nd viremi urve prmeters in pigs infeted with either the NVSL or KS06 PRRSV isolte. Lest squre mens of the WUR genotype for VL (A), WG42 (B), TP (C), PV (D), Tmx (E), nd Vmx (F) when fitting the Isolte * WUR intertion into the niml model in ASReml [28] using the full G-mtrix. All trils, exept tril 13, were used for the nlysis. Estimtes with different letter ssignments re signifintly different (P 0.05) F Mximl Dey Rte (Log 10 (Viremi)/Dy) n=1079 b n=374 b n=44 b n=44 n=573 n=573 d n=97 KS06 AA AB BB AA AB BB AA AB BB AA AB BB n=97 n=6 d n=6 for NVSL, the differene in viremi levels between AA nd AB nimls first ppered round pek viremi nd beme lrger during the primry stges of infetion due to fster lerne rte for AB nimls. In generl, WUR genotype hd lower ssoition with response to infetion in KS06-infeted pigs thn in NVSL-infeted pigs, whih suggests tht the mgnitude of the effet of QTL on hromosome 4 depends on virulene of the PRRSV isolte. Impt of the WUR region on heritbilities nd geneti orreltions Heritbilities of response trits were estimted by inluding ll SNPs in the full G-mtrix (Tble 4) nd lso by exluding SNPs in the 5 Mb region surrounding the WUR SNP (G W ; Tble 7) in order to ssess how muh of the estimtes of heritbility were ttributed to the WUR genotype. In the NVSL trils, estimted heritbilities were lower for ll trits when the G W ws used, exept for

13 Pge 13 of 20 Tble 7 Estimtes of geneti orreltions of response to infetion between PRRSV isoltes when exluding the 5 Mb WUR region from the G mtrix Trit Full (G W ) Blok digonl (G B W ) Pired blok digonl (G P W ) Heritbility Genet or. Heritbility Genet or. Heritbility Genet or. NVSL KS06 NVSL KS06 NVSL KS06 VL 0.25 (0.06) 0.49 (0.09) 0.76 (0.22) 0.34 (0.06) 0.49 (0.08) 0.44 (0.26) 0.45 (0.08) 0.49 (0.09) 0.51 (0.24) WG 0.28 (0.06) 0.30 (0.09) 0.89 (0.29) 0.33 (0.06) 0.31 (0.07) 0.93 (0.35) 0.36 (0.08) 0.38 (0.11) 0.90 (0.32) TP 0.21 (0.05) 0.20 (0.09) 0.18 (0.34) 0.27 (0.06) 0.28 (0.10) 0.36 (0.37) 0.31 (0.08) 0.29 (0.12) 0.37 (0.37) PV 0.13 (0.05) 0.40 (0.08) 0.79 (0.34) 0.22 (0.06) 0.39 (0.08) 0.81 (0.37) 0.51 (0.10) 0.55 (0.36) 0.77 (0.36) Tmx 0.19 (0.05) 0.14 (0.09) 0.80 (0.54) 0.24 (0.06) 0.16 (0.10) 0.90 (0.62) NE NE NE Vmx 0.10 (0.05) 0.23 (0.09) 0.70 (0.53) 0.13 (0.05) 0.21 (0.09) 0.57 (0.70) 0.06 (0.07) 0.22 (0.11) 0.75 (1.03) WG weight gin (kg), VL virl lod (re under the Wood s urve of log 10 serum viremi from 0 to 21 dys post infetion; viremi * dys), TP time to pek viremi (dys), PV pek viremi (log 10 serum viremi), Tmx time to mximl rte of viremi dey (dys), Vmx mximl rte of viremi dey (log 10 serum viremi/dy) G W = full G-mtrix onstruted exluding the 5 Mb region ontining WUR with ll reltionships inluded; G B W = blok digonl G-mtrix onstruted exluding the 5 Mb region ontining WUR, with the reltionships between nimls from different geneti bkgrounds set to zero; G P W = pired blok digonl G-mtrix onstruted exluding the 5 Mb region ontining WUR, with the reltionships between nimls from different geneti bkgrounds set to zero, nd only inluded trils in whih pigs from the sme geneti bkground were infeted with both NVSL nd KS06 NE: were not estimted beuse the model did not hieve onvergene in ASReml Vmx, whih remined the sme. Heritbility estimtes for WG nd Tmx were not ffeted by using G W, while the estimtes dropped for ll other trits when G W ws used, with the lrgest drops for PV nd VL. Geneti orreltions for the response trits between isoltes were lso estimted using G (Tble 6) nd G W (Tble 7). Estimtes of geneti orreltions between isoltes for WG nd Vmx were slightly lrger when using G W ompred to G, while the estimtes for ll other response trits deresed, with the lrgest dereses for PV nd VL. Geneti orreltion estimtes between isoltes were signifintly different from 0 nd not signifintly different from 1 for VL, WG nd PV when using either G or G W (Tbles 4, 7). The high geneti orreltion estimtes for these trits when using G W indites tht the onserved host geneti response between isoltes for VL nd PV did not solely depend on the WUR genotype but hs lrge polygeni omponent. The inrese in the geneti orreltion between isoltes for WG when using G W ompred to G is onsistent with the observed effet of the WUR genotype in pigs infeted with NVSL nd the muh smller effet in pigs infeted with KS06. Estimtes of geneti orreltions between isoltes for Tmx nd Vmx hd lrge stndrd errors when using either G or G W, so no onlusions ould be drwn beuse estimtes were not signifintly different from 0 or 1. Estimtes of the geneti orreltions between isoltes for TP were signifintly different from 1 but not signifintly different from 0 for either G or G W, inditing tht the host s geneti ontrol of TP is not highly onserved ross isoltes. Mtries G B nd G P were lso onstruted without the 5-Mb WUR region (G B W nd G P W, respetively). The results obtined using these mtries were s expeted bsed on the differenes between G nd G W nd were lso onsistent with the differenes in the estimtes when ompring the use of G B nd G P to the use of G (Tbles 4, 7). Disussion Our results suggest tht the KS06 PRRSV isolte is less virulent thn NVSL but, importntly, tht geneti seletion for pigs with improved weight gin nd redued virl lod under either PRRSV infetion is expeted to be effetive ross these PRRSV isoltes. This study lso ffirmed the importnt influene of the WUR genomi region on pig hromosome 4 on host response to PRRSV. The effet of this genomi region ws onsistent between isoltes for trits relted to viremi. While AB nimls gined slightly more weight thn AA nimls during infetion with KS06, signifint differene between WUR genotypes ws only observed for weight gin with NVSL, suggesting tht the influene of this genomi region on weight gin my depend on virulene of the PRRSV isolte. Consistent with the previously onduted studies of the PHGC, the nimls used in this study were inoulted with PRRSV both intrmusulrly nd intrnslly. The infetion protool is stndrd hllenge protool designed to give every pig onsistent mount of virus. It lso simultes the most likely routes for infetion through needle stiks nd intrnsl exposure. Previous studies ompring routes of exposure hve shown tht the dosge used to inoulte pigs impts the level of viremi in the pigs independently of the route of exposure, resulting in similr levels of ntibody prodution [32]. Similrly, growth nd ntibody responses were similr between

14 Pge 14 of 20 intrnslly nd simultneously intrnslly/intrmusulrly vinted pigs [33]. Given tht no differenes in host response re expeted between these methods, the pproh used in this study ws the most pproprite to ensure onsistent levels of infetion between pigs to provide more power to distinguish between nimls tht differ in their geneti merit for response to PRRSV infetion. Modelling viremi using the Wood s urve This study demonstrtes the utility of mthemtil funtions to ssess the impt of host genetis nd virus isolte on PRRS viremi kinetis. The Wood s urve uses three prmeters,, b, nd, whih re relted to the overll level of viremi () nd desribe the shpe of the urve (b, whih is dominnt pre-pek, nd, whih is dominnt post-pek) [27]. While other mthemtil funtions my more dequtely model PRRS viremi during infetion, the number of dt points olleted during these trils limited the use of more omplex models. The Wood s urve is more useful method for ompring viremi kinetis thn the LOESS smoothed fit used in previous nlyses of these dt [9 11]. The LOESS smoothed fit uses prmeter tht indites the degree of polynomil to fit to the dt nd smoothing prmeter for urve fitting [34], with the primry intent of filtering out noise from the dt. The limittion of the LOESS fit, however, is tht it does not lend itself to extrting fitted prmeters tht speify prtiulr biologil properties of system tht hve importnt implitions in understnding the dynmis of PRRSV infetion. Although both methods dequtely fitted the dt, Wood s urve prmeters desribe both the mgnitude nd shpe of the urve, whih n be used to explore different hrteristis of the viremi urves. Exploring Wood s urve hrteristis n provide insight into importnt biologil questions, suh s whih spets of host response re under strongest geneti ontrol nd how seletion for one urve hrteristi my ffet others, nd thus the entire profile. The Wood s funtion n lso be used to explore the reltionship between urve hrteristis nd other phenotypes, suh s growth under infetion. Furthermore, omprison of the extended Wood s nd Wood s urve funtions llowed for n objetive method to seprte primry infetion from rebound infetion viremi urves [14]. While the dvntges of fitting Wood s urve to model the dynmis of PRRS viremi re ler, re must be tken in the interprettion of orreltions between urve hrteristis beuse strong orreltions between these urve hrteristis re in prt n rtift of the Wood s funtion nd prtly reflet true orreltions between urve hrteristis tht re independent of the Wood s funtion. For exmple, Tmx nd Vmx re expeted to hve high geneti orreltion beuse both rely hevily on the b prmeter of the Wood s funtion. Geneti prmeter estimtes using different G mtries Three different G mtries were onstruted for both the full G mtrix nd the G W mtrix. G B only ontined reltionships between nimls from the sme geneti bkground, with zeros for reltionships between nimls from different ompnies. Thus, while G ontins informtion bout geneti vrine tht exists within geneti bkground s well s between geneti bkgrounds, G B only ontins informtion bout geneti vrine within geneti bkground nd is, therefore, more similr to the pedigree-bsed reltionship mtrix beuse there ws no pedigree informtion between nimls from different geneti bkgrounds. G P ws blok digonl mtrix tht used only pigs from the sme geneti bkground tht were pired ross isoltes nd ws used to void bises in estimtes tht ould result from inluding different breeds in the nlyses for eh isolte. In generl, estimtes using G P, whih onsidered only nimls from trils tht were pired ross isoltes, were onsistent with estimtes using G B. This suggests tht, while the geneti orreltion between isoltes for VL my be moderte within geneti bkgrounds, some geneti bkgrounds hve high VL under infetion with both NVSL nd KS06, while some geneti bkgrounds hve low VL under infetion with both NVSL nd KS06, suh tht when the reltionships between geneti bkgrounds re onsidered (using G), the geneti orreltion between isoltes for VL inreses. Seleting for improved VL during infetion with one PRRSV isolte is likely to improve VL during infetion with nother PRRSV isolte, but the extent to whih suh seletion is suessful ross isoltes will likely differ between geneti bkgrounds. Comprison of geneti prmeter estimtes for virl lod nd weight gin to previous estimtes Estimtes of heritbility of VL nd WG during NVSL infetion were slightly different from previously reported estimtes using these dt [11] (VL: 0.31 ± 0.06 vs ± 0.13; WG: 0.33 ± 0.06 vs ± 0.11) (Tble 3). Differenes between estimtes n be ttributed to the use of genomi rther thn pedigree-bsed reltionships, the inlusion of tril 15, nd the ddition of ge nd weight t infetion s ovrites in the model used in this study. Age nd weight t infetion re importnt to inlude in the nlysis beuse pigs tht re older or hevier t infetion tend to be ble to mount stronger immune response [35, 36]. The use of genomi insted of pedigree-bsed reltionships hlved the stndrd errors of estimtes beuse the G mtrix ptures reltionships