## 4 Discussion  
Here, data are presented showing that ZIKV derived from the human placenta can infect both the BeWo and HUVEC cell lines and replicate without causing excessive CPE. Other studies have documented CPE in ZIKV-infected placental cell lines, which could be due to experimental differences in the cell lines and virus strains [11; 61]. ZIKV has been known to infect placental tissue in humans as well as in animal models, and it can cross the placental barrier to infect a developing fetus to cause congenital syndromes [11; 28; 62; 63; 64]. The data in this study show that within 72 h p.i., ZIKV effectively crosses two monolayers, a basement membrane, and infects an EB. The detection of ZIKV in cells located on each side of the membrane as a model for the maternal-fetal axis supports reports of the isolation of ZIKV from placentas and fetuses [28; 50; 65; 66]. However, the permissiveness of the cells observed in this study may not reflect the cellular tropism of ZIKV in actual placentas [67; 68; 69; 70]. Of interest, the interactions of the YFV antisera varied depending on the cell line infected. The addition of the Late antiserum resulted in ZIKV neutralization in the co-culture model but did not neutralize when the EBs were directly infected. Further, the Early antiserum enhanced ZIKV infection when the EBs were directly infected but not in the co-culture model. When Vero cells were infected  
Figure 7: Immunofluorescentence of EBs (blue = DAPI, red = ZIKV-4G2). EBs were stained with D1-4G2-4-15 antibody and mounted on glass slides. Coverslips were pressed down on the EBs to flatten them, producing a ring effect in most images. Images were obtained under 4\(\times\) magnification. Scale bar represents 100 \(\upmu\)M.  
with virus/antiserum, the ANOVA results were not significant, which agrees with other research where YFV/ZIKV cross-reactivity was explored [71; 72; 73]. The Pre-Vacination serum was collected prior to vaccination with the 17D YFV vaccine, the Early serum was collected 30 days p.i., and the Late serum was collected at 30-day intervals between 120 and 420 days p.i. It has been shown that broadly neutralizing IgM antibodies quickly appear after vaccination with the 17D vaccine, typically 4-7 days p.i., and have been found to circulate anywhere between 2 and 11 years p.i. [74; 75; 76]. Other research has shown that the IgM response is donor-specific, with antibody maturation taking 6-9 months post-vaccination to complete [77]. [PERSON] et al. also showed that classical IgM waned rapidly over time, while swig + IgM were stable for at least a year post-vaccination [77]. It could be that the secondary antibody used in our studies did not detect swig + IgM. While IgM was not detected in any of the antisera, enhancement occurred when the EBs were directly infected with serum collected 30 days post-vaccination. Young IgG antibodies tend to exhibit increased cross-reactivity in relation to older IgG antibodies and could explain the enhancement we observed when the EBs were directly infected [78; 79]. It is possible that some of the phenotypic effects observed were due to serum factors that are not necessarily altered by vaccination. These differences are important to consider when analyzing why some sera in this study enhanced or neutralized while others did not. IgG antibodies have Fc regions and can readily cross the placenta due to the FcRn receptor on placental syncyotrophoblasts and endothelial cells and have already been found to be endocytosed by the BeWo cell line [80; 81; 82; 83]. By the time Late antiserum was collected, IgG antibodies would have been abundant, thus suggesting why ZIKV neutralization was observed in the co-cultured EBs while enhancement was observed in the Vero cells and directly infected EBs. Anti-DENV antibodies have been shown to enhance ZIKV infection in multiple model systems and case studies [84; 85; 83; 9; 9; 11].