Balb c mice

Six-week-old female BALB/c mice (Samtako, Seoul, Korea) were used for all infection experiments. Ten mice per group were inoculated intranasally with 10²–10⁷ EID₅₀/50 mL of MDk/W452(H5N8) and EM/W149(H5N1). Body weights and survival patterns were recorded daily for 14 days. For virological and pathological examinations, groups of mice were inoculated intranasally with 10⁵ EID₅₀/50 mL of virus, and six mice per group were euthanised at 1, 3, 5, 7 and 9 dpi to examine the growth kinetics of the virus in mouse lungs.

Six-week-old female BALB/c mice were purchased from Samtako (Osan, Korea). The experiments were performed in accordance with the guidelines for the care and use of laboratory animals under the approval of animal ethics committee at Seoul National University (SNU-130415–1). The mice, maintained under standard pathogen-free conditions, were provided with free access to food and water during the experiments. Mice (n = 5) were immunized subcutaneously with 10 μg of purified proteins three times at 2-week intervals. Complete Freund’s adjuvant (CFA) and incomplete Freund’s adjuvant (IFA) were mixed with antigens for the priming and boosting of animals. Sera were collected from the mice at 0, 14, 28 and 42 days after first immunization for the antibody detection and serum neutralization assay.
The induction of antigen specific serum IgG level was measured by ELISA. Plates were coated and blocked using the same method described in the indirect ELISA section. After blocking, 2 fold serial diluted mouse sera were added to the wells and incubated for 1 h at room temperature. HRP conjugated goat anti-mouse IgG secondary antibody diluted at 1:5000 was added and incubated for 1 h at room temperature. Other assay procedures are same as described in the indirect ELISA section.

BALB/c mice (male, 6 weeks) were obtained from Samtako (Osan, Korea), and mice were randomly assigned to four per cage (a 12 h light/dark cycle, 55% humidity, and 22 ± 2 °C). Each group (n = 14) was divided into 6 groups, and each consisted of a normal control (NC) group, a PM_2.5-exposed group (negative control group; PM_2.5), and WEE groups (50, 100, and 200 mg/kg of body weight, respectively; WEE 50, WEE 100, and WEE 200). The WEE was dissolved into drinking water and was orally fed using a stomach tube once a day for 12 weeks. WHO Air quality guidelines for fine particulate matter recommend expositions to PM_2.5 < 25 μg/m³ 24 h mean in human, and the annual PM_2.5 concentration in the most polluted cities in the world is around 170 μg/m³ [56 ,57 ]. The mice were exposed to PM_2.5 at a 500 μg/m³ concentration in the whole-body exposure chamber (5 h/day and 5 days/week for 12 weeks).