Figure 4.

Infectious cell-associated ZIKV is excreted in murine breast milk. (A): Female dams were MOCK or ZIKV-infected with the Brazil/2016 strain as described in Figure 1. Breast milk samples were collected at 4 days post-infection and cell pellets were purified by three spin-wash cycles in PBS to eliminate free viral particles. (B): The viral titers in the same breast milk samples were determined by plaque assay on Vero cells. (C): For each sample (n = 6), duplicates of one million cells were serially diluted and co-cultured with Vero cells for 2 h at 37 °C. The cells were then co-cultured in medium containing carboxymethylcellulose for 6 days at 37 °C. After three PBS washes and PFA 4% fixation, the cell layers were stained with crystal violet and plaques were counted manually. * and **: Significantly different (B: p = 0.0015; C: p = 0.023) by Mann–Whitney test (p < 0.05).