1. Enteric bacteria promote human and
mouse norovirus infection of B cells
Melissa K. Jones,1* Makiko Wantanbe,1* Shu Zhu,1
Christina L. Graves,2,3 Lisa R. Keyes,1 Katrina R.
Grau,1 Mariam B. Gonzalez-Hernandez,4 Nicole M.
Iovine,5 Christina E. Wobus,4 Jan Vinjé,6 Scott A.
Tibbetts,1 Shannon M. Wallet,2,3 Stephanie M.
Karst1†
Presented by: Katherin Portwood

2. 1
• Easily transmitted in vomit aerosols, person to person contact,
and fecal-oral contamination
• Small infection dose: 10-20 viral particles
• Cruise ship diarrhea
• Dehydration, vomiting, nausea, diarrhea
• Duration: 24-48 hours
• Most prevalent strain today is GII.4 Sydney

3. Norovirus is a lytic virus
• Attaches to host cell and inserts its genome
• Utilizes host machinery and one viral protein (RNA dependent
RNA polymerase) to replicate virons
• Host cells are destroyed to release virus descendants

4. Norovirus is an +ssRNA virus
• RNA dependent
RNA polymerase
(RdRp) replicates
the genome
– +ssRNA
– -ssRNA
– +ssRNA

5. Norovirus is a non-enveloped virus
• Norovirus binds to Histo-
Blood Group Antigens
(HBGAs)
• Every virus has capsid
proteins that protect the
genome
– VP1 is the major capsid
protein in norovirus.
– VP1 encodes for a P
domain capable of
binding to HBGAs

6. Histo-Blood Group Antigens
7

7. Norovirus genes
3

8. Mouse Norovirus (MuNoV)
• Acute
infection
• Protective
immunity
– short lived
• Persistent
infections
• Attenuated
Infect
macrophages and
Dendritic Cells of
the host immune
system
Time course of
infection
MNV-1 MNV-3

9. Measurement of Virus infectivity
• Cytopathic effects (CPE)
– Structural changes in the host cells that are caused by viral
invasion
– Evidence of cell lysis (plaques)
– Represents actively replicating norovirus
• Plaque forming units (PFU)
– Visual detections to determine the amount of virus particles from
dead host cells
• TCID-50
– Represents the viral concentration necessary to induce cell death/
pathological changes in 50% of inoculated cells
– Determined by a specific calculation

10. Figure 1A
• RAW246.7-
mouse
macrophage
• M12- Mature
mouse B cell
• WEHI.231-
immature
mouse B cell
• CMT-93-
intestinal
epithelial cells
MuNoVs infect B cells in culture

11. Determining cell viability
• Propidium Iodide
staining
– Red fluorescent stain
– Only permeates the
membranes of dead cells
– Binds to DNA

12. Figure 1B
MuNoVs infect B cells in culture

13. Immunofluorescence
4

14. Figure 1C
MuNoVs infect B cells in culture

15. Figure 1D
MuNoVs infect B cells in culture

16. Figure 2A
MuNoVs target Peyer’s patch B cells

17. Figure 2B
MuNoVs target Peyer’s patch B cellsWhy was there no significant difference in infection of the colon tissue?

18. Peyer’s Patches
• lymphatic follicles that sample the
contents of the small intestine
• “waiting room” for B cells who will
soon interact with their antigen
• Once the interaction is made, the B
cells travel to the mesenteric lymph
nodes to continue immune
responses
11

19. Peyer’s Patches
8
NoV
B

20. Peyer’s Patches
8

21. Peyer’s Patches
8

22. Peyer’s Patches
8

23. Peyer’s Patches
8

24. Peyer’s Patches
8

25. Peyer’s Patches
8

26. Peyer’s Patches
8

27. Peyer’s Patches
8

28. Peyer’s Patches
8

29. Peyer’s Patches
8

30. Peyer’s Patches
8

31. Peyer’s Patches
8

32. Peyer’s Patches
8

33. Peyer’s Patches
8

34. Peyer’s Patches
8

35. Peyer’s Patches
8

36. Peyer’s Patches
8

37. Peyer’s Patches
8

38. Peyer’s Patches
8

39. Peyer’s Patches
8

40. Peyer’s Patches
8

41. Reverse Transcriptase-Polymerase
Chain Reaction
5

42. Figure 2C
• RT-PCR detected
the presence of
viral genome in
CD19 marked B
cells and bulk cells
collected from
Peyer’s Patches.
MuNoVs target Peyer’s patch B cells

43. Flow Cytometry
6

44. Figure 2D
• N-term is a non-
structural protein
that activates host
cell apoptosis
• CD19 and B220 are
B cell surface
markers
• Flow cytometry
detected the
presence of viral
replication in
diverse types of B
cells
MuNoVs target Peyer’s patch B cells

45. Figure 3A
• GII.4 Sydney is
the current
dominate HuNoV
strain
• Human Burrkits
lymphoma cells
(BJABs) were the
test B cells
• viral genome
growth in B cells
at 3 and 5 dpi
HuNoVs productively infect B cells in culture

46. Figure 3B
• UV light acts as a
mutagen -inactivates
the viral replication
process
HuNoVs productively infect B cells in culture

47. Figure 3C-D
HuNoVs productively infect B cells in culture

48. Figure 3E-F
HuNoVs productively infect B cells in culture

49. Figure 4 A
Intestinal bacteria facilitate NoV infections

50. Figure 4 B
Intestinal bacteria facilitate NoV infections

51. Figure 4 C
Intestinal bacterial facilitate NoV infections

52. Figure 4 D
Intestinal bacteria facilitate NoV infections

53. Conclusion
• Murine Norovirus infects B cells in culture
• MuNoVs target Peyer’s Patch B cells
• HuNoVs productively infect B cells in culture
• Intestinal bacteria facilitate NoV infections

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58. Questions?