niman

Sequences producing significant alignments:Select:AllNone Selected:0 AlignmentsDownloadGenBankGraphicsDistance tree of resultsShow/hide columns of the table presenting sequences producing significant alignmentsSequences producing significant alignments:Select for downloading or viewing reportsDescriptionMax scoreTotal scoreQuery coverE valueIdentAccessionSelect seq gb|KU509998.1|Zika virus strain Haiti/1225/2014, complete genome183951839599%0.099%KU509998.1Select seq gb|KJ776791.1|Zika virus strain H/PF/2013 polyprotein gene, complete cds183901839099%0.099%KJ776791.1Select seq gb|KU321639.1|Zika virus strain ZikaSPH2015, complete genome183811838199%0.099%KU321639.1Select seq gb|KU647676.1|Zika virus strain MRS_OPY_Martinique_PaRi_2015 polyprotein gene, complete cds183721837299%0.099%KU647676.1Select seq gb|KU365779.1|Zika virus strain BeH819966 polyprotein gene, complete cds183721837299%0.099%KU365779.1Select seq gb|KU365780.1|Zika virus strain BeH815744 polyprotein gene, complete cds183591835999%0.099%KU365780.1Select seq gb|KU365777.1|Zika virus strain BeH818995 polyprotein gene, complete cds183541835499%0.099%KU365777.1Select seq gb|KU501217.1|Zika virus strain 8375 polyprotein gene, complete cds183501835099%0.099%KU501217.1Select seq gb|KU501216.1|Zika virus strain 103344 polyprotein gene, complete cds183451834599%0.099%KU501216.1Select seq gb|KU365778.1|Zika virus strain BeH819015 polyprotein gene, complete cds183411834199%0.099%KU365778.1Select seq gb|KU312312.1|Zika virus isolate Z1106033 polyprotein gene, complete cds183411834199%0.099%KU312312.1Select seq gb|KU501215.1|Zika virus strain PRVABC59, complete genome183321833299%0.099%KU501215.1Select seq gb|JN860885.1|Zika virus isolate FSS13025 polyprotein gene, partial cds177531775399%0.098%JN860885.1Select seq gb|KF993678.1|Zika virus strain PLCal_ZV from Canada polyprotein gene, partial cds177151771598%0.099%KF993678.1Select seq gb|EU545988.1|Zika virus polyprotein gene, complete cds176031760399%0.098%EU545988.1Select seq gb|HQ234499.1|Zika virus isolate P6-740 polyprotein gene, partial cds164111641199%0.095%HQ234499.1

OCUS 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

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"http://www.ncbi.nlm.nih.gov/nuccore/KU497555

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

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ResultsSerum, 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 1Comparative 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 2Maximum 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 4Maximum 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. DiscussionDetection 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.

MethodsCase historiesThe 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 collectionFollowing 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 analysisA 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 analysisPhylogenetic 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 sourceThe 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.

IntroductionSince 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.

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.

SummaryBackgroundThe 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. MethodsIn 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. FindingsWe 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. InterpretationThese 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. FundingConsellho Nacional de Desenvolvimento e Pesquisa (CNPq), Fundação de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ).

Detection and sequencing of Zika virus from amniotic fluid of fetuses with microcephaly in Brazil: a case studyGuilherme Calvet, PhD*, Renato S Aguiar, PhD*, Adriana S O Melo, PhD, Simone A Sampaio, BSc, Ivano de Filippis, PhD,Allison Fabri, BSc, Eliane S M Araujo, BSc, Patricia C de Sequeira, PhD, Marcos C L de Mendonça, PhD, Louisi de Oliveira, PhD, Diogo A Tschoeke, PhD, Carlos G Schrago, PhD, Fabiano L Thompson, PhD, Patricia Brasil, PhD, Flavia B dos Santos, PhD, Rita M R Nogueira, PhD, Prof Amilcar Tanuri, PhD†, Dr Ana M B de Filippis, PhD†*Contributed equally†Contributed equallyPublished Online: 17 February 2016 http://www.thelancet.com/journals/laninf/article/PIIS1473-3099(16)00095-5/fulltext DOI: http://dx.doi.org/10.1016/S1473-3099(16)00095-5

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9 Canadians infected with Zika virus, Health Minister Jane Philpott saysVirus was contracted outside of Canada in all cases, with no local transmission, minister saysBy Ramisha Farooq, CBC News Posted: Feb 17, 2016 3:00 PM ET Last Updated: Feb 17, 2016 3:00 PM ET Health Minister Jane Philpott warns against travelling to countries that have been shown to have local transmission of the Zika virus. (Sean Kilpatrick/Canadian Press) Health Minister paying close attention to spread of Zika virus 6:59 2 shares Facebook Twitter Reddit Google Share Email Related StoriesZika virus: modified mosquitoes, bacteria use encouraged by WHOZika virus renews calls to eradicate disease-carrying mosquitoesBrazil deploys military to battle mosquito spread of Zika virusZika virus linked to 'vision-threatening lesions' in babies with birth defectZika virus has infected around 3,100 pregnant Colombians, health officials sayThe number of Canadians infected with the Zika virus has risen to nine, Health Minister Jane Philpott said Wednesday. The minister said there have been no cases of locally transmitted Zika virus in Canada. "We can assume that they were most likely mosquito-borne, but they were all contracted [outside] the country, not local transmission," Philpott said. The minister could not confirm whether the virus was or could be sexually transmitted. One of the cases has been confirmed in Alberta, and another two in B.C. that were contracted in El Salvador and Columbia, according to the B.C. Centre for Disease control. Health officials in both provinces have said there is no risk to public health in the province. Philpott stressed people should be cautious when travelling abroad. Brazilian officials still say they believe there's a sharp increase in cases of microcephaly and strongly suspect the Zika virus, which first appeared in the country last year, is to blame. (Felipe Dana/Associated Press) "We encourage people if they are pregnant or could become pregnant that they consider postponing their trip, and if they feel that it's necessary to go ahead with their trip that they take all precautions to avoid daytime mosquito bites," said Philpott. According to the World Health Organization, vaccines to protect against Zika virus are at least 18 months away from large-scale trials. The mosquito-borne Zika virus has been linked to microcephaly, a rare birth defect that sees babies born with unusually small heads and can cause lasting developmental problems. The mosquitoes that carry and transmit the virus do not live in Canada, due to the climate, says Alberta Health, and human-to-human transmission is rare. The World Health Organization has said that Canada and continental Chile are the only countries in the Americas where the Zika virus is unlikely to spread. More than 3,100 pregnant Columbian women are now infected with the Zika virus.

Media reports cite 9 confirmed Zika cases in Canada. http://www.cbc.ca/news/politics/zika-virus-canadians-1.3452016

Brazil says 'most' of confirmed microcephaly cases linked to ZikaPosted 18 Feb 2016 03:15 1 Email More A A RIO DE JANEIRO: Brazil's Health Ministry on Wednesday said that "most" of the 508 confirmed cases of microcephaly reported in the country are likely related to the ongoing outbreak of Zika virus infections. The ministry earlier in the day reported a total of 4,443 suspected and confirmed cases of the rare defect, up from 4,314 a week earlier. It did not, however, update its total of 41 cases in which it said that microcephaly had been linked by laboratory tests to Zika infections. In a clarification, it said that the government would cease to update the confirmed number of linked cases because "the Health Ministry considers that there were Zika virus infections in most of the mothers whose babies have been diagnosed" with the condition. Of the total reported on Wednesday, Brazil said 508 cases of microcephaly have been confirmed, while 3,935 are still being investigated. Microcephaly is a condition marked by abnormally small head size that can result in developmental problems. Brazil is investigating a surge in the number of cases across the country in conjunction with the outbreak of Zika, but have not yet proven that the virus can cause microcephaly. Zika has now spread to more than 30 countries and has led the World Health Organization to declare a global health emergency. (Reporting by Paulo Prada) http://www.channelnewsasia.com/news/health/brazil-says-most-of-con/2525006.html - Reuters

New case of travel-related Zika virus confirmed in FloridaCredit: APAn Aedes aegypti mosquito is photographed through a microscope at the Fiocruz institute in Recife, Pernambuco state, Brazil, Wednesday, Jan. 27, 2016. The mosquito is a vector for the proliferation of the Zika virus currently spreading throughout Latin America. (AP Photo/Felipe Dana)By Liz Freeman of the Naples Daily NewsPosted: 2:45 p.m.SHARE 0TOPOne more case of travel-related Zika virus has been confirmed, bringing the statewide number to 22, according to the Florida Department of Health on Wednesday. The new case was reported in Brevard County. The state’s public health emergency spans nine counties, including Lee County which has had three travel-related Zika cases. All of the cases are travel-related, where the infected individuals had traveled out of the country and were diagnosed with the mosquito-borne virus after their return. About one in five people infected become symptomatic, which generally involves a low-grade fever, rash and joint pain. Pregnant women are at risk if their unborn babies are exposed to the virus, which can lead to birth defects. Researchers with the U.S. Centers for Disease Control are examining the link between Zika and birth defects. The state health department encourages residents to drain standing water in any containers, which is the breeding source for mosquitoes; cover exposed skin with long-sleeved shirts and pants, and to wear mosquito repellent outdoors. For more information, go to www.FloridaHealth.gov. http://www.naplesnews.com/news/health/new-case-of-travel-related-zika-virus-confirmed-in-florida-2bfd0d50-8e7e-677d-e053-0100007f716d-369150171.html

Last Updated: Wednesday, February 17, 2016, 2:36 PM0 BREVARD COUNTY -- Florida health officials say one person has been diagnosed with the Zika virus. This is the 22nd case diagnosed in the state. Health officials say all the cases in Florida are travel-associated and there have been no locally-acquired cases of Zika in the state. CountyNumber of Cases (all travel related)Alachua1Brevard1Broward4Hillsborough3Lee3Miami-Dade7Osceola1Santa Rosa1St. Johns1Total22This is a developing story. Check back and refresh this article for the latest updates. http://www.mynews13.com/content/news/cfnews13/news/article.html/content/news/articles/cfn/2016/2/17/_1_person_diagnosed_.html?cid=rss&utm_source=dlvr.it&utm_medium=twitter

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Surgeon General Dr. John Armstrong’s Daily Zika Update: One New Case Confirmed in Brevard CountyBy Florida Department of Health, Office of Communications February 17, 2016 Press ReleaseSHARE THIS PAGEFacebookTwitter Feb. 16, 2016 SURGEON GENERAL DR. JOHN ARMSTRONG'S DAILY ZIKA UPDATE: ONE NEW CASE CONFIRMED IN BREVARD COUNTY Contact:Communications [email protected](850) 245-4111 Tallahassee, Fla.—In an effort to keep Florida residents and visitors safe and aware about the status of the Zika virus, State Surgeon General and Secretary of Health Dr. John Armstrong will issue a Zika virus update each week day at 2 p.m. Updates will include a CDC-confirmed Zika case count by county and information to better keep Floridians prepared. As of today, one new Zika case has been confirmed in Brevard County. Dr. Armstrong’s Declaration of Public Health Emergency has been expanded to include Brevard County. County Number of Cases (all travel related) Alachua 1 Brevard 1 Broward 4 Hillsborough 3 Lee 3 Miami-Dade 7 Osceola 1 Santa Rosa 1 St. Johns 1 Total 22 Last week, Governor Rick Scott directed State Surgeon General Dr. John Armstrong to activate a Zika Virus Information Hotline for current Florida residents and visitors, as well as anyone planning on traveling to Florida in the near future. The hotline, managed by the Department of Health, has assisted 439 callers since Friday. The number for the Zika Virus Information Hotline is 1-855-622-6735. All cases are travel-associated. There have been no locally-acquired cases of Zika in Florida. None of the confirmed cases involve pregnant women. For more information on the Zika virus, click here. State Surgeon General and Secretary of Health Dr. John Armstrong urges Floridians to drain standing water weekly, no matter how seemingly small. A couple drops of water in a bottle cap can be a breeding location for mosquitoes. Residents and visitors also need to use repellents when enjoying the Florida outdoors. More Information on DOH action on Zika: On Feb. 3, Governor Scott directed State Surgeon General and Secretary of Health Dr. John Armstrong to issue a Declaration of Public Health Emergency for the counties of residents with travel-associated cases of Zika.The Declaration currently includes the eight effected counties – Alachua, Broward, Hillsborough, Lee, Miami-Dade, Osceola, Santa Rosa and St. Johns – and will be updated as needed. DOH encourages Florida residents and visitors to protect themselves from all mosquito-borne illnesses by draining standing water; covering their skin with repellent and clothing; and covering windows with screens.DOH has a robust mosquito-borne illness surveillance system and is working with the CDC, the Florida Department of Agriculture and Consumer Services and local county mosquito control boards to ensure that the proper precautions are being taken to protect Florida residents and visitors.Florida currently has the capacity to test 4,963 people for active Zika virus and 1,341 for Zika antibodies.Federal Guidance on Zika: According to the CDC, Zika illness is generally mild with a rash, fever and joint pain. CDC researchers are examining a possible link between the virus and harm to unborn babies exposed during pregnancy.Yesterday, the FDA released guidance regarding donor screening, deferral and product management to reduce the risk of transfusion-transmission of Zika virus. Additional information is available on the FDA websitehere.For more information on Zika virus, click here. About the Florida Department of Health The department works to protect, promote and improve the health of all people in Florida through integrated state, county and community efforts. Follow us on Twitter at @HealthyFla and on Facebook. For more information about the Florida Department of Health, please visit www.FloridaHealth.gov. http://www.floridahealth.gov/newsroom/2016/02/021716-zika-update-10.html

County Number of Cases (all travel related) Alachua 1 Brevard 1 Broward 4 Hillsborough 3 Lee 3 Miami-Dade 7 Osceola 1 Santa Rosa 1 St. Johns 1 Total 22 http://www.floridahealth.gov/_documents/newsroom/press-releases/2016/02/021716-zika-update-10.pdf

Zika Virus – Feb. 17, 2016 Texas has 12 cases of Zika virus disease. 11 are travelers who were infected abroad and diagnosed after they returned home. One case involves a Dallas County resident who had sexual contact with someone who acquired the Zika infection while traveling abroad. Case counts by county: Bexar County – 3Dallas County – 2Harris County – 7 http://www.texaszika.org/

Zika: The current epidemic, research agenda and public health responseWednesday at 12:00pm

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ReferencesDick GWA, Kitchen SF, Haddow AJ. Zika virus. I. Isolations and serological specificity. Trans R Soc Trop Med Hyg.1952;46:509–20 . DOIDuffy MR, Chen TH, Hancock WT, Powers AM, Kool JL, Lanciotti RS, Zika virus outbreak on Yap Island, Federated States of Micronesia. N Engl J Med. 2009;360:2536–43 . DOIPubMedCao-Lormeau VM, Roche C, Teissier A, Robin E, Berry ALT, Mallet HP, Zika virus, French Polynesia, South Pacific, 2013. Emerg Infect Dis. 2014;20:1085–6 . DOIMusso D, Nilles EJ, Cao-Lormeau VM. Rapid spread of emerging Zika virus in the Pacific area. Clin Micobiol Infect.2014; .DOIOehler E, Watrin L, Larre P, Leparc-Goffart I, Lastere S, Valour F, Zika virus infection complicated by Guillain-Barre syndrome–case report, French Polynesia, December 2013. Euro Surveill. 2014;19:7–9 .PubMedHirayama T, Mizuno Y, Takeshita N, Kotaki A, Tajima S, Omatsu T, Detection of dengue virus genome in urine by real-time reverse transcriptase PCR: a laboratory diagnostic method useful after disappearance of the genome in serum. J Clin Microbiol. 2012;50:2047–52 and. DOIPubMedHayes EB. Zika virus outside Africa. Emerg Infect Dis. 2009;15:1347–50 . DOIPubMedBesnard M, Lastère S, Teissier A, Cao-Lormeau VM, Musso D. Evidence of perinatal transmission of Zika virus, French Polynesia, December 2013 and February. Euro Surveill. 2014;19:20751[cited 2014 Dec 17].http://www.eurosurveillance.org/ViewArticle.aspx?ArticleId=20751PubMedMusso D, Nhan T, Robin E, Roche C, Bierlaire D, Zizou K, Potential for Zika virus transmission through blood transfusion demonstrated during an outbreak in French Polynesia, November 2013 to February 2014. Euro Surveill.2014;19:20771 .PubMedFoy BD, Kobylinski KC, Chilson Foy JL, Blitvich BJ, Travassos da Rosa A, Haddow AD, Probable non-vector-borne transmission of Zika virus, Colorado, USA. Emerg Infect Dis. 2011;17:880–2 . DOIPubMedStefanovic KB, Gregg PC, Soung M. Evaluation and treatment of hematospermia. Am Fam Physician. 2009;80:1421–7.PubMedHabu A, Murakami Y, Ogasa A, Fujisaki Y. Disorder of spermatogenesis and viral discharge into semen in boars infected with Japanese encephalitis virus. Uirusu. 1977;27:21–6 . DOIPubMedBarzon L, Pacenti M, Franchin E, Pagni S, Martello T, Cattai M, Excretion of West Nile virus in urine during acute infection. J Infect Dis. 2013;208:1086–92 . DOIPubMedTonry JH, Xiao SY, Siirin M, Chen H, da Rosa AP, Tesh RB, Persistent shedding of West Nile virus in urine of experimentally infected hamsters. Am J Trop Med Hyg. 2005;72:320–4 .PubMedDomingo C, Yactayo S, Agbenu E, Demanou M, Schulz AR, Daskalow K, Detection of yellow fever 17D genome in urine. J Clin Microbiol. 2011;49:760–2 . DOIPubMed Suggested citation for this article: Musso D, Roche C, Robin E, Nhan T, Teissier A, Cao-Lormeau VM. Potential sexual transmission of Zika virus. Emerg Infect Dis. 2015 Feb [date cited]. http://dx.doi.org/10.3201/eid2102.141363 DOI: 10.3201/eid2102.141363

ConclusionsThe ZIKV natural transmission cycle involves mosquitoes, especially Aedes spp. (7), but perinatal transmission (8) and potential risk for transfusion-transmitted ZIKV infections has also been demonstrated (9). Moreover, ZIKV transmission by sexual intercourse has been suggested by Foy et al. (10), who described a patient who was infected with ZIKV in southeastern Senegal in 2008. After returning to his home in Colorado, United States, he experienced common symptoms of ZIKV infection and symptoms of prostatitis. Four days later, he observed signs of hematospermia, and on the same day, his wife had symptoms of ZIKV infection. Because the wife of the patient had not traveled out of the United States during the previous year and had sexual intercourse with him 1 day after he returned home, transmission by semen was suggested. ZIKV infection of the patient and his wife was confirmed by serologic testing, but the presence of ZIKV in the semen of the patient was not investigated. Infectious organisms, especially sexually transmitted microorganisms including viruses (human papillomavirus or herpes simplex virus), are known to be etiologic agents of hematospermia (11). To our knowledge, before the report of Foy et al. (10) and this study, arbovirus infections in humans had not been reported to be associated with hematospermia, and no arboviruses had been isolated from human semen. We detected a high ZIKV RNA load and replicative ZIKV in semen samples, but ZIKV remained undetectable by rRT-PCR in the blood sample collected at the same time. These results suggest that viral replication may have occurred in the genital tract, but we do not know when this replication started and how long it lasted. The fact that the patient had no common symptoms of ZIKV acute infection concomitantly to hematospermia suggests that the viremic phase occurred upstream, probably during the first or second episode of mild fever, headache, and arthralgia. The detection of ZIKV in both urine and semen is consistent with the results obtained in a study of effects of Japanese encephalitis virus, another flavivirus, on boars. The virus was isolated from urine and semen of experimentally infected animals, and viremia developed in female boars that artificially inseminated with the infectious semen (12). Flaviviruses have also been detected in urine of persons infected with West Nile virus (WNV). WNV RNA was detected in urine for a longer time and with higher RNA load than in plasma (13). WNV antigens were detected in renal tubular epithelial cells, vascular endothelial cells, and macrophages of kidneys from infected hamsters (14), suggesting that persistent shedding of WNV in urine was caused by viral replication in renal tissue. Dengue virus (DENV) RNA and DENV nonstructural protein 1 antigen were also detected in urine samples for a longer time than in blood, but infectious DENV has not been isolated in culture. Hirayama et al. concluded that the detection of DENV by rRT-PCR was useful to confirm DENV infections after the viremic phase (6). Also, yellow fever virus RNA was isolated from the urine of vaccinated persons (15), and Saint Louis encephalitis viral antigens, but not infective virus, have been detected in urine samples from infected patients (10). Our findings support the hypothesis that ZIKV can be transmitted by sexual intercourse. Furthermore, the observation that ZIKV RNA was detectable in urine after viremia clearance in blood suggests that, as found for DENV and WNV infections, urine samples can yield evidence ofZIKV for late diagnosis, but more investigation is needed. Dr. Musso is a medical doctor and director of the Diagnosis Medical Laboratory and the Unit of Emerging Infectious Diseases of the Institut Louis Malardé, Papeete, Tahiti, French Polynesia. His research programs target endemic infectious diseases, especially arbovirus infections, leptospirosis, tuberculosis, and lymphatic filariasis. AcknowledgmentWe obtained written informed consent from the patient for publication of this report, and publication of data related to ZIKV infections have been approved by the Ethics Committee of French Polynesia under reference 66/CEPF.