Quantitative proteomics by 2‐DE, ¹⁶O/¹⁸O labelling and linear ion trap mass spectrometry analysis of lymph nodes from piglets inoculated by porcine circovirus type 2

Abstract

Porcine circovirus type 2 (PCV2) has been identified as the essential causal agent of postweaning multisystemic wasting syndrome. However, little is known regarding the mechanism(s) underlying the pathogenesis of PCV2-induced disease and the interaction of the virus with the host immune system. Here, we present a proteomics study on inguinal lymph nodes of piglets inoculated with PCV2, in order to better understand the pathogenesis of postweaning multisystemic wasting syndrome and the pathways might be affected after infection. We used two proteomics strategies, 2-DE and 1-DE followed by (16)O/(18)O peptide labelling and peptide identification and quantification by MS. More than 100 proteins were found to be differentially regulated and the results obtained by the two strategies were fairly concordant but also complementary, the (18)O labelling approach being a more robust alternative. Analysis of these proteins by systems biology tools revealed the implication of acute phase response and NrF2-mediated oxidative stress, suggesting a putative role for these pathways in the pig immune response. Besides, CD81 was found to be up-regulated, suggesting a possible role in the internalization of the virus. The use of proteomics technologies together with biology analysis systems opens up the way to gain more exhaustive and systematic knowledge of virus-pathogen interactions.

Proteomic analysis of porcine alveolar macrophages infected with porcine circovirus type 2

“…Studies have analyzed the interplay between PCV2 and host cells using proteomics analysis, which include the interactions of PCV2 and PK15 cells [31] and inguinal lymph nodes of piglets inoculated with PCV2 [35]. However, interactions of target cells of PCV2 and the virus have not been studied.…”

Comparative proteomic analysis reveals different responses in porcine lymph nodes to virulent and attenuated homologous African swine fever virus strains

“…Thus, proteomic strategies have been used to study the differences observed after in vitro infection of Vero cells with virulent or attenuated strains of porcine epidemic diarrhoea virus [19], or PK-15 cells infected with classical swine fever virus [20]. Additionally, our group has previously described the protein profile of lymph nodes of piglets inoculated with Porcine circovirus type 2 (PCV2) [21]. Two proteomic studies have been published so far for ASFV infection: the first one focussed on proteins differentially expressed after ASFV-macrophage infection, although they were not specifically identified [17] and the second one, using Vero cells infected with the 608VR13 ASFV isolate, identifying proteins involved in apoptosis or in the transcription modulation [10].…”

Pig proteomics: A review of a species in the crossroad between biomedical and food sciences

“…Ramirez-Boo et al have mapped the porcine monocyte proteome using 2DE combined to MS/MS based identification, resulting in the characterization of 76 proteins of which the majority related to basic metabolic pathways and cell structure functions, and only very few proteins were of direct relevance to porcine immune response [89]. A recent mass spectrometry based study of porcine lymph nodes did however contribute a more detailed characterization of also immune related proteins [90]. Also a 2DE study of porcine alveolar macrophages, which are the primary phagocytes in lung, was presented by the same research group [91].…”

“…Some of the protein changes were confirmed by transcript analyses, and data indicate that regulation of T-helper cells differentiation actively takes part in this host response model. A different approach was presented [90] studying the host response in lymph nodes sampled from in vivo infected piglets. Also these authors used a stable isotope labeling technique to provide relative quantification of proteome changes, using 16O/ 18O labeling combined with LC-MS/MS analyses.…”