Supplementary Appendix

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1 Supplementary Appendix This appendix has been provided by the authors to give readers additional information about their work. Supplement to: Carteaux G, Maquart M, Dessap AM, et al. Zika virus associated with meningoencephalitis. N Engl J Med 2016;374: DOI: /NEJMc

2 SUPPLEMENTARY APPENDIX Title Zika virus associated with meningoencephalitis Table of content ADDITIONAL METHODS... 2 ADDITIONAL CLINICAL INVESTIGATIONS... 4 REFERENCES... 5 FIGURE LEGEND... 6 Figure S Table S

3 ADDITIONAL METHODS DETAILED DESCRIPTION OF MOLECULAR METHODS USED FOR DETECTION OF ZIKV, DENV, AND CHIKV RNA extraction from samples 140µL of cerebrospinal fluid (CSF) and plasma were added to 560µL of lysis buffer. Extraction quality was validated by the addition of 10µL of a commercial internal control Rico Extra R-gene to the lysis buffer. Internal control PCR was processed as described by the manufacturer (Biomerieux, Marcy l Etoile). Extraction was processed using the RNeasy Mini Kit (Qiagen, Hilden, Germany). RNA extraction and RT-PCRs were repeated 3 times separately and independently. Real time RT-PCR for detection of ZIKV RNA using the RealStar Zika Virus RT- PCR Kit 1.0 (Altona Diagnostics EN) The amplification was performed in a LightCycler 2.0 Instrument (Roche Diagnostics GmbH, Switzerland). The RealStar Zika Virus RT-PCR Kit 1.0 (Altona Diagnostics EN) was used with a 20 μl reaction mixture under the following conditions: 3.5 μl of Master A, 10.5 μl of master B and 6 μl of the extracted RNA from sample. The negative control consisted of blank reagent and water. The positive control consisted of a Zika virus RNA confirmed by sequencing at the National Reference Center for arboviruses. The cycling conditions were as follows: 55 c- 20 min for reverse transcription, 95 C 2 min, then 45 cycles of 95 C 15 sec / 55 C 45 sec / 72 C 15 sec. Real time RT-PCR for detection of DENV and CHIKV using in-house methods 2

4 The amplification was performed in a LightCycler 2.0 Instrument (Roche Diagnostics GmbH, Switzerland). DENV and CHIKV were analyzed using the methods described respectively by Leparc-Goffart et al., 1 and Pastorino et al. 2 DESCRIPTION OF THE SEROLOGIC ASSAYS USED FOR INVESTIGATION OF PLASMA AND CEREBROSPINAL FLUID In-house immunoglobulin (Ig) M-capture enzyme immunoassays (MAC-ELISA) and IgG indirect ELISA were used to detect serum IgM and IgG antibodies against Zika virus (ZIKV), Dengue virus (DENV), Chikungunya virus (CHIKV), West Nile virus (WNV) and Ross River virus (RRV). Briefly, IgM antibodies were captured with rabbit anti-human IgM antibodies (Interchim, Montluçon, France). ZIKV, DENV, CHIKV, WNV, RRV antigens, prepared on Vero cells, precipitated and inactivated by betapropiolactone (Sigma-Aldrich, St Quentin Fallavier, France), was added. Specific binding was demonstrated by using a ZIKV, DENV, CHIKV, WNV, RRV mouse hyperimmune ascitic fluid virus and a goat anti-mouse peroxidase-labeled conjugate (Interchim). For IgG detection, ZIKV, DENV, CHIKV, WNV, RRV antigens were coated, followed by test sera; and specific binding was demonstrated by using a peroxidase-labeled goat anti-human IgG conjugate. Serum samples were considered positive if the optical density at 450 nm was >3-fold the mean of negative sera with a Sunrise spectrophotometer (Tecan, Lyon, France). 3 VIRAL ISOLATION Vero cell cultures were performed at 37 C in humidified atmosphere containing 5% CO2 in Dulbecco s modified Eagle s medium (DMEM) supplemented with 5% heat-inactivated fetal calf serum together with Sodium Pyruvate (1mM) and L-glutamine (5mM) (Dutscher, Brumath, France). The CSF detected positive for Zika virus by RT-PCR was prepared for isolation as follows: 200 µl of CSF was diluted in 2 ml of cell culture medium with 20 µl of 3

5 heparin and filtered with a 0.22µ filter. Subconfluent Vero cells were infected with 2 ml of the filtered preparation in a 25 cm 2 flask. Cell culture medium was changed 24 hours after infection with 8 ml of cell culture medium. Second passage was performed at Day 5 post infection on Vero cells in a 25cm 2 flask (Figure S1) and 140µl of supernatant was collected and analyzed by real-time RT-PCR as described previously. ADDITIONAL INVESTIGATIONS Common causes of multiple ischemic foci were search for by initial investigations. Computed tomography (CT) angiogram showed some atheromatous disease on carotids and vertebral arteries, but without ulceration or obstruction. Interestingly, the CT angiogram also revealed an irregular narrowing of the right anterior cerebral artery. Electrocardiogram was in sinus rhythm. Transesophageal echocardiography showed normal left systolic ventricular function without cardiac dilatation, intracardiac thrombosis, valvular vegetation or aortic atheroma. A moderate right-to-left shunting across patent foramen ovale was observed on contrast study under positive pressure ventilation. A Doppler ultrasound scan of the lower limbs showed no deep venous thrombosis. Infectious and immune investigations: Three lumbar punctures were performed, on days 1, 3, and 8 after ICU admission (see Table S1). The first lumbar puncture showed meningitis with normal glucose level on cerebrospinal fluid (CSF). Our initial conclusion was therefore a meningoencephalitis associated with cerebral vasculitis and the patient was treated with amoxicillin (12 g/day), cefotaxime (15 g/day), gentamicin (5 mg/kg/day), and acyclovir (10 mg/kg three times a day). These antimicrobials were stopped at day five on account of negative cultures and two consecutive (on the first and second lumbar punctures) negative PCR for Herpes Simplex virus and Varicella Zoster Virus in the CSF. Additionally, the following tests were found negative in 4

6 the first CSF: Human Herpesvirus 6, JC virus and Enterovirus PCR, Mycobacterium tuberculosis PCR, Cryptococcus antigen, Pneumococcus antigen. In the plasma, serological tests for human immunodeficiency virus, hepatitis B and C, Borrelia, and syphilis were negative. Antineutrophil cytoplasmic antibodies (ANCA) test was negative and antinuclear antibodies test was positive at a non-clinically relevant level (1/80). Several biological samples were sent to the French national reference center for Arboviruses that latter provided the following results (see methods above): - Day-1 samples: the RT-PCR for the ZIKV was negative in the blood, but positive in the CSF (cycle threshold of 34). In addition, the ZIKV was isolated from the CSF on Vero cell line (Figure S1). The serological tests for West-Nile, Ross River virus, Dengue, and Chikungunya viruses were negative in the blood and CSF. The RT-PCR for the two latter viruses were also negative in the blood and CSF. - Latter samples: the serological tests for ZIKV, Ross River, Dengue, and Chikungunya viruses were negative on day-8 CSF sample and on day-17 blood sample. REFERENCES: 1. Leparc-Goffart I, Baragatti M, Temmam S, et al. Development and validation of real-time one-step reverse transcription-pcr for the detection and typing of dengue viruses. J Clin Virol Off Publ Pan Am Soc Clin Virol 2009;45(1): Pastorino B, Bessaud M, Grandadam M, Murri S, Tolou HJ, Peyrefitte CN. Development of a TaqMan RT-PCR assay without RNA extraction step for the detection and quantification of African Chikungunya viruses. J Virol Methods 2005;124(1-2): Peyrefitte CN, Pastorino BAM, Bessaud M, et al. Dengue type 3 virus, Saint Martin, Emerg Infect Dis 2005;11(5):

7 FIGURE LEGEND Figure S1: Isolation of the Zika virus from cerebrospinal fluid on Vero cell culture. Zika virus cytopathic effect was observed at day 5 post-infection of Vero cell culture with a sample of the patient s cerebrospinal fluid. Typical pattern of Zika virus cytopathic effect was observed, with the two following main features: the presence of adherent dead cells (black circle); and the absence of heaps, all dead cells being scattered. Figure S1 6

8 Table S1 Cerebro-spinal fluid findings Date Normal values Day-1 Day-3 Day-8 Leukocyte count (cells/μl) < 10 41* 3 <1 Red cells count (cells/ μl) < 1 9 <1 <1 Protein (mg/dl) < Glucose (mmol/l) CSF/ blood glucose ratio > Culture - Sterile Sterile Sterile CSF=cerebrospinal fluid; * Differential count: polymorphonuclears 98%, Lymphocytes 1%, other cells 1%. 7

Supplementary Appendix

Supplementary Appendix This appendix has been provided by the authors to give readers additional information about their work. Supplement to: de Laval F, Matheus S, Labrousse T, Enfissi A, Rousset D, Briolant