Characterization of homologous and heterologous adaptive immune responses in porcine reproductive and respiratory syndrome virus infection

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1 Díz et l. Veterinry Reserch 212, 43:3 VETERINARY RESEARCH RESEARCH Open Access Chrcteriztion of homologous nd heterologous dptive immune responses in porcine reproductive nd respirtory syndrome virus infection Ivn Díz 1, Mrion Gimeno 1,2, Lil Drwich 1,2, Nuri Nvrro 1, Liudmil Kuzemtsev 1,2, Sergio López 1,2, Ivn Glindo 1,2, Joquim Seglés 1,2, Mrgrit Mrtín 1,2, Jon Pujols 1,3 nd Enric Mteu 1,2* Astrct The present study chrcterized the homologous nd heterologous immune response in type-i porcine reproductive nd respirtory syndrome virus (PRRSV) infection. Two experiments were conducted: in experiment 1, eight pigs were inoculted with PRRSV strin 3262 nd 84 dys post-inocultion (dpi) they were chllenged with either strin 3262 or strin 3267 nd followed for the next 14 dys (98 dpi). In experiment 2, eight pigs were inoculted with strin 3267 nd chllenged t 84 dpi s ove. Clinicl course, viremi, humorl response (neutrlizing nd non-neutrlizing ntiodies, NA) nd virus-specific IFN-γ responses (ELISPOT) were evluted ll throughout the study. Serum levels of IL-1, IL-6, IL-8, TNF-α nd TGF-β were determined (ELISA) fter the second chllenge. In experiment 1 primo-inocultion with strin 3262 induced viremi of 28 dys, low titres of homologous NA ut strong IFN-γ responses. In contrst, strin 3267 induced longer viremis (up to 56 dys), higher NA titres ( 6 log 2 ) nd lower IFN-γ responses. Inocultion with 3267 produced higher serum IL-8 levels. After the re-chllenge t 84 dpi, pigs in experiment 1 developed mostly one week viremi regrdless of the strin used. In experiment 2, neither the homologous nor the heterologous chllenge resulted in detectle viremi lthough PRRSV ws present in tonsils of some nimls. Homologous re-inocultion with 3267 produced elevted TGF-β levels in serum for 7 14 dys ut this did not occur with the heterologous re-inocultion. In conclusion, inocultion with different PRRSV strins result in different virologicl nd immunologicl outcomes nd in different degrees of homologous nd heterologous protection. Introduction One of the min ostcles for the development of new vccines of greter efficcy ginst porcine reproductive nd respirtory syndrome virus (PRRSV) is the limited understnding of the mechnisms involved in protection [1-4]. Up to now, most studies hve focused in the development of neutrlizing ntiodies (NA) nd to virus-specific interferon-γ secreting cells (IFN-γ-SC) s the min correltes of protection [5 1] lthough the precise role * Correspondence: enric.mteu@u.ct Equl contriutors 1 Centre de Recerc en Snitt Animl (CReSA), UAB-IRTA, Cmpus de l Universitt Autònom de Brcelon, 8193, Bellterr, Brcelon, Spin 2 Deprtment de Snitt i Antomi Animls, Universitt Autònom de Brcelon, 8193, Bellterr, Brcelon, Spin Full list of uthor informtion is ville t the end of the rticle of these elements is not well understood. Cross neutrliztion experiments hve shown tht cross rectivity etween different PRRSV strins cn e low nd even some PRRSV strins seem not to induce neutrlizing response t ll [11,12]. Moreover, little is known out cell medited responses in heterologous chllenge models [8,1]. As result, t present it is very difficult or impossile- to predict the pnel of strins or the chrcteristics of PRRSV isoltes ginst which one pig is effectively protected fter immuniztion. As mtter of fct, the common ssumption is tht immunity ginst homologous strin is sterilizing or lmost completewhile immunity ginst other strins will depend, genericlly, on the degree of genetic/ntigenic similrity etween the immunizing nd the infecting strins [1,13]. 212 Diz et l.; licensee BioMed Centrl Ltd. This is n Open Access rticle distriuted under the terms of the Cretive Commons Attriution License ( which permits unrestricted use, distriution, nd reproduction in ny medium, provided the originl work is properly cited.

2 Díz et l. Veterinry Reserch 212, 43:3 Pge 2 of 15 However, sequencing of ORF5 of given strin nd vccine is scrcely predictive of protection [8,14]. It is worth to note tht fter creful review of the ville scientific literture, there re very few chrcterized models of homologous/heterologous chllenge considering simultneously potentil correltes of protection (NA nd IFN-γ-SC), the development of clinicl signs nd the virologicl outcome of the chllenge model. In the present study, we chrcterized the clinicl nd virologicl course nd the evolution of neutrlizing ntiodies nd interferon responses fter inocultion with two PRRSV strins previously reported to e different [15,16]. We evluted the neutrlizing nd IFN-γ-SC responses ginst heterologous strin fter immuniztion nd we lso tested the immunologicl responses fter the homologous nd heterologous chllenges of previously immunized pigs. Mterils nd Methods Viruses Two genotype I PRRSV strins designted s 3262 nd 3267 were used in the present study. Strin 3262 ws isolted in 25 from the lung of pneumonic piglet of Spnish frm nd strin 3267 ws isolted in 26 from serum of or of Portuguese frm. Virl stocks were produced from pssge n = 3 in porcine lveolr mcrophges (PAM). Virl titrtions were performed y inocultion in PAM nd confirmtion y immunoperoxidse monolyer ssy (IPMA) [17]. Virl stocks were produced in n mount enough to llow the use of single tch of virus for ll experiments nd ssys reported elow. Donor nimls for PAM were free of PRRSV, Aujeszky s disesevirusndother mjor swine diseses including ll interntionlly notifile diseses plus Mycoplsm hyopneumonie nd swine influenz virus s shown y their serologicl sttus. The nimls hd mternl ntiodies ginst porcine circovirus type 2 (PCV2) ut were free of virus s determined y PCR using lood smples. PAM nd virl stocks used were shown to e free of PCV2, Mycoplsm hyopneumonie, heptitis E virus nd Torque teno sus virus (TTSuV) y PCR s reported efore [18-21]. Strins used in the present study hve een chrcterized in vitro efore with regrds to their impct on the phenotype of ntigen presenting cells nd hve een sequenced from ORF1 to ORF7 [15,16] (Gennk ccession numers: JF nd JF276435). Overll genetic similrity etween strins ws 88.5 % [15]. Briefly, strin 3262 is known to induce IL-1 nd TNF-α relese in dendritic cells while strin 3267 is unle to induce ny of those cytokine in the sme system [16]. Strin 3262 lso hrours 74 deletion in in nsp2 [15]. Experimentl design nd nimls All experiments involving pigs were done under the pprovl of the Ethics Commission for Humn nd Animl Experimenttion of the Universitt Autònom de Brcelon (pprovl n 665). Animls were kept in pproved experimentl fcilities nd were sujected to veterinry supervision for helth nd welfre. Hndling of pigs ws done y veterinrins or trined personnel llowed to do so under the Spnish nd Europen Union regultions. If necessry, nimls were sedted for decresing hndling stress. Euthnsi ws performed y pentoritl overdose ccording to Europen Union nd Spnish regultions. The study ws developed in two consecutive experiments (Tle 1) crried out in iosfety level 3 fcilities of CReSA one immeditely fter the other. Experiments were crried out using pigs of the sme origin (sme frm, sme genetic ckground, etc.) nd were housed under the sme controlled environmentl conditions. Source frm ws historiclly free of PRRSV nd pigs were re-confirmed to e free of PRRSV y ELISA (Herdcheck 2XR, Idexx Lortories) nd RT-PCR [22] nd free of PCV2 y PCR [19]. In oth experiments, upon rrivl t the experimentl fcilities, pigs were er-tgged, weighed, led nd rndomly distriuted (rndom numers) in two pens; then, piglets were left to cclimtize for one week. Inocultions were lwys done y the intrnsl route using 2 ml (1 ml/nostril) of the pproprite PRRSV strin t dose of TCID 5 /ml. Control nimls received 2 ml of sterile RPMI y the intrnsl route when necessry. Also, t the strt nd t the end of the experiment serum smples were exmined y ELISA for ntiodies Tle 1 Experimentl design Groups 1 st Chllenge* ( pi) Experiment 12 pigs Experiment 211 pigs 2 nd Chllenge* (84 pi) A strin 3262 Group A + A (n =4) Homologous chllenge: strin pigs Group A + B (n =4) Heterologous chllenge: strin 3267 C1 Plceo Group C1 + B (n =2) 6 pigs nïve controls: strin 3267 B strin 3267 Group B + B (n =4) Homologous chllenge: strin pigs Group B + A (n = 3**) Heterologous chllenge: strin 3262 C2 Plceo Group C 2 +A(n =3) 3 pigs nïve controls: strin 3262 * Inocultion ws performed intrnslly (2 ml; 1 ml/nostril) with dosis of TCID 5 /ml of the corresponding strin ** One pig died t 17 pi in the first inocultion. Six pigs were euthnized; three t 15 pi nd three t 35 pi. Four pigs were euthnized: two t 15 pi nd two t +35 pi.

3 Díz et l. Veterinry Reserch 212, 43:3 Pge 3 of 15 ginst PCV2 (Ingezim PCV2, Ingens, Mdrid, Spin) nd heptitis E virus [23]. Presence of TTSuV ws ssessed y PCR s reported [21]. Other exmined pthogens were Aujeszky s disese virus (Herdcheck PRV ge, Idexx Lortories, Brcelon, Spin), Mycoplsm hyopneumonie (Idei Mycoplsm Hyopneumonie EIA KIT, Oxoid, Cmridge, UK) nd swine influenz virus (Civtest suis influenz, Hipr Lortories, Amer, Spin). All nimls were seronegtive to those pthogens when the experiments ended. In experiment 1, 2 three-week-old piglets were divided in two groups: A (n = 14) nd C1 (n = 6). Group A ws inoculted with strin 3262 nd group C1 received RPMI s plceo. At 15 dpi, three piglets of group A nd two nimls of group C1 were euthnized nd sujected to complete necropsy. On dy 35 post-inocultion (pi), three further A piglets nd two control nimls (C1) were euthnized nd sujected to necropsy. Remining nimls (eight in group A nd two in C1) were followed up until dy 84 pi. At tht time, four A piglets were rndomly selected (rndom numers), seprted in different isoltion ox nd inoculted with strin 3262 (group A + A corresponding to the homologous chllenge (HoC) for experiment 1). The other four A pigs were inoculted with strin 3267 (group A + B; nmely heterologous chllenge (HeC) for experiment 1). C1 pigs were chllenged with PRRSV strin 3267 (from now on C1 + B). All nimls were monitored for the following two weeks nd then were euthnized nd sujected to necropsy (dy 98 pi, 14 dys post-second chllenge). In experiment 2, 11 three-week-old nimls were used. Initilly two groups were formed: B (n = 8) nd C2 (n = 3). B nimls were inoculted with strin 3267 nd nimls in C2 received RPMI. In this cse sequentil necropsies t dys 15 nd 35 pi were not done. Eighty four dys lter (dy 84 pi), four B pigs were inoculted gin with strin 3267 (B + B group, HoC for experiment 2) nd 3 piglets (one piglet died t dy 17 pi fter the initil chllenge) received strin 3262 (B + A, HeC for experiment 2). C2 nimls were lso chllenged then with PRRSV strin Animls were monitored for the following two weeks s ove. Clinicl follow-up nd smple tking In oth experiments, nimls were cliniclly exmined on rrivl nd then were monitored for the development of clinicl signs including fever: 18 dys in experiment 1 or for 36 dys in experiment 2. This difference in the periods for which rectl tempertures were recorded ws ttriutle to the fct tht feverish pigs were still oserved t 14 pi in experiment 2. Weights were recorded weekly from dy to 42 pi. Blood smples were tken (siliconized nd heprinized tues) weekly. Pthology At necropsy, smples were systemticlly tken from lungs (portions of ll loes of the right lung plus the ccessory loe), sumndiulr, trcheoronchil, mesenteric nd inguinl superficil lymph nodes, tonsils, thymus nd spleen. If relevnt gross lesions were found esides those orgns, dditionl d hoc smples were tken. All orgns were smpled in duplicte; one smple ws fixed in 1 % neutrl uffered formlin nd emedded in prffin nd the other ws frozen t 8 C until needed for further nlysis. Tissues were exmined histopthologiclly (hemtoxylin/eosin stining) for evluting the nture nd severity of lesions. In order to void ises, this exmintion ws done in linded fshion y three pthologists. For interstitil pneumoni, 4 grde score ws used: (no lesion), 1 (mild), 2 (moderte) nd 3 (severe). Similr grding ws used for the evlution of lymphoid hyperplsi (white pulp hyperplsi in spleen nd folliculr hyperplsi in lymph nodes nd tonsil). For thymus, the rtio cortex to medull ws clculted nd the presence of tingile ody mcrophges ws evluted in thymus ( to 4 scle, = none; 1 = low, 2 = moderte, 3 = high, 4 = very high). Also, lymphoid tissues were nlysed y in situ hyridiztion (ISH) to ssess presence of PCV2 s previously descried [25]. In cse tht in the course of necropsies gross lesions indictive of cteril infections were found, pproprite microiologicl cultures were performed. Virologicl nlysis The evolution of viremi ws initilly ssessed y virl isoltion in PAM nd y nested RT-PCR (nrt-pcr). For this, lood smples were serilly diluted from 1 to 1-4 nd inoculted in PAM cultures (5 μl in qudruplictes). Isoltion ws verified t dy 3 pi in PAM y mens of IPMA s reported ove. For ech niml, serum smples were re-tested for the presence of PRRSV y nrt-pcr [6] strting in the lst smple (in chronologicl order) producing positive virl isoltion. For formlin fixed tissues, PRRSV presence ws determined initilly y mens of n immunohistochemicl technique (IHC) [25]. For lungs, tonsils nd spleen, smples producing negtive IHC were then re-tested using the sme nrt-pcr s ove, dpting RNA extrction procedure to frozen tissues. In order to exmine if viremi t lte times fter the different chllenges corresponded to the predominnce of neutrliztion escpe mutnts, RNA ws extrcted from selected smples nd nlysed for ORF4 nd ORF5 specific sequences ccording to previously reported methods [15]. In order to ssess if virus present in tissues fter the second chllenge corresponded to persistent infection or not, sequencing for ORF5 ws

4 Díz et l. Veterinry Reserch 212, 43:3 Pge 4 of 15 performed on nrt-pcr positive tonsils collected from HeC pigs. Humorl response Serum smples were nlysed y mens of commercil ELISA (HerdCHeck 2XR, Idexx Lortories). Results were reported s rtio (S/P) of opticl densities etween the results of given smple nd the positive control included in the kit (cut-off: S/P.4). All serum smples were tested the sme dy nd in the sme tch of pltes. Also, levels of NA were determined. Since the used PRRSV strins could not e dpted to grow in MARC- 145 cells t low pssges (n 4), virl neutrliztion tests (VNT) were done in PAMs. In order to test the cross-rectivity of ntiodies ginst strins 3262 or 3267, VNT were performed in prllel with ech virl strin. Briefly, ser were diluted (in duplicte) in seril dilution (log 2 ) from 1/2 to 1/256, inctivted t 56 C 3 min nd incuted overnight 1:1 vol/vol t 4 C with one or the other of the ovementioned strins (2 TCID 5 /ml). PAMs were cultured in 96 wells pltes (5 /well) with the virus-serum mixture. After 72 h of incution, PAMs were fixed in solute ethnol t 2 C until needed. The rection ws reveled y mens of peroxidse conjugte using non-neutrlizing monoclonl ntiody ginst ORF5 (3AH9; Ingens, Mdrid, Spin) nd insolule TMB (TMB/h; Milipore, Billeric, USA) s chromogen. In order to hve n ssessment of the ccurcy of the mesurement, testing ws done twice for dys 42 pi, 84 pi nd 98 pi (14 post second chllenges). In ddition, to test if negtive neutrliztion results t 72 h post-inocultion of PAM could e due to selection nd growth of neutrliztion escpe mutnt vrints or qusispecies present in the originl virus stock, pnel of selected ser yielding negtive results in the VNT were used in second VNT tht ws performed s ove ut reveled fter 12 h of incution of PAM with the virus/ serum mixture in order to void propgtion of the hypotheticl neutrliztion escpe mutnts. IFN-γ ELISPOT Evlution of the cell-medited immune response (CMI) ws done y the IFN-γ ELISPOT using peripherl lood mononucler cells (PBMC) [6,26] t dys, 21, 42, 63, 7, 77 nd 84 pi plus on dys 91 nd 98 (7 nd 14 post second chllenges, respectively). In order to evlute the in vitro homologous nd heterologous responses in the IFN-γ ELISPOT, PBMC were stimulted in prllel (5 PBMC/well, 3 wells per pig nd stimulus) with strins 3262 nd 3267 t multiplicity of infection (m.o.i.) of.1. Unstimulted cells nd phytohemgglutinin-stimulted controls (1 μg/ml) were lso included. PRRSV-specific corrected frequencies of IFN-γ-SC were clculted y sutrcting counts of spots in unstimulted wells from counts in virus-stimulted wells. Frequencies of IFN-γ-SC were expressed s responding cells in 1 6 PBMC. Cytokine ELISAs In oth experiments serum smples were tken t dys, 1, 2, 3, 7 nd 14 post second chllenges (first chllenge for control groups C1 nd C2) nd tested y ELISA for determining serum levels of TNF-α, IL-1, IL-6, IL-8, IL- 1 nd TGF-β. ELISAs were performed using commercil pirs of monoclonl ntiodies ccording to mnufcturer s instructions: TNF-α, IL-1, IL-6 nd IL-8 (Porcine TNF-α, IL-1, IL-6 nd IL-8 DuoSet, respectively; R&D Systems, Minnepolis, USA) nd IL-1 nd TGF-β (IL-1 Swine Antiody pir nd TGF-β1 Multispecies ELISA Kit; Invitrogen). The cut-off of ech ELISA ws clculted to e equl to the men opticl density (OD) of negtive controls plus three stndrd devitions (cutoff = 32 pg/ml for IL-6, IL-1, TNF-α, TGF-β1; cut-off = 62 pg/ml for IL-1 nd IL-8). For given smple, the OD ws then trnsformed to concentrtion y pplying liner regression formul clculted from the results of the stndrds provided in ech kit. In order to ccount for the pre-2nd chllenge levels of given cytokine, vlues (pg/ml) otined with smples of dy (84 pi) were sutrcted to the vlues otined t dys 1, 2, 3, 7 or 14 producing thus corrected vlue (Corrected vlue for cytokine X = Concentrtion of cytokine X dy n - Concentrtion of cytokine X dy ). Sttisticl nlysis Sttistics were performed using SttsDirect v Kruskl-Wllis test (Conover-Inmn method for multiple comprisons) ws used for comprisons of mens etween groups nd the Log Rnk & Wilcoxon test ws used for compring the durtion of viremis. Comprison of the proportion of infected tissues ws determined y the χ 2 test (Fisher s exct test). EpiClc 2 ws used for clcultion of confidence intervls of proportions. Results Clinicl follow-up nd weight gins Figure 1 shows the evolution of ody tempertures nd weight gins in experiments 1 nd 2. In oth experiments inocultion of PRRSV induced moderte fever, up to 4.4 C, during t lest the first 14 dys pi. In the cse of experiment 2, fever ws more prolonged nd ody tempertures of inoculted nimls were significntly higher (p <.5) compred to controls until dy 22 pi. In experiment 1, respirtory signs were sent or very mild while in experiment 2, dullness nd respirtory signs were slightly more evident including occsionl coughing nd loured reth. In the second experiment

5 Díz et l. Veterinry Reserch 212, 43:3 Pge 5 of 15 c Averge weekly weight gin (Kg) d1-d14* Experiment 1 Experiment 1 * c n.s. n.s. 2nd chllenge d1-d16 nd d19-d22* d 2nd chllenge Figure 1 Rectl temperture nd ody weight gins fter inocultion with PRRSV. Evolution of verge rectl tempertures nd ody weight gins in experiments 1 (figure -c) nd 2 (-d). In experiment 1, nimls received initilly (dy pi) PRRSV strin 3262 (A; lck crosses, light grey rs) or were mock-inoculted (Control; empty circles, empty rs). At dy +84 pi, four pigs were chllenge with strin 3262 gin (A + A; lck tringles, lck rs) or received heterologous chllenge with strin 3267 (A + B; grey rhomus or drk grey rs). Nïve pigs were chllenged with strin 3267 (C1 + B; empty squres or digonl line rs). In experiment 2, pigs were initilly inoculted with strin 3267 (B) nd lter chllenged with 3267 (B + B) or 3262 (B + A). Controls were included (Control nd C2 + A). Significnt (p <.5) differences in ody tempertures re mrked with n sterisk. For weight gins (right side of the figure), rs with letter ove show significnt (p <.5) differences. one niml died t 17 pi showing gross lesions of firinous to firous pleuritis, ctrrhl ronchopneumoni nd interstitil pneumoni, suggesting cteril compliction tht could not e confirmed in the microiologicl nlysis. Regrding weight gins, in Experiment 1 inoculted nimls showed decresed weight gins (p <.5) compred to controls until dy 21 pi nd in experiment 2 differences were noticele until 42 pi. For the first 42 dys pi, weight gins of control pigs were 33.3 Kg in experiment 1 nd 31.3 Kg in experiment 2, while weight gins for virus inoculted nimls in the sme period were 3.5 Kg in experiment 1 nd 16.3 Kg in experiment 2 (p <.5). After the second chllenge, clinicl signs were sent in oth experiments except in control nimls (C1 nd C2) tht were immunologiclly nive nd showed fever occsionlly. After the second chllenge weight gins were significntly different mong groups (p <.5) nd lwys nimls sujected to HoC hd etter gins thn nimls sujected to HeC or thn nïve pigs inoculted for the first time. Pthology In experiment 1, necropsies performed t dy 15 pi showed tht inoculted nimls developed gross interstitil pneumoni tht microscopiclly rnged from slight

6 Díz et l. Veterinry Reserch 212, 43:3 Pge 6 of 15 (1) to severe (3). No gross lesions were seen eyond those oserved in lungs. At tht time (15 pi) PRRSV ws detected y IHC in lungs of ll inoculted nimls. Uninoculted controls did not develop gross or microscopic lesions nd were free of PRRSV y IHC. At dy 35 pi, inoculted pigs did not show mcroscopic or microscopic signs of interstitil pneumoni nd were negtive to PRRSV y IHC. Regrding the development of lesions fter the second chllenge in experiment 1 -tht took plce t dy 84 pinone of the pigs showed mcroscopic evidence of interstitil pneumoni, except for one in A + B group. Microscopiclly (Tle 2), oth the HoC nd the HeC groups showed similr lesions of interstitil pneumoni with scores rnging from slight to severe depending on the lung loe exmined. Control nimls infected with strin 3267 for the first time (C1 + B) hd similr lung lesions thn the HoC or the HeC group. In experiment 2, necropsies performed two weeks fter the second chllenge showed tht only one pig (B + A group) hd signs of gross interstitil pneumoni. No differences were oserved etween groups regrding lymphoid hyperplsi in spleen, tonsils or lymph nodes in none of the experiments. However, density of tingile ody mcrophges ws pprently higher in the B + A group compred to the others (not shown). Virologicl nlysis Results of the evolution of viremi re shown in Tles 3 nd 4. Inocultion of nive pigs with strin 3262 (experiment 1) resulted in viremi (nrt-pcr) y dy 7 pi in ll nimls. The proportion of viremic pigs declined fterwrd to 11/14 y dy 14 pi (78.6 %; CI 95% : %), 4/11 t 21 pi (36.4 %; CI 95% : %) nd 1/11 t 28 pi (9.1 %; CI 95% : %). By dy 35 pi ll inoculted Tle 2 Results of the histopthologicl exmintion of lungs Experiment Group Interstitil pneumoni* 1 C1+B 5. ± 2.8 A+A A+B 6.5 ± ± C2+A 8.7 ± 2.3 B+B B+A 6.5 ± ±.6 Different superscript letters indicte significnt differences (p <.5) etween the groups in given experiment ccording to the Kruskl-Wllis test (Conover-Inmn test for multiple comprisons). No significnt differences were seen for the doule chllenged groups etween experiment 1 nd 2. * Microscopic interstitil pneumoni ws evluted in 4 portion of lung corresponding to the picl, crdic, diphrgmtic nd intermedite loes, scored in 4-grde scle ( = sent to 3 = severe). The vlues shown correspond to the verge of the summtory of scores for ech pig in given group nd the stndrd devition of the summtories. With this clcultion, mximum possile verge is 12. In the cse of group C2 + A; p =.6. nimls hd ecome negtive in lood nd tissues (nrt- PCR nd IHC). Virl lod in serum peked t 7 pi (verge virl titre of TCID 5 /ml) nd declined lter on. After the second chllenge t dy 84 pi, ll nimls, regrdless of the previous sttus of immuniztion or the strin used for this second chllenge, ecme infected gin s shown y results of ser y nrt-pcr (Tle 4); however, virl isoltion from lood ws only chieved for PRRSV-nïve pigs. Exmintion of tissues y IHC nd nrt-pcr (Tle 5) reveled tht 14 dys fter the second inocultion, PRRSV could e detected in lungs of 2/ 4 pigs in oth the HoC nd HeC groups. In contrst, trend for significntly higher presence of virus in tonsils ws oserved in the HeC compred to the HoC (p =.7). In experiment 2, ll nimls were viremic (nrt-pcr) for the first 35 dys pi nd y dy 56 pi 3/7 pigs were still positive (42.9 %; CI 95% : %). Regrding virl isoltion nd titrtion, viremi peked t dy 7 pi with n verge virl lod in serum of TCID 5 /ml nd virl lod remined ove 1 3. TCID 5 /ml for 21 dys pi. From dy 14 pi onwrds, virl lods were higher in nimls infected with strin 3267 compred to nimls infected with strin 3262 in experiment 1 (p <.5) nd durtion of viremi ws lso sttisticlly longer (p <.5). To test if the longer viremi seen for strin 3267 ws ttriutle to the genertion nd expnsion of neutrliztion escpe mutnts in the course of infection, virl isoltes of dy 49 pi were sequenced for the ORF4 nd ORF5. Results of sequencing showed tht the NE in GP4 chnged during the course of the infection nd tht GP5 gined one potentil glycosyltion t N37 (DSS!NSS). After the second chllenge of dy 84 pi only PRRSVnïve pigs developed detectle viremi tht lsted until the end of the experiment (Tle 4). Exmintion of tissues indicted however tht t lest two nimls in the HoC group nd one niml in the HeC group hd ecome infected s demonstrted y the presence of the virus in tonsils (Tle 5). Sequencing demonstrted tht the virus found in the HeC experiments lwys corresponded to the heterologous virus nd ws not remnnt of the initil inocultion. Humorl response After inocultion with either one or the other PRRSV strin, pigs rpidly developed ntiodies detectle in ELISA lthough S/P rtios were different mong groups. Thus, for the first 84 dys pi (except t dy 35 pi) S/P rtios were higher (p <.5) in nimls inoculted with experiment 2- thn in nimls inoculted with experiment 1- (Figure 2). After the second chllenge (dy 84 pi), re-inoculted nimls ecme similr

7 Díz et l. Veterinry Reserch 212, 43:3 Pge 7 of 15 Tle 3 Detection of virus in ser y isoltion in porcine lveolr mcrophges nd y nested RT-PCR fter first chllenge with PRRSV Dys post 1st inocultion (pi) Experiment Assy Virl isoltion 14/14 11/14 4/11 1/11 /11 /8 /8 /8 /8 /8 /8 /8 (titre) 3.1±.6 2.9±.5 2.3±.2 2.3± nrt-pcr 14/14 11/14 4/11 1/11 /11 /8 /8 /8 /8 /8 /8 /8 Virl isoltion 8/8 8/8 7/7 * 7/7* 7/7* 3/7* 3/7* 1/7* /7 /7 /7 /7 (titre) 3.7 ± ± ± ± ±.2 2. ±.3 2. ± ± nrt-pcr N.D. N.D. N.D. N.D. N.D. 4/7 4/7 3/7 /7 /7 /7 /7 Results of virl titrtion of ser in porcine lveolr mcrophges (PAM) using the immunoperoxidse monolyer ssy re expressed s numer of positive pigs over the numer of pigs in the group nd s the men ± stndrd devition (log 1 ) of titres (TCID 5 /ml) of positive pigs. *p <.5 for the comprison of infected groups etween experiment 1 (strin 3262) nd 2 (strin 3267). For the nrt-pcr results only show the numer of positive pigs in the second round of the PCR. Results of control (uninoculted) pigs re not shown ecuse they were lwys negtive from dy to 84 post-inocultion. Three pigs were euthnized t dy 15 pi nd three pigs more were killed t dy 35 pi. One pig died t dy 17 pi. N.D. Not done in terms of S/P rtios within ech one of the experiments (Figures 2 nd 2c). Development of NA ginst the homologous nd the heterologous strins ws exmined in VNT using PAM (Tle 6). In experiment 1, fter the first inocultion none of the pigs developed NA ginst the inocultion strin 3262 (homologous VNT). After chllenge t dy 84 pi, serum of pigs re-inoculted with either 3262 or 3267 did not show neutrlizing ctivity ginst strin In contrst, in experiment 2 (inocultion with strin 3267) NA ginst the homologous strin were Tle 4 Detection of virus in ser y isoltion in porcine lveolr mcrophges nd y nested RT-PCR fter the second chllenge with PRRSV Dys post-2 nd chllenge Positive/totl Virl titre ± stndrd devition Experiment Group Assy +7 (91 pi) +14 (98 pi) 1 2 C1 + B Virl isoltion 2/2 2/2 3.2 ±.6 2. ±. nrt-pcr 2/2 2/2 A + A Virl isoltion /4 /4 nrt-pcr 4/4 1/4 A + B Virl isoltion /4 /4 nrt-pcr 4/4 /4 C2 + A Virl isoltion 3/3 2/3 3.4 ± ±.1 nrt-pcr 3/3 3/3 B + B Virl isoltion /4 /4 nrt-pcr /4* /4 B + A Virl isoltion /4 /4 nrt-pcr /4* /4 *Asterisks indicte sttisticl differences in the proportion of pigs found to e positive y RT-PCR in comprle groups of experiments 1 nd 2 (i.e. homologous A + A versus homologous B + B). demonstrted in some pigs lredy y dy 21 pi nd y dy 42 ll inoculted pigs were positive for NA. At dy 84 pi, titres of homologous (3267) NA rnged from 3 log 2 to 6 log 2. After the second chllenge (dy 84 pi), most nimls showed n increse of the homologous (3267) VNT titres y 1 2 log 2. Nïve pigs inoculted with 3267 for the first time t 4 months of ge in experiment 1 lredy showed NA titres in the rnge of 2 3 log 2 ginst tht strin 14 dys fter the inocultion (dy 98). When cross-neutrliztion tests were performed, the picture ws different. Thus, in experiment 1 y dy 84 pi ser of 6/8 nimls (75 %; CI 95% : %) showed neutrlizing ctivity ginst 3267 (heterologous in this cse) ut with low titres (1 3 log 2 ). After the second chllenge, oost in the VNT titres ginst 3267 (3 6 Tle 5 Distriution of PRRSV in tissues fter the homologous or heterologous chllenge y nested RT-PCR or immunohistochemistry (IHC) Nº positives/nº exmined Experiment Group Assy Lung Tonsil Spleen 1 2 *p =.7 C1 + B nrt-pcr /2 2/2 1/2 IHC /2 1/2 /2 A+A nrt-pcr 2/4 1/4 1/4 IHC /4 1/4 1/4 A+B nrt-pcr 2/4 4/4* 3/4 IHC /4 /4 1/4 C2 + A nrt-pcr 1/3 1/3 /3 IHC /3 /3 /3 B+B nrt-pcr /4 2/4 /4 IHC /4 /4 /4 B+A nrt-pcr /3 1/3 /3 IHC /3 1/3 /3

8 Díz et l. Veterinry Reserch 212, 43:3 Pge 8 of 15 ) ISA EL o (E rtio /P S/ 2,4 2,2 2 1,8 1,6 1,4 1,2 1,8,6,4,2 n.s Dys post-inocultion A B C1 C2 ISA A) (E ELI os tio S/P r S Experiment 1 Experiment ,4 2,4 2,2 22 2,2 22 ns n.s 2 2 1,8 18 1,8 16 1,6 16 1,6 1,4 14 1,4 12 1,2 1,2 1 1,8 8,8,6,6 4,4,4 2,2 2,2 7 Dys post-chllenge 14 7 Dys post-chllenge 14 A+A A+B C1+B ISA A) (EL LI os ( tio r /P S/ B+B B+A C2+A Figure 2 Evolution of the humorl response (ELISA) ginst PRRSV in experiments 1 nd Averge smple to positive (S/P) rtios of opticl densities from dy +15 to +84 post-inocultion. Empty rs correspond to pigs in experiment 1 (strin 3262: A); grey rs correspond to pigs in experiment 2 (strin 3267: B). Lines show results of control (un-inoculted pigs) in oth experiments.2. Serologicl evolution (S/P rtios) of pigs in experiment 1 fter the second chllenge (84 pi). Empty rs correspond to the homologous chllenge (group A + A), grey rs to the heterologous chllenge (A + B) nd lck rs depict results of nïve pigs inoculted for the first time with strin 3267 (C1 + B)2c. Serologicl evolution (S/P rtios) of pigs in experiment 2 fter the second chllenge (84 pi). Empty rs: (B + B); grey rs: B + A; lck rs: nïve pigs inoculted for the first time with strin 3262 (C2 + A).In ll cses, different superscript letters indicted sttisticlly significnt differences (p <.5) s determined in the Kruskl-Wllis test; n.s. = non-significnt. c log 2 ) ws seen regrdless of the strin used for tht second chllenge. In experiment 2, ser of pigs inoculted with 3267 showed wek neutrlizing ctivity ginst 3262 (heterologous) y dy 84 pi (1 4 log 2 ). After the second chllenge, heterologous titres ginst 3262 did not sustntilly increse except for one pig (receiving 3267 gin). Evolution of the IFN-γ ELISPOT fter the initil chllenge For ech of the experiments, the IFN-γ ELISPOT ws performed using in prllel ech of the strins included in the study in order to exmine the homologous nd heterologous response (Figures 3 nd ). In experiment 1, inocultion with strin 3262, the use of the sme 3262 strin in the ELISPOT reveled pek of virus-specific IFN-γ secreting cells (SC) y dy 21 pi; therefter frequencies decresed to low y dy 63 pi. Using the strin 3267 s stimulus (heterologous stimultion in this cse), the trend ws similr ut with significntly (p <.5) lower frequencies of responding cells in ll exmined dys. In experiment 2, inocultion with strin 3267, the ELI- SPOT using the strin 3267 produced similr temporl trend with the lowest frequencies t dys 42 nd 63 pi. In this cse, the use of strin 3262 (heterologous stimultion in ELISPOT), resulted in much higher frequencies (p <.5) for the first 7 dys pi nd lmost significnt differences (p =.6) were still determined y dy 77 pi. When equivlent series of IFN-γ ELISPOT results were compred etween experiments; nmely, homologous stimultion in experiment 1 versus homologous stimultion

9 Díz et l. Veterinry Reserch 212, 43:3 Pge 9 of 15 Tle 6 Virl neutrliztion test results using PRRSV strins 3262 nd 3267 Nº of responding pigs; men titre (log 2 ) nd stndrd devitionrnge of titres (log 2 ) for positive pigs Strin used in the virl neutrliztion test Dys post-inocultion Dys post-inocultion Chllenge group A+A /4 /4 /4 /4 /4 3/4* 4/4* 4/4* ( ) ±.6 2. ± ± 1. (1 2) (1 3) (3 5) A+B /4 /4 /4 /4 /4 2/4 2/4 4/4* ( ) ±. 2. ±. 5.3 ±.5 (1 1) (2 2) (5 6) C1 + B /2 /2 /2 /2 /2 /2 /2 2/2* (C+3267) ±.5 (2 3) B+B /4 2/4 4/4 4/4 4/4 4/4 4/4 4/4 ( ) /4 1. ±. 2.2 ± ± ± ± ± ±.8 - (1 1) (1 4) (1 5) (1 3) (2 4) (4 6) (5 7) B+A /3 /3 3/3 3/3 1/3 3/3 3/3 3/3 ( ) 2. ± ±.6 1. ±. 3. ± ± ± (1 3) (1 2) (1 1) (2 4) (3 5) (5 6) C2 + A /3 /3 /3 /3 /3 /3 /3 /3 (C+3262) Chronologicl sequence of strins used for inocultion is shown in prentheses. * The sterisk show sttisticl differences (p <.5) for the proportion of responding pigs using one or the other PRRSV strin. Superscript letters indicte sttisticl differences (p <.5; p =.8) in the verge titres etween different dys using given strin. The symol indictes sttisticlly significnt differences for the proportion of responding pigs for similr groups etween experiments (i.e. homologous A + A vs. homologous B + B) using the sme strin. in experiment 2 nd heterologous stimultion in experiment 1 versus heterologous stimultion in experiment 2, differences were lso seen. Thus, inocultion with 3262 produced higher frequencies of IFN-γ cells (p <.5) for dys 21, 42 nd 77 pi compred to the inocultion with 3267 (14 ±73 vs. 38 ± 3; 54 ± 24 vs. 9 ± 9 nd 16 ± 69 vs. 34 ± 25, respectively). Compring results of heterologous stimultion in ELISPOT, the use of strin 3262 resulted in higher responses (p <.5) t dys 21, 42, 63 nd 7 pi (181 ± 12 vs. 29 ± 19; 68 ± 37 vs. 14 ± 12; 91 ± 22 vs. 2 ± 3 nd 11 ± 34 vs. 22 ± 2). Evolution of the IFN-γ ELISPOT fter the second chllenge Figures 4 nd summrize this prt of the experiment. After the chllenge of dy 84 pi, the ehviour of the IFN-γ-SC response vried depending on the experiment. Thus, for experiment 1 (initil immuniztion ginst strin 3262), re-inocultion of PRRSV resulted in significnt oost of the IFN-γ ELISPOT responses oth in the HoC nd in the HeC groups y dy 91 except for nimls tht hd reched frequencies of IFN-γ SC 1 efore the chllenge. In this cse gin, use of strin 3267 in the ELISPOT resulted in lower frequencies regrdless of the strin used for chllenge. In experiment 2, re-inocultion of PRRSV did not produce cler pttern. Cytokine (IL-1, IL-6, IL-8, TNF-α, IL-1, nd TGF-β) ELISAs Cytokine levels in serum were exmined only fter the second chllenge of ech experiment nd in nïve control pigs. Regrding IL-1, ll smples were elow the ssy detection limit in oth experiments. For IL-6, in experiment 1 two nimls in ech group (A + A nd A + B) hd detectle levels of this cytokine from dy 1 to 7 post-chllenge; nïve pigs inoculted for the first time produced negtive results. In contrst, ll smples yielded negtive results for IL-6 in experiment 2. For IL-8 (Tle 7), differences were noticed mong groups nd tretments. In experiment 1, nïve pigs inoculted for the first time t dy 84 with strin 3267 were positive for IL-8 (corrected concentrtion) t dys 1, 2, 3 nd 7 post-chllenge. In contrst, in the HeC model IL-8 ws only detected t dys 1 nd 2. In the HoC, t lest one niml produced positive result every exmined dy ut no constnt pttern ws seen. In experiment 2, the picture ws the opposite to tht of experiment 1. Nïve nimls

10 Díz et l. Veterinry Reserch 212, 43:3 Pge 1 of 15 PRRSV-specific IFN- secreting cells per million PBMC PRRSV-specific IFN- secreting cells per million PBMC ** 8/8 7/8 8/8 ** 7/8 8/8 ** 8/8 Homologous Heterologous,c,c DPI ** 7/7 6/7 5/7 ** 7/7 7/7 ** 4/8,,,, DPI * 7/7 7/7 7/7 7/8 8/8 * * 7/7 7/8 8/8 ** 5/8 Homologous Heterologous n.s. 7/7 7/7 6/7 Figure 3 PRRSV-specific cell-medited immune response s determined y the IFN-γ ELISPOT, dys 21 to 84 pi. Peripherl lood mononucler cells (PBMC) were stimulted with either of the PRRSV strins (3262 or 3267) used in the study. Results re shown (columns) s verge frequencies of virus-specific IFN-γ secreting cells per million of PBMC otined in the ELISPOT with the corresponding stndrd devitions (rs). For ech experiment the results of ELISPOT re shown in two series: drker shdowed columns indicte results otined fter in vitro stimultion with the sme PRRSV strin with which hd een immunized initilly (homologous) while lighter coloured rs show the results of in vitro stimultion with other PRRSV isolte used in the present study (heterologous). Asterisks indicte significnt differences etween results otined in the homologous nd the heterologous ELISPOT for ech experiment (*p <.5; **p <.1; p =.7) t given time point. Letters indicte significnt differences (p <.5) mong different time points in given series. For ech time point nd series, the numer of responding pigs over the totl numer of exmined pigs is shown. 3A = Experiment 1 (inocultion with strin 3262); 3B = Experiment 2 (inocultion with strin 3267). dpi = dys post-inocultion. inoculted for the very first time with 3262 produced IL-8 results elow the ssy detection limit t ll points. In contrst, t lest one niml hd detectle IL-8 levels t ny given time fter the HoC or HeC. For TNF-α, no significnt differences were noticed nd for IL-1, in oth experiments positive results were spordic. For TGF-β (Tle 8), different outcome ws seen depending on the experiment nd group. Thus, in experiment 1, nïve pigs inoculted with 3267 were spordiclly positive similrly to wht occurred in nïve pigs inoculted with 3262 in experiment 2. In experiment 1, oth in the HoC nd in the HeC, re-inocultion with PRRSV induced incresed levels of TGF-β tht peked t dy 7 or 14 post chllenge. In experiment 2, differences were seen depending on the strin used for re-inocultion. Thus, while in the HoC

11 Díz et l. Veterinry Reserch 212, 43:3 Pge 11 of 15 A Experiment 1 (initil immuniztion with strin 3262): Homologous chllenge (strin 3262) ) IFN- ELISPOT with strin 3262 ) IFN- ELISPOT with strin nº 3 nº 7 nº 9 nº 12 nº 3 nº 7 nº 9 nº 12 Experiment 1 (initil immuniztion with strin 3262): Heterologous chllenge (strin 3267) ) IFN- ELISPOT with strin 3262 ) IFN- ELISPOT with strin nº 2 nº 6 nº 8 nº 13 nº 2 nº 6 nº 8 nº 13 B Experiment 2 (initil immuniztion with strin 3267): Homologous chllenge (strin 3267) ) IFN- ELISPOT with strin ) IFN- ELISPOT with strin 3262 nº 51 nº 52 nº 55 nº 56 nº 51 nº 52 nº 55 nº 56 Experiment 2 (initil immuniztion with strin 3267): Heterologous chllenge (strin 3262) ) IFN- ELISPOT with strin 3267 ) IFN- ELISPOT with strin nº 49 nº 5 nº 53 nº 49 nº 5 nº 53 Figure 4 Individul responses in the IFN-γ ELISPOT fter second chllenge tht took plce t dy 84 pi. Brs depict individul frequencies of virus-specific IFN-γ secreting cells fter the homologous nd heterologous chllenge for this experiment. ELISPOTs were performed with oth strins used in the present study.

12 Díz et l. Veterinry Reserch 212, 43:3 Pge 12 of 15 Tle 7 Serum levels of IL-8 fter chllenge with PRRSV t 84 dys fter the initil inocultion Corrected IL-8 levels in serum (pg/ml) N responding pigs; Averge ± stndrd devition Experiment 1 Dys post-chllenge Group 1* 2* 3 7} 14 C1 + B 2/2 27 ± 18 A+A /4 A+B 2/4 644 ± 479 Experiment 2 Group C2 + A 2/2 424 ± 255 1/4 44 ± 3/4 194 ± 98 2/2 44 ± 434 2/4 272 ± 363 /4 Dys post-chllenge 2/2 47 ± 66 1/4 822 ± /4 1/2 711 ± 1/4 124 ± / } /3 B+B 3/4 249 ± 192 B+A 2/3 38 ± 2 /3 1/4 526 ± 1/3 667 ± /3 2/4 212 ± 211 2/3 188 ± 47 /3 2/4 262 ± 247 1/3 577 ± N.A /3 3/4 297 ± 273 3/3 291 ± 117 In order to ccount the pre-2nd chllenge levels of given cytokine, vlues (pg/ml) otined with smples of dy (84 pi) (C1 + B:653 ± 66; A + A:796 ± 36; A + B:162 ± 157; C2 + A:53 ± 133; B + B:187 ± 198; B + A:41.3 ± ) were sutrcted to the vlues otined t dys 1, 2, 3, 7 or 14, respectively for producing corrected vlue (Corrected vlue for cytokine X = Concentrtion of cytokine X dy n - Concentrtion of cytokine X dy ). not pplicle. Symols (* p =.5; } p <.5) show dys for which sttisticl differences were oserved etween groups of given experiment with regrds to the proportion of positive pigs. Letters indicte sttisticlly significnt differences for the proportion of responding pigs for similr groups when compring etween experiments; i.e. nïve pigs inoculted with 3267 (C1 + B) vs. nïve pigs inoculted with 3262 (C2 + A). In this cse,, correspond to p.1. model ll nimls hd sustined serum levels of TGFβ from dy 1 to 14 post re-inocultion, in the HeC model TGF-β ws detected only spordiclly. Discussion The present study shows tht two different PRRSV isoltes of genotype I sutype I cn produce two different models of infection sed on the clinicl, virologicl nd immunologicl prmeters exmined. Although oth models were sent of very overt clinicl signs, in one of them (strin 3262) fever nd the decrese in weekly weight gins were very mild compred to the other (strin 3267). Differences in the outcome of the inocultion ttriutle to the reed, genetics, to the origin of pigs or to different environmentl conditions were controlled y using nimls from single frm with common genetic nd helth ckground. The use of controlled environment BSL3 fcilities reduced vriility ttriutle to environmentl conditions. Regrding the potentil is cused for concomitnt secondry pthogens, the results mke evident tht some pigs in experiment 2 developed cteril infection, fct tht ws Tle 8 Serum levels of TGF-β fter chllenge with PRRSV t 84 dys fter the initil inocultion Corrected TGF-β levels in serum (pg/ml) Nº responding pigs; Averge ± stndrd devition Experiment 1 Dys post-chllenge Group C1 + B /2 /2 1/2 1/2 1/2 74 ± 113 ± 23 ± 18 A+A 3/4 3/4 4/4 4/4 4/4 5 ± 1 67 ± ± 6 93 ± ± 11 A+B 3/4 2/4 2/4 4/4 4/4 57 ± ± 6 19 ± 5 99 ± ± 9 Experiment 2 Dys post-chllenge Group 1 2 3* 7* 14 C2 + A 1/3 1/3 /3 1/3 1/3 57 ± 149 ± 17 ± 33 ± B+B 4/4 4/4 4/4 4/4 4/4 42 ± ± ± ± ± 14 B+A 2/3 2/3 /3 /3 1/3 16 ± ± 61 N.A 37 ± In order to ccount the pre-2nd chllenge levels of given cytokine, vlues (pg/ml) otined with smples of dy (84 pi) (C1 + B:141 ± 2; A + A:58 ± 29; A + B:86 ± 63; C2 + A:124 ± 17; B + B:137 ± 14; B + A:136 ± 171) were sutrcted to the vlues otined t dys 1, 2, 3, 7 or 14, respectively for producing corrected vlue (Corrected vlue for cytokine X = Concentrtion of cytokine X dy n - Concentrtion of cytokine X dy ). not pplicle. Symols (*p.5) show dys for which sttisticl differences etween groups with regrds to the proportion of positive pigs were oserved. Letters indicte sttisticlly significnt differences for the proportion of responding pigs for similr groups etween experiments; i.e. nïve pigs inoculted with 3267 (C1 + B) vs. Nïve pigs inoculted with 3262 (C2 + A). In this cse,, correspond to p.5. not seen in experiment 1. This could hve ffected the clinicl outcome of the infection fter some dys of clinicl course ut is difficult to elucidte the precise impct of secondry infections on the virologicl course or on virus-specific immune responses. Regrding the virologicl outcome of the initil chllenge, it ws evident tht strin 3267 produced longer nd sustined viremis in piglets compred to wht strin 3262 did. Although cuses for those different outcomes cnnot e precisely defined t present, those oservtions will e coherent with the higher repliction rtes of strin 3267 oserved in vitro in PAM cultures compred to those of 3262 ( versus TCID 5 /ml fter 48 h of incution t similr m.o.i., dt not shown). Also, persistence of the virus cn hve n individul component s evidenced y the fct tht hlf of the pigs infected with strin 3267 resolved viremi y dy 42 pi. For the humorl response, s fr s S/P vlues in ELISA re concerned, strin 3267 induced higher nd

13 Díz et l. Veterinry Reserch 212, 43:3 Pge 13 of 15 more sustined S/P vlues thn strin Although the nture of this fct is difficult to scertin with the ville dt; it cn e hypothesized tht the higher sustined repliction levels of 3267 could e the cuse. With regrds to NA, in the present study it ecme evident tht their role in clering viremi during the course of the infection cn e limited. In experiment 1 viremi cesed in sence of detectle NA ginst the homologous isolte nd in experiment 2, viremi coexisted for weeks with NA. Glycosyltion of neutrlizing epitopes hve een reported to result in poorer development of NA or in incresed difficulties for virl neutrliztion to occur [27]. Strin 3262 for which development of NA did not occur -or could not e demonstrted- hd six potentil glycosyltion sites in GP3, the neutrliztion epitope (NE) in GP4 hd deletion nd the ssumed NE in GP5 contined three potentil glycosyltion sites (N-37, N-46 nd N-53). Strin 3267 hrour six potentil glycosyltion sites in GP3, hd n intct GP4 very close to tht of the reference Lelystd virus- nd hrour two glycosyltion sites in GP5 (N-46 nd N-53) [15]. Additionlly, when the virus 3267 present in lood of viremic pigs t dy 49 pi ws sequenced, third glycosyltion site in N-37 of GP5 ws detected nd GP4 hd suffered some vritions in comprison with the prentl strin (dt not shown). Thus differences in the levels of NA my rise from the different chrcteristics in the sequence nd numer of glycosyltions of the NE of GP3 GP4 nd GP5. On the other hnd, when cross-neutrliztion experiments were performed, it ws seen tht ser of nimls primo-inoculted with strin 3267 were lso cple of neutrlizing strin 3262 lthough with much lower efficiency. Ser rised ginst 3262 were devoid of neutrlizing cpilities even ginst the homologous Considered glolly, these results suggest tht in PRRSV, under some circumstnces heterologous NA could e more efficient thn the homologous ones. This hs een oserved recently y Mrtínez-Loo et l. [12]. The nture of this phenomenon is unknown. Clssiclly, these differences would hve een ttriuted to chnges in the sequence of the NE nd to the numer of glycosyltions present in those NE nd ctully, these chnges occurred in the present study. Nevertheless, in the study y Mrtínez- Loo et l. [12] differences in cross-neutrliztion in ser rised ginst different genotype I strins could not e relted strictly to the sequence nd numer of glycosyltions of the known GP3, GP4 or GP5 NE nd those uthors suggested tht mye the conformtionl chrcteristics of the epitopes could hve role on the crossrectivity or, lterntively, tht other NE unknown yet exist. In ny cse, tking in considertion the ovementioned fcts, if pre-formed NA ply role in protection ginst PRRSV re-infection s indicted y previous ppers [5,9,27], nimls infected/immunised initilly y strin 3262 should e more likely infected upon the HoC or HeC thn nimls infected/immunized initilly with This is wht occurred nd s shown in Tles 4 nd 5: nimls immunized with strin 3262 developed viremi fter either the HoC or the HeC, lthough viremi ws of low intensity. When the cell-medited comprtment of the immune response ws exmined, it ws evident tht strin 3262 induced stronger virus-specific response mesured s IFN-γ-SC. Interestingly, this sitution ws not reversed y the second chllenge t dy 84 pi, even for nimls primo-immunized with 3267 nd chllenged gin with In this cse the higher levels of repliction of isolte 3267 did not seem to contriute to higher cell-medited response. In previous study [16] it ws shown tht infection of dendritic cells with strin 3267 resulted in n increse of SLA-II + /CD8/86 - nd SLA-II - /CD886 + cells while strin 3262 induced decrese in the proportion of SLA-II + /CD8/86 + cells. Both 3262 nd 3267 down regulted SLA-I in infected cells. Also, strins 3267 nd 3262 induced different TNF-α nd IL-1 responses in dendritic cells. These differences my lie ehind the different immune responses developed fter the first chllenge. Besides tht, strin 3262 produced higher frequencies of IFN-γ SC when used either s homologous or heterologous recll stimulus in ELISPOT. This fct points to the potentil differences in the respective cpilities of the exmined strins to inhiit IFN-γ responses or to different ntigenicity of T-epitopes. Interestingly, fter the second chllenge, oost of IFN-γ SC responses were only seen for pigs showing frequencies lower thn 1 15 cells per million oth in experiment 1 nd experiment 2, fct tht suggest tht this vlue indictes sturtion of the IFN-γ responses. To our knowledge this is the first report showing differences in cell-medited responses ginst different PRRSV field strins. When protection ginst second chllenge ws exmined, results showed tht weight gins were lwys etter in the HoC scenrio. However, in virologicl terms, the picture ws different. In experiment 1 (isolte 3262), in which pigs hd low or non-detectle levels of neutrlizing ntiodies, the outcome of the HoC or HeC ws similr regrding viremi ut homologous protection ws etter when distriution of virus in tissues ws considered (1/4 positive results in tonsil of pigs in the HoC versus 4/4 for the HeC). Since NA ginst 3262 were not present, or were not detected, this would indicte tht cell-medited immunity most proly ws responsile for limiting the spred of the infection nd the durtion of viremi. In