Zika virus induces abnormal cranial osteogenesis by negatively affecting cranial neural crest development

Zika virus (ZIKV) infection during gestation is deemed to be coupled to birth defects through direct impairment of the nervous system during neurogenesis. However, in this study, our data showed that ZIKV infection dramatically suppressed cranial osteogenesis, shown by Safranin O/Fast Green and alizarin red staining, in chick embryos, which provides another possibility that craniofacial bone malformation caused by ZIKV may be a major cause of ZIKV-mediated birth defects. By immunofluorescent staining and electron microcopy, we confirmed ZIKV infection in chick embryo neural tubes and sites of neural crest. Next, in vivo (chick embryos) and in vitro [primary culture of neural crest cells (NCC)] ZIKV and HNK-1 double immunofluorescent staining demonstrated that ZIKV infection inhibited the production of migratory NCC. The reduction of both AP-2α- and Pax7-positive NCC in HH10 chick embryos infected by ZIKV confirmed that abnormal development of cranial NCC also occurred in the migratory process. Whole mount in situ hybridization demonstrated that cadherin 6B expression was elevated and Slug, FoxD3, and BMP4/Msx1 expressions decreased in ZIKV-infected HH10 chick embryos, implying that epithelial-mesenchymal transition (EMT) of neural crest production was blocked by ZIKV infection. Moreover, in vivo and in vitro pHIS3 and Pax7 double immunofluorescent staining showed that NCC proliferation was repressed by ZIKV infection. C-caspase-3 and AP-2α double immunofluorescent staining in HH10 chick embryos and western blotting showed that NCC apoptosis increased following ZIKV infection. Finally, electron microscopy showed multiple autophagosomes in ZIKV-infected embryos, and western blot and LC3B immunofluorescent staining demonstrated that autophagy-related genes were activated by ZIKV infection. Taken together, our data first showed that ZIKV infection during embryogenesis could interfere with cranial neural crest development, which in turn causes aberrant cranial osteogenesis. Our results provided new insights into brain malformations induced by ZIKV infection.