C57BL/6 nulliparous female 6-week-old mice were obtained from the Animal Care Facility of the Microbiology Institute of the Federal University of Rio de Janeiro. They were divided into two groups according to the consumed diet. Control females were fed a standard diet with the recommended amount of macro- and micronutrients (TD91352, Harlan Teklad, Madison). In the control diet, protein content was 20.3%. In contrast, the LP group received a diet with 6.1% of protein in the form of casein and dl-methionine. These diets are isocaloric (3.8 kcal/g), having the same percentage of fat (5.5%) and a different proportion of carbohydrates (control, 61.6%; LP, 75.6%).

Administration of both diets started 7 to 10 days before mating and continued until the harvesting dates. Animals had ad libitum access to water and food. Throughout the whole experiment, we assessed food intake daily. No significant differences between groups were found regarding the amount of consumed food. Females were housed in single cages throughout the whole experiment. Pregnancy was confirmed through observation of the postcoital vaginal plug to have an accurate estimation of embryonic age.

On E12, we performed intraperitoneal injections in control and LP dams of ZIKV (infected) or Mock (noninfected). Dams were injected with 106 PFU of a Brazilian ZIKV strain obtained from a patient presenting typical symptoms of ZIKV infection (Recife/Brazil, ZIKV PE/243, accession no. KX197192.1) (41). For experimental comparisons with noninfected animals, we injected control and LP dams with the supernatant of A. albopictus C6/36 cells (Mock). The viral titer of the stock was 3.4 × 106 PFU/ml, and the injected volume was 295 μl. These treatments yielded four experimental groups: control/Mock (control diet, noninfected), control/ZIKV (control diet, ZIKV infected), LP/Mock (LP, noninfected), and LP/ZIKV (LP/ZIKV infected). Harvesting of samples was carried out during embryonic life in E15 and on the first day of postnatal life (P0). In the case of E15 sample harvesting, dams were culled using cervical dislocation. Neonates at P0 were decapitated. Embryos and neonates were weighed on a standard laboratory scale with a definition of 1 mg (OHAUS, USA) before decapitation.

Weight in E15 embryos was measured in specimens of each litter giving a total sample of n = 33 (Co/Mock), n = 33 (Co/ZIKV), n = 27 (LP/Mock), and n = 50 (LP/ZIKV). For Co/Mock, five independent litters in E15 were used (number of embryos for each litter = 3, 7, 7, 8, and 8), while for the Co/ZIKV embryos, weights derived from six independent litters (number of embryos for each litter = 2, 3, 5, 6, 8, and 9) were measured. For LP/Mock, four independent litters were used (number of embryos for each litter = 5, 7, 7, and 8), and last, in LP/ZIKV, embryos from seven litters were weighed (number of embryos for each litter = 5, 6, 6, 6, 8, 9, and 10). In P0, we reported body weights from those specimens that were CT-scanned to obtain brain size (Co/Mock, n = 9 neonates from three independent litters; Co/ZIKV, n = 6 neonates from two independent litters; LP/Mock, n = 6 neonates from three independent litters; and LP/ZIKV, n = 20 from four independent litters).

ZIKV was propagated in C6/36 cells. Briefly, C6/36 cells were infected at a multiplicity of infection of 0.01 and incubated at 37°C. After 1 hour, the inoculum was removed and replaced with growth media supplemented with 2% fetal bovine serum. After 6 days, the conditioned medium was harvested, centrifuged at 300g, and filtered through 0.22 μm to remove cells and cellular debris. Supernatants were collected and stored at −80°C. Virus titers were determined by plaque assay performed on Vero cells.

All animals used in this study were housed in the Animal Care Facility of the Microbiology Institute of the Federal University of Rio de Janeiro. All procedures were approved by institutional ethical committee, protocol CEUA-CCS/UFRJ 01200.001568/2013-87.

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