Flaviviruses are mosquito- and tick-borne viruses that include Zika, dengue, yellow fever, and hepatitis C viruses. They have positive-sense RNA genomes and enveloped virions. Flaviviruses are transmitted via arthropod bites or bodily fluids and can cause diseases like hepatitis, encephalitis, Guillain-Barré syndrome, and microcephaly in fetuses. Diagnosis involves virus isolation, antibody detection, nucleic acid testing and neutralization assays. Promising approaches for Zika vaccines include mRNA packaged in lipid nanoparticles.

1. Sirohi et al 2016 Science Mar 31 DOI: 10.1126/science.aaf5316
Chapter 12: Flaviviruses

2. Some hosts of Flaviviridae ….
Humans
Mosquitoes Ticks
Cattle Horses Chimpanzees
Bats
Pigs

3. Transmission Routes
• Arthropod (mosquitoes & ticks)
• Blood transfusion (HCV)
• IV drug use (HCV)
*needle sticks
• Sexual intercourse
• Bodily fluids ** (tears/sweat: Zika)
• Mother to child

7. Flavivirus Symptoms
Hepatitis C Virus (HCV)
http://www.healthline.com/health/hepatitis-c/facts-statistics-infographic
Zika virus
http://www.cdc.gov/zika/symptoms/symptoms.html

8. Disease resulting from Flavivirus infection
Hepatitis C
• hepatitis: inflammation of the liver
• cirrhosis: formation of scar tissue
• hepatocellular carcinoma:
third leading cause of cancer deaths worldwide
http://cvr.academicblogs.co.uk/viral-hepatitis-causes-4000-preventable-deaths-every-day/
https://www.slideshare.net/harinduudapitiya/cirrhosis-harindu-print

9. Disease resulting from Flavivirus infection
Zika
• fetus: microcephaly
o seizures
o developmental delays
o vision problems
o intellectual disabilities
o feeding problems (swallowing) https://www.britannica.com/in-the-news/zika-proven-to-cause-microcephaly

11. Figure 2E: Effects of select cytokines on neurospheres
Bayless et al. Cell Host Microbe. 2016 Oct 12;20(4):423-428. doi: 10.1016/j.chom.2016.09.006.

12. Figure 2F, 2G, 2H: Effects of select cytokines on neurospheres
Independent variable:
Dependent variable(s):
Controls:
type of cytokine added to media
no cellular projections (Fig 2F)
neuronal projections with >15 neurons (Fig 2G)
normalized apoptotic activity (Fig 2H)
negative control (no added cytokines)
Bayless et al. Cell Host Microbe. 2016 Oct 12;20(4):423-428. doi: 10.1016/j.chom.2016.09.006.

13. Disease resulting from Flavivirus infection
http://www.alwaysayurveda.net/2016/08/guillain-barre-syndrome-herbal-remedies.html
Willison et al 2016 388(10045): 717-727
Zika
• adult: Guillain-Barre syndrome (autoimmune disease)
o immune system attacks peripheral nervous system
o weakness/tingling in legs, spreads to arms/
upper body, can lead to total paralysis
o may need to be put on ventilator, watched
for abnormal heart beat and clots
https://www.youtube.com/watch?v=kDspLPFhkS4

14. Disease resulting from Flavivirus infection
Zika
•Guillain-Barre syndrome treatment options:
 intravenous immunoglobulin
 plasma exchange
 supportive care
 “symptoms peak within 4 weeks”
 “recovery period can last months/years”
http://www.ntinfusioncenters.com/iviginfusionsdallas.php
https://www.youtube.com/watch?v=kDspLPFhkS4

15. Brief history of Flaviviruses
• Yellow Fever
o Origin: West Africa
o Introduced to the Americas via
the slave trade
o Stages of the virus:
1. high fever, muscle pain, headaches, vomiting
2. fever dissipates, appearance of remission
3. Repeat stage 1 but more severe:
-acute hepatitis
-multiple organ failure
-20-50% mortality
The Foundations of Virology Discoverers and Discoveries, Inventors and Inventions, Developers and Technologies Copyright ˝ 2011/2014 by Frederick A. Murphy

16. Brief history of Flaviviruses
• Yellow Fever
o Philadelphia 1793
1. Death total ~5000
2. ~20,000 fled the city
3. New York: doctors organize
to prevent ships from
Philadelphia from entering
New York Harbor
o Philadelphia 1799
1. Another severe epidemic
2. All but 7000 residents flee city

17. Brief history of Flaviviruses
• Yellow Fever
o Carlos Juan Finlay
1. Hypothesis: mosquito transmits
Yellow fever from
person to person
o Henry Rose Carter
1. Noticed pattern to infections in
Mississippi
2. Period of extrinsic
incubation
The Foundations of Virology Discoverers and Discoveries, Inventors and Inventions, Developers and Technologies Copyright ˝ 2011/2014 by Frederick A. Murphy

18. Brief history of Flaviviruses
The Foundations of Virology Discoverers and Discoveries, Inventors and Inventions, Developers and Technologies Copyright ˝ 2011/2014 by Frederick A. Murphy
• Yellow Fever
o Walter Reed Studies
Havana, Cuba
o Infected Clothing experiment
-3 Trials: 20 days each
-7 subjects
-does clothing/bedding from
infected individuals pass
disease?
o Infected Mosquito experiment
-4 trials: 2 groups control vs exposed
-mosquitoes obtained from infected
patients rooms
-Controls: no infection

19. Brief history of Flaviviruses
The Foundations of Virology Discoverers and Discoveries, Inventors and Inventions, Developers and Technologies Copyright ˝ 2011/2014 by Frederick A. Murphy
• Yellow Fever (Vaccine)
o Sample from Ghana patient Asibi 1927
o Passaged in rhesus macaques
o 1932: passage in mouse embryonic
tissue (17 passages)
o Then passaged 58 times in whole
chicken embryonic tissue
o Then passaged in denervated chicken embryonic
tissue (passage 114: inject into rhesus macaque)
-reduction in viscero & neurotropism
o Then passage until 227/229 passages
o Use to immunize 8 volunteers (17D virus)

20. Brief history of Flaviviruses

21. Family: Flaviviridae
Baltimore Group IV: (+) ss RNA genome
Genus:
• Flavivirus
o Yellow Fever virus, Dengue virus, Zika virus
• Hepacivirus
o Hepatitis C virus (HCV)
• Pegivirus
o Pegivirus B, human pegivirus (HPgV)
• Pestivirus
o Bovine diarrhea virus 1 & 2,
o Classical swine fever virus

22. Screaton et al 2015 Nature Reviews Immunology 15: 745-759

23. Structure: Viral proteins
• Lipid envelope (orange)
• Envelope protein E (green): anchored at C-terminus
o 3 domains: DI (central), DII (fusion), DIII (Ig-like fold)
• Membrane protein M (blue): prM stabilizes E protein’s DII
o Following prM cleavage, pr peptide still associated
• Capsid protein C (red)
• ssRNA genome (black)
Cruz-Oliveira et al. 2015. FEMS Microbiology Reviews. 39(2):155–170,
https://doi.org/10.1093/femsre/fuu004

24. Structure: Triangulation number
• Triangulation number = 3
• 90 dimers of glycoprotein E (180 subunits)
• Herringbone pattern
• “Bumpy form” of virion found at 37˚C
-intermediate between smooth mature & fusogenic
Dengue virion at 25˚C Dengue virion at > 33˚C
Zhang et al 2013 PNAS 110(17): 6795-6799
Zhang et al 2013 PNAS 110(17): 6795-6799
Immature
Screaton et al 2015 Nature Reviews Immunology 15: 745-759

25. Genomes of Class IV viruses: single stranded (ss) (+) RNA
ViralZone www.expasy.ch/viralzone, Swiss Institute of Bioinformatics

26. Genome
• The genome is (+) ss RNA: Baltimore classification IV
• There is one type of viral mRNA and it is identical to
the genome.
• The viral mRNA is translated by the host ribosome
to produce 1 polyprotein
• The 5’ UTR has a methylated cap
• Three tandem stop codons at end of coding frame

27. Proteins: polyprotein processed by proteases

28. Apte-Sengupta et al 2014 Curr Opin Virol. 0:134-142 doi: 10.1016/j.coviro.2014.09.020
Proteins: arrangement of viral proteins
ER Lumen
Cytoplasm

29. Proteins: roles

30. Dengue viral entry: Putative receptors
• suggested use of various receptors
o variety of hosts & variety of host cells infected
Cruz-Oliveira et al 2015 FEMS Microbiology Reviews 39: 155-170

31. Dengue entry mechanisms
Cruz-Oliveira et al 2015 FEMS Microbiology Reviews 39: 155-170

32. Dengue entry mechanisms
Cruz-Oliveira et al 2015 FEMS Microbiology Reviews 39: 155-170

33. Dengue exiting the endosome: pH trigger
Alen & Schols 2012 Carbohydrates- Comprehensive Studies on Glycobiology and Glycotechnology Chapter 8
Conformational changes in E protein

34. Dengue: antibody dependent enhancement (ADE) of viral fusion
Flipse et al 2016 Sci Rep. 6:29201 doi: 10.1038/srep29201

35. Dengue genome uncoating is independent
of proteasome degradation but requires ubiquitination
Inhibiting Ubiquitination blocks uncoating
Byk et al 2016 mBio 7(3): e00804-16

36. Treat with UBEI41 (host E1 inhibitor): Luciferase assay
Cells infected in presence of UBEI41
Translation measured at 3 & 5 hours
post infection (p.i.)
Cells transfected with
viral RNA in presence or
absence of UBI41
Byk et al 2016 mBio 7(3): e00804-16 Byk et al 2016 mBio 7(3): e00804-16

37. Treat with UBEI41 (host E1 inhibitor): Luciferase assay
• Treat with UBEI41 2 hours after initial infection
• Measure translation 4 hours following UBEI41
Byk et al 2016 mBio 7(3): e00804-16

38. Treat with UBEI41: Time series luciferase assay
• Treat with UBEI41 starting at select time points
• Measure translation 6 hours post-infection
Byk et al 2016 mBio 7(3): e00804-16

39. Treat with UBEI41: Block viral genome release
• RNA levels stay the same in UBEI41 experiment
Byk et al 2016 mBio 7(3): e00804-16

40. Screaton et al 2015 Nature Reviews Immunology 15: 745-759
Translation of viral genome using host ribosomes
• Thought to occur on cytoplasmic side of ER

41. Genome synthesis occurs in ER-derived vesicles
(replication complexes)
• NS5: RNA dependent RNA polymerase
& methyltransferase
• NS3: Helicase & Nucleotide tri-phosphatase (capping)
Bidet & Garcia-Blanco 2014 Biochem J. 462: 215-230

42. Protease cleavage
by host Furin
Form immature
particles in ER lumen
Transported to Golgi
Screaton et al 2015 Nature Reviews Immunology 15: 745-759
Exiting the cell

43. Exiting the cell Drop in pH results in conformational change
that exposes furin cleavage site
pRM protein is
cleaved
pR stays
associated
to prevent
fusion in
Golgi
Yu et al J Virol. 2009 Dec; 83(23): 12101–12107. doi: 10.1128/JVI.01637-09

44. Diagnosis: Zika
Sharma & Lai 2017 Front. Microbiol 8:100 doi: 10.3389/fmicb.2017.00110

45. Diagnosis: Zika
Sharma & Lai 2017 Front. Microbiol 8:100 doi: 10.3389/fmicb.2017.00110

46. http://eng.bioneer.com/diagnostic/HumanMDxkits/Accupower-ZIKV-Multiplex-technical.aspx
Quantitative Real Time RT-PCR

47. http://eng.bioneer.com/diagnostic/HumanMDxkits/Accupower-ZIKV-Multiplex-technical.aspx

48. MAC-ELISA: Example using Dengue
http://www.scielo.org.co/scielo.php?script=sci_arttext&pid=S0120-00112014000400014

49. Zika MAC-ELISA
(IgM Antibody
Capture Enzyme-
Linked
Immunosorbent
Assay)
Significant cross-reactivity
to anti-dengue virus
IgM antibodies
https://www.cdc.gov/zika/pdfs/zika-mac-elisa-instructions-for-use.pdf

50. Zika Plaque Reduction Neutralization Test (PRNT)
• Measures level of protective antibodies an individual has
toward a virus
• virus + antibody = inactivation of virus, inability to infect cells and
produce plaques
• Incubate known virus suspension with serum of patient,
perform dilution series and plate samples as you would with
typical plaque assay
https://www.cdc.gov/mmwr/volumes/65/wr/mm6521e1.htm?s_cid=mm6521e1_w

51. Zika paper-based sensor
Pardee et al 2016 Cell 165(4): 1255-1266

52. Zika paper-based sensor
Pardee et al 2016 Cell 165(4): 1255-1266

53. Zika paper-based sensor
Pardee et al 2016 Cell 165(4): 1255-1266

54. Zika vaccine candidate (mRNA-LNP)
• packaged in lipid nanoparticles (LNPs)
• expressing both pre-membrane and envelope proteins
will result in assembly & secretion of subviral particles
Pardi et al 2017 Nature 543: 248-251 doi: 10.1038/nature21428

55. Zika vaccine candidate (mRNA-LNP)
Figure 2: A single immunization of nucleoside-modified ZIKV prM–E mRNA–LNP provides
rapid and durable protection against ZIKV challenge in mice.
Pardi et al 2017 Nature 543: 248-251 doi: 10.1038/nature21428

56. Zika vaccine candidate (mRNA-LNP)
Figure 4: A single immunization of nucleoside-modified ZIKV prM–E
mRNA–LNP protects rhesus macaques against ZIKV challenge at 5
weeks after immunization.
Pardi et al 2017 Nature 543: 248-251 doi: 10.1038/nature21428