Intracellular Spread of Rabies Virus Is Reduced in the Paralytic Form of Canine Rabies Compared to the Furious Form.

Studies of the furious and paralytic forms of canine rabies at the early stage of disease have shown a more rapid viral colonization of the cerebral hemispheres in the furious form, as measured by viral antigen within neuronal cell bodies and viral RNA levels. Measurement of cellular processes separate from neuronal cell body provides a visual record of the spread of rabies virus which occurs across synapses. In this study, the amount of rabies viral antigen within cell processes was quantitatively assessed by image analysis in a cohort of naturally rabies infected non-vaccinated dogs (5 furious and 5 paralytic) that were sacrificed shortly after developing illness. Measurements were taken at different levels of the spinal cord, brain stem, and cerebrum. Results were compared to the amount of rabies viral antigen in neuronal cell bodies. Generally, the amount of rabies viral antigen in cell processes decreased in a rostral direction, following the pattern for the amount of rabies viral antigen in neuronal cell bodies and the percentage of involved cell bodies. However, there was a delay in cell process involvement following cell body involvement, consistent with replication occurring in the cell body region and subsequent transport out to cell processes. Greater amounts of antigen were seen in cell processes in dogs with the furious compared to paralytic form, at all anatomic levels examined. This difference was even evident when comparing (1) neurons with similar amounts of antigen, (2) similar percentages of involved neurons, and (3) anatomic levels that showed 100% positive neurons. These findings suggest that intracellular transport of the virus may be slower in the paralytic form, resulting in slower viral propagation. Possible mechanisms might involve host-specific differences in intracellular virus transport. The latter could be cytokine-mediated, since previous studies have documented greater inflammation in the paralytic form

Graph of the percentage of areas in cellular processes positive for rabies antigen (Y axis) vs. the percentage of neuronal cell bodies positive for rabies antigen (X axis).

<p>Data points and linear regression line from dogs with furious rabies are shown in red and paralytic rabies in blue. RABV = Rabies virus, Ag = antigen.</p

Image analysis for determination of rabies antigen within cellular processes (A and B) and the hippocampal dentate fascia (C and D).

<p>For all CNS regions (except the dentate fascia of hippocampus), following immunostaining for rabies antigen (A), all neuronal cell bodies were manually outlined. The antigen signals within the cell body were then deleted to allow only the antigen in cellular processes to be detected and quantified by computer software (B). For the hippocampal dentate fascia (C), detection of the antigen was done by computer software after the area had been manually outlined (D). (A-D, immunoperoxidase using anti-rabies nucleocapsid antibody).</p

Graph of the area in cellular processes positive for rabies antigen (Y axis) vs. the antigen-positive area per neuron (X axis).

<p>Data points and linear regression line from dogs with furious rabies are shown in red and paralytic rabies in blue. RABV = Rabies virus, Ag = antigen.</p

% RABA positive area outside the neuronal cell body is shown for individual dogs at each CNS regions.

<p>P1-P5 and F1-F5 in the X axis represent 5 paralytic and 5 furious canines, respectively. Spinal cord samples were not available in P3, P5, F1, and F2, while medulla oblongata was not available in F1. Values in the Y axis of all graphs are % RABA positive area outside the neuronal cell body. An asterisk following the label of a specific anatomical region indicates a significantly higher percentage of RABA area in furious as compared to paralytic dogs. CA = cornu ammonis; and DF = dentate fascia.</p

Bar graphs comparing dogs with furious rabies (red) and paralytic rabies (blue) with respect to percentage of neuronal cell bodies positive for rabies antigen (upper row), and percentage of areas in cellular processes positive for rabies antigen (lower row).

<p>Results are shown for each of five different levels of the CNS (spinal cord, brain stem, cerebellum, cerebral midline structures and cerebrum). The data on percentage of neuronal cell bodies positive for rabies antigen have been previously reported [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004748#pntd.0004748.ref002" target="_blank">2</a>] and are shown here for comparison to the data on cellular processes.</p