Virulent Pseudorabies Virus Infection Induces a Specific and Lethal Systemic Inflammatory Response in Mice

Abstract

Pseudorabies virus (PRV) is an alphaherpesvirus that infects the peripheral nervous system (PNS). The natural host of PRV is the swine, but it can infect most mammals, including cattle, rodents, and dogs. In these nonnatural hosts, PRV always causes a severe acute and lethal neuropathy called the "mad itch," which is uncommon in swine. Thus far, the pathophysiological and immunological processes leading to the development of the neuropathic itch and the death of the animal are unclear. Using a footpad inoculation model, we established that mice inoculated with PRV-Becker (virulent strain) develop a severe pruritus in the foot and become moribund at 82 h postinoculation (hpi). We found necrosis and inflammation with a massive neutrophil infiltration only in the footpad and dorsal root ganglia (DRGs) by hematoxylin and eosin staining. PRV load was detected in the foot, PNS, and central nervous system tissues by quantitative reverse transcription-PCR. Infected mice had elevated plasma levels of proinflammatory cytokines (interleukin-6 [IL-6] and granulocyte colony-stimulating factor [G-CSF]) and chemokines (Gro-1 and monocyte chemoattractant protein 1). Significant IL-6 and G-CSF levels were detected in several tissues at 82 hpi. High plasma levels of C-reactive protein confirmed the acute inflammatory response to PRV-Becker infection. Moreover, mice inoculated with PRV-Bartha (attenuated, live vaccine strain) did not develop pruritus at 82 hpi. PRV-Bartha also replicated in the PNS, and the infection spread further in the brain than PRV-Becker. PRV-Bartha infection did not induce the specific and lethal systemic inflammatory response seen with PRV-Becker. Overall, we demonstrated the importance of inflammation in the clinical outcome of PRV infection in mice and provide new insights into the process of PRV-induced neuroinflammation.IMPORTANCE Pseudorabies virus (PRV) is an alphaherpesvirus related to human pathogens such as herpes simplex virus 1 and varicella-zoster virus (VZV). The natural host of PRV is the swine, but it can infect most mammals. In susceptible animals other than pigs, PRV infection always causes a characteristic lethal pruritus known as the "mad itch." The role of the immune response in the clinical outcome of PRV infection is still poorly understood. Here, we show that a systemic host inflammatory response is responsible for the severe pruritus and acute death of mice infected with virulent PRV-Becker but not mice infected with attenuated strain PRV-Bartha. In addition, we identified IL-6 and G-CSF as two main cytokines that play crucial roles in the regulation of this process. Our findings give new insights into neuroinflammatory diseases and strengthen further the similarities between VZV and PRV infections at the level of innate immunity.

Keywords: cytokines; immunopathogenesis; inflammation; mice; nervous system; pseudorabies virus.

FIG 1

Clinical observations of PRV infection in mice. (A and B) Body weight and temperature (A) and clinical score (B) for B57BL/6 mice following PRV infection with the Becker strain (8 × 10⁶ PFU) (red), the Bartha strain (10⁸ PFU) (blue), or controls (black). (n = 10 per group). *, P < 0.05. (C) Representative images of mouse right hind paws at 82 hpi after PRV inoculation. Black arrows indicate the sites of abrasion.

FIG 2

PRV-Becker-infected mice showed signs of severe inflammation in the footpad and DRGs 82 h after footpad inoculation. (A and B) H&E staining of mouse inoculated footpads (A) and ipsilateral DRGs (B) from control (a), PRV-Becker-infected (b), and PRV-Bartha-infected (c) mice at 82 hpi. Histopathological manifestations observed in PRV-Becker-infected animal tissues (epidermal and neuronal necrosis and neutrophil infiltration) were absent from all examined mock-infected and PRV-Bartha-infected mice. The results are representative of three biological replicates for a given type of tissue. Black arrows indicate representative areas of inflammation with immune cell infiltration. Scale bars (50 μm) are indicated for each image.

FIG 3

Quantitation of the PRV genome in mouse tissues after footpad inoculation. At the indicated time points, PRV DNA was quantitated in mouse tissues by qRT-PCR using UL54 primers. PRV-Becker (red) and PRV-Bartha (blue) loads are expressed as PFU per mg of tissue. PRV-Becker and PRV-Bartha loads were detected only in the foot, DRGs, spinal cord, and brain (n = 10 per group). Dotted lines indicate the detection limit.

FIG 4

Plasma cytokine and chemokine levels in PRV-infected and control mice. (A) Relative levels of 12 proinflammatory cytokines measured in the plasma of mock-infected (black), PRV-Becker-infected (red), and PRV-Bartha-infected (blue) mice at 82 hpi as determined by ELISA. (B) Concentrations of six proinflammatory chemokines measured in the plasma of these three groups. The concentration is expressed in pg per ml (n = 10 per group). ****, P < 0.0001; ns, not significant. (C) Kinetics of IL-6, G-CSF, Gro-1, and MCP-1 expression in the plasma of mock-infected, PRV-Becker-infected, and PRV-Bartha-infected mice. The concentration is expressed in pg per ml (n = 5 per group). *, P < 0.05; **, P < 0.01; ****, P < 0.0001; ns, not significant.

FIG 5

Local cytokine and chemokine protein levels in PRV-Becker-infected and control mouse tissues. IL-6 (A), G-CSF (B), Gro-1 (C), and MCP-1 (D) levels were significantly upregulated in PRV-Becker-infected (red) and control (black) mouse tissues at 82 hpi. Protein levels were quantified by ELISA and are expressed as pg per mg of tissue (n = 10 per group). *, P < 0.05. The dotted line indicates the detection limit.

FIG 6

Plasma level of CRP in PRV-infected and control mice. The plasma level of CRP was measured by ELISA in PRV-Becker-infected (red), PRV-Bartha-infected (blue), and control (black) mice at 82 hpi. Protein levels are expressed in mg per liter. ****, P < 0.0001 (n = 10 per group).