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1 Supplementary appendix This appendix formed part of the original submission and has been peer reviewed. We post it as supplied by the authors. Supplement to: Baud D, Gubler DJ, Schaub B, Lanteri MC, Musso D. An update on Zika virus infection. Lancet 2017; published online June 21. S (17)

2 Supplemental table 1: animal models of ZIKV infections, main results Date Animal model Zika strain Major findings References ADVERSE PREGNANCY OUTCOMES May A129 mice H/PF/2013 (French ZIKV infection early in pregnancy results in infection of (1) Polynesia, 2013) placental cells, IUGR and injury of the fetal brain PFU injection at day of gestation ZIKV RNA load in placenta superior than in maternal blood, suggesting active viral replication in the placenta. May WT C57BL/6 mice (2) Asian ZIKV strain SZ01 (KU866423) isolated from a Chinese patient returning from Samoa in 10 5 PFU injection at day 13.5 of gestation June SJL mice ZIKV isolate from a clinical case in Brazil PFU injection at day 10 of gestation July ICR Mice Asian ZIKV strain SZ PFU injection at day 13.5 of gestation August WT C57BL/6 mice Asian ZIKV isolated in 2010 (Cambodian ZIKV Vertical transmission to fetuses Infection of NPCs is responsible for cortex development leading to microcephaly of the pups Changes in expression of microcephaly or cell cyclerelated genes ZIKV crosses the placenta and cause IUGR and microcephaly. ZIKV infects human cortical progenitor cells in vitro, leading to an increase in cell death by disregulating genes involved in apoptosis and autophagy. Infection of human brain organoids results in a reduction of proliferative zones and disrupted cortical layers. After injection in fetal brain, ZIKV replicates efficiently, infects NPCs and causes microcephaly ZIKV infection leads to NPC cell-cycle arrest and defects in differentiation ZIKV infection induces immune response in brain and apoptosis of post-mitotic neurons ZIKV replicates in the vagina of WT mice. Vaginal ZIKV infection in early pregnancy leads to IUGR (3) (4) (5) 1

3 February 2013 Adult FVB/NJ (JAX#1800) and C57BL/6J (JAX#664) mice strain FSS13025) 2.5x10 4 PFU injection at day of gestation ZIKV HS-2015-BA-01, Brazil 205 and fetal brain infection. Mice deficient in IFN pathways had higher levels of vaginal ZIKV replication leading to abortion when pregnant. ZIKV is responsible for hydrocephalus and arthrogryposis in mice born from ZIKV infected mothers. (6) ADVERSE NEUROLOGICAL OUTCOMES March A129 mice Asian ZIKV (Cambodian Disease and mortality in an age-dependent manner ZIKV strain FSS13025) PFU injection ZIKV detected in the brain (day 3) and caused signs of neurologic disease (day 6) Robust replication noted in the testis. April AG129 mice ZIKV strain H/PF/2013 Systemic dissemination, severe brain pathology and lethal disease (day 7-8) regardless of dose ( ) or age of mice May IRF-TKO Mice H/PF/2013 (French ZIKV injection leads to neurological disease and death (other mice Polynesia, 2013) + other High viral loads in the brain, spinal cord and testes strains not older strains susceptible to 10 3 PFU injection ZIKV) May A129 mice ZIKV strain MP1751, isolated in 1962 from pools of Aedes africanus 10 6 PFU injection September A129 mice H/PF/2013 (French Polynesia, 2013) & ZIKV strain Paraiba PFU injection Severe symptoms (day 6) with ZIKV detection in the blood, brain, spleen, liver and ovaries Conjunctivitis, pan-uveitis and infection of the cornea, iris, optic nerve and retina. Infection is independent of the entry receptors Axl or Mertk, (7) (8) (9) (10) (11) 2

4 November October November December December Janvier 2017 IRF-TKO Mice WT C57BL/6 treated with anti-ifnar1 antibody (antagonize type I IFN signalling) BALB/c mice + dexamethasone 50mg/kg AG129 mice AG129 mice AG129 mice Asian lineage ZIKV strain (FSS13025, 2010 Cambodian isolate) Mouse-adapted African ZIKV (Dakar 41519) Puerto Rico strain PRVABC PFU intraperitoneal infection ZIKA-SMGC PFU intraperitoneal infection ZIKV strain FSS13025 (2010 clinical isolate from Cambodia) 10 5 PFU vaginal infection ZIKV strain (PRVABC59; Puerto Rico 2015) No evidence of congenital ocular disease After retro-orbital injection, ZIKV can infect NPC in the adult mouse brain, leading to cell death and reduced cell proliferation. Persistence of ZIKV in the testis and epididymis of male mice, associated with tissue injury that caused diminished testosterone, inhibin B levels and oligospermia. ZIKV preferentially infected spermatogonia, primary spermatocytes, and Sertoli cells in the testis, resulting in cell death and destruction of the seminiferous tubules. Less damage observed with a contemporary Asian ZIKV strain (H/PF/2013) Disseminated infection and clinical deterioration occurred during viral clearance by host immune response. Inflammatory infiltrates in multiple organs. CD45+ & CD8+ cells in the testis. Type I interferon treatments improved clinical outcome of mice ZIKV infects testis/epididymis, but not prostate/seminal vesicle, leading to male infertility Hormonal staging influences susceptibility to ZIKV vaginal infection, transgenital transmission and persistence of viral replication in female Mice infected during estrus-like phase were resistant, whereas death occurs after infection during diestrus-like phase ZIKV sexual transmission from male that shed ZIKV in semen to female (12) (13) (14) (15) (16) (17) 3

5 June September December November December Pregnant / nonpregnant Rhesus macaques Rhesus macaques Cynomolgus and Rhesus macaques Pregnant pigtail macaque at 28 weeks gestation H/PF/2013 (French Polynesia, 2013) PFU injection GZ01/ (KU820898) isolated from a Chinese male patient returned from Venezuela 10 5 PFU injection Thai and Puerto Rican origin (KF and KU501215) 10 6 PFU injection FSS13025 was isolated in Cambodia in PFU injection Rhesus macaque East African ZIKV strain MR PFU injection RESEARCH ON PRIMATES ZIKV detected in blood, saliva, urine and cerebrospinal fluid. Non-pregnant and pregnant animals remain viremic for 21 versus 57 days, respectively. Neutralizing antibodies detected by 21 days post infection. Rechallenge after 10 weeks suggests protective immunity against homologous strains. Fever and viremia, with robust excretion of ZIKV in urine, saliva, and lacrimal fluid. Systematic infections involving central nervous system and visceral organs ZIKV detected in blood, saliva, urine, cerebrospinal fluid, semen, vaginal secretions, brain, and male and female reproductive tracts. ZIKV cleared from blood and urine within 10 d. Persistence in saliva and seminal fluids. Re-infection with a heterologous strain suggests protective immunity. Identification of ZIKV in fetal brain + periventricular lesions developed within 10 days Arrested fetal brain growth, white matter injury and detection of ZIKV RNA in multiple fetal organs (brain, liver and placenta). Animals were completely protected from subsequent infection with heterologous Asian ZIKV January Rhesus macaque Brazilian ZIKV strain Animals developed short-lived plasma viremias that cleared (23) (18) (19) (20) (21) (22) 4

6 2017 as neutralizing anti- body developed ZIKV RNA could be detected longer in whole blood than in plasma IUGR : Intra Uterine Growth Restriction ; NPC : neural progenitor cells; WT: Wild type; IFN: Interferon; A129 mice strain: IFN-a/b receptor knockout mice leading to lacking type I interferon signalling (Ifnar1-/-). Background: 129/Sv mice AG129 mice strain: IFN-a/b and -g receptor knockout leading to lacking type I interferon signalling; Background: 129/Sv IRF-TKO mice strain: mice triply deficient in INF regulatory factor. Background: C57BL/6 mice. SJL mice strain: immuno-competent mice used in autoimmune encephalomyelitis models. Lower circulating B cells but elevated circulating T cells. References 1. Miner JJ, Cao B, Govero J, Smith AM, Fernandez E, Cabrera OH, Garber C, Noll M, Klein RS, Noguchi KK, Mysorekar IU, Diamond MS.. Zika Virus Infection during Pregnancy in Mice Causes Placental Damage and Fetal Demise. Cell 165: Wu K-Y, Zuo G-L, Li X-F, Ye Q, Deng Y-Q, Huang X-Y, Cao W-C, Qin C-F, Luo Z-G.. Vertical transmission of Zika virus targeting the radial glial cells affects cortex development of offspring mice. Cell Res. 3. Cugola FR, Fernandes IR, Russo FB, Freitas BC, Dias JLM, Guimarães KP, Benazzato C, Almeida N, Pignatari GC, Romero S, Polonio CM, Cunha I, Freitas CL, Brandão WN, Rossato C, Andrade DG, Faria D de P, Garcez AT, Buchpigel CA, Braconi CT, Mendes E, Sall AA, Zanotto PM de A, Peron JPS, Muotri AR, Beltrão-Braga PCB.. The Brazilian Zika virus strain causes birth defects in experimental models. Nature 534: Li C, Xu D, Ye Q, Hong S, Jiang Y, Liu X, Zhang N, Shi L, Qin C-F, Xu Z.. Zika Virus Disrupts Neural Progenitor Development 5

7 and Leads to Microcephaly in Mice. Cell Stem Cell pii: S (16) doi: /j.stem Yockey LJ, Varela L, Rakib T, Khoury-Hanold W, Fink SL, Stutz B, Szigeti-Buck K, Van den Pol A, Lindenbach BD, Horvath TL, Iwasaki A.. Vaginal Exposure to Zika Virus during Pregnancy Leads to Fetal Brain Infection. Cell 166: e4. 6. Xavier-Neto J, Carvalho M, Pascoalino B dos S, Cardoso AC, Costa ÂMS, Pereira AHM, Santos LN, Saito Â, Marques RE, Smetana JHC, Consonni SR, Bandeira C, Costa VV, Bajgelman MC, Oliveira PSL de, Cordeiro MT, Gonzales Gil LHV, Pauletti BA, Granato DC, Paes Leme AF, Freitas-Junior L, Holanda de Freitas CBM, Teixeira MM, Bevilacqua E, Franchini K Hydrocephalus and arthrogryposis in an immunocompetent mouse model of ZIKA teratogeny: A developmental study. PLoS Negl Trop Dis 11:e Rossi SL, Tesh RB, Azar SR, Muruato AE, Hanley KA, Auguste AJ, Langsjoen RM, Paessler S, Vasilakis N, Weaver SC.. Characterization of a Novel Murine Model to Study Zika Virus. Am J Trop Med Hyg. 8. Aliota MT, Caine EA, Walker EC, Larkin KE, Camacho E, Osorio JE.. Characterization of Lethal Zika Virus Infection in AG129 Mice. PLoS Negl Trop Dis 10:e Lazear HM, Govero J, Smith AM, Platt DJ, Fernandez E, Miner JJ, Diamond MS.. A Mouse Model of Zika Virus Pathogenesis. Cell Host Microbe. 10. Dowall SD, Graham VA, Rayner E, Atkinson B, Hall G, Watson RJ, Bosworth A, Bonney LC, Kitchen S, Hewson R.. A Susceptible Mouse Model for Zika Virus Infection. PLoS Negl Trop Dis 10:e Miner JJ, Sene A, Richner JM, Smith AM, Santeford A, Ban N, Weger-Lucarelli J, Manzella F, Rückert C, Govero J, Noguchi KK, Ebel GD, Diamond MS, Apte RS.. Zika Virus Infection in Mice Causes Panuveitis with Shedding of Virus in Tears. Cell Rep. 12. Li H, Saucedo-Cuevas L, Shresta S, Gleeson JG.. The Neurobiology of Zika Virus. Neuron 92: Govero J, Esakky P, Scheaffer SM, Fernandez E, Drury A, Platt DJ, Gorman MJ, Richner JM, Caine EA, Salazar V, Moley KH, Diamond MS.. Zika virus infection damages the testes in mice. Nature. 14. Chan JF-W, Zhang AJ, Chan CC-S, Yip CC-Y, Mak WW-N, Zhu H, Poon VK-M, Tee K-M, Zhu Z, Cai J-P, Tsang JO-L, Chik KK-H, Yin F, Chan K-H, Kok K-H, Jin D-Y, Au-Yeung RK-H, Yuen K-Y.. Zika Virus Infection in Dexamethasoneimmunosuppressed Mice Demonstrating Disseminated Infection with Multi-organ Involvement Including Orchitis Effectively Treated by 6

8 Recombinant Type I Interferons. EBioMedicine. 15. Ma W, Li S, Ma S, Jia L, Zhang F, Zhang Y, Zhang J, Wong G, Zhang S, Lu X, Liu M, Yan J, Li W, Qin C, Han D, Qin C, Wang N, Li X, Gao GF.. Zika Virus Causes Testis Damage and Leads to Male Infertility in Mice. Cell. 16. Tang WW, Young MP, Mamidi A, Regla-Nava JA, Kim K, Shresta S.. A Mouse Model of Zika Virus Sexual Transmission and Vaginal Viral Replication. Cell Rep 17: Duggal NK, Ritter JM, Pestorius SE, Zaki SR, Davis BS, Chang G-JJ, Bowen RA, Brault AC Frequent Zika Virus Sexual Transmission and Prolonged Viral RNA Shedding in an Immunodeficient Mouse Model. Cell Rep 18: Dudley DM, Aliota MT, Mohr EL, Weiler AM, Lehrer-Brey G, Weisgrau KL, Mohns MS, Breitbach ME, Rasheed MN, Newman CM, Gellerup DD, Moncla LH, Post J, Schultz-Darken N, Schotzko ML, Hayes JM, Eudailey JA, Moody MA, Permar SR, O Connor SL, Rakasz EG, Simmons HA, Capuano S, Golos TG, Osorio JE, Friedrich TC, O Connor DH.. A rhesus macaque model of Asian-lineage Zika virus infection. Nat Commun 7: Li XF, Dong HL, Huang XY, Qiu YF, Wang HJ, Deng YQ, Zhang NN, Ye Q, Zhao H, Liu ZY, Fan H, An XP, Sun SH, Gao B, Fa YZ, Tong YG, Zhang FC, Gao GF, Cao WC, Shi PY, Qin CF.. Characterization of a Clinical Isolate of Zika Virus in Nonhuman Primates. - PubMed - NCBI. EBioMedicine pii: S (16) doi: /j.ebiom Osuna CE, Lim S-Y, Deleage C, Griffin BD, Stein D, Schroeder LT, Omage R, Best K, Luo M, Hraber PT, Andersen-Elyard H, Ojeda EFC, Huang S, Vanlandingham DL, Higgs S, Perelson AS, Estes JD, Safronetz D, Lewis MG, Whitney JB.. Zika viral dynamics and shedding in rhesus and cynomolgus macaques. Nat Med. 21. Adams Waldorf KM, Stencel-Baerenwald JE, Kapur RP, Studholme C, Boldenow E, Vornhagen J, Baldessari A, Dighe MK, Thiel J, Merillat S, Armistead B, Tisoncik-Go J, Green RR, Davis MA, Dewey EC, Fairgrieve MR, Gatenby JC, Richards T, Garden GA, Diamond MS, Juul SE, Grant RF, Kuller L, Shaw DWW, Ogle J, Gough GM, Lee W, English C, Hevner RF, Dobyns WB, Gale M, Rajagopal L.. Fetal brain lesions after subcutaneous inoculation of Zika virus in a pregnant nonhuman primate. Nat Med Aliota MT, Dudley DM, Newman CM, Mohr EL, Gellerup DD, Breitbach ME, Buechler CR, Rasheed MN, Mohns MS, Weiler AM, Barry GL, Weisgrau KL, Eudailey JA, Rakasz EG, Vosler LJ, Post J, Capuano S, Golos TG, Permar SR, Osorio JE, Friedrich TC, O Connor SL, O Connor DH.. Heterologous Protection against Asian Zika Virus Challenge in Rhesus Macaques. 7

9 PLoS Negl Trop Dis 10:e Coffey LL, Pesavento PA, Keesler RI, Singapuri A, Watanabe J, Watanabe R, Yee J, Bliss-Moreau E, Cruzen C, Christe KL, Reader JR, von Morgenland W, Gibbons AM, Allen AM, Linnen J, Gao K, Delwart E, Simmons G, Stone M, Lanteri M, Bakkour S, Busch M, Morrison J, Van Rompay KKA Zika Virus Tissue and Blood Compartmentalization in Acute Infection of Rhesus Macaques. PLoS One 12:e

10 Supplemental Table 2: Prevention of non vector-borne ZIKV infections Prevention of ZIKV sexual transmission People living in areas with active ZIKV circulation People living in areas without active ZIKV circulation but returning from areas with active ZIKV circulation Information about risk of sexual transmission (Counseling on safe sex Men and women should use barriers against infection or consider practices, use of condoms, use of contraceptives) safe sex practice or abstinence for at least 6 months for men or 8 Pregnant women should use barrier methods against infection or practice weeks (CDC) or 6 months (WHO) for women after return safe sex or abstain from sexual activity Couples planning pregnancy should wait at least 6 months (men) and Couples in which a partner had confirmed ZIKV infection or clinical illness 8 weeks (women) before trying to conceive consistent with ZIKV infection should consider use of condoms or safe sex Sexual partners of pregnant women (men and women) should practice or abstinence for at least 6 months for men and 8 weeks for women consider safe sex practice or abstinence Couples who reside might consider using barrier methods against infection Couples in which a partner had confirmed ZIKV infection or clinical or abstaining from sex while active transmission persists. illness consistent with ZIKV infection should consider use of Women should have access to emergency contraceptive services and condoms or safe sex practice or abstinence for at least 6 months for counseling men and 8 weeks for women References 1-3 Prevention of ZIKV transfusion-transmission WHO recommendations (February ) Reinforcing blood collection in non endemic areas Donors deferral 28 days after visiting an area with active ZIKV Donors deferral 28 days after confirmed or suspected ZIKV infection and circulation confirmed or suspected ZIKV infection and for sexual for sexual partner of men with confirmed or suspected ZIKV infection in the partner of men with confirmed or suspected ZIKV infection in the last 3 months last 3 months Post donation information Nucleic acid testing of blood donations from blood donors returning 4

11 Nucleic acid testing of blood donations from affected areas is an alternative to deferral Implementation of pathogen inactivation Implementation of pathogen inactivation Quarantine of blood components Updated FDA recommendations in the US and its territories (August ) Donors deferral 120 days after resolution of symptoms or date of a reactive test Testing all blood donations with a nucleic acid test for ZIKV or Implement pathogen inactivation/reduction for platelets and plasma using a FDA-approved technology. Implement pathogen inactivation/reduction for whole blood or red blood cells when an FDA-approved technology becomes available 5-6 Prevention of ZIKV transmission by substances of human origin (other than blood): FDA recommendations in the US and its territories Living donors are considered as ineligible if they have any of the following risk factors o ZIKV infection within the past 6 months o Residence or travel in area with active ZIKV transmission within the past 6 months o Sex in the past 6 months with a men with the above risk factors For donors of umbilical cord blood, placenta or other gestational tissues are considered as ineligible if they had any of the following risk factors at any point of the pregnancy o Medical diagnosis of ZIKV infection or residence or travel 7

12 to an area of active transmission or sex with men with the above risk factors Cadaveric donors o ZIKV infection within the past 6 months References 1 World Health Organization. Prevention of sexual transmission of Zika virus. Interim guidance update 6 September. ; WHO/ZIKV/MOC/16.1 Rev.3 2 Petersen EE, Meaney-Delman D, Neblett-Fanfair R, et al. Update: Interim Guidance for Preconception Counseling and Prevention of Sexual Transmission of Zika Virus for Persons with Possible Zika Virus Exposure - United States, September. MMWR Morb Mortal Wkly Rep ; 65: Brooks JT, Friedman A, Kachur RE, LaFlam M, Peters PJ, Jamieson DJ. Update: Interim Guidance for Prevention of Sexual Transmission of Zika Virus - United States, July. MMWR Morb Mortal Wkly Rep ; 65: World Health Organization. Maintaining a safe and adequate blood supply during Zika virus outbreaks. ; 5 American Association of Blood Banks. Association bulletin # Updated recommendations for Zika, dengue, and chikungunya viruses. AABB ; : 6 Food and Drug Administration. Revised recommendations for reducing the risk of Zika virus transmission by blood and blood components. Guidance for industry. ; https://www.fda.gov/biologicsbloodvaccines/guidancecomplianceregulatoryinformation/guidances/blood/ 7 Food and Drug Administration. Donor screening recommendations to reduce the risk of transmission of Zika virus by human cells, tissues, and cellular and tissue-based products. ; https://www.fda.gov/biologicsbloodvaccines/guidancecomplianceregulatoryinformation/guidances/tissue/

13 Supplemental table 3 Reported complications of ZIKV infections in adults Complications References Neurological complications Guillain-Barré syndromes 1-6 Encephalitis 7 Myelitis 8 Meningoencephalitis 9 Reversible sensory polyneuropathy 10 Papilledema 6 Uveitis 11,12 Maculopathy 13 Non neurological complications Myocarditis 14 Severe thrombocytopenia ZIKV related death 7,19,20 References 1 Oehler E, Watrin L, Larre P, et al. Zika virus infection complicated by Guillain--Barre syndrome-case report, French Polynesia, December Euro Surveill 2014; 19: pii: Brasil P, Sequeira PC, Freitas AD, et al. Guillain-Barré syndrome associated with Zika virus infection. Lancet ; 387: Dos Santos T, Rodriguez A, Almiron M, et al. Zika Virus and the Guillain-Barré Syndrome - Case Series from Seven Countries. N Engl J Med ; 375: Parra B, Lizarazo J, Jiménez-Arango JA, et al. Guillain-Barré Syndrome Associated with Zika Virus Infection in Colombia. N Engl J Med ; 375:

14 5 Cao-Lormeau V, Blake A, Mons S, et al. Guillain-Barré Syndrome outbreak caused by Zika virus infection in French Polynesia: a case control study. Lancet ; 387: Dirlikov E, Major CG, Mayshack M, et al. Guillain-Barré Syndrome During Ongoing Zika Virus Transmission - Puerto Rico, January 1- July 31,. MMWR Morb Mortal Wkly Rep ; 65: Soares CN, Brasil P, Carrera RM, et al. Fatal encephalitis associated with Zika virus infection in an adult. J Clin Virol ; 83: Mécharles S, Herrmann C, Poullain P, et al. Acute myelitis due to Zika virus infection. Lancet ; 378: Carteaux G, Maquart M, Bedet A, et al. Zika Virus Associated with Meningoencephalitis. N Engl J Med ; 374: Medina MT, England JD, Lorenzana I, et al. Zika virus associated with sensory polyneuropathy. J Neurol Sci ; 369: Furtado JM, Espósito DL, Klein TM, Teixeira-Pinto T, da Fonseca BA. Uveitis Associated with Zika Virus Infection. N Engl J Med ; 375: Kodati S, Palmore TN, Spellman FA, Cunningham D, Weistrop B, Sen HN. Bilateral posterior uveitis associated with Zika virus infection. Lancet ; 389: Parke DW, Almeida DRP, Albini TA, Ventura C V, Berrocal AM, Mittra RA. Serologically Confirmed Zika-Related Unilateral Acute Maculopathy in an Adult. Ophthalmology ; 123: Aletti M, Lecoules S, Kanczuga V, et al. Transient myocarditis associated with acute Zika virus infection. Clin Infect Dis ; doi: /cid/ciw Chraïbi S, Najioullah F, Bourdin C, et al. Two cases of thrombocytopenic purpura at onset of Zika virus infection. J Clin Virol ; 83: Sharp TM, Muñoz-Jordán J, Perez-Padilla J, et al. Zika Virus Infection Associated with Severe Thrombocytopenia. Clin Infect Dis ; 63: Boyer Chammard T, Schepers K, Breurec S, et al. Severe Thrombocytopenia after Zika Virus Infection, Guadeloupe,. Emerg Infect Dis 2017; 23. doi: /eid Karimi O, Goorhuis A, Schinkel J, et al. Thrombocytopenia and subcutaneous bleedings in a patient with Zika virus infection. Lancet

15 ; 387: Azevedo RSS, Araujo MT, Martins Filho AJ, et al. Zika virus epidemic in Brazil. I. Fatal disease in adults: Clinical and laboratorial aspects. J Clin Virol ; 85: Arzuza-Ortega L, Polo A, Pérez-Tatis G, et al. Fatal Sickle Cell Disease and Zika Virus Infection in Girl from Colombia. Emerg Infect Dis ; 22:

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