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Toll-like receptor 3 mediates West Nile virus entry into the brain causing lethal encephalitis

Abstract

West Nile virus (WNV), a mosquito-borne single-stranded (ss)RNA flavivirus, causes human disease of variable severity. We investigated the involvement of Toll-like receptor (Tlr) 3, which recognizes viral double-stranded (ds)RNA, on WNV infection. Tlr3-deficient (Tlr3−/−) mice were more resistant to lethal WNV infection and had impaired cytokine production and enhanced viral load in the periphery, whereas in the brain, viral load, inflammatory responses and neuropathology were reduced compared to wild-type mice. Peripheral WNV infection led to a breakdown of the blood-brain barrier and enhanced brain infection in wild-type but not in Tlr3−/− mice, although both groups were equally susceptible upon intracerebroventricular administration of the virus. Tumor necrosis factor-α receptor 1 signaling is vital for blood-brain barrier compromise upon Tlr3 stimulation by dsRNA or WNV. Collectively, WNV infection leads to a Tlr3-dependent inflammatory response, which is involved in brain penetration of the virus and neuronal injury.

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Figure 1: Tlr3−/− mice are resistant to lethal WNV infection.
Figure 2: Tlr3−/− mice have increased WNV viral load and reduced cytokine production in the circulation.
Figure 3: Attenuated viral load and cytokine responses in Tlr3−/− brains after WNV infection.
Figure 4: Reduced neuronal and inflammatory pathology in Tlr3−/− mouse brains after WNV infection.
Figure 5: BBB permeability is increased after WNV infection or poly(I:C) injection in wild-type but not Tlr3−/− mice.
Figure 6: TLR3-mediated disruption of blood-brain-barrier integrity is affected by TNF-α receptor signaling.

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Acknowledgements

We thank F. Manzo for assistance in preparing the manuscript and P. Cresswell for the use of the Zeiss Axioplan 2 fluorescence microscope. All experiments have been approved by the Yale University Animal Care Committee. These studies were supported by grants from the United States National Institutes of Health (AIO55749) and the United States Department of Agriculture. R.A.F. is an investigator of the Howard Hughes Medical Institute. E.F. is the recipient of a Burroughs Wellcome Clinical Scientist Award in Translational Research. T.W. is supported by a J. Kempner Postdoctoral Fellowship from University of Texas Medical Branch at Galveston. T.T. is supported by a Ruth L. Kirschstein NIH/NRSA Postdoctoral Fellowship.

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Correspondence to Erol Fikrig.

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Supplementary information

Supplementary Fig. 1

WNV infection in spleens from wild-type and Tlr3−/− mice. (PDF 332 kb)

Supplementary Fig. 2

Brain anatomical localization of WNV infection. (PDF 130 kb)

Supplementary Fig. 3

Conditioned media from WNV-infected wild-type, but not Tlr3−/− mouse microglia, induce neuronal apoptosis in vitro. (PDF 61 kb)

Supplementary Fig. 4

Blood-brain barrier permeability is increased after WNV infection in wild-type, but not Tlr3−/− or Tnf-r1−/− mice. (PDF 784 kb)

Supplementary Table 1

Summarized pathological observations of WNV-infected brains from wild-type or Tlr3−/− mice (PDF 14 kb)

Supplementary Methods (PDF 9 kb)

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Wang, T., Town, T., Alexopoulou, L. et al. Toll-like receptor 3 mediates West Nile virus entry into the brain causing lethal encephalitis. Nat Med 10, 1366–1373 (2004). https://doi.org/10.1038/nm1140

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