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Zika Linked Miscarriage Netherlands ex-Suriname - NEJM

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Annemiek A. van der Eijk, M.D., Ph.D.
Erasmus Medical Center, Rotterdam, the Netherlands 

Perry J. van Genderen, M.D., Ph.D.
Harbour Hospital, Rotterdam, the Netherlands

Rob M. Verdijk, M.D., Ph.D.
Chantal B. Reusken, Ph.D.
Ramona Mögling, Ph.D.
Jeroen J.A. van Kampen, M.D., Ph.D.
Widagdo Widagdo, M.D.
Georgina I. Aron, B.Sc.
Corine H. GeurtsvanKessel, M.D., Ph.D.
Suzan D. Pas, Ph.D.
V. Stalin Raj, Ph.D.
Bart L. Haagmans, Ph.D.
Marion P.G. Koopmans, D.V.M., Ph.D.
Erasmus Medical Center, Rotterdam, the Netherlands

Supported by the Horizon 2020 research and innovation program of the European Union (grant agreement no. 643476) and by the European Virus Archive Goes Global project, which received a grant (653316) from the European Union Horizon 2020 Framework Program for Research and Innovation.

Disclosure forms provided by the authors are available with the full text of this letter at

Drs. van der Eijk, van Genderen, and Verdijk contributed equally to this letter.

This letter was published on July 27, 2016, at

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To the Editor:

Data linking Zika virus (ZIKV) infection to fetal death have been reported in only a handful of cases to date.1,2 Here, we present a case of ZIKV infection in a woman who had a miscarriage. ZIKV was detected in the fetal tissue, and ZIKV viremia lasted for at least 21 days.

In January 2016, a 31-year-old woman who was 10 weeks pregnant visited an outpatient clinic in Rotterdam, the Netherlands, because of a 2-day history of headache, mild arthralgia in both wrists and the left knee, and a pruritic, macular rash. The symptoms had begun the day after her return from a 3.5-week trip to Suriname, which borders on northern Brazil. During her visit, she had not used malaria chemoprophylaxis or personal protective measures, such as insect repellents.

The patient’s medical history was uneventful, as was her pregnancy. When she was a child, she had received vaccines according to the regular vaccination program in Suriname, where she had lived until her early twenties. She was vaccinated against yellow fever at the age of 5 years.

A physical examination revealed normal vital-sign measurements and an absence of fever. A discrete macular rash was noted on the patient’s trunk and both arms. The joints were not visibly swollen or warm but mildly painful with movement. Blood and urine samples were obtained at regular intervals after the physical examination.

The patient’s symptoms resolved spontaneously after 6 days. On day 14 after the onset of symptoms, ultrasonography performed at a routine prenatal screening visit (estimated gestational age, 11 weeks and 4 days) showed no fetal heartbeat.

One week later, 21 days after the onset of ZIKV-associated disease, amniocentesis was performed, followed by dilation and curettage. Amniotic fluid and fetal and placental tissue were obtained for further laboratory analysis. (A detailed description of the methods is provided in theSupplementary Appendix, available with the full text of this letter at

The amniotic fluid and fetal and placental tissue tested positive for ZIKV on semiquantitative real-time reverse-transcriptase–polymerase-chain-reaction (RT-PCR) assays and cell-culture isolation; these results were confirmed by means of whole-genome sequencing (GenBank accession number, KU937936) (Fig. S1 through S4 in the Supplementary Appendix). The viral isolate (reference number, 011V-01621) can be obtained through the European Virus Archive Goes Global catalogue (

Serum and urine samples obtained from the patient were positive for ZIKV on semiquantitative RT-PCR at multiple time points. A serum specimen was still positive for ZIKV on semiquantitative RT-PCR 21 days after the onset of clinical symptoms, although no ZIKV was detected in urine specimens after day 12. A more than quadrupling of neutralizing antibody titers against ZIKV was observed between days 2 and 12. On day 28, ZIKV RNA was no longer detected in serum specimens (see Table S1 in the Supplementary Appendix). Possible genetic disorders and other congenital infections were not identified (see Sections 8 and 9 in the Supplementary Appendix).

The findings on histopathological analysis of placental tissue specimens were consistent with intrauterine fetal death 1 week before curettage was performed. Histopathological and immunohistochemical investigation with the use of CD45 and CD3 antibodies to detect inflammation did not show evidence of increased inflammatory-cell infiltration. In situ hybridization with the use of ZIKV-specific probes, as compared with control probes, revealed that placental amniotic epithelium was positive for ZIKV RNA, whereas fetal chorion and trophoblasts and maternal decidua did not show evidence of ZIKV infection. In addition, positive staining of fetal mesenchymal cells with affinity for perichondrium was observed (Figure 1FIGURE 1nejmc1605898_f1.gifZika Virus (ZIKV) Infection of Amniotic Epithelial Cells and Fetal Mesenchymal Cells Detected with the Use of a Specific ZIKV RNA Probe.). Because of autolysis, no recognizable fetal brain tissue was present. Other fetal tissues, including those from the cartilage, kidneys, adrenal glands, gut, and eyes, were negative for ZIKV. These observations were confirmed on immunostaining of double-stranded RNA in fetal tissues (Fig. S5 in theSupplementary Appendix).

We found evidence of ZIKV infection in amniotic epithelial cells and in fetal mesenchymal cells with affinity for perichondrium. Our observation indicates that ZIKV replicates in pluripotent (amniotic stem) cells involved in early-stage embryo development. The observation of prolonged viremia until day 21 in the patient is in concordance with the findings of Driggers et al.3 and provides further data for consideration in the ongoing development of testing algorithms in pregnant women. These algorithms are currently based on the assumption that ZIKV viremia can be detected only up to 7 days.

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3 References

  1. 1

    Sarno MSacramento GAKhouri R, et al. Zika virus infection and stillbirths: a case of hydrops fetalis, hydranencephaly and fetal demise. PLoS Negl Trop Dis2016;10:e0004517-e0004517
    CrossRef | Web of Science

  2. 2

    Martines RBBhatnagar JKeating MK, et al. Notes from the field: evidence of Zika virus infection in brain and placental tissues from two congenitally infected newborns and two fetal losses — Brazil, 2015. MMWR Morb Mortal Wkly Rep 2016;65:159-160
    CrossRef | Web of Science | Medline

  3. 3

    Driggers RWHo C-YKorhonen EM, et al. Zika virus infection with prolonged maternal viremia and fetal brain abnormalities. N Engl J Med 2016;374:2142-2151
    Free Full Text | Medline

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LOCUS       KU937936               10542 bp    RNA     linear   VRL 02-MAY-2016
DEFINITION  Zika virus isolate ZIKVNL00013 polyprotein gene, complete cds.
VERSION     KU937936.1  GI:1013874252
SOURCE      Zika virus
  ORGANISM  Zika virus
            Viruses; ssRNA viruses; ssRNA positive-strand viruses, no DNA
            stage; Flaviviridae; Flavivirus.
REFERENCE   1  (bases 1 to 10542)
  AUTHORS   Stalin Raj,V., van der Eijk,A., van Genderen,P.J., Verdijk,R.,
            Dijkman,A., Reusken,C.B., van Kampen,J.J.A., Aron,G.I.,
            Geurtsvankessel,C.H., Pas,S.D., Haagmans,B.L. and Koopmans,M.P.G.
  TITLE     Direct Submission
  JOURNAL   Submitted (18-MAR-2016) Department of Viroscience, Erasmus Medical
            Center, Dr Molewaterplein 50, Rotterdam 3015 GE, the Netherlands
COMMENT     ##Assembly-Data-START##
            Assembly Method       :: CLC Bio v. 4.9
            Sequencing Technology :: Sanger dideoxy sequencing; 454
FEATURES             Location/Qualifiers
     source          1..10542
                     /organism="Zika virus"
                     /mol_type="genomic RNA"
                     /host="Homo sapiens"
                     /note="passage details: Vero cells"
     CDS             88..10359
        1 cagactgcga cagttcgagt ttgaagcgaa agctagcaac agtatcaaca ggttttattt
       61 tggatttgga aacgagagtt tctggtcatg aaaaacccaa aaaagaaatc cggaggattc
      121 cggattgtca atatgctaaa acgcggagta gcccgtgtga gcccctttgg gggcttgaag
      181 aggctgccag ccggacttct gctgggtcat gggcccatca ggatggtctt ggcgattcta
      241 gcctttttga gattcacggc aatcaagcca tcactgggtc tcatcaatag atggggttca
      301 gtggggaaaa aagaggctat ggaaataata aagaagttca agaaagatct ggctgccatg
      361 ctgagaataa tcaatgctag gaaggagaag aagagacgag gcgcagatac tagtgtcgga
      421 attgttggcc tcctgctgac cacagctatg gcagcggagg tcactagacg tgggagtgca
      481 tactatatgt acttggacag aaacgatgct ggggaggcca tatcttttcc aaccacattg
      541 gggatgaata agtgttatat acagatcatg gatcttggac acatgtgtga tgccaccatg
      601 agctatgaat gccctatgct ggatgagggg gtggaaccag atgacgtcga ttgttggtgc
      661 aacacgacgt caacttgggt tgtgtacgga acctgccatc acaaaaaagg tgaagcacgg
      721 agatctagaa gagctgtgac gctcccctcc cattccacta ggaagctgca aacgcggtcg
      781 caaacctggt tggaatcaag agaatacaca aagcacttga ttagagtcga aaattggata
      841 ttcaggaacc ctggcttcgc gttagcagca gctgccatcg cttggctttt gggaagctca
      901 acgagccaaa aagtcatata cttggtcatg atactgctga ttgccccggc atacagcatc
      961 aggtgcatag gagtcagcaa tagggacttt gtggaaggta tgtcaggtgg gacttgggtt
     1021 gatgttgtct tggaacatgg aggttgtgtc accgtaatgg cacaggacaa accgactgtc
     1081 gacatagagc tggttacaac aacagtcagc aacatggcgg aggtaagatc ctactgctat
     1141 gaggcatcaa tatcagacat ggcttcggac agccgctgcc caacacaagg tgaagcctac
     1201 cttgacaagc aatcagacac tcaatatgtc tgcaaaagaa cgttagtgga cagaggctgg
     1261 ggaaatggat gtggactttt tggcaaaggg agcctggtga catgcgctaa gtttgcatgc
     1321 tccaagaaaa tgaccgggaa gagcatccag ccagagaatc tggagtaccg gataatgctg
     1381 tcagttcatg gctcccagca cagtgggatg atcgttaatg acacaggaca tgaaactgat
     1441 gagaatagag cgaaagttga gataacgccc aattcaccaa gagccgaagc caccctgggg
     1501 ggttttggaa gcctaggact tgattgtgaa ccgaggacag gccttgactt ttcagatttg
     1561 tattacttga ctatgaataa caagcactgg ttggttcaca aggagtggtt ccacgacatt
     1621 ccattacctt ggcacgctgg ggcagacacc ggaactccac actggaacaa caaagaagca
     1681 ctggtagagt tcaaggacgc acatgccaaa aggcaaactg tcgtggttct agggagtcaa
     1741 gaaggagcag ttcacacggc ccttgctgga gctctggagg ctgagatgga tggtgcaaag
     1801 ggaaggctgt cctctggcca cttgaaatgt cgcctgaaaa tggataaact tagattgaag
     1861 ggcgtgtcat actccttgtg tactgcagcg ttcacattca ccaagatccc ggctgaaaca
     1921 ctgcacggga cagtcacagt ggaggtacag tacgcaggga cagatggacc ttgcaaggtt
     1981 ccagctcaga tggcggtgga catgcaaact ctgaccccag ttgggaggtt gataaccgct
     2041 aaccccgtaa tcactgaaag cactgagaac tctaagatga tgctggaact tgatccacca
     2101 tttggggact cttacattgt cataggagtc ggggagaaga agatcaccca ccactggcac
     2161 aggagtggca gcaccattgg aaaagcattt gaagccactg tgagaggtgc caagagaatg
     2221 gcagtcttag gagacacagc ctgggacttt ggatcagttg gaggcgctct caactcattg
     2281 ggcaagggca tccatcaaat ttttggagca gctttcaaat cattgtttgg aggaatgtcc
     2341 tggttctcac aaattctcat tggaacgttg ctgatgtggt tgggtctgaa cacaaagaat
     2401 ggatctattt cccttatgtg cttggcctta gggggagtgt tgatcttctt atccacagcc
     2461 gtctctgctg atgtggggtg ctcggtggac ttctcaaaga aggagacgag atgcggtaca
     2521 ggggtgttcg tctataacga cgttgaagcc tggagggaca ggtacaagta ccatcctgac
     2581 tccccccgta gattggcagc ggcagtcaag caagcctggg aagatggtat ctgcgggatc
     2641 tcctctgttt caagaatgga aaacatcatg tggagctcag tagaagggga gctcaacgca
     2701 atcctggaag agaatggagt tcaactgacg gtcgttgtgg gatctgtaaa aaaccccatg
     2761 tggagaggtc cacagagatt gcccgtgcct gtgaacgagc tgccccacgg ctggaaggct
     2821 tgggggaaat cgtacttcgt cagagcagca aagacaaata acagctttgt cgtggatggt
     2881 gacacactga aggaatgccc actcaaacat agagcatgga acagctttct tgtggaggat
     2941 catgggttcg gggtatttca cactagtgtc tggctcaagg ttagagaaga ttactcatta
     3001 gagtgtgatc cagccgttat tggaacagct gttaagggaa aggaggctgt acacagtgat
     3061 ctaggctact ggattgagag tgagaagaat gacacatgga ggctgaagag ggcccatctg
     3121 atcgagatga aaacatgtga atggccaaag tcccacacat tgtggacaga tggaatagaa
     3181 gagagtgatc tgatcatacc caagtcttta gctgggccac tcagccatca caataccaga
     3241 gagggctaca ggacccaaat gaaagggcca tggcacagtg aagagcttga aattcggttt
     3301 gaggaatgtc caggcactaa ggtccacgtg gaggaaacat gtggaacaag aggaccatct
     3361 ctgagatcaa ccactgcaag cggaagggtg atcgaggaat ggtgctgcag ggagtgcaca
     3421 atgcccccac tgtcgttccg ggctaaagat ggctgttggt atggaatgga gataaggccc
     3481 aggaaagaac cagaaagcaa cttagtaagg tcaatggtga ctgcaggatc aactgatcac
     3541 atggaccact tctcccttgg agtgcttgtg atcctgctca tggtgcagga agggctgaag
     3601 aagagaatga ccacaaagat catcataagc acatcaatgg cagtgctggt agctatgatc
     3661 ctgggaggat tttcaatgag tgacctggct aagcttgcaa ttttgatggg tgccaccttc
     3721 gcggaaatga acactggagg agatgtagct catctggcgc tgatagcggc attcaaagtc
     3781 agaccagcgt tgctggtatc tttcatcttc agagctaatt ggacaccccg tgaaagcatg
     3841 ctgctggcct tggcctcgtg tcttttgcaa actgcgatct ccgccttgga aggcgacctg
     3901 atggttctca tcaatggttt tgctttggcc tggttggcaa tacgagcgat ggttgttcca
     3961 cgcactgata acatcacctt ggcaatcctg gctgctctga caccactggc ccggggcaca
     4021 ctgcttgtgg cgtggagagc aggccttgct acttgcgggg ggtttatgct cctctctttg
     4081 aagggaaaag gcagtgtgaa gaagaactta ccatttgtca tggccctggg actaaccgct
     4141 gtgaggctgg tcgaccccat caacgtggtg ggactgctgt tgctcacaag gagtgggaag
     4201 cggagctggc cccctagcga agtactcaca gctgtcggcc tgatatgcgc attggctgga
     4261 gggttcgcca aggcagatat agagatggct gggcccatgg ccgcggtcgg tctgctaatt
     4321 gtcagttacg tggtctcagg aaagagtgtg gacatgtaca ttgaaagagc aggtgacatc
     4381 acatgggaaa aagatgcgga agtcactgga aacagtcccc ggctcgatgt ggcgctagat
     4441 gagagtggtg atttctccct ggtggaggat gacggtcccc ccatgagaga gatcatactc
     4501 aaggtggtcc tgatgaccat ctgtggcatg aacccaatag ccataccctt tgcagctgga
     4561 gcgtggtacg tatacgtgaa gactggaaaa aggagtggtg ctctatggga tgtgcctgct
     4621 cccaaggaag taaaaaaggg ggagaccaca gatggagtgt acagagtaat gactcgtaga
     4681 ctgctaggtt caacacaagt tggagtggga gttatgcaag agggggtctt tcacactatg
     4741 tggcacgtca caaaaggatc cgcgctgaga agcggtgaag ggagacttga tccatactgg
     4801 ggagatgtca agcaggatct ggtgtcatac tgtggtccat ggaagctaga tgccgcctgg
     4861 gacgggcaca gcgaggtgca gctcttggcc gtgccccccg gagagagagc gaggaacatc
     4921 cagactctgc ccggaatatt taagacaaag gatggggaca ttggagcggt tgcgctggat
     4981 tacccagcag gaacttcagg atctccaatc ctagacaagt gtgggagagt gataggactt
     5041 tatggcaatg gggtcgtgat caaaaatggg agttatgtta gtgccatcac ccaagggagg
     5101 agggaggaag agactcctgt tgagtgcttc gagccctcga tgctgaagaa gaagcagcta
     5161 actgtcttag acttgcatcc tggagctggg aaaaccagga gagttcttcc tgaaatagtc
     5221 cgtgaagcca taaaaacaag actccgtact gtgatcttag ctccaaccag ggttgtcgct
     5281 gctgaaatgg aggaggccct tagagggctt ccagtgcgtt atatgacaac agcagtcaat
     5341 gtcacccact ctggaacaga aatcgtcgac ttaatgtgcc atgccacctt cacttcacgt
     5401 ctactacagc caatcagagt ccccaactat aatctgtata ttatggatga ggcccacttc
     5461 acagatccct caagtatagc agcaagagga tacatttcaa caagggttga gatgggcgag
     5521 gcggctgcca tcttcatgac cgccacgcca ccaggaaccc gtgacgcatt tccggactcc
     5581 aactcaccaa tcatggacac cgaagtggaa gtcccagaga gagcctggag ctcaggcttt
     5641 gattgggtga cggatcattc tggaaaaaca gtttggtttg ttccaagcgt gaggaacggc
     5701 aatgagatcg cagcttgtct gacaaaggct ggaaaacggg tcatacagct cagcagaaag
     5761 acttttgaga cagagttcca gaaaacaaaa catcaagagt gggactttgt cgtgacaact
     5821 gacatttcag agatgggcgc caactttaaa gctgaccgtg tcatagattc caggagatgc
     5881 ctaaagccgg tcatacttga tggcgagaga gtcattctgg ctggacccat gcctgtcaca
     5941 catgccagcg ctgcccagag gagggggcgc ataggcagga atcccaacaa acctggagat
     6001 gagtatctgt atggaggtgg gtgcgcagag actgacgaag accatgcaca ctggcttgaa
     6061 gcaagaatgc tccttgacaa tatttacctc caagatggcc tcatagcctc gctctatcga
     6121 cctgaggccg acaaagtagc agccattgag ggagagttca agcttaggac ggagcaaagg
     6181 aagacctttg tggaactcat gaaaagagga gatcttcctg tttggctggc ctatcaggtt
     6241 gcatctgccg gaataaccta cacagataga agatggtgct ttgatggcac gaccaacaac
     6301 accataatgg aagacagtgt gccggcagag gtgtggacca gacacggaga gaaaagagtg
     6361 ctcaaaccga ggtggatgga cgccagagtt tgttcagatc atgcggccct gaagtcattc
     6421 aaggagtttg ccgctgggaa aagaggagcg gcttttggag tgatggaagc cctgggaaca
     6481 ctgccaggac acatgacaga gagattccag gaagccattg acaacctcgc tgtgctcatg
     6541 cgggcagaga ctggaagcag gccttacaaa gccgcggcgg cccaattgcc ggagacccta
     6601 gagaccataa tgcttttggg gttgctggga acagtctcgc tgggaatctt cttcgtcttg
     6661 atgaggaaca agggcatagg gaagatgggc tttggaatgg tgactcttgg ggccagcgca
     6721 tggctcatgt ggctctcgga aattgagcca gccaggattg catgtgtcct cattgttgtg
     6781 ttcctattgc tggtggtgct catacctgag ccagaaaagc aaagatctcc ccaggacaac
     6841 caaatggcaa tcatcatcat ggtagcagta ggtcttctgg gcttgattac cgccaatgaa
     6901 ctcggatggt tggagagaac aaagagtgac ctaagccatc taatgggaag gagagaggag
     6961 ggggcaacca taggattctc aatggacatt gacctgcggc cagcctcagc ttgggccatc
     7021 tatgctgcct tgacaacttt cattacccca gccgtccaac atgcagtgac cacctcatac
     7081 aacaactact ccttaatggc gatggccacg caagctggag tgttgtttgg tatgggcaaa
     7141 gggatgccat tctacgcatg ggactttgga gtcccgctgc taatgatagg ttgctactca
     7201 caattaacac ccctgaccct aatagtggcc atcattttgc tcgtggcgca ctacatgtac
     7261 ttgatcccag ggctgcaggc agcagctgcg cgtgctgccc agaagagaac ggcagctggc
     7321 atcatgaaga accctgttgt ggatggaata gtggtgactg acattgacac aatgacaatt
     7381 gacccccaag tggagaaaaa gatgggacag gtgctactca tagcagtagc cgtctccagc
     7441 gccatactgt cgcggaccgc ctgggggtgg ggggaggctg gggccctgat cacagccgca
     7501 acttccactt tgtgggaagg ctctccgaac aagtactgga actcctctac agccacttca
     7561 ctgtgtaaca tttttagggg aagttacttg gctggagctt ctctaatcta cacagtaaca
     7621 agaaacgctg gcttggtcaa gagacgtggg ggtggaacag gagagaccct gggagagaaa
     7681 tggaaggccc gcttgaacca gatgtcggcc ctggagttct actcctacaa aaagtcaggc
     7741 atcaccgagg tgtgcagaga agaggcccgc cgcgccctca aggacggtgt ggcaacggga
     7801 ggccatgctg tgtcccgagg aagtgcaaag ctgagatggt tggtggagcg gggatacctg
     7861 cagccctatg gaaaggtcat tgatcttgga tgtggcagag ggggctggag ttactacgcc
     7921 gccaccatcc gcaaagttca agaagtgaaa ggatacacaa aaggaggccc tggtcatgaa
     7981 gaacccgtgt tggtgcaaag ctatgggtgg aacatagtcc gtcttaagag tggggtggac
     8041 gtctttcata tggcggctga gccgtgtgac acgttgctgt gtgacatagg tgagtcatca
     8101 tctagtcctg aagtggaaga agcacggacg ctcagagtcc tctctatggt gggggattgg
     8161 cttgaaaaaa gaccaggagc cttttgtata aaagtgttgt gcccatacac cagcactatg
     8221 atggaaaccc tggagcgact gcagcgtagg tatgggggag gactggtcag agtgccactc
     8281 tcccgcaact ctacacatga gatgtactgg gtctctggag cgaaaagcaa caccataaaa
     8341 agtgtgtcca ccacgagcca gctcctcttg gggcgcatgg acgggcctag gaggccagtg
     8401 aaatatgagg aggatgtgaa tctcggctct ggcacgcggg ctgtggtaag ctgcgctgaa
     8461 gctcccaaca tgaagatcat tggtaaccgc attgaaagga tccgcagtga gcacgcggaa
     8521 acgtggttct ttgacgagaa ccacccatat aggacatggg cttaccatgg aagctatgag
     8581 gcccccacac aagggtcagc gtcctctcta ataaacgggg ttgtcaggct cctgtcaaaa
     8641 ccctgggatg tggtgactgg agtcacagga atagccatga ccgacaccac accgtatggt
     8701 cagcaaagag ttttcaagga aaaagtggac actagggtgc cagaccccca agaaggcact
     8761 cgtcaggtta tgagcatggt ctcttcctgg ttgtggaaag agctaggcaa acacaaacgg
     8821 ccacgagtct gtaccaaaga agagttcatc aacaaggttc gtagcaatgc agcattaggg
     8881 gcaatatttg aagaggaaaa agagtggaag actgcagtgg aagctgtgaa cgatccaagg
     8941 ttctgggctc tagtggacaa ggaaagagag caccacctga gaggagagtg ccagagttgt
     9001 gtgtacaaca tgatgggaaa aagagaaaag aaacaagggg aatttggaaa ggccaagggc
     9061 agccgcgcca tctggtatat gtggctaggg gctagatttc tagagttcga agccctcgga
     9121 ttcttgaacg aggatcactg gatggggaga gagaactcag gaggtggtgt tgaagggctg
     9181 ggattacaaa gactcggata tgtcctagaa gagatgagtc gtataccagg aggaagaatg
     9241 tatgcagatg acactgctgg ctgggacacc cgcattagca ggtttgatct ggagaatgaa
     9301 gctctaatca ccaaccaaat ggagaaaggg cacagggcct tggcattggc cataatcaag
     9361 tacacatacc aaaacaaagt ggtaaaggtc cttagaccag ctgaaaaagg gaaaacagtt
     9421 atggacatta tttcgagaca agaccaaagg gggagcggac aagttgtcac ttacgctctt
     9481 aacacattta ccaacctagt ggtgcaactc attcggaata tggaggctga ggaagttcta
     9541 gagatgcaag acttgtggct gctgcggagg tcagagaaag tgaccaactg gttgcagagc
     9601 aacggatggg ataggctcaa acgaatggca gtcagtggag atgattgcgt tgtgaagcca
     9661 attgatgata ggtttgcaca tgccctcagg ttcttgaatg atatgggaaa agttaggaag
     9721 gacacacaag agtggaaacc ctcaactgga tgggacaact gggaagaagt tccgttttgc
     9781 tcccaccact tcaacaagct ccatctcaag gacgggaggt ccattgtggt tccctgccgc
     9841 caccaagatg aactgattgg ccgggcccgc gtctctccag gggcgggatg gagcatccgg
     9901 gagactgctt gcctagcaaa atcatatgcg caaatgtggc agctccttta tttccacaga
     9961 agggacctcc gactgatggc caatgccatt tgttcatctg tgccagttga ctgggttcca
    10021 actgggagaa ctacctggtc aatccatgga aagggagaat ggatgaccac tgaagacatg
    10081 cttgtggtgt ggaacagagt gtggattgag gagaacgacc acatggaaga caagacccca
    10141 gttacgaaat ggacagacat tccctatttg ggaaaaaggg aagacttgtg gtgtggatct
    10201 ctcatagggc acagaccgcg caccacctgg gctgagaaca ttaaaaacac agtcaacatg
    10261 gtgcgcagga tcataggtga tgaagaaaag tacatggact acctatccac ccaagttcgc
    10321 tacttgggtg aagaagggtc tacacctgga gtgctgtaag caccaatctt aatgttgtca
    10381 ggcctgctag tcagccacag cttggggaaa gctgtgcagc ctgtgacccc cccaggagaa
    10441 gctgggaaac caagcctata gtcaggccga gaacgccatg gcacggaaga agccatgctg
    10501 cctgtgagcc cctcagagga cactgagtca aaaaacccca cg

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