The study was approved by the local animal welfare authority (Landesamt für Gesundheit und Soziales, Berlin, Germany) under the registration number G 0116/12. German Landrace piglets (n = 72) of both sexes from a PRRSV-free herd were weaned at the age of 28 days, moved to a biosafety level 2 experimental facility (Bundesinstitut für Risikobewertung, Berlin, Germany), and randomly allocated to six pens (n = 12 per pen). Piglets were assigned to three different diets (2 pens per diet). At the age of 63 days, the animals receiving the Znhigh diet were switched to the Znmed diet, in order to avoid toxic effects of Zn. One week after commencing the different diets, one pen per diet was chosen randomly and the animals were vaccinated intramuscularly with inactivated LV (accession M96262; kindly provided by Prof. H. Nauwynck (Ghent University, Ghent, Belgium)). Four weeks after vaccination, at the age of 63 days, all pigs were challenged with PRRSV field strain CReSA 3267 (accession JF276435; kindly provided by Prof. J. Segalés and Prof. E. Mateu (CReSA, Barcelona, Spain). Animals were infected by intranasal application of 1 ml of virus suspension with a titer of 5 × 106 TCID50/ml to each nostril using a spray nebulizer.
Pigs were monitored daily for the presence of clinical signs and body weights were recorded weekly. Blood samples were collected weekly after vaccination and at 0, 4, 7, 14, 21, 28, and 35 days post infection (dpi). Nasal swabs were taken on the same dpi as blood samples for quantification of virus shedding. All pigs were necropsied on day 35 pi. Lung and lymphoid tissues were evaluated by visual inspection for macroscopic lesions, and samples from lungs, lymph nodes, tonsils, and spleen were taken and immediately frozen in liquid nitrogen and stored at −70°C.
For virological analysis, serum samples (4, 7, 14, 21, 28 dpi), nasal swabs (4, 7, 14 dpi), and tonsil, lung and tracheobronchial lymph node samples (35 dpi) were examined by qPCR to determine PRRSV copy numbers. RNA extraction was performed using a viral RNA/DNA purification kit (Stratec) applying 200 μl of serum or 10 mg of tissue each. RNA yields and quality were determined with a NanoDrop® ND-100 spectrophotometer (NanoDrop Technologies). Reverse transcription (RT) was performed using the DyNAmo™ cDNA Synthesis Kit (Thermo Fisher Scientific). Viral loads were quantified using a TaqMan fluorescent probe-based real-time qPCR assay in an iCycler iQ™5 detection system (Bio-Rad) with primers described elsewhere
PRRSV-specific IgM and IgG antibodies were measured by ELISA (Ingezim PRRS DR, Ingenasa) according to the manufacturer’s instructions. VN antibodies against PRRSV were quantified by a viral neutralization test as previously described
. Neutralization of PRRSV strain CReSA 3267 was examined using PRRSV GP5 specific monoclonal antibody 3H4 (Ingenasa) and Alexa Fluor™ 488 conjugated anti-mouse IgG (H + L) secondary antibody (Invitrogen).
Peripheral blood mononuclear cells (PBMC) were isolated using density centrifugation through a Ficoll gradient (LSM1077, PAA Laboratories). Samples were treated with erythrocyte lysis buffer for 5 min on ice, PBMC were washed with 10 ml of PBS with 0.2% BSA and centrifuged for 15 min at 700 × g at 4°C. In all samples, PBMC viability was confirmed using standard procedures.
The cell-mediated PRRSV-specific immune response was measured by using ELISpot for the enumeration of IFN-gamma-SC in PBMC (Mabtech). In order to compare homologous and heterologous responses, PBMC were stimulated in parallel (2.5 × 105 PBMC/well, 3 wells per pig and stimulus) with CReSA 3267 or LV at a multiplicity of infection of 0.1. Unstimulated and PHA-stimulated cells (10 μg/ml) were used as negative and positive controls, respectively. IFN-gamma-SC numbers were counted using an ELISpot Reader System (A.EL.VIS GmbH).
Flow cytometry analysis was performed as described before
 using a BD FACSCanto™ flow cytometer (BD Biosciences). Data were analyzed with FlowJo™ software (TreeStar).