Pathogenesis of Two Western Mediterranean West Nile Virus Lineage 1 Isolates in Experimentally Infected Red-Legged Partridges (Alectoris rufa).

West Nile virus (WNV) is the most widespread flavivirus in the world with a wide vertebrate host range. Its geographic expansion and activity continue to increase with important human and equine outbreaks and local bird mortality. In a previous experiment, we demonstrated the susceptibility of 7-week-old red-legged partridges (Alectoris rufa) to Mediterranean WNV isolates Morocco/2003 and Spain/2007, which varied in virulence for this gallinaceous species. Here we study the pathogenesis of the infection with these two strains to explain the different course of infection and mortality. Day six post-inoculation was critical in the course of infection, with the highest viral load in tissues, the most widespread virus antigen, and more severe lesions. The most affected organs were the heart, liver, and spleen. Comparing infections with Morocco/2003 and Spain/2007, differences were observed in the viral load, virus antigen distribution, and lesion nature and severity. A more acute and marked inflammatory reaction (characterized by participation of microglia and CD3+ T cells) as well as neuronal necrosis in the brain were observed in partridges infected with Morocco/2003 as compared to those infected with Spain/2007. This suggests a higher neurovirulence of Morocco/2003, probably related to one or more specific molecular determinants of virulence different from Spain/2007.

Natural Bagaza virus infection in game birds in southern Spain.

In late summer 2010 a mosquito born flavivirus not previously reported in Europe called Bagaza virus (BAGV) caused high mortality in red-legged partridges (Alectoris rufa) and ring-necked pheasants (Phasianus colchicus). We studied clinical findings, lesions and viral antigen distribution in naturally BAGV infected game birds in order to understand the apparently higher impact on red-legged partridges. The disease induced neurologic signs in the two galliform species and, to a lesser extent, in common wood pigeons (Columba palumbus). In red-legged partridges infection by BAGV caused severe haemosiderosis in the liver and spleen that was absent in pheasants and less evident in common wood pigeons. Also, BAGV antigen was present in vascular endothelium in multiple organs in red-legged partridges, and in the spleen in common wood pigeons, while in ring-necked pheasants it was only detected in neurons and glial cells in the brain. These findings indicate tropism of BAGV for endothelial cells and a severe haemolytic process in red-legged partridges in addition to the central nervous lesions that were found in all three species.

Antibodies to West Nile virus and related flaviviruses in wild boar, red foxes and other mesomammals from Spain.

Red foxes (Vulpes vulpes), wild boar (Sus scrofa) and Iberian pigs (Sus scrofa domestica) that are raised extensively outdoors, as well as other wild mesomammals from south central Spain and wild boar from Doñana National Park (DNP), were tested for antibodies against related flaviviruses by ELISA and for antibodies against WNV by VNT. Mean flavivirus seroprevalence according to ELISA was 20.4 ± 7.8% (21 out of 103) in red foxes, 12.6 ± 2.8% (69 out of 545) in wild boars, and 3.3±2.7% (6 out of 177) in Iberian pigs. A stone marten (Martes foina) also tested positive. Flavivirus seroprevalence in wild boar was significantly higher in DNP, and increased with age. Haemolysis of the serum samples limited interpretation of VNT to 28 samples, confirming WNV seroprevalence in one red fox, four Iberian pigs and nine wild boars. ELISA positive, microVNT negative samples suggest presence of non-neutralizing antibodies against WNV or antibodies to other antigenically related flaviviruses. Despite the importance of wetlands for flavivirus maintenance and amplification, WNV/flavivirus seroprevalence in wild boar and red foxes was not associated to wetland habitats. This is the first report of exposure of red foxes to WNV. With view to use of the tested species as sentinels for flavivirus activity, limited exposure of Iberian pigs that would be available for regular sampling, low numbers of foxes collected and concentration of wild boar harvest in the winter season are major drawbacks.

Do wild ungulates allow improved monitoring of flavivirus circulation in Spain?

As a response to the need for improved and cost-efficient West Nile virus (WNV) and other flavivirus surveillance tools, we tested 887 juvenile free-living red deer, 742 free-living juvenile wild boar, and 327 farmed deer, to detect temporal variability in exposure to these viruses. Thirty of 742 wild boar samples (4%; 95% CI 2.8,5.7) yielded a positive ELISA result. Antibody-positive individuals had been sampled between 2003 and 2011 in localities from central and southern Spain. No wild boar from the northern half of Spain (n=120) tested positive. Regarding juvenile wild red deer, only two out of 887 samples yielded a positive ELISA result (0.2%; 95% CI 0.1,0.8). These two samples came from the same site and sampling year. The likelihood of detecting contact with WNV or cross-reacting flaviviruses was 18 times higher among juvenile wild boar than among juvenile red deer. ELISA positivity among farmed deer increased 10-fold after local flavivirus outbreaks recorded in the summer and autumn of 2010. This survey demonstrated the potential usefulness of juvenile wild ungulates, particularly wild boar, as suitable flavivirus sentinels in southwestern Europe, and that systematic serum banking of samples from hunter-harvested wildlife or from individual farmed ungulates provides valuable material for retrospective epidemiological surveys and future disease monitoring.

Pathogenicity of two recent Western Mediterranean West Nile virus isolates in a wild bird species indigenous to Southern Europe: the red-legged partridge.

West Nile virus (WNV) is an emerging zoonotic pathogen whose geographic spread and incidence in humans, horses and birds has increased significantly in recent years. WNV has long been considered a mild pathogen causing self-limiting outbreaks. This notion has changed as WNV is causing large epidemics with a high impact on human and animal health. This has been particularly noteworthy since its introduction into North America in 1999. There, native bird species have been shown to be highly susceptible to WNV infection and disease with high mortalities. For this reason, the effect of WNV infection in North American bird species has been thoroughly studied by means of experimental inoculations in controlled trials. To a lesser extent, European wild birds have been shown to be affected clinically by WNV infection. Yet experimental studies on European wild bird species are lacking. The red-legged partridge (Alectoris rufa) is a gallinaceous bird indigenous to the Iberian Peninsula, widely distributed in South Western Europe. It plays a key role in the Mediterranean ecosystem and constitutes an economically important game species. As such it is raised intensively in outdoor facilities. In this work, red-legged partridges were experimentally infected with two recent WNV isolates from the Western Mediterranean area: Morocco/2003 and Spain/2007. All inoculated birds became viremic and showed clinical disease, with mortality rates of 70% and 30%, respectively. These results show that Western Mediterranean WNV variants can be pathogenic for some European bird species, such as the red-legged partridge.