Figure 1. Eye malformation and neural tube defect after influenza B virus infection in the chick embryo model. A, E, G, I, and K were from sham-infected control embryos. B, C, and D were from an infected embryo. F, H, J, and L were from another infected embryo. A-D were processed through in situ hybridization with a RNA probe containing influenza B virus hemagglutinin segment. The presence of viral RNA was indicated by blue or purple signals. E-H were processed through in situ apoptosis assay on transverse tissue sections, with apoptotic cells indicated by green fluorescence signals. J-L were also processed through in situ apoptosis assay on transverse tissue sections, but without adding terminal deoxynucleotidyl transferase. A: A sham-infected control embryo. B: Dorsal view of an infected embryo exhibited abnormal optic vesicle development (indicated by arrows and dotted line), as compared to the control embryo showing normal optic vesicle development in A (indicated by arrows and dotted line). The viral RNA was detected in the head neuroepithelium, optic vesicle, periocular mesenchyme, and in the heart. C: Ventral view of the same embryo in B, showing evident presence of viral RNA in the head neuroepithelium. D: Coronal section of the embryo in B, showing the presence of viral RNA in the neural tube (indicated by arrowhead) and the forming ventricles of the developing brain enclosed by twisted neuroepithelium (indicated by arrows). E and G: Midbrain and hindbrain sections of a sham-infected control embryo showing a few apoptotic cells normally observed during early chick embryogenesis. F and H: Extensive apoptotic cells in the midbrain and hindbrain sections of a virus-infected embryo. I and K: Negative controls for TUNEL assay on sham-infected embryos corresponding to E and G and showing no background signals. J and L: Negative controls for TUNEL assay on virus-infected embryos corresponding to F and H and showing no background signals. Abbreviations: fb, forebrain; hb, hindbrain; ht, heart; mb, midbrain; mes, mesenchyme; nep, neuroepithelium; opv, optic vesicle. Scale bar: 50 µm.