Zika Sequences Amniotic Fluid Microcephaly Fetuses - Paraiba Brazil - Lancet

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).

niman · February 18, 2016

Research in context

Evidence before this study

Many cases of microcephaly in newborn babies in Brazil have occurred in regions where infections of Zika virus and other arboviruses have also been detected. We searched PubMed with the search terms “Zika”, and “microcephaly” for articles published up to Feb 5, 2016. We found 11 articles that suggested a possible relation between Zika virus and microcephaly in neonates. A short case report by our group, reporting the ultrasound evidence of the two fetal microcephaly cases reported here, has been published previously. Our search found no other clear evidence that Zika virus could cross the placental barrier and infect the human fetus.

Added value of this study

This study presents the virological and genetic data implicating Zika virus in the two cases of fetal malformation that we described briefly in our previous case report. We used quantitative reverse transcription PCR and viral metagenomics technology applied to samples of amniotic fluid obtained from the two pregnant women carrying fetuses with microcephaly, and obtained sequences of the Zika virus genome. The study of these cases provides empirical evidence for the association between Zika virus infection during pregnancy and fetal microcephaly. Furthermore, we isolated the whole genome of Zika virus directly from the amniotic fluid of two pregnant women, and provided evidence to support previous findings indicating that the origin of the virus is French Polynesia.

Implications of all the available evidence

On the basis of our findings, Zika virus should be regarded as a possible causative agent in cases of microcephaly, especially during Zika virus outbreaks in endemic regions. Our work emphasises not only the importance of controlling the Aedes aegypti mosquito population while no vaccine or antiviral is available, but also the need for further studies to understand the mechanisms of immunopathogenicity that lead to congenital malformation due to Zika virus infection.

niman · February 18, 2016

Introduction

Since 2015, Brazil has been facing a public health emergency regarding the dramatic increase in the number of newborn babies with microcephaly. Epidemiological data indicate that up to Jan 6, 2016, 4783 cases were reported in 21 states in the North, Northeast, South, and Southeast Regions of Brazil.1 This incidence of microcephaly is 20 times higher than in previous years, reaching 99·7 per 100 000 livebirths, and including 76 deaths of neonates as of Jan 6, 2016.1

When diagnosed prenatally by ultrasound imaging, congenital microcephaly is a strong predictor of adverse neurological outcomes.2 As defined by WHO, microcephaly occurs whenever the occipital frontal circumference of the head of the newborn child or fetus is 2 standard deviations smaller than the mean for the same age and sex.3 A brain size that is significantly different to that in the normal range is an important risk factor for cognitive and motor delay.4 Microcephaly is associated with several causes, including genetic disorders (eg, autosomal recessive microcephaly, Aicardi-Goutières syndrome, chromosomal trisomy, Rett syndrome, and X-chromosomal microcephaly); drug and chemical intoxication of the pregnant mother (eg, use of alcohol, cocaine, or antiepileptic drugs, lead or mercury intoxication, or radiation); maternal malnutrition; and transplacental infections with viruses or bacteria.5 Maternal viral infections, including rubella, cytomegalovirus, herpes simplex, varicella zoster virus, HIV, and arboviruses such as chikungunya, have also been associated with microcephaly in neonates.5, 6

Epidemiological evidence suggests that Zika virus infection of pregnant women in Brazil might be associated with the increasing numbers of congenital microcephaly cases reported in the country. Several mosquito species have been found to be naturally infected with Zika virus, including Aedes africanus, Aedes luteocephalus, Aedes hensilli, Aedes polynesiensis, Aedes dalzielii, Aedes albopictus, Aedes apicoargenteus, and Aedes aegypti among others, but little is known about their vector competence.7, 8, 9, 10 A aegypti is the overwhelmingly predominant mosquito species found in Brazil, and is also associated with other arboviruses already reported in Brazil, such as the dengue and chikungunya viruses.

Zika virus was first isolated from human beings in Nigeria7 during studies undertaken in 1954. Further cases were reported in other African countries11 (Uganda, Tanzania, Egypt, Sierra Leone, Gabon, Nigeria, Côte d'Ivoire, Cameroon, Senegal, and Central African Republic), in Asian countries (India, Pakistan, Malaysia, Philippines, Thailand, Cambodia, Vietnam, and Indonesia), in several islands of the Pacific region since 2007 (Federated States of Micronesia, Cook Islands, French Polynesia, New Caledonia, Guam, Samoa, Vanuatu, and Solomon Islands), and since about early 2015 in the Americas (Chile, Colombia, El Salvador, Guatemala, Mexico, Paraguay, Suriname, Venezuela, Canada, and the USA).9, 10, 11, 12, 13, 14, 15 Outbreaks of Zika virus infection on Yap Island (in 2007) and in French Polynesia (2013–14), with further spread to New Caledonia, the Cook Islands, and Easter Island, have shown the propensity of this arbovirus species to spread outside its usual geographical range and to cause large outbreaks.

The first autochthonous cases of Zika virus in Brazil were confirmed in May, 2015.16 Since then, as of Jan 6, 2015, 21 states have confirmed virus circulation, with a higher prevalence in the Northeast Region.17 Reports of microcephaly incidence in Brazil geographically overlap with Zika virus reports; most of the mothers whose infants were diagnosed with microcephaly complained during their pregnancies of clinical manifestations, such as low-grade fever, headache, and cutaneous rashes, that might have been symptoms of Zika virus infection or infection with any other arbovirus species that is prevalent in the region.

In the face of this potential association between Zika virus infection and the increasing number of cases of microcephaly, the Brazilian Ministry of Health and WHO have recommended that pregnant women should take precautions to avoid contact with all potential vectors, since no specific antiviral treatment for Zika virus infection exists.1

Small fragments of the genome of the Zika virus strain circulating in Brazil have been sequenced and phylogenetic analysis has indicated that the virus is similar to the one that circulated in French Polynesia in 2013.16, 17 However, evidence linking the high incidence of microcephaly to the presence of Zika virus is scarce. In January, 2016, our group reported ultrasound image evidence of two cases of fetal microcephaly in women who had complained of Zika-like virus symptoms during pregnancy, and we reported brief preliminary PCR findings, confirming the presence of Zika virus in their amniotic fluid.18 In this case study, we expand upon these previously reported findings, and describe how we used quantitative reverse transcription PCR (RT-qPCR) and viral metagenomics to detect and sequence the Zika virus genome in the amniotic fluid samples of these two pregnant women with microcephalic fetuses.

niman · February 18, 2016

Methods

Case histories

The first case in our study was of a 27-year-old woman in her first pregnancy, from an inner city in the state of Paraíba, in the Northeast Region of Brazil (patient 1). Her prenatal care was uneventful until early September, 2015, when, at 18 weeks of gestation, the woman developed a cutaneous rash with itching of the hands and back. On the basis of her clinical status, she was diagnosed at an emergency service unit with allergic reaction, and was prescribed intravenous hydrocortisone. The next day, her symptoms worsened as she developed a fever and myalgia. She had a normal fetal ultrasound at 16 weeks. The patient had not travelled outside the state of Paraíba during the previous few years, and she had not had contact with any ill individuals. She had no immunodeficiency or autoimmune diseases. At 21 weeks of gestation, a further ultrasound indicated a fetal microcephaly diagnosis with moderate ventriculomegaly and partial agenesis of the cerebellar vermis. A third ultrasound done at 27 weeks confirmed the microcephaly diagnosis with relevant dilation of ventricles, asymmetry of hemispheres, and hypoplastic cerebellum with complete absence of the cerebellar vermis. The patient was healthy and stable during the ultrasound and amniocentesis procedures. Results of all laboratory examinations showed no diabetes and blood-pressure-related disorders. Additionally, the patient did not report taking any medication (other than hydrocortisone), recreational drug use, alcohol consumption, or smoking during the pregnancy. Patient 1 is still being monitored by the physicians in our group. At 40 weeks of gestation the fetus presented microcephaly with calcification areas and head circumference of 29 cm assessed by ultrasonography before birth. The baby was born at 40 weeks of gestation and had an actual head circumference of 30 cm.

The second case in our study was of a 35-year-old woman in her first pregnancy, also from the state of Paraíba (patient 2). The patient, with no relevant past medical history, sought care when she developed mild Zika virus disease-like symptoms at 10 weeks of gestation. She was prescribed symptomatic treatment. A morphological ultrasound at 22 weeks of gestation revealed mild hypoplasia of the cerebellar vermis. The fetal head circumference on the 22nd week of gestation was below the 10th percentile. A second ultrasound done at 25 weeks of gestation revealed more severe hypoplasia of the cerebellar vermis, enlargement of the posterior fossa, and microcephaly, yielding a head circumference below the third percentile. The brain parenchyma was normal. The patient was healthy and stable during the ultrasound and amniocentesis procedures. All the laboratory examinations showed no evidence of diabetes or blood-pressure-related disorders. Additionally, she did not report taking any medication, recreational drug use, alcohol consumption, or smoking during the pregnancy. Patient 2 is still being monitored by the physicians in our group. She delivered on Feb 3, 2016, and the neonate presented severe ventriculomegaly, microphthalmia, cataract, and severe arthrogryposis in the legs and arms.

Sample collection

Following Brazilian health public recommendations, amniocentesis was done at gestational week 28 in both women to investigate the cause of microcephaly. Ultrasound-guided transabdominal amniocentesis was done and about 5 mL of amniotic fluid was aspirated and immediately stored at −80°C.

Viral metagenomics and sequence analysis

A 0·5 mL sample of the amniotic fluid from each patient was filtered through 0·45 μm filters to remove residual host cells. The samples were then centrifuged at 21 130 × g and 15 000 rpm (rotor FA-45–24–11, Eppendorf, Hamburg, Germany) for 90 min at 4°C to concentrate virus particles. Pelleted virus particles were treated with deoxyribonuclease and ribonuclease A at 37°C for 90 min according to previously reported protocols.19 RNA was isolated using the QIAamp MinElute Virus Spin Kit (Qiagen, Hilden, Germany), omitting carrier RNA. Double-stranded cDNA libraries were prepared using the TruSeq Stranded Total RNA LT Sample Preparation Kit (Illumina, San Diego, CA, USA). Library size distribution was assessed using the 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA) and the High Sensitivity DNA Kit (Agilent). Accurate quantification of the libraries was accomplished with the 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) and the KAPA Library Quantification Kit (Kapa Biosystems, Wilmington, MA, USA). Paired-end sequencing (2 × 210 bp) was done using a MiSeq sequencing system (Illumina).

The sequences obtained were preprocessed using the PRINSEQ software to remove reads smaller than 35 bp and sequences with scores of lower quality than a Phred quality score of 20. Fast length adjustment of short reads (FLASH) software was used to merge and extend the paired-end Illumina reads using the default parameters, with a maximum overlap of 400 bp. The extended reads were analysed with basic local alignment search tool (BLAST), against the Human Transcriptome Database (RefSeq, Annotation Release 107; 162 916 sequences), with e-value cutoff of 1e-5, to remove human RNA sequences. Non-human reads were analysed against all GenBank viral genomes (65 052 sequences), and reads that were similar to the Zika virus were collected and used for genomic assembly. The Zika virus genome (Brazil strain) was assembled de novo using the CAP3 assembly software, using the parameters overlap length cutoff (-o) of 16, and overlap percent identity cutoff (-p) of 85. The Atlas genome was constructed using BRIG (BLAST Ring Image Generator) software. We used the Zika virus genome sequence H/PF/2013 (KJ776791.1) as the reference. This strain was isolated in French Polynesia, and we compared it with a strain from Uganda, MR 766 (accession: NC_012532.1), another strain isolated in Senegal, ArD157995 (accession: KF383118), and our assembled Zika virus genome.

Phylogenetic analysis

Phylogenetic reconstruction was completed using both maximum likelihood and Bayesian inference methods on alignments of the envelope and NS5 regions of the polyprotein coding sequence. The best choice of substitution model used in the maximum likelihood and Bayesian inference analyses was determined with the likelihood-ratio test, implemented using HyPhy software. The generalised time-reversible (GTR) model with gamma-distributed evolutionary rates (G) and invariable sites (I), GTR + G + I, was chosen. We undertook maximum likelihood analysis with PhyML 3.0 phylogeny software, using the approximate likelihood-ratio test as a means of assigning statistical significance to internal branches. Bayesian inference was run on MrBayes 3.2 software with default Markov chain Monte Carlo (MCMC) algorithm settings—ie, two independent runs with four chains each were sampled every 500th generation until 1 000 000 samples were obtained. 25% of the MCMC samples were discarded as a burn-in step. Chain convergence was measured by the Gelman-Rubin statistic, using the potential scale reduction factor, or PSRF, which was close to 1 for all parameters. Maximum likelihood and Bayesian inference topologies were identical. We therefore report the results from the maximum likelihood analysis.

To investigate recombination breakpoints along the Zika virus genome, a sliding window strategy was implemented using an in-house script. By building a stand-alone BLAST database containing all reference flavivirus genomes, we scanned the Zika virus genome every 50 bp regions and registered their BLAST hits using a cutoff e-value of 0·0001. We did genome-wide multiple alignments using the Multi-LAGAN algorithm as implemented in the VISTA database. Phylogeny of whole genomes was also inferred by maximum likelihood and Bayesian inference methods.

Role of the funding source

The funder of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. The corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication.

niman · February 18, 2016

Results

Serum, urine, and amniotic fluid samples from both patients (all taken at week 28 of gestation) were tested for dengue virus, chikungunya virus, and Zika virus. The RT-qPCR for dengue virus²⁰ and the RT-qPCR for chikungunya virus²¹ were negative in all samples. The RT-qPCRs for Zika virus²²confirmed Zika virus infection in the amniotic fluids of patients 1 and 2, but were negative in urine and serum samples in both patients. Serology tests of serum, urine, and amniotic fluid samples using anti-dengue-virus IgM, anti-dengue-virus IgG, anti-chikungunya-virus IgM, and anti-chikungunya-virus IgG yielded negative results by ELISA. However, ELISA for Zika virus was positive for anti-Zika-virus IgM in amniotic fluid, and negative in serum and urine in both patients 1 and 2. TORCH (toxoplasmosis, HIV, syphilis, measles, rubella, cytomegalovirus, and herpes simplex) panels of both women were also negative, as well as specific HIV, syphilis, cytomegalovirus, and parvovirus B19 screens.

Virus particles were filtrated and concentrated from the amniotic fluid samples. After cellular contaminants and human sequences were eliminated, 288 904 sequences showed similarity with virus sequences through BLAST analysis of the GenBank viral genome database. 683 sequences matched the Zika virus, comprising 167 143 bp, used to assemble the genome. Two different fragments corresponding to Zika virus genome positions 1641–1763 and 6466–6566 were sequenced from samples of patient 2. Metagenomic analysis of samples of patient 1 covered 10 793 bases of the Zika virus genome with 19 × coverage. The complete sequence (10 793 nucleotides) was deposited at the GenBank database (accession number ID: KU497555).

Figure 1 shows the whole Zika virus genome isolated from the amniotic fluid of patient 1 with viral gene annotation. The Brazilian Zika virus shares 97–100% of its genomic identity with the Zika virus sequence KJ776791.1 isolated in French Polynesia. The comparison with African strains such as NC_012532.1 (Zika virus Uganda) and KF383118.1 (Zika virus Senegal) yielded 87–90% identity. The proportion of GC content in the Brazilian Zika virus was 51·2% (figure 1).

Figure 1

Comparative genome BLAST Atlas diagram of Zika virus

The green outer circle corresponds to the complete Brazilian Zika virus genome isolated from the amniotic fluid of patient 1. 10 793 bases were sequenced. The red circle corresponds to the Senegal (KF383118.1) strain of Zika virus and the blue circle corresponds to the Uganda strain (NC_012532.1). The percentage deviation in GC content between the Brazilian Zika virus and the reference Zika virus is represented along the Zika virus genome by the varying heights of the black bars. The innermost (black) circle corresponds to the reference genome (French Polynesia, KJ776791.1). Genome shared identity between each strain and the reference genome are shown as percentages. BLAST=basic local alignment search tool.

We compared the viral sequences from patient 1 with previously released sequence data from Zika virus outbreaks in Asia and Africa and other flaviviruses, including dengue virus serotypes 1–4, West Nile virus, and yellow fever virus. Phylogenetic analyses were done using the coding region for the envelope (figure 2) and NS5 genes (figure 3). The geographical origin of the Brazilian Zika virus strain could not be determined because of sampling limitations, but Brazilian Zika virus is probably more closely related to French Polynesia sequences than to African strains. Maximum likelihood and Bayesian inference methods applied to the alignment of the envelope and NS5 regions of the polyprotein coding sequence yielded identical estimates of phylogenetic topologies. In both envelope (figure 2) and NS5 (figure 3) genomic regions, the new genome clustered with sequences from North and South America, southeast Asia, and the Pacific.

Figure 2

Maximum likelihood topologies of envelope genomic region from Brazilian Zika virus

Brazilian Zika virus (in red text) isolated from the amniotic fluid of patient 1, whose fetus was diagnosed with microcephaly, was compared with previously released sequence data. Approximate likelihood-ratio test support values greater than 0·5 are shown at nodes. Zika virus Brazil shares the same origin as those of Asian, Pacific, and American lineages (red branches). For most strains, the country of isolation is shown, in some cases followed by the date of isolation. Burkina=Burkina Faso. Central=Central African Republic. Cook=Cook Islands.

Figure 3

Maximum likelihood topologies of NS5 genomic region from Brazilian Zika virus

Brazilian Zika virus (in red text) isolated from the amniotic fluid of patient 1, whose fetus was diagnosed with microcephaly, was compared with previously released sequence data. Approximate likelihood-ratio test support values greater than 0·5 are shown at nodes. Zika virus Brazil shares the same origin as those of Asian, Pacific, and American lineages (red branches). For most sequences, the country of isolation is shown, in some cases followed by the date of isolation.

The geographical and chronological distributions of Zika virus lineages also indicate that southeast Asian strains could have remained genetically isolated from African strains for about 50 years, because Malaysian sequence HQ234449, collected in 1966, is the sister group of the remaining New World and Pacific strains. This pattern was further confirmed by the genomic distance between the newly sequenced Brazilian Zika virus and the Ugandan Zika virus genome available in GenBank (ZIKV LC002520; figure 4).

Figure 4

Maximum likelihood phylogeny of Brazilian Zika virus, other Flaviviridae genomes, and an alphavirus genome

Brazilian Zika virus (in red) was isolated from the amniotic fluid of patient 1, whose fetus was diagnosed with microcephaly. Approximate likelihood-ratio test and Bayesian inference support values are shown at nodes. Chikungunya is an alphavirus; all other viruses are from the Flaviviridae family. DENV=dengue virus. JEV=Japanese encephalitis virus. YFV=yellow fever virus. ZIKV=Zika virus.

We assessed the possibility of recombination events between the Zika virus and other flaviviruses by scanning the Zika virus genome every 50 bp using as references the genomes from dengue virus serotypes 1–4, West Nile virus, yellow fever virus, and chikungunya virus (an alphavirus that is transmitted by the same vector). The sliding window strategy with local alignments of genomic fragments ruled out the hypothesis that the newly sequenced Brazilian Zika virus genome is a recombinant strain with other mosquito-borne flaviviruses. All genomic regions consistently presented best hits and significant e-values with previously reported Zika virus genomes, ruling out the hypothesis of genomic recombination.

Discussion

Detection of the Zika virus genome and anti-Zika-virus IgM in the amniotic fluid of pregnant women with microcephalic fetuses has not been previously reported in detail in the scientific literature. This finding shows that the Zika virus can cross the placental barrier and, possibly, infect the fetus. A previous report23 suggested that fragments of Zika virus genome were identified in saliva, breastmilk, urine, and serum of two mothers and their newborn babies within 4 days of delivery. However, our group is the first, to our knowledge, to isolate the whole genome of Zika virus directly from the amniotic fluid of a pregnant woman before delivery, supporting the hypothesis that Zika virus infection could occur through transplacental transmission.

Some neglected tropical diseases have well known neurological effects. Many distinct clinical syndromes, from mild fever and arthralgia to severe haemorrhagic and encephalitic manifestations, are known to be associated with flavivirus infections.24 Other severe neurological complications such as Guillain-Barré syndrome have been reported in patients infected with Zika virus.25 Two key properties allow these viruses to affect the neural system: the ability to enter the CNS (neuroinvasiveness) and the capacity to infect neural cells through a process known as neurovirulence. A connection between Zika virus infections and poor CNS outcomes remains presumptive, and is based on a temporal association. New studies should be done to investigate whether the Zika virus can infect either neurological precursor cells or final differentiated cells.

Congenital microcephaly is a descriptive diagnosis. It can be caused by various factors, such as genetic disorders, exposure to chemicals, brain injury, consumption of teratogenic drugs, and intrauterine infections.26 Here, we focused on viral infection to explain these two cases of microcephaly. However, other possible causes or contributing factors should continue to be pursued as new cases arise in Brazil.

In these two patients, fetal microcephaly was detected early during gestation and a severe outcome was expected. Ultrasound tests revealed the presence of malformation, including ventriculomegaly and cerebellar hypoplasia. Fetal brain malformation can often result from viral infections during pregnancy. Cytomegalovirus infection occurring before 18 weeks of gestation is frequently associated with lissencephaly with a thin cerebral cortex, cerebellar hypoplasia, and ventriculomegaly, among other malformations.27 However, in the two cases presented here, serological and RT-PCR tests for cytomegalovirus were negative, ruling out cytomegalovirus infection. The viral metagenomic approach used here does not exclude either DNA or RNA viruses; nevertheless, no cytomegalovirus sequence was identified in the amniotic fluid in our analyses. An increase in the incidence of CNS malformations in fetuses and neonates was reported after a Zika virus outbreak in French Polynesia; however, the occurrence of microcephaly associated with these previous outbreaks was not documented.1

Our previous image findings18 and our results shown here of the presence of viral genomic material in both patients, several weeks after the acute phase of Zika virus disease, suggest that the intrauterine viral load results from persistent replication. In turn, this persistence could be partly explained by the reduced immune system response of the fetus, as described in the pathogenesis of congenital cytomegalovirus.28, 29

The Zika virus could have undergone several recombination events, and the recurrent loss and gain of the N-linked glycosylation site in the E protein could be related to mosquito-cell infectivity.30 We found no evidence of recombination events in the Zika virus genomes that we tested. The role of recombination in Zika virus virulence warrants further study.

Our results provide insight into the origin of the Zika virus circulating in Brazil, and suggest that a causal relation might exist between Zika virus infection and fetal microcephaly. New studies coordinated by the Brazilian Ministry of Health and other institutions are underway to further test this hypothesis, and hopefully elucidate the cellular and molecular mechanisms of Zika virus infection.

We recommend that Zika virus infection should be regarded as a possible causative agent in cases of microcephaly, especially during Zika virus outbreaks in endemic regions. Early diagnosis of Zika virus infection, supportive care, symptomatic treatment, and referral of children with microcephaly to specialised care are all necessary measures to improve neurodevelopment of affected children.

Contributors

AMBdF, RSA, AT, and FLT designed the study. ASOM did the ultrasound and collected the amniotic fluid samples. SAS, AF, ESMA, RSA, PCdS, MCLdM, and LdO did the laboratory studies. AMBdF, RMRN, FBdS, RSA, CGS, AT, FLT, DAT, PB, and IdF analysed the data. GC, AMBdF, and RSA wrote and edited initial drafts. All authors reviewed the final draft.

Declaration of interests

We declare no competing interests.

Acknowledgments

We thank David O'Connor and Dawn Dudley for helping with the viral metagenomics protocols. We thank Consellho Nacional de Desenvolvimento e Pesquisa (CNPq) and Fundação de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ) for funding.

Edited February 18, 2016 by Admin

niman · February 18, 2016

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Edited February 18, 2016 by Admin

niman · February 18, 2016

LOCUS       KU497555               10793 bp    RNA     linear   VRL 18-FEB-2016
DEFINITION  Zika virus isolate Brazil-ZKV2015, complete genome.
ACCESSION   KU497555
VERSION     KU497555.1  GI:985578255
KEYWORDS    .
SOURCE      Zika virus
  ORGANISM  Zika virus
            Viruses; ssRNA viruses; ssRNA positive-strand viruses, no DNA
            stage; Flaviviridae; Flavivirus.
REFERENCE   1  (bases 1 to 10793)
  AUTHORS   Calvet,G., Aguiar,R.S., Melo,A.S.O., Sampaio,S.A., de Filippis,I.,
            Fabri,A., Araujo,E.S.M., de Sequeira,P.C., de Mendonca,M.C.L., de
            Oliveira,L., Tschoeke,D.A., Schrago,C.G., Thompson,F.L., Brasil,P.,
            dos Santos,F.B., Nogueira,R.M.R., Tanuri,A. and de Filippis,A.M.B.
  TITLE     Detection and sequencing of Zika virus from amniotic fluid of
            fetuses with microcephaly in Brazil: a case study
  JOURNAL   Lancet Infect Dis (2016) In press
  REMARK    Publication Status: Available-Online prior to print
REFERENCE   2  (bases 1 to 10793)
  AUTHORS   Tanuri,A., Bispo,A., Thompson,F., Santana,R., Tschoeke,D., de
            Oliveira,L. and Guerra,C.
  TITLE     Direct Submission
  JOURNAL   Submitted (06-JAN-2016) UFRJ, UFRJ, Avenida Carlos Chagas Filho,
            373, Rio de Janeiro, Rio de Janeiro 21040-900, Brazil
COMMENT     ##Assembly-Data-START##
            Assembly Method       :: SPAdes v. 3.6
            Assembly Name         :: ZKV Brazil
            Coverage              :: 19 X
            Sequencing Technology :: Illumina
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..10793
                     /organism="Zika virus"
                     /mol_type="genomic RNA"
                     /isolate="Brazil-ZKV2015"
                     /isolation_source="anminiotic liquid"
                     /host="Homo sapiens"
                     /db_xref="taxon:64320"
                     /country="Brazil"
                     /collection_date="30-Nov-2015"
     CDS             101..10372
                     /codon_start=1
                     /product="polyprotein"
                     /protein_id="AMD16557.1"
                     /db_xref="GI:985578256"
                     /translation="MKNPKKKSGGFRIVNMLKRGVARVSPFGGLKRLPAGLLLGHGPI
                     RMVLAILAFLRFTAIKPSLGLINRWGSVGKKEAMEIIKKFKKDLAAMLRIINARKEKK
                     RRGADTSVGIVGLLLTTAMAAEVTRRGSAYYMYLDRNDAGEAISFPTTLGMNKCYIQI
                     MDLGHMCDATMSYECPMLDEGVEPDDVDCWCNTTSTWVVYGTCHHKKGEARRSRRAVT
                     LPSHSTRKLQTRSQTWLESREYTKHLIRVENWIFRNPGFALAAAAIAWLLGSSTSQKV
                     IYLVMILLIAPAYSIRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVTVMAQDKPTVDIE
                     LVTTTVSNMAEVRSYCYEASISDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWG
                     NGCGLFGKGSLVTCAKFACSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIVNDTGHET
                     DENRAKVEITPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWF
                     HDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGTQEGAVHTALAGALEAE
                     MDGAKGRLSSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKIPAETLHGTVTVEVQYAG
                     TDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPFGDSYIVIGVG
                     EKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFGSVGGALNSLGKGIHQIFG
                     AAFKSLFGGMSWFSQILIGTLLMWLGLNTKNGSISLMCLALGGVLIFLSTAVSADVGC
                     SVDFSKKETRCGTGVFVYNDVEAWRDRYKYHPDSPRRLAAAVKQAWEDGICGISSVSR
                     MENIMWRSVEGELNAILEENGVQLTVVVGSVKNPMWRGPQRLPVPVNELPHGWKAWGK
                     SYFVRAAKTNNSFVVDGDTLKECPLKHRAWNSFLVEDHGFGVFHTSVWLKVREDYSLE
                     CDPAVIGTAVKGKEAVHSDLGYWIESEKNDTWRLKRAHLIEMKTCEWPKSHTLWTDGI
                     EESDLIIPKSLAGPLSHHNTREGYRTQMKGPWHSEELEIRFEECPGTKVHVEETCGTR
                     GPSLRSTTASGRVIEEWCCRECTMPPLSFRAKDGCWYGMEIRPRKEPESNLVRSMVTA
                     GSTDHMDHFSLGVLVILLMVQEGLKKRMTTKIIISTSMAVLVAMILGGFSMSDLAKLA
                     ILMGATFAEMNTGGDVAHLALIAAFKVRPALLVSFIFRANWTPRESMLLALASCFLQT
                     AISALEGDLMVLINGFALAWLAIRAMVVPRTDNITLAILAALTPLARGTLLVAWRAGL
                     ATCGGFMLLSLKGKGSVKKNLPFVMALGLTAVRLVDPINVVGLLLLTRSGKRSWPPSE
                     VLTAVGLICALAGGFAKADIEMAGPMAAVGLLIVSYVVSGKSVDMYIERAGDITWEKD
                     AEVTGNSPRLDVALDESGDFSLVEDDGPPMREIILKVVLMTICGMNPIAIPFAAGAWY
                     VYVKTGKRSGALWDVPAPKEVKKGETTDGVYRVMTRRLLGSTQVGVGVMQEGVFHTMW
                     HVTKGSALRSGEGRLDPYWGDVKQDLVSYCGPWKLDAAWDGHSEVQLLAVPPGERARN
                     IQTLPGIFKTKDGDIGAVALDYPAGTSGSPILDKCGRVIGLYGNGVVIKNGSYVSAIT
                     QGRREEETPVECFEPSMLKKKQLTVLDLHPGAGKTRRVLPEIVREAIKTRLRTVILAP
                     TRVVAAEMEEALRGLPVRYMTTAVNVTHSGTEIVDLMCHATFTSRLLQPIRVPNYNLY
                     IMDEAHFTDPSSIAARGYISTRVEMGEAAAIFMTATPPGTRDAFPDSNSPIMDTEVEV
                     PERAWSSGFDWVTDHSGKTVWFVPSVRNGNEIAACLTKAGKRVIQLSRKTFETEFQKT
                     KHQEWDFVVTTDISEMGANFKADRVIDSRRCLKPVILDGERVILAGPMPVTHASAAQR
                     RGRIGRNPNKPGDEYLYGGGCAETDEDHAHWLEARMLLDNIYLQDGLIASLYRPEADK
                     VAAIEGEFKLRTEQRKTFVELMKRGDLPVWLAYQVASAGITYTDRRWCFDGTTNNTIM
                     EDSVPAEVWTRHGEKRVLKPRWMDARVCSDHAALKSFKEFAAGKRGAAFGVMEALGTL
                     PGHMTERFQEAIDNLAVLMRAETGSRPYKAAAAQLPETLETIMLLGLLGTVSLGIFFV
                     LMRNKGIGKMGFGMVTLGASAWLMWLSEIEPARIACVLIVVFLLLVVLIPEPEKQRSP
                     QDNQMAIIIMVAVGLLGLITANELGWLERTKSDLSHLMGRREEGATIGFSMDIDLRPA
                     SAWAIYAALTTFITPAVQHAVTTSYNNYSLMAMATQAGVLFGMGKGMPFYAWDFGVPL
                     LMIGCYSQLTPLTLIVAIILLVAHYMYLIPGLQAAAARAAQKRTAAGIMKNPVVDGIV
                     VTDIDTMTIDPQVEKKMGQVLLIAVAVSSAILSRTAWGWGEAGALITAATSTLWEGSP
                     NKYWNSSTATSLCNIFRGSYLAGASLIYTVTRNAGLVKRRGGGTGETLGEKWKARLNQ
                     MSALEFYSYKKSGITEVCREEARRALKDGVATGGHAVSRGSAKLRWLVERGYLQPYGK
                     VIDLGCGRGGWSYYAATIRKVQEVKGYTKGGPGHEEPVLVQSYGWNIVRLKSGVDVFH
                     MAAEPCDTLLCDIGESSSSPEVEEARTLRVLSMVGDWLEKRPGAFCIKVLCPYTSTMM
                     ETLERLQRRYGGGLVRVPLSRNSTHEMYWVSGAKSNTIKSVSTTSQLLLGRMDGPRRP
                     VKYEEDVNLGSGTRAVVSCAEAPNMKIIGNRIERIRSEHAETWFFDENHPYRTWAYHG
                     SYVAPTQGSASSLINGVVRLLSKPWDVVTGVTGIAMTDTTPYGQQRVFKEKVDTRVPD
                     PQEGTRQVMSMVSSWLWKELGKHKRPRVCTKEEFINKVRSNAALGAIFEEEKEWKTAV
                     EAVNDPRFWALVDKEREHHLRGECQSCVYNMMGKREKKQGEFGKAKGSRAIWYMWLGA
                     RFLEFEALGFLNEDHWMGRENSGGGVEGLGLQRLGYVLEEMSRIPGGRMYADDTAGWD
                     TRISRFDLENEALITNQMEKGHRALALAIIKYTYQNKVVKVLRPAEKGKTVMDIISRQ
                     DQRGSGQVVTYALNTFTNLVVQLIRNMEAEEVLEMQDLWLLRRSEKVTNWLQSNGWDR
                     LKRMAVSGDDCVVKPIDDRFAHALRFLNDMGKVRKDTQEWKPSTGWDNWEEVPFCSHH
                     FNKLHLKDGRSIVVPCRHQDELIGRARVSPGAGWSIRETACLAKSYAQMWQLLYFHRR
                     DLRLMANAICSSVPVDWVPTGRTTWSIHGKGEWMTTEDMLVVWNRVWIEENDHMEDKT
                     PVTKWTDIPYLGKREDLWCGSLIGHRPRTTWAENIKNTVNMVRRIIGDEEKYMDYLST
                     QVRYLGEEGSTPGVL"
ORIGIN      
        1 ccaatctgtg aatcagactg cgacagttcg agtttgaagc gaaagctagc aacagtatca
       61 acaggtttta ttttggattt ggaaacgaga gtttctggtc atgaaaaacc caaaaaagaa
      121 atccggagga ttccggattg tcaatatgct aaaacgcgga gtagcccgtg tgagcccctt
      181 tgggggcttg aagaggctgc cagccggact tctgctgggt catgggccca tcaggatggt
      241 cttggcgatt ctagcctttt tgagattcac ggcaatcaag ccatcactgg gtctcatcaa
      301 tagatggggt tcagtgggga aaaaagaggc tatggaaata ataaagaagt tcaagaaaga
      361 tctggctgcc atgctgagaa taatcaatgc caggaaggag aagaagagac gaggcgcaga
      421 tactagtgtc ggaatcgttg gcctcctgct gaccacagct atggcagcgg aggtcactag
      481 acgtgggagt gcatactata tgtacttgga cagaaacgat gctggggagg ccatatcttt
      541 tccaaccaca ttggggatga ataagtgtta tatacagatc atggatcttg gacacatgtg
      601 tgatgccacc atgagctatg aatgccctat gctggatgag ggggtggaac cagatgacgt
      661 cgattgttgg tgcaacacga cgtcaacttg ggttgtgtac ggaacctgcc atcacaaaaa
      721 aggtgaagca cggagatcta gaagagctgt gacgctcccc tcccattcca ctaggaagct
      781 gcaaacgcgg tcgcaaacct ggttggaatc aagagaatac acaaagcact tgattagagt
      841 cgaaaattgg atattcagga accctggctt cgcgttagca gcagctgcca tcgcttggct
      901 tttgggaagc tcaacgagcc aaaaagtcat atacttggtc atgatactgc tgattgcccc
      961 ggcatacagc atcaggtgca taggagtcag caatagggac tttgtggaag gtatgtcagg
     1021 tgggacttgg gttgatgttg tcttggaaca tgggggttgt gtcaccgtaa tggcacagga
     1081 caaaccgact gtcgacatag agctggttac aacaacagtc agcaacatgg cggaggtaag
     1141 atcctactgc tatgaggcat caatatcaga catggcttcg gacagccgct gcccaacaca
     1201 aggtgaagcc taccttgaca agcaatcaga cactcaatat gtctgcaaaa gaacgttagt
     1261 ggacagaggc tggggaaatg gatgtggact ttttggcaaa gggagcctgg tgacatgcgc
     1321 taagtttgca tgctccaaga aaatgaccgg gaagagcatc cagccagaga atctggagta
     1381 ccggataatg ctgtcagttc atggctccca gcacagtggg atgatcgtta atgacacagg
     1441 acatgaaact gatgagaata gagcgaaggt tgagataacg cccaattcac caagagccga
     1501 agccaccctg gggggttttg gaagcttagg acttgattgt gaaccgagga caggccttga
     1561 cttttcagat ttgtattact tgactatgaa taacaagcac tggttggttc acaaggagtg
     1621 gttccacgac attccattac cttggcacgc tggggcagac accggaactc cacactggaa
     1681 caacaaagaa gcactggtag agttcaagga cgcacatgcc aaaaggcaaa ctgtcgtggt
     1741 tctagggact caagaaggag cagttcacac ggcccttgct ggagctctgg aggctgagat
     1801 ggatggtgca aagggaaggc tgtcctctgg ccacttgaaa tgtcgcctga aaatggataa
     1861 acttagattg aagggcgtgt catactcctt gtgtaccgca gcgttcacat tcaccaagat
     1921 cccggctgaa acactgcacg ggacagtcac agtggaggta cagtacgcag ggacagatgg
     1981 accttgcaag gttccagctc agatggcggt ggacatgcaa actctgaccc cagttgggag
     2041 gttgataacc gctaaccccg taatcactga aagcactgag aactctaaga tgatgctgga
     2101 acttgatcca ccatttgggg actcttacat tgtcatagga gtcggggaga agaagatcac
     2161 ccaccactgg cacaggagtg gcagcaccat tggaaaagca tttgaagcca ctgtgagagg
     2221 tgccaagaga atggcagtct tgggagacac agcctgggac tttggatcag ttggaggcgc
     2281 tctcaactca ttgggcaagg gcatccatca aatttttgga gcagctttca aatcattgtt
     2341 tggaggaatg tcctggttct cacaaattct cattggaacg ttgctgatgt ggttgggtct
     2401 gaacacaaag aatggatcta tttcccttat gtgcttggcc ttagggggag tgttgatctt
     2461 cttatccaca gccgtctctg ctgatgtggg gtgctcggtg gacttctcaa agaaggagac
     2521 gagatgtggt acaggggtgt tcgtctataa cgacgttgaa gcctggaggg acaggtacaa
     2581 gtaccatcct gactctcccc gtagattggc agcagcagtc aagcaagcct gggaagatgg
     2641 tatctgcggg atctcctctg tttcaagaat ggaaaacatc atgtggagat cagtagaagg
     2701 ggagcttaac gcaatcctgg aagagaatgg agttcaactg acggtcgttg tgggatctgt
     2761 aaaaaacccc atgtggagag gtccacagag attgcccgtg cctgtgaacg agctgcccca
     2821 cggctggaag gcttggggga aatcgtactt cgtcagagca gcaaagacaa ataacagctt
     2881 tgtcgtggat ggtgacacac tgaaggaatg cccactcaaa catagagcat ggaacagctt
     2941 tcttgtggag gatcatgggt tcggggtatt tcacactagt gtctggctca aggttagaga
     3001 agattattca ttagagtgtg atccagccgt tattggaaca gctgttaagg gaaaggaggc
     3061 tgtacacagt gatctaggct actggattga gagtgagaag aatgacacat ggaggctgaa
     3121 gagggcccat ctgatcgaga tgaaaacatg tgaatggcca aagtcccaca cattgtggac
     3181 agatggaata gaagagagtg atctgatcat acccaagtct ttagctgggc cactcagcca
     3241 tcacaatacc agagagggct acaggaccca aatgaaaggg ccatggcaca gtgaagagct
     3301 tgaaattcgg tttgaggaat gcccaggcac taaggtccac gtggaggaaa catgtggaac
     3361 aagaggacca tctctgagat caaccactgc aagcggaagg gtgatcgagg aatggtgctg
     3421 cagggagtgc acaatgcccc cactgtcgtt ccgggctaaa gatggctgtt ggtatggaat
     3481 ggagataagg cccaggaaag aaccagaaag caacttagta aggtcaatgg tgactgcagg
     3541 atcaactgat cacatggatc acttctccct tggagtgctt gtgattctgc tcatggtgca
     3601 ggaagggctg aagaagagaa tgaccacaaa gatcatcata agcacatcaa tggcagtgct
     3661 ggtagctatg atcctgggag gattttcaat gagtgacctg gctaagcttg caattttgat
     3721 gggtgccacc ttcgcggaaa tgaacactgg aggagatgta gctcatctgg cgctgatagc
     3781 ggcattcaaa gtcagaccag cgttgctggt atctttcatc ttcagagcta attggacacc
     3841 ccgtgaaagc atgctgctgg ccttggcctc gtgttttttg caaactgcga tctccgcctt
     3901 ggaaggcgac ctgatggttc tcatcaatgg ttttgctttg gcctggttgg caatacgagc
     3961 gatggttgtt ccacgcactg acaacatcac cttggcaatc ctggctgctc tgacaccact
     4021 ggcccggggc acactgcttg tggcgtggag agcaggcctt gctacttgcg gggggtttat
     4081 gctcctctct ctgaagggaa aaggcagtgt gaagaagaac ttaccatttg tcatggccct
     4141 gggactaacc gctgtgaggc tggtcgaccc catcaacgtg gtgggactgc tgttgctcac
     4201 aaggagtggg aagcggagct ggccccctag cgaagtactc acagctgttg gcctgatatg
     4261 cgcattggct ggagggttcg ccaaggcaga tatagagatg gctgggccca tggccgcggt
     4321 cggtctgcta attgtcagtt acgtggtctc aggaaagagt gtggacatgt acattgaaag
     4381 agcaggtgac atcacatggg aaaaagatgc ggaagtcact ggaaacagtc cccggctcga
     4441 tgtggcgcta gatgagagtg gtgacttctc cctggtggag gatgacggtc cccccatgag
     4501 agagatcata ctcaaggtgg tcctgatgac catctgtggc atgaacccaa tagccatacc
     4561 ctttgcagct ggagcgtggt acgtatacgt gaagactgga aaaaggagtg gtgctctatg
     4621 ggatgtgcct gctcccaagg aagtaaaaaa gggggagacc acagatggag tgtacagagt
     4681 aatgactcgt agactgctag gttcaacaca agttggagtg ggagttatgc aagagggggt
     4741 ctttcacact atgtggcacg tcacaaaagg atccgcgctg agaagcggtg aagggagact
     4801 tgatccatac tggggagatg tcaagcagga tctggtgtca tactgtggtc catggaagct
     4861 agatgccgcc tgggacgggc acagcgaggt gcagctcttg gccgtgcccc ccggagagag
     4921 agcgaggaac atccagactc tgcccggaat atttaagaca aaggatgggg acattggagc
     4981 ggttgcgctg gattacccag caggaacttc aggatctcca atcctagaca agtgtgggag
     5041 agtgatagga ctttatggca atggggtcgt gataaaaaat gggagttatg ttagtgccat
     5101 cacccaaggg aggagggagg aagagactcc tgttgagtgc ttcgagcctt cgatgctgaa
     5161 gaagaagcag ctaactgtct tagacttgca tcctggagct gggaaaacca ggagagttct
     5221 tcctgaaata gtccgtgaag ccataaaaac aagactccgt actgtgatct tagctccaac
     5281 cagggttgtc gctgctgaaa tggaggaagc ccttagaggg cttccagtgc gttatatgac
     5341 aacagcagtc aatgtcaccc actctggaac agaaatcgtc gacttaatgt gccatgccac
     5401 cttcacttca cgtctactac agccaatcag agtccccaac tataatctgt atattatgga
     5461 tgaggcccac ttcacagatc cctcaagcat agcagcaaga ggatacattt caacaagggt
     5521 tgagatgggc gaggcggctg ccatcttcat gaccgccacg ccaccaggaa cccgtgacgc
     5581 atttccggac tccaactcac caattatgga caccgaagtg gaagtcccag agagagcctg
     5641 gagctcaggc tttgattggg tgacggatca ttctggaaaa acagtttggt ttgttccaag
     5701 cgtgaggaac ggcaatgaga tcgcagcttg tctgacaaag gctggaaaac gggtcataca
     5761 gctcagcaga aagacttttg agacagagtt ccagaaaaca aaacatcaag agtgggactt
     5821 tgtcgtgaca actgacattt cagagatggg cgccaacttt aaagctgacc gtgtcataga
     5881 ttccaggaga tgcctaaagc cggtcatact tgatggcgag agagtcattc tggctggacc
     5941 catgcctgtc acacatgcca gcgctgccca gaggaggggg cgcataggca ggaatcccaa
     6001 caaacctgga gatgagtacc tgtatggagg tgggtgcgca gagactgacg aagaccatgc
     6061 acactggctt gaagcaagaa tgctccttga caatatttac ctccaagatg gcctcatagc
     6121 ctcgctctat cgacctgagg ccgacaaagt agcagccatt gagggagagt tcaagcttag
     6181 gacggagcaa aggaagacct ttgtggaact catgaaaaga ggagatcttc ctgtttggct
     6241 ggcctatcag gttgcatctg ccggaataac ctacacagat agaagatggt gctttgatgg
     6301 cacgaccaac aacaccataa tggaagacag tgtgccggca gaggtgtgga ccagacacgg
     6361 agagaaaaga gtgctcaaac cgaggtggat ggacgccaga gtttgttcag atcatgcggc
     6421 cctgaagtca ttcaaggagt ttgccgctgg gaaaagagga gcggcttttg gagtgatgga
     6481 agccctggga acactgccag gacacatgac agagagattc caggaagcca ttgacaacct
     6541 cgctgtgctc atgcgggcag agactggaag caggccttac aaagccgcgg cggcccaatt
     6601 gccggagacc ctagagacca ttatgctttt ggggttgctg ggaacagtct cgctgggaat
     6661 ctttttcgtc ttgatgagga acaagggcat agggaagatg ggctttggaa tggtgactct
     6721 tggggccagc gcatggctca tgtggctctc ggaaattgag ccagccagaa ttgcatgtgt
     6781 cctcattgtt gtgttcctat tgctggtggt gctcatacct gagccagaaa agcaaagatc
     6841 tccccaggac aaccaaatgg caatcatcat catggtagca gtaggtcttc tgggcttgat
     6901 taccgccaat gaactcggat ggttggagag aacaaagagt gacctaagcc atctaatggg
     6961 aaggagagag gagggggcaa ccataggatt ctcaatggac attgacctgc ggccagcctc
     7021 agcttgggcc atctatgctg ccttgacaac tttcattacc ccagccgtcc aacatgcagt
     7081 gaccacttca tacaacaact actccttaat ggcgatggcc acgcaagctg gagtgttgtt
     7141 tggtatgggc aaagggatgc cattctacgc atgggacttt ggagtcccgc tgctaatgat
     7201 aggttgctac tcacaattaa cacccctgac cctaatagtg gccatcattt tgctcgtggc
     7261 gcactacatg tacttgatcc cagggctgca ggcagcagct gcgcgtgctg cccagaagag
     7321 aacggcagct ggcatcatga agaaccctgt tgtggatgga atagtggtga ctgacattga
     7381 cacaatgaca attgaccccc aagtggagaa aaagatggga caggtgctac tcatagcagt
     7441 agccgtctcc agcgccatac tgtcgcggac cgcctggggg tggggggagg ctggggccct
     7501 gatcacagcc gcaacttcca ctttgtggga aggctctccg aacaagtact ggaactcctc
     7561 tacagccact tcactgtgta acatttttag gggaagttac ttggctggag cttctctaat
     7621 ctacacagta acaagaaacg ctggcttggt caagagacgt gggggtggaa caggagagac
     7681 cctgggagag aaatggaagg cccgcttgaa ccagatgtcg gccctggagt tctactccta
     7741 caaaaagtca ggcatcaccg aggtgtgcag agaagaggcc cgccgcgccc tcaaggacgg
     7801 tgtggcaacg ggaggccatg ctgtgtcccg aggaagtgca aagctgagat ggttggtgga
     7861 gcggggatac ctgcagccct atggaaaggt cattgatctt ggatgtggca gagggggctg
     7921 gagttactac gccgccacca tccgcaaagt tcaagaagtg aaaggataca caaaaggagg
     7981 ccctggtcat gaagaacccg tgttggtgca aagctatggg tggaacatag tccgtcttaa
     8041 gagtggggtg gacgtctttc atatggcggc tgagccgtgt gacacgttgc tgtgtgacat
     8101 aggtgagtca tcatctagtc ctgaagtgga agaagcacgg acgctcagag tcctctccat
     8161 ggtgggggat tggcttgaaa aaagaccagg agccttttgc ataaaagtgt tgtgcccata
     8221 caccagcact atgatggaaa ccctggagcg actgcagcgt aggtatgggg gaggactggt
     8281 cagagtgcca ctctcccgca actctacaca tgagatgtac tgggtctctg gagcgaaaag
     8341 caacaccata aaaagtgtgt ccaccacgag ccagctcctc ttggggcgca tggacgggcc
     8401 taggaggcca gtgaaatatg aggaggatgt gaatctcggc tctggcacgc gggctgtggt
     8461 aagctgcgct gaagctccca acatgaagat cattggtaac cgcattgaaa ggatccgcag
     8521 tgagcacgcg gaaacgtggt tctttgacga aaaccaccca tataggacat gggcttacca
     8581 tggaagctat gtggccccca cacaagggtc agcgtcctct ctaataaacg gggttgtcag
     8641 gctcctgtca aaaccctggg atgtggtgac tggagtcaca ggaatagcca tgaccgacac
     8701 cacaccgtat ggtcagcaaa gagttttcaa ggaaaaagtg gacactaggg tgccagaccc
     8761 ccaagaaggc actcgtcagg ttatgagcat ggtctcttcc tggttgtgga aagagctagg
     8821 caaacacaaa cgaccacgag tctgtaccaa agaagagttc atcaacaagg ttcgtagcaa
     8881 tgcagcatta ggggcaatat ttgaagagga aaaagagtgg aagactgcag tggaagctgt
     8941 gaacgatcca aggttctggg ctctagtgga caaggaaaga gagcaccacc tgagaggaga
     9001 gtgccagagt tgtgtgtaca acatgatggg aaaaagagaa aagaaacaag gggaatttgg
     9061 aaaggccaag ggcagccgcg ccatctggta tatgtggcta ggggctagat ttctagagtt
     9121 cgaagccctt ggattcttga acgaggatca ctggatgggg agagagaact caggaggtgg
     9181 tgttgaaggg ctgggattac aaagactcgg atatgtccta gaagagatga gtcgcatacc
     9241 aggaggaagg atgtatgcag atgacactgc tggctgggac acccgcatca gcaggtttga
     9301 tctggagaat gaagctctaa tcaccaacca aatggagaaa gggcacaggg ccttggcatt
     9361 ggccataatc aagtacacat accaaaacaa agtggtaaag gtccttagac cagctgaaaa
     9421 agggaaaaca gttatggaca ttatttcgag acaagaccaa agggggagcg gacaagttgt
     9481 cacttacgct cttaacacat ttaccaacct agtggtgcaa ctcattcgga atatggaggc
     9541 tgaggaagtt ctagagatgc aagacttgtg gctgctgcgg aggtcagaga aagtgaccaa
     9601 ctggttgcag agcaacggat gggataggct caaacgaatg gcagtcagtg gagatgattg
     9661 cgttgtgaag ccaattgatg ataggtttgc acatgccctc aggttcttga atgatatggg
     9721 aaaagttagg aaggacacac aagagtggaa accctcaact ggatgggaca actgggaaga
     9781 agttccgttt tgctcccacc acttcaacaa gctccatctc aaggacggga ggtccattgt
     9841 ggttccctgc cgccaccaag atgaactgat tggccgggcc cgcgtctctc caggggcggg
     9901 atggagcatc cgggagactg cttgcctagc aaaatcatat gcgcaaatgt ggcagctcct
     9961 ttatttccac agaagggacc tccgactgat ggccaatgcc atttgttcat ctgtgccagt
    10021 tgactgggtt ccaactggga gaactacctg gtcaatccat ggaaagggag aatggatgac
    10081 cactgaagac atgcttgtgg tgtggaacag agtgtggatt gaggagaacg accacatgga
    10141 agacaagacc ccagttacga aatggacaga cattccctat ttgggaaaaa gggaagactt
    10201 gtggtgtgga tctctcatag ggcacagacc gcgcaccacc tgggctgaga acattaaaaa
    10261 tacagtcaac atggtgcgca ggatcatagg tgatgaagaa aagtacatgg actacctatc
    10321 cacccaagtt cgctacttgg gtgaagaagg gtctacacct ggagtgctgt gagcaccaat
    10381 cttaatgttg tcaggcctgc tagtcagcca cagcttgggg aaagctgtgc agcctgtgac
    10441 ccctccagga gaagctgggt aaccaagcct atagtcaggc cgagaacgcc atggcacgga
    10501 agaagccatg ctgcctgtga gcccctcaga ggacactgag tcaaaaaacc ccacgcgctt
    10561 ggaggcgcag gatgggaaaa gaaggtggcg accttcccca cccttcaatc tggggcctga
    10621 actggagatc agctgtggat ctccagaaga gggactagtg gttagaggag accccccgga
    10681 aaacgcaaaa cagcatattg acgctgggaa agaccagaga ctccatgagt ttccaccacg
    10741 ctggccgcca ggcacagatc gccgaatagc ggcggccggt gtggggaaat cca

niman · February 18, 2016

Sequence map updated

https://www.google.com/maps/d/u/0/edit?mid=zv94AJqgUct4.kI8kcFySb4J0&hl=en

niman · February 18, 2016

Zika virus: Study supports link to microcephaly

By Smitha MundasadHealth reporter

10-year-old Elison holds his two-month-old brother Jose Wesley at their house in Poco Fundo, Pernambuco state, Brazil (23 December 2015) — Image captionA 10-year-old Brazilian boy holds his two-month-old brother, who was born with microcephaly

Scientists say a study involving pregnant women in Brazil "strengthens" the theory that Zika is linked to microcephaly birth defects in babies.

The research confirmed the presence of Zika virus in the amniotic fluid of two women who had had Zika-like symptoms during their pregnancies.

Brazilian experts say this suggests the virus can infect the foetus.

But WHO experts caution the link is not proven and expect to release more information in the next few weeks.

Urgent investigations

Brazil has seen a rise in microcephaly - babies born with abnormally small heads and, in some cases, problems with brain development - in the last year, at the same time as a rise in the number of people infected with Zika virus.

This has led to a number of studies investigating whether the virus is behind the rise.

The research, published in the journal Lancet Infectious Diseases, involved two women who had fever, rash and muscle aches during their pregnancies.

After ultrasound scans revealed their developing foetuses had microcephaly, scientists ran further amniocentesis checks.

This involved taking a small sample of the amniotic fluid that surrounds the foetus in the womb.

Genetic analysis of this fluid confirmed the presence of Zika virus - discounting similar viruses that may have been responsible.

Lead scientist, Dr Ana de Filippis, from the Oswaldo Cruz Institute in Rio de Janeiro, Brazil, said: "This study reports details of the Zika virus being identified directly in the amniotic fluid of a woman during her pregnancy, suggesting the virus could cross the placental barrier and potentially infect the foetus."

She added: "This study cannot determine whether the Zika virus identified in these two cases was the cause of microcephaly in the babies.

"Until we understand the biological mechanism linking Zika to microcephaly we cannot be certain that one causes the other, and further research is urgently needed."

Prof Jimmy Whitworth, at the London School of Hygiene and Tropical Medicine added that while the research cannot prove the link: "This study does strengthen the body of evidence that Zika virus is the cause of foetal microcephaly in Brazil."

Separately the paper suggests that the virus looks genetically very similar to the Zika virus circulating in French Polynesia in 2013.

But scientists say despite growing research, a lot remains unknown and a number of questions still need urgent answers - including how big the risk of microcephaly is if a woman has Zika virus infection in pregnancy and whether the timing of the infection makes a difference.

Brazil, the country hardest-hit by Zika, has about 508 confirmed cases of microcephaly and is investigating about 3,935 suspected cases.

The ministry said last week that 41 of the confirmed cases of microcephaly had shown links to Zika infection.

Microcephaly can be caused by a range of factors, including genetic conditions, infections and drugs.

Experts say women who are pregnant are most at risk from mosquito-borne Zika and should try to protect themselves from mosquito bites.

http://www.bbc.com/news/health-35597465

Zika Sequences Amniotic Fluid Microcephaly Fetuses - Paraiba Brazil - Lancet

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Detection and sequencing of Zika virus from amniotic fluid of fetuses with microcephaly in Brazil: a case study

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Summary

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Introduction

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Viral metagenomics and sequence analysis

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Zika virus: Study supports link to microcephaly

Urgent investigations

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