Gene expression of peripheral blood mononuclear cells and CD8+ T cells from gilts after PRRSV infection

Porcine reproductive and respiratory syndrome virus (PRRSV) is a positive-stranded RNA virus, which emerged in Europe and U.S.A. in the late 1980s and has since caused huge economic losses. Infection with PRRSV causes mild to severe respiratory and reproductive clinical symptoms in pigs. Alteration of the host immune response by PRRSV is associated with the increased susceptibility to secondary viral and bacterial infections resulting in more serious and chronic disease. However, the expression profiles underlying innate and adaptive immune responses to PRRSV infection are yet to be further elucidated. In this study, we investigated gene expression profiles of PBMCs and CD8+ T cells after PRRSV AUT15-33 infection. We identified the highest number of differentially expressed genes in PBMCs and CD8+ T cells at 7 dpi and 21 dpi, respectively. The gene expression profile of PBMCs from infected animals was dominated by a strong innate immune response at 7 dpi which persisted through 14 dpi and 21 dpi and was accompanied by involvement of adaptive immunity. The gene expression pattern of CD8+ T cells showed a strong adaptive immune response to PRRSV, leading to the formation of highly differentiated CD8+ T cells starting from 14 dpi. The hallmark of the CD8+ T-cell response was the increased expression of effector and cytolytic genes (PRF1, GZMA, GZMB, GZMK, KLRK1, KLRD1, FASL, NKG7), with the highest levels observed at 21 dpi. Temporal clustering analysis of DEGs of PBMCs and CD8+ T cells from PRRSV-infected animals revealed three and four clusters, respectively, suggesting tight transcriptional regulation of both the innate and the adaptive immune response to PRRSV. The main cluster of PBMCs was related to the innate immune response to PRRSV, while the main clusters of CD8+ T cells represented the initial transformation and differentiation of these cells in response to the PRRSV infection. Together, we provided extensive transcriptomics data explaining gene signatures of the immune response of PBMCs and CD8+ T cells after PRRSV infection. Additionally, our study provides potential biomarker targets useful for vaccine and therapeutics development

Pathologische, immunologische und virologische Untersuchungen nach experimenteller Infektion vier Wochen alter Ferkel mit einem europäischen Porzinen Reproduktiven und Respiratorischen Syndrom Virus - Isolat mit und ohne Applikation einer attenuierten Lebendvakzine am ersten Lebenstag

Dissertation - Veterinärmedizinische Universität Wien - 2021 Aus rechtlichen Gründen sind nicht alle Teile dieser Arbeit frei zugänglich. Der Zugriff auf den elektronischen Volltext ist auf Angehörige der Veterinärmedizinischen Universität Wien beschränkt. Bitte einloggen!PRRSV ist aus wirtschaftlichen Gesichtspunkten einer der verheerendsten viralen Krankheitserreger in der Schweineproduktion. Die frühe Zirkulation des Virus in Ferkeln nach dem Absetzen hat zur Entwicklung von MLV-Impfstoffen geführt, welche bereits für die Verwendung ab dem ersten Lebenstag zugelassen sind. Im Rahmen dieser Studie sollte das protektive Ausmaß dieses frühen Impfzeitpunktes nach vierwöchiger Säugezeit mittels eines Infektionsmodelles evaluiert werden. Zu diesem Zweck wurden PRRSV-seronegative Ferkel innerhalb der ersten 24 Lebensstunden geimpft (n = 21; Suvaxyn® PRRS MLV) oder verblieben als Kontrollgruppe (n = 20). Alle Ferkel wurden zum Absetzzeitpunkt mit einem heterologen PRRSV-1-Isolat (PRRSV-1 Subtyp 1 „AUT15-33“) infiziert. Neben der täglichen klinischen Untersuchung wurden in regelmäßigen Abständen Blut- und Tupferproben entnommen sowie die Tiere gewogen. Zwei Wochen später wurden die Ferkel unter Einhaltung tierschutzrechtlicher Bestimmungen euthanasiert, anatomisch-pathologisch untersucht und Gewebeproben entnommen. Diese Studie bestätigt die hohe Virulenz des PRRSV-1 Feldisolats „AUT15-33“. Bei allen nicht-geimpften Ferkeln konnte eine hohe Viruslast im Serum und im Nasensekret nachgewiesen werden. Außerdem traten bei diesen Tieren häufiger ein vermindertes Allgemeinbefinden und Atemwegssymptome auf, gleichzeitig war die durchschnittliche tägliche Gewichtszunahme im Vergleich zu den geimpften Ferkeln deutlich geringer. Bei den nicht-geimpften Ferkeln konnten zudem deutlich ausgeprägte PRRSV-assoziierte histologische Läsionen festgestellt werden, während bei den geimpften Ferkeln alle fünf untersuchten histologischen Lungenläsionen kaum ausgeprägt waren. Anhand dieser Ergebnisse kann geschlussfolgert werden, dass unter gegebenen Versuchsbedingungen die Ferkel bereits am ersten Lebenstag impffähig waren. Es konnte eine wirksame Immunität aufgebaut werden, welche die Ferkel vor einer frühen PRRSV-Infektion beim Absetzen schützte und die Viruslast im Serum und die nasale Ausscheidung des Virus deutlich reduzierte. Diese Studie zeigt außerdem, dass die Viruslast im Serum negativ mit der Anzahl der PRRSV-spezifischen IFN-γ-sezernierenden Zellen in den PBMCs korreliert und lässt annehmen, dass die T-Zell-gesteuerte Immunantwort in neugeborenen Ferkeln bereits sehr effektiv ist.Dissertation - Veterinärmedizinische Universität Wien - 2021 Aus rechtlichen Gründen sind nicht alle Teile dieser Arbeit frei zugänglich. Der Zugriff auf den elektronischen Volltext ist auf Angehörige der Veterinärmedizinischen Universität Wien beschränkt. Bitte einloggen!PRRSV ist aus wirtschaftlichen Gesichtspunkten einer der verheerendsten viralen Krankheitserreger in der Schweineproduktion. Die frühe Zirkulation des Virus in Ferkeln nach dem Absetzen hat zur Entwicklung von MLV-Impfstoffen geführt, welche bereits für die Verwendung ab dem ersten Lebenstag zugelassen sind. Im Rahmen dieser Studie sollte das protektive Ausmaß dieses frühen Impfzeitpunktes nach vierwöchiger Säugezeit mittels eines Infektionsmodelles evaluiert werden. Zu diesem Zweck wurden PRRSV-seronegative Ferkel innerhalb der ersten 24 Lebensstunden geimpft (n = 21; Suvaxyn® PRRS MLV) oder verblieben als Kontrollgruppe (n = 20). Alle Ferkel wurden zum Absetzzeitpunkt mit einem heterologen PRRSV-1-Isolat (PRRSV-1 Subtyp 1 „AUT15-33“) infiziert. Neben der täglichen klinischen Untersuchung wurden in regelmäßigen Abständen Blut- und Tupferproben entnommen sowie die Tiere gewogen. Zwei Wochen später wurden die Ferkel unter Einhaltung tierschutzrechtlicher Bestimmungen euthanasiert, anatomisch-pathologisch untersucht und Gewebeproben entnommen. Diese Studie bestätigt die hohe Virulenz des PRRSV-1 Feldisolats „AUT15-33“. Bei allen nicht-geimpften Ferkeln konnte eine hohe Viruslast im Serum und im Nasensekret nachgewiesen werden. Außerdem traten bei diesen Tieren häufiger ein vermindertes Allgemeinbefinden und Atemwegssymptome auf, gleichzeitig war die durchschnittliche tägliche Gewichtszunahme im Vergleich zu den geimpften Ferkeln deutlich geringer. Bei den nicht-geimpften Ferkeln konnten zudem deutlich ausgeprägte PRRSV-assoziierte histologische Läsionen festgestellt werden, während bei den geimpften Ferkeln alle fünf untersuchten histologischen Lungenläsionen kaum ausgeprägt waren. Anhand dieser Ergebnisse kann geschlussfolgert werden, dass unter gegebenen Versuchsbedingungen die Ferkel bereits am ersten Lebenstag impffähig waren. Es konnte eine wirksame Immunität aufgebaut werden, welche die Ferkel vor einer frühen PRRSV-Infektion beim Absetzen schützte und die Viruslast im Serum und die nasale Ausscheidung des Virus deutlich reduzierte. Diese Studie zeigt außerdem, dass die Viruslast im Serum negativ mit der Anzahl der PRRSV-spezifischen IFN-γ-sezernierenden Zellen in den PBMCs korreliert und lässt annehmen, dass die T-Zell-gesteuerte Immunantwort in neugeborenen Ferkeln bereits sehr effektiv ist.Dissertation - University of Veterinary Medicine Vienna - 2021 The full text is only available to university members. Please log in!PRRSV is one of the most devastating viral pathogens in pig production worldwide. Potential early circulation of PRRSV in piglets after weaning has led to the development of MLV vaccines with an approval for usage from the first day of life onwards. The main objective of the present study was the evaluation of the level of protective immunity built up after PRRSV-1 vaccination during a four-week suckling period against heterologous challenge. For this purpose, PRRSV-seronegative piglets were vaccinated within the first 24 hours of life (n = 21; Suvaxyn® PRRS MLV) or sham-treated (n = 20) and exposed to a heterologous PRRSV-1 isolate (PRRSV-1 field strain AUT15-33) at weaning. In addition to daily clinical examination, blood and swab samples were taken at regular intervals and body weight was recorded in the course of the study. Necropsy was performed two weeks post-challenge. Hence, this study confirmed the phenotypic virulence of the PRRSV-1 field isolate AUT15-33. All non-vaccinated piglets were viremic and shed virus in nasal swabs. In addition, the incidence of depression and respiratory distress was higher in these piglets, while the average daily weight gain was decreased compared to the vaccinated ones. Severe histologic lesions could be found in non-vaccinated piglets, whereas in vaccinated piglets all five investigated histologic lung lesions were less pronounced. Under the conditions of this trial, piglets were already vaccinable on the first day of life. An effective immunity could be established, which protected the piglets against early PRRSV infection at weaning and significantly reduced the amount of viral load in serum and nasal shedding of the virus. This study clearly showed that the viral load in serum is negatively correlated with the number of PRRSV-specific IFN-γ secreting cells in PBMC. This suggests that the T-cell driven immune response in newborn piglets is already highly effective

Litters of Various-Sized Mummies (LVSM) and Stillborns after Porcine Reproductive and Respiratory Syndrome Virus Type 1 Infection—A Case Report

Diverse origins and causes are described for papyraceous mummifications of porcine foetuses, but the porcine reproductive and respiratory syndrome virus (PRRSV) is not one of them. In contrast, PRRSV is unlikely to cause mid-term placental transmission but may cause late-term abortions and weakness of piglets. This case report describes a sudden occurrence of mummified foetuses of various sizes and stillborns and delayed birth (>115 days) in more than 50% of sows from one farrowing batch, while newborn piglets were mostly vital. Neither increased embryonic death nor infertility was reported. Three litters with mummies, autolysed piglets and stillborn piglets were investigated, and infections with porcine parvoviruses, porcine teschoviruses, porcine circoviruses, encephalomyocarditis virus, Leptospira spp. and Chlamydia spp. were excluded. Instead, high viral loads of PRRSV were detected in the thymus pools of piglets at all developmental stages, even in piglets with a crown–rump length between 80 and 150 mm, suggesting a potential mid-term in utero transmission of the virus. Genomic regions encoding structural proteins (ORF2–7) of the virus were sequenced and identified the virulent PRRSV-1 strain AUT15-33 as the closest relative. This case report confirms the diversity of PRRSV and its potential involvement in foetal death in mid-gestation

A Conserved Stem-Loop Structure within ORF5 Is a Frequent Recombination Hotspot for Porcine Reproductive and Respiratory Syndrome Virus 1 (PRRSV-1) with a Particular Modified Live Virus (MLV) Strain

The emergence of recombinant PRRSV strains has been observed for more than a decade. These recombinant viruses are characterized by a genome that contains genetic material from at least two different parental strains. Due to the advanced sequencing techniques and a growing number of data bank entries, the role of PRRSV recombinants has become increasingly important since they are sometimes associated with clinical outbreaks. Chimeric viruses observed more recently are products of PRRSV wild-type and vaccine strains. Here, we report on three PRRSV-1 isolates from geographically distant farms with differing clinical manifestations. A sequencing and recombination analysis revealed that these strains are crossovers between different wild-type strains and the same modified live virus vaccine strain. Interestingly, the recombination breakpoint of all analyzed isolates appears at the beginning of open reading frame 5 (ORF5). RNA structure predictions indicate a conserved stem loop in close proximity to the recombination hotspot, which is a plausible cause of a polymerase template switch during RNA replication. Further research into the mechanisms of the stem loop is needed to help understand the PRRSV recombination process and the role of MLVs as parental strains

Short-Chain Fatty Acids Modulate Permeability, Motility and Gene Expression in the Porcine Fetal Jejunum Ex Vivo

Postnatally, short-chain fatty acids (SCFA) are important energetic and signaling agents, being involved in host nutrition, gut imprinting and immune and barrier function. Whether SCFA exert similar effects during the late fetal phase has been insufficiently elucidated. This study aimed to evaluate whether the fetal jejunum senses SCFA and whether SCFA modify the muscle tension and epithelial permeability and related signaling in jejunal tissue from the porcine fetus in late gestation. Exposure of fetal jejunal tissue to a mix of SCFA (70 µmol/mL) in an organ bath for 20 min lowered the muscle tension. Moreover, SCFA decreased the transepithelial conductance while increasing the short-circuit current in the Ussing chamber, indicating reduced permeability and increased SCFA absorption. Gene expression in the tissues harvested from the Ussing chamber after 30 min indicated downregulation of the expression of receptors (i.e., FFAR2 and TLR2), MCT1 and tight-junction and adherens proteins, which may be a negative feedback response to the applied high SCFA concentration compared with the micromolar concentration detected in fetal gastric fluid. Taken together, our data demonstrate that the fetal jejunum senses SCFA, which trigger electrophysiological, muscle contraction and related gene transcription responses. Hence, SCFA may play a role in prenatal gut nutrition and imprinting

Efficacy of a Modified Live Virus Vaccine against Porcine Reproductive and Respiratory Syndrome Virus 1 (PRRSV-1) Administered to 1-Day-Old Piglets in Front of Heterologous PRRSV-1 Challenge

PRRSV is one of the most important viruses in the global swine industry and is often controlled by the use of modified live virus (MLV) vaccines. This study assessed the impact of a PRRSV-1 MLV vaccine applied to 1-day-old piglets challenged on day 28 of life with a PRRSV-1 field isolate (AUT15-33). Twenty-one piglets were vaccinated within 24 h of birth (T02), whereas 20 piglets were left unvaccinated (T01). Necropsy was performed two weeks post-challenge. Comparing the two groups, T02 piglets showed significantly higher (p = 0.017) average daily weight gain. In addition, significantly lower (p < 0.0001) PRRSV RNA loads were measured in serum of T02 piglets at all investigated time points. All T01 piglets were viremic and shed virus in nasal swabs, whereas only 71.4% and 38.1% of the T02 group were viremic or shed virus, respectively. Piglets from T02 had significantly higher numbers (p < 0.0001) of IFN-γ producing lymphocytes compared to T01. At necropsy, differences in gross and histologic lung lesions were statistically significant (p = 0.012 and p < 0.0001, respectively) between the two groups. Hence, this MLV vaccine administered to 1-day-old piglets was able to protect piglets against PRRSV infection at weaning

T-Cell Cytokine Response in Salmonella Typhimurium-Vaccinated versus Infected Pigs

Vaccination with the live attenuated vaccine Salmoporc is an effective measure to control Salmonella Typhimurium (STM) in affected swine populations. However, the cellular immune response evoked by the Salmoporc vaccine including differences in vaccinated pigs versus non-vaccinated pigs upon STM infection have not been characterized yet. To investigate this, tissue-derived porcine lymphocytes from different treatment groups (vaccination-only, vaccination and infection, infection-only, untreated controls) were stimulated in vitro with heat-inactivated STM and abundances of IFN-γ, TNF-α and/or IL-17A-producing T-cell subsets were compared across organs and treatment groups. Overall, our results show the induction of a strong CD4+ T-cell response after STM infection, both locally and systemically. Low-level induction of STM-specific cytokine-producing CD4+ T cells, notably for the IFN-γ/TNF-α co-producing phenotype, was detected after vaccination-only. Numerous significant contrasts in cytokine-producing T-cell phenotypes were observed after infection in vaccinated and infected versus infected-only animals. These results suggest that vaccine-induced STM-specific cytokine-producing CD4+ T cells contribute to local immunity in the gut and may limit the spread of STM to lymph nodes and systemic organs. Hence, our study provides insights into the underlying immune mechanisms that account for the efficacy of the Salmoporc vaccine

DataSheet_1_Characterization of the immune system of Ellegaard Göttingen Minipigs - An important large animal model in experimental medicine.docx

Interest in Ellegaard Göttingen Minipigs (EGMs) as a model in experimental medicine is continuously growing. The aim of this project is to increase the knowledge of the immune system of EGMs as information is still scarce. Therefore, we studied the postnatal maturation of their immune system from birth until 126 weeks of age. For the first 26 weeks of the study, animals were kept under pathogen-reduced conditions (SPF) and afterwards under conventional housing conditions. The development of the immune system was analyzed by monitoring changes in total numbers of leukocytes and lymphocytes of ten individuals and the composition of leukocyte populations by multi-color flow cytometry (FCM). We followed the presence of monocytes using monoclonal antibodies (mAbs) against CD172a+ and CD163+ and B cells based on the expression of CD79a. NK cells were distinguished as CD3-CD16+CD8α+/dim cells and further subdivided using NKp46 (CD335) expression into NKp46-, NKp46+, and NKp46high NK cells. T-cell receptor (TCR) γδ T cells were defined by the expression of TCR-γδ and different subsets were determined by their CD2 and perforin expression. TCR-αβ T cells were classified by their CD8β+ or CD4 expression. For monitoring their differentiation, expression of CD27 and perforin was investigated for CD8β++ T cells and CD8α together with CD27 for CD4+ T cells. We clearly detected a postnatal development of immune cell composition and identified phenotypes indicative of differentiation within the respective leukocyte subsets. Examination of the development of the antigen-specific immune system after transfer to different distinct housing conditions and after vaccination against common porcine pathogens such as porcine circovirus 2 (PCV2) revealed a markedly increased presence of more differentiated CD8+ and CD4+ T cells with central and effector memory T-cell phenotypes. To complement the findings, a PCV2 vaccine-specific antigen was used for in vitro restimulation experiments. We demonstrated antigen-specific proliferation of CD4+CD8α+CD27+ central and CD4+CD8α+CD27- effector memory T cells as well as antigen-specific production of TNF-α and IFN-γ. This study of postnatal immune development defines basic cellular immune parameters of EGMs and represents an important milestone for the use of EGMs for immunological questions in experimental medicine.</p

DataSheet_1_Influence of PRRSV-1 vaccination and infection on mononuclear immune cells at the maternal-fetal interface.docx

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most devastating viruses for the global swine industry. Infection during late gestation causes reproductive failure but the local immune response in utero remains poorly understood. In this study, an experimental PRRSV-infection model with two different PRRSV-1 field isolates was used to investigate the immune cell phenotypes at the maternal-fetal interface during late gestation. In addition, phenotypic changes induced by a modified live virus (MLV, ReproCyc® PRRS EU) vaccine were studied. Vaccinated (n = 12) and non-vaccinated pregnant gilts (n = 12) were challenged with either one of the PRRSV-1 field isolates (low vs. high virulent, LV or HV) or sham-inoculated at day 84 of gestation. Twenty-one days post infection all gilts were euthanized and the fetal preservation status for all fetuses per litter was assessed. Leukocytes from the maternal-fetal interface were isolated and PRRSV-induced changes were investigated using ex vivo phenotyping by flow cytometry. PRRSV load in tissue from the maternal endometrium (ME) and fetal placenta (FP) was determined by RT-qPCR. In the ME, a vast increase in CD8β T cells with CD8αposCD27dim early effector phenotype was found for fetuses from the non-vaccinated LV and HV-challenged gilts, compared to non-treated and vaccinated-only controls. HV-challenged fetuses also showed significant increases of lymphocytes with effector phenotypes in the FP, including NKp46pos NK cells, CD8αhigh γδ T cells, as well as CD8αposCD27pos/dim CD4 and CD8 T cells. In vaccinated animals, this common activation of effector phenotypes was more confined and the fetal preservation status significantly improved. Furthermore, a negative correlation between the viral load and CD163highCD169pos mononuclear phagocytic cells was observed in the FP of HV-infected animals. These results suggest that the strong expansion of effector lymphocytes in gilts that were only infected causes immune-pathogenesis rather than protection. In contrast, the attenuated MLV seems to dampen this effect, yet presumably induces memory cells that limit reproductive failure. This work provides valuable insights into changes of local immune cell phenotypes following PRRSV vaccination and infection.</p