Herpes simplex virus type 1 infection of the rat enteric nervous system evokes small-bowel neuromuscular abnormalities.

BACKGROUND & AIMS: Infectious agents, such as neurotropic viruses, are proposed to disrupt the enteric neuromuscular system, leading to dysmotility, although the mechanisms are unknown. Our purpose was to assess whether herpes simplex virus type-1 (HSV-1) establishes an enteric-neuronal infection and induces gut dysmotility. METHODS: Rats were inoculated with HSV-1 intranasally and after 4 weeks intragastrically. After 1-10 weeks, infection was determined by molecular analysis whereas neuromuscular function was evaluated by pharmacologic/electrical stimulation of longitudinal ileal segments and by gastrointestinal transit and by [(3)H]acetylcholine release measurements. Inflammation in the neuromuscular layer was assessed by myeloperoxidase and cytokine levels and by anti-CD3(+) immunohistochemistry. RESULTS: After 1-10 weeks of intragastric inoculation, HSV-1 latency-associated messenger RNA transcripts were detected in the brain and in ileal neurons with no signs of illness or histologic gut abnormalities. By using a recombinant HSV-1 carrying the lacZ gene, HSV-1 virions were localized in myenteric ganglia by in situ X-gal staining. Interleukin-2 and IFN-gamma levels were increased significantly 1 and 6 weeks after inoculation. CD3(+) cells were found around the myenteric ganglia 6 weeks after inoculation. Smooth muscle responses to carbachol, CaCl(2), and gut transit were increased significantly after 1 and 6 weeks, whereas KCl- and electrical field stimulation-mediated contractions were modified significantly only 1-2 weeks after HSV-1 administration. The release of [(3)H]acetylcholine was reduced significantly in ileum segments after 1 and 6 weeks. CONCLUSIONS: After intragastric inoculation, HSV-1 establishes a latent infection in the rat myenteric ganglia, which leads to gut dysmotility.

Rabies virus entry at the neuromuscular junction in nerve-muscle cocultures.

Early events in rabies virus entry into neurons were investigated in chick spinal cord-muscle cocultures. Rabies virus (CVS strain) was adsorbed to the surface of cells in the cold. At times up to 10 min of warming to 37 degrees C, virus was most intensely localized to dense swellings on the myotube surface. Texas Red-labeled alpha-bungarotoxin, which binds to nicotinic acetylcholine receptors, colocalized precisely with virus at the densities identifying these regions as neuromuscular junctions. Rabies virus also colocalized in the junctions with synapsin I, a marker for synaptic vesicles. The endosome tracers Lucifer Yellow, Texan Red-dextran, and rhodamine-wheat germ agglutinin were added to the cultures at the end of the virus adsorption period and the cultures were warmed. At 10 min, rabies virus and tracers colocalized at neuromuscular junctions and nerve terminals. At 30 min, rabies virus and tracers showed more intense fluorescence over nerve fibers and nerve cell bodies. At 60 min, nerve terminals, nerve fibers, and nerve cell bodies showed intense fluorescence and colocalization for rabies virus and tracers. LysoTracker Red, a marker for acidic compartments, colocalized with rabies virus at nerve-muscle contacts. These findings show that in nerve-muscle cocultures, the neuromuscular junction is the major site of entry into neurons. Colocalization of virus and endosome tracers within nerve terminals indicates that virus resides in an early endosome compartment, some of which are acidified. The progressive increase of virus and tracers in nerve fibers and nerve cell bodies over time is consistent with retrograde transport of endocytosed virus from the motor nerve terminal.