key: cord-266571-qbskh1uu authors: de Arriba, M.L; Carvajal, A; Pozo, J; Rubio, P title: Lymphoproliferative responses and protection in conventional piglets inoculated orally with virulent or attenuated porcine epidemic diarrhoea virus date: 2002-06-04 journal: J Virol Methods DOI: 10.1016/s0166-0934(02)00063-0 sha: doc_id: 266571 cord_uid: qbskh1uu Lymphocyte proliferative responses were evaluated in mucosal (mesenteric lymph nodes) and systemic (spleen and blood) lymphoid tissues of conventional piglets inoculated with the virulent or attenuated isolates of porcine epidemic diarrhoea virus (PEDV) strain CV-777 and challenged 21 days later with the virulent isolate of the same virus. A lymphoproliferative assay was developed in which mononuclear cells isolated from lymphoid tissues at different postinoculation and postchallenge days underwent a secondary in vitro stimulation with semipurified antigen obtained from PEDV-infected cell cultures. Vigorous lymphocyte proliferative responses were detected in the pigs inoculated with the virulent PEDV at postinoculation days 4–21, especially in the mesenteric lymph nodes and the blood; however, in the spleen this response was lower and less regular. The pigs inoculated with the attenuated virus showed a less intense response, the higher lymphocyte proliferation also corresponded to the mononuclear cells from mesenteric lymph nodes. Lymphocyte proliferation responses showed high correlations with protection against homologous challenge with virulent PEDV, and this correlation was higher in the gut associated lymphoid tissues (mesenteric lymph nodes). The cell proliferation response detected in blood mirrored that detected in the mesenteric lymph nodes, and showed also good correlation with protection. The results confirm that T-cell-helper function, assessed by lymphocyte proliferation responses, contributes to establishing a protective immune response against PEDV infections. Porcine epidemic diarrhoea is an enteric disease in pigs caused by a member of the Coronaviridae family, porcine epidemic diarrhoea virus (PEDV) (Cavanagh et al., 1994; Murphy et al., 1999) . Because of the high prevalence and the severity of clinical symptoms, porcine epidemic diarrhoea represents the most important viral infection of swine intestinal tract in Spain and in many other European and Asean countries (Carvajal et al., 1995b; Pensaert, 1999; Sueyoshi et al., 1995; Van Reeth and Pensaert, 1994) , although not in the USA where the PEDV is not present but a high prevalence of transmissible gastroenteritis virus infections is still present (Saif, 1998; Saif and Wesley, 1999) . The disease is characterized by acute watery diarrhoea, depression and anorexia. Although morbidity is high and most of the animals in the herd can be affected within a week, mortality is usually low in adult animals (3%), which can recover in 7-10 days. However, when piglets less than 4-5 weeks are infected, mortality is usually 50% and can reach 90% in severe outbreaks, as usually happens in transmissible gastroenteritis. Besides the mortality, PEDV infection causes important economic loses due to the diminishing of the productive indexes (Pensaert, 1999) . As in many other viral infections of food animals, the lack of effective treatment makes immunity the main key for prevention and control of the disease. However, the development of candidate vaccines needs previous knowledge of immunological aspects related to the infection. Due to the enteric nature of the disease and the special configuration of the mucosal immune system, protection depends mostly on the local immune response (Corthesy and Kraehenbuhl, 1999; Kagnoff, 1996; Saif et al., 1994; Van Cott et al., 1994) , and is the reason why these studies cannot be limited to blood but require organs containing gut associated lymphoid tissues, in which local immune response can be measured. In spite of the fact that the importance of humoral immune response in gastroenteric viral infections of porcine is well recognized (Corthesy and Kraehenbuhl, 1999; Saif et al., 1994; Tô et al., 1998; Van Cott et al., 1994; Yuan et al., 1996) , little is known about cell-mediated immunity, particularly in PEDV infections. However, it has been proposed that cellular immune response may play an important role in the protection and recovery from infection, besides the fact that production of antibodies is regulated by cytokines produced by activated T lymphocytes and other mononuclear cells (Corthesy and Kraehenbuhl, 1999; Kraehenbuhl and Neutra, 1992; Saif, 1999; Totterdell et al., 1988) . The aim of this study was to assess the cellular immune response following natural infection with PEDV and also after inoculation of an attenuated virus, and the contribution to the establishment of a protective response. Virus-specific lymphoproliferative responses of systemic tissues (spleen and blood) and mesenteric lymph nodes were studied in conventional piglets after primary inoculation with the virulent, wild type, strain CV-777 of PEDV or its cell culture attenuated form and after challenge, 3 weeks later, with a high dose of the virulent virus. Vero cells were grown with Eagle's minimum essential medium (Gibco, Life Technologies) buffered with bicarbonate and supplemented with 5% (v/v) fetal calf serum (Gibco), 0.04% (w/v) yeast extract (Difco, MI, USA), streptomycin (10 mg/l) and penicillin (10,000 UI/l) (Penicillin-Streptomycin, Gibco). The cell culture adapted strain of PEDV CV-777, attenuated by many passages, was propagated in Vero cells as described by Hofmann and Wyler (1989) , infecting confluent monolayers of cells after removing the growth medium and adding the viral inoculum diluted in medium without fetal calf serum but containing 10 ml/ml of trypsin (Difco). A PEDV-infected cell lysate was used as attenuated PEDV inoculum. The wild type isolated of the CV-777 strain of PEDV, kindly provided by Dr Peansert (Gent, Belgium), was amplified by passages in conventional 1-week-old piglets without antibodies against PEDV and prepared in PBS for use as virulent PEDV inoculum. After oral inoculation, the animals were killed in the acute phase of diarrhoea, and the intestinal contents and the small intestine were collected at necropsy. The small intestine from each animal was macerated in PBS (1:2 (w/v)) and, like the intestinal contents, clarified by centrifugation at 5000× g for 20 min at 4°C. The richest fractions were pooled and stored at − 70°C. A total of 62 conventional 11-day-old piglets, seronegative to PEDV and from a herd with no previous history of the disease were assigned to three different experimental groups which were maintained in isolation facilities to prevent virus circulation. Group 1 (n = 29) was inoculated with a low dose of the virulent isolate of PEDV strain CV-777 that was adjusted in a previous experiment to produce a high morbidity without causing severe disease in the animals. Pigs from group 2 (n =20) were inoculated with 2.55×10 5 fluorescent focus-forming units per pig of the attenuated isolate of the same PEDV strain. The third group (n = 13) was mock-inoculated and served as control. Twenty-one days after inoculation, the three groups of pigs were challenged with the virulent PEDV, using a two times higher dose than that used to inoculate the group 1. Animals were observed daily for clinical symptoms and rectal swabs were taken for 11 days after inoculation and for 9 days after challenge. Faecal scores were recorded as normal faeces, pasty faeces, semiliquid (moderate diarrhoea) and liquid (watery diarrhoea). At different postinoculation and postchallenge days (postinoculation days 4, 7 and 14, and postinoculation/postchallenge days 21/0, 25/4 and 33/12) subsets of each group of pigs (n= 2-5) were killed by injection of barbiturate overdose (Eutalender, Normon, Madrid, Spain). Hundred millilitre of blood were collected from cardiac cavities in 25% (v/v) acid citrate glucose. Spleen and mesenteric lymph nodes were also collected aseptically and placed in ice-cold wash medium (RPMI 1640 containing 10 mM HEPES and 200 mg of gentamicin and 20 mg ampicillin per ml). Three PEDV-seronegative, unexposed pigs served as negative control and were killed to obtain the background values for the lymphoproliferative assay. Mononuclear cells from blood and tissues were isolated as described previously (De Arriba et al., 2001a) . Briefly, peripheral blood lymphocytes were obtained by density gradient centrifugation in Ficoll-Paque (Ficoll-Paque Research Grade, Pharmacia Biotech, Upsala, Sweden). Lymphocytes collected from the interface were washed twice in Hanks' balanced salt solution and suspended in RPMI 1640 containing 8% fetal calf serum, 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM nonessential aminoacids, 20 mM HEPES and 20 mg of ampicillin and 100 mg of gentamicin per ml (enriched medium). Mononuclear cells from spleen and mesenteric lymph nodes were obtained by pressing the tissues through stainless steel screens (80 mesh) of a cell collector (Cellecter; E-C Apparatus Corp., FLA, USA). Cell suspensions were centrifuged, and the mononuclear cells were removed from the pellet by continuous and discontinuous gradient centrifugation in Percoll (Pharmacia Biotech.), washed twice with wash medium and resuspended in enriched medium. Viability of all mononuclear cells preparations was confirmed by the trypan blue exclusion test, in every case being \ 95%. The lymphoproliferative assay for detection of PEDV-specific T cells was adapted from methods published previously (Brim et al., , 1995 Ward et al., 1996) . Semipurified PEDV antigen for in vitro stimulation of mononuclear cells cultures was obtained from lysates of PEDV-infected cell cultures that were concentrated 50 times by ultracentrifugation at 100,000×g for 2 h at 4°C and then semipurified by ultracentrifugation through 20% sucrose under the same conditions. The most favourable concentration of antigen for optimal antigenic stimulation of the mononuclear cells was established by dose-response curves. A PEDVcontrol antigen was obtained giving the same treatment to mock-infected cultures. The T-cell mitogen phytohaemagglutinin (Gibco) was used as positive control at a final concentration of 10 ml/ml, following the manufacturer's instructions. Optimal conditions of number of cells and duration of incubation were determined by preliminary studies. Mononuclear cells at a concentration of 5×10 5 cells per 100 ml and per well were placed in 96-well culture plates and stimulated in triplicate with the PEDV antigen or the control antigen or the phytohaemagglutinin. Cells were incubated for 72 h at 37°C in 5% CO 2 and 18 h prior to harvest each well was labelled by pulsing 1 mCi of [ 3 H]thymidine (Amersham Pharmacia Biotech). Harvesting of cells was carried out on glass fiber filters (FilterMAT, Skatron Inc., Va, USA) and [ 3 H]thymidine incorporation was determined by liquid scintillation spectrophotometry. The lymphocyte proliferative responses for each mononuclear cells sample assayed was expressed as the stimulation index (SI), calculated as SI= mean cpm of PEDV stimulated wells/mean cpm of control antigen stimulated wells, being cpm counts per minute. Viral antigen was detected in faecal samples by the double antibody sandwich ELISA described by Carvajal et al. (1995a) . This ELISA is based on the use of two monoclonal antibodies (Lelsytad CVI-66.31 and Lelystad CVI-66.49) directed specifically against the S protein of the virus. A blocking step with rabbit-anti PEDV hyperimmune serum was included to increase the specificity. Twofold serial dilutions of the samples were assayed starting at 1:2 and titres were expressed as the inverse of the lowest positive dilution. For calculation of the geometric mean titre (GMT), negative samples were given a titre of 1. One-way analysis of variance followed by the paired Student's t-test was used to determine the nature of differences observed in virus shedding and lymphocyte proliferation responses among inoculated groups, tissues and days. Correlation between proliferation responses at challenge day and protection against the infection was established by Spearman's correlation coefficient (z). Significance was assessed at PB 0.05. For the analysis the SYSTAT for Windows v.5.03 (SYS-TAT Inc.) and the spreadsheet Microsoft EXCEL v.7.0 (Microsoft Comp.) were used. A summary of faecal virus shedding and clinical disease after inoculation and challenge is given in Table 1 . After primary inoculation of pigs from group 1 with virulent PEDV, moderate to severe diarrhoea was observed in 33% of the animals and virus shedding in 100%. The onset of diarrhoea was observed between postinoculation days 2 and 4 and the average duration was 1.7 days. Virus shedding was detected in some of the pigs at postinoculation day 1 but most shed PEDV in faeces from postinoculation day 2 to 6. The average duration of the shedding period was 5.4 days. The GMT of viral antigen in faecal samples was measured using ELISA; it increased strongly from postinoculation day 2 and reached a peak at postinoculation day 5. Conversely, pigs from group 2 inoculated with the attenuated PEDV did not show typical signs of the disease and only one pig had moderate diarrhoea for 1 day and virus shedding was detected in only one sample at postinoculation day 5 and with a low titre. Differences between the GMT of antigen in the faeces of the two groups were significant statistically. On the challenge day, at postinoculation day 21, pigs from group 1 were protected against infection and disease, none developed diarrhoea and not viral antigen was detected in the samples taken after challenge. Diarrhoea was not observed in pigs from group 2 after challenge either, but viral detection in rectal swab samples revealed that protection against the infection was only partial (25%), and the antigen was detected in 75% of the challenged animals (9 out of 12). In the control group moderate diarrhoea was seen in 46% of the pigs starting between postchallenge days 3 and 4 and with an average duration of 1.5 days. Viral antigen was detected in faeces of all of the challenged pigs from this group in which the average duration of viral shedding (4.6 days) was significantly higher than in group 2 (2.6 days). The GMT of viral antigen detected in the control group was also significantly higher than in group 2. In order to obtain the optimal secondary antigenic stimulation for the lymphoproliferative assay, two different doses of antigen were used, 25.5 ng of the semipurified antigen were added to each 5× 10 5 mononuclear cells from spleen and blood, whereas in the mesenteric lymph nodes a minor dose, 12.8 ng, yielded the optimal stimulation of the cells. Mean cpm obtained after stimulation of each tissue with the different antigens and the mitogen are shown in Table 2 . Background cpm, obtained after stimulation of each mononuclear cells culture with the control antigen, were low in mesenteric lymph nodes and blood (usually B 4000) but not in spleen, where these counts sometimes reached values close to 22,000. After inoculation of group 1, a specific lymphocyte proliferation response was detected for the first time in the mesenteric lymph nodes at postinoculation day 4 (Fig. 1) and was maintained until challenge day (postinoculation day 21), when the maximum value of SI was found (18.82). SI in this tissue was significantly higher from postinoculation day 4 to 21 than that observed in unexposed pigs. The lymphoproliferative responses in mesenteric lymph nodes of pigs from group 2 increased significantly at postinoculation day 14 compared with their responses in previous days ( Fig. 1) and SI reached its peak that day at 5.73. Values of the SI between postinoculation days 14 and 21 were significantly higher compared with responses for mononuclear cells from mesenteric lymph nodes in unexposed pigs. Group 1 SI was greater than group 2 at any PID, however statistical significance was only detected at postinoculation day 4. Group 3 was mock inoculated and served as control at challenge. Statistically significant differences among groups are denoted by the letters: 'a' when differences are between groups 1 and 2, 'b' when differences are between groups 1 and 3 and 'c' for differences between groups 2 and 3. Level of significance is defined by *P50.5, **P50.01. a PID: postinoculation days. b PCD: postchallenge days. c GMT: geometric mean titre of antigen detected in faeces by ELISA. Mononuclear cells were stimulated with positive, negative antigen or the T-cell mitogen phytohaemagglutinin (PHA). Background values correspond to pigs with no previous contact to PEDV. PID, postinoculation day; PCD, postchallenge day. Fig. 2 . Correlations between lymphocyte proliferative responses in mononuclear cells collected from mesenteric lymph nodes, blood and spleen from pigs inoculated with virulent or attenuated PEDV or mock-inoculated and protection against challenge 21 days later with virulent PEDV. Correlations were assessed by Spearman rank correlation test. Lymphocyte proliferative responses were expressed as mean cpm of PEDV stimulated wells versus mean cpm of control antigen stimulated wells, being cpm counts per minute. Fig. 1 . Course of the virus-specific lymphocyte proliferative responses represented by SI for mononuclear cells from mesenteric lymph nodes, spleen and blood from pigs after inoculation with virulent (group 1) or attenuated (group 2) PEDV or mock-inoculation (group 3) and after challenge with virulent PEDV. The SI are the mean cpm of virus-specific stimulated wells versus mean cpm of control antigen stimulated wells, being cpm counts per minute. The mean value of the SI obtained from the group of unexposed pigs is represented by a line crossing the Y-axis. Statistically significant differences (P 50.05) with values obtained in nonexposed pigs are noted as *. Differences between groups are nodded as: 'a' when differences are between groups 1 and 2, 'b' between groups 1 and 3 and 'c' for differences between groups 2 and 3. Mononuclear cells purified from blood of group 1 showed a vigorous proliferative response after inoculation starting at postinoculation day 7 with significant increases over the following days. The SI obtained for this group in blood were significantly higher than values in blood of unexposed pigs between postinoculation days 7 and 21 and as in the mesenteric lymph nodes, the peak value occurred at postinoculation day 21 (Fig. 2) . Virusspecific lymphoproliferative responses in blood from group 2 occurred at postinoculation day 14, the only day in which SI value was significantly higher than that in unexposed animals (SI= 9.58, PB0.001). Likewise in mesenteric lymph nodes, the SI of group 2 were minor than the indexes of group 1, although the difference was statistical significant only at postinoculation day 21. The magnitude of the virus-specific proliferation in the spleen of group 1 after inoculation was lower than in the other tissues, being also less regular. At postinoculation days 4 and 7, the response of this group was low and similar to the proliferation shown by unexposed pigs. In the following days there was an increase in the SI that peaked at postinoculation day 14, however, the value of SI was not significantly higher than the background values at any postinoculation time. Group 2 did not show a virus-specific proliferative response in mononuclear cells from spleen after inoculation, with an SI similar to that in unexposed pigs. After challenge at postinoculation day 21, the lymphoproliferative responses in mesenteric lymph nodes of group 1 underwent an important increase and even though at postchallenge day 4 (postinoculation day 25) the SI was lower than on challenge day (although not significantly), this value again reached its peak at postchallenge day 7 (postinoculation day 28) with a value of 29.25 (Fig. 1) . In group 2 lymphocyte proliferation responses after challenge were low and only at postchallenge day 7 (postinoculation day 28) was the SI significantly higher than that in unexposed animals. Responses in group 3, the mock-inoculated control group, after challenge were similar to responses described in mesenteric lymph nodes of pigs from group 1 after inoculation with virulent PEDV, but showed a higher intensity. The SI in this group was significantly higher than in unexposed animals from postchallenge day 7 (postinoculation day 28) and reached 5 days later (at postchallenge day 12, postinoculation day 33) the highest value detected in the mesenteric lymph nodes of all the groups. When the SI of the three groups were compared, statistically significant differences were found at postchallenge day 4 (postinoculation day 25) between group 1 and groups 2 and 3 and at postchallenge day 12 (postinoculation day 33) the SI of groups 1 and 3 were significantly higher than index in group 2. Lymphocyte proliferative responses in blood after challenge in group 1 were significantly lower at postchallenge day 4 (postinoculation day 25) compared to the challenge day, however, from postchallenge day 7 (postinoculation day 28) there were significant increases, reaching maximum value at postchallenge day 12 (postinoculation day 33) (Fig. 2) , the only day that this value could be demonstrated significantly higher than that in unexposed pigs. Similarly to mesenteric lymph nodes, the response detected after challenge in blood from group 2 pigs was low, only the SI at postchallenge day 12 (postinoculation day 33) was significantly higher than unexposed animals index. In the control group, lymphoproliferative responses were low up to postchallenge day 12 (postinoculation day 33) and there was not statistical significance in the differences observed with regard to the unexposed animals. Comparisons between the SI in the blood after challenge in the different groups showed a higher response in group 1, although statistical significance was only shown at postchallenge day 12 (postinoculation day 33). The SI was lower in group 3 than in group 2, but not significantly. In the spleen, responses after challenge of group 1 increased significantly at postchallenge day 7 (postinoculation day 28) with regard to the previous day, similar to the mesenteric lymph nodes. Group 2 response after challenge was maximum at postchallenge day 4 (postinoculation day 25), this being the only time in which the SI of mononuclear cells of spleen from this group was significantly higher than the SI of unexposed animals. The control group underwent for the first time a specific proliferation response at postchallenge day 12 (postinoculation day 33). The SI obtained in each group at each point in time were compared and no statistically significant differences were found. Correlations between lymphoproliferative responses detected in each tissue and group at the challenge day and protection against challenge, represented by the protection rate against infection, were established by the Spearman rank correlation test and are shown in Fig. 2 . The magnitude of the response in all tissues examined at postinoculation day 21 correlated positively with protection against challenge, although statistical significance were not attained. The highest correlation was detected in the mesenteric lymph nodes (z= 0.99, P= 0.08). Protection in swine gastroenteric viral infections, as PED, has been related almost exclusively to the antibody immune responses. However, cell-mediated immunity must play an important role in protecting and recovery from infection, besides the control function of the B cell-humoral responses carried out by T cell populations (Corthesy and Kraehenbuhl, 1999; Kraehenbuhl and Neutra, 1992; McGhee et al., 1992; Saif, 1999; Totterdell et al., 1988) . Thus, without any B cell population deficiency in humans the lack of antibody and specific T-cell responses, resulting in rotavirus persistent infection with viral excretion in faeces for 15 months (Totterdell et al., 1988) . Moreover, Welch et al. (1988) , in pigs inoculated with transmissible gastroenteritis virus, related peaks of lymphoproliferative responses ending up with final virus shedding in faeces and the beginning of recovery from the disease. In this study, an in vitro virus-specific proliferation assay was carried out as a method to estimate the cell-mediated immune response since this antigen-induced proliferation has been recognised as a property of CD4 +(T helper) cells in studies undertaken on pigs, mice and humans with rotavirus and coronavirus Offit et al., 1992; Ward et al., 1996) . The specific proliferative response after inoculation of pigs with virulent PEDV was detected immediately in the mesenteric lymph nodes, the organs directly associated with the mucosal immune system. The maximum values were found around postinoculation day 21, just when a strong response of virus-specific antibody-secreting cells was detected in this organ and also in the duodenum and ileum lamina propria (De Arriba et al., 2001b) . In pigs inoculated with the attenuated strain of PEDV this specific lymphoproliferative response was detected later, at postinoculation day 14 and it was lower that in pigs inoculated with the wild virus. This minor response of group 2 also corresponded to a low response of PEDV-specific antibody-secreting cells (De Arriba et al., 2001b) . The difference observed between the lymphoproliferative responses of the two inoculated groups has also been described by other researchers (Brim et al., , 1995 Ward et al., 1996) in other gastroenteric viruses of swine, describing that the lymphocyte proliferative responses induced by attenuated strains of transmissible gastroenteritis and rotavirus were significantly lower than that induced by the homologous virulent virus. These results suggest that a protective antibody response to the virulent PEDV could be associated with previous development of a strong specific cell-mediated immune response. This consideration could be reinforced by the fact that antibody production by specialised B cells requires T cell help (Corthesy and Kraehenbuhl, 1999) . The virus-specific lymphocyte proliferative response in the systemic lymph tissues (blood and spleen) was observed later than in mesenteric lymph nodes, as well as being considerably lower in the spleen than in other tissues. This delay could be explained if it is considered that the PEDV-specific T cells located in blood and spleen originate in the inductive sites from the gut-associated lymphoid tissues, like the mesenteric lymph nodes, and its presence in systemic tissues is due to the homing process necessary for its maturation (Corthesy and Kraehenbuhl, 1999; Kagnoff, 1996; Kantele et al., 1997; Salmi and Jalkanen, 1997) . The specific cell proliferation response in the blood was more similar to that observed in the mesenteric lymph nodes than in the spleen, especially in the group 1, in spite of both, blood and spleen, being linked to the systemic immune system. However, the blood, together with the lymphatic system, is the main vehicle for lymphocyte migration (Salmi and Jalkanen, 1997) , and its lymphoid population may reflect primed T cells migrating to the gut for some time after an infection. The lymphocyte proliferative responses at the challenge day showed high correlation with protection against challenge. Pigs from group 1 inoculated initially with the virulent PEDV, were 100% protected against infection 21 days later with a higher dose of the same virus whereas protection in group 2, inoculated with the attenuated PEDV, was just partial and only 25% of pigs were protected against infection with the virulent virus. The highest correlation was observed in mesenteric lymph nodes. This result again suggests that T-cell response, especially in the gut associated lymphoid tissues, contributes in an important way to the development of a protective immune response in PEDV infections. The highly attenuated PEDV conferred partial protection against challenge with virulent virus in conventional pigs, this protection is related to the inoculated dose and increases when a higher dose is used (De Arriba et al., 2001b) . Kweon et al. (1999) also described the induction of protective immunity by a attenuated strain of PEDV inoculated intramuscularly. After the challenge, there was an increase in the lymphocyte proliferative response in pigs from group 1, however this increase was not reflected either by an enhancement of the virus-specific antibody secreting cell response or the GMT of PEDV-specific serum IgG and IgA (De Arriba et al., 2001b) . Thus, Ward et al. (1996) also reported that pigs inoculated and challenged with virulent rotavirus strains showed after challenge lymphoproliferative responses similar or poorer than after inoculation. Although there is no clear explanation in this, the possibility remains that this secondary response after challenge could be related to the proliferation of cell clones involved in immune regulatory functions different to providing help for antibody production, such as T suppressor populations. In this study the development of cell-mediated immunity occurred in systemic and lymphoid tissues after inoculation with virulent and attenuated strains of the PEDV. The results suggest that cell-mediated immune responses contribute significantly to the instauration of protective immune status against homologous virulent virus challenge. The lymphoproliferative responses both in gut associated lymphoid tissues and systemic tissues had a higher magnitude when virulent PEDV was used versus attenuated PEDV to inoculate pigs. However, higher doses and administration methods have to be assayed in order to develop vaccines. 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Pensaert for providing the wild type isolated of the PEDV strain CV-777 and Dr L.J. Saif and Dr L.A. Ward for laboratory training. We also wish to thank G.F. Bayó n and B. Escudero for their excellent techni-cal assistance. This work was funded by the Comisió n Interministerial de Ciencia y Tecnología (CICYT) project No. AGF-960486. Salaries were provided by the Excelentisima Diputació n Provincial de Leó n.