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Cytokine Expression in the Central Nervous System and Periphery during Infection with Rabies Virus

Nicholas JohnsonRabies and Wildlife Zoonoses Group

WHO Collaborating Centre for the Characterization of Rabies and Rabies-Related Viruses

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Interferon induction following infection with a fixed strain of Rabies virus (Challenge Virus Standard)

RV nucleoprotein

Interferon-

Interferon –

Interferon-

603bp

x RV negative RV positive

310bp

603bp

310bp

310bp

310bp

-actin

TNF-

Johnson et al., 2006 JGV 87, 2663

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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

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The Model

00 2 4 6 8 10 12

Inoculate Antibodies Signs Death

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

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Anti-RABV immunostaining in the Dorsal Root Ganglion & Spinal Cord

Dorsal Root Ganglion (d11) Spinal cord (d11)

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Inflammatory changes observed in the DRG and Spinal Cord

DRG (Media only) DRG (RABV d11)

Spinal cord (Media only) Spinal cord (RABV d11)

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T- and B-Cell Lymphocyte Immunostaining

CD45R/220

CD3

Dorsal Root Ganglia Spinal cord

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Inflammatory Cytokine Detection in the Brain

CCL3

M

RABV - RABV + Day O Day 5 Day 11

GAPDH

CCL2

CCL4

CCL5

Overt disease

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Chemokine transcript response in the brain

0

30

60

90

120

0 5 110

100

200

300

400

0 5 11

0

100

200

300

400

0 5 11

0

80

160

240

320

0 5 11

0

600

1200

1800

2400

0 5 110

1200

2400

3600

4800

0 5 11

INF- CCL2 (MCP-1) CCL3 (MIP-1)

CCL4 (MIP 1) CCL5 (Rantes) CXCL10 (IP10)

Tra

nscr

ipti

on f

old

incr

ease

Time post inoculation (days)

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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-, IL-2, IL-6, IL-10, TNF-)

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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.

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Acknowledgements

Co-authors

Veterinary Laboratories Agency

Anthony FooksSharon BrookesKaren MansfieldDerek HealyDaniel HicksAlex Nunez

University of Edinburgh

John FazakerleyClive McKimmie

Funding

Defra (UK): grant SE0524