Highly pathogenic H5N1 influenza virus can enter the central nervous system and induce neuroinflammation and neurodegeneration

Abstract

One of the greatest influenza pandemic threats at this time is posed by the highly pathogenic H5N1 avian influenza viruses. To date, 61% of the 433 known human cases of H5N1 infection have proved fatal. Animals infected by H5N1 viruses have demonstrated acute neurological signs ranging from mild encephalitis to motor disturbances to coma. However, no studies have examined the longer-term neurologic consequences of H5N1 infection among surviving hosts. Using the C57BL/6J mouse, a mouse strain that can be infected by the A/Vietnam/1203/04 H5N1 virus without adaptation, we show that this virus travels from the peripheral nervous system into the CNS to higher levels of the neuroaxis. In regions infected by H5N1 virus, we observe activation of microglia and alpha-synuclein phosphorylation and aggregation that persists long after resolution of the infection. We also observe a significant loss of dopaminergic neurons in the substantia nigra pars compacta 60 days after infection. Our results suggest that a pandemic H5N1 pathogen, or other neurotropic influenza virus, could initiate CNS disorders of protein aggregation including Parkinson's and Alzheimer's diseases.

Fig. 1.

Localization of A/Vietnam/1203/04 (H5N1) influenza virus in the nervous system. (A) Three days p.i. with H5N1, the virus is detected in Auerbach's plexus of the enteric nervous system (arrow) by using an antibody to the viral NP. (B) In the thoracic DRG, viral NP is first detected on day 3 p.i.; by day 10 p.i., virtually all large and medium-sized neurons (arrows), but few small neurons, are NP-positive. (C) At day 3 p.i., NP protein (red) is first detected in the neurons (white arrows) of the brainstem solitary nucleus. Blue stain is DAPI. (D) At day 7p.i., NP protein (green; arrows) is detected in the dopaminergic neurons of the SNpc (red, anti-TH). (E and F) At day 7 p.i., NP protein (brown) is found in the periglomerular cells (arrows, E) and mitral cells (arrow, F) of the olfactory bulb. (G) At day 10 p.i., NP protein is found in the spinal cord, primarily at the thoracic level. High levels of NP are seen around Clarke's column and the intermediomedial cell column (arrowhead). (H) NP protein (red) is found in the nuclei of neurons (blue, DAPI; green, anti-beta3-tubulin). (I) NP protein (red) is found in the nuclei and cytoplasm of microglia (blue, DAPI; green, anti-Iba-1; areas of colocalization are yellow). (J) NP protein (red; arrows) is not observed in astrocytes (green, anti-GFAP). (K) Transmission electron micrograph of a neuron in the brainstem shows a virus particle of the shape and size characteristic of influenza virus in the cytoplasm (arrow). The mitochondrion shows relative size. (L) Transmission electron micrograph of an axon traversing the brainstem shows virus particles of the characteristic shape and size of influenza virus in the axoplasm (arrow). Arrowheads indicate the myelin sheath surrounding the axon. [Scale bars: 20 μm (A);50 μm (B) ; 25 μm (C); 45 μm (D); 20 μm (E); 5 μm (F); 200 μm (G); 10 μm (H); 6 μm (I); 12 μm (J); 0.200 μm (K); 0.120 μm (L).]

Fig. 2.

Transport of H5N1 virus through axons in microfluidic chambers. (A) A/Vietnam/1203/04 (H5N1) influenza virus (2 × 10⁶ PFU suspended in 20 μL serum-free medium) was added to the process compartments of microfluidic chambers containing freshly dissociated DRG (55). After 1 h, the process compartment was washed twice by adding 130 μL serum-free medium to the top reservoir and collecting the flow-through in the bottom reservoir. One hundred thirty microliters medium containing serum was then added to the top and bottom reservoirs of the process compartments to maintain hydrostatic balance. Twenty-four hours after addition of H5N1 virus to the process compartment (bottom), H5N1 NP is detected in neuronal bodies within the somal compartment (white arrows). Yellow arrow shows axons (green, beta3-tubulin) traversing the series of 150 μm-wide grooves between the compartments. (B) High power photomicrograph of DRG axons (green, beta3-tubulin) growing through the 150-μm grooves. Virus particles (arrows) within the axons appear yellow because of colocalization with neuronal beta3-tubulin. (C) Neuronal bodies (green) are infected after direct exposure to H5N1 virus (red, viral NP protein; arrows). Yellow indicates colocalization of proteins. [Scale bars: 160 μm (A); 6 μm (B); 60 μm (C).]

Fig. 3.

A/Vietnam/1203/04 (H5N1) influenza virus induces a parkinsonian phenotype. (A) In uninfected C57BL/6J mice, no pSer129SYN is seen in the dopaminergic neurons of the SNpc (green). (B) At day 60 p.i., pSer129SYN (white arrows) is seen in the nuclei of dopaminergic neurons (green) in the SNpc. (blue, DAPI nuclear stain.) (C) At day 90 p.i., aggregated alpha synuclein (arrows) is seen in swollen neurites in the hippocampus. (D) At day 10 p.i., activated caspase-3 (arrows) is seen in brainstem regions that contain NP protein (red). The cells expressing activated caspase-3 do not appear to be NP-positive. (E) At day 10 p.i., activated microglia marked by anti-Iba-1 (green; yellow arrow) surround infected cells (red; white arrow). (F) At day 60 p.i., activated microglia (arrows) persist in midbrain regions (and all other regions) infected by H5N1. Findings were similar at day 90 p.i. [Scale bars: 30 μm (A and B); 50 μm (C); 40 μm (D); 35 μm (E); 50 μm (F).]