XXXI In vitro and in vivo infection of porcine monocyte-macrophages-lineage cells with Porcine Reproductive and Respiratory Syndrome Virus ( PRRSV ) isolates with different pathogenicity

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) represents one of the most important viruses affecting the swine population with a huge impact on pig production in many countries. Moreover, the recent emergence of some highly pathogenic isolates (HP-PRRSV-1) has caused more severe economic losses. The effects of PRRSV on the immune system is poorly understood, indeed, the infection and persistence of the virus is related to a complex interaction with the immune system that determines a dysregulation on the mechanisms and the secretion of important cytokines of the host. The virus shows a restricted tropism for cells from the monocyte-macrophage-lineage, the main in vivo target cells is represented by porcine alveolar macrophages (PAM). The aim of the present thesis is to analysing the response of the macrophages population to the infection of different PRRSV isolates, both in vitro and in vivo. In the first in vitro study, we used as a model, monocytes derived macrophages (MDMs) pre-treated with different cytokines as IFNγ, IL-4 and IFNβ. Nine PRRSV-1 isolates were analyzed: two Italian strains, five Eastern European strains, and Lena and Lelystad as reference-strains for HP and low pathogenic PRRSV, respectively. The different strains were able to infect MDMs, with the best efficiency in unpolarized MØ and IL-4 treated MDMs, while the IFN-treatment determined an antiviral state, this evidence was most pronounced with IFNβ. In general, the high pathogenicity isolates infected a higher percentage of MDMs and replicated to higher titers. Regarding the cytokines measurement, IFNα and IL-10 were not detected in the supernatant of infected MDMs; moreover, the Italian PR40 strain was the only one that induced a significant release of TNF-α and IL1-β. The second study was performed by using as a model the polarized-porcine alveolar macrophages (PAMs) pre-treated with IFNγ, IL-4 and IFNβ, in order to compare two PRRSV-1.1 Italian strains, the normal pathogenic PR11 strain and the HP-PRRSV PR40. Interesting, we did not see difference between the two isolates, and any difference in the infectivity among Mock, IL-4 and IFNγ treatments, that evidence could found explanation in a re-polarization from the M1 to M2 phenotype after the infection. While, the infection of both viruses was completely blocked by IFNβ-treatment, highlighting the antiviral state induced by Interferon. Moreover, both strains did not induce the production of the cytokines tested: IFNα, IL-10 and TNFα. The in vivo study consisted in two different experiments, the aim of the first one was to analyse the impact of two Italian PRRSV-1 subtype 1 strains (PR11 and PR40), with different in vivo pathogenicity, on the macrophages population of the thymus, as target cell for both PRRSV replication and response of the host immune system. The second experiment aimed at evaluating the effect of a heterologous vaccination on macrophages population of the thymus after a challenge infection with a HP-PRRSV. In order to define the macrophages population, immunohistochemistry for the N protein of the virus, TUNEL labelling and immunolabeling for CD163, CD172a, CD107a and BA4D5 was performed. The thymus of the animals infected with PRRSV strains, PR11 and PR40 dead at 10-14 dpi showed more severe lesions and the higher number of macrophages in all the compartments of the thymus gland compared with the others groups. This last result suggests a strong and early inflammatory process, without any difference between the normal and highly pathogenic strain.