1 Centre de Recerca en Sanitat Animal (CReSA), Campus UAB, 08193 Bellaterra, Spain
2 Departament de Sanitat i Anatomia Animals, Facultat de Veterinària, Edifici V, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain
3 Institut de Recerca i Tecnologia Agroalimentària, Barcelona, Spain
Correspondence
I. Díaz
ivan.diaz{at}uab.es
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Immune response to PRRSV is poorly understood but, in spite of this, some vaccines made from European- or American-type strains are being commercialized. Several studies have reported controversial results about the efficacy of vaccination (Meng, 2000). It seems that American-type vaccines are more effective to protect against infections caused by American-type strains than against European-type infections (van Woensel et al., 1998
; Lager et al., 1999
). However, some degree of protection against the heterologous genotype is observed (Lager et al., 1999
; Labarque et al., 2003
). This fact indicates that critical determinants of the immune response against each type of strains are similar, but not equal. As a consequence, at present there is no single vaccine that can claim full protection against all strains of PRRSV.
One of the reasons for the lack of development of newer and more efficacious vaccines against PRRSV is the scarce knowledge on the immune response of pigs after infection by field strains. Available data have mostly been obtained by using American-type strains and have indicated that the adaptative immune response against American-type PRRSV is unique in its features. Infected piglets can be viraemic for up to 612 weeks, but circulating antibodies can be easily detected much earlier (Yoon et al., 1995; Batista et al., 2004
; Johnson et al., 2004
). Nevertheless, neutralizing antibodies (NA) do not appear until 810 weeks post-infection (p.i.) and their role in protection is not clear (Murtaugh et al., 2002
). Besides, cell-mediated immune responses, measured as virus-specific gamma interferon-secreting cells (IFN-
-SC), are erratic during the first weeks p.i. Later, there is a sudden increase in IFN-
-SC that is maintained, despite a progressive fading of PRRSV-specific antibodies. However, the IFN-
-SC response seems to be delayed, compared with other pathogens (Meier et al., 2003
).
To date, there has been no report showing the humoral and cell-mediated evolution of the immune response after infection with a wild PRRSV strain of the European genotype. The aim of the present study was to characterize the immune responses of pigs after infection with a European-type field strain of PRRSV.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Virus and challenge.
The virulent PRRSV VP21 strain used for the challenge was isolated in porcine alveolar macrophages (PAM) from sera of naturally infected pigs during an outbreak of PRRS affecting a breeding farm located in the north of Spain in December 1991. That outbreak was characterized by abortions, stillbirths and, later on, piglet mortality. The virus was shown to be of the European genotype by sequencing of ORFs 27. Viral stock was done by passage (n=3) in PAM and titrated by means of the immunoperoxidase monolayer assay (IPMA). Before use, the viral stock was checked for bacterial contamination and for the presence of other viruses. Challenge was done when pigs were 17 weeks old. Pigs were divided randomly in two groups (A and B). Pigs in group A were inoculated intranasally with 2 ml viral suspension containing 106 TCID50 PRRSV VP21 strain ml1, whilst pigs in group B (controls) received 2 ml sterile PBS.
Clinical examination and production parameters.
Experimental pigs were clinically examined daily and rectal body temperatures were recorded from days 0 to 14 p.i. Every week, pigs were weighed and the amount of feedstuff consumed was recorded.
Samples.
Blood samples were collected in duplicate (heparinized and siliconized blood-collecting tubes) immediately before challenge and then at days 7, 14, 21, 28, 56 and 70 p.i. Sera were used for PRRSV-specific RT-nested PCR (nPCR), determination of humoral responses and evaluation of haptoglobin and interleukin 8 (IL8), IL10 and transforming growth factor beta (TGF-) levels. Peripheral blood mononuclear cells (PBMC) were obtained from heparinized blood samples and used in flow-cytometry assays and for in vitro experiments.
Detection of viraemia.
Serum samples were inoculated on MARC-145 and PAM cultures and incubated at 37 °C in 5 % CO2 for 90 min. Then, sera were removed and minimal essential medium (Sigma) was added. Cultures were incubated for 3 days at 37 °C in 5 % CO2. Infection of inoculated cells was determined by IPMA.
Besides viral isolation, sera were examined by nPCR. Briefly, total RNA was extracted from 150 µl serum by using a commercial system (Nucleospin RNA virus; Macherey-Nagel). Total RNA was transcribed and cDNA was used in a first PCR round directed to viral ORF5 (Mateu et al., 2003). This first-round PCR had a sensitivity of about 102 TCID50 (ml serum)1. PCR products were used in an nPCR (forward primer, 5'-TCTTGCTTCTGGTGGCTTTT-3'; reverse primer, 5'-CATGTTTGATGGTGACGAGG-3') that produced a 499 bp amplicon. Cycling parameters for both PCRs were: 94 °C for 45 s; 55 °C for 45 s; 72 °C for 45 s for a total of 35 cycles. Under these conditions, the nPCR was considered to be able to detect <10 viral copies (ml serum)1.
Humoral immune response.
A commercially available ELISA (HerdChek PRRS 2XR; Idexx Laboratories) was used to measure PRRSV-specific antibodies. According to the manufacturer, sample to positive control (S/P) ratios of >0·4 were considered positive. PRRSV antibody titres were also determined in sera by using IPMA as described by Wensvoort et al. (1991), using MARC-145 cells infected with the PRRSV VP21 strain. Tests were done in duplicate.
NA were measured by the technique described by Yoon et al. (1994) and Jusa et al. (1996)
. Tests were done in triplicate by using whole serum, inactivated serum and serum plus 10 % fresh guinea-pig complement. Briefly, 50 µl of each serum to be tested was diluted serially from 1/20 to 1/160 in cell-culture medium. Dilutions were mixed with 50 µl viral suspension containing 200 TCID50 of the PRRSV strain VP21 (with or without complement). Virusserum mixtures were incubated for 1 h at 37 °C and then added to MARC-145 cultures in duplicate (96-well plates) and incubated for 3 days at 37 °C in 5 % CO2. Infection of cell cultures was revealed by using IPMA.
Total serum antibodies (IgG and IgM) were analysed by means of a commercial capture ELISA (Pig IgG ELISA quantification kit and Pig IgM ELISA quantification kit; Bethyl Laboratories), done according to the recommendations of the manufacturer. Serum-antibody concentrations were calculated by using a regression line calculated after measurement of ODs of the standards provided with the kit.
Isolation and culture of PBMC.
PBMC were separated from whole blood by density-gradient centrifugation with Histopaque 1077 (Sigma). For PBMC cultures, RPMI 1640 medium supplemented with 10 % fetal calf serum (Invitrogen), 1 mM non-essential amino acids (Invitrogen), 1 mM sodium pyruvate (Invitrogen), 5 mM 2-mercaptoethanol (Sigma), 50 000 IU penicillin l1 (Invitrogen), 50 mg streptomycin l1 (Invitrogen) and 50 mg gentamicin l1 (Sigma) was used. Trypan blue was used to assess viability.
Flow-cytometry assays.
Phenotypic analysis of PBMC subsets was done by flow cytometry using the following mAbs: anti-CD4PE (phycoerythrin) (clone 74-12-4, provided by Dr J. Domínguez, INIA, Madrid, Spain), anti-CD8FITC (fluorescein isothiocyanate) (clone 76-2-11, INIA), anti-CD21FITC (4530-02; Southern Biotechnologies), anti-CD25 (K231-3B2, INIA), anti-SWC3PE (4525-09, Southern Biotechnologies) and anti-SLA-II DR (clone 1F12, INIA).
When needed, goat F(ab')2 anti-mouse IgG2a R-PE-conjugated antibody and goat F(ab')2 anti-mouse IgG1 FITC-conjugated antibody (Southern Biotechnologies) were used as secondary antibodies. Irrelevant, isotype-matched antibodies were included as background controls. Analyses were done by using an EPICS XL-MLC cytometer (Coulter) to an excitation wavelength of 488 nm and with 580 and 630 nm filters.
ELISPOT: PRRSV-specific IFN--SC and IL4-secreting cells (IL4-SC).
Frequencies of PRRSV-specific IFN--SC and IL4-SC in PBMC were analysed by an ELISPOT assay using commercial mAbs (Swine IFN-
and Swine IL4 Cytosets; Biosource Europe) according to a previously reported method (Díaz & Mateu, 2005
). Briefly, for IFN-
, ELISA plates (Costar 3590; Corning) were coated overnight with 8·3 µg IFN-
capture antibody ml1 diluted in carbonate/bicarbonate buffer (pH 9·6). Plates were then washed and blocked for 1 h at 37 °C with 150 µl PBS with 1 % BSA. After removal of the blocking solution, 5x105 PBMC were dispensed per well (50 µl) and stimulated with the VP21 strain at an m.o.i. of 0·1 as the recall antigen. After 20 h incubation at 37 °C in a 5 % CO2 atmosphere, cells were removed and the biotinylated detection antibody was added at 2·5 µg ml1 (50 µl) and incubated for 1 h at 37 °C. The reaction was revealed by sequential incubation of plates with streptavidinperoxidase (1 h) and insoluble TMB blue (Calbiochem). For IL4, the protocol was similar, but working dilutions of the mAbs were 9·6 µg ml1 (capture antibody diluted in PBS, pH 7·2) and 5·0 µg ml1 (biotinylated detection antibody). In both cases, unstimulated cells and phytohaemagglutinin (PHA)-stimulated controls (10 µg ml1) were also included.
To calculate the PRRSV-specific frequencies of IFN--SC and IL4-SC, counts of spots in unstimulated wells were subtracted from counts in virus-stimulated wells. Frequencies of cytokine-producing cells were expressed as responding cells in 106 PBMC.
Cytokine (IL2, IL4, IL8, IL10 and TGF-) and haptoglobin ELISAs.
PBMC were seeded at a density of 2x106 cells per well (250 µl) in 96-well plates and were mock-stimulated or stimulated with either PRRSV strain VP21 (m.o.i. of 0·1) or PHA (10 µg ml1). After 24 h incubation at 37 °C in 5 % CO2, cell-culture supernatants were collected and frozen at 80 °C until needed. Capture ELISAs for IL2, IL4 and IL10 were performed as reported previously (Darwich et al., 2003; Díaz & Mateu, 2005
), using commercial pairs of mAbs (Swine IL2, IL4 and IL10 cytosets; Biosource Europe). For IL8, an ELISA kit was used (IL8 Immunoassay kit; Biosource Europe). For TGF-
, an ELISA was developed by using commercial antibodies (TGF-
cytosets; Biosource Europe). The cut-off point of each ELISA was calculated as the mean+3SD OD of negative controls. Cytokine concentrations were calculated by using the linear-regression formula from ODs of the cytokine standards provided by the manufacturer. IL8, IL10 and TGF-
were also measured in sera of experimental animals by using the above-mentioned ELISAs. Serum-haptoglobin levels were determined by means of ELISA (Haptoglobin assay phase range; Tridelta) according to the manufacturer's instructions.
Statistical analysis.
The regression line of cytokine standards in ELISA was calculated by using SPSS v. 12.0 (SPSS Inc.). Statistical comparisons between groups (MannWhitney test) were done by using StatsDirect v. 2.4.1. All tests done in the study, as well as the statistical analysis, were performed blind.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
Humoral immune response
All pigs were seronegative to PRRSV at day 0. By using ELISA, two infected pigs seroconverted by day 7 (mean S/P ratio, 0·51) and all were clearly positive by day 14 (mean S/P ratio, 2·12). ELISA titres remained high throughout the 70 days of observation and a slow decline was seen from day 56 p.i. onwards (mean S/P ratio at day 70 p.i., 1·81). IPMA was slightly less sensitive than ELISA. With IPMA, positive results were only obtained from day 14 p.i. onwards. The highest mean IPMA titres were seen at day 56 p.i. [log10(titre)=3·05].
NA were first detected at day 56 p.i. and only in three of five infected pigs. The remaining two pigs had not developed NA by day 70. By using whole serum, the highest neutralizing titre was log10(titre)=1·80. Addition of fresh complement increased titres by one dilution. All uninoculated controls remained seronegative to PRRSV in all tests throughout the study. Table 2 summarizes these results.
|
Flow-cytometry assays
At day 7 p.i., infected pigs had a significant increase in the proportion of peripheral blood CD8+ cells and a decrease in CD21+ cells (P<0·05). At day 14 p.i., these differences disappeared. Infected pigs again had a decrease in CD21+ cells at 28 days p.i. No differences were seen among groups regarding expression of CD4, CD25, SWC3 or SLA-II in PBMC. Table 3 shows the complete results of the flow-cytometry analysis.
|
|
In PRRSV-challenged pigs, serum-haptoglobin levels were increased significantly at days 7 and 14 p.i. (P<0·05), returning to values similar to those of uninfected pigs by day 21 p.i. (Fig. 3).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Our experiment was deliberately conducted with relatively aged pigs (17 weeks of age). This selection was done firstly because we wished to have a clear model of infection in which maturation of the immune system was not a source of confusion and, secondly, because we tried to avoid development of a severe disease that could kill animals before the planned end of the experiment.
After experimental infection with the PRRSV VP21 strain, all infected pigs developed only mild signs of disease in the first 24 h p.i. These signs, mainly fever, anorexia and weight loss, lasted until 14 days p.i., when viraemia ceased. This course of infection is similar to that described in other reports using European-type strains (van der Linden et al., 2003).
Interestingly, viraemia was low and was only detectable after nPCR was used. These facts suggest that clinical signs are related to the presence of virus in blood. As PRRSV does not replicate in monocytes or other blood cells (Duan et al., 1997), it would be expected that viraemia reflected the intensity of virus replication in tissues. Other experimental PRRSV infections or vaccinations have also shown short viraemias, even in pigs younger than ours (López Fuertes et al., 1999
; Meier et al., 2003
; Sipos et al., 2003
).
By using ELISA, development of specific antibodies could be seen as early as 7 days p.i. and lasted until the end of the study at detectable levels. The performance of IPMA was somewhat poorer but, by day 14 p.i., all infected animals were detected as seropositive. It is known that detection of early antibodies is useful as a diagnostic tool, but it seems that they do not play a role in protection against the infection (reviewed by Murtaugh et al., 2002; Lopez & Osorio, 2004
).
In our experiment and as reported by others (Vezina et al., 1996; Meier et al., 2003
), NA appeared late (from day 56 p.i. onwards) and not in all animals. The existence of decoy immunodominant epitopes in GP5 has been suggested as the cause of such delayed production (Ostrowski et al., 2002
).
The role of NA in protection against PRRSV is controversial. Osorio et al. (2002) showed that NA could be protective against PRRSV if administered exogenously at high doses. However, most studies showed that the production of NA after an experimental infection or vaccination is low and sporadic, with considerable individual variability (Loemba et al., 1996
; Meier et al., 2003
). In our case, clearance of viraemia was observed before NA could be detected. We cannot rule out the possibility that very small amounts of NA, undetectable in neutralization assays, could have existed at that time and played a role in the cessation of viraemia but, as three different methods of examining serum NA were used, it seems more reasonable to think that viraemia disappeared in the absence of NA. In immunodeficient mice infected with Lactate dehydrogenase-elevating virus, a related arterivirus, viraemia can also disappear without noticeable NA (Onyekaba et al., 1989
).
One of the effects attributed to PRRSV is the induction of a polyclonal B activation (Lamontagne et al., 2001). In our case, neither total IgM nor IgG levels rose significantly after infection. Also, the proportion of neither CD21+ nor CD25+ cells increased. Moreover, the proportion of CD21+ cells in blood decreased at 1 week p.i. Mobilization of B cells to tissues could explain these results and would be consistent with the suggestions of other authors, which have indicated that, in PRRS, polyclonal activation of B cells occurs in lymphoid organs, but not in blood (Kawashima et al., 1999
; Lamontagne et al., 2001
).
In contrast, CD8+ cells increased in blood in the first week p.i. Samsom et al. (2000) observed that CD8+ cells also increased in bronchoalveolar fluid of pigs after a PRRSV infection. However, this increase is not likely to be attributable to a clonal expansion of PRRSV-specific T cells. If this was the case, other evidence, such as IL2 production, IFN-
increase or an upshift of CD25+ cells, should have been observed. Other reports (Shimizu et al., 1996
; Albina et al., 1998
) indicated that this was not a mitogenic effect of the virus, but a response to some physiological stimulus. As we will discuss later, this increase in CD8+ cells correlated with the detection of IL10 in PBMC-culture supernatants and with the raise of haptoglobin levels in serum. Moore et al. (2001)
showed that IL10 can promote growth and differentiation of CD8+ cells.
Regarding the cellular immune response, our results showed that PRRSV-specific IFN--SC appeared at week 2 p.i. Levels of IFN-
-SC were undulant in the first 70 days p.i. This evolution agrees with the results of Meier et al. (2003)
although, in our case, the strength of the response, in terms of the number of IFN-
-SC, was lower. Whether these differences were caused by the use of different mAbs in ELISPOT or reflect a true difference cannot be known at this time.
Interestingly, although the initial raise of PRRSV-specific IFN--SC was delayed for 14 days after the challenge, the last detection of viraemia in infected pigs corresponded to the appearance of these cells. This fact may be incidental, but suggests some role of IFN-
-SC in the control of the infection. Regarding IL4-SC, no significant responses against PRRSV were detected in infected pigs. This is surprising, considering the fast and high humoral response against the virus, but might be an explanation for the delayed development of NA.
In our study, results for IL2, IL4 and IL8 did not show significant differences between control and infected animals. These observations reinforce the notion that some type of immunomodulation takes place in the course of infection (Molitor et al., 1997; Lager & Mengeling, 2000
). We also evaluated IL10 responses of infected pigs. Other authors showed that this cytokine can play a significant role in PRRS (Chung & Chae, 2003
; Suradhat & Thanawongnuwech, 2003
; Suradhat et al., 2003
). In our case, the ability of PBMC to produce IL10 in response to viral stimulation was a significant fact in the first week p.i., but IL10 was not detected significantly in cell-culture supernatants afterwards. This pattern is difficult to explain.
It is known that IL10 is produced mainly by cells of the monocyte/macrophage lineage, by regulatory T cells or, less frequently, by B cells. At this time, none of these populations can be ruled out as the source of this cytokine. Besides, TGF- levels were not significantly different in serum or cell-culture supernatants of infected and uninfected pigs and, as we discussed before, IL4 was not detected in response to PRRSV. Natural regulatory T cells are thought to belong to the CD4+CD25+ subset (McGuirk & Mills, 2002
). We did not see any change in the relative proportion of these two markers in PBMC throughout the study and, under this perspective, it seems unlikely that natural regulatory T cells were involved in IL10 production. It is tempting to think of monocytes as being responsible for this production. However, this needs further research to be clarified.
The increased ability of PBMC to produce IL10 against PRRSV at the first week p.i. correlated with the rise of serum-haptoglobin levels. Haptoglobin is an acute-phase protein produced by the liver and related to IL6 (Asai et al., 1999). It seems to play a major role in modulating immune responses through a complex network of interactions. Thus, haptoglobin release has been related to the secretion of anti-inflammatory cytokines, particularly IL10, through the interaction with CD163, a haemoglobin-scavenger receptor that is solely present in cells of the monocyte/macrophage lineage (Moestrup & Moller, 2004
; Philippidis et al., 2004
). Interestingly, other pig viruses, such as African swine fever virus, replicate mainly in CD163+ macrophages (Sánchez-Torres et al., 2003
).
In summary, comparison of our results with previous studies performed with American strains (Meier et al., 2003; Royaee et al., 2004
) suggests that the immune response in all PRRSV infections is similar. Main traits are a strong, non-neutralizing humoral response with a delayed development of NA, a low IFN-
-SC frequency, very low or undetectable IL2 and IL4 responses and, in the first stages of infection, a significant involvement of IL10 and haptoglobin.
As suggested by Xiao et al. (2004), the results presented in this work are consistent with a hypothetical model in which the outcome of PRRSV infection is related more to the dynamics of permissive macrophages and the early events of the natural response than to the development of specific immunity. In such a model, the earliest events in the course of the infection would involve release of IL10 and haptoglobin. This IL10, either released by infected macrophages as an anti-inflammatory response or induced by viral proteins, would probably impair, but not abolish, the development of cell-mediated immunity (Royaee et al., 2004
). This fact would be reflected in the erratic levels of virus-specific IFN-
-SC during the first weeks p.i. (Meier et al., 2003
) and in the lack of virus-specific IL2- and IL4-producing cells. As infection progresses, the cytolytic cycles of virus replication would decrease the number of permissive macrophages (Xiao et al., 2004
). As seen by others (Duan et al., 1997
), permissive cells should account for only a small proportion of total tissue macrophages. The stronger the cytolytic cycle, the faster the resolution of the viraemia. At this point, the weak cell-mediated immunity would be able to confine PRRSV to certain tissues (i.e. lymph nodes) where the proportion of permissive cells could be still relatively high (Xiao et al., 2004
). In consequence, viraemia should cease or become inconstant and of low level. This would occur regardless of whether or not NA had developed. However, as long as the virus persisted in tissues, local IL10 release (or other cytokines) would keep T-cell responses low. As far as the infection can be confined to certain tissues and permissive macrophages are not replaced at a high rate, the number of infected cells will decline steadily and the immune response will finally be able to clear the infection. This moment would correspond with the rise of blood IFN-
-SC and the final development of NA. From then onwards, the pig would be protected against homologous challenge (Mengeling et al., 2003
). Such a model would explain why no clear correlation between the immune response and the clearing of infection can be determined, as well as making sense of the delayed T-cell responses observed in PRRSV infection. In our opinion, this hypothesis deserves further study, as it can give a whole image of the events taking place in PRRSV infection and can contribute to understanding how to develop new and more efficient vaccines.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Albina, E., Piriou, L., Hutet, E., Cariolet, R. & L'Hospitalier, R. (1998). Immune responses in pigs infected with porcine reproductive and respiratory syndrome virus (PRRSV). Vet Immunol Immunopathol 61, 4966.[CrossRef][Medline]
Asai, T., Mori, M., Okada, M., Uruno, K., Yazawa, S. & Shibata, I. (1999). Elevated serum haptoglobin in pigs infected with porcine reproductive and respiratory syndrome virus. Vet Immunol Immunopathol 70, 143148.[CrossRef][Medline]
Batista, L., Pijoan, C., Dee, S., Olin, M., Molitor, T., Joo, H. S., Xiao, Z. & Murtaugh, M. (2004). Virological and immunological responses to porcine reproductive and respiratory syndrome virus in a large population of gilts. Can J Vet Res 68, 267273.[Medline]
Chung, H.-K. & Chae, C. (2003). Expression of interleukin-10 and interleukin-12 in piglets experimentally infected with porcine reproductive and respiratory syndrome virus (PRRSV). J Comp Pathol 129, 205212.[CrossRef][Medline]
Darwich, L., Balasch, M., Plana-Durán, J., Segalés, J., Domingo, M. & Mateu, E. (2003). Cytokine profiles of peripheral blood mononuclear cells from pigs with postweaning multisystemic wasting syndrome in response to mitogen, superantigen or recall viral antigens. J Gen Virol 84, 34533457.
Dea, S., Gagnon, C. A., Mardassi, H., Pirzadeh, B. & Rogan, D. (2000). Current knowledge on the structural proteins of porcine reproductive and respiratory syndrome (PRRS) virus: comparison of the North American and European isolates. Arch Virol 145, 659688.[CrossRef][Medline]
Díaz, I. & Mateu, E. (2005). Use of ELISPOT and ELISA to evaluate IFN-, IL-10 and IL-4 responses in conventional pigs. Vet Immunol Immunopathol (in press).
Drew, T. W., Lowings, J. P. & Yapp, F. (1997). Variation in open reading frames 3, 4 and 7 among porcine reproductive and respiratory syndrome virus isolates in the UK. Vet Microbiol 55, 209221.[CrossRef][Medline]
Duan, X., Nauwynck, H. J. & Pensaert, M. B. (1997). Virus quantification and identification of cellular targets in the lungs and lymphoid tissues of pigs at different time intervals after inoculation with porcine reproductive and respiratory syndrome virus (PRRSV). Vet Microbiol 56, 919.[CrossRef][Medline]
Forsberg, R., Storgaard, T., Nielsen, H. S., Oleksiewicz, M. B., Cordioli, P., Sala, G., Hein, J. & Bøtner, A. (2002). The genetic diversity of European type PRRSV is similar to that of the North American type but is geographically skewed within Europe. Virology 299, 3847.[CrossRef][Medline]
Gagnon, C. A. & Dea, S. (1998). Differentiation between porcine reproductive and respiratory syndrome virus isolates by restriction fragment length polymorphism of their ORFs 6 and 7 genes. Can J Vet Res 62, 110116.[Medline]
Goldberg, T. L., Lowe, J. F., Milburn, S. M. & Firkins, L. D. (2003). Quasispecies variation of porcine reproductive and respiratory syndrome virus during natural infection. Virology 317, 197207.[CrossRef][Medline]
Johnson, W., Roof, M., Vaughn, E., Christopher-Hennings, J., Johnson, C. R. & Murtaugh, M. P. (2004). Pathogenic and humoral immune responses to porcine reproductive and respiratory syndrome virus (PRRSV) are related to viral load in acute infection. Vet Immunol Immunopathol 102, 233247.[CrossRef][Medline]
Jusa, E. R., Inaba, Y., Kouno, M., Hirose, O., Shibata, I., Kubota, M. & Yasuhara, H. (1996). Slow-reacting and complement-requiring neutralizing antibody in swine infected with porcine reproductive and respiratory syndrome (PRRS) virus. J Vet Med Sci 58, 749753.[Medline]
Kawashima, K., Narita, M. & Yamada, S. (1999). Changes in macrophage and lymphocyte subpopulations of lymphoid tissues from pigs infected with the porcine reproductive and respiratory syndrome virus (PRRSV). Vet Immunol Immunopathol 71, 257262.[CrossRef][Medline]
Labarque, G., Van Gucht, S., Van Reeth, K., Nauwynck, H. & Pensaert, M. (2003). Respiratory tract protection upon challenge of pigs vaccinated with attenuated porcine reproductive and respiratory syndrome virus vaccines. Vet Microbiol 95, 187197.[CrossRef][Medline]
Lager, K. M. & Mengeling, W. L. (2000). PRRS: nature of the RNA virus and how it causes disease (a keynote paper). In Proceedings of the 16th International Pig Veterinary Society Congress, pp. 538543. 1720 September 2000, Melbourne, Australia.
Lager, K. M., Mengeling, W. L. & Brockmeier, S. L. (1999). Evaluation of protective immunity in gilts inoculated with the NADC-8 isolate of porcine reproductive and respiratory syndrome virus (PRRSV) and challenge-exposed with an antigenically distinct PRRSV isolate. Am J Vet Res 60, 10221027.[Medline]
Lamontagne, L., Page, C., Larochelle, R., Longtin, D. & Magar, R. (2001). Polyclonal activation of B cells occurs in lymphoid organs from porcine reproductive and respiratory syndrome virus (PRRSV)-infected pigs. Vet Immunol Immunopathol 82, 165182.[CrossRef][Medline]
Loemba, H. D., Mounir, S., Mardassi, H., Archambault, D. & Dea, S. (1996). Kinetics of humoral immune response to the major structural proteins of the porcine reproductive and respiratory syndrome virus. Arch Virol 141, 751761.[Medline]
Lopez, O. J. & Osorio, F. A. (2004). Role of neutralizing antibodies in PRRSV protective immunity. Vet Immunol Immunopathol 102, 155163.[CrossRef][Medline]
López Fuertes, L., Doménech, N., Alvarez, B., Ezquerra, A., Domínguez, J., Castro, J. M. & Alonso, F. (1999). Analysis of cellular immune response in pigs recovered from porcine respiratory and reproductive syndrome infection. Virus Res 64, 3342.[CrossRef][Medline]
Mateu, E., Martín, M. & Vidal, D. (2003). Genetic diversity and phylogenetic analysis of glycoprotein 5 of European-type porcine reproductive and respiratory virus strains in Spain. J Gen Virol 84, 529534.
McGuirk, P. & Mills, K. H. G. (2002). Pathogen-specific regulatory T cells provoke a shift in the Th1/Th2 paradigm in immunity to infectious diseases. Trends Immunol 23, 450455.[CrossRef][Medline]
Meier, W. A., Galeota, J., Osorio, F. A., Husmann, R. J., Schnitzlein, W. M. & Zuckermann, F. A. (2003). Gradual development of the interferon- response of swine to porcine reproductive and respiratory syndrome virus infection or vaccination. Virology 309, 1831.[CrossRef][Medline]
Meng, X. J. (2000). Heterogeneity of porcine reproductive and respiratory syndrome virus: implications for current vaccine efficacy and future vaccine development. Vet Microbiol 74, 309329.[CrossRef][Medline]
Meng, X. J., Paul, P. S., Halbur, P. G. & Lum, M. A. (1995). Phylogenetic analyses of the putative M (ORF 6) and N (ORF 7) genes of porcine reproductive and respiratory syndrome virus (PRRSV): implication for the existence of two genotypes of PRRSV in the U.S.A. and Europe. Arch Virol 140, 745755.[CrossRef][Medline]
Mengeling, W. L., Lager, K. M., Vorwald, A. C. & Koehler, K. J. (2003). Strain specificity of the immune response of pigs following vaccination with various strains of porcine reproductive and respiratory syndrome virus. Vet Microbiol 93, 1324.[CrossRef][Medline]
Meulenberg, J. J., Hulst, M. M., de Meijer, E. J., Moonen, P. L., den Besten, A., de Kluyver, E. P., Wensvoort, G. & Moormann, R. J. (1994). Lelystad virus belongs to a new virus family, comprising lactate dehydrogenase-elevating virus, equine arteritis virus, and simian hemorrhagic fever virus. Arch Virol Suppl 9, 441448.[Medline]
Moestrup, S. K. & Moller, H. J. (2004). CD163: a regulated hemoglobin scavenger receptor with a role in the anti-inflammatory response. Ann Med 36, 347354.[CrossRef][Medline]
Molitor, T. W., Bautista, E. M. & Choi, C. S. (1997). Immunity to PRRSV: double-edged sword. Vet Microbiol 55, 265276.[CrossRef][Medline]
Moore, K. W., de Waal Malefyt, R., Coffman, R. L. & O'Garra, A. (2001). Interleukin-10 and the interleukin-10 receptor. Annu Rev Immunol 19, 683765.[CrossRef][Medline]
Murtaugh, M. P., Elam, M. R. & Kakach, L. T. (1995). Comparison of the structural protein coding sequences of the VR-2332 and Lelystad virus strains of the PRRS virus. Arch Virol 140, 14511460.[CrossRef][Medline]
Murtaugh, M. P., Xiao, Z. & Zuckermann, F. (2002). Immunological responses of swine to porcine reproductive and respiratory syndrome virus infection. Viral Immunol 15, 533547.[CrossRef][Medline]
Nielsen, J. & Bøtner, A. (1997). Hematological and immunological parameters of 4-month old pigs infected with PRRS virus. Vet Microbiol 55, 289294.[CrossRef][Medline]
Onyekaba, C. O., Harty, J. T., Even, C., Hu, B.-G. & Plagemann, P. G. W. (1989). Persistent infection of mice by lactate dehydrogenase-elevating virus: effects of immunosuppression on virus replication and antiviral immune responses. Virus Res 14, 297315.[CrossRef][Medline]
Osorio, F. A., Galeota, J. A., Nelson, E., Brodersen, B., Doster, A., Wills, R., Zuckermann, F. & Laegreid, W. W. (2002). Passive transfer of virus-specific antibodies confers protection against reproductive failure induced by a virulent strain of porcine reproductive and respiratory syndrome virus and establishes sterilizing immunity. Virology 302, 920.[CrossRef][Medline]
Ostrowski, M., Galeota, J. A., Jar, A. M., Platt, K. B., Osorio, F. A. & Lopez, O. J. (2002). Identification of neutralizing and nonneutralizing epitopes in the porcine reproductive and respiratory syndrome virus GP5 ectodomain. J Virol 76, 42414250.
Philippidis, P., Mason, J. C., Evans, B. J., Nadra, I., Taylor, K. M., Haskard, D. O. & Landis, R. C. (2004). Hemoglobin scavenger receptor CD163 mediates interleukin-10 release and heme oxygenase-1 synthesis: antiinflammatory monocyte-macrophage responses in vitro, in resolving skin blisters in vivo, and after cardiopulmonary bypass surgery. Circ Res 94, 119126.
Royaee, A. R., Husmann, R. J., Dawson, H. D., Calzada-Nova, G., Schnitzlein, W. M., Zuckermann, F. A. & Lunney, J. K. (2004). Deciphering the involvement of innate immune factors in the development of the host response to PRRSV vaccination. Vet Immunol Immunopathol 102, 199216.[CrossRef][Medline]
Samsom, J. N., de Bruin, T. G. M., Voermans, J. J. M., Meulenberg, J. J. M., Pol, J. M. A. & Bianchi, A. T. J. (2000). Changes of leukocyte phenotype and function in the broncho-alveolar lavage fluid of pigs infected with porcine reproductive and respiratory syndrome virus: a role for CD8+ cells. J Gen Virol 81, 497505.
Sánchez-Torres, C., Gómez-Puertas, P., Gómez-del-Moral, M., Alonso, F., Escribano, J. M., Ezquerra, A. & Domínguez, J. (2003). Expression of porcine CD163 on monocytes/macrophages correlates with permissiveness to African swine fever infection. Arch Virol 148, 23072323.[CrossRef][Medline]
Shimizu, M., Yamada, S., Kawashima, K., Ohashi, S., Shimizu, S. & Ogawa, T. (1996). Changes of lymphocyte subpopulations in pigs infected with porcine reproductive and respiratory syndrome (PRRS) virus. Vet Immunol Immunopathol 50, 1927.[CrossRef][Medline]
Sipos, W., Duvigneau, C., Pietschmann, P. & 7 other authors (2003). Parameters of humoral and cellular immunity following vaccination of pigs with a European modified-live strain of porcine reproductive and respiratory syndrome virus (PRRSV). Viral Immunol 16, 335346.[CrossRef][Medline]
Suradhat, S. & Thanawongnuwech, R. (2003). Upregulation of interleukin-10 gene expression in the leukocytes of pigs infected with porcine reproductive and respiratory syndrome virus. J Gen Virol 84, 27552760.
Suradhat, S., Thanawongnuwech, R. & Poovorawan, Y. (2003). Upregulation of IL-10 gene expression in porcine peripheral blood mononuclear cells by porcine reproductive and respiratory syndrome virus. J Gen Virol 84, 453459.
van der Linden, I. F. A., Voermans, J. J. M., van der Linde-Bril, E. M., Bianchi, A. T. J. & Steverink, P. J. G. M. (2003). Virological kinetics and immunological responses to a porcine reproductive and respiratory syndrome virus infection of pigs at different ages. Vaccine 21, 19521957.[CrossRef]
van Woensel, P. A., Liefkens, K. & Demaret, S. (1998). European serotype PRRSV vaccine protects against European serotype challenge whereas an American serotype vaccine does not. Adv Exp Med Biol 440, 713718.[Medline]
Vezina, S. A., Loemba, H., Fournier, M., Dea, S. & Archambault, D. (1996). Antibody production and blastogenic response in pigs experimentally infected with porcine reproductive and respiratory syndrome virus. Can J Vet Res 60, 9499.[Medline]
Wensvoort, G., Terpstra, C., Pol, J. M. & 19 other authors (1991). Mystery swine disease in the Netherlands: the isolation of Lelystad virus. Vet Q 13, 121130.[Medline]
Xiao, Z., Batista, L., Dee, S., Halbur, P. & Murtaugh, M. P. (2004). The level of virus-specific T-cell and macrophage recruitment in porcine reproductive and respiratory syndrome virus infection in pigs is independent of virus load. J Virol 78, 59235933.
Yoon, I. J., Joo, H. S., Goyal, S. M. & Molitor, T. W. (1994). A modified serum neutralization test for the detection of antibody to porcine reproductive and respiratory syndrome virus in swine sera. J Vet Diagn Invest 6, 289292.[Medline]
Yoon, K. J., Zimmerman, J. J., Swenson, S. L. & 7 other authors (1995). Characterization of the humoral immune response to porcine reproductive and respiratory syndrome (PRRS) virus infection. J Vet Diagn Invest 7, 305312.[Medline]
Received 9 February 2005;
accepted 11 April 2005.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |