2.6. Immunization and RSV Challenge in Cotton Rats

Six-week-old inbred female cotton rats (Sigmodon hispidus) were used. Three cotton rats for each group were immunized with 1 × 10⁵ TCID₅₀ of MVAIK and MV/RSV intramuscularly (i.m.) or intranasally (i.n.). Serum samples were obtained immediately before and 1, 3, 5, 8, 9, 12, and 15 weeks after immunization. The immunization experiment was performed in duplicate. They were re-immunized at 8 weeks after the first dose and were challenged with RSV four weeks after reimmunization (12 weeks after the first dose). Briefly, cotton rats were anesthetized and challenged with 10⁶ PFU/0.5 mL of RSV/Long through intranasal administration. They were sacrificed four days after the challenge and lung tissues were obtained. Lung samples were divided into two portions, one for pathological examination and another for recovering the infectious viral particles. Experimental protocol was approved by the Committee of Experimental Animal Study of the Kitasato Institute for Life Sciences (No. 16-026, 17-027, and 18-030).

Tissues were homogenized and 0.1 mL volume of serial 10-fold dilutions of homogenized samples were placed on HEp-2 cells and were then overlaid with MEM 5% FBS and 0.5% agar. Plaque numbers were counted after incubation for six days at 37 °C and infectivity was expressed as the number of plaques adjusted to 50 mg of lung tissue.

Lungs were inflated with 4% formalin and submerged in formalin for overnight fixation. The fixed tissue was embedded in paraffin, sectioned, and stained with hematoxylin–eosin. Microscopic figures were taken at more than 6 different sites in each group and were evaluated in a blind manner.