Cells were washed twice with PBS-HEPES between all blocking and staining steps and resuspended in PBS with 1% formalin prior to analysis. selleck compound To confirm the specificity of lectins, cells were pretreated with 1 U per ml of neuraminidase (NA) from Clostridium perfringens (Sigma) for 1 h prior to the avidin/biotin block. Samples were analyzed on a BD Facscalibur or BD LSR II (BD Biosciences), and a minimum of 5,000 events were recorded. Viruses and viral titration. Stocks of A/New Caledonia/20/99 (H1N1) and A/Wisconsin/67/05 (H3N2) viruses were kindly provided to San Raffaele Hospital (HSR) by Nadia Naffakh, Pasteur Institute, CNR Virus Influenza (Paris, France), and were serially expanded 2 times in MDCK cells prior to use.

To propagate IAV, monolayer-cultured MDCK cells were washed twice with PBS and infected with A/NewCaledonia/20/99 (H1N1) or A/Wisconsin/67/05 (H3N2) at a multiplicity of infection (MOI) of 0.001. After virus adsorption for 1 h at 35��C, the cells were washed twice and incubated at 35��C with Dulbecco’s modified Eagle’s medium (DMEM) without serum supplemented with tosylsulfonyl phenylalanyl chloromethyl ketone (TPCK)-treated trypsin (1 ��g/ml; Worthington Biomedical Corporation, Lakewood, NJ). Supernatants were recovered 48 h postinfection. LPAI H7N1 A/turkey/Italy/3675/1999 and H7N3 A/turkey/Italy/2962/2003 viruses isolated during epidemics in Italy were grown in 9-day-old embryonated SPF chicken eggs as described above. The sequences for A/New Caledonia/20/99 (H1N1), A/Wisconsin/67/05 (H3N2), and A/turkey/Italy/3675/1999 (H7N1) isolates were previously published.

For viral titration, plaque assays were performed as previously described (28). Briefly, MDCK monolayer cells, plated in 24-well plates at 2.5 �� 105 cells/well, were washed twice with DMEM without serum, and serial dilutions of virus were adsorbed onto cells for 1 h. The cells were covered with 2�� minimal essential Brefeldin_A medium (MEM)-Avicel (FMC Biopolymer, Philadelphia, PA) mixture supplemented with TPCK-treated trypsin (1 ��g/ml). Crystal violet staining was performed 48 h postinfection, and visible plaques were counted. Virus replication kinetics in pancreatic-cell lines. Semiconfluent monolayers of HPDE6 and hCM cells seeded on 24-well plates were washed twice with PBS and then infected with human viruses (H1N1 and H3N2) at an MOI of 0.001 and with avian viruses (H7N1 and H7N3) at an M.O.I of 0.01 using 200 ��l of inoculum per well. The inoculum was removed after 1 h of absorption and replaced with 1 ml of serum-free medium containing 0.05 ��g/ml TPCK-trypsin (Sigma).