BACKGROUND: The incidence of microcephaly in Brazil in 2015 was 20 times higher 
than in previous years. Congenital microcephaly is associated with genetic 
factors and several causative agents. Epidemiological data suggest that 
microcephaly cases in Brazil might be associated with the introduction of Zika 
virus. We aimed to detect and sequence the Zika virus genome in amniotic fluid 
samples of two pregnant women in Brazil whose fetuses were diagnosed with 
microcephaly.
METHODS: In this case study, amniotic fluid samples from two pregnant women from 
the state of Paraíba in Brazil whose fetuses had been diagnosed with 
microcephaly were obtained, on the recommendation of the Brazilian health 
authorities, by ultrasound-guided transabdominal amniocentesis at 28 weeks' 
gestation. The women had presented at 18 weeks' and 10 weeks' gestation, 
respectively, with clinical manifestations that could have been symptoms of Zika 
virus infection, including fever, myalgia, and rash. After the amniotic fluid 
samples were centrifuged, DNA and RNA were extracted from the purified virus 
particles before the viral genome was identified by quantitative reverse 
transcription PCR and viral metagenomic next-generation sequencing. Phylogenetic 
reconstruction and investigation of recombination events were done by comparing 
the Brazilian Zika virus genome with sequences from other Zika strains and from 
flaviviruses that occur in similar regions in Brazil.
FINDINGS: We detected the Zika virus genome in the amniotic fluid of both 
pregnant women. The virus was not detected in their urine or serum. Tests for 
dengue virus, chikungunya virus, Toxoplasma gondii, rubella virus, 
cytomegalovirus, herpes simplex virus, HIV, Treponema pallidum, and parvovirus 
B19 were all negative. After sequencing of the complete genome of the Brazilian 
Zika virus isolated from patient 1, phylogenetic analyses showed that the virus 
shares 97-100% of its genomic identity with lineages isolated during an outbreak 
in French Polynesia in 2013, and that in both envelope and NS5 genomic regions, 
it clustered with sequences from North and South America, southeast Asia, and 
the Pacific. After assessing the possibility of recombination events between the 
Zika virus and other flaviviruses, we ruled out the hypothesis that the 
Brazilian Zika virus genome is a recombinant strain with other mosquito-borne 
flaviviruses.
INTERPRETATION: These findings strengthen the putative association between Zika 
virus and cases of microcephaly in neonates in Brazil. Moreover, our results 
suggest that the virus can cross the placental barrier. As a result, Zika virus 
should be considered as a potential infectious agent for human fetuses. 
Pathogenesis studies that confirm the tropism of Zika virus for neuronal cells 
are warranted.
FUNDING: Consellho Nacional de Desenvolvimento e Pesquisa (CNPq), Fundação de 
Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ).