niman Posted July 28, 2016 Report Posted July 28, 2016 CORRESPONDENCEMiscarriage Associated with Zika Virus InfectionJuly 27, 2016DOI: 10.1056/NEJMc1605898http://www.nejm.org/doi/full/10.1056/NEJMc1605898?query=featured_home
niman Posted July 28, 2016 Author Report Posted July 28, 2016 Annemiek A. van der Eijk, M.D., Ph.D.Erasmus Medical Center, Rotterdam, the Netherlands [email protected]Perry J. van Genderen, M.D., Ph.D.Harbour Hospital, Rotterdam, the NetherlandsRob 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 NetherlandsSupported 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 NEJM.org.Drs. van der Eijk, van Genderen, and Verdijk contributed equally to this letter.This letter was published on July 27, 2016, at NEJM.org.
niman Posted July 28, 2016 Author Report Posted July 28, 2016 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 NEJM.org.)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 (www.european-virus-archive.com/virus/zika-virus-strain-suriname-2016).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 1Zika 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.
niman Posted July 28, 2016 Author Report Posted July 28, 2016 3 References1Sarno M, Sacramento GA, Khouri R, et al. Zika virus infection and stillbirths: a case of hydrops fetalis, hydranencephaly and fetal demise. PLoS Negl Trop Dis2016;10:e0004517-e0004517CrossRef | Web of Science2Martines RB, Bhatnagar J, Keating 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;65:159-160CrossRef | Web of Science | Medline3Driggers RW, Ho C-Y, Korhonen EM, et al. Zika virus infection with prolonged maternal viremia and fetal brain abnormalities. N Engl J Med 2016;374:2142-2151Free Full Text | Medline
niman Posted July 28, 2016 Author Report Posted July 28, 2016 LOCUS KU937936 10542 bp RNA linear VRL 02-MAY-2016 DEFINITION Zika virus isolate ZIKVNL00013 polyprotein gene, complete cds. ACCESSION KU937936 VERSION KU937936.1 GI:1013874252 KEYWORDS . 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 ##Assembly-Data-END## FEATURES Location/Qualifiers source 1..10542 /organism="Zika virus" /mol_type="genomic RNA" /isolate="ZIKVNL00013" /host="Homo sapiens" /db_xref="taxon:64320" /country="Suriname" /collection_date="11-Feb-2016" /note="passage details: Vero cells" CDS 88..10359 /note="C-prM/M-E-NS1-NS2a-NS2b-NS3-NS4A-NS4B-NS5" /codon_start=1 /product="polyprotein" /protein_id="AMU04506.1" /db_xref="GI:1013874253" /translation="MKNPKKKSGGFRIVNMLKRGVARVSPFGGLKRLPAGLLLGHGPI RMVLAILAFLRFTAIKPSLGLINRWGSVGKKEAMEIIKKFKKDLAAMLRIINARKEKK RRGADTSVGIVGLLLTTAMAAEVTRRGSAYYMYLDRNDAGEAISFPTTLGMNKCYIQI MDLGHMCDATMSYECPMLDEGVEPDDVDCWCNTTSTWVVYGTCHHKKGEARRSRRAVT LPSHSTRKLQTRSQTWLESREYTKHLIRVENWIFRNPGFALAAAAIAWLLGSSTSQKV IYLVMILLIAPAYSIRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVTVMAQDKPTVDIE LVTTTVSNMAEVRSYCYEASISDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWG NGCGLFGKGSLVTCAKFACSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIVNDTGHET DENRAKVEITPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWF HDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAE MDGAKGRLSSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKIPAETLHGTVTVEVQYAG TDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPFGDSYIVIGVG EKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFGSVGGALNSLGKGIHQIFG AAFKSLFGGMSWFSQILIGTLLMWLGLNTKNGSISLMCLALGGVLIFLSTAVSADVGC SVDFSKKETRCGTGVFVYNDVEAWRDRYKYHPDSPRRLAAAVKQAWEDGICGISSVSR MENIMWSSVEGELNAILEENGVQLTVVVGSVKNPMWRGPQRLPVPVNELPHGWKAWGK SYFVRAAKTNNSFVVDGDTLKECPLKHRAWNSFLVEDHGFGVFHTSVWLKVREDYSLE CDPAVIGTAVKGKEAVHSDLGYWIESEKNDTWRLKRAHLIEMKTCEWPKSHTLWTDGI EESDLIIPKSLAGPLSHHNTREGYRTQMKGPWHSEELEIRFEECPGTKVHVEETCGTR GPSLRSTTASGRVIEEWCCRECTMPPLSFRAKDGCWYGMEIRPRKEPESNLVRSMVTA GSTDHMDHFSLGVLVILLMVQEGLKKRMTTKIIISTSMAVLVAMILGGFSMSDLAKLA ILMGATFAEMNTGGDVAHLALIAAFKVRPALLVSFIFRANWTPRESMLLALASCLLQT 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 SYEAPTQGSASSLINGVVRLLSKPWDVVTGVTGIAMTDTTPYGQQRVFKEKVDTRVPD PQEGTRQVMSMVSSWLWKELGKHKRPRVCTKEEFINKVRSNAALGAIFEEEKEWKTAV EAVNDPRFWALVDKEREHHLRGECQSCVYNMMGKREKKQGEFGKAKGSRAIWYMWLGA RFLEFEALGFLNEDHWMGRENSGGGVEGLGLQRLGYVLEEMSRIPGGRMYADDTAGWD TRISRFDLENEALITNQMEKGHRALALAIIKYTYQNKVVKVLRPAEKGKTVMDIISRQ DQRGSGQVVTYALNTFTNLVVQLIRNMEAEEVLEMQDLWLLRRSEKVTNWLQSNGWDR LKRMAVSGDDCVVKPIDDRFAHALRFLNDMGKVRKDTQEWKPSTGWDNWEEVPFCSHH FNKLHLKDGRSIVVPCRHQDELIGRARVSPGAGWSIRETACLAKSYAQMWQLLYFHRR DLRLMANAICSSVPVDWVPTGRTTWSIHGKGEWMTTEDMLVVWNRVWIEENDHMEDKT PVTKWTDIPYLGKREDLWCGSLIGHRPRTTWAENIKNTVNMVRRIIGDEEKYMDYLST QVRYLGEEGSTPGVL" ORIGIN 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
niman Posted July 28, 2016 Author Report Posted July 28, 2016 MC Map Updatehttps://www.google.com/maps/d/u/0/edit?mid=1RcVTrkYW6hax_iITjKUkEcBCVeI
Recommended Posts
Please sign in to comment
You will be able to leave a comment after signing in
Sign In Now