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Acta Neuropathologica

, Volume 130, Issue 2, pp 233–245 | Cite as

Capillaries in the olfactory bulb but not the cortex are highly susceptible to virus-induced vascular leak and promote viral neuroinvasion

  • Clayton W. Winkler
  • Brent Race
  • Katie Phillips
  • Karin E. Peterson
Original Paper

Abstract

Viral neuroinvasion is a critical step in the pathogenesis of viral encephalitis. Multiple mechanisms of neuroinvasion have been identified, but their relative contribution to central nervous system (CNS) infection remains unclear for many viruses. In this study, we examined neuroinvasion of the mosquito-borne bunyavirus La Crosse (LACV), the leading cause of pediatric viral encephalitis in the USA. We found that the olfactory bulb (OB) and tract were the initial areas of CNS virus infection in mice. Removal of the OB reduced the incidence of LACV-induced disease demonstrating the importance of this area to neuroinvasion. However, we determined that infection of the OB was not due to axonal transport of virus from olfactory sensory neurons as ablation of these cells did not affect viral pathogenesis. Instead, we found that OB capillaries were compromised allowing leakage of virus-sized particles into the brain. Analysis of OB capillaries demonstrated specific alterations in cytoskeletal and Rho GTPase protein expression not observed in capillaries from other brain areas such as the cortex where leakage did not occur. Collectively, these findings indicate that LACV neuroinvasion occurs through hematogenous spread in specific brain regions where capillaries are prone to virus-induced activation such as the OB. Capillaries in these areas may be “hot spots” that are more susceptible to neuroinvasion not only for LACV, but other neurovirulent viruses as well.

Keywords

Bunyavirus Neuroinvasion Olfactory bulb Brain capillary endothelial cells Olfactory sensory neurons Blood brain barrier 

Notes

Acknowledgments

This study was performed at Rocky Mountain Laboratories (RML) and funded by the Division of Intramural Research (DIR), as part of the Nation Institute of Allergy and Infectious Disease (NIAID) within the National Institutes of Health (NIH). We thank Suzette A. Priola, Byron Caughey, Lara M. Myers, Sonja M. Best, Roger A. Moore, Burhan A. Khan, Tyson A. Woods and Paul F. Policastro for critical reading of the manuscript. Also, we thank Dan Long, Vinod Nair, Nancy Kurtz and Aaron B. Carmody for technical assistance with experiments. Figure preparation and image presentation assistance were provided by Anita Mora and Ryan Kissinger.

Conflict of interest

All authors declare no conflict of interest.

Supplementary material

401_2015_1433_MOESM1_ESM.tif (3.4 mb)
Supplementary material 1 (TIFF 3513 kb). Supplemental Fig. 1. LACV does not infect BCECs. Representative images from the OB (a) and cerebellum (b) of weanling mice infected i.p. with 103 PFU LACV at 6 dpi demonstrate BCECs (CD31, red) are not infected with LACV (green) despite significant infection of neighboring neurons (See Fig. 2c for verification of neuronal infection). These observations are consistent with findings from i.n. infected animals (data not shown). Scale bars represent 200 μm
401_2015_1433_MOESM2_ESM.tif (9.9 mb)
Supplementary material 2 (TIFF 10119 kb). Supplemental Fig. 2. Peripheral LACV infection alters expression of BBB integrity specific proteins in the OB. CyDye labeled 2D-DIGE gels containing protein lysates from mock infected olfactory bulb (left column), LACV infected OB (middle column) and LACV infected cortex (right column). Spots for top seven proteins (A-G) within each gel are indicated by the red outline, with the center dot representing peak of signal intensity. Directly below each gel are 3D signal intensity plots obtained using DeCyder software (see Methods and Materials) for each spot demonstrating the area of each outline and the peak height and volume within the outline. Numerical representations of each plot are shown directly below
401_2015_1433_MOESM3_ESM.tif (653 kb)
Supplementary material 3 (TIFF 653 kb). Supplemental Fig. 3. LACV neuroinvasion is mechanistically distinct between i.n. and i.p. routes of inoculation. Weanling (a) and adult (b) C57/B6 mice were infected i.n. (filled circles) or i.p. (filled boxes) with 103 PFU of LACV and monitored for the development of terminal neurological symptoms. Resulting survival curves were compared using a Log-rank test (n = 7-18 mice per group,*p < 0.05, ***p < 0.005). A portion of mice included in the i.p. survival curves were previously published by our lab [44]
401_2015_1433_MOESM4_ESM.tif (288 kb)
Supplementary material 4 (TIFF 287 kb). Supplemental Fig. 4. Bulbectomized mice are resistant to neuroinvasion when inoculated i.n. Weanling (a) or adult (b) C57BL/6 mice were given BulbX or Sham surgery (see Materials and Methods) to remove the olfactory bulb. Three days post-surgery, 103 PFU LACV was administered i.n. to Sham (filled circles) and BulbX (filled boxes) animals and resulting survival curves compared using a Long-rank test (n = 7-14 mice per group, **p < 0.01)
401_2015_1433_MOESM5_ESM.pdf (56 kb)
Supplementary material 5 (PDF 56 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg (outside the USA) 2015

Authors and Affiliations

  • Clayton W. Winkler
    • 1
  • Brent Race
    • 1
  • Katie Phillips
    • 1
  • Karin E. Peterson
    • 1
  1. 1.Laboratory of Persistent Viral Diseases, Rocky Mountain LaboratoriesNational Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH)HamiltonUSA

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