Coronavirus neurovirulence correlates with the ability of the virus to induce proinflammatory cytokine signals from astrocytes and microglia

Abstract

The molecular and cellular basis of coronavirus neurovirulence is poorly understood. Since neurovirulence may be determined at the early stages of infection of the central nervous system (CNS), we hypothesize that it may depend on the ability of the virus to induce proinflammatory signals from brain cells for the recruitment of blood-derived inflammatory cells. To test this hypothesis, we studied the interaction between coronaviruses (mouse hepatitis virus) of different neurovirulences with primary cell cultures of brain immune cells (astrocytes and microglia) and mouse tissues. We found that the level of neurovirulence of the virus correlates with its differential ability to induce proinflammatory cytokines (interleukin 12 [IL-12] p40, tumor necrosis factor alpha, IL-6, IL-15, and IL-1beta) in astrocytes and microglia and in mouse brains and spinal cords. These findings suggest that coronavirus neurovirulence may depend on a novel discriminatory ability of astrocytes and microglia to induce a proinflammatory response in the CNS.

Figures

FIG. 1.

Virus titers in astrocytes and microglial cultures at 24 h postinfection with MHV-A59 and MHV-2, detected by plaque assay.

FIG. 2.

Analysis of mRNA levels of cytokines 24 h following infection of astrocytes with a neurotropic (MHV-A59) and a nonneurotropic (MHV-2) virus compared with an uninfected control culture. The blots of mouse cytokine array assays are shown. The cytokine key is as follows: A, colony-stimulating factor granulocyte; B, gamma interferon; C, IL-1α; D, IL-1β; E, IL-2; F, IL-3; G, IL-4; H, IL-5; I, IL-6; J, IL-7; K, IL-9; L, IL-10; M, IL-11; N, IL-12 p35; O, IL-12 p40; P, IL-13; Q, IL-15; R, IL-16; S, IL-17; T, IL-18; U, lymphotoxin B; V, TNF-α; W, TNF-β; X, GAPDH; Y, β-actin; Z, bacterial plasmid (pUC18).

FIG. 3.

The blot assays shown in Fig. 2 were scanned and analyzed for relative quantitative levels of mRNA. The results are shown in a graphical form as arbitrary units based on the cytokine levels relative to the level of the housekeeping genes as explained in the text. The results for two alpha interferon (IFN-α) genes from another kit were also included in this figure. LT-b, lymphotoxin B; G-CSF, colony-stimulating factor granulocyte.

FIG. 4.

Analysis of mRNA levels of 5 cytokines (TNF-α, IL-12 p40, IL-6, IL-15, and IL-1β) following infection with a neurotropic (MHV-A59) and a nonneurotropic (MHV-2) virus compared with an uninfected control. Cytokine levels were examined in astrocytes (24 h postinfection), microglia (24 h postinfection), brains during acute encephalitis (5 days postinfection), and spinal cords during chronic demyelinating disease (30 days postinfection). Cytokine mRNA was expressed in relative strength, as evaluated against the expression of a housekeeping gene, β-actin (arbitrarily designated as 1.0 U of strength).

FIG. 5.

Cytokine protein levels in astrocytes at 24 h postinfection with MHV-A59 and MHV-2 compared with an uninfected control group. The levels of cytokine proteins are expressed in picograms per milliliter.

FIG. 6.

Analysis of mRNA levels of 5 cytokines (TNF-α, IL-12 p40, IL-6, IL-15, and IL-1β) in astrocytes following infection with neurotropic viruses (MHV-A59 and MHV-JHM) and a nonneurotropic virus (MHV-2) compared with an uninfected control. Cytokine mRNA levels were examined by 24 h postinfection and were expressed in relative strength, as evaluated against the expression of a housekeeping gene, β-actin (arbitrarily designated as 1.0 U of strength).

FIG. 7.

A schematic diagram of the proposed sequence of events of proinflammatory cytokine secretion from astrocytes following a neurotropic viral infection with MHV-A59 at 2 PFU/cell (multiplicity of infection = 2).