Respiratory syncytial virus (RSV) infects neuronal cells and processes that innervate the lung by a process involving RSV G protein

Abstract

Respiratory syncytial virus (RSV) is a primary cause of morbidity and life-threatening lower respiratory tract disease in infants and young children. Children with acute RSV bronchiolitis often develop respiratory sequelae, but the disease mechanisms are poorly understood. Mounting evidence suggests that RSV may mediate persistent infection. Using immunohistochemistry to identify RSV and RSV-infected cell types, we show that RSV infects primary neurons and neuronal processes that innervate the lungs through a process that involves RSV G protein and the G protein CX3C motif. These findings suggest a mechanism for disease chronicity and have important implications for RSV disease intervention strategies.

FIG. 1.

Expression of RSV G and N proteins in primary cortical neurons at day 5 post-RSV infection. Mouse primary neurons were infected (MOI of 1) with wild-type RSV strain A2 (RSV/A2), recombinant RSV derived from strain A2 (6340WT), 6340WT lacking the G protein gene (6340ΔG), or with a RSV strain A2 point mutant having a CYS→ARG amino acid change at position 186 (A2/ΔCX3C). At 5 day p.i., neurons were fixed with 4% paraformaldehyde and viral antigens were detected using anti-RSV N monoclonal antibodies. RSV N protein expression was detected in only cells infected with RSV/A2 and 6340WT, not in cells infected with mutant RSV, i.e., 6340ΔG and A2/ΔCX3C. Magnification, ×32.

FIG. 2.

Codetection of RSV N antigen and neuronal markers in primary neurons at day 5 post-RSV infection. Primary neurons infected with RSV/A2 were fixed at 5 day p.i. RSV N protein was detected together with neuronal markers Nissl (A), MAP2 (B), PGP9.5 (C), and neurofilament (D). RSV N antigen staining was merged with each of the neuronal markers. RSV N antigen staining overlapped with each of the neuronal markers, indicating that RSV infects primary neurons (Magnification, ×32). No staining was detected in negative control neuronal cells (Fig. 1).

FIG. 3.

Detection of RSV N antigen in neuronal processes in lung tissue. Lung tissues from mice infected with 10⁶ PFU RSV/A2, 6340WT, 6340ΔG, or A2/ΔCX3C virus were fixed and subjected to immunohistochemistry using monoclonal antibodies to RSV N protein or neurofilament. RSV N protein staining was merged with neurofilament staining. Overlapping staining was observed in only the lungs of mice infected with wild-type RSV (RSV/A2 and 6340WT), not in lungs of mice infected with mutant RSV (6340ΔG and A2/ΔCX3C) (Magnification, ×16).

FIG. 4.

Codetection of RSV N protein and neuronal marker PGP9.5 in RSV-infected lung tissue. (A) RSV N protein was detected primarily in the epithelial walls surrounding small bronchioles (longitudinal section), and Hoechst staining shows only a few RSV-positive cells (most likely epithelial cells). (B) RSV N protein staining overlapped with neuronal marker PGP9.5 in nervous fibers in a small bronchiole (transversal section). These nerve fibers may come from one neuron (Magnification, ×32).