title: Detection and sequencing of Zika virus from amniotic fluid of fetuses with microcephaly in Brazil: a case study.
abstract: 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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
  
  
  BACKGROUND
  Consellho Nacional de Desenvolvimento e Pesquisa (CNPq), Fundação de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ).
authors.affiliation: Instituto Nacional de Infectologia Evandro Chagas, Laboratório de Pesquisa Clínica em Doenças Febris Agudas, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil.
authors.forename: Guilherme
authors.initials: G
authors.lastname: Calvet
authors.affiliation: Departamento de Genética, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Renato S
authors.initials: RS
authors.lastname: Aguiar
authors.affiliation: Instituto de Pesquisa Professor Joaquim Amorim Neto (IPESQ), Campina Grande, Brazil.
authors.forename: Adriana S O
authors.initials: ASO
authors.lastname: Melo
authors.affiliation: Laboratório de Flavivírus, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil.
authors.forename: Simone A
authors.initials: SA
authors.lastname: Sampaio
authors.affiliation: Instituto Nacional de Controle e Qualidade, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil.
authors.forename: Ivano
authors.initials: I
authors.lastname: de Filippis
authors.affiliation: Laboratório de Flavivírus, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil.
authors.forename: Allison
authors.initials: A
authors.lastname: Fabri
authors.affiliation: Laboratório de Flavivírus, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil.
authors.forename: Eliane S M
authors.initials: ESM
authors.lastname: Araujo
authors.affiliation: Laboratório de Flavivírus, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil.
authors.forename: Patricia C
authors.initials: PC
authors.lastname: de Sequeira
authors.affiliation: Laboratório de Flavivírus, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil.
authors.forename: Marcos C L
authors.initials: MCL
authors.lastname: de Mendonça
authors.affiliation: Departamento de Genética, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Louisi
authors.initials: L
authors.lastname: de Oliveira
authors.affiliation: Instituto de Biologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Diogo A
authors.initials: DA
authors.lastname: Tschoeke
authors.affiliation: Departamento de Genética, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Carlos G
authors.initials: CG
authors.lastname: Schrago
authors.affiliation: Instituto de Biologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil; Laboratório de Sistemas Avançados de Gestão de Produção-SAGE-COPPE, Centro de Gestão Tecnológica-CT2, UFRJ, Rio de Janeiro, Brazil.
authors.forename: Fabiano L
authors.initials: FL
authors.lastname: Thompson
authors.affiliation: Instituto Nacional de Infectologia Evandro Chagas, Laboratório de Pesquisa Clínica em Doenças Febris Agudas, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil.
authors.forename: Patricia
authors.initials: P
authors.lastname: Brasil
authors.affiliation: Laboratório de Flavivírus, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil.
authors.forename: Flavia B
authors.initials: FB
authors.lastname: Dos Santos
authors.affiliation: Laboratório de Flavivírus, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil.
authors.forename: Rita M R
authors.initials: RMR
authors.lastname: Nogueira
authors.affiliation: Departamento de Genética, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Amilcar
authors.initials: A
authors.lastname: Tanuri
authors.affiliation: Laboratório de Flavivírus, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil. Electronic address: abispo@ioc.fiocruz.br.
authors.forename: Ana M B
authors.initials: AMB
authors.lastname: de Filippis
mesh_terms: D000653:Amniotic Fluid / Q000821:virology*; D001938:Brazil / Q000453:epidemiology; D004196:Disease Outbreaks; D005260:Female; D016679:Genome, Viral / Q000235:genetics; D005865:Gestational Age; D006801:Humans; D007231:Infant, Newborn; D008831:Microcephaly / Q000453:epidemiology* / Q000235:genetics; D010802:Phylogeny; D010920:Placenta / Q000821:virology; D011247:Pregnancy; D011251:Pregnancy Complications, Infectious / Q000821:virology; D012367:RNA, Viral / Q000302:isolation & purification; D000071244:Zika Virus / Q000302:isolation & purification*; D000071243:Zika Virus Infection / Q000175:diagnosis* / Q000821:virology
chemical_list: D012367:RNA, Viral