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

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

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

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

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

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 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

Viral burden in the brains of mice infected with WNV-FIN, Ita09 and 578/10.

<p>Mice were euthanized within 6 to 14 days upon reaching humane endpoints and viral burden was measured in terms of (<b>A</b>) RNA copies per gram of brain, and (<b>B</b>) TCID₅₀ per gram of brain. (<b>C</b>) Viral RNA copies per gram of brain in mice infected with WNV-FIN, Ita09 and 578/10, and euthanized on day 20 in the absence of clinical signs of disease. * <i>P</i> <0.05, ** <i>P</i> <0.01.</p

Characterization of the Mouse Neuroinvasiveness of Selected European Strains of West Nile Virus

<div><p>West Nile virus (WNV) has caused outbreaks and sporadic infections in Central, Eastern and Mediterranean Europe for over 45 years. Most strains responsible for the European and Mediterranean basin outbreaks are classified as lineage 1. In recent years, WNV strains belonging to lineage 1 and 2 have been causing outbreaks of neuroinvasive disease in humans in countries such as Italy, Hungary and Greece, while mass mortality among birds was not reported. This study characterizes three European strains of WNV isolated in Italy (FIN and Ita09) and Hungary (578/10) in terms of <i>in vitro</i> replication kinetics on neuroblastoma cells, LD₅₀ values in C57BL/6 mice, median day mortality, cumulative mortality, concentration of virus in the brain and spinal cord, and the response to infection in the brain. Overall, the results indicate that strains circulating in Europe belonging to both lineage 1 and 2 are highly virulent and that Ita09 and 578/10 are more neurovirulent compared to the FIN strain.</p> </div