Heterogeneous pathological outcomes after experimental pH1N1 influenza infection in ferrets correlate with viral replication and host immune responses in the lung

Vidaña, Beatriz; Martínez, Jorge; Martínez-Orellana, Pamela; García Migura, Lourdes; Montoya, María; Martorell, Jaime; Majó, Natàlia

For detection of influenza A virus IAV antigen by immunohistochemistry (IHC), the tissues were stained with a primary antibody against the influenza A nucleoprotein (NP) as previously described [26] . Briefly, an antigen retrieval step was performed using protease XIV (Sigma-Aldrich, UDA) for 10 min at 37°C and then blocked for 1 h with 2% bovine serum albumin (85040C, Sigma-Aldrich Química, S.A., Spain) at room temperature (RT). Samples were then incubated with a commercially available mouse-derived monoclonal antibody (ATCC, HB-65, H16L-10-4R5) concentration (343 mg/mL) as primary antibody at a dilution of 1:100 at 4°C overnight followed by 1 h incubation with biotinylated goat antimouse IgG secondary antibody (Dako, immunoglobulins As, Denmark). Finally, an avidin-biotin-peroxidase complex (ABC) system (thermo Fisher Scientific, Rockjord, IL, USA) was used and the antigen -antibody reaction was visualized with 3.3′-diaminobenzidine tetrahydrochloride (DAB) such as chromogen. Sections were counterstained with Mayer's Haematoxylin. Positive control consisted of formalin-fixed paraffin-embedded heart of chicken experimentally infected with influenza. The same sections in which the specific primary antibodies were substituted with PBS were used as negative controls. Semiquantitative assessments of influenza virus antigen expression in the lungs were performed, and the results were correlated with the lesional patterns. The positive cells in 6 arbitrarily chosen 20× objective fields in alveolar areas and 5 arbitrarily chosen 20× objective fields in bronchial or bronchiolar areas were quantified separately in each lung lobe (cranial, medial and caudal) for every animal. The mean of the total cell counts per field across the three lobes was calculated for each animal. The viral loads in the lung tissues were assessed by quantitative Reverse Transcription quantitative Polymerase Chain Reaction (RT-qPCR) following a Taq-Man one-step RT-qPCR performed with Fast 7500 equipment (Applied Biosystems, Foster city, CA, USA) as described previously [27] . Viral RNA was extracted with QIAamp viral mini kit (Qiagen, Valencia, CA, USA) obtaining 60 μL of eluted viral RNA. The matrix gene (M) fragment amplification was carried out using the primers showed in Table 1 and One-Step RT-PCR Master Mix Reagents (Applied Biosystems) following the manufacturer's instructions using 5 μL of eluted RNA in a total volume of 25 μL. The amplification conditions were as follows: reverse transcription at 48°C for 30 min; initial denaturation reaction at 95°C for 15 min and 40 PCR-cycles of 95°C 15 s and 60°C 1 min. Standard curves and quantification were achieved by prior amplification of a 99 bp fragment of the M gene using One Step RT-PCR reagents (Qiagen) following manufacturer's instructions. The M gene fragment amplicon obtained was cloned into pGEMT vector (Promega, Madison, WI, USA) and transformed by heat shock in Escherichia coli competent cells (Invitrogen, Paisley, UK). The recombinant plasmid was purified using the QIA prep Spin kit (Qiagen) and spectrophotometrically quantified (Qubit, Invitrogen) as described previously [28] . Serial 10fold dilutions of both plasmids of known concentration were made and the standard curves were generated. The genome equivalent copies (GEC) of plasmid from the collected samples were determined based on these standard curves and by taking their volumes into account.