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Stable spatial distribution of porcine reproductive and respiratory syndrome (PRRSV)-1 subtypes in Europe is accompanied by a strong population immunity induced by local PRRSV strains. In the present study, it was examined if the immunity induced by three West European subtype 1 PRRSV strains (2007 isolate 07V063 and 2013 isolates 13V091 and 13V117) offers protection against the highly virulent East European subtype 3 PRRSV strain Lena. The number of fever days was greater (p p p

The spatial distribution of PRRSV-1 subtypes in Europe is quite stable, most probably due to a strong population immunity induced by the local PRRSV strains. In this study, we evaluated the potential of the immunity induced by several West European subtype 1 PRRSV strains (2007 isolate 07V063 and 2013 isolates 13V091 and 13V117) to provide a protection against the highly virulent East European subtype 3 PRRSV strain Lena. Eleven-week-old pigs were inoculated with subtype 1 PRRSV strains (07V063, 13V091 or 13V117). Seven weeks later, the pigs were challenged with PRRSV strain Lena. Clinical, virological and serological parameters were monitored upon challenge. Number of fever days was higher (P < 0.05) in the non-immune control group (7.6 ± 1.7 days) compared to animals from immune groups (07V063-immune: 4.0 ± 1.2 days, 13V091-immune: 4.6 ± 1.1 days, 13V117-immune: 4.0 ± 2.9 days). In all groups, protection was characterized by reduction (P < 0.05) of AUC values of nasal shedding (control: 14.6 ± 5.6, 07V063-immune: 3.4 ± 3.4, 13V091-immune: 8.9 ± 6.1, 13V117-immune: 8.0 ± 6.1) and viremia (control: 28.1 ± 11.0, 07V063-immune: 5.4 ± 4.4, 13V091-immune: 9.0 ± 1.5, 13V117-immune: 8.3 ± 4.8). Reduction of respiratory disease, nasal shedding (mean AUC and mean peak values) and viremia (mean AUC and mean peak values) was more pronounced in 07V063-immune (P < 0.05) than in 13V091-immune and 13V117-immune animals. Inoculation of animals with subtype 1 PRRSV strains caused a priming of Lena-specific VN antibody response. Upon challenge with PRRSV Lena a serological booster effect was observed for neutralizing antibodies against strains used for the first inoculation. Our results indicate that immunity elicited by inoculation with subtype 1 PRRSV strains can partially protect against antigenically divergent subtype 3 strains. We conclude that the lower protection level elicited by recently isolated subtype 1 PRRSV strains may facilitate spatial expansion of subtype 3 strains from East Europe to West Europe.

Porcine reproductive and respiratory syndrome (PRRS) is one of the most devastating diseases causing huge economic losses in swine industry. Only in the US the losses reached $664 million in the year 2013. The disease is caused by the PRRS virus (PRRSV). The virus affects pigs of all ages, resulting in reproductive failures in sows and respiratory disorders in piglets. A high genetic heterogeneity divided PRRSV into the European type 1 and the American type 2. The virus shows a high mutation rate and evolves rapidly, resulting in the appearance of new highly pathogenic variants of both genotypes in Europe, North America and Asia over the last decade. Specifically in Europe, highly pathogenic PRRSV strains with increased respiratory disorders and more severe clinical signs have emerged recently. Therefore, the main goal of the present thesis was to examine the pathogenesis of the newly emerged isolates and identify the factors that contribute to the increased virulence. In Chapter 1, an introduction is given based on the current literature on PRRSV heterogeneity, pathogenesis, immune response and vaccination, and the applications of explant culture systems. Fianlly, the histology and the immune system of the respiratory mucosae are presented. In Chapter 2 the general aim of the thesis is stated, and the specific goals are presented. In Chapter 3, the different clinical, virological, serological and tissue tropism outcome of two new PRRSV strains (13V091 and 13V117), isolated in 2013 from two different Belgian farms with enzootic respiratory problems shortly after weaning in the nursery, were compared with the Belgian strain 07V063 isolated in 2007. 13V091 was the most pathogenic type 1 subtype 1 strain isolated up till now inducing fever, respiratory problems, high virus titers in nasal secretions, an increased percentage of lung lesions in young animals and an expanded cell tropism to Sn- cells in nasal mucosa and other lymphoid tissues. Despite the fact that 13V117 and 07V063 have only eight amino acid differences, they showed contrasting clinical and virological outcomes. 13V117 was able to induce fever, high viremia, and respiratory problems at the early stages of infection and it was able to replicate in the nasal mucosa but not in the lymph nodes, whereas 07V063 showed only fever, a 10-fold lower viremia, low virus titers in nasal excretions, but higher virus titers in lymphoid tissues. In Chapter 4.1, a new polarized nasal mucosa explant system was used to study the invasion of the low virulent subtype 1 PRRSV strain Lelystad (LV) and the highly virulent subtype 3 PRRSV strain Lena at the portal of entry. LV was mainly restricted to CD163+Sn+ cells, which were located in the lamina propria. Only 2.5 LV-infected cells/mm2 were observed at 72 hpi, all of them located in the lamina propria and the 97% of them had a CD163+Sn+ phenotype. On the other hand, Lena was found to replicate 10 to 100 times more efficiently than LV and appears to hijack both CD163+Sn+ and CD163+Sn- cells lying within or close to the epithelium in order to gain access to deeper parts of the mucosa. In Chapter 4.2, the explant system was used to study the replication characteristics in the nasal mucosa of four type 1 (LV, 07V063, 08VA, 13V091), three type 2 (VR2332, MN-184, VN) and two attenuated (MLV-DV, MLV-VR2332) PRRSV strains. Results showed that (i) based on virus shedding in the respiratory explants, PRRSV strains can be categorized as poor (MLV-DV, MLV-VR2332, LV, 08VA), moderate (Lena, VR2332) and strong (07V063, 13V091, MN-184, VN) secretors, and (ii) based on the number of infected cells isolates can be categorized as low (MLV-DV, MLV-VR2332, LV), moderately (08VA, VR2332), highly (07V063, Lena, 13V091) and hyper (MN-184, VN) virulent in the nasal mucosa. Finally, a general discussion of the main findings of this thesis and the future perspectives are presented in Chapter 5.

Although cities can be characterised as sources of economic, environmental and social challenges, they can also be part of the solution for healthy and sustainable societies. While most cities are situated close to water, whether inland waterways, lakes, or the sea, these blue spaces are not integrated into urban planning to their full potential and their public health impacts are not always recognised by planning authorities. Furthermore, cities face future challenges regarding climate change, economic developments like tourism, urbanisation, and rising social inequalities. The development of healthy blue spaces can support cities in their pursuit of ways to confront these challenges. Interdisciplinary and transdisciplinary analyses of the local impacts of these trends and promising interventions have been scarce to date. The BlueHealth project has explored the use of such methodology by presenting experiences related to five European cities: Amsterdam, Barcelona, Plymouth, Tallinn and Thessaloniki, using an interactive and participative approach with local experts and stakeholders (Wuijts, De Vries et al. under review). This report encompasses the future outlooks or scenarios that have been developed for each of these cities. The scenarios are based on the question: what contributory role could healthy blue spaces play in enhancing the impacts of long term trends? The results highlight the importance of addressing the local context when seeking sustainable solutions for cities. The future outlooks deliver information that could serve as useful input for local planning processes.

Although cities can be characterised as sources of economic, environmental and social challenges, they can also be part of the solution for healthy and sustainable societies. While most cities are situated close to water, whether inland waterways, lakes, or the sea, these blue spaces are not integrated into urban planning to their full potential and their public health impacts are not always recognised by planning authorities. Furthermore, cities face future challenges regarding climate change, economic developments like tourism, urbanisation, and rising social inequalities. The development of healthy blue spaces can support cities in their pursuit of ways to confront these challenges. Interdisciplinary and transdisciplinary analyses of the local impacts of these trends and promising interventions have been scarce to date. The BlueHealth project has explored the use of such methodology by presenting experiences related to five European cities: Amsterdam, Barcelona, Plymouth, Tallinn and Thessaloniki, using an interactive and participative approach with local experts and stakeholders (Wuijts, De Vries et al. under review). This report encompasses the future outlooks or scenarios that have been developed for each of these cities. The scenarios are based on the question: what contributory role could healthy blue spaces play in enhancing the impacts of long term trends? The results highlight the importance of addressing the local context when seeking sustainable solutions for cities. The future outlooks deliver information that could serve as useful input for local planning processes.