1. Cytokine Expression in the Central Nervous System and Periphery during Infection with Rabies Virus
Nicholas Johnson
Rabies and Wildlife Zoonoses Group
WHO Collaborating Centre for the Characterization of Rabies and Rabies-Related Viruses

2. Interferon induction following infection with a fixed strain of Rabies virus (Challenge Virus Standard)
RV nucleoprotein
Interferon-a
Interferon –b
Interferon-g
603bp Fx
RV negative RV positive
310bp
b-actin
TNF-a
Johnson et al., 2006 JGV 87, 2663

3. Objectives
Measure inflammatory changes in response to infection with a non-fixed strain
Observe inflammation within the Central Nervous System
Identify possible mediators of inflammation (cytokines / chemokines)
Compare the CNS to the periphery during infection

4. The Model Isolate RV 61 (UK human, ex-India) CD1 outbred mice
Subdermal inoculation / hind footpad
11d course (sampled at 0, 5, 11)
Brain / salivary glands: Transcript
Spinal cord: Histopathology
Blood: Cytokine / Antibody 2 4 6 8 10 12
Inoculate
Antibodies
Signs
Death

5. Anti-RABV immunostaining in the Dorsal Root Ganglion & Spinal Cord
Dorsal Root Ganglion (d11)
Spinal cord (d11)

6. Inflammatory changes observed in the DRG and Spinal Cord
DRG (Media only)
DRG (RABV d11)
Spinal cord (Media only)
Spinal cord (RABV d11)

7. T- and B-Cell Lymphocyte Immunostaining
Dorsal Root Ganglia
Spinal cord
CD45R/220
CD3

8. Inflammatory Cytokine Detection in the Brain
RABV RABV +
Day O
Day 5
Day 11 M a
GAPDH
CCL2 b
CCL3
CCL4 g
CCL5
Overt disease

9. Chemokine transcript response in the brain
INF-g
CCL2 (MCP-1)
CCL3 (MIP-1a)
Transcription fold increase
CCL4 (MIP 1b)
CCL5 (Rantes)
CXCL10 (IP10)
Time post inoculation (days)

10. Peripheral Changes Salivary glands: No change in TLR-3 transcripts.
Increases observed in levels of a number of inflammatory transcripts (IL-6, CCL5, CXCL10, 2’-5’ OAS1)
Serum:
Neutralising antibodies detected as early as day 5.
No change in a range of cytokines (INF-g, IL-2, IL-6, IL-10, TNF-a)

11. Conclusions
Inflammatory changes are observed in the CNS during neurovirulent
infection with RABV.
Infiltrating lymphocytes are predominantly CD3+ (but not observed in the brain)
Interferons and chemokines are up-regulated in response to infection.
Chemokine responses proceed detection of RABV in the brain.
Chemokines that attract T cells show the greatest up-regulation.
These changes are not reflected in the periphery.

12. Acknowledgements Co-authors Veterinary Laboratories Agency
Anthony Fooks
Sharon Brookes
Karen Mansfield
Derek Healy
Daniel Hicks
Alex Nunez
University of Edinburgh
John Fazakerley
Clive McKimmie
Funding
Defra (UK): grant SE0524