niman

REFERENCES 1 Dick GW, Kitchen SF, Haddow AJ. Zika virus. I. Isolations and serological specificity.Trans R Soc Trop Med Hyg 1952;46:509-520 CrossRef | Web of Science | Medline 2 Cao-Lormeau VM, Roche C, Teissier A, et al. Zika virus, French Polynesia, South Pacific, 2013. Emerg Infect Dis 2014;20:1085-1086 Web of Science | Medline 3 Duffy MR, Chen T-H, Hancock WT, et al. Zika virus outbreak on Yap Island, Federated States of Micronesia. N Engl J Med 2009;360:2536-2543 Free Full Text | Web of Science | Medline 4 Musso D, Nilles EJ, Cao-Lormeau VM. Rapid spread of emerging Zika virus in the Pacific area. Clin Microbiol Infect 2014;20:O595-6 CrossRef | Web of Science | Medline 5 Zika virus outbreaks in the Americas. Wkly Epidemiol Rec 2015;90:609-610 Medline 6 Lanciotti RS, Kosoy OL, Laven JJ, et al. Genetic and serologic properties of Zika virus associated with an epidemic, Yap State, Micronesia, 2007. Emerg Infect Dis 2008;14:1232-1239 CrossRef | Web of Science | Medline 7 Lanciotti RS, Lambert AJ, Holodniy M, Saavedra S, del Carmen Castillo SL. Phylogeny of Zika virus in western hemisphere, 2015. Emerg Infect Dis (in press). 8 Besnard M, Lastere S, Teissier A, Cao-Lormeau V, Musso D. Evidence of perinatal transmission of Zika virus, French Polynesia, December 2013 and February 2014. Euro Surveill 2014;19 CrossRef | Medline 9 Oliveira Melo AS, Malinger G, Ximenes R, Szejnfeld PO, Alves Sampaio S, Bispo de Filippis AM. Zika virus intrauterine infection causes fetal brain abnormality and microcephaly: tip of the iceberg? Ultrasound Obstet Gynecol 2016;47:6-7 CrossRef | Web of Science | Medline 10 Mlakar J, Korva M, Tul N, et al. Zika virus associated with microcephaly. N Engl J Med2016;374:951-958 Free Full Text | Web of Science | Medline 11 Brasil P, Pereira JP Jr, Raja Gabaglia C, et al. Zika virus infection in pregnant women in Rio de Janeiro — preliminary report. N Engl J Med. DOI: 10.1056/NEJMoa1602412. Medline 12 Interim guidelines for pregnant women during a Zika virus outbreak — United States, 2016.MMWR Morb Mortal Wkly Rep 2016;65:30-33 CrossRef | Web of Science | Medline 13 Zika virus infection: global update on epidemiology and potentially associated clinical manifestations. Wkly Epidemiol Rec 2016;91:73-81 Medline 14 Rapid risk assessment: Zika virus epidemic in the Americas: potential association with microcephaly and Guillain-Barre syndrome. Stockholm: European Center for Disease Prevention and Control, 2015. 15 Zika travel information. Atlanta: Centers for Disease Control and Prevention, January 2016 (http://wwwnc.cdc.gov/travel/page/zika-travel-information). 16 Update: interim guidance for health care providers caring for women of reproductive age with possible Zika virus exposure — United States, 2016. MMWR Morb Mortal Wkly Rep2016 March 25 (http://www.cdc.gov/mmwr/volumes/65/wr/mm6512e2er.htm?s_cid=mm6512e2er). Web of Science 17 CDC health advisory: recognizing, managing, and reporting Zika virus infections in travelers returning from Central America, South America, the Caribbean and Mexico. Atlanta: Centers for Disease Control and Prevention, 2016 (http://emergency.cdc.gov/han/han00385.asp). 18 Table 24. In: Kline-Fath B, Bahado-Singh R, Bulas DI. Fundamental and advanced fetal imaging: ultrasound and MRI. Philadelphia: Wolters Kluwer Health, 2015:860-863. 19 Harreld JH, Bhore R, Chason DP, Twickler DM. Corpus callosum length by gestational age as evaluated by fetal MR imaging. AJNR Am J Neuroradiol 2011;32:490-494 CrossRef | Web of Science | Medline 20 Means and standard deviations of organ weights and measurements of live-born infants. In: Gilbert-Barness E, Debich-Spicer DE. Handbook of pediatric autopsy pathology. . New York: Humana Press, 2005:54. 21 About Zika virus disease. Atlanta: Centers for Disease Control and Prevention, 2016 (http://www.cdc.gov/zika/about/index.html). 22 Gourinat AC, O’Connor O, Calvez E, Goarant C, Dupont-Rouzeyrol M. Detection of Zika virus in urine. Emerg Infect Dis 2015;21:84-86 CrossRef | Web of Science | Medline 23 Korhonen EM, Huhtamo E, Smura T, Kallio-Kokko H, Raassina M, Vapalahti O. Zika virus infection in a traveller returning from the Maldives, June 2015. Euro Surveill 2016;21 CrossRef | Medline 24 Musso D, Roche C, Robin E, Nhan T, Teissier A, Cao-Lormeau VM. Potential sexual transmission of Zika virus. Emerg Infect Dis 2015;21:359-361 CrossRef | Web of Science | Medline 25 Foy BD, Kobylinski KC, Chilson Foy JL, et al. Probable non-vector-borne transmission of Zika virus, Colorado, USA. Emerg Infect Dis 2011;17:880-882 CrossRef | Web of Science | Medline 26 McCarthy M. Zika virus was transmitted by sexual contact in Texas, health officials report.BMJ 2016;352:i720-i720 CrossRef | Web of Science | Medline 27 Venturi G, Zammarchi L, Fortuna C, et al. An autochthonous case of Zika due to possible sexual transmission, Florence, Italy, 2014. Euro Surveill 2016;21 28 Transmission of Zika virus through sexual contact with travelers to areas of ongoing transmission — continental United States, 2016. MMWR Morb Mortal Wkly Rep2016;65:215-216 CrossRef | Web of Science | Medline 29 Zika virus: clinical evaluation and disease. Atlanta: Centers for Disease Control and Prevention (http://www.cdc.gov/zika/hc-providers/clinicalevaluation.html). 30 Tang H, Hammack C, Ogden SC, et al. Zika virus infects human cortical neural progenitors and attenuates their growth. Cell Stem Cell2016 March 3 (Epub ahead of print)

DISCUSSIONThe current recommendations for ZIKV diagnostic practices are based on the understanding that ZIKV viremia lasts for less than a week after the onset of infection.15 During the week of symptomatic infection, RNA detection in serum or blood is considered to be the diagnostic method of choice. ZIKV RNA can be detected in urine for some days longer.22,23 ZIKV is also present in semen for an unknown length of time, and scattered reports of sexual transmission of ZIKV have emerged.24-28 ZIKV RNA testing is not recommended for pregnant women after the first week after the onset of clinical disease. The diagnosis is usually based on a ZIKV-specific antibody response with higher IgM and neutralizing-antibody responses to ZIKV than to other flaviviruses.13 However, we have detected ZIKV RNA in the serum of a pregnant woman at 4 weeks and 10 weeks after the clinical onset of ZIKV infection but not after delivery. We suspect that the persistent ZIKV viremia in the patient described here was a consequence of viral replication in the fetus or placenta, which had high viral loads. Therefore, in addition to current ZIKV diagnostics, the use of quantitative RT-PCR methods may be a potential diagnostic approach for ongoing placental or fetal infections in pregnant women. Notably, in this patient, the ZIKV RNA levels were slightly higher in the maternal serum than in the amniotic fluid. The dynamics of ZIKV RNA in the serum of infected pregnant women are not well understood and will need to be assessed in larger studies. It is estimated that 80% of ZIKV infections are asymptomatic.29 Although the evidence of the association between the presence of ZIKV in pregnant women and fetal brain abnormalities continues to grow, the timing of infection during fetal development and other factors that may have an effect on viral pathogenesis and their effects on the appearance of brain abnormalities on imaging are poorly understood. Oliveira Melo et al.9 described two cases of ZIKV intrauterine infection associated with microcephaly and brain calcifications that were diagnosed by means of ultrasonography during the third trimester. Similar to the fetus in our report, the two fetuses in that study showed abnormal development of the corpus callosum and decreased brain parenchymal volume. In the case described by Mlakar et al.,10 the results of ultrasonography that was performed at 14 weeks and 20 weeks of gestation were normal, but microcephaly, ventriculomegaly, and calcifications were seen on ultrasonography at 29 weeks of gestation.10 In the larger Brazilian cohort, cerebellar atrophy was seen in a fetus at 20 weeks of gestation, but microcephaly was not diagnosed until 27 to 35 weeks in their cohort.11 In our study, a review of three sequential ultrasonographic images between 16 and 20 weeks showed a decrease in the fetal head circumferences from the 47th percentile to the 24th percentile, which suggests a reduction in the rate of brain growth during that period (Fig. S3 in the Supplementary Appendix). We suspect these reductions in brain growth would have eventually met the criteria for microcephaly. As this case shows, the latency period between ZIKV infection of the fetal brain and the detection of microcephaly and intracranial calcifications on ultrasonography is likely to be prolonged. Negative ultrasonographic studies during this period would be falsely reassuring and might delay critical time-sensitive decision making. Serial ultrasonographic measurements of head circumference may provide useful predictive information. The superior soft-tissue resolution of fetal brain MRI might be more sensitive to developmental and encephaloclastic changes, thereby expediting the detection of evolving fetal brain anomalies. This case is an early foray into the histopathological findings associated with ZIKV in the midgestational fetal brain. The overwhelming findings were of loss of intermediately differentiated postmigratory neurons through an apoptotic mechanism. There appeared to be preservation of more differentiated neurons in basal ganglia, limbic region, and dorsal spinal cord. The germinal matrix cells also appeared to be spared. Of note, the germinal matrix consists predominantly of glioblasts at midgestation with the majority of the neuroblasts having already migrated out of the zone. Although we could not evaluate neuronal precursor subtypes other than calretinin-expressing interneuron lineage cells, selective neuronal vulnerability to ZIKV injury requires further investigation. The successful isolation of infectious ZIKV from human fetal brain fulfills Koch’s second postulate regarding the isolation of pathogens from a diseased organism and strengthens the association between congenital ZIKV infection and fetal brain damage. Although ZIKV RNA was found in several fetal organs and the placenta, the virus could be isolated only from brain tissue. The rapid isolation in a human neuroblastoma cell line suggests a predilection of the ZIKV strain for human neural lineage cells. This hypothesis is in line with the histopathological findings and the results of a recent study showing a high rate of ZIKV infection in cortical neural progenitor cells but not in embryonic or pluripotent stem cells.30 The close genetic relationship between the isolate in our report and Guatemalan ZIKV strains was consistent with the anamnestic knowledge on the likely geographical origin of the infection. We found a relatively high frequency of nonsynonymous mutations between the FB-GWUH-2016 genome and the Guatemalan ZIKV genome (Fig. S4 in theSupplementary Appendix), a finding that could indicate viral adaptation to growth in the fetal brain. However, no amino acid changes were identical to previously reported alterations in the ZIKV genome sequenced from fetal brain tissue.10 In conclusion, our study highlights the possible importance of ZIKV RNA testing of serum obtained from pregnant women beyond the first week after symptom onset, as well as a more detailed evaluation of the fetal intracranial anatomy by means of serial fetal ultrasonography or fetal brain MRI. The isolation of ZIKV from fetal brain provides additional evidence for the association between congenital ZIKV infection and fetal brain damage and provides tools for further studies of the pathogenesis of ZIKV-induced microcephaly. Future studies at various gestational ages will offer better insight into the role of ZIKV infection in abnormal brain development and provide markers for its detection. Disclosure forms provided by the authors are available with the full text of this article at NEJM.org. Drs. Driggers and Ho, Ms. Korhonen, and Ms. Kuivanen and Drs. du Plessis and Vapalahti contributed equally to this article. This article was published on March 30, 2016, at NEJM.org. We thank the patient and her husband for their support of our study; Irina Suomalainen, Inkeri Luoto, and Sanna Mäki of the University of Helsinki for their technical assistance; Tamas Baszinka and Kirsi Aaltonen for their help in preparing samples; Dr. Eili Huhtamo for guidance in setting up the nested RT-PCR assay; the Finnish IT Center for Science for the allocation of computational resources; and Yvette Veloso, Mandy Field, and Laurie King for their technical support.

RESULTSFetal Neurologic AbnormalitiesA postmortem examination was performed with materials collected for additional study. Gross examination showed normal fetal anatomy and severe autolysis. The brain weighed 30 g (reference weight, 49±1520) and showed no apparent gross abnormalities. Microscopic analysis revealed abundant apoptosis primarily affecting the intermediately differentiated postmigratory neurons in the neocortex (Figure 4FIGURE 4Neuropathological Features of Fetal ZIKV Infection., and Fig. S2 in the Supplementary Appendix). Early mineralization was seen in association with apoptotic neurons focally. In contrast, the well-differentiated neurons of the basal ganglia and limbic regions as well as primitive cells in the germinal matrix appeared to be unaffected. In addition to the cortical neuronal abnormalities, the subventricular zone and white matter showed severe volume loss with extensive axonal rarefaction and macrophage infiltrates (Figure 4). This pattern correlates with the atrophy of the subplate seen on prenatal imaging. There was diffuse infiltration of macrophages in the cerebral cortex, subventricular zone, white matter, and leptomeninges but not in the germinal matrix of the ganglionic eminence. Scattered loose microglial aggregates were observed in the deep gray matter and brain stem, but there was no evidence of well-formed microglial nodules or other classic histologic features of viral encephalitis, such as perivascular inflammatory infiltrates, viral inclusions, or ventriculitis. Ultrastructural examination of fixed cortical tissue showed a rare aggregate of intracellular electron-dense, viral-like particles that measured 39 to 41 nm in diameter (mean, 40.26). Our ability to specifically localize the cellular compartment housing the particles was limited by poor tissue preservation, but the morphologic features and size of this structure were similar to those reported by Mlakar et al.10 and the CDC.21 The choroid plexus was focally enlarged and edematous, with scant hemosiderin deposits, which may appear to be similar to intraventricular hemorrhage on prenatal imaging. Histologic examination of the eyes, spinal cord gray matter, dorsal-root ganglia, and spinal nerves did not reveal overt microscopic abnormalities. Spinal white-matter tracts were not well visualized. A detailed pathological description of the brain and other organs is provided in the Methods section of the Supplementary Appendix. Fetal and Maternal ZIKV Viral LoadsThe highest ZIKV viral loads were found in fetal brain, with substantial viral loads in the placenta, fetal membranes, and umbilical cord, as studied on quantitative RT-PCR (Table S2 in theSupplementary Appendix). Lower amounts of ZIKV RNA were found in fetal muscle, liver, lung, and spleen. Amniotic fluid that was obtained at the time of termination was positive for ZIKV RNA with low viral counts. On PCR assays to detect DNA, the amniotic fluid was negative for parvovirus B19, herpes simplex virus types 1 and 2, cytomegalovirus (CMV), and Toxoplasma gondii, and the fetal brain tissue was negative for herpes simplex virus types 1 and 2 and varicella–zoster virus. Maternal serum that was obtained on the day before termination was also positive for ZIKV RNA with a low viral count (2.1×103 copies per milliliter). No ZIKV RNA was detected in the serum, peripheral-blood mononuclear cells, saliva, or urine in samples obtained 11 days and 13 days after termination. On IgM analysis, the mother had no evidence of serum antibodies indicating acute infection with CMV, parvovirus B19, T. gondii, or rubella virus. Samples obtained from her spouse were all negative for ZIKV RNA, including urine (obtained 11 weeks after travel), serum (obtained 5 and 11 weeks after travel), and semen (obtained 10 and 12 weeks after travel), although results of testing for ZIKV IgG (titer 320) and IgM (titer 20) were positive. Virus IsolationZIKV replication was detected as an increase in ZIKV RNA on quantitative RT-PCR assay of SK-N-SH and Vero E6 cells inoculated with the fetal brain sample. The quantities of ZIKV RNA increased rapidly in the SK-N-SH cells after the first day of inoculation, whereas in the Vero E6 cells, viral RNA loads started to increase on day 4 after inoculation. Viral replication was not detected in cells inoculated with other samples. The tissue-inoculated SK-N-SH and Vero E6 cells were further shown to express ZIKV antigens by reactivity with human convalescent anti-ZIKV serum (obtained from the father of the fetus) on immunofluorescence staining and to produce flavivirus-like particles, as seen on electron microscopy (Figure 5FIGURE 5Isolation of ZIKV from Fetal Brain Tissue, ZIKV Growth in Fetal Tissues, Electron Microscopy of a Flavivirus-like Particle, and Amino Acid Differences in the Newly Isolated Strain.). A complete ZIKV genome was sequenced from supernatant of SK-N-SH cells on day 5 after inoculation. Phylogenetic analysis indicated that the viral strain (designated ZIKV_FB-GWUH-2016; GenBank number, KU870645) was a member of the Asian genotype and closely related to two ZIKV sequences obtained from Guatemalan patients who presented with mild illness (Figure 6FIGURE 6Phylogenetic Tree Showing Newly Isolated ZIKV Strain., and Fig. S6 in the Supplementary Appendix).7 The FB-GWUH-2016 strain had 23 to 51 nucleotide differences and 8 to 14 amino acid differences as compared with the ZIKV strains detected previously in the Americas (99.6 to 99.8% identities) (Figure 5D). Five of the eight differences in amino acids between FB-GWUH-2016 and the Guatemalan strains were specific for the FB-GWUH-2016 strain (i.e., differences that were not detected in other ZIKV strains sequenced so far). One amino acid substitution was a reversion toward the African ZIKV genotype. Three amino acid substitutions were common for FB-GWUH-2016 and the Guatemalan strains but distinct from all other reported ZIKV strains

METHODSWe tested samples obtained from the patient, her spouse, and the fetus and from viral isolation trials for ZIKV RNA using nested pan-flavivirus RT-PCR and quantitative RT-PCR for ZIKV. Levels of ZIKV IgM, IgG, and neutralizing-antibody titers were determined by means of standard methods. We performed immunohistochemical and electron microscopic analyses to study fetal brain tissue. Viral isolation trials using the patient’s serum and fetal tissues were performed with the use of SK-N-SH human neuroblastoma cells, Vero E6 green monkey kidney cells, and C6/36 Aedes albopictus mosquito cells. We used next-generation sequencing and Bayesian analysis to study the genetics of the ZIKV strain isolate. Additional details about the analyses are provided in the Methods section of the Supplementary Appendix.

CASE REPORTA 33-year-old Finnish woman who was in the 11th week of gestation was on holiday in Mexico, Guatemala, and Belize with her husband in late November 2015. (Details are provided in Section 1.0 of the Supplementary Appendix, available with the full text of this article at NEJM.org.) During their travels, she and her husband recalled being bitten by mosquitoes, particularly in Guatemala. One day after her arrival at her current residence in Washington, D.C., she became ill with ocular pain, myalgia, and mild fever (maximum, 37.5°C), which lasted for 5 days. On the second day of fever, a rash developed (Figure 1FIGURE 1Timeline of Symptoms and Radiographic and Laboratory Studies., and Fig. S5 in the Supplementary Appendix). Her husband was concomitantly reporting similar symptoms. Serologic analysis that was performed 4 weeks after the onset of illness while she was on a trip to her native Finland was positive for IgG antibodies and negative for IgM antibodies against dengue virus. Subsequent serologic analysis was positive for both IgG and IgM antibodies against ZIKV, findings that were compatible with acute or recent ZIKV infection. Serologic analysis for the presence of chikungunya virus was negative. The patient had been vaccinated against tick-borne encephalitis and yellow fever more than 10 years earlier. Fetal ultrasonography that was performed at 13, 16, and 17 weeks of gestation (1, 4, and 5 weeks after the resolution of symptoms) showed no evidence of microcephaly or intracranial calcifications. However, there was a decrease in the fetal head circumference from the 47th percentile at 16 weeks to the 24th percentile at 20 weeks. At 16 weeks of gestation, the presence of flavivirus in serum was detected on nested reverse-transcriptase–polymerase-chain-reaction (RT-PCR) assay, and sequencing showed identity to Central American epidemic strains of ZIKV. The finding was confirmed with a specific ZIKV quantitative RT-PCR assay (Table S2 in the Supplementary Appendix). The Division of Vector-Borne Diseases Arbovirus Diagnostic Laboratory at the CDC reported serologic evidence of infection at 17 weeks of gestation, with serum positivity for ZIKV IgM and a titer of more than 1:2560 on a plaque-reduction neutralization test. On the basis of these results, the patient sought more thorough assessment of the fetus. Fetal ultrasonography at 19 weeks of gestation showed abnormal intracranial anatomy (Figure 2FIGURE 2Fetal Ultrasonography at 19 Weeks of Gestation., and Fig. S1 in the Supplementary Appendix). The cerebral mantle appeared to be thin with increased extra-axial spaces. Both frontal horns were enlarged with heterogeneous, predominantly echogenic material present in the frontal horn and body of the left lateral ventricle, a finding that raised concern about intraventricular hemorrhage. Dilation and upward displacement of the third ventricle, dilation of the frontal horns of the lateral ventricles, concave medial borders of the lateral ventricles, and the absence of the cavum septum pellucidum suggested agenesis of the corpus callosum. No parenchymal calcifications were seen. The head circumference measured in the 24th percentile for gestational age. The remainder of the fetal anatomy was normal. Fetal MRI at 20 weeks of gestation showed diffuse atrophy of the cerebral mantle, which was most severe in the frontal and parietal lobes, with the anterior temporal lobes least affected (Figure 3FIGURE 3Magnetic Resonance Imaging of the Fetal Brain at 19 Weeks of Gestation.). The normal lamination pattern of the cerebral mantle was absent, and the subplate zone was largely undetectable. The corpus callosum was significantly shorter than expected for gestational age, with an anterior–posterior length of 14 mm (expected range, 18 to 22).18,19 The cavum septum pellucidum was very small. The lateral ventricles were mildly enlarged, as was the third ventricle, with a transverse diameter measuring 2.5 mm (average measurement at gestational age, 1.75 mm [range, 1.1 to 2.3]).18 The fourth ventricle was normal. The volume of the choroid plexus was unusually prominent, without evidence of hemorrhage. No focal destructive lesions were identified within the cerebral cortex or white matter. The cerebellum was normal in appearance and size. Given the grave prognosis, the patient elected to terminate the pregnancy at 21 weeks of gestation.

Zika virus (ZIKV), a mosquito-borne flavivirus and member of the Flaviviridae family, was originally isolated from a sentinel primate in Uganda in 1947.1 ZIKV was associated with mild febrile disease and maculopapular rash in tropical Africa and some areas of Southeast Asia. Since 2007, ZIKV has caused several outbreaks outside its former distribution area in islands of the Pacific: in 2007 on Yap island in Micronesia, in 2013 and 2014 in French Polynesia, and in 2015 in South America, where ZIKV had not been identified previously.2-5 There are separate African and Asian lineages of the virus,6 and the latter strains have caused the outbreaks in the Pacific and the Americas.7 As in the transmission of dengue and chikungunya viruses, the main transmission cycle of ZIKV occurs between urban aedes mosquitoes and humans. One striking feature of the current ZIKV outbreak is the apparent increased risk of intrauterine or perinatal transmission of the virus as well as the marked increase in the number of newborns with microcephaly reported in Brazil.8-17 A recent prospective study showed fetal ultrasonographic abnormalities in 12 of 42 women (29%) with ZIKV infection during pregnancy; 7 of the 42 fetuses (17%) that were studied had microcephaly, cerebral atrophy, or brain calcifications.11 Because of the association between ZIKV infection and microcephaly and other neurologic disorders, the World Health Organization has declared the ZIKV epidemic a public health emergency of international concern.13 Early in this particular outbreak, investigations into the viral pathogenesis, vertical transmission rates, potential viral cofactors, and sensitivity and specificity of diagnostic testing have presented more questions than answers. Nevertheless, the Centers for Disease Control and Prevention (CDC) has issued a travel advisory for pregnant women,15 as well as guidelines for health providers caring for all travelers from affected regions.16,17 The CDC recommends that pregnant women with a history of travel to an area in which ZIKV is endemic should undergo ZIKV serologic testing and fetal ultrasonography to screen for microcephaly or intracranial calcifications.16 For a diagnosis of fetal ZIKV infection, RNA detection in amniotic fluid may be considered in pregnant women with positive results on ZIKV serologic testing.16 Here we present a report of a case of congenital ZIKV infection and subsequent findings in a pregnancy that was terminated at 21 weeks of gestation.

The current outbreak of Zika virus (ZIKV) infection has been associated with an apparent increased risk of congenital microcephaly. We describe a case of a pregnant woman and her fetus infected with ZIKV during the 11th gestational week. The fetal head circumference decreased from the 47th percentile to the 24th percentile between 16 and 20 weeks of gestation. ZIKV RNA was identified in maternal serum at 16 and 21 weeks of gestation. At 19 and 20 weeks of gestation, substantial brain abnormalities were detected on ultrasonography and magnetic resonance imaging (MRI) without the presence of microcephaly or intracranial calcifications. On postmortem analysis of the fetal brain, diffuse cerebral cortical thinning, high ZIKV RNA loads, and viral particles were detected, and ZIKV was subsequently isolated.

Zika Virus Infection with Prolonged Maternal Viremia and Fetal Brain AbnormalitiesRita W. Driggers, M.D., Cheng-Ying Ho, M.D., Ph.D., Essi M. Korhonen, M.Sc., Suvi Kuivanen, M.Sc., Anne J. Jääskeläinen, Ph.D., Teemu Smura, Ph.D., Avi Rosenberg, M.D., Ph.D., D. Ashley Hill, M.D., Roberta L. DeBiasi, M.D., Gilbert Vezina, M.D., Julia Timofeev, M.D., Fausto J. Rodriguez, M.D., Lev Levanov, Ph.D., Jennifer Razak, M.G.C., C.G.C, Preetha Iyengar, M.D., Andrew Hennenfent, D.V.M., M.P.H., Richard Kennedy, M.D., Robert Lanciotti, Ph.D., Adre du Plessis, M.B., Ch.B., M.P.H., and Olli Vapalahti, M.D., Ph.D. March 30, 2016DOI: 10.1056/NEJMoa1601824 SOURCE INFORMATIONFrom the Department of Gynecology and Obstetrics, Division of Maternal Fetal Medicine (R.W.D., J.T.), and the Department of Pathology (F.J.R.), Johns Hopkins University School of Medicine, Baltimore; the Division of Maternal Fetal Medicine, Sibley Memorial Hospital (R.W.D., J.T., J.R.), the Division of Pathology and Center for Genetic Medicine Research (C.-Y.H., A.R., D.A.H.), Division of Pediatric Infectious Diseases (R.L.D.), Department of Diagnostic Radiology and Imaging (G.V.), and the Fetal Medicine Institute, Division of Fetal and Transitional Medicine (A.P.), Children’s National Health System, the Departments of Integrative Systems Biology (C.-Y.H., D.A.H.), Pediatrics and Microbiology, Immunology and Tropical Medicine (R.L.D.B.), and Radiology and Pediatrics (G.V.), George Washington University School of Medicine and Health Sciences, the Center for Policy, Planning and Evaluation (P.I.) and Centers for Disease Control and Prevention (CDC)–Council of State and Territorial Epidemiologists (CSTE) Applied Epidemiology Fellowship (A.H.), District of Columbia Department of Health, and One Medical Group (R.K.) — all in Washington, DC; the Departments of Virology (E.M.K., S.K., T.S., L.L., O.V.) and Veterinary Biosciences (E.M.K., O.V.), University of Helsinki, and the Department of Virology and Immunology, University of Helsinki and Helsinki University Hospital (A.J.J., O.V.), Helsinki; and the Arboviral Diseases Branch, Division of Vector-Borne Diseases, National Center for Emerging Zoonotic Infectious Diseases, CDC, Atlanta (R.L.). Address reprint requests to Dr. Driggers at [email protected], to Dr. du Plessis at [email protected], or to Dr. Vapalahti at [email protected]. http://www.nejm.org/doi/full/10.1056/NEJMoa1601824#t=article

Patient B detailed in NEJM: Zika Virus Infection with Prolonged Maternal Viremia and Fetal Brain AbnormalitiesRita W. Driggers, M.D., Cheng-Ying Ho, M.D., Ph.D., Essi M. Korhonen, M.Sc., Suvi Kuivanen, M.Sc., Anne J. Jääskeläinen, Ph.D., Teemu Smura, Ph.D., Avi Rosenberg, M.D., Ph.D., D. Ashley Hill, M.D., Roberta L. DeBiasi, M.D., Gilbert Vezina, M.D., Julia Timofeev, M.D., Fausto J. Rodriguez, M.D., Lev Levanov, Ph.D., Jennifer Razak, M.G.C., C.G.C, Preetha Iyengar, M.D., Andrew Hennenfent, D.V.M., M.P.H., Richard Kennedy, M.D., Robert Lanciotti, Ph.D., Adre du Plessis, M.B., Ch.B., M.P.H., and Olli Vapalahti, M.D., Ph.D. March 30, 2016DOI: 10.1056/NEJMoa1601824 SOURCE INFORMATIONFrom the Department of Gynecology and Obstetrics, Division of Maternal Fetal Medicine (R.W.D., J.T.), and the Department of Pathology (F.J.R.), Johns Hopkins University School of Medicine, Baltimore; the Division of Maternal Fetal Medicine, Sibley Memorial Hospital (R.W.D., J.T., J.R.), the Division of Pathology and Center for Genetic Medicine Research (C.-Y.H., A.R., D.A.H.), Division of Pediatric Infectious Diseases (R.L.D.), Department of Diagnostic Radiology and Imaging (G.V.), and the Fetal Medicine Institute, Division of Fetal and Transitional Medicine (A.P.), Children’s National Health System, the Departments of Integrative Systems Biology (C.-Y.H., D.A.H.), Pediatrics and Microbiology, Immunology and Tropical Medicine (R.L.D.B.), and Radiology and Pediatrics (G.V.), George Washington University School of Medicine and Health Sciences, the Center for Policy, Planning and Evaluation (P.I.) and Centers for Disease Control and Prevention (CDC)–Council of State and Territorial Epidemiologists (CSTE) Applied Epidemiology Fellowship (A.H.), District of Columbia Department of Health, and One Medical Group (R.K.) — all in Washington, DC; the Departments of Virology (E.M.K., S.K., T.S., L.L., O.V.) and Veterinary Biosciences (E.M.K., O.V.), University of Helsinki, and the Department of Virology and Immunology, University of Helsinki and Helsinki University Hospital (A.J.J., O.V.), Helsinki; and the Arboviral Diseases Branch, Division of Vector-Borne Diseases, National Center for Emerging Zoonotic Infectious Diseases, CDC, Atlanta (R.L.). Address reprint requests to Dr. Driggers at [email protected], to Dr. du Plessis at [email protected], or to Dr. Vapalahti at [email protected]. http://www.nejm.org/doi/full/10.1056/NEJMoa1601824#t=article

Zika Virus Infection with Prolonged Maternal Viremia and Fetal Brain AbnormalitiesRita W. Driggers, M.D., Cheng-Ying Ho, M.D., Ph.D., Essi M. Korhonen, M.Sc., Suvi Kuivanen, M.Sc., Anne J. Jääskeläinen, Ph.D., Teemu Smura, Ph.D., Avi Rosenberg, M.D., Ph.D., D. Ashley Hill, M.D., Roberta L. DeBiasi, M.D., Gilbert Vezina, M.D., Julia Timofeev, M.D., Fausto J. Rodriguez, M.D., Lev Levanov, Ph.D., Jennifer Razak, M.G.C., C.G.C, Preetha Iyengar, M.D., Andrew Hennenfent, D.V.M., M.P.H., Richard Kennedy, M.D., Robert Lanciotti, Ph.D., Adre du Plessis, M.B., Ch.B., M.P.H., and Olli Vapalahti, M.D., Ph.D. March 30, 2016DOI: 10.1056/NEJMoa1601824 http://www.nejm.org/doi/full/10.1056/NEJMoa1601824#t=article

Zika Virus Infection with Prolonged Maternal Viremia and Fetal Brain AbnormalitiesRita W. Driggers, M.D., Cheng-Ying Ho, M.D., Ph.D., Essi M. Korhonen, M.Sc., Suvi Kuivanen, M.Sc., Anne J. Jääskeläinen, Ph.D., Teemu Smura, Ph.D., Avi Rosenberg, M.D., Ph.D., D. Ashley Hill, M.D., Roberta L. DeBiasi, M.D., Gilbert Vezina, M.D., Julia Timofeev, M.D., Fausto J. Rodriguez, M.D., Lev Levanov, Ph.D., Jennifer Razak, M.G.C., C.G.C, Preetha Iyengar, M.D., Andrew Hennenfent, D.V.M., M.P.H., Richard Kennedy, M.D., Robert Lanciotti, Ph.D., Adre du Plessis, M.B., Ch.B., M.P.H., and Olli Vapalahti, M.D., Ph.D. March 30, 2016DOI: 10.1056/NEJMoa1601824 SOURCE INFORMATIONFrom the Department of Gynecology and Obstetrics, Division of Maternal Fetal Medicine (R.W.D., J.T.), and the Department of Pathology (F.J.R.), Johns Hopkins University School of Medicine, Baltimore; the Division of Maternal Fetal Medicine, Sibley Memorial Hospital (R.W.D., J.T., J.R.), the Division of Pathology and Center for Genetic Medicine Research (C.-Y.H., A.R., D.A.H.), Division of Pediatric Infectious Diseases (R.L.D.), Department of Diagnostic Radiology and Imaging (G.V.), and the Fetal Medicine Institute, Division of Fetal and Transitional Medicine (A.P.), Children’s National Health System, the Departments of Integrative Systems Biology (C.-Y.H., D.A.H.), Pediatrics and Microbiology, Immunology and Tropical Medicine (R.L.D.B.), and Radiology and Pediatrics (G.V.), George Washington University School of Medicine and Health Sciences, the Center for Policy, Planning and Evaluation (P.I.) and Centers for Disease Control and Prevention (CDC)–Council of State and Territorial Epidemiologists (CSTE) Applied Epidemiology Fellowship (A.H.), District of Columbia Department of Health, and One Medical Group (R.K.) — all in Washington, DC; the Departments of Virology (E.M.K., S.K., T.S., L.L., O.V.) and Veterinary Biosciences (E.M.K., O.V.), University of Helsinki, and the Department of Virology and Immunology, University of Helsinki and Helsinki University Hospital (A.J.J., O.V.), Helsinki; and the Arboviral Diseases Branch, Division of Vector-Borne Diseases, National Center for Emerging Zoonotic Infectious Diseases, CDC, Atlanta (R.L.). Address reprint requests to Dr. Driggers at [email protected], to Dr. du Plessis at [email protected], or to Dr. Vapalahti at [email protected]. http://www.nejm.org/doi/full/10.1056/NEJMoa1601824#t=article

Zika Virus Infection with Prolonged Maternal Viremia and Fetal Brain AbnormalitiesRita W. Driggers, M.D., Cheng-Ying Ho, M.D., Ph.D., Essi M. Korhonen, M.Sc., Suvi Kuivanen, M.Sc., Anne J. Jääskeläinen, Ph.D., Teemu Smura, Ph.D., Avi Rosenberg, M.D., Ph.D., D. Ashley Hill, M.D., Roberta L. DeBiasi, M.D., Gilbert Vezina, M.D., Julia Timofeev, M.D., Fausto J. Rodriguez, M.D., Lev Levanov, Ph.D., Jennifer Razak, M.G.C., C.G.C, Preetha Iyengar, M.D., Andrew Hennenfent, D.V.M., M.P.H., Richard Kennedy, M.D., Robert Lanciotti, Ph.D., Adre du Plessis, M.B., Ch.B., M.P.H., and Olli Vapalahti, M.D., Ph.D. March 30, 2016DOI: 10.1056/NEJMoa1601824 SOURCE INFORMATIONFrom the Department of Gynecology and Obstetrics, Division of Maternal Fetal Medicine (R.W.D., J.T.), and the Department of Pathology (F.J.R.), Johns Hopkins University School of Medicine, Baltimore; the Division of Maternal Fetal Medicine, Sibley Memorial Hospital (R.W.D., J.T., J.R.), the Division of Pathology and Center for Genetic Medicine Research (C.-Y.H., A.R., D.A.H.), Division of Pediatric Infectious Diseases (R.L.D.), Department of Diagnostic Radiology and Imaging (G.V.), and the Fetal Medicine Institute, Division of Fetal and Transitional Medicine (A.P.), Children’s National Health System, the Departments of Integrative Systems Biology (C.-Y.H., D.A.H.), Pediatrics and Microbiology, Immunology and Tropical Medicine (R.L.D.B.), and Radiology and Pediatrics (G.V.), George Washington University School of Medicine and Health Sciences, the Center for Policy, Planning and Evaluation (P.I.) and Centers for Disease Control and Prevention (CDC)–Council of State and Territorial Epidemiologists (CSTE) Applied Epidemiology Fellowship (A.H.), District of Columbia Department of Health, and One Medical Group (R.K.) — all in Washington, DC; the Departments of Virology (E.M.K., S.K., T.S., L.L., O.V.) and Veterinary Biosciences (E.M.K., O.V.), University of Helsinki, and the Department of Virology and Immunology, University of Helsinki and Helsinki University Hospital (A.J.J., O.V.), Helsinki; and the Arboviral Diseases Branch, Division of Vector-Borne Diseases, National Center for Emerging Zoonotic Infectious Diseases, CDC, Atlanta (R.L.). Address reprint requests to Dr. Driggers at [email protected], to Dr. du Plessis at [email protected], or to Dr. Vapalahti at [email protected]. http://www.nejm.org/doi/full/10.1056/NEJMoa1601824#t=article

TABLE I. Provisional* cases of selected† infrequently reported notifiable diseases (<1,000 cases reported during the preceding year), United States, week ending March 26, 2016 (WEEK 12) Disease Total cases reported for previous years Current weekCum 20165-year weekly average§20152014201320122011States reporting cases during current week (No.12) Anthrax-------1 Arboviral diseases ¶,**: Chikungunya virus ††-161797NNNNNNNN Eastern equine encephalitis virus--0688154 Jamestown Canyon virus §§---8112223 La Crosse virus §§---55808578130 Powassan virus---7812716 St. Louis encephalitis virus--01910136 Western equine encephalitis virus-------- Zika virus ¶¶1274038NNNNNNNNMS (1 ) ¶¶ This table does not include cases from the U.S. territories. There may be some delay between identification of a case and reporting to CDC. All cases reported are travel related. Office of Management and Budget approval of the NNDSS Revision #0920-0728 on January 21, 2016, authorized CDC to receive data for these conditions. CDC is in the process of soliciting data for these conditions †† Data for Chikungunya virus and Hantavirus infection, non-HPS. Office of Management and Budget approval of the NNDSS Revision #0920-0728 on January 21, 2016, authorized CDC to receive data for these conditions. CDC is in the process of soliciting data for these conditions

Zika virus ¶¶1274038NNNNNNNNMS (1 ) http://wonder.cdc.gov/mmwr/mmwr_2016.asp?mmwr_year=2016&mmwr_week=12&mmwr_table=1&request=Submit&mmwr_location=

Map Update https://www.google.com/maps/d/edit?hl=en&hl=en&authuser=0&authuser=0&mid=zv94AJqgUct4.kT4qLMXp3SLU

March 30, 2016 DEPARTMENT OF HEALTH DAILY ZIKA UPDATE: ONE NEW TRAVEL-RELATED CASE TODAY IN PALM BEACH 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, the Florida Department of Health 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. There is one new case today in Palm Beach County. Palm Beach County has been added to the Declaration of Public Health Emergency. Of the cases confirmed in Florida, three cases are still exhibiting symptoms. According to the CDC, symptoms associated with the Zika virus last between seven to 10 days. Based on CDC guidance, several pregnant women who have traveled to countries with local-transmission of Zika have received antibody testing, and of those, four have tested positive for the Zika virus. The CDC recommends that a pregnant woman with a history of Zika virus and her provider should consider additional ultrasounds. It is recommended that women who are pregnant or thinking of becoming pregnant postpone travel to Zika affected areas. County Number of Cases (all travel related) Alachua 4 Brevard 2 Broward 11 Clay 1 Collier 1 Hillsborough 3 Lee 3 Miami-Dade 32 Orange 5 Osceola 4 Palm Beach 1 Polk 2 Santa Rosa 1 Seminole 1 St. Johns 1 Cases involving pregnant women* 4 Total 76 *Counties of pregnant women will not be shared. On Feb. 12, Governor Scott directed the State Surgeon General 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 1,216 callers since it launched. 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. For more information on the Zika virus, click here. The department 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 the State Surgeon General to issue a Declaration of Public Health Emergency for the counties of residents with travel-associated cases of Zika.The Declaration currently includes the 15 affected counties – Alachua, Brevard, Broward, Clay, Collier, Hillsborough, Lee, Miami-Dade, Orange, Osceola, Palm Beach, Polk, Santa Rosa, Seminole 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 4,028 people for active Zika virus and 1,698 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.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 website here.The CDC has put out guidance related to the sexual transmission of the Zika virus. This includes the CDC recommendation that if you have traveled to a country with local transmission of Zika you should abstain from unprotected sex.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/2016/03/033016-zika-update.html

County Number of Cases (all travel related) Alachua 4 Brevard 2 Broward 11 Clay 1 Collier 1 Hillsborough 3 Lee 3 Miami-Dade 32 Orange 5 Osceola 4 Palm Beach 1 Polk 2 Santa Rosa 1 Seminole 1 St. Johns 1 Cases involving pregnant women* 4 Total 76 *Counties of pregnant women will not be shared.

Study explores mechanism linking Zika virus, birth defectsResearchers say a protein the Zika virus uses to infect skin cells and cause a rash is also present in stem cells of the developing brain and retina of a fetus. By HealthDay News | March 30, 2016 at 12:07 PM 0 Comments Baby with microcephaly. Photo: U.S. Centers for Disease Control and PreventionWEDNESDAY, March 30, 2016 -- Scientists say they've discovered how the Zika virus might cause severe brain and eye birth defects.The Zika outbreak in Brazil and other parts of Latin American and the Caribbean has coincided with a sharp increase in the number of babies born with microcephaly, which results in abnormally small heads and brains. There has also been a rise in other brain and eye birth defects in countries affected by the Zika outbreak. But firm evidence of a link between the virus and these birth defects has been lacking. ADVERTISINGinRead invented by TeadsIn a new study, researchers at the University of California, San Francisco (UCSF), found that a protein the Zika virus uses to infect skin cells and cause a rash is also present in stem cells of the developing brain and retina of a fetus. The so-called AXL protein sits on the surface of cells and can provide an entry point for Zika. Learning more about the link between Zika and AXL could lead to drugs to block Zika infection, according to the researchers. The brain and eye birth defects occurring in areas with Zika outbreaks are "precisely the kind of damage we would expect to see from something that was destroying neural and retinal stem cells during development," said study senior author Dr. Arnold Kriegstein. He is director of UCSF's Center of Regeneration Medicine and Stem Cell Research. "If we can understand how Zika may be causing birth defects, we can start looking for compounds to protect pregnant women who become infected," Kriegstein said in a university news release. The study was published online March 30 in the journal Cell Stem Cell. A mosquito-borne virus, Zika has been suspected of causing thousands of cases of microcephaly in Brazil. While the bulk of Zika cases leading to microcephaly may occur via maternal infection during pregnancy, cases of sexual transmission from a man to his female partner have come to light, according to the U.S. Centers for Disease Control and Prevention. Zika infection is usually a mild illness in adults, and many cases may occur without symptoms, experts say. However, because of the risk to babies, the CDC is advising that men with known or suspected infection with Zika refrain from sex -- or only have sex with a condom -- for six months after a diagnosis. The agency also advises that, for couples involving a man who has traveled to or resides in an area endemic for Zika: The couple refrain from sex, or use condoms during sex, throughout the duration of a pregnancy.They refrain from sex, or use condoms during sex, for eight weeks if the man has returned from travel to a Zika-endemic area but has not shown signs of infection.For couples living in a Zika-endemic area, they refrain from sex or engage in sex only with a condom for as long as active Zika transmission persists in that area.The latest guidelines also recommend that women who know they've been infected, or who have no symptoms but have recently been to a Zika-endemic area, or think they might have been exposed via sex, should wait at least eight weeks before trying to get pregnant. The CDC has also advised that all pregnant women consider postponing travel to any area where Zika virus transmission is ongoing. If a pregnant woman must travel to or live in one of these areas, she should talk to her health-care provider first and strictly follow steps to prevent mosquito bites. In the majority of Zika infections, symptoms included rash (97 percent of cases), fever and joint pain. "Zika virus disease should be considered in patients with acute onset of fever, rash, arthralgia [joint pain], or conjunctivitis [pink eye] who traveled to areas with ongoing Zika virus transmission or who had unprotected sex with someone who traveled to one of those areas and developed compatible symptoms within two weeks of returning," according to the CDC. First discovered in Uganda in 1947, the Zika virus wasn't thought to pose major health risks until last year, when it became clear that it posed potentially devastating threats to pregnant women. The Zika virus has now spread to over 38 countries and territories, most in Latin America and the Caribbean. The World Health Organization estimates there could be up to 4 million cases of Zika in the Americas in the next year. More informationFor more on Zika virus, visit the U.S. Centers for Disease Control and Prevention. To see the CDC list of sites where Zika virus is active and may pose a threat to pregnant women, click here. http://www.upi.com/Health_News/2016/03/30/Study-explores-mechanism-linking-Zika-virus-birth-defects/3461459353864/

ReferencesAuthorsTitleSourceAhlfors, K., Ivarsson, S.A., and Bjerre, I.Microcephaly and congenital cytomegalovirus infection: a combined prospective and retrospective study of a Swedish infant population.Pediatrics. 1986; 78: 1058–1063Barkovich, A.J., Guerrini, R., Kuzniecky, R.I., Jackson, G.D., and Dobyns, W.B.A developmental and genetic classification for malformations of cortical development: update 2012.CrossRef | PubMed | Scopus (198)Brain. 2012; 135: 1348–1369Bell, T.M., Field, E.J., and Narang, H.K.Zika virus infection of the central nervous system of mice.CrossRef | PubMed | Scopus (6)Arch. Gesamte Virusforsch. 1971; 35:183–193Brasil, P., Pereira, J.P. Jr., Raja Gabaglia, C., Damasceno, L., Wakimoto, M., Ribeiro Nogueira, R.M., Carvalho de Sequeira, P., Machado Siqueira, A., Abreu de Carvalho, L.M., Cotrim da Cunha, D. et al.Zika Virus Infection in Pregnant Women in Rio de Janeiro - Preliminary Report.CrossRef | PubMedN. Engl. J. Med. 2016;DOI: http://dx.doi.org/10.1056/NEJMoa1602412Calvet, G., Aguiar, R.S., Melo, A.S., Sampaio, S.A., de Filippis, I., Fabri, A., Araujo, E.S., de Sequeira, P.C., de Mendonça, M.C., de Oliveira, L. et al.Detection and sequencing of Zika virus from amniotic fluid of fetuses with microcephaly in Brazil: a case study.Abstract | Full Text | Full Text PDFLancet Infect. Dis. 2016;DOI: http://dx.doi.org/10.1016/S1473-3099(16)00095-5Carteaux, G., Maquart, M., Bedet, A., Contou, D., Brugières, P., Fourati, S., Cleret de Langavant, L., de Broucker, T., Brun-Buisson, C., Leparc-Goffart, I., and Mekontso Dessap, A.Zika Virus Associated with Meningoencephalitis.CrossRefN. Engl. J. Med. 2016;DOI: http://dx.doi.org/10.1056/NEJMc1602964Conboy, T.J., Pass, R.F., Stagno, S., Britt, W.J., Alford, C.A., McFarland, C.E., and Boll, T.J.Intellectual development in school-aged children with asymptomatic congenital cytomegalovirus infection.PubMedPediatrics. 1986; 77: 801–806de Paula Freitas, B., de Oliveira Dias, J.R., Prazeres, J., Sacramento, G.A., Ko, A.I., Maia, M., and Belfort, R. Jr.Ocular Findings in Infants With Microcephaly Associated With Presumed Zika Virus Congenital Infection in Salvador, Brazil.CrossRef | PubMedJAMA Ophthalmol. 2016;DOI:http://dx.doi.org/10.1001/jamaophthalmol.2016.0267Dick, G.W.Zika virus. II. Pathogenicity and physical properties.PubMedTrans. R. Soc. Trop. Med. Hyg. 1952; 46:521–534Dick, G.W., Kitchen, S.F., and Haddow, A.J.Zika virus. I. Isolations and serological specificity.PubMedTrans. R. Soc. Trop. Med. Hyg. 1952; 46:509–520Fietz, S.A., Kelava, I., Vogt, J., Wilsch-Bräuninger, M., Stenzel, D., Fish, J.L., Corbeil, D., Riehn, A., Distler, W., Nitsch, R., and Huttner, W.B.OSVZ progenitors of human and ferret neocortex are epithelial-like and expand by integrin signaling.CrossRef | PubMed | Scopus (222)Nat. Neurosci. 2010; 13: 690–699Fowler, K.B., Stagno, S., Pass, R.F., Britt, W.J., Boll, T.J., and Alford, C.A.The outcome of congenital cytomegalovirus infection in relation to maternal antibody status.CrossRef | PubMedN. Engl. J. Med. 1992; 326: 663–667Gérardin, P., Sampériz, S., Ramful, D., Boumahni, B., Bintner, M., Alessandri, J.L., Carbonnier, M., Tiran-Rajaoefera, I., Beullier, G., Boya, I. et al.Neurocognitive outcome of children exposed to perinatal mother-to-child Chikungunya virus infection: the CHIMERE cohort study on Reunion Island.CrossRef | PubMed | Scopus (7)PLoS Negl. Trop. Dis. 2014; 8: e2996Gruber, R., Zhou, Z., Sukchev, M., Joerss, T., Frappart, P.O., and Wang, Z.Q.MCPH1 regulates the neuroprogenitor division mode by coupling the centrosomal cycle with mitotic entry through the Chk1-Cdc25 pathway.CrossRef | PubMed | Scopus (82)Nat. Cell Biol. 2011; 13: 1325–1334Hamel, R., Dejarnac, O., Wichit, S., Ekchariyawat, P., Neyret, A., Luplertlop, N., Perera-Lecoin, M., Surasombatpattana, P., Talignani, L., Thomas, F. et al.Biology of Zika Virus Infection in Human Skin Cells.CrossRef | PubMed | Scopus (8)J. Virol. 2015; 89: 8880–8896Hansen, D.V., Lui, J.H., Parker, P.R., and Kriegstein, A.R.Neurogenic radial glia in the outer subventricular zone of human neocortex.CrossRef | PubMed | Scopus (336)Nature. 2010; 464: 554–561Heymann, D.L., Hodgson, A., Sall, A.A., Freedman, D.O., Staples, J.E., Althabe, F., Baruah, K., Mahmud, G., Kandun, N., Vasconcelos, P.F. et al.Zika virus and microcephaly: why is this situation a PHEIC?.Abstract | Full Text | Full Text PDF | PubMed | Scopus (3)Lancet. 2016; 387: 719–721Ji, R., Tian, S., Lu, H.J., Lu, Q., Zheng, Y., Wang, X., Ding, J., Li, Q., and Lu, Q.TAM receptors affect adult brain neurogenesis by negative regulation of microglial cell activation.CrossRef | PubMed | Scopus (20)J. Immunol. 2013; 191: 6165–6177Ji, R., Meng, L., Jiang, X., Cvm, N.K., Ding, J., Li, Q., and Lu, Q.TAM receptors support neural stem cell survival, proliferation and neuronal differentiation.CrossRef | Scopus (5)PLoS ONE. 2014; 9: e115140Kawasaki, H., Kosugi, I., Sakao-Suzuki, M., Meguro, S., Arai, Y., Tsutsui, Y., and Iwashita, T.Cytomegalovirus initiates infection selectively from high-level β1 integrin-expressing cells in the brain.Abstract | Full Text | Full Text PDF | PubMedAm. J. Pathol. 2015; 185: 1304–1323Lanari, M., Capretti, M.G., Lazzarotto, T., Gabrielli, L., Rizzollo, S., Mostert, M., and Manzoni, P.Neuroimaging in CMV congenital infected neonates: how and when.Abstract | Full Text PDF | PubMed | Scopus (10)Early Hum. Dev. 2012; 88: S3–S5Lemke, G. and Burstyn-Cohen, T.TAM receptors and the clearance of apoptotic cells.CrossRef | PubMed | Scopus (49)Ann. N Y Acad. Sci. 2010; 1209: 23–29Lizarraga, S.B., Margossian, S.P., Harris, M.H., Campagna, D.R., Han, A.P., Blevins, S., Mudbhary, R., Barker, J.E., Walsh, C.A., and Fleming, M.D.Cdk5rap2 regulates centrosome function and chromosome segregation in neuronal progenitors.CrossRef | PubMed | Scopus (86)Development. 2010; 137: 1907–1917Lukaszewicz, A., Savatier, P., Cortay, V., Giroud, P., Huissoud, C., Berland, M., Kennedy, H., and Dehay, C.G1 phase regulation, area-specific cell cycle control, and cytoarchitectonics in the primate cortex.Abstract | Full Text | Full Text PDF | PubMed | Scopus (149)Neuron. 2005; 47: 353–364Martines, R.B., Bhatnagar, J., Keating, M.K., Silva-Flannery, L., Muehlenbachs, A., Gary, J., Goldsmith, C., Hale, G., Ritter, J., Rollin, D. 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.CrossRefMMWR Morb. Mortal. Wkly. Rep. 2016;65: 159–160Mécharles, S., Herrmann, C., Poullain, P., Tran, T.H., Deschamps, N., Mathon, G., Landais, A., Breurec, S., and Lannuzel, A.Acute myelitis due to Zika virus infection.Abstract | Full Text | Full Text PDFLancet. 2016;DOI: http://dx.doi.org/10.1016/S0140-6736(16)00644-9Meertens, L., Carnec, X., Lecoin, M.P., Ramdasi, R., Guivel-Benhassine, F., Lew, E., Lemke, G., Schwartz, O., and Amara, A.The TIM and TAM families of phosphatidylserine receptors mediate dengue virus entry.Abstract | Full Text | Full Text PDF | PubMed | Scopus (68)Cell Host Microbe. 2012; 12: 544–557Miner, J.J., Daniels, B.P., Shrestha, B., Proenca-Modena, J.L., Lew, E.D., Lazear, H.M., Gorman, M.J., Lemke, G., Klein, R.S., and Diamond, M.S.The TAM receptor Mertk protects against neuroinvasive viral infection by maintaining blood-brain barrier integrity.CrossRef | Scopus (1)Nat. Med. 2015; 21: 1464–1472Mlakar, J., Korva, M., Tul, N., Popović, M., Poljšak-Prijatelj, M., Mraz, J., Kolenc, M., Resman Rus, K., Vesnaver Vipotnik, T., Fabjan Vodušek, V. et al.Zika Virus Associated with Microcephaly.CrossRef | Scopus (12)N. Engl. J. Med. 2016; 374: 951–958Nakao, T. and Chiba, S.Cytomegalovirus and microcephaly.Pediatrics. 1970; 46: 483–484O’Leary, D.R., Kuhn, S., Kniss, K.L., Hinckley, A.F., Rasmussen, S.A., Pape, W.J., Kightlinger, L.K., Beecham, B.D., Miller, T.K., Neitzel, D.F. et al.Birth outcomes following West Nile Virus infection of pregnant women in the United States: 2003-2004.CrossRef | PubMed | Scopus (53)Pediatrics. 2006; 117: e537–e545Oliveira Melo, A.S., Malinger, G., Ximenes, R., Szejnfeld, P.O., Alves Sampaio, S., and Bispo de Filippis, A.M.Zika virus intrauterine infection causes fetal brain abnormality and microcephaly: tip of the iceberg?.CrossRef | PubMed | Scopus (19)Ultrasound Obstet. Gynecol. 2016; 47: 6–7Ostrem, B.E., Lui, J.H., Gertz, C.C., and Kriegstein, A.R.Control of outer radial glial stem cell mitosis in the human brain.Abstract | Full Text | Full Text PDF | PubMed | Scopus (12)Cell Rep. 2014; 8: 656–664Perera-Lecoin, M., Meertens, L., Carnec, X., and Amara, A.Flavivirus entry receptors: an update.CrossRef | Scopus (16)Viruses. 2014; 6: 69–88Pollen, A.A., Nowakowski, T.J., Chen, J., Retallack, H., Sandoval-Espinosa, C., Nicholas, C.R., Shuga, J., Liu, S.J., Oldham, M.C., Diaz, A. et al.Molecular identity of human outer radial glia during cortical development.Abstract | Full Text | Full Text PDF | PubMed | Scopus (6)Cell. 2015; 163: 55–67Rothlin, C.V., Ghosh, S., Zuniga, E.I., Oldstone, M.B., and Lemke, G.TAM receptors are pleiotropic inhibitors of the innate immune response.Abstract | Full Text | Full Text PDF | PubMed | Scopus (318)Cell. 2007; 131: 1124–1136Sakaguchi, H., Kadoshima, T., Soen, M., Narii, N., Ishida, Y., Ohgushi, M., Takahashi, J., Eiraku, M., and Sasai, Y.Generation of functional hippocampal neurons from self-organizing human embryonic stem cell-derived dorsomedial telencephalic tissue.CrossRef | Scopus (1)Nat. Commun. 2015; 6: 8896Schuler-Faccini, L., Ribeiro, E.M., Feitosa, I.M., Horovitz, D.D., Cavalcanti, D.P., Pessoa, A., Doriqui, M.J., Neri, J.I., Neto, J.M., Wanderley, H.Y...., and Brazilian Medical Genetics Society–Zika Embryopathy Task Force.Possible Association Between Zika Virus Infection and Microcephaly - Brazil, 2015.CrossRef | PubMedMMWR Morb. Mortal. Wkly. Rep. 2016;65: 59–62Sinha, S.K., Kaveggia, E., and Gordon, M.C.The incidence of cytomegalovirus among mentally retarded and microcephalic children in a state institution.J. Ment. Defic. Res. 1972; 16: 90–96Tang, H., Hammack, C., Ogden, S.C., Wen, Z., Qian, X., Li, Y., Yao, B., Shin, J., Zhang, F., Lee, E.M. et al.Zika Virus Infects Human Cortical Neural Progenitors and Attenuates Their Growth.Abstract | Full Text | Full Text PDFCell Stem Cell. 2016;DOI: http://dx.doi.org/10.1016/j.stem.2016.02.016Teissier, N., Fallet-Bianco, C., Delezoide, A.L., Laquerrière, A., Marcorelles, P., Khung-Savatovsky, S., Nardelli, J., Cipriani, S., Csaba, Z., Picone, O. et al.Cytomegalovirus-induced brain malformations in fetuses.CrossRef | Scopus (8)J. Neuropathol. Exp. Neurol. 2014; 73:143–158von der Hagen, M., Pivarcsi, M., Liebe, J., von Bernuth, H., Didonato, N., Hennermann, J.B., Bührer, C., Wieczorek, D., and Kaindl, A.M.Diagnostic approach to microcephaly in childhood: a two-center study and review of the literature.CrossRef | PubMed | Scopus (12)Dev. Med. Child Neurol. 2014; 56: 732–741Wang, J., Zhang, H., Young, A.G., Qiu, R., Argalian, S., Li, X., Wu, X., Lemke, G., and Lu, Q.Transcriptome analysis of neural progenitor cells by a genetic dual reporter strategy.CrossRef | PubMed | Scopus (7)Stem Cells. 2011; 29: 1589–1600Woods, C.G., Bond, J., and Enard, W.Autosomal recessive primary microcephaly (MCPH): a review of clinical, molecular, and evolutionary findings.Abstract | Full Text | Full Text PDF | PubMed | Scopus (187)Am. J. Hum. Genet. 2005; 76: 717–728

Main TextIn February 2016, the World Health Organization declared the 2015 outbreak of the Zika virus (ZIKV) in Central and South America a global health emergency (Heymann et al., 2016) following a strong correlation between cases of ZIKV infection and a dramatic increase in microcephaly cases in Brazil (Oliveira Melo et al., 2016, Schuler-Faccini et al., 2016). Subsequent reports have now established the ability of ZIKV to cross the human fetal-placental barrier to infect the developing central nervous system (Calvet et al., 2016, Martines et al., 2016,Mlakar et al., 2016). The neurotropism and neurovirulence of ZIKV has been appreciated in model systems since the earliest description of the virus (Bell et al., 1971, Dick, 1952, Dick et al., 1952), but it has only recently been described in human neural stem and progenitor cells using in vitro systems (Tang et al., 2016; P.P. Garcêz, E.C. Loiola, R.M. da Costa, L.M. Higa, P. Trindade, R. Delvecchio, J.M. Nascimento, R. Brindeiro, A. Tanuri, and S.K. Rehen, 2016,PeerJ, preprint). Although pathology data is currently limited, the first imaging studies and cases with confirmed ZIKV infection in the prenatal brain showed devastating consequences, including severe microcephaly, lissencephaly, hydrocephaly, necrosis, periventricular and cortical calcification, diffuse astrogliosis, and activated microglia (Mlakar et al., 2016, Schuler-Faccini et al., 2016). The findings of massive cell death and necrosis reflect a far more destructive process than occurs in many genetic forms of microcephaly. Primary microcephaly is thought to result from a depletion of the founder population of radial glia, the neural stem cells in developing brain, either through cell death or premature differentiation (Barkovich et al., 2012). Infrequent cases of neurodevelopmental brain malformations including microcephaly have been reported in association with viral infections, including cytomegalovirus (CMV), rubella virus, West Nile Virus, HIV, herpes simplex, and chikungunya (Ahlfors et al., 1986, Gérardin et al., 2014, Lanari et al., 2012, Nakao and Chiba, 1970,O’Leary et al., 2006, Sinha et al., 1972, Teissier et al., 2014, von der Hagen et al., 2014). Of the few viruses known to cross the placental barrier, CMV infection causes similar neurodevelopmental brain abnormalities to those caused by ZIKV (Conboy et al., 1986, Fowler et al., 1992, Teissier et al., 2014). CMV neuroinvasiveness is mediated by a variety of entry factors, including integrins and EGFR, which are highly expressed by radial glia, a neural stem cell population. Higher expression of these entry proteins determines the initial susceptible cell population (Kawasaki et al., 2015). Based on the role of neural stem cells in other forms of microcephaly, we hypothesized that human radial glia may selectively express proteins promoting ZIKV entry and infectivity during neurogenesis. In support of this hypothesis, two recent papers demonstrated the vulnerability of neural stem and progenitor cells to ZIKV using in vitro cultures derived from pluripotent stem cells (Tang et al., 2016; P.P. Garcêz, E.C. Loiola, R.M. da Costa, L.M. Higa, P. Trindade, R. Delvecchio, J.M. Nascimento, R. Brindeiro, A. Tanuri, and S.K. Rehen, 2016,PeerJ, preprint). Many surface proteins facilitate flavivirus entry into cells (Perera-Lecoin et al., 2014), but the precise mechanism remains largely unknown and additional factors may also contribute to infection. Several of these proteins are sufficient to support ZIKV entry into HEK293T cells that normally have low infectivity, including DC-SIGN (encoded by CD209), TIM1 (encoded byHAVCR1), TYRO3, and AXL. Furthermore, blocking or silencing AXL reduces infectivity in cultured fibroblasts and alveolar epithelial cells by as much as 90% (Hamel et al., 2015). Understanding the expression patterns of putative flavivirus receptors could strengthen the possible link between ZIKV infection and microcephaly and support the discovery of a mechanism of ZIKV neurovirulence. To identify cell populations that may be particularly vulnerable to ZIKV infection, we analyzed the expression of candidate genes mediating flavivirus entry across single cells from the developing human cerebral cortex (Figure 1A). We previously classified single cells from developing cortex as astrocytes, radial glia, intermediate progenitor cells, and immature excitatory and inhibitory neurons using patterns of genome-wide gene expression (Pollen et al., 2015). To survey additional cell types, we also analyzed cells from developing cortex that express markers of microglia and endothelial cells (Table S1). Importantly, while many candidate entry receptors and attachment factors have been described, other unknown factors may mediate ZIKV entry, and we also include a global table of gene expression across single cells (Table S2). Across cell types, we found that multiple putative flavivirus entry receptor genes, including AXL and heat shock protein genes, showed a strong pattern of enrichment in radial glia cells, astrocytes, endothelial cells, and microglia, suggesting that these cell types may be particularly vulnerable to ZIKV infection (Figures 1A and 1B). AXL, known to mediate ZIKV and dengue virus entry in human skin cells (Hamel et al., 2015), showed particularly high expression in radial glia (78/96 radial glia displayed expression greater than 6 log2 normalized read counts). In contrast, other candidate genes known to permit ZIKV entry showed more limited expression at this threshold including TYRO3 (7/418 cells and 5/96 radial glia) and CD209 (DC-SIGN, 0/418 cells, Figure S1). Based on these observations, we further investigated the expression pattern of AXL protein in primary human tissue samples using immunohistochemistry. At mid-neurogenesis, AXL is expressed in a highly reproducible pattern throughout the cortex, with strong expression bordering the lateral ventricle and in the outer subventricular zone (OSVZ) (Figures 2C, 2D, and S1). Closer examination revealed that staining along the ventricle resulted from specific localization of AXL to radial glia apical end-feet (Figures 2D and S1). AXL was also detected at the pial end-feet of radial glia near the meninges (Figure 2B). In recent years a second population of radial glial cells, known as outer radial glia (oRG), has been identified in the OSVZ of the developing human brain (Fietz et al., 2010, Hansen et al., 2010). We observed high levels of AXL in the cell bodies of oRG cells, accounting for the pattern of AXL labeling in the OSVZ (Figures 2C and S1). In addition, pronounced AXL immunostaining outlined brain capillaries (Figures 2A and S1), consistent with AXL expression observed in endothelial cells by single-cell analysis. We further examined AXL expression from stages of early neurogenesis (GW13.5) to term. We found that AXL expression persisted in radial glia throughout the period of neurogenesis and in capillaries and astrocytes to term but remained largely absent from SATB2-expressing neurons, even at later developmental stages (Figure S1). A recent report of 29 infants with presumed ZIKV microcephaly reported that 10 (34.5%) had severe ocular abnormalities. The ocular lesions consisted of focal pigment mottling and chorioretinal atrophy that was particularly severe in the macula (de Paula Freitas et al., 2016). Therefore, we examined AXL expression in developing human retina. We dissected two human neural retina samples at GW10 and GW12 and captured single cells for mRNA sequencing. AXL was highly expressed in cells that had a stem cell gene signature (Figures 1G andS1). To confirm this finding, we immunostained tissue sections of developing retina. AXL was expressed along the outer margin of the neural retina, where it was co-expressed with SOX2, a marker of neural stem cells. In addition, AXL was highly enriched in cells of the ciliary marginal zone, adjacent to the neural retina (Figure 1G). We next investigated the possible conservation of AXL expression across model systems that could be used to study the mechanism of ZIKV infection and pathogenicity. Public repositories of in situ hybridization data indicate AXL expression in mouse radial glia (Figure 2A). In addition, previous studies of mouse cortex reported enriched Axl expression in the apical end-feet of radial glia cells (Wang et al., 2011). We examined the expression pattern of AXL in developing ferret cortex and found that, similar to human cortex, AXL is expressed in the end-feet of radial glia cells at the ventricular edge and in oRG cells in the OSVZ (Figure 2B). Finally, we generated human iPSC-derived cerebral organoids and observed AXL expression along the lumen of neuroepithelial-like rosette regions in the organoids, which resemble the VZ of primary human cortex, and in SOX2-expressing cells away from the lumen (Figure S2). The specific expression of AXL in radial glia-like and oRG-like cells in the organoids and limited expression in neurons is consistent with observations from single-cell mRNA-seq analysis of similarly derived cerebral organoids (Figures 2C and S2). Interestingly, human cerebral organoids also contain cells that resemble early choroid plexus cells (Sakaguchi et al., 2015), and these cells strongly express AXL (Figures 2C and S2), consistent with the expression pattern in embryonic mouse (Figure 2A). Here we report that the candidate ZIKV receptor AXL is highly enriched in radial glia, the neural stem cells of the human fetal cerebral cortex, providing a hypothesis for why these cells are particularly vulnerable to ZIKV infection and providing a candidate mechanism for ZIKV-induced microcephaly. This finding supports recent suggestions that ZIKV preferentially targets in-vitro-derived progenitor cells rather than immature neurons (Tang et al., 2016). Furthermore, we show that AXL is expressed by cortical astrocytes, blood microcapillaries, microglia, and progenitors in the neural retina and ciliary marginal zone. The latter finding could help explain how ZIKV causes ocular lesions (de Paula Freitas et al., 2016). The specificity of AXL expression in radial glia neural stem cells is also conserved in mouse and ferret cerebral cortex and in human PSC-derived cerebral organoids. We suggest that these diverse systems may support studies of ZIKV infectivity in radial glia and the downstream consequences that may mediate disease pathogenesis. Transgenic mouse models of microcephaly mutations often show less severe phenotypes than human patients with the same mutation (Barkovich et al., 2012,Gruber et al., 2011, Lizarraga et al., 2010, Woods et al., 2005). Differences in brain development that include massively expanded OSVZ and increased diversity of cortical progenitors in the human cortex likely contribute to this difference. For example, the contribution of oRG cells to brain malformations such as microcephaly or lissencephaly is largely unknown, although this cell type becomes the predominant neural stem cell population in the developing primate and human cortex toward mid-gestation when OSVZ proliferation dramatically increases (Lukaszewicz et al., 2005, Hansen et al., 2010). Our results indicate that oRG cells express AXL at very high levels and are likely targets for ZIKV infectivity. Involvement of oRG cells, which have been linked to developmental and evolutionary cortical expansion (Hansen et al., 2010, Ostrem et al., 2014, Pollen et al., 2015), may make a significant contribution to the severe phenotype of ZIKV microcephaly and agyria. Signaling through AXL suppresses the innate immune response (Rothlin et al., 2007). In dengue virus infection, AXL not only supports virus entry, but its kinase domain also enhances virus infectivity following entry (Meertens et al., 2012). If ZIKV binds AXL during entry, it may similarly activate AXL signaling and suppress the innate immune response, enabling the virus to better establish an infection and prevent viral clearance (Mlakar et al., 2016). These features suggest that a small-molecule inhibitor of AXL function may be protective against ZIKV infectivity. However, signaling through Axl normally supports neural stem cell survival, proliferation, and neurogenesis (Ji et al., 2014, Lemke and Burstyn-Cohen, 2010), and Axl also maintains the blood-brain barrier, protecting against the neurotropism of other viruses (Miner et al., 2015). Interference with normal AXL has been shown to stimulate production of inflammatory cytokines, promote microglia activation, and eventually lead to the loss of neural stem cells (Ji et al., 2013). Therefore, while blocking AXL may protect against cellular infection or viral replication, perturbation of AXL function may also have multiple adverse consequences. We propose a testable hypothesis: after breaching the placental-fetal barrier, ZIKV reaches the developing brain by hematogenous spread or via the cerebrospinal fluid (CSF) and invades radial glia cells as the first target population with highest AXL expression, either through their processes that often make contact with blood vessels, or via their apical end-feet that make direct contact with the CSF. By preferentially destroying radial glia cells, the founder cell population that generates all cortical neurons, ZIKV can produce severe microcephaly. Future studies will be needed to test this hypothesis and particularly whether AXL expression alone determines the cellular population with enhanced neurotropism for ZIKV in the developing human brain or whether other binding factors, including genes expressed at low levels, may be involved. In addition, further studies are urgently needed to determine (1) how the virus crosses the placenta to infect fetal brain and causes generalized growth restriction (Brasil et al., 2016) and (2) whether the virus infects adult human brain, as ZIKV has recently been detected in the CSF of adults (Carteaux et al., 2016, Mécharles et al., 2016). Finally, other flaviviruses that use similar entry receptors have not been strongly associated with fetal brain abnormalities, and future work must examine potential changes in recent strains of ZIKV. The current manuscript constitutes an initial step toward the understanding of how ZIKV might cause developmental brain malformations. Author ContributionsT.J.N. and E.D.L performed expression studies. T.J.N., A.A.P., and C.S.E. performed bioinformatic analysis. E.D.L. and M.B. generated cerebral organoids. T.J.N, A.A.P., and A.R.K. conceived of the project and wrote the manuscript with input from all authors. AcknowledgmentsWe are grateful to Hanna Retallack, Shaohui Wang, Anne Leyrat, Joe Shuga, Aaron Diaz, Mercedes Paredes, Joseph LoTurco, Melanie Bedolli, Lillian Adame, Joe DeRisi, and Jeremy Reiter for helpful comments, suggestions, and technical help. A.A.P. is supported by a Damon Runyon Cancer Research Foundation postdoctoral fellowship (DRG-2166-13). This research was supported by NIH awards U01 MH105989, R37 NS35710, and R01NS075998 to A.R.K. and CIRM award GCIR-06673-A, and by gifts from Helen Ford and Bernard Osher. All work using human tissues and cells was conducted in strict observance of the protocols approved by the Human Gamete, Embryo, and Stem Cell Research Committee (institutional review board), and work involving animal tissues was performed in accordance with protocols approved by the University of California San Francisco Institutional Animal Care and Use Committee. Accession NumbersThe accession number for the single cell sequencing data reported in this paper is dbGaP: phs000989.v2.p1.

Graphical Abstract Highlights

SummaryThe recent outbreak of Zika virus (ZIKV) in Brazil has been linked to substantial increases in fetal abnormalities and microcephaly. However, information about the underlying molecular and cellular mechanisms connecting viral infection to these defects remains limited. In this study we have examined the expression of receptors implicated in cell entry of several enveloped viruses including ZIKV across diverse cell types in the developing brain. Using single-cell RNA-seq and immunohistochemistry, we found that the candidate viral entry receptor AXL is highly expressed by human radial glial cells, astrocytes, endothelial cells, and microglia in developing human cortex and by progenitor cells in developing retina. We also show that AXL expression in radial glia is conserved in developing mouse and ferret cortex and in human stem cell-derived cerebral organoids, highlighting multiple experimental systems that could be applied to study mechanisms of ZIKV infectivity and effects on brain development.

Highlights •Single-cell analysis reveals expression and specificity of candidate Zika receptors•AXL shows strong expression in human radial glia, brain capillaries, and microglia•Developing human retina progenitors also show high AXL expression•AXL expression is conserved in rodents and human cerebral organoid model systems

Tomasz J. Nowakowski3, Alex A. Pollen3, Elizabeth Di Lullo, Carmen Sandoval-Espinosa, Marina Bershteyn,Arnold R. Kriegstein3Co-first authorPublication stage: In Press Corrected Proof DOI: http://dx.doi.org/10.1016/j.stem.2016.03.012 Article Info