This document discusses the Zika virus and its potential link to microcephaly. It begins with background on brain development and microcephaly. Studies are presented that found Zika virus in the brains of microcephalic fetuses and linked Zika infection in pregnant women to fetal abnormalities. Research suggests Zika infects neural progenitor cells, reducing brain size in model systems. The protein AXL is investigated as a potential receptor for Zika entry into brain cells, including progenitor cells, and is expressed in the expected regions. While evidence links Zika to microcephaly, outstanding questions remain regarding transmission and the virus's effects on the developing brain.

1. 3/14/2016
Zika Virus and Microcephaly
Preparedness for the Zika Virus:
A Public Health Emergency
Alex Pollen, Kriegstein Lab
University of California, San Francisco
March 7th 2016

2. Development of the Human Brain
• Cerebral cortex neurons generated during fetal
development (GW8 – GW29)

3. Radial Glia Generate Cortical Neurons
• Radial glia neural stem cells line the ventricular zone
• Generate neurons that migrate to the cortical plate
Lateral
ventricle
VZ
CP

4. Microcephaly is a Rare Neurodevelopmental
Disorder
Centers for Disease Control and Prevention
U.S. Department of Health & Human Services
2-12 cases per 10,000 live births in the US
3 standard deviations
below average
2 standard deviations
below average

5. • Intellectual Disability
• Developmental Delay
• Motor Skills Defects
• Vision Problems
• Hearing Loss
Symptoms Associated with Microcephaly
Centers for Disease Control and Prevention
U.S. Department of Health & Human Services

6. Microcephaly Genes Affect Radial Glia Cell Division
Thornton and Woods, Trends in Genetics 2009, Cell Press

7. Can Viruses Cause Microcephaly?

8. Can Viruses Cause Microcephaly?
Newborn infant with congenital CMV infection
Swanson Pediatr. Clin. North. Am. 2013, W.B.Saunders
• Ventricular calcifications (arrows), pachygyria and
lissencephaly, thinning of cortex, and hydrocephalous

9. Does Zika Virus Cause Microcephaly? – What do we
Know?
Associated Press/Felipe Dana
Percio Campos, EPA Associated Press/Felipe Dana
Associated Press/Felipe Dana

10. Study of 42 Pregnant Women With Zika Virus
• Enrolled symptomatic pregnant women, tested for virus using PCR
Brasil et al NEJM 2016
• Fetal abnormalities in 12/42 vs. 0/16 in control group
• Death (2), growth defects/microcephaly (5), ventricular calcifications
(7), abnormal amniotic fluid or arterial flow

11. Zika Virus Detected in Microcephalic Fetal Brain
Mlakar et al NEJM 2016, Massachusetts Medical Society
• Calcifications (arrows)
• Smooth brain (arrowheads)
• Flavivirus particles in brain
• Zika virus genome detected

12. Ocular Defects Associated with Congenital Zika Virus
Infection
Paramacular chorioretinal
atrophy
De Paula Freitas JAMA Ophthalmology 2016
Optic disc hypoplasia

13. What Cells Does Zika Target During Development?
• We can model aspects of human neurogenesis in vitro
VZ
CP

14. Zika Virus Likely Infects Neural Progenitors
Tang et al. Cell Stem Cell 2016
Immature neurons exhibited lower levels of infection

15. Zika Virus Likely Infects Neural Progenitors
and Induces Cell Death
Tang et al. Cell Stem Cell 2016
Infected progenitor cells release infectious viral particles

16. Infection Reduces the Size of Cerebral
Organoid Models
Garcez et al., 2016, PeerJ Preprints

17. Possible Routes for ZIKV Neuroinvasiveness –
Why does virus selectively target neural stem cells?
Associated Press/Felipe Dana
Percio Campos, EPA Associated Press/Felipe Dana
Associated Press/Felipe Dana

18. Flavivirus Entry Receptors
Perera-Lecoin et al., 2014, MDPI

19. Several Proteins Promote Zika Virus Entry
Hamel et al, 2015 , J. Virology; Meertens et al., 2012, Cell Host and Microbe
AXL
TYRO3
HAVCR1
(TIM1)
Virus

20. Analyzing Gene Expression in Single Cells
Virus

21. Cellular Resolution “Map” of Gene Expression
Highlights Vulnerable Cell Populations

22. Cellular Resolution “Map” of Gene Expression
Highlights Vulnerable Cell Populations
Hamel et al, 2015 , J. Virology
AXL is necessary and sufficient for Zika virus entry in skin cells

23. AXL-Expressing Cells in the Fetal Brain
Tom Nowakowski, unpublished

24. AXL-Expressing Cells in the Fetal Brain
Tom Nowakowski, unpublished

25. AXL-Expressing Cells in the Fetal Brain
Tom Nowakowski, unpublished

26. AXL is expressed in the retinal stem cells.
CMZ
Tom Nowakowski, unpublished

27. Does in vitro System Recapitulate AXL Expression?

28. Does in vitro System Recapitulate AXL Expression?
Liz Di Lullo, unpublished

29. Conclusion
• Strong published evidence that Zika virus causes
microcephaly and other abnormalities
• Zika virus may target radial glia neural stem cells
• AXL is a strong candidate entry receptor in brain and eye,
based on results in skin and CNS expression pattern

30. Outstanding Questions
• What are consequences of virus in larger cohort,
including asymptomatic pregnant women?
• Why is Zika virus associated with microcephaly, when
Dengue virus is not?
• Is this new strain of Zika virus different from previous
strains?
• How does virus cross placenta and blood-brain barrier?
• Is AXL necessary for viral entry and infectivity in neural
stem cells?

31. Tom Nowakowski Elizabeth Di Lullo Hanna Retallack
Joe DeRisi LabArnold Kriegstein Lab
Jeremy Reiter Lab