Virological kinetics and immunological responses to a porcine reproductive and respiratory syndrome virus infection of pigs at different ages

Vaccine 21 (2003) 1952–1957

Virological kinetics and immunological responses to a porcine reproductive and respiratory syndrome virus infection of pigs at different ages I.F.A. van der Linden a,∗ , J.J.M. Voermans b,1 , E.M. van der Linde-Bril b,1 , A.T.J. Bianchi b,1 , P.J.G.M. Steverink a a

Department of Infectious Diseases and Chain Quality, ID-Lelystad, P.O. Box 65, 8200 AB Lelystad, The Netherlands b CIDC-Lelystad, Houtribweg 39, 8221 RA Lelystad, The Netherlands Received 9 April 2002; received in revised form 5 August 2002; accepted 6 December 2002

Abstract The objective of this study was to measure the effect of two variables (pig age and virus strain) on selected responses (clinical signs, viraemia, virus excretion and seroconversion) of pigs following exposure to porcine reproductive and respiratory syndrome (PRRS) virus. Therefore, young (6 till 8 weeks old) and old (6 months old) pigs were infected with 3 different PRRSV strains, i.e. LV ter Huurne (LVTH), LV4.2.1 and SDSU#73. Regardless of the strain used for exposure, young pigs were more susceptible to infection as shown by a higher number of viraemic and virus excreting pigs. Strain differences were also evident. LV ter Huurne induced virus excretion in a higher number of pigs and with a higher virus titre, whereas SDSU#73 induced most severe clinical signs. LV4.2.1 induced viraemia and virus excretion in a low number of pigs. The kinetics of the antibody response differed per virus strain. The results presented here are useful in developing a less expensive standardised infection model, consisting of young pigs intranasally infected with a virulent PRRSV strain, to study the efficacy of new vaccine strains. © 2003 Elsevier Science Ltd. All rights reserved. Keywords: PRRSV; Age; Strain

1. Introduction The disease described as porcine reproductive and respiratory syndrome (PRRS) was first recognised in the United States (US) in 1987 [1,2] and was in Europe recognised in 1990 [3]. The syndrome is characterised by abortions, birth of stillborn and mummified piglets, and respiratory problems in young pigs. The causative agent, firstly isolated in 1991 in the Netherlands [3], belongs to the family of the Arteriviridae which includes lactate dehydrogenase elevating virus (LDV), equine arthritis virus (EAV) and simian haemorrhagic fever virus (SHFV) [4,5]. The family consists of positive stranded RNA viruses that have a specific tropism for cells of the monocyte/macrophage lineage and a discontinuous replication mechanism [5,6]. PRRSV is divided ∗ Corresponding author. Tel.: +31-320-238364/238238; fax: +31-320-238668. E-mail address: [email protected] (I.F.A. van der Linden). 1 Tel.: +31-320-238800; fax: +31-320-238668.

into European and American subtypes based on genetic and antigenic differences. The clinical signs also differ between strains: the American strains often cause respiratory and reproductive problems, while the European strains mainly cause reproductive problems [7]. Respiratory signs and the extent of lung involvement may vary per strain and are difficult to reproduce under experimental conditions [8,9]. Therefore, the reproductive model is readily used to study the clinical effects of a PRRSV infection [10]. However, this is a costly model, which justifies the need for a well-defined, standardised (for age and strain) and less costly PRRSV infection model to study infection and assess vaccine quality. Such an infection model should include parameters to study and quantify the virulence of the virus based on frequency and severity of clinical signs, viral parameters (viraemia and virus excretion) and seroconversion. Furthermore, the kinetics of viraemia and viral excretion could also be important parameters in studying vaccination efficacy, since reduction in shedding and transmission of virus among pigs is an important goal in the control of PRRSV [8,11,12].

0264-410X/03/$ – see front matter © 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0264-410X(02)00822-8

I.F.A. van der Linden et al. / Vaccine 21 (2003) 1952–1957

2. Materials and methods 2.1. Animals and experimental design Conventionally-raised Landrace pigs, obtained from a PRRSV-free farm in Denmark, born from PRRSV-unvaccinated sows and PRRSV-free as tested by an ELISA (IDEXX, Maine USA) were used in two experiments. Each experiment used two age groups; pigs of 6–8 weeks old and pigs of around 24 weeks old, referred to hereafter as young and old pigs, respectively. In the first experiment, six young and five old pigs were inoculated intranasally with 2 ml of 105 TCID50 ml−1 of a European wild-type PRRSV strain (LV ter Huurne (LVTH)). Clinical signs (general health status, rectal temperature and diarrhoea) were recorded daily, prior to feeding. Serum samples for measuring antibody response and viraemia were collected at 0, 4, 7, 11, 14, 18, 21, 25, 28, 32, 35, 39 and 42 days post-infection (dpi). Tonsillar swab specimens were taken daily until 11 dpi and again at 14 dpi to monitor virus excretion. In the second experiment, thirty young and twelve old pigs were used. The young pigs were divided into five groups each consisting of 6 pigs inoculated either intranasally with 2 ml of 105 TCID50 ml−1 of the European wild-type virus strain LV ter Huurne (LVTH), PRRSV-free cell lysate prepared in a similar fashion as LV ter Huurne virus (sham-PAM), a cell-line adapted European strain (LV4.2.1), an American PRRSV strain (SDSU#73), or inoculated intramuscularly with a sham cell-line inoculum (sham-CL) prepared in the same way as LV4.2.1 and SDSU#73. The old pigs were divided in two groups each consisting of 6 pigs inoculated intranasally with either LV4.2.1 or SDSU#73. The LV ter Huurne infected group in the second experiment was included in order to allow comparison between experiments 1 and 2. Clinical signs, serum samples and tonsillar swabs were taken as described in experiment 1. The time points of the serum samples were the same as experiment 1 except for the following differences where blood was collected on days 3, 10, 17, 24, 31 and 38 instead of days 4, 11, 18, 25, 32 and 39. The time points of experiment 1 will be referred to in this article. Experimental and animal management procedures were undertaken in accordance with the requirements of the animal care and ethics committee of ID-Lelystad in accordance with the Dutch legislation on animal experiments. 2.2. Sample handling Serum samples and tonsillar swab specimens remained on ice after collection and during handling. Swab specimens were extracted by incubating the swab in swab holders with 4 ml RPMI supplemented with 5% foetal calf serum (FCS) and 10% antibiotics (ABII) and subsequently spinning the fluid down. To determine the virus titre present in the fluid collected in the swab, the samples were titrated and corrected for the amount of fluid present in the swab before adding

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the RPMI [13,14]. All samples (serum and swab specimens) were stored at −70 ◦ C and tested in parallel at the same time in a particular test. 2.3. Cells and viruses CL2621-cells were propagated in Eagle’s minimal essential medium supplemented with Hank’s salts, 10% FCS, 5% ABII, 1.5% sodium bicarbonate and 1% l-glutamine. Porcine alveolar macrophages (PAMs) were isolated by a pulmonary lavage after pigs were necropsied as previously described [3]. Before use, they were tested on propagation of two representatives of PRRSV (LV4.2.1 (EU) and US17 (US)), as shown by CPE and positive immunostaining by PRRSV-specific antibodies. If the PAM were not able to fulfil the criteria, they were not used. The LV ter Huurne strain is a virulent European wild-type of PRRSV and was isolated on PAMs from a clinical case of PRRS [3]. The inoculum used was the 6th passage of the LV ter Huurne strain propagated and titrated on PAMs as described earlier [3]. The LV4.2.1 strain is a derivative of wild-type strain LV ter Huurne that was adapted for growth on CL2621-cells by serial passages. The virus was propagated on CL2621-cells and the inoculum was titrated on PAMs as described earlier [3]. Dr. E.Vaughn (Boehringer Ingelheim, Animal Health) kindly provided the SDSU#73 strain (passage 3 on CL2621-cells). It is known as one of the most virulent American wild-type strains of PRRSV. The virus was propagated and titrated in the same way as described for LV4.2.1. 2.4. Virus isolation and virus titration To detect virus in serum and tonsillar swabs, PAMs were seeded with a concentration of 105 cells per well into 96-wells plastic plates about 16–24 h prior to incubation. Ten-fold serial dilutions were prepared for each sample, in a 96-well plastic dummy plate and 50 ␮l of the dilutions was transferred in duplicate to the corresponding well of the test plate-containing PAMs. After 2 days, 25 ␮l of the growth medium was transferred to corresponding wells of 96-wells plastic plates, containing fresh macrophages plated 16–24 h prior to incubation. After 1 day, the medium was discarded and the cells were washed with 0.15 M NaCl, dried and frozen. Subsequently, the cells were fixed with a cold solution of paraformaldehyde (4% (w/v)) for 10 min and rinsed with wash buffer (0.15 M NaCl + 0.5% Tween 80). PRRSV-infected cells were labelled using a PRRSV-specific monoclonal antibody (122.17) in a 10−1 dilution in buffer containing 0.5 M NaCl, 4% horse serum and 0.05% Tween 80 (pH 7.2). The plates were incubated 1 h at 37 ◦ C and then washed three times in wash buffer. A rabbit anti-mouse (HRPO) conjugate (1/250 in dilution buffer) was used for staining. After incubation for 1 h at 37 ◦ C and washing as previously, 10 ␮g per well chromogen substrate (AEC) dissolved in 0.05 M sodium-acetate (pH 5.0) and 0.05% H2 O2 was added to the plates. Plates were

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I.F.A. van der Linden et al. / Vaccine 21 (2003) 1952–1957

incubated for at least 30 min at room temperature. Finally, the AEC was replaced by 50 ␮l 0.05 M sodium-acetate (pH 5.0) per well. Presence of virus was confirmed when the cytoplasm of macrophages was stained deeply red. Unstained cytoplasm indicated absence of virus. Negative and positive control samples were included in the assay. To determine the virus titre, PAMs were seeded in the same way as for virus isolation. Ten-fold serial dilutions were prepared 6 times in parallel for each sample in a 96-well plastic dummy plate and 50 ␮l were transferred to the corresponding wells of the test plate-containing PAMs. After 5–7 days, the macrophages were examined for CPE. Titres were calculated according to the method of Reed and Muench [15] and expressed as log10 TCID50 ml−1 . The detection limit of the virus titration is log10 titre 1.8. All samples with a log10 titre lower than 1.8 were considered to be negative, although low amounts of virus may be present. 2.5. Antibody detection by immuno peroxidase monolayer assay (IPMA) To detect antibodies in serum, macrophages were seeded as described by virus isolation. The cells were infected with 0.1 multiplication of infection (m.o.i.) of either the LV4.2.1 or the US17 (American strain) and incubated for 36 h in a humid atmosphere containing 5% CO2 . The plates were washed with 0.15 M NaCl, dried and frozen. Basically, the IPMA was performed in the same way as was described by virus isolation. Virus-positive cells were labelled with four-fold serum dilutions prepared in duplicate. For staining, rabbit-anti-swine HRPO was used at a concentration of 26 ng per well. 2.6. Statistical analysis Since variance within the groups was large, statistical analysis was performed per age independent of PRRSV strain and per PRRSV strain, independent of age. Viraemia and virus excretion were analysed by calculating the probability (P ≤ 0.05) per time point of the frequency of virus-positive pigs with the aid of the logit equation. The model comprised main effects for experiments (experiments 1 and 2), age and strain. All calculations were performed with the statistical software package LogXact (Windows, Version 2.1, 1996, CYTEL Software Corporation, Cambridge, USA). Seroconversion was analysed by log transformed titres. Separate analyses were performed for each time point with an analysis of variance model (P ≤ 0.05). The model comprised main effects for experiments (experiments 1 and 2) and main effects and interaction terms for age of the pigs (6–8 weeks old and 6 months old) and strains (LV ter Huurne, LV4.2.1 and SDSU#73). All calculations were performed with the statistical programming language Genstat

5 (Release 3, Reference Manual, Clarendon Press, Oxford, 1993).

3. Results 3.1. Clinical signs In experiment 1, the effect of age differences between young and old pigs infected with LV ter Huurne is examined, while in experiment 2 the effect of age differences between young and old LV4.2.1 or SDSU#73 infected pigs is examined. To be able to compare the results of experiments 1 and 2, young animals infected with LV ter Huurne were included in both experiments. The results of experiments 1 and 2 were comparable, except for viraemia at 21 dpi, virus excretion at 8 and 9 dpi and seroconversion at 7, 11, 25 and 28 dpi. Therefore, LV ter Huurne infected pigs were excluded from comparison with LV4.2.1 and SDSU#73 infected pigs at these days. Clinical signs were comparable in both experiments. Diarrhoea and fever were seen in all groups, including the group inoculated with sham-PAM, but not or less severe in the group inoculated with sham-CL (Table 1). Respiratory signs, abnormal behaviour and loss of appetite occurred more often in the old groups. More pigs inoculated with SDSU#73 developed clinical signs than pigs inoculated with the European strains (Table 1). 3.2. Viraemia Viraemia was first detected in all pigs at 4 dpi, except for the old group infected with LV4.2.1 where it was first detected at 7 dpi and not at all in one animal of this group (data not shown). In young pigs viraemia was detected until 42 dpi with a maximum frequency of positive animals of 88% (Fig. 1a) and a log10 virus titre ranging from 2.1 to 2.6 at 4, 7, 11 and 14 dpi. In old pigs, viraemia was detected until 28 dpi with a maximum frequency of 65% positive animals per time point (Fig. 1a) and a log10 virus titre ranging from 0.7 to 1.7. Pigs exposed to LV ter Huurne showed significantly different viraemia at 14 dpi than pigs exposed to SDSU#73 (Fig. 1b). After 25 dpi, both LV ter Huurne and SDSU#73 were intermittently detected in the serum of pigs. LV4.2.1-infected pigs showed a shorter duration of viraemia (until 21 dpi), a significantly lower frequency of positive pigs (41%) at 7 and 11 dpi (P ≤ 0.05) (Fig. 1b) and a lower log10 virus titre at 4, 7, 11 and 14 dpi as compared to LV ter Huurne and SDSU#73. 3.3. Virus excretion Virus excretion was detected from 1 dpi until the end of the measurement (14 dpi). A higher percentage of young than old pigs (maximum of positive animals per time point was 50 and 37%, respectively) excreted virus with a higher

I.F.A. van der Linden et al. / Vaccine 21 (2003) 1952–1957

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Table 1 Clinical signs of pigs infected with PRRSV Inoculae

Diarrhoea

Temperaturea

Respiratory signsb

Abnormal behaviourc

Loss of appetite

Young

Old

Young

Old

Young

Old

Young

Old

Young

Old

6/6d 3–21e

5/5 1

6/6 1–4

3/5 1–8

0/6 0

1/5 2

0/6 0

2/5 1–5

0/6 0

2/5 1–5

LVTH (experiment 2)

5/6 2–8

n.i.f n.i.

6/6 1–9

n.i. n.i.

0/6 0

n.i. n.i.

0/6 0

n.i. n.i.

0/6 0

n.i. n.i.

Sham-PAM

2/6 2

n.i. n.i.

5/6 1–4

n.i. n.i.

0/6 0

n.i. n.i.

0/6 0

n.i. n.i.

0/6 0

n.i. n.i.

LV4.2.1

3/6 2–6

0/6 0

6/6 1–4

4/6 1–9

0/6 0

1/6 1

0/6 0

2/6 1–6

0/6 0

1/6 5

SDSU#73

6/6 3–8

0/6 0

6/6 5–9

6/6 1–13

1/6 2

4/6 1–2

0/6 0

6/6 8–10

0/6 0

6/6 9–11

Sham-CL

0/6 0

n.i. n.i.

1/6 1

n.i. n.i.

0/6 0

n.i. n.i.

0/6 0

n.i. n.i.

0/6 0

n.i. n.i.

LVTH (experiment 1)

Higher than 40 ◦ C was defined as fever. Characterised by tachypnoe or excessive nose fluid. c Was characterised namely by lethargic behaviour. d Number of pigs showing the clinical signs per total number of pigs. e Number of days that pigs display the clinical signs. f Group was not included in the experiments. a

b

log10 virus titre (between 0.2 and 1.9, and 0 and 0.3, respectively) (Fig. 2a, P ≤ 0.05). All pigs exposed to LV ter Huurne, but not all pigs exposed to LV4.2.1 and SDSU#73, excreted virus (58 and 67%, respectively). Per time point, more pigs infected with LV ter Huurne excreted virus (maximum 100%) with a higher log10 virus titre (range 0–3.7) at days 4, 7, 11 and 14 when compared to LV4.2.1 (maximum 17%, log10 virus titre of 0) and SDSU#73 (maximum 50%, log10 virus titre ranging from 0 to 2.7) (Fig. 2b).

Fig. 2. Virus excretion as measured by titrating fluids of tonsillar swabs of young and old pigs infected with LV ter Huurne, LV4.2.1 or SDSU#73: (a) frequency of virus excreting pigs independent of PRRSV strain; (b) frequency of virus excreting pigs independent of age of the pigs. Three open symbols per time point: significant difference (P ≤ 0.05) between all strains. Two open symbols per time point: significantly different with regard to the other strain or the other age group with an open symbol (P ≤ 0.05). One open symbol per time point: significantly different with regard to the other strains (P ≤ 0.05). Asterisk (∗) means experiments 1 and 2 were significantly different (P ≤ 0.05), therefore strain comparison was not relevant. Fig. 1. Viraemia of young or old pigs infected with LV ter Huurne, LV4.2.1 or SDSU#73: (a) frequency of viraemic pigs independent of PRRSV strain; (b) frequency of viraemic pigs independent of age of the pigs. Three open symbols per time point: significant difference (P ≤ 0.05) between all strains. Two open symbols per time point: significantly different with regard to the other strain or the other age group with an open symbol (P ≤ 0.05). One open symbol per time point: significantly different with regard to the other strains (P ≤ 0.05). Asterisk (∗) means experiments 1 and 2 were significantly different (P ≤ 0.05), therefore strain comparison was not relevant.

3.4. Seroconversion All pigs seroconverted after inoculation, confirming infection in all pigs. There was no significant difference (P ≤ 0.05) between young and old pigs (Fig. 3a). The antibody response of pigs infected with LV ter Huurne was slower than after infection with SDSU#73 and LV4.2.1 (P ≤ 0.05). The

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I.F.A. van der Linden et al. / Vaccine 21 (2003) 1952–1957

Fig. 3. Seroconversion was measured in serum by end-point titration with IPMA, after infection of young and old pigs with LV ter Huurne, LV4.2.1 or SDSU#73: (a) the antibody response per age independent of PRRSV strain; (b) the antibody response per strain independent of age of the pigs. Three open symbols: significant difference (P ≤ 0.05) between all strains. Two open symbols: significantly different with regard to the other strain with an open symbol at that time point (P ≤ 0.05) or between the age groups. One open symbol: significantly different with regard to the other strains at that time point (P ≤ 0.05). Asterisk (∗) means experiments 1 and 2 were significantly different (P ≤ 0.05), therefore strain comparison was not relevant.

response was first detected at 7 dpi, and increased slowly to a plateau of a titre between 3.2 and 4.0 at 21 dpi (Fig. 3b). Seroconversion of pigs infected with LV4.2.1 was initially detected at 7 dpi and rose slowly to a plateau at 21 dpi with a log10 titre between 2.8 and 3.8. Seroconversion of SDSU#73 infected pigs showed a significantly different pattern (P ≤ 0.05); it rose quickly to a peak at 11 dpi with log10 titre between 4.0 and 4.3 and declined slowly to a plateau at 21 dpi with log10 titre between 3.1 and 3.7 (Fig. 3b). 4. Discussion The objective of this work was to study the response of pigs of different ages to PRRSV infection caused by different virus strains, in order to find an inexpensive standardised model to study efficacy of vaccines. Currently, the reproductive model for PRRS is used for vaccine studies since it is reproducible and allows optimal monitoring of clinical signs and comparison of the virulence of PRRSV strains. However, this model is expensive and laborious. The infection model presented here, serves as an attractive alternative, featuring reproducibility (since experiments 1 and 2 were comparable) and allowing the classification of PRRSV strains on virulence for target pigs. LV ter Huurne and SDSU#73 appear to be the most virulent as a result of the occurrence of a high level of virus-positive pigs and a long duration of viraemia (both virus strains), more severe clinical signs (SDSU#73), and a high level of virus excretion (LV ter Huurne). LV4.2.1 was less virulent, since it showed fewer clinical signs, low

viraemia and low virus excretion. The low virulence of LV 4.2.1 was confirmed by impairment in its ability to cause reproductive problems for gestation sows when compared to LV ter Huurne [16]. Sows infected with LV ter Huurne and LV4.2.1 resulted in litters with the same number of piglets, but the piglets of the sows infected with the higher virulence strain were weaker. In addition, the group infected with the higher virulence virus LV ter Huurne, showed a higher frequency of virus-positive piglets until 28 days post-farrowing [16]. Apparently, the cause of the lower virulence of LV4.2.1 is not related to macrophage infection, since the growth curve of LV4.2.1 in vitro on macrophages is comparable to the growth curve of LV ter Huurne [17]. The difference between the European and American strains, has resulted in a different onset of the immune response as measured by a difference in kinetics of the serological response. Such a difference in kinetics has not been described before. Whether this difference has any direct association with subtype of virus strains (EU or US) or difference in virulence or both remains to be determined. To standardise our PRRSV infection model, young pigs were compared to old pigs. The young pigs showed higher levels of viraemia and excreted more virus, possibly related to the more efficacious viral replication in macrophages of young pigs. This was shown in vitro in macrophages of 4 weeks old pigs producing more infectious virus than macrophages of 4 months old pigs [18]. Another difference between young and old pigs, is the cellular condition in the lymphoid organs, for example the T-cell distribution (presence of CD4+ CD8+ in lungs and tonsil of old pigs) [19]. The difference between young and old pigs in viraemia and virus excretion is therefore possibly caused by a combination of an altered macrophage response and a difference in cell subsets present in lymphoid organs and lungs. In our experiments, pigs from a Danish PRRSV-free farm were used. The pigs were tested for the presence of PRRSV, but not for other diseases. Since diarrhoea and fever were also seen in the negative control group, these clinical signs cannot be linked solely to PRRSV infection. Respiratory signs, abnormal behaviour and loss of appetite were not seen in the control pigs, therefore those clinical signs were attributed to PRRSV. The negative control group showed no antibody response against PRRSV therefore we conclude that the pigs of the control group remained PRRSV-free throughout the experiment. Clinical signs, viraemia, virus excretion and seroconversion were used as parameters to characterise this PRRSV infection model. In previous studies, clinical signs were often not reproducible [8,20,21], while viraemia, virus excretion and seroconversion are more consistent [11,22]. Our model is able to distinguish between PRRSV strains by using viraemia, virus excretion and seroconversion. Consequently, these parameters are useful tools to study PRRSV infection or vaccination.

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Acknowledgements This study could not have been performed without the help of the animal caretakers of the institute. We also wish to thank Bas Engel and Willem Buist for statistical assistance and Eugène van Rooij and Willem van Eden for critical reading of the manuscript. Eric Vaughn kindly provided the US strain SDSU#73. References [1] Keffaber KK. Reproductive failure of unknown etiology. Am Assoc Swine Pract Newslett 1989;1:1–10. [2] Hill H. Overview and history of mystery swine disease (swine infertility and respiratory syndrome). Proc. Myst. Swine Dis. Comm. Meet. 1990;29–31. [3] Wensvoort G, Terpstra C, Pol JM, et al. Mystery swine disease in The Netherlands: the isolation of Lelystad virus. Vet Q 1991;13(3):121– 30. [4] Meulenberg JJ, Hulst MM, de Meijer EJ, et al. Lelystad virus belongs to a new virus family, comprising lactate dehydrogenase-elevating virus, equine arteritis virus, and simian hemorrhagic fever virus. Arch Virol Suppl 1994;9:441–8. [5] Plagemann PG, Moennig V. Lactate dehydrogenase-elevating virus, equine arteritis virus, and simian hemorrhagic fever virus: a new group of positive-strand RNA viruses. Adv Virus Res 1992;4192–9. [6] Meulenberg JJM, den Besten Petersen A, De Kluyver EP, Moormann RJM, Schaaper WMM, Wensvoort G. Characterization of proteins encoded by ORFs 2–7 of Lelystad virus. Virology 1995;206(1):155– 63. [7] Meng XJ. Heterogeneity of porcine reproductive and respiratory syndrome virus: implications for current vaccine efficacy and future vaccine development. Vet Microbiol 2000;74(4):309–29. [8] van Woensel PA, Liefkens K, Demaret S. Effect on viraemia of an American and a European serotype PRRSV vaccine after challenge with European wild-type strains of the virus. Vet Rec 1998;142(19):510–2. [9] Zimmerman JJ, Yoon KJ, Wills RW, Swenson SL. General overview of PRRSV: a perspective from the United States. Vet Microbiol 1997;55(1–4):187–96. [10] Mengeling WL, Vorwald AC, Lager KM, Brockmeier SL. Comparison among strains of porcine reproductive and respiratory syndrome virus for their ability to cause reproductive failure. Am J Vet Res 1996;57(6):834–9.

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