Turkey red blood cells

Based on the Hallym University IACUC permission, Hallym 2012-95, 108 nasopharyngeal samples were collected from pigs between December 2011 and May 2012 in Gyeongsangnam-do, a southeastern province of Korea. Carried in universal viral transport media (BD, Franklin Lakes, NJ), samples were prepared for the virus isolation. The prepared samples were inoculated into the Madin-Darby canine kidney (MDCK) cell monolayer for 1 h at 37°C. The cells were washed and maintained with growth media for 48–72 h. Viral growth was determined by the morphological changes (cytopathic effects) in the MDCK cells and by a hemagglutination assay of cell supernatants using 0.5% turkey red blood cells (Rockland Immunochemicals Inc.; Gilbertville, PA). The double-positive sample was purified by a standard plaque assay in the MDCK cells and prepared after propagation in fertile chicken eggs.

We diluted amounts of virus equivalent to 64 hemagglutinating units in minimal essential medium supplemented with 0.3% bovine serum albumin and heat treated the diluted virus samples at 55°C for the indicated times. We then determined the hemagglutination activity of the heat-treated viruses by using HA assays with 0.5% turkey red blood cells (Rockland Immunochemicals Inc., Limerick, PA, USA). Virus infectivity was determined by performing plaque assays in MDCK cells.

HAI titres were used to determine vaccine-specific antibody titres based on a standard WHO protocol, as previously described^{57 (link)}. Briefly, plasma samples were treated with receptor-destroying enzyme (RDE) (Denka Seiken, Tokyo, Japan) by adding one-part plasma to three-parts RDE and incubating at 37 °C overnight. The next day, RDE was inactivated by incubating the samples at 56 °C for 1 h. Then, the samples were serially diluted with phosphate-buffered saline (PBS) in 96-well V-bottom plates (Nalge Nunc International Corporation, Rochester, NY, USA), and 4 HA-units each of H1N1 (California strain), H3N2 (Switzerland strain), or influenza-B virus (Phuket strain) was added to each well. After 30 min at room temperature, 50 µl of 0.5% turkey red blood cells (Rockland Immunochemicals, Philadelphia, PA, USA) suspended in PBS with 0.5% bovine serum albumin (BSA) was added to each well, and the plates were manually agitated. After an additional 30 min at room temperature, the plasma titres were read. Negative and positive control plasma samples for each virus were used for reference. “Sero-protection” was defined as an HAI titre >1:40, and a “sero-response” was defined as a minimum four-fold increase in antibody titre, 30 days post-vaccination³³ .

The HAI assay was performed with A/Shanghai/2/2013 (H7N9)-PR8-IDCDC-RG32A. For HAI, 25 μL of 4 HA units of virus were incubated for 1 hour at room temperature with 25 μL 2-fold serial dilutions of antibodies starting at 10 μg/mL in PBS. The 50 μL of antibody-virus mixture was incubated for 45 minutes at 4°C with 50 μL of turkey red blood cells (Rockland Immunochemicals) diluted in PBS. The IC₁₀₀ value was defined as the lowest antibody concentration that inhibited hemagglutination of red blood cells.