Viruses were propagated in either Madin-Darby Canine Kidney Epithelial Cells (MDCK) or primary swine kidney cell cultures. Virus isolation was achieved from routine diagnostic samples, nasal swabs or lung tissue samples, sent to the Danish National Veterinary Laboratory for routine diagnostic purposes. Details on all viruses included in the study are summarised in Table 1.
Sequencing and phylogenetic analysis
Sequencing of the HA and NA genes was performed as previously described by Bragstad et al. 2005  and full genome sequencing of all 8 gene segments was performed as described in Bragstad et al. 2006 . Full genome sequencing was performed on the following virus isolates: A/swine/Denmark/13608/2004(H1N2), A/swine/Denmark/12687/2003(H1N2), A/swine/Denmark/14348-9/2003(H3N2), and A/swine/Denmark/14348-3/2003(H1N1).
For phylogenetic analyses, gene sequences were aligned with MUSCLE using CLC DNA Workbench version 6.8.1. Neighbour Joining trees were calculated with a bootstrap value of 1000.
Amino acid sequences were deduced from nucleotide sequences using CLC DNA Workbench version 6.8.1 and aligned with MUSCLE. N-glycosylation sites were predicted with NetNGlyc 1.0 (CBS, DTU, Denmark).
Landrace/Yorkshire (LY) pigs, both males and females, two months of age and free from more than 30 known swine pathogens including the important respiratory pathogens porcine circovirus 2 (PCV2), porcine reproductive and respiratory syndrome virus (PRRSV), swine influenza virus (SIV), porcine respiratory coronavirus, Actinobacillus pleuropneumonia and mycoplasmas , were used for the experiment. The pigs were randomly divided into two groups of 6 pigs, denoted group 1 and group 2. Each group was housed in separate isolation units (biosafety level 3). The pigs in group 1 were inoculated intranasally in the left nostril on PID 0 with 4 ml of A/swine/Denmark/19126/1993 (H1N1) with a titer of 105.7 tissue culture infectious dose 50% (TCID50) per ml. Two pigs were euthanized on PID 4. On PID 28, the remaining four pigs were re-inoculated with 4 ml of A/swine/Denmark/10074/2004 (H1N2) with a titer of 105.9 TCID50 per ml and they were euthanized on PID 56. For the pigs in group 2, the experimental design was similar except that the pigs were inoculated on PID 0 with the H1N2 isolate and then on PID 28 with the H1N1 isolate. Euthanization was performed by intravenous injection of pentobarbiturate (50 mg/kg) followed by exsanguination by cutting the arteria axillaris.
Body temperature was measured rectally before further handling of the pigs on PID -3, 0–9, and 28–37. Clinical signs and well-being of the pigs was monitored twice daily during the experiment. Blood samples were taken on PID -3, 0, 7, 11, 14, 18, 22, 28, 35, 39, 42, 46, 50, and 56 from vena jugulars from all pigs and from animals when euthanized on PID 4. Blood samples obtained on PID 0 and 28 were collected prior to inoculation. The blood samples were left on ice to coagulate for 15 minutes and centrifuged at 3500 rpm for 10 minutes at 4°C. Sera were transferred to new vials and stored at -80°C.
Nasal swab samples were collected on PID -3, 0–9, 11, 14, 18, 22, 28–37, 39, 42, 46, 50, and 56 from the right nostril. The swabs were placed in 1 ml phosphate buffered saline (PBS) (pH 7.5) and stored at -80°C until analysis. Nasal swabs taken on PID 0 and 28 were collected prior to inoculation. At post mortem, samples were collected and frozen at -80°C from the following nine parts of the lung, lu1: cranioventral part of the left cranial lobe; lu2: caudoventral part of the left cranial lobe; lu3: dorsal part of the left cranial lobe; lu4: ventral part of the right cranial lobe; lu5: middle part of the right cranial lobe; lu6: dorsal part of the right cranial lobe; lu7: middle part of the right middle lobe; lu8: middle part of the accessory lobe; and lu9: middle part of the right caudal lobe.
The study was carried out in strict accordance with the Danish legislation on animal experiments (LBK nr 1306 – 23/11/2007) and EU regulations on the use of laboratory animals for research.
Acute phase proteins
Haptoglobin concentrations were determined by a sandwich ELISA as described previously . The detection limit was 66 μg/ml.
A commercially available sandwich ELISA assay (Phase SAA assay, Tridelta Development Ltd., Kildare, Ireland) was used for determination of Serum Amyloid A (SAA) concentrations. This assay was based on anti-human monoclonal antibodies in a sandwich set-up as originally described by McDonald et al.. The detection limit of the assay was 125 μg/ml (porcine SAA equivalents).
C – Reactive Protein (CRP) was analyzed by a sandwich type ELISA using dendrimer-coupled cytidine diphosphocholine (a CRP-binding ligand) in the coating layer as previously described  employing polyclonal rabbit anti-human antibodies with cross-reactivity towards porcine CRP followed by peroxidase-conjugated goat anti rabbit antibody for detection (both antibodies from DAKO, Glostrup, Denmark). The cross-reactivity of the anti human CRP antibody with porcine CRP was demonstrated previously . The detection limit was 1416 ng/mL (human equivalents).
Antibodies were detected with the Hemagglutination Inhibition (HI) – test. Serum samples were pre-treated as follows: 100 μl of serum was inactivated at 56°C for 30 minutes. 400 μl of a 25% kaolin suspension in PBS was added and incubated for 20 minutes at room temperature and regularly shaken. The samples were centrifuged at 3000 rpm for 10 min, the supernatant mixed with 60 μl of packed chicken erythrocytes and incubated with the erythrocytes in a water bath at 37°C for 1 hour and regularly shaken. Finally, the samples were centrifuged at 3000 rpm for 10 min and the supernatant was used in the HI-test.
In the HI-test, 25 μl PBS was added to each well of a microtitre plate, then 25 μl of the sample was added and diluted two-fold. Then 25 μl of the antigen (4 HA units) were added to each well. The plate was shaken for 10 sec and incubated for 1 hour at room temperature. After incubation, 25 μl of 0.6% chicken erythrocyte dilution was added to the wells, shaken for 10 sec and incubated for 30 minutes at room temperature. The HI-titre of the serum samples was determined as the highest dilution showing complete inhibition of agglutination.
Chicken erythrocytes were used as standard blood cells. During testing it was noted that the H1N2 isolate used for inoculation (A/swine/Denmark/10074/2004 (H1N2)) did not have the ability to agglutinate chicken erythrocytes. Instead guinea pig erythrocytes were used for this isolate. The procedure for the HI-test using guinea pig erythrocytes was similar except that a 1% suspension of erythrocytes in PBS with 0.005% gelatine was used.
RNA was purified from nasal swabs and lung tissue samples with RNeasy Minikit (Qiagen, GmbH, Germany). 200 μl nasal swab sample was lysed in 400 μl RLT-buffer containing 1% ß-mercaptoethanol while 30 mg of lung tissue were homogenized and lysed with 600 μl RLT-buffer containing ß-mercaptoethanol by bead-beating. After lysis, the RNA was purified according to the kit instructions. RNA was stored at -80°C until analysis. RNA was extracted from all nasal swabs and lung tissue samples and tested by a real time RT-PCR screenings assay. All positive samples were further tested in a quantitative real time RT-PCR assay.
Real time RT-PCR screening assay
A previously designed general influenza A virus RT-PCR assay with specific primers for a conserved region of the nucleoprotein (NP) gene described by Munch et al. was modified into a real time assay using SYBR green chemistry detection. This assay was used to screen the nasal swabs and tissue samples for influenza A virus specific RNA. The real time RT-PCR was performed in a Rotor-Gene 3000 machine (Corbett Research, Australia) in a total volume of 25 μl using the Qiagen OneStep RT-PCR kit (Qiagen, GmbH, Germany) with 2 μl of extracted RNA, 0.6 μM of each primer (Forward Primer MMU39 and Reverse Primer MMU19), and 6.25× SYBR green (Invitrogen SYBR® Green I Nucleic acid stain 10000×). The amplification temperature profile was 50°C for 30 min for reverse transcription followed by 95°C for 15 min and 45 cycles of 94°C for 20 s, 60°C for 20 s and 72°C for 30 s. This was immediately followed by a melting point analysis: 50°C for 60 s, ramping from 50°C to 99°C in increments of 1°C and holding for 15 s at each step. The fluorescence signal was measured at 72°C during each PCR cycle and at each temperature increment step during the melting analysis. All fluorescence measurements were analysed with the Rotor-Gene Software Version 6.0. with non template control (NTC) threshold set at 10% and the normalised fluorescence threshold limit set at 0.05 for cycle threshold (Ct values) determination.
Relative quantification by real time RT-PCR
For quantification of virus load in nasal swabs and in lung tissue a quantitative real time RT-PCR assay based on Primer Probe Energy Transfer technology (PriProET) targeting a conserved region of the matrix gene was used [30, 43]. The real time RT-PCR was performed in a Rotor-Gene 3000 machine (Corbett Research, Australia) with a total reaction volume of 25 μl using the RNA Ultrasense™ One-Step Quantitative RT-PCR System (Invitrogen), 5 μl of extracted RNA, 0.4 μM of forward primer, 1.0 μM of FAM-labelled reverse primer, and 1.0 μM of Cy5-labelled probe. The amplification temperature profile was 50°C for 30 min for reverse transcription followed by 95°C for 5 min and 45 cycles of 95°C for 15 s, 55°C for 30 s and 72°C for 20 s and finally by 72°C for 5 min. This was immediately followed by a melting point analysis: 95°C for 30 s, ramping from 40°C to 99°C in increments of 1°C and holding for 30 s at each step. The fluorescence signal was measured at 55°C during each PCR cycle and at each temperature increment step during the melting analysis. Signals were generated by excitation of the donor fluorophor (FAM) followed by collection of emission spectra from the acceptor fluorophor (Cy5). All fluorescence measurements were analysed with the Rotor-Gene Software Version 6.0. In each real time RT-PCR run, RNA purified from cell cultured A/swine/Denmark/19126/1993 (H1N1) with 103.8 TCID50 per ml was used as positive control and RNase free water as negative control. The NTC threshold was set at 10% and the normalised fluorescence threshold limit at 0.02 for cycle threshold (Ct) value determination. A standard curve for the real time RT-PCR assay was obtained using a ten-fold dilution series made from an initial concentration of 107.8 TCID50 per ml of the virus A/swine/Denmark/19126/1993 (H1N1). Ct-values from samples were calibrated towards the standard curve to obtain a relative quantification of TCID50 equivalents. The efficiency of the PCR was 92% and the range of quantification spanned 6 log10 dilutions from dilution 10-1 to 10-7.
To determine whether there was a statistical significant difference of nasal virus excretion, virus load in lungs, and acute phase proteins between the two groups of pigs an un-paired t-test with a 95% confidence interval was applied .