Histopathology of 8-week old BALB/c mice infected i.m. with 10⁶ TCID₅₀ of RABV-PV.

<p>(A) Purkinje cell layer of the cerebellum stained with anti-NP rabies antibody, (B) Extensive rabies virus antigen expression in the spinal cord (anti-NP rabies staining; 10× objective). (C) CD3+ staining in a perivascular cuff of the spinal cord. (D) astrocytosis in the cerebrum of infected animals (GFAP staining; 40× objective). Examples of positively stained cells are indicated by block arrows.</p

In vivo replication of DUVV-NL07.

<p>Viral RNA detected in the brain of animals (mean values expressed in copies/ml) infected with high, intermediate or low dose of DUVV-NL07 via peripheral route. The number of DUVV-NL07 RNA positive animals per total number of animals inoculated is indicated within brackets. Numbers in superscript indicate the amount of animals that developed paralysis in the respective groups.</p

Histopathology of 8-week old BALB/c mice infected i.m. with 10⁶ TCID₅₀ of DUVV-NL07.

<p>(A) Neo-cortical neurons stained with anti-NP rabies antibody, (B) HE staining of spinal cord section illustrating perivascular cuffing (objective 40×), (C) CD3+ cells infiltrating the neuropil of the spinal cord, (D) activated microglia (Iba1 staining; 40× objective) in the brainstem. Examples of positively stained cells are indicated by block arrows.</p

Histopathology of 8-week old BALB/c mice infected i.m. with 10⁶ TCID₅₀ of SHBRV-18.

<p>(A) The dentate nucleus of the cerebellum stained with anti-NP rabies antibody, (B) necrotic neurons in the spinal cord (HE staining; 40× objective). (C) CD3+ cells infiltrating the neuropil of the spinal cord. (D) astrocytosis in the brainstem of infected animals (GFAP staining; 40× objective). Examples of positively stained cells are indicated by block arrows.</p

Development of RABV-specific antibodies over time in three- and eight-week old mice.

<p>BALB/c mice were inoculated i.m or s.c with (a) 10² TCID₅₀, (b) 10⁴ TCID₅₀ (c) 10⁶ TCID₅₀ of DUVV-NL07 or DUVV-NL07 BPL control. Dotted lines indicate threshold level of the assay.</p

Virulence of lyssaviruses in eight-week old BALB/c mice.

<p>(A): survival curves of animals infected with DUVV-NL07 (n = 25), RABV-PV (n = 25) and SHBRV-18 (n = 20). Animals were infected i.m. with 10⁶ TCID₅₀ of DUVV-NL07 (squares, dotted line), RABV-PV (triangles, straight line) and SHBRV-18 (circles, dotted line) and followed for clinical signs for 20 days. (B): Infectious viral titres recovered from the brains of mice infected i.m with 10⁶ TCID₅₀ of DUVV-NL07, RABV-PV or SHBRV-18. *: statistically different (two-tailed, Mann-Whitney test).</p

Virulence of DUVV-NL07, SHBRV-18 and RABV-PV in 8-week old mice inoculated i.m. with 10⁶ TCID₅₀.

<p>The number of positive animals (viral RNA) per total number of animals tested is indicated.</p>1<p>Day of euthanasia varied between the three viruses (day 9–17 for DUVV-NL07, day 8–17 for RABV-PV and day 6–9 for SHBRV-18). See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002682#ppat-1002682-g004" target="_blank">Figure 4</a>, survival curves.</p

Replication characteristics of DUVV-NL07 in mouse or human neuroblastoma cells.

<p>N2a cells (left panels) and SK-N-SH cells (right panels) were inoculated with m.o.i = 0.01 (a) and m.o.i. = 10 (b) Experiments were performed in triplicate and data represent mean ± standard error of the mean (SE). Replication kinetics of DUVV-NL07 (purple) were compared with RABV-PV (green) and SHBRV-18 (blue).</p

Histopathology rabies encephalitis due to Duvenhage virus.

<p>Rabies encephalitis (lyssavirus; genotype 4) brain pathology: cerebral cortex and anti-RNP Immunohistochemistry. (A) (HE staining): frontal cortex: extensive neuropil vacuolization and neuronal cell loss. (B) Few residual cortical neurons (arrows). (C) (HE staining): perivascular lymphocytic inflammatory infiltrates (arrows). (D and E) Anti-RNP immunoreactivity within the frontal cortex. Several neuronal cells show immunopositivity (arrows and insert in [E]) with multiple antigenic masses present in the neuronal cytoplasm, as well as in dendrites and axon. Scale bar: (A, B) 100 µm; (C, D) 60 µm; (E) 40 µm; insert in (E): 12 µm.</p

Immunofluorescence tests with antirabies nucleocapside conjugate.

<p>Immunofluorescence of murine neuroblastoma cells using antirabies nucleocapside conjugate (Bio-Rad). The arrow points to cells with positive green immunofluorescence. The right picture is a magnification of the left with the scale bar.</p