Minimum essential medium (mem)

Influenza virus A/Puerto Rico/8/1934 (PR8) (H1N1) was propagated in 10-day-old embryonated chicken eggs at 35 °C for 48 h, and the collected allantoic fluid was stored at −80 °C until use. Madin–Darby canine kidney (MDCK) cells were maintained in minimum essential medium (MEM) (Nissui Pharmaceutical, Tokyo, Japan) supplemented with 10% nonimmobilized fetal calf serum (FCS; SAFC Biosciences, Lenexa, KS, USA), 0.3 mg/mL L-glutamine (Wako Chemicals, Tokyo, Japan), 100 U/mL penicillin G (Meiji Seika Pharma, Tokyo, Japan), 0.1 mg/mL streptomycin (Meiji Seika Pharma, Tokyo, Japan), and 8 μg/mL gentamicin (Takata Pharmaceutical, Saitama, Japan) in an incubator at 37 °C with 5% CO₂.

MO/15 was kindly provided by Dr. Alicia Janas-Martindale (United States Department of Agriculture, Raleigh, NC, USA). A/duck/Czechoslovakia/1956 (H4N6) (Dk/Cz), A/budgerigar/Hokkaido/1/1977 (H4N6) (Budge/Hok), A/duck/Hokkaido/491003/2014 (H4N2) (Dk/Hok/491003), A/duck/Mongolia/769/2015 (H4N6) (Dk/Mon), A/duck/Hokkaido/138/2007 (H4N6) (Dk/Hok/138), A/swan/Hokkaido/481102/2017 (H4N6) (Swan/Hok), and A/mallard/Alberta/223/1979 (H4N2) (Mal/Alb) were isolated from birds. All viruses used in this study were propagated in 10-day-old embryonated chicken eggs at 35 °C for 48 h, and the allantoic fluid was collected and stored at −80 °C until use. Madin–Darby canine kidney (MDCK) cells were maintained in minimum essential medium (MEM; Nissui Pharmaceutical, Tokyo, Japan) supplemented with 10% non-immobilized fetal calf serum (FCS; Sigma-Aldrich, St. Louis, MO, USA), 0.3 mg/mL L-glutamine (Nacalai Tesque, Kyoto, Japan), 100 U/mL penicillin G (Meiji Seika Pharma, Tokyo, Japan), 0.1 mg/mL streptomycin (Meiji Seika Pharma, Tokyo, Japan), and 8 μg/mL gentamicin (Takata Pharmaceutical, Saitama, Japan) in an incubator at 37 °C with 5% CO₂.

An MDCK cell line (obtained from Dr Nakajima, Nagoya City University School of Medicine, Nagoya, Japan) was grown in Eagle’s minimum essential medium (MEM; Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) containing 5% fetal bovine serum. Influenza virus A/PR/8/34 (H1N1) (obtained from Dr Nakajima) was used throughout the experiments. The viruses were propagated in MDCK cells cultured in MEM supplemented with 0.1% bovine serum albumin (BSA) and acetylated trypsin (4 μg/ml). The viruses were stored at −80°C until required for further use. The amount of infectious virus was measured using a plaque assay on the MDCK cells, as described previously (13 (link)).

Colon homogenates that tested positive for the PRV genome in RT-PCR were inoculated onto Vero E6 cell cultures, followed by 1 h incubation at 37°C in 5% CO₂ for virus adsorption. After removal of the inocula, the cells were washed twice with PBS and maintained in Eagle’s minimum essential medium (MEM; Nissui Pharmaceutical Co., Tokyo, Japan) containing 5 μg/mL trypsin, 0.3% bovine serum albumin, 2 mM L-glutamine, 4% antibiotic–antimycotic solution (Anti-Anti; Gibco, Waltham, MA, USA), and 1.0 mg/mL NaHCO₃ at 37°C in 5% CO₂. When the cells displayed a cytopathic effect (CPE), the culture medium was harvested, centrifuged at 1,750 × g for 5 min, and stored at -80°C.

Pulmonary viral titer of infected mouse was determined by focus-forming assay. Briefly, a confluent monolayer culture of MDCK cells in a 12-well tissue culture plate containing cover slips (4.0 × 10⁵ cells/well) was washed with serum-free Minimum essential medium (MEM; Nissui) and then was infected with homogenized lung supernatants containing viruses in serum free MEM. After incubating for 1 h at 37°C for virus adsorption, the cells were washed with serum-free MEM and then incubated for 4 h in DMEM containing 10% bovine fetal calf serum. Cells were fixed with 4% PFA for 10 min and were subjected to indirect immunofluorescence assay using anti-NP antibody as described previously described (Kawaguchi et al., 2011 (link)). The focus forming units (FFU) was determined by counting cells labeled with the fluorescent antibody.

The swine kidney cell line SK-L [15 (link)] was propagated in Eagle’s minimum essential medium (MEM) (Nissui Pharmaceutical, Tokyo, Japan) supplemented with 0.295% tryptose phosphate broth (Becton Dickinson, San Jose, CA, USA), 10 mM N,N-bis-(2-hydroxyethyl)-2-aminoethanesulfonic acid (Sigma-Aldrich, St. Louis, MO, USA) and 10% horse serum (Life Technologies, Carlsbad, CA, USA). The SK6-MxLuc cell line carrying a Mx/Luc reporter gene [16 (link)] was propagated in MEM supplemented with 0.295% tryptose phosphate broth and 7% horse serum. The human embryonic kidney cell line 293T was maintained in Dulbecco’s MEM (Life Technologies) and 10% fetal calf serum (Cambrex, Grand Island, NY, USA). All cells were incubated at 37 °C in the presence of 5% CO₂.