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


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.


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



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)


  1. 1.
    Abbott NJ, Ronnback L, Hansson E (2006) Astrocyte-endothelial interactions at the blood-brain barrier. Nat Rev Neurosci 7:41–53PubMedCrossRefGoogle Scholar
  2. 2.
    Allavena RE, Desai B, Goodwin D, Khodai T, Bright H (2011) Pathologic and virologic characterization of neuroinvasion by HSV-2 in a mouse encephalitis model. J Neuropathol Exp Neurol 70:724–734PubMedCrossRefGoogle Scholar
  3. 3.
    Bennett RS, Cress CM, Ward JM, Firestone CY, Murphy BR, Whitehead SS (2008) La Crosse virus infectivity, pathogenesis, and immunogenicity in mice and monkeys. Virol J 5:25PubMedCentralPubMedCrossRefGoogle Scholar
  4. 4.
    Bentz GL, Jarquin-Pardo M, Chan G, Smith MS, Sinzger C, Yurochko AD (2006) Human cytomegalovirus (HCMV) infection of endothelial cells promotes naive monocyte extravasation and transfer of productive virus to enhance hematogenous dissemination of HCMV. J Virol 80:11539–11555PubMedCentralPubMedCrossRefGoogle Scholar
  5. 5.
    Brandt I, Brittebo EB, Feil VJ, Bakke JE (1990) Irreversible binding and toxicity of the herbicide dichlobenil (2,6-dichlorobenzonitrile) in the olfactory mucosa of mice. Toxicol Appl Pharmacol 103:491–501PubMedCrossRefGoogle Scholar
  6. 6.
    Butchi NB, Woods T, Du M, Morgan TW, Peterson KE (2011) TLR7 and TLR9 trigger distinct neuroinflammatory responses in the CNS. Am J Pathol 179:783–794PubMedCentralPubMedCrossRefGoogle Scholar
  7. 7.
    Curmi PA, Andersen SS, Lachkar S, Gavet O, Karsenti E, Knossow M, Sobel A (1997) The stathmin/tubulin interaction in vitro. J Biol Chem 272:25029–25036PubMedCrossRefGoogle Scholar
  8. 8.
    Daniels BP, Holman DW, Cruz-Orengo L, Jujjavarapu H, Durrant DM, Klein RS (2014) Viral pathogen-associated molecular patterns regulate blood-brain barrier integrity via competing innate cytokine signals. MBio 5:e01476–e014714PubMedCentralPubMedCrossRefGoogle Scholar
  9. 9.
    de Vries HE, Blom-Roosemalen MC, Van OM, de Boer AG, van Berkel TJ, Breimer DD, Kuiper J (1996) The influence of cytokines on the integrity of the blood-brain barrier in vitro. J Neuroimmunol 64:37–43PubMedCrossRefGoogle Scholar
  10. 10.
    Dohgu S, Ryerse JS, Robinson SM, Banks WA (2012) Human immunodeficiency virus-1 uses the mannose-6-phosphate receptor to cross the blood-brain barrier. PLoS ONE 7:e39565PubMedCentralPubMedCrossRefGoogle Scholar
  11. 11.
    Dups J, Middleton D, Yamada M, Monaghan P, Long F, Robinson R, Marsh GA, Wang LF (2012) A new model for Hendra virus encephalitis in the mouse. PLoS ONE 7:e40308PubMedCentralPubMedCrossRefGoogle Scholar
  12. 12.
    Ferrari G, Langen H, Naito M, Pieters J (1999) A coat protein on phagosomes involved in the intracellular survival of mycobacteria. Cell 97:435–447PubMedCrossRefGoogle Scholar
  13. 13.
    Fletcher NF, Wilson GK, Murray J, Hu K, Lewis A, Reynolds GM, Stamataki Z, Meredith LW, Rowe IA, Luo G, Lopez-Ramirez MA, Baumert TF, Weksler B, Couraud PO, Kim KS, Romero IA, Jopling C, Morgello S, Balfe P, McKeating JA (2012) Hepatitis C virus infects the endothelial cells of the blood-brain barrier. Gastroenterology 142:634–643PubMedCentralPubMedCrossRefGoogle Scholar
  14. 14.
    Gaensbauer JT, Lindsey NP, Messacar K, Staples JE, Fischer M (2014) Neuroinvasive arboviral disease in the United States: 2003 to 2012. Pediatrics 134:e642–e650PubMedCrossRefGoogle Scholar
  15. 15.
    Haddow AD, Odoi A (2009) The incidence risk, clustering, and clinical presentation of La Crosse virus infections in the eastern United States, 2003–2007. PLoS ONE 4:e6145PubMedCentralPubMedCrossRefGoogle Scholar
  16. 16.
    Harberts E, Yao K, Wohler JE, Maric D, Ohayon J, Henkin R, Jacobson S (2011) Human herpesvirus-6 entry into the central nervous system through the olfactory pathway. Proc Natl Acad Sci USA 108:13734–13739PubMedCentralPubMedCrossRefGoogle Scholar
  17. 17.
    Harkema JR, Carey SA, Wagner JG (2006) The nose revisited: a brief review of the comparative structure, function, and toxicologic pathology of the nasal epithelium. Toxicol Pathol 34:252–269PubMedCrossRefGoogle Scholar
  18. 18.
    Hollidge BS, Nedelsky NB, Salzano MV, Fraser JW, Gonzalez-Scarano F, Soldan SS (2012) Orthobunyavirus entry into neurons and other mammalian cells occurs via clathrin-mediated endocytosis and requires trafficking into early endosomes. J Virol 86:7988–8001PubMedCentralPubMedCrossRefGoogle Scholar
  19. 19.
    Janssen R, Gonzalez-Scarano F, Nathanson N (1984) Mechanisms of bunyavirus virulence. Comparative pathogenesis of a virulent strain of La Crosse and an avirulent strain of Tahyna virus. Lab Invest 50:447–455PubMedGoogle Scholar
  20. 20.
    Johnson RT (1983) Pathogenesis of La Crosse virus in mice. Prog Clin Biol Res 123:139–144PubMedGoogle Scholar
  21. 21.
    Kalinke U, Bechmann I, Detje CN (2011) Host strategies against virus entry via the olfactory system. Virulence 2:367–370PubMedCrossRefGoogle Scholar
  22. 22.
    Kraus J, Oschmann P (2006) The impact of interferon-beta treatment on the blood-brain barrier. Drug Discov Today 11:755–762PubMedCrossRefGoogle Scholar
  23. 23.
    Lamalice L, Le BF, Huot J (2007) Endothelial cell migration during angiogenesis. Circ Res 100:782–794PubMedCrossRefGoogle Scholar
  24. 24.
    Lebrun L, Junter GA (1994) Diffusion of dextran through microporous membrane filters. J Membrane Sciences 88:253–261CrossRefGoogle Scholar
  25. 25.
    Liou ML, Hsu CY (1998) Japanese encephalitis virus is transported across the cerebral blood vessels by endocytosis in mouse brain. Cell Tissue Res 293:389–394PubMedCrossRefGoogle Scholar
  26. 26.
    Liu P, Woda M, Ennis FA, Libraty DH (2009) Dengue virus infection differentially regulates endothelial barrier function over time through type I interferon effects. J Infect Dis 200:191–201PubMedCrossRefGoogle Scholar
  27. 27.
    McJunkin JE, de los Reyes EC, Irazuzta JE, Caceres MJ, Khan RR, Minnich LL, Fu KD, Lovett GD, Tsai T, Thompson A (2001) La Crosse encephalitis in children. N Engl J Med 344:801–807PubMedCrossRefGoogle Scholar
  28. 28.
    Miller F, Afonso PV, Gessain A, Ceccaldi PE (2012) Blood-brain barrier and retroviral infections. Virulence 3:222–229PubMedGoogle Scholar
  29. 29.
    Mukherjee P, Woods TA, Moore RA, Peterson KE (2013) Activation of the innate signaling molecule MAVS by bunyavirus infection upregulates the adaptor protein SARM1, leading to neuronal death. Immunity 38:705–716PubMedCrossRefGoogle Scholar
  30. 30.
    Neal JW (2014) Flaviviruses are neurotropic, but how do they invade the CNS? J Infect 69:203–215PubMedCrossRefGoogle Scholar
  31. 31.
    Olofsson B (1999) Rho guanine dissociation inhibitors: pivotal molecules in cellular signalling. Cell Signal 11:545–554PubMedCrossRefGoogle Scholar
  32. 32.
    Pate M, Damarla V, Chi DS, Negi S, Krishnaswamy G (2010) Endothelial cell biology: role in the inflammatory response. Adv Clin Chem 52:109–130PubMedCrossRefGoogle Scholar
  33. 33.
    Pekosz A, Phillips J, Pleasure D, Merry D, Gonzalez-Scarano F (1996) Induction of apoptosis by La Crosse virus infection and role of neuronal differentiation and human bcl-2 expression in its prevention. J Virol 70:5329–5335PubMedCentralPubMedGoogle Scholar
  34. 34.
    Phares TW, Kean RB, Mikheeva T, Hooper DC (2006) Regional differences in blood-brain barrier permeability changes and inflammation in the apathogenic clearance of virus from the central nervous system. J Immunol 176:7666–7675PubMedCrossRefGoogle Scholar
  35. 35.
    Ressler KJ, Sullivan SL, Buck LB (1993) A zonal organization of odorant receptor gene expression in the olfactory epithelium. Cell 73:597–609PubMedCrossRefGoogle Scholar
  36. 36.
    Rippe B, Rosengren BI, Carlsson O, Venturoli D (2002) Transendothelial transport: the vesicle controversy. J Vasc Res 39:375–390PubMedCrossRefGoogle Scholar
  37. 37.
    Roe K, Kumar M, Lum S, Orillo B, Nerurkar VR, Verma S (2012) West Nile virus-induced disruption of the blood-brain barrier in mice is characterized by the degradation of the junctional complex proteins and increase in multiple matrix metalloproteinases. J Gen Virol 93:1193–1203PubMedCentralPubMedCrossRefGoogle Scholar
  38. 38.
    Salinas S, Schiavo G, Kremer EJ (2010) A hitchhiker’s guide to the nervous system: the complex journey of viruses and toxins. Nat Rev Microbiol 8:645–655PubMedCrossRefGoogle Scholar
  39. 39.
    Schafer A, Brooke CB, Whitmore AC, Johnston RE (2011) The role of the blood-brain barrier during Venezuelan equine encephalitis virus infection. J Virol 85:10682–10690PubMedCentralPubMedCrossRefGoogle Scholar
  40. 40.
    Sotir MJ, Glaser LC, Fox PE, Doering M, Geske DA, Warshauer DM, Davis JP (2007) Endemic human mosquito-borne disease in Wisconsin residents, 2002-2006. WMJ 106:185–190PubMedGoogle Scholar
  41. 41.
    Stamatovic SM, Keep RF, Andjelkovic AV (2008) Brain endothelial cell-cell junctions: how to “open” the blood brain barrier. Curr Neuropharmacol 6:179–192PubMedCentralPubMedCrossRefGoogle Scholar
  42. 42.
    Talmon Y, Prasad BV, Clerx JP, Wang GJ, Chiu W, Hewlett MJ (1987) Electron microscopy of vitrified-hydrated La Crosse virus. J Virol 61:2319–2321PubMedCentralPubMedGoogle Scholar
  43. 43.
    Taylor KG, Peterson KE (2014) Innate immune response to La Crosse virus infection. J Neurovirol 20:150–156PubMedCrossRefGoogle Scholar
  44. 44.
    Taylor KG, Woods TA, Winkler CW, Carmody AB, Peterson KE (2014) Age-dependent myeloid dendritic cell responses mediate resistance to la crosse virus-induced neurological disease. J Virol 88:11070–11079PubMedCentralPubMedCrossRefGoogle Scholar
  45. 45.
    Tsao N, Hsu HP, Wu CM, Liu CC, Lei HY (2001) Tumour necrosis factor-alpha causes an increase in blood-brain barrier permeability during sepsis. J Med Microbiol 50:812–821PubMedGoogle Scholar
  46. 46.
    Tuszynski JA, Brown JA, Sept D (2003) Models of the collective behavior of proteins in cells: tubulin, actin and motor proteins. J Biol Phys 29:401–428PubMedCentralPubMedCrossRefGoogle Scholar
  47. 47.
    van den Pol AN, Ding S, Robek MD (2014) Long-distance interferon signaling within the brain blocks virus spread. J Virol 88:3695–3704PubMedCentralPubMedCrossRefGoogle Scholar
  48. 48.
    Van RD, Verdijk R, Kuiken T (2015) The olfactory nerve: a shortcut for influenza and other viral diseases into the central nervous system. J Pathol 235:277–287CrossRefGoogle Scholar
  49. 49.
    Winkler CW, Foster SC, Matsumoto SG, Preston MA, Xing R, Bebo BF, Banine F, Berny-Lang MA, Itakura A, McCarty OJ, Sherman LS (2012) Hyaluronan anchored to activated CD44 on central nervous system vascular endothelial cells promotes lymphocyte extravasation in experimental autoimmune encephalomyelitis. J Biol Chem 287:33237–33251PubMedCentralPubMedCrossRefGoogle Scholar
  50. 50.
    Wojciak-Stothard B, Tsang LY, Paleolog E, Hall SM, Haworth SG (2006) Rac1 and RhoA as regulators of endothelial phenotype and barrier function in hypoxia-induced neonatal pulmonary hypertension. Am J Physiol Lung Cell Mol Physiol 290:L1173–L1182PubMedCrossRefGoogle Scholar

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

Personalised recommendations