Figure 1.
Phylogenetic tree showing the relationship of DUVV-NL07 strain with the different lyssaviruses based on complete genome sequence.
Phylogenetic tree (unrooted) was constructed with Tree Puzzle version 5.2 software [31] using the maximum likelihood principle and the Quartet Puzzling algorithm to determine the substitution models that would best fit our data sets. Numbers above the branches indicate the percentage of 10000 puzzling steps. Puzzling support values above the 70% are significant. Accession numbers of all isolates used for the construction of the tree are given in Table S1.
Figure 2.
Replication characteristics of DUVV-NL07 in mouse or human neuroblastoma cells.
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).
Figure 3.
Development of RABV-specific antibodies over time in three- and eight-week old mice.
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.
Table 1.
In vivo replication of DUVV-NL07.
Figure 4.
Virulence of lyssaviruses in eight-week old BALB/c mice.
(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).
Table 2.
Virulence of DUVV-NL07, SHBRV-18 and RABV-PV in 8-week old mice inoculated i.m. with 106 TCID50.
Figure 5.
Histopathology of 8-week old BALB/c mice infected i.m. with 106 TCID50 of DUVV-NL07.
(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.
Figure 6.
Histopathology of 8-week old BALB/c mice infected i.m. with 106 TCID50 of RABV-PV.
(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.
Figure 7.
Histopathology of 8-week old BALB/c mice infected i.m. with 106 TCID50 of SHBRV-18.
(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.
Table 3.
Overview of histopathological changes observed in the brain and spinal cord of animals infected with DUVV-NL07, RABV-PV or SHBRV-18 virus at the time of paralysis.