Replication characteristics of porcine reproductive and respiratory syndrome virus (PRRSV) European subtype 1 (Lelystad) and subtype 3 (Lena) strains in nasal mucosa and cells of the monocytic lineage: indications for the use of new receptors of PRRSV (Lena)

Recently, it has been demonstrated that subtype 3 strains of European type porcine reproductive and respiratory syndrome virus (PRRSV) are more virulent/pathogenic than subtype 1 strains. This points to differences in the pathogenesis. In the present study, 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. Different cell types of the monocytic lineage (alveolar macrophages (PAM), cultured blood monocytes and monocyte-derived dendritic cells (moDC)) were enclosed to examine replication kinetics of both strains in their putative target cells. At 0, 12, 24, 48 and 72 hours post inoculation (hpi), virus production was analyzed and the infected cells were quantified and identified. Lena replicated much more efficiently than LV in the nasal mucosa explants and to a lesser extent in PAM. Differences in replication were not found in monocytes and moDC. Confocal microscopy demonstrated that for LV, almost all viral antigen positive cells were CD163+Sialoadhesin (Sn)+, which were mainly located in the lamina propria of the respiratory mucosa. In Lena-infected nasal mucosa, CD163+Sn+, CD163+Sn- and to a lesser extent CD163-Sn- monocytic subtypes were involved in infection. CD163+Sn- cells were mostly located within or in the proximity of the epithelium. Our results show that, whereas LV replicates in a restricted subpopulation of CD163+Sn+ monocytic cells in the upper respiratory tract, Lena hijacks a broader range of subpopulations to spread within the mucosa. Replication in CD163+Sn- cells suggests that an alternative entry receptor may contribute to the wider tropism of Lena

Comparison of in/ex vivo replication characteristics of different porcine reproductive and respiratory syndrome virus strains

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

The immunity raised by recent European subtype 1 PRRSV strains allows a better replication of East European subtype 3 PRRSV strain Lena than the immunity raised by an older strain -increased risk for spatial expansion of PRRSV Lena-like strains

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

Immunity raised by recent European subtype 1 PRRSV strains allows better replication of East European subtype 3 PRRSV strain Lena than that raised by an older strain

International audienceAbstractStable 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 < 0.05) in the control group (7.6 ± 1.7 days) compared to the 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, 07V063-immune: 3.4, 13V091-immune: 8.9, 13V117-immune: 8.0) and viremia (control: 28.1, 07V063-immune: 5.4, 13V091-immune: 9.0, 13V117-immune: 8.3). 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 with subtype 1 PRRSV strains caused priming of the Lena-specific virus neutralization antibody response. Upon challenge with Lena, we observed a very strong serological booster effect for neutralizing antibodies against strains used for the first inoculation. Our results indicate that inoculation with subtype 1 PRRSV strains can partially protect against antigenically divergent subtype 3 strains. The lower protection level elicited by recently isolated subtype 1 PRRSV strains may impair the outcome of the spatial expansion of subtype 3 strains from East Europe to West Europe

The health potential of urban water: Future scenarios on local risks and opportunities

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, socio-economic developments like tourism, urbanization, 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. This study explores 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. Future scenarios have been developed based on the question: How can blue spaces contribute to a healthier city population, given the long term trends? The results highlight the importance of addressing the local context when seeking sustainable solutions for cities. The future scenarios deliver information that could serve as useful input for local planning processes

The health potential of urban water: Future scenarios on local risks and opportunities

This is the final version. Available on open access from Elsevier via the DOI in this recordAlthough 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, socio-economic developments like tourism, urbanization, 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. This study explores 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. Future scenarios have been developed based on the question: How can blue spaces contribute to a healthier city population, given the long term trends? The results highlight the importance of addressing the local context when seeking sustainable solutions for cities. The future scenarios deliver information that could serve as useful input for local planning processes.European Union Horizon 202

In/ex vino σύγκριση χαρακτηριστικών πολλαπλασιασμού διαφορετικών στελεχών του ιού του αναπαραγωγικού και αναπνευστικού συνδρόμου του χοίρου

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), which is a single-stranded positive RNA virus, classified in the Arteriviridae family. The virus affects pigs of all ages, resulting in reproductive failures in sows and gilts 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 the first part of chapter 1, an introduction is given based on the current literature on virus history, taxonomy, heterogeneity, pathogenesis, immune response and vaccination. In the second part, a brief introduction on the history and the applications of explant culture systems is presented, and the histology and immune system of the respiratory mucosae. 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. Full genome sequencing was performed to identify the origin of the new isolates. Twelve weeks-old pigs were inoculated intranasally (IN) with 13V091, 13V117 or 07V063 (9 pigs/group). At 10 days post inoculation (dpi), 4 animals from each group were euthanized and tissues were collected for pathology, virological and serological analysis. 13V091 infection resulted in the highest respiratory disease scores and longest period of fever. Gross lung lesions were more pronounced for 13V091 (13%) than for 13V117 (7%) and 07V063 (11%). The nasal shedding and viremia was also most extensive with 13V091. The 13V091-inoculated group showed the highest virus replication in conchae, tonsils and retropharyngeal lymph nodes. 13V117 infection resulted in the lowest virus replication in lymphoid tissues. 13V091 showed higher numbers of sialoadhesin- infected cells/mm2 in conchae, tonsils and spleen than 13V117 and 07V063. Neutralizing antibody response with 07V063 was stronger than with 13V091 and 13V117. In summary: (1) two new PRRSV strains designated 13V091 and 13V117 were isolated from pigs showing respiratory distress. Pathogenic and genetic studies showed that 13V091 is the most pathogenic type 1 subtype 1 strain isolated up till now inducing fever, breathing 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. (2) Despite the fact that 13V117 and 07V063 show 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. The increased respiratory disorders compared to contemporary PRRSV isolates and the expanded cell tropism observed in the results of chapter 3 triggered us to study the virus replication kinetics in the nasal mucosa more extensively. Thus, 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. Different cell types of the monocytic lineage (alveolar macrophages (PAM), cultured blood monocytes and monocyte-derived dendritic cells (moDC)) were enclosed to examine replication kinetics of both strains in their putative target cells. At 0, 12, 24, 48 and 72 hours post inoculation (hpi), virus production was analyzed and the infected cells were quantified and identified. Differences in replication were not found in monocytes and moDC. Confocal microscopy demonstrated that for LV, almost all viral antigen positive cells were CD163+Sialoadhesin (Sn)+, which were mainly located in the lamina propria of the respiratory mucosa. In Lena-infected nasal mucosa, CD163+Sn+, CD163+Sn- and to a lesser extent CD163-Sn- monocytic subtypes were involved in infection. CD163+Sn- cells were mostly located within or in the proximity of the epithelium. In summary, we proposed a model for the different mechanisms of invasion that were observed between these two PRRSV strains in nasal mucosa. On the one hand, LV susceptibility was mainly restricted to CD163+Sn+ cells, which were located in the lamina propria. The small number of Sn+ cells (5.2/mm2) that are present within the epithelial cell layer likely does not allow LV to reach high levels of infection. 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. After 48 hpi, clusters of mainly CD163+Sn- Lena-infected cells were observed within or close to the epithelium and clusters of CD163+Sn+ cells were observed in the lamina propria. These clusters expanded at 72 hpi, and to a lesser extent than the other phenotypes, CD163-Sn- cells were also detected at the periphery of them. In chapter 4.2, the explant system was used to expand our research and 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. After 72 hpi mean virus titers reached 104.5 to 4.8 TCID50/ml for LV and 08V204, 105.2 to 5.4 TCID50/ml for Lena and VR2332, and 105.8 to 6.3 TCID50/ml for 07V063, 13V091, MN-184 and VN strains, whereas attenuated strains remained around the detection limit (0.9 TCID50/ml). The mean number of PRRSV-positive cells/mm2 at 72 hpi was 1.1 and 1.3 for the attenuated strains and LV, 13.3 for 08VA, 23.5 and 29.3 for VR2332 and 07V063, 31.1 and 33.8 for 13V091 and Lena, and, 39.1 and 59.2 for MN-184 and VN respectively. All the LV and MLV-DV infected cells were Sn+, whereas all other strains also infected Sn- macrophages. In conclusion, (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.Το αναπαραγωγικό και αναπνευστικό σύνδρομο των χοίρων (PRRS) είναι μια από τις πιο καταστροφικές ασθένειες που προκαλεί τεράστιες οικονομικές απώλειες στη βιομηχανία των χοίρων. Μόνο στις ΗΠΑ οι απώλειες έφτασαν τα 664 εκατομμύρια δολάρια το έτος 2013. Η ασθένεια προκαλείται από τον ιό PRRS (PRRSV), ο οποίος είναι ένας μονόκλωνος θετικός ιός RNA, ταξινομημένος στην οικογένεια των Arteriviridae. Ο ιός προσβάλλει χοίρους όλων των ηλικιών, με αποτέλεσμα αναπαραγωγικές ανεπάρκειες σε χοιρομητέρες και θηλυκούς χοίρους και αναπνευστικές διαταραχές στα χοιρίδια. Μια υψηλή γενετική ετερογένεια διαίρεσε τον PRRSV στον ευρωπαϊκό τύπο 1 και τον αμερικανικό τύπο 2. Ο ιός εμφανίζει υψηλό ποσοστό μετάλλαξης και εξελίσσεται γρήγορα, με αποτέλεσμα την εμφάνιση νέων εξαιρετικά παθογόνων παραλλαγών και των δύο γονότυπων στην Ευρώπη, τη Βόρεια Αμερική και την Ασία κατά την τελευταία δεκαετία. Συγκεκριμένα στην Ευρώπη, πρόσφατα εμφανίστηκαν στελέχη PRRSV υψηλής παθογονικότητας με αυξημένες αναπνευστικές διαταραχές και πιο σοβαρά κλινικά σημεία. Ως εκ τούτου, κύριος στόχος της παρούσας διατριβής ήταν η εξέταση της παθογένειας των νεοεμφανισθέντων απομονώσεων και ο εντοπισμός των παραγόντων που συμβάλλουν στην αυξημένη λοιμογόνο δράση. Στο πρώτο μέρος του κεφαλαίου 1, γίνεται μια εισαγωγή με βάση την τρέχουσα βιβλιογραφία για το ιστορικό ιών, την ταξινόμηση, την ετερογένεια, την παθογένεση, την ανοσολογική απόκριση και τον εμβολιασμό. Στο δεύτερο μέρος, παρουσιάζεται μια σύντομη εισαγωγή για την ιστορία και τις εφαρμογές των συστημάτων καλλιέργειας μοσχευμάτων, καθώς και την ιστολογία και το ανοσοποιητικό σύστημα των αναπνευστικών βλεννογόνων. Στο κεφάλαιο 2 αναφέρεται ο γενικός στόχος της διπλωματικής εργασίας και παρουσιάζονται οι συγκεκριμένοι στόχοι. Στο κεφάλαιο 3, η διαφορετική κλινική, ιολογική, ορολογική και ιστική έκβαση τροπισμού δύο νέων στελεχών PRRSV (13V091 και 13V117), που απομονώθηκαν το 2013 από δύο διαφορετικά βελγικά αγροκτήματα με ενζωοτικά αναπνευστικά προβλήματα λίγο μετά τον απογαλακτισμό στο φυτώριο, συγκρίθηκαν με τα βελγικά στέλεχος 07V063 που απομονώθηκε το 2007. Πραγματοποιήθηκε πλήρης αλληλουχία γονιδιώματος για να προσδιοριστεί η προέλευση των νέων απομονώσεων. Χοίροι ηλικίας δώδεκα εβδομάδων εμβολιάστηκαν ενδορινικά (ΙΝ) με 13V091, 13V117 ή 07V063 (9 χοίροι/ομάδα). Στις 10 ημέρες μετά τον εμβολιασμό (dpi), 4 ζώα από κάθε ομάδα υποβλήθηκαν σε ευθανασία και συλλέχθηκαν ιστοί για παθολογική, ιολογική και ορολογική ανάλυση. Η λοίμωξη 13V091 είχε ως αποτέλεσμα τις υψηλότερες βαθμολογίες αναπνευστικής νόσου και τη μεγαλύτερη περίοδο πυρετού. Οι μικτές βλάβες στους πνεύμονες ήταν πιο έντονες για το 13V091 (13%) από ό,τι για το 13V117 (7%) και το 07V063 (11%). Η ρινική έκκριση και η ιαιμία ήταν επίσης πιο εκτεταμένες με το 13V091. Η ομάδα που εμβολιάστηκε με το στέλεχος 13V091 έδειξε τον υψηλότερο πολλαπλασιασμό του ιού σε κόγχες, αμυγδαλές και οπισθοφάρυγγα λεμφαδένες. Η λοίμωξη με το στέλεχος 13V117 είχε ως αποτέλεσμα τη χαμηλότερη αντιγραφή του ιού στους λεμφικούς ιστούς. Το 13V091 έδειξε υψηλότερους αριθμούς μολυσμένων με σιαλοαδεσίνη κυττάρων/mm2 σε κόγχες, αμυγδαλές και σπλήνα από το 13V117 και το 07V063. Η απόκριση εξουδετερωτικού αντισώματος με 07V063 ήταν ισχυρότερη από ό,τι με τα 13V091 και 13V117. Συνοπτικά: (1) δύο νέα στελέχη PRRSV που ονομάζονται 13V091 και 13V117 απομονώθηκαν από χοίρους που εμφάνιζαν αναπνευστική δυσχέρεια. Παθογόνες και γενετικές μελέτες έδειξαν ότι το 13V091 είναι το πιο παθογόνο στέλεχος τύπου 1 υποτύπου 1 που έχει απομονωθεί μέχρι τώρα προκαλώντας πυρετό, αναπνευστικά προβλήματα, υψηλούς τίτλους ιού στις ρινικές εκκρίσεις, αυξημένο ποσοστό πνευμονικών βλαβών σε νεαρά ζώα και διευρυμένο κυτταρικό τροπισμό σε Sn- κύτταρα στο ρινικό βλεννογόνο και σε άλλους λεμφικούς ιστούς. (2) Παρά το γεγονός ότι τα 13V117 και 07V063 εμφανίζουν μόνο οκτώ διαφορές αμινοξέων, εμφάνισαν αντίθετα κλινικά και ιολογικά αποτελέσματα. Το 13V117 μπόρεσε να προκαλέσει πυρετό, υψηλή ιαιμία και αναπνευστικά προβλήματα στα αρχικά στάδια της μόλυνσης και ήταν σε θέση να αναπαραχθεί στον ρινικό βλεννογόνο αλλά όχι στους λεμφαδένες, ενώ το 07V063 έδειξε μόνο πυρετό, 10 φορές χαμηλότερη ιαιμία, χαμηλή τίτλους ιού στις ρινικές εκκρίσεις, αλλά υψηλότερους τίτλους ιού στους λεμφικούς ιστούς. Οι αυξημένες αναπνευστικές διαταραχές σε σύγκριση με τις σύγχρονες απομονώσεις PRRSV και ο διευρυμένος κυτταρικός τροπισμός που παρατηρήθηκε στα αποτελέσματα του κεφαλαίου 3 μας ώθησαν να μελετήσουμε εκτενέστερα την κινητική αντιγραφής του ιού στον ρινικό βλεννογόνο. Έτσι, στο κεφάλαιο 4.1, χρησιμοποιήθηκε ένα νέο πολωμένο σύστημα μοσχευμάτων ρινικού βλεννογόνου για τη μελέτη της εισβολής του στελέχους Lelystad (LV) υποτύπου 1 PRRSV χαμηλής λοιμογόνου δράσης και του στελέχους Lena του υποτύπου 3 PRRSV υψηλής λοιμογόνου δράσης στην πύλη εισόδου. Διαφορετικοί τύποι κυττάρων της μονοκυτταρικής γενεαλογίας (κυψελιδικά μακροφάγα (PAM), καλλιεργημένα μονοκύτταρα αίματος και δενδριτικά κύτταρα προερχόμενα από μονοκύτταρα (moDC)) περιλήφθηκαν για να εξεταστούν οι κινητικές αντιγραφής και των δύο στελεχών στα υποτιθέμενα κύτταρα-στόχους τους. Στις 0, 12, 24, 48 και 72 ώρες μετά τον εμβολιασμό (hpi), η παραγωγή του ιού αναλύθηκε και τα μολυσμένα κύτταρα ποσοτικοποιήθηκαν και ταυτοποιήθηκαν. Διαφορές στην αντιγραφή δεν βρέθηκαν στα μονοκύτταρα και στο moDC. Η ομοεστιακή μικροσκοπία έδειξε ότι για την LV, σχεδόν όλα τα θετικά για το αντιγόνο ιικά κύτταρα ήταν CD163+Sialoadhesin (Sn)+, τα οποία εντοπίζονταν κυρίως στο lamina propria του αναπνευστικού βλεννογόνου. Στον ρινικό βλεννογόνο μολυσμένο με Λένα, οι μονοκυτταρικοί υποτύποι CD163+Sn+, CD163+Sn- και σε μικρότερο βαθμό CD163-Sn- ενεπλάκησαν στη μόλυνση. Τα κύτταρα CD163+Sn- βρίσκονταν κυρίως εντός ή κοντά στο επιθήλιο. Συνοπτικά, προτείναμε ένα μοντέλο για τους διαφορετικούς μηχανισμούς εισβολής που παρατηρήθηκαν μεταξύ αυτών των δύο στελεχών PRRSV στον ρινικό βλεννογόνο. Από τη μία πλευρά, η ευαισθησία του LV περιοριζόταν κυρίως στα κύτταρα CD163+Sn+, τα οποία βρίσκονταν στο lamina propria. Ο μικρός αριθμός κυττάρων Sn+ (5,2/mm2) που υπάρχουν εντός της επιθηλιακής κυτταρικής στιβάδας πιθανότατα δεν επιτρέπει στο LV να φτάσει σε υψηλά επίπεδα μόλυνσης. Μόνο 2,5 μολυσμένα με LV κύτταρα/mm2 παρατηρήθηκαν στους 72 hpi, όλα εντοπίζονται στο lamina propria και το 97% από αυτά είχαν φαινότυπο CD163+Sn+. Από την άλλη πλευρά, η Lena βρέθηκε να αναπαράγει 10 έως 100 φορές πιο αποτελεσματικά από το LV και φαίνεται να πειράζει τα κύτταρα CD163+Sn+ και CD163+Sn- που βρίσκονται μέσα ή κοντά στο επιθήλιο για να αποκτήσει πρόσβαση σε βαθύτερα μέρη του βλεννογόνου. Μετά από 48 hpi, συστάδες κυττάρων κυρίως μολυσμένων με CD163+Sn-Lena παρατηρήθηκαν εντός ή κοντά στο επιθήλιο και συστάδες κυττάρων CD163+Sn+ στο lamina propria. Αυτά τα συμπλέγματα επεκτάθηκαν στους 72 hpi και σε μικρότερο βαθμό από τους άλλους φαινοτύπους, ανιχνεύθηκαν κύτταρα CD163-Sn- στην περιφέρειά τους. Στο κεφάλαιο 4.2, το σύστημα μοσχευμάτων χρησιμοποιήθηκε για την επέκταση της έρευνάς μας και τη μελέτη των χαρακτηριστικών αντιγραφής στον ρινικό βλεννογόνο τεσσάρων τύπου 1 (LV, 07V063, 08VA, 13V091), τριών τύπου 2 (VR2332, MN-184, VN) και δύο εξασθενημένα (MLV-DV, MLV-VR2332) στελέχη PRRSV. Μετά από 72 hpi, οι μέσοι τίτλοι του ιού έφτασαν τα 104,5 έως 4,8 TCID50/ml για LV και 08V204, 105,2 έως 5,4 TCID50/ml για Lena και VR2332, και 105,8 έως 6,3 TCID50/ml για 078, 07V3, TCID50/ml για 070, 07N3, 07, 10, 10, 3, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 40 μετριασμένη στελέχη παρέμειναν γύρω από το όριο ανίχνευσης (0,9 TCID50/ml). Ο μέσος αριθμός θετικών σε PRRSV κυττάρων/mm2 στους 72 hpi ήταν 1,1 και 1,3 για τα εξασθενημένα στελέχη και LV, 13,3 για 08VA, 23,5 και 29,3 για VR2332 και 07V063, 31,1 και 33,29 και 33,10V και 33,1,9 και 33,1,9 και 33,1,9 και 31,5 και 1,5 και 1,5 για MN-184 και VN αντίστοιχα. Όλα τα μολυσμένα με LV και MLV-DV κύτταρα ήταν Sn+, ενώ όλα τα άλλα στελέχη μόλυναν επίσης Sn-μακροφάγους. Συμπερασματικά, (i) με βάση την αποβολή του ιού στα αναπνευστικά εκφυτεύματα, τα στελέχη PRRSV μπορούν να κατηγοριοποιηθούν ως φτωχά (MLV-DV, MLV-VR2332, LV, 08VA), μέτρια (Lena, VR2332) και ισχυρά (07V063, 13V091, MN -184, VN) εκκρίτες και (ii) με βάση τον αριθμό των μολυσμένων κυττάρων απομόνωσης μπορούν να κατηγοριοποιηθούν ως χαμηλά (MLV-DV, MLV-VR2332, LV), μέτρια (08VA, VR2332), υψηλά (07V063, Lena, 13V091 ) και υπερ (MN-184, VN) λοιμογόνος στον ρινικό βλεννογόνο. Τέλος, μια γενική συζήτηση για τα κύρια ευρήματα της παρούσας διατριβής και τις μελλοντικές προοπτικές παρουσιάζονται στο κεφάλαιο 5

Changing receptor use of PRRSV leads to different virological, immunological and clinical outcome: impact on diagnosis and control

A new disease characterized by reproductive and respiratory problems emerged in Northern America and Western Europe in the late eighties, early nineties. It was caused by a porcine arterivirus, which based on the symptoms was called porcine reproductive and respiratory syndrome virus (PRRSV)(Meulenberg et al., 1993). On the two continents, two clearly different genetic/antigenic viruses were circulating: an American type (amPRRSV) and a European type (euPRRSV). Based on serological examinations, it was shown that amPRRSV circulated already earlier in Northern America and by genetic analysis, more genetic variation was detected for amPRRSV in Northern America than for euPRRSV in Western Europe. This latter finding could be attributed to the earlier circulation and/or multiple introductions of the virus in the American pig population. Western Europe was confronted with a single introduction, starting in Western Germany. As a consequence, the early euPRRSV strains were genetically closely related (Stadejek et al., 2002). In the early nineties, amPRRSV was proven to be more pathogenic than euPRRSV. Indeed, whereas both virus types had the same power to give reproductive problems upon infection during late gestation, amPRRSV was giving more general clinical signs (fever, anorexia) and respiratory problems than euPRRSV. Only in combinations with other pathogens/toxins euPRRSV was able to induce overt general and respiratory clinical signs (Van Reeth et al., 1996). By recombination and genetic drift, American strains evolved fast, giving rise to new strains that were even more virulent and extremely difficult to control by commercial vaccines (atypical PRRSV, Sow Abortion and Mortality Syndrome (SAMS))(Mengeling et al., 1998). In 2006, extremely aggressive variants of amPRRSV appeared in China, which are now damaging the whole Asian pig population (long lasting high fever, respiratory and reproductive problems, high mortality) and which represent a real threat to other continents (Tian et al., 2007). In Eastern Europe, a surprisingly large variation was found for euPRRSV isolates leading to the identification of new subtypes (2, 3 and 4) that were quite different from subtype 1 (Stadejek et al., 2006). At present, it is hypothesized that euPRRSV was circulating in Eastern Europe a long time before the entrance of subtype 1 in Western Europe. When the whole group of Western and Eastern European euPRRSV strains are considered, a genetic variation of euPRRSV was found that was even larger than the one found with amPRRSV in the US, leading to a very complex genetic world picture of PRRSV. Eastern European PRRSV strains of subtype 2 (prototype Bor) and 3 (prototype Lena) have been shown to be more virulent than the Western European strains (subtype 1 (prototype Lelystad))(Karniychuk et al., 2009). The euPRRSV strain Lena is even as virulent and pathogenic as the high fever disease amPRRSV in Asia. Over the years in Western Europe, euPRRSV remained mainly linked with reproductive problems. Fever and respiratory problems were absent upon experimental single inoculations. However, starting from mid 2013, PRRSV is responsible for flu-like problems in nurseries in Belgium and most probably also neighboring countries (unpublished data). Upon experimental inoculation with one of these isolates, Flanders 13, fever and respiratory problems were reproduced. Genetically, this virus is quite different from other circulating PRRS viruses. The pathogenesis of PRRSV is fully determined by differentiated macrophages. During the past 20 years, the euPRRSV strains of subtype 1 (prototype Lelystad) replicated very similarly in a pig (Duan et al., 1993). Targets are differentiated macrophages that are carrying the sialoadhesin receptor (Duan et al., Vanderheijden et al., 2010, Karniychuk et al., 2013). These cells can easily be found in tonsils and lungs, lymph nodes, spleen, maternal endometrium and fetal placenta and at lower levels in all other tissues of the pig (Karniychuk et al. 2009). Because the virus does not replicate well in the upper respiratory tract, the virus is difficult to isolate from nasal swabs and the virus does not spread fast between pigs (Albina, 1997). Due to the rather restricted number of differentiated cells that are infected, virus titers of 102-104 TCID50/ml are generally found in serum. EuPRRSV strains of subtype 3 (prototype Lena) differ from LV-like strains because they are able to infect a new subset of differentiated macrophages that do not possess the sialoadhesin receptor (Frydas et al. 2013). An additional receptor is most probably responsible for this. By experiments in nasal mucosa explants it was found that the additional subset is present at high concentrations in and under the respiratory epithelial cells of the upper respiratory tract, allowing a much stronger replication in respiratory tissues (up to 10-100x higher) and giving rise to a strong viral shedding and a fulminant viremia (100x higher; virus titers up to 104-106 TCID50/ml). Based on the localisation of this new subset of susceptible macrophages, it is hypothesized that they represent nasal macrophages. These cells are forming a dense network and are taking care of the first line of defense against pathogens (Vareille et al., 2011). Destroying both nasal and alveolar lung macrophages is most probably the reason why euPRRSV Lena has been associated with secondary bacterial infections and sepsis (Karniychuk et al., 2010). The new Flanders 13-like strains are euPRRSV subtype 1 strains that are also evolving in the same direction as Lena. By using the nasal mucosa explants, this virus also replicated in non-sialoadhesin positive macrophages. The virus titers in nasal secretions from euPRRSV infected animals are in line with the replication of the virus in nasal mucosa explants. Whereas it is difficult to detect LV-like euPRRSV subtype 1 in nasal secretions, it is very easy to do so with the more virulent euPRRSV subtype 3 and Flanders 13-like strains. Transmission experiments with these different strains are ongoing in order to find out if the power of the virus to replicate in the nasal macrophages may be related to its aerogenic spread. The increase of macrophage subsets that are infected with euPRRSV in time is a dangerous evolution. A ten- to hundredfold increase of replication gives rise to viral mutants with the same magnitude. Knowing that mutagenesis is helping the virus to escape from immunity and is increasing the risk that highly virulent strains emerge is bringing Europe in a very dangerous situation. In addition, because of the close relationship of the porcine receptors sialoadhesin and CD163 with their human homologs, one should consider the risk of a species jump to humans (Van Breedam et al., 2013). With these interesting findings with euPRRSV strains, we have recently performed experiments with amPRRSV strains in nasal mucosa explants. It was found that both old (VR2332) and more recent amPRRSV strains (SDSU-73, NADC, MN-184) from the US easily replicate to high levels in both sialoadhesin positive and sialoadhesin negative macrophages in the nasal mucosa (unpublished data). This finding is explaining several things. AmPRRSV behaved differently from euPRRSV from the very beginning; it replicated in more subsets of macrophages than the old subtype 1 euPRRSV LV-like strains. Its high replication in the upper respiratory tract differs from the low level of replication of the old subtype 1 euPRRSV strains. This also explains why it was and still is very easy for amPRRSV to spread via airborne transmission (Otake et al., 2010). In addition, the higher number of macrophage subsets that are infected with amPRRSV explains why it is more virulent/pathogenic than the old subtype 1 euPRRSV strains and give more rise to secondary infections. It also explains why amPRRSV is able to replicate in sialoadhesin-negative pigs (Prather et al., 2013). All these findings are very important in function of the diagnosis. An etiological diagnosis of PRRS during reproductive failure is straightforward for all PRRSV strains. PRRSV is replicating in the macrophages of the fetal placenta when they become sialoadhesin positive at late gestation (Karniychuk et al., 2009). This results in a severe placentitis and viral spread to the fetus. The placentitis is the main cause of fetal pathology and death (Karniychuk et al., 2013). Due to the huge size of the placenta and the regularly localized PRRSV replication, it is difficult to make the diagnosis from placental tissues (which part to take?). Because fetuses do not have the time to develop antibodies before they die, it is impossible to diagnose PRRS by serological examinations on fetal fluids. Taking all these pathogenetic aspects into account, it is advised to do the diagnosis of PRRS during reproductive problems by qRT-PCR on umbilical cords (connected with fetal placenta) and organ pools (lungs/spleen) of aborted fetuses. Diagnosis of euPRRS in the context of respiratory problems is very difficult with LV-like euPRRSV strains. They are always causing long-lasting infections in young piglets, independent of their health status (diseased or healthy). Therefore, what is the meaning of a positive result (demonstrating the presence of the virus in lungs or blood). In the same context, it is difficult to interpret a seroconversion. It is not because an animal is seroconverting to LV-like euPRRSV strains that the virus is responsible for problems. This diagnostic problem is one of the main reasons why most farmers did not vaccinate their piglets up till now in Western Europe. However, in pigs with flu-like problems caused by amPRRSV, subtype 2 and 3 euPRRSV and the new Flanders 13-like euPRRSV, it is easy to demonstrate the high replication of PRRSV in the upper respiratory tract by taking nasal swabs and blood and quantitating the high viral load (up to 105 TCID50/ml in nasal secretions and 104-6 TCID50/ml in blood) by virus titration or qRT-PCR. PRRSV is a difficult target for the immunity. Several branches of the immunity have been shown not to be induced or not to be functional. Low levels of interferons are induced (Van Reeth et al., 1999). Antibodies are raised starting from 8 days post infection, but it takes several weeks before a weak neutralization can be demonstrated (Labarque et al., 2000). Natural killer cells and cytotoxic T-lymphocytes are not sufficiently effective (Cao et al., 2013; Costers et al., 2009). Only neutralizing antibodies, which appear after one month and at low levels together with a not yet identified porcine killer cell are the two branches that still can do the job and should be activated by vaccination. The drift of the virus makes it a moving target and complicates the whole vaccination strategy (Labarque et al, 2004). In the near future, it is important to have access to vaccines that are adaptable, enclosing strains that are closely related to the strains circulating in the field (Nauwynck et al., 2012). The technologies for making effective adaptable inactivated (Geldhof et al., 2012) and attenuated vaccines (unpublished data) became available and should be urgently implemented in the field. The ultimate dream is the development of an adaptable marker vector vaccine (cassette system) that induces a local immunity in the respiratory tract. As long as PRRSV does not persist such as its sister-arterivirus, lactate dehydrogenase elevating virus (LDV), we should keep on going with the investment to develop vaccines. In conclusion, it is extremely important to better control PRRS in the near future and not to wait till a complete catastrophe occurs. There is an urgent need for adaptable inactivated and attenuated marker vaccines and improved biosafety measures in order to fully control PRRSV circulation. Pig producers, PRRS researchers and pharmaceutical companies should take their responsibility and join forces to come up with solutions to eradicate this ever-changing enemy that may turn into a real nightmare. In this context, funding PRRS research should be prioritized by all agencies all over the world. Not doing this is an unforgivable error not only for animal health but possibly also for human health