Fig. 9
Pathogenicity and immunogenicity of m6A-mutated rgRSVs in cotton rats. a RSV titer in lungs. Four-week-old SPF cotton rats were inoculated intranasally with 2.0 × 105 TCID50 of each rgRSV. At day 4 post-infection, the cotton rats were killed, and lungs and nasal turbinates were collected for virus titration by TCID50 assay. Viral titers are the geometric mean titer (GMT) of 5 animals ± standard deviation. Detection limit is 2.0 log TCID50/g tissue. The P value (Student’s t-test) for rgRSV-G1, rgRSV-G3, and rgRSV-G12 is ****P = 9.99 × 10−6, ****P = 4.06 × 10−5, and ****P = 7.89 × 10−7 respectively. b RSV titer in nasal turbinates. The P value (Student’s t-test) for rgRSV-G1, rgRSV-G2, rgRSV-G3, and rgRSV-G12 is ***P = 0.000740, *P = 0.0460, **P = 0.00623, and ****P = 2.88 × 10−7 respectively. c m6A mutated rgRSVs had less lung histopathological changes compared to rgRSV. Representative pathological changes from each group are shown. Right lung lobe of each cotton rat was fixed in 4% neutral buffered formaldehyde, embedded in paraffin, sectioned at 5 µm, and stained with hematoxylin-eosin (HE) for the examination of histological changes by light microscopy. Micrographs with ×20 magnification (scale bar of 500 μm) are shown. d m6A mutated rgRSV provides complete protection against RSV challenge. Four-week-old SPF cotton rats were inoculated intranasally with 2.0 × 105 TCID50 of each rgRSV. At week 4 post-immunization, cotton rats were challenged with 2.0 × 105 TCID50 rgRSV. At day 4 post-challenge, the cotton rats were killed, and lungs and nasal turbinates were collected for virus titration by TCID50 assay. Viral titers are the geometric mean titer (GMT) of 5 animals ± standard deviation. The detection limit is 2.0 log TCID50/g tissue. e rgRSV induced a high level of neutralizing antibody. Blood samples were collected from each rat weekly by retro-orbital bleeding. The RSV-neutralizing antibody titer was determined using a plaque reduction neutralization assay, as described in Materials and Methods. The P value (Student’s t-test) for rgRSV-G1 at week 3 and 4 is *P = 0.0166 and *P = 0.0490, respectively
