Zika virus (ZIKV) is associated with brain development abnormalities such as primary microcephaly, a severe reduction in brain growth. Here we demonstrated in vivo the impact of congenital ZIKV infection in blood vessel development, a crucial step in organogenesis. ZIKV was injected intravenously in the pregnant type 2 interferon (IFN)-deficient mouse at embryonic day (E) 12.5. The embryos were collected at E15.5 and postnatal day (P)2. Immunohistochemistry for cortical progenitors and neuronal markers at E15.5 showed the reduction of both populations as a result of ZIKV infection. Using confocal 3D imaging, we found that ZIKV infected brain sections displayed a reduction in the vasculature density and vessel branching compared to mocks at E15.5; altogether, cortical vessels presented a comparatively immature pattern in the infected tissue. These impaired vascular patterns were also apparent in the placenta and retina. Moreover, proteomic analysis has shown that angiogenesis proteins are deregulated in the infected brains compared to controls. At P2, the cortical size and brain weight were reduced in comparison to mock-infected animals. In sum, our results indicate that ZIKV impairs angiogenesis in addition to neurogenesis during development. The vasculature defects represent a limitation for general brain growth but also could regulate neurogenesis directly.

The northeast (NE) region of Brazil commonly goes through drought periods, which favor cyanobacterial blooms, capable of producing neurotoxins with implications for human and animal health. The most severe dry spell in the history of Brazil occurred between 2012 and 2016. Coincidently, the highest incidence of microcephaly associated with the Zika virus (ZIKV) outbreak took place in the NE region of Brazil during the same years. In this work, we tested the hypothesis that saxitoxin (STX), a neurotoxin produced in South America by the freshwater cyanobacteria Raphidiopsis raciborskii, could have contributed to the most severe Congenital Zika Syndrome (CZS) profile described worldwide. Quality surveillance showed higher cyanobacteria amounts and STX occurrence in human drinking water supplies of NE compared to other regions of Brazil. Experimentally, we described that STX doubled the quantity of ZIKV-induced neural cell death in progenitor areas of human brain organoids, while the chronic ingestion of water contaminated with STX before and during gestation caused brain abnormalities in offspring of ZIKV-infected immunocompetent C57BL/6J mice. Our data indicate that saxitoxin-producing cyanobacteria is overspread in water reservoirs of the NE and might have acted as a co-insult to ZIKV infection in Brazil. These results raise a public health concern regarding the consequences of arbovirus outbreaks happening in areas with droughts and/or frequent freshwater cyanobacterial blooms.

31The northeast (NE) region of Brazil commonly goes through drought periods, which 32 favor cyanobacterial blooms, capable of producing neurotoxins with implications for human 33 and animal health. The most severe dry spell in the history of Brazil occurred between 2012 34 and 2016. Coincidently, the highest incidence of microcephaly associated with the Zika virus 35 (ZIKV) outbreak was described in the NE region of Brazil during the same years. In this 36 work, we tested the hypothesis that saxitoxin (STX), a neurotoxin produced in South America 37 by the freshwater cyanobacteria Raphidiopsis raciborskii, could have contributed to the most 38 severe Congenital Zika Syndrome (CZS) profile described worldwide. Quality surveillance 39 showed higher cyanobacteria amounts and STX occurrence in human drinking water supplies 40 of NE compared to other regions of Brazil. Experimentally, we described that STX doubled 41 the amount of ZIKV-induced neural cell death in progenitor areas of human brain organoids, 42 while the chronic ingestion of water contaminated with STX before and during gestation 43 caused brain abnormalities in offspring of ZIKV-infected immunocompetent C57BL/6J mice. 44Our data indicate that saxitoxin-producing cyanobacteria is overspread in water reservoirs of 45 the NE and might have acted as a co-insult to ZIKV infection in Brazil. These results raise a 46 public health concern regarding the consequences of arbovirus outbreaks happening in areas 47 with droughts and/or frequent freshwater cyanobacterial blooms. 48Author summary 49 The uncontrolled spreading of cyanobacteria in drinking water reservoirs has been the 50 cause of serious public health problems worldwide. Toxin-producing cyanobacterial blooms 51 commonly occur during drought periods in the northeast (NE) region of Brazil. During Zika 52Virus (ZIKV) outbreak in 2015-16, Brazilian NE showed disproportionately higher 53 microcephaly incidence. Here, we test the hypothesis that the cyanotoxin saxitoxin (STX) 54 3 may act as a co-insult for ZIKV. Water quality surveillance data showed increased 55 cyanobacteria population and higher STX amount in NE region during 2014-2018 we observed that neural progenitor cell death was doubled after STX exposure to ZIKV-57 infected brain organoids. In vivo, chronic ingestion of STX during gestational period 58 potentiated ZIKV-derived brain abnormalities in newborn mice. Our study provides new 59 insights that may explain the discrepancies among Brazilian regions regarding CZS severity. 60Moreover, the data highlight the importance of cyanobacteria and cyanotoxin freshwater 61 monitoring for future arbovirus outbreaks. 62